Methods and compositions for modulating the activity of dimerizer-regulated immunomodulatory complexes - Patents.com

Abstract

Methods and compositions are described for inducing multimerization of at least a first fusion protein and a second fusion protein to form a dimerizer-regulated immunomodulatory complex, thereby priming the dimerizer-regulated immunomodulatory complex for inducing signal transduction. The methods and compositions of the invention utilize a dosing schedule of a dimerizer that is designed to (i) maintain a predetermined blood trough concentration of the dimerizer, (ii) activate the immunomodulatory complex, (iii) reduce or avoid potential immunosuppressive effects of the dimerizer, (iv) reduce or avoid immune cell exhaustion, and / or (v) reduce or avoid side effects associated with activation of the immunomodulatory complex.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 288,468, filed December 10, 2021, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing Reference The sequence listing accompanying this application is provided in XML format rather than hard copy, and is incorporated herein by reference. The XML file containing this sequence listing is named S281-0037PCT.xml. This text file is 64KB, was created on December 9, 2022, and was submitted electronically via the Patent Center.

[0003] The present disclosure provides methods and compositions for inducing signal transduction by forming a dimerizer-regulated immunomodulatory complex by inducing multimerization of at least a first fusion protein and a second fusion protein, thereby priming the dimerizer-regulated immunomodulatory complex. The methods and compositions of the present invention utilize a dosing schedule of the dimerizer that is designed to one or more of: (i) maintain a predetermined blood trough concentration of the dimerizer; (ii) activate the immunomodulatory complex; (iii) reduce or avoid potential immunosuppressive effects of the dimerizer; (iv) reduce or avoid exhaustion of immune cells; and / or (v) reduce or avoid side effects associated with activation of the immunomodulatory complex. [Background technology]

[0004] Genetic engineering has made great advances in activating immune system cells to kill cancer cells or infected cells. For example, T cells have been engineered to express molecules that have an extracellular portion that binds to a specific target antigen and an intracellular portion that induces the action of the T cell when the extracellular portion binds to the target antigen. As an example, the extracellular portion can be designed to bind to a target antigen found on cancer cells or infected cells, and when the target antigen of the cancer cell or infected cell binds to the extracellular portion, the intracellular portion activates the T cell to destroy the cell that has bound to the extracellular portion. An example of such a molecule is the chimeric antigen receptor (CAR), which is used in adoptive cellular immunotherapy (June et al., Nat. Biotechnol. 30:611, 2012; Restifo et al., Nat. Rev. Immunol. 12:269, 2012). Antigen binding stimulates a signaling domain on the intracellular segment of the CAR, which transmits signals that trigger inflammatory and cytotoxic mechanisms. CAR-based adoptive cellular immunotherapy is being used to treat cancer patients with tumors that are refractory to conventional standard therapies (see Grupp et al., N. Engl. J. Med. 368:1509, 2013; Kalos et al., Sci. Transl. Med. 3:95ra73, 2011).

[0005] Despite the success of CAR T cell therapy, safety and efficacy issues are often present, including concerns about cytokine release syndrome and neurotoxicity (Mirzaei, et al., Frontiers in immunology. 2017, 8, 1850; and Srivastava and Riddell, J Immunol. 2018, 200(2):459-468), as well as myeloid cell hypoplasia and other toxicities due to the expression of some antigen targets (e.g., CD33) on normal tissues. Efficacy issues include relapse due to antigen escape and T cell exhaustion (Gardner et al., Blood, 2016, 127(20): p. 2406-10; Ruella and Maus, Comput Struct Biotechnol J. 2016, 14:357-362; Haneen et al., Haematologica. 2018, 103(5):e215-e218). Next-generation CAR T cell design should address these concerns by providing a platform for controllable T cell activation. Summary of the Invention [Means for solving the problem]

[0006] The present disclosure utilizes methods and compositions for in vivo priming of DARIC for inducing signal transduction by inducing multimerization of at least a first fusion protein and a second fusion protein to form a dimerizing agent regulated immunomodulatory complex (DARIC). More specifically, the present disclosure utilizes the use of rapamycin or an analog thereof to regulate the multimerization of a first fusion protein comprising a first multimerization domain (e.g., FKBP-rapamycin binding (FRB) domain or FK506 binding protein (FKBP) domain) and a second fusion protein comprising a second multimerization domain (e.g., FKBP-rapamycin binding (FRB) domain or FK506 binding protein (FKBP) domain) to form DARIC.

[0007] In certain embodiments, methods and compositions of the invention utilize a dosing schedule of a dimerizer designed to one or more of: (i) maintain a predetermined blood trough concentration of the dimerizer; (ii) activate an immunomodulatory complex; (iii) reduce or avoid potential immunosuppressive effects of the dimerizer; (iv) reduce or avoid immune cell exhaustion; and / or (v) reduce or avoid side effects associated with activation of an immunomodulatory complex.

[0008] In various embodiments, the blood trough concentration of the dimerizer is maintained within the range of 1-5 ng / mL. In certain embodiments, the blood trough concentration of the dimerizer is maintained within the range of 1.5-3 ng / mL. In various embodiments, the target blood trough concentration is 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, or 5 ng / mL. In certain embodiments, the target trough blood concentration is 2 ng / mL.

[0009] In various embodiments, the subject (subject's body surface area) is 1.5 m 2In certain embodiments, the subject is administered a dimerizer (e.g., rapamycin or an analog thereof) in a dose range of 0.75 to 4 mg. 2 and the dimerizer (e.g., rapamycin or an analog thereof) is administered to a subject in need thereof at a dose of 0.75 mg, 1.0 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3.0 mg, 3.25 mg, 3.5 mg, 3.75 mg or 4 mg.

[0010] In various embodiments, the target is 1.5 m 2 and the dimerizer is administered to a subject in need thereof at a dose of 0.75 mg / m 2 In certain embodiments, the subject is administered a dose of less than 1.5 m 2 and the dimerizer is administered to a subject in need thereof at a dose of 0.30 mg / m 2 , 0.40 mg / m 2 , 0.50 mg / m 2 , 0.60 mg / m 2 or 0.70 mg / m 2 is administered at a dose of

[0011] In various embodiments, the dimerizer (e.g., rapamycin or an analog thereof) is administered daily from at least 16 hours after the subject in need thereof has in vivo cells expressing DARIC. In certain embodiments, the dimerizer is administered from the first, second, third, fourth or fifth day after the subject in need thereof has in vivo cells expressing DARIC. In certain embodiments, the dimerizer is administered daily for 17, 18, 19, 20, 21, 22, 23 or 24 days after the first administration of the dimerizer. In certain embodiments, the dimerizer is administered daily from the second day to the 21st day after the subject in need thereof has in vivo cells expressing DARIC.

[0012] In certain embodiments, the first course of daily administration of the dimerizer is followed by a washout period during which the dimerizer is not administered to the subject.In certain examples, the washout period is 12 days, 13 days, 14 days, 15 days or 16 days.

[0013] In certain embodiments, the next course of daily administration of dimerizer is administered to the subject after the drug holiday.Usually, the next course is not started less than 30 days, less than 31 days, less than 32 days, less than 33 days, less than 34 days, less than 35 days, less than 36 days, less than 37 days, less than 38 days, less than 39 days, less than 40 days, less than 41 days, less than 42 days, less than 43 days, less than 44 days or less than 45 days after the subject has in vivo cells expressing DARIC.

[0014] In certain embodiments, a subject exhibiting persistent disease is administered a next course of dimerizer. In certain embodiments, the disease comprises leukemia. In certain embodiments, a subject in remission is administered a next course of dimerizer. In certain embodiments, a subject not experiencing grade 3 or higher toxicity is administered a next course of dimerizer. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 500 cells / μL is administered a next course of dimerizer. In certain embodiments, a next course of dimerizer is administered 42 days after the subject is infused with cells engineered to express DARIC. In certain embodiments, a next course of dimerizer is administered 14 days after the withdrawal of the previous dimerizer administration. [Brief description of the drawings]

[0015] Some of the drawings submitted in this application may be more easily understood in color, and applicants hereby contemplate color versions of these drawings as part of the original application and reserve the right to submit color images of such drawings in subsequent proceedings.

[0016] [Figure 1A-1C](Figure 1A) Lentiviral construct of Dimerizing Agent Regulated Immunomodulatory Complex (DARIC33) with anti-CD33 VHH antibody. In DARIC33, the antigen-binding and signaling functions of the chimeric antigen receptor (CAR) are separated. To improve the modularity of the design, the lentiviral construct shown in this figure incorporates a humanized camelid nanobody (VHH) that targets a novel epitope within the juxtamembrane domain of CD33. Thus, DARIC33 is a CD33 CAR with controllable and reversible activity that targets a juxtamembrane epitope within CD33. (Figure 1B) Priming mechanism of DARIC33 to induce signaling. By adding rapamycin (RAPA), the antigen-binding chain and the signaling chain form a heterodimer through the FK506-binding protein (FKBP) domain and the FKBP-rapamycin-binding (FRB) domain embedded in the cell membrane, which reconstitutes the immunomodulatory receptor complex and allows it to exert its action. In the presence of the target antigen, the signaling domain of 41bb and the signaling domain of CD3z activate the effector function of T cells. Upon removal of rapamycin, DARIC33 reverts to an inactive state, and DARIC33 can be reactivated by adding rapamycin again. (Figure 1C) Progression of MV4-11 tumors after treatment with SC-DARIC33 or control T cells. The antitumor activity of SC-DARIC33 in vivo was compared to control T cells with or without rapamycin. Tumor cell lines were engineered to express the luciferase gene, and the light flux (photons / second) over 36 days was graphed.

[0017] [Diagram 2]To comparatively evaluate the activity of DARIC33 in vitro, we used CD19 / CD33 dual-expressing Raji cells as tumor xenografts. After implantation of dual-expressing Raji cells into NOD Sicdγ (NSG) mice, the mice were treated with control mock T cells, CD19 CAR T cells, or DARIC33 T cells in the presence or absence of rapamycin.

[0018] [Diagram 3] Mice were treated with CD33+MV4-11 AML cells on day -7 and received SC-DARIC33 on day 1. Graph shows flux (photons / sec) over the indicated time periods following tumor injection followed by treatment with mock T cells, rapamycin only, 1x107 DARIC33 cells, or rapamycin and 1x107 DARIC33 cells.

[0019] [Figure 4A-4F]Activation of SC-DARIC33 is reversible. (Fig. 4A) Cytokine response of DARIC33 cells to antigen at various time points after washing out the rapamycin-containing medium. DARIC33 cells pre-cultured in rapamycin-free medium or replaced with rapamycin-containing medium were used as control. t1 / 2 was determined by curve fitting of a single-phase exponential decay. (Fig. 4B) 7 days after implantation of 1x106 MV4-11.ff / luc leukemia cells into NSG mice, 107 SC-DARIC33+ cells or the same number of untransduced (UTD) control cells were injected intravenously (IV). After T cell injection, mice were treated with 0.1 mg / kg rapamycin three times a week for the time periods indicated on the graph and observed. (Fig. 4C) Tumor progression monitored by bioluminescence in n=5 mice per group. Images taken during the rapamycin “withdrawal” period are shown (5th column, rows 4–13 and 6th column, rows 4–5). (Figure 4D) Quantification of tumor growth. Points represent measurements from individual mice, and best-fit tumor growth curves are shown. (Figure 4E) Tumor growth rates. Points represent growth rates in individual mice, box plots show mean and standard deviation, and asterisks indicate **p<0.01 using t-tests followed by Benjamini-Hochberg correction for multiple comparisons. (Figure 4F) Survival rates after treatment with various rapamycin dosing schedules following injection of DARIC33 or UTD cells. Uncorrected p-values ​​from the Mantel-Cox test (log-rank test) are shown.

[0020] [Figure 5A-5E]Modeling rapamycin responsiveness in SC-DARIC33 in vitro allows targeted dosing of rapamycin in vivo. (Figure 5A) Cytokine release upon stimulation of DARIC33 cells with MV4-11 AML cells in the presence of increasing concentrations of rapamycin in medium or whole blood is shown. IFNγ responsiveness was normalized for each donor, and a four-parameter logistic dose-response curve was used to determine the apparent EC50. (Figure 5B) Pharmacokinetics of rapamycin in mice is shown. The concentration of rapamycin in whole blood is shown at the top, and the timing of intraperitoneal (IP) injection of rapamycin is indicated by the bars at the bottom, after administration of various doses of rapamycin three times a week. The upper limit of quantification (ULOQ = 200 ng / mL) and the lower limit of quantification (LLOQ = 1 ng / mL) are also shown on the graph. (Figure 5C, Figure 5D) AML tumor progression in mice treated with DARIC33 and various dose schedules of rapamycin on days 0-18 after T cell injection. (Figure 5C) Schematic showing the experimental design. (Figure 5D) Quantification of tumor growth kinetics. Each point represents a bioluminescence measurement of an individual mouse (n=5-10 per group) and each line represents a tumor growth curve modeled using a linear mixed effects model. (Figure 5E) Tumor progression in control plots and Kaplan-Meier survival plots.

[0021] [Figure 6] Rapamycin concentrations in whole blood of treated mice after xenografting are shown.

[0022] [Figure 7]Figure 1 shows a comparison of rapamycin concentrations in whole blood and time course in pediatric patients. By integrating pediatric patients' pharmacokinetic (PK) data from population models, exposure to typical immunosuppressants, EC50 values ​​of rapamycin to activate SC-DARIC33 in vitro, and data on rapamycin's involvement in efficacy in mouse xenografts, the predicted dose of rapamycin to prime SC-DARIC33 to induce signaling in pediatric patients was estimated. The recommended starting daily dose of rapamycin is 0.50 mg / m2 (for patients with a body surface area of ​​1.5 m2 or less) or up to 4.0 mg (for patients with a body surface area of ​​more than 1.5 m2). In some embodiments, the starting daily dose is 0.75 mg or 1.5 mg depending on the age of the patient. Patients include pediatric patients, adult patients, and patients in the pediatric-adult transition (e.g., patients aged 18-28 years). Estimated trough concentrations of rapamycin in whole blood were found to be between 1.5 and 3 ng / mL in the majority of patients.

[0023] [Figure 8A-8B]A protocol for administering DARIC33 and rapamycin to lymphocyte-depleted patients is shown. The study design (Figure 8A) shows apheresis, SC-DARIC33 T cell production, and bridging therapy before lymphocyte depletion on day -2. On day 0, the subject is infused with SC-DARIC33 T cells. On day 2, rapamycin is administered and continues to be administered until day 21 after SC-DARIC33 T cell infusion. At this point, rapamycin administration can be suspended. On day 42, rapamycin administration can be resumed or kept suspended depending on the subject's response to treatment. Rapamycin can be administered in cycles. During the course of this administration protocol, bone marrow aspirates and / or biopsy specimens are taken at various times, such as days -5, 14, 28, and / or 42. (Figure 8B) An alternative protocol is shown. In this protocol, pediatric patients with relapsed or refractory AML receive lymphodepleting chemotherapy with fludarabine and cyclophosphamide followed by escalating doses of DARIC33 cells, with rapamycin administered on days 3-21.

[0024] [Figure 9] Several sirolimus dose levels (0.5 mg, 0.75 mg, 1.0 mg, 1.25 mg, and 1.5 mg) were simulated over a 19-21 day period on a once-daily dosing schedule.

[0025] [Figure 10] Exposure profiles were generated showing the geometric mean (solid line) and the 10th and 90th percentiles of expected sirolimus concentrations (shaded areas) for a starting dose of 1.5 mg administered daily. A starting dose of 1.5 mg administered daily will achieve target sirolimus concentrations of 1.5-3 ng / mL in most patients. Dose adjustments are also possible.

[0026] [Figure 11]Pharmacokinetic (PK) data from patients (body surface area >1.5 m2) following administration of rapamycin demonstrate dose-exposure relationships for peak and trough concentrations and indicate that dose can be adjusted to obtain target ranges. Rapamycin administration was initiated at a dose of 0.75 mg orally, and peak and trough concentrations were monitored after each dose using a clinical LC-MS / MS assay as described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Genetic engineering has made great advances in activating immune system cells to kill cancer cells or infected cells. For example, T cells have been engineered to express molecules that have an extracellular portion that binds to a specific target antigen and an intracellular portion that induces the action of the T cell when the extracellular portion binds to the target antigen. As an example, the extracellular portion can be designed to bind to a target antigen found on cancer cells or infected cells, and when the target antigen of the cancer cell or infected cell binds to the extracellular portion, the intracellular portion activates the T cell to destroy the cell that has bound to the extracellular portion. An example of such a molecule is the chimeric antigen receptor (CAR), which is used in adoptive cellular immunotherapy (June et al., Nat. Biotechnol. 30:611, 2012; Restifo et al., Nat. Rev. Immunol. 12:269, 2012). Antigen binding stimulates a signaling domain on the intracellular segment of the CAR, which transmits signals that trigger inflammatory and cytotoxic mechanisms. CAR-based adoptive cellular immunotherapy is being used to treat cancer patients (or subjects) whose tumors are refractory to conventional standard therapies (see Grupp et al., N. Engl. J. Med. 368:1509, 2013; Kalos et al., Sci. Transl. Med. 3:95ra73, 2011).

[0028] Despite the success of CAR T cell therapy, safety and efficacy issues are often present, including cytokine release syndrome and neurotoxicity (Mirzaei, et al., Frontiers in immunology. 2017, 8, 1850; and Srivastava and Riddell, J Immunol. 2018, 200(2):459-468), as well as concerns about myeloid cell hypoplasia, given that some antigen targets (e.g., CD33) are expressed on normal tissues. Efficacy issues include relapse due to antigen escape and T cell exhaustion (Gardner et al., Blood, 2016, 127(20): p. 2406-10; Ruella and Maus, Comput Struct Biotechnol J. 2016, 14:357-362; Haneen et al., Haematologica. 2018, 103(5):e215-e218).

[0029] The present disclosure addresses these concerns by providing a platform that allows control of immune cell activation. Controlling the activity of engineered immune-modulating molecules (e.g., engineered receptors (CARs)) provides benefits such as improved toxicity profiles and prevention of exhaustion. The ability to control the activity of engineered immune-modulating molecules is of further importance when targeting myeloid antigens that are of concern in bone marrow aplasia. The present disclosure provides methods and compositions for inducing multimerization of at least a first fusion protein and a second fusion protein to form a dimerizer-regulated immune-modulating complex (DARIC), thereby priming the DARIC to induce signaling. More specifically, the present disclosure relates to regulating the multimerization of a first fusion protein comprising an FKBP-rapamycin binding multimerization domain and a second fusion protein comprising an FK506 binding protein multimerization domain using rapamycin or an analog thereof to form a DARIC, and priming the DARIC to induce signaling.

[0030] In certain embodiments, a subject in need of treatment is administered a dose of cells expressing DARIC comprising a first fusion protein comprising a first multimerization domain and a second fusion protein comprising a second multimerization domain, followed by administration of a dimerization agent that binds to the first multimerization domain and the second multimerization domain.

[0031] In certain embodiments, a subject in need of treatment is administered a dose of cells expressing DARIC comprising a first fusion protein comprising an FKBP-rapamycin binding (FRB) multimerization domain and a second fusion protein comprising an FK506 binding protein (FKBP) multimerization domain, followed by administration of rapamycin or an analog thereof that binds to the first multimerization domain and the second multimerization domain.

[0032] In certain embodiments, a subject in need of treatment is administered a dose of cells expressing DARIC comprising a first fusion protein comprising an FK506 binding protein (FKBP) multimerization domain and a second fusion protein comprising an FKBP-rapamycin binding (FRB) multimerization domain, followed by administration of rapamycin or an analog thereof that binds to the first multimerization domain and the second multimerization domain.

[0033] In certain embodiments, a subject in need of treatment is administered a composition that edits the subject's cells to express DARIC comprising a first fusion protein comprising a first multimerization domain and a second fusion protein comprising a second multimerization domain, and then a dimerization agent that binds to the first multimerization domain and the second multimerization domain is administered.

[0034] In a particular embodiment, a subject in need of treatment is administered a composition that edits the subject's cells to express DARIC comprising a first fusion protein comprising an FKBP-rapamycin binding (FRB) multimerization domain and a second fusion protein comprising an FK506 binding protein (FKBP) multimerization domain, and then a dimerization agent that binds to the first multimerization domain and the second multimerization domain is administered.

[0035] In a particular embodiment, a subject in need of treatment is administered a composition that edits the subject's cells to express DARIC comprising a first fusion protein comprising an FK506 binding protein (FKBP) multimerization domain and a second fusion protein comprising an FKBP-rapamycin binding (FRB) multimerization domain, and then a dimerization agent that binds to the first multimerization domain and the second multimerization domain is administered.

[0036] In certain embodiments, DARIC is primed to induce signal transduction by administering a dimerizing agent. In certain embodiments, the dimerizing agent is rapamycin. In certain embodiments, the dimerizing agent is an analog of rapamycin.

[0037] In certain embodiments, DARIC comprises a first fusion protein comprising a first multimerization domain and a second fusion protein comprising a second multimerization domain. In certain embodiments, the first fusion protein comprises a binding domain, a first transmembrane domain, and a first multimerization domain, and the second fusion protein comprises a second multimerization domain, a second transmembrane domain, and an intracellular portion. In certain embodiments, the first fusion protein comprises a first transmembrane domain, a first multimerization domain, and a first intracellular signaling portion, and the second fusion protein comprises a second multimerization domain, a second transmembrane domain, and a second intracellular portion. In certain embodiments, when both fusion proteins are expressed, both multimerization domains are localized extracellularly. In certain embodiments, when both fusion proteins are expressed, both multimerization domains are localized intracellularly.

[0038] In certain embodiments, the first multimerization domain is a FRB multimerization domain and the second multimerization domain is a FKBP multimerization domain. In certain embodiments, the first multimerization domain is a FKBP multimerization domain and the second multimerization domain is a FRB multimerization domain. In certain embodiments, the binding domain comprises an anti-CD33 VHH antibody and / or an anti-CLL1 VHH antibody. In certain embodiments, the first transmembrane domain comprises a transmembrane domain of CD4 or a transmembrane domain of CD8α. In certain embodiments, the second transmembrane domain comprises a transmembrane domain of CD4 or a transmembrane domain of CD8α. In certain embodiments, the intracellular portion comprises an intracellular primary signaling domain of CD3ζ. In certain embodiments, the intracellular portion comprises a costimulatory domain of 4-1BB. In certain embodiments, the intracellular portion comprises a costimulatory domain of OX40 or TNFR2.

[0039] In certain embodiments, the object is 1.5 m 2 and the dimerizer is administered to a subject in need thereof at a dose of 0.75 mg, 1.0 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3.0 mg, 3.25 mg, 3.5 mg, 3.75 mg, or 4 mg. In certain embodiments, the subject is 2 and the dimerizer is administered to a subject in need thereof at a dose of 0.75 mg / m 2 In certain embodiments, the subject is administered 1.5 mg of 2 and the dimerizer is administered to a subject in need thereof at a dose of 0.30 mg / m 2 , 0.40 mg / m 2 , 0.50 mg / m 2 , 0.60 mg / m 2 or 0.70 mg / m 2 In certain embodiments, the subject is administered a dose of 1.5 2 and the dimerizer is administered to a subject in need thereof at a dose of 0.50 mg / m2 is administered at a dose of

[0040] In certain embodiments, the dimerizer is administered to a subject in need thereof at a dose that maintains a target trough blood concentration in the range of 1.5-3 ng / mL. In certain embodiments, the dimerizer is administered to a subject in need thereof at a dose that maintains a target trough blood concentration in the range of 1-4.5 ng / mL, 1-4 ng / mL, 1-3.5 ng / mL, 1-3 ng / mL, 1.5-5 ng / mL, 1.5-4.5 ng / mL, 1.5-4 ng / mL, 1.5-3.5 ng / mL, or 1.5-3 ng / mL. In certain embodiments, the dimerizer is administered to a subject in need thereof at a dose that maintains a target trough blood concentration of 2 ng / mL.

[0041] In certain embodiments, the object is 1.5 m 2 In certain embodiments, the subject has a 1.5 mg dose of rapamycin or an analog thereof administered to the subject in need thereof. 2 and the rapamycin or analogue is administered to a subject in need thereof at a dose of 0.50 mg / m 2 In certain embodiments, rapamycin or an analog thereof is administered to a subject in need thereof at a dose that maintains a target trough blood concentration of 2 ng / mL. In certain embodiments, rapamycin or an analog thereof is administered to a subject in need thereof at a dose that maintains a target trough blood concentration in the range of 1.5-3 ng / mL.

[0042] In certain embodiments, the dimerizer (e.g., rapamycin or an analog thereof) is administered daily from at least 16 hours after the subject in need thereof has in vivo cells expressing DARIC. In certain embodiments, the dimerizer is administered from the first, second, third, fourth or fifth day after the subject in need thereof has in vivo cells expressing DARIC. In certain embodiments, the dimerizer is administered daily for 17, 18, 19, 20, 21, 22, 23 or 24 days after the first administration of the dimerizer. In certain embodiments, the dimerizer is administered daily from the second to the 21st day after the subject in need thereof has in vivo cells expressing DARIC. In certain embodiments, the dimerizer is administered daily from the third to the 21st day after the subject in need thereof has in vivo cells expressing DARIC.

[0043] In certain embodiments, the drug holiday period after the first course of daily administration of dimerizer is not administered to the subject.In certain examples, the drug holiday period is 12 days, 13 days, 14 days, 15 days or 16 days.In certain examples, the drug holiday period is at least 12 days, at least 13 days, at least 14 days, at least 15 days or at least 16 days.In certain examples, the drug holiday period is 21 days or less.In certain examples, the drug holiday period is 28 days or less.In certain examples, the drug holiday period is 1 month or less, 2 months or less or 3 months or less.

[0044] In certain embodiments, the subject after the drug holiday is administered a next course of daily administration of dimerizer.Usually, the next course is not started less than 36 days, less than 37 days, less than 38 days, less than 39 days, less than 40 days, less than 41 days, less than 42 days, less than 43 days, less than 44 days or less than 45 days after the subject has in vivo cells expressing DARIC.

[0045] In certain embodiments, a subject exhibiting persistent disease is administered a next course of dimerizer. In certain embodiments, the disease comprises leukemia. In certain embodiments, a subject in remission is administered a next course of dimerizer. In certain embodiments, a subject not experiencing grade 3 or higher toxicity is administered a next course of dimerizer. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 500 cells / μL is administered a next course of dimerizer. In certain embodiments, a next course of dimerizer is administered 42 days after the subject is infused with cells engineered to express DARIC. In certain embodiments, a next course of dimerizer is administered 14 days after the withdrawal of the previous dimerizer administration.

[0046] In certain embodiments, a subject exhibiting persistent disease is administered a next course of rapamycin or an analog thereof. In certain embodiments, the disease comprises leukemia. In certain embodiments, a subject in remission is administered a next course of rapamycin or an analog thereof. In certain embodiments, a subject in the absence of grade 3 or higher toxicity is administered a next course of rapamycin or an analog thereof. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 500 cells / μL is administered a next course of dimerizer. In certain embodiments, a next course of rapamycin or an analog thereof is administered 42 days after the subject is infused with cells engineered to express DARIC. In certain embodiments, a next course of rapamycin or an analog thereof is administered 14 days after the previous dimerizer administration holiday.

[0047] Various aspects of the disclosure are described in more detail below with further options. Various aspects of the disclosure are described under the following headings: (i) Dimerizer-Controlled Immunomodulatory Complex (DARIC); (ii) Dimerizer; (iii) Fusion Protein; (iii-a) Multimerization Domain; (iii-b) Binding Domain; (iii-c) Intracellular Part; (iii-d) Transmembrane Domain; (iii-e) Linker; (iii-f) Tags and Selectable Markers; (iv) Polynucleotides; (v) Recombinant Cells; (vi) Formulations; (vii) Methods of Treatment; (viii) Exemplary Embodiments; (ix) Examples; (x) Supporting Sequences of the Disclosure; and (xi) Conclusion. These headings are provided for organizational purposes only and are not intended to limit the scope or interpretation of the disclosure.

[0048] (i) Dimerizer-regulated immunomodulatory complex (DARIC)

[0049] The present disclosure provides methods and compositions for priming dimerizer-regulated immunomodulatory complex (DARIC) to induce signal transduction by multimerizing the components of DARIC. In certain embodiments, DARIC comprises a first fusion protein comprising a first multimerization domain and a second fusion protein comprising a second multimerization domain and an intracellular portion, and a dimerizer binds to the first multimerization domain and the second multimerization domain, thereby multimerizing the first fusion protein and the second fusion protein to form a DARIC capable of activation (i.e., priming DARIC to induce signal transduction).

[0050] The temporal control achieved through the multimerization mechanism described herein only primes the mechanism for inducing signaling. Unlike conventional CARs and other recombinant receptors that are already primed for activation, DARIC requires administration of a dimerization agent to prime for activation. In certain embodiments, the multimerization induced by the dimerization agent reconstitutes the receptor with enhanced signaling, but downstream signaling is not activated because there is no assembly of the intracellular signaling moiety. Thus, spatial control is achieved based on the presence or absence of a target recognized by the binding domain of one of the fusion proteins. Because the binding domain of the fusion protein is secreted outside the cell (or added externally), it accumulates only at the site where the target is present, and the cell is activated only when both the target (e.g., a cell surface antigen) and the dimerization agent are present. In certain embodiments, when both fusion proteins are expressed, both multimerization domains are localized outside the cell. In certain embodiments, when both fusion proteins are expressed, both multimerization domains are localized inside the cell.

[0051] In certain embodiments, the terms "priming to induce signal transduction," "priming for induction of signal transduction," and "priming for induction of signal transduction," as well as similar terms (e.g., "priming DARIC for induction of signal transduction") refer to reconstituting components of DARIC to functionally link a fusion protein containing a binding domain to another fusion protein containing an intracellular portion, such that activation or downstream signal transduction occurs in a recombinant cell when the formed DARIC binds to a target antigen. In some places herein, primed DARIC to induce signal transduction is described as "activated" or "active." In some embodiments, DARIC does not contain a binding domain. In some embodiments, DARIC that does not contain a binding domain contains an intracellular portion in each of the first fusion protein and the second fusion protein, and signal transduction occurs when the first fusion protein and the second fusion protein multimerize.

[0052] As used herein, "recombinant" refers to a cell, microorganism, organism, nucleic acid molecule or vector that has been genetically modified or engineered by the introduction of a heterologous nucleic acid molecule, or to a cell that has been modified such that expression of an endogenous nucleic acid molecule or endogenous gene can be controlled.

[0053] As used herein, a "heterologous" nucleic acid molecule, "heterologous" construct or "heterologous" sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule sequence that does not naturally occur in the cell in which it is expressed, or a nucleic acid molecule or portion thereof that naturally occurs in the host cell into which an alteration or mutation is introduced, or a nucleic acid molecule whose expression under similar conditions as compared to native expression is altered.

[0054] In certain embodiments, DARIC may be a dimer, trimer or higher multimer formed by at least two proteins, at least one of which has a binding domain specific to a target, and / or at least one of which has an intracellular portion, such as an intracellular signaling domain, a co-stimulatory domain, or a co-receptor domain. When at least two of the proteins are associated with a related protein by a dimerizing agent, DARIC is primed to induce signal transduction. In certain embodiments, DARIC comprises at least one intracellular portion responsible for the propagation or transmission of intracellular signals. In another embodiment, DARIC comprises a binding domain.

[0055] (ii) Dimerizing Agent

[0056] A "dimerizer" refers to any molecule that can bind to a first multimerization domain and a second multimerization domain, bridging the two multimerization domains together, thereby bridging the moieties attached to each multimerization domain.

[0057] In certain embodiments, the dimerizer is rapamycin (sold under the trade name Rapamune® (Amgen, Thousand Oaks, Calif.) and also known as sirolimus). Analogs of rapamycin (rapalogs) may also be used. Examples of rapamycin analogs include those disclosed in U.S. Pat. No. 6,649,595, which describes the structures of various rapalogs. In certain embodiments, the dimerizer is a rapalog that has a significantly reduced immunosuppressive effect compared to rapamycin. "Substantially reduced immunosuppressive effect" refers to a rapalog that has an immunosuppressive effect that is at least 0.1-fold to 0.005-fold less than that observed or expected for an equimolar amount of rapamycin, as measured in clinical trials to determine immunosuppressive activity in humans, appropriate in vitro surrogates (e.g., inhibition of T-cell proliferation), or in vivo surrogates. Alternatively, "sub ... EC20 value less than that observed for rapamycin in an in vitro assay. 50 EC at least 10-250 times higher than the value 50 Other exemplary rapalogs include everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, rimiducid (AP1903), AP20187 (also known as 2,2'-[[2-[(dimethylamino)methyl]-1,3-propanediyl]bis[imino(2-oxo-2,1-ethanediyl)oxy-3,1-phenylene[(1R)-3-(3,4-dimethoxyphenyl)propylidene]]] ester; (2S,2'S)-1-[(2S)-1-oxo-2-(3,4,5-trimethoxyphenyl)butyl]-2-piperidinecarboxylic acid; B / B Homodimerizer), AP21967 (also known as C16-(S)-7-methylindolerapamycin; C16-AiRap), and BPC015.

[0058] In certain embodiments, the dimerizing agent includes rapamycin (sirolimus) or a rapalog thereof, coumermycin or a derivative thereof, gibberellin or a derivative thereof, abscisic acid (ABA) or a derivative thereof, methotrexate or a derivative thereof, cyclosporin A or a derivative thereof, FKCsA or a derivative thereof, trimethoprim (Tmp)-FKBP synthetic ligand (SLF) or a derivative thereof, or any combination thereof.

[0059] In another specific embodiment, the anti-dimerization agent inhibits the binding of at least two first fusion proteins by the dimerization agent. For example, cyclosporine or FK506 can be used as the anti-dimerization agent, and the dimerization agent can be titrated to bind only one of the multimerization domains, thereby stopping signaling. In a specific embodiment, the anti-dimerization agent (e.g., cyclosporine or FK506) is an immunosuppressant. For example, an immunosuppressive anti-dimerization agent can be used to prevent or minimize the function of the fusion protein of the present disclosure, while simultaneously preventing or preventing clinically undesirable or pathological inflammatory responses.

[0060] (iii) Fusion protein

[0061] "Fusion protein" refers to a protein that contains multiple polypeptide moieties (e.g., fusion polypeptides) derived from one or more parent proteins or parent polypeptides and does not naturally occur in a host cell. A fusion protein contains two or more naturally occurring amino acid sequences linked in a manner that does not occur in nature. For example, a fusion protein may contain two or more moieties from the same protein linked in a manner not normally found in cells, or may contain multiple moieties from two, three, four, five or more proteins linked in a manner not normally found in cells. A fusion protein can be encoded by a nucleic acid molecule in which a nucleotide sequence encoding one protein or portion thereof is added in frame to a nucleic acid molecule encoding another protein or portions thereof, and the nucleotide sequences and nucleic acid molecules may be separated by nucleotides encoding linkers, spacers or linking amino acids. In certain embodiments, a nucleic acid molecule encoding a fusion protein is introduced into a host cell and expressed.

[0062] As used herein, "host" refers to a cell (e.g., a T cell) or microorganism that may be genetically engineered with an exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., a binding or signaling portion of DARIC). In certain embodiments, the host cell may already have another genetic modification that confers a desired characteristic related to the biosynthesis of a fusion protein or a desired characteristic not related to the biosynthesis of a fusion protein (e.g., deletion, modification, or truncation of a TCR, checkpoint protein, or other gene; increased expression of a costimulatory factor, etc.), or may be engineered to include such a genetic modification. In certain embodiments, the host cell is a human T cell, or a human T cell in which the TCR alpha chain or TCR beta chain, or both, have been knocked out with a site-specific nuclease (e.g., LAGLIDADG homing endonuclease (LHE)).

[0063] In certain embodiments, the DARIC comprises a first fusion protein comprising a first multimerization domain and a second fusion protein comprising a second multimerization domain. In certain embodiments, the first fusion protein comprises a binding domain, a first transmembrane domain, and a first multimerization domain, and the second fusion protein comprises a second multimerization domain, a second transmembrane domain, and an intracellular portion. In certain embodiments, the first fusion protein and / or the second fusion protein may comprise a linker, a tag, or a selection marker.

[0064] The fusion protein to which the fusion protein is fused may contain two or more multimerization domains, including a multimerization domain that promotes the formation of homodimers in the presence of a homobivalent dimerization agent. In such an embodiment, administration of the dimerization agent promotes some basal signaling under conditions in which no extracellular target is bound, for example, promoting cell proliferation in vitro or in vivo before activation. It is known that T cells are proliferated when activated at a low level, but increased multimerization (which may occur due to high density of antigen on target cells and heterodimerization of the fusion protein by the dimerization agent) induces activation of cytotoxicity.

[0065] In certain embodiments, the fusion protein may have multiple binding domains. For example, the recombinant cell can express a third fusion protein comprising a binding domain and a second multimerization domain, the third fusion protein may comprise a transmembrane domain or may comprise a transmembrane domain with an intracellular portion, and the third fusion protein is localized extracellularly when expressed. In related embodiments, the fusion protein comprises one binding domain, two binding domains, three binding domains, or four binding domains, the one binding domain, two binding domains, three binding domains, or four binding domains being specific for one target or up to four different targets. In certain embodiments, DARIC comprises binding domains that bind to CD33 and CLL1.

[0066] A fusion protein may contain one or more polypeptide domains or segments, including signal peptides, cell-penetrating peptide domains (CPPs), binding domains, signaling domains, and the like, as well as epitope tags (e.g., maltose-binding protein ("MBP"), glutathione-S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G, HA, and the like), polypeptide linkers, and polypeptide cleavage signals. Fusion proteins and fusion polypeptides are usually linked in a C-terminal to N-terminal direction, but may also be linked C-terminal to C-terminal, N-terminal to N-terminal, or N-terminal to C-terminal direction. In certain embodiments, the polypeptides contained in the fusion protein may be in any order. Fusion polypeptides or fusion proteins may further contain conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and cross-species homologs, so long as the desired activity of the fusion polypeptide is retained. Fusion polypeptides may be produced by chemical synthesis or by chemical conjugation between two moieties, and generally may be prepared using other standard techniques. The DNA sequences ligated together, such as the fusion polypeptide, are operably linked to suitable transcriptional or translational regulatory elements, as otherwise disclosed herein.

[0067] Fusion proteins and fusion polypeptides may include one or more linkers that can be used to link one or more polypeptides or domains within a polypeptide. Peptide linker sequences may be used to separate two or more polypeptide moieties from each other by a sufficient distance to ensure that each polypeptide folds into the appropriate secondary and tertiary structures to enable each polypeptide domain to perform its desired function. Such peptide linker sequences are incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences may be selected based on factors such as (1) being able to adopt a flexible extended conformation, (2) not adopting secondary structures that may interact with the functional epitopes of the first and second polypeptides, and (3) not having hydrophobic or charged residues that may react with the functional epitopes of the first and second polypeptides. In certain embodiments, the peptide linker sequence preferably includes glycine, asparagine and serine residues. Additionally, other near-neutral amino acids such as Thr and Ala may be used in the linker sequence. Amino acid sequences that may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258-8262, 1986; U.S. Pat. Nos. 4,935,233 and 4,751,180. If a particular fusion polypeptide segment contains a non-essential N-terminal amino acid region that can be used to separate functional domains to prevent steric hindrance, a linker sequence is not required. In certain embodiments, flexible amino acid subsequences that are typically synthesized as part of a recombinant fusion protein are preferred as linkers. Linker polypeptides may be 1-200 amino acids long, 1-100 amino acids long, or 1-50 amino acids long, or any integer value of amino acid length between these ranges.

[0068] Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites, such as protease cleavage sites; nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), viral oligopeptide self-cleavage sites (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0069] Suitable protease cleavage sites and autocleaving peptides are known to those of skill in the art (see, e.g., Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include the cleavage site of the potyvirus NIa protease (e.g., the cleavage site of the tobacco etch virus protease), the cleavage site of the potyvirus HC protease, the cleavage site of the potyvirus P1 (P35) protease, the cleavage site of the byovirus NIa protease, the cleavage site of the byovirus RNA2-encoded protease, the cleavage site of the aphthovirus L protease, and the cleavage site of the enterovirus 2A protease. , the cleavage site of the rhinovirus 2A protease, the cleavage site of the picornavirus 3C protease, the cleavage site of the comovirus 24K protease, the cleavage site of the nepovirus 24K protease, the cleavage site of the RTSV (Rice Tungro Spherical Virus) 3C-like protease, the cleavage site of the PYVF (Parsnip Yellow Fleck Virus) 3C-like protease, the cleavage site of heparin, the cleavage site of thrombin, the cleavage site of factor Xa, and the cleavage site of enterokinase.

[0070] In certain embodiments, the polypeptide cleavage signal is a viral autocleaving peptide or a ribosomal skipping sequence.

[0071] Specific examples of ribosomal skipping sequences include a 2A site domain or sequence thereof, or a 2A-like site domain or sequence thereof (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In certain embodiments, the viral 2A peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

[0072] In one embodiment, the viral 2A peptide is selected from the group including a 2A peptide of foot and mouth disease virus (FMDV), a 2A peptide of equine rhinitis A virus (ERAV), a 2A peptide of Thosea asigna virus (TaV), a 2A peptide of porcine teschovirus 1 (PTV-1), a 2A peptide of a theilovirus, and a 2A peptide of encephalomyocarditis virus.

[0073] (iii-a) Multimerization domain

[0074] As used herein, a "multimerization domain" refers to a molecule that selectively interacts or associates with another molecule, either directly or via a dimerizer, and where the interaction between different types of multimerization domains significantly contributes to or efficiently promotes the formation of multimers (i.e., the formation of dimers, trimers, or complexes consisting of multiple moieties, which may be homodimers, heterodimers, homotrimers, heterotrimers, homomultimers, or heteromultimers).

[0075] In certain embodiments, the multimerization domains can be associated by using a dimerization agent. In certain embodiments, the dimerization agent is rapamycin or an analog thereof. For example, the first and second multimerization domains are a pair selected from FK506 binding protein (FKBP) multimerization domain and FKBP-rapamycin binding (FRB) multimerization domain, or variants thereof. The FRB domain is a polypeptide region (protein "domain") that can form a ternary complex with the FKBP protein and rapamycin or its rapalog. The FRB domain is present in a variety of natural proteins, including, for example, the mTOR protein (also referred to in the literature as FRAP, RAPT1 or RAFT) from humans and other species; yeast proteins such as Tor1 and Tor2; and FRAP homologs from the Candida genus. Information regarding the nucleotide sequences, cloning and other aspects of these proteins is known in the art. For example, the accession number for the protein sequence of human mTOR is GenBank Accession No. L34075.1 (Brown et al., Nature 369:756, 1994).

[0076] In certain embodiments, when the first fusion protein and the second fusion protein are expressed, the first multimerization domain and the second multimerization domain are localized extracellularly. In certain embodiments, when the first fusion protein and the second fusion protein are expressed, the first multimerization domain and the second multimerization domain are localized intracellularly.

[0077] In certain embodiments, the term "FKBP-rapamycin binding (FRB) multimerization domain" refers to an FRB polypeptide. The FRB domain used in the fusion protein of the present disclosure typically contains at least 85 to 100 amino acid residues. In certain embodiments, the FRB amino acid sequence used in the fusion protein of the present disclosure contains an amino acid sequence consisting of 93 residues from Ile at position 2021 to Lys at position 2113, and has a T2098L mutation (corresponding to T82L in the amino acid sequence of the 93-residue long FRB polypeptide) compared to GenBank Accession No. L34075.1. The FRB domain used in the fusion protein of the present disclosure can bind to a complex consisting of an FKBP protein and a rapamycin or analog thereof of the present disclosure bound thereto. In certain embodiments, the peptide sequence of the FRB domain includes (a) a naturally occurring peptide sequence spanning at least the aforementioned 93 amino acid region of human mTOR or the corresponding region of a homologous protein; (b) a variant of a naturally occurring FRB peptide in which up to 10 amino acids, 1-5 amino acids, or 1-3 amino acids, or depending on the embodiment, only one amino acid, is deleted, inserted, or substituted; or (c) a peptide encoded by a nucleic acid molecule that can selectively hybridize to a DNA molecule encoding a naturally occurring FRB domain, or a peptide encoded by a DNA sequence that can selectively hybridize to a DNA molecule encoding a naturally occurring FRB domain due to the degeneracy of the genetic code. In certain embodiments, the FRB polypeptide binds to an FKBP polypeptide via a cross-linking agent to form a ternary complex.

[0078] In certain embodiments, ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDL L QAWDLYYHVFRRISK (SEQ ID NO:55) In a particular embodiment, the FRB sequence shown in ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDL T QAWDLYYHVFRRISK (SEQ ID NO:56) The sequence shown in is utilized.

[0079] In certain embodiments, "FK506 binding protein (FKBP) multimerization domain" refers to an FKBP polypeptide. FKBP is a cytosolic receptor for macrolide antibiotics such as FK506, FK520, and rapamycin, and is highly conserved among species. For purposes of this disclosure, FKBP is a protein or protein domain that can bind to rapamycin or its analogs to form a ternary complex with an FRB-containing protein or an FRB-containing fusion protein. An FKBP domain is also referred to as a "rapamycin binding domain." Information regarding the nucleotide sequences, cloning, and other aspects of various FKBP species are known in the art (see, for example, Staendart et al., Nature 346:671, 1990 (human FKBP12); Kay, Biochem. J. 314:361, 1996). Homologous FKBP proteins from other mammalian species, yeast, and other organisms are also known in the art and may be used in the fusion proteins disclosed herein. The size of the FKBP domain used in this disclosure varies depending on which FKBP protein is used. The FKBP domain of the fusion protein of this disclosure can bind to rapamycin or an analog thereof and participate in the formation of a ternary complex with the FRB-containing protein (the formation of this complex may be determined by any means of directly or indirectly detecting such binding).

[0080] Peptide sequences of the FKBP domain of the FKBP fusion proteins of the present disclosure include (a) a naturally occurring FKBP peptide sequence, preferably a naturally occurring FKBP peptide sequence from the human FKBP12 protein (GenBank Accession No. AAA58476.1) or a peptide sequence derived from this naturally occurring FKBP peptide sequence, from another human FKBP, from a mouse or other mammalian FKBP, or from an FKBP of another animal, yeast or fungus; (b) a variant of a naturally occurring FKBP sequence in which up to 10 amino acids, 1-5 amino acids or 1-3 amino acids, or depending on the embodiment only a single amino acid, of a naturally occurring FKBP peptide have been deleted, inserted or substituted; or (c) a peptide sequence encoded by a nucleic acid molecule capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FKBP, or a peptide sequence encoded by a DNA sequence capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FKBP due to the degeneracy of the genetic code. In certain embodiments, the FKBP polypeptide is an FKBP12 polypeptide or an FKBP12 polypeptide comprising an F36V mutation. In certain embodiments, the FKBP polypeptide contemplated herein binds to an FRB polypeptide via a cross-linking agent to form a ternary complex.

[0081] In certain embodiments, GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKK F DSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:57) In a particular embodiment, the sequence shown in GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKK V DSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO:58) The sequence shown in is utilized.

[0082] A "cross-linking agent" refers to a molecule that associates with and is positioned between two or more multimerization domains. In certain embodiments, the multimerization domains contribute significantly to or efficiently promote the formation of a polypeptide complex only in the presence of the cross-linking agent. In certain embodiments, the multimerization domains do not contribute to or efficiently promote the formation of a polypeptide complex in the absence of the cross-linking agent. Specific examples of cross-linking agents suitable for use in certain embodiments contemplated herein include AP21967, rapamycin (sirolimus) or its rapalogs, coumermycin or its derivatives, gibberellin or its derivatives, abscisic acid (ABA) or its derivatives, methotrexate or its derivatives, cyclosporin A or its derivatives, FKCsA or its derivatives, synthetic ligand of trimethoprim (Tmp)-FKBP (SLF) or its derivatives, or any combination thereof.

[0083] Other multimerization domain pairs include FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1, or GIB1 and GAI, or variants thereof.

[0084] In certain embodiments, the first multimerization domain is an FRB multimerization domain, and the second multimerization domain is an FKBP multimerization domain. In certain embodiments, the first multimerization domain is an FKBP multimerization domain, and the second multimerization domain is an FRB multimerization domain. In certain embodiments, the dimerization agent / crosslinking agent is rapamycin and / or its analogue.

[0085] In certain embodiments, the first multimerization domain and the second multimerization domain are the same or different.

[0086] (iii-b) Binding domain

[0087] "Binding domain" refers to a protein, polypeptide, oligopeptide, peptide or other molecule that has the ability to specifically recognize and bind to a target (e.g., CD19, CD20, CD33, CLL1 and / or other target antigens).

[0088] Binding domains of fusion proteins useful in the present disclosure include binding domains known in the art or described herein, or binding domains made by various methods known in the art (see, e.g., U.S. Pat. Nos. 6,291,161 and 6,291,158). For example, binding domains of fusion proteins may be identified by screening Fab phage libraries for Fab fragments that specifically bind to the target of interest (see, Hoet et al., Nat. Biotechnol. 23:344, 2005). Additionally, binding domains of fusion proteins may be identified by screening Fab phage libraries for Fab fragments that specifically bind to the target of interest (see, e.g., mice, HuMAb mice, TC mice, etc.), for example, in a convenient system using the target antigen as an immunogen (e.g., mice, HuMAb mice, TC mice, etc.). TM Anti-target antibodies having target-specific binding domains can be produced using conventional methods for producing hybridomas in various animal models (eg, mouse, KM Mouse®, llama, sheep, chicken, rat, hamster, rabbit, etc.).

[0089] Further binding domains can be derived from target-specific antibody variable domains from various species, such as human, rodent, avian, ovine (which may be in the form of an antibody, sFv, scFv, Fab, soluble VH domain antibody or domain antibody). Additionally, binding domains may be derived from variable domains of antibodies from other species, such as camelids (camel, dromedary or llama) (Ghahroudi et al., FEBS Letters 414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; Hamers-Casterman et al., Nature 363:446, 1993; and Nguyen et al., J. Mol. Biol. 275:413, 1998), nurse shark (Roux et al., Proc. Nat'l. Acad. Sci. (USA) 95:11804, 1998), spotted ratfish (Nguyen et al., Immunogenetics 54:39, 2002), or lamprey (Herrin et al., Proc. Nat'l. Acad. Sci. (USA) 105:2040, 2008 and Alder et al., Nature Immunol. 9:319, 2008). These antibodies appear to be capable of forming antigen-binding regions using only the heavy chain variable regions, i.e., these functional antibodies are homodimers of only heavy chains (referred to as "heavy chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006 and Barthelemy et al., J. Biol. Chem. 283:3639, 2008).

[0090] Additionally, target-specific binding domains can be derived from sequences encoding random peptide libraries or recombinant variants of amino acids in the loop regions of other non-antibody scaffolds, such as, for example, fibrinogen domains (see, e.g., Weisel et al. (1985) Science 230:1388), Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498), ankyrin repeat proteins (also known as DARPins; Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004), fibronectin binding domains (also known as adnectins or monobodies; Richards et al., J. Mol. Biol. 326:1475, 2003; Parker et al., Protein Eng. Des. Sel. 18:435, 2005 and Hackel et al., J. Mol. Biol. 381:1238, 2008), cysteine ​​knot miniproteins (Vita et al., Proc. Nat'l. Acad. Sci. (USA) 92:6404, 1995; Martin et al., Nat. Biotechnol. 21:71, 2002 and Huang et al., Structure 13:755, 2005), tetratricopeptide repeat domains (Main et al., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008), leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471, 2003), anticalins (Skerra, FEBS J. 275:2677, 2008), lipocalin domains (see, e.g., PCT Publication WO2006 / 095164, Beste et al., Proc. Nat'l. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et al., Proc. Nat'l. Acad.Sci. (USA) 106:8198, 2009), armadillo repeat proteins (ArmRPs; Varadamsetty et al., J. Mol. Biol. 424:68, 2012), diabodies (Manzke et al., Int. J. Cancer 82:700, 1999), repebodies (Lee et al., Proc. Nat'l. Acad. Sci. USA 109: 3299, 2012), minibodies (Hu et al., Cancer Res. 56:3055, 1996), cyclotides (Craik et al., J. Mol. Biol. 294:1327, 1999), V-like domains (see, e.g., U.S. Patent Application Publication No. 2007 / 0065431), C-type lectin regions (Zelensky and Gready, FEBS J. 272:6179, 2005; Beavil et al.I, Proc. Nat'l. Acad. Sci. (USA) 89:753, 1992 and Sato et al., Proc. Nat'l. Acad. Sci. (USA) 100:7779, 2003), mAbs. 2 or Fcab TM (see, e.g., PCT Publications WO2007 / 098934; WO2006 / 072620), and others (Nord et al., Protein Eng. 8:601, 1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Eur. J. Biochem. 268:4269, 2001; and Binz et al. (2005) Nat. Biotechnol. 23:1257, 2005).

[0091] In further embodiments, the binding domain is specific for a target that is an antigen associated with cancer (e.g., solid malignancies, hematological malignancies), inflammatory disease, autoimmune disease, or graft-versus-host disease. Exemplary target antigens include alpha folate receptor (FRα), alpha vβ6 integrin, ADGRE2, BACE2, B cell maturation antigen (BCMA), B7-H3 (CD276), B7-H4, B7-H6, CA19.9, carbonic anhydrase IX (CAIX), CCR1, CD7, CD16, CD19, CD20, CD22, CD30, CD33, CD37, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD133, CD138, CD171, CD244, carcinoembryonic antigen (CEA), C-type lectin-like molecule 1 (CLL1), CD2 subset 1 (CS-1), CLDN6, cMET, chondroitin sulfate proteoglycan 4 (CSPG4), CLDN18.2, cutaneous T-cell lymphoma-associated antigen 1 (CTAGE1), DLL3, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), EGFR806, epithelial glycoprotein 2 (EGP2), epithelial glycoprotein 40 (EGP40), EPHB2, ERBB4, epithelial cell adhesion molecule (EPCAM), ephrin type A receptor 2 (EPHA2), fibroblast activation protein (FAP), Fc receptor like 5 (FCRL5), fetal acetylcholinesterase receptor (AchR), FLT3, FN, FN-EDB, FRβ, ganglioside G2 (GD2), ganglioside G3 (GD3), glypican 3 (GPC3), EGFR family (including ErbB2 (HER2)), HER2p95, EGFRv3, IL-10Rα, IL-13Rα2, κ, cancer / testis antigen 2 (LAGE-1A), K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, λ, Lewis-Y (LeY), L1 cell adhesion molecule (L1-CAM), LILRB2, LY6G6GD, melanoma antigen recognized by T cells 1 (MelanA or MART1), mesothelin (MSLN), MMP10, MUC1, MUC16, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), neural cell adhesion molecule (NCAM), prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), transmembrane activator and CAML interactor (TACI), tumor endothelial marker 1 (TEM1 / CD248), tumor These include endothelial marker 7-related (TEM7R), TIM3, trophoblast glycoprotein G (TPBG), UL16-binding protein (ULBP) 1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and vascular endothelial growth factor receptor 2 (VEGFR2).

[0092] In some embodiments, the one or more antigen binding domains bind to CD19, CD20, CD22, CD33, CD79A, CD79B, B7H3, Muc16, Her2, EGFR, FN-EDB, CLDN18.2, DLL3, FLT3, CLL1, CD123 or BCMA. In some embodiments, the one or more antigen binding domains bind to CD33, CLL1, CD19, CD20, CD22, CD79A, CD79B or BCMA. In some embodiments, the one or more antigen binding domains bind to CD33 and / or CLL1. In certain embodiments, the binding domain is an anti-CD33 VHH antibody. In certain embodiments, the binding domain is an anti-CLL1 VHH antibody.

[0093] In various embodiments, the one or more antigen binding domains are selected from the group consisting of alpha-fetoprotein (AFP), ASCL2, B melanoma antigen (BAGE) family members, Brother of the regulator of imprinted sites (BORIS), cancer testis antigen, cancer testis antigen 83 (CT-83), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), cytomegalovirus (CMV) antigen, melanoma cytotoxic T cell (CTL) recognition antigen (CAMEL), Epstein-Barr virus (EBV) antigen, EPHB2, G antigen 1 (GAGE-1), GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, glycoprotein 100 (GP100), hepatitis B virus (HBV) antigen, hepatitis C virus (HCV) nonstructural protein 3 (NS3), human papillomavirus (HPV)-E6, HPV-E7, human telomerase reverse transcriptase (hTERT), IGF2BP3 / A3, IGF2BP1, K-Ras, K-Ras G12C, K-Ras G12D, K-Ras G12V, latent membrane protein 2 (LMP2), LY6G6D, melanoma antigen family A1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, melanoma antigen recognized by T cells (MART-1), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), New York esophageal squamous cell carcinoma-1 (NYESO-1), P53, P antigen (PAGE) family member, PAP, PIK3CA, PIK3CA H1047R, placenta specific protein 1 (PLAC1), selectively expressed antigen in melanoma (PRAME), prostate specific antigen PSA, survivin, synovial sarcoma X1 (SSX1), synovial sarcoma X2 (SSX2), synovial sarcoma X3 (SSX3), synovial sarcoma X4 (SSX4), synovial sarcoma X5 (SSX5), synovial sarcoma X8 (SSX8), thyroglobulin, TP53The antibody binds to a target polypeptide derived from a protein selected from the group including R175H, tyrosinase, tyrosinase-related protein (TRP) 1, TRP2, UBD, Wilms tumor protein (WT-1), Wnt10A, X antigen family member 1 (XAGE1), and X antigen family member 2 (XAGE2).

[0094] (iii-c) Intracellular part

[0095] The intracellular portion of the fusion protein comprises one or more intracellular signaling domains, costimulatory domains, or co-receptor domains responsible for the transmission or propagation of intracellular signals. In certain embodiments, the intracellular portion generates a signal that promotes immune effector functions of the fusion protein-modified cell. In certain embodiments, the intracellular portion generates a stimulatory and / or costimulatory signal upon binding of a ligand. Examples of immune effector functions include cytolytic and helper activities, including secretion of cytokines. Signals generated by the intracellular portion can also induce proliferation, activation, differentiation, etc. of immune cells.

[0096] A "signaling domain" refers to a functional portion of a protein that regulates cellular activity by transmitting intracellular information through a defined signaling pathway, either by generating second messengers or by functioning as an effector that responds to second messengers. "Stimulation" refers to the primary response induced by the binding of a stimulatory molecule (e.g., a fusion protein) or a costimulatory molecule with its cognate ligand, which results in a signaling event, such as signal transduction through the appropriate signaling domain of a fusion protein. Stimulation can result in altered expression of a particular molecule.

[0097] The intracellular signaling domain may comprise the entire intracellular region of the signaling domain or a functional fragment thereof. In certain embodiments, the intracellular signaling domain may comprise an intracellular primary signaling domain. In certain embodiments, the intracellular primary signaling domain comprises an intracellular signaling domain derived from a molecule responsible for primary or antigen-dependent stimulation. In certain embodiments, the intracellular signaling domain may comprise an intracellular costimulatory domain.

[0098] The intracellular primary signaling domain may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of intracytoplasmic primary signaling sequences that contain ITAMs include those derived from CD3zeta, common FcRgamma (FCER1G), FcgammaRIIa, FcRβ (FcεR1b), CD3gamma, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0099] In certain embodiments, the CD3ζ (CD247) stimulatory domain may comprise sufficient amino acid residues from the intracytoplasmic domain of the T cell receptor ζ chain, or a functional fragment thereof, to functionally transmit an initial signal required for cell activation. In certain embodiments, the CD3ζ stimulatory domain may comprise a human CD3ζ stimulatory domain, or a functional fragment thereof. In certain embodiments, in the case of an intracellular signaling domain derived from a CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure to be able to generate a signal under appropriate conditions.

[0100] In certain embodiments, the intracellular signaling domain may comprise an intracellular costimulatory domain. In certain embodiments, the intracellular costimulatory signaling domain comprises an intracellular costimulatory signaling domain derived from a molecule responsible for costimulatory signals or antigen-independent stimulation. In certain embodiments, the intracellular costimulatory signaling domain may be the intracellular portion of a costimulatory molecule. A "costimulatory molecule" is a binding partner present on an immune cell that is recognized by and specifically binds to a costimulatory ligand, thereby mediating the induction of a costimulatory response (e.g., proliferation) by the immune cell. Costimulatory molecules include cell surface molecules that contribute to an efficient immune response, other than antigen receptors and their ligands. Costimulatory molecules may be represented in the TNF receptor protein family, the immunoglobulin-like protein family, the cytokine receptor family, the integrin family, the signaling lymphocyte activation molecule (SLAM protein) family, and the activation-induced NK cell receptor family.Examples of such costimulatory molecules include MHC class I molecules, B-lymphocyte-T-lymphocyte attenuator (BTLA, CD272), Toll ligand receptor, CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS (CD278), BAFFR, HVEM (LIGHTR), ICAM-1, lymphocyte function-associated antigen 1 (LFA-1; CD11a / CD18), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80(KLRF1), NKp30, NKp44, NKp46, CD160( BY55), B7-H3(CD276), CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, IA4, CD49d, ITGA 6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, C These include ligands that specifically bind to RTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0101] In certain embodiments, the intracellular costimulatory signaling domain (also referred to as costimulatory domain) comprises 4-1BB (CD137, TNFRSF9). "4-1BB" refers to a member of the tumor necrosis factor receptor (TNFR) superfamily. In certain embodiments, the costimulatory domain of 4-1BB comprises the costimulatory domain of human 4-1BB or a functional fragment thereof.

[0102] In certain embodiments, the intracellular costimulatory signaling domain comprises CD28. CD28 is a T cell specific glycoprotein that is involved in T cell activation, induction of cell proliferation, production of cytokines and promotion of T cell survival. In certain embodiments, the costimulatory domain of CD28 comprises the costimulatory domain of human CD28 or its functional fragment.

[0103] In certain embodiments, the intracellular portion comprises a combination of one or more stimulatory domains and one or more costimulatory domains described herein. In certain embodiments, the intracellular portion comprises a costimulatory domain of 4-1BB and a stimulatory domain of CD3zeta. In certain embodiments, the intracellular portion comprises a costimulatory domain of 4-1BB and a stimulatory domain of CD3zeta.

[0104] In certain embodiments, the intracellular portion comprises the intracellular primary signaling domain of CD3ζ and the intracellular costimulatory domain of OX40. In certain embodiments, the intracellular portion comprises the intracellular primary signaling domain of CD3ζ and the intracellular costimulatory domain of TNFR2.

[0105] (iii-d) Transmembrane domain

[0106] The fusion protein can be designed to include a transmembrane domain. The transmembrane domain can anchor the fusion protein to the cell membrane. The transmembrane domain can include one or more additional amino acids adjacent to its transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids from the extracellular region of the protein from which the transmembrane domain is derived) and / or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acids from the intracellular region of the protein from which the transmembrane domain is derived). In certain embodiments, the transmembrane domain may be derived from the same protein as the intracellular portion, the signaling domain, the costimulatory domain, the hinge domain, or the co-receptor. In certain embodiments, the transmembrane domain is derived from a different protein than the protein from which the other domains of the fusion protein are derived. In certain embodiments, the transmembrane domain can be selected or modified by amino acid substitution to prevent binding to or minimize interaction with the other domains of the fusion protein.

[0107] In certain embodiments, the transmembrane domain has a three-dimensional structure, typically 15-30 amino acids long, that is thermodynamically stable in the cell membrane. The structure of the transmembrane domain can include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof.

[0108] The transmembrane domain may be of natural or recombinant origin. If of natural origin, the transmembrane domain may be of membrane-bound or transmembrane protein origin. In certain embodiments, the transmembrane domain can transmit a signal to the intracellular portion when the target binds to the extracellular ligand-binding domain of the fusion protein. In certain embodiments, the transmembrane domain may include at least the transmembrane region of the α, β or ζ chain of the T cell receptor; CD28; CD27; CD3ε; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64; CD80; CD86; CD134; CD137; and / or CD154. In certain embodiments, the transmembrane domain is selected from the group consisting of KIRDS2; OX40; CD2; LFA-1; ICOS; 4-1BB; GITR; CD40; BAFFR; HVEM; SLAMF7; NKp80; NKp44; NKp30; NKp46; CD160; CD19; IL2Rβ; IL2Rγ; IL7Ra; ITGA1; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6; VLA-6; CD49f; ITGAD; CD11d; ITGAE; CD103; ITGAL; CD11a; ITGAM; CD11b; ITGAX; CD11c; ITGB1; CD29; ITGB2; CD18; ITGB7; TNFR2; DNAM1; SLAMF4; CD84; CD96; CEACAM1; CRT and / or NKG2C. In certain embodiments, the transmembrane domain may comprise at least the transmembrane domain from the CD28 chain, the CD4 chain, or the CD8α chain.

[0109] In certain embodiments, the transmembrane domain may be composed primarily of hydrophobic residues such as leucine and valine. In certain embodiments, the transmembrane domain may contain a triplet of phenylalanine, tryptophan and valine at each end. In certain embodiments, the hinge of CD28, CD4 or CD8 is located on the extracellular side of the transmembrane domain.

[0110] In some embodiments, the fusion protein (e.g., the binding portion of DARIC) comprises a transmembrane domain or a GPI signal sequence. In further embodiments, the fusion protein (e.g., the binding portion of DARIC) comprises a GPI molecule from which the GPI signal sequence has been removed or which has been modified to add a GPI signal sequence.

[0111] In certain embodiments, the first fusion protein and / or the second fusion protein comprises a transmembrane domain that comprises the transmembrane domain of CD4. In certain embodiments, the first fusion protein and / or the second fusion protein comprises a transmembrane domain that comprises the transmembrane domain of CD8α.

[0112] (iii-e) Linker

[0113] Herein, the linker contained in the fusion protein may be a part of the fusion protein that serves to connect two subcomponents or domains of the fusion protein. In certain embodiments, the linker can provide flexibility to various parts of the fusion protein. The linker that connects the VH and VL of the antibody-derived binding domain in the scFv is also described above. The linker may further include a spacer region and a linking amino acid.

[0114] A spacer region is a type of linker region used to provide appropriate distance from and / or flexibility relative to other linked moieties.

[0115] In certain embodiments, the length of the spacer region can be customized for individual purposes. For example, the spacer region can be customized for individual cell markers on the target cell to optimize the recognition and destruction by the cell upon binding of the fusion protein. In certain instances, the length of the spacer can be such that the responsiveness of the fusion protein expressing cells upon binding to the antigen is enhanced compared to the absence of the spacer. In certain embodiments, the length of the spacer region can be selected according to the location of the epitope of the cell marker, the affinity of the binding domain for the epitope, and / or the ability of the fusion protein recombinant cells to destroy the target cells in response to recognition of the cell marker ex vivo and / or in vivo. The spacer region can induce high expression in the fusion protein recombinant cells. In certain embodiments, the extracellular spacer region of the fusion protein can be located between the transmembrane domain and the extracellular binding domain.

[0116] Exemplary spacers include spacers having 10-250 amino acids, 10-200 amino acids, 10-150 amino acids, 10-100 amino acids, 10-50 amino acids, or 10-25 amino acids. In certain embodiments, the spacer region is 12, 20, 21, 26, 27, 45, or 50 amino acids long. In certain embodiments, a long spacer is greater than 119 amino acids long, a medium length spacer is 13-119 amino acids long, and a short spacer is 10-12 amino acids long.

[0117] In certain embodiments, the spacer region comprises an immunoglobulin hinge region. The immunoglobulin hinge region may be a wild-type immunoglobulin hinge region or a modified wild-type immunoglobulin hinge region. In certain embodiments, the immunoglobulin hinge region is a human immunoglobulin hinge region. The immunoglobulin hinge region may be an IgG, IgA, IgD, IgE or IgM hinge region. The IgG hinge region may be an IgG1, IgG2, IgG3 or IgG4 hinge region. In certain embodiments, the spacer region may comprise the entire or a portion of the hinge region sequence from IgG1, IgG2, IgG3, IgG4, or IgD, or a combination of the entire or a portion of the hinge region sequence from IgG1, IgG2, IgG3, IgG4, or IgD with the entire or a portion of the CH2 region; the entire or a portion of the CH3 region; or a combination of the entire or a portion of the CH2 region with the entire or a portion of the CH3 region. As used herein, the term "wild-type immunoglobulin hinge region" refers to the amino acid sequence of the hinge located between and linking the CH1 and CH2 domains of the heavy chain in the upper and middle parts of the naturally occurring antibody (in the case of IgG, IgA, and IgD), or the amino acid sequence of the hinge located between and linking the CH1 and CH3 domains of the heavy chain (in the case of IgE and IgM).

[0118] Exemplary spacers include a spacer consisting of only an IgG4 hinge, a spacer consisting of an IgG4 hinge linked to a CH2 domain and a CH3 domain, or a spacer consisting of an IgG4 hinge linked to a CH3 domain. The hinge region can be engineered to prevent undesired structural interactions, such as dimerization with unintended partners. Other examples of hinge regions that can be used in the fusion proteins described herein include the hinge regions present in the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, CD7, and the like, and these type 1 membrane proteins can be wild type or variants thereof.

[0119] In certain embodiments, the spacer region comprises the hinge region of the interdomain region (stalk region) of a type II C-type lectin or the hinge region of the stalk region of a cluster of differentiation (CD) molecule. The "stalk region" of a type II C-type lectin or CD molecule refers to a portion of the extracellular domain that is located between the C-type lectin-like domain (CTLD) (e.g., similar to the CTLD of the natural killer cell receptor) and the hydrophobic portion (transmembrane domain) in a type II C-type lectin or CD molecule. For example, the extracellular domain of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, whereas the CTLD corresponds to amino acid residues 61-176, and the stalk region of the human CD94 molecule includes amino acid residues 34-60, and is therefore located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:15, 1999; for a description of other stalk regions, see further Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:153, 1992; and Figdor et al., Nat. Rev. Immunol. 2:11, 2002). These type II C-type lectins or CD molecules may further have linking amino acids between the stalk region and the transmembrane domain or between the stalk region and the CTLD. In another example, the 233 amino acid long human NKG2A protein (UniProt ID P26715.1) has a hydrophobic portion (transmembrane domain) consisting of amino acids 71-93 and an extracellular domain consisting of amino acids 94-233. The CTLD of the human NKG2A protein comprises amino acids 119-231, and the stalk region comprises amino acids 99-116, which may be flanked by additional linking amino acids.Other type II C-type lectins or CD molecules, or their extracellular ligand-binding domains, stalk regions and CTLDs, are also known in the art (see, e.g., GenBank Accession Nos. NP 001993.2; AAH07037.1; NP 001773.1; AAL65234.1; and CAA04925.1 for sequences and descriptions of human CD23, human CD69, human CD72, human NKG2A and human NKG2D, respectively).

[0120] (iii-f) Tags and selection markers

[0121] In certain embodiments, the fusion protein may include one or more tags and / or express one or more selectable markers. Exemplary tags include His-tag, Flag-tag, Xpress-tag, Avi-tag, calmodulin-binding peptide (CBP)-tag, polyglutamic acid-tag, HA-tag, Myc-tag, Strep-tag (previously called STREP®-tag or STREP-tag II (IBA (Institut fur Bioanalytik) GmbH, Germany); see, e.g., U.S. Patent Publication No. 7,981,632), Softag 1, Softag 3 and V5. See FIG. 6 for exemplary sequences.

[0122] Binding molecules that specifically bind to the tag sequences disclosed herein to form complexes are commercially available. For example, His tag antibodies are commercially available from manufacturers such as Life Technologies, Pierce Antibodies, GenScript, etc. Flag tag antibodies are commercially available from manufacturers such as Pierce Antibodies, GenScript, Sigma-Aldrich, etc. Xpress tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Life Technologies, GenScript, etc. Avi tag antibodies are commercially available from manufacturers such as Pierce Antibodies, IsBio, Genecopoeia, etc. Calmodulin tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, Pierce Antibodies, etc. HA tag antibodies are commercially available from manufacturers such as Pierce Antibodies, Cell Signal, Abcam, etc. Myc tag antibodies are commercially available from manufacturers such as Santa Cruz Biotechnology, Abcam, Cell Signal, etc. Strep tag antibodies are commercially available from manufacturers such as Abcam, Iba, Qiagen, etc.

[0123] In certain embodiments, one or more transduction markers can be co-expressed with the fusion protein, for example, using a skipping sequence or IRES site that allows the transduction marker and other portions of the fusion protein to be expressed as separate molecules. Exemplary self-cleaving polypeptides include the 2A peptide (P2A) from porcine teschovirus-1, the 2A peptide (T2A) from Thosea asigna virus, the 2A peptide (E2A) from equine rhinitis A virus, and the 2A peptide (F2A) from foot and mouth disease virus, which are described elsewhere herein.

[0124] In certain embodiments, the transduction marker may include a cell surface-presented marker detectable with an antibody capable of binding to the marker and selecting cells bearing the marker. In certain embodiments, the transduction marker may include a magnetically selectable streptavidin-binding peptide (SBP) marker presented on the cell surface by a truncated low affinity nerve growth receptor (LNGFRF) and one-step selection with streptavidin-labeled magnetic beads (Matheson et al. (2014) PloS one 9(10): e111437); or a truncated human epidermal growth factor receptor (EGFR) (tEGFR; see Wang et al., Blood 118: 1255, 2011).

[0125] In some embodiments, the transduction marker is truncated EGFR (EGFRt), truncated Her2 (Her2), truncated Her2 (Her2tG), truncated CD19 (CD19t) or the transduction marker DHFRdm.

[0126] The transduction marker may comprise a suitable fluorescent protein, including blue fluorescent protein (e.g., BFP, eBFP, eBFP2); cyan fluorescent protein (e.g., eCFP, cerulean, CyPet); green fluorescent protein (e.g., GFP-2, tagGFP, turboGFP, eGFP); orange fluorescent protein (e.g., mOrange, mKO, Kusabira-Orange); red fluorescent protein (e.g., mKate, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express); yellow fluorescent protein (e.g., YFP, eYFP, Citrine, Venus); and other suitable fluorescent proteins (e.g., firefly luciferase, etc.).

[0127] (iv) polynucleotide

[0128] In certain embodiments, polynucleotides encoding DARIC, one or more DARIC components, a signaling portion of DARIC and / or a binding portion of DARIC are provided.

[0129] As used herein, "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and DNA / RNA hybrids. Polynucleotides may be single-stranded or double-stranded, and may be recombinant, synthetic or isolated polynucleotides. Polynucleotides include pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA or recombinant DNA. Polynucleotide refers to ribonucleotides or deoxyribonucleotides, modified forms of these types of nucleotides, and any intermediate length nucleotides, in the polymerized form of nucleotides of at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, at least 40 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 1000 nucleotides, at least 5000 nucleotides, at least 10000 nucleotides, or at least 15000 nucleotides, or longer.In this context, it is easily understood that "intermediate length" refers to lengths between the values ​​described herein, such as 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.In certain embodiments, a polynucleotide or variant has at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence.

[0130] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from adjacent sequences in the natural state, e.g., a DNA fragment that has been removed from sequences that are normally adjacent to it. In certain embodiments, an "isolated polynucleotide" refers to an artificially produced complementary DNA (cDNA), recombinant DNA, or other polynucleotide that does not exist in nature. In certain embodiments, an "isolated polynucleotide" is a synthetic polynucleotide, a semi-synthetic polynucleotide, or a polynucleotide obtained or derived from a recombinant source.

[0131] In various embodiments, the polynucleotide comprises an mRNA that encodes a polypeptide contemplated herein. In certain embodiments, the mRNA comprises a cap, one or more nucleotides, and a poly(A) tail.

[0132] In certain embodiments, a polynucleotide encoding one or more DARIC components may be codon optimized. As used herein, "codon optimization" refers to substituting codons in a polynucleotide encoding a polypeptide to increase expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include one or more of the following: (i) variation in codon bias between two or more organisms, between two or more genes, or between two or more synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons in adjacent sequences, etc.; (iv) variation of codons due to the tRNA that decodes the codon; (v) variation of codons due to GC content throughout the sequence or at one position of a triplet; (vi) variation in the degree of similarity to a reference sequence, e.g., a natural sequence; (vii) variation in codon frequency cutoff values; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the codon substitution set is designed; (x) systematic variation of the codon set for each amino acid; and / or (xi) isolated removal of pseudo translation start sites.

[0133] As used herein, "nucleotide" refers to a heterocyclic nitrogenous base to which a phosphorylated sugar is N-glycosidically linked. Nucleotides include natural bases and a variety of modified bases known in the art. Such bases are usually located at the 1' position of the nucleotide sugar moiety. Nucleotides usually include a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, i.e., a sugar that does not have the hydroxyl group present in ribose.

[0134] In various specific embodiments, polynucleotides contemplated herein include polynucleotides encoding one or more DARIC components, recombinant antigen receptors or fusion polypeptides, as well as expression vectors, viral vectors and transfer plasmids comprising polynucleotides contemplated herein.

[0135] As used herein, terms such as "polynucleotide variant" and "variant" refer to polynucleotides that exhibit sufficient sequence identity with a reference polynucleotide sequence or hybridize to a reference sequence under stringent conditions, as defined below. These terms also include polynucleotides that are distinct from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Thus, the terms "polynucleotide variant" and "variant" include those in which one or more nucleotides are added to another nucleotide, one or more nucleotides are deleted, one or more nucleotides are modified, or one or more nucleotides are replaced by another nucleotide. In this regard, it is well recognized in the art that certain modifications can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, and that the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0136] As used herein, "nucleic acid cassette" or "expression cassette" refers to a genetic sequence within a vector capable of expressing RNA to produce a polypeptide. In one embodiment, the nucleic acid cassette comprises a single gene or multiple genes of interest, e.g., a single polynucleotide or multiple polynucleotides of interest. In another embodiment, the nucleic acid cassette comprises one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, a gene of interest (e.g., a polynucleotide of interest). A vector may comprise one, two, three, four, five, six, seven, eight, nine, or ten or more nucleic acid cassettes. The nucleic acid cassette is oriented in the proper position and order within the vector so that the nucleic acid therein can be transcribed into RNA, translated into a protein or polypeptide as necessary, subjected to the appropriate post-translational modification required for activity in the transformed cell, and targeted to the appropriate intracellular compartment for transport to a compartment suitable for biological activity or secreted into an extracellular compartment. The nucleic acid cassette preferably has 3' and 5' ends configured for easy insertion into a vector, e.g., has restriction endonuclease sites at both ends, and can be removed from or inserted into a plasmid or viral vector as a single unit.

[0137] A polynucleotide includes a single polynucleotide or multiple polynucleotides of interest. As used herein, a "polynucleotide of interest" refers to a polynucleotide that encodes a polypeptide or a fusion polypeptide, as described herein, or a polynucleotide that serves as a template for transcription of an inhibitory polynucleotide.

[0138] The polynucleotides contemplated herein may be combined with additional DNA sequences, such as, for example, promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP sites, FRT sites and Att sites), stop codons, transcription termination signals, polynucleotides encoding self-cleaving polypeptides, epitope tags, etc., as described elsewhere herein or as known in the art, regardless of the length of the coding sequence itself, and the total length of the combined sequence may vary significantly. Thus, it is contemplated that polynucleotide fragments of almost any length may be used, with the total length being preferably limited by the ease of preparation and the recombinant DNA protocol used.

[0139] Polynucleotides can be prepared, engineered, expressed and / or delivered using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0140] Examples of vectors include plasmids, autonomously replicating sequences, and transposable elements (eg, Sleeping Beauty and PiggyBac).

[0141] Other specific examples of vectors include plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and P1-derived artificial chromosomes (PACs); bacteriophages, such as λ phage and M13 phage; and animal viruses.

[0142] Examples of viruses useful as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (eg, herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (eg, SV40).

[0143] Specific examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST for lentivirus-mediated gene transfer and expression in mammalian cells. TM , pLenti6 / V5-DEST TM and pLenti6.2 / V5-GW / lacZ (Invitrogen). In certain embodiments, the coding sequences for the polypeptides disclosed herein can be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

[0144] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, "episomal" refers to a vector that can replicate autonomously without being integrated into the chromosomal DNA of the host, and the introduced gene is not gradually lost with the division of the host cell, which means that the vector can replicate extrachromosomally, i.e., can replicate as an episome.

[0145] "Expression control sequences," "control elements," or "regulatory sequences" present in an expression vector are non-translated regions of the expression vector, including replication origins, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, polyadenylation sequences, and 5' and 3' non-translated regions, all of which interact with host cell proteins to mediate transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, such as ubiquitous and inducible promoters, may be used.

[0146] In certain embodiments, the polynucleotide comprises a vector, including expression vectors and viral vectors. The vector may comprise one or more exogenous, endogenous or heterologous control sequences, such as, for example, promoters and / or enhancers. An "endogenous control sequence" is a sequence that is naturally linked to a particular gene in a genome. An "exogenous control sequence" is a sequence that is placed in juxtaposition to a gene by genetic engineering (i.e., molecular biology techniques), such that the transcription of the gene is induced by the enhancer / promoter linked in this way. A "heterologous control sequence" is an exogenous sequence that originates from a different organism than the cell that is genetically engineered. A "synthetic" control sequence may comprise one or more elements of an endogenous and / or exogenous sequence, and / or may comprise elements of a sequence that has been demonstrated in vitro or in silico to provide optimal promoter and / or enhancer activity for a particular treatment.

[0147] As used herein, a "promoter" refers to a recognition site in a polynucleotide (DNA or RNA) to which an RNA polymerase binds. The RNA polymerase initiates transcription of a polynucleotide operably linked to the promoter. In certain embodiments, promoters that operate in mammalian cells include an AT-rich region 25-30 bases upstream from the transcription start site, and / or a CNCAAT region (where N is any nucleotide), which is another sequence 70-80 bases upstream from the transcription start site.

[0148] "Enhancer" refers to a DNA segment that contains a sequence that can enhance transcription, and in some cases can function regardless of orientation relative to another control sequence. Enhancers can function cooperatively or additively with a promoter and / or other enhancer elements. "Promoter / enhancer" refers to a DNA segment that contains a sequence that can function both as a promoter and as an enhancer.

[0149] "Operably linked" refers to an ordered arrangement of the listed components in a relationship permitting each to function in its intended manner. In one embodiment, "operably linked" refers to a functional linkage between a sequence that controls expression of a nucleic acid (e.g., a promoter and / or enhancer) and a second polynucleotide sequence (e.g., a polynucleotide of interest), where the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0150] As used herein, a "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for sustained or continuous transcription of an operably linked sequence. A constitutive expression control sequence may be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows for expression in a wide variety of cell and tissue types, or may be a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows for expression in a limited variety of cell and tissue types.

[0151] Specific examples of ubiquitous expression control sequences suitable for use in certain embodiments include the immediate early promoter of cytomegalovirus (CMV), the Simian Virus 40 (SV40) (e.g., the early or late promoter), the LTR promoter of Moloney Murine Leukemia Virus (MoMLV), the LTR of Rous Sarcoma Virus (RSV), the (thymidine kinase) promoter of Herpes Simplex Virus (HSV), the H5 promoter, the P7.5 promoter, and the P11 promoter of Vaccinia virus. ter, elongation factor 1 alpha (EF1a) promoter, early growth response protein 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), 70 kDa heat shock protein 5 (HSPA5), 90 kDa heat shock protein beta member 1 (HSP90B1), 70 kDa heat shock protein (HSP70), beta-kinesin (beta-KIN), human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter / myeloproliferative sarcoma virus enhancer, and the (MND) U3 promoter with the negative control region deleted and the dl587rev primer binding site replaced (Haas et al. Journal of Virology. 2003;77(17): 9439-9450).

[0152] In one embodiment, the vector comprises the MNDU3 promoter.

[0153] In one embodiment, the vector comprises the EF1a promoter including the first intron of the human EF1a gene.

[0154] In one embodiment, the vector comprises the EF1a promoter lacking the first intron of the human EF1a gene.

[0155] In a particular embodiment, it may be desirable to use cell-type, cell lineage, or tissue-specific expression control sequences to effect cell-type, cell lineage, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a nucleic acid encoding a particular polypeptide only in a subset of cells of a particular type, a subset of cell lineages, or a subset of tissues, or at a particular developmental stage).

[0156] In certain embodiments, it may be desirable to express the polynucleotide using a T cell specific promoter.

[0157] As used herein, "conditional expression" may refer to any type of conditional expression, such as induced expression, repressed expression, expression in cells or tissues in a particular physiological, biological or disease state. This definition does not exclude cell type-specific or tissue-specific expression. In certain embodiments, conditional expression of a polynucleotide of interest is provided, for example, expression is controlled by exposing a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide or increases or decreases the expression of the polynucleotide encoded by the polynucleotide of interest.

[0158] Specific examples of inducible promoters / systems include steroid-inducible promoters such as promoters for genes encoding glucocorticoid receptors or estrogen receptors (inducible by treatment with the corresponding hormones), metallothionein promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the mifepristone-regulated system of "GeneSwitch" (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switch (WO2002 / 088346), tetracycline-dependent regulatory systems, etc. Inducing agents include glucocorticoids, estrogens, mifepristone (RU486), metals, interferons, small molecules, cumate, tetracycline, doxycycline, and derivatives thereof.

[0159] As used herein, "internal ribosome entry site" or "IRES" refers to an element that allows an internal ribosome to directly enter the initiation codon (e.g., ATG) of a cistron (a protein coding region) to initiate cap-independent gene translation. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83 and Jackson and Kaminski. 1995. RNA 1(10):985-1000. Examples of IRES commonly used by those skilled in the art include those described in U.S. Patent No. 6,692,736. Further examples of "IRES" known in the art include IRESs from picornaviruses (Jackson et al., 1990) and IRESs from viral or cellular mRNAs, such as the IRESs from immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18(11):6178-6190), fibroblast growth factor 2 (FGF-2), insulin-like growth factor (IGFII), translation initiation factor eIF4G, yeast transcription factors TFIID and HAP4, and encephalomyocarditis virus (EMCV) commercially available from Novagen (Duke et al., 1992. J. Virol 66(3):1602-9), as well as the VEGF IRES (Huez et al., 1998. Mol. Cell. Biol. 18(11):6178-6190). 18(11):6178-90). IRES has also been reported to be present in the viral genomes of Picornaviridae, Dicistroviridae, and Flaviviridae, as well as HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV).

[0160] In one embodiment, the IRES used in the polynucleotides contemplated herein is the IRES of EMCV.

[0161] In certain embodiments, the polynucleotide contemplated herein is a consensus Kozak sequence. As used herein, "Kozak sequence" refers to a short nucleotide sequence that significantly promotes the initial binding of mRNA to the small subunit of ribosome, thereby increasing translation. The consensus Kozak sequence is represented by (GCC)RCCATGG (SEQ ID NO:59), where R is purine (A or G) (Kozak, 1986. Cell. 44(2):283-92 and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48).

[0162] Expression of heterologous genes can be increased by factors capable of inducing efficient transcription termination and polyadenylation of heterologous nucleic acid transcripts. Transcription termination signals are usually found downstream of polyadenylation signals. In certain embodiments, vectors contain a polyadenylation sequence at the 3' end of the polynucleotide encoding the polypeptide to be expressed. As used herein, "polyA site" or "polyA sequence" refers to a DNA sequence that induces both transcription termination and polyadenylation of the nascent RNA transcript transcribed by RNA polymerase II. Polyadenylation sequences can improve mRNA stability by adding a polyA tail to the 3' end of the coding sequence, thereby contributing to improved translation efficiency. Cleavage and polyadenylation are induced by poly(A) sequences in the RNA. In the core poly(A) sequence of mammalian pre-mRNA, two recognition factors are present adjacent to the site where cleavage and polyadenylation occur. Typically, the nearly undiverse 6-mer AAUAAA is 20-50 bases upstream of a highly diverse factor rich in U or GU residues. Cleavage of the new transcript occurs between this AAUAAA sequence and the GU-rich or U-rich sequence, with up to 250 adenosines being added to the 5' cleavage product. In certain embodiments, the core poly(A) sequence is a typical polyA sequence (e.g., AATAAA, ATTAAA, AGTAAA). In certain embodiments, the poly(A) sequence is the SV40 polyA sequence, the bovine growth hormone polyA sequence (BGHpA), the rabbit β-globin polyA sequence (rβgpA), variants thereof, or another suitable heterologous or endogenous polyA sequence known in the art. In certain embodiments, the poly(A) sequence is a synthetic sequence.

[0163] In certain embodiments, a polynucleotide encoding one or more polypeptides or fusion polypeptides may be introduced into an effector immune cell (e.g., a T cell) by non-viral or viral methods. In certain embodiments, delivery of one or more polynucleotides may be by the same or different methods and / or by the same or different vectors.

[0164] As used herein, "vector" refers to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The nucleic acid to be transferred is usually linked to the nucleic acid molecule of the vector, e.g., inserted into the nucleic acid molecule of the vector. The vector may contain a sequence that induces the cell to replicate autonomously, or may contain a sequence sufficient to achieve integration into the DNA of the host cell. In certain embodiments, a non-viral vector is used to deliver one or more polynucleotides contemplated herein to T cells.

[0165] Examples of non-viral vectors include plasmids (eg, DNA plasmids and RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0166] Specific methods of non-viral delivery of polynucleotides contemplated in certain embodiments include electroporation, sonoporation, lipofection, microinjection, particle bombardment, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid complexes, naked DNA, artificial virions, DEAE-dextran-based transfer, gene guns, and heat shock.

[0167] Specific examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include those provided by Amaxa Biosystems, Maxcyte, BTX Molecular Delivery Systems, and Copernicus Therapeutics. Lipofection reagents are commercially available (e.g., Transfectam TM or Lipofectin TM(See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12.) Suitable cationic and neutral lipids for efficient transfer of polynucleotides by receptor-recognizing lipofection have been described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. Delivery using antibody-targeted, non-biological nanocells derived from bacteria is also contemplated in certain embodiments.

[0168] As can be easily understood by those skilled in the art, the term "viral vector" is generally used to mean a nucleic acid molecule (e.g., transfer plasmid) that contains a virus-derived nucleic acid element that facilitates the transfer or integration of a nucleic acid molecule into a cell genome, or a viral particle that mediates the transfer of nucleic acid. A viral particle usually contains various viral components, and in some cases, may contain host cell components other than nucleic acid. A "viral vector" or a "lentiviral vector" refers to a virus or viral particle that can transfer a nucleic acid into a cell, or the transferred nucleic acid itself. A viral vector and a transfer plasmid contain structural and / or functional genetic elements that are mainly derived from a virus.

[0169] Viral vectors comprising polynucleotides according to certain embodiments can be delivered in vivo to individual patients by administration, typically systemically (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial injection) or topically, as described below. Alternatively, vectors can be delivered to ex vivo cells, such as cells explanted from individual patients (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy specimens, etc.) or hematopoietic stem cells obtained from a universal donor, which can then be reimplanted into the patient.

[0170] In one embodiment, the viral vectors containing the polynucleotides contemplated herein are directly administered to an organism or subject to transduce cells in vivo. Alternatively, naked DNA can be administered. Administration can be by any route that is typically used to introduce molecules to ultimately contact blood or tissue cells, including injection, infusion, topical application, and electroporation. Suitable methods of administration of such nucleic acids are available and will be familiar to those skilled in the art. Depending on the type of composition, more than one route of administration may be used, although certain routes often provide more immediate and effective responses than others.

[0171] Specific examples of viral vector systems suitable for use in certain embodiments described herein include adeno-associated viral (AAV) vectors, retroviral vectors, herpes simplex viral vectors, adenoviral vectors and vaccinia viral vectors.

[0172] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into effector immune cells (e.g., T cells) by transduction with a recombinant adeno-associated virus (rAAV) containing the one or more polynucleotides.

[0173] AAV is a small (26 nm), non-enveloped virus that exists mainly as an episome and is replication-defective. AAV can infect both dividing and non-dividing cells, and may integrate its genome into the genome of a host cell. Recombinant AAV (rAAV) is usually composed of at least a transgene and its regulatory sequence, and AAV inverted repeats (ITRs) at the 5' and 3' ends. The length of the ITR sequence is 145 bp. In a specific embodiment, rAAV comprises the ITR and capsid sequences isolated from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10.

[0174] In some embodiments, a chimeric rAAV is used in which the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from another AAV serotype. For example, a rAAV with an ITR sequence from AAV2 and a capsid sequence from AAV6 is called AAV2 / AAV6. In certain embodiments, a rAAV vector may contain an ITR from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In a preferred embodiment, the rAAV contains an ITR sequence from AAV2 and a capsid sequence from AAV6. In a preferred embodiment, the rAAV contains an ITR sequence from AAV2 and a capsid sequence from AAV2.

[0175] In some embodiments, recombination and selection methods can be applied to the AAV capsid to obtain AAV capsids that are readily amenable to transduction into cells of interest.

[0176] The construction of rAAV vectors and their production and purification are disclosed, for example, in U.S. Pat. No. 9,169,494; U.S. Pat. No. 9,169,492; U.S. Pat. No. 9,012,224; U.S. Pat. No. 8,889,641; U.S. Pat. No. 8,809,058; and U.S. Pat. No. 8,784,799, which are incorporated by reference in their entireties.

[0177] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into effector immune cells (e.g., T cells) by transduction with a retrovirus (e.g., a lentivirus) containing the one or more polynucleotides.

[0178] As used herein, a "retrovirus" is an RNA virus whose genomic RNA is reverse transcribed to produce a double-stranded linear DNA copy, and the genomic DNA is integrated into the host genome via covalent linkage.Specific examples of retroviruses suitable for use in certain embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV) and lentivirus.

[0179] As used herein, "lentivirus" refers to the complex retrovirus group (or complex retrovirus genus). Specific examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalomyelitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, a vector backbone based on HIV (i.e., HIV cis-acting sequence elements) is preferred.

[0180] In various embodiments, lentiviral vectors contemplated herein include as accessory elements all or one or more of cPPT / FLAP, Ψ packaging signal, transport factor, and poly(A) sequence, as well as one or more LTRs, and optionally a WPRE or HPRE, an insulator factor, a selection marker, and a cell suicide gene, as described elsewhere herein.

[0181] In certain embodiments, the lentivirus vector contemplated herein can be integrated lentivirus, non-integrated lentivirus, or integration-defective lentivirus.In this specification, "integration-defective lentivirus" or "IDLV" refers to the lentivirus that has integrase that cannot integrate viral genome into the genome of host cell.Non-integrated viral vectors are described in patent application WO2006 / 010834 (this document is incorporated herein in its entirety).

[0182] Specific examples of HIV-1 pol gene mutations that are suitable for suppressing integrase activity include H12N, H12C, H16C, H16V, and S81. R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116 N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E 157A, K159E, K159A, K160A, R166A, D167A, E170A, H171A, K173A, K186Q, K1 86T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221 L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264H.

[0183] "Long terminal repeat (LTR)" refers to the base-paired domains at both ends of the DNA of a retrovirus which, in the native sequence, are direct repeats and include the U3, R and U5 regions.

[0184] As used herein, "FLAP elements" or "cPPT / FLAP" refer to nucleic acids that contain a central polypurine tract (cPPT) and a central termination sequence (CTS) of a retrovirus (e.g., HIV-1 or HIV-2) in their sequence. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173.

[0185] As used herein, "packaging signal" or "packaging sequence" refers to the Ψ sequence present in the genome of retroviruses, which is required for the insertion of viral RNA into viral capsids or viral particles.See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109.

[0186] "Transport factor" refers to the cis-acting post-transcriptional regulatory factor that regulates the transport of RNA transcripts from cell nucleus to cytoplasm.Examples of RNA transport factors include the rev response element (RRE) of human immunodeficiency virus (HIV) (see, for example, Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the post-transcriptional regulatory element (HPRE) of hepatitis B virus.

[0187] In certain embodiments, the expression of heterologous sequence in viral vector can be increased by incorporating post-transcriptional regulators and efficient polyadenylation sites into viral vector, and transcription termination signals can also be incorporated into viral vector.Various post-transcriptional regulators can increase the expression of heterologous nucleic acid in said protein, such as woodchuck hepatitis virus post-transcriptional regulator (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); Hepatitis B virus post-transcriptional regulator (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and other post-transcriptional regulators (Liu et al., 1995, Genes Dev., 9:1766).

[0188] Lentiviral vectors are preferably made safer in several ways by the LTR being engineered. "Self-inactivating (SIN)" vectors refer to replication-deficient vectors, e.g., retroviral and lentiviral vectors, in which the right (3') LTR enhancer promoter region, known as the U3 region, is engineered (e.g., deleted or replaced) to prevent viral transcription beyond one round of viral replication. Self-inactivation is preferably achieved by introducing a deletion in the U3 region of the 3'LTR of the vector DNA, i.e., a deletion in the DNA utilized in the production of vector RNA. Thus, upon reverse transcription, this deletion is replicated in the 5'LTR of the proviral DNA. In certain embodiments, it is desirable to remove the U3 sequence sufficiently to substantially reduce or completely eliminate the transcriptional activity of the LTR, thereby substantially reducing or completely eliminating the production of full-length vector RNA in transduced cells. In the case of HIV-based lentivectors, it has been found that large deletions of the U3 region including the TATA box of the LTR (e.g., deletion of -418 to -18) are possible without significantly reducing vector titer.

[0189] Safety can be further improved by replacing the U3 region of 5'LTR, which induces the transcription of the viral genome in the production of viral particles, with a heterologous promoter. Examples of heterologous promoters that can be used include, for example, Simian Virus 40 (SV40) (e.g., early or late) promoter, Cytomegalovirus (CMV) (e.g., immediate early) promoter, Moloney Murine Leukemia Virus (MoMLV) promoter, Rous Sarcoma Virus (RSV) promoter and Herpes Simplex Virus (HSV) (thymidine kinase) promoter.

[0190] As used herein, "pseudotype" or "pseudotyping" refers to a virus in which a viral envelope protein has been replaced with an envelope protein of another virus that has favorable properties. For example, the envelope protein of HIV (encoded by the env gene) is normally encoded by the CD4 + Although HIV is targeted to presentation cells, pseudotyping it with the envelope protein G protein (VSVG) of the vesicular stomatitis virus allows HIV to infect a broad range of cells.

[0191] In certain embodiments, lentiviral vectors are produced by known methods, see, e.g., Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186 / 1472-6750-9-10; Kutner et al. Nat. Protoc. 2009;4(4):495-505. doi: 10.1038 / nprot.2009.22.

[0192] According to certain specific embodiments described herein, all or most of the viral vector backbone sequences are derived from lentiviruses (e.g., HIV-1). However, various retroviral and / or lentiviral sequences may be used or combined, or numerous substitutions and modifications may be incorporated into a particular lentiviral sequence without impairing the ability of the transfer vector to perform the functions described herein. Additionally, various lentiviral vectors are known in the art, see Naldini et al. (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998; U.S. Patent No. 6,013,516; U.S. Patent No. 5,994,136, many of which may be employed in the manufacture of viral vectors or transfer plasmids contemplated herein.

[0193] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into effector immune cells by transduction with an adenovirus containing the one or more polynucleotides.

[0194] Adenovirus-based vectors can achieve very high transduction efficiency in many types of cells without the need for cell division. High titers and high expression can be obtained by using such vectors. Such vectors can be mass-produced in a relatively simple system. Most adenovirus vectors are engineered to replace the adenovirus E1a, E1b and / or E3 genes with a transgene, and after replacement with the transgene, the replication-deficient vector is propagated in human 293 cells that complement the function of the defective gene as a trans-factor. Adenovirus vectors can transduce various types of in vivo tissues, including non-dividing differentiated cells found in the liver, kidney, muscle, etc. Conventional adenovirus vectors have a large packaging capacity.

[0195] The production and propagation of the replication-deficient adenoviral vector of the present invention may be achieved by using a special helper cell line called 293T cells, which are transformed from human embryonic kidney cells with Ad5 DNA fragments and constitutively express E1 protein (Graham et al., 1977). Since the E3 region is not essential for the adenoviral genome (Jones and Shenk, 1978), the adenoviral vector of the present invention can insert foreign DNA into the E1 region or D3 region or both with the help of 293T cells (Graham and Prevec, 1991). Adenoviral vectors have been used for gene expression in eukaryotic cells (Levrero et al., 1991; Gomez-Foix et al., 1992) and for vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies of recombinant adenovirus administration to various tissues include intratracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation (Le Gal La Salle et al., 1993). An example of the use of adenovirus vectors in clinical trials is polynucleotide therapy by intramuscular injection for antitumor immunization (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0196] In various embodiments, one or more polynucleotides encoding one or more DARIC components and / or other polypeptides contemplated herein are introduced into effector immune cells by transduction with a herpes simplex virus (e.g., HSV-1 or HSV-2) containing the one or more polynucleotides.

[0197] Mature HSV virions have an enveloped icosahedral capsid, containing a viral genome that contains a linear double-stranded DNA molecule of 152 kb. In one embodiment, the HSV-based viral vector is defective in one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions that remove one or more immediate early, early or late HSV genes to prevent replication. For example, the HSV vector may be defective in an immediate early gene selected from the group including ICP4, ICP22, ICP27, ICP47 and combinations thereof. Advantages of HSV vectors include the ability to enter a latent phase of long-term DNA expression and the large viral DNA genome that can accommodate up to 25 kb of foreign DNA inserts. HSV-based vectors are described, for example, in U.S. Pat. No. 5,837,532, U.S. Pat. No. 5,846,782 and U.S. Pat. No. 5,804,413, and International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637 and WO99 / 06583, which are incorporated by reference in their entireties.

[0198] (v) Genetically modified cells

[0199] The present disclosure includes cells genetically engineered to express a dimerizer-regulated immunomodulatory complex (DARIC). As used herein, "genetic modification," "editing," or "genetic engineering" refers to the introduction of additional genetic material in the form of DNA or RNA into a cell. Also, the terms "genetically engineered cell" and "recombinant cell" are used interchangeably. In certain embodiments, cells genetically engineered to express DARIC or components thereof include effector immune cells. "Effector immune cells" include immune system cells that have one or more effector functions (e.g., cytotoxic and killing activity, secretion of cytokines, induction of antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC)). Effector immune cells are a subtype of immune cells.

[0200] "Express" or "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene.

[0201] Immune cells of the present disclosure may be autologous / autologous cells ("self" cells) or non-autologous cells ("non-self" cells, e.g., allogeneic, syngeneic or xenogeneic cells). An "autologous" cell refers to a cell obtained from the same subject. An "allogeneic" cell refers to a cell that is genetically different from a comparison cell, but originates from the same species. A "syngeneic" cell refers to a cell derived from another subject that is genetically identical to a comparison cell. A "xenogeneic" cell refers to a cell that originates from a different species than a comparison cell. In certain embodiments, recombinant cells of the present disclosure may be autologous or allogeneic cells.

[0202] In certain embodiments, the genetically modified cells comprise lymphocytes. In certain embodiments, the genetically modified cells comprise T cells, B cells, natural killer (NK) cells, monocytes / macrophages, or HSPCs.

[0203] The majority of T cells have a T cell receptor (TCR) that consists of two different peptide chains (α- and β-TCR chains). γδ T cells are a small subset of T cells that have a different type of T cell receptor (TCR) that consists of one γ chain and one δ chain.

[0204] CD3 is expressed on all mature T cells. T cells can be further classified into cytotoxic T cells (CD8+ T cells, also called CTLs) and helper T cells (CD4+ T cells).

[0205] Cytotoxic T cells can destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells recognize their targets by binding to antigens bound to MHC class I molecules, which are present on the surface of almost all cells in the body.

[0206] "Central memory T cells (TCM)" refer to CTLs that have experienced an antigen and, compared to naive cells, express CD62L or CCR7 and CD45RO, but do not express CD45RA or have reduced expression of CD45RA.

[0207] "Effector memory T cells (TEM)" refer to T cells that have experienced an antigen and do not express CD62L or have reduced expression of CD62L compared to central memory cells, and do not express CD45RA or have reduced expression of CD45RA compared to naive cells. In certain embodiments, effector memory cells are negative for expression of CD62L and CCR7, and positive or negative for expression of CD28 and CD45RA compared to naive or central memory cells. Effector T cells are positive for granzyme B and perforin compared to memory or naive T cells.

[0208] Helper T cells support other immune cells through functions such as activating cytotoxic T cells and macrophages and promoting B cell maturation. Helper T cells are activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, helper T cells are responsible for regulating or supporting an active immune response by dividing rapidly and secreting cytokines.

[0209] Natural killer T (NKT) cells are a T cell subset that co-express the αβ T cell receptor and express various molecular markers typically associated with natural killer cells, such as NK1.1 (CD161), CD16 and / or CD56.

[0210] Natural killer cells (also known as K cells and killer cells) express CD8, CD16 and CD56, but not CD3. NK cells also express activating receptors (such as NKp46) and inhibitory receptors (such as NKG2A) that regulate the cytotoxic function of NK cells against tumor cells and virus-infected cells.

[0211] Tumor-infiltrating lymphocytes (TILs) are immune cells that can migrate from the blood into tumors and recognize and kill cancer cells. Bone marrow-infiltrating lymphocytes (MILs) are antigen-experienced immune cells that migrate to the bone marrow and persist there. Mucosal-associated invariant T (MAIT) cells are found in mucosa, blood, and secondary lymphoid organs (SLOs) and are innate immune-like T cells that exhibit an effector phenotype. MAIT cells display a semi-invariant T cell receptor (TCR) and are restricted by the major histocompatibility complex-related molecule MR1.

[0212] Macrophages (and their precursor cells, monocytes) are present in all tissues of the body and phagocytose apoptotic cells, pathogens and other non-self components. Monocytes / macrophages express CD11b, F4 / 80, CD68, CD11c, IL-4Rα and / or CD163.

[0213] Immature dendritic cells (i.e., preactivated dendritic cells) phagocytose peripheral antigens and other non-self components, become activated, migrate to T cell areas of lymphoid tissues, and present antigens to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21, CD35, CD39, CD40, CD86, CD101, CD148, CD209, and DEC-205.

[0214] Hematopoietic stem cells (HSCs) refer to undifferentiated hematopoietic cells that are capable of self-renewal and differentiation into all other types of hematopoietic cells. HSCs are CD34+.

[0215] Hematopoietic progenitor cells (HPCs) originate from HSCs and can further differentiate into mature cell types. HPCs are capable of self-renewal or can differentiate into (i) myeloid progenitors that eventually differentiate into monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets or dendritic cells, or (ii) lymphoid progenitors that eventually differentiate into T cells, B cells and NK cells. HPCs express CD24 lo Lin - CD117 + It is.

[0216] HSPC refers to a cell population that includes HSC and HPC. The HSPC cell population may be positive for CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof.

[0217] Induced pluripotent stem cells (iPSCs) refer to a type of pluripotent stem cell that is artificially created from a non-pluripotent cell, usually an adult somatic cell or a terminally differentiated cell, such as a fibroblast, hematopoietic cell, muscle cell, neuron, or epidermal cell, by the introduction or contact of a reprogramming factor.

[0218] Cells can be genetically modified ex vivo and in vivo using methods known in the art, hi certain embodiments, cells are genetically modified using cell-targeted delivery methods.

[0219] In certain embodiments, lymphocytes are isolated from a sample, such as blood or a blood-derived sample, or a sample obtained by apheresis or leukapheresis. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), bone marrow, thymus, cancer tissue, lymphoid tissue, spleen, or other suitable sources.

[0220] Sources of HSPCs include, for example, peripheral blood (see U.S. Pat. No. 5,004,681; U.S. Pat. No. 7,399,633; and U.S. Pat. No. 7,147,626; as well as Craddock, et al., 1997, Blood 90(12):4779-4788; Jin, et al., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292; Papayannopoulou, et al., 1998, Blood 91(7):2231-2239; Tricot, et al., 2008, Haematologica 93(11):1739-1742; and Weaver et al., 2001, Bone Marrow Transplantation 27(2):S23-S29).

[0221] Methods for collecting, anticoagulating, and processing blood samples are described, for example, in Alsever, et al., 1941, NY St. J. Med. 41:126; De Gowin, et al., 1940, J. Am. Med. Ass. 114:850; Smith, et al., 1959, J. Thorac. Cardiovasc. Surg. 38:573; Rous and Turner, 1916, J. Exp. Med. 23:219; and Hum, 1968, Storage of Blood, Academic Press, New York, pp. 26-160.

[0222] In certain embodiments, the recovered cells may be washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, or to lyse or remove cells sensitive to a particular reagent. Isolation may include one or more of a variety of cell preparation and separation steps, including separation based on one or more properties, such as size, density, sensitivity or resistance to a particular reagent, and / or affinity for an antibody or other binding partner (e.g., immunoaffinity).

[0223] In certain embodiments, one or more cell populations enriched, isolated and / or selected from a sample by the methods provided herein are comprised of cells positive for one or more particular markers (e.g., cell surface markers) (e.g., markers + ) or cells that highly express one or more specific markers (markers hi ), or cells negative for one or more markers (markers - ) or cells with relatively low expression of one or more markers (markers lo ).

[0224] In certain embodiments, T cells lyse red blood cells and then bind to, for example, PERCOLL TMMonocytes can be isolated from peripheral blood mononuclear cells (PBMCs) by removing monocytes, such as by centrifugation using a density gradient. In certain embodiments, specific T cell subpopulations expressing CD3, CD28, CD4, CD8, CD45RA and CD45RO are further isolated by positive or negative selection techniques. In certain embodiments, cell sorting and / or selection is performed by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells to be negatively selected. For example, CD4 + When cells are enriched by negative selection, a monoclonal antibody cocktail can generally be used that includes CD14, CD20, CD11b, CD16, HLA-DR and CD8 antibodies.

[0225] After the cells are isolated and / or enriched, they can be expanded to increase their numbers. In certain embodiments, the cells can be grown to increase their numbers using methods such as those described in, for example, U.S. Patent Publication Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; and U.S. Patent Publication Nos. 6,103,316, 6,111,315, 6,112,317, and 6,123,711. T cells can be activated and expanded using methods described in U.S. Patent Publication No. 7,232,566; U.S. Patent Publication No. 7,175,843; U.S. Patent Publication No. 5,883,223; U.S. Patent Publication No. 6,905,874; U.S. Patent Publication No. 6,797,514; U.S. Patent Publication No. 6,867,041; or U.S. Patent Publication No. 2006 / 0121005, either before or after genetic modification to express an activity-inducible fusion protein.

[0226] Typically, T cells are expanded by contacting them with a surface bound to an agent that stimulates signals associated with the CD3-TCR complex and a ligand that stimulates costimulatory molecules on the surface of the T cells. In certain embodiments, PBMCs or isolated T cells are contacted with stimulatory and costimulatory agents, e.g., anti-CD3 and anti-CD28 antibodies, typically bound to beads or other surfaces, in culture medium containing appropriate cytokines (see Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999). In certain embodiments, T cells may be activated and stimulated to expand with feeder cells and appropriate antibodies and cytokines, such as using methods described in U.S. Patent Publication No. 6,040,177; U.S. Patent Publication No. 5,827,642; or WO2012 / 129514.

[0227] In certain embodiments, K562 cells, U937 cells, 721.221 cells, T2 cells or C1R cells can be engineered to generate artificial APCs (aAPCs) that can be used to induce stable expression and secretion of various costimulatory molecules and cytokines. aAPCs are described in WO03 / 057171 and US Patent Publication No. 2003 / 0147869.

[0228] In certain embodiments, HSPCs can be isolated from or differentiated from HSPCs as described, e.g., in U.S. Patent Publication No. 7,399,633; U.S. Patent Publication No. 5,004,681; U.S. Patent Publication No. 2010 / 0183564; WO2006 / 047569; WO2007 / 095594; WO2011 / 127470; or WO2011 / 127472; Vamum-Finney, et al., 1993, Blood 101:1784-1789; Delaney, et al., 2005, Blood 106:2693-2699; Ohishi, et al., 2002, J. Clin. Invest. 110:1165-1174; Delaney, et al., 2010, Nature Med. 16(2): or Chapter 2 of Regenerative Medicine, Department of Health and Human Services, August 2006, or in the references cited therein. The harvesting and processing of other types of cells described herein will be known to those of skill in the art.

[0229] In certain embodiments, the steps of isolating, incubating, growing and / or recombining are performed in a sterile or sealed environment and / or in an automated manner, e.g., under the control of a computer linked to the device in which each step is performed. The final formulation of recombinant cells to prepare a preparation for administration to a subject is known to those skilled in the art, and aspects relevant to this process are described elsewhere herein.

[0230] Also, immune cells can be genetically modified in vivo using targeting viral vectors and / or nanoparticles.The viral vectors that can be used to deliver genes encoding fusion proteins to cells, and various targeting viral vectors (e.g., pseudotyped viral vectors) are known to those skilled in the art.

[0231] Exemplary cell-targeting nanoparticles include nanoparticles with cell-targeting ligands (e.g., CD3, CD4, CD8, CD34) attached to their surface that are selectively taken up by a selected cell type due to the cell-targeting ligands attached to their surface, and then the nanoparticles deliver the genetically modified moiety to express DARIC.

[0232] Exemplary nanoparticles include liposomes (tiny vesicles with at least one lipid bilayer surrounding an aqueous core forming a concentric sphere), liposomal nanoparticles (liposomal structures used to encapsulate smaller nanoparticles within their cores), and lipid nanoparticles (liposome-like structures that do not have the continuous lipid bilayer characteristic of liposomes). Other polymer-based nanoparticles can also be used, as well as porous nanoparticles composed of materials capable of forming a porous network. Exemplary materials include metals, transition metals, and metalloids (e.g., lithium, magnesium, zinc, aluminum, and silica).

[0233] Nanoparticles intended for in vivo delivery and cellular uptake may have an uncharged or negatively charged coating and may be 130 nm or smaller in size. Nanoparticle dimensions can be measured using conventional techniques such as, for example, dynamic light scattering and / or electron microscopy.

[0234] (vi) Preparation

[0235] The formulations described herein may include ex vivo recombinant cells, vectors for ex vivo or in vivo transduction, and dimerizing agents (e.g., rapamycin and / or its analogs).

[0236] A "pharmaceutical" formulation or composition comprises an active compound (e.g., a genetically modified cell, a viral vector, a nanoparticle, a drug molecule, or a dimerizing agent) formulated for administration in a pharmaceutically acceptable carrier.

[0237] "Pharmaceutically acceptable" refers to compounds, materials and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without causing undue toxicity, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. In certain cases, pharma- ceutically acceptable carriers are those approved by the relevant regulatory authorities (e.g., the U.S. Food and Drug Administration (US FDA)).

[0238] Depending on the active compound (i.e., active ingredient) to be delivered and the context, the "pharmaceutical acceptable carrier" includes adjuvants, excipients, glidants, diluents, preservatives, dyes / colorants, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, isotonicity agents, solvents, surfactants or emulsifiers in accordance with the above requirements. Exemplary pharmaceutical acceptable carriers are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, the formulations and compositions can be prepared to meet the standards of sterility, pyrogenicity, general safety and purity as required by the Office of Biologics Standards of the U.S. FDA and / or other relevant regulatory authorities of other countries.

[0239] Exemplary pharma- ceutically acceptable carriers include saline, buffered saline, saline, water, Hank's solution, Ringer's solution, Nonnosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, Ill.), glycerol, ethanol, and combinations thereof. In certain embodiments, human serum albumin (HSA) or other human serum components or fetal bovine serum can be added to the carrier. In certain embodiments, the carrier for infusion comprises buffered saline with 5% HSA or dextrose. Alternative tonicity agents include polyhydric sugar alcohols, including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, and the like.

[0240] Furthermore, pharma- ceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art and are described, for example, in the Physicians Desk Reference, 62nd edition. Oradell, NJ: Medical Economics Co., 2008; Goodman & Gilman's The Pharmacological Basis of Therapeutics, Eleventh Edition. McGraw-Hill, 2005; Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000; and The Merck Index, Fourteenth Edition. Whitehouse Station, NJ: Merck Research Laboratories, 2006.

[0241] The carrier may contain a buffer such as a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a gluconate buffer, an oxalate buffer, a lactate buffer, an acetate buffer, a phosphate buffer, a histidine buffer and / or a trimethylamine salt.

[0242] Stabilizers refer to a wide variety of excipients that can have various functions, from additives that can prevent cell adhesion to the walls of the container to bulking agents. Typical stabilizers include polyhydric sugar alcohols, amino acids, organic sugars, sugar alcohols, PEG, sulfur-containing reducing agents, bovine serum albumin, gelatin, immunoglobulins, polyvinylpyrrolidone, and sugars.

[0243] Where necessary or beneficial, the formulation may include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0244] Exemplary preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides, hexamethonium chloride, alkylparabens, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0245] The therapeutically effective amount of the active ingredient contained in the formulation may be in the range of 0.1-5 μg / kg or 0.5-1 μg / kg. In another example, the dosage may be 1 μg / kg, 30 μg / kg, 90 μg / kg, 150 μg / kg, 500 μg / kg, 750 μg / kg, 0.1-5 mg / kg, or 0.5-1 mg / kg. In another example, the dosage may be 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0246] The therapeutically effective amount of the dimerizer contained in the formulation per body surface area of ​​the subject is 0.1 to 5 mg or 0.1 to 5 mg / m2 , or 0.5-1 mg or 0.5-1 mg / m 2 In another example, the dose may be in the range of 0.1 mg / m 2 , 0.3 mg / m 2 , 0.75mg, 0.9mg, 1.5mg, 0.1-5mg or 0.1-5mg / m 2 , or 0.5-1 mg or 0.5-1 mg / m 2 In another example, the dose is 0.1 mg / m 2 , 0.2 mg / m 2 , 0.3 mg / m 2 , 0.4 mg / m 2 , 0.5 mg / m 2 , 0.6 mg / m 2 , 0.7 mg / m 2 , 0.75 mg or 0.75 mg / m 2 In certain embodiments, the therapeutically effective amount of dimerizer in the formulation may be 1.5 mg, 0.8 mg, 0.9 mg, 1 mg or more. 2 For subjects larger than 1.5 mg, the therapeutically effective amount of the dimerizer in the formulation may be 0.75 mg to 5.0 mg. 2 0.75 mg / m for the following subjects: 2 Less than (e.g. 0.50 mg / m 2 ) are mentioned.

[0247] The therapeutically effective amount of the dimerizer contained in the formulation should be a dose that maintains the target trough blood concentration of the dimerizer per unit blood volume at 1 to 4 ng / mL (e.g., 1.5 to 3 ng / mL). In another example, the dose may be such that the target trough blood concentration is 1 ng / mL, 1.1 ng / mL, 1.2 ng / mL, 1.3 ng / mL, 1.4 ng / mL, 1.5 ng / mL, 1.6 ng / mL, 1.7 ng / mL, 1.8 ng / mL, 1.9 ng / mL, 2.0 ng / mL, 2.1 ng / mL, 2.2 ng / mL, 2.3 ng / mL, 2.4 ng / mL, 2.5 ng / mL, 2.6 ng / mL, 2.7 ng / mL, 2.8 ng / mL, 2.9 ng / mL, or 3.0 ng / mL. In another example, the dose may be such that the target trough blood concentration is in the range of 1 ng / mL to 5 ng / mL. In certain embodiments, the therapeutically effective amount of the dimerizer contained in the formulation should be a dose such that the target trough blood concentration of the dimerizer per unit blood volume is maintained at 2 ng / mL. In certain embodiments, the therapeutically effective amount of the dimerizer contained in the formulation should be a dose such that the target trough blood concentration of the dimerizer per unit blood volume is maintained at 1.5-3 ng / mL.

[0248] In certain embodiments, administration of the formulation causes a dimerizer to bind to the multimerization domain of the first fusion protein and the multimerization domain of the second fusion protein, thereby multimerizing the first fusion protein and the second fusion protein to form a DARIC.

[0249] The formulation and composition can be prepared for administration, for example, by injection, infusion, perfusion, lavage or oral ingestion.The formulation and composition can be formulated for bone marrow injection, intravenous injection, intradermal injection, intraarterial injection, intralymph node injection, intralymphatic injection, intraperitoneal injection, intralesional injection, intraprostatic injection, intravaginal injection, intrarectal injection, external injection, intrathecal injection, intratumoral injection, intramuscular injection, intravesical injection and / or subcutaneous injection.In certain embodiments, the administration of the dimerizer utilizes oral liquid formulations and / or oral solid tablets.

[0250] (vii) Treatment method

[0251] In certain embodiments, the present disclosure provides a method of modulating immune cell activation, comprising: Administering to a subject in need of regulating immune cell activation an effective amount of cells expressing DARIC comprising a first fusion protein comprising a first multimerization domain and a second fusion protein comprising a second fusion domain and an intracellular portion, and an agent that induces expression of DARIC in the cells, or a pharmaceutical composition comprising the same; administering a dimerizer; Including, The dimerizing agent binds to the first multimerization domain and the second multimerization domain, thereby priming DARIC to induce signal transduction. In certain embodiments, the first fusion protein further comprises a binding domain that binds to a target cell antigen. When the binding domain of DARIC binds to the target cell antigen, the immune cell is activated.

[0252] In another embodiment, the first fusion protein does not include a binding domain, but further includes an intracellular portion. When a dimerizer binds to the first multimerization domain and the second multimerization domain, DARIC may be primed to induce signal transduction, resulting in the generation of a signal.

[0253] "Administer," "administering," or "administration" refers to the administration of a therapy or the application of a therapy.

[0254] The present disclosure provides a method and composition for priming DARIC to induce signal transduction, and the method and composition comprises inducing the multimerization of at least a first fusion protein and a second fusion protein to form DARIC.More specifically, the present disclosure relates to using rapamycin or its analogue to regulate the multimerization of a first fusion protein comprising FKBP-rapamycin binding multimerization domain and a second fusion protein comprising FK506 binding protein multimerization domain to form DARIC, and priming the DARIC to induce signal transduction.

[0255] In another aspect, the disclosure provides a method for inhibiting the growth, metastasis or metastatic growth of a malignant tumor (e.g., a solid malignant tumor or a hematological malignant tumor), comprising administering to a subject in need of inhibiting the growth, metastasis or metastatic growth of a malignant tumor an effective amount of cells encoding DARIC and a dimerizing agent provided herein, or a composition comprising the same.

[0256] A "subject in need" refers to a subject at risk for or suffering from a disease, disorder or condition that would benefit from treatment or mitigation with a non-naturally occurring cell, non-naturally occurring polypeptide complex, or composition thereof provided herein. In certain embodiments, the subject is a human. In certain embodiments, the subject is a pediatric patient. In certain embodiments, the subject is 18 years of age or younger. In certain embodiments, the subject is at least 18 years of age. In certain embodiments, the subject is a late adolescent subject, typically defined as 18-21 years of age. In certain embodiments, the subject is 18, 19, 20, 21, 22, 23, 23, 25, 26, 27, or 28 years of age. In certain embodiments, the subject is 16-30 years of age. In further embodiments, the subject is 31 years of age or younger, 30 years of age or younger, or 26 years of age or younger. In further embodiments, the subject is an adult (greater than 31 years of age).

[0257] In certain embodiments, the subject has or has been diagnosed with cancer, an infectious disease, an autoimmune disease, an inflammatory disease, an immune deficiency, or a condition related thereto.

[0258] Treatment with the compositions and methods disclosed herein is believed to be effective against a variety of cancers, including solid and hematological malignancies. The types of cancer that may be treated with the compositions and methods disclosed herein include adenocarcinomas of the breast, prostate, pancreas, colon and rectum; all forms of bronchogenic lung cancer (including squamous cell carcinoma, adenocarcinoma, small cell lung cancer and non-small cell lung cancer); myeloid cancer; melanoma; hepatoma; neuroblastoma; papilloma; apdoma; stratoma; branchiocarcinoma; malignant carcinoid syndrome; carcinoid heart disease; and carcinomas (e.g. Walker's carcinoma, basal cell carcinoma, basosquamous cell carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich's tumor, Krebs 2 carcinoma, Merkel cell carcinoma, mucinous carcinoma, non-small cell lung carcinoma, oat cell carcinoma, papillary carcinoma, scirrhous carcinoma, bronchiolar carcinoma, bronchogenic, squamous cell carcinoma, and transitional cell carcinoma). Other types of cancer that may be treated include histiocytic diseases; leukemia; malignant histiocytosis; Hodgkin's disease; non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; renal cell carcinoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumor; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma; mesenchymoma; mesonephroma; sarcoma; ameloblastoma; cementoma; odontoma; teratoma; thymoma; and trophoblastic tumor.

[0259] In certain embodiments, the subject has or has been diagnosed with a solid cancer. In certain embodiments, the solid cancer is selected from the group including lung cancer (e.g., non-small cell lung cancer), squamous cell carcinoma (e.g., head and neck squamous cell carcinoma), colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, and brain cancer (e.g., glioma, glioblastoma, or oligodendroglioma).

[0260] "Treatment," "treat," or "treated" refers to therapeutic or prophylactic treatment. A "treatment" is a therapeutic treatment if at least one symptom of the disease of the individual receiving the treatment is ameliorated, or the treatment slows the progression of the individual's disease or prevents the onset of another related disease.

[0261] Additionally, cancer types that may benefit from treatment include: adenoma; cholangiomas; cholesteatomas; cystadenocarcinomas; cystadenomas; granulosa cell tumors; ginandroblastomas; hepatomas; hidradenomas; islet cell tumors; Leydig cell tumors; papillomas; Sertoli cell tumors; theca cell tumors; leiomyomas; leiomyosarcomas; myoblastomas; fibroids; myosarcomas; rhabdomyoma; rhabdomyosarcomas; ependymomas; gangliocytoma; gliomas; medulloblastomas; meningiomas; schwannoma; neuroblastomas; neuroepithelioma; neurofibroma; neuroma; paraganglioma; nonchromaffin paraganglioma; and glioblastoma multiforme. Additionally, other types of cancer that may be treated include angiokeratoma; eosinophilic angiolymphocytic hyperplasia; sclerosing hemangioma; hemangiomatosis; glomus hemangiomatosis; hemangioendothelioma; hemangioma; hemangiopericytoma; angiosarcoma; lymphangioma; lymphangioleiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; phyllodes cystosarcoma; fibrosarcoma; angiosarcoma; leiomyosarcoma; leukemia sarcoma; liposarcoma; lymphangiosarcoma; myxosarcoma; ovarian cancer; rhabdomyosarcoma; sarcoma; neoplasm; neurofibromatosis; and cervical dysplasia.

[0262] Further examples of cancers that may be effectively treated with the compositions and methods disclosed herein include B-cell cancers, including B-cell lymphomas (various forms of B-cell lymphoma, such as Hodgkin's disease, non-Hodgkin's lymphoma (NHL), central nervous system lymphoma, etc.), leukemias (acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myeloblastic leukemia, etc.), and myelomas (such as multiple myeloma). Additionally, B-cell cancers include small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, isolated bone plasmacytoma, extraskeletal plasmacytoma, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma / leukemia, B-cell proliferations with malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

[0263] In certain embodiments, the subject has or has been diagnosed with a hematological malignancy. In certain embodiments, the hematological malignancy is leukemia, lymphoma, or multiple myeloma. In certain embodiments, the hematological malignancy is acute myeloid leukemia (AML).

[0264] In another aspect, the disclosure provides a method for treating an autoimmune or inflammatory disease, disorder, or condition, comprising administering to a subject in need of treatment for an autoimmune or inflammatory disease, disorder, or condition an effective amount of a cell comprising a DARIC as described herein and a dimerizing agent, or a composition comprising the same.

[0265] Exemplary autoimmune or inflammatory diseases, disorders, or conditions that may be treated by the fusion proteins, compositions, or unit dosage forms thereof of the invention include inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), diabetes (e.g., type 1 diabetes), dermatomyositis, polymyositis, pernicious anemia, primary biliary cirrhosis, acute disseminated encephalomyelitis (ADEM), Addison's disease, ankylosing spondylitis, antiphospholipid syndrome (APS), autoimmune hepatitis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), and others. ), Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, lupus nephritis, neuropsychiatric manifestations of systemic lupus erythematosus, multiple sclerosis (MS), myasthenia gravis, pemphigus vulgaris, asthma, psoriatic arthritis, rheumatoid arthritis, Sjögren's syndrome, temporal arteritis (also known as "giant cell arteritis"), autoimmune hemolytic anemia, bullous pemphigoid, vasculitis, celiac disease, chronic obstructive pulmonary disease, endometriosis, hidradenitis suppurativa, interstitial cystitis, morphea, scleroderma, narcolepsy, neuromyotonia, vitiligo, and autoimmune inner ear disease.

[0266] In certain embodiments, the method of treating a hyperproliferative disease, an inflammatory disease, an autoimmune disease, or a graft-versus-host disease comprises: (a) administering a recombinant cell comprising a first nucleic acid molecule encoding a first fusion protein comprising a first multimerization domain, a transmembrane domain and an intracellular portion, and a second nucleic acid molecule encoding a second fusion protein comprising a binding domain and a second multimerization domain; (c) administering a dimerizer; Including, the dimerization agent associates with and is positioned between the multimerization domains of the first fusion protein and the multimerization domain of the second fusion protein, thereby promoting the formation of DARIC on the surface of the recombinant cell; The binding domain of DARIC specifically binds to targets on the surface of diseased cells of a hyperproliferative disease, inflammatory disease, autoimmune disease or graft-versus-host disease, thereby promoting an immunomodulatory response and thereby treating the disease.

[0267] In certain embodiments, the method of treating a hyperproliferative disease, an inflammatory disease, an autoimmune disease, or a graft-versus-host disease comprises: (a) administering one or more recombinant cells comprising a first nucleic acid molecule encoding a first fusion protein comprising a first multimerization domain and a second nucleic acid molecule encoding a second fusion protein comprising a second multimerization domain; (c) administering a dimerizer; Including, the dimerization agent associates with and is positioned between the multimerization domains of the first fusion protein and the multimerization domain of the second fusion protein, thereby promoting the formation of a DARIC (e.g., a BiTE) primed for signaling; The binding domain of DARIC specifically binds to targets on the surface of diseased cells of a hyperproliferative disease, inflammatory disease, autoimmune disease or graft-versus-host disease, thereby promoting an immunomodulatory response and thereby treating the disease.

[0268] In certain embodiments, the cells are genetically engineered to express components of DARIC. In certain embodiments, the cells are genetically engineered to express a first fusion protein comprising a first multimerization domain and a second fusion protein comprising a second multimerization domain. In certain embodiments, the cells can be genetically engineered in vivo or ex vivo. In certain embodiments, the genetically engineered cells are administered to the subject at a cell dose per subject's body weight, the dose being 1×10 5 pieces / kg~2000×10 6 pieces / kg, 1×10 6 pieces / kg~1000×10 6 pieces / kg, 1×10 6 pieces / kg~100×10 6 pieces / kg, 5×10 6 pieces / kg~500×10 6 pieces / kg, 10×10 6 pieces / kg~1000×10 6 pieces / kg, 1×106 pieces / kg~2×10 6 pieces / kg, 3×10 6 pieces / kg~5×10 6 Pieces / kg, or 7.5 x 10 6 pieces / kg~10×10 6 The range is 1 / kg.

[0269] In certain embodiments, the subject is lymphodepleted prior to administration of the genetically modified cells or prior to genetically modifying the cells. In certain embodiments, lymphodepletion is performed with 30 mg / m 2 of fludarabine administered intravenously once daily for 4 days at 500 mg / m 2 of cyclophosphamide administered intravenously once daily for two days. In certain embodiments, cyclophosphamide is administered on days 3 and 4 of administration of fludarabine. In certain embodiments, lymphodepletion is initiated 5 days prior to administering the cells or 5 days prior to genetically modifying the cells.

[0270] A dose of 1m 2 When listed in grams per square meter, the dose is calculated based on the subject's body surface area. For example, "0.50 mg / m 2 " is a dose equivalent to 1 m of body surface area. 2 This means that a dose of 0.50 mg will be administered to each patient.

[0271] In certain embodiments, the dimerizer is administered orally. The dimerizer can be administered orally, for example, as a liquid or a solid (e.g., a solid tablet). In certain embodiments, the dimerizer is administered orally in a dose of 1.5 mg. 2 In certain embodiments, the dimerizer is administered orally to subjects greater than 1.5 mg at a dose of 0.75 mg, 1.0 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2.0 mg, 2.25 mg, 2.5 mg, 2.75 mg, 3.0 mg, 3.25 mg, 3.5 mg, 3.75 mg, or 4 mg. 2 In certain embodiments, the dimerizer is administered orally to subjects greater than 1.5 mg at a dose of 0.75 mg or 3.0 mg.2 In certain embodiments, the dimerizer is administered orally to a subject greater than 1.5 mg at a dose of at least 0.75 mg. 2 In certain embodiments, the dimerizer is administered orally to a subject greater than 1 mg in a dose of at least 1 mg. 2 In certain embodiments, the dimerizer is administered orally to a subject greater than 1.5 mg at a dose of at least 1.25 mg. 2 In certain embodiments, the dimerizer is administered orally to a subject greater than 1.5 mg in a dose of at least 1.5 mg. 2 In certain embodiments, the dimerizer is administered orally to a subject greater than 1.5 mg at a dose of at least 1.75 mg. 2 In certain embodiments, the dimerizer is administered orally to subjects greater than 1.5 mg at a dose of at least 2 mg. 2 In certain embodiments, the dimerizer is administered orally to subjects greater than 1.5 mg at a dose of at least 2.25 mg. 2 In certain embodiments, the dimerizer is administered orally to subjects greater than 1.5 mg at a dose of at least 2.5 mg. 2 In certain embodiments, the dimerizer is administered orally to a subject greater than 1.5 mg at a dose of at least 2.75 mg. 2 To subjects greater than 2.5 mm in diameter, a dose of 3.0 mg is administered orally.

[0272] In certain embodiments, the dimerizer is 1.5m 2 0.50 mg / m 2 In certain embodiments, the dimerizer is administered orally at a dose of 1.5 mg. 2 0.1-2.0mg / m for the following subjects: 2In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration at 2 ng / mL. In various embodiments, the dosing should be adjusted to maintain a target trough blood concentration within a target range of 1-4 ng / mL. In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration within a target range of 1.5-3 ng / mL. In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration within a target range of 3-9 ng / mL. In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration within a target range of 1-2 ng / mL.

[0273] In certain embodiments, rapamycin or an analog thereof is 1.5 mM 2 In certain embodiments, rapamycin or an analog thereof is administered orally to subjects greater than 1.5 mg at a dose of 0.75 mg or more. 2 0.50 mg / m 2 In certain embodiments, the rapamycin or analogue is administered orally at a dose of 0.1 to 2.0 mg / m 2 In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration at 2 ng / mL. In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration within a target range of 1.5-3 ng / mL. In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration within a target range of 3-9 ng / mL. In certain embodiments, the dosing should be adjusted to maintain a target trough blood concentration within a target range of 1-2 ng / mL.

[0274] In certain embodiments, administration of the dimerizing agent is initiated on day 2 after infusion of the recombinant cell product. In certain embodiments, administration of the dimerizing agent is initiated on day 1 after infusion of the recombinant cell product. In certain embodiments, administration of the dimerizing agent is initiated on day 3 after infusion of the recombinant cell product. In certain embodiments, administration of the dimerizing agent is initiated on day 4 after infusion of the recombinant cell product. In certain embodiments, the dimerizing agent is administered daily after initiation of administration. In certain embodiments, the dimerizing agent is administered twice daily. In certain embodiments, the dimerizing agent is administered daily on days 2-21 (or days 3-21) after infusion of the recombinant cell product. In certain embodiments, the dimerizer is administered daily for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of administration. In certain embodiments, the dimerizer is administered at least 16 hours after administration of the cells. In certain embodiments, the dimerizer is administered at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, or at least 84 hours after administration of the cells. In certain embodiments, the dimerizer is administered 1-4 days, 1-3 days, 1-2 days, 2-4 days, 3-4 days, or 2-3 days after administration of the cells. In certain embodiments, administration of the dimerizer includes a drug holiday before administering the next course of the dimerizer, hi certain embodiments, the drug holiday is at least 10-45 days.

[0275] In certain embodiments, the rapamycin or analogue thereof is administered on the second day after injection of the recombinant cell product. In certain embodiments, the rapamycin or analogue thereof is administered on the first day after injection of the recombinant cell product. In certain embodiments, the rapamycin or analogue thereof is administered on the third day after injection of the recombinant cell product. In certain embodiments, the rapamycin or analogue thereof is administered on the fourth day after injection of the recombinant cell product. In certain embodiments, the rapamycin or analogue thereof is administered daily. In certain embodiments, the rapamycin or analogue thereof is administered twice daily. In certain embodiments, the rapamycin or analogue thereof is administered daily from day 2 to day 21 after injection of the recombinant cell product. In certain embodiments, the rapamycin or analogue thereof is administered for 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days. In certain embodiments, the dimerizer and dose of cells are administered simultaneously. In certain embodiments, the rapamycin or analogue thereof is administered on the same day as the dose of cells. In certain embodiments, the rapamycin or analogue thereof is administered at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, or at least 84 hours after administration of the cells. In certain embodiments, the rapamycin or analogue thereof is administered 1-4 days, 1-3 days, 1-2 days, 2-4 days, 3-4 days, or 2-3 days after administration of the cells. In certain embodiments, administration of rapamycin or an analog thereof includes a drug holiday prior to administration of the next course of rapamycin or an analog thereof, hi certain embodiments, the drug holiday is at least 10-45 days.

[0276] "Course" refers to the administration of a drug, compound or treatment in one or more separate doses, as it refers to a treatment or method of treatment. For example, a course may include a dose of a drug, compound or treatment once a day for 10 days. After a rest period, another course of treatment can be administered.

[0277] In certain embodiments, a bone marrow aspirate and / or biopsy is performed and disease is analyzed. In certain embodiments, a bone marrow aspirate and / or biopsy is performed on day 28. In certain embodiments, subjects with less than 1% morphological remission of disease as measured by multiparameter flow cytometry continue to have a dimerizer off-dose. In certain embodiments, subjects with evidence of persistent disease at greater than 1% on bone marrow evaluation may be administered a subsequent course of dimerizer. In certain embodiments, the disease is leukemia. In certain embodiments, the disease comprises leukemia. In certain embodiments, subjects in remission are administered a subsequent course of dimerizer. In certain embodiments, subjects without grade 3 or higher toxicity are administered a subsequent course of dimerizer. In certain embodiments, subjects in remission with absolute phagocyte counts (APC) greater than 500 cells / μL are administered a subsequent course of dimerizer. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 400 cells / μL is administered a subsequent course of a dimerizer. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 300 cells / μL is administered a subsequent course of a dimerizer. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 300 cells / μL is administered a subsequent course of a dimerizer. In certain embodiments, a subsequent course of a dimerizer is administered 42 days after the subject is infused with cells engineered to express DARIC. In certain embodiments, the next course of dimerizer is administered 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days after injecting cells engineered to express DARIC into the subject. In certain embodiments, the next course of dimerizer is administered on any day after injecting cells engineered to express DARIC into the subject. In certain embodiments, the next course of dimerizer is administered 14 days after the withdrawal of the previous dimerizer.In certain embodiments, the next course of dimerizer is administered 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more days after the cessation of administration of the previous dimerizer.

[0278] In certain embodiments, a bone marrow aspirate and / or biopsy is performed and analysis of disease is performed. In certain embodiments, a bone marrow aspirate and / or biopsy is performed on day 28. In certain embodiments, a holiday from rapamycin or an analogue is continued in subjects with less than 1% morphological remission of disease as measured by multi-parameter flow cytometry. In certain embodiments, a next course of rapamycin or an analogue may be administered to subjects with evidence of persistent disease greater than 1% in bone marrow evaluations. In certain embodiments, the disease is leukemia. In certain embodiments, the disease comprises leukemia. In certain embodiments, a next course of rapamycin or an analogue is administered to subjects in remission. In certain embodiments, a next course of rapamycin or an analogue is administered to subjects in the absence of grade 3 or higher toxicity. In certain embodiments, a next course of rapamycin or an analogue is administered to subjects in remission with an absolute phagocyte count (APC) greater than 500 cells / μL. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 400 cells / μL is administered a next course of rapamycin or an analog thereof. In certain embodiments, a subject in remission with an absolute phagocyte count (APC) of greater than 300 cells / μL is administered a next course of rapamycin or an analog thereof. In certain embodiments, a next course of rapamycin or an analog thereof is administered 42 days after the subject is infused with cells engineered to express DARIC. In certain embodiments, a next course of rapamycin or an analogue thereof is administered 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 days after infusion of cells recombinantly engineered to express DARIC into the subject.In certain embodiments, the next course of rapamycin or an analog thereof is administered any day after the cells engineered to express DARIC are injected into the subject. In certain embodiments, the next course of rapamycin or an analog thereof is administered 14 days after the cessation of administration of the previous rapamycin or an analog thereof. In certain embodiments, the next course of rapamycin or an analog thereof is administered 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or more days after the cessation of administration of the previous rapamycin or an analog thereof.

[0279] The non-native cells, fusion proteins, dimerizers and other accessory molecules described above may be used in the therapeutic methods of the present disclosure.

[0280] (viii) Exemplary embodiments Exemplary embodiments - set 1 1. Identifying a subject having cells expressing a dimerizer-regulated immunomodulatory complex (DARIC); administering to said subject a course of rapamycin or an analogue thereof; A method comprising: the DARIC comprises a first fusion protein comprising a CD33 single domain heavy chain variable (VHH) binding domain, an FK506 binding protein (FKBP) multimerization domain, and a transmembrane domain, and a second fusion protein comprising an FKBP-rapamycin binding (FRB) multimerization domain, a transmembrane domain, and an intracellular portion; said rapamycin or analogue thereof being bound to and positioned between the multimerization domains of a first fusion protein and the multimerization domain of a second fusion protein; The course is: achieving a blood trough concentration of the rapamycin or analogue thereof of 1.5 ng / mL to 3 ng / mL; Beginning 2 or 3 days after the subject has in vivo cells expressing DARIC; by daily administration of said rapamycin or analogue thereof for 18, 19 or 20 days; The target is 1.5m 2 or administering a daily dose of 0.75 mg or more when the subject is greater than 1.5 m 2 including administering a daily dose of less than 0.75 mg, and and including a washout period at the end of said course during which rapamycin or an analog thereof is not administered to said subject. A method comprising: 2. Identifying a subject having cells expressing a dimerizer-regulated immunomodulatory complex (DARIC); administering to said subject a course of rapamycin or an analogue thereof; A method comprising: The DARIC comprises a first fusion protein comprising an extracellular portion, an FK506 binding protein (FKBP) multimerization domain or an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof, and a transmembrane domain, and a second fusion protein comprising an FK506 binding protein (FKBP) multimerization domain or an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof, a transmembrane domain, and an intracellular portion; said rapamycin or analogue thereof being bound to and positioned between the multimerization domains of a first fusion protein and the multimerization domain of a second fusion protein; The course is: achieving a blood trough concentration of the rapamycin or analogue thereof of 1.5 ng / mL to 3 ng / mL; Beginning 0-4 days after the subject has in vivo cells expressing the DARIC; by daily administration of said rapamycin or analogue thereof for at least 14 days; and and including a washout period at the end of said course during which rapamycin or an analog thereof is not administered to said subject. The method, characterized by any one or more of the following: 3. Identifying a subject having cells expressing a dimerizer-regulated immunomodulatory complex (DARIC); administering to said subject a course of rapamycin or an analogue thereof; A method comprising: The DARIC comprises a first fusion protein comprising an extracellular portion, an FK506 binding protein (FKBP) multimerization domain or an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof, and a transmembrane domain, and a second fusion protein comprising an FK506 binding protein (FKBP) multimerization domain or an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof, a transmembrane domain, and an intracellular portion; said rapamycin or analogue thereof being bound to and positioned between the multimerization domains of a first fusion protein and the multimerization domain of a second fusion protein; The course is: achieving a blood trough concentration of the rapamycin or analogue thereof of 1.5 ng / mL to 3 ng / mL; The target is 1.5m 2 or administering a daily dose of 0.75 mg or more when the subject is greater than 1.5 m 2 including administering a daily dose of less than 0.75 mg, and and including a washout period at the end of said course during which rapamycin or an analog thereof is not administered to said subject. The method, characterized by any one or more of the following: 4. Identifying a subject having cells expressing a dimerizer-regulated immunomodulatory complex (DARIC); administering to said subject a course of rapamycin or an analogue thereof; A method comprising: the DARIC comprises a first fusion protein comprising a multimerization domain and a second fusion protein comprising a multimerization domain; The method, wherein the rapamycin or analogue thereof is bound to and positioned between the multimerization domain of a first fusion protein and the multimerization domain of a second fusion protein. 5. The method of embodiment 4, wherein the course achieves a blood trough concentration of 1.0 ng / mL to 3 ng / mL of the rapamycin or analogue thereof. 6. The method of embodiment 4, wherein the course begins 0 to 4 days after the subject has in vivo cells expressing the DARIC. 7. The method of any one of embodiments 4 to 6, wherein the course begins 2 or 3 days after the subject has in vivo cells expressing the DARIC. 8. The method of any one of embodiments 4 to 7, wherein the course is administered for at least 14 days. 9. The method of any one of embodiments 4 to 8, wherein the course is administered daily for 18 days, 19 days, 20 days, 21 days or 22 days. 10. The course is for a subject with a body surface area of ​​1.5 m 2 or administering a daily dose of at least 0.75 mg to a subject having a body surface area greater than 1.5 m 2 The method of any one of embodiments 4-9, comprising administering a daily dose of less than 0.75 mg if: 11. The subject's body surface area is 1.5 m 2 and said daily dose is between 0.75 mg and 3.5 mg. 12. The subject's body surface area is 1.5 m 2 and said daily dose is 0.25 mg / m or less. 2 ~0.74mg / m 2 11. The method of embodiment 10, wherein 13. The subject's body surface area is 1.5 m 2and said daily dose is 0.50 mg / m or less. 2 11. The method of embodiment 10, wherein 14. The method of embodiment 10, wherein the course includes a course of daily administration followed by a drug holiday during which rapamycin or an analogue thereof is not administered to the subject. 15. The method of embodiment 14, wherein the drug holiday is 13 days, 14 days, or 15 days. 16. The method of embodiment 14, wherein the drug holiday is at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, or at least 22 days. 17. The method of embodiment 14, wherein the drug holiday period is at least 14 days. 18. The method of embodiment 14, wherein the drug holiday period is 14 days. 19. The method of embodiment 14, further comprising administering a second course of rapamycin or an analog thereof after the drug holiday period. 20. The method of embodiment 19, wherein the second course achieves a blood trough concentration of 1.0 ng / mL to 3 ng / mL of the rapamycin or analogue thereof. 21. The method of embodiment 19 or 20, wherein the second course is administered for at least 14 days. 22. The method of embodiment 19 or 20, wherein the second course is carried out for 18 days, 19 days, 20 days, 21 days or 22 days by daily administration of said rapamycin or analogue thereof. 23. The second course is for the subject's body surface area to be 1.5 m 2 or administering a daily dose of at least 0.75 mg to a subject having a body surface area greater than 1.5 m 2 The method of any one of embodiments 19-22, comprising administering a daily dose of less than 0.75 mg if: 24. The multimerization domain of the first fusion protein comprises an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof, and the multimerization domain of the second fusion protein comprises an FK506 binding protein (FKBP) multimerization domain or a variant thereof; or The multimerization domain of the first fusion protein comprises an FK506 binding protein (FKBP) multimerization domain or a variant thereof, and the multimerization domain of the second fusion protein comprises an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof; The method according to any one of embodiments 4 to 23. 25. The method of embodiment 24, wherein the FKBP multimerization domain or variant thereof is FKBP12. 26. The method of embodiment 24, wherein the FKBP multimerization domain or a variant thereof has a sequence as set forth in SEQ ID NO: 55 or 56, or has at least 90% sequence identity with the sequence as set forth in SEQ ID NO: 55 or 56. 27. The method of embodiment 24, wherein the FKBP multimerization domain or a variant thereof has a sequence as set forth in SEQ ID NO: 55 or 56, or has at least 95% sequence identity with the sequence as set forth in SEQ ID NO: 55 or 56. 28. The method of embodiment 24, wherein the FKBP multimerization domain or a variant thereof has a sequence as set forth in SEQ ID NO: 55 or 56, or has at least 98% sequence identity with the sequence as set forth in SEQ ID NO: 55 or 56. 29. The method of embodiment 24, wherein the FRB multimerization domain or a variant thereof is FRB T2098L. 30. The method of embodiment 24, wherein the FRB multimerization domain or a variant thereof has a sequence set forth in SEQ ID NO: 57 or 58, or has at least 90% sequence identity with the sequence set forth in SEQ ID NO: 57 or 58. 31. The method of embodiment 24, wherein the FRB multimerization domain or a variant thereof has a sequence set forth in SEQ ID NO: 57 or 58, or has at least 95% sequence identity with the sequence set forth in SEQ ID NO: 57 or 58. 32. The method of embodiment 24, wherein the FRB multimerization domain or a variant thereof has a sequence set forth in SEQ ID NO: 57 or 58, or has at least 98% sequence identity with the sequence set forth in SEQ ID NO: 57 or 58. 33. The method of embodiment 24, wherein, when the first fusion protein and the second fusion protein are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly. 34. The method of embodiment 24, wherein when the first fusion protein and the second fusion protein are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized intracellularly. 35. The method of any one of embodiments 4 to 34, wherein the first fusion protein and / or the second fusion protein further comprises a binding domain. 36. The method of embodiment 35, wherein the binding domain binds to a cancer antigen. 37. The method of embodiment 35 or 36, wherein the binding domain is a single-domain heavy chain variable region (VHH) or a single-chain variable region fragment (scFv). 38. The method of embodiment 35 or 36, wherein the binding domain comprises the extracellular domain of a receptor or a ligand. 39. The method of any one of embodiments 35 to 38, wherein the binding domain comprises a binding domain of a CD33 antibody. 40. The method of embodiment 39, wherein the binding domain of the CD33 antibody is a VHH. 41. The method of embodiment 40, wherein the VHH has an array as shown in any of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21. 42. The method of embodiment 40, wherein the VHH has at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 and specifically binds to CD33. 43. The method of embodiment 40, wherein the VHH has at least 95% sequence identity to any of the sequences set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 and specifically binds to CD33. 44. The method of embodiment 40, wherein the VHH has at least 98% sequence identity to any of the sequences set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 and specifically binds to CD33. 45. The method of any one of embodiments 4 to 44, wherein the first fusion protein and / or the second fusion protein comprises a binding domain that binds to C-type lectin-like molecule 1 (CLL1). 46. ​​The method of embodiment 45, wherein the binding domain that binds to CLL1 has an array as set forth in any of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54. 47. The method of embodiment 45, wherein the binding domain that binds to CLL1 has at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54 and specifically binds to CLL1. 48. The method of embodiment 45, wherein the binding domain that binds to CLL1 has at least 95% sequence identity to any of the sequences set forth in SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54 and specifically binds to CLL1. 49. The method of embodiment 45, wherein the binding domain that binds to CLL1 has at least 98% sequence identity to any of the sequences set forth in SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54 and specifically binds to CLL1. 50. The method of any one of embodiments 4 to 49, wherein the first fusion protein and / or the second fusion protein further comprises an intracellular portion. 51. The method of embodiment 50, wherein the intracellular portion comprises an intracellular primary signaling domain. 52. The method of embodiment 51, wherein the intracellular primary signaling domain comprises CD3ζ or a fragment thereof. 53. The method of embodiment 50 or 51, wherein the intracellular portion comprises a coreceptor domain. 54. The method of any one of embodiments 50 to 53, wherein the intracellular portion comprises a costimulatory domain. 55. The costimulatory domain is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell receptor 55. The method of embodiment 54, comprising associated protein kinase 70 (ZAP70), or a fragment or combination thereof. 56. The method of embodiment 54, wherein the costimulatory domain comprises CD137 (4-1BB) or a fragment or combination thereof. 57. The method of any one of embodiments 4 to 56, wherein the first fusion protein and / or the second fusion protein further comprises a transmembrane domain. 58. The method of embodiment 57, wherein the transmembrane domain of the first fusion protein and / or the second fusion protein is the transmembrane domain of CD4 or the transmembrane domain of CD8α. 59. The method of any one of embodiments 4 to 58, wherein the first fusion protein and / or the second fusion protein further comprises a spacer. 60. (a) a first fusion protein comprising an FRB multimerization domain or a variant thereof; a transmembrane domain of CD8α or a transmembrane domain of CD4; a costimulatory domain of CD137; and / or a primary signaling domain of CD3ζ; (b) the second fusion protein comprises a CD33 VHH having an amino acid sequence as set forth in any one of SEQ ID NOs: 2 to 21; an FKBP multimerization domain or a variant thereof; and a CD4 transmembrane domain or a CD8α transmembrane domain; The method according to any one of embodiments 4 to 59. 61. The method of any one of embodiments 4 to 60, wherein the first fusion protein comprises a signal peptide, a transmembrane domain of CD8α, a costimulatory domain of CD137, and a primary signaling domain of CD3ζ. 62. The method of any one of embodiments 4 to 61, wherein the second fusion protein comprises a signal peptide and a transmembrane domain of CD4. 63. The method of any one of embodiments 4 to 62, wherein the first fusion protein and / or the second fusion protein has a sequence as set forth in any one of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41. 64. The method of any one of embodiments 4 to 63, wherein the first fusion protein and / or the second fusion protein has at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41. 65. A method according to any one of embodiments 4 to 64, wherein the first fusion protein and / or the second fusion protein has the sequence shown in SEQ ID NO: 40. 66. A method according to any one of embodiments 4 to 65, wherein the first fusion protein and / or the second fusion protein has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 40. 67. A method according to any one of embodiments 4 to 66, wherein the first fusion protein and / or the second fusion protein has at least 95% sequence identity to the sequence set forth in SEQ ID NO: 40. 68. A method according to any one of embodiments 4 to 67, wherein the first fusion protein and / or the second fusion protein has at least 98% sequence identity to the sequence set forth in SEQ ID NO: 40. 69. The method of any one of embodiments 4 to 68, wherein the rapamycin or analogue thereof is rapamycin, AP1903, AP20187, AP21967, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus or zotarolimus. 70. A method according to any one of embodiments 4 to 69, wherein at least one subset of cells expressing the DARIC in the identified subject's body are hematopoietic cells. 71. A method according to any one of embodiments 4 to 70, wherein at least one subset of cells expressing the DARIC in the identified subject's body is a T cell. 72. A method according to any one of embodiments 4 to 71, wherein at least one subset of cells expressing the DARIC in the identified subject's body is αβ T cells or γδ T cells. 73. A method according to any one of embodiments 4 to 72, wherein at least one subset of cells expressing the DARIC in the identified subject's body is CD3+ cells, CD4+ cells or CD8+ cells. 74. A method according to any one of embodiments 4 to 73, wherein at least one subset of cells expressing the DARIC in the identified subject's body are effector immune cells. 75. A method according to any one of embodiments 4 to 74, wherein at least one subset of cells expressing the DARIC in the body of the identified subject is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL) or a helper T cell. 76. A method according to any one of embodiments 4 to 75, wherein at least one subset of cells expressing the DARIC in the body of the identified subject is a natural killer (NK) cell or a natural killer T (NKT) cell. 77. The method of any one of embodiments 4 to 76, wherein the cells are isolated from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, splenic tissue or a tumor. 78. The method of embodiment 77, wherein the cells are produced ex vivo to express the DARIC. 79. The cells are administered at a concentration of 1 x 10 per body weight of the subject. 5 pieces / kg~2000×10 6 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 80. The cells are administered at a concentration of 1 x 10 per body weight of the subject. 6 pieces / kg~1000×10 6 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 81. The cells are administered at a concentration of 1 x 10 per body weight of the subject. 6 pieces / kg~100×10 6 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 82. The cells are administered at a concentration of 5×10 per body weight of the subject. 6 pieces / kg~500×106 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 83. The cells are administered at a concentration of 10 x 10 per body weight of the subject. 6 pieces / kg~1000×10 6 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 84. The cells are administered at a concentration of 1 x 10 per body weight of the subject. 6 pieces / kg~2×10 6 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 85. The cells are administered at a concentration of 3×10 per body weight of the subject. 6 pieces / kg~5×10 6 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 86. The cells are administered at a concentration of 7.5 x 10 per unit weight of the subject. 6 pieces / kg~15×10 6 The method of embodiment 78, wherein the subject is administered a dose of 100 mg / kg. 87. The dose of the cells per body weight of the subject is 10 x 10 6 79. The method of embodiment 78, wherein the dose is 1 / kg. 88. The method of any one of embodiments 4 to 87, wherein the cells are engineered to express the DARIC in a subject in vivo. 89. The method of any one of embodiments 4 to 88, wherein the subject is a pediatric patient. 90. The method of any one of embodiments 4 to 88, wherein the subject is 28 years of age or younger. 91. The method of any one of embodiments 4 to 88, wherein the subject is 18 years of age or younger. 92. The method of any one of embodiments 4 to 88, wherein the subject is 18 to 28 years old. 93. The method of any one of embodiments 4 to 88, wherein the subject is an adult patient. 94. The method of any one of embodiments 4 to 88, wherein the subject is at least 18 years of age. 95. The method of any one of embodiments 4 to 94, wherein the subject has or has been diagnosed with cancer, an infectious disease, an autoimmune disease, an inflammatory disease, an immune deficiency, or a condition related thereto. 96. The method of any one of embodiments 4 to 95, wherein the subject has or has been diagnosed with a solid cancer. 97. The method of embodiment 96, wherein the solid cancer comprises lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer or brain cancer. 98. The method of embodiment 97, wherein the lung cancer is non-small cell lung cancer. 99. The method of embodiment 97, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma. 100. The method of embodiment 97, wherein the brain tumor comprises a glioma, a glioblastoma or an oligodendroglioma. 101. The method of any one of embodiments 4 to 100, wherein the subject has or has been diagnosed with a hematological malignancy. 102. The method of embodiment 101, wherein the hematological malignancy is leukemia, lymphoma or multiple myeloma. 103. The method of any one of embodiments 4 to 102, wherein the hematological malignancy is acute myeloid leukemia (AML). 104. A method according to any one of embodiments 4 to 103, wherein the subject is lymphodepleted prior to administration of the course. 105. The method of embodiment 104, wherein said lymphodepletion comprises administering a dose of fludarabine and a dose of cyclophosphamide. 106. The fludarabine is administered at 30 mg / m 2 once daily for four days, and the cyclophosphamide is administered intravenously at a dose of 500 mg / m 2 The method of embodiment 105, wherein the compound is administered intravenously at a dose of 107. The method of any one of embodiments 104 to 106, wherein the cyclophosphamide is administered on the third and fourth days after the administration of the fludarabine. 108. The method of any one of embodiments 104 to 107, wherein the lymphocyte depletion is initiated 7 days prior to administration of the course. Exemplary Embodiments - Set 2 1. A method of treating a subject, comprising: a) administering a dose of cells expressing a dimerizer-regulated immunomodulatory complex (DARIC) comprising a first fusion protein comprising a multimerization domain and a second fusion protein comprising a multimerization domain; b) administering a course of rapamycin or an analogue thereof; Including, The method, wherein the rapamycin or analogue thereof is bound to and positioned between the multimerization domain of a first fusion protein and the multimerization domain of a second fusion protein. 2. A method of treating a subject, comprising: a) editing a cell obtained from the subject to express a dimerizer-regulated immunomodulatory complex (DARIC) comprising a first fusion protein comprising a multimerization domain and a second fusion protein comprising a multimerization domain; b) administering a course of rapamycin or an analogue thereof; Including, The method, wherein the rapamycin or analogue thereof is bound to and positioned between the multimerization domain of a first fusion protein and the multimerization domain of a second fusion protein. 3. A method of priming a dimerizer-regulated immunomodulatory complex (DARIC) to induce signaling in a subject, comprising: a) administering a dose of cells expressing DARIC comprising a first fusion protein comprising a multimerization domain and a second fusion protein comprising a multimerization domain; b) administering a course of rapamycin or an analogue thereof; Including, The method, wherein the rapamycin or analogue thereof is bound to and positioned between the multimerization domain of a first fusion protein and the multimerization domain of a second fusion protein. 4. A method of priming a dimerizer-regulated immunomodulatory complex (DARIC) to induce signaling in a subject, comprising: a) genetically modifying a cell obtained from a subject to express a DARIC comprising a first fusion protein comprising a multimerization domain and a second fusion protein comprising a multimerization domain; b) administering a course of rapamycin or an analogue thereof; Including, The method, wherein the rapamycin or analogue thereof is bound to and positioned between the multimerization domain of a first fusion protein and the multimerization domain of a second fusion protein. 5. The method of any one of embodiments 1 to 4, wherein the second fusion protein further comprises a binding domain. 6. The method of any one of embodiments 1 to 4, wherein the first fusion protein further comprises a binding domain. 7. The method of embodiment 5 or 6, wherein the binding domain binds to a cancer antigen. 8. The method of any one of embodiments 5 to 7, wherein the binding domain is a heavy chain variable region (VHH) or a single chain variable region fragment (scFv). 9. The method of any one of embodiments 5 to 7, wherein the binding domain is a receptor. 10. The method of embodiment 9, wherein the receptor is an extracellular portion of the receptor. 11. The method of any one of embodiments 1 to 10, wherein the second fusion protein further comprises a spacer. 12. The method of any one of embodiments 1 to 11, wherein the first fusion protein further comprises an intracellular portion comprising an intracellular signaling domain. 13. The method of any one of embodiments 1 to 12, wherein the multimerization domain of the first fusion protein comprises an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof, and the multimerization domain of the second fusion protein comprises an FK506 binding protein (FKBP) multimerization domain or a variant thereof. 14. The method of any one of embodiments 1 to 12, wherein the multimerization domain of the first fusion protein comprises an FK506 binding protein (FKBP) multimerization domain or a variant thereof, and the multimerization domain of the second fusion protein comprises an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof. 15. The method of embodiment 13 or 14, wherein the FKBP multimerization domain or variant thereof is FKBP12. 16. The method of embodiment 13 or 14, wherein the FRB multimerization domain or a variant thereof is FRB T2098L. 17. A method according to any one of embodiments 13 to 16, wherein, when the first fusion protein and the second fusion protein are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized outside the cell. 18. A method according to any of embodiments 1 to 17, wherein the administration achieves a target trough blood concentration of rapamycin or an analogue thereof in the range of 0.5 ng / mL to 5 ng / mL. 19. The method of any of embodiments 1-17, wherein the administration achieves a target trough blood concentration of rapamycin or an analogue thereof in the range of 1 ng / mL to 5 ng / mL. 20. A method according to any of embodiments 1 to 17, wherein the administration achieves a target trough blood concentration of rapamycin or an analogue thereof in the range of 1 ng / mL to 4 ng / mL. 21. A method according to any one of embodiments 1 to 17, wherein the administration achieves a target trough blood concentration of rapamycin or an analogue thereof in the range of 1 ng / mL to 3 ng / mL. 22. A method according to any one of embodiments 1 to 17, wherein the administration achieves a target trough blood concentration of rapamycin or an analogue thereof in the range of 1.5 ng / mL to 3 ng / mL. 23. The method of any of embodiments 1-22, wherein said administering achieves a target trough blood concentration of rapamycin or an analogue thereof of 0.5 ng / mL, 1 ng / mL, 1.1 ng / mL, 1.2 ng / mL, 1.3 ng / mL, 1.4 ng / mL, 1.5 ng / mL, 1.6 ng / mL, 1.7 ng / mL, 1.8 ng / mL, 1.9 ng / mL, 2.0 ng / mL, 2.1 ng / mL, 2.2 ng / mL, 2.3 ng / mL, 2.4 ng / mL, 2.5 ng / mL, 2.6 ng / mL, 2.7 ng / mL, 2.8 ng / mL, 2.9 ng / mL or 3.0 ng / mL. 24. The method of any one of embodiments 1 to 22, wherein the administration achieves a target trough blood concentration of rapamycin or an analogue thereof of 2 ng / mL. 25. The subject's body surface area is 1.5 m 2 25. The method of any one of the preceding claims, wherein the 26. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.75 mg to 4 mg. 27. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analogue thereof during the course is between 0.5 mg and 4 mg. 28. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.75 mg to 3.5 mg. 29. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is between 0.75 mg and 3.5 mg. 30. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.75 mg to 1.5 mg. 31. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is between 0.75 mg and 1.5 mg. 32. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.75 mg to 2.0 mg. 33. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is between 0.75 mg and 2.0 mg. 34. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.75 mg to 2.5 mg. 35. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is between 0.75 mg and 2.5 mg. 36. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.5 mg. 37. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 0.5 mg. 38. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.75 mg. 39. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 0.75 mg. 40. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 1.0 mg. 41. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 1.0 mg. 42. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 1.5 mg. 43. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 1.5 mg. 44. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 2.0 mg. 45. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 2.0 mg. 46. ​​The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 2.5 mg. 47. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 2.5 mg. 48. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 3.0 mg. 49. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 3.0 mg. 50. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 3.5 mg. 51. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 3.5 mg. 52. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.8 mg. 53. Each dose or most doses of rapamycin or an analogue thereof during said course is 0.8 mg / m 2 26. The method of embodiment 25, wherein 54. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.7 mg. 55. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 0.7 mg. 56. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 0.6 mg. 57. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 0.6 mg. 58. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is greater than 0.75 mg. 59. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is in an amount greater than 0.75 mg. 60. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is greater than 1.0 mg. 61. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is in an amount greater than 1.0 mg. 62. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is greater than 1.5 mg. 63. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is in an amount greater than 1.5 mg. 64. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is greater than 2.0 mg. 65. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is in an amount greater than 2.0 mg. 66. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is greater than 2.5 mg. 67. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is in an amount greater than 2.5 mg. 68. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is greater than 3.0 mg. 69. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is in an amount greater than 3.0 mg. 70. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is greater than 3.5 mg. 71. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is in an amount greater than 3.5 mg. 72. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 3.0 to 4.0 mg. 73. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 3.0 to 4.0 mg. 74. The method of embodiment 25, wherein the dose of rapamycin or an analogue thereof during the course is 3.5 to 4.5 mg. 75. The method of embodiment 25, wherein each dose or the majority of doses of rapamycin or an analog thereof during the course is 3.5 to 4.5 mg. 76. The subject's body surface area is 1.5 m 2 25. The method according to any one of embodiments 1 to 24, wherein: 77. The dose of rapamycin or an analogue thereof during the course is 0.2 mg / m 2 ~0.75mg / m 2 77. The method of embodiment 76, wherein 78. Each or most of the doses of rapamycin or its analogues during the course of treatment is 0.2 mg / m 2 ~0.75mg / m 2 77. The method of embodiment 76, wherein 79. The dose of rapamycin or an analogue thereof during the course is 0.3 mg / m 2 ~0.7mg / m 2 77. The method of embodiment 76, wherein 80. Each or most of the doses of rapamycin or its analogues during said course is 0.3 mg / m 2 ~0.7mg / m 2 77. The method of embodiment 76, wherein 81. The dose of rapamycin or an analogue thereof during the course is 0.4 mg / m 2 ~0.6mg / m 2 77. The method of embodiment 76, wherein 82. Each or most of the doses of rapamycin or its analogues during the course of treatment is 0.4 mg / m 2 ~0.6mg / m 2 77. The method of embodiment 76, wherein 83. The dose of rapamycin or an analogue thereof during the course is 0.1 mg / m 2 ~0.5mg / m 2 77. The method of embodiment 76, wherein 84. Each or most of the doses of rapamycin or its analogues during the course of treatment is 0.1 mg / m 2 ~0.5mg / m 2 77. The method of embodiment 76, wherein 85. The dose of rapamycin or an analogue thereof during the course is 0.5 mg / m 2 ~0.7mg / m 2 77. The method of embodiment 76, wherein 86. Each or most of the doses of rapamycin or its analogues during the course of treatment is 0.5 mg / m 2 ~0.7mg / m 2 77. The method of embodiment 76, wherein 87. The dose of rapamycin or an analogue thereof during the course is 0.1 mg / m 2 77. The method of embodiment 76, wherein 88. Each or most of the doses of rapamycin or its analogues during the course of treatment is 0.1 mg / m 2 77. The method of embodiment 76, wherein 89. The dose of rapamycin or an analogue thereof during the course is 0.2 mg / m 2 77. The method of embodiment 76, wherein 90. Each or most of the doses of rapamycin or its analogues during the course of 2 77. The method of embodiment 76, wherein 91. The dose of rapamycin or an analogue thereof during the course is 0.3 mg / m 2 77. The method of embodiment 76, wherein 92. Each or most of the doses of rapamycin or its analogues during the course of treatment is 0.3 mg / m 2 77. The method of embodiment 76, wherein 93. The dose of rapamycin or an analogue thereof during the course is 0.4 mg / m 2 77. The method of embodiment 76, wherein 94. Each or most of the doses of rapamycin or its analogues during the course of treatment is 0.4 mg / m 2 77. The method of embodiment 76, wherein 95. The dose of rapamycin or an analogue thereof during the course is 0.5 mg / m 2 77. The method of embodiment 76, wherein 96. Each or most of the doses of rapamycin or an analogue thereof during said course is 0.5 mg / m 2 77. The method of embodiment 76, wherein 97. The dose of rapamycin or an analogue thereof during the course is 0.6 mg / m 2 77. The method of embodiment 76, wherein 98. Each or most of the doses of rapamycin or an analogue thereof during said course is 0.6 mg / m 2 77. The method of embodiment 76, wherein 99. The dose of rapamycin or an analogue thereof during the course is 0.7 mg / m 2 77. The method of embodiment 76, wherein 100. Each dose or most doses of rapamycin or an analogue thereof during said course is 0.7 mg / m 2 77. The method of embodiment 76, wherein 101. The dose of rapamycin or an analogue thereof during the course is 0.75 mg / m 2 77. The method of embodiment 76, wherein 102. The method of any of embodiments 26-101, wherein the dose of rapamycin or an analog thereof is administered once a day. 103. The method of any of embodiments 26-101, wherein the dose of rapamycin or an analog thereof is administered twice daily. 104. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 10 to 28 days. 105. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 11 to 27 days. 106. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 12 to 26 days. 107. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 13 to 25 days. 108. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 14 to 24 days. 109. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 15 to 23 days. 110. A method according to any of embodiments 1 to 102, wherein the course is daily administration for 16 to 22 days. 111. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 17 to 21 days. 112. A method according to any one of embodiments 1 to 102, wherein the course is daily administration for 18 to 20 days. 113. The method of any of embodiments 1 to 102, wherein the course is daily administration for 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days. 114. A method according to any one of embodiments 1 to 113, wherein the course is daily administration for 19 days. 115. A method according to any of embodiments 1 to 114, wherein the course is initiated at least 16 hours after the subject first has in vivo cells that express DARIC. 116. A method according to any of embodiments 1 to 114, wherein the course is not initiated for at least 24 hours after the subject first has in vivo cells expressing DARIC (e.g., by injection of cells expressing DARIC). 117. A method according to any of embodiments 1 to 114, wherein the course is not initiated until at least 36 hours after the subject first has in vivo cells that express DARIC. 118. A method according to any of embodiments 1 to 114, wherein the course is not initiated until at least 48 hours after the subject first has in vivo cells that express DARIC. 119. A method according to any one of embodiments 1 to 114, wherein the course is not initiated until at least 60 hours after the subject first has in vivo cells that express DARIC. 120. A method according to any one of embodiments 1 to 114, wherein the course is not initiated until at least 72 hours after the subject first has in vivo cells that express DARIC. 121. A method according to any one of embodiments 1 to 114, wherein the course is not initiated until at least 84 hours after the subject first has in vivo cells that express DARIC. 122. A method according to any of embodiments 1 to 114, wherein the course is initiated 1 to 4 days after the subject first has in vivo cells that express DARIC. 123. A method according to any of embodiments 1 to 114, wherein the course is initiated 1 to 3 days after the subject first has in vivo cells that express DARIC. 124. A method according to any of embodiments 1 to 114, wherein the course is initiated 1 to 2 days after the subject first has in vivo cells that express DARIC. 125. A method according to any of embodiments 1 to 114, wherein the course is initiated 2 to 4 days after the subject first has in vivo cells expressing DARIC. 126. A method according to any of embodiments 1 to 114, wherein the course is initiated 3 to 4 days after the subject first has in vivo cells expressing DARIC. 127. A method according to any of embodiments 1 to 114, wherein the course is initiated 2 to 3 days after the subject first has in vivo cells expressing DARIC. 128. The method of any of embodiments 1 to 127, comprising following the course a rest period during which rapamycin or an analogue thereof is not administered. 129. The method of embodiment 128, wherein the drug holiday period is 10 to 45 days. 130. The method of embodiment 128, wherein the drug holiday period is 14 to 25 days. 131. The method of embodiment 128, wherein the drug holiday period is 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days. 132. The method of embodiment 128, wherein the drug holiday period is at least 14 days. 133. The method of any of embodiments 128-132, wherein after the drug holiday, a next course of rapamycin or an analogue thereof is administered to the subject. 134. The method of embodiment 133, wherein the next course is the same as the course described in any of the preceding embodiments. 135. The method of embodiment 134, wherein the subsequent course is followed by a second drug holiday. 136. The method according to embodiment 135, wherein the drug holiday period is the same as the drug holiday period described in any one of embodiments 128 to 135. 137. The method of embodiment 135 or 136, wherein after a second drug holiday, a second subsequent course of rapamycin or an analog thereof is administered to the subject. 138. The method of embodiment 137, wherein the second subsequent course is the same as the course described in any of the preceding embodiments. 139. The method of any of embodiments 1 to 138, wherein the rapamycin or analog thereof is rapamycin, AP1903, AP20187, AP21967, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, or BPC015. 140. The method of any of embodiments 1-4 and 11-139, wherein the first fusion protein and / or the second fusion protein comprises a binding domain. 141. The method of any one of embodiments 5, 6, 7, and 140, wherein the binding domain comprises a binding domain of a CD33 antibody. 142. The method of embodiment 141, wherein the binding domain of the CD33 antibody is a VHH. 143. The method of embodiment 142, wherein the VHH has an array as shown in any of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21. 144. The method of embodiment 142, wherein the VHH has at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, and specifically binds to CD33. 145. The method of any of embodiments 1-142, wherein the first fusion protein and / or the second fusion protein further comprises a binding domain that binds to C-type lectin-like molecule 1 (CLL1). 146. The method of embodiment 145, wherein the binding domain that binds to CLL1 has a sequence set forth in any of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 and 54. 147. The method of embodiment 145, wherein the binding domain that binds to CLL1 has at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54, and specifically binds to CLL1. 148. A method according to any of embodiments 1 to 147, wherein the first fusion protein comprises the transmembrane domain of CD8a and the second fusion protein comprises the transmembrane domain of CD4. 149. A method according to any one of embodiments 1 to 148, wherein the first fusion protein comprises an intracellular portion. 150. The method of embodiment 149, wherein the intracellular portion comprises a major signaling domain. 151. The method of embodiment 150, wherein the intracellular primary signaling domain comprises CD3ζ or a fragment thereof. 152. The method of any one of embodiments 149 to 151, wherein the intracellular portion comprises a costimulatory domain. 153. The costimulatory domain is selected from the group consisting of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, and zeta chain of T cell 153. The method of embodiment 152, comprising receptor associated protein kinase 70 (ZAP70), or a fragment or combination thereof. 154. The method of embodiment 152, wherein the costimulatory domain is OX40 or TNFR2, or a fragment or combination thereof. 155. (a) A first fusion protein comprising: an FRB multimerization domain or a variant thereof; a transmembrane domain of CD8α or a transmembrane domain of CD4; a costimulatory domain of CD137; and / or a primary signaling domain of CD3ζ; (b) the second fusion protein comprises a CD33 VHH having an amino acid sequence as set forth in any one of SEQ ID NOs: 2 to 21; an FKBP multimerization domain polypeptide or a variant thereof; and a transmembrane domain of CD4 or a transmembrane domain of CD8α; The method according to any one of embodiments 1 to 154. 156. A method according to any of embodiments 1 to 155, wherein the first fusion protein and / or the second fusion protein has a sequence as set forth in any of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41. 157. A method according to any of embodiments 1 to 155, wherein the first fusion protein and / or the second fusion protein has at least 90% sequence identity to any of the sequences set forth in SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41. 158. A method according to any of embodiments 1 to 155, wherein the first fusion protein and / or the second fusion protein has the sequence shown in SEQ ID NO:40. 159. A method according to any of embodiments 1 to 155, wherein the first fusion protein and / or the second fusion protein has at least 90% sequence identity to the sequence set forth in SEQ ID NO:40. 160. A method according to any one of embodiments 1 to 159, wherein at least one subset of the cells is a hematopoietic cell. 161. A method according to any one of embodiments 1 to 160, wherein at least one subset of the cells is T cells. 162. A method according to any of embodiments 1 to 161, wherein at least one subset of the cells is αβ T cells or γδ T cells. 163. At least one subset of the cells is CD3 + cells, CD4 + Cells or CD8 + The method of any one of embodiments 1 to 162, wherein the cell is a cell. 164. A method according to any one of embodiments 1 to 163, wherein at least one subset of the cells is an effector immune cell. 165. The method of any of embodiments 1-164, wherein at least one subset of the cells is a cytotoxic T lymphocyte (CTL), a tumor-infiltrating lymphocyte (TIL), or a helper T cell. 166. A method according to any of embodiments 1 to 165, wherein at least one subset of the cells is natural killer (NK) cells or natural killer T (NKT) cells. 167. The method of any of embodiments 1 to 166, wherein the cells are isolated from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, splenic tissue or a tumor. 168. The dose of the cells is 1 x 10 per body weight of the subject. 5 pieces / kg~2000×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 169. The dose of the cells is 1 x 10 per body weight of the subject. 6 pieces / kg~1000×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 170. The dose of the cells is 1 x 10 per body weight of the subject. 6 pieces / kg~100×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 171. The dose of the cells is 5×10 per body weight of the subject. 6 pieces / kg~500×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 172. The dose of the cells is 10 x 10 per body weight of the subject. 6 pieces / kg~1000×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 173. The dose of the cells is 1 x 10 per body weight of the subject. 6 pieces / kg~2×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 174. The dose of the cells is 3 x 10 per body weight of the subject. 6 pieces / kg~5×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 175. The dose of the cells is 7.5 x 10 per body weight of the subject. 6 pieces / kg~15×10 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 176. The dose of the cells is 10 x 10 per body weight of the subject. 6 The method according to any one of embodiments 1, 3 and 5 to 167, wherein the amount of the administered dose is 1 / kg. 177. The method of any one of embodiments 1 to 176, wherein the subject is a pediatric patient. 178. The method of any one of embodiments 1 to 176, wherein the subject is 28 years of age or younger. 179. The method of any one of embodiments 1 to 176, wherein the subject is 18 years of age or younger. 180. The method of any one of embodiments 1 to 176, wherein the subject is 18 to 28 years old. 181. The method of any of embodiments 1 to 176, wherein the subject has or has been diagnosed with cancer, an infectious disease, an autoimmune disease, an inflammatory disease, an immune deficiency, or a condition related thereto. 182. A method according to any one of embodiments 1 to 181, wherein the subject has or has been diagnosed with a solid cancer. 183. The method of embodiment 182, wherein the solid cancer comprises lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer or brain cancer. 184. The method of embodiment 183, wherein the lung cancer is non-small cell lung cancer. 185. The method of embodiment 183, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma. 186. The method of embodiment 183, wherein the brain tumor comprises a glioma, glioblastoma, or oligodendroglioma. 187. The method of any one of embodiments 1 to 186, wherein the subject has or has been diagnosed with a hematological malignancy. 188. The method of embodiment 187, wherein the hematological malignancy is leukemia, lymphoma or multiple myeloma. 189. The method of embodiment 188, wherein the hematological malignancy is acute myeloid leukemia (AML). 190. A method according to any of embodiments 1 to 189, wherein the subject is lymphodepleted prior to administering the cells or prior to genetically modifying the cells. 191. The method of embodiment 190, wherein said lymphodepletion comprises administering a dose of fludarabine and a dose of cyclophosphamide. 192. The fludarabine is administered at 30 mg / m 2 once daily for four days, and the cyclophosphamide is administered intravenously at a dose of 500 mg / m 2 The method of embodiment 191, wherein the compound is administered intravenously at a dose of 193. The method of embodiment 190 or 191, wherein the cyclophosphamide is administered on the third and fourth days after administration of the fludarabine. 194. The method of any of embodiments 191 to 193, wherein lymphodepletion is initiated 5 days prior to administering the cells or 5 days prior to genetically modifying the cells. 195. The method of any of embodiments 1 to 194, comprising an FRB multimerization domain and an FKBP multimerization domain (which may be, but are not limited to, the "said" FRB multimerization domain and "said" FKBP multimerization domain of the preceding embodiment), wherein, when the first fusion protein and the second fusion protein are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized intracellularly. EXAMPLES

[0281] (ix) Experimental Example

[0282] Example 1: Preparation of a dimerizer-regulated immunomodulatory complex (DARIC33) with anti-CD33 VHH antibodies

[0283] Despite the success of CAR T cell therapy, safety and efficacy issues are often present. Safety issues include cytokine release syndrome and neurotoxicity, as well as concerns about myeloid cell hypoplasia due to CD33 expression in normal hematopoietic tissues. Efficacy issues include relapse due to antigen escape and T cell exhaustion. Designing next-generation cell therapies may address these concerns by providing platforms with controllable cell activation.

[0284] In this experimental example, DARIC binding and signaling moieties with anti-CD33 VHH antibodies were designed, constructed, and validated. A CD33-specific VHH antibody-containing DARIC lentiviral vector was constructed (Figure 1A; SEQ ID NO: 40) that includes a MNDU3 promoter operably linked to a polynucleotide encoding a DARIC signaling moiety (signal peptide of CD8α, FRB variant (T82L), transmembrane domain of CD8α, intracellular costimulatory domain of 4-1BB, and signaling domain of CD3ζ); a P2A sequence; and a DARIC binding moiety (signal peptide of Igκ, CD33-specific VHH binding domain (camelid or humanized), G4S linker, FKBP12 domain, and transmembrane domain and truncated intracellular signaling domain from CD4). See, for example, SEQ ID NOs: 32-41. See, for example, WO2015 / 017214 and WO2020 / 227474. FIG. 1B shows the SC-DARIC33 construct and its activation by rapamycin.

[0285] To further demonstrate the efficacy of tumor control by SC-DARIC33, we established two in vivo models to treat tumor cell line-derived xenografts by adoptive cell therapy with genetically engineered cells. The tumor cell lines were engineered to express a luciferase transgene so that tumor progression could be monitored by bioluminescence.

[0286] In the first model, immunodeficient NSG mice were transfected with the acute myeloid leukemia (AML) cell line MV4-11 (CD33+ ) were transplanted into the mice in the presence or absence of rapamycin, and 10 × 10 6 10 × 10 SC-DARIC33 cells were treated with 10 × 10 non-lentiviral transduced SC-DARIC33 cells. 6 Control T cells were included in the study as controls. Mice were treated with control T cells in the presence or absence of rapamycin and 10 6 Mice treated with SC-DARIC33 T cells showed a rapid increase in photoluminescence in the tumors, whereas mice treated with SC-DARIC33 in the presence of rapamycin showed suppressed tumor growth over the 36-day study period. Additionally, mice treated with SC-DARIC33 and administered rapamycin did not show a statistically significant increase in photoluminescence in the tumors over the study period following adoptive transfer of SC-DARIC33 cells (compared to the maximum time point before treatment, Figure 1C).

[0287] Example 2: Determining the dose of CAR-T

[0288] To determine the appropriate starting dose of the SC-DARIC33 T-cell product, we sought to compare the efficacy of SC-DARIC33 with that of previously developed CD19 CAR T-cell products.

[0289] To design a comparative model for CD19- and CD33-targeted T-cell therapy, we transduced the non-Hodgkin's lymphoma Raji cell line with vectors driving expression of CD33 and eGFP:ff / luc to generate Raji.CD33-expressing cells. The resulting cell line expresses CD19 and CD33 at similar antigen densities and, when injected into NSG mice, develops disseminated, progressive disease that typically results in death within 30 days if untreated. We then generated matched clinical-scale T-cell products and mock T-cell products derived from the same donors.

[0290] Immunodeficient mice were treated with 5 × 10 5 Raji.CD33 cells were transplanted, and tumor growth was confirmed 7 days later, 10 × 10 6pcs or 30 x 10 6 Patients were treated with SC-DARIC33 T cells (with or without rapamycin), CD19 CAR T cells, or mock T cells. Tumor progression was monitored by bioluminescence for 33 days.

[0291] As shown in Figure 2, mice treated with rapamycin alone or with control (mock-transduced) T cells after tumor cell injection rapidly increased the bioluminescence expressed by the tumor. Also, mice treated with SC-DARIC33 without rapamycin showed a similar rate and magnitude of increase in tumor flux. Meanwhile, 30 × 10 6 In mice treated with SC-DARIC33 T cells and rapamycin, 10 × 10 6 Mice treated with 10x10 CD19 CAR T cells showed similar inhibition of tumor growth. 6 Mice treated with SC-DARIC33 T cells plus rapamycin did not inhibit tumor growth, suggesting that this dose of SC-DARIC33 was less effective.

[0292] Example 3: Rapamycin-induced activity of DARIC33 against tumor xenografts

[0293] To determine the optimal clinical dose of rapamycin to activate DARIC33, it was necessary to determine the concentration of rapamycin required to activate DARIC33 in vivo.

[0294] An AML cell line (MV-4-11) (engineered to express luciferase for in vivo bioluminescence tracking) was adoptively transferred into NSG mice. Tumor-implanted mice were then cultured for 10 7Mice were treated with DARIC33 T cells or the same number of non-transduced T cells as a control. After T cell injection, mice were treated daily with intraperitoneal (IP) injections of 0.01 mg / kg rapamycin on days 1–20. Tumor growth was tracked twice weekly by bioluminescence. Tumor growth was inhibited in six of eight mice treated with DARIC33 T cells and rapamycin, whereas mice not treated with rapamycin, mice treated with rapamycin but not with DARIC33 T cells, and mice treated with non-transduced control T cells all exhibited rapid tumor growth (Figure 3).

[0295] Example 4: Assessment of the reversibility of rapamycin-induced activation of DARIC33 T cells

[0296] Transiently halting the effector function of DARIC33 T cells in patients after cell administration could alleviate toxicity and control hematopoietic recovery. Furthermore, intermittent quiescence of therapeutic T cells would reduce functional exhaustion and allow repopulation of the memory cell compartment. With this in mind, to examine the kinetic effects of rapamycin removal, 50,000 DARIC33 T cells were cultured with rapamycin for 24 h, washed with rapamycin-free medium, and then cultured with 50,000 CD33 T cells in rapamycin-free medium. + As a control, 50,000 DARIC33 T cells were continuously maintained in the presence of rapamycin and cocultured with the same MV-4-11 AML tumor line as above at a 1:1 ratio. After initiation of coculture, incubation was performed at 37°C and samples were taken at various time points (0, 2, 4, 6, 24, 48, 72, 96, and 120 hours) to measure activity. Preincubated SC-DARIC33 T cells were highly active at early time points, as measured by IFNγ release, but then activity as measured by IFNγ release gradually decreased, returning to baseline within 96 hours and exhibiting a half-life (t 1 / 2 ) was first-order kinetics over 17 h (Figure 4A). 1 / 2was determined by curve fitting a single-phase exponential decay.

[0297] To demonstrate the reversibility of SC-DARIC33 T cell activation in vivo, mice bearing recombinant MV4-11-derived AML xenografts were cultured for 10 min at 4 °C for bioluminescence imaging. 7 SC-DARIC33 + T cells or the same number of untransduced (UTD) control cells. 6 Seven days after implantation of MV4-11.ff / luc cells into NSG mice, T cells were injected intravenously (IV). Following T cell injection, mice were treated with 0.1 mg / kg rapamycin three times per week for the time periods indicated on the graph. Specifically, rapamycin was delivered on a continuous dosing schedule (days 1–150), an intermittent dosing schedule (days 1–14 and 28–150), or an abbreviated schedule (days 1–14) (see dosing regimens in Figure 4B). While mice treated with UTD control cells (with or without rapamycin) showed tumor growth and tumor-associated symptoms by day 50, mice treated with SC-DARIC33 cells and rapamycin showed delayed tumor progression (Figure 4C) and extended symptom-free survival (Figure 4F). Four of five mice treated with the abbreviated rapamycin dosing schedule experienced tumor recurrence 3 weeks after rapamycin administration was discontinued. In contrast, resumption of rapamycin treatment resulted in tumor suppression in four of five mice by the end of the observation period. A linear mixed-effects model was used to estimate tumor growth kinetics, which showed similar tumor growth rates in inactively treated mice and similar tumor growth suppression in mice treated with rapamycin on a continuous and intermittent dosing schedule (Figure 4D and Figure 4E). Suppression of AML growth by SC-DARIC33 cells correlated with increased survival (Figure 4E). These data are consistent with a model in which withdrawal of rapamycin temporarily abrogates the antitumor activity of SC-DARIC33, which can be restored by resuming exposure to rapamycin.

[0298] Example 5: Modeling DARIC33 T cell responses to rapamycin in vitro

[0299] To select a dose of rapamycin for first-in-human testing of SC-DARIC33, we sought to determine the concentration of rapamycin required for activation of SC-DARIC33 in situ. We first measured cytokine release upon overnight stimulation of DARIC33 cells with MV4-11 AML cells in culture medium (X-VIVO), human whole blood, or mouse whole blood in the presence of a series of increasing concentrations of rapamycin.

[0300] Specifically, the antigen positive for human whole blood or mouse whole blood (CD33 + ) after co-culture with target cells or antigen-positive (CD33 + ) The amount (concentration) of rapamycin that resulted in half-maximal release of IFNγ cytokine from DARIC33 T cells after co-culture with target cells was quantified.

[0301] Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, activated with anti-CD3 and anti-CD28 antibodies, transduced with DARIC33 A07-LV or control vectors, and expanded in G-Rex24 multiwell culture plates. On day 10 after the start of T cell production, T cells were harvested, washed, and the resulting 50,000 DARIC T cells were plated with acute myeloid leukemia (AML) cell line MV-4-11 at an effector:target ratio of 1:1. T cells and tumor cells were immediately centrifuged and resuspended in 200 μl of a serial dilution series of rapamycin prepared in heparinized human whole blood from healthy volunteers or mouse whole blood from NSG mice. Co-cultures were incubated at 37°C for 24 hours, then centrifuged to separate plasma and IFNγ release was assessed using a Meso Scale Discovery (MSD) cytokine assay.

[0302] IFNγ levels were used to determine the EC of rapamycin that releases IFNγ from DARIC33 T cells. 50 For each of the six total T cell and whole blood donor combinations in the two experiments, replicate measurements were averaged and each value was individually normalized to a maximum IFNγ concentration of 1. The normalized data points were then plotted and fitted to a three-parameter dose-response curve with a Hill coefficient of 1.0 to calculate the EC 50 The calculated EC values ​​for rapamycin were 50 The EC value was 2.6 nM, ranging from 1.5 to 4.3 nM, and at this rapamycin concentration, IFNγ production from DARIC33 T cells when cocultured with target tumor cells was half-maximal (Figures 5A-5E). 50 The value is the CD33 present in the blood of a healthy human. + Cellular activation and exogenous CD33 + Consistency was observed between activation in human blood supplemented with target cells. A similar data conversion was performed for IFNγ values ​​obtained from co-cultures of T cells and tumor cells in mouse blood. ECs determined from T cells from three PBMC donors 50 The value was 2.8 nM, ranging from 1.4 to 4.1 nM.

[0303] The rapamycin-dependent increase in IFNγ release was similar between samples (EC 50 The EC value was 2.6 nM, which is the EC value for IFNγ release in mouse blood. 50 The EC value was 2.8 nM) and was similar between human T cell donors (EC 50 Values ​​ranged from 1.5 nM to 6.3 nM (tested in duplicate, n = 12 total). See Figure 5A. From these data, we determined the target concentration range of rapamycin in whole blood that allows heterodimerization of DARIC33.

[0304] Next, we measured the exposure of rapamycin to tumor-naive mice following single and repeated administration of rapamycin using a quantitative LC-MS / MS whole blood assay (J Chromatogr B Biomed Sci Appl, 1998 Nov 6;718(2)251-7). The whole blood concentrations of rapamycin following administration of various doses of rapamycin three times per week are shown in the graph, with the time of intraperitoneal (IP) injection of rapamycin indicated by the bars at the bottom (see Figure 5B). The upper limit of quantification (ULOQ=200ng / mL) and lower limit of quantification (LLOQ=1ng / mL) are also shown in the graph. Rapamycin blood concentrations were generally dose-proportional, peaking within 2 hours of IP administration and declining with an elimination half-life of 16-24 hours. Peak rapamycin concentrations ranged from 10 ng / mL at the 0.02 mg / kg dose to approximately 100 ng / mL at the 0.1 mg / kg dose (Table 1). [Table 1]

[0305] To examine the effect of different dose levels and administration schedules of rapamycin on the anti-AML activity of SC-DARIC33 T cells, tumor-bearing mice were treated with 10 × 10 7 (10 million) SC-DARIC33 T cells or 10 × 10 6Mice were treated with untransduced (UTD) T cells followed by rapamycin (0.02 mg / kg qMWF, 0.05 mg / kg qMWF, 0.1 mg / kg qMWF, or 0.01 mg / kg daily for 21 days, all IP injections; see dosing regimens in Figure 5C). Mice treated with regimens predicted to be inactive (e.g., no treatment, rapamycin only, UTD cells only, UTD cells followed by rapamycin, or SC-DARIC33 cells only) did not experience a reduction in tumor growth (log[luminous flux] / day = 0.26-0.27, Figure 5E). On the other hand, treatments predicted to be active (e.g., administration of SC-DARIC33 cell products followed by rapamycin) delayed tumor growth, with tumor growth slowest in mice treated with 0.01 mg / kg rapamycin administered daily intraperitoneally (IP) (log[luminous flux] / day = 0.058). Tumor growth correlated with survival; control mice showed tumor-related symptoms around day 45, whereas no mice treated with active treatment (DARIC33 + rapamycin) showed signs of tumor progression at the same time point. All doses of rapamycin tested prolonged survival (p < 0.001, log-rank test). Specifically, at the end of the 90-day observation period, tumor growth was continuously suppressed in 5 of 10 mice treated with SC-DARIC33 and 0.01 mg / kg rapamycin daily (Figure 5E).

[0306] To determine the minimal concentration of rapamycin associated with DARIC33 efficacy in tumor-bearing mice, we measured the concentration of rapamycin in blood in mice treated with the minimal dose of rapamycin that showed antitumor activity in vivo (0.01 mg / kg rapamycin administered intraperitoneally (ip) daily) and SC-DARIC33 T cells. Blood samples taken 2 h after rapamycin administration on days 1 and 15 and 24 h after rapamycin administration on day 20 contained 1.4 mg / mL to 3.3 ng / mL rapamycin (LC MS / MS quantitative whole blood assay, n = 5 mice). Interestingly, in vitro mouse and human whole blood assays showed similar rapamycin-dependent activation of DARIC33, although differences were observed between species for rapamycin translocated to red blood cells (RBCs) and plasma protein bound (PPB) (Tables 2 and 3). [Table 2] [Table 3]

[0307] In humans, 94.5% of rapamycin is bound to red blood cells (RBCs) and only 3.1% is present in plasma. In human plasma, the majority of rapamycin is protein bound (92%). In contrast, in mice, only 5.5% of rapamycin is transferred to RBCs and >99% is plasma protein bound (PPB) (Table 4), suggesting that species-specific differences in rapamycin distribution may result in similar differences in the concentration of bioavailable rapamycin in vitro. Collectively, these data indicate that the target trough blood concentration of rapamycin to induce DARIC heterodimerization in humans is in the range of 1.5-3 ng / mL. [Table 4]

[0308] Example 6: Measurement of whole blood rapamycin concentrations in treated mice

[0309] To measure the concentration of rapamycin in the mice that showed inhibition of tumor growth, peripheral blood was collected by retro-orbital bleeding and frozen at -80 °C until analysis. The concentration of rapamycin in whole blood samples was measured using quantitative liquid chromatography tandem mass spectrometry (LS-MS / MS). As shown in Figure 6, samples collected on day 1 were found to contain 0.916 ± 0.896 ng / mL of rapamycin in whole blood 2 hours after rapamycin injection. Samples collected on day 15 were found to contain 2.65 ± 1.983 ng / mL of rapamycin in whole blood 2 hours after rapamycin injection. These findings were consistent with the accumulation of rapamycin in the mice. Additionally, the timing of sample collection was consistent with the timing of peak rapamycin concentrations observed in previous experiments. Samples taken on day 21 revealed a mean rapamycin concentration of 0.56±0.771ng / mL in whole blood 24 hours after rapamycin administration, which was below the lower limit of quantification in three of five mice. Collectively, these data suggest that a rapamycin dose resulting in a peak concentration of 2.65±1.983ng / mL and a trough concentration of 0.56±0.771ng / mL was sufficient to activate DARIC33 T cells and exert anti-AML tumor suppression in mice.

[0310] Example 7: In silico modeling of rapamycin responsiveness of DARIC33

[0311] To determine the recommended starting dose of rapamycin (sirolimus) in pediatric patients (also referred to as subjects), a population pharmacokinetic model was constructed using whole blood exposure of sirolimus for pediatric patients reported in published papers (Goyal et al, Biol Blood Marrow Transplant 2013, 569-575; Wu et al, CPT: Pharmacometrics & Systems Pharmacol 2012, 1, e17, doi:10.1038 / psp.2012.18). Several sirolimus dose levels were simulated over 19-21 days on a once-daily dosing schedule. Exposure profiles were generated showing the geometric mean (dark blue line) and the 10th and 90th percentiles of expected sirolimus concentrations (light blue areas) (see Figure 7). Typical immunosuppressive trough concentrations of sirolimus are in the range of 5-15 ng / mL (dashed red line). In a daily dosing schedule (body surface area 1.5 m 2 For patients with: 2 Sirolimus dose or (body surface area 1.5 m 2 The best exposure range was obtained when simulating a 0.75 mg sirolimus dose (for patients above 0.75 mg), with the majority of patients achieving a constant sirolimus exposure in the range of 1.5-3 ng / ml (target trough concentration range, green area).

[0312] The target trough concentration range of rapamycin was determined based on the EC 50 This EC value is calculated. 50 The value is 2.6 nM (equivalent to 2.6 ng / mL).

[0313] Furthermore, this target trough concentration range of rapamycin was supported by an analysis of efficacy-related whole blood concentrations of rapamycin in tumor-bearing immunodeficient mice injected with SC-DARIC33, which showed that concentrations of free rapamycin in the mice were below the target trough concentration of an equivalent dose of free rapamycin in pediatric patients.

[0314] Furthermore, a simulation was performed with a daily dosing schedule for 19 days (body surface area 1.5 m2). 2 For patients with 2 Sirolimus dose or (body surface area 1.5 m 2 Using a sirolimus dose of 0.75 mg (for the above patients), we were able to simulate the clearance of sirolimus after a drug holiday that could turn off activation of SC-DARIC33.

[0315] Example 8: Specific Example of a Clinical Protocol

[0316] Rapamycin (or rapalog) dosing and target concentration Rapamycin is 2 0.75 mg / m for patients with 2 or (body surface area 1.5 m 2 0.50 mg / m for the following patients: 2 Dosing should be adjusted to maintain a target trough blood concentration of 2 ng / mL within the target range of 1.5 to 3 ng / mL.

[0317] In the absence of evidence of DARIC activation, target concentrations can be greater than 3 ng / mL, with a maximum target concentration of 9 ng / mL.

[0318] Initial rapamycin schedule Rapamycin is administered daily starting on day +2 after infusion of the SC-DARIC T cell product to activate SC-DARIC33. Rapamycin administration continues until day +21. Bone marrow aspirate and / or biopsy is performed on day 28, followed by flow cytometric analysis of multiple parameters, and if the subject shows morphological remission of disease <1%, rapamycin rest is continued. The next course of rapamycin may begin after day +42 as shown below.

[0319] If bone marrow evaluation on or before day 42 shows evidence of persistent leukemia greater than 1%, the next course of rapamycin can be started immediately on day 42.

[0320] Figure 8A shows a protocol for cell infusion, administration of rapamycin, and bone marrow aspiration and / or biopsy, while Figure 8B shows an alternative protocol for administration of rapamycin on days 3-21.

[0321] Next course of rapamycin (Tables 5 and 6) For subjects who, in the clinical judgment of the investigator, are believed to be benefiting from activation of DARIC T cells with rapamycin, a next course of rapamycin may be initiated any time after day +42 post-T cell infusion for patients in remission or immediately for patients with evidence of persistent leukemia, and rapamycin may be continued until benefit from DARIC T cell activation ceases. Requirements for initiating a next course of rapamycin include the absence of grade 3 or higher toxicity associated with DARIC T cells that is assessed as clinically significant.

[0322] For the next course of rapamycin in subjects in sustained remission, treatment should begin at least 14 days after the previous rapamycin dose was discontinued, and the patient's absolute phagocytic cell (APC) count should exceed 500 cells / μL.

[0323] [Table 5]

[0324] [Table 6]

[0325] Long-term follow-up As required by the FDA, subjects will be followed up for up to 15 years after infusion of the DARIC T cell product to investigate any late adverse events associated with the use of lentiviral-transduced T cells and to obtain additional information, including disease evaluation. If a hematopoietic cell transplant (HCT) donor is identified and deemed most appropriate for the patient's goals, HCT may be initiated after conditioning at any time after DARIC T cell infusion, according to the physician's discretion. Information regarding HCT, including the conditioning regimen administered, stem cell origin, and date of HCT, will be collected. In the group of subjects with persistent myeloid cell aplasia, information regarding infectious complications will also be collected. Follow-up information may be provided by the subject's primary care physician.

[0326] Example 9 To determine the recommended starting dose of rapamycin (sirolimus) in adult patients (also referred to as subjects), a population pharmacokinetic model was constructed using the whole blood exposure of sirolimus for adult patients reported in a published paper (Wu et al, CPT Pharmacometrics Syst Pharmacol 2012, 1, e17). Several sirolimus dose levels (0.5 mg, 0.75 mg, 1.0 mg, 1.25 mg, and 1.5 mg) were simulated over a 19-21 day period on a once daily dosing schedule (Figure 9).

[0327] An exposure profile was generated showing the geometric mean (solid line) and the 10th and 90th percentiles (shaded areas) of expected sirolimus concentrations using a daily starting dose of 1.5 mg (Figure 10). A daily starting dose of 1.5 mg will achieve target sirolimus concentrations of 1.5-3 ng / mL in most patients. Dose adjustments are also possible.

[0328] Example 10 Patients after rapamycin administration (body surface area 1.5 m 2Pharmacokinetic (PK) data for sirolimus (> 0.75 mg) showed dose-exposure relationships for peak and trough concentrations that could be adjusted to obtain the target range (Figure 11). Rapamycin administration was initiated by oral administration at a dose of 0.75 mg, and peak and trough concentrations were monitored using a clinical LC-MS / MS assay as described above. Peak exposure (2 hours after administration) was within the target range of 1.5-3 ng / ml, while trough concentrations (24 hours after treatment with sirolimus) were below the detection limit of the assay (< 1 ng / ml). After three doses of sirolimus at an initial dose of 0.75 mg, the dose was adjusted to 1.5 mg so that the target trough concentrations were within the horizontal dotted boundary. After four doses, trough values ​​were not within the target range, so the dose was adjusted again to 3 mg to obtain the target trough values. After two doses of 3 mg sirolimus, trough concentrations were detected within the target range, and this sirolimus dose was maintained. After eight doses of 3 mg sirolimus, trough concentration measurements exceeded the target range. At this point, sirolimus was temporarily suspended for 2 days. The sirolimus dose was reduced to 2 mg for three doses, and sirolimus was discontinued after the 20th day of treatment. Sirolimus concentrations were monitored after cessation of sirolimus (D / C), and sirolimus was undetectable at the last measurement on day 28. Importantly, the data support the preclinical model described above by showing how the dose can be adjusted after monitoring to achieve a target trough concentration range of 1.5 to 3 ng / mL.

[0329] (x) Sequences Supporting the Disclosure [Table 7] TIFF2024545163000009.tif247164TIFF2024545163000010.tif246164TIFF20245451630 00011.tif246164TIFF2024545163000012.tif245163TIFF2024545163000013.tif196164

[0330] SEQ ID NO: 1 shows the amino acid sequence of full-length human CD33. SEQ ID NOs: 2-21 show the amino acid sequences of anti-CD33 VHH domains. SEQ ID NOs: 22-31 show the amino acid sequences of anti-CD33 VHH DARIC binding domains. SEQ ID NOs: 32-41 show the amino acid sequences of anti-CD33 VHH DARIC fusion proteins comprising a binding domain and an intracellular signaling portion separated by a self-cleavable 2A peptide. SEQ ID NO: 42 shows the amino acid sequence of anti-CD33 VHH DARIC comprising an intracellular signaling portion and a multimerization domain but not a binding domain.

[0331] (xi) Conclusion

[0332] The nucleic acid and amino acid sequences provided herein are represented by the abbreviations used for nucleotide bases and amino acid residues as set forth in 37 CFR 1.822 and as set forth in Tables 1 and 3 of WIPO Standard ST.25 (1998) Supplement 2. Only one strand is shown for each nucleic acid sequence, although the complementary strand, if appropriate, is also included in the embodiments.

[0333] As used herein, the terms "specific binding affinity," "specifically binds," "specific binding," or "specific target" refer to one molecule binding to another molecule with a binding affinity higher than background binding. 5 M -1 A binding domain "specifically binds" to a target molecule if it binds to or associates with the target molecule with an affinity or Ka (i.e., the equilibrium binding constant of a particular binding interaction, expressed in units of 1 / M) of 10 or more. 6 M -1 That's it, 10 7 M -1 That's it, 10 8 M -1 That's it, 10 9 M-1 That's it, 10 10 M -1 That's it, 10 11 M -1 That's it, 10 12 M -1 Greater than or equal to 10 13 M -1 A "high affinity" binding domain binds to a target with a Ka of at least 10 7 M -1 At least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or higher.

[0334] Alternatively, affinity can be expressed as the equilibrium dissociation constant (Kd) (units: M) of a particular binding interaction (e.g., 10 -5 M~10 -13 M or less). The affinity of the binding domain and CAR protein according to the present disclosure can be readily measured using conventional techniques, such as, for example, competitive ELISA (enzyme-linked immunosorbent assay); binding (association); displacement assays using labeled ligands; surface plasmon resonance instruments such as the Biacore T100 available from Biacore, Inc. (Piscataway, NJ); optical biosensor technology such as the EPIC system available from Corning and EnSpire available from PerkinElmer (see, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; U.S. Patent Publication No. 5,283,173; U.S. Patent Publication No. 5,468,614).

[0335] In certain embodiments, the affinity of specific binding is 2 times background binding, 5 times background binding, 10 times background binding, 20 times background binding, 50 times background binding, 100 times background binding, 1000 times background binding, or more.

[0336] As will be appreciated by those of skill in the art, each embodiment disclosed herein comprises, consists essentially of, or consists of the particular components, steps, materials, or ingredients described. Thus, the terms "comprise" or "comprising" should be interpreted to mean "comprise, consist essentially of, or consist of." The transitional phrase "comprise" means, but is not limited to, that a component, step, material, or ingredient not described is included, even if it is in a greater amount. The transitional phrase "consisting of" excludes all components, steps, materials, or ingredients not described. The transitional phrase "consisting essentially of" limits the scope of the embodiment to the components, steps, materials, or ingredients described and those components, steps, materials, or ingredients that do not significantly affect the embodiment. A significant effect refers to an effect of administering rapamycin or an analog thereof to a statistically significant reduction in cancer or a statistically significant increase in the activity of CAR-T cells, as described herein.

[0337] Unless otherwise indicated, all numerical values ​​expressing quantities or properties of materials, such as molecular weight and reaction conditions, in the specification and claims are to be construed in all instances as modified by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Without intending to limit the scope of the doctrine of equivalents to the scope of the claims, each numerical parameter should, at the very least, be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. For clarity, the term "about," when used in conjunction with a stated value or range, has a meaning that would be reasonably interpreted by one of ordinary skill in the art, i.e., within ±20% of the stated value; within ±19% of the stated value; within ±18% of the stated value; within ±17% of the stated value; within ±16% of the stated value; within ±15% of the stated value; within ±14% of the stated value; within ±13% of the stated value; within ±12% of the stated value; within ±11% of the stated value; within ±10% of the stated value; within ±9% of the stated value; within ±8% of the stated value; within ±7% of the stated value; within ±6% of the stated value; within ±5% of the stated value; within ±4% of the stated value; within ±3% of the stated value; within ±2% of the stated value; or within ±1% of the stated value.

[0338] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations and approximate ranges, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain certain errors necessarily resulting from the standard deviation associated with their respective testing measurements.

[0339] In the description of the present invention (particularly in the description of the claims below), the terms "a", "an", "the" and similar modifiers are intended to include both the singular and the plural unless otherwise indicated or the context clearly indicates otherwise. Numerical ranges described herein are intended to be a shorthand way of referring to each numerical value falling within the range individually. Unless otherwise indicated, each numerical value is described herein as if it were individually described herein. Any method described herein can be performed in any suitable order unless otherwise indicated or the context clearly indicates otherwise. The use of any examples or language of examples (e.g., "etc.") provided herein is intended to be for the purpose of illustrating the invention only and does not limit the scope of the invention as described in the claims. No term described herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0340] Groupings of other elements of the invention disclosed herein or of various embodiments of the invention should not be construed as limiting the invention. Members of each group may be described herein or in the claims individually or in combination with other members of the group or other elements described herein. It is anticipated that for reasons of convenience and / or patentability, one or more members of a group may be added to another group, or one or more members may be deleted from a group. When such additions or deletions are made, the specification includes groups that are constructed to satisfy the recitation of all Markush groups set forth in the appended claims.

[0341] Specific embodiments of the present invention are described herein, including those embodiments known to the inventors to be the best mode for carrying out the invention. Of course, those skilled in the art will readily appreciate that the embodiments described herein may be modified in various ways upon review of the above detailed description. The inventors anticipate that such modifications may be adopted by those skilled in the art, and intend that the present invention may be practiced in other ways than as specifically described herein. Accordingly, the present invention includes all modifications of the subject matter recited in the appended claims and all equivalents of the subject matter of the present invention to the extent permitted within the scope of applicable law. Moreover, the present invention includes all combinations of the above-described elements in any and all variations thereof, unless otherwise indicated or the context clearly dictates otherwise.

[0342] Additionally, throughout this specification, various patents, publications, journal articles and other documents are cited (references herein). Each reference cited herein is individually incorporated herein by reference for the teachings thereof as if it were a part of this specification.

[0343] Finally, the embodiments of the invention disclosed herein are to be considered as illustrative of the principles of the invention. Other modifications may be adopted within the scope of the invention. Thus, by way of example, but not by way of limitation, alternative configurations of the invention may be utilized in accordance with the teachings herein. Thus, the invention is not to be limited to what has been precisely shown and described herein.

[0344] The details described herein are by way of example and are presented solely for the purpose of illustrating preferred embodiments of the present invention, to provide what is believed to be the most useful, and to facilitate an understanding of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no structural details of the present invention are described in more detail than is necessary for a basic understanding of the present invention, and those skilled in the art will be able to easily understand how to actually embody some forms of the present invention by reading the description of the present invention in conjunction with the drawings and / or examples.

[0345] The definitions and explanations used in this disclosure are intended to control future interpretations, unless clear and unambiguous changes are made in the examples, or the meaning of the terms makes the interpretation meaningless or substantially meaningless. If the definition of a term does not make sense or substantially meaningless from the interpretation of the term, please refer to the definition of the term from a dictionary known to those skilled in the art, such as Webster's Dictionary (3rd Edition) or Oxford Dictionary of Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford, 2006).

Claims

1. 1. A pharmaceutical composition for treating a subject having cells expressing a dimerizer-regulated immunomodulatory complex (DARIC), comprising rapamycin or a rapalog thereof; the DARIC comprises a first fusion protein comprising a multimerization domain and a second fusion protein comprising a multimerization domain; the treatment comprises administering a course of rapamycin or a rapalog thereof; the rapamycin or a rapalog thereof is bound to and positioned between the multimerization domains of the first fusion protein and the multimerization domain of the second fusion protein; and A pharmaceutical composition wherein the course achieves a blood trough concentration of the rapamycin or its rapalog of 1.0 ng / mL to 3 ng / mL.

2. The pharmaceutical composition of claim 1, wherein the course is initiated 0 to 4 days after the subject has in vivo cells that express the DARIC.

3. The pharmaceutical composition of claim 1, wherein the course is initiated two or three days after the subject has in vivo cells that express the DARIC.

4. 10. The pharmaceutical composition of claim 1, wherein the course is administered for at least 14 days.

5. 10. The pharmaceutical composition of claim 1, wherein the course is administered daily for 18, 19, 20, 21, or 22 days.

6. The course is designed to cover a body surface area of ​​the subject of 1.5 m 2 or administering a daily dose of at least 0.75 mg when said subject has a body surface area greater than 1.5 m 2 10. The pharmaceutical composition of claim 1, comprising administering a daily dose of less than 0.75 mg when:

7. The subject's body surface area is 1.5 m 2 7. The pharmaceutical composition of claim 6, wherein the daily dose is greater than 0.75 mg to 3.5 mg.

8. The subject's body surface area is 1.5 m 2 and the daily dose is 0.25 mg / m or less. 2 ~0.74 mg / m 2 The pharmaceutical composition according to claim 6, wherein

9. The subject's body surface area is 1.5 m 2 and said daily dose is 0.50 mg / m or less. 2 The pharmaceutical composition according to claim 6, wherein

10. 10. The pharmaceutical composition of claim 1, wherein the course comprises a course of daily administration followed by a washout period during which rapamycin or a rapalog thereof is not administered to the subject.

11. The drug holiday period is (a) 13, 14 or 15 days; (b) at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, or at least 22 days; (c) for at least 14 days; or (d) 14 days; The pharmaceutical composition of claim 10.

12. The method of claim 1, wherein the course further comprises administering a second course of rapamycin or a rapalog thereof after the withdrawal period; (a) the second course is characterized by achieving a blood trough concentration of the rapamycin or rapalog thereof of 0.5 ng / mL to 3 ng / mL; (b) the second course is administered for at least 14 days, or for 18, 19, 20, 21, or 22 days by daily administration of the rapamycin or rapalog thereof; and / or (c) the second course comprises administering a daily dose of at least 0.75 mg if the subject has a body surface area greater than 1.5 m2, or administering a daily dose of less than 0.75 mg if the subject has a body surface area of ​​1.5 m2 or less; The pharmaceutical composition of claim 10.

13. The multimerization domain of the first fusion protein comprises an FKBP-rapamycin binding (FRB) multimerization domain or a variant thereof, and the multimerization domain of the second fusion protein comprises an FK506 binding protein (FKBP) multimerization domain or a variant thereof; or the multimerization domain of the first fusion protein comprises an FK506-binding protein (FKBP) multimerization domain or a variant thereof, and the multimerization domain of the second fusion protein comprises an FKBP-rapamycin-binding (FRB) multimerization domain or a variant thereof; The pharmaceutical composition of claim 1.

14. The pharmaceutical composition of claim 13, wherein the FKBP multimerization domain or a variant thereof is FKBP12 and the FRB multimerization domain or a variant thereof is FRB T2098L. (a) the FKBP multimerization domain or a variant thereof has a sequence as set forth in SEQ ID NO: 55 or 56, or a sequence having at least 90%, at least 95%, or at least 98% sequence identity to the sequence as set forth in SEQ ID NO: 55 or 56; and / or (b) the FRB multimerization domain or a variant thereof has a sequence set forth in SEQ ID NO: 57 or 58, or a sequence having at least 90%, at least 95%, or at least 98% sequence identity to the sequence set forth in SEQ ID NO: 57 or 58; The pharmaceutical composition of claim 13.

16. The method of claim 16, wherein when the first fusion protein and the second fusion protein are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized extracellularly; or When the first fusion protein and the second fusion protein are expressed, the FRB multimerization domain and the FKBP multimerization domain are localized intracellularly. The pharmaceutical composition according to claim 6.

17. The pharmaceutical composition of claim 1, wherein the first fusion protein and / or the second fusion protein further comprise one or more binding domains.

18. 18. The pharmaceutical composition of claim 17, wherein the one or more binding domains bind to a cancer antigen.

19. the one or more binding domains are single-domain heavy chain variable domains (VHH) or single-chain variable domain fragments (scFv); or the one or more binding domains comprise the extracellular domain of a receptor or a ligand; 18. The pharmaceutical composition of claim 17.

20. the one or more binding domains comprise a binding domain for a CD33 antibody and / or a binding domain that binds to C-type lectin-like molecule 1 (CLL1); and the binding domain of the CD33 antibody is a VHH; the VHH has a sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21; or a sequence having at least 90%, at least 95%, or at least 98% sequence identity to a sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, and specifically binds to CD33.

18. The pharmaceutical composition of claim 17.

21. The one or more binding domains comprise a binding domain for a CD33 antibody and / or a binding domain that binds to CLL1; and the binding domain that binds to CLL1 has a sequence set forth in any of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54; or has a sequence that has at least 90%, at least 95%, or at least 98% sequence identity to a sequence set forth in any of SEQ ID NOs: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54, and specifically binds to CLL1; 18. The pharmaceutical composition of claim 17.

22. The first fusion protein and / or the second fusion protein further comprises an intracellular portion, wherein the intracellular portion is (a) Contains a key intracellular signaling domain; (b) comprises a co-receptor domain; and / or (c) containing a costimulatory domain; The pharmaceutical composition of claim 1.

23. The pharmaceutical composition of claim 22, wherein the intracellular major signaling domain comprises CD3ζ, CD3ε, or a fragment thereof.

24. The costimulatory domain may be a domain of Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, caspase recruitment domain family member 11 (CARD11), CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD94, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DNAX-activation protein 10 (DAP10), Linker for activation of T-cells family member 1 (LAT), SH2 Domain-Containing Leukocyte Protein Of 76 kD (SLP76), T cell receptor associated transmembrane adaptor 1 (TRAT1), TNFR2, TNFRS14, TNFRS18, TNRFS25, or zeta chain of T cell receptor associated protein kinase. 70 (ZAP70), or a fragment or combination thereof; or the costimulatory domain comprises CD137 (4-1BB) or a fragment thereof; 23. The pharmaceutical composition of claim 22.

25. The pharmaceutical composition of claim 1, wherein the first fusion protein and / or the second fusion protein further comprise a transmembrane domain.

26. The pharmaceutical composition described in claim 25, wherein the transmembrane domain of the first fusion protein and / or the second fusion protein is the transmembrane domain of CD4 or the transmembrane domain of CD8α.

27. The pharmaceutical composition of claim 1, wherein the first fusion protein and / or the second fusion protein further comprise a spacer.

28. (a) the first fusion protein comprises an FRB multimerization domain or a variant thereof; a transmembrane domain of CD8α or a transmembrane domain of CD4; a costimulatory domain of CD137; and / or a primary signaling domain of CD3ζ; and (b) the second fusion protein comprises a CD33 VHH having an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 21; an FKBP multimerization domain or a variant thereof; and a CD4 transmembrane domain or a CD8α transmembrane domain; The pharmaceutical composition of claim 1.

29. The pharmaceutical composition described in claim 1, wherein the first fusion protein and / or the second fusion protein has a sequence set forth in any of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41: or has a sequence having at least 90% sequence identity to a sequence set forth in any of SEQ ID NOs: 32, 33, 34, 35, 36, 37, 38, 39, 40 and 41.

30. The pharmaceutical composition described in claim 1, wherein the first fusion protein and / or the second fusion protein have a sequence as set forth in SEQ ID NO: 40; or have a sequence having at least 90%, at least 95%, or at least 98% sequence identity to the sequence as set forth in SEQ ID NO:

40.

31. 2. The pharmaceutical composition of claim 1, wherein the rapamycin or rapalog thereof is rapamycin, AP1903, AP20187, AP21967, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, or zotarolimus.

32. At least one subset of cells in the subject's body that express DARIC, (a) Hematopoietic cells; (b) are T cells; (c) are αβ T cells or γδ T cells; (d) are CD3+ cells, CD4+ cells, or CD8+ cells; (e) are effector immune cells; (f) is a cytotoxic T lymphocyte (CTL), tumor-infiltrating lymphocyte (TIL), or helper T cell; and / or (g) natural killer (NK) cells or natural killer T (NKT) cells; The pharmaceutical composition of claim 1.

33. 2. The pharmaceutical composition of claim 1, wherein the cells are isolated from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor.

34. The pharmaceutical composition of claim 33, wherein the cells are produced ex vivo to express the DARIC.

35. The cells (a) 1 x 10 per body weight of the subject 5 pieces / kg~2000×10 6 administered to the subject at a dose of 100 mg / kg; (b) administered to the subject at a dose of 1×10 6 cells / kg to 1000×10 6 cells / kg of the subject's body weight; (c) administered to the subject at a dose of 1×10 6 cells / kg to 100×10 6 cells / kg of the subject's body weight; (d) administered to the subject at a dose of 5×10 6 cells / kg to 500×10 6 cells / kg of the subject's body weight; (e) administered to the subject at a dose of 10×10 6 cells / kg to 1000×10 6 cells / kg of the subject's body weight; (f) administered to the subject at a dose of 1×10 6 cells / kg to 2×10 6 cells / kg of the subject's body weight; (g) administered to the subject at a dose of 3×10 6 cells / kg to 5×10 6 cells / kg of the subject's body weight; or (h) administered to the subject at a dose of 7.5×10 6 cells / kg to 15×10 6 cells / kg of the subject's body weight; 35. The pharmaceutical composition of claim 34.

36. The dose of the cells per body weight of the subject is 10×10 6 The pharmaceutical composition of claim 35, wherein the amount is 1 / kg.

37. The pharmaceutical composition of claim 33, wherein the cells are engineered to express the DARIC in a subject in vivo.

38. The object is (a) Pediatric patients; (b) is under 28 years of age; (c) is under 18 years of age; (d) are between 18 and 28 years old; (e) is an adult patient; and / or (f) are at least 18 years of age; The pharmaceutical composition of claim 1.

39. 10. The pharmaceutical composition of claim 1, wherein the subject has or has been diagnosed with cancer, an infectious disease, an autoimmune disease, an inflammatory disease, an immune deficiency, or a condition related thereto.

40. the subject has or has been diagnosed with a solid cancer, including lung cancer, squamous cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, thyroid cancer, bladder cancer, cervical cancer, esophageal cancer, ovarian cancer, gastric cancer, endometrial cancer, or brain cancer; and (a) the lung cancer is non-small cell lung cancer; (b) the squamous cell carcinoma is head and neck squamous cell carcinoma; and / or (c) the brain tumor comprises a glioma, a glioblastoma, or an oligodendroglioma; The pharmaceutical composition of claim 1.

41. the subject has or has been diagnosed with a hematological malignancy, wherein the hematological malignancy is leukemia, lymphoma, or multiple myeloma; or the hematological malignancy is acute myeloid leukemia (AML); The pharmaceutical composition of claim 1.

42. 2. The pharmaceutical composition of claim 1, wherein the subject is lymphodepleted before administering the course.

43. 43. The pharmaceutical composition of claim 42, wherein the lymphocyte depletion comprises administering a dose of fludarabine and a dose of cyclophosphamide and is initiated 7 days prior to administration of the course.

44. The fludarabine is administered at a dose of 30 mg / m 2 once daily for 4 days, and the cyclophosphamide is administered intravenously at a dose of 500 mg / m 2 44. The pharmaceutical composition of claim 43, wherein the composition is administered intravenously at a dose of

45. 44. The pharmaceutical composition of claim 43, wherein the cyclophosphamide is administered on days 3 and 4 of the administration of the fludarabine.

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