Conditional control of universal CAR T cells by stimulus-responsive adaptors

Abstract

Disclosed herein are compositions and methods related to the construction and use of conditional universal synthetic Notch (synNotch) receptors and conditional chimeric antigen receptor (CAR) T cells. Also disclosed herein are methods of using synNotch receptors in the construction of engineered cells. It is contemplated herein that the disclosed synNotch receptors and engineered cells can be used in regenerative medicine. Additionally, disclosed herein are methods of using the disclosed synNotch, CAR T cells, and engineered cells in the treatment of cancer, autoimmune diseases, autoinflammatory diseases, and infectious diseases.

Description

[Technical Field]

[0001] This invention was made with government support under RO1 GM142007, R35 CA210039 and R21 AI130815 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 110,194, filed November 5, 2020, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Despite the promise of CAR T cells in treating cancer patients, several limitations exist in the generalized clinical application of CAR T cells. First, because single tumor antigens are not universally expressed by all cancer types, it is necessary to construct an scFv in the CAR for each tumor antigen to be targeted. Second, the financial and labor-intensive work associated with identifying and engineering scFvs for various tumor antigens poses significant challenges. Third, tumor antigens targeted by CARs may downregulate or mutate in response to treatment, resulting in tumor escape. Current CAR T cells recognize only one target antigen, and such tumor alterations render the treatment ineffective. Therefore, the generation of CAR T cells that can recognize multiple tumor antigens is highly desirable. Finally, their implementation is limited by the difficulty of achieving cancer cell specificity through single antigen targeting, which leads to undesirable on-target / off-disease toxicity and toxicity from overactive cells. Therefore, existing CAR T cell systems need to be modified to address and overcome the obstacles currently preventing their development into effective in vivo treatments. Summary of the Invention

[0004] Disclosed are methods and compositions relating to a conditional universal chimeric antigen receptor (CAR) system, a conditional universal synthetic Notch (synNotch) receptor, and methods of their use.

[0005] In one aspect, disclosed herein is a conditional universal chimeric antigen receptor (CAR) system comprising: i) a conditional adapter molecule, a hinge domain (e.g., a CD8α domain, a CD28 hinge, or a modified IgG hinge with a deletion or modification of the CH2 and / or CH3 domain), and a signaling domain (e.g., a DAP10, NKG2D, NKG2C, NKp44, CD28, CD27, Meg10, CD32, CD16, 2B4, or CD3ζ signaling domain, etc.), coupled to a tag ligand (e.g., benzylguanine (BG), benzylcytosine (BC), chloroalkane, fluorescein (FITC), SpyTag, leucine zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc and ii) a conditional adapter molecule comprising an antigen recognition element (e.g., an antibody, an antigen-recognizing fragment thereof, a protein-binding domain, a lectin, a DNA aptamer, an RNA aptamer, a small molecule ligand for a cell surface receptor, or a peptide / protein ligand of a natural protein receptor), a stimuli-reactive group, and a tag ligand (e.g., benzylguanine (BG), benzylcytosine (BC), a chloroalkane, fluorescein (isothiocyanate), SpyTag, leucine zipper, La-SS-B, CD19, an anti-folate receptor antibody, an Fc domain, a peptide neoepitope (PNE), or biotin).

[0006] Also disclosed herein, in one aspect, is a conditional universal CAR system of any preceding aspect, wherein the conditional adapter molecule comprises NHS-ester conjugation, disulfide restapling approaches (including, but not limited to, bis-sulfone conjugation and other disulfide restapling approaches), glycan conjugate chemistry, recombinant antibodies with tagged ligands via one or more short peptide tags (such as, for example, incorporation of BG or BC), sortase-mediated ligation, chemical ligation, split intein, THIOMAB, and / or unnatural amino acids. In some aspects, the antigen recognition element is covalently linked to the universal adapter molecule.

[0007] In one aspect, disclosed herein is the conditional universal CAR system of any preceding aspect, wherein the antigen recognition element comprises rituximab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacizumab, or an antigen-binding fragment of any one thereof.

[0008] Also disclosed herein is the conditional universal CAR system of any preceding embodiment, wherein the stimulus to which the stimulus-responsive group responds is light (e.g., 365, 405, 544, 780 nm, etc.), an enzyme (e.g., legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase A (KHK-A), hexokinase (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase), small molecules (e.g., phosphines or tetrazines), pH, hypoxia, HO, and / or ROS.

[0009] In one aspect, disclosed herein is a conditional universal CAR system of any preceding aspect, wherein the stimulus-reactive group comprises a cleavable linker (e.g., a photocleavable or phosphine-cleavable linker, etc.) or a stimulus-reactive caging group (e.g., a photoreactive caging group comprising nitrobenzyl, coumarin, BODIPY, or cyanine), which blocks the CAR from binding to the conditional adapter molecule.

[0010] Also disclosed herein is a conditional universal CAR system of any preceding aspect, further comprising one or more costimulatory domains (e.g., CD27, CD28, ICOS, 4-1BB, or OX40, etc.).

[0011] In one aspect, disclosed herein is the conditional universal CAR system of any preceding aspect, wherein the tag ligand that targets the CAR is comprised on a CAR T cell, a CAR NK cell, a CAR NK T cell, a CAR B cell, or a CAR macrophage.

[0012] In one aspect, disclosed herein is a conditional universal synthetic Notch (synNotch) receptor system comprising a conditional adaptor molecule, the conditional adaptor molecule comprising an antigen-binding domain, a stimulatory reactive group, and a tag (e.g., benzylguanine (BG), benzylcytosine (BC), chloroalkane, FITC, SpyTag, leucine zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc domain, peptide neoepitope (PNE), biotin, etc.), a synthetic Notch receptor with a tag-reactive domain, a notch core comprising one or more cleavage sites, and one or more transcription factors.

[0013] Also disclosed herein is a conditional universal synNotch receptor system of any preceding aspect, wherein the adapter molecule comprises NHS-ester conjugation, disulfide restapling approaches (such as, but not limited to, bis-sulfone conjugation and other disulfide restapling approaches), glycan conjugate chemistry, recombinant antibodies with tag incorporation via one or more short peptide tags (such as, for example, incorporation of BG or BC), sortase-mediated ligation, chemical ligation, split intein, THIOMAB, and / or unnatural amino acids.

[0014] In one aspect, disclosed herein is the conditional universal synNotch receptor of any preceding aspect, wherein the stimulus to which the stimulus-responsive group responds is light (e.g., 365, 405, 544, 780 nm, etc.), an enzyme (e.g., legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member A1, (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase A (KHK-A), hexokinase (HK), nucleoside diphosphate phosphate (NPP), or a combination thereof. phosphate kinase (NDPK or NDK), and 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase), small molecules (e.g., phosphines or tetrazines), pH, hypoxia, HO, and / or reactive oxygen species (ROS).

[0015] Also disclosed herein is a conditional universal synNotch receptor of any preceding embodiment, wherein the stimulus-responsive group comprises a cleavable linker (such as, for example, a photocleavable or phosphine-cleavable linker) or a stimulus-responsive caging group (such as, for example, a photoreactive caging group comprising nitrobenzyl, coumarin, BODIPY, or cyanine), which blocks binding of the CAR to the conditional adapter molecule.

[0016] In one aspect, disclosed herein is the conditional universal synNotch receptor of any preceding aspect, wherein the transcription factor comprises Gal4-VP64, Gal4-VP16, TetR-VP64, or LacI-VP64.

[0017] Also disclosed herein is a conditional universal synNotch receptor of any preceding aspect, further comprising an antigen recognition element (e.g., an antibody, an antigen-recognizing fragment thereof, a protein-binding domain, a lectin, a DNA aptamer, an RNA aptamer, a small molecule ligand of a cell surface receptor, or a peptide / protein ligand of a natural protein receptor, etc.), wherein the antigen recognition element is, or can be covalently linked to, the conditional universal adaptor molecule.

[0018] In one aspect, disclosed herein is the conditional universal synNotch receptor of any preceding aspect, wherein the antigen recognition element comprises rituximab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacizumab, or an antigen-binding fragment of any one thereof.

[0019] Also disclosed herein are engineered cells (e.g., immune cells (such as, but not limited to, T cells, NK cells, NK T cells, B cells, and macrophages), neurons, epithelial cells, and endothelial cells, or stem cells) comprising the conditional universal CAR system of any preceding embodiment and / or the conditional universal synNotch of any preceding embodiment.

[0020] In one aspect, disclosed herein is the engineered cell of any preceding aspect, further comprising a vector comprising a transcription response element operably linked to a promoter driving expression of one or more cellular response genes (e.g., IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin, etc.), wherein one or more of the transcription factors on the synNotch receptor are specific for the transcription response element.

[0021] Also disclosed herein is the engineered cell of any preceding embodiment, wherein one or more transcription factors of the conditional universal synNotch receptor activate expression of one or more natural cellular response genes (e.g., IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin, etc.).

[0022]

[0013] In one aspect, disclosed herein is a method of treating, reducing, reducing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis in a subject, the method comprising administering to the subject the conditional universal chimeric antigen receptor (CAR) system of any preceding aspect and / or the engineered cells of any preceding aspect.

[0023] Also disclosed herein are methods of treating, reducing, reducing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis of any of the preceding aspects, the method comprising administering to a subject a first CAR system of any preceding aspect and a second conditional universal CAR system of any preceding aspect; wherein the first conditional universal chimeric antigen receptor CAR system comprises a stimulus-responsive group that includes a stimulus-cleavable linker, and the second CAR system comprises a stimulus-responsive caging group that blocks binding of the CAR to the conditional adapter molecule. In one aspect, the first and second conditional universal CAR systems are responsive to the same stimulus. In another aspect, the first and second conditional universal CAR systems are responsive to different stimuli.

[0024] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments and, together with the description, illustrate the disclosed compositions and methods. [Brief explanation of the drawings]

[0025] [Figure 1]Schematic diagrams of antigen receptors are shown. A shows a chimeric antigen receptor (CAR) that binds to an antigen cluster receptor, which activates the T cell signaling pathway. B shows that synNotch receptor binding to antigen results in receptor cleavage, releasing transcription factors (purple) into the nucleus and turning on custom gene programs. C shows a universal CAR that binds to a tag-ligand molecule (red) on an antibody adaptor, allowing the same receptor to target multiple antigens on a target cell. [Figure 2] This figure shows an outline of OFF switch adaptors generated via photocleavable and small molecule cleavable benzylguanine (BG) groups to deactivate SNAP receptor activity. The ON switch adaptor allows activation of receptor signaling through BG uncaging in response to light, small molecules, low pH, proteases, or reactive oxygen species (ROS). Two adaptors bind to different antigens on target cells, and in AND logic, one adaptor enzymatically uncages the BG on the other adaptor, whereas in NOT logic, it cleaves the BG. [Figure 3] A schematic diagram of the functional assembly of the covalent adapters SNAP-CAR and SNAP-synNotch receptor is shown. Benzylguanine (BG) was chemically conjugated to the antibody using BG-NHS ester. The BG-antibody conjugate was then fused to the SNAP tag on the cell surface via a self-labeling reaction. This allows for universal retargeting of CAR and synNotch signaling to any antigen of interest. [Figure 4]The engineered SNAP-CAR demonstrates efficacy against primary human T cells. (A) Schematic of SNAP-CAR and BG adaptor-mediated activation. (B) Flow cytometry analysis of CD62L T cell activation marker on SNAP-CAR T cells co-incubated with target cell lines (x-axis) and the indicated antibodies (1.0 μg / mL), reported as mean fluorescence intensity (MFI). (C) ELISA data for IFNγ production from primary human T cells after incubation with (B). (D) Specific lysis of target cells by primary human SNAP-CAR T cells and BG-conjugated antibodies. Multiple ANOVA comparisons were performed for B, C, and D. Tukey's HSD was used for post-hoc analysis between antibody conditions. * indicates p<0.0001, n=3 independent experiments ± standard error. [Figure 5] Figure 5A shows the structure of photocleavable biotin NHS carbonate. Figure 5B shows that the photocleavable OFF switch adaptor deactivates the function of mSA2CAR T cells. Primary human mSA2-CAR T cells were incubated with RajiCD20+ target cells and various concentrations of anti-CD20 OFF switch adaptor antibody exposed to 365 nm light for 24 hours. T cell activation markers CD69 (increased upon activation) and CD62L (decreased upon activation) were measured by flow cytometry. n=3 biological replicates. [Figure 6] Figure 1 shows that small molecule cleavable OFF switch adaptors deactivated mSA2 CAR T cell function. A shows an aryl azide-containing biotin adaptor that triggers cleavage via 1,6-elimination in response to phosphine-induced Staudinger reduction. B shows primary human mSA2-CAR T cells incubated with Raji CD20+ target cells for 24 hours and exposed to increasing concentrations of anti-CD20 OFF switch adaptor with 2DPBM as indicated. T cell activation markers CD69 and CD62L were measured by flow cytometry. [Figure 7A] Photoactivated biotin is shown. [Figure 7B]The light-triggered ON-switch adaptor anti-CD20 antibody was shown to display biotin on the target cell surface, which was measured by streptavidin-APC cell staining in a light-dependent manner (365 nm). [Figure 7C] We show that a light-triggered OFF switch adaptor anti-CD20 antibody displays light-triggered biotin cleavage on the surface of CD20+ Raji target cells in a light exposure time-dependent manner (365 nm) as measured by streptavidin-APC cell staining by flow cytometry. [Figure 8A] Phosphine-activated biotin is shown. [Figure 8B] 2 shows the ON switch adaptor anti-CD20 antibody triggered by a small molecule in a DPBM-dependent manner (same staining protocol as in Figure 7). [Figure 8C] We show by flow cytometry that a small molecule-triggered OFF switch adaptor anti-CD20 antibody displays 2DPBM-triggered biotin cleavage on the surface of CD20+Raji target cells in a 2DPBM concentration-dependent manner as measured by streptavidin-APC cell staining. [Figure 9] We demonstrate the clinical utility of OFF switch adaptor control: a light-triggered OFF switch can provide spatiotemporal anatomical site protection from CAR T cell toxicity and treat the primary tumor and distant metastatic sites, while a small molecule drug-triggered OFF switch provides rapid systemic cessation of treatment. [Figure 10] A shows the proposed synthesis of a BG adapter capable of site-specific antibody conjugation via a bis-sulfone. B shows the proposed synthesis of a BG adapter capable of site-specific antibody conjugation via a dibromopyrazidinedione. [Figure 11]Site-specific labeling of antibodies by disulfide re-crosslinking is shown. Disulfides can be reduced using tris-(2-carboxyethyl)phosphine (TCEP) and re-crosslinked using established dibromopyridazinedione or bis-sulfone reagents. Antibodies contain four disulfides. Therefore, four conjugates can be added site-specifically (only one is shown here for clarity). [Figure 12] Figure 1 shows mTGase-mediated enzyme-antibody conjugation. A shows the synthesis of a substrate for mTG-mediated conjugation. PEG spacer 13 is commercially available (n = 0-6). B shows the site-specific introduction of two conjugates (one is shown here for clarity). [Figure 13] A shows the synthesis of BG with a phosphine-cleavable linker (green). B shows the synthesis of a photocleavable linker (blue). C shows an alternative red-shifted chromophore introduced in place of N3 of 16, inducing linker cleavage by 1,6-elimination. X is a conjugation group. [Figure 14] The clinical utility of ON switch adaptor control is shown. A. Light-triggered ON switches enable external spatial control over activity. B. Small molecule trigger switches enable tunable dosing of CAR T activation, avoiding nonspecific toxicity due to overactivation of CAR T cells. C. TME trigger switches avoid ON-targeted OFF tumor toxicity. [Figure 15A] The mechanism of action of the ON switch adaptor is shown. [Figure 15B] The conditionally activated adaptors and their role in mediating CAR T cell targeting shown in Figure 2B are based on three components: a conjugating group X, a caging group R, and a benzylguanine BG. [Figure 16]The crystal structure of BG (turquoise) interacting with SNAPtag (tan) is shown. There are five residues (purple) with atoms within 4 Å of the exocyclic amine of BG, demonstrating the ability to generate a conditionally activatable (caged) BG. [Figure 17] A indicates that the photoremovable caging group R comprises a coumarin. B indicates that the photoremovable caging group R comprises a BODIPY. C indicates that the photoremovable caging group R comprises a cyanine. [Figure 18] A shows the mechanism for the release of the caging group R in response to a phosphine. B shows the mechanism for the release of the caging group R in response to a tetrazine. [Figure 19] The figures show caging groups that are sensitive to TME. A is acid, B is ROS-sensitive caging group, C is legumain peptide structure, and D is MMP-catalyzed BG release. [Figure 20] We present a strategy for designing combinatorial antigen adaptors that result in the rational activation of receptor signaling and cell lysis in response to antigen combinations. [Figure 21] Figure 1 shows the proposed structures of two NTR-activating adaptors. Synthesis can follow the pathway developed herein. X = conjugate group. [Figure 22] This demonstrates that the SNAP-synNotch receptor can be targeted to an antigen of interest by a BG-conjugated antibody. (A) SNAP-synNotch receptor and activation. In this system, Gal4-VP64 transcription factor-driven TagBFP expression is triggered. (B) Flow cytometry analysis of activation of SNAP-synNotch cells incubated with the target cell line and the indicated amount of antibody. TagBFP levels are reported as mean fluorescence intensity (MFI). n=3 biologically independent experiments ± standard deviation. [Figure 23]Figure 1 shows an antibody adapter OFF switch that allows for stimulus-controlled presentation of an adapter tag molecule on the cell surface. A shows a diagram of a cell surface biotin assay for measuring accessible tags on the surface of target cells bound by an adapter OFF switch. B shows cells stained with the indicated amount of OFF switch adapter targeting HER2 (Herceptin) or the adapter CD20 (Rituximab) and then exposed to 365 nm light for the indicated time. C shows results with the indicated amount of 2DPBM drug. Cells were incubated at 37°C for 24 hours before staining with streptavidin-APC and evaluation by flow cytometry. (Note: The antibody amounts in the legend correspond to staining concentrations in μg / mL.) [Figure 24] We demonstrate that the phosphine-cleavable OFF switch can be triggered by an additional phosphine drug. Cells were stained with 5 μg / ml of an OFF switch adaptor targeting HER2 (Herceptin) and exposed to the indicated amounts of small phosphine molecules (bis(p-sulfonatophenyl)phenylphosphine, Tris(3-sulfonatophenyl)phosphine, 2(diphenylphosphanyl)benzamide [2DPBM]). After incubation at 37°C for 2 hours, cells were stained with streptavidin-APC and evaluated by flow cytometry. [Figure 25]Figure 1 shows that the OFF switch adaptor mediates conditional lysis of target cells by universal CAR T cells. (A) shows mSA2 universal CAR T cells co-incubated with K562+HER2 or K562+CD20 target cells at an E:T ratio of 10:1 and pre-stained with the indicated concentrations of adaptor. The co-cultures were then exposed to 365 nm light for the indicated times and incubated at 37°C for 24 hours. The co-cultures were then evaluated for target cell lysis by flow cytometry. (B) shows testing of the phosphine-controlled OFF switch; co-culture assays were seeded similarly to panel "25A," except that the indicated concentrations of 2DPBM drug were added to the wells and no light exposure was performed. "PEG2" indicates a control adaptor made with a non-cleavable, inactive PEG linker. CD20-CAR (ON-target) indicates anti-CD20CAR T cells incubated with K562+CD20 target cells, and CD20-CAR (OFF-target) indicates anti-CD20CAR T cells incubated with K562 (CD20-negative) target cells. (Note: The antibody amounts in the legend correspond to the staining concentration in μg / mL.) [Figure 26] MTGase enzyme antibody-adapter conjugation. (A) SDS-PAGE of rituximab-BG adapter (RTX) generated via MTGase conjugation, co-incubated with SNAPtag (2 equivalents per BG), and visualized using Coomassie staining reveals near-complete conjugation. (B) Primary human SNAP-CAR T cells co-incubated with MTGase-generated site-specific adapter in the presence or absence of CD20+ Raji target cells. Expression of the CD69 T cell activation marker on CAR T cells was assessed after 24 hours. n=3 biological replicates. [Figure 27]Engineered SNAP-CARs are effective in an in vivo mouse model. (A) NSG mice were injected intravenously (iv) with SNAP CAR T cells and intraperitoneally (ip) with either PBS or rituximab-BG adaptor. Blood was collected 24 hours later, and T cells were assessed by flow cytometry for CAR expression and surface-bound rituximab-BG adaptor (anti-human IgG-APC). n=3 mice per group. (B) NSG mice were challenged with 10 Raji CD20+ human leukemia cells expressing firefly luciferase, followed by adapter injections at the indicated doses on days 4 and 9, and SNAP CAR T cell or anti-CD20 CAR T cell injections ("aCD20") on day 5. Raji tumor burden was assessed by IVIS luminescence imaging on days 4, 9, and 14. DETAILED DESCRIPTION OF THE INVENTION

[0026] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to particular synthetic methods or to particular recombinant biotechnology methods, unless otherwise specified, or to particular reagents, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0027] A.Definition As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0028] Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant in relation to the other endpoint, and independently of the other endpoint. A number of numerical values ​​are disclosed herein, and it is understood that each numerical value is also disclosed herein as "about" that particular value in addition to the numerical value itself. For example, if the numerical value "10" is disclosed, "about 10" is also disclosed. When a numerical value is disclosed, it is understood that "less than or equal to," "greater than or equal to," and possible ranges between values ​​are also disclosed, as appropriately understood by one of ordinary skill in the art. For example, if the numerical value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of the data points. For example, if a specific data point "10" and a specific data point 15 are disclosed, it is understood that greater than, greater than, less than, less than, less than, and equal to 10 and 15, as well as 10 to 15, are considered disclosed. It is also understood that each unit between two specified units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0029] In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings.

[0030] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0031] "Increase" can refer to any change that increases the amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, or composition by a statistically significant amount. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, so long as the increase is statistically significant.

[0032] "Reduction" can refer to any change that reduces the amount of a symptom, disease, composition, condition, or activity. A substance is also understood to reduce the genetic production of a gene when the genetic production of a gene product containing the substance is reduced compared to the production of a gene product without the substance. A reduction can also be, for example, a change in the symptoms of a disorder such that the symptoms are less than those previously observed. A reduction can be any individual, median, or average decrease in a condition, symptom, activity, or composition by a statistically significant amount. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, so long as the reduction is statistically significant.

[0033] "Inhibit," "inhibiting," and "inhibition" mean a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% decrease in the activity, response, condition, or disease compared to native or control levels. Thus, the decrease can be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any decrease in between, compared to native or control levels.

[0034] "Reduce" or other forms of the word, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., tumor growth). This is usually understood to be relative to some standard or expected value, in other words, a relative value, although reference to a standard or relative value is not necessarily required. For example, "reducing tumor growth" means a decrease in the rate of tumor growth compared to a standard or control.

[0035] "Prevent" or other forms of the word, such as "preventing" or "prevention," means to stop a particular event or characteristic, to stabilize or delay the occurrence or progression of a particular event or characteristic, or to minimize the likelihood of a particular event or characteristic occurring. Prevent is usually absolute compared to, for example, reduce and therefore does not require a comparison to a control. As used herein, something may be reduced but not prevented, or something may be reduced and prevented. Similarly, something may be prevented but not reduced, or something may be prevented and reduced. Where reduce or prevent is used, it is understood that the use of other language is expressly disclosed unless specifically indicated otherwise.

[0036] The term "subject" refers to any individual that is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one embodiment, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or an animal patient. The term "patient" refers to a subject receiving treatment from a clinician, e.g., a physician.

[0037] The term "comprising" is intended to mean that the composition, method, etc., includes the recited elements, but does not exclude other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean including the recited elements, but excluding any other elements that are essential to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers, e.g., phosphate-buffered saline, preservatives, etc. "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0038] A "control" is another subject or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0039] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon.

[0040] B. Composition The disclosed compositions and the components used to prepare the compositions themselves used in the methods disclosed herein are disclosed. When these and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each of the various individual and collective combinations and permutations of these compounds is specifically contemplated and described herein, although specific reference may not be explicitly disclosed. For example, when a specific conditional universal CAR system or conditional universal SynNotch receptor is disclosed and discussed, and a number of modifications that can be made to multiple molecules including the universal CAR system or conditional universal SynNotch receptor are discussed, it is specifically contemplated that each and every combination and permutation of the universal CAR system or conditional universal SynNotch receptor, and possible modifications, unless otherwise specified. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of combination molecule A-D is disclosed, each is contemplated individually and collectively to mean the combination, even if each is not individually recited, and A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the subgroups A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that can be performed, it is understood that each of these additional steps can be performed in any specific embodiment or combination of embodiments of the disclosed methods.

[0041] Adoptive cell therapy using antigen receptor-engineered T cells is a highly promising therapeutic approach in which T cells are genetically modified to express antigen receptor proteins and then adoptively transferred into patients. These cells act as "herbal medicines" that can elicit potent therapeutic effects in response to sensing target antigens on neighboring cells anywhere in the body.

[0042] The most clinically advanced antigen receptor technology is the chimeric antigen receptor (CAR), which consists of an antigen-binding region, an extracellular spacer domain, a transmembrane domain, and a T cell signaling domain (Figure 1A). Upon antigen binding, CARs cluster and activate the T cell receptor signaling pathway, which leads to target cell lysis, cell proliferation, and cytokine production that can amplify the immune response. CAR T cell therapy targeting the CD19 protein, an antigen found on the surface of leukemic and normal B cells, has been approved by the FDA and has shown significant levels of cancer remission in 85% of patients. CAR T cells targeting novel antigens are being developed to treat a variety of diseases, including solid tumors, viral infections, and autoimmune disorders.

[0043] An even more versatile class of antigen receptors under development is the synthetic Notch (synNotch) receptor, which can regulate the expression of one or more therapeutic genes of interest upon binding to a target antigen without affecting endogenous cell signaling pathways. Engineered from the Notch / Delta signaling pathway, synNotch receptors consist of an extracellular antigen-binding domain, a Notch core protein, and an intracellular transcription factor. Upon antigen binding, the receptor is stretched by mechanical force, exposing enzyme (e.g., protease) cleavage sites, which upon proteolysis release transcription factors and regulate gene expression (Figure 1B). SynNotch receptors can sense a wide range of antigens and can be engineered to express numerous therapeutic-yielding genes in response, including cytokines, toxins, chemokines, other receptors, and entire gene circuits. This highly programmable platform technology has been applied to various cell types in addition to T cells and has attracted great interest in the fields of immunotherapy and tissue engineering.

[0044] Recently, we have created a universal adaptor CAR system, as well as the first universal adaptor synNotch system using a SNAPtag targeting domain. Universal adaptor receptors are a new generation of antigen receptors that, instead of directly binding to antigens on target cells, bind to a common tag molecule fused or conjugated to an antigen-specific antibody, called an "adapter." These systems are designed to inject the adaptor, which forms a physical link between the target cell and the receptor T cell, into the patient (Figure 1C). Adapter receptors are called "universal" because, by simply administering different adaptors sequentially or simultaneously, a single receptor can target multiple tumor antigens within a single patient or across different disease indications. Universal SNAP receptors are unique because they form a covalent bond rather than just transiently interacting with the adaptor. Through the bioorthogonal benzylguanine (BG) tag and SNAP receptor, several different antigens can be targeted, achieving highly potent receptor activation and signaling. The importance of adapter CAR systems has been recognized with the development of antibody-based adapters modified with biotin, fluorescein, peptide neoepitopes, Fcγ, and leucine zippers, with the first adapter CAR systems currently undergoing clinical trials.

[0045] Here, we utilize a synthetic adapter approach to gain additional (photo)chemical or biological control over these systems. This control is achieved by inserting several stimulus-responsive OFF->ON and ON->OFF switches into the adapter that respond to inputs such as small molecules (for user-defined temporal control), light (for user-defined temporal and spatial control), pH, hydrogen peroxide, proteases, and other enzymes (for highly specific activation in the tumor microenvironment, TME). The conditional ON switch system described herein consists of an adapter with a stimulus-responsive group, often referred to as a "caging group," that blocks the tag-ligand molecule on the adapter. In the absence of a stimulus, the caging group blocks recognition by CAR T cells; however, in the presence of a stimulus, the caging group is removed, allowing recognition and receptor activation by CAR T cells. In contrast, the OFF switch consists of an adapter with a stimulus-responsive chemical cleavage group between the tag-ligand and the antibody. Thus, in the absence of a stimulus, the adapter can activate CAR T cells, but in the presence of a stimulus, the tag-ligand is cleaved from the adapter, preventing CAR T cell activation.

[0046] The unprecedented level of conditional control enabled by our light and small molecule switch system, along with the proposed physiological signal response system, may improve patient outcomes by reducing toxicity through improved targeting and granting the ability to treat new disease indications. While methods for conditional control of CAR T cells, such as activation by a combination of small molecules, light, proteases, and antigens, have been developed, these methods have been implemented at the level of receptor protein engineering, and implementing them for every new antigen(s) targeted requires extensive genetic modification. By focusing engineering on synthetic adapter molecules in the context of a universal receptor system, this technology leverages technological advances in the field of conditional linker control chemistry to provide a fundamentally new solution to the problem of antigen receptor specificity. Based on the flexibility offered by synthetic adapters, including 1) both an ON and OFF switch, 2) several conditional control inputs (e.g., light, small molecules, etc.), and 3) targeting various antigens of interest, this approach creates superior reagents that can be used with the same universal cell receptor without further genetic engineering for all modes of implementation. These innovations represent the first conditionally regulated universal CAR and universal synNotch receptor with improved signaling activity and flexible programmability through covalent cell surface modifications. The disclosed synNotch receptor and CAR systems achieve their universality through the use of adapter molecules on the extracellular portion of the synNotch receptor or CAR and that form covalent bonds with antigen recognition elements. This system for generating universal CARs and synNotch receptors using adapter molecules to form covalent bonds with antigen recognition elements represents a significant improvement over existing adapter CAR T cells and is the first successful adapter synNotch system ever generated.

[0047] However, this universality does not address the issues of specificity and off-site toxicity. The present disclosure solves this problem by creating a conditional universal CAR system and a conditional universal SynNotch receptor. Thus, in one aspect, disclosed herein are: i) a CAR comprising a receptor that targets a tag ligand on a conditional adapter molecule, a hinge domain (e.g., a CD8α domain, a CD28 hinge, or a modified IgG hinge with deletion or modification of the CH2 and / or CH3 domain, etc.), and a signaling domain (e.g., a DAP10, NKG2D, NKG2C, NKp44, CD28, CD27, Meg10, CD32, CD16, 2B4, or CD3ζ signaling domain, etc.); and ii) an antigen recognition element (e.g., an antibody, an antigen-recognizing fragment thereof, a protein, etc.). The conditional universal chimeric antigen receptor (CAR) system comprises a conditional adaptor molecule containing a protein-binding domain, a lectin, a DNA aptamer, an RNA aptamer, a small molecule ligand for a cell surface receptor, or a peptide / protein ligand for a natural protein receptor, a stimuli-responsive group, and a tag ligand (e.g., benzylguanine (BG), fluorescein (FITC), chloroalkane, SpyTag, leucine zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc domain, peptide neoepitope (PNE), biotin, or benzylcytosine (BC)). Also disclosed herein are conditional universal synthetic Notch (synNotch) receptors comprising a conditional adaptor molecule, wherein the conditional adaptor molecule comprises a stimulus-responsive group and a tag ligand (e.g., benzylguanine (BG), FITC, SpyTag, leucine zipper, La-SS-B, CD19, anti-folate receptor antibody, Fc domain, peptide neoepitope (PNE), biotin, chloroalkane, and benzylcytosine (BC)), and the notch core comprises one or more cleavage sites and one or more transcription factors.

[0048] Both the universal synNotch receptor and universal CAR systems disclosed herein include adapter molecules that facilitate the formation of binding interactions with antigen recognition elements (such as antibodies or antibody fragments). The binding interactions can be covalent, non-covalent, or other interactions, such as receptor-ligand or antibody-antigen / peptide / protein binding interactions (such as FITC and anti-FITC or biotin / avidin). When covalently attached, the covalent bond can occur via pi-clamp; ligand-directed tosyl chemistry; recombinant antibodies with short peptide tags, tag-ligand incorporation (e.g., BG or BC incorporation) via sortase-mediated labeling; unnatural amino acid mutagenesis followed by "click" chemistry, [3+2] cycloaddition, split intein, THIOMAB, tetrazine ligation, Staudinger ligation, imine formation, thiol-ene reaction, native chemical ligation; biotin ligase-mediated labeling; lipoic acid ligase-mediated labeling; NHS-ester conjugation, conjugation to cysteine, disulfide restapling via bis-sulfone or other reagents, glycan conjugate chemistry, or formylglycine conversion. Formation of the covalent bond can also occur through the use of an adapter molecule comprising a polypeptide tag covalently linked to the target modification. Examples of polypeptide adapter molecules include, but are not limited to, the SNAP-tag (an O-tag capable of insertion into an antigen recognition element). 6 -benzylguanine covalently bound), CLIP-tag (an O-tag that can be inserted into an antigen recognition element) 2-benzylcytosine), Halo-tag (covalently linked to a chloroalkane linker that can be inserted into an antigen recognition element), SpyTag (covalently linked to a Spy catcher peptide sequence that can be inserted into an antigen recognition element), SnoopTag (covalently linked to a Snoop catcher peptide sequence that can be inserted into an antigen recognition element), or Isopep-tag (covalently linked to its binding partner that can be inserted into an antigen recognition element). The formation of a covalent bond is an important improvement over other adaptor CAR T cell systems that rely on weak interactions; the covalent bond is strong enough that it is not cleaved upon antigen binding, thereby revealing the Notch cleavage site(s) in the synNotch receptor.

[0049] Also disclosed herein are conditional universal CAR systems and synNotch receptors, wherein the stimuli to which the stimulus-responsive group is responsive include light (including, but not limited to, a laser light source that delivers incident light to the patient's tissue, including light in the visible light spectrum (400-650 nm), light in the 650-790 nm range, and light in the 800-840 nm range (e.g., light with wavelengths of 365, 405, 544, or 780 nm)), enzymes (e.g., legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase-A (KHK-A), hexokine, and the like). The enzymes that can be used include mitochondrial kinases (HK), nucleoside diphosphate kinase (NDPK or NDK) and 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase, small molecules (e.g., phosphines or tetrazines), pH, hypoxia (e.g., to use hypoxia-reactive motifs such as nitroaromatic compounds), H2O2, and / or reactive oxygen species (ROS) (e.g., ROS-labile linkers: N 1 -(4-Bromobenzyl)-N 3 -(4-bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -tetramethylpropane-1,3-diaminium (TSPBA) because they use ROS-reactive motifs.

[0050] In one aspect, disclosed herein is a conditional universal CAR system, wherein the stimulus-reactive group comprises a cleavable linker (e.g., a photocleavable or phosphine-cleavable linker) or a stimulus-reactive caging group (e.g., a photoreactive caging group comprising nitrobenzyl, coumarin, BODIPY, or cyanine), which blocks the CAR from binding to the tag ligand.

[0051] It is understood and contemplated herein that the antigen recognition element can be an antibody or any antigen-recognizing fragment thereof (e.g., Fab, Fab'2, scFv, Fv, etc.). In one aspect, the antigen recognition element can comprise an anti-cancer drug-based monoclonal antibody, such as cetuximab (anti-EGFR), nimotuzumab (anti-EGFR), panitumumab (anti-EGFR), retuximab (anti-CD20), omalizumab (anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-Her2), herceptin (anti-Her2), gemtuzumab (anti-CD33), alemtuzumab (anti-CD52), FMC63 (anti-CD19), and bevacuzimab (anti-VEGF), or an antigen-recognizing fragment thereof. In some embodiments, antigen recognition elements can include protein binding domains (e.g., nanobodies and single domain antibodies (e.g., monobodies), lectins, DNA aptamers, RNA aptamers, any small molecule ligand for a cell surface receptor (e.g., folate bound by the folate receptor), peptide / protein ligands of natural protein receptors (e.g., cytokines capable of binding to NKG2D and / or their natural receptors, etc.).

[0052] In one aspect, it is understood and contemplated herein that for T cell activation of a CAR T cell to occur, additional cell signaling events must occur beyond the CAR T cell antigen recognition element binding its target. Costimulation is also required. Costimulation occurs during the activation of any T cell and can occur through native interactions already present on any CAR T cell, such as stimulation of CD28 and 4-1BB through interaction with their respective ligands B7 and 4-1BBL on the surface of the target cell. Alternatively, a universal CAR can further comprise one or more costimulatory domains (e.g., signaling domains of CD27, CD28, ICOS, 4-1BB, or OX40) such that costimulation occurs on the antigen recognition element binding its target without the need for an additional target cell to provide the necessary costimulatory signal (Figures 5A and 5B). Thus, in one aspect, disclosed herein is a universal CAR comprising an adaptor molecule, a hinge domain (such as, for example, a CD8α domain, a CD28 hinge, or a modified IgG hinge with a deletion or modification of the CH2 and / or CH3 domain), and a signaling domain (such as, for example, a DAP10, NKG2D, NKG2C, NKp44, CD28, CD27, Meg10, CD32, CD16, 2B4, or CD3ζ signaling domain), wherein the CAR further comprises one or more costimulatory domains (such as, for example, a signaling domain of CD27, CD28, ICOS, 4-1BB, or OX40). Also disclosed herein are universal CAR T cells expressing any of the universal CARs disclosed herein that further comprise one or more costimulatory domains. Thus, in one aspect, disclosed herein is a conditional universal CAR system that further comprises one or more costimulatory domains (such as, for example, CD27, CD28, ICOS, 4-1BB, or OX40).

[0053] The disclosed CARs and synNotch receptors are generated from and / or ultimately expressed on stem cells of T cells, NK cells, NK T cells, B cells, and / or macrophages and any immune cells (e.g., T cells, B cells, memory T cells, memory B cells, NK T cells, monocytes, natural killer cells, dendritic cells, macrophages, regulatory T cells, helper T cells, γδ T cells, or cytotoxic T cells) directed against the CAR, neurons, epithelial cells, and endothelial cells, or synNotch receptors. The cells used to generate and express the universal CARs and synNotch receptors disclosed herein, as well as any cells containing these receptors, can be derived from autologous, syngeneic, or allogeneic sources, with the selection depending on the disease being treated and the means available for treatment. In the case of T cells, suitable populations of effector cells that can be used in the present methods include any immune cell with cytolytic activity, such as T cells. Exemplary subpopulations of T cells include, but are not limited to, those expressing CD3+, e.g., CD3+CD8+ T cells, CD3+CD4+ T cells, and NK T cells. In one aspect, T cells are derived from peripheral blood mononuclear cells (PBMCs) of any HLA background and are utilized in autologous, syngeneic, or allogeneic systems. T cells can also be isolated from any source, including, but not limited to, tumor explants or intratumoral T cells of the subject being treated. For convenience, effector cells will be generally referred to herein as T cells; however, unless otherwise indicated, it should be understood that any reference to T cells is a reference to all effector cell types as defined herein. Accordingly, disclosed herein are engineered T cells comprising a universal CAR (universal CAR T cells) and / or a universal synNotch as disclosed herein (engineered universal synNotch T cells). It is understood and contemplated herein that, in one aspect, a synNotch receptor and a CAR can be expressed on the same T cell. In such a situation, the antigen recognition elements may be the same, allowing both synNotch transactivation of cytokines and T cell activation to occur.Alternatively, the synNotch receptor and the CAR can comprise different antigen recognition elements. To ensure that the antigen recognition elements of the CAR and synNotch are different, when both are present on the same cell, the synNotch receptor and the CAR can comprise different adaptor molecules that enable different covalent interactions. Thus, in one aspect, disclosed herein are engineered T cells comprising any of the universal CARs and universal synNotch receptors disclosed herein, wherein the CAR and the synNotch receptor comprise different adaptor molecules.

[0054] As described above, the effector action of the universal synNotch receptor occurs through transcriptional activation of intracellular response genes. One or more transcription factors (e.g., Gal4-VP64, Gal4-VP16, TetR-VP64, LacI-VP64, etc.) can be specifically designed to activate transcription in T cells, where the response genes can be T cell effector molecules such as, but not limited to, IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin. Transcriptional activation can occur using native or designer transcription factors (Crispr / Cas9, TALEN, or zinc finger). Thus, in one aspect, disclosed herein are engineered T cells that comprise a universal synthetic Notch (synNotch) receptor, wherein one or more transcription factors of the universal synNotch receptor activate the expression of one or more natural cellular response genes (e.g., T cell effector molecules IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin, etc.). Alternatively, it is contemplated that a transcription factor (e.g., Gal4-VP64, Gal4-VP16, TetR-VP64, LacI-VP64, etc.) can be specific for a transcription response element (e.g., Gal4-VP64, Gal4-VP16, TetR-VP64, LacI-VP64, etc.) on a vector expressing a transgene system, allowing for a unique, non-native interaction and expression of one or more response genes (e.g., IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin). The one or more response genes can be encoded on the vector along with a promoter driving expression of the transcription response element and effector molecule.Thus, in one aspect, disclosed herein is an engineered cell comprising a universal synthetic Notch (synNotch) receptor, further comprising a vector comprising a transcription response element operably linked to a promoter driving expression of one or more response genes (e.g., T cell effector molecules IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin), wherein the transcription response element is specific for one or more of the transcription factors on the synNotch receptor.

[0055] In some embodiments, the conditional universal synNotch receptor can further comprise an antigen recognition element (e.g., an antibody, an antigen-recognition fragment thereof, a protein-binding domain, a lectin, a DNA aptamer, an RNA aptamer, a small molecule ligand of a cell surface receptor, or a peptide / protein ligand of a natural protein receptor); wherein the antigen recognition element is or can be covalently linked to the conditional universal adaptor molecule. In some embodiments, the antigen recognition element comprises rituximab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacizumab, or an antigen-binding fragment of any one thereof.

[0056] Also disclosed herein are engineered cells (e.g., immune cells, neurons, epithelial cells, and endothelial cells, or stem cells) comprising any of the conditional universal synNotch receptors disclosed herein. In one aspect, disclosed herein are engineered cells further comprising a vector comprising a transcription response element operably linked to a promoter driving expression of one or more cellular response genes (e.g., IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin), wherein one or more of the transcription factors on the synNotch receptor are specific for the transcription response element. Also disclosed herein are engineered cells, wherein one or more transcription factors of the conditional universal synNotch receptor activate expression of one or more natural cellular response genes (e.g., IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin).

[0057] 1.Homology / Identity It is understood that one way to define any known variants and derivatives or those that can arise from the genes and proteins disclosed herein is by defining the variants and derivatives in terms of their homology to a specific known sequence. Generally, specifically disclosed are variants of these and other genes and proteins disclosed herein that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology with the described sequence. Those skilled in the art will readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, homology can be calculated after aligning the two sequences to maximize homology.

[0058] Alternative methods for calculating homology can be performed using published algorithms. Optimal alignment of sequences for comparison may be performed using the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0059] The same type of homology can be obtained for nucleic acids, for example, by the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989, which are incorporated herein by reference, at least for material related to nucleic acid alignment.

[0060] 2. Delivery of the Composition to Cells There are several compositions and methods that can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can be broadly divided into two classes: viral-based delivery systems and non-viral-based delivery systems. For example, nucleic acids can be delivered to cells or carriers, such as cationic liposomes, via several direct delivery systems, or via transfer of genetic material, including electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, and cosmids. Suitable means for transfection, such as viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described, for example, by Wolff, J.A., et al., Science, 247, 1465-1468, (1990); and Wolff, J.A. Nature, 352, 815-818, (1991). Such methods are well known in the art and can be easily adapted for use with the compositions and methods described herein. In some cases, methods can be modified to function specifically with large DNA molecules. Additionally, these methods can be used to target specific diseases and cell populations by using the targeting properties of the carriers.

[0061] a) Nucleic Acid-Based Delivery Systems A transfer vector may be any nucleotide construct (e.g., a plasmid) used to deliver genes to cells or as part of a general strategy for delivering genes, such as a recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).

[0062] As used herein, a plasmid or viral vector is an agent that transports the disclosed nucleic acid, such as a chimeric antigen receptor or synNotch, into cells without degradation and contains a promoter that drives gene expression in the cells to which it is delivered. Viral vectors include adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, neurotrophic virus, lentivirus, Sindbis, and other RNA viruses, such as those with an HIV backbone. Any viral family that shares the properties of these viruses that make them suitable for use as vectors is also preferred. Retroviruses include murine moloney leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as vectors. Retroviral vectors are commonly used vectors because they can carry larger gene payloads, i.e., transgenes or marker genes, than other viral vectors. However, they are not useful in non-proliferating cells. Adenoviral vectors are relatively stable, easy to handle, have high titers, can be delivered in aerosol formulations, and can transfect non-dividing cells. Poxvirus vectors are large, have several sites for inserting genes, are thermostable, and can be stored at room temperature. A preferred embodiment is a viral vector engineered to suppress the immune response of the host organism elicited by viral antigens. Preferred vectors of this type can carry the coding regions for interleukin 8 or 10.

[0063] Viral vectors can have a higher transfection capacity (ability to introduce genes) than chemical or physical methods for introducing genes into cells. Typically, viral vectors contain nonstructural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and encapsidation, and a promoter that controls transcription and replication of the viral genome. When designed as a vector, viruses typically have one or more of their early genes removed, and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral DNA. This type of construct can carry up to approximately 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically provided by cell lines engineered to express the gene products of the early genes in trans.

[0064] (1) Retroviral vector Retroviruses are animal viruses belonging to the viral family Retroviridae, of any type, subfamily, genus, or tropism, including, but not limited to, lentiviruses (such as HIV-based lentiviral vectors and gammaretroviral vectors). Retroviral vectors are generally described by Verma, IM, Retroviral Vectors for Gene Transfer.

[0065] Retroviruses are essentially packages loaded with nucleic acid cargo. The nucleic acid cargo contains a packaging signal, which ensures efficient packaging of replicated daughter molecules within the package coat. In addition to the packaging signal, there are several other molecules required for cis replication and packaging of the replicated virus. A typical retroviral genome contains the gag, pol, and env genes, which are involved in the production of the protein coat. It is the gag, pol, and env genes that are typically replaced by the foreign DNA introduced into target cells. Retroviral vectors typically contain a packaging signal for incorporation into the package coat, a sequence signaling the initiation of the gag transcription unit, elements required for reverse transcription, such as a primer binding site for binding to a tRNA primer for reverse transcription, long terminal repeats that direct RNA strand switching during DNA synthesis, a purine-rich sequence from the 5' to 3' long terminal repeat (LTR) that serves as a priming site for synthesis of the second strand of DNA, and specific sequences near the end of the long terminal repeat (LTR) that allow insertion of the retroviral DNA into the host genome. Removal of the gag, pol, and env genes results in approximately 8 kb of foreign sequence being inserted into the viral genome, reverse transcribed, and packaged into new retroviral particles during replication. This amount of nucleic acid is sufficient to deliver one to many genes, depending on the size of each transcript. It is preferable to include either a positive or negative selectable marker along with other genes in the insert.

[0066] Because the replication machinery and packaging proteins (gag, pol, and env) of most retroviral vectors have been removed, vectors are typically generated by placing them in packaging cell lines. Packaging cell lines are cell lines transfected or transformed with a retrovirus that contains the replication and packaging machinery but does not contain any packaging signals. When a vector carrying a DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles by machinery provided in cis by the helper cells. The genome for the machinery is not packaged because it does not contain the necessary signals.

[0067] (2) Adenovirus vector The construction of replication-deficient adenoviruses has been reported (Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang, "Generation and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysis," BioTechniques 15:868-872 (1993)). The advantage of using these viruses as vectors is that they can replicate within the initially infected cells but are unable to form new infectious viral particles, thereby limiting their potential spread to other cell types.Recombinant adenoviruses have been shown to achieve highly efficient gene transfer after direct in vivo delivery to airway epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and multiple other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); LaSalle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476(1993);Zabner,Nature Genetics 6:75-83(1994);Guzman,Circulation Research 73:1201-1207(1993);Bout,Human Gene Therapy 5:3-10(1994);Zabner,Cell 75:207-216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)).Recombinant adenoviruses achieve gene transfer by binding to specific cell surface receptors in the same manner as wild-type or replication-deficient adenoviruses, and the virus is then internalized by receptor-mediated endocytosis (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell. 73:309-319(1993)).

[0068] Viral vectors can be based on adenoviruses from which the E1 gene has been removed, and these virions are produced in cell lines such as the human 293 cell line. In another preferred embodiment, both the E1 and E3 genes are removed from the adenoviral genome.

[0069] (3) Adeno-associated virus vector Another type of viral vector is based on adeno-associated virus (AAV). This defective parvovirus is capable of infecting many cell types and is nonpathogenic to humans, making it a preferred vector. AAV-type vectors can transport approximately 4-5 kb, and wild-type AAV is known to stably integrate into chromosome 19. Vectors that contain this site-specific integration property are preferred. A particularly preferred embodiment of this type of vector is the P4.1C vector produced by Avigen, San Francisco, CA. The P4.1C vector may contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as a gene encoding green fluorescent protein (GFP).

[0070] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) flanking at least one cassette containing a promoter that directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene that is not native to AAV or B19 parvovirus.

[0071] Typically, the AAV and B19 coding regions are deleted, resulting in a safe, non-cytotoxic vector. The AAV ITRs or variants thereof confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. U.S. Patent No. 6,261,834 is incorporated herein by reference for material related to AAV vectors.

[0072] Thus, the disclosed vectors provide DNA molecules that can be integrated into mammalian chromosomes without substantial toxicity.

[0073] Genes inserted into viruses and retroviruses usually contain promoters and / or enhancers that help control the expression of the desired gene product. A promoter is generally one or more DNA sequences that function when in a relatively fixed location with respect to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors and may contain upstream elements and response elements.

[0074] (4) Large payload viral vectors Molecular genetic experiments using large human herpesviruses have provided a means by which large heterologous DNA fragments can be cloned, propagated, and established in cells permissive to herpesvirus infection (Sun et al., Nature Genetics 8:33-41, 1994; Cotter and Robertson, Curr Opin Mol Ther 5:633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV)) have the potential to deliver fragments of human heterologous DNA exceeding 150 kb to specific cells. EBV recombinants can maintain large DNA fragments as episomal DNA in infected B cells. Individual clones have harbored human genomic inserts of up to 330 kb that appear to be genetically stable. Maintenance of these episomes requires a specific EBV nucleoprotein, EBNA1, which is constitutively expressed during EBV infection. Furthermore, these vectors can be used for transfection and transiently produce large amounts of protein in vitro. The herpesvirus amplicon system has also been used to package pieces of DNA in excess of 220 kb and to infect cells that are able to stably maintain the DNA as an episome.

[0075] Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.

[0076] b) Non-nucleic acid based systems The disclosed compositions can be delivered to target cells in a variety of ways. For example, the compositions can be delivered by electroporation, by lipofection, or by calcium phosphate precipitation. The delivery mechanism selected will depend in part on the type of cell being targeted and whether delivery is occurring, for example, in vivo or in vitro.

[0077] Thus, the composition can include a vector such as a lipid, such as a liposome, for example, a cationic liposome (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposome. Liposomes can further include proteins, if desired, to facilitate targeting to specific cells. A composition comprising a compound and cationic liposomes can be administered via afferent blood flow to the target organ or inhaled into the airways to target cells in the airways. For liposomes, see, for example, Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No. 4,897,355. Furthermore, the compound can be administered as a component of a microcapsule that can target a specific cell type, such as a macrophage, or where the diffusion or delivery of the compound from the microcapsule is designed for a specific rate or dosage.

[0078] In the methods described above, which involve the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transfer or transfection), delivery of the composition to the cells can be via various mechanisms. By way of example, delivery can be via liposomes using commercially available liposome preparations, such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc., Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to standard procedures in the art. Additionally, the disclosed nucleic acids or vectors can be delivered in vivo by electroporation (technology available from Genetronics, Inc., San Diego, CA) or using a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

[0079] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). These techniques can also be used for a variety of other specific cell types. Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Generally, receptors are involved in pathways of endocytosis, either constitutive or ligand-induced. These receptor clusters in clathrin-coated pits enter the cell via clathrin-coated vesicles and pass through acidified endosomes, where the receptors are sorted and then recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and control of receptor levels. Many receptors follow multiple intracellular pathways depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0080] The nucleic acid that is delivered to cells and integrated into the host cell genome typically comprises integration sequences.These sequences are often virus-related sequences, especially when virus-based systems are used.These viral integration systems can also be incorporated into the nucleic acid that is delivered using non-nucleic acid-based delivery systems such as liposomes, so that the nucleic acid that is contained in the delivery system can be integrated into the host genome.

[0081] Other common techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequences flanking the nucleic acid to be expressed that have sufficient homology with the target sequence in the host cell genome for recombination to occur between the vector nucleic acid and the target nucleic acid, thereby integrating the delivered nucleic acid into the host genome. These systems and the methods necessary to promote homologous recombination are known to those skilled in the art.

[0082] c) in vivo / ex vivo As described above, the compositions can be administered in a pharmaceutically acceptable carrier, and the cells can be delivered to the subject in vivo and / or ex vivo by a variety of mechanisms known in the art (e.g., naked DNA uptake, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis, etc.).

[0083] When using ex vivo methods, cells or tissues can be removed and maintained outside the body according to standard protocols known in the art. The composition can be introduced into cells via any gene transfer mechanism, such as calcium phosphate-mediated gene delivery, electroporation, microinjection, or proteoliposomes. The transduced cells can then be injected (e.g., in a pharmaceutically acceptable carrier) or orthotopically transplanted into a subject according to standard methods for the cell or tissue type. Standard methods for transplanting or injecting various cells into a subject are known.

[0084] 3. Expression system Nucleic acids delivered to cells typically contain expression control systems. For example, genes inserted into viral and retroviral systems usually contain promoters and / or enhancers that help control the expression of the desired gene product. A promoter is generally one or more DNA sequences that function when located in a relatively fixed position relative to the transcription start site. A promoter contains core elements required for basic interaction between RNA polymerase and transcription factors and may contain upstream elements and response elements.

[0085] a) Viral promoters and enhancers Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, including the genomes of viruses such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably, cytomegalovirus, or from heterologous mammalian promoters (e.g., the beta-actin promoter). The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature, 273:113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ et al., Gene 18:355-360 (1982)). Of course, promoters from host cells or related species are also useful herein.

[0086] Enhancers generally refer to DNA sequences that function at random distances from the transcription start site and can be 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3' (Lusky, M. L. et al., Mol. Cell Bio. 3:1108 (1983)) relative to the transcription unit. Enhancers can also be located within introns (Banerji, J. Lett. et al., Cell 33:729 (1983)) and within the coding sequence itself (Osborne, T. F. et al., Mol. Cell Bio. 4:1293 (1984)). They are typically 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers often also contain response elements that mediate transcriptional regulation. Promoters can also contain response elements that mediate transcriptional regulation. Enhancers often determine the regulation of gene expression. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, -fetoprotein, and insulin), but enhancers from eukaryotic cell viruses are typically used for general expression. Preferred examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0087] The promoter and / or enhancer can be specifically activated either by light or by specific chemical events that trigger their function. The system can be regulated by agents such as tetracycline and dexamethasone. There are also methods to enhance gene expression in viral vectors by exposure to radiation, such as gamma irradiation, or alkylating chemotherapy drugs.

[0088] In certain embodiments, the promoter and / or enhancer region can function as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain constructs, the promoter and / or enhancer region is activated in all eukaryotic cell types, even if it is only expressed in certain cell types at certain times. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, cytomegalovirus (full-length promoter), and retroviral vector LTR.

[0089] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types, such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0090] Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells) may also contain sequences necessary for transcription termination, which can affect mRNA expression. These regions are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also includes the transcription termination site. The transcription unit preferably also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcription unit will be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that a homologous polyadenylation signal be used in the transgene construct. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of approximately 400 bases. It is also preferred that the transcription unit contain other standard sequences, alone or in combination with the above sequences, to improve expression from or stability of the construct.

[0091] b) Marker Viral vectors can contain nucleic acid sequences encoding marker products, which are used to determine whether the gene has been delivered to a cell and is being expressed after delivery. Preferred marker genes are the E. coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein.

[0092] In some embodiments, the marker can be a selectable marker. Examples of selectable markers suitable for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive when placed under selective pressure. There are two widely used categories of selection regimens. The first category is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow independently of supplemented media. Two examples are CHO DHFR cells and mouse LTK cells. These cells lack the ability to grow without added nutrients such as thymidine or hypoxanthine. Because these cells lack certain genes required for a complete nucleotide synthesis pathway, the cells cannot survive unless the missing nucleotides are provided in a supplemented media. Instead of supplementing the media, an intact DHFR or TK gene is introduced into cells lacking the respective gene. This alters growth requirements: individual cells not transformed with the DHFR or TK genes cannot survive in unsupplemented media.

[0093] The second category is dominant selection. This refers to a selection scheme that can be used with any cell type and does not require the use of mutant cell lines. These schemes typically use drugs to inhibit host cell growth. Those cells carrying the novel gene will express a protein that conveys drug resistance and survive selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid (Mulligan, R.C. and Berg, P., Science 209:1422 (1980)), or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985)). Three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug, G418, or neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively. Others include the neomycin analogue G418 and pramycin.

[0094] 4. Antibodies (1) Antibodies in general The term "antibody" is used broadly herein and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers of these immunoglobulin molecules, as well as human or humanized forms of immunoglobulin molecules or fragments thereof, so long as they are selected for their ability to interact with a given antigen target. Antibodies can be tested for their desired activity using the in vitro assays described herein or by similar methods, and then their in vivo therapeutic and / or prophylactic activity is tested according to known clinical trial methods. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Those skilled in the art will recognize the equivalent classes in mice. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

[0095] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of antibody molecules. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired antagonist activity.

[0096] The disclosed monoclonal antibodies can be produced using any procedure that produces monoclonal antibodies. For example, the disclosed monoclonal antibodies can be prepared using the hybridoma method, such as that described in Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

[0097] Monoclonal antibodies may also be produced by recombinant DNA methods. DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Libraries of antibodies or active antibody fragments can also be produced and screened using phage display techniques, as described, for example, in U.S. Pat. No. 5,804,440 (Burton et al.) and U.S. Pat. No. 6,096,441 (Barbas et al.).

[0098] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to generate fragments thereof, particularly Fab fragments, can be achieved using routine techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO94 / 29348, published December 22, 1994, and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically generates two identical antigen-binding fragments, called Fab fragments, each containing a single antigen-binding site and a remaining Fc fragment. Pepsin treatment generates a fragment that contains two antigen-binding sites and is still capable of cross-linking antigens.

[0099] As used herein, the term "antibody or fragment thereof" encompasses fragments such as F(ab')2, Fab', Fab, Fv, and scFv, including chimeric and hybrid antibodies with dual or multiple antigen or epitope specificities, as well as hybrid fragments. Thus, fragments of antibodies that retain the ability to bind to their specific antigens are provided. For example, antibody fragments that retain target binding activity are included within the meaning of the term "antibody or fragment thereof." Such antibodies and fragments can be produced and screened for specificity and activity by techniques known in the art, according to methods described in the Examples and general methods for producing antibodies and screening antibodies for specificity and activity (see Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, (1988)).

[0100] Antibody fragments and conjugates of antigen-binding proteins (single chain antibodies) are also included within the meaning of "antibody or fragment thereof."

[0101] Fragments may also contain insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not linked to other sequences. However, the activity of the antibody or antibody fragment may not be significantly altered or may be impaired compared to the unmodified antibody or antibody fragment. These modifications may provide additional properties, such as removing / adding amino acids capable of disulfide bonding, extending biological lifespan, or altering secretion characteristics. In either case, the antibody or antibody fragment must retain biologically active properties, such as specific binding to its cognate antigen. Functional or active regions of an antibody or antibody fragment may be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to those skilled in the art and may include site-directed mutagenesis of nucleic acids encoding the antibody or antibody fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).

[0102] As used herein, the term "antibody" or "antibodies" can also refer to human antibodies and / or humanized antibodies. Many non-human antibodies (e.g., those derived from mice, rats, or rabbits) are naturally antigenic in humans and, therefore, can provoke an unwanted immune response when administered to humans. Therefore, the use of human or humanized antibodies in the methods helps to reduce the likelihood that antibodies administered to humans will provoke an unwanted immune response.

[0103] (2) Human antibodies The disclosed human antibodies can be prepared using any technique. The disclosed human antibodies can also be obtained from transgenic animals. For example, transgenic mutant mice capable of producing a full repertoire of human antibodies in response to immunization have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)). Specifically, homozygous deletion of the antibody heavy chain linking region (J(H)) gene in these chimeric and germline mutant mice results in complete suppression of endogenous antibody production, and successful introduction of the human germline antibody gene array into such germline mutant mice results in the production of human antibodies upon antigen challenge. Antibodies with the desired activity are selected using the Env-CD4-coreceptor complexes described herein.

[0104] (3) Humanized antibodies Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule. Thus, humanized forms of non-human antibodies (or fragments thereof) are chimeric antibodies or antibody chains (or fragments thereof, such as sFv, Fv, Fab, Fab', F(ab')2, or other antigen-binding portions of antibodies) that contain part of the antigen-binding site from the non-human (donor) antibody incorporated into the framework of a human (recipient) antibody.

[0105] To generate a humanized antibody, residues from one or more complementarity-determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule known to have the desired antigen-binding characteristics (e.g., a particular level of specificity and affinity for the target antigen). In some instances, Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues. Humanized antibodies may also contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, humanized antibodies have one or more amino acid residues introduced from a source that is non-human. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. A humanized antibody generally comprises at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

[0106] Methods for humanizing non-human antibodies are well known in the art. For example, humanized antibodies can be produced by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Methods that can be used to produce humanized antibodies are also described in U.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332 (Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No. 5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.), U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377 (Morgan et al.).

[0107] 5. Aptamers Aptamers are molecules that interact with target molecules in a specific manner. Typically, aptamers are small nucleic acids, 15-50 bases in length, that fold into defined secondary and tertiary structures, such as stem-loops and G-quartets. Aptamers can bind small molecules, such as ATP (U.S. Pat. No. 5,631,146) and theophylline (U.S. Pat. No. 5,580,737), as well as large molecules, such as reverse transcriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Aptamers can bind to 10 -12 k of the target molecule less than M d Aptamers can bind very strongly at 10 -6 , 10 -8 , 10 -10 , or 10 -12 Less than k dAptamers can bind to target molecules with a very high degree of specificity. For example, aptamers have been isolated that have binding affinities greater than 10,000-fold between a target molecule and another molecule that differs at only a single position on the molecule (U.S. Patent No. 5,543,293). Aptamers are characterized by a k d k with the target molecule that is at least 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold lower d For example, when the comparison is made with respect to a polypeptide, the background molecule is preferably a different polypeptide. Representative examples of methods for making and using aptamers that bind to a variety of different target molecules can be found in the following non-limiting list of U.S. Patents: 5,476,766; 5,503,978; 5,631,146; 5,731,424; 5,780,228; 5,792,613; 5,795,721; 5,84 Nos. 6,713, 5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698.

[0108] 6. Pharmaceutical Carriers / Drug Delivery As mentioned above, the composition can also be administered in vivo in a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the material is not biologically or otherwise undesirable, i.e., the material can be administered to a subject together with a nucleic acid or vector without causing any undesirable biological effects or interacting in any adverse manner with any of the other components of the pharmaceutical composition in which the material is contained. Of course, the carrier will be selected to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0109] Compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, externally, topically, etc., including topical administration or administration by inhalation. As used herein, "topical intranasal administration" refers to delivery of a composition to the nose and nasal cavity via one or both nostrils and may include delivery by a spray or droplet mechanism, or by aerosolization of the nucleic acid or vector. Administration of a composition by inhalation may be via the nose or mouth via delivery by a spray or droplet mechanism. Delivery may also be directly to any region of the respiratory system (e.g., the lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, the mode of administration, etc. Thus, it is not possible to specify an exact amount for every composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0110] Parenteral administration of compositions when used is generally characterized by injection.Injectable agents can be prepared in conventional form, either as liquid solution or suspension, solid form suitable for the solution of suspension in liquid before injection, or emulsion.The approach that has been revised recently for parenteral administration includes the use of sustained release or sustained release system, so that a constant dosage is maintained.For example, see U.S. Patent No. 3,610,795 (incorporated herein by reference).

[0111] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). Generally, receptors are involved in pathways of endocytosis, either constitutive or ligand-induced. These receptor clusters in clathrin-coated pits enter the cell via clathrin-coated vesicles and pass through acidified endosomes, where the receptors are sorted and then recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and control of receptor levels. Many receptors follow multiple intracellular pathways depending on the cell type, receptor concentration, ligand type, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0112] a) Pharmaceutically acceptable carrier The compositions containing the antibodies can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0113] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and concentration of the administered composition.

[0114] Pharmaceutical carriers are known to those skilled in the art. These are most commonly standard carriers for administering drugs to humans, including solutions such as sterile water, physiological saline, and buffered solutions at physiological pH. The composition can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0115] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surfactants, etc. Pharmaceutical compositions may also include one or more active ingredients such as antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0116] Pharmaceutical compositions may be administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including ophthalmically, vaginally, rectally, or intranasally), oral, by inhalation, or parenterally, for example, by intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0117] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases.

[0118] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable.

[0119] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0120] Some of the compositions may optionally be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl and aryl amines, and substituted ethanol amines.

[0121] b) Therapeutic use The effective dosage and schedule for administering the composition may be determined empirically, and making such determinations is within the skill of the art. The dosage range for administering the composition is sufficient to produce the desired effect in which the symptoms of the disorder are addressed. The dosage should not be so high as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, etc. In general, the dosage will vary depending on the patient's age, condition, sex, and extent of disease, the route of administration, or whether other drugs are included in the dosage regimen, and can be determined by one of ordinary skill in the art. The dosage can be adjusted by an individual physician if there are any adverse indications. The dosage can vary and can be administered in one or more doses per day for one or several days. Guidance regarding appropriate dosages for a given class of pharmaceuticals can be found in the literature. For example, guidance in selecting an appropriate dose of an antibody can be found in the literature on the therapeutic use of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Typical daily dosages of antibodies used alone can range from about 1 μg / kg body weight to up to 100 mg / kg body weight or more per day, depending on the factors mentioned above.

[0122] 7. Kit Disclosed herein are kits that link reagents that can be used in carrying out the methods disclosed herein. The kits can include any reagent or combination of reagents discussed herein or that would be understood to be necessary or beneficial in carrying out the disclosed methods and for generating the disclosed universal CAR, universal synNotch, universal CAR T cells, and / or universal synNotch cells. For example, the kits can include antibodies or fragments thereof and expression vectors discussed in certain embodiments of the methods and compositions, as well as necessary buffers and enzymes.

[0123] C. Treatment of Disease The disclosed conditional universal synNotch cells and conditional universal CAR systems disclosed herein can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer, autoimmune diseases, autoinflammatory diseases, and infectious diseases. Accordingly, in one aspect, disclosed herein is a method of treating, reducing, reducing, inhibiting, ameliorating, and / or preventing cancer and / or metastasis, autoimmune disorders, autoinflammatory disorders, and infectious diseases in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the engineered universal CAR systems and / or engineered conditional universal synNotch cells disclosed herein.

[0124] As used herein, "treat," "treating," "treatment," and grammatical variations thereof include administering a composition with the intent or purpose of partially or completely preventing, delaying, curing, curing, attenuating, mitigating, altering, repairing, ameliorating, stabilizing, alleviating, and / or reducing the intensity or frequency of one or more diseases or conditions, symptoms of a disease or condition, or underlying causes of a disease or condition. Treatment according to the present invention may be applied preventatively, prophylactically, palliatively, or therapeutically. Prophylactic treatment is administered to a subject pre-onset (e.g., before overt signs of cancer), at early onset (e.g., at early signs and symptoms of cancer), or after established onset of cancer. Prophylactic administration can occur days to years before the appearance of symptoms of disease or infection.

[0125] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. The term includes active treatment, i.e., treatment specifically aimed at ameliorating a disease, condition, or disorder, and also includes causal treatment, i.e., treatment aimed at eliminating the cause of the associated disease, condition, or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, pathological condition, or disorder; prophylactic treatment, i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of the associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment employed to complement another specific therapy aimed at ameliorating the associated disease, pathological condition, or disorder.

[0126] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, such as oral, topical, intravenous, subcutaneous, transdermal, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, intracerebroventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, intravaginal, by inhalation, via an implanted reservoir, or parenterally (e.g., subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques). As used herein, "concurrent administration," "co-administration," "simultaneous administration," or "administering simultaneously" means that compounds are administered at the same time or essentially immediately after each other. In the latter case, the two compounds are administered sufficiently close in time that the results observed are indistinguishable from those obtained if the compounds were administered at the same time in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, via an entry into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to an area or areas immediately adjacent to the point of administration, and does not introduce the agent into the subject's body in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the point of administration, but is undetectable or detectable in negligible amounts in distal portions of the subject's body. Administration includes self-administration and administration by another.

[0127] The term "therapeutically effective" refers to the amount of composition used being sufficient to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration need only reduce or alter, not necessarily eliminate, one or more of the causes or symptoms of a disease or disorder.

[0128] As noted above, the disclosed CAR systems and / or synNotch cells can be used to treat, reduce, decrease, inhibit, ameliorate, and / or prevent cancer and / or metastasis in a subject. A non-limiting list of various types of cancer that can be treated through administration of the disclosed universal CAR T cells and / or universal synNotch cells is as follows: lymphoma (Hodgkin's and non-Hodgkin's), leukemia, carcinoma, carcinoma of solid tissue, squamous cell carcinoma, adenocarcinoma, sarcoma, glioma, high-grade glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, adenoma, hypoxic tumor, myeloma, AIDS-related lymphoma or sarcoma, metastatic cancer, or cancer in general.

[0129] A representative, but non-limiting list of cancers that the disclosed compositions can be used to treat are lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, including small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck carcinoma, large bowel cancer, hematopoietic cancer; testicular cancer; colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0130] The compounds disclosed herein may also be used to treat precancerous conditions such as cervical and anal dysplasia, other dysplasias, severe dysplasias, hyperplasias, atypical hyperplasias, and neoplasias.

[0131] It is contemplated herein that the disclosed methods of inhibiting, alleviating, and / or treating cancer can include administering any anti-cancer agent known in the art, including, but not limited to, abemaciclib, abiraterone acetate, avitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, trastuzumab emtansine (Ado-Trastuzumab Emtansine), Adriamycin (doxorubicin hydrochloride), afatinib maleate, Afinitor (everolimus), Aquinzeo (netupitant palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alectinib), alectinib, alemtuzumab, Alimta (pemetrexed disodium), Alicopa (copanlisib hydrochloride), Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alunbrig (brigatinib), Ambochlorin (chlorambucil), Amifostine, Aminolevulinic acid, Anastrozole, aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Alanon (nelarabine), arsenic trioxide, Arzera (ofatumumab), asparaginase erwinia chrysanthemum, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Besenam (carmustine), Beleodac (belinostat), belinstat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I) 131 Tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Bilincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, BuMel, busulfan, Busulfex (busulfan), cabazitaxel,Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Campus (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carlac (topical fluorouracil), carboplatin, carboplatin-taxol, carfilzomib, Carumbris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Chloral (clofarabine), CMF, cobimetinib, Cometriq (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), Crizolib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab) , cytarabine, cytarabine liposome, Cytosar U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darazalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposome, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox, denosumab, DepoCyt (cytarabine liposome), dexamethasone Tazone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposomal), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, Dox-SL (doxorubicin hydrochloride liposomal), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil-topical), ERYTECH (rasburicase), Elence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab),Enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinase (asparaginase erwinia chrysanthemum), Ethiol (amifostine), Etopofos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride) Ran), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Fairston (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, FOLOTYN (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nonvalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Giotrif (afatinib dimaleate), Gleevec (imatinib mesylate) acid salt), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemandiol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV nonvalent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride),Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, ifosfamide (ifosfamidum), IL-2 (aldesleukin), imatinib mesylate, Imbruvica (ibrutinib), Imfinzi (durvalumab), imiquimod, Imlijiq (talimogene laherparepvec), Inlyta (axitinib), inotuzumab ozogamicin, interferon alfa-2b, recombinant, interleukin-2 (aldesleukin), Intron A (recombinant interferon alfa-2b), iodine I 131 Tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposomal, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakavi (ruxolitinib), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine) Syn), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Raltruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate) Leuprolide acetate), Letrozole, Leucovorin calcium, Leukeran (chlorambucil), Leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), Lipodox (doxorubicin hydrochloride liposome), Lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Leupron Depot (leuprolide acetate), Leupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marquibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, mesna,Mesnex (Mesna), Metazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone Bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone Hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Ilotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant and palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Neulasta (pegfilgrastim), Neupogen (filgrastim) stim), Nexavar (sorafenibut tosylate), Nilandrone (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), N-Plate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepesuxinate, Oncaspar (peguaspargase), ondansetron hydrochloride, Onibide (irinotecan hydrochloride liposomal), Onta Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgrastim, peginterferon alfa-2b,PEG-Intron (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portraza (necitumumab), , pralatrexate, prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Pralia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenzi (sipuleucel-T), Purintoll (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human Human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonvalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenamidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hycela (rituximab and human hyaluronidase), rituximab, rituximab and human hyaluronidase, rolapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), rucaparib camsylate, ruxolitinib phosphate, Rydapt (midostaurin), Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon alfa-2b), Silvant (Siltuximab), Synribo (Omacetaxine Mepesuxinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib) Nib), Tagrisso (osimertinib), talc, talimogene laherparepvec, tamoxifen citrate, Tarabin PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide),Temozolomide, temsirolimus, thalidomide, Thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (fluorouracil-topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab, and iodine I 131 Tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosilate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Varubi (rolapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban ( Vinblastine sulfate), Velcade (bortezomib), Versal (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasal PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triphosphate) triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposomal), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabeculopeptide Ctedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zedula (niraparibut tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib),Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). Also contemplated herein are chemotherapeutic agents that are PD1 / PDL1 blockade inhibitors (e.g., lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab, or avelumab).

[0132] As described above, the disclosed conditional universal synNotch cells and / or conditional universal CAR systems can be used to treat autoimmune diseases (i.e., a range of diseases, disorders, or conditions resulting from an adaptive immune response (T cell and / or B cell response) against a host organism). Examples of autoimmune diseases include, but are not limited to, achalasia, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposity dolorosa, adult Still's disease, agammaglobulinemia, alopecia areata, Alzheimer's disease, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, aplastic anemia, autoimmune angioedema, autoimmune autonomic failure, autoimmune encephalomyelitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, and autoimmune inner ear disease (AIDS). ED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune polyendocrine syndrome, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN), Balo's disease, Behçet's disease, benign mucous membrane pemphigoid, Bickerstaff's brainstem encephalitis, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas' disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multiple myelitis (CRMO), Churg-Strauss disease, Cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), diabetes mellitus type 1, discoid lupus, Dressler's syndrome, endometriosis, enthesitis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, fibromyalgia, pulmonary fibrosis fibrosis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne inverse), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC),Inflammatory bowel disease (IBD), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus nephritis, lupus vasculitis, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, Ord's thyroiditis thyroiditis), relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), facial hemifacial atrophy, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II Type III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis These include: rheumatic fever, rheumatoid arthritis, rheumatoid vasculitis, sarcoidosis, Schmidt syndrome, Schnitzler syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm & testicular autoimmunity, stiff-person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, Sydenham chorea, sympathetic ophthalmia (SO), systemic lupus erythematosus, systemic scleroderma, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), urticaria, urticarial vasculitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)). ,

[0133] Also disclosed herein is a method of treating an autoinflammatory disease (i.e., a disorder in which the innate immune response attacks host cells), comprising administering the universal CAR T cells and / or universal synNotch cells disclosed herein to a subject with the autoinflammatory disease. Examples of autoinflammatory diseases include asthma, graft-versus-host disease, allergies, transplant rejection, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), neonatal-onset multisystem autoinflammatory disease (NOMID) (also known as chronic infantile neurological, cutaneous, and articular syndrome (CINCA)), familial Mediterranean fever (FMF), tumor necrosis factor (TNF)-associated periodic syndrome (TRAPS), TNFRSF11A-associated hereditary fever disorder (TRAPS11), hyperimmunoglobulinemia with periodic fever syndrome D (HIDS), mevalonic aciduria (MA), mevalonate kinase deficiency (MKD), interleukin-1β (IL-1β) receptor antagonist (DIRA) deficiency (osteomyelitis, pustular periostitis), and inflammatory bowel disease (periostitis). (also known as sterile multifocal with pustulosis), Majeed syndrome, chronic nonbacterial osteomyelitis (CNO), early-onset inflammatory bowel disease, diverticulitis, interleukin-36 receptor antagonist deficiency (DITRA), familial psoriasis (PSORS2), pustular psoriasis (15), suppurative sterile arthritis, pyoderma gangrenosum, and acne syndrome (PAPA), hereditary sideroblastic anemia with immunodeficiency, fever, and developmental delay (SIFD), pediatric granulomatous arthritis These include inflammatory bowel disease (PGA), familial Behçet's-like autoinflammatory syndrome, NLRP12-associated periodic fever syndrome, proteasome-associated autoinflammatory syndrome (PRAAS), spondylochondroplasia with immune dysregulation (SPENCDI), STING-associated vasculitis of infantile onset (SAVI), Aicardi-Goutières syndrome, acute febrile neutrophilic dermatosis, X-linked familial hemophagocytic lymphohistiocytosis, and Lyn kinase-associated autoinflammatory disease (LAID).

[0134] As described above, the disclosed conditional universal CAR systems and conditional universal synNotch cells can be used to treat diseases resulting from bacterial, viral, fungal and / or parasitic infections.

[0135] In one embodiment, the infection to be treated is herpes simplex virus-1, herpes simplex virus-2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus-6, smallpox virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus (e.g., avian coronavirus (IBV), porcine epidemic diarrhea virus (PEDV), porcine respiratory coronavirus), or the like. Pregnancy-related pulmonary virus (PRCV), transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV), feline infectious peritonitis virus (FIPV), feline enteric coronavirus (FECV), canine coronavirus (CCoV), rabbit coronavirus (RaCoV), mouse hepatitis virus (MHV), rat coronavirus (RCoV), rat sialoadenitis virus (SDAV), bovine coronavirus (BCoV), bovine enterovirus (BEV), porcine coronavirus HKU15 (PorCoV) HKU15), porcine epidemic diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus (HEV), turkey bluecomb coronavirus (TCoV), human coronavirus (HCoV)-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, severe acute respiratory syndrome (SARS)-coronavirus (CoV) (SARS-CoV), severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 (SARS-CoV-2) (including, but not limited to, the B1.351 variant, B.1.1.7 variant, USA-WA1 / 2020, or P.1 variant), or may be the result of infection with a virus selected from the group consisting of Middle East Respiratory Syndrome (MERS) coronavirus (CoV) (MERS-CoV), influenza virus A, influenza virus B, measles virus, polyomavirus, human papillomavirus, respiratory syncytial virus, adenovirus, coxsackievirus, dengue virus, mumps virus, poliovirus, rabies virus, Rous sarcoma virus, reovirus, yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type-1, and human immunodeficiency virus type-2.

[0136] Manufacturers include Mycobaterium tuberculosis, Mycobaterium bovis, Mycobaterium bovis for BCG, BCG for Mycobaterium avium. Mycobaterium intracellular. Mycobaterium africanum. Mycobaterium kansasii. Mycobaterium marinum. Mycobaterium ulcerans pneumophila, some Legionella species, Salmonella typhi, Salmonella enterica, some Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri , some Shigella , Yersinia pestis , Pasteurella haemolytica , Pasteurella multocida , some Pasteurella , Actinobacillus pleuropneumoniae , Listeria monocytogenes , Listeria ivanovii , Brucella abortus, some Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii, a Bordetella agent, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, ChlamydiaThe infection may be the result of an infection with a bacterium selected from the group consisting of: Staphylococcus psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species. In one aspect, the bacterium is not Bacillus anthracis.

[0137] In another aspect, the infectious disease being treated can be the result of an infection with a fungus selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata.

[0138] In another embodiment, the infectious disease to be treated is selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma It may be the result of infection with a parasite selected from the group of parasites consisting of Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.

[0139] D. Regenerative Medicine It is understood and contemplated herein that the disclosed conditional universal SynNotch receptors have uses that are not limited to the treatment of cancer, autoimmune diseases, autoinflammatory diseases, or infectious diseases. In one aspect, the disclosed conditional universal SynNotch can be used in cell and tissue engineering efforts (i.e., regenerative medicine) to restore or establish normal cell, tissue, and / or organ function. As noted above, in one aspect, disclosed herein are engineered cells (e.g., immune cells, neurons, epithelial cells, and endothelial cells, or stem cells, etc.) that comprise any of the conditional universal synNotch receptors disclosed herein. In one aspect, disclosed herein are engineered cells that further comprise a vector comprising a transcription response element operably linked to a promoter that drives expression of one or more cellular response genes (e.g., IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin, etc.), wherein one or more of the transcription factors on the synNotch receptor are specific for the transcription response element. Also disclosed herein are engineered cells, wherein one or more transcription factors of a conditional universal synNotch receptor activate the expression of one or more natural cellular response genes (e.g., IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin). In one aspect, disclosed herein is the use of such engineered cells to restore or establish normal cell, tissue, and / or organ function. Various embodiments of the present invention are described below. 1. A conditional universal chimeric antigen receptor (CAR) system comprising: i) a CAR comprising a receptor that targets a tag ligand, a hinge domain, and a signaling domain on a conditional adapter molecule; and ii) a conditional adapter molecule comprising an antigen recognition element, a stimulatory response group, and a tag ligand. 2. The conditional universal CAR system of claim 1, wherein the tag ligand on the conditional adapter molecule comprises benzylguanine (BG), benzylcytosine (BC), a chloroalkane, fluorescein (FITC), SpyTag, leucine zipper, La-SS-B, CD19, an anti-folate receptor antibody, an Fc domain, a peptide neoepitope (PNE), or biotin. 3. The conditional universal CAR system of claim 1, wherein the conditional adapter molecule comprises NHS-ester conjugation, disulfide restapling, glycan conjugation chemistry, recombinant antibodies with tagged ligands incorporation via one or more short peptide tags, sortase-mediated ligation, THIOMAB, chemical ligation, split inteins, and / or unnatural amino acids. 4. The conditional universal CAR system described in 3 above, wherein the antigen recognition element comprises an antibody or an antigen-recognizing fragment thereof. 5. The conditional universal CAR system of claim 4, wherein the antigen recognition element comprises rituximab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacizumab, or an antigen-binding fragment of any one thereof. 6. The conditional universal CAR system of claim 3, wherein the antigen recognition element comprises a protein binding domain, a lectin, a DNA aptamer, an RNA aptamer, a small molecule ligand of a cell surface receptor, or a peptide / protein ligand of a natural protein receptor. 7. The stimulus to which the stimuli-reactive group is reactive is light, enzyme activity, small molecules, pH, H 2 O 2 7. A conditional universal CAR system according to any one of 1 to 6 above, comprising hypoxia, and / or ROS. 8. A conditional universal CAR system according to any one of 1 to 7 above, wherein the stimuli-reactive group comprises a cleavable linker. 9. The conditional universal CAR system according to claim 8, wherein the stimuli-reactive group comprises a photocleavable linker or a phosphine-cleavable linker. 10. A conditional universal CAR system described in any one of 1 to 9 above, wherein the stimulus-responsive group comprises a stimulus-responsive caging group that blocks the CAR from binding to the tag ligand. 11. The conditionally universal CAR system of claim 10, wherein the stimuli-reactive group comprises a photoreactive caging group comprising nitrobenzyl, coumarin, BODIPY, or cyanine. 12. A conditional universal CAR system described in any one of 1 to 10 above, wherein the stimulus-reactive group comprises a photoreactive group and the wavelength of the light stimulus comprises 365, 405, 544, or 780 nm. 13. The stimulatory reactive group comprises an enzyme reactive group, and the enzyme to which the reactive group is reactive is legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase A (KHK-A), hexokinase (HK), nucleoside diphosphate kinase (NDPK or NDK), and A conditional universal CAR system described in any of 1-7 above, comprising 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase. 14. A conditional universal CAR system according to any one of 1 to 7 above, wherein the stimuli-reactive group is reactive to small molecules including phosphines or tetrazines. 15. A conditional universal CAR system according to any one of 1 to 14 above, further comprising one or more costimulatory domains. 16. The conditional universal CAR system according to claim 15, wherein the one or more costimulatory domains comprise a signaling domain of CD27, CD28, ICOS, 4-1BB, or OX40. 17. A conditional universal CAR system according to any of 1 to 15 above, wherein the tag ligand that targets the CAR is comprised on a CAR T cell, a CAR NK cell, a CAR NK T cell, a CAR B cell, or a CAR macrophage. 18. A conditional universal synthetic Notch (synNotch) receptor comprising a conditional adaptor molecule comprising a stimulus-responsive group and a tag, a notch core comprising one or more cleavage sites, and one or more transcription factors. 19. The conditional universal synNotch of claim 18, wherein the conditional adapter molecule comprises a tag ligand comprising benzylguanine (BG), benzylcytosine (BC), a chloroalkane, fluorescein (FITC), SpyTag, a leucine zipper, La-SS-B, CD19, an anti-folate receptor antibody, an Fc domain, a peptide neoepitope (PNE), or biotin. 20. The conditional universal synNotch of claim 18 or 19, wherein the adapter molecule comprises NHS-ester conjugation, disulfide restapling, glycan conjugate chemistry, recombinant antibodies with tag incorporation via one or more short peptide tags, sortase-mediated ligation, chemical ligation, split intein, THIOMAB, and / or unnatural amino acids. 21. The stimulus to which the stimulus-reactive group is reactive is light, an enzyme, a small molecule, pH, hypoxia, H 2 O 2 21. The conditional universal synNotch according to any one of 18 to 20 above, comprising: 22. The conditional universal synNotch described in any one of 18 to 21 above, wherein the stimulus-responsive group comprises a stimulus-cleavable linker. 23. The conditional universal synNotch according to claim 22, wherein the stimuli-responsive group comprises a photocleavable linker or a phosphine-cleavable linker. 24. A conditional universal synNotch according to any one of claims 18 to 21, wherein the stimulus-responsive group comprises a stimulus-responsive caging group that blocks the receptor from binding to the tag ligand. 25. The conditional universal synNotch described in any of 18 to 21 above, wherein the stimuli-responsive group comprises a photoreactive caging group comprising nitrobenzyl, coumarin, BODIPY, or cyanine. 26. The conditional universal synNotch according to claim 25, wherein the stimulus-responsive group comprises a photoreactive group and the wavelength of the light stimulus comprises 365, 405, 544, or 780 nm. 27. The stimulatory reactive group includes an enzyme reactive group, and the enzyme with which the reactive group reacts is legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase A (KHK-A), hexokinase (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphodiesterase (6-phosphodiesterase). A conditional universal synNotch as described in any of 18-21 above, including phosphofructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase. 28. A conditional universal synNotch according to any one of 18 to 21 above, wherein the stimuli-responsive group is reactive to a small molecule comprising a phosphine or tetrazine. 29. The conditional universal synNotch according to any one of 18 to 28 above, wherein the transcription factor comprises Gal4-VP64, Gal4-VP16, TetR-VP64, or LacI-VP64. 30. A conditional universal synNotch according to any one of claims 18 to 29, further comprising an antigen recognition element, said antigen recognition element being capable of becoming covalently linked to said conditional universal adaptor molecule. 31. The conditional universal synNotch according to any one of 18 to 30 above, wherein the antigen recognition element comprises an antibody or an antigen-recognizing fragment thereof. 32. The conditional universal synNotch described in any one of 18 to 31 above, wherein the antigen recognition element comprises rituximab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacizumab, or an antigen-binding fragment of any one thereof. 33. The conditional universal synNotch according to any of claims 18 to 32, wherein the antigen recognition element comprises a protein binding domain, a lectin, a DNA aptamer, an RNA aptamer, a small molecule ligand of a cell surface receptor, or a peptide / protein ligand of a natural protein receptor. 34. An engineered cell comprising a conditional universal CAR according to any one of 1 to 17 above and / or a conditional universal synNotch according to any one of 18 to 33 above. 35. The engineered cell of claim 34, further comprising a vector comprising a transcription response element operably linked to a promoter that drives expression of one or more cellular response genes, wherein one or more of the transcription factors on the synNotch receptor are specific for the transcription response element. 36. The engineered cell of claim 34 or 35, wherein the one or more response genes include IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin. 37. The engineered cell according to any one of claims 34 to 36, wherein one or more transcription factors of said conditional universal synNotch receptor activate expression of one or more natural cellular response genes. 38. The engineered cell of claim 37, wherein the one or more natural cellular response genes comprise IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin. 39. The engineered cell according to any one of 34 to 36 above, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. 40. A method of treating a disease or disorder in a subject, said method comprising administering to said subject a conditional universal chimeric antigen receptor (CAR) system as described in any one of 1 to 17 above, a conditional SynNotch as described in any one of 18 to 33 above, and / or an engineered cell as described in any one of 34 to 39 above, wherein said disease or disorder comprises cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection. 41. A method for treating a disease or disorder according to claim 40, wherein the disease is a cancer selected from the group consisting of lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck carcinoma, large bowel cancer, hematopoietic cancer; testicular cancer; colon cancer, rectal cancer, prostate cancer, and pancreatic cancer. 42. The disease is achalasia, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposity dolorosa, adult Still's disease, agammaglobulinemia, alopecia areata, Alzheimer's disease, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, aplastic anemia, autoimmune angioedema, autoimmune autonomic failure, autoimmune encephalomyelitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, or autoimmune polymyelitis. Endocrine syndromes, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN), Balo's disease, Behçet's disease, benign mucous membrane pemphigoid, Bickerstaff's brainstem encephalitis, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas' disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multiple myelitis (CRMO), Churg-Strauss syndrome (CSS), eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multiple myelitis (CRMO), M.D. disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), diabetes mellitus type 1, discoid lupus, Dressler's syndrome, endometriosis, enthesitis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, fibromyalgia, pulmonary fibrosis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), Herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (acne inverse), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), inflammatory bowel disease (IBD), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease (LAD), lupus nephritis, lupus vasculitis, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA),Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, Ord's thyroiditis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), facial hemifacial atrophy, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome types I, II, and III, rheumatoid arthritis Polymyalgia machinosum, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, rheumatoid arthritis 41. A method for treating a disease or disorder according to claim 40, wherein the disease or disorder is an autoimmune disease selected from the group consisting of: thyroid vasculitis, sarcoidosis, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff-person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, Sydenham's chorea, sympathetic ophthalmia (SO), systemic lupus erythematosus, systemic scleroderma, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), urticaria, urticarial vasculitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)). 43. The disease is asthma, graft-versus-host disease, allergy, transplant rejection, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), neonatal-onset multisystem autoinflammatory disease (NOMID) (also known as chronic infantile neurological, cutaneous, and articular syndrome (CINCA)), familial Mediterranean fever (FMF), tumor necrosis factor (TNF)-associated periodic syndrome (TRAPS), TNFRSF11A-associated hereditary fever disorder (TRAPS11), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), mevalonic aciduria (MA), mevalonate kinase deficiency (MKD), interleukin-1β (IL-1β) receptor antagonist (DIRA) deficiency (also known as sterile multifocal osteomyelitis, pustular periostitis), Majeed syndrome, chronic nonbacterial osteomyelitis (CNO), early 41. A method for treating the disease or disorder described in claim 40, wherein the autoinflammatory disease is selected from the group consisting of onset inflammatory bowel disease, diverticulitis, interleukin-36 receptor antagonist deficiency (DITRA), familial psoriasis (PSORS2), pustular psoriasis (15), suppurative sterile arthritis, pyoderma gangrenosum, and acne syndrome (PAPA), hereditary sideroblastic anemia with immunodeficiency, fever, and developmental delay (SIFD), childhood granulomatous arthritis (PGA), familial Behcet's-like autoinflammatory syndrome, NLRP12-associated periodic fever syndrome, proteasome-associated autoinflammatory syndrome (PRAAS), spondylochondroplasia with immune dysregulation (SPENCDI), STING-associated vasculitis of infantile onset (SAVI), Aicardi-Goutieres syndrome, acute febrile neutrophilic dermatosis, X-linked familial hemophagocytic lymphohistiocytosis, and Lyn kinase-associated autoinflammatory disease (LAID). 44. The disease is caused by herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus-6, smallpox virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus, influenza A virus, influenza B virus, measles virus, polyomavirus, human papillomavirus, respiratory syncytial virus, adenovirus, coxsackievirus, dengue virus, mumps virus, poliovirus, or rabies virus. 41. A method for treating a disease or disorder described in claim 40, wherein the viral infection is selected from the group consisting of: rabies virus, Rous sarcoma virus, reovirus, yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1, and human immunodeficiency virus type 2. 45. The above diseases are caused by Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG subspecies, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, Legionella pneumophila, Salmonella typhi, Salmonella enterica, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, Yersinia pestis, Pasteurella haemolytica、Pasteurella multocida、Actinobacillus pleuropneumoniae、Listeria monocytogenes、Listeria ivanovii、Brucella abortus、Cowdria ruminantium、Borrelia burgdorferi、Bordetella avium、Bordetella pertussis、Bordetella bronchiseptica、Bordetella trematum、Bordetella hinzii、Bordetella pteri、Bordetella parapertussis、Bordetella ansorpii, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, Clostridium tetani, and Yersinia 41. A method for treating a disease or disorder as described in claim 40, wherein the disease or disorder is a bacterial infection selected from the group consisting of: Bacillus subtilis, Bacillus casei, Bacillus subtilis, Bacillus subtilis, Bacillus spp. ... 46. ​​A method for treating a disease or disorder according to claim 40, wherein the disease is a fungal infection selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata. 47. A method for treating a disorder or disease described in any of claims 40 to 46, wherein the method comprises administering to the subject a first conditional universal CAR system described in any of claims 1 to 17 and a second conditional universal CAR system described in any of claims 1 to 17, wherein the first conditional universal CAR system comprises a stimulus-responsive group that includes a stimulus-cleavable linker, and the second CAR system comprises a stimulus-responsive caging group that blocks binding of the CAR to the tag. 48. A method for treating a disorder or disease according to any one of claims 40 to 46, wherein the first conditional universal CAR system and the second conditional universal CAR system are responsive to the same stimulus. 49. A method for treating a disorder or disease according to any one of claims 40 to 46, wherein the first conditional universal CAR system and the second conditional universal CAR system are responsive to different stimuli. [Example]

[0140] E. Working Example The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices, and / or methods claimed herein, and are intended to be purely illustrative and are not intended to limit the present disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

[0141] 1. Example 1: SNAP CAR T cells and SNAP synNotch receptor Recently, we developed SNAP CARs and SNAP synNotch universal receptors, which can post-translationally target BG-conjugated antibody adaptors to multiple antigens of interest (Figure 3). These universal receptors form covalent bonds with antibodies, enabling highly potent retargeting of receptor activity at low concentrations of antibody adaptors. The development of SNAP receptors has been directly integrated into our work to create high-affinity biotin-conjugated receptor CARs using monomeric streptavidin 2 protein (mSA2), which is activated by biotinylated antibodies bound to their target antigens. Compared to previous biotin-conjugated CARs, the stronger activity due to the higher affinity of mSA2 led us to reason that even higher affinity interactions, particularly covalent conjugation, would yield superior results. Indeed, SNAP receptors demonstrated significantly improved levels of T cell-induced lysis and signaling. Furthermore, we obtained functional SNAP-synNotch systems, whereas mSA2 was ineffective, further supporting the importance of covalent attachment.

[0142] a) Adaptor targeting of SNAP CAR T cell activation. We constructed stably transduced primary human T cells with a universal SNAP-CAR that activates T cell signaling when labeled with a BG-conjugated adaptor antibody on the surface of target cells (Figure 4A). To generate the adaptor antibody, BG-N-hydroxysuccinimidyl (NHS) ester was conjugated to the lysine and / or N-terminus of several clinically relevant antibodies, such as rituximab, FMC63, Herceptin, and cetuximab. Without positional control, an average of two BG motifs per antibody was determined from the labeling reaction.

[0143] To test the functionality of the CAR, we co-incubated SNAP-CAR T cells with various antigen-positive or -negative target tumor cell lines and the BG antibody adaptor for 24 hours. We found that the antibody-BG adaptor induced activation of SNAP-CAR T cells (e.g., T cell activation markers), interferon gamma cytokine production, and CAR cell-specific lysis of antigen-positive target cells (Figure 4B, C, D). These outputs depended on the precise antibody-target cell combination, demonstrating the ability to reprogram T cell specificity. Importantly, we also found that SNAP CAR T cells can simultaneously target multiple antigens from different cell populations. This could be used to prevent cancer recurrence due to antigen heterogeneity or antigen loss in experiments where SNAP CAR T cells were co-cultured with a mixture of CD20+ and EGFR+ target cells and cell lysis was assayed with a single anti-CD20 or anti-EGFR antibody, or a combination thereof.

[0144] b) Function of SNAP-CAR T cells in animal models To demonstrate the feasibility of in vivo therapy using SNAP-CAR T cells, we conducted experiments to confirm the covalent attachment of the SNAP receptor to the antibody adapter in mice and evaluated its antitumor function. The adapter and SNAP CAR T cells were injected into NOD-SCID g-chain-deficient (NSG) immunodeficient mice, which can receive human cells, by various routes (intraperitoneal adapter and intravenous CAR T cells). One day later, we analyzed SNAP CAR T cells in the blood of the mice and observed efficient chemical labeling of SNAP-CAR T cells with the adapter (Figure 28A). Next, we performed the first human tumor xenograft model experiment to examine tumor regression. At day 14, we observed significant regression or delay of tumor growth in mice receiving SNAP CAR T cells and the adapter compared with SNAP-CAR T cells without the adapter, which was comparable to or better than the anti-CD20 CAR positive control (Figure 28B).

[0145] c) SNAP synadaptor targeting of Notch receptor activation. We engineered a universal adaptor, synNotch, that directs receptor activation and generates transcriptional gene output in response to interaction with a BG-conjugated adaptor antibody on the surface of target cells (Figure 22). We cloned a SNAP-synNotch receptor consisting of a SNAPtag domain fused to the Notch core protein and the Gal4 transcription factor (to turn on the TagBFP gene driven by the Gal4 promoter) and transduced it into Jurkat cells. Next, we co-incubated these cells with a BG-conjugated anti-CD19 antibody and CD19-(+) or -(-) tumor cells, and observed significant upregulation of TagBFP in CD19(+) target cells. Receptor activation was highly sensitive and tunable, showing a significant response at antibody concentrations as low as 0.04 μg / mL and peaking at 0.25 μg / mL. Subsequently, response gene activation decreased with increasing antibody concentrations, demonstrating a "hook effect," as expected for ternary complex formation. BG-conjugated antibodies targeting other antigens could also activate the SNAP-synNotch receptor in an antigen-specific manner, demonstrating the universality of the cell reprogramming approach. They also revealed the modularity of the SNAP-synNotch output, as it could express IL-7-responsive genes, candidate therapeutic genes of interest, for their ability to promote T cell proliferation.

[0146] d) Photocleavable and small molecule cleavable OFF switch adaptors. To create a prototype OFF-switch adaptor (see Figure 2), photocleavable biotin 1 was conjugated to an anti-CD20 antibody via NHS ester chemistry to form a carbamate with an amino group on the protein (Figure 5). The assembled adaptor was fully active and deactivated mSA2 CAR T cell signaling in coculture with antigen (+) tumor cells after only 30 seconds of 365 nm exposure, as assessed by CD69 and CD62L activation marker expression.

[0147] To demonstrate small-molecule-controlled OFF-switching, we developed a nine-step synthesis of phosphine-cleavable biotin NHS ester 2 containing an aryl azide linker (Figure 6A). Next, we conjugated linker 2 to an anti-CD20 antibody. To test the adaptor's ability to function on target cells, we co-incubated CD20+ cells with varying amounts of the OFF-switch antibody and the small-molecule trigger 2DPBM ((diphenylphosphino)-2-benzamide). 2DPBM was chosen based on previous work using a phosphine as a small-molecule trigger. In the presence of the adaptor and CD20+ target cells, we observed robust activation of primary mSA2 CAR T cells, and importantly, inhibition of this signal was observed upon addition of 2DPBM (Figure 6B). This demonstrates that adaptor CAR activity conditionally switches OFF in response to light and small-molecule treatment.

[0148] Cleavage of the biotin motif was tested in CD20+ target cells in response to 365 nm light titration and staining with fluorescently labeled streptavidin (Figure 7C). In the absence of light exposure, the biotin adapter bound to streptavidin. Furthermore, exposure for as little as 30 seconds was sufficient to completely deactivate the adapter. We also observed a robust decrease in biotin staining that correlated with small molecule drug and antibody concentrations, demonstrating tunable cell labeling (Figure 8C). This demonstrates that adapter CAR activity can be conditionally switched off in response to light and small molecule treatment.

[0149] e) Light and small molecule activated ON switch adaptor. To fabricate a prototype light-triggered ON-switch adapter (see Figure 2), we synthesized photoactivatable biotin sulfo-NHS ester 3 (Figure 7A). The caging group was prepared in one step from 4,5-(methylenedioxy)-2-nitrobenzaldehyde in 86% yield, followed by assembly of 1 in three steps with an average yield of 63%. The sulfonate moiety was incorporated to enhance water solubility for subsequent conjugation to an anti-CD20 antibody. Uncaging of the biotin motif was tested on CD20+ target cells in response to 365 nm light titration and staining with fluorescently labeled streptavidin (Figure 7B). Without light exposure, the caged biotin adapter failed to bind to streptavidin, and flow cytometry results were indistinguishable from those of an antibody control without the adapter. Furthermore, only 30 seconds of exposure was required to fully activate the adapter.

[0150] Here, we again constructed a small molecule triggered ON switch based on the Staudinger reduction, synthesizing aryl azide-caged biotin NHS ester 4 (Figure 8A). The caging group was prepared in two steps from 4'-aminoacetophenone in an average yield of 93%, followed by the synthesis of phosphine-activated biotin NHS ester 4 in three steps. While only the ortho isomer is shown, the para isomer was also synthesized. Interestingly, when incubated at room temperature in PBS at pH 7.4, the carbamate of the para compound was completely hydrolyzed within 24 hours, while the ortho analog remained stable (>90%) for 1 week and was therefore selected.

[0151] We next conjugated the stable caged biotin 3 to an anti-CD20 antibody and tested its response to 2DPBM. We observed a robust increase in biotin staining that correlated with the small molecule drug and antibody concentrations, demonstrating tunable cell labeling (Figure 8B). This indicates that the adapter activity is conditionally switched on in response to light and small molecule treatment.

[0152] 2. Example 2: Conditional control of universal antigen receptor signaling by an OFF switch adaptor We have developed a general method for creating adaptor safety switches that can turn off CAR or synNotch receptor signaling using small molecules or light, thereby enabling transient and localized mitigation of toxicity caused by unwanted receptor activation. Light is a dynamically controlled stimulus that can be restricted to target tissues by several methods, including fiber-optic probes in standard endoscopic procedures, fluorescence-based systems (used in image-guided tumor resection), and irradiation of circulating blood (without affecting the organ site). Light-controlled OFF switches can be implemented to protect sensitive anatomical sites known to be positive for the target antigen by light exposure to these areas, while still treating distant metastatic lesions. HER2 and EGFR are examples of tumor antigens that are also expressed on various normal epithelial subsets and are candidates for OFF-tumor / ON-target toxicity, which has resulted in severe toxicity for CAR T cells. Small-molecule-triggered OFF switches have the complementary advantage of being routinely administered and allowing rapid, complete systemic receptor deactivation, including tissue sites inaccessible by optical probes, providing additional safety switch functionality (Figure 9). This universal adapter format allows some control of CAR activity through antibody administration. However, the average half-life of IgG antibodies is 10–21 days, and while different antibody engineering approaches can shorten or extend this time, it still does not allow for the rapid termination required to mitigate toxicity, which can occur as quickly as 15–30 minutes after injection. Unlike cell suicide switches, the most clinically advanced control approach for CAR T cell therapy, OFF-switch adapters allow for reversible control by administering additional (possibly different) adapter antibodies, without eliminating engineered cells for potential reactivation. We found that SNAP receptor and antibody conjugates can be recycled from the cell surface and covalently attached to new adapters over 1–2 days (even more rapidly) after receptor activation. The adapter OFF-switch is constructed on a photocleavable and phosphine-cleavable linker, a finding supported by results for the biotin OFF-switch adapter in mSA2-CAR.In addition to the OFF switch, two different approaches for generating site-specific antibody conjugates can be tested, with the goal of creating homogeneous adapters for predictable reagent behavior and suitable for structure-activity relationship (SAR) studies. Homogeneous adapters will facilitate the optimization of these approaches and provide a pathway toward the long-term goal of clinically testing conditionally controlled universal antigen receptor methodologies.

[0153] a) Synthesis and validation of site-specific adapter conjugates. To date, adapter antibodies have been generated by the reaction of protein surface lysines, resulting in a heterogeneous mixture of antibody conjugates that differ in the number and location of modifications. Because the synaptic distance between CAR T cells and target cells has been shown to be a critical factor in the signaling strength of both CAR and synNotch receptors, heterogeneous adapters may lead to suboptimal receptor activation. Using the rigorously tested chemistry described below, we engineered site-specific introduction of adapter molecules. Based on the results, we tested enzyme-conjugated BGs via PEG spacer arms of various lengths, including two, four, or six PEGs, to optimize the spacer length for each antibody / antigen. Each approach was evaluated using four clinically approved antibodies targeting the antigens CD19, CD20, EGFR, and HER2. The synthesis and implementation of these reagents are described below. After conjugation to each antibody, the antibody conjugates were verified by mass spectrometry and purified using a buffer exchange column. Conjugation yields for bis-sulfones average 30%, while conjugation yields for more recent dibromopyrazidinedione are typically quantitative. The selected conjugation approach allows for the use of commercially available FDA-approved antibodies without the need to engineer new sites for conjugation, thereby eliminating potential manufacturing / scalability issues.

[0154] In disulfide relinking conjugates, antibodies undergo bis-alkylation to conjugate both thiols from the two cysteine ​​residues of the reduced native disulfide bond. These reagents react with each of the four interchain disulfides of the antibody to generate four conjugates. The products of this conjugation are stable in serum, retain antibody structure, and have been used in antibody-drug conjugates (ADCs).

[0155] We have already prepared 5 as a common intermediate for attaching different linkers for site-specific antibody conjugation. Compound 6 can be generated by amide coupling of a PEG chain to a bis-sulfone-NHS ester for cell-cell synapse modulation, which can then be coupled to 5 after acid-mediated deprotection to deliver 7 (Figure 10A). Furthermore, the known dibromopyrazidinedione intermediate 8 can be synthesized in four steps from methylhydrazine and then coupled to 5 in two steps to give 9 (Figure 10B). These reagents enable site-selective, reproducible conjugation to antibody disulfides (Figure 11).

[0156] In a complementary enzyme conjugation approach, terminal amine 11 was synthesized from commercially available PEG10 (Figure 12A) and selectively modified with the enzyme MTGase to target conserved glutamines within each of the heavy chains of a deglycosylated IgG1 antibody (Figure 12B). This produced a uniform conjugate in quantitative yield with exactly two modifications added, using the conserved glutamine as the sole γ-carbonyl amide donor on the deglycosylated IgG1. This method has been used in dozens of clinical ADCs. This process produces uniform conjugates with exactly two modifications and has been used in dozens of clinical ADCs. The feasibility of this approach was demonstrated by generating a PEG2-BG-labeled rituximab (RTX-BG) adapter, which showed nearly complete BG labeling upon incubation with SNAP protein (Figure 26A). Furthermore, the site-specific adapter was efficient in inducing T cell activity (Figure 26B).

[0157] To evaluate the assembled adaptor-antibody conjugates for optimal activation, they can be tested in receptor activation assays using SNAP-CAR and SNAP synNotch T cells using the co-incubation methodology described herein, such as target lysis, T cell activation, and cytokine production (SNAP-CAR) and TagBFP production (SNAP-synNotch). Based on optimal receptor activation, selected site-specific chemistries matched to the antibody / antigen pair can be used to create OFF and ON switches for subsequent purposes. One or more site-specific BG incorporation approaches can result in improved receptor activation compared to current heterologous adaptors. It is possible that a single optimal conjugation strategy can be identified, or that the optimal strategy may depend on the adaptor and antigen due to differences in synaptic length.

[0158] b) Synthesis and validation of cleavable linker OFF switch adaptor conjugates. Encouraged by our results on the creation of biotin-adapter OFF switches (Figures 5 & 6), similar syntheses can be performed by replacing the biotin motif with BG and applying the site-specific conjugation chemistry established here. The use of phosphine (Staudinger reduction)-cleavable BGs via aryl azide linkers (Figure 13A) and caged aniline linkers allows the synthesis of photocleavable BGs (Figure 13B). Staudinger ligation has been reported in vivo, and related applications of azides and aryl phosphines have been shown to be nontoxic and orthogonal in cells and animals. While cleavage of BGs by Staudinger reduction is very slow (less than 90% within 4 h), a fast (t<5 min) inverse electron-demand Diels-Alder (IEDDA) reaction can synthesize trans-cyclooctene linkers capable of fragmentation when reacted with tetrazines (following the chemistry shown in Figure 18).

[0159] To synthesize the former, 12 can be hydrolyzed under basic conditions and then coupled to an amine-bearing antibody conjugate group (e.g., bis-sulfone) to form amide 13. The alcohol can be activated and coupled to 5 to generate phosphine-cleavable BG 14. To synthesize photocleavable BG, the known intermediate 15 is coupled to a PEG spacer to generate intermediate 16. The alcohol can be activated and coupled to the amine of 5 to form BG 17, which is cleavable with 365 nm light. Additionally, niclobenzyl, coumarin, BODIPY, and cyanine-based photolabile linkers can also be synthesized. These chromophores are cleaved at longer wavelengths (405–690 nm, Figure 13C), reducing potential phototoxicity and improving tissue penetration. After conjugation to each antibody (anti-CD19, anti-CD20, anti-EGFR, anti-HER2), the conjugates can be verified by mass spectrometry and purified via buffer exchange columns as ADC standards.

[0160] c) Analysis of SNAP-CAR OFF switch regulation and synNotch receptor signaling. OFF switch adaptors can be first evaluated for their ability to conditionally interact with SNAP receptors, and then, using a co-incubation method, assessed for their ability to modulate universal SNAP-CAR and SNAP-synNotch receptor signaling. For small molecule inducible OFF switches, different amounts of 2DPBM (0-100 μM) can be applied, and for photocleavable OFF switches, various exposure times to light (0, 30, 60, and 120 seconds) can be applied at wavelengths matching the chromophore. Cells can then be stained with a fluorescently labeled secondary antibody that recognizes the antibody constant region of the OFF switch adaptor. Intact adaptors are expected to be retained on the surface of SNAP receptor cells, while cleaved antibodies without BGs will not bind. PEG-BG antibody conjugates. After BG cleavage is confirmed, deactivation of receptor signaling can be confirmed following the addition of the relevant triggering stimulus.

[0161] The spatiotemporal control of toxicity can be directly tested using a light-triggered OFF-switch adaptor. Specifically, the ability of light to protect a population of antigen-positive cells (antigen-mimicking, normal cells) from killing by primary human SNAP-CAR T cells or SNAP-synNotch cells can be assayed. In the case of synNotch cells, the TagBFP response gene can be replaced with TRAIL bound to the cell surface, which has been shown to lead to target cell death. Cell death can be monitored in real time by fluorescence microscopy, and target cell death can be quantified in light-exposed and non-exposed regions. Adherent target cells, SKOV-3 (HER2+ and EGFR+) cells, can be stained with a caspase-activating dye (CellEvent) to monitor apoptosis. Next, cells can be labeled with the OFF-switch adaptor, a defined region of the cells is exposed to light, and SNAP receptor cells can be added. This is expected to efficiently cleave the adaptor and inhibit target cell death. We can also examine the kinetics of small molecules' ability to switch off adaptor CAR signaling by performing a time course assay of calcium signaling by fluorescence microscopy and staining with Fluo-4AM dye. Calcium signaling is one of the earliest and fastest acting signals in CAR T cell activation. Beyond this proposal, the functional reagents we generate can undergo preclinical development in mouse disease models. We are currently conducting animal studies of a constitutively active SNAP-CAR T cell line to develop protocols that address these long-term goals.

[0162] We sought to determine whether switching off an antibody would enable stimulus-controlled presentation of an adapter-tagged molecule on the cell surface. Using a cell surface biotin assay, we were able to measure accessible tags on the surface of target cells bound by the adapter OFF switch (Figure 23A). We tested cells using OFF switch adapters targeting HER2 (Herceptin) or the adapter CD20 (rituximab) and then exposed them to 365 nm light for the indicated times (Figure 23B). We also examined the effects after exposure to 2DPBM. We also demonstrated that the effects observed with 2DPBM are not unique, confirming similar effects using small phosphine molecules (bis(p-sulfonatophenyl)phenylphosphine, tris(3-sulfonatophenyl)phosphine, 2(diphenylphosphanyl)benzamide [2DPBM]).

[0163] Next, we investigated the ability of the OFF-switch adapter to mediate conditional lysis of target cells by universal CAR T cells. Figure 25A shows that mSA2 universal CAR T cells were co-incubated with K562+HER2 or K562+CD20 target cells that were pre-stained with the indicated concentrations of adapter, exposed to light, and evaluated for lysis by flow cytometry (Figure 25A). Figure 25B shows the same experiment without light exposure, but in the presence of 2DPBM.

[0164] 3. Example 3: Conditional control of universal antigen receptor signaling using an ON switch adaptor In addition to antigen sensing, we are developing universal adaptor ON switches that require a second input trigger to activate CAR and synNotch receptor signaling (see Figure 2B). Small-molecule- and light-controlled ON switches can provide spatiotemporal control over receptor signaling, complementing the conditional OFF switches generated here, enabling controlled dose escalation and toxicity monitoring by clinicians, either systemically with small molecules or site-specifically using light (Figure 14A, B). ON switches triggered by TME stimulation can introduce an additional autonomous activation filter, thereby increasing disease specificity for disease-associated but not disease-specific target antigens (most cancer antigens), thereby eliminating ON-target / OFF-disease toxicity (Figure 14C).

[0165] The TME of solid tumors is characterized by abnormal features that also contribute to cancer progression and are shared among other disease manifestations. Acidosis (intracellular and extracellular pH 6.0–7.2) commonly occurs due to excessive glycolysis and hypoxia by tumors, leading to increased lactate production and amplifying tumor growth and metastatic potential. Increased secreted proteases, such as matrix metalloproteinases (MMP-2, -9, and -14) by tumor cells and / or increased secreted proteases by tumor-associated cells (e.g., legumain), contribute to TME remodeling, cancer cell growth, metastasis, and cell survival. Hydrogen peroxide and other reactive oxygen species (ROS) are also found at elevated levels in tumor cells and in the extracellular space due to upregulation of superoxide dismutase (SOD), increasing the rate of H2O2 generation (up to 0.5 nmol / 10 4 These triggers have already found clinical use in prodrugs and ADCs.

[0166] a) Synthesis and validation of ON switch adaptors Based on these results, we can generate self-labeling enzyme substrates that can be conditionally activated using exogenous triggers or conditions specific to the tumor microenvironment. To achieve this, we synthesized molecules bearing three components: 1) a site-specific antibody conjugation group X (e.g., bis-sulfone or dibromopyrazine), 2) the SNAPtag substrate BG, and 3) a stimuli-responsive caging group R (Figure 15B). Optimal PEG and conjugation groups are identified herein. To cage the SNAPtag substrate and thereby (temporarily) block its interaction with the SNAPtag protein, we chose to modify the exocyclic 2-amino group of BG with carbamate and amide. Crystal structure analysis of the BG-SNAPtag interaction shows that there are five residues within 4 Å of the 4-amino group (Figure 16). Thus, the caging group can sterically prohibit BG from fitting into the SNAPtag active site.

[0167] Caging groups can be synthesized that can be removed using exogenous triggers or conditions imposed by the tumor microenvironment. For photoactivatable SNAPtag substrates, coumarin (Figure 17A), BODIPY (Figure 17B), and cyanine (Figure 17C)-based caging groups can be utilized to enable activation at various wavelengths. Two small molecule cleavable moieties are available. A phosphine trigger caging group (Figure 18A) can be synthesized in a similar manner using the Sandmeyer reaction as the key step. An additional small molecule trigger caging group, TCO (Figure 18B), is commercially available and can cleave BG when reacted with an optimized tetrazine via an inverse demand Diels-Alder reaction. A hydrazone-based caging group (Figure 19A) designed to release BG in the acidic tumor microenvironment can be synthesized using imine formation as the key step. Arylboronic esters (Figure 19B) are commercially available and are reduced to alcohols in the presence of peroxides (ROS) in the tumor microenvironment. Furthermore, caging groups sensitive to legumain (Figure 19C) and matrix metalloproteinases (MMPs) (Figure 19D) can be generated by solid-phase peptide synthesis. After MMP cleavage, additional cell surface proteases cleave the remaining peptide, leaving no peptide scar on the adapter.

[0168] b) Analysis of ON switch adaptor regulation of SNAP-CAR and synNotch signaling The ability of anti-EGFR and anti-HER2ON switch adaptors to modulate universal SNAP-CAR and SNAP-synNotch receptor signaling can be tested using the methodology outlined herein. Here, uncaging can be assessed by staining of SNAP receptor cells in response to triggers, as well as CAR T cell activation and synNotch signaling in response to triggers and antigen-positive target cells. In addition to light and small molecule triggers, TME stimuli can be tested, such as the addition of recombinant proteases (MMP-2, MMP-9, MMP-14, and legumain), exogenous H2O2, and lactic acid (to lower the medium pH to 6.0). We expect that triggers that efficiently uncage the BG moiety on the adaptor will lead to high levels of SNAP staining and receptor activation by antigen-matched target cells. Spatiotemporal induction of receptor signaling can be tested for light-triggered ON switch adaptors using the light-controlled microscopy assay established here. Tunable regulation by the 2DPBM small molecule (0-100 μM) can also be assayed. The photo-activated ON-switch adapter mediates potent tumor cell lysis by CAR T cells and TRAIL-producing synNotch cells only in areas exposed to specific wavelengths of light, and cell killing is expected to correlate with the 2DPBM dose of the small molecule adapter. Tumor microenvironment gating control of CAR T cells can be assessed for each ON-switch adapter using an established TME cell co-culture system that mimics naturally occurring conditions and antigen-positive and -negative target cells (HER2 and EGFR), primary human SNAP CAR T cells, and the ON-switch adapter. All co-cultures can be assessed for tumor cell killing and T cell activation by flow cytometry and cytokine production by ELISA. MMP-triggered adapters can be tested on SKOV-3 cells, which naturally secrete MMPs 2, 9, and 14 and express high levels of EGFR and HER2 antigens. Commercially available selective MMP inhibitors can be used to demonstrate the MMP-specific activity of each adapter.For legumain protease, coculture assays can be performed again using SKOV-3 target cells, which are negative for legumain secretion but administered with different numbers of M2THP-1 macrophage cells according to established methods, to mimic tumor-associated macrophage production of legumain. To model acidosis, pH can be adjusted (decreased) by titrating with various levels of glucose and culturing CACO-2 colon cancer cells (EGFR(+) and HER2(+)) in control medium, as described. For HO2, the SKOV-3 cell line, which naturally produces high levels of HO2, can again be used, and spiking in recombinant catalase, which normal cells use to remove ROS, can reveal dependence on HO2. TME remodeling using synNotch cells. Low levels of TME infiltration by endogenous or engineered T cells (CAR or TCR transgenic) are a major negative prognostic factor for cancer outcome. Combining SNAP-synNotch cells engineered to secrete the CXCL9 and CXCL10 chemokines, known to selectively recruit cytotoxic T cells, with a TME ON switch adaptor can remodel the TME by increasing T cell infiltration. T cell recruitment can be assayed using a transwell migration assay, in which supernatants from synNotch-target co-incubations can be assessed for their ability to recruit primary human T cells through a porous membrane. We have previously generated chemokine-synNotch response constructs and tested them in anti-CD19 synNotch cells stably integrated via lentivirus. Here, we expect to observe high levels of chemokines expressed in response to CD19+ target cells and high levels of CD8+ T cell recruitment in response to antigen recognition and TME stimuli consistent with the ON switch adaptor.

[0169] 4. Example 4: Combinatorial control of universal adaptor cells To generate combinatorial adapters that can activate SNAP receptor signaling in response to a combination of antigens on the cell surface (antigen A AND antigen B) or (antigen A AND (NOT antigen B)) (Figure 20), adapters with caging groups or enzymes adapted from the nfsB gene or other enzymes (including, but not limited to, enzymes adapted from directed enzyme prodrug therapy (DEPT), such as antibody DEPT (ADEPT), gene DEPT (GDEPT), viral DEPT (VDEPT), lectin DEPT (LDEPT), polymer DEPT (PDEPT), and clostridial DEPT (DEPT)) can be used. Linkers can be engineered that are substrates for bacterial nitroreductases (NTRs) encoded by PTs (CDEPTs), including enzymes such as, but not limited to, carboxypeptidase G2 (CPG2), β-D-glucosidase, carboxylesterase (CE), horseradish peroxidase (HRP), purine nucleoside phosphorylase (PNP), cytochrome P450 (CYP450) / oxazaphosphorine, cytosine deaminase / 5-fluorocytosine, and human carboxylesterase (hCE-2). NTRs selectively reduce nitroaromatics in a manner orthogonal to the eukaryotic environment and are widely used as tools to activate molecular probes in cells and animals, and in clinical conjunction with cancer prodrug therapy. Both logic systems can consist of two adaptor-antibody conjugates: one fused to the NTR and the other carrying either a caged BG 20 that is activated by a reductase or an NTR-cleavable linker 21 that is deactivated (by a self-sacrificial 1,6 elimination) near the NTR (Figure 21). For therapeutic applications, antibodies can be administered sequentially, first with the NTR-conjugated enzyme, followed by a reactive antibody. The NTR-fused antibody reaches a higher local concentration on antigen A-positive cells, triggering the cleavage or activation of an antibody conjugate targeting B on the same cell surface. The SNAP receptor can then react with either the uncaged BG or the uncleaved BG. Overall, this system can dramatically increase the specificity of ON and OFF targets, enhancing the localized targeting of disease-related cells based on two antigens and expanding the range of diseases that can be safely treated by antigen receptor therapy.Finally, taking advantage of the universal receptor morphology that allows for rapid screening of novel CAR antigens and targeting domains by simply combining the universal CAR with new antibodies, a combinatorial system can be applied to validate 20 predicted clinically relevant antigen combinations for targeting breast, brain, liver, and colorectal cancers with AND and NOT logic.

[0170] a) Design and validation of AND-gate and NOT-gate adaptors for universal SNAP receptors Nitroreductase-antibody fusions can be generated by expressing recombinant NTR protein as a fusion with the SNAPtag enzyme in E. coli, following the design of previously reported fusion proteins (e.g., containing fluorescent proteins) that retain NTR function. To maximize the decaging kinetics of the nitroimidazole group, the recently reported extended NTR (eNTR), which exhibits an approximately 100-fold increase in cellular activity over wild-type NTR, can be used. Synthetic adapter molecules 10, 12, or 14 can be used to conjugate eNTR-SNAPtag to any commercially available antibody to generate an antigen A-antibody-NTR system. Experience with the generation of antibody-protein (e.g., T cell receptor) conjugates suggests that this will not present any problems. Adapters 20 and 21 can be synthesized and used as disclosed herein. For both, nitroimidazole carbamate is used as the caging group, as it is a well-established substrate for rapid removal by NTR. 20 and 21 can then be conjugated to anti-HER2 and anti-EGFR antibodies. This is because we have cell lines that express none, one, or both of the antigen combinations to evaluate them.

[0171] b) Analysis of multi-antigen Boolean logic gated SNAP-CAR and synNotch signaling and effector functions First, we pre-treat the adapter conjugate with or without recombinant NTR and assay the staining of SNAP-CAR T cells to assess BG uncaging and BG-containing linker cleavage. To assess the ability of the adapter to activate the AND and NOT logic gates of the SNAP CAR and SNAP-synNotch receptors, we perform co-incubation assays with SNAP cells, ON and OFF target cells (no antigen, EGFR only, HER2 only, or both EGFR and HER2), and antibody combinations, followed by assays of receptor activation after 24 hours. Regarding antibody addition, to mimic the sequential antibody administrations performed in vivo, including diffusion of the initial antibody away from antigen-negative sites, we first stain the target cells with the NTR-antibody fusion protein, wash the cells, and then add the BG-reactive adapter antibody and SNAP receptor cells. Experiments can also be performed using different doses of adapter. This suggests that specificity of antigen combinations can be observed at low levels of NTR adapters, but that this specificity is lost at higher levels, where the unbound NTR can uncage or cleave the BG adapter conjugate. After confirming potent activation using model antigens, the logic switch adapter system can be applied to screen for novel cancer-targeting antigen combinations. Mackay et al. recently applied a systematic computational approach to identify novel clinically relevant combinations targeting currently untested cancers. Screening for novel antigen combinations is an ideal application for universal receptors, as commercially available antibodies can be easily mixed and matched with SNAP-CAR T cells or SNAP-synNotch cells. On the other hand, creating CAR logic receptors using traditional methods requires the laborious process of generating novel receptors for each antigen pair. Using antigen (+) cell lines identified from a cell line expression database, the 10 AND and 10 (A) AND (NOT B) antigen combinations listed in Table 1 can be evaluated. Combinatorial adaptors can be generated and co-incubation lysis assays with SNAP-CAR T cells can be performed to assess lysis of target cell lines.To mimic single-antigen negative conditions, as a first pass, cell lines can be pre-stained with unlabeled antibodies to block antigen recognition before the co-incubation assay. Then, CRISPR / Cas9 can be used to generate antigen-negative cell lines, and stable double-antigen positive cell lines can be generated by lentiviral transduction in the absence of combined antigen controls. [Table 1]

Claims

1. A conditional universal chimeric antigen receptor (CAR) combination, comprising: i) a CAR comprising a receptor that targets a tag ligand on a conditional adapter molecule, a hinge domain, and a signaling domain; and ii) a conditional adapter molecule comprising an antigen recognition element, a stimulus-responsive group, and a tag ligand, wherein the tag ligand comprises benzylguanine (BG) and the conditional adapter molecule comprises SNAPtag, or the tag ligand comprises biotin and the conditional adapter molecule comprises mSA2, and the antigen recognition element comprises an antibody or an antigen-recognizing fragment thereof.

2. 2. The conditional universal CAR combination of claim 1, wherein the tag ligand on the conditional adapter molecule comprises benzylguanine (BG) and the conditional adapter molecule comprises a SNAPtag.

3. The conditional universal CAR combination described in claim 1, wherein the tag ligand comprises biotin and the conditional adapter molecule comprises mSA2.

4. 2. The conditional universal CAR combination of claim 1, wherein the conditional adapter molecule comprises NHS-ester conjugation, disulfide restapling, glycan conjugate chemistry, recombinant antibodies with incorporation of tagged ligands via one or more short peptide tags, sortase-mediated ligation, THIOMAB, chemical ligation, split inteins, and / or unnatural amino acids.

5. 2. The conditional universal CAR combination of claim 1, wherein the antigen recognition element comprises rituximab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab, or an antigen-binding fragment of any one thereof.

6. The stimulus to which the stimuli-reactive group is reactive is light, enzyme activity, small molecules, pH, H ２ O ２ 6. The conditional universal CAR combination of any of claims 1 to 5, comprising: hypoxia, and / or ROS.

7. The conditional universal CAR combination of any of claims 1 to 6, wherein the stimuli-responsive group comprises a cleavable linker.

8. 8. The conditional universal CAR combination of claim 7, wherein the stimuli-reactive group comprises a photocleavable linker or a phosphine-cleavable linker.

9. The conditional universal CAR combination of any of claims 1 to 8, wherein the stimulus-responsive group comprises a stimulus-responsive caging group that blocks the CAR from binding to the tag ligand.

10. 10. The conditionally universal CAR combination of any of claims 1 to 9, wherein the stimuli-reactive group comprises a photoreactive caging group comprising nitrobenzyl, coumarin, BODIPY, or cyanine.

11. 11. The conditional universal CAR combination of any of claims 1 to 10, wherein the stimulus-responsive group comprises a photoreactive caging group and the wavelength of the photostimulation comprises 365, 405, 544, or 780 nm.

12. The stimuli-reactive group includes an enzyme-reactive group, and the enzymes to which the reactive group is reactive include legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase A (KHK-A), hexokinase (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphatase.

7. The conditional universal CAR combination of any one of claims 1 to 6, comprising phosphofructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase.

13. 7. The conditional universal CAR combination of any one of claims 1 to 6, wherein the stimuli-reactive group is reactive to small molecules comprising phosphines or tetrazines.

14. 14. The conditional universal CAR combination of any of claims 1 to 13, further comprising one or more costimulatory domains.

15. 15. The conditional universal CAR combination of claim 14, wherein the one or more costimulatory domains comprise a signaling domain of CD27, CD28, ICOS, 4-1BB, or OX40.

16. The conditional universal CAR combination of any one of claims 1 to 15, wherein the CAR is comprised on a CAR T cell, a CAR NK cell, a CAR NK T cell, a CAR B cell, or a CAR macrophage.

17. A conditional universal synthetic Notch (synNotch) receptor comprising a conditional adapter molecule comprising a stimulus-responsive group and a tag ligand, a notch core comprising one or more cleavage sites, and one or more transcription factors, wherein the tag ligand comprises benzylguanine (BG) and the conditional adapter molecule comprises SNAPtag, or the tag ligand comprises biotin and the conditional adapter molecule comprises mSA2.

18. 18. The conditional universal synNotch of claim 17, wherein the conditional adapter molecule comprises a tag ligand comprising benzylguanine (BG), and wherein the conditional adapter molecule comprises a SNAPtag.

19. The conditional universal synNotch of claim 17, wherein the conditional adapter molecule comprises a tag ligand comprising biotin, and the conditional adapter molecule comprises mSA2.

20. The stimulus to which the stimuli-reactive group is reactive is light, an enzyme, a small molecule, pH, hypoxia, H ２ O ２ 20. A conditionally universal synNotch according to any one of claims 17 to 19, comprising:

21. 21. The conditionally universal synNotch of any of claims 17 to 20, wherein the stimuli-responsive group comprises a stimuli-cleavable linker.

22. 22. The conditionally universal synNotch of claim 21, wherein the stimuli-responsive group comprises a photocleavable linker or a phosphine-cleavable linker.

23. 23. The conditionally universal synNotch of any of claims 17 to 22, wherein the stimulus-responsive group comprises a stimulus-responsive caging group that blocks the receptor from binding to the tag ligand.

24. 24. The conditionally universal synNotch of any of claims 17-23, wherein the stimuli-reactive group comprises a photoreactive caging group comprising nitrobenzyl, coumarin, BODIPY, or cyanine.

25. 25. The conditionally universal synNotch of claim 24, wherein the stimulus-responsive group comprises a photoreactive caging group and the wavelength of the light stimulus comprises 365, 405, 544, or 780 nm.

26. The stimuli-reactive group includes an enzyme-reactive group, and examples of the enzymes with which the reactive group reacts include legumain, matrix metalloproteinase, pyridoxal kinase (PDXK), aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), lipase C, liver type (LIPC), poly(ADP-ribose) polymerase 1 (PARP1), pyruvate kinase M2 (PKM2), phosphoglycerate kinase 1 (PGK1), ketohexokinase A (KHK-A), hexokinase (HK), nucleoside diphosphate kinase (NDPK or NDK), and 6-phosphophosphorylamine.

21. The conditional universal synNotch of any of claims 17-20, comprising fructo-2-kinase / fructose-2,6-biphosphatase 4 (PFKFB4), mitochondrial α-ketoglutarate dehydrogenase (α-KGDH), lysine acetyltransferase 2A (KAT2A), acetyl-CoA synthetase short chain family member 2 (ACSS2), ATP-citrate lyase (ACLY), pyruvate dehydrogenase complex (PDC), α-ketoglutarate dehydrogenase (α-KGDH), CD39, CD73, or fumarase.

27. 21. A conditionally universal synNotch according to any of claims 17 to 20, wherein the stimuli-reactive group is reactive towards small molecules comprising phosphines or tetrazines.

28. 28. The conditional universal synNotch of any of claims 17 to 27, wherein the transcription factor comprises Gal4-VP64, Gal4-VP16, TetR-VP64, or LacI-VP64.

29. A conditional universal synNotch described in any of claims 17 to 28, further comprising an antigen recognition element comprising an antibody or an antigen-recognition fragment thereof, wherein the antigen recognition element can become covalently linked to the conditional universal adapter molecule.

30. 30. The conditional universal synNotch of any of claims 17-29, wherein the antigen recognition element comprises rituximab, FMC63, herceptin, cetuximab, nimotuzumab, panitumumab, omalizumab, tositumomab, trastuzumab, gemtuzumab, alemtuzumab, bevacuzimab, or an antigen-binding fragment of any one thereof.

31. 31. An engineered cell comprising a conditional universal CAR combination according to any one of claims 1 to 16 and / or a conditional universal synNotch according to any one of claims 17 to 30.

32. 32. The engineered cell of claim 31 , further comprising a vector comprising a transcriptional response element operably linked to a promoter that drives expression of one or more cellular response genes, wherein one or more of the transcription factors on the synNotch receptor are specific for the transcriptional response element.

33. 33. The engineered cell of claim 31 or 32, wherein the one or more response genes comprise IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin.

34. 34. The engineered cell of any of claims 31-33, wherein one or more transcription factors of the conditional universal synNotch receptor activate expression of one or more natural cellular response genes.

35. 35. The engineered cell of claim 34, wherein the one or more natural cellular response genes comprise IL-4, IL-10, FASL, IFN-γ, TNF-α, granzyme A, granzyme B, granulysin, and / or perforin.

36. 36. The engineered cell of any one of claims 31 to 35, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

37. 10. A composition for treating a disease or disorder in a subject, the composition comprising a conditional universal chimeric antigen receptor (CAR) combination according to any one of claims 1 to 16, a conditional SynNotch according to any one of claims 17 to 30, and / or an engineered cell according to claims 31 to 36, wherein the disease or disorder comprises cancer, an autoimmune disease, an autoinflammatory disease, a viral infection, a bacterial infection, or a fungal infection.

38. The disease may include lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer. cancer), squamous cell carcinoma of the head and neck, lung cancer, small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, larynx, and lungs, cervical cancer, cervical cancer breast cancer, epithelial cancer, renal cancer, genitourinary tract cancer, lung cancer, esophageal cancer, head and neck cancer, large bowel cancer, hematopoietic cancer; testicular cancer; colon cancer 38. The composition of claim 37, wherein the cancer is selected from the group consisting of colon cancer, prostate cancer, and pancreatic cancer.

39. The disease may be achalasia, acute disseminated encephalomyelitis, acute motor axonal neuropathy, Addison's disease, adiposity dolorosa, adult Still's disease, agammaglobulinemia, alopecia areata, Alzheimer's disease, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, aplastic anemia, autoimmune angioedema, autoimmune autonomic failure, autoimmune encephalomyelitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune polyendocrine Syndrome, Autoimmune Retinopathy, Autoimmune Urticaria, Axonal & Neuropathic Neuropathy (AMAN), Balo's Disease, Behçet's Disease, Benign Mucous Membrane Pemphigoid, Bickerstaff's Brainstem Encephalitis, Bullous Pemphigoid, Castleman's Disease (CD), Celiac Disease, Chagas' Disease, Chronic Fatigue Syndrome, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Relapsing Multiple Osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS), Eosinophilic Granulomatosis with Polyneuropathy (EGPA), Cicatricial Pemphigoid, Cogan's Syndrome, Cold Agglutinin Disease, Congenital Heart Block, Coxsackie Myocarditis, CREST Syndrome, Crohn's Disease Disease, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), diabetes mellitus type 1, discoid lupus, Dressler's syndrome, endometriosis, enthesitis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, Felty's syndrome, fibromyalgia, pulmonary fibrosis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalopathy, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), pregnancy Herpes gestationis or pemphigoid of gestationis (PG), hidradenitis suppurativa (HS) (inverse acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), inflammatory bowel disease (IBD), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear IgA disease (LAD), lupus nephritis, lupus vasculitis, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA),Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, Ord's sthyroiditis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), facial hemifacial atrophy, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, type II, type III, Polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis Rheumatoid vasculitis, sarcoidosis, Schmidt's syndrome, Schnitzler's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's syndrome, Sydenham's chorea, sympathetic ophthalmia (SO), systemic lupus erythematosus, systemic scleroderma, Takayasu's arteritis, temporal arteritis / giant cell arteritis, platelets 38. The composition of claim 37, wherein the autoimmune disease is selected from the group consisting of thyroid purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), urticaria, urticarial vasculitis, uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, and Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

40. The disease may be asthma, graft-versus-host disease, allergies, transplant rejection, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), neonatal-onset multisystem autoinflammatory disease (NOMID) (also known as chronic infantile neurological cutaneous and articular syndrome (CINCA)), familial Mediterranean fever (FMF), tumor necrosis factor (TNF)-associated periodic syndrome (TRAPS), TNFRSF11A-associated hereditary fever disorder. (TRAPs11), hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), mevalonic aciduria (MA), mevalonate kinase deficiency (MKD), interleukin-1β (IL-1β) receptor antagonist (DIRA) deficiency (also known as sterile multifocal osteomyelitis, pustular periostitis), Majeed syndrome, chronic nonbacterial osteomyelitis (CNO) , early-onset inflammatory bowel disease, diverticulitis, interleukin-36 receptor antagonist deficiency (DITRA), familial psoriasis (PSORS2), pustular psoriasis (15), suppurative sterile arthritis, pyoderma gangrenosum, and acne syndrome (PAPA), hereditary sideroblastic anemia with immunodeficiency, fever, and developmental delay (SIFD), childhood granulomatous arthritis (PGA), familial Behçet-like autoinflammatory syndrome, and NLRP12-associated periodic fever syndrome.

38. The composition of claim 37, wherein the autoinflammatory disease is selected from the group consisting of proteasome-associated autoinflammatory syndrome (PRAAS), spondylochondrodysplasia with immune dysregulation (SPENCDI), STING-associated vasculitis of infantile onset (SAVI), Aicardi-Goutieres syndrome, acute febrile neutrophilic dermatosis, X-linked familial hemophagocytic lymphohistiocytosis, and Lyn kinase-associated autoinflammatory disease (LAID).

41. The disease is herpes simplex virus type 1, herpes simplex virus type 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus-6, smallpox virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus, influenza A virus, influenza B virus, measles virus, polyomavirus, human papillomavirus, respiratory syncytial virus, adenovirus, coxsackievirus, dengue virus, mumps virus, poliovirus, or rabies.

38. The composition of claim 37, wherein the viral infection is selected from the group consisting of: rabies virus, Rous sarcoma virus, reovirus, yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1, and human immunodeficiency virus type 2.

42. The disease is Mycobaterium tuberculosis, Mycobaterium bovis, Mycobaterium bovis strain BCG, BCG substrain, Mycobaterium avium, Mycobaterium intracellular, Mycobaterium africanum, Mycobaterium kansasii, Mycobaterium marinum, Mycobaterium ulcerans, Mycobaterium avium subspecies paratuberculosis, Nocardia asteroides, Legionella pneumophila, Salmonella typhi, Salmonella enterica, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpi, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, Clostridium tetani, and Yersinia enterolitica, the composition according to claim 37, which is a bacterial infection selected from the group consisting of

43. 38. The composition of claim 37, wherein the disease is a fungal infection selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermititidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata.

44. 44. The composition of any of claims 37-43, comprising a first conditionally universal CAR combination of any of claims 1-16 and a second conditionally universal CAR combination of any of claims 1-16, wherein the first conditionally universal CAR combination comprises a stimulus-responsive group that comprises a stimulus-cleavable linker, and the second CAR combination comprises a stimulus-responsive caging group that blocks binding of the first CAR combination to the tag ligand.

45. 45. The composition of claim 44, wherein the first conditional universal CAR combination and the second conditional universal CAR combination are responsive to the same stimulus.

46. 45. The composition of claim 44, wherein the first conditional universal CAR combination and the second conditional universal CAR combination are responsive to different stimuli.

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