Use of brain-specific antigens to home, block, and deliver cell-based therapies to the brain

Abstract

It provides a method for delivering therapeutic payloads (e.g., cytokines, antibodies, or chimeric antigen receptors) to the brain while avoiding off-site effects. [Solution] Cells are provided that contain a recombinant nucleic acid encoding a transmembrane protein having an extracellular binding domain that specifically binds to a brain-selective extracellular antigen, such as MOG, CDH10, PTPRZ1 or NRCAM, but do not contain a nucleic acid encoding an antigen-specific therapeutic agent that binds to a killing antigen expressed by glioblastoma.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 980,885, filed February 24, 2020. The benefit of which is claimed, and that application is incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made possible through grant number R01 CA196277 awarded by the National Institutes of Health. This invention was made with government support under the terms of the Federal Register. The government has certain rights in this invention. [Background technology]

[0003] Some cell therapies have demonstrated remarkable therapeutic responses and benefits for patients with several diseases. While this research has demonstrated promising results, the development of effective cell-based therapies for other diseases has largely relied on specific cell types. This remains a challenge due to the difficulty of delivering therapeutic agents only to the tissue. Some treatments for toxic effects may be harmful to non-brain tissues. Administering treatments that target diseased cells in other tissues often leads to side effects. This issue is often particularly problematic for cell-based therapies, The reason is that many of these treatments are very powerful. The clinical use of these treatments is limited by off-site effects, not on-site effects. There are many.

[0004] To avoid off-site effects, the therapeutic payload (e.g., cytokines, antibodies, or It may be desirable to deliver a specific antigen receptor (e.g., a chimeric antigen receptor) to a specific tissue. This article addresses this issue regarding the brain. Summary of the Invention

[0005] Specific for brain-selective extracellular antigens, such as MOG, CDH10, PTPRZ1, or NRCAM The method includes the step of: providing a recombinant nucleic acid encoding a transmembrane protein having an extracellular binding domain that specifically binds to the and an antigen-specific therapeutic agent that binds to a killing antigen expressed by glioblastoma. Provided herein are cells that do not contain nucleic acids encoding MOG, CDH10, PTPR. Z1 or NRCAM are brain selective, therefore binding to either of these antigens For example, in some embodiments, transmembrane proteins may be used to limit or concentrate the effects of the cells in the brain. The protein localizes the therapeutic cells to the brain, thereby facilitating the transfer of therapeutic cells from the brain to other tissues. In other embodiments, the transmembrane protein may be, but is not limited to, a s Receptors that contain binding-triggered transcriptional switches, such as those exemplified by the ynNotch receptor In these embodiments, the cells may contain (i) a coding sequence encoding a therapeutic protein. and (ii) a regulatory sequence, wherein the regulatory sequence operably links the coding sequence to the nucleic acid. These embodiments are operably linked and responsive to activation of the binding-triggered transcriptional switch. So, because the binding-triggered transcriptional switch is preferentially activated in the brain, therapeutic proteins It is expressed or delivered in brain tissue, but not in other tissues. [Brief explanation of the drawings]

[0006] [Figure 1A-D] 1A-1D show examples of brain-specific therapeutic circuits employing antigen recognition and therapeutic targeting using brain-specific antigens with or without diffusible components and target antigens expressed by diseased cells.

[0007] [Figure 2A] 2A-2B demonstrate the selective activation of synNotch receptors targeting various antigens in the presence of target GBM cells in a synNotch→CAR T cell GBM circuit as described herein. [Figure 2B] 2A-2B demonstrate the selective activation of synNotch receptors targeting various antigens in the presence of target GBM cells in a synNotch→CAR T cell GBM circuit as described herein.

[0008] [Figure 3A-B] Figures 3A-3D demonstrate selective synNotch activation and cell killing in the presence of target GBM cells by a synNotch→CAR T circuit as described herein. [Figure 3C-D] Figures 3A-3D demonstrate selective synNotch activation and cell killing in the presence of target GBM cells by a synNotch→CAR T circuit as described herein.

[0009] [Figure 4] FIG. 4 shows cells containing IF / THEN circuits with and without OR gate function in the associated binding-triggered transcriptional switch, antigen-specific therapeutic agent, or both.

[0010] [Figure 5] Figure 5 shows the design of a combinatorial antigen "prime and kill" circuit to overcome the dual challenges of antigen heterogeneity and off-tumor toxicity, for example, where antigen A is MOG, CDH10, PTPRZ1, or NRCAM and antigen B is a disease-specific antigen. This diagram shows an example of a "kill" circuit using a chimeric antigen receptor. However, these circuits can be easily adapted to produce other molecules, such as antibodies, enzymes, or cytokines.

[0011] (a) Restriction of standard CAR T cells in heterogeneous tumors. Ideal for glioblastoma. One of the main challenges in identifying suitable CAR antigens is to generate CAR T cells that are highly specific and uniform. The goal is to target tumor-specific antigens (e.g., CAR T cells) that are expressed on tumors. , EGFRvIII), but the expression of this antigen is heterogeneous (all If the antigen is not expressed in the tumor cells, the non-antigen-expressing tumor cells may escape from the treatment. On the other hand, CAR T cells can be expressed uniformly in tumors but can also express specific Targeting tumor antigens that are poorly expressed (but also expressed on some normal cells) If this is the case, this will result in on-target off-tumor toxicity. The dual challenge of gender inherently limits the therapeutic window of CAR T cells.

[0012] (b) The T cell circuit consists of two incomplete but complementary antigens: one specific antigen and one The present inventors' approach can be designed to combine recognition of multiple homogenous antigens. The key is to engineer T cells to use a sequential prime-and-kill mechanism: Induce the expression of a CAR targeting antigen B, which is primarily tumor-specific but not necessarily absolutely tumor-specific. To induce T cells, they first target antigen A (either a highly specific antigen or a tissue-specific antigen). (or) are primed by specificity prefilters across less constrained killing mechanisms. By applying these combinatorial antigen recognition circuits, engineered T cells can target tumors. It may be possible to overcome antigenic heterogeneity while also eliminating on-target extratumoral toxicity.

[0013] [Figure 6]FIG. 6 shows that CD8+ T cells with an α-EGFRvIII synNotch→α-EphA2 / IL13α2 CAR prime-and-kill circuit can effectively kill a U87 GBM population with heterogeneous EGFRvIII expression in vitro.

[0014] (a) Primary human CD8+ T cells were transfected with the α-EGFRvIII synNotch receptor. , corresponding to α-EphA2 / IL13α2 4-1BBζ CAR expression, which controls expression of CAR. response element ("EphA2 / IL13 mutein CAR", Figure 11a) IL13 muteins are mutant forms of IL13 (E) that preferentially bind to IL13Rα2. 13K, K105R) (Krebs et al., 2014). Primary T cells initiate CAR expression. To achieve this, they must first recognize EGFRvIII via their synNotch receptors. Prime-and-kill CAR T cells must be exposed to EGFRvIII-positive cells. It is activated to kill EphA2+ or IL13Rα2+ target cells only when exposed to It should be sexualized.

[0015] (b) We used an engineered U87 GBM cell line to demonstrate the phenotype observed in GBM. U87 cells express two target antigens, EphA2 and IL13Rα2, to mimic the heterogeneity of the IL-13Rα subunits. naturally expresses EGFRvIII but not EGFRvIII (U87-EGFRvIII-negative cells) -also referred to herein as "target" cells). U87 cells were engineered to also express the antigen (U87-EGFRvIII positive cells - here (Also called "priming" cells in the literature.) We have used these U87 cells in different By mixing ratios, different levels of heterogeneity can be systematically generated. Tumor cells can be labeled with different fluorescent proteins to allow tracking of cell survival by individual cell types. It was made into a Noh play.

[0016] (c) “Prime-and-kill” T cell circuits can overcome tumor heterogeneity. The prime-and-kill circuit utilizes the synNotch receptor to target priming antigens (gold ), thereby inducing the expression of a CAR that recognizes a different killing antigen (blue). We demonstrate that local priming of T cells within tumors occurs in adjacent tumors that lack the priming antigen. We hypothesize that this may allow specific but heterogeneous killing of cells (blue). While the antigens expressed can serve as good priming antigens, the antigens expressed uniformly but not necessarily In this model, tumor-specific antigens that are not specific to the tumor can act as good killing antigens. Unless the antigen is expressed, other normal tissues that express the killing antigen are spared.

[0017] (d) Time course analysis of killing at different heterogeneous tumor cell ratios. Primary CD8+ human T cells bearing nNotch→α-EphA2 / IL13α2 CAR were cultured with the indicated U87 cell mixture at an E:T ratio of 5:1 and analyzed by IncuCyte system. The cells were imaged over a 3-day period using a fluorochrome imaging system. EGFRvIII-positive primed cells were yellow. EGFRvIII-negative target cells are colored blue (T cells) (Unlabeled). The dotted black line indicates 100% proliferation of target cells as a reference only. Data from these experiments (cell survival measured by fluorescence) are shown in the time course plots below. Quantitative analysis was performed using a 3D CT scanner (n=3, error bars are SEM).

[0018] (e) Cytotoxicity assay using primary CD8+ prime-and-kill CAR T cells Primary CD8+ prime-and-kill CAR T cells as described in Figure 6a were treated with U 87 cells. Forward and side scatter flow cytometry plots (72 h) Viable U87 cells fall within the red gate. Prime-and-kill CAR T The cells kill the U87 population only if priming cells are found within the population, which As indicated by the decrease in cells in the U87 gate (representative of three experiments). Quantification of prime-and-kill CAR T cell killing as a function of the CAR / target cell ratio (n=3 , error bars are SEM).

[0019] [Figure 7a-b] Figure 7 shows that T cells with an α-EGFRvIII synNotch→α-EphA2 / IL13α2 prime-and-kill circuit mediate effective and localized antitumor responses against U87 GBM, which heterogeneously express EGFRvIII in the brain.

[0020] (a) U87 GBM xenografts were orthotopically implanted into the brains of immunodeficient NCG mice. contained one of the following three priming / target cell ratios: i) 100% U87 (EGFRvIII-negative) target tumor cells, ii) 50% / 50% U87-EGF EGFRvIII-positive (priming) tumor cells and EGFRvIII-negative (target) tumor cells, and iii) 100% U87-EGFRvIII positive (primed) tumor cells. The cells were engineered to express luciferase to allow for tracking of tumor size. Six days after tumor implantation, mice were intravenously injected with 3 million CD4+ and 3 million CD8+ T cells. T cells were infused with either i) no construct (untransduced control) or ii) α-EGFRvII I. SynNotch → α-EphA2 / IL13α2 CAR circuit Ta.

[0021] (b) Tumor size was determined by longitudinal bioluminescence imaging. Traces are shown as thin lines and the average as thick lines. Prime-and-kill CAR T cell treatment is shown in pink. Kill CAR T cells have no effect on tumors lacking EGFRvIII priming ( Left panel, n=5) shows significant improvement in tumor size reduction (**** p<0 .0001, t-test).

[0022] [Figure 7c-f] (c) NCG mice were simultaneously implanted with two tumors: i) a heterogeneous tumor containing EGFRvIII-positive U87 and EGFRvIII-negative U87 cells in the brain at a 1:1 ratio, and ii) subcutaneous EGFRvIII-negative U87 tumor cells in the flank. Thus, both tumors express killing antigens (EphA2 and IL13Rα2) but differ in their expression of the EGFRvIII priming antigen. Mice were treated once (6 days after tumor implantation) with an intravenous infusion of untransduced T cells (n = 6) or prime-and-kill CAR T cells (n = 6).

[0023] (d) Tumor size was measured by luciferase luminescence. Prime-and-Kill CAR T cells are shown in pink, and untransduced control T cells are shown in gray. A significant suppression of inflammatory bowel disease was observed in mice treated with prime-and-kill CAR T cells. Although it was observed that the flank tumors were expressed by non-transduced T cells (**** p<0.00001; t-test), the flank tumors were expressed by non-transduced T cells. They grew at the same rate as mice treated with erythrocytes.

[0024] (e) Heterogeneous U87(EGFRv) treated with prime-and-kill CAR T cells. Time course of mice bearing EGFRvIII-positive and EGFRvIII-negative tumor cells (1:1 mixture) Each column represents one mouse; each row represents an imaging time point.

[0025] (f) Tumor-bearing mice were euthanized 2 days after prime-and-kill CAR T cell infusion. Flow cytometry was performed on isolated cells from intracranial heterogeneous tumors, spleen, and control flank tumors. The results were analyzed on engineered T cells primed with EGFRvIII antigen. The T cells that were transfected were positive for GFP (CAR was fused to GFP). Upregulation of GFP expression in CD3+ cells was observed only in brain xenografts. was observed in T cells isolated from the flank tumor and spleen, but not in T cells isolated from the flank tumor and spleen.

[0026] [Figure 8a-c] Figure 8 shows that T cells harboring an α-EGFRvIII synNotch→α-EphA2 / IL13α2 CAR prime-and-kill circuit durably clear patient-derived GBM6 xenograft tumors in mice despite heterogeneous EGFRvIII expression.

[0027] (a) Endogenous expression of EGFRvIII in patient-derived xenograft GBM6 cells is heterogeneous. It is one.

[0028] (b) GBM6 patient-derived xenograft cells were orthotopically transplanted into the brains of immunodeficient NCG mice. Tumor cells were transfected with mCherry and luciferase to allow for tracking of tumor size. EGFRvIII expression on GBM6 cells is heterogeneous. Ten days after tumor implantation, mice were intravenously injected with 3 million CD4+ and CD8+ T cells. T cells were injected with either i) no construct (untransduced control) (n=5), ii) α-EGF RvIII synNotch→α-EphA2 / IL13α2 CAR circuit (n=7) or iii) a constitutively expressed α-EGFRvIII CAR (n=6). was expressed.

[0029] (c) Tumor size (top row) and survival (bottom row) over time. Tumor size was measured by longitudinal bioluminescence Individual traces for each animal are shown as thin lines, and the average is shown as thick lines. Negative control treatment with non-transduced T cells is shown in black, and prime-and-kill CAR Circuit treatment is shown in pink, and conventional α-EGFRvIII CAR treatment (only clear The mean of the α-EGFRvIII CAR T cells is shown as the purple dotted line. resulted in early tumor regression followed by recurrence in all mice (n=6), Two mice died of tumor induction by day 17. In contrast, the prime-and-kill CAR All mice treated with T cells showed complete tumor clearance (p<0.00 01 t-test untransduced vs prime-and-kill CAR T cells). These mice All mice survived for more than 125 days, except for two mice that were euthanized due to related infections. An independent replicate of this experiment is shown in Figure 13e.

[0030] [Figure 8d-h] (d) Longitudinal bioluminescence imaging of GBM6-bearing mice treated with prime-and-kill CAR T cells and conventional α-EGFRvIII CAR T cells. Each column represents one mouse; each row represents an imaging time point.

[0031] (e) Representative fluorescence microscopy of GBM6 xenografts reveals tumor inoculation (mCherry Heterogeneous expression of EGFRvIII (red) is shown 10 days after tumor growth.

[0032] (f) Fluorescence microscopy reveals that engraftment G after systemic administration of synNotch-CAR T cells Clearance of BM6 xenografts (lack of mCherry tumor cells) is demonstrated. Retention of prime-and-kill CAR T cells in the brain parenchyma and meninges (stained for CD45) color, red).

[0033] (g) Tumor recurrence for conventional α-EGFRvIII CAR T cell treated xenografts Representative images of tumor cells expressing EGFRvIII (mCherry-positive tumor cells) and showing loss of EGFRvIII expression (red). image.

[0034] (h) Representative confocal fluorescence of prime-and-kill CAR T cell-treated GBM6 xenografts. Light microscopy revealed primed GFP+ T cells (yellow) (white arrows) in the tumor bed. The red stained hCD45 (colocalized with hCD45, indicated by the red dots) is revealed. Mundokil CAR T cells express GFP upon priming. Right panel. Splenic Prime-and-kill CAR T cells (red) do not express GFP.

[0035] [Figure 9a-f] Figure 9 shows that local brain-specific prime-and-kill CAR T cells mediate effective anti-GBM responses.

[0036] (a) Primary human CD8+ T cells were transfected with the anti-brain antigen synNotch receptor and α-EphA 2 / IL13α2 4-1BBζ Corresponding response element controlling the expression of CAR expression ("EphA2 / IL13 mutein CAR", Figure 11a). The cells express brain antigens via their synNotch receptor to initiate CAR expression. First, we must recognize that the prime-and-kill circuitry is activated only when exposed to the brain. IL13Rα2+ target cells. be.

[0037] (b) CDH10 and MOG across a subset of tissue samples in GTEx v7 Box plots showing tissue-specific expression. Units shown are obtained from the GTEx Portal v7. Log-scaled normalized RNAseq counts (transcripts / million) do.

[0038] (c) Primary CD8+α-CDH10 or α-MOG synNotch→GFP PG K BFP T cells were cultured with murine CDH10 or MOG, or human CDH10 or Co-cultured with either parental K562 or K562 transduced to express MOG T cell priming after 48 hours of exposure was measured by induction of the GFP reporter. FACS histograms show mouse CDH10+ or ​​MOG+, or human CDH10+ or MOG+K562, showing induction of the GFP reporter. Not shown in the presence of vesicles (representative of three experiments).

[0039] (d) GBM6 patient-derived xenograft cells were orthotopically transplanted into the brains of immunodeficient NCG mice. Tumor cells were transfected with mCherry and luciferase to allow for tracking of tumor size. Ten days after tumor implantation, mice were each injected with 3 million CD4 CD8+ and CD8+ T cells were infused intravenously. T cells were: i) without construct (untransduced control); , ii) α-MOG synNotch→α-EphA2 / IL13α2 CAR circuit ( n = 6), or iii) α-CDH10 synNotch → α-EphA2 / IL13 α2 CAR circuit (n=7).

[0040] (e) Tumor size (top row) and survival (bottom row) over time. Tumor size was measured using longitudinal bioluminescence Negative control treatment with non-transduced T cells is shown in grey, and Lime and kill CAR circuit treatment is shown in pink. Compared to the non-transduced treatment group, α- Mice treated with MOG-prime and kill CAR T cells (4 out of 6 mice) (animals) showed a strong antitumor response (t-test and Holm-Sidak correction for multiple comparisons). p<0.001) and 60-day survival (p=0.05 Log-rank (Man tel-Cox) test).

[0041] (f) Tumor size (top row) and survival (bottom row) over time. Tumor size was measured by longitudinal bioluminescence Negative control treatment with non-transduced T cells is shown in grey, and Lime and kill CAR circuit treatment is shown in purple. Compared to the non-transduced treatment group, α-CD Mice treated with H10-prime and kill CAR T cells (5 out of 7 mice) (animals) demonstrated a durable antitumor response (t-test and Holm-Sid for multiple comparisons). p<0.001 with log-rank (Mantel) correction -Cox) test).

[0042] [Figure 9g-h](g) GBM6 PDX tumor cells were implanted into the brain and flank of NCG mice. Both tumors express killing antigens (EphA2 and IL13Rα2), but expression of the priming antigen MOG / CDH10 is restricted to the brain. Mice were treated once (10 days after tumor implantation) with an intravenous infusion of untransduced CAR T cells (n=5), or α-MOG prime-and-kill CAR T cells (n=6), or (iii) α-CDH10 prime-and-kill CAR T cells (n=5).

[0043] (h) Tumor size was measured by luciferase luminescence. Prime-and-kill CAR T cells are shown in pink, and α-CDH10-prime-and-kill CAR T cells are shown in purple, and untransduced control T cells are shown in gray. Significant reduction in brain tumor size. was observed in mice treated with prime-and-kill CAR T cells (** **p<0.0001; t-test), flank tumors were It grew at the same rate as the

[0044] [Figure 10] Figure 10 shows that multi-antigen T cell circuits can be used to flexibly engineer tumor recognition.

[0045] (a) and (b). The prime-and-kill circuit developed here is a 3-input AND-OR Gates represent T cells expressing the priming antigen EGFRvIII (a) or MOG (b). When the cells encounter either a killing antigen (EphA2 or IL13Rα2), killing activity is induced. Be guided.

[0046] (c) The prime-and-kill circuit primes cells (here, EGFRvIII-positive cells) It is hypothesized that this allows the killing of target cells in a "killing radius" around the target cell. Once the cells leave the tumor and no longer receive the continuous priming signal, CAR expression declines over time. This attenuation occurs over time, preventing sustained killing responses in other tissues (Roybal et al., 2016a ;Roybal et al., 2016b).

[0047] (d) The prime-and-kill CAR circuit targets all tumors while avoiding cross-reactivity. Surgically create an antigen space to optimize cell capture, where the antigen space is a 3-dimensional The three axes are represented by the original EGFRvIII or MOG as priming antigens, killing The prime-and-kill CAR cycle is shown in Fig. 1. Selecting tumors within the pale pink volume: Engineered T cells expressed within the brain tumor environment EGFRvIII or MOG, and EphA2 or IL13R on GBM cells Expression of either α2 must be encountered.

[0048] [Figure 11] Figure 11 shows the design and testing of α-EGFRvIII synNotch→α-EphA2 / IL13Rα2 CAR T cells against U87 GBM.

[0049] (a) α-EphA2 / IL13α2 CAR, α-EphA2 CAR, and α-I Domain structure of IL13Rα2 CAR. IL13 muteins preferentially bind to IL13Rα2. It is a mutant form of IL13 (E13K, K105R) that binds to IL13 ( Krebs et al., 2014 ).

[0050] (b) U87 wild-type cells (EphA2 / IL13α2) with the novel α-EphA2 / IL13α2 CAR EphA2+IL13Rα2+) was killed by α-EphA2 CAR or α-IL13Rα2 Total fluorescence (live cells) was measured over time and compared with that of T cells expressing either CAR. IncuCyte killing assays measure killing using fluorescently labeled U87 cells. (n=3, error bars are SEM). Tandem CARs were 24, 48 and 7 Kills faster and more effectively than either of the individual target CARs at 2 hours (p≦0.16 4; Tukey's multiple comparison test).

[0051] (c)α-EGFRvIII synNotch→α-EphA2 / IL13Rα2 CAR inhibited 50 / 50% EGFRvIII-positive / EGFRvIII-negative responses in U87 cells. The α-EphA2 / IL13α2 CAR was effective in killing the cultures for 48 hours. Later, they were as effective as T cells with similar circuits in inducing α-EphA2 CAR. Killing (n=3, error bars are SEM, p=not significant; t-test).

[0052] (d) Primary CD8+ synNotch CAR T cells as described in Figure 7a were cultured in a 24-well platelet-free medium containing 1000 ng / ml of CAR T cells as described in Figure 7b. T cell priming after 24 and 48 hours of exposure was Tracking the induction of α-EphA2 / IL13α2 CAR fused to a GFP reporter FACS histograms show the results of induction in the absence of priming cells. There was no significant induction by approximately 10% of primed cells (EGFRvIII positive). The results are shown (representative of at least three independent experiments).

[0053] (e) After 48 hours of exposure, α-EGFRvIII synNotch → α-EphA 2 / IL13Rα2 CAR co-cultured with the U87-EGFRvIII negative 50% population Compare relative cell survival to the U87-EGFRvIII negative 90% population. Fluorescently labeled U87 cells were used in an IncuCyte assay to measure the number of cells over time. Survival was measured using the U87-EGFRvIII (n=3, error bars are SEM). The negative 90% population had a higher relative cell viability compared to the U87-EGFRvIII negative 50% population. The survival rate was higher (p=.0149; t-test).

[0054] (f) α-EGFRvIII synNot in primary CD4+ and CD8+ T cells ch Gal4VP64 receptor and α-EphA2 / IL13Rα2 CAR 4-1B Bζ CAR GFP pGK showing the expression of the corresponding response element regulating BFP Representative contour plots. T cells positive for the synNotch receptor are shown as synNo Staining was performed via the myc tag present on the tch receptor, and response elements were identified based on BFP expression. T cells within the quadrant circled in red were selected for in vitro and in vivo experiments. Selected for.

[0055] [Figure 12] Figure 12 shows representative immunofluorescence images of 50 / 50% EGFRvIII-positive / EGFRvIII-negative U87 xenografts 6 days after tumor cell inoculation. U87-EGFRvIII-positive and U87-EGFRvIII-negative cells are tagged with GFP and mCherry, respectively, and nuclei are stained with DRAQ7. Scale bar, 100 μm. See Methods for cell line generation.

[0056] [Figure 13] Figure 13 shows the results of testing α-EGFRvIII synNotch→α-EphA2 / IL13Rα2 CAR T cells against GBM6.

[0057] (a) Primary CD8+α-EGFRvIII synNotch→α-EphA2 / IL 13Rα2 CAR T cells were co-cultured with GBM6 cells at a 1:1 ET ratio. SynNotch killing assay. Relative cell survival was quantified over 72 hours. demonstrated the cytotoxic ability of CAR T cells to overcome GBM6 cell populations (n=3, error Bars are SEM).

[0058] (b) Primary CD8+α-EGFRvIII synNotch→α-EphA2 / IL 13Rα2 CAR T cells were co-cultured with GBM6 cells at a 1:1 ET ratio or with GBM6 cells. T cell priming after 48 hours of exposure was assessed using a GFP reporter assay. This was measured by tracking the induction of α-EphA2 / IL13Rα2 CAR fused to The FACS histogram showed no induction in the absence of GBM6 cells, but Significant induction by cells is shown (representative of at least three independent experiments).

[0059] (c) GBM6 cells were sorted for various levels of EGFRvIII expression and analyzed by flow cytometry. The cells were assessed by sorting after cytometry. Grey represents the unstained control.

[0060] (d) Primary cells co-cultured with either no EGFRvIII expression, high EGFRvIII expression, or unsorted. CD 8+α-EGFRvIII synNotch→α-EphA2 / IL13Rα 2 CAR T cells, α-EGFRvIII CAR T cells, or α-EphA2 / I Killing assay using L13Rα2 CAR T cells. Relative cell viability over 72 hours. quantified the presence of α-EGFRvIII synNotch→α-EphA2 / IL13R α2 CAR T cells and α-EGFRvIII CAR T cells are It was shown that the GBM6 cell population, which does not express any antigen, cannot be killed (n = 3, error bar = 0.05). (This is SEM).

[0061] (e) Independent repeats of the mouse experiment shown in Figure 8c (GBM6 cells with different T cell circuits) PDX tumor treatment).

[0062] [Figure 14] Figure 14 shows the results of testing α-CDH10 synNotch→α-EphA2 / IL13Rα2 CAR T cells and α-MOG synNotch→α-EphA2 / IL13Rα2 CAR T cells against GBM6.

[0063] (a) First generation CD8+α-CDH10 synNotch→α-EphA2 / IL13R α2 CAR T cells were transfected with U87 cells and K562 cells expressing mouse CDH10 or The cells were co-cultured with K562 cells that do not express mouse CDH10. Cell survival was quantified and only when the primed cells expressed mouse CDH10. α-CDH10 synNotch→α-EphA2 / IL13Rα2 CAR T cells The results showed that the cytotoxicity of α-glucan in ... Cell population ratio: 1:1:1, 10K cells each.

[0064] (b) First generation CD8+α-MOG synNotch→α-EphA2 / IL13Rα2 CAR T cells were cultured in GBM6 cells and L9 cells expressing or not expressing mouse MOG. Relative cell survival was quantified over 72 hours and the effect of α-MOG on cell viability was assessed. Priming cells for ynNotch→α-EphA2 / IL13α2 CAR T cells showed cytotoxicity in killing GBM6 cells only when they expressed mouse MOG (n=3, error bars are SD). Cell population ratio: 1:1:1, 10K cells each.

[0065] (c) Representative fluorescence microscopy images of α-MOG synNotch-CAR T cells. Clearance of implanted GBM6 xenografts after systemic administration of cytochrome P450 (mCherry tumor cell deficiency) The retention of prime-and-kill CAR T cells (CD4 5 staining).

[0066] definition As used herein, the terms "treatment," "treating," "treating," and the like mean to obtain the desired pharmacological and / or physiological effects and / or responses associated with the treatment. The effect is preventative in that it completely or partially prevents the disease or its symptoms. and / or partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, "treatment" refers to the treatment of a mammal, particularly a human. (a) any treatment of a disease in a person who may be predisposed to the disease but has not yet developed it; (b) preventing the disease from occurring in a subject who has not been diagnosed with the disease; (c) inhibiting, i.e., arresting its development; and (c) alleviating the disease. That is, it involves causing regression of the disease.

[0067] A "therapeutically effective amount" or "effective amount" refers to the amount of a compound administered to a mammal or other subject to treat a disease. When administered, the drug (biological agent, e.g., cell A "therapeutically effective amount" refers to the amount of (one or more) The dosage may vary depending on the drug, the disease and its severity, and the age, weight, etc. of the subject being treated. do.

[0068] The terms "individual," "subject," "host," and "patient" are used interchangeably herein. The term includes, but is not limited to, murines (e.g., rats, mice), non-human primates, Humans, dogs, cats, ungulates (e.g., horses, cattle, sheep, pigs, goats), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is A non-human primate. Optionally, the individual is a rodent, e.g., a rat or mouse. In some cases, the individual is a lagomorph, such as a rabbit.

[0069] As used herein, the term "refractory" refers to a disease or condition that does not respond to treatment. As used herein, with respect to cancer, "refractory cancer" refers to a cancer that does not respond to treatment. Refractory cancers may be resistant at the start of treatment or may become resistant during treatment. Refractory cancers are also called resistant cancers.

[0070] As used herein, the terms "histology" and "histological" refer generally and without limitation to The cytoanatomical structure and / or structure of cells from multicellular organisms, including plants and animals, but not from animals. Or refers to the microscopic analysis of morphology.

[0071] As used herein, the terms "cytology" and "cytological" generally refer to the analysis of individual cells. refers to a subclass of histology that involves the microscopic analysis of dissociated cells, free cells, clusters of cells, etc. The cells in a cytological sample are cells in, or obtained from, one or more body fluids. The cells may be cells obtained from tissue dissociated into a liquid cell sample.

[0072] The terms "chimeric antigen receptor" and "CAR" are used interchangeably herein. Typically, but not exclusively, an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain, domain and one or more intracellular signaling domains, which activate immune cells. The term CAR refers to an artificial multi-module molecule that can be induced or inhibited. It is not specifically limited to the CAR molecule, but also includes CAR mutants. CAR mutants include: The extracellular portion (e.g., ligand-binding portion) and the intracellular portion (e.g., intracellular signaling portion) of the CAR are This includes split CARs, in which the CAR fragment (the nucleic acid transfer moiety) is present on two separate molecules. Variants also exist for conditionally activatable CARs, e.g., the two parts of a split CAR. ON-Swipe containing split CAR whose conditional heterodimerization is pharmacologically controlled including CAR (e.g., PCT Publication No. WO 2014 / 127261 A1 and U.S. Patent Application Publication No. 2015 / 0368342 A1, the disclosure of which is incorporated herein by reference. (The text is incorporated herein by reference in its entirety.) CAR variants may contain the activity of the primary CAR. Also included are bispecific CARs that contain a secondary CAR binding domain that can amplify or suppress activity. CAR mutants may also be used, for example, where binding of a secondary CAR binding domain inhibits primary CAR activity. Chimeric inhibitory antigens that can be used as components of bispecific CAR systems leading to suppression of immunization The CAR molecule and its derivatives (i.e., CAR variants) are See, e.g., PCT Application No. US 2014 / 016527; Fedorov et al. Sci Tr ansl Med(2013);5(215):215ra172;Glienke et al.Front Pharmacol(2015)6:21;Kakarla&G ottschalk 52 Cancer J(2014)20(2):151-5;Riddell et al.Cancer J(2014)20(2):141-4;P egram et al.Cancer J(2014)20(2):127-33;Cheadle et al.Immunol Rev(2014)257(1):91- 106;Barrett et al.Annu Rev Med(2014)65:333-47;Sadelain et al.Cancer Discov(2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; The disclosures of which are incorporated herein by reference in their entireties. Useful CARs include No. Lentiviral-loaded CTL 019 commercially available from vartis (Basel, Switzerland) (Tisagenlecleucel-T) Anti-CD1 expressed by CAR-T cells This also includes 9-4-1BB-CD3 ζCAR.

[0073] The terms "T cell receptor" and "TCR" are used interchangeably and generally refer to major tissue-specific receptors. It is responsible for recognizing fragments of antigens as peptides bound to MHC molecules. The TCR complex is usually a molecule found on the surface of a T cell or T lymphocyte. Highly variable alpha (α) and beta chains expressed as part of a complex with a three-chain molecule It is a disulfide-linked membrane-anchored heterodimeric protein consisting of (β) chains. CR exists in heterodimeric αβ or γδ forms. The heterodimeric αβ form is completely endogenous. The reactive TCR complex consists of eight chains: the alpha chain (referred to herein as TCRα or TCRα) and the alpha chain (referred to herein as TCRα). the beta chain (referred to herein as TCRβ or TCR beta), It contains a delta chain, a gamma chain, two epsilon chains, and two zeta chains. Although TCRs are generally referred to by reference to only the TCR α and TCR β chains, The combined TCR complex is composed of endogenous delta, gamma, epsilon, and / or zeta chains. As such, the skilled artisan will understand that reference to a TCR present on the cell membrane is, in its entirety, as necessary, It will be readily understood that the term "TCR complex" may include reference to a partially assembled TCR complex.

[0074] Recombinant or engineered individual TCR chains and TCR complexes have been developed for therapeutic use. Reference to the use of TCRs in the present invention may refer to individual recombinant TCR chains. The resulting TCR is composed of individual modified TCR α chains or modified TCR β chains and a linking polypeptide. modified and / or unmodified TCR α chain and TCR β chain joined into a single polypeptide The TCR may comprise a single chain TCR comprising:

[0075] As used herein, the term "binding-triggered transcriptional switch" or "BTTS" The researchers then analyzed specific binding events outside the cell (e.g., binding of the extracellular domain of BTTS) to identify the specific binding events. any polypeptide or polypeptides that can be transformed into activation of a recombinant promoter in the nucleus of a cell Many BTTSs release transcription factors that activate promoters. In these embodiments, the BTTS acts by forming a protein upon binding to an antigen. One or more of the following undergo proteolytic cleavage to release gene expression regulators that activate the recombinant promoter: For example, BTTS is composed of (i) an antigen-specific antibody (ii) an extracellular domain containing an antigen-binding region; and (ii) one or more proteolytic cleavage sites. and (iii) an intracellular domain; and wherein binding of the antigen-binding region to an antigen is via the one or more proteolytic cleavage sites. Inducing cleavage of the sequence at the cytoplasm, thereby releasing the intracellular domain, The main promoter activates the transcription of the expression cassette. BTTS is a transcription factor that activates the transcription of the expression cassette, e.g., synNotch, A2, MESA, or force receptor based, although others are known or may be constructed. It can be built.

[0076] As used herein, a "chimeric bispecific binding member" refers to a chimeric bispecific binding member that binds two different Chimeric polypeptides with dual specificity for binding partners (e.g., two different antigens) A non-limiting example of a chimeric bispecific binding member is a bispecific antibody. antibody, bispecific conjugate monoclonal antibody (mab)2, bispecific antibody fragment ( For example, F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific Bispecific T cell engagers (BiTEs), bispecific conjugates (bi-conjugates), and Examples include adjuvanted single domain antibodies, micabodies and their mutants. Non-limiting examples of bispecific binding members include those described in Kontermann. MAbs. (2012) 4(2):182-197; Sta mova et al.Antibodies 2012,1(2),172-198;Farhadfar et al.Leuk Res.(2016)49:13-21; Benjamin et al.Ther Adv Hematol.(2016)7(3):142-56;Kiefer et al.Immunol Rev.(2016 )270(1):178-92;Fan et al.J Hematol Oncol.(2015)8:130;May et al.Am J Health Syst Pharm. (2016) 73(1):e6-e13; and the disclosures therein are incorporated by reference. is incorporated herein by reference in its entirety.

[0077] "Biological sample" encompasses a variety of sample types obtained from an individual or population of individuals, including, for example, cells. They can be used in a variety of methods, including isolation of cells or biomolecules, diagnostic assays, etc. The definition includes blood and other liquid samples of biological origin, solid tissue samples, e.g., biopsy specimens or tissue cultures. This definition also includes any individual or group of cells derived therefrom and their progeny. Mixing or pooling of samples, treatment with reagents, solubilization, or separation of specific components, e.g., cells, polymerase chain reaction (PCR) Concentration of oligonucleotides, polypeptides, etc., can be manipulated in some way after their procurement. The term "biological sample" encompasses clinical samples, including cells in culture, cells Also included are cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions such as plasma and serum, etc. The term "biological sample" includes solid tissue samples, tissue culture samples (e.g., biopsy samples), and ), and cell samples. Thus, a biological sample may be a cell sample or an acellular sample. obtain.

[0078] The terms "antibody" and "immunoglobulin" refer to an antibody or immunoglobulin of any isotype. globulin, antigens including but not limited to Fab, Fv, scFv, and Fd fragments Antibody fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, nanobodies, etc. that retain specific binding to antibodies, single domain antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Contains proteins.

[0079] An "antibody fragment" is a portion of an intact antibody, such as the antigen-binding region of an intact antibody or a fragment thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; Diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062(1995)); single chain This includes antibody molecules; as well as multispecific antibodies formed from antibody fragments. The antibody fragments are synthesized by cleaving two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site. The resulting fragment is a soluble "Fc" fragment and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment results in a protein that has two antigen-binding sites and is still capable of cross-linking antigens. The resulting F(ab')2 fragment is

[0080] "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, These domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide The peptides comprise the VH and VL domains which enable the sFv to form the desired structure for antigen binding. It further comprises a polypeptide linker between the domains. For a review of sFvs, see Pluckt, hun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburg and Moore eds ., Springer-Verlag, New York, pp. 269-315 (1994).

[0081] As used herein, the term "nanobody" (Nb) refers to a naturally occurring heavy chain antibody. The smallest antigen-binding fragment or single variable domain (V HH ), which is known to those skilled in the art They are derived from heavy-chain-only antibodies found in camelids (Hamers-Casterman et al.(1993)Nature 363:446;Desmyter et al.(2015)Curr.Opin.Struct.Biol.32:1). " In the "camelid" family, immunoglobulins lacking light polypeptide chains are found. "Camelids" refers to the Old World camelids (Camelus bactrianus and Dromedary (Camelus dromedarius), and New World camelids (e.g., Alpine camels) Pacas (Llama paccos), llamas (Llama glama), guanacos (Llama guanicoe) and vicuñas ( Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH are called antibodies.

[0082] As used herein, the term "affinity" refers to the equilibrium of reversible binding of two drugs. The affinity is expressed as the dissociation constant (Kd) for an unrelated amino acid sequence. at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold the affinity of the antibody At least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, At least 10 times, at least 20 times, at least 30 times, at least 40 times, at least At least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times , at least 100-fold, or at least 1000-fold or more. The affinity of the antibody for the protein is, for example, about 100 nanomolar (nM) to about 0.1 nM, about 1 00 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM), or As used herein, the term "binding activity" refers to the amount of avidity of a solution after dilution. "Immunoreactivity" and "preferential" refer to the resistance of a complex of two or more drugs to isolation. The terms "specifically bind" and "specifically bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments. Used for.

[0083] The term "bond" includes, for example, covalent bonds, electrostatic bonds, hydrophobic bonds, and salt and water bridges. between two molecules by ionic and / or hydrogen bonding interactions, including Refers to direct association. Non-specific binding is approximately 10 -7 Binding with an affinity less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity such as M.

[0084] One or more "orthogonal" or "orthogonalizing" members of a binding pair are those that bind specifically to one another. and binds to the unmodified or wild-type member of the pair, but does not specifically or substantially bind to the unmodified or wild-type member of the pair. Any binding partner / specific binding pair may be modified from their original or wild-type form. Orthogonalization may be performed, for example, but not limited to, the binding moieties described herein. Contains a ner / specific binding pair.

[0085] The terms "domain" and "motif" are used interchangeably herein and refer to one or more structured domains with one or more specific functions and unstructured domains with one or more specific functions It refers to both unstructured segments of a polypeptide that retain a specific function. For example, but not limited to, a structured domain may be a structural domain that contributes to a particular function of a polypeptide. A plurality of consecutive or non-consecutive amino acids or amino acids in a folded polypeptide, including a three-dimensional structure. In other instances, the domain may be unfolded or disordered. A plurality of two or more amino acids or portions thereof that maintain a specific function of the polypeptide. This definition may include unstructured segments of polypeptides that contain disordered or unstructured polypeptides. However, there are also domains that become structured or ordered upon association with a target or binding partner. Essentially unstructured domains and unstructured domains of essentially unstructured proteins are included. Non-limiting examples include, for example, Dyson & Wright, Nature Reviews Molecular Cell Biology 6:197-2 It is described in 08.

[0086] As used herein, the terms "synthetic," "chimeric," and "engineered" generally refer to refers to one or more artificially derived polypeptides that are encoded by non-naturally occurring nucleic acids. Synthetic polypeptides and / or nucleic acids may be composed of, for example, single amino acids, single nucleotides, etc. They may be constructed de novo from basic subunits including The modification of an existing polypeptide or polynucleotide, whether derived from, for example, recombinant means, The nucleic acid encoding the chimeric and engineered polypeptide or polypeptides may be derived from a nucleotide sequence. A peptide generally comprises two or more nucleic acids encoding one or more different polypeptides. a combination or junction of one or more polypeptide domains, or nucleic acids encoding the polypeptide domains or constructed by fusion. Chimeric and engineered nucleic acids encoding one or more polypeptides. A peptide may have two or more joined polypeptide or nucleic acid "portions" that are different in type. When derived from a protein (or a nucleic acid encoding a different protein), as well as when conjugated Although the portions comprise different regions of the same protein (or nucleic acid encoding the protein), the portions This includes cases where the moieties are joined in ways that do not occur in nature.

[0087] The term "recombinant" as used herein refers to a nucleic acid molecule, e.g., a genome, Polynucleotides of DNA, viral, semisynthetic, and / or synthetic origin, All or part of the polynucleotide sequence with which it is associated in nature, by source or manipulation It refers to a polynucleotide that is not accompanied by a The term recombinant refers to a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant as used in reference to a host cell or virus means a recombinant A recombinant refers to a host cell or virus into which a recombinant polynucleotide has been introduced. With respect to a material (e.g., a cell, a nucleic acid, a protein, or a vector), the material is heterologous material. modified by the introduction of (e.g., a cell, nucleic acid, protein, or vector) is used herein to refer to

[0088] The term "operably linked" means that the components so described are intended to be For example, a promoter is a molecule that is involved in transcription. A promoter is operably linked to a coding sequence if it affects the transcription or expression of the gene. Operably linked nucleic acid sequences can be, but do not have to be, contiguous. For example, in some instances, a coding sequence operably linked to a promoter In some instances, the promoter may be operably linked to the promoter. The ligated coding sequence may be bounded by one or more intervening sequences, including coding and non-coding sequences. In some instances, three or more sequences may be operably linked. For example, but not limited to, two or more coding sequences may be linked together. This includes when the gene is operably linked to a single promoter.

[0089] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to ribonucleotides. of any length of nucleotides, either ribonucleotides or deoxyribonucleotides Thus, the term includes, but is not limited to, single-chain, double-chain, Single-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA Hybrids, or purine and pyrimidine bases or other natural, chemical or biochemical The present invention also includes polymers containing modified, non-natural, or derivatized nucleotide bases. It can be enjoyed.

[0090] "Polypeptide," "peptide," and "protein" are used interchangeably herein. The term refers to polymeric forms of amino acids of any length, including genetically encoded amino acids. amino acids and non-genetically encoded amino acids, chemically or biochemically modified or derivatized The term may include polypeptides with modified amino acids, as well as polypeptides with modified peptide backbones. These include, but are not limited to, fusion proteins with heterologous amino acid sequences, heterologous and homologous Fusion proteins with or without an N-terminal methionine residue, proteins; immuno-tagged proteins; etc.

[0091] A "vector" or "expression vector" is a vector that contains another DNA segment, or "insert." " can be combined to bring about replication of the combined segments within a cell, e.g., a replicon, e.g., Examples include plasmids, phages, viruses, and cosmids.

[0092] As used herein, the term "heterologous" refers to a heterologous (e.g., naturally occurring) means a nucleotide or polypeptide sequence not found in any nucleic acid or protein A heterologous nucleic acid or polypeptide is a nucleic acid or polypeptide that is present or expressed in a The organism or cell may be derived from a different species. Thus, a heterologous nucleic acid or polypeptide may be derived from a Generally, it has a different evolutionary origin compared to the cell or organism in which it resides.

[0093] As used herein, the term "brain-selective extracellular antigen" refers to an antigen that is selectively expressed in brain cells. "Selectively expressed" refers to extracellular antigens (i.e., antigens expressed on the outer surface of a cell) that are expressed by a specific antigen. The term "antigen" refers to the antigen or its encoding mRNA being analyzed by a method (RNA-seq, RT-PCR, It is more abundant in brain cells than in other non-CNS tissues tested (as measured by PCR or array). Other structures can be selected from those shown in Figure 9b. , CDH10, PTPRZ1, and NRCAM are examples of brain-selective extracellular antigens. The target extracellular antigen is at least 5-fold higher in the brain than in the next highest expression non-CNS tissues. , at least 10-fold higher, at least 20-fold higher, or at least 50-fold higher The extracellular antigen may be selected from those shown in Figure 9b. Thus, MOG is a brain-specific antigen, but other antigens can be identified. The expression of brain-specific antigens is found in neurons (motor neurons, sensory neurons, and interneurons). cells (including neurocytes) or glial cells (including oligodendrocytes, microglia and / or astrocytes) may be limited to

[0094] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, but It is therefore to be understood that variations may occur, of course. The terminology used herein refers to a particular embodiment, limited only by the scope of the claim. It is also understood that this is for illustrative purposes only and is not intended to be limiting. I want to be.

[0095] Where a range of values ​​is provided, the difference between the upper and lower limits of the range shall be Unless otherwise specified, each intervening value to the tenth of the lower unit, and any other value within that stated range, It will be understood that any stated or intervening value is encompassed within the present invention. The upper and lower limits of a range may independently be included in the smaller range, and within the stated range Any ranges within the ranges set forth are encompassed by the present invention, subject to any specifically excluded limits. Where one or both of the limits are included, ranges excluding either or both of those included limits are also included. Included in the present invention.

[0096] Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present invention. The term "term" has the same meaning as commonly understood by a person skilled in the art to which the present disclosure pertains. Any methods and materials similar or equivalent to those described herein may also be used in the practice of the present invention. Although any of the methods and materials described herein may be used for any of the above tests, the preferred methods and materials are described herein. All publications mentioned in this document are to be construed as including the relevant methods and / or materials to which they are cited. No. 6,023,799, filed Dec. 1, 2003, incorporated herein by reference for purposes of disclosure and explanation.

[0097] As used in this specification and the appended claims, the singular forms "a," "an," and " "The" includes plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" refers to a plurality of such cells. Reference to "the cell" includes reference to one or more cells and their related substances known to those skilled in the art. The claims may be amended to exclude any optional element. It is further noted that the description may be drafted as follows: In connection with a list of elements or the use of a "negative" limitation, exclusionary terms such as "solely" and "only" are It is intended to serve as a prerequisite for the use of generic terms.

[0098] Certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be incorporated into a single It will be understood that these may be provided in combination in various embodiments. Various features of the invention that are, for purposes of illustration, described in the context of a single embodiment, may also be used separately or optionally. Any combination of the embodiments related to the present invention may be provided. Combinations are specifically embraced by the present invention and are intended to be illustrative and not restrictive. Each of the various embodiments is disclosed herein as if each such embodiment were individually and expressly disclosed. All subcombinations of the embodiments and elements thereof are more specifically encompassed by the present invention. and as if each and every such subcombination were individually and expressly incorporated herein. The present invention is hereby incorporated by reference as if fully set forth in the specification.

[0099] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention antedates such publication by virtue of prior invention. Further, the publication dates provided are not intended to be construed as an admission that no right exists. Actual publication dates may vary and may need to be independently confirmed. DETAILED DESCRIPTION OF THE INVENTION

[0100] As summarized above, the present disclosure provides methods for detecting brain-selective extracellular antigens, such as MOG, CDH10, P A transmembrane protein with an extracellular binding domain that specifically binds to TPRZ1 or NRCAM A cell containing a recombinant nucleic acid encoding a glioblastoma killing protein expressed by the glioblastoma. Disclosed herein are cells that do not contain a nucleic acid encoding an antigen-specific therapeutic that binds to the antigen. The cells express ephrin type A receptor 2 (EphA2), ephrin type A receptor 3 (EphA3), Interleukin-13 receptor subunit alpha-1 (IL13RA1), interleukin Interleukin-13 receptor subunit alpha-2 (IL13RA2), epidermal growth factor receptor (EGFR ) or antigen-specific therapy that binds to erb-b2 receptor tyrosine kinase 2 (ERBB2) In some embodiments, the extracellular binding domain is a nucleic acid encoding a brain-selective agent. specifically bind to a selective extracellular antigen, such as MOG, CDH10, PTPRZ1, or NRCAM. The variable domain of the antibody to be combined (e.g., nanobody or single-chain Fv)

[0101] Transmembrane proteins can have a variety of structures. Proteins consist of an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. In these embodiments, the transmembrane protein may not include a transmembrane protein that converts the cell into a brain cell. It may function to tether, thereby preventing the cell from migrating to another tissue.

[0102] In other embodiments, the transmembrane protein is characterized in that the extracellular binding domain binds to its cognate antigen (i.e., MOG, CDH10, PTPRZ1 or NRCAM) inside the cell. In these embodiments, the transmembrane protein is capable of signal transduction. It may comprise a binding domain, a transmembrane domain and an intracellular signaling domain. In their embodiments, the transmembrane protein is a chimeric antigen receptor (or T cell receptor) or a In some embodiments, the transmembrane protein may be a repressor-triggered transcriptional switch. and inhibitory immune cell receptors (iICRs), such as interferon chimeric antigen receptors (iCARs), which are expressed in the brain. Binding of iICR to selective extracellular antigens, MOG, CDH10, PTPRZ1 or NRCAM Such iICR proteins suppress the activation of immune cells in which iICR is expressed. The quality can be determined, for example, by the methods described in International Publication No. 2017087723 and Fedorov et al. (Sci.Transl.Med. 2013 5:215ra17) and other references cited above, which The description and examples thereof are incorporated by reference. Such inhibitory immunoreceptors contain intracellular immunoreceptor tyrosine-dependent inhibitory motifs (ITIMs), Immunoreceptor tyrosine-dependent switch motif (ITSM), NpxY motif, or YX An exemplary intracellular domain of such a molecule may include, for example, PD1 , CTLA4, BTLA, CD160, KRLG-1, 2B4, Lag-3, Tim-3 and other immune checkpoints. See, for example, Odorizzi and Wherry (2012) ) J. Immunol. 188:2957; and Baitsch et al. (2012) PLoSOne 7:e30852.

[0103] In some embodiments, the transmembrane protein is a target protein, e.g., a therapeutic protein. It may be part of a molecular circuitry that confines protein expression to the brain. The protein may optionally be antigen-specific. In these embodiments, the protein may be a transmembrane protein. The protein may be a binding-triggered transcriptional switch, as described in more detail below. In these embodiments, the cells contain (i) a coding sequence encoding a therapeutic protein and (ii) a regulatory sequence, wherein the regulatory sequence is operably linked to the coding sequence. In some embodiments, the transcriptional switch is responsive to activation of the binding-triggered transcriptional switch. The binding-triggered transcriptional switch is a SynNotch polypeptide, e.g., (i) a brain-selective cell Extracellular antigen (e.g., MOG-, CDH10-, PTPRZ1-, or NRCAM-) specific (ii) an extracellular domain containing the antigen-binding region of an antibody; (iii) a proteolytically cleavable Notch receptor polypeptide comprising the cleavage site; and In these embodiments, the polypeptide may comprise a surface of a brain cell; to brain-selective extracellular antigens such as MOG, CDH10, PTPRZ1 or NRCAM The binding of the extracellular domain in (i) may be followed by one or more Induces cleavage of the synNotch polypeptide at the proteolytic cleavage site. In embodiments of the present invention, the released intracellular domain is operably linked to its coding sequence. The expression of the therapeutic protein is induced via the regulatory sequences provided.

[0104] In these embodiments, the therapeutic protein, once expressed, can be secreted by the cell. In embodiments where the therapeutic protein is secreted, the therapeutic protein may be secreted or may be present on the surface of the cell. Therapeutic proteins include, for example, antibodies (e.g., PD1, PD-L1, PD-L2, CTLA 4. Antibodies that bind to TIM3 or LAG3, or, for example, another immune checkpoint ), enzymes (e.g., superoxide dismutase for scavenging reactive oxygen species, or protease proteases that can expose cytokines) or biologically active peptides such as cytokines tides (e.g., IL-1ra, IL-4, IL-6, IL-10, IL-11, among others) , IL-13, or TGF-β).

[0105] In embodiments where the therapeutic protein is localized to the surface of a cell, the therapeutic protein may be, e.g., The antibody comprises an extracellular binding domain (e.g., an antibody variable domain), a transmembrane domain, and an intracellular sequence. The protein may be a signal transduction protein containing a signal transduction domain, the protein being expressed outside of a cell. It transmits signals generated by the binding of the binding domain to antigens on the cell interior. For example, a therapeutic protein may be expressed on the surface of an immune cell and, upon binding to an antigen, induce the immune cell The protein may be a protein that activates immune cells or suppresses immune cell activation. The therapeutic protein may be an immune cell receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor). In these embodiments, the transmembrane protein may be a brain-selective extracellular receptor (TCR). The antigen, e.g., MOG, CDH10, PTPRZ1, or NRCAM, is associated with its extracellular binding domain. Upon binding via the ATP-binding domain, the intracellular domain is released from the transmembrane protein, thereby In these embodiments, the expression of immune cell receptors is induced in glioblastoma cells. They do not have an extracellular binding domain that binds to cancer-specific antigens expressed by tumors. In this study, immune cell receptors bind to disease-specific antigens on diseased cells that are not glioblastoma cells. The binding of an immune cell to such an antigen may activate the immune cell. This should activate the ATP-dependent ATP synthesis, thereby killing the diseased cells.

[0106] In some embodiments, a therapeutic delivered by activation of a binding-triggered transcriptional switch The protein may prevent the therapy from being delivered to the brain. For example, in some embodiments, Therapeutic drugs target inhibitory immune cell receptors (iICRs), such as inhibitory chimeric antigen receptors (iCARs). ) (which may also be referred to as "inhibitory immunoreceptors"). In these embodiments, transmembrane The protein is a brain-selective extracellular antigen (e.g., MOG, CDH10, PTPRZ1, or NRC Upon binding to iI, the intracellular domain is released from the transmembrane protein. In these embodiments, the expression of the iICR is induced, e.g., on non-diseased cells. The extracellular binding domain may bind to an antigen present in the immune system. The binding suppresses immune cell activation, thereby allowing cell therapy to be activated in non-diseased areas of the brain. Such iICR proteins may provide a method for preventing the spread of viruses. International Publication No. 2017087723, Fedorov et. al. (Sci.Transl.Med.2013 5:215ra17) and in the other references cited above.

[0107] Embodiments in which the transmembrane protein is a binding-triggered transcriptional switch that activates expression of CAR In this state, a binding-induced transcriptional switch activates the binding of brain-selective extracellular antigens (MOG, CDH10, PTPR) Upon binding to the CAR itself, the CAR binds to one or more non-glioma tumors in the brain. Cancer-associated killing antigens (i.e., antigens that are also expressed on other normal cells outside the brain) These antibodies can be activated by binding to disease-specific antigens that can be detected by these antibodies. In embodiments, the CAR is a CAR whose extracellular binding region is capable of targeting one or more pediatric brain tumors (e.g., Medulloblastoma, diffuse midline glioma (formerly known as DIPG), ependymoma, craniopharyngioma, embryonal tumor (formerly known as P NET), pineoblastoma, brainstem glioma, choroid plexus carcinoma or germ cell tumor, or one or More adult brain tumors, such as pituitary adenomas and acoustic neuromas (also known as vestibular schwannomas) meningioma, oligodendroglioma, hemangioblastoma, CNS lymphoma, non-GBM (or low-grade malignant tumors) ) Associated with astrocytoma or tumors of unknown cell types (i.e., gliomas of unspecified origin) They can be activated by binding to cancer-specific antigens, which are associated with many of these tumors. The cancer-specific antigens are known or may become known.

[0108] A transmembrane protein is a binding-triggered transcriptional switch that activates the expression of secreted proteins In embodiments, the binding-triggered transcriptional switch binds to a brain-selective extracellular antigen (e.g., MOG, CDH 10, PTPRZ1 or NRCAM), secreted proteins are Although there is no evidence that Infections, ataxia, amyotrophic lateral sclerosis (ALS), Huntington's disease, and other conditions that affect the brain Inherited genetic errors in children, Parkinson's disease, multiple sclerosis, and brain cancer (non-pleomorphic any of a variety of diseases and conditions, including glioblastoma and other brain cancers listed above Many treatments for such diseases and conditions are known or may be specific to the treatment. Or it may become publicly known.

[0109] In some embodiments, the circuitry includes at least two BTTSs (e.g., two, three, or four BTTS) and antigen-specific therapeutic agents, the BTTSs being in series (one One BTTS activates another BTTS) or can be connected in parallel, and one of the BTTS One of the BTTS binds to a brain-selective antigen, and at least one of the other BTTS binds to a different antigen, e.g., another brain-specific antigen. The use of multiple BTTSs makes treatment more specific and reduces side effects. It could be less.

[0110] The circuit detects brain-selective extracellular antigens (e.g., MOs) on brain cells (which may be diseased or normal). G, CDH10, PTPRZ1, or NRCAM) (referred to herein as a "priming antigen"). and the expression of at least a second antigen expressed on a second diseased cell in the brain. In some instances, the present invention may be integrated with the present invention to produce a desired result for a second cell. The circuitry involves the expression of a brain-selective extracellular antigen present on normal brain cells and a second one expressed on diseased cells. and at least a second antigen to produce a desired result with respect to the second cell. The targeting events can be achieved by using a gene that is expressed by different cells of a heterogeneous cell population. The integration of two antigens can be referred to herein as "transtargeting."

[0111] In some embodiments, brain-selective extracellular antigens (e.g., MOG, CDH10, PTP RZ1 or NRCAM) may be expressed on primary brain cells (which may be normal) and may be expressed by the cells. The therapeutic protein produced thereby can have a therapeutic effect on the second cell. In this form, brain-selective extracellular antigens may be expressed on diseased brain cells and produced by the cells. Therapeutic proteins may have a therapeutic effect on the same cells.

[0112] For comparison, in this context, sister targeting refers to targeting a priming antigen (e.g., For example, MOG, CDH10, PTPRZ1, or NRCAM) and a target antigen (e.g., a disease-specific targeting a single cell that expresses both a specific marker and a specific target cell, and Therefore, in sister targeting, the target Target cells express the priming antigen and the target antigen in cis-expression. In transtargeting, the target cell expresses both the antigen and the target cell. Express only the target antigen and the priming antigen so that they are expressed in trans to the cells. Therefore, transtargeting does not express the priming antigen. In some examples, the circuits of the present disclosure can be used to target cells. Both primary and secondary targeting may be used, i.e., primary targeting and secondary targeting may be used. Targeting and transtargeting may be combined in a single circuit. Alternatively, the disclosed circuits may use transtargeting only, e.g., sister Targeting may be excluded.

[0113] In some embodiments, the therapeutic cells are derived from brain-selective extracellular antigens (e.g., MOG, CD H10, PTPRZ1, or NRCAM) The binding-triggerable transcriptional switch may be expressed in the plasma membrane of the cell. Binding of the binding-triggered transcriptional switch to a priming antigen can activate the binding-triggered transcriptional switch. In some embodiments, the expression of the protein in the HIV-1-expressing cell can be induced. The protein may be a heterologous antigen-specific protein, such as a second binding-triggered transcriptional switch or The antigen-specific therapeutic agent may be a heterologous antigen-specific therapeutic agent, examples of which are described above and below. In the context of priming, binding of the binding-triggered transcriptional switch to a priming antigen is essential for the transcription of priming antigens. Induce the expression of antigen-specific proteins specific to the target antigen that are further expressed by the target cells (i.e., this cell is the priming cell and the target cell). In the context of targeting, binding-induced transcriptional switching to a priming antigen expressed on the cell The binding of the antibody is specific to a target antigen expressed on a different cell line that does not express the priming antigen. Induce the expression of antigen-specific proteins.

[0114] Thus, transtargeting may target the presence of brain cells expressing brain-selective extracellular antigens. Only under these conditions will it be possible to target cells with therapeutic proteins, such as antigen-specific therapeutics. Correspondingly, transtargeting allows target cells to be selectively extracellular in the brain, such as in heterogeneous cancers. In heterogeneous cell populations that do not express an antigen, antigen-specific proteins, such as antigen-specific therapeutic agents, can be expressed. Therefore, such target priming antigens ( -) cells are priming antigen positive ("priming antigen (+)"), i.e., The antigen may be spatially associated with cells expressing the antigen.

[0115] method As summarized above, some embodiments of the present disclosure provide a method for treating diseased cells in the brain. In some embodiments, diseased cells can be targeted in trans. The method comprises administering to a subject in need thereof a brain-selective extracellular antigen (e.g., MOG, CDH10, Transmembrane tag with an extracellular binding domain that specifically binds to PTPRZ1 or NRCAM Therapeutic cells containing a recombinant nucleic acid encoding a protein expressed by glioblastoma. administering cells that do not contain a nucleic acid encoding an antigen-specific therapeutic agent that binds to the killing antigen to be treated; As noted above, transmembrane proteins can have many different structures. In some embodiments, the transmembrane protein drives expression of a therapeutic protein. It may be a binding-triggered transcriptional switch.

[0116] Treatment method As summarized above, the methods of the present disclosure can be used to treat brain diseases or disorders, including but not limited to: However, medulloblastoma, diffuse midline glioma (formerly known as DIPG), ependymoma, craniopharyngioma, and embryonal tumor (formerly known as PNET), pineoblastoma, brainstem glioma, choroid plexus carcinoma, or is a germ cell tumor, or one or more adult brain tumors, such as pituitary adenoma, acoustic neuroma ( vestibular schwannoma), meningioma, oligodendroglioma, hemangioblastoma, CNS lymphoma tumors with unknown cell types (i.e., unspecified) gliomas), Alzheimer's disease, stroke, brain and spinal cord injuries, brain cancer, HIV infection in the brain Infections, ataxia, amyotrophic lateral sclerosis (ALS), Huntington's disease, and other conditions that affect the brain Brain diseases or disorders, including childhood congenital genetic errors, Parkinson's disease, and multiple sclerosis Such treatment finds use in treating subjects for at least one of the following: It may involve obtaining a desired effect on one disease cell type (or a subpopulation thereof).

[0117] In some embodiments, the disease is cancer of non-brain or non-CNS tissue origin that has metastasized to the brain. Brain metastases can arise from any type of cancer. The most common types of cancer that spread to the brain are , breast cancer, lung cancer, kidney cancer, melanoma, colon cancer, and thyroid cancer.

[0118] The subject methods can include introducing into a subject in need thereof a population of cells as described above. The introduced cells can be immune cells, including, for example, myeloid or lymphoid cells. In some cases, the introduced cells are not immune cells.

[0119] In some examples, the method can include contacting the cell with one or more nucleic acids. Such contacting step is sufficient to introduce the nucleic acid(s) into the cell. Any convenient method of introducing the compound into a cell may find use herein, including but not limited to Although not intended to be used for transfection, viral transfection, electroporation, lipofection, transfection, bombardment, chemical transformation, transducing vehicles (e.g., transduction Nucleic acids can be delivered in vitro or ex vivo. The nucleic acid can be introduced into maintained or cultured cells. by the use of one or more vectors (e.g., viral vectors) that deliver It can be introduced into living cells of a subject in vivo without the need to isolate, culture, or maintain cells outside the body. You can enter.

[0120] The introduced nucleic acid may be maintained within the cell or may be transiently present. In some instances, the introduced nucleic acid is capable of being maintained in the cell, e.g., incorporated into the genome. Any convenient method of incorporating nucleic acids can be used in the subject method. These include, but are not limited to, viral-based integration, transposons, Some examples include integration based on homologous recombination, and integration based on homologous recombination. The introduced nucleic acid can be transient, e.g., extrachromosomal, in the cell. Transiently present nucleic acids can be, for example, transferred into any convenient transient medium. It can remain as part of the injected vector.

[0121] The introduced nucleic acid encoding the circuit drives expression of one or more components of the circuit. The vector can be introduced so that it is operably linked to a regulatory sequence such as a promoter. The source of such regulatory sequences can vary, for example, if the regulatory sequences are used in conjunction with the nucleic acid, e.g., If introduced as part of a construct, or if the regulatory sequences are present in the cell prior to introduction of the nucleic acid or introduced after the nucleic acid. Useful regulatory sequences can include, for example, endogenous promoters and heterologous promoters. For example, in some instances, the nucleic acid comprises a heterologous promoter operably linked to the nucleic acid sequence. In some instances, the nucleic acid may be introduced as part of an expression construct comprising the nucleic acid. As part of an expression construct containing a copy of a promoter endogenous to the cell in which it is introduced In some instances, the nucleic acid may be introduced without any regulatory sequences, resulting in a cellular Once incorporated into the genome, the nucleic acid is operably linked to endogenous regulatory sequences already present in the cell. Depending on the confirmation and / or regulatory sequences used, recovery from the nucleic acid may occur. Expression of each component of the pathway is constitutive, inducible, tissue-specific, and cell-type-specific. A combination of these may also be included.

[0122] Any convenient method of delivering the circuit code components may find use in the subject methods. In some instances, the subject circuits can be administered to a subject by administering cells expressing the circuits to the subject. In some instances, the target circuit may be delivered by one or more The delivery can be by administering to a subject a nucleic acid comprising one or more of the nucleotide sequences. The step of administering to a subject a nucleic acid encoding the and the nucleic acid may or may not be expressed. In examples, administering to a subject a nucleic acid encoding a circuit comprises administering the nucleic acid to a subject so as to deliver the nucleic acid to a cell. The method may include administering the designed vector to a subject.

[0123] Thus, the subject therapeutic methods involve in vitro administration of nucleic acids encoding the circuit or its components. In some instances, cells may be administered ex vivo or in vivo. The cells are harvested from the culture medium, transfected with a nucleic acid, and the transfected cells are subjected to a limiting However, the subject may be a mammalian cell line, with or without further manipulation, including, for example, in vitro propagation. In some instances, the nucleic acid can be administered, for example, with or without a delivery vector. Regardless, it can be administered directly to the subject.

[0124] The priming and target cells of the circuit generally contain at least a priming antigen and In some instances, the priming cells and the target cells differ in their expression of at least one antigen. At least one surface-expressed epitope, e.g., a surface-expressed protein, presented in the context of MHC The expression of antigens expressed may differ, e.g., surface-expressed epitopes may be priming antigens and / or In some cases, the target antigen may be a molecule other than the target antigen. The cells contain at least one surface-expressed epitope, e.g., a surface-expressed protein, associated with MHC. The expression of antigens and the like that are presented may differ.

[0125] Differential expression between two cells or two cell types can vary. For example, in some instances In some cases, a cell expresses one surface epitope that is not expressed by other cells. In this example, a cell may express one surface epitope in a manner that is incompatible with a surface epitope expressed by another cell. The cells express the surface epitope more highly than the surface epitope. When the levels are different, the differences in the levels may vary but are generally substantially different, e.g., The expression difference between cells is sufficiently different to allow practical targeting of one cell to the other. , for example, but not limited to, 1 digit, 2 digits, 3 digits, 4 digits, 5 digits, 6 digits, 7 digits, 8 digits ranging from less than one order of magnitude to ten or more orders of magnitude, including 9 orders of magnitude, 10 orders of magnitude, etc. In some instances, two cell types that differ in the expression level of a particular epitope may These are sometimes referred to as "high" and "low" for that epitope, respectively. Low expression can be distinguished using conventional methods known to those skilled in the art.

[0126] In some instances, the methods of the present disclosure may be used to treat minimal residual disease (MRD) remaining after a previous treatment. used to target, treat, or clear a subject from multiple myeloma (MRD) Targeting, treatment and / or clearance of MRD can be used to treat MRD at a level that is comparable to the previous level. Regardless of whether the disease is refractory to treatment or has been determined to be refractory, In some examples, the methods of the present disclosure can be carried out using Previous treatment or one or more available treatments other than those using the circuit described herein targeting and treating the subject's MRD after determination that the patient is refractory to treatment options; and and / or can be used for clearance.

[0127] In some instances, the methods can be used prophylactically for monitoring. If the elephant does not have detectable disease but is at risk of developing the disease, it is required The subject can be administered a treatment that includes one or more of the circuits described herein. In some instances, a preventative approach is used when a subject is at particularly high risk of developing a disease. In some instances, the subject may have been previously treated for the disease and may be at risk of recurrence. If there is a risk, a preventative approach can be used. Any combination of priming antigens and targeting antigens can be used for prophylactic treatment, including those described in It can be used.

[0128] The treatment methods described herein may, in some instances, be administered in combination with one or more conventional treatments. For example, in oncology, the methods described herein may be used in subjects who have previously undergone The methods described above may, in some instances, be used in combination with conventional chemotherapy, for example and without limitation. , following conventional cancer treatments, including conventional radiation therapy, conventional immunotherapy, and surgery. In some instances, the methods described herein can be used to treat a variety of conditions, including: It can be used when the condition is severe or refractory.

[0129] With respect to the disease as a whole, the desired effect of the described treatment is a reduction in the number of diseased cells, This may result in a decrease in size, a reduction in one or more symptoms, and the like.

[0130] Immune cell activation as a result of some embodiments of the methods described herein includes, but is not limited to, For example, but not limited to, measuring the expression level of one or more markers of immune cell activation. Useful markers of immune cell activation can be measured by a variety of methods, including measuring Examples of the target antigen include, but are not limited to, CD25, CD38, and CD40L (CD 154), CD69, CD71, CD95, HLA-DR, and CD137. For example, in some instances, immune cells become activated upon antigen binding by immune cell receptors. and elevated levels of markers of immune cell activation (e.g., CD69) (e.g., antigen-responsive). The activated immune cells of the present disclosure may express at least one of the following levels: The increase in expression levels varied, with a ≥1-fold increase in marker expression compared to non-activated controls. For example, but not limited to, a 1-fold increase, a 2-fold increase, a 3-fold increase, a 4-fold increase, This may include increases, including additions.

[0131] In some examples, immune cells engineered to encode the circuits of the present disclosure can be used to target antigens. Upon binding to the antibody, the antibody may have increased cytotoxic activity, for example, compared to non-activated control cells. In some instances, activated immune cells encoding the target circuitry exhibited significantly higher levels of ATP than non-activated control cells. In some instances, activated immune cells may exhibit cell killing of 10% or more of antigen-expressing target cells. The level of increased cell killing of cells varies and may be 10% or more compared to appropriate controls. However, for example, 20% or more, 30% or more, 40% or more, 50% or more, 60% The range may include greater than or equal to 70%, greater than or equal to 80%, greater than or equal to 90%, etc.

[0132] In some examples, the treatment involves administering to a subject an immune cell comprising a nucleic acid sequence encoding a circuit described herein. This may include modulation, including induction, of cytokine expression and / or secretion by cells. Non-limiting examples of cytokines whose secretion can be regulated include, but are not limited to, e.g., For example, interleukins and related substances (e.g., IL-1-like, IL-1α, IL-1β, IL-1RA, IL-18, IL-2, IL-4, IL-7, IL-9, IL-13, I L-15, IL-3, IL-5, GM-CSF, IL-6-like, IL-6, IL-11, G -CSF, IL-12, LIF, OSM, IL-10-like, IL-10, IL-20, IL -14, IL-16, IL-17, etc.), interferons (e.g., IFN-α, IF IFN-β, IFN-γ, etc.), TNF family (e.g., CD154, LT-β, ​​TNF -α, TNF-β, 4-1 BBL, APRIL, CD70, CD153, CD178, GITRL, LIGHT, OX40L, TALL-1, TRAIL, TWEAK, TRA NCE, etc.), TGF-β family (e.g., TGF-β1, TGF-β2, TGF- β3, etc.

[0133] In some instances, activation of immune cells via the circuits of the present disclosure occurs in the absence of the circuits. Increased cytokine expression and / or secretion over that of otherwise inactive equivalent cells The amount of increase may vary and may be greater than 10%. The increase may be in the range of, but is not limited to, 10% or more, 25% or more, 5% or more, or more. 0% or more, 75% or more, 100% or more, 150% or more, 200% or more, 250% or more, 3 This includes increase ranges such as 00% or more, 350% or more, and 400% or more.

[0134] Conventional and combined treatments As will be readily appreciated, the methods of treatment described herein may, in some instances, involve the administration of a single or more conventional treatments, such as those described herein. In some examples, therapies include, but are not limited to, drug treatments, conventional chemical treatments, and the like. Combined with conventional therapies including chemotherapy, conventional radiation therapy, conventional immunotherapy, and surgery. This can be done.

[0135] In some instances, the methods described herein may be used before or after conventional therapy. For example, the methods described herein can be used to treat a variety of conditions, e.g., when a subject experiences improvement from conventional therapy. It can be used as an adjuvant therapy after administration of the compound or when a subject has not responded to conventional therapy. In some instances, the methods described herein can be used in further For example, the subject may be administered an additional therapy, e.g., a conventional therapy, as described herein, prior to the therapy. It can be used before preparing for the test.

[0136] Antigen-specific therapeutic agents As summarized above, in some embodiments, the method involves the step of generating a priming antigen in response to the priming antigen. The binding-triggered transcriptional switch (BTTS) regulates antigen-specific responses to one or more target antigens. Useful antigen-specific therapeutic agents vary in surface expression and Examples of such therapeutic agents include secretory antigen-specific therapeutic agents. The antigen-specific therapeutic agent used in the method is capable of targeting immune cells, i.e., BTTS, in response to their activation. Immune cells genetically engineered to encode the priming / targeting circuits described herein. In some examples, the antigen-specific therapeutic used in the methods of the present disclosure may be expressed on the surface of a cell. The drug induces immune cells in response to BTTS activation, i.e., the priming described herein. / Can be secreted from immune cells genetically modified to encode the targeting circuitry.

[0137] Generally, unless otherwise stated, the antigen-specific therapeutics of the circuits described herein In addition, the expression of BTTS is not expressed in the absence of activation of BTTS, which induces the expression of BTTS. Except as noted above, the antigen-specific therapeutics of the circuits described herein are directed to therapeutics that bind to antigens in the absence of the antigen to which they bind. ie, they are not active without binding to the antigen for which they are specific. The binding of the respective antigen, or antigens in the case of multispecific or bispecific agents, is Activation of antigen-specific therapeutic agents. Expressed by immune cells or otherwise interacting with immune cells. Upon association with immune cells and binding to the antigen(s), the antigen-specific therapeutic activates the immune cells. Activated immune cells can have the beneficial effects described herein, such as limited immune response. Although it does not target the disease in the target brain, it does target the disease in the target brain, including killing cancer cells and releasing cytokines. It may mediate one or more beneficial effects on the cell.

[0138] With respect to the antigen-specific binding domains described herein, the term "antigen" refers to an antigen-specific The term "specific binding partner" is used broadly to refer to essentially any specific binding partner to which a therapeutic agent binds. Thus, any convenient specific binding pair, including but not limited to, Specific binding members, including, for example, antigen-antibody pairs, ligand-receptor pairs, scaffold protein pairs, etc. and specific binding partner pairs may find use in antigen-specific therapeutics of the present methods. In some examples, the specific binding member may be an antibody and the binding partner may be an antibody. In some instances, the specific binding member may be a receptor, an antigen to which the body specifically binds. and the binding partner may be a ligand to which the receptor specifically binds. In some instances, the specific binding member may be a ligand and its binding partner may be a ligand. The receptor to which the peptide specifically binds may be a receptor.

[0139] In some cases, useful ligand-receptor specific binding pairs include those that specifically bind The members may have at least one mutation, including but not limited to, a mutation in the wild-type ligand. For example, one or more mutations, two or more mutations, three or more mutations, four or more Muteins of the ligand having mutations containing more than one mutation, such as five or more mutations. In some instances, useful muteins may be At least 90% sequence identity with the amino acid sequence, for example, but not limited to, related A sequence identical to the wild-type amino acid sequence by at least 95%, at least 96%, at least 97%, or It is believed that the sequence identity of the sequence is at least 98%, at least 99%, etc. In some instances, the mutein used in the subject polypeptide is a polypeptide that is a cross-link between the receptor and the wild-type ligand. The agonist may have a higher affinity for the receptor compared to the affinity of the agonist.

[0140] Antigen-specific therapeutic agents useful in the methods of the present disclosure include a variety of agents, including but not limited to: For example, chimeric antigen receptors (CARs), T cell receptors (TCRs), chimeric bispecific antibodies, It may include joint members, etc.

[0141] Useful CARs include single-chain and multi-chain CARs directed against one or more target antigens. The present invention includes essentially any CAR useful in the treatment of cancer, including CARs that are capable of inhibiting the growth of cancer cells. The CARs generally contain, at a minimum, an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The CARs used are likely to contain one or more costimulatory domains. The composition may further include a

[0142] Non-limiting examples of CARs that can be used include CARs directed against one or more appropriate target antigens. or modified to target one or more appropriate target antigens. These include those used in commercially available CAR T cell (CART) therapies. CARs used in the present invention include, but are not limited to, EphA2, EphA3, IL-1, and IL-2. 13R (e.g., IL13RA1 or IL13RA2), EGFR, and ERBB2 Does not target glioblastoma antigens.

[0143] For example, useful CARs that can be engineered to target appropriate target antigens, or e.g. Useful domains that can be used in CARs directed to appropriate target antigens include: In some examples, see U.S. Patent Nos. 9,914,909; 9,821,012; No. 9,815,901; No. 9,777,061; No. 9,662,405; No. 9 ,657,105; Same No. 9,629,877; Same No. 9,624,276; Same No. 9, No. 598,489; No. 9,587,020; No. 9,574,014; No. 9,5 No. 73,988; No. 9,499,629; No. 9,446,105; No. 9,39 No. 4,368; No. 9,328,156; No. 9,233,125; No. 9,175 ,308 and 8,822,647; the disclosures of which are The entire contents of which are incorporated herein by reference. In some instances, useful CARs include Contains heterodimeric (also called dimerizable or switchable) CAR and / or may include or exclude one or more of its domains. Useful heterodimeric CARs and / or useful domains thereof include, in some examples: Nos. 9,587,020 and 9,821,012, and U.S. patent application Ser. Publication No. 20170081411A1, U.S. Patent Application Publication No. 20160311901A1 No. 20160311907A1, U.S. Patent Application Publication No. 20150 266973A1, and PCT Publication Number International Publication No. 2014127261A1, International Publication No. 2015142661A1, International Publication No. 2015090229A1 and International Examples include those described in Publication No. 2015017214A1, the disclosure of which is incorporated herein by reference. The entirety of which is incorporated herein by reference.

[0144] As summarized above, in some instances, the antigen-binding domain of the CAR (e.g., the limiting (such as those described in any one of the documents referenced above, but which do not For use in the methods described herein, different antigens (such as, but not limited to, alternative antigen binding directed to a target antigen (e.g., one or more of the antigens described herein, but not limited to the target antigen) The antigen-binding domain may be replaced or modified with an additional antigen-binding domain. In this case, the intracellular portion of the antigen domain-substituted CAR (i.e., the intracellular signaling domain or The co-stimulatory domain(s) may be modified or unmodified.

[0145] Useful CARs and / or useful domains thereof include, in some instances, one or more of: These include those that have been studied in clinical trials or are currently being studied. This includes, but is not limited to, CARs directed against the following antigens (examples include: Clinical trials are listed with their corresponding clinical trial numbers and further information relating thereto can be found at www.clin can be found by visiting icaltrials(dot)gov): e.g., NCT03 AFP in 349255; e.g., BCMA in NCT03288493; e.g., CD10 in NCT03291444; CD in NCT03291444 117; CD123 in e.g., NCT03114670; e.g., NCT025413 CD133 in 70; e.g., CD138 in NCT01886976; e.g., CD171 in NCT02311621; e.g., C in NCT02813252 D19; e.g., NCT03277729; CD20; e.g., NCT0324430 CD22 in e.g., NCT02917083; CD30 in e.g., NCT0 CD33 in 3126864; e.g., CD34 in NCT03291444; e.g., For example, CD38 in NCT03291444; for example, CD38 in NCT03081910 CD5; e.g., NCT03291444; CD56; e.g., NCT0274272 CD7 in 7; CD70 in e.g. NCT02830724; CD80 in 356808; e.g., CD86 in NCT03356808; e.g., For example, CEA in NCT02850536; C in NCT03159819 LD18; e.g., NCT03312205; CLL-1; e.g., NCT01837 cMet in 602; e.g., EGFR in NCT03182816; e.g., NC EGFRvIII in T02664363; e.g., in NCT03013712 EpCAM; e.g., NCT02575261; EphA2; e.g., NCT018 GD-2 in 22652; e.g., glypican 3 in NCT02905188; e.g., For example, GPC3 in NCT02723942; for example, GPC4 in NCT02547961 HER-2; e.g., kappa immunoglobulin in NCT00881920; e.g., N LeY in CT02958384; LMP1 in NCT02980315 mesothelin in NCT02930993; mesothelin in NCT02862704 MG7 in e.g., NCT02587689; MUC1 in e.g., NCT02 NKG2D ligands in 203825; PD in e.g. NCT03330834 -L1; e.g., PSCA in NCT02744287; e.g., NCT0335679 PSMA in 5; ROR1 in NCT02706392; ROR1R, e.g., in 02194374; TAC, e.g., in NCT03287804 I; and VEGFR2, e.g., in NCT01218867.

[0146] Useful TCRs include single-chain and multi-chain TCRs directed against target antigens, and are useful in the treatment of cancer. The TCRs used in the present methods generally include essentially any TCR useful in At a minimum, the antigen-binding domain and modified or unmodified TCR chain or part thereof, but not limited to The T used includes, but is not limited to, modified or unmodified α chains, modified or unmodified β chains, etc. The CR may further comprise one or more costimulatory domains. The TCR used in this document contains alpha and beta chains and is a major histocompatibility complex (MHC) Thus, they recognize antigens when presented.

[0147] Essentially, for example, epitopes expressed on the surface of cancer cells, peptides on the surface of cancer cells, Any TCR containing specific epitopes, including peptide-MHC complexes, Any TCR can be induced by BTTS using the methods of the present disclosure. Thus, the TCR is an engineered TCR.

[0148]

[0033] A method for the preparation of a medicament for the treatment of a cancer, comprising administering to a patient a therapeutic agent, a medicament for the treatment of a cancer, a cancer cell, a cancer cell activating agent, a medicament for the treatment of a cancer ... Non-engineered TCRs, including those that can be modified to contain specific antigen-binding domains. Non-limiting examples include, for example, antigen-specific TCRs, monoclonal TCRs (MTCRs), single Chain MTCR, high affinity CDR2 mutant TCR, CD1-binding MTCR, high affinity NY-ES O TCRs, VYG HLA-A24 telomerase TCR, for example, PCT published Nos. WO 2003 / 020763, WO 2004 / 033685, and WO 2004 / 033685. Publication No. 2004 / 044004, International Publication No. 2005 / 114215, International Publication No. 006 / 000830, WO 2008 / 038002, WO 2008 / 039818, WO 2004 / 074322, WO 2005 / 1135 No. 95, International Publication No. 2006 / 125962; Strommes et al. Immunol Rev. 2014; 257 ( 1):145-64;Schmitt et al.Blood.2013;122(3):348-56;Chapuls et al.Sci Transl Med.20 13;5(174):174ra27;Thaxton et al.Hum Vaccin Immunother.2014;10(11):3313-21(PMID:2 5483644);Gschweng et al.Immunol Rev.2014;257(1):237-49(PMID:24329801);Hinrichs e t al.Immunol Rev.2014;257(1):56-71(PMID:24329789);Zoete et al.Front Immunol.2013 ;4:268(PMID:24062738);Marr et al.Clin Exp Immunol.2012;167(2):216-25(PMID:222359 97);Zhang et al.Adv Drug Deliv Rev.2012;64(8):756-62(PMID:22178904);Chhabra et a l.Scientific World Journal.2011;11:121-9(PMID:21218269);Boulter et al.Clin Exp I mmunol.2005;142(3):454-60(PMID:16297157);Sami et al.Protein Eng Des Sel.2007;20( 8):397-403;Boulter et al.Protein Eng.2003;16(9):707-11;Ashfield et al.IDrugs.200 6;9(8):554-9;Li et al.Nat Biotechnol.2005;23(3):349-54;Dunn et al.Protein Sci.20 06;15(4):710-21;Liddy et al.Mol Biotechnol.2010;45(2);Liddy et al.Nat Med.2012;1 8(6):980-7;Oates,et al.Oncoimmunology.2013;2(2):e22891;McCormack,et al.Cancer Im munol Immunother.2013 Apr;62(4):773-85;Bossi et al.Cancer Immunol Immunother.201 4;63(5):437-48 and Oates, et al. Mol Immunol. 2015 Oct;67(2 Pt A):67-74; and US Pat. No. 6,239,999, the disclosures of which are incorporated herein by reference in their entireties.

[0149] Useful TCRs include those with wild-type affinity for their respective antigens, as well as those These include those with enhanced affinity for each of these antigens. TCRs with enhanced affinity due to their affinity are called "affinity-enhanced" or "enhanced affinity" TCRs. The affinity of TCRs can be improved by, but not limited to, binding site engineering (e.g., (i.e., rational design), screening (e.g., TCR display), etc. The affinity of TCRs can be enhanced by any convenient means. Non-limiting examples of methods for preparing include, but are not limited to, those described in PCT Publication No. No. 20150118208, 2013256159, 2016008344 No. 9, No. 20140349855, No. 20100113300, No. 20140371 No. 085, No. 20060127377, No. 20080292549, No. 201602 No. 80756, No. 20140065111, No. 20130058908, No. 2011 Nos. 0038842, 20110014169, 2003276403, etc. and JP 2004-102666 A, the disclosure of which is incorporated herein by reference in its entirety. and targeting an appropriate target antigen that can be expressed in response to shedding of the intracellular domain of the BTTS. Additional engineered TCRs that have been modified to produce the desired TCRs include, for example, those described in PCT Application No. US 2017 / 048040, the disclosure of which is incorporated herein by reference in its entirety. incorporated herein.

[0150] Useful TCRs that can be engineered to target appropriate target antigens include, but are not limited to: See U.S. Patent Nos. 9,889,161; 9,889,160; and 9,868, No. 765; No. 9,862,755; No. 9,717,758; No. 9,676,8 No. 67; No. 9,409,969; No. 9,115,372; No. 8,951,51 No. 0; No. 8,906,383; No. 8,889,141; No. 8,722,048 No. 8,697,854; No. 8,603,810; No. 8,383,401 ;8,361,794;Same No.8,283,446;Same No.8,143,376;Same No. No. 8,003,770; No. 7,998,926; No. 7,666,604; No. 7 , No. 456,263; No. 7,446,191; No. 7,446,179; No. 7, 329,731; 7,265,209; and 6,770,749. Further examples include: It can be enjoyed.

[0151] As noted above, in some instances, the antigen-binding domain of a TCR, including but not limited to, Although not specified, those described or referenced above and the like are intended for use in the methods described herein. To achieve this, different antigens, including but not limited to one or more of the antigens described herein, may be used. , or by substitution or addition of an alternative or additional antigen-binding domain directed against In such cases, the antigen domain can be modified by replacing other parts of the TCR (i.e. , transmembrane domains, any intracellular signaling domains, etc.) may or may not be modified. That's fine.

[0152] As summarized above, in some instances, useful antigen-specific therapeutics may involve activating BTTS. These include those expressed and secreted from producing cells upon induction by IL-1, and these secreting cells are immune cells. For example, the binding of BTTS expressed by immune cells may induce BT TS induces the expression and secretion of an encoded antigen-specific therapeutic agent specific for the target antigen. Secretory antigen-specific therapeutic agents target target antigen-expressing cancer cells in trans, This can mediate the killing of target cells. As described herein, in some examples Therefore, secretory antigen-specific therapeutic agents are used in combination with non-secretory (e.g., membrane-expressed) antigen-specific therapeutic agents. The target or kill area of ​​the target circuit may be enlarged compared to a similar circuit that is being loaded.

[0153] Useful secretory antigen-specific therapeutic agents are varied and include, but are not limited to, However, they may include, for example, chimeric bispecific binding members. Bispecific binding members include, but are not limited to, T cell receptors. A receptor on immune cells that contains components of a T cell receptor (TCR), e.g., one or more T cell co-receptors These include those that target proteins expressed on the TCR surface. A chimeric bispecific binding member that binds to a TCR is referred to herein as a TCR-targeted bispecific binding member. Chimeric bispecific binding members useful in the present methods can generally be referred to as They are specific for the target antigen and, in some cases, are expressed on the surface of immune cells that are related to the target antigen. It is specific for proteins that are involved in the TCR (e.g., components of the TCR, such as the CD3 co-receptor). obtain.

[0154] In some instances, useful chimeric bispecific binding members include bispecific T cells BiTEs are generally used to target immune cell antigens. A specific binding member (e.g., scFv) that binds to a cancer antigen (e.g., tumor-associated antigen) is The antibody is fused to a specific binding member (e.g., scFv) that binds to an antigen (e.g., a tumor-specific antigen, etc.). For example, exemplary BiTEs include a short peptide linker (e.g., For example, an anti-tumor-associated antigen (e.g., Ep) can be linked via a five amino acid linker, e.g., GGGGS. Anti-CD3 scFv fused to an anti-CAM, CD19, etc. scFv.

[0155] As summarized above, in some instances, the antigen-binding domain of the chimeric bispecific binding member The present invention relates to the use of the present invention in a variety of applications, including, but not limited to, those described or referenced above. For use in the methods, different antigens, including but not limited to those described herein, may be used. Alternatively or additional antigen binding domains directed to one or more of the above antigens may be used. In one example, the antigen domain can be replaced or modified by substitution of a binding domain, etc. Other portions of the chimeric bispecific binding member (i.e., linker domain, any immunocytochemical The domains (e.g., cell targeting domain) may be modified or unmodified.

[0156] In some instances, to its respective priming antigen in the methods described herein The payload induced by the binding of BTTS to a secreted bioorthogonal adaptor molecule Such bioorthogonal adapter molecules may, in some instances, target a target antigen. and also binds or is bound by heterologous polypeptides expressed by immune cells. It can be configured to be able to do so.

[0157] For example, in some instances, the titular circuits used in the methods described herein are BTTS responds to priming antigens within the target antigen; and a therapeutic agent or portion thereof that binds to a bioorthogonal adaptor molecule. In such circuits, the expression and secretion of bioorthogonal adaptor molecules is driven by BTTS. Then, (1) the target antigen-expressing cancer cells and (2) 2) In the presence of both a therapeutic agent and a bioorthogonal adaptor molecule, the therapeutic agent binds to the bioorthogonal adaptor molecule. The bioorthogonal adaptor molecule then binds to the target antigen, The therapeutic agent is then activated. The activated therapeutic agent then reacts with the target antigen to prime it. against diseased cells expressing the target antigen, including when the antigen is expressed in trans As described herein, some In some instances, the secreted bioorthogonal adapter molecule may be coupled to a non-secreted (e.g., membrane-expressed) antigen. Expand the target or kill area of ​​a circuit of interest compared to a similar circuit encoding a specific therapeutic agent. It can be made bigger.

[0158] Bioorthogonal adapter molecules can be used in a variety of contexts within the methods described herein. For example, in some instances, the diffusible antigen-binding portion of the antigen-specific therapeutic, e.g., CA Bioorthogonal adaptor molecules containing the diffusible antigen-binding portion of R and the diffusible antigen-binding portion of TCR. In some instances, such diffusible antibodies of antigen-specific therapeutics can be used. The original binding moiety includes, for example, a "diffusible CAR head" and a "diffusible TCR head." This can be called a "diffusive head."

[0159] In some instances, the therapeutic agent may be directly conjugated to the bioorthogonal adapter molecule. Strategies for direct attachment of therapeutic agents to peptide molecules can vary. For example, some examples include In this study, the therapeutic agent is a binding moiety (e.g., an antibody targeting a target molecule) covalently attached to a bioorthogonal adaptor. a binding domain (e.g., an orthogonal antibody or fragment thereof) that binds to the orthogonal epitope As a non-limiting example, the therapeutic agent may comprise a non-naturally occurring epitope, e.g., an anti-flu and a bioorthogonal adaptor molecule, which may comprise a binding domain to an orescein antibody or fragment thereof. may contain an epitope, e.g., fluorescein, covalently attached to it. In examples, the composition and therapeutic interaction of the bioorthogonal adaptor molecule can be achieved by, for example, covalently binding a therapeutic agent. the bioorthogonal adaptor molecule comprises a binding domain for the epitope. The useful epitopes are varied and may be reversed compared to those described above, including cases where Examples of suitable epitopes include, but are not limited to, small molecule-based epitopes, peptide-based epitopes, and the like. epitopes (e.g., peptide neoepitopes), oligonucleotide-based epitopes, etc. Epitope binding domains can vary accordingly and include, but are not limited to: However, for example, small molecule binding domains, peptide binding domains, oligonucleotide binding domains Domains can be mentioned.

[0160] Non-limiting examples of useful bioorthogonal adapter molecules and their binding domains include: For example, but not limited to, see Rodgers et al. Proc Natl Acad Sci USA. (2016) 113(4 ):E459-68 and Cao et al., Angew Chem Int Ed Engl. 2016 Jun 20;55(26):7520-4 and P Switchable C, including those described in CT Publication No. WO 2016168773 Peptide neoepitopes used in AR (sCAR) T cells and antibody binding to them and the like, the disclosure of which is incorporated herein by reference in its entirety. do.

[0161] In some instances, the therapeutic agent can be indirectly conjugated to a bioorthogonal adaptor molecule. , for example, when binding is mediated by a diffusible dimerizing agent. Non-limiting examples of suitable dimerizers and dimerization domains attached thereto include: includes protein dimerizers.

[0162] Protein dimers are generally prepared, for example, in the presence of or upon exposure to a dimerizing agent. The dimerized polypeptides of the protein dimerization product contain a pair of polypeptides that dimerize when exposed to light. The peptide pairs may be homodimerized or heterodimerized (i.e., A dimerization polypeptide pair can be two identical polypeptides forming homodimers or heterodimers. (It may contain two different polypeptides that form a dimer.) Non-limiting examples of protein dimers Suitable pairs (with the associated dimerizer in parentheses) include, but are not limited to, For example, FK506-binding protein (FKBP) and FKBP (rapamycin); FKBP and rapamycin Lucineurin catalytic subunit A (CnA) (rapamycin); FKBP and cyclophyrins Phosphorus (rapamycin); FKBP and FKBP-rapamycin-related protein (FRB) ( rapamycin); gyrase B (GyrB) and GyrB (coumermycin); dihydro Folate reductase (DHFR) and DHFR(methotrexate); DmrB and DmrB(AP 20187); PYL and ABI (abscisic acid); Cry2 and CIB1 (blue light); G AI and GID1 (gibberellin); etc. Further description, including amino acid sequences, can be found in U.S. Patent Application Publication No. 2015-0368342 A No. 1, the disclosure of which is incorporated herein by reference in its entirety.

[0163] Useful protein dimers include those described in PCT Publication No. WO 2017 / 12054 6 and U.S. Patent Application Publication No. 2017 / 0306303 A1. Further examples include nuclear hormone receptor-derived protein dimers that dimerize in the presence of The disclosure of such nuclear hormone receptors is incorporated herein by reference in its entirety. The dimers derived from the body are generally the first member of a dimerization pair that is a coregulator of a nuclear hormone receptor. a member of the dimerization pair, and a second member of the dimerization pair comprises the LBD of a nuclear hormone receptor.

[0164] If a bioorthogonal adapter molecule is used in the circuit, bind to the bioorthogonal adapter molecule. The expression of therapeutic agents that mediate target antigen recognition may or may not be controlled by this circuit. In other words, the expression of the therapeutic agent is mediated by the activation of BTTS in the circuit (e.g., the activation of BTTS). The antigen may or may not be bound to a specific antigen (priming antigen or binding to another antigen). In some examples, the circuit may be configured to detect binding of the BTTS to its antigen via a bioorthogonal adapter. In some instances, the therapeutic agent may be configured to induce expression of a therapeutic agent that binds to the therapeutic molecule. The BTTS that induces the expression of the drug is the same BTTS that induces the expression of the bioorthogonal adaptor molecule. In some instances, the therapeutic agent induces expression of a bioorthogonal adaptor molecule, BTT. It is induced by a BTTS that is different (i.e., distinct) from S.

[0165] In some instances, expression of therapeutic agents that bind bioorthogonal adaptor molecules is mediated by BTTS. For example, in some instances, such therapeutic agents may not be induced by BTT. The expression of therapeutic agents is not induced by S, but is instead induced by, for example, but not limited to, Distinct regulation, including constitutive, inducible, conditional, tissue-specific, and cell-type-specific In some instances, the expression is under the control of an element or sequence, e.g., an introduced immune response. Independent expression of a therapeutic agent by a cell (e.g., constitutive expression, inducible expression, etc.) is not a diffusible product. Orthogonal adaptor molecules activate therapeutics in immune cells distal to the priming site Makes it possible to mediate.

[0166] In some instances, expression of a bioorthogonal adaptor molecule bound by a therapeutic agent is associated with a corresponding Therapeutic drugs that are used may be induced by BTTS, including those that are not induced by BTTS. For example, in some instances, such bioorthogonal adapter molecules may be BTTS The present invention is not limited to the use of a bioorthogonal adapter, but is instead induced by, for example, but not limited to, Expression of the molecule may be constitutive, inducible, conditional, tissue-specific, or cell-type-specific. In some instances, the bioorthogonal polypeptide is expressed under the control of separate regulatory elements or sequences. The adaptor molecule may be provided externally.

[0167] In some instances, the antigen-specific therapeutic agent is a specific antibody that binds to a second member of a specific binding pair. The extracellular domain may comprise a first member of a hetero-binding pair, The antibody does not include any additional first or second members of a second specific binding pair. In some instances, the antigen-specific therapy comprises a first antigen-binding domain that binds to the antigen. The extracellular domain may comprise an extracellular domain containing any additional antigen-binding domain. It does not contain any other antigens and does not bind to any other antigens. In some instances, it may contain only a single extracellular domain. Therapeutic agents may be specific for a single antigen or may be specific for only a single antigen. Such antigen-specific therapeutics can be referred to as "single-antigen antigen-specific therapeutics."

[0168] In some instances, the antigen-specific therapy comprises the first or second of two or more specific binding pairs. The second member can have an extracellular domain comprising the second member. For example, in some instances, Antigen-specific therapeutics are therapeutics that use different extracellular domains, each specific for two different antigens. and an extracellular domain comprising a first antigen-binding domain and a second antigen-binding domain. In some instances, antigen-specific therapeutics can be directed to two different specific binding sites. It may have two or more extracellular domains comprising the first or second member of the pair. For example, in some instances, the antigen-specific therapy comprises a first cell comprising a first antigen-binding domain. and a second extracellular domain comprising a second antigen-binding domain. The two different antigen-binding domains are each specific for a different antigen. An antigen-specific therapeutic agent can be specific for two different antigens.

[0169] Either two extracellular domains or one extracellular domain specific for two different antigens Antigen-specific therapeutic agents specific for two or more different antigens, including The antigen-specific therapeutic agent is constructed such that binding of either antigen to the therapeutic agent is sufficient to activate the antigen-specific therapeutic agent. Such antigen-specific therapeutics can be produced by targeting any of two or more antigens. It is described as a component of a logic gate containing an OR function that can be activated by In some instances, the antibodies specific for two different antigens may find use in a variety of different circuits. Such antigen-specific therapeutics can be referred to as "two-headed antigen-specific therapeutics." Antigen-specific therapeutic agents specific to multiple antigens are not limited to only two antigens, but can also be used, for example, For more than two antigens, including three or more, four or more, five or more, etc. It may be specific and / or activated by more than two antigens.

[0170] An example of an antigen-specific therapeutic agent specific for two or more different antigens is a tandem CAR ( "Tandem CAR" is also called "tan CAR". A tandem CAR is a bispecific CAR that contains two or more non-identical antigen recognition domains. Non-limiting examples of CARs are described in U.S. Patent Nos. 9,447,194; 10,155 ,038; 10,189,903; and 10,239,948; U.S. Patents. Publication No. 20130280220 and PCT Publication No. WO 2013 / 12 3061, the disclosures of which are incorporated herein by reference in their entireties. Tandem CARs include, but are not limited to, those described herein. Two or more of the antigens and / or ,038; 10,189,903; and 10,239,948; U.S. Patents. Publication No. 20130280220 and PCT Publication No. WO 2013 / 12 3061. can be achieved

[0171] Binding-triggered transcriptional switch (BTTS) The disclosed methods involve using BTTS to induce expression of an encoded antigen-specific therapeutic. As used herein, a "binding-triggered transcriptional switch" or "binding-triggered transcriptional switch" may include the use of a circuit that BTTS generally comprise an extracellular domain containing the first member of a specific binding pair, the binding transporter. A synthetic modular polypeptide or interacting polypeptide having a transducer and an intracellular domain. When the second member of the specific binding pair binds to the BTTS, the binding system The signal is the intracellular domain that activates the signal and is not activated in the absence of a binding signal. The binding-triggered transcriptional switch is a transcription factor that is transmitted to the intracellular domain to perform some function. See, for example, PCT Publication No. WO 2016 / 138034, and U.S. Pat. Nos. 670,281 and 9,834,608, the disclosures of which are incorporated herein by reference. is incorporated herein by reference in its entirety.

[0172] The specific binding member of the extracellular domain generally determines the specificity of the BTTS. In some instances, BTTSs have specificity determined based on their specific binding members. For example, a specific binding member having a binding partner "X" can be , X-BTTS or anti-X BTTS.

[0173] Any convenient specific binding pair, i.e., but not limited to, antigen-antibody Specific binding members and specific bindings, including antibody pairs, ligand-receptor pairs, scaffold protein pairs, etc. Partner pairs may find use in the BTTS of the present method. In some examples: The specific binding member may be an antibody, and the binding partner may be a molecule to which the antibody specifically binds. In some instances, the specific binding member may be a receptor, the binding partner of which may be an antigen. The receptor may be a ligand to which the receptor specifically binds. The binding member may be a scaffold protein, and the binding partner may be a protein for which the scaffold protein is specific. Useful specific binding pairs include those described herein. priming antigen and / or one or more target antigens / killing antigens, This includes things like:

[0174] In some cases, the specific binding member is an antibody. An antibody can be any antigen-binding antibody-based The polypeptide may be a specific polypeptide, a variety of which are known in the art. Is the specific binding member a monoclonal antibody, a single chain Fv (scFv), a Fab, etc.? or containing other antibody-based recognition domains (cAb VHH (camel antibody variable heterodimers) domain) and humanized version, IgNAR VH (shark antibody variable domain) and human sdAb VH (single domain antibody variable domain) and "camelized" antibodies Variable domains are suitable for use. In some instances, single chain TCRs (scTv, VαV T cell receptor (TCR)-based recognition domains, such as β-containing single chain two-domain TCRs is also suitable for use.

[0175] When the specific binding member of the BTTS is an antibody-based binding member, the BTTS may be For example, antibody-based binding members, including antigens specifically bound by antibody-based binding members. In some instances, the binding member of the The antibody-based binding member, as commonly practiced in the relevant art, may be an antibody-based binding member. can be defined based on the antigen bound by the antibody, e.g., antibody-based binding The members may be "anti" antigen antibodies, such as anti-priming antigen antibodies (e.g., anti-IL13RA 2 antibody, anti-IL13RA1 antibody, anti-Neuroligin antibody, anti-NRXN1 antibody, anti-P TPRZ1 antibody, anti-NRCAM antibody, anti-CDH10 antibody, anti-PCDHGC5 antibody, anti-CD 70 antibody, anti-CSPG5 antibody, anti-BCAN antibody, anti-GRM3 antibody, anti-CRB1 antibody, anti-G AP43 antibody, anti-ATP1B2 antibody, anti-PTPRZ1-MET fusion antibody, etc.) Therefore, the BTTS or antigen-specific therapeutic agent of this method includes Suitable antibody-based binding members can have a variety of antigen-binding specificities.

[0176] The components of the BTTS and the switch used in the described method correspond to each other. The arrangement may include, but is not limited to, a desired binding trigger, an intracellular domain, Many of these findings include the activity of BTTS, the overall function of BTTS, and the broader arrangement of molecular circuits that include BTTS. The first binding member may be, for example, but not limited to, The intracellular domain may include, but is not limited to, an agent that binds to an antigen as described herein. However, for example, regulatory sequences that activate or repress transcription, e.g., downstream of a particular circuit Examples include intracellular domains that induce or repress the expression of components.

[0177] The coupling transducers in the BTTS also vary depending on the desired delivery method of the coupling signal. Generally, coupling transducers are, for example, coupled by receptors of various signal transduction pathways. A polypeptide and / or a polypeptide that transduces an extracellular signal into an intracellular signal. The transmission of the binding signal may include, but is not limited to, a domain of a polypeptide. For example, binding-induced proteolytic cleavage, binding-induced phosphorylation, binding-induced conformational change This can be achieved by a variety of mechanisms, including coupling transducers, etc. Upon binding, the binding signal is transmitted by cleavage of BTTS, e.g., the intracellular domain The polypeptide may contain a ligand-inducible proteolytic cleavage site from which the polypeptide is released. In some examples, the BTTS may be a chimeric Notch receptor, e.g., as described herein. The polypeptide may comprise a Notch-derived cleavable bond transducer such as a polypeptide.

[0178] In other examples, the binding signal can be transmitted in the absence of inducible proteolytic cleavage. Any one or more signaling components of a signaling pathway may be proteolytically cleaved. Whether or not it is necessary for the propagation of a signal, it may find use in BTTS. For example, in some instances, including but not limited to, Jak-Sta A phosphorylation-based coupling transduction pathway comprising one or more signaling components of the t-pathway. The user may find use in non-proteolytic BTTS.

[0179] Briefly, BT polypeptides, including but not limited to chimeric Notch receptor polypeptides, TS has been described primarily as a single polypeptide chain. However, chimeric TSs are not The BTTS, which contains the ch receptor polypeptide, is composed of two or more separate polypeptide chains. It may be partitioned or split, and functional BTTS, e.g., chimeric Notch receptor polypeptides. The joining of two or more polypeptide chains to form a bond can be constitutive or conditional. For example, constitutive joining of the two parts of a split BTTS can be achieved by heterodimerization. The first portion of the split polypeptide is fused to the second portion of the split polypeptide so that the split portions are functionally joined upon merization. This is achieved by inserting a constitutive heterodimerization domain between the first and second portions. It can be done.

[0180] Useful BTTS that can be used in the subject method include, but are not limited to, modular Modular extracellular sensor architecture (MES) A) A MESA polypeptide includes a) a ligand-binding domain; a) a transmembrane domain; c) a protease cleavage site; and d) a functional domain. The target domain can be a transcriptional regulator (e.g., a transcriptional activator, a transcriptional repressor). In some cases, the MESA receptor comprises two polypeptide chains. The MESA receptor comprises a single polypeptide chain. Non-limiting examples of MESA polypeptides This is described, for example, in U.S. Patent Application Publication No. 2014 / 0234851. The disclosure is incorporated herein by reference in its entirety.

[0181] Useful BTTS that can be used in the subject method include, but are not limited to, TAN The polypeptides used in the TANGO assay are included. The polypeptide comprises a Tobacco Virus (Tev) protease, and the second polypeptide It is a heterodimer containing a Tev proteolytic cleavage site (PCS) fused to a transcription factor. A TANGO polypeptide is used. Two polypeptides are close to each other, and this If proximity is mediated by natural protein-protein interactions, Tev binds to the PCS Non-limiting examples of TANGO polypeptides include, for example, Ba rnea et al. (Proc Natl Acad Sci USA.2008 Jan.8;105(1):64-9), The disclosure of which is incorporated herein by reference in its entirety.

[0182] Useful BTTS that can be used in the subject method include, but are not limited to, von Will vWF cleavage domain-based BTTS, including but not limited to The vWF cleavage domains include unmodified and modified vWF A2 domains. The base BTTS generally consists of: an extracellular domain containing the first member of the binding pair; von Willebrand factor (vWF) cleavage domain containing a proteolytic cleavage site; cleavable transmembrane domain and intracellular domain; vWF cleavage domain and A non-limiting example of a BTTS based on BTTS is described by Langridge & Struhl (Cell (2017) 171(6):1383-1396). No. 6,239,999, the disclosure of which is incorporated herein by reference in its entirety.

[0183] Useful BTTS that can be used in the subject method include, but are not limited to, chimeric Notc receptor polypeptides, such as, but not limited to, synNotch polypeptides Non-limiting examples of which include PCT Publication No. WO 2016 / 138034 No. 9,670,281, U.S. Pat. No. 9,834,608, Roybal et al. .Cell(2016)167(2):419-432, Roybal et al.Cell(2016)164(4):770-9, and Morsut et al Cell (2016) 164(4):780-91; the disclosure of which is incorporated herein by reference in its entirety. It will be incorporated into the specification.

[0184] SynNotch polypeptides generally comprise: a) an extracellular domain that contains a specific binding member; b) proteolytically cleavable containing one or more proteolytic cleavage sites and c) a protein component generally comprising a Notch receptor polypeptide; and c) an intracellular domain. a specific binding member with its binding partner being an catalytically cleavable chimeric polypeptide; Binding of the synNotch protein generally occurs via the cleavage of the synNotch protein at one or more proteolytic cleavage sites. In some instances, the method further comprises: inducing cleavage, thereby releasing the intracellular domain. Release of the intracellular domain triggers (i.e., induces) production of the encoded payload Depending on the particular situation, the encoding nucleic acid sequence may be contained within the cell. The delivered payload is then typically expressed on the cell surface or secreted. Notch polypeptides generally have an extracellular domain that binds to, for example, a Notch receptor. If the domain is heterologous, if the intracellular domain is heterologous, the extracellular domain and the intracellular domain heterologous to the Notch receptor polypeptide, including when both the polypeptide and the target polypeptide are heterologous. The nucleic acid sequence comprises at least one sequence which is (ie, is not derived from a Notch receptor).

[0185] Useful synNotch BTTSs are based on the domains used and the structure of such domains. SynNotch polypeptides generally have one or more Notch receptor polypeptides containing one or more ligand-inducible proteolytic cleavage sites. Notch receptor polypeptides vary in length, ranging from about 50 amino acids to It can range in length from less to about 1000 amino acids or more.

[0186] In some cases, the Notch receptor polypeptide present in the synNotch polypeptide The peptide may be 50 amino acids (aa) to 1000 aa, for example, 50 aa to 75 aa, 75 aa a~100aa, 100aa~150aa, 150aa~200aa, 200aa~25 0aa, 250a~300aa, 300aa~350aa, 350aa~400aa, 4 00aa~450aa, 450aa~500aa, 500aa~550aa, 550aa ~600aa, 600aa~650aa, 650aa~700aa, 700aa~750 aa, 750aa~800aa, 800aa~850aa, 850aa~900aa, 9 It has a length of 00 aa to 950 aa, or 950 aa to 1000 aa. The Notch receptor polypeptide present in the synNotch polypeptide is 300 aa~400aa, 300aa~350aa, 300aa~325aa, 350aa~4 The length may be 0.00aa, 750aa to 850aa, or 50aa to 75aa. The Notch receptor polypeptide is 310 aa to 320 aa, for example, 310 aa, 3 11aa, 312aa, 313aa, 314aa, 315aa, 316aa, 317aa , 318 aa, 319 aa, or 320 aa in length. The receptor polypeptide has a length of 315 aa. The peptide is 360aa to 370aa, for example, 360aa, 361aa, 362aa, 363aa 364aa, 365aa, 366aa, 367aa, 368aa, 369a Optionally, the Notch receptor polypeptide has a length of 100 aa, or 370 aa. It is 367 aa in length.

[0187] Optionally, the Notch receptor polypeptide has the amino acid sequence of a Notch receptor. and at least 50%, at least 55%, at least 60%, at least 65%, or less At least 70%, at least 75%, at least 80%, at least 85%, at least 90% %, at least 95%, at least 98%, at least 99% or 100% of the amino acid sequence In some instances, the Notch receptor polypeptide comprises an amino acid sequence having sequence identity with the Notch receptor polypeptide. The Notch regulatory region of the gene is, but is not limited to, a mouse Notch (e.g., For example, mouse Notch1, mouse Notch2, mouse Notch3 or mouse Notc h4) Regulatory region, rat Notch regulatory region (e.g., rat Notch1, rat No tch2 or rat Notch3), human Notch regulatory region (e.g., human Notch 1, human Notch2, human Notch3, or human Notch4) Notch regulatory region, or derived from a mammalian Notch regulatory region, and a mammalian Notch receptor The amino acid sequence of the mammalian Notch regulatory region is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 7 5%, at least 80%, at least 85%, at least 90%, at least 95%, N having at least 98%, at least 99%, or 100% amino acid sequence identity otch regulatory region.

[0188] The Notch regulatory region is shown to be a subset of its various components (e.g., domains, cleavage sites, etc.). It may include or exclude the following: It may be wholly or partially present or absent Examples of such components of the Notch regulatory region that may be used, as appropriate, include, for example: one or more EGF-like repeat domains, one or more Lin12 / Notch repeats domain, one or more heterodimerization domains (e.g., HD-N or HD-C) , a transmembrane domain, one or more proteolytic cleavage sites (e.g., furin-like proteinases), ase site (e.g., S1 site), ADAM family protease site (e.g., S2 site) and / or γ-secretase protease site (e.g., S3 site)). In some instances, the Notch receptor polypeptide may be, for example, a Delta-binding one or more Notch cells comprising a Notch ligand binding domain, such as a Notch domain The Notch receptor polypeptide may be a polypeptide of any of several types, including a polypeptide of any of several types, and may be a polypeptide of any of several types. Examples include a Notch ligand binding domain, such as a Delta binding domain. containing one or more non-functional versions of one or more Notch extracellular domains. In some instances, the Notch receptor polypeptide may be, for example, Notch R bp-associated molecular domain (i.e., RAM domain), Notch ankyrin repeat domain Contains the Notch main domain, Notch transactivation domain, and Notch PEST domain. One or more Notch intracellular domains may be omitted in whole or in part. The receptor polypeptide may, in some instances, be, for example, a non-functional Notch Rbp-related molecule. domain (i.e., RAM domain), non-functional Notch ankyrin repeat domain nonfunctional Notch transactivation domain, nonfunctional Notch PEST domain One or more non-functional variants of one or more Notch intracellular domains, including It may include a section.

[0189] Non-limiting examples of specific synNotch BTTSs, their domains, and appropriate domain arrangements. Typical examples are PCT Publication Nos. WO 2016 / 138034, WO 2017 / No. 193059, International Publication No. 2018 / 039247, U.S. Patent No. 9,670,281 and U.S. Pat. No. 9,834,608, the disclosures of which are incorporated by reference in their entireties. and is incorporated herein by reference.

[0190] Useful BTTS domains, e.g., extracellular domains, binding-transducer domains, The domains, intracellular domains, etc. may be directly joined, i.e., joined with no intervening amino acid residues. It may be free of a group or may contain a peptide linker joining the two domains. The peptide linker may be synthetic or, for example, a naturally occurring polypeptide. It may be of natural origin, including fragments.

[0191] The peptide linker may be from about 3 amino acids (aa) or less to about 200 aa or more. For example, but not limited to, 3aa to 10aa, 5aa to 15aa, 10 aa~25aa, 25aa~50aa, 50aa~75aa, 75aa~100aa, 1 00aa~125aa, 125aa~150aa, 150aa~175aa, or 175 The peptide linker may vary in length from 3 aa to 30 aa, e.g. For example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 The peptide linker may have a length of 5 aa to 50 aa, 5 aa to 4 aa, 0aa, 5aa~35aa, 5aa~30aa, 5aa~25aa, 5aa~20aa, It may have a length of 5 aa to 15 aa or 5 aa to 10 aa.

[0192] In some instances, the BTTS binds to a specific binding pair that binds to a second member of the specific binding pair. The extracellular domain can comprise a first member of the pair, the extracellular domain comprising a second member of the pair. The present invention does not include any additional first or second members of two specific binding pairs. In some instances, the BTTS comprises an extracellular domain that includes a first antigen-binding domain that binds to an antigen. wherein the extracellular domain does not include any additional antigen-binding domain; The subject BTTS does not bind to any other antigens. In some instances, the subject BTTS binds to a single extracellular The BTTS used may contain only the nucleotide sequence of the BTTS. Therefore, the BTTS used is specific for a single antigen. Such a BTTS may be a "single-antigen BTTS." In some instances, a "double antigen BTTS" can be used. do.

[0193] In some instances, the BTTS comprises a first or second member of two or more specific binding pairs. For example, in some instances, BTTS may have an extracellular domain that includes a cytoplasmic domain. The first antigen-binding domain and the second antigen-binding domain are different so that the outer domains are specific for two different antigens. and a second antigen-binding domain. S is a two or more molecule complex, each of which comprises a first or second member of two different specific binding pairs. For example, in some instances, the BTTS may have one or more extracellular domains. a first extracellular domain containing an antigen-binding domain and a second extracellular domain containing a second antigen-binding domain. and an extracellular domain, and two different antigen-binding domains each having a different antigen. Therefore, a BTTS can be specific for two different antigens.

[0194] Either two extracellular domains or one extracellular domain specific for two different antigens BTTS specific for two or more different antigens, including Binding of the antigen activates BTTS, e.g., by proteolytic cleavage of the cleavage domain of BTTS. The shedding, for example, is sufficient to induce the release of the intracellular domain of BTTS. Such BTTS can be induced by either two or more antigens. It can be used in the described circuit as a component of a logic gate containing an OR function. In some instances, BTTS specific for two different antigens can be found as "bivalent" antibodies. It can be called "two-headed BTTS" or tandem BTT (or tanBTTS). For example, in some instances, the antibody can be configured to bind to two or more different antigens. The synNotch BTTS is a tandem SynNotch or tanSynNo Multi-antigen specific BTTS can be called tch. two, including, for example, three or more, four or more, five or more, etc. may be specific for more than one antigen and / or elicited by more than two antigens Good too.

[0195] Preparation method The present disclosure provides methods for preparing the nucleic acids, circuits, and cells used in the methods described herein. Further included are the manipulation of nucleic acids in preparing the subject nucleic acids and circuits and their components; Using any convenient method of modification and amplification (e.g., collectively referred to as "cloning"). In preparing target cells containing nucleic acids encoding the described circuits, Any convenient method such as transfection, transduction, or culture can be used.

[0196] Nucleotide sequences encoding all or part of the components of the circuits of the present disclosure can be expressed in expression vectors. The subject circuit or its components may be present in a cloning vector and / or a cloning vector. If the polypeptide is divided between two or more separate polypeptides, the two or more The nucleotide sequences encoding the polypeptides may be cloned into the same vector or into separate vectors. Expression vectors contain a selection marker, an origin of replication, and a vector They may contain other features that provide for replication and / or maintenance. Suitable expression vectors include, for example, Examples include plasmids and viral vectors.

[0197] Many suitable vectors and promoters are known to those of skill in the art, many of which are suitable for recombinant The following vectors are provided as examples: Bacteria: pB s, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Strategy ne, La Jolla, Calif., USA);pTrc99 A, pKK223- 3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Upps ala, Sweden). Eukaryotes: pWLneo, pSV2cat, pOG44, PX R1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL( Pharmacia).

[0198] Expression vectors generally provide for the insertion of nucleic acid sequences encoding heterologous proteins. , with convenient restriction sites located near the promoter sequence. Suitable expression vectors include, but are not limited to, those containing a selectable marker. However, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus) Viral vectors based on (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 254 9,1994;Borras et al.,Gene Ther 6:515 524,1999:Li and Davidson,PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649 ;WO 93 / 03769;WO 93 / 19191, WO 94 / 2 See WO 95 / 00655, WO 95 / 11984, and WO 95 / 00655. adeno-associated virus-based viral vectors (e.g., Ali et al., Hum G ene Ther 9:81 86,1998, Flannery et al.,PNAS 94:6916 6921,1997;Bennett et al.,In vest Opthalmol Vis Sci 38:2857 2863,1997;Jomary et al.,Gene Ther 4:683 690,1997 , Rolling et al.,Hum Gene Ther 10:641 648,1999;Ali et al.,Hum Mol Genet 5:591 5 94,1996;Srivastava, WO 93 / 09239, Samulski et al., J. Vir. (1989)6 3:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (19 93)90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus Viral vectors based on viruses (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; See Akahashi et al., J Virol 73:7812-7816, 1999); retroviral vectors - (e.g., murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Bay sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus viral vectors derived from retroviruses such as rabies, mammary tumor virus, and other retroviruses can be done.

[0199] As noted above, in some embodiments, a method for encoding the circuits or components thereof of the present disclosure may be implemented. The nucleic acid comprising the nucleotide sequence may, in some embodiments, be DNA or RNA, e.g., These include in vitro synthesized DNA, recombinant DNA, in vitro synthesized RNA, and recombinant RNA. Methods for in vitro synthesis of DNA / RNA are known in the art and can be prepared using any known method. By transfecting the DNA / RNA into the host cells, it is possible to synthesize DNA / RNA containing the desired sequence. Methods for introducing DNA / RNA into host cells are known in the art. It can be carried out in vitro, ex vivo or in vivo. For example, in a host cell (e.g., N and inducing a nucleic acid encoding all or part of the circuit of the present disclosure into cells (e.g., K cells, cytotoxic T lymphocytes, etc.). DNA / RNA containing the nucleic acid sequence is transduced or transferred in vitro or ex vivo. The cells can be transfected or electroporated.

[0200] The disclosed method includes culturing cells genetically modified to encode the disclosed circuitry. Such methods include, but are not limited to, culturing cells prior to administration, in vitro or in vitro methods. and culturing the cells ex vivo (e.g., in the presence or absence of one or more antigens). Any convenient method for cell culture can be used, but such Such methods may vary depending on, for example, but not limited to, the type of cells being cultured, the intended use of the cells (e.g., Various factors, including, for example, whether the cells are cultured for research or therapeutic purposes, In some instances, the methods of the present disclosure are not limiting. However, for example, steps of seeding a cell culture, feeding a cell culture, and passaging the cell culture, splitting the cell culture, analyzing the cell culture, The general process of cell culture, including drug treatment, cell culture harvesting, etc. It may further include a step.

[0201] The disclosed methods, in some instances, involve receiving and / or collecting cells for use in the subject methods. In some examples, the cells are obtained from a subject. The step of harvesting the cells may include obtaining a tissue sample from the subject and concentrating the cells from the tissue sample; The isolation and / or enrichment of cells can include, for example, culturing. isolation / enrichment by cell sorting (e.g., adherent culture, suspension culture, etc.), cell sorting (e.g., FACS, This can be done using any convenient method, including microfluidics, etc. Cells may be derived from, but are not limited to, blood (e.g., peripheral blood, umbilical cord blood, etc.) Collect from any convenient tissue sample, including bone marrow, biopsy, skin sample, cheek swab, etc. In some instances, the cells can be obtained from a source, including, for example, a blood bank, a tissue bank, etc. The cells received may be previously isolated or may be from a tissue sample. The cells may be received as part of the sample, and therefore isolation / enrichment may be performed at the time of receipt of the cells. In certain instances, the received cells may be cultured cells, e.g., a cultured cell line. Cells suitable for use in the methods described herein may be non-primary cells, including cells of the , as further detailed herein.

[0202] nucleic acid As summarized above, the present disclosure provides a circuit for treating a subject with a brain disease or disorder. The nucleic acid to be used is provided.

[0203] Such nucleic acids may be used to target antigen-specific therapeutics in response to BTTS activation. The first gene may be configured to be operably linked to a regulatory sequence that responds to the target antigen. Uses transtargeting, which utilizes recognition of priming antigens expressed on brain cells Essentially any circuit that performs the Nucleic acids encoding the subject circuits are provided. Isolated nucleic acids encoding the subject circuits, as well as vectors Various constructs containing such nucleic acids, such as expression cassettes, recombinant expression vectors, Virus vectors and the like are included.

[0204] Recombinant expression vectors of the disclosure include those containing one or more of the described nucleic acids. Nucleic acids comprising nucleotide sequences encoding all or part of the components of the circuits of the present disclosure. In some embodiments, the vector is DNA, including, for example, a recombinant expression vector. Nucleic acids containing nucleotide sequences encoding all or part of the circuit components are In embodiments, the RNA is an in vitro synthesized RNA.

[0205] As summarized above, in some instances, the target circuitry includes a transcriptional control element (e.g., a coding nucleic acid operably linked to a regulatory sequence such as a promoter; an enhancer; etc. (eg, nucleic acids encoding BTTS or antigen-specific therapeutics) can be used. In some cases, the transcriptional control element is inducible. In some cases, the promoter is functional in eukaryotic cells. In some cases, the promoter is a cell type specific promoter. The promoter is a tissue-specific promoter.

[0206] Constitutive and inducible promoters, depending on the host / vector system utilized. Some suitable transcription and transcription factors include transcription enhancer elements, transcription terminators, etc. Any of the translational control elements can be used in the expression vector (e.g., Bitt See er et al. (1987) Methods in Enzymology, 153:516-544).

[0207] The promoter may be a constitutively active promoter (i.e., constitutively active / "ON" state). It can be an inducible promoter (i.e., a promoter in its active state) and an inducible promoter (i.e., a promoter in its active state). "ON" or inactive / "OFF" is determined by an external stimulus, e.g., a specific temperature, a compound or a protein a promoter controlled by the presence of a gene that is spatially restricted - (i.e., transcriptional control elements, enhancers, etc.) (e.g., tissue-specific promoters) promoters, cell type specific promoters, etc.), and temporally restricted promoters. (i.e., promoters may be used to express genes during specific stages of embryonic development or in biological processes, e.g., in the mouse The hair follicle may be in an "ON" state or an "OFF" state during a particular stage of the hair follicle cycle.

[0208] Suitable promoter and enhancer elements are known in the art. For expression in the vector, suitable promoters include, but are not limited to, lacI, Examples include lacZ, T3, T7, gpt, lambda P, and trc. For this purpose, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulins. Immunoglobulin gene promoter and enhancer elements; Cytomegalovirus front Early promoter; Herpes simplex virus thymidine kinase promoter; Early and late SV40 promoter; a promoter found in the long terminal repeat sequence of retrovirus origin the mouse metallothionein-I promoter; and various art-known tissue-specific A promoter is included.

[0209] In some instances, the transcriptional control elements of the nucleic acids described herein are cis-acting regulatory sequences. Any suitable cis-acting regulatory sequence may be used in the nucleic acids described herein. For example, in some instances, the cis-acting regulatory sequence may be an upstream activating upstream activating sequence e) (UAS). In some examples, the nucleic acids described herein may be or may include The UAS may be a Gal4-responsive UAS.

[0210] Suitable reversible promoters, including reversibly inducible promoters, are known in the art. Such reversible promoters have been isolated from many organisms, including eukaryotes and prokaryotes. A reversible promoter from a first organism for use in a second organism can be generated and induced. Modification of a target, e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote Such reversible promoters and methods for producing such reversible promoters are well known in the art. Systems based on a promoter but also including additional regulatory proteins include, but are not limited to: However, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) Gene promoter, pro- Page 11 11 response to alcohol transactivator protein (AlcR) motors), tetracycline-regulated promoters (e.g., TetActivator rs, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysosome promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g. , metallothionein promoter system, etc.), pathogenicity-related regulated promoters (e.g., sal Thiamin-regulated promoter, ethylene-regulated promoter, benzothiadiazole-regulated promoter temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., , HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoter motors, synthetic inducible promoters, etc.

[0211] Inducible promoters suitable for use include those described herein or known to those of skill in the art. Any inducible promoter is included. Examples of inducible promoters include, but are not limited to: However, chemically / biochemically regulated promoters and physically regulated promoters are not promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g. For example, anhydrotetracycline (aTc) responsive promoters and other tetracycline tetracycline-responsive promoter system (which includes the tetracycline repressor protein (tet R), tetracycline operator sequence (tetO) and tetracycline transactivation activator fusion protein (tTA), steroid-regulated promoters (e.g. For example, rat glucocorticoid receptor, human estrogen receptor, and mosquito ecdysone receptor promoters based on the steroid / retinoid / thyroid receptor superfamily promoters from yeast, mouse, and human origin), metal-regulated promoters (e.g., promoters from yeast, mouse, and human origin), Derived from the tarothionine gene (a protein that binds to and captures metal ions) promoters regulated by salicylic acid, ethylene or benzoyltransferase; zothiadiazole (BTH)-induced), temperature / heat-induced promoters (e.g. heat shock promoters) and light-regulated promoters (e.g., light-regulated promoters derived from plant cells). responsive promoters).

[0212] In some cases, the promoter is an immune cell promoter, e.g., a CD8 cell-specific promoter. promoter, CD4 cell-specific promoter, neutrophil-specific promoter, or NK-specific promoter Heterologous promoters. For example, the CD4 gene promoter can be used; See, e.g., Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739; and Marodon et al. (2003 ) Blood 101:3416. As another example, the CD8 gene promoter is used NK cell-specific expression can be achieved by using the Ncr1(p46) promoter. This can be achieved; see, for example, Eckelhart et al. (2011) Blood 117:1565.

[0213] In some examples, the immune cell-specific promoter of the nucleic acids of the disclosure is a promoter of the B29 gene promoter. Motor, CD14 gene promoter, CD43 gene promoter, CD45 gene Promoter, CD68 gene promoter, IFN-β gene promoter, WASP Gene promoter, T cell receptor β chain gene promoter, V9γ (TRGV9) gene promoter, such as the V2δ (TRDV2) gene promoter. do.

[0214] In some cases, a nucleic acid encoding the circuit of the present disclosure or one or more components thereof The nucleic acid comprising the nucleotide sequence is a recombinant expression vector or is contained in a recombinant expression vector. In some embodiments, the recombinant expression vector is a viral construct, e.g., a recombinant Recombinant adeno-associated virus (AAV) constructs, recombinant adenovirus constructs, recombinant lentivirus constructs viral constructs, recombinant retroviral constructs, etc. A nucleic acid comprising a nucleotide sequence encoding a tract, or one or more components thereof, Optionally, the circuit of the present disclosure or one or more thereof is a recombinant lentiviral vector. A nucleic acid comprising a nucleotide sequence encoding one or more components is used as a recombinant AAV vector. It is Tar.

[0215] Suitable expression vectors include, but are not limited to, viral vectors (e.g., Viral vectors based on vaccinia virus; poliovirus; adenovirus (e.g. For example, Li et al.,Invest Opthalmol Vis Sci 35:2543 2549,1994;Borras et al.,Gene Ther 6:515 524,1999:Li and Davidson,PNAS 92:7700 7704,1995;Sakamoto et al.,Hum Gene Ther 5:1088 1097,1999;WO 94 / 12649;WO 93 / 03769 No. WO 93 / 19191, WO 94 / 28938, WO 95 / 11984 and WO 95 / 00655); adeno-associated viruses Viral vectors based on the ribosomal RNA (e.g., Ali et al., Hum Gene Ther 9:81-86, 1998; ery et al.,PNAS 94:6916 6921,1997;Bennett et al.,Invest Opthalmol Vis Sci 38:285 7 2863,1997;Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum Gene Ther 10:641 648,1999;Ali et al.,Hum Mol Genet 5:591 594,1996;Srivastava, International Publication No. No. 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Vi rol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617. );SV40; herpes simplex virus; human immunodeficiency virus-based viral vectors (For example, Miyoshi et al., PNAS 94:10319 23,1997; Takahashi et al., J Virol 73:7812 7816, 1999); retroviral vectors (e.g., murine leukemia virus , spleen necrosis virus, as well as Rous sarcoma virus, Harvey sarcoma virus, and avian leukosis virus Viruses, lentiviruses, human immunodeficiency viruses, myeloproliferative sarcoma viruses, and mammary tumors (viral vectors derived from retroviruses such as tumor viruses). In other words, the vector is a lentiviral vector. Also, the vector is a transposon-mediated vector. For example, the piggyback vector and the sleeping beauty vector Tar is also suitable.

[0216] In some instances, the nucleic acids of the disclosure may comprise a single polypeptide encoding two or more polypeptides. a single sequence, wherein expression of the two or more polypeptides is This is possible by the presence of sequence elements between the individual coding regions that facilitate their individual expression. Such sequence elements are referred to herein as binary sequences. This can be called a bicistronic promoting sequence, and it is The presence of a facilitating sequence allows the production of distinct polypeptides from each coding region present in a single nucleic acid sequence. In some instances, the nucleic acid allows for expression of two polypeptides present in a single nucleic acid. Two coding regions encoding ribonucleotides and a bicistronic promoter sequence between the coding regions. For individually expressing multiple individual polypeptides from a single nucleic acid sequence, Any suitable method can be used, as can any suitable method of bicistronic expression. The method can be used.

[0217] In some instances, the bicistronic enhancing sequence is linked by a cleavable linking polypeptide. This allows for the expression of two polypeptides from a single transiently joined nucleic acid sequence. In such cases, the bicistronic enhancing sequence may be a promoter for one or more of the encoded peptides. Suitable peptide cleavage sites include those of self-cleaving peptides, as well as those of In some instances, bicistronic amino acids are cleaved by separate enzymes. The peptide cleavage site of the lock-promoting sequence may include a furin cleavage site (i.e., a bicistronic sequence). The ronic promoting sequence may encode a furin cleavage site).

[0218] In some instances, the bicistronic enhancing sequence encodes a self-cleaving peptide sequence. Useful self-cleaving peptide sequences include, but are not limited to, T peptides containing the 2A sequence, such as, but not limited to, the 2A sequence. .

[0219] In some instances, the bicistronic enhancing sequence may comprise one or more spacer sequences. The spacer coding sequence generally comprises a peptide tag. Useful spacer coding sequences include, for example, For example, but not limited to, V5 peptides, including sequences encoding the V5 peptide tag. Coding sequences are included.

[0220] Multi- or bicistronic expression of multiple coding sequences from a single nucleic acid sequence is limiting. Although not intended to be a method for the detection of furin cleavage, methods using T2A and V5 peptide tag sequences have been used. For example, in some instances, an internal ribosome entry site (IRES) may be used. Systems based on, for example, but not limited to, Yang et al. (2008) ) Gene Therapy. 15(21): 1411-1423; Martin et al. (2006) BMC Biotechnology. 6: 4; Any suitable method of bicistronic expression can be used, including those described in The disclosure is incorporated herein by reference in its entirety.

[0221] cell As summarized above, the present disclosure also provides immune cells. The immune cells of the present disclosure include any of the immune cells described herein. and / or a nucleic acid, expression vector, or the like, encoding the circuit. The immune cells of the present disclosure include, for example, cells that are genetically engineered to produce components of the circuits of the present disclosure. Mammalian immune cells, including those genetically modified or those into which such nucleic acids have been separately introduced. In some instances, the target immune cells comprise one or more components of the circuits of the present disclosure. transfected with one or more nucleic acids and / or expression vectors to express the components are.

[0222] Suitable mammalian immune cells include primary cells and immortalized cell lines. Cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) In some instances, the cells are not immortalized cell lines but are derived from an individual. For example, in some cases, the cells are obtained from an individual (e.g., primary cells). In one example, the cells are derived from an individual. The resulting lymphoid cells are, for example, lymphocytes or their precursor cells. In another example, the cell is a cytotoxic cell, or a precursor cell thereof, obtained from the individual. The stem cells are obtained from an individual, or their progenitor cells.

[0223] As used herein, the term "immune cells" generally refers to cells produced in the bone marrow. Contains white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs). The "cells" include, for example, lymphoid cells, namely lymphocytes (T cells, B cells, natural killer NK cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages "T cells" include helper T cells (CD4+ cells), cytotoxic T cells (CD4+ cells), and including regulatory T cells (CD8+ cells), regulatory T cells (Treg) and gamma delta T cells, All types of immune cells that express CD3 are included. "Cytotoxic cells" include CD8+ These cells include T cells, natural killer (NK) cells, and neutrophils, which are responsible for cytotoxicity. "B cells" include, for example, cells that express CD19, e.g., B cells, such as pre-B cells, immature B cells, mature B cells, memory B cells, and plasmablasts. Immune cells include mature and immature cells of the cell lineage. B cells, including pro-B cells, Also included are B cell lineage derivatives such as progenitor cells and plasma cells.

[0224] Immune cells encoding the circuits of the present disclosure can be generated by any convenient method. Nucleic acid encoding one or more components of the target circuit may be used in a manner such that the target nucleic acid is only transiently expressed. Targets may be present, maintained extrachromosomally, or integrated into the host genome. The nucleic acid may be stably or transiently introduced into the immune cells. The genetic modification can be performed in vivo, in vitro, or ex vivo.

[0225] In some cases, the introduction and / or genetic modification of the subject nucleic acid is performed ex vivo. For example, T lymphocytes, stem cells, or NK cells may be obtained from an individual, and the cells obtained from the individual may be are modified to express components of the circuits of the present disclosure. is determined by one or more antigen-binding domains present on the introduced components of the circuit. As defined, the antibody can be redirected to one or more selected antigens. In some cases, the modified cells are conditioned ex vivo. In other cases, the cells are conditioned ex vivo (e.g., For example, the cells may be introduced into an individual (e.g., the individual from which the cells were obtained) and / or may already be present in the individual and the cells In vivo, for example, by administering a nucleic acid or vector to said individual in vivo. It is adjusted.

[0226] circuit As summarized above, the present disclosure also provides, in some instances, a method for producing a nucleic acid sequence encoded by the nucleic acid sequence. These circuits, also called molecular circuits, are in some instances expression-based. The present invention also provides a circuit for detecting a gene that is a target of the present invention. The target nucleic acid may, in some instances, be a vector, including, for example, viral vectors and non-viral vectors. Such circuits may be contained within cells, such as immune cells, in some instances. The vector may be present in the cell or may be introduced into the cell by various means, including, for example, the use of a viral vector. The cells may, in some instances, be genetically modified to encode the circuit of interest. Such modifications can be effectively permanent (e.g., embedded) or may be transient.

[0227] The coded components of the circuits of the present disclosure generally include at least one coded BT TS and at least one encoded therapeutic protein. The expression of a protein may depend on the state (i.e., active / inactive) of other components of the circuit. For example, the expression of a therapeutic agent may depend on the activation of BTTS, which is a specific In some cases, one of the pathways The dependency of one component on another can be mediated by a regulatory sequence. The sequence encoding the second component comprises a regulatory sequence that responds to activation of the first component of the circuit. can be operably linked to the first component, thereby controlling the expression of the second component. can be linked to the activation of

[0228] The use of BTTS in the circuits of the present disclosure allows for the coupling of expression and / or activity to molecular binding events. The binding-triggered transcriptional switch and the system comprising its components are disclosed in PCT Publication No. International Patent Publication No. 2016 / 138034, U.S. Patent Application Publication No. 2016-0264665A1 No. 9,670,281 and U.S. Pat. No. 9,834,608. No. 6,239,799, the disclosure of which is incorporated herein by reference in its entirety.

[0229] The circuits of the present disclosure can be configured in a variety of ways. In some examples, two or more Independent activities and / or induced effects of more than one polypeptide or domain of a single polypeptide The resulting expression may produce a logic gate circuit. Such a logic gate circuit may include, but is not limited to, However, for example, "AND gate", "OR gate", "NOT gate", and so on These can include combinations of, for example, higher order AND gates, higher order OR gates, etc. gates, higher order NOT gates, higher order combinational gates (i.e. AND, OR , and / or gates using some combination of NOT gates) In some instances, useful circuits may further include IF / THEN gates. This can be done.

[0230] An "AND" gate represents the case where two or more inputs are required for a signal to propagate. For example, in some instances, the AND gate includes a first polypeptide or a second polypeptide. A signaling pathway via a first input of a peptide domain and a second input that depends on the output of the first input. An AND gate allows for signaling through the circuit using two inputs, e.g. For example, two antigens are required.

[0231] An "OR" gate is a gate where either of two or more inputs can allow a signal to propagate. For example, in some instances, an OR gate may be used to detect either binding of two different antigens. In an OR gate, any one input, e.g., two Any of the antigens can induce the signaling output of the circuit. In one embodiment, an OR gate can be achieved by the use of two separate molecules or constructs. The gates are constructed using a single construct that recognizes two antigens, e.g., each binding to a different antigen. , two different antigen binding events capable of independently propagating a signal Using domain-containing TCRs or antigen-specific therapeutic agents (e.g., CARs or TCRs) This can be achieved by inducing the expression of downstream components of the circuit, e.g., by activating immune cells. activation, etc.).

[0232] A "NOT" gate contains inputs that can prevent the signal from propagating. For example, In some examples, the NOT gate inhibits signaling through the circuit of the present disclosure. In this case, the NOT gate is a manifestation of a circuit component, or a specific component of a circuit, e.g., C It can prevent activation of the AR or TCR.

[0233] An "IF / THEN" gate contains a case where the output of the gate depends on the first input. For example, in some instances, if a first input is present, THEN signaling occurs when a second input In the absence of the first input, signal transduction cannot proceed. A non-limiting example of a circuit containing an IF / THEN gate is a circuit having at least two receptors. In this circuit, a first receptor responds to an input and induces the expression of a second receptor, and the second input It is a circuit that has some output in response to a force. Therefore, the first receptor of the IF When input 1 is present, THEN the second receptor is expressed and signaling begins via the second input It can then be passed through a second receptor to generate an output. A receptor may or may not contain an OR component (e.g., a receptor with OR function). Good too.

[0234] Non-limiting examples of IF / THEN gates, including examples with OR functionality, are shown in FIG. The circuit shown in the first (top) cell is a BTTS that responds to antigen "A" and a BTTS that responds to antigen "C." and antigen-specific therapeutic agents that bind to the antigen. The antigen-specific therapeutic agents are referred to as CARs. However, the disclosure is not so limited and is readily substituted with other antigen-specific therapeutics. Note that in the first (top) circuit, the presence of IF antigen A results in TH EN cell killing is induced based on the presence of antigen C.

[0235] In various embodiments, one or more components of the target circuit may include an OR function. Therefore, OR function can be used. As shown, OR functions have specificity for two or more antigens and BTTS induced or activated by one or more antigens, antigen-specific therapeutic agents, or It can be provided by both

[0236] For example, the second cell (from the top) shown in Figure 4 has BTTS that respond to antigen "A" and Antibody that binds to antigen "C" or antigen "D" and is activated by antigen "C" or antigen "D" In such circuits, antigen A of the IF is present. THEN cell killing is induced based on the presence of antigen C or (OR) antigen D. Killing of cells expressing antigen C and antigen D, and killing of cells expressing antigen C alone or antigen D alone Note that cell killing can also be induced.

[0237] In the third cell (from the top) shown in Figure 4, BTT responds to antigen "A antigen" or "B". and an antigen-specific therapeutic agent that binds to and is activated by antigen "C." In such circuits, either antigen A of IF or antigen B of (OR) is present. THEN cell killing is induced based on the presence of antigen C. Immune cells are killed by cells expressing only antigen A, only antigen B, or both antigens A and B. Note that the wound may be primed.

[0238] In the fourth (bottom) cell shown in Figure 4, BTTS responding to antigens "A" or "B" , binds to antigen "C" or antigen "D" and is activated by antigen "C" or antigen "D" In such circuits, antigen A or ( OR) antigen B is present, THEN cell killing is based on the presence of antigen C or antigen D Immune cells encoding the target circuit are induced by antigen A alone, antigen B alone, or antigen A and B alone. Note that killing can be primed by cells expressing both β and β. Furthermore, killing of cells expressing antigen C and antigen D, and killing of cells expressing antigen C alone or antigen D alone Note that killing of cells that are involved in the activation of the receptor may also be induced.

[0239] In some instances, the use of an OR function can have certain advantages. For example, an OR gate function The above-mentioned circuits (i.e., the second, third, and fourth cells in Figure 4) and their transformations Without being bound by theory, the shape is , comprising cells that express neither of the two priming and / or killing antigens, or The need for generation / production leads to heterogeneous cancer escape resistance and improved Provides effectiveness.

[0240] In some instances, multiple antigen-binding domains present on the BTTS or antigen-specific therapeutic agent may be present. The nucleotides can provide OR gate capabilities to the molecular circuits described herein. For example, some examples In the present invention, a BTTS having two different antigen-binding domains binds a first antigen (e.g., a first priming antigen) or (OR) a second antigen (e.g., a second priming antigen) For example, in some instances, an antibody having two different antigen-binding domains may be used. The antigen-specific therapeutic agent (e.g., CAR, TCR, etc.) targets a first antigen (e.g., a first target antigen). The antigen may be responsive to a primary antigen (primary antigen) or a secondary antigen (e.g., a secondary target antigen).

[0241] In some instances, such OR gates are combined with other gates, including AND gates. For example, an antigen of an OR gate having two different antigen-binding domains can be combined. The nucleic acid encoding the specific therapeutic agent is a promoter that responds to the BTTS in response to the priming antigen. Thus, upon binding to the priming antigen, the BT TS drives the expression of antigen-specific therapeutics in response to two different antigens, resulting in AND-OR This brings about

[0242] In some instances, the OR gate may be configured to select two or more target antigens (or two or more The disclosed circuit may find use in generating an OR gate for a specific antigen (or antigens). For example, in some instances, the circuitry can be The cells are expressed by the targeted cancer cells (or by two different targeted cancer cells). antigen specificity that binds to the first target antigen / killing antigen or the second target antigen / killing antigen (expressed) The nucleic acid sequence encoding the specific therapeutic agent is used to target the first target antigen / killing antigen or the second target antigen. Activated by either of two target / killing antigens, e.g., active against cell killing In some instances, the circuits of the present disclosure can be configured to produce cells that are A first antigen-specific therapeutic agent and a second antigen-specific therapeutic agent, each binding to a different target antigen / killing antigen. It may include a nucleic acid sequence that encodes a therapeutic agent.

[0243] In some instances, OR gates are used to simultaneously target cells in both trans and cis. For example, in some cases, the second OR gate can be The killing antigen can be expressed by the priming cells. In some instances, in brain cells OR gate for targeting to target two antigens that are not mutually exclusively expressed can be used.

[0244] kit The present disclosure provides methods for performing the methods described herein and / or for one or more A method for constructing a pathway, its components, nucleic acids encoding a circuit or its components, etc. In some cases, the subject kits include the circuit or one or more of the disclosed circuits. A vector, such as an expression vector or delivery vector, containing a nucleotide sequence encoding the above moiety. The delivery vector may be provided in a delivery device or may be delivered separately, e.g., For example, as a kit containing a delivery vector and a delivery device as separate components of the kit. may be provided.

[0245] In some cases, the subject kits include nucleotide sequences encoding the disclosed circuits or portions thereof. A cell, such as a host cell or a cell that has been or will be genetically modified with a nucleic acid comprising the sequence. In some cases, the subject kits include a host cell line. Cells that have been genetically modified or will be genetically modified with a recombinant expression vector containing a nucleotide sequence The kit components may be in the same container or in separate containers. It's okay to have one.

[0246] Any of the above kits may further comprise one or more additional reagents, Additional reagents such as dilution buffer; reconstitution solution; wash buffer; control reagent; control expression vector ter; encoding a negative control (e.g., a circuit lacking one or more critical elements) a nucleic acid; a nucleic acid encoding a positive control polypeptide; and the like.

[0247] In addition to the components described above, the subject kits may include any of the components of the kit for practicing the subject methods. The instructions for practicing the subject methods generally include: The instructions may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. As such, the instructions may be printed on the kit container or The label of that component (i.e., associated with the packaging or subpackaging) In other embodiments, the instructions may be present on a suitable computer-readable storage medium, e.g. Electronic storage data files, such as those found on CD-ROMs, diskettes, flash drives, etc. In yet other embodiments, the actual instructions are not present in the kit. However, means are provided for obtaining instructions from a remote source, for example via the Internet. An example of this embodiment is a device that allows users to view and / or download instructions from the device. The kit includes a web address where the user can download the instructions. This means for obtaining the writing is recorded on a suitable substrate.

[0248] Embodiment In any of the above or below embodiments, MOG, CDH10, PTPRZ1 or NRCA Instead of M, brevican core protein (BCAN) or chondroitin sulfate proteolytic enzyme Alternative brain-selective extracellular antigens can be used, including CSPG5 (Complex Cardiovascular System Glycan 5).

[0249] Embodiment 1. A transmembrane antibody having an extracellular binding domain that specifically binds to a brain-selective extracellular antigen A cell comprising a recombinant nucleic acid encoding a protein expressed by a glioblastoma. The cells do not contain a nucleic acid encoding an antigen-specific therapeutic agent that binds to a killing antigen.

[0250] Embodiment 2. The brain-selective extracellular antigen is MOG, CDH10, PTPRZ1, or NR 2. The cell of embodiment 1, which is a CAM.

[0251] Embodiment 3. The extracellular binding domain specifically binds to the brain-selective extracellular antigen. 3. The cell of embodiment 1 or 2, wherein the cell is a variable domain of an antibody.

[0252] Embodiment 4. The transmembrane protein is a binding-triggered transcriptional switch, and the cell is and (ii) a nucleic acid comprising a coding sequence encoding a therapeutic protein and a regulatory sequence. the regulatory sequence is operably linked to the coding sequence, and the binding-induced transcription sequence 4. The cell of any one of embodiments 1 to 3, wherein the cell is responsive to activation of a switch.

[0253] Embodiment 5. The binding-triggered transcriptional switch activates a transcription factor upon binding to the brain-selective extracellular antigen. The gene expression regulator is released upon proteolytic cleavage to activate the transcription of a therapeutic protein. 5. The system according to any one of embodiments 1 to 4, wherein the system is one or more polypeptides that express the

[0254] Embodiment 6. The binding-triggered transcriptional switch is a SynNotch receptor, an A2 receptor, 6. The method of claim 5, wherein the receptor is MESA or another receptor that undergoes binding-induced proteolytic cleavage. The cells described.

[0255] Embodiment 7. The binding-triggered transcriptional switch

[0256] (i) an extracellular domain that binds to the brain-selective extracellular antigen;

[0257] (ii) a proteolytically cleavable sequence containing one or more proteolytic cleavage sites; column; and

[0258] (iii) an intracellular domain; Including,

[0259] (i) the binding of the extracellular domain to the brain-selective extracellular antigen is Inducing cleavage of the proteolytic cleavable sequence at the proteolytic cleavage site above and releasing the intracellular domain, wherein the released intracellular domain is a regulatory sequence of (ii). 7. The cell of embodiment 6, wherein the expression of said therapeutic protein of (i) is induced via:

[0260] Embodiment 8. The therapeutic protein of (i) is, upon expression, by the cell or 8. The cell of any of embodiments 4 to 7, wherein the protein is secreted onto the surface of the cell. .

[0261] Embodiment 9. The therapeutic protein, when expressed on the surface of an immune cell, From embodiment 4, the protein is a protein that activates or suppresses the activation of said immune cells. 9. A cell according to any one of 8.

[0262] Embodiment 10. The method of any one of embodiments 4 to 9, wherein the therapeutic protein is an antigen-specific therapeutic. The cell according to any one of the above.

[0263] Embodiment 11. The antigen-specific therapy is a chimeric antigen receptor (CAR) or a T cell receptor. (TCR), and the binding of said transmembrane protein to said brain-selective extracellular antigen is 11. The cell of embodiment 10, which induces expression of R or TCR.

[0264] Embodiment 12. The antigen-specific therapy is an inhibitory chimeric antigen receptor (iCAR); Binding of the transmembrane protein to the extracellular antigen induces expression of the iCAR. The cell of Form 10.

[0265] Embodiment 13. The antigen-specific therapeutic agent is another binding-triggered transcriptional switch. 11. The cell according to aspect 10.

[0266] Embodiment 14. The therapeutic protein of (i) is, upon expression, an intracellular protein. 8. The cell of any one of embodiments 4 to 7.

[0267] Embodiment 15. Any of embodiments 1 to 8 or 10, wherein the antigen-specific therapy is an antibody. The cell described in any one of the above.

[0268] Embodiment 16. Any of embodiments 1 to 10, wherein the therapeutic protein is an inhibitory immunoreceptor. The cell according to any one of the preceding claims.

[0269] Embodiment 17. The method of any one of embodiments 1 to 17, wherein the therapeutic protein is a secreted peptide or enzyme. 11. A cell according to any one of claims 10 to 19.

[0270] Embodiment 18. The transmembrane protein is an inhibitory chimeric antigen receptor (iCAR), Binding of the brain-selective extracellular antigen inhibits activation of immune cells expressing the iCAR. , The cell described in embodiments 1 to 10.

[0271] Embodiment 19. A cell according to any one of embodiments 1 to 18, which is an immune cell.

[0272] Embodiment 20. The cell of any one of embodiments 1 to 19, which is a myeloid cell or a lymphoid cell. Cells on the plate.

[0273] Embodiment 21. The lymphocyte cells are T lymphocytes, B lymphocytes, or natural killer cells. 21. The cell of embodiment 20, which is a cell.

[0274] Embodiment 22. A cell according to any one of embodiments 1 to 18, which is not an immune cell.

[0275] Embodiment 23. A method of treating a subject for a disease, comprising:

[0276] 23. A method comprising administering to said subject a cell of any of embodiments 1 to 22. .

[0277] Embodiment 24. The method of embodiment 23, wherein the disease is a disease of the brain and / or central nervous system. The method described.

[0278] Embodiment 25. The subject is a patient with medulloblastoma, diffuse midline glioma, ependymoma, craniopharyngioma, embryonal glioma, or glioma. blastoma, pineoblastoma, brainstem glioma, choroid plexus carcinoma, germ cell tumor, pituitary adenoma, acoustic neuroma, Have meningioma, oligodendroglioma, hemangioblastoma, CNS lymphoma, or non-GBM astrocytoma 25. The method of embodiment 24.

[0279] Embodiment 26. The subject is diagnosed with Alzheimer's disease, stroke, brain and spinal cord injury, brain cancer, HIV infection in the brain, ataxia-onset disorder, amyotrophic lateral sclerosis (ALS), Huntington's disease have a genetic disorder that affects the brain, a childhood genetic error, Parkinson's disease, or multiple sclerosis. 24. The method of embodiment 23, wherein

[0280] Embodiment 27. The method of claim 27, wherein the disease is a cancer originating from a non-brain or non-CNS tissue that has metastasized to the brain. 24. The method of embodiment 23.

[0281] Embodiment 28. The brain-selective extracellular antigen is MOG. Any of embodiments 1 to 27.

[0282] Embodiment 29. The brain-selective extracellular antigen is CDH1 29. The embodiment of any one of embodiments 1 to 28, wherein N is 0.

[0283] Embodiment 30. The brain-selective extracellular antigen is PTPR Any of the preceding embodiments, wherein Z1.

[0284] Embodiment 31. The brain-selective extracellular antigen is NRCA The embodiment of any one of embodiments 1 to 30, wherein M is M.

[0285] Embodiment 32. The brain-selective extracellular antigen is an antigen-specific extracellular antigen. The embodiment of any of embodiments 1 to 31, wherein the antibody is a bican core protein (BCAN).

[0286] Embodiment 33. The brain-selective extracellular antigen is chondroitin. 33. The method of any one of embodiments 1 to 32, wherein the CSPG is cysteine ​​sulfate proteoglycan 5 (CSPG5). Embodiment. [Example]

[0287] The following examples provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. It is set forth for the purpose of illustration only and is not intended to limit the scope of what the inventors regard as their invention. This does not represent that the experiments below are all or the only experiments performed. The accuracy of numbers used (e.g., amounts, temperatures, etc.) Efforts have been made to ensure accuracy, but some experimental error and deviation should be considered. Unless otherwise indicated, parts are parts by weight and molecular weights are weight average molecular weights. , temperatures are in degrees Celsius, and pressures are at or near atmospheric. Standard abbreviations are used, e.g., bp, kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; i .m., intramuscular (in); ip, intraperitoneal (in); sc, subcutaneous (in); etc. can be used. do.

[0288] Example 1: Therapy using brain-specific circuits As noted above, the cells, in some embodiments, activate expression of a therapeutic protein. They express a transmembrane protein that may be a binding-triggered transcriptional switch (BTTS). In some of these embodiments, the therapeutic protein binds to an antigen-specific immune cell receptor (e.g., These latter embodiments are described in more detail below. However, transmembrane proteins do not need to be BTTS, and therapeutic proteins can be expressed as immune cell receptors. Therefore, the following description is not intended to limit the present disclosure in any way. stomach.

[0289] In this example, a brain-selective antigen (MOG, CDH10, PTPRZ1 or NRCAM) using disease-specific antigens (which may be "killing" or "deleting"), depending on how the circuit is used. In this example, MOG, CDH10, PTPR on diseased cells using a brain-specific priming antigen selected from Z1 or NRCAM A second antigen that targets diseased cells based on the second antigen (or combination of antigens) expressed. This approach primes the expression of a therapeutic molecule. Without being bound by theory, this is effective even if the gene is not completely disease-specific and is expressed in non-brain tissues. Although not intended to be a generalized approach, this approach allows for the synthesis of two or more incomplete antigens (in this case, the brain High selectivity and heterogeneity of antigen expression are achieved by utilizing the antigen-specific and disease-specific antigens. It is thought that combinatorial T cells that exhibit both sensitivity and insensitivity will be developed.

[0290] In this example, therapeutic cells are primed against brain-specific antigens, thereby providing a therapeutic Induce expression of a therapeutic protein (e.g., CAR, BiTE, etc.) and then target it We designed a circuit to deliver antigens to nearby cells that express them (see Figure 1A). The circuit is primed with brain-specific antigens and targets antigens on diseased cells. Thus, cells in the "killing zone" surrounding the priming antigen cells are killed (see Figure 1B). The size of the wound area depends on, but is not limited to, the stability or use of extracellularly diffusible drugs as killing payloads (e.g., bispecific adapters) can be adjusted based on various factors (see Figures 1C and 1D).

[0291] As shown in FIGS. 1A-1D, a cell operated using a circuit such as that shown in FIG. Priming of therapeutic cells, such as cells, can be achieved by using a method in which such cells do not express the priming antigen. Create an area around the therapeutic cells so that cells expressing the target antigen are targeted even when the target antigen is not present. An example of this scenario is the priming of cells with tissue-specific, i.e., brain-specific, antigens. This is diagrammed in Figure IB, which shows the therapeutic cells, designated as T cells. The therapeutic cells selected include cells expressing target antigens and brain-specific antigens, as well as cells expressing target antigens. targeting (e.g., killing) cells in its vicinity, including cells that express brain-specific antigens but do not express brain-specific antigens In this way, the therapeutic cells are primed to have a biological effect on the cells. It has an effect on cells surrounding the targeted cells (i.e., only in the brain) and affects all diseased cells. This results in effective treatment or clearance of the cells.

[0292] In this example, the size of the region may vary depending on, for example, the use of a diffusible payload, the therapeutic Depending on the stability of the drug (e.g., CAR stability), this can be expanded or adjusted as desired. For example, Figure 1C shows the effect of priming antigens (circles) on diffusible CAR head expression. 1 shows a circuit comprising a synNotch binding-triggered transcriptional switch configured to bind. The diffusible CAR head is specific for a disease-specific target antigen (triangle) and binds to T cells upon antigen binding. The "split CAR" in Figure 1C contains the intracellular signaling components required for cytoplasmic activation. The CAR is bound by a part of the CAR called the CAR-binding domain. By spreading, the diffusible CAR head expresses split CAR, but does not necessarily spread. Mediates antigen recognition and target cell processing in more distal T cells that do not express the diffuse CAR head. It helps to mediate.

[0293] As shown in the left panel of Figure 1D, a conventional CAR (i.e., an antigen recognition domain and synNotch, which drives the expression of a single continuous chain containing intracellular signaling components Killing of non-primed cancer cells expressing a killing antigen by using a circuit comprising In contrast, as shown in the right panel of Figure 1D, the diffusive C By using circuits containing diffusible orthogonal bispecific adaptors such as the AR head, The killing radius of non-primed cancer cells expressing tumor antigens is increased, thus achieving the desired killing effect. The radius can be controlled as desired. In some instances, for example, the killing antigen is When expressed in cancerous tissue (i.e., bystander tissue), a short killing radius is desirable In other instances, for example, a relatively small number of cells expressing the priming antigen may be targeted. If the cancer is diffuse throughout the cancerous area, a wide kill radius may be desirable .

[0294] The following examples describe circuits for the treatment of glioblastoma. The concepts can be applied to other diseases and conditions.

[0295] Example 2: Testing the antigen target of SynNotch receptor for glioblastoma In this example, circuits using synNotch receptors for various target antigens were developed using G T cells were tested for BM targeting. Specifically, human primary CD8+ T cells were , the antigen target of synNotch receptor for glioblastoma, namely EGFRvIII; NRCAM, EphA2, EphA3, IL13Ra2, Her2, EGFR and PTR By selecting Z1 and the corresponding response element that controls the expression of the reporter (eGFP), These CD8+ synNotch AND gated T cells were first When detected, the respective surface GBM antigens are sensed via Notch receptors. , constructed to express the eGFP reporter. Primary CD8+ synNotch A ND gate T cells cultured alone ("T cells only") or co-cultured with GBM cells ("T cells"). The GBM cells used were derived from human patient-derived xenografts (PDX) of adult human liver. The target cells were GBM6 cells, a glioblastoma cell line. Figure 2A shows the synNo against various antigens. A histogram of reporter (eGFP) expression levels showing tch receptor activation is provided. do.

[0296] Figure 2B provides quantification related to Figure 2A. Specifically, CD8+synNotc h Quantification of AND-gated primary T cell activation reveals synNotch receptors to their target antigens. These data were obtained by subtracting the basal leakage of GFP expression, which is independent of somatic binding. The various levels of activation of the constructs tested in specific GBM6 cell lines were shown, e.g., activity the desired level of immunosuppression and / or the presence of a particular antigen in the target cell population and / or It has been demonstrated that different antigens can be targeted depending on the level.

[0297] Circuits using IL13Ra2 and EphA2 antigen targeting were further evaluated. Specifically: Human primary CD8+ T cells were transfected with anti-IL13Ra2 synNotch receptor or anti-Eph A2 synNotch receptor and anti-IL13Ra2 / EphA2-4-1 BBz C The AR gene was engineered with the corresponding response element that controls the expression of the GFP receptor. CD8+ synNotch AND gated T cells were first identified as synNotch receptor The cells then sense surface EphA2 or IL13Ra2 via anti-IL13 Express the Ra2 / EphA2 CAR and prime it for activation in response to CAR antigen binding. Figure 3A shows the CD8+ synNotch AND gated primary T cells and primary G Forward (FSC) and side scatter ( SSC) flow cytometry plots are provided. The target SF11411 is located within the circular gate. The IL13Ra2 synNotch panel showed a significant increase in expression of the IL13Ra2 synNotch gene compared to non-transduced controls. Decrease in cells in the SF11411 gate in the EphA2 synNotch panel As shown, synNotch targeting either antigen AND gated T cells The cells resulted in killing of the targeted SF11411 GBM cells.

[0298] CAR expression, measured via a GFP reporter, was measured in SF11411-targeted GBM cells. The results were evaluated in the presence ("T cells + SF11411") and absence ("T cells only") of the cells. Figure 3B shows the effect of a-IL13Ra2 / EphA2 CAR GFP recipients in these situations. A histogram of receptor expression levels is provided, showing that engineered T cells were co-cultured with SF11411. When the engineered T cells are cultured in vitro, the CAR is expressed compared to when the engineered T cells are cultured alone. and / or increased expression.

[0299] Figure 3C provides quantification related to Figure 3A, specifically, IL13Ra2 synN Replicating CD8+ cells induced by the Notch and EphA2 synNotch circuits Figure 3D shows quantification of cytotoxicity of ynNotch and gated primary T cells. B provides quantification related to CD8+synNotch AND gated first. Quantification of pro-T cell activation revealed that synNotch receptor binding to its target antigen was independent of the activation. The basal leakage of GFP expression is subtracted. CAR expression is induced in the presence of GBM target cells (SF11411).

[0300] Taken together, these data demonstrate that antigen-specific therapeutics such as CARs can be effectively used in the presence of target GBM cells. This demonstrates that various antigens can be used to target the circuitry to drive the expression of therapeutic agents. Furthermore, targeted therapeutic drugs are effective in targeting GBM cells because there is no antigen to induce the expression of the therapeutic drug. In the absence of ATP, it is essentially not expressed. We demonstrate targeted and effective killing of GBM cells by the described circuit.

[0301] Example 3: Multi-antigen CAR T cells precisely and sustainably treat heterogeneous glioblastoma The following example demonstrates that multi-antigen CAR T cells precisely and sustainably treat heterogeneous glioblastoma. This example is applicable to other therapeutic proteins and The results can be extrapolated to the treatment of solid cancers and other diseases. Treatment is difficult due to the lack of tumor-specific and uniformly expressed antigens. Epidermal growth factor receptor variant III neoantigen is tumor-specific but heterogeneously expressed and is involved in tumor proliferation. In contrast, glioblastoma antigens are more uniformly expressed than other normal This is not ideal for expression in organs that may result in potential cross-reactive toxicity. Therefore, it is expected that a multi-antigen recognition circuit will have the flexibility and precision to overcome these dual challenges. As a specificity prefilter (recognizing neoantigens or tissue-specific antigens) Use of synNotch receptors restricts CAR T cell cytotoxic activity to local sites This allows for controlled killing via antigens that are not absolutely tumor-specific. By incorporating multiple incomplete but complementary antigens, T cells against glioblastoma This improves both the specificity and durability of the method, providing a general recognition strategy that can be applied to other solid tumors. It is possible.

[0302] This approach involves a "prime-and-kill" dual antigen-recognizing T cell circuit, resulting in tumor-specific A common but heterogeneous antigen (EGFRvIII) or myelin oligodendrocyte glycoprotein synN recognizes either a priming antigen or a brain-specific antigen such as protein (MOG). otch receptors to identify tumor-specific genes that are relatively uniform throughout the tumor but not necessarily tumor-restricted recognizes tumor-associated antigens (e.g., killing antigens: EphA2 or IL13Rα2) that are not T cells with these types of circuits are primed to induce CAR expression locally. specific cells that are locally activated by killing antigens and target neighboring cells that express killing antigens Such cytotoxicity can be mediated by the localized area surrounding the priming cell. They act in a local "blast radius" and target distal normal cells that express killing antigens but lack priming antigens. This type of circuit is thought to avoid indiscriminate killing in tissues. Spatially integrates the recognition of two imperfect but complementary antigen targets across the cell. The binding antigen provides specificity, while the killing antigen ensures uniformity of the therapeutic attack.

[0303] T cells expressing this type of prime-and-kill circuitry are expressed in heterogeneous GBM in mice. Showing significantly improved efficacy and durability in treating PDX tumors, but killing antigen alone It has been shown that there is no cross-reactivity with the tissues in which they are expressed. This type of T cell circuit It can integrate information across multiple neighboring cells within a tumor, allowing for the recognition and rejection of solid tumors. These circuits provide powerful tools for overcoming fundamental challenges in power removal. By combining individually incomplete antigens, we can create multi-antigen tumor signatures that are more accurately and effectively recognized. This study demonstrates that cell-based therapy can provide a nuanced, multi-parameter in that it can be programmed to recognize and treat diseases based on the characteristics of , clearly demonstrating how unique it is among therapeutic platforms.

[0304] Results of Example 3 Dual antigen prime-and-release fusions that maintain high specificity but overcome antigen heterogeneity Kill Circuit Design

[0305] Conventional single-antigen-targeted CAR T cells escape tumor cells that do not express the target antigen. and therefore, in some cases, may be ineffective against antigens with heterogeneous expression. (Figure 5a). Dual antigen prime-and-kill CAR T cell circuit (Morsut et al., 2016; Roybal et al., 2016b) can overcome heterogeneous expression of tumor antigens (Figure 5b). These circuits recognize tumor-specific antigen A (called the priming antigen) The constitutively expressed synNotch receptor is used. Antigen A is recognized by the synNotch receptor. This results in the activation of the Ch receptor and subsequent proteolytic cleavage of antigen B (called the killing antigen). This releases an intracellular transcriptional activation domain that can drive the expression of a CAR that recognizes the target gene (known as a CAR). In this type of circuit, T cells are first primed by synNotch receptor activation. Unless the cells are tagged, they do not express a CAR against the killing antigen.

[0306] One flexible feature of this circuit is the selection of priming and killing antigens. The priming antigen should be highly tumor-specific, but in principle, it can be expressed by tumors. It does not have to be uniformly expressed. Conversely, killing antigens (like priming antigens) provide specificity. It does not need to be absolutely tumor-specific (as it is expressed uniformly throughout the tumor cells) but Therefore, in this type of circuit, T cells must be highly specific. They are locally primed by heterogeneous antigens, and then the priming signal is released. can be activated to mediate killing of tumor cells expressing the killing antigen within a surrounding local radius. In short, two defective antigens with different defects can be synthesized by such a double antigen circuit. The following describes two methods for designing such a circuit. One is primed by tumor-specific neoantigens, and the other is tissue-specific The following describes those primed by specific antigens (Figure 5b).

[0307] Designing a circuit for heterogeneous GBM recognition: priming with EGFRvIII and and killing by EphA2 or IL13Rα2 The T cell circuit shown in Figure 6a recognizes and kills EGFRvIII-positive GBM. Designed to target the GBM-specific neoantigen EGFRvIII as a priming antigen Although potential cross-reactivity is not an issue, the EGFRvIII neoepitope is Heterogeneously expressed in tumors (10–95% of cells in tumors express EGFRvIII) (O'Rourke et al., 2017). Evaluating CAR T cells targeting EGFRvIII. In a recent clinical trial, tumors were found to be resistant to EGFRvIII despite the reduction of EGFRvIII-positive GBM cells. The patient relapsed, likely due to survival and regrowth of EGFRvIII-negative cells. (O'Rourke et al., 2017). In summary, EGFRvIII has excellent specificity. Although it is a target, its heterogeneous expression throughout the tumor makes it less than ideal as a killing antigen. To make something that doesn't exist.

[0308] To kill the target, we used ephrin type A receptor 2 (EphA2) and IL-1. We focused on IL13 receptor α2 (IL13Rα2). Both antigens are expressed by the majority of GBM cells. It is expressed in the brain and is absent in normal brain, but is also expressed at low levels in some non-tumor tissues. (i.e., these are GBM-associated antigens rather than GBM-specific antigens) (Bielamowicz et al., 2018; Hegde et al., 2013b; Wykosky et al., 2005). These GBM-associated antigens are not a reasonable fit for traditional single-target CAR T-cell therapy approaches. However, these are not ideal targets, and tumor selectivity is mediated by priming antigens. If provided, it can serve as an effective killing antigen.

[0309] Therefore, we manipulated the prime-and-kill circuit to inhibit EGFRvIII. Recognition by synNotch receptor followed by recognition of both EphA2 and IL13Rα2 CAR (α-EGFRvIII synNotchα-EphA2 / IL13α2 IL13 mutein (IL13Rα2 rather than IL13Rα1) was induced. A single-chain antibody against EphA2 fused to a mutant of the IL13 ligand with high affinity for EphA2 was developed. We used a tandem CAR with an extracellular domain containing (for details of the circuit design, see Figure 1). 11a) (Kahlon et al., 2004). Targeting IL13Rα2 or IL13Rα2 inhibits tumor escape via loss of the killing antigen. It was inferred that this would further reduce the risk of pulmonary embolism.

[0310] Target cells were positive for both EphA2 and IL13Rα2, but not E We used the U87 GBM tumor cell line, which is negative for GFRvIII (Chow et al. ,2013; Krenciute et al.,2016). The present inventors stably transfected EGFRvIII By injecting the EGFRvIII-positive version of the U87 cell line (U87-E We constructed U87-EGF (GFRvIII) (Johnson et al., 2015; Ohno et al., 2013). EGFRvIII-positive and U87-EGFRvIII-negative cells were mixed at various ratios to investigate the effects of EGFRvIII on GBM. We were able to reproduce the different levels of heterogeneity observed in patients (primed cells 1 (0-100%) (Figure 6b). In vitro cytotoxicity assay was performed using α-EGFRvIII. Primary engineered with synNotch→α-EphA2 / IL13Rα2 CAR circuit This model was performed with human CD8+ T cells. In this model, T cells were transduced into EGFRvIII-positive tumors. Primed by sex cells and adjacent tumor cells, including those lacking EGFRvIII expression They were induced to express a CAR capable of killing (Fig. 6c).

[0311] α-EGFRvIII synNotch→α-EphA2 / IL13Rα2 CAR CD8+ T cells engineered using a prime-and-kill circuit exhibited as low as 10% EGF Even RvIII-positive primed cells were able to differentiate heterogeneous U87 GBM cell populations in vitro. In contrast, the absence of priming cells was In these assays, induction of CAR expression and two The killing kinetics of different tumor cell populations (EGFRvIII-positive and EGFRvIII-negative) The cells were followed for 72 hours (see Figure 11d for CAR induction). Significant clearance (p=0.0149; t-test) was observed with as few as 10% of primed cells. Although killing was observed, killing was somewhat slower compared to 50% primed cells (Fig. 2d and S1e). Taken together, these in vitro killing studies demonstrate that the prime-and-kill circuitry is essential for the regulation of This represents a promising strategy that can significantly reduce the likelihood of tumor escape due to homogeneity. It is suggesting.

[0312] α-EGFRvIII synNotch→α-EphA2 / IL13Rα2 CAR Circuited T cells affect co-transplanted flank tumors lacking priming antigens effectively inhibit the growth of heterogeneous EGFRvIII-positive GBM tumors in the brain without Based on these in vitro data, these prime-and-kill CAR T cells Antitumor activity was evaluated in a GBM xenograft mouse model. To systematically investigate GFRvIII heterogeneity, U87-EGFRvIII-negative cells were U87-EGFRvIII positive cells and various ratios (0:100; 50:50; and 10 The mixture was mixed at a ratio of 0:0 and the mixed population was injected into the brains of immunodeficient NCG mice (Figure 7a). -EGFRvIII-negative cells were transfected with wild-type EGFR to produce EGFRvII EGFRvIII-negative partner cell lines proliferated at the same rate as I-positive cells (see Figure 12). EGFRvIII-negative cells and EGFRvIII-positive cells were prepared (Bonavia et al., 2012). The cell-cell mixture ratio was confirmed to be maintained in vivo by histological examination on day 6. These tumor-bearing mice were then either untransduced (negative control) or treated with PG-1. Prime-and-kill circuit treated with transduced human primary T cells. Neither CAR nor control T cells demonstrated clearance in 0% of EGFRvIII-positive tumors Prime-and-kill CAR T cells did not induce 50% and 100% EGFRv showed equally effective tumor clearance in both III-positive tumors (controls showed clearance Therefore, in this context, the prime Andkill CAR T cells recognize tumors with heterogeneous EGFRvIII expression and show efficacy can be effectively overcome.

[0313] Evaluate whether the function of prime-and-kill CAR T cells is localized at the GBM site (thus avoiding cross-reactivity with other distant normal tissues that express the killing antigen) Therefore, T cells primed by EGFRvIII within the GBM tumor site are distributed throughout the body. Determine whether the antibodies can specifically mediate any anti-EphA2 or IL13Rα2 activity. For this purpose, as specificity controls, two tumors were identified: 50% EGFRvIII-positive U87 tumors and U87-EGFRvIII-negative tumors in the flank A parallel experiment was performed using mice inoculated with flank tumors (Fig. 7c). Represents potentially cross-reactive normal tissues that express the antigen but do not express the priming antigen.

[0314] Six days after tumor inoculation, mice were treated with prime-and-kill CAR T cells or control untransduced CAR T cells. All mice treated with control T cells were given i.v. injection of transfected T cells (n=6 / group). The patients showed tumor growth at both sites and were rapidly euthanized with a median survival of 25.5 days. In contrast, mice treated with prime-and-kill CAR T cells achieved a There was a significant suppression of intracranial tumor growth compared to control mice (p<0.001; t-test). Importantly, however, mice treated with prime-and-kill CAR T cells , did not show a statistically significant inhibition of flank tumors compared with the control group (p = 0.4; t 7d and e). The selective lack of killing in non-primed flank tumors was consistent with the The cytotoxic activity of priming and killing CAR T cells is enhanced by both priming and killing antigens. It is shown to be spatially restricted to the intracranial tumor in which it occurs.

[0315] Prime-and-kill CAR T cells show locally induced expression of CAR EGFRvIII-induced expression of α-EphA2 / IL13Rα2 CAR improves intracranial GB To further confirm that T cells were restricted to M tumors (EGFRvIII positive), CAR-GFP construct to express GFP upon priming of the Notch receptor We engineered mice with dual tumors (EGFRvIII-positive intracranial tumor and EGFRvIII-negative intracranial tumor). From patients with flank tumors, we administered prime-and-kill CAR T cells i.v. Two days after administration, human T cells were isolated from intracranial and flank tumors and the spleen. T cells were recovered from intracranial tumors, but not from splenic or flank tumors. These findings suggest that expression of the killing CAR is essential for the activation of the priming antigen. These data suggest that the ATP-dependent agonist is localized to the surrounding local environment. Safe and effective treatment that reduces systemic on-target off-tumor toxicity associated with targeting tumor-associated antigens Supporting the development of CAR T therapy based on the synNotch priming system as a strategy .

[0316] Prime-and-kill CAR T cells heterogeneously express EGFRvIII in GBM6 Resulting in complete remission of PDX tumors Primer in tumor models showing naturally occurring heterogeneity of EGFRvIII expression The efficacy of Indo-Kil CAR T cells was evaluated (Figure 8a). GBM6 patient-derived xenografts ( PDX) tumors exhibited intrinsic EGFRvIII heterogeneity (Fig. 8a), and most importantly and show a reproducible ability to avoid treatment with EGFRvIII single-antigen CARs When implanted intracranially, GBM6 tumors grow rapidly and develop within 40 days. The mice died within 2 days (Fig. 8c). The tumors shrink dramatically, but recur slowly and steadily with high reproducibility (Figure 8c, purple dotted line). , and Fig. 13e). These recurrent tumors show loss of EGFRvIII expression (Fig. 8g Further in vitro studies showed that GBM6 cultures express undetectable levels of EGFRvII. I antigen-bearing cells in the population, and these cells are not related to the conventional EGFRvIII C These results demonstrate that GB T cells are resistant to killing by AR T cells (Fig. 13c, d). M6 overcomes the heterogeneity-based escape observed in EGFRvIII CAR clinical trials To evaluate alternative T cell circuits that can mimic and therefore overcome these issues, represents an ideal model.

[0317] α-EGFRvIII synNotch→α-EphA2 / IL13Rα2 CAR Circuit-engineered CD8+ T cells eradicate heterogeneous GBM6 populations in vitro NCG mice bearing GBM6 tumors in the brain showed a similar pattern to control non-cancerous tumors (Fig. 13a). When receiving an iv injection of transduced T cells, all of the mice ( n = 5) died of tumor progression (Figures 8b and c). Treatment with EGFRvIII consistently (n=6) prevented recurrence of EGFRvIII-negative tumors after initial regression. (3 of 6 mice died of tumor progression by day 125), GBM6 This demonstrates the clinical relevance of the model (Figures 8c and g) (O'Rourke et al., 2017). In this study, we demonstrated that i.v.-injected EGFRvIII CAR T cells significantly improved tumor survival in recurrent tumors. In marked contrast, no T cell persistence was detected in the prime-antigen (Fig. 8g). All mice (n=6) treated with Dokil CAR T cells demonstrated long-term progression of GBM6 tumors. This more durable and complete tumor clearance was highly reproducible. Postmortem immunofluorescence analysis revealed that tumor cells in the brain parenchyma and meninges were non- specifically Although the CAR T cells were present, they were persistent (Fig. 8f). To visualize the induction of EphA2 / Il13Rα2 CAR expression and the subsequent transcriptional regulation of CAR expression, Remarkably, 6 days after T cell injection, we observed In the tumor bed, GFP-positive prime-and-kill CAR T cells (also known as human CD45) staining) was detected in the spleen but not in the liver (Fig. 8h).

[0318] In summary, prime-and-kill CAR T cells are a promising candidate for persistent or persistent CAR T cell therapy in the heterogeneous GBM6 model. Compared with conventional α-EGFRvIII CAR T cells in terms of their ability to induce more complete remissions Therefore, these prime-and-kill CAR T cells are better than EGF. Maintaining EGFRvIII-directed tumor specificity (as shown in the previous section) and suppressing EGFRvIII heterogeneity can be overcome (as shown in this section).

[0319] Prime-and-kill GBM circuits using brain-specific antigens These results demonstrate that prime-and-kill CAR T cells act uniformly on all tumor cells. This clearly demonstrates that priming can be efficiently achieved by antigens that are not expressed. Furthermore, prime-and-kill CAR T cells share a transpriming / killing mechanism—different but related. It has been demonstrated that priming off of a single cell can be achieved to induce killing of adjacent cells. Therefore, by recognizing tissue-specific antigens expressed only on non-malignant cells, It was hypothesized that it might also be possible to engineer T cells that are locally primed by For example, in the case of GBM, the cells are primed by recognizing brain-specific antigens. This then induces local killing of T cells based on the GBM antigens EphA2 and IL-13Rα2. It may be possible to engineer a cellular circuit whereby anti-brain synNotch → Eph A2 / IL-13Rα2 CAR prime-and-kill CAR T cells express EGFRvII It appears to offer a potential solution for treating I-negative GBM patients.

[0320] Two brain-restricting surface proteins, namely, brain-specific cadherin, cadherin 10 ( CDH10) and a surface protein on the myelin sheath of neurons (involved in multiple sclerosis) Myelin oligodendrocyte glycoprotein (MOG), which is also an autoantigen, is a virulent protein. The predicted tissue distribution of these antigens was bioinformatically identified. Expression is shown in Figure 9b. Antibodies that bind to these antigens were identified and used to identify cognate sy We constructed nNotch receptors. We screened some of these synNotch receptors. and activated by cells expressing the mouse isoforms of CDH10 or MOG. We identified versions of these receptors that could be activated (Fig. 9c) and therefore used these receptors to target endogenous This allows for the priming of the brain-specific synNo protein from adult mouse brain tissue. The pathway by which tch receptor induces the expression of α-EphA2 / IL-13Rα2 tandem CAR (Fig. 9d) α-CDH10 or α-MOG synNotch → α-Eph CD8+ T cells engineered with the A2 / IL-13Rα2 CAR circuit were shown to be U87 cells only in the presence of transprimed cells (MOG or CDH10 cells) It was found that it was possible to eradicate GBM or GBM6 cell populations (Fig. 14a, b).

[0321] To test the efficacy of these brain antigen priming circuits in vivo, GBM6 P DX tumors were implanted into the brains of NCG mice and primed based on the recognition of MOG or CDH10. In both cases, prime-and-kill T cells were treated with T cells capable of activating the prime-and-kill circuit. The majority of mice treated with cells showed effective GBM6 tumor clearance (Fig. 9e , f). These findings suggest that brain prime-and-kill T cells are indeed the tissues responsible for the development of T cells in the mouse brain. Recognition of specific antigens effectively primes the cells, thereby activating the killing CAR receptor. These results suggest that expression can be induced locally. Mice treated with either circuit showed showing significantly improved survival compared to mice treated with control T cells (Fig. 9e, f).

[0322] Whether the effects of MOG or CDH10 primed CAR T cells are restricted to brain tumors To assess whether GBM6 tumors are amenable to endothelial growth factor receptor 6 (EGFR) therapy, these experiments were performed using GBM6 tumors implanted simultaneously in the brain and flank. This was repeated by intravenously injecting prime-and-kill CAR T cells (Figure 9g). Consistent with the last experiment, MOG-primed CAR T cells and CDH10-primed CAR In the flank, both MOG- and T cells demonstrated effective anti-brain tumor responses (Fig. 9h). Immunokine-kill CAR T cells had no effect on tumor growth compared to control, non-transduced T cells This suggests that MOG-prime-and-kill CAR T cells function specifically in the brain. On the other hand, CDH10-primed CAR T cells showed a significant reduction in flank tumor size. The anti-CDH10 scFV cross-reacts with epitopes on other antigens present outside the brain. RNA-s showing possible or non-CNS organ CDH10 mRNA expression CDH10 expression may not be sufficiently brain-restricted as suggested by the eq data This suggested that (Figure 9b).

[0323] Overall, these data support the notion that integrative priming from normal tissue-specific antigens is essential for the development of inflammatory cytokines. Demonstrating the versatility of prime-and-kill CAR T cells.

[0324] Method of Example 3 Design of SynNotch receptor and response element constructs The SynNotch receptor was expressed as EGFRvIII 139 scFv (Johnson et al., 2014). 15), MOG M26 scFv (von Budingen et al., 2002) and CDH10 (Sidh (gift from the u lab) was cloned into the mouse Notch 1 (NM_008714) minimal regulatory region ( By fusing the α-Gal4 DBD to VP64, All synNotch receptors were constructed using the N-terminal CD8a sequence for membrane targeting. signal peptide and a-myc A647 (cell-signaling #2233 ) or a-flag A647 (RND systems #IC8529R) Contains a myc tag or flag tag for easy determination of expression. For the ch receptor peptide sequence, see Morsut et al. (Morsut et al., 2016). The receptor was selected from the PGK or SFFV promoter-containing vectors for all primary T cell experiments. The modified pHR'SIN:CSW vector was cloned into the pHR'SIN:CSW vector. The vector was also modified to prepare a plasmid containing a response element. The DNA binding domain target sequence was cloned into a minimal CMV promoter. The ment plasmid contains mCherry or B for easy identification of transduced T cells. The PGK promoter, which constitutively drives FP expression, is also included. hA2 scFv (Goldgur et al., 2014), IL 13 mutein [E13K, K105R ] (Krebs et al., 2014) or IL13 mutein [E13K, K105R]-G4Sx4- EphA2 scFv (Goldgur et al., 2014) was cloned into the human CD8 α-chain hinge region and human The transmembrane and cytoplasmic regions of 4-1BB and the CD3z signaling endodomain are fused together. The inducible CAR construct was constructed by merging the CAR-CDNA fragments with the CAR-CDNA fragments into the multiple cloning site 3′. The GAL4 response element was cloned into the BamHI site of the . For possible CAR vectors, CAR can be tagged at the c-terminus with GFP / BFP or or myc / flag tags to verify surface expression. Cloned by fusion cloning (Clontech #ST0345) did.

[0325] Isolation and culture of primary human T cells Primary CD4+ and CD8+ T cells were obtained from anonymous donor blood after apheresis with negative selection. The lysates were isolated from the lysate (STEMCELL Technologies #15062 and #1 5063). University Institutional Review Bo As approved by the Blood Centers of the Pac Blood was obtained from Fisci. T cells were cultured in 20% human AB serum (Valley Biome). RPMI-1640 (UCS, #HP1022) and 10% DMSO After thawing, T cells were cryopreserved in a 100% PBS-F cell culture core. For all experiments, X-VIVO 15 (Lonza #04-418Q), 5% Human AB serum, and 10 mM neutralized N-acetyl-L-cysteine ​​(Sigma-Aldrich) ch #A9165) and 30 units / mL IL-2 (NCI BRB Prec The cells were cultured in human T cell medium supplemented with 100% ethanol (Institute of Clinical Repository). uCyte experiments were performed in vitro with 30 units / mL of IL-2 (NCI BRB Preclinical 5% human AB serum (Valley Biomed Repository) supplemented with 5% AB serum (Valley Biomed Repository). Cultured in RPMI-1640 (UCSF Cell Culture Core) containing 1000 kJ / ml of PBS containing 100 kJ / ml of PBS (Cell Culture Core, #HP1022). I raised them.

[0326] Lentiviral transduction of human T cells pHR' to Lenti-X 293 T cells (Clontech #11131D) SIN:CSW transgene expression vector and viral packaging plasmid pCMV dR8.91 and pMD2.G were transfected with Fugene HD (Promega #E231 2) by transfection with pantropic VSV-G sequences. Primary T cells were thawed on the same day and cultured for 24 hours. Human T-Activator CD3 / CD28 Dynab at a cell:bead ratio of 1:3 The cells were stimulated with Life Technologies #11131D eads. Between assays, viral supernatant was collected and, in some assays, concentrator-based Lenti-X (Clon Primary T cells were enriched using a 100% ELISA kit (Etech #631231). Primary T cells were exposed to virus for 24 hours. On the fourth day after T cell stimulation, the Dynabeads were removed, and the T cells were rested and then up-regulated. T cells were cultured at Beckton Dickinson University Hospital and expanded until day 9 when they could be used for clinical trials. Kinson (BD) FACs were selected for assay using ARIA Fusion. AND-gated T cells exhibiting basal CAR expression were gated out during sorting.

[0327] Cancer cell lines The cancer cell lines used were K562 myeloid leukemia cells (ATCC #CCL-243), L929 mouse fibroblast cells (ATCC# CCL-1), U87 MG GBM cells (ATCC #HTB-14) and GBM6 PDX cells (Dr. Frank Furn ari, a kind gift of the Ludwig Institute and UCSD) U87-EGFRvIII luciferase negative (Ohno et al., 2013) and U87 MG was expressed as GFP under the control of the spleen focus forming virus (SFFV) promoter, respectively. The cells were transduced with lentivirus to stably express mCherry or mCherry. After 2 hours, cells were sorted based on GFP expression using an Aria Fusion cell sorter (BD Bios Cells were sorted using a 100% GFP or mCherry positive cell sorter (sciences). All cell lines were screened for transgene expression. U87-luciferase and U87-luciferase-mCherry cells were transfected with the retroviral constructs ( (gift from Matthew Myerson; Addgene plasmid #11011) The unmutated EGFR was stably transduced using EGFRvIII-positive U87 cell line. We prepared an EGFRvIII-negative cell line that proliferated at a rate of 1000 kJ / s (see Figure S2). Overexpression of live EGFR is associated with human GBM with EGFR amplification (Bonavia et al., (2012). Both GBM6 cells were lentivirally transduced with mCherry and firefly luciferase. These cells were cultured in a medium containing EGF (20 μg / mL), FGF (30 μg / mL), and IFN-γ. DMEM F12 medium supplemented with GF (20 μg / mL) and heparin (5 μg / mL) K562 cells were lentivirally transduced to stably express surface CDH10. (CDH10 extracellular domain was fused to the PDGF transmembrane domain). 9 was lentivirally transduced to stably express full-length MOG.

[0328] In vitro stimulation of SynNotch T cells For all in vitro synNotch T cell stimulations, co-culture with U87, 1x 104 U87 cells were cultured overnight in a flat-bottom 96-well tissue culture plate. The next morning, 1x1 Add 0.4–5 × 10 T cells to a flat-bottom 96-well tissue culture plate and co-culture with the target tumor. The activation and specific lysis of tumor cells were analyzed from 24 to 96 hours. For all in vitro synNotch T cell stimulations, co-culture with 1 x 1 T cells. GBM6 cells were cultured overnight in a flat-bottom 96-well tissue culture plate. The next morning, 1x1 04 T cells were added to flat-bottom 96-well tissue culture plates, and the co-cultures were used to measure the activity of target tumor cells. The lysis and specific lysis were analyzed at 24-96 hours. For all in vitro synNotch T cell stimulations cultured, target cells (GB M6) at 1×104 cells, and priming cells (either K562 or L929) were used. ) were cultured overnight at 1 × 10 cells in a flat-bottom 96-well tissue culture plate. 104 T cells were added to a flat-bottom 96-well tissue culture plate, and the co-culture was performed with the target tumor cells. Activation and specific lysis were analyzed at 24-96 hours. Measurement was performed using a BD LSR II or Attune NxT flow cytometer. and analysis was performed with FlowJo software (TreeStar).

[0329] Assessment of cytotoxicity of SynNotch AND-gated T cells Gate CD8+ synNotch AND - T cells as above with the indicated antigens. The level of specific lysis of target cancer cells was assessed by stimulation with non-specific cytotoxic T cells for 24 to 96 hours. The percentage of surviving target cells in culture was compared to treatment with transduced T cell controls. Cell death was determined by comparing side and forward scatter, which are normally occupied by target cells. Alternatively, cell viability was monitored by measuring the percentage of target cells in the situ culture medium. Analysis was performed using the uCyte Zoom system (Essen Bioscience). Tumor cells were plated overnight in triplicate in 96-well plates at a density of 1.0 × 104 cells / well. The next day, T cells were added to each well in a final volume of 200 μl / well. and T cells were co-cultured as described above. Two fields per well were taken every 15 minutes. To determine cell viability, mean fluorescence intensity (MFI) was measured using the IncuCyte Zoom™. The data were calculated using software (Essen BioScience). Results are summarized as ±SEM.

[0330] Statistical analysis and curve fitting Statistical significance was determined by specific tests and means ± standard errors are shown as indicated in the figure legends. Results are presented as mean (SEM) or mean ± standard deviation (SD). Kaplan-Meier estimates Use quantification to create survival curves and use the Log-Rank test to assess differences in survival distributions All p values ​​are shown in the figures or their legends. All statistical analyses were performed using P This was performed using rism software version 7.0 (GraphPad).

[0331] Mouse model All mouse experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee (IACUC). For the heterogeneous model using U87, 1.5 × 10 4 U87-lu c-mCherry cells and 1.5 × 10 4 U87-luc-EGFRvIII-negative The mixture of GFP cells was cultured in 6- to 8-week-old female NCG mice (Charles River). Six to ten mice per group were intracranially implanted with homogeneous U87-luc-GFP-EGFR For the vIII-positive model, 3 × 10 cells were injected into the brains of NCG mice. For the orthotopic heterogeneous model using 6, 1.0 × 10 GBM6-luc-mche Rry cells were implanted intracranially into 6-8 week old female NCG mice, 6-10 mice per group. Stereotaxic surgery for tumor cell inoculation was performed at a distance of 2 mm to the right and 1 mm in front of bregma and 3 mm into the brain. The injection site was coordinated in mm. Mice were analgesic before and for 3 days after surgery. The mice were treated with drugs and monitored for adverse symptoms according to IACUC guidelines. G mice were injected with 1.0 × 106 U87-Luc-mcherry+ or 1.2 × 105 On day 0, either GBM6-luc-mcherry cells were cultured in 100 μl of HBSS. Tumor progression was monitored by intraperitoneal D-lymphocyte injection according to the manufacturer's instructions (GoldBio). After injection of luciferin, the cells were evaluated by luminescence in Xenogen IVIS Spectrum. Before treatment, mice were placed in a 100-well tube to ensure that the initial tumor burden in the control and treatment groups was comparable. Mice were randomized to receive 6 doses of 100 μl of PBS intravenously via the tail vein. Each patient was treated with 0x10 engineered T cells or a corresponding number of non-transduced T cells. Survival was assessed by Predetermined IACUC-approved endpoints (hunching, neurological deficits, e.g., circling, ataxia) , paralysis, lameness, head tilt, balance problems, seizures) were assessed over time (n = 6–10 mice / group).

[0332] Immunofluorescence After euthanasia, the mice were transcardially perfused with cold PBS. The brains were then removed and analyzed at 4% After fixation overnight in PFA-PBS, the cells were transferred to 30% sucrose and submerged (1–2 days). The brains were then placed in OCT compound (Tissue-Tek; 4583; Sakura F The tissue was embedded in PET (Inetek). Serial 10 μm coronal sections were then cut using a freezing microtome. The sections were then thawed and stained overnight at 4°C. The primary antibodies used were , CD45(D9M8I)XP® Rabbit mAb (Cell Signaling g Technologies, 1:100), anti-EGFRvIII, clone DH8. 3 (Millipore Sigma, 1:100), human EphA2 Alexa F Luor 700 conjugated antibody (R&D Systems, 1:100) and anti-I The L13 receptor alpha 2 antibody (Abcam, 1:100) was produced in donkey and Immunofluorescence was performed using a secondary antibody conjugated with lexFluor 647 for 2 hours at 4°C. The sections were stained with the nuclear dye DRAQ7 (Abcam) or DAPI5 (Thermo Fisher Scientific). The images were stained using TissueFAXS scanning software (Ti A Zeiss Axio Imager 2 microscope equipped with a ssueGnostics 20x magnification) or Zeiss LSM with Zeiss Zen imaging software The images were acquired using either a 780 microscope (magnification 20x). The exposure time and threshold were adjusted according to the image quality. The results were kept consistent between samples within a sampling session. [Table 1] JPEG2026041745000002.jpg251169JPEG2026041745000003.jpg234169

[0333] Here, we demonstrate that a more sophisticated T cell recognition circuit that integrates the recognition of multiple antigens can be used to detect G It has been shown that these problems can be overcome with BM (Figure 10). To address this issue, we constructed a series of prime-and-kill circuits. EGFRvIII, a neoantigen expressed in EGFRvIII, was targeted as a priming antigen (Fig. 10a ), and then extended the strategy to target brain-specific antigens expressed on non-tumor cells in the brain ( Figure 10b).

[0334] In the circuit described here, the synNotch receptor mediates the expression of two GBM-associated antigens, Eph This induces gene expression of a tandem CAR targeting IL13Rα2 and IL13Rα2. As therapeutic targets, these two antigens are expressed in several other normal non-brain tissues. However, these prime and kill circuits may be incomplete. This interaction is important because expression is induced only in the vicinity of cells expressing EGFRvIII or MOG. The differential reactivity is reduced. The prime-and-kill cycle combines these incomplete antigens. and integrate them to optimize how they each contribute to the overall perception. EGFRvIII and MOG are highly specific for GBM tumors and the brain, respectively. As they are highly potent, they represent excellent priming antigens. α2 is less specific but is more uniformly expressed throughout the tumor, making this Targeting these antigens may facilitate killing of a broader population of GBM cells. Therefore, these antigens may be involved in the killing of HIV-1-associated viruses, as long as their killing is controlled by local priming signals. Furthermore, these killing antigens are more suited to killing antigen targets individually and completely. Even if not uniform, the use of tandem CARs in killing may result in higher tumor clearance. This provides a new possibility (the tandem CAR functions as an OR gate) (Hegde et al., 2016 ) Therefore, these circuits, even if presented on different cells within the tumor, The signals from the three antigens are integrated to produce a highly localized but sufficient signal to be effective. The circuit generates a killing "explosive" around the primed cells. Other recent studies have hypothesized that EGFR doublets essentially create a “diameter” (Figure 10c). Locally enhancing alternative, less specific therapeutic responses, such as secretion of specific engagers To address this issue, researchers are also exploring how EGFRvIII recognition can be utilized (Choi, 2019). .

[0335] In vivo data demonstrate that α-EphA2 / IL-13Rα2 CAR inhibits EGFRvIII or MOG-induced expression and their cytotoxic activity were confirmed by both priming and killing antigens. This is confined to intracranial tumors expressing the killing antigen, thereby limiting the killing antigen expression to tissues at distal sites. This demonstrates that the α-amyloid β ... In the priming and killing pathway, T cells expressing similar prime-and-kill circuits (i.e., "AND gate") for highly selective killing of transplanted tumor cells uniformly expressing However, the tumor cells transplanted on the opposite side, which express only the killing antigen, were not killed. The fact that no killing occurred in the contralateral control tumors suggests that the tumors were not killed in one organ. The priming of T cells in the body leads to the formation of primed T cells that can then kill at long range. (Once the priming stimulus is removed, CAR expression is reduced within a few hours.) These observations are consistent with previous findings from the study, which showed that the immune system is attenuated within 24 hours and prevents the initiation of a sustained immune response. Although synNotch-mediated T cell priming can induce short-range killing of target cells, long-range This is consistent with the current model, which shows that it is not possible to induce abscission.

[0336] Considering EGFRvIII as a priming antigen, approximately 20% of GBM patients respond to EGF positive for RvIII ( Heimberger et al., 2005 ; Moscatello et al., 1995 ; Thorn (e et al., 2016; Wikstrand et al., 1997). Furthermore, between 10 and 95% of patients had EGFR O'Rourke et al., 2017), which may indicate heterogeneity in VIII expression. Killing also resulted in a parallel reduction of both non-primed and primed tumor cells. So what percentage of tumors do we need to achieve sufficient priming to eliminate the tumor? It remains unclear whether EGFRvIII-positive cells are necessary in vivo. In some cases, it may be possible that priming cells are eliminated too early. First, based on our data using the GBM6 model (Figure 8), The priming strategy is promising as all mice showed long-term tumor clearance This compares favorably with other similar treatments (Johnson et al., 2015). Elimination of priming signals disrupts the brain antigen priming circuit (e.g., those against MOG) This is less of a concern because prime-and-kill CAR T cells deliver the necessary killing. Normal brain cells expressing MOG lack the antigen (EphA2 or IL-13Rα2). Because it doesn't kill.

[0337] References for Example 3 Bielamowicz et al.(2018).Trivalent CAR T cells overcome interpatient antigenic Variability in glioblastoma.Neuro Oncol 20,506-518.

[0338] Bonavia et al.(2012).EGFRvIII promotes glioma angiogenesis and growth through the NF-kappaB,interleukin-8 pathway.Oncogene 31,4054-4066.

[0339] Brown et al.(2016).Regression of Glioblastoma after Chimeric Antigen Receptor T-Cell Therapy.N Engl J Med 375,2561-2569.

[0340] Cameron et al.(2013).Identification of a Titin-derived HLA-A1-presented peptid e as a cross-reactive target for engineered MAGE A3-directed T cells.Sci Transl Med 5,197ra103.

[0341] Choi et al.(2019).CAR-T cells secreting BiTEs circumvent antigen escape withou t detectable toxicity.Nat.Biotechnol.37:1049-1058

[0342] Drumm et al.(2019).Extensive brainstem infiltration,not mass effect,is a commo n feature of end-stage cerebral glioblastomas.Neuro Oncol.

[0343] Goldgur et al.(2014).Generation and characterization of a single-chain anti-Ep hA2 antibody.Growth Factors 32,214-222.

[0344] Hegde et al.(2013a)Combinational targeting offsets antigen escape and enhances effector functions of adoptively transferred t cells in glioblastoma Mol Ther.2 013 21:2087-101.

[0345] Hegde et al.(2013b).Combinational targeting offsets antigen escape and enhance s effector functions of adoptively transferred T cells in glioblastoma.Mol Ther 21,2087-2101.

[0346] Heimberger et al.(2005).Prognostic effect of epidermal growth factor receptor and EGFRvIII in glioblastoma multiforme patients.ClinCancer Res 11,1462-1466.

[0347] Johnson et al.(2009).Gene therapy with human and mouse T-cell receptors mediat es cancer regression and targets normal tissues expressing cognate antigen.Blood 114,535-546.

[0348] Johnson et al.(2015).Rational development and characterization of humanized an ti-EGFR variant III chimeric antigen receptor T cells for glioblastoma.Sci Trans l Med 7,275ra222.

[0349] June et al.(2018).Chimeric Antigen Receptor Therapy.N Engl J Med 379,64-73.

[0350] Kahlon et al.(2004).Specific recognition and killing of glioblastoma multiform e by interleukin 13-zetakine redirected cytolytic T cells.Cancer Res 64,9160-916 6.

[0351] Krebs et al.(2014).T cells redirected to interleukin-13Ralpha2 with interleuki n-13 mutein--chimeric antigen receptors have anti-glioma activity but also recog nize interleukin-13Ralpha1.Cytotherapy 16,1121-1131.

[0352] Morgan et al.(2013).Cancer regression and neurological toxicity following anti -MAGE-A3 TCR gene therapy.J Immunother 36,133-151.

[0353] Morgan et al.(2010).Case Report of a Serious Adverse Event Following the Admin istration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERB B2.Mol Ther 18,843-851.

[0354] Morsut et al.(2016).Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors.Cell 164,780-791.

[0355] Moscatello et al.(1995).Frequent expression of a mutant epidermal growth facto r receptor in multiple human tumors.Cancer Res 55,5536-5539.

[0356] O’Rourke et al.(2017).A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients w ith recurrent glioblastoma.Sci Transl Med 9.

[0357] Ohno et al.(2013).Expression of miR-17-92 enhances anti-tumor activity of T-ce lls transduced with the anti-EGFRvIII chimeric antigen receptor in mice bearing human GBM xenografts.Journal for immunotherapy of cancer 1,21.

[0358] Parkhurst et al.(2011).T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis.M ol Ther 19,620-626.

[0359] Roybal et al.(2016a).Precision Tumor Recognition by T Cells With Combinatorial Antigen-Sensing Circuits.Cell 164,770-779.

[0360] Roybal et al.(2016b).Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors.Cell 167,419-432 e416.

[0361] Sampson et al.(2010).Immunologic Escape After Prolonged Progression-Free Survi val With Epidermal Growth Factor Receptor Variant III Peptide Vaccination in Pat ients With Newly Diagnosed Glioblastoma.Journal of Clinical Oncology 28,4722-472 9.

[0362] Shahideh et al.(2012).Systematic review of primary intracranial glioblastoma m ultiforme with symptomatic spinal metastases,with two illustrative patients.J Cl in Neurosci 19,1080-1086.

[0363] Thorne et al(2016).Epidermal growth factor receptor targeting and challenges i n glioblastoma.Neuro Oncol 18,914-918.

[0364] Watanabe et al.(2018).Expanding the Therapeutic Window for CAR T Cell Therapy in Solid Tumors:The Knowns and Unknowns of CAR T Cell Biology.Front Immunol 9,24 86.

[0365] Wikstrand et al.(1997).Cell surface localization and density of the tumor-asso ciated variant of the epidermal growth factor receptor,EGFRvIII.Cancer Res 57,41 30-4140.

[0366] Wykosky et al.(2005).EphA2 as a novel molecular marker and target in glioblast oma multiforme.Mol Cancer Res 3,541-551.

[0367] Although the present invention has been described with reference to specific embodiments thereof, it is to be understood that the true spirit and scope of the invention may be varied in various ways. Various modifications may be made and equivalents may be substituted without departing from the spirit and scope of the invention. It should be understood by one of ordinary skill in the art that variations in the nature of the materials, compositions of matter, methods, and / or compositions of matter may occur. Many modifications may be made to the method, one or more method steps to adapt it to the objective, spirit and scope of the present invention. All such modifications are intended to fall within the scope of the appended claims. It is intended.

Claims

1. Transmembrane proteins with extracellular binding domains that specifically bind to brain-selective extracellular antigens a cell comprising a recombinant nucleic acid encoding a killing antigen expressed by glioblastoma; The cells do not contain nucleic acid encoding the antigen-specific therapeutic agent to which they bind.

2. The brain-selective extracellular antigen is MOG, CDH10, PTPRZ1, or NRCAM. The cell of claim 1.

3. The extracellular binding domain is a variable domain of an antibody that specifically binds to the brain-selective extracellular antigen. The cell according to claim 1 or 2, which is a main

4. the transmembrane protein is a binding-triggered transcriptional switch, and the cell is and (ii) a nucleic acid comprising a coding sequence encoding a protein, and (iii) a regulatory sequence, a sequence operably linked to the coding sequence, the sequence inhibiting the activity of the binding-triggered transcriptional switch A cell according to any one of claims 1 to 3, which is responsive to stimulation.

5. The binding-triggered transcriptional switch induces proteolysis upon binding to the brain-selective extracellular antigen. 1 or 2, which undergo cleavage to release a gene expression regulator that activates transcription of a therapeutic protein.

5. A system according to any one of claims 1 to 4, which is a polypeptide of any one of the above types.

6. The binding-triggered transcriptional switch is a SynNotch receptor, an A2 receptor, MESA, or is another receptor that undergoes binding-induced proteolytic cleavage.

7. the binding-triggered transcriptional switch (i) an extracellular domain that binds to the brain-selective extracellular antigen; (ii) a proteolytically cleavable sequence containing one or more proteolytic cleavage sites; column; and (iii) an intracellular domain; Including, (i) the binding of the extracellular domain to the brain-selective extracellular antigen is Inducing cleavage of the proteolytic cleavable sequence at the proteolytic cleavage site above and releasing the intracellular domain, wherein the released intracellular domain is a polypeptide of the regulatory sequence (ii).

7. The cell of claim 6, wherein the expression of the therapeutic protein of (i) is induced via

8. (i) the therapeutic protein, upon expression, is expressed by or on the surface of the cell; The cell according to any one of claims 4 to 7, wherein the protein is secreted into the host.

9. The therapeutic protein activates immune cells when expressed on the surface of the immune cells. or a protein that suppresses activation of the immune cells. The cells described.

10. 10. The method of claim 4, wherein the therapeutic protein is an antigen-specific therapeutic. cell.

11. The antigen-specific therapeutic agent is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). and wherein the binding of the transmembrane protein to the brain-selective extracellular antigen is The cell of claim 10, wherein expression is induced.

12. The antigen-specific therapeutic agent is an inhibitory chimeric antigen receptor (iCAR), and the transmembrane protein 11. The method of claim 10, wherein binding of a protein to the extracellular antigen induces expression of the iCAR. cell.

13. The cell of claim 10, wherein the antigen-specific therapeutic agent is another binding-triggered transcriptional switch. Cell.

14. 10. The method of claim 4, wherein the therapeutic protein of (i) is an intracellular protein when expressed.

8. A cell described in any one of the above 7.

15. The cell of any one of claims 1 to 8 or 10, wherein the antigen-specific therapeutic agent is an antibody. 。

16. 11. The method of claim 1, wherein the therapeutic protein is an inhibitory immunoreceptor. cells.

17. 11. Any of claims 1 to 10, wherein the therapeutic protein is a secreted peptide or an enzyme. The cell described in

18. The transmembrane protein is an inhibitory chimeric antigen receptor (iCAR), and the brain-selective cells Binding of an extracellular antigen to an iCAR suppresses activation of an immune cell expressing the iCAR.

11. The cell described in 10.

19. 19. The cell of any one of claims 1 to 18, which is an immune cell.

20. 20. The cell of any one of claims 1 to 19, which is a myeloid cell or a lymphoid cell.

21. The lymphocyte cells are T lymphocytes, B lymphocytes, or natural killer cells.

21. The cell according to item 20.

22. 19. The cell of any one of claims 1 to 18, which is not an immune cell.

23. 1. A method of treating a subject for a disease, comprising:

23. A method comprising administering to the subject a cell according to any one of claims 1 to 22.

24. 24. The method of claim 23, wherein the disease is a disease of the brain and / or central nervous tissue.

25. The subject is a patient with a tumor of the following type: medulloblastoma, diffuse midline glioma, ependymoma, craniopharyngioma, embryonal tumor, pineal bud tumor, tumor, brainstem glioma, choroid plexus carcinoma, germ cell tumor, pituitary adenoma, acoustic neuroma, meningioma, oligodendroma 25. The method of claim 24, wherein the patient has a glioma, a hemangioblastoma, a CNS lymphoma, or a non-GBM astrocytoma. The method described.

26. The subject is diagnosed with Alzheimer's disease, stroke, brain and spinal cord injury, brain cancer, HIV infection in the brain, Infection, ataxia, amyotrophic lateral sclerosis (ALS), Huntington's disease, and other conditions that affect the brain 2. The patient has a childhood congenital genetic error, Parkinson's disease, or multiple sclerosis that affects the immune system.

3. The method according to claim 3.

27. 24. The method of claim 23, wherein the disease is a cancer originating from a non-brain or non-CNS tissue that has metastasized to the brain. How to do it.

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