Drug delivery using targeted ligand-payloads for cell therapy

Abstract

To provide a drug delivery platform for cell therapy that can control cell activity after transplantation in order to avoid concerns such as tumorigenesis of transplanted cells, in treatment or regenerative medical treatment using chimeric antigen receptor T cells and stem cells.SOLUTION: Provided is a drug delivery platform for cell therapy, comprising: a. an engineered protein on a target cell for transplant consisting of a fusion protein with a first component and a second component, the first component and the second component being bound by a peptide linker, the first component being a non-membrane protein, the second component being a membrane anchored peptide or protein; b. at least one small ligand bound to a linker, and having intrinsic high affinity to at least one component of the engineered protein; and c. at least one payload of drug bound to the linker, and associated with the target cell when the small ligand binds to at least one component of the engineered protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application was filed on February 17, 2017, under Section 119(e) of the United States Patent Act. We claim the benefit of priority under Provisional Patent Application 62 / 460,118, the details of which are specified herein. It will be fully integrated.

[0002] This invention provides a drug delivery platform for cell therapy. Specifically, it provides a drug delivery platform for human Functional proteins are linked to high-affinity ligands that carry at least one drug payload. The drug payload binds and is taken up into the transplanted cell via the artificial functional protein. , to adjust the effectiveness of transplanted cell therapy. [Background technology]

[0003] Over the past few decades, in terms of cell types, delivery methods, and appropriate disease models, the field has... It is progressing very rapidly. In terms of cell types, current cell therapy uses chimeric antigen receptors ( They are generally classified into CARs (Cardiac Adrenal Cells), cells for tumor models, and stem cells used in regenerative medicine.

[0004] CAR T is also known as a chimeric T cell receptor, chimeric immune receptor, or artificial T cell receptor. However, immune effector cells (usually T cells or NK cells) can detect the corresponding antigen. It enables the recognition of target cells and the manifestation of cytotoxicity. This is the foundation of CAR T technology. Head and developmental cancers show promise against certain types of cancer, and are being studied in biological and clinical research. CAR T technology is attracting considerable attention in both fields.

[0005] Regenerative medicine is revolutionizing the medical field, aiming to completely heal damaged tissues and organs. For patients with conditions that are considered incurable but still have potential, solutions and hope. This provides advances in developmental and cell biology, immunology, and other fields. This presents an opportunity to improve existing regenerative therapies and develop new ones.

[0006] Stem cells undergo a process called differentiation, which leads to the development of various different types of cells, such as skin cells, brain cells, and lung cells. Stem cells have the ability to evolve into other cells. Stem cells are enabling new clinical applications for regenerative medicine. It is an important element in medical treatment.

[0007] Various stem cells, including adult stem cells and embryonic stem cells, are used in regenerative medicine. Furthermore, the umbilical cord Various types of progenitor cells, including cells found in cord blood, and what are called induced pluripotent stem cells. Biotechnology-generated cells are being used in regenerative medicine. Some cells possess unique properties, and some are more versatile than others.

[0008] Many regenerative medicine treatments currently under development begin with the patient's own cells. For example, Patient skin cells are collected and then processed in a laboratory to assign specific characteristics to these cells. They are reprogrammed and returned to patients to cure their diseases. [Overview of the project] [Problems that the invention aims to solve]

[0009] Anti-CD19 CAR T therapies have achieved great success in clinical application of leukemia treatment, but the rapid progression of the disease Cytokine storms caused by the lysis of tumor cells, and anti-CD19 CAR T cells Because rapid proliferation can lead to fatal side effects such as the death of normal CD19+ B cells, There is a need to more precisely control CAR T cells. In stem cell-based regenerative medicine, there is an effort to gain a deeper understanding regarding the differentiation process and the trophic role of transplanted cells in the target tissue. On the other hand, these processes may be replaced by some small molecule drugs specifically delivered to stem cells that can further contribute to the regeneration of target cells.

[0010] In addition, a long-standing concern regarding CAR T cells and stem cell-based regenerative medicine is the tumorigenic ability of transplanted cells. In short, it is desirable to endow either CAR T cells or stem cells with a secret pathway to control the activity of transplanted cells after transplantation.

Means for Solving the Problems

[0011] The present invention provides a drug delivery platform that enables fine-tuning of cell therapy. The drug delivery system a. consists of a fusion protein comprising a first component and a second component, the first component and the second component are linked by a peptide linker, the first component is a non-membrane protein, and the second component is a membrane anchor peptide or protein, an artificial functional protein on target cells for transplantation, b. at least one small ligand that binds to the linker and has an inherent high affinity for at least one component of the artificial functional protein, c. at least one drug payload that binds to the linker and is related to the target cells when the small ligand binds to at least one component of the artificial functional protein,

[0012] In some embodiments, the drug delivery platform is an imager It has a drug payload of imaging agents. Such imaging agents include the fluorescent dyes rhodamine and fluorine. Selected from the group consisting of Recein and S0456. Alternatively, such imaging The agent consists of a radioisotope chelate imaging portion, an EC 20 chelate head, and a NOTA. The group is selected from the group consisting of DOTA.

[0013] In some embodiments, the drug delivery platform delivers cytotoxic drugs It has a drug payload. Such cytotoxic drugs include tubulicin, DM1, DM4, and The group is selected from the group consisting of and auristatin.

[0014] In some embodiments, the drug delivery platform is used for gene expression It has a drug payload of modified substances.

[0015] In some embodiments, the drug delivery platform is used for cell activity It has a drug payload of modified substances.

[0016] In some embodiments, the modified substance is dasatinib, a MEK1 / 2 inhibitor, and a group consisting of PI3K inhibitors, HDAC inhibitors, kinase inhibitors, and metabolic inhibitors A group consisting of GSK3-beta inhibitors, MAO-B inhibitors, and Cdk5 inhibitors. Selected from the group.

[0017] In some embodiments, the modified substance is a phosphatase inhibitor, RORγt It is an agonist or siRNA mi181a1.

[0018] In some embodiments, the drug payload is SHP1 / 2, TC-PTP Phosphatase inhibitors include, but are not limited to, inhibitors of [specific enzymes].

[0019] In some embodiments, the drug in the drug delivery platform The payload is such that the small ligand binds to at least one component of the artificial functional protein. If the combination is correct, it is taken up by the target cells.

[0020] In some embodiments, the drug delivery platform is located in the small riga It has a release linker that connects the end and the drug payload. The linker is shown below. Selected from the group.

[0021] [ka]

[0022] In some embodiments, the artificial functional protein component is the folate receptor ALF α (FRa), folate receptor beta (FRb), urokinase receptor (uPAR), FK5 O6 binding protein (FKBP), dihydrofolate reductase (DHFR), fluorescein Single-chain fragment variable component for inisothiocyanate (scF for FITC) v), and single-chain fragment variable components for dinitrophenol (for DNP) Selected from the group consisting of scFv).

[0023] In some embodiments, the small ligand is selected from the group shown below.

[0024] [ka] JPEG0007860924000003.jpg78165

[0025] In some embodiments, the drug delivery platform includes a first component and FKBP, as the second component, contains glycosylphosphatidylinositol as the first component. (GPI) It has a peptide that affixes an anchor, and the small ligand is FK506 or It is a derivative of . In some embodiments, the FK506 derivative is calcium Disable the -lin binding site.

[0026] In some embodiments, the second component is a full-length or truncated folate receptor (F R) is the answer.

[0027] In some embodiments, the drug delivery platform is the artificial machine Flexible peptide linker SG for binding the first and second components of potency proteins. It has at least one segment of GGS.

[0028] In some embodiments, the drug delivery platform is SEQ I D NOS:1-2 (respectively, the amino acid sequences of mouse FKBP-FRa and human FK It comprises an artificial functional protein selected from the group consisting of the amino acid sequence of BP-FRa. .

[0029] In some embodiments, the drug delivery platform is SEQ I It contains artificial functional proteins selected from D NOS:12-15.

[0030] In some embodiments, the target cells for transplantation are immune cells. For example, Immune cells can be NK cells or chimeric antigen receptor T (CAR T) cells. These CAR T cells are selected from amino acid sequences of SEQ ID NOS:3-4. It expresses.

[0031] In some embodiments, the drug delivery platform is represented by formula I It has a small ligand complex.

[0032] [ka]

[0033] In some embodiments, the drug delivery platform is SEQ I D NO:3 (amino acid sequence of mouse anti-CD19 CAR T component) or SEQ I CAR expressing D NO:4 (amino acid sequence of human anti-CD19 CAR T component) It possesses target cells for transplantation as T cells.

[0034] In some embodiments, the small ligand is fluorescent to track drug absorption. It is further bound to a dye or radioactive probe.

[0035] In some embodiments, the drug delivery platform utilizes endogenous genetics. Small rigors that further bind to the regulators of the expression of the introduced transgene or the regulators of the expression of the introduced transgene. It is equipped with a hood.

[0036] In some embodiments of the drug delivery platform, the transplant mark Target cells are stem cells and progenitor cells designed to synthesize biochemicals that are lacking in the patient. , or transplanted cells.

[0037] This invention expresses an amino acid sequence selected from SEQ ID NOS:12-15. Provides CAR T cells equipped with the necessary components.

[0038] The present invention relates to an amino acid sequence selected from the group consisting of SEQ ID NOS: 12-15. It provides DNA constructs that encode [the specified character].

[0039] This invention relates to a SEQ ID operably bound to the EF1a promoter in an expression vector. DN encoding an FKBP-FRa fusion receptor possessing either NOS:1-2 A component is provided. In some embodiments, such an expression vector is SEQ This is a pWPI with ID NO:5.

[0040] This invention provides a DNA construct comprising any of SEQ ID NOS:6-8. .

[0041] This invention relates to the inserted gene hFKBP-FR (SEQ ID NO:7) and human anti-CD syndrome. We provide transplant cells equipped with 19 CAR (SEQ ID NO:9).

[0042] This invention relates to the inserted gene mFKBP-FR (SEQ ID NO:8) and mouse anti-C We provide transplantable cells equipped with D19 CAR (SEQ ID NO: 10).

[0043] This invention provides a method for adjusting the effects of cell therapy. The method is a. Identify target cells for transplantation that possess cellular therapy functions upon transplantation. b. The surface of the target cell comprises a first component and a second component, the first component and the second component They are linked by a peptide linker, the first component is a non-membrane protein, and the second component is glycan. It is a cosylphosphatidylinositol (GPI) anchored peptide or protein. , provides artificial functional proteins, c. A drug in which the drug payload is bound to a small ligand via a linker in the target cells. The complex is provided, and the small ligand is at least one of the artificial functional proteins It binds with high affinity to the components and, together with the drug payload, to the target cells absorbed, d. To modulate the cell therapy function of the target cells, the drug is administered within the target cells. Release the drug from the drug payload. It includes a process.

[0044] In some embodiments, the cell therapy function provides the patient with optically guided surgery. .

[0045] In some embodiments, the cell therapy function controls the proliferation of the target cells. .

[0046] In some embodiments, the cell therapy function involves cancer cells in which the target cells are involved. It induces cytotoxicity against [the target organism].

[0047] In some embodiments, the target cells are immune cells. These cells are CAR T cells.

[0048] In some embodiments, the target cells combine the biochemicals that are deficient in the patient. These are stem cells, progenitor cells, or transplanted cells that are configured to perform a specific function.

[0049] In some embodiments, the drug payload is rhodamine and FITC Imaging agents selected from fluorescent dyes, or EC 20 chelate heads, NOT A is a radioisotope imaging agent selected from DOTA.

[0050] In some embodiments, the drug payload is tubulicin, DM1, D It is a cytotoxic drug selected from the group consisting of M4 and auristatin.

[0051] In some embodiments, the drug payload is dasatinib, MEK1 / 2 inhibitor. Harmful drugs, and kinase inhibitors including PI3 kinase inhibitors, or mi181a1 s It is a gene expression modifier selected from iRNA.

[0052] In some embodiments, the target cells are SEQ ID NOS:12-15 It comprises a fusion protein selected from the group consisting of the following.

[0053] In some embodiments, the artificial functional protein component is FRa, FRb, scFv for uPAR, FKBP, DHFR, FITC, and sc for DNP Selected from the group consisting of Fv.

[0054] In some embodiments, the small ligand is selected from the group shown below.

[0055] [ka] JPEG0007860924000006.jpg78165

[0056] In some embodiments, the small ligand and the drug payload are used to bind the small ligand and the drug payload. The linker is selected from the following group.

[0057] [ka]

[0058] In some embodiments, the target cells are SEQ ID NO:1 and SE The artificial functional FKBP-Linker-FRa fusion is selected from the group consisting of Q ID NO:2. It contains a combination of proteins.

[0059] In some embodiments, the target cells are SEQ ID NO:3 and SE Artificial functional anti-CD19 CAR T components selected from the group consisting of Q ID NO:4 These are CAR T cells that possess [certain characteristics].

[0060] In some embodiments, the drug complex comprises FK506-releasing liVitamin I. Equipped with a linker, the binding domain of FK506 is approximately 4 pM to 100 pM of FKBP. It has affinity.

[0061] [ka]

[0062] In some embodiments, the target cells are CAR T cells, and the drug conjugate The body uses GSK to control the excessive hypercytokinemia of the CAR T cells after transplantation. The system includes an inhibitor selected from the group consisting of 3b inhibitors and MAPK inhibitors.

[0063] In some embodiments, the target cells are CAR T cells, and the drug conjugate The body comprises a modifying substance configured to control the unnecessary proliferation of the CAR T cells.

[0064] In some embodiments, the target cells are stem cells or progenitor cells, and the drug The agent conjugate contains a GSK3b inhibitor to promote fracture repair.

[0065] In some embodiments, the target cells synthesize biochemicals that are deficient in the patient. Stem cells, progenitor cells, or transplanted cells configured to do so, and the drug conjugate is MAO-B inhibitors and cdk5 inhibitors for the treatment of neurodegenerative diseases, including Kinson's disease. It includes an inhibitor selected from a group of drugs.

[0066] In some embodiments, the target cells are NK cells, and the drug conjugate is It contains a RORγt agonist for controlling the cellular immune response by Th17 cells.

[0067] The above and other features of the present invention, as well as its embodiments and effects, are shown in the following drawings and related documents. This is made clear by the description and claims. [Brief explanation of the drawing]

[0068] [Figure 1A] Figure 1A is a schematic diagram of a drug delivery platform using FKBP-FRa and FK506 payloads for cell therapy. [Figure 1B] Figure 1B illustrates a secret pathway platform for delivering payloads to CAR T cells. [Figure 2A] The left panel of Figure 2A shows the chemical structure of FK506 with the highlighted FKBP binding site (yellow) and derivation site (red). The right panel of Figure 2A shows the cocrystal structure of the ternary complex of calcineurin A fragment (green), calcineurin B (blue-green), FKBP12 (purple), and FK506 (yellow), and PDB:1TCO. [Figure 2B]Figure 2B shows various combinations of two modules in an artificial functional fusion protein, along with their respective ligand selections, highlighting potential induction sites. [Figure 3] The left panel of Figure 3 shows the negative and positive regulation of CAR T cell activity, taken from "The quest for spatio-temporal control of CAR T cells" by Sun J. et al. in 2015. The right panel of Figure 3 shows the mechanism of apoptosis induced by AP1903 (FK506 dimer) and FKBP-caspase 9, as well as the structure of AP1903, taken from "Inducible Apoptosis as a Safety Switch for Adoptive Cell Therapy" by Malcolm KB et al. in 2011. [Figure 4A] Figure 4A is a pWPI expression vector map containing the FKBP-FRa insert (hFRa1-24: red, FKBP: yellow, hFRa25-258: red). [Figure 4B] Figure 4B shows FKBP-FRa-transduced K562 cells appearing in a higher-order band around 50 kDa in a comparison with FRa-positive KB cells and untransduced K562 cells. [Figure 4C] Figure 4C shows the design of the payload carrier components and the CAR T components. [Figure 4D] Figure 4D shows the structural design of FKBPFR3GS (denoted as FF3). From the N-terminal to the C-terminal, it contains the signal peptide, human FKBP protein, and human FRa 1-24aa and human FRa 25-258aa, represented as three Gly-Ser linkers. In FKBPFR1GS (denoted as FF1), the three Gly-Ser linkers of FF3 are replaced with one Gly-Ser linker, without changing the other parts. [Figure 4E]Figure 4E shows the component design of 4m5.3FR. From the N terminal to the C terminal, it contains the hCD8 signal peptide, the scFv of the 4M5.3 antibody against FITC, the GS linker, and 25-258aa of human FRa. [Figure 5] Figure 5 shows the interference between FR and FKBP in the FKBPFR1GS fusion receptor. Folic acid binding in the FKBPFR1GS fusion protein inhibits FK506-rhodamine binding at a low concentration of 0.01 nM and completely eliminates FK506-rhodamine binding at 50 nM. [Figure 6] Figure 6 shows the decrease in FK506-rhodamine intensity after binding to OTL38 in FKBPFR1GS Jarcut cells. FRET (fluorescence resonance energy transfer) from FK506-rhodamine (donor) to OTL38 (FA-S0456, acceptor, ex / em: 774 / 794 nm) within the fusion receptor indicates the interaction between FR and FKBP. [Figure 7] Figure 7 shows that increasing the linker length between FKBP and FR significantly reduces interference between FKBP and FR. Compared to FF1 (1GS between FKBP and FR), FF3 (3GS between FKBP and FR) retains the binding of FK506-rhodamine in the presence of 10 nM FA (comparable to the physiological concentration of FA in the human body). [Figure 8] Figure 8 shows the release of the GPI-anchored fusion receptor FF3 upon PI-PLC treatment. Jarcut T cells possessing the FF3 fusion receptor show saturated binding to 20 nM FA-FITC (EC17), but after treatment with 5 mU or 50 mU of PI-PLC, FA-FITC loses its binding to the cells. This indicates the release of the GPI-anchored FF3 fusion receptor. [Figure 9]Figure 9 shows the FA-rhodamine binding curve at the FKBPFR3GS fusion receptor. The FKBPFR3GS fusion receptor, stably expressed on human T cells, can bind to the folic acid derivative (FA-rhodamine) with high affinity (Kd = 0.95 nM), comparable to the affinity of FA-rhodamine in FR+KB cells (Kd around 1 nM). Therefore, the binding characteristics of FR at the fusion receptor are preserved. [Figure 10] Figure 10 shows the FK506-rhodamine binding curve at the FKBPFR3GS fusion receptor. The FKBPFR3GS fusion receptor, which is stably expressed on human T cells, can bind to the FK506 derivative (FK506-rhodamine) with high affinity (Kd = 3.93 nM), thus preserving the FKBP binding properties at the fusion receptor. [Figure 11] Figure 11 shows that SLF-FITC binds to the FKBPFR3GS fusion receptor with relatively high binding affinity (Kd=62nM), but competition with free SLF (100x, pre-incubation) inhibits this binding. SLF, a mimic of FK506, has a 10-fold lower binding affinity to the FKBPFR fusion receptor compared to its parent ligand, FK506, which is consistent with previous reports. [Figure 12] Figure 12 shows the FA-rhodamine binding curve at the 4M5.3FR fusion receptor. FA-rhodamine can bind to the 4M5.3FR fusion receptor stably expressed on human T cells with high affinity (Kd = 2.25 nM), comparable to the affinity of FA-rhodamine in FR+KB cells (Kd around 1 nM). Therefore, the FR binding properties at the 4M5.3FR fusion receptor are preserved. [Figure 13] Figure 13 shows the FITC-AF647 binding curve at the 4M5.3FR fusion receptor. FITC-AF647 can bind to the 4M5.3FR fusion receptor, which is stably expressed on human T cells, with high affinity (Kd = 8.03 nM). 100x comp represents free FITC sodium. The binding properties of scFv 4M5.3 with FITC are retained at the 4M5.3FR fusion receptor. [Figure 14]Figure 14 shows that FA-tubulicin can exert receptor-specific toxic effects on FF3+ human T cells. This effect is inhibited by compensation with FA (100x pre-incubation of FA). This indicates that the free drug tubulicin is absorbed and released without problems via the FF3 fusion receptor system. [Figure 15] Figure 15 shows that FA-tubulosin specifically kills hFF3+ cells in a mixed human T cell culture medium. The absolute number of hFF3+ cells decreases as the FA-tubulosin concentration increases, and hFF3- cells are also killed by the released drug, although a bystander effect is observed at high concentrations. [Figure 16] Figure 16 shows that SLF-Tub specifically causes lethality of hFF3+ Jarcut cells with an IC50 (50% inhibitory concentration) of 138 nM. This indicates that SLF-Tub was successfully taken up by the FKBPFR3GS fusion receptor, and tubulicin was released within the cells. [Figure 17] Figure 17 shows that both FITC-DM4 and FITC-Tub specifically lethal 4M5.3FR+ human T cells, but FITC-Tub has a higher IC50. Compensation with free FITC sodium (100x, pre-incubation) inhibits the receptor-mediated lethal effect. This indicates that FITC-cytotoxic agents are taken up and released by T cells without problems via the 4M5.3FR fusion receptor. [Figure 18] Figure 18 shows that FITC-tubulicin specifically kills the 4M5.3FR+ group in mixed human T cell cultures. As the FITC-Tub concentration increases, the absolute number of 4M5.3FR+ cells decreases, while 4M5.3FR- cells are also killed by the released drug, although a bystander effect is observed at high concentrations. [Figure 19]Figure 19 shows that FITC-DM4 specifically kills the 4M5.3FR+ group in mixed human T cell cultures. As the FITC-DM4 concentration increases, the absolute number of 4M5.3FR+ cells decreases, while 4M5.3FR- cells are also killed by the released drug, although a bystander effect is observed at high concentrations. [Figure 20] Figure 20 shows that 10 nM concentrations of dasatinib (Lck inhibitor) and ibrutinib (ITK inhibitor) reduce the lytic effect of anti-CD19 CAR T cells (FMC63 CAR T, effector) against CD19+ large tumor cells (target). Two effector:target ratios (E:T) were tested. Normal T cells and anti-CD19 CAR T cells with CD19-K562 were used as negative controls. [Figure 21] Figure 21 shows that FITC-dasatinib can reduce the lytic effect of FMC63+4M5.3FR+hT cells on large cells. This indicates that FITC-dasatinib was absorbed and released into T cells without problems via the 4M5.3FR fusion receptor, and that dasatinib was released into the T cells. [Figure 22] Figure 22 shows that a 100 nM TC-PTP inhibitor reduces co-inhibitor molecules in exhausted anti-CD19 CAR T cells (generated by 7x stimulation with CD19+ large cells; see the process described below for details). PD-1, LAG3, and double-positive individuals are all reduced by treatment. This suggests that phosphatase inhibitors like TC-PTP inhibitors can be used as payloads for a secret gateway platform to restore exhausted CAR T cells.

[0069] Array List SEQ ID NO:1 Amino acid sequence of mouse FKBP-FRa SEQ ID NO:2 Amino acid sequence of human FKBP-FRa SEQ ID NO:3 Amino acid sequence of mouse anti-CD19 CAR T component SEQ ID NO:4 Amino acid sequence of human anti-CD19 CAR T components SEQ ID NO:5 Vector pWPI for human T cell transduction SEQ ID NO:6 pMP71 gb NotIEc for mouse T cell transduction oRI mouse anti-CD19 SEQ ID NO:7 pWPI-FRa 1-24 FKBP FRa SEQ ID NO:8 pWPImFKBP-mFRa SGGGS SEQ ID NO:9 pHR EcorI hAnti cd19 1D3 my c hinge cd28 cd3zeta SEQ ID NO:10 pWPI pmei mAnti cd19 1D3 m yc hinge cd28 cd3zeta SEQ ID NO:11 FKBP-1SG- has a GPI anchor amino acid sequence. FR SEQ ID NO:12 FKBP-3SG- has a GPI anchor amino acid sequence. FR SEQ ID NO:13 4M5.3-FR with GPI anchor amino acid sequence SEQ ID NO:14 FMC63-T2A-FKBP3SGFR SEQ ID NO:15 FMC63-T2A-4M5.3SGFR SEQ ID NO:16 FRb containing signal peptide SEQ ID NO:17 uPAR containing signal peptide SEQ ID NO:18 DHFR SEQ ID NO:19 FITC:4M5.3(Kd=200pM) Fv SEQ ID NO:20 FITC 4D5Flu(Kd=10nM) Fv SEQ ID NO:21 scFv for DNP SPE7 [Modes for carrying out the invention]

[0070] The concept of the present invention has been described in detail in the drawings and specification, but the drawings and specification do not describe the concept of the present invention. The results shown are illustrative and do not limit the features of the present invention. Exemplary embodiments are shown. As such, all changes and modifications that fall within the scope of the present invention are protected. It should.

[0071] Unless otherwise defined, chemical and technical terms refer to terms used by those skilled in the art in the field of the present invention. It shall have the same meaning as the commonly understood meaning.

[0072] This invention involves genetically incorporating fusion receptors onto the surface of transplanted cells, thereby promoting the activity of transplanted cells. This provides a novel platform for controlling sex. These transplanted cells are small molecule ligands. By utilizing the inherent high affinity between the fusion receptor on the surface of the transplanted cell and the drug payload, It is specifically targeted by the bound small molecule ligand. By some fusion receptors, The fusion is taken up by the transplanted cell, and once the payload is taken up by the transplanted cell, the release linker The payload is released via the following: the type of transplanted cells and the desired adjustments imposed on the transplanted cells. Based on the structure, the drug payload exhibits various functions. By altering the process, for example by using cytotoxic drugs or kinase inhibitors The drug payload is applied to multiple aspects of transplanted cells, such as proliferation, differentiation, or cytokine release properties. It can be used to control surfaces.

[0073] Peptidylproline isomerase (PPIases) are a group of FK506-binding proteins. It consists of (FKBP), cyclophylline, and parburin. In humans, 18 FK These include BPs, 24-cyclophylline, and 3-palbrin. Of these, FKBP5 1 and FKBP52 are FKBP12(KD FK506 Compared to (≒0.2nM), it is high. It is moderate, KD FK506 ≈10⁴nM, and KD FK506 Approximately 23 nM Furthermore, each of these two FKBPs has binding affinity to FK506. Neither of these are expressed on the cell membrane, and in the human system, they have almost no cross-binding activity. i. FKBP WT FKBP12 F36V FKBP51 F67V Affinity to Efforts have also been made to construct synthetic ligands that maintain the overall structure of the wild-type protein. All homologs and mutant proteins having these ligands are applicable to the present invention. It is possible.

[0074] Specifically, in one embodiment, an exemplary pair of a small molecule ligand and a fusion receptor are used. The FK506-FKBP is selected. For all processes, see Figure 1A for general information. The following was shown. First, the FK506 payload binds to the FKBP-FRa artificial functional cells. And, through this binding, the transmembrane fusion protein incorporates the payload-linker-FK506. Next, the captured FK506 payload is cut by the linker, and the payload is finely divided. It is released into the cell. Depending on the cell type and payload type, the released drug payload The dot performs the desired function.

[0075] Figure 1B illustrates cell therapy using specific chimeric antigen receptor T cells. In this model, C AR T cells have a structure from the N-terminal to the C-terminal and possess appropriate signal peptides. A protein containing a fusion protein and bound to the GPI-anchored protein module 1. Module 2 is expressed in cancer cells. In some embodiments, cancer cells express CD19 It has a surface protein, and the target ligand is at least one module of the fusion receptor When bound, this CD19 surface protein is recognized by CAR T cells. It engages with the payload possessed by CAR T cells. Typically, the target ligand is one of these modules. It has high affinity for one of the ligands, and the payload bound to the ligand is the target molecule. It is taken up into the cell, released, and engages with cancer cells via chimeric antigen receptors.

[0076] As shown in Figure 2B, there are many different combinations of GPI-anchored proteins. Module 1 and its ligand, the target cell surface protein of Module 2 and It is presented as a ligand. Either a protein from Module 1 or a protein from Module 2. Alternatively, both may be high affinity targets to facilitate ligand-bound payload delivery. It engages with a ligand. For example, a fusion protein having an FRa-linker-FKBP structure. The quality is feasible, FRa engages with FA derivatives, and simultaneously FKBP is FK506 derivative. The FA derivative or FK506 derivative, or both, engage with the body and act as cytotoxic agents. It can be bound to a payload such as an imaging agent or a modifying substance. Because it has the flexibility to carry the same or different payloads, different or the same payload Unexpected effects, synergistic effects, or adjustments can be obtained through loading, and the payload is image When used as a aging agent, it becomes possible to observe target cells. The payload of this system The advantages of the flexibility and versatility of code delivery will be further illustrated by additional examples.

[0077] Similarly, other embodiments of ligands paired with GPI anchor proteins include uP The ligands that pair with AR are shown below.

[0078] [ka]

[0079] Furthermore, as for ligand structures, for example, FITC or its derivatives are FITC-based The structure (Chemical Formula 5) that binds to a single-chain fragment of the antibody variant (scFv), FKBP and Pairing ligand structure (Chemical Formula 6), ligand structure that pairs with DHFR (Chemical Formula 7), or It has a ligand structure (Chemical Formula 8) that pairs with scFV for DNP.

[0080] [ka]

[0081] [ka]

[0082] [ka]

[0083] [ka]

[0084] FK506 is an exemplary ligand-protein pair in this delivery system. - Let's mention some of the advantages of choosing FKBP. Advantage 1: FKBP is suitable for mammals Because it is not a membrane protein that naturally exists on the cell membrane, the FK506-payload conjugate , it specifically binds to target cells. Advantage 2: The FKBP protein has a molecular weight of 12 kDa. Because it is a relatively small protein, it is most susceptible to perturbations that affect the structure and absorption properties of the receptor. It can be reduced in size and easily fused with other receptors. Advantage 3: FK506 is used in the human body Because it does not naturally exist, the fusion receptor is not inhibited. Advantage 4: FK506-FKBP and Since the binding affinity is approximately 4 pM, the drug payload is transported with high affinity. Point 5: The co-crystal structure of FK506-FKBP is available, and stable induction of FK506 is possible. The somatic site disables unwanted binding between FK506 and calcineurin, while FK5 The 06-FKBP bond is maintained (see Figure 2A).

[0085] The sequence of FKBP is thought to be modifiable, and the corresponding FK506 ligand is considered to be available. Furthermore, the modified FKBP and modified FK506 achieve the desired affinity exemplified herein. It can be modified within the range that maintains a higher level of affinity.

[0086] Other parts of the fusion protein have a good process for incorporating monomers. Folate receptors (FRs) are selected. According to previous research, they bind to "magic carbonates". The folate conjugate is taken up via FR and cleaved by the reducing environment within the cell. Using this mechanism, the drug payload in the FK506-FKBP conjugate is delivered to FR. Therefore, it is taken up and released into the cytoplasm.

[0087] The great potential of CAR T therapy, and its severe side effects, has led to the development of several controlled CAR T therapies. T cell designs have been reported. The vast majority of them are Boolean for T cell activity. The focus is on ON / OFF switches by incorporating one of the cascaded paths. (Sun J. et al.'s "The quest for spatio-temporal control of CAR T") The negative regulation and positive changes in CAR T cell activity, as quoted from "cells" (2015) (See Figure 3, left, for an example of the idiomatic adjustment.) Malcolm KB's group, FK Designing a BP-caspase9 fusion protein to induce apoptosis in target cells For this purpose, the FK506 dimer was used (by Malcolm KB et al. in 'Inducible Ap Quoted from "Optosis as a Safety Switch for Adoptive Cell Therapy" (2011). FKBP-caspase9, induced by AP1903 (FK506 dimer) (See Figure 3, right panel, which shows the mechanism of apoptosis and the structure of AP1903).

[0088] The present invention has several advantages over these reported methods. Advantage 1: Bina Instead of an ON / OFF switch, the present invention platform has multiple types of adjustable pedals. Because it can deliver iroad and modify multiple aspects of target cells, Compared to an ON / OFF switch, it is extremely flexible. Advantage 2: In the control section Because the FK506 payload is a small molecule, it is linear when compared to pre-artificial functional cells. Control and optimization of drug dosage are possible. Advantage 3: The platform is CAR T cell It can be used not only within the body but also in regenerative medicine based on multiple other stem cells. ru.

[0089] The latest and most important aspect of the platform according to the present invention is the potential side effects or It lies in a multi-functional payload that can be selected to address any need for improving the efficiency of cell therapy. Under the following conditions, cytotoxic drugs are delivered to transplanted cells: Condition 1: Anti-CD19 C Like AR T cells, cells proliferate excessively and affect normal organs or systems. Case 2: Due to the lentivirus-based genetic manipulation and the unique characteristics of stem cells, If the cyst becomes tumor-forming.

[0090] On the other hand, some cell therapies have been largely unsuccessful due to the suppressive microenvironment of the target tissue. No. For example, in CAR T-cell therapy for solid tumors, a low penetrability is not the case. Separately, MDSCs and tumor cells severely suppress the proliferation and activity of CAR T cells. This is due to T cells induced by RORrt agonists or MAP kinase inhibitors. Cellular activity, TLR8 agonist-induced granzyme B expression in CAR T cells The current situation may be mitigated. The payload of the present invention includes intracellular targets such as RORrt. Although this is more preferable, due to proximity on the cell membrane, membrane receptors such as TLR8 also act It is possible.

[0091] In stem cell regenerative therapy, the payload becomes more diverse depending on the disease model. A pre-fixed gene developed using stem cells as a delivery platform. Instead of transporting offspring, this invention allows for fine-tuning of the transplanted cells and their microenvironment. This makes it possible to obtain desired phenotypes using a variety of small molecule payloads. This small molecule specifically targets transplanted cells that overexpress FKBP-FRa. Because it is bound to 506, nonspecific targeting of normal tissue by small molecules is avoided. It can be done.

[0092] As one example, mesenchymal stem cells (MSCs) for fracture repair in bone regeneration therapy are used. One example is the induction of BMP2 overexpression. BMP2 and / or VEGF expression The level is achieved by the drug payload delivery system of the present invention, which delivers GSK3 to transplanted cells. This can be improved by introducing GSK3-beta inhibitors. GSK3-beta inhibitors are used in bone It is a desirable drug for fracture repair. Therefore, GSK3 beta inhibitors are used within the fracture site. Available payloads to further refine the functionality of the ported MSC and microenvironment. It is ideal as such.

[0093] Other examples of drug payload delivery systems include those for Alzheimer's disease and Parkinson's disease. There are neurodegenerative diseases, including Sonnson's disease. In these neurodegenerative diseases, MSC-based treatments are used. Therapy is key to future success. MSC uses GDNF, VEGF, and several other substances. It is manipulated to overexpress itokines. On the other hand, small molecules like MAO-B inhibitors The offspring can increase the expression of GDNF, NGF, and BDNF in astrocytes. Confirmed. PI3K inhibitors (BEZ235), Cdk5 inhibitors (roscovitine), And several kinase inhibitors, such as GSK3b inhibitors (NP-12), also help with Alzheimer's disease. These have been proposed as a treatment for Mars disease. By using the FK506-FKBP pair, which specifically transports small molecules, the regeneration efficiency is improved. As the effectiveness of these potent inhibitors and agonists improves in other non-target tissues, This will help avoid the side effects.

[0094] [material and method] (Compounds and synthesis process) The target ligand binds to the payload via the linker. Linker optimization options. The following are the payloads: I. Imaging, II. Cytotoxic drugs, III. Regulatory small They are classified into three types of molecular drugs.

[0095] (Linker optimization) [ka]

[0096] (Compound classification) Category Function Example I. Imaging Fluorescent dyes: Rhodamine, FITC Radioisotope imaging: EC 20 chelate head, NOTA, DOTA II. Cytotoxic drugs: Microtubule inhibitors: tubulosin, DM1, DM4 III. Modifiers Kinase inhibitors: Dasatinib, MEK1 / 2i, PI3Ki siRNA:mi181a1

[0097] [Detailed compound structure and synthesis route] (FK506-Rhodamine) [ka]

[0098] Process: Rhodamine-NHS ester (1.0 equivalent) in dimethylformamide is added to Boc -NH-PEG3-NH2 (1.2 equivalents) and diisopropylethylamine (3.0 equivalents) The mixture was reacted with (amount) at room temperature for 2 hours. The product was then subjected to preparative reverse-phase HPLC equipped with a UV detector. Therefore, it was purified. Purified rhodamine-PEG3-NH-Boc conjugate (1.0 equivalent) ) is stirred for 2 hours in a 1:10 TFA-dichloromethane system, which results in Bo c was deprotected. The crude free amine product was dissolved in dimethylformamide and diisopropyl alcohol was released. In the presence of propylethylamine (3.0 equivalents), EDC (2.0 equivalents), HOBT It was activated by (2.0 equivalents). After 15 minutes, FK506-CO2H (1.2 equivalents, This information is found on pages 5763-5768 of Bioorg. Med. Chem., Vol. 17 (2009). The synthesized product (using the procedure) was added, and the reaction mixture was stirred overnight. A UV detector was used. After purification by preparative reverse-phase HPLC (monitored at a wavelength of 280 nM), the final FK506 - The rhodamine conjugate was isolated. The crude product was subjected to preparative HPLC on Xterra RP18. Loaded onto a column (water), 95% 5 mM sodium phosphate (mobile phase A, pH 7.4) The process starts with 5% acetonitrile (mobile phase B), and the flow rate is 12 mL / min for 35 minutes. Elution was performed under gradient conditions where phase A reached 0% and mobile phase B reached 50%. The product peak was The retention time is within the gradient (mobile phase B is 0-50%) in the 7-minute HPLC-MS analysis. The time was 2.5 minutes (ESIm / z = 1539.6, abbreviation: PEG = polyethylene glycoside EDC = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HO BT = hydroxybenzotriazole; HPLC = high-performance liquid chromatography).

[0099] (FK506-NIR dye)

change

[0100] Synthetic Engineering:

change

[0101] (SLF-FITC)

change

[0102] Synthetic Engineering:

change

[0103] (SLF-EC20)

change

[0104] Synthetic Engineering:

change

[0105] (SLF-チューブリシン)

change

[0106] Synthetic Engineering:

change

[0107] (FITC-AF647)

change

[0108] Synthesis process: [ka]

[0109] (FITC-DM4) [ka]

[0110] Synthesis process: [ka]

[0111] Process: Add DM4 (1.0 equivalent) in dimethyl sulfoxide to 2-(pyridine-2-yl) Disulfanyl)ethane-1-amine (1.0 equivalent) and diisopropylethylamine (3.0 equivalents) was reacted with FITC (1.0 equivalent). The reaction mixture was stirred for 1 hour. Preparative reverse-phase HPLC (28) equipped with a UV detector. After purification (monitored at a wavelength of 0 nM), the final FITC-DM4 conjugate was isolated. The crude product was loaded onto an Xterra RP18 preparative HPLC column (Waters). Then, 95% 5 mM sodium phosphate (mobile phase A, pH 7.4) and 5% acetonitrile (mobile Starting in mobile phase B), at a flow rate of 12 mL / min for 10 minutes, mobile phase A is 0% and mobile phase B Elution occurred under gradient conditions where the gradient reached 100%. The retention time of the product peak was 7 minutes in H The gradient in the PLC-MS analysis (mobile phase B from 0-100%) was 4.23 minutes. (ESIm / z=1244.8, abbreviation: FITC = fluorescein isothiocyanate; HPLC (High-Performance Liquid Chromatography).

[0112] (FITC-Tub)

Chemistry

[0113] Synthetic engineering:

Chemistry

[0114] (FITC-EC20)

Chemistry

[0115] Synthetic engineering:

Chemistry

[0116] (FITC-Dasatinib)

Chemistry

[0117] Synthetic engineering:

Chemistry

[0118] [Experimental procedure] (Cell culture) 293TN cells were cultured in DMEM with 10% FBS (without antibiotics for lentivirus packaging). Large cells and Jurkat cells were cultured in RPMI-1640 with 10% FBS, 10% penicillin / streptomycin. Primary human T cells were isolated from hPBMC using Ficoll and negative selection with the EasySep™ Human T Cell Enrichment Kit (19051, Stemcell Tech). to). Large cells and Jurkat cells were cultured in RPMI-1640 with 10% FBS, 10% penicillin / streptomycin. Primary human T cells were isolated from hPBMC using Ficoll and the EasySep™ Human T Cell Enrichment Kit (19051, Stemcell Tech) by negative selection Concentrated by Dynabeads CD3 / CD28 (11161D, Therm Activated for 1 day by Fisher (130-097) TexMACS medium supplemented with -745 (Miltenyi Biotec Inc.) The T cells were cultured in 20% human AB serum (HP1022, Valley Biom). The samples were cryopreserved in RPMI-1640 (containing edical) and 10% DMSO.

[0119] (Lentivirus packaging) Using lipofectamine 2000, the transgene expression vector and packaging Rasmid mixture (CPCP-K2A, Cellecta) 293TN cells Pantropic VSV-G jade typification via linguistics (transfer of genes). A lentivirus was produced. After 24 hours, the viral supernatant was obtained, concentrated, and then... It was added to specific cell lines or primary T cells that were thawed on the same day. For input, after adding the viral supernatant and 8 ug / ml polyblen, the cells were divided into 2500 cells. Centrifugation was performed at rpm and 37°C for 90 minutes.

[0120] (Binding assay) For binding assays, either ligand-dye only, or ligand-dye and free ligatures can be used. The cells were cultured at 4°C for 30 minutes with a 100x pre-cultured culture medium. Next, wash the cells three times, resuspend them in 2% FBS PBS, and use 7-A to remove dead cells. AD was added. FRET imaging of FK506-rhodamine and FA-S0456. G: To determine the occupancy rate of the fusion receptor, use FKBP-FR in the prescribed procedure and concentration. a+ Jarcut cells FA / FA-S0456, FK506 / FK506-Rhodami It was cultured together with FRET. FRET is found to affect FA-OTL38 on the same or nearby receptors. As energy is transferred, this is visualized by the loss of FA-rhodamine's intensity, BD Detected by a Fortessa® flow cytometer. The result is Flow The analysis was performed using Jo software.

[0121] (PI-PLC treatment to release GPI-anchored proteins) 1 x 10 5 Dissolve the cells in digestion buffer (2% BSA) at a dose of 5 mU or 50 mU. The cells were incubated in PI-PLC (P5542-5UN, Sigma) at 37°C for 30 minutes. After incubation, the cells were washed three times with PBS and cultured on ice with ligand dye for 30 minutes.

[0122] (Cell viability assay) The cells were seeded in 96% well plates and given free ligand (100x pre-incubated). For both cases where competition by (synthesis) is present and where it is not, specific ligands at different concentrations - Cells were seeded in a 96-well plate with a cytotoxic agent and cultured for 2 hours. After culturing, the cells were washed three times with warm medium and replenished with fresh medium. After 72 hours, Cel The lTiter-Glo® assay (G7570, Promega) The number of cells is examined, or the receptor-positive cells are quantified by staining them with ligand dyes. Ta.

[0123] (CAR T cell lysis effect) 1 x 10 5 CAR T cells and a certain number of large cells, with or without therapeutic agents. , Co-cultured in a 96-well plate based on the E:T ratio. After 24 hours, 100 ul of the supernatant was taken out for the LDH assay. The lysis rate was calculated as (treatment group - CAR T only) / (maximum lysis - CAR T only)%. The supernatant was taken out for the LDH assay. The lysis rate was calculated as (treatment group - CAR T only) / (maximum lysis - CAR T only)%. The supernatant was taken out for the LDH assay. The lysis rate was calculated as (treatment group - CAR T only) / (maximum lysis - CAR T only)%.

[0124] (Exhaustion of CAR T cells) Without changing the medium, 1×10 large cells were repeatedly added to 1×10 CAR T cells in a 24-well plate every 12 hours. The exhaustion status was characterized by a lower lysis effect and higher expression of inhibitory molecules (PD-1, LAG-3 and Tim-3). 6 Without changing the medium, 1×10 large cells were repeatedly added to 1×10 CAR T cells in a 24-well plate every 12 hours. The exhaustion status was characterized by a lower lysis effect and higher expression of inhibitory molecules (PD-1, LAG-3 and Tim-3). Without changing the medium, 1×10 large cells were repeatedly added to 1×10 CAR T cells in a 24-well plate every 12 hours. The exhaustion status was characterized by a lower lysis effect and higher expression of inhibitory molecules (PD-1, LAG-3 and Tim-3). 6 Without changing the medium, 1×10 large cells were repeatedly added to 1×10 CAR T cells in a 24-well plate every 12 hours. The exhaustion status was characterized by a lower lysis effect and higher expression of inhibitory molecules (PD-1, LAG-3 and Tim-3). Without changing the medium, 1×10 large cells were repeatedly added to 1×10 CAR T cells in a 24-well plate every 12 hours. The exhaustion status was characterized by a lower lysis effect and higher expression of inhibitory molecules (PD-1, LAG-3 and Tim-3). Without changing the medium, 1×10 large cells were repeatedly added to 1×10 CAR T cells in a 24-well plate every 12 hours. The exhaustion status was characterized by a lower lysis effect and higher expression of inhibitory molecules (PD-1, LAG-3 and Tim-3).

[0125] (In vivo elimination of fusion receptor-positive CAR T cells by ligand-cytotoxic drug) 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 8th day, the ligand-cytotoxic drug (0.5 umole / kg, 1 umole / kg) was intravenously injected again. Serum samples collected every 3 days after the injection of CAR T cells were used to measure IL2 and INF by ELISA. The CAR T cells in peripheral blood were counted using flow cytometry (flow cytometry method). 5 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 8th day, the ligand-cytotoxic drug (0.5 umole / kg, 1 umole / kg) was intravenously injected again. Serum samples collected every 3 days after the injection of CAR T cells were used to measure IL2 and INF by ELISA. The CAR T cells in peripheral blood were counted using flow cytometry (flow cytometry method). 7 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 8th day, the ligand-cytotoxic drug (0.5 umole / kg, 1 umole / kg) was intravenously injected again. Serum samples collected every 3 days after the injection of CAR T cells were used to measure IL2 and INF by ELISA. The CAR T cells in peripheral blood were counted using flow cytometry (flow cytometry method). 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 8th day, the ligand-cytotoxic drug (0.5 umole / kg, 1 umole / kg) was intravenously injected again. Serum samples collected every 3 days after the injection of CAR T cells were used to measure IL2 and INF by ELISA. The CAR T cells in peripheral blood were counted using flow cytometry (flow cytometry method). 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 8th day, the ligand-cytotoxic drug (0.5 umole / kg, 1 umole / kg) was intravenously injected again. Serum samples collected every 3 days after the injection of CAR T cells were used to measure IL2 and INF by ELISA. The CAR T cells in peripheral blood were counted using flow cytometry (flow cytometry method). 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 8th day, the ligand-cytotoxic drug (0.5 umole / kg, 1 umole / kg) was intravenously injected again. Serum samples collected every 3 days after the injection of CAR T cells were used to measure IL2 and INF by ELISA. The CAR T cells in peripheral blood were counted using flow cytometry (flow cytometry method). 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 8th day, the ligand-cytotoxic drug (0.5 umole / kg, 1 umole / kg) was intravenously injected again. Serum samples collected every 3 days after the injection of CAR T cells were used to measure IL2 and INF by ELISA. The CAR T cells in peripheral blood were counted using flow cytometry (flow cytometry method).

[0126] (In vivo regulation of fusion receptor-positive CAR T cells by ligand-drug) 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 7th day, the ligand-drug (0.5 um 5 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 7th day, the ligand-drug (0.5 um 7 4×10 luc+ large cells were intravenously injected into NSG mice. Six days later, 1×10 fusion receptor-positive CAR T cells were intravenously injected. On the 7th day, the ligand-drug (0.5 um The ole / kg (1 umole / kg) was injected intravenously again. CAR T cell exhaustion model In the case of ru, 1 × 10 5 CAR T cells were injected intravenously on day 6, and CAR T cells were introduced into the peripheral blood. If a cell population appears and the tumor volume remains unstable and continues to increase, a ligand-drug (0.5µg) may be used. The dose (mol / kg, 1 umole / kg) was administered intravenously.

[0127] (In vivo imaging and tracking of fusion receptor-positive CAR T cells) 2 x 10 6 Large cells were subcutaneously injected into the right shoulder of NSG mice. After 14 days, 1 × 10⁶ cells were found. 7 Melt Synergistic receptor-positive CAR T cells were intravenously injected. On day 6, 99m of the cells were injected intravenously into mice. A conjugate containing Tc (10 nMol, 150 μCi) was administered, followed by SPECT imaging. The images were captured using a imaging device. [Examples]

[0128] 1. Design of fusion proteins and fusion protein expression 1.1 Design of the FKBP-FR fusion receptor (SEQ ID NO:2) Synthesis of FK506 derivatives: FK506-rhodamine and FK506-tubulicine The synthesis is described in the Materials and Methods section.

[0129] hFRa is a GPI-anchored membrane protein, and as a signal peptide, it has 24 at its N-terminus. It contains the amino acids. In order to maintain membrane presence and uptake characteristics, it is a full-length FR. Using a, the hFKBP sequence and the flexible peptide between T24 and R25 of hFRa The drinker (SGGGS) is incorporated (Figure 4A). The flexible linker is one of the human body They are selected to be resistant to general enzymatic digestion. They produce proteins in transduced T cells. Along with EF1a as the desired promoter to represent the entire sequence, the entire sequence is pWPI Renam Insert it into the thivirus expression vector.

[0130] 1.2 Expression of FKBP-FR fusion receptor in transduced cells FKBP-FRa expression is observed in K562 transduced by lentivirus. Confirmed by tamblotting. Lysis of transduced K562 cells is confirmed by hFRa antibody. In contrast to non-transduced cells, it shows a specific band around 50 kDa (Figure 4B). ).

[0131] 1.3 Construction of the FKBPFR3GS fusion protein (denoted as FF3) Refer to Figure 4D (SEQ ID NO: 12). From terminal N to terminal C, signal peptide Human FRa 1-24aa as a component, human FKBP protein, and three Gly-Ser It contains a linker and human FRa 25-258aa. FKBPFR1GS(FF In the construction design of FF3 (referred to as 1), the three Gly-Ser linkers of FF3 are the other It can be replaced with a single Gly-Ser linker without changing any parts. As shown in (i), by increasing the length of the linker, the two components of the fusion protein Interference between them is reduced.

[0132] 1.4 Construction of a FITC-svFv-FR fusion protein with a GS linker See Figure 4E. This component is also called 4M5.3FR (SEQ ID NO: 13). From the N terminal to the C terminal, the hCD8 signal peptide (sp), 4M5.3 (against FI) It contains TC) svFv, GS linker, and human FRa 25-258aa.

[0133] 1.5 FK506-99mTc PET imaging, in vivo, F Non-invasive tracking of KBP-FRa / FKBPtFRa-positive cells For CAR T cell models, 1 × 10 6 KB cells were introduced into severely immunodeficient mice (NSG, Ja It was subcutaneously transplanted to the ckson Laboratory. The tumor was 100 mm. 3 After exceeding that, 15 million anti-FITC CAR+ FKBP- FRa+ human cells, or anti- Human T cells of FITC CAR+ FKBP- FRa- were intravenously injected into the mice in question. FITC-FA was injected for the specified number of days to induce proliferation of CAR T cells. The mice were imaged every two days after transplanting CAR T cells using the following procedure. On the day of the event, based on previous reports, 99mTc (technetium-99) was also present. The FK506-EC20 head was incorporated into the solution. 200uCi(my) in 100ul solution 99mTc (Crocury) was intravenously injected into each mouse, and the tumor area, spleen, and Focusing on the lymph nodes, a whole-body image was acquired using MiLab PET / CT. 3D images were created using MOD software. Approximately 10 days after CAR T cell transplantation. After imaging, the mice were euthanized, and the 99mTc levels in each organ were measured. The distribution was counted using a gamma counter.

[0134] For hematopoietic stem cell transplantation models, we created humanized NSG mice as previously reported. 10 million CD34+ FKBP- FRa+ hHSCs were introduced into humanized NSG mice. It was administered by intravenous injection. Four months later, as described above, FK506-99mTc was used to treat the bone marrow and spinal cord. We focused on the vertebrae and took a full-body image.

[0135] 2. FKBP-FRa fusion for specific binding and incorporation into the FK506 payload. receptor 2.1 FKBP-FRa fusion receptor that specifically binds to FK506-rhodamine For the binding assay, either ligand dye alone, or ligand dye and free ligand ( The cells were cultured at 4°C for 30 minutes along with (pre-cultured at 100x for 30 minutes). After culturing, Wash the cells three times, resuspend them in 2% FBS PBS, and use 7-AAD to remove dead cells. [The substance] was added. A BD Fortessa® flow cytometer was used. The results were: The analysis was performed using FlowJo software.

[0136] 2.2. Binding of FK506-rhodamine by the FKBP-FRa fusion receptor (FK5 06-FRET imaging of rhodamine and FA-S0456) To determine the occupancy rate of the fusion receptor, use the specified procedure and concentration: FKBP- FRa+ Jarcut cells with FA / FA-S0456, FK506 / FK506-rhodamine They were cultured together. FRET (fluorescence resonance energy transfer) was performed on the same or nearby receptors. As energy is transferred to FA-OTL38, the strength of FA-rhodamine is lost. Therefore, it is visualized and detected by the BD Fortessa™ flow cytometer. The results were analyzed using FlowJo software.

[0137] Figure 5 shows that the binding of folate to the FKBPFR1GS fusion protein is 0.01 nM. Even at low doses, it inhibits the binding of FK506-rhodamine, and at 50 nM, it inhibits the binding of FK506-rhodamine. This demonstrates the complete disappearance of the nucleotide bond.

[0138] Figure 6 shows that FKBPFR1GS Jarcut cells react to the folate receptor target dye OTL3 This shows a decrease in FK506-rhodamine strength after binding to 8. FK506-rhodamine ( (Donor) OTL38 (FA-S0456, acceptor, ex / em:774 / 794 FRET (fluorescence resonance energy transfer) to nm) is performed on FR and F within the fusion receptor. This shows the interaction between KBPs.

[0139] Figure 7 shows how the two components of the fusion protein, FKBP, are separated by lengthening the linker. This indicates a significant reduction in interference between FRs. FF1 (between FKBP and FRs) Compared to 1GS, FF3 (3GS between FKBP and FR) has a 10nM FA FK506-rhodamine binding in the presence of (a concentration comparable to the physiological concentration of FA in the human body) It holds.

[0140] 2.3. Release of GPI-anchored FF3 fusion receptor By using PI-PLC treated T cells containing FF3 fusion protein, GPI The anchor receptor FF3 was released. Jarcat T cells possessing the FF3 fusion receptor... It shows saturated coupling with 20nM FA-FITC(EC17), but not with 5mU PI-PLC. Alternatively, after treatment with 50mU of PI-PLC, FA-FITC loses its binding to cells. This means the release of the GPI-anchored FF3 fusion receptor.

[0141] 2.4 Retention of FR binding properties of the fusion protein FKBPFR3GS in human T cells This shows the FA-rhodamine binding curve at the FKBPFR3GS fusion receptor. (Human T cells) The FKBPFR3GS fusion receptor, which is stably expressed above, is found in FR+KB cells (around 1 nM). High affinity (Kd=0.95nM) comparable to that of FA-rhodamine in terms of Kd. ) allows it to bind to the folic acid derivative (FA-rhodamine). Therefore, fusion The binding properties of FR at the receptor are preserved. See Figure 9.

[0142] 2.5 FKBP binding properties of the fusion protein FKBPFR3GS in human T cells retention This shows the FK506-rhodamine binding curve at the FKBPFR3GS fusion receptor. (Human) The FKBPFR3GS fusion receptor, which is stably expressed on T cells, exhibits high affinity (Kd=3). At 93nM, it can bind to the FK506 derivative (FK506-rhodamine). Therefore, the binding properties of FKBP at the fusion receptor are preserved. See Figure 10.

[0143] 2.6 FK of SLF-FITC due to relatively high binding affinity (Kd=62nM) Binding to the BPFR3GS fusion receptor Binding is inhibited due to competition with free SLF (100x, pre-incubation). SLF, which mimics FK506, is different from FKBPFR when compared to its parent ligand FK506. The binding affinity to the fusion receptor is 10 times lower. See Figure 11 and compare it with Figure 10.

[0144] 2.7 Binding curve of FA-rhodamine at the 4M5.3FR fusion receptor FA-rhodamine is found in FR+KB cells (Kd around 1 nM). With high affinity (Kd=2.25nM) comparable to that of [another substance], it can stably form on human T cells. It can bind to the expressed 4M5.3FR fusion receptor. Therefore, 4M5.3 The FR binding properties of the FR fusion receptor are preserved. See Figure 12.

[0145] 2.8 FITC-AF647 binding curve at the 4M5.3FR fusion receptor FITC-AF647 is stable in human T cells due to its high affinity (Kd=8.03nM). It can bind to the 4M5.3FR fusion receptor that is expressed as a result. "p" represents free FITC sodium. The 4M5.3 bond of scFv containing FITC. The properties are retained in the 4M5.3FR fusion receptor. See Figure 13.

[0146] 3.1. Lethal effect of FA-tubulosin on FF3+ human T cells FA-tubulosin exerts a receptor-specific lethal effect against FF3+ human T cells. Compensation by FA (FA's 100x pre-incubation) does not hinder such effects. This means that the free drug tubulicin can enter the FF3 fusion receptor system without any problems. This means that substances are taken in and released. See Figure 14.

[0147] 3.2 Efficacy of FA-tubulosin on hFF3+ cells in mixed human T cell cultures Death effect FA-tubulosin specifically induces lethality in the hFF3+ group in mixed human T cell cultures. The percentage of hFF3+ cells decreases as the concentration of FA-tubulosin increases. See Figure 15.

[0148] 3.3 IC 50 S = 138nM specifically kills hFF3+ Jarcut cells. LF-tubulosin SLF-tubulosin was cultured with hFF3 jarcut cells for 2 hours. This allows for the lethalation of receptor-positive cells. This is because SLF-tubulicin, It is taken up without issue by the FKBPFR3GS fusion receptor and enters the cell via tubes. This indicates the release of ricin. See Figure 16.

[0149] 3.4 FITC-DM4 and FITC-Turn on 4M5.3FR+ human T cells - The lethal effect of brisin FITC-tubulosine has a higher IC 50 It has FITC-DM4 and F Both ITC-tubulicin specifically lethal 4M5.3FR+ human T cells. Compensation with free FITC sodium (100x pre-incubation) is effective for receptors. It inhibits the mediating lethal effect. This is because FITC- This demonstrates that cytotoxic drugs are taken up and released by T cells without any problems. See Figure 17.

[0150] 3.5 FITC-tubulosin 4M5.3FR+ group in mixed human T cell cultures Specific lethal effect against As the concentration of FITC-tubulicin increases, the absolute number of 4M5.3FR+ cells increases. While the number of 4M5.3FR- cells decreases, 4M5.3FR- cells are also lethal due to the released drug, but at high concentrations... The bystander effect is observed. See Figure 18.

[0151] 3.6 Equivalent of FITC-DM4 in the 4M5.3FR+ group in mixed human T cell cultures Specific lethal effect As FITC-DM4 concentration increases, the absolute number of 4M5.3FR+ cells decreases. On the other hand, 4M5.3FR- cells are also lethal by the released drug, but at high concentrations, Bystander The Dar effect appears. See Figure 19.

[0152] 3.7 Kinase inhibition of anti-CD19 CAR T cells against CD19+ large cells Drug adjustment effect Dasatinib (Lck inhibitor) and ibrutinib (ITK inhibitor) at 10 nM concentrations are, Anti-CD19 CAR T cells (FMC) against CD19+ large tumor cells (target) Reduces the dissolving effect of 63 CAR T (effector). Two effectors: The E:T ratio was tested using normal T cells and CD19-K562 cells. Anti-CD19 CAR T cells possessing [specific trait] were used as a negative control. See Figure 20. Light.

[0153] 3.8 FITC-dasatinib-induced changes in large cells: FMC63+4MFR+h Reduction of the lytic effect of T cells This means that FITC-dasatinib can reach T cells without problems via the 4M5.3FR fusion receptor. This shows that dasatinib is taken up and released, and then released into T cells. See Figure 21. .

[0154] 3.9 Exhausted anti-CD19 CARs treated with a 100 nM TC-PTP inhibitor Reduction of co-inhibitor molecules in T cells Exhausted anti-CD19 CAR T cells respond to 7 times the stimulation from CD19+ large cells. This is how it is produced. See the Materials and Methods section for details. PD-1 positive, LAG3 positive The digestive and double-positive groups decrease with treatment. See Figure 22.

[0155] 4. Other FK506-payloads for controlling the activity of cell therapy A technically beneficial feature of this drug payload delivery system is its multifunctionality. The applicable payloads and their respective effects are shown in Table 1 below. The small molecule payload was selected based on the following parameters: 1. Free movement in and out of the body. The functionality of the drug must be confirmed by previously published literature or by tests conducted at our research laboratory. 2 The chemical structure of the drug has a relatively easily accessible free amine for derivatization. 3. It is either an FDA (U.S. Food and Drug Administration) approved drug or is commercially available at an affordable price. It is preferable that the FK506-payload be first tested in vitro, and multiplexed Swimnoassays are used to observe T cell activity and cytokine release from stem cells. For in vivo disease models, established and stable CAR T is used in medical and fracture mouse models. The lab is owned by the applicant's laboratory and has several neurodegenerative mouse models. There are co-owners.

[0156] Applicability of the FKBP-FRa cell therapy platform and corresponding payloads [Table 1]

Claims

1. A composition comprising a drug conjugate, The aforementioned drug conjugate is used to inhibit and / or kill CAR T cells in patients. The CAR T cells, in the peripheral blood of the patient, have a fusion protein on the surface of the CAR T cells. The aforementioned fusion protein comprises a first component and a second component linked by a peptide linker, The first component is a single-chain fragment variable component (scFv relative to FITC) for fluorescein isothiocyanate, and, The second component is folate receptor alpha (FRa) or folate receptor beta (FRb), The drug conjugate comprises a payload bound to a small molecule ligand via a linker, The aforementioned small molecule ligand is FITC or folic acid. The FITC binds to the first component of the fusion protein, the folic acid binds to the second component, and The payload comprises a cytotoxic agent, or a gene expression modifier or cell activity modifier, that inhibits and / or kills the proliferation of the CAR T cells in the patient, and is released into the CAR T cells bound by the small molecule ligand. composition.

2. The composition according to claim 1, wherein the CAR T cells comprise an artificial functional anti-CD19 CAR T component having an amino acid sequence selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:

4.

3. The composition according to claim 1, wherein the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NOS: 12-15.

4. The composition according to claim 1, wherein the payload is a phosphatase inhibitor as an inhibitor of SHP1 / 2 or TC-PTP.

5. The payload is The cytotoxic agent selected from the group consisting of tubulicin, DM1, DM4, and auristatin, or A gene expression modifier or cell activity modifier selected from the group consisting of dasatinib, MEK1 / 2 inhibitors, PI3K inhibitors, HDAC inhibitors, kinase inhibitors, metabolic inhibitors, GSK3 beta inhibitors, MAO-B inhibitors, Cdk5 inhibitors, and RORγt agonists. A composition according to claim 1, selected from the following.

6. The composition according to claim 1, wherein the payload is a gene expression modifier selected from dasatinib, a MEK1 / 2 inhibitor, a PI3K inhibitor, or a kinase inhibitor consisting of mi181a1 siRNA.

7. The composition according to claim 1, wherein the cell activity modifier is a GSK3 beta inhibitor and / or a phosphatase inhibitor that acts on SHP1 / 2 and TC-PTP.

8. The composition according to claim 1, wherein the linker that binds the small molecule ligand and the payload is selected from the group shown below.

9. The composition according to claim 1, wherein the peptide linker is at least one segment of SGGGS.

10. The composition according to claim 1, wherein the small molecule ligand is further bound to a fluorescent dye, a radioactive probe, an endogenous gene expression regulator, or an introduced transgene expression regulator.

11. The composition according to claim 1, wherein the drug conjugate controls hypercytokinemia induced by the CAR T cells.

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