Modular synthesis receptor and method of use thereof

Abstract

Providing modular synthesis receptors. [Solution] A modular synthetic receptor is provided. The synthetic receptor may include an extracellular domain that can bind to one or more ligand molecules and be released from the synthetic receptor after binding, a transmembrane domain derived from a Notch receptor, and an intracellular domain that, upon release from the synthetic receptor, can have one or more functional activities. A method of using the synthetic receptor is also provided, in which, when the extracellular domain binds to a specific ligand, the synthetic receptor undergoes protein cleavage to release both or either the extracellular domain and the intracellular domain. The extracellular binding domain, upon release, can remain bound to a congener ligand and exert one or more further functional activities. The intracellular domain, upon release, can stimulate or inhibit one or more intracellular activities.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority based on U.S. Provisional Application No. 63 / 005,739, filed on April 6, 2020, the entire content of which is incorporated herein by reference.

[0002] Technical Field This application relates to novel receptors, specifically to Notch synthetic receptors or CTLA - 4 synthetic receptors designed for transplantation, oncology, and autoimmune therapies.

Background Art

[0003] Mammalian cells have transmembrane receptors that can recognize extracellular molecules and trigger intracellular responses. The Notch receptor is an evolutionarily conserved family of signaling receptors that, in response to binding to cognate ligands, releases the extracellular domain and the intracellular domain by using protein cleavage. The ability to cause protein cleavage is contained within a limited region of the Notch receptor, which includes the transmembrane domain and the recognition site involved in the cleavage phenomenon. The ability to cause cleavage in response to ligand binding is transmissible and enables the production of synthetic receptors with various ligand specificities that can release various modified extracellular and intracellular subdomains upon binding.

Summary of the Invention

[0004] The present disclosure provides a synthetic receptor comprising at least three domains. In certain aspects of the present disclosure, the synthetic receptor may include: a) at least one domain comprising an extracellular domain that specifically binds to one or more ligands and is configured to optionally dissociate from the synthetic receptor after binding to the ligand; b) at least one domain comprising a transmembrane domain that includes a Notch receptor or is derived from a Notch receptor; and c) at least one domain comprising an intracellular domain that is configured to optionally activate one or more functional activities when released from the synthetic receptor.

[0005] In some embodiments, when the extracellular domain binds to a specific ligand, the synthetic receptor may undergo protein cleavage, releasing both or either the extracellular domain and the intracellular domain. The extracellular binding domain may remain bound to a homologous ligand and, even after release, may continue to exert one or more functional activities. These functional activities include at least one of antibody-dependent cytotoxicity, complement-dependent cytotoxicity, apoptosis, or enzymatic activity. Furthermore, and without limitation, the activity may be at least one of blocking or inducing protein-protein interactions and secretion of extracellular functional molecules. In some embodiments, the extracellular functional molecule may be at least one of cytokines or chemokines.

[0006] The intracellular domain of the synthetic receptor may, upon release, stimulate or inhibit one or more intracellular activities. In certain aspects of this disclosure, the intracellular domain is a secreted protein that, upon release, stimulates or inhibits one or more extracellular activities.

[0007] These extracellular activities include, but are not limited to, at least one of signal transduction, transport, adhesion, blockade of protein-protein interactions, and / or stability. The one or more intracellular activities include at least one of signal transduction, gene expression, transport, and / or stability.

[0008] The extracellular domain may contain an antibody or a fragment thereof. For example, in some embodiments, the extracellular domain may be a single-chain variable region (scFV) molecule of an antibody that binds to a glycolipid disialoganglioside (GD2) fused to the Fc region of human IgG1.

[0009] The intracellular activity may also include at least one secreted fusion protein of a human CTLA4 extracellular domain fused to the wild-type Fc region or modified Fc region of human immunoglobulin G (IgG). In certain embodiments, the human IgG may be, for example, at least one of IgG1, IgG2, or IgG4.

[0010] The intracellular domain activity may also include at least one transgene of human interleukin.

[0011] For example, in a particular embodiment, the intracellular activity includes at least one transgene encoding human interleukin 2. In another embodiment, the at least one transgene may encode human interleukin 12. In some embodiments, the at least one transgene is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 , IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.

[0012] In certain embodiments of this disclosure, the Notch receptor of the synthetic receptor may be a member of the human Notch receptor family. In other embodiments of the synthetic receptor, the Notch receptor is a member of the Notch receptor family derived from at least one of flies, insects, pigs, and mice.

[0013] The receptor may include a human CD3-specific single-stranded Fv molecule fused to the Fc region of human IgG1. In these embodiments, the receptor may include a polypeptide sequence having at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3. The receptor may also include a mouse CD3-specific single-stranded Fv molecule fused to the Fc region of human IgG1. In these embodiments, the transgene includes a polypeptide sequence having at least 85% amino acid sequence identity with SEQ ID NO: 3.

[0014] In some embodiments, the synthetic receptor may also include a single-stranded Fv molecule derived from dinutuximab fused to the Fc region of human IgG1. In these embodiments, the receptor includes a polypeptide sequence having at least 85% amino acid sequence identity with respect to SEQ ID NO: 2.

[0015] In some embodiments, the receptor further comprises a transgene encoding a fusion protein containing a human CTLA4 extracellular domain fused to the Fc region of human IgG1.

[0016] In certain other embodiments, the receptor further comprises a transgene encoding a human interleukin 2 molecule fused to the Fc region of human IgG1. In these embodiments, the transgene comprises a polypeptide sequence having at least 85% amino acid sequence identity with SEQ ID NO: 4.

[0017] In further embodiments, the receptor includes a transgene encoding a modified single-stranded human interleukin 12 molecule fused to the Fc region of human IgG1. In these embodiments, the transgene includes a polypeptide sequence having at least 85% amino acid sequence identity with SEQ ID NO: 5.

[0018] This specification also provides a method for regulating the activity of target cells using a synthetic receptor. The method may include contacting the synthetic receptor with a receptor on a target cell, enabling the cleavage of the synthetic receptor while the extracellular binding domain is bound to the target cell, and releasing the intracellular domain into the nucleus where the intracellular domain induces gene expression. In some aspects of this disclosure, the extracellular domain may be an antibody or a fragment thereof. In certain embodiments, the extracellular domain may be a single-chain Fv molecule of an antibody bound to a glycolipid disialoganglioside fused to the Fc region of human IgG1.

[0019] The intracellular activity of the above method may comprise at least one fusion protein of a human CTLA4 extracellular domain fused to the wild-type Fc region or modified Fc region of human IgG. In some embodiments, this IgG may comprise IgG1, IgG2, or IgG4. The intracellular activity may also comprise at least one transgene encoding a human interleukin. In some embodiments, the human interleukin is human IL2. In certain other embodiments, the intracellular activity may comprise at least one transgene encoding human IL12. In some embodiments, the at least one transgene is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 , IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.

[0020] Contacting a synthetic receptor with a receptor on a target cell may include administering the synthetic receptor to a subject with cancer. The contact may further include administering the synthetic receptor to a subject with autoimmune disorder. The contact may further include administering the synthetic receptor to a subject after allogeneic or xenotransplantation. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows a schematic diagram of the synthetic receptor of this disclosure. [Figure 2A-2E] Figures 2A-2E show polyacrylamide gel electrophoresis results demonstrating the production of correctly sized products from the synthetic construct. Figure 2A shows a synthetic receptor containing a human CD3-specific single-stranded Fv molecule fused to the Fc region of human IgG1. Figure 2B shows a synthetic receptor containing a single-stranded Fv molecule derived from dinutuximab (marketed by United Therapeutics Corporation under the trademark Unituxin® (Dinutuximab)) fused to the Fc region of human IgG1. Figure 2C shows a transgene encoding belatacept, a fusion protein containing a human CTLA4 extracellular domain fused to the Fc region of human IgG1. Figure 2D shows a transgene encoding two human interleukin molecules fused to the Fc region of human IgG1. Figure 2E shows a transgene encoding twelve modified single-stranded human interleukin molecules fused to the Fc region of human IgG1. [Figure 3] Figure 3 graphically shows flow cytometry analysis demonstrating that belatacept encoded by a transgene containing a human CTLA4 extracellular domain fused to the Fc region of human IgG1 binds to porcine CD80 / CD86 molecules. [Figure 4]Figure 4 graphically illustrates the function of a synthetic receptor having an extracellular domain containing a human CD3-specific single-stranded Fv molecule fused to the Fc region of human IgG1, which recognizes CD3 and responds with belatacept expression and secretion. Porcine aortic endothelial cells modified with the anti-hCD3 synthetic receptor and a responsive transgene encoding belatacept were exposed to human Jerkat T cells that spontaneously express CD3 for 48 hours, and belatacept expression was analyzed. The results showed that only cells expressing the synthetic receptor induced belatacept expression in the presence of human T cells, which is thought to have a dual advantage of blocking both CD3 and CD80 / 86. [Figure 5A-5B] Figure 5A shows a graph of IL-2Fc production when porcine aortic endothelial cells expressing the dinutuximab (marketed under the trademark Unituxin®) were co-cultured for 48 hours with CHP-134 cells spontaneously expressing the target glycolipid disialoganglioside (GD2). Figure 5B shows a graph of scIL-12Fc production levels when porcine aortic endothelial cells expressing the Unituxin synthesis receptor were co-cultured for 48 hours with CHP-134 cells spontaneously expressing the target glycolipid disialoganglioside (GD2). The levels of IL-2Fc protein and scIL12-Fc protein were quantified by ELISA. [Figure 6A] Figure 6A graphically shows that the modified human IL2-Fc protein encoded by the transgene functions at a similar level to undenatured human IL2 in the CTLL-2 proliferation response assay (relative fluorescence units). Figure 6B shows the results of the CTLL-2 proliferation response assay, in which supernatant derived from porcine aortic endothelial cells modified with an anti-GD2 synthesis receptor and a responsive transgene encoding human IL2-Fc was cultured for 48 hours in or without human CHP-134 cells that spontaneously express the target glycolipid disialoganglioside (GD2). "Negative" refers to CTLL-2 cells alone, and "Positive" refers to CTLL-2 cells cultured with purified human IL2-Fc protein. [Figure 6B]Figure 6A graphically shows that the modified human IL2-Fc protein encoded by the transgene functions at levels similar to native human IL2 in the CTLL-2 proliferation assay (relative fluorescence units). Figure 6B shows the results of the CTLL-2 proliferation assay, in which supernatants from porcine aortic endothelial cells modified with an anti-GD2 synthetic receptor and a responsive transgene encoding human IL2-Fc were cultured for 48 hours in the presence or absence of human CHP-134 cells that naturally express the target glycolipid disialoganglioside (GD2). "Negative" refers to CTLL-2 cells alone, and "positive" refers to CTLL-2 cells cultured with purified human IL2-Fc protein. [Figure 7] Figure 7 shows a sequence comparison of the transmembrane domains and adjacent first and second cleavage recognition sequences of Notch receptors from Drosophila (dNotch), Caenorhabditis elegans (GLP-1), pig (pNotch1), mouse (mNotch1), and human (hNotch1-4).

Mode for Carrying Out the Invention

[0022] Definition: As used in this specification and the appended claims, singular articles such as "a," "an," "the," and similar demonstrative terms shall be construed to include both the singular and the plural, unless otherwise indicated herein or clearly negated by the context, in light of the recitation of each element.

[0023] As used herein, "about" shall be understood as that understood by those skilled in the art and may vary somewhat depending on the context in which it is used. Where the context in which the term "about" is used is such that the term is used unclearly to those skilled in the art, "about" shall mean from 10% minus to 10% plus of that particular term.

[0024] As will be understood by those skilled in the art, for all purposes, the entire scope disclosed herein also encompasses any possible partial scope or combination of such partial scopes. Furthermore, as will be understood by those skilled in the art, the scope encompasses the individual components.

[0025] As used herein, the term “exemplary” means “acting as an example, instance, or illustration,” and does not mean “preferred” or “advantageous over other embodiments.”

[0026] As used herein, "antibody-dependent cell-mediated cytotoxicity" (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, refers to a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells, the target cells being those to which specific antibodies are bound to membrane surface antigens. It is one of the mechanisms by which antibodies act to limit and suppress infection as part of the humoral immune response.

[0027] As used herein, "abatacept" may refer to a soluble fusion protein in which the extracellular domain of human cytotoxic T lymphocyte antigen 4 (CTLA4) is linked to the modified Fc (hinge, CH2, and CH3 domains) of human immunoglobulin G1 (IgG1). Structurally, abatacept is a glycosylated fusion protein with a MALDI-MS molecular weight of 92,300 Da, and is a homodimer of two homologous polypeptide chains, each having 357 amino acids. It is produced in mammalian CHO cells by recombinant DNA technology. This agent acts as a selective costimulatory modifier with inhibitory activity against T lymphocytes.

[0028] As used herein, “complement-dependent cell-mediated cytotoxicity” (CDC) may refer to the effector function of immunoglobulins, typically IgG and IgM antibodies. When these antibodies bind to surface antigens on target cells (e.g., bacterial or viral infected cells), the classical complement pathway is triggered by the binding of protein C1q to these antibodies, resulting in the formation of membrane invasion complexes (MACs) and the lysis of target cells.

[0029] As used herein, “Dinutuximab” (marketed by United Therapeutics Corporation under the trademark Unituxin® (Dinutuximab)) is a GD2-conjugated monoclonal antibody that requires concomitant use with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid (RA), and is intended for the treatment of pediatric patients with high-risk neuroblastoma who respond at least partially to prior first-line multi-drug combination therapy.

[0030] As used herein, unless otherwise specified, “human interleukin” may refer to any one of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40. Interleukins play an essential role in the activation and differentiation of immune cells, as well as in the proliferation, maturation, migration, and adhesion of immune cells. It also has pro-inflammatory and anti-inflammatory properties.

[0031] Synthetic receptors: The synthetic receptor comprises: a) at least one domain including an extracellular domain configured to specifically bind to one or more ligands and optionally be released from the synthetic receptor after binding to the ligands; b) at least one domain including a transmembrane domain derived from a Notch receptor; and c) at least one domain including an intracellular domain configured to optionally activate one or more functional activities when released from the synthetic receptor.

[0032] In some embodiments, when the extracellular domain binds to a specific ligand, the synthetic receptor may undergo protein cleavage, releasing both or either the extracellular and intracellular domains. The extracellular binding domain may continue to bind to a homologous ligand and, even after release, may continue to exert one or more functional activities. These functional activities include at least one of antibody-dependent cytotoxicity, complement-dependent cytotoxicity, or enzymatic activity. The activity may be at least one of blocking or inducing protein-protein interactions and secretion of extracellular functional molecules. Antibody-dependent cell-mediated cytotoxicity (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is a mechanism of cell-mediated immune defense in which effector cells of the immune system actively lyse target cells, which are membrane-surface antigens to which specific antibodies are bound. It is one mechanism by which antibodies act to limit and suppress infection as part of the humoral immune response. In some embodiments, the extracellular functional molecule may be at least one of cytokines or chemokines.

[0033] The intracellular domain of the synthetic receptor, when released, may stimulate or inhibit one or more intracellular or extracellular activities.

[0034] In certain aspects of this disclosure, the intracellular domain may be a secreted protein that stimulates or inhibits extracellular activity. These extracellular activities include, but are not limited to, at least one of signaling, transport, adhesion, blockade of protein-protein interactions, and / or stability. The one or more intracellular activities include at least one of signaling, gene expression, transport, and / or stability.

[0035] The extracellular domain may contain an antibody or a fragment thereof. For example, in some embodiments, the extracellular domain may be a single-chain Fv molecule of an antibody that binds to a glycolipid disialoganglioside (GD2) fused to the Fc region of human IgG1.

[0036] The intracellular activity may also include at least one secreted fusion protein of a human CTLA4 extracellular domain fused to the wild-type Fc region or modified Fc region of human immunoglobulin G (IgG). In certain embodiments, the human IgG may be at least one of, for example, IgG1, IgG2, or IgG4. Referring to Figure 3, the graph shows flow cytometry analysis demonstrating that betalacept encoded by the transgene, containing a human CTLA4 extracellular domain fused to the Fc region of human IgG1, binds to the porcine CD80 / CD86 molecule.

[0037] The intracellular domain activity may also include at least one transgene of human interleukin.

[0038] For example, in a particular embodiment, the intracellular activity includes at least one transgene encoding human interleukin 2. In another embodiment, the at least one transgene may encode human interleukin 12. In some embodiments, the at least one transgene is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 , IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.

[0039] In certain embodiments of this disclosure, the Notch receptor subdomain of the synthetic receptor may be a member of the human Notch receptor family. In other embodiments of the synthetic receptor, the Notch receptor is a member of the Notch receptor family derived from at least one of flies, insects, pigs, mice, or humans.

[0040] The receptor may comprise a human CD3-specific single-stranded Fv molecule fused to the Fc region of human IgG1. In some embodiments, as disclosed in Table 1, the receptor may comprise a polypeptide sequence having at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with respect to SEQ ID NO: 1. The receptor may also comprise a mouse CD3-specific single-stranded Fv molecule fused to the Fc region of human IgG1. In some embodiments, as disclosed in Table 3, the receptor may comprise a polypeptide sequence having at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with respect to SEQ ID NO: 3. The Notch receptor may be derived from further members of the human Notch receptor family (as shown in Figure 7) or from members of the Notch receptor family from other species that have different sequences but possess protein cleavage functionality. Figure 7 shows a partial sequence comparison of the first and second cleavage regions when the extracellular domain is bound to a specific ligand. The sequences of Notch receptors from flies (dNotch), insects (GLP-1), pigs (pNotch1), mice (mNotch1), and humans (hNotch1-4) are shown.

[0041] In some embodiments, the synthetic receptor may also comprise a single-stranded Fv molecule derived from dinutuximab fused to the Fc region of human IgG1. In these embodiments, as disclosed in Table 2, the receptor comprises a polypeptide sequence having at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with respect to SEQ ID NO: 2.

[0042] In some embodiments, the receptor further comprises a transgene encoding a fusion protein containing a human CTLA4 extracellular domain fused to the Fc region of human IgG1.

[0043] In certain other embodiments, the receptor further comprises a transgene encoding a human interleukin-2 molecule fused to the Fc region of human IgG1. In these embodiments, as disclosed in Table 4, the transgene comprises a polypeptide sequence having at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with respect to SEQ ID NO: 4.

[0044] In further embodiments, the receptor comprises a transgene encoding a modified single-stranded human interleukin 12 molecule fused to the Fc region of human IgG1. In these embodiments, as disclosed in Table 5, the transgene comprises a polypeptide sequence having at least 85%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with respect to SEQ ID NO: 5. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0045] Method of use and treatment: This specification also provides a method for regulating the activity of target cells using a synthetic receptor. The method may include contacting the synthetic receptor with a receptor on a target cell, enabling the cleavage of the synthetic receptor while the extracellular binding domain is bound to the target cell, and releasing the intracellular domain into the nucleus where the intracellular domain induces gene expression.

[0046] Referring to Figure 1, a schematic diagram of the synthetic receptor of this disclosure is provided in relation to the possibility of first and second protein cleavage after a congener ligand has bound to the extracellular binding domain. Here, in some embodiments, it is shown that the extracellular domain of the synthetic receptor binds to a receptor on a target cell. This binding may induce cleavage of the synthetic receptor. The extracellular binding domain may remain bound to the target cell. The intracellular transcriptional activation domain may be released into the cytoplasm of the cell. The intracellular domain may then move into the nucleus, where it may induce gene expression, and the gene product may subsequently be secreted from the cell.

[0047] In some aspects of this disclosure, the extracellular domain may be an antibody or a fragment thereof. In certain embodiments, the extracellular domain may be a single-chain Fv molecule of an antibody that binds to a glycolipid disialoganglioside (GD2) fused to the Fc region of human IgG1.

[0048] The intracellular activity of the above method may comprise at least one fusion protein of a human CTLA4 extracellular domain fused to the wild-type Fc region or modified Fc region of human IgG. In some embodiments, this IgG may comprise IgG1, IgG2, or IgG4. The intracellular activity may also comprise at least one transgene encoding a human interleukin. In some embodiments, the human interleukin is human IL2. In certain other embodiments, the intracellular activity may comprise at least one transgene encoding human IL12. In some embodiments, the at least one transgene is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 , IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, or IL-40.

[0049] Contacting a synthetic receptor with a receptor on a target cell may include administering the synthetic receptor to a subject with cancer. The contact may further include administering the synthetic receptor to a subject with autoimmune disorder. The contact may further include administering the synthetic receptor to a subject after allogeneic or xenotransplantation.

[0050] Possible uses: The synthetic receptors of this invention have many potential applications. Exemplary, but not limited to, these synthetic receptors may be useful in allogeneic and xenotransplant patients by recognizing allogeneic and xenotransmitters and inducing immune tolerance responses. The receptors of this disclosure may also act as therapeutic agents in oncology, recognizing tumor antigens and inducing immunogenic responses (i.e., immune activation). The receptors of this disclosure may also be useful in modulating autoimmunity by recognizing pro-inflammatory or immunomodulatory mediators and inducing anti-inflammatory responses (i.e., immunosuppression). [Examples]

[0051] Example 1: Design of the demonstration experiment Figures 2A-2E show polyacrylamide gel electrophoresis results demonstrating the production of correctly sized products from the synthetic constructs. Figure 2A shows a synthetic receptor containing a human CD3-specific single-chain Fv molecule fused to the Fc region of human IgG1. Figure 2B shows a synthetic receptor containing a single-chain Fv molecule derived from dinutuximab, marketed under the trademark Unituxin®, fused to the Fc region of human IgG1. Figure 2C shows belatacept, a fusion protein containing a human CTLA4 extracellular domain fused to the Fc region of human IgG1. Figure 2D shows human interleukin-2 fused to the Fc region of human IgG1. Figure 2E shows a modified single-chain human interleukin-12 molecule fused to the Fc region of human IgG1.

[0052] Example 2: Anti-hCD3 synthesis receptor Referring to Figure 4, the results of measuring a synthetic receptor with an extracellular domain capable of binding to human CD3 and inducing a response involving belatacept expression and secretion are shown. Here, porcine aortic endothelial cells modified with an anti-hCD3 synthetic receptor and a responsive transgene encoding belatacept were exposed to human Jercutt T cells for 48 hours. The supernatant from the cells was collected and analyzed for belatacept expression. Only the aforementioned synthetic receptor induced belatacept expression in the presence of human T cells. This suggests that it has the dual advantage of blocking both CD3 and CD80 / CD86.

[0053] Example 3: scFV dinutuximab (marketed under the trademark Unituxin®) fused with Fc-IgG1 A synthetic receptor was created by fusing a single-stranded Fv derived from dinutuximab with the Fc portion of an IgG1 antibody. This synthetic receptor binds to GD2 on tumor cells and elicits a response involving the expression and secretion of human IL-2Fc or human scIL-12Fc. Porcine aortic endothelial cells modified with the anti-GD2 synthetic receptor and a responsive transgene encoding human IL-2Fc or human scIL-12Fc were contacted with human CHP134 cells for 48 hours. The supernatant was collected from the cells and analyzed for the expression of human IL-2Fc or human scIL-12Fc. Figures 5A and 5B show the results of the protocol, with IL-2Fc shown graphically in Figure 5A and scIL-12Fc shown graphically in Figure 5B. Only the aforementioned synthetic receptor induced the expression of human IL-2-Fc or human scIL-12-Fc in the presence of GD2-expressing CHP-134 cells. This is thought to have a dual advantage: blocking GD2 and producing antitumor cytokines.

[0054] Example 4: Measurement of CTLL-2 proliferation response CTLL is a subclone of T cells derived from C57BL / 6 mice. These cells require IL-2 for proliferation and are used to measure the presence or absence of IL-2 in conditioned medium; therefore, the presence or absence of T-cell cytokines can be confirmed by measuring the proliferation of CTLL-2 cells. In this example, supernatant from porcine aortic endothelial cells modified with an anti-GD2 synthesis receptor and a responsive transgene encoding human IL-2Fc was co-cultured in the presence or absence of human CHP-134 cells and tested for 48 hours in the CTLL-2 proliferation response measurement. The measurement results are shown in Figures 6A and 6B. Figure 6A is a graph showing that the purified modified human IL2-Fc protein encoded by the transgene functions at a level similar to undenatured human IL2 in the CTLL-2 proliferation response measurement (RFU refers to relative fluorescence units). Figure 6B shows the IL-2-Fc activity in the CTLL-2 proliferation response measurement. Figure 6B is a graph showing the results of culturing porcine aortic endothelial cells modified with an anti-GD2 synthetic receptor and a responsive transgene encoding human IL2-Fc for 48 hours in the presence or absence of GD2-expressing CHP134 cells. In Figure 6B, CHP-134(-) refers to culture in the absence of CHP-134 cells, and CHP-134(+) refers to culture in the presence of CHP-134 cells. "Negative" refers to CTLL-2 cells alone, and "Positive" refers to CTLL-2 cells to which purified human IL2-Fc protein has been added. As shown in Figure 6B, only the aforementioned synthetic receptor induced IL2-Fc expression in co-culture with GD2-expressing human CHP-134 cells. This is demonstrated by the ability of the CHP-134 supernatant to stimulate CTLL-2 proliferation.

[0055] Equal parts: This specification provides a broad and comprehensive description of the Technology. Any narrower classifications and subcategories included in that comprehensive disclosure also form part of the Technology, including any conditions or negative limitations in the comprehensive description of the Technology that exclude certain content from the comprehensive classification, regardless of whether the excluded content is specifically described herein.

[0056] In addition, if any feature or aspect of the present technology is described in terms of the Markush group, a person skilled in the art will recognize that the present technology is also described in terms of any individual member or any multiple members of any subgroup of the Markush group.

[0057] All publications, patent applications, patents, and other references referred to herein are expressly invoked by reference in whole to the same extent that each is invoked by reference individually. In case of any conflict, including definitions, this specification shall prevail. Appendices are invoked herein by reference.

Claims

1. A modular synthesis receptor comprising at least three domains, a) comprising an extracellular domain configured to specifically bind to one or more ligands and optionally be released from the synthetic receptor after binding to the ligands, b) At least one domain comprises a transmembrane domain derived from the Notch receptor, and c) A modular synthetic receptor comprising at least one intracellular domain configured to optionally activate functional activity when released from the synthetic receptor.

2. The synthetic receptor according to claim 1, wherein when the extracellular domain binds to a specific ligand, the synthetic receptor undergoes protein cleavage to release either or both of the extracellular domain and the intracellular domain.

3. The synthetic receptor according to claim 1, wherein the extracellular binding domain continues to bind to a homologous ligand and continues to exert one or more functional activities even after being released.

4. The synthetic receptor according to claim 3, wherein the functional activity includes at least one of antibody-dependent cytotoxicity, complement-dependent cytotoxicity, or enzymatic function.

5. The synthetic receptor according to claim 4, wherein the activity comprises at least one of blocking or inducing protein-protein interactions, or secreting extracellular functional molecules.

6. The synthetic receptor according to claim 5, wherein the extracellular functional molecule comprises at least one cytokine or chemokine.

7. The synthetic receptor according to claim 1, wherein the intracellular domain, when released, stimulates or inhibits one or more intracellular activities.

8. The synthetic receptor according to claim 1, wherein the intracellular domain is a secreted protein that, when released, stimulates or inhibits one or more extracellular activities.

9. The synthetic receptor according to claim 8, wherein the extracellular activity comprises at least one of signal transduction, transport, adhesion, blockage of protein-protein interactions, and / or stability.

10. The synthetic receptor according to claim 7, wherein one or more intracellular activities include at least one of signal transduction, gene expression, transport, and / or stability.

11. The synthetic receptor according to claim 1, wherein the extracellular domain comprises an antibody or a fragment thereof.

12. The synthetic receptor according to claim 11, wherein the extracellular domain comprises a single-chain Fv molecule of an antibody that binds to a glycolipid disialoganglioside (GD2) fused to the Fc region of human IgG1.

13. The synthetic receptor according to claim 1, wherein the intracellular activity comprises at least one secreted fusion protein of a human CTLA4 extracellular domain fused to the wild-type Fc region or modified Fc region of human immunoglobulin G (IgG).

14. The synthetic receptor according to claim 13, wherein the human IgG comprises IgG1, IgG2, or IgG4.

15. The synthetic receptor according to claim 1, wherein the intracellular domain activity comprises at least one transgene of human interleukin.

16. The synthetic receptor according to claim 7, wherein the intracellular activity comprises at least one transgene encoding human interleukin 2.

17. The synthetic receptor according to claim 7, wherein the intracellular activity comprises at least one transgene encoding human interleukin 12.

18. The synthetic receptor according to claim 1, wherein the Notch receptor is a member of the human Notch receptor family.

19. The synthetic receptor according to claim 1, wherein the Notch receptor is a member of the Notch receptor family derived from at least one of flies, insects, pigs, mice, or humans.

20. The synthetic receptor according to claim 1, wherein the receptor comprises a human CD3-specific single-stranded Fv molecule fused to the Fc region of human IgG1.

21. The synthetic receptor according to claim 20, wherein the receptor comprises a polypeptide sequence having at least 85% amino acid sequence identity with respect to SEQ ID NO: 1 or SEQ ID NO:

3.

22. The synthetic receptor according to claim 1, wherein the receptor comprises a single-stranded Fv molecule derived from dinutuximab fused to the Fc region of human IgG1.

23. The synthetic receptor according to claim 22, wherein the receptor comprises a polypeptide sequence having at least 85% amino acid sequence identity with respect to SEQ ID NO:

2.

24. The synthetic receptor according to claim 1, further comprising a transgene encoding a fusion protein consisting of a human CTLA4 extracellular domain fused to the Fc region of human IgG1.

25. The synthetic receptor according to claim 1, further comprising a transgene encoding two human interleukin molecules fused to the Fc region of human IgG1.

26. The synthetic receptor according to claim 25, wherein the introduced gene comprises a polypeptide sequence having at least 85% amino acid sequence identity with respect to SEQ ID NO:

4.

27. The synthetic receptor according to claim 1, further comprising a transgene encoding a modified single-stranded human interleukin 12 molecule fused to the Fc region of human IgG1.

28. The synthetic receptor according to claim 27, wherein the introduced gene comprises a polypeptide sequence having at least 85% amino acid sequence identity with respect to SEQ ID NO:

5.

29. A method for regulating the activity of a target cell using the synthetic receptor of claim 1, comprising: contacting the synthetic receptor with a receptor on the target cell; enabling the cleavage of the synthetic receptor while the extracellular binding domain is bound to the target cell; and releasing the intracellular domain into the nucleus where the intracellular domain induces gene expression.

30. The method according to claim 29, wherein the extracellular domain comprises an antibody or a fragment thereof.

31. The method according to claim 29, wherein the extracellular domain comprises a single-chain Fv molecule of an antibody that binds to a glycolipid disialoganglioside (GD2) fused to the Fc region of human IgG1.

32. The method according to claim 29, wherein the intracellular activity comprises at least one fusion protein of a human CTLA4 extracellular domain fused to the wild-type Fc region or modified Fc region of human immunoglobulin.

33. The method according to claim 32, wherein the human immunoglobulin comprises at least one of IgG1, IgG2, or IgG4.

34. The method according to claim 29, wherein the intracellular activity comprises at least one transgene encoding a human interleukin.

35. The method according to claim 29, wherein the intracellular activity comprises at least one transgene encoding at least one of human interleukin 2 and human interleukin 12.

36. The method according to claim 29, wherein contact includes administering the synthetic receptor to a subject suffering from cancer.

37. The method according to claim 29, wherein contact is provided by administering the synthetic receptor to a subject suffering from an autoimmune disorder.

38. The method according to claim 29, wherein contact includes administering the synthetic receptor to the subject after the subject has received at least one allograft or xenograft.

Images