Modular synthetic receptors and methods of use

Abstract

Modular synthetic receptors are provided. The synthetic receptors can include an extracellular domain capable of binding to one or more ligand molecules and which can be released from the synthetic receptor upon binding, a transmembrane domain derived from a Notch receptor, and an intracellular domain which can have one or more functional activities upon release from the synthetic receptor. Methods of using the synthetic receptors are also provided, wherein upon binding of the extracellular domain to a particular ligand, the synthetic receptor undergoes proteolytic cleavage to release one or both of the extracellular domain, which if released, can continue to bind to its cognate ligand and can exert one or more additional functional activities, and the intracellular domain, which if released, can stimulate or inhibit one or more intracellular activities.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 005,739, filed April 6, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to novel receptors, particularly Notch or CTLA-4 synthetic receptors designed for transplantation, oncology, and autoimmune therapy. Background Technology

[0004] Mammalian cells possess transmembrane receptors capable of recognizing extracellular molecules and inducing intracellular responses. Notch receptors are an evolutionarily conserved family of signaling receptors that utilize proteolytic cleavage to release extracellular and intracellular domains in response to binding with homologous ligands. The ability to perform proteolytic cleavage is contained within a limited region of the Notch receptor, which includes the transmembrane domains and recognition sites for cleavage events. This ability to cleave in response to ligand binding is transferable, allowing for the creation of synthetic receptors with diverse ligand specificity. These synthetic receptors, upon binding, can release a variety of engineered extracellular and intracellular subdomains.

[0005] Invention Summary

[0006] This disclosure provides a synthetic receptor comprising at least three domains. In certain aspects of this disclosure, the synthetic receptor may include: a) at least one domain comprising an extracellular domain configured to specifically bind to one or more ligands and selectively release from the synthetic receptor upon binding to said ligands; b) at least one domain comprising a transmembrane domain comprising or derived from a Notch receptor; and c) at least one domain comprising an intracellular domain configured to selectively initiate one or more functional activities upon release from the synthetic receptor.

[0007] In some embodiments, after the extracellular domain binds to a specific ligand, the synthetic receptor may undergo proteolytic cleavage to release one or both of the extracellular and intracellular domains. Despite release, the extracellular binding domain may continue to bind to a homologous ligand and exercise one or more functional activities. These functional activities may include at least one of antibody-dependent cytotoxicity, complement-dependent cytotoxicity, apoptosis, or enzyme function. Furthermore, by way of illustration and not limitation, the activity may be at least one of blocking or inducing protein-protein interactions or secreting extracellular functional molecules. In some embodiments, the extracellular functional molecule may be at least one of cytokines or chemokines.

[0008] The intracellular domain of a synthetic receptor, if released, can stimulate or inhibit one or more intracellular activities. In some aspects of this disclosure, the intracellular domain is a secretory protein that, if released, stimulates or inhibits one or more extracellular activities.

[0009] These extracellular activities may include, but are not limited to, at least one of signal transduction, transport, adhesion, blocking 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.

[0010] 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 fragment (scFV) molecule of an antibody fused to the Fc region of human IgG1 and binding glycolipid disialic acid ganglioside (GD2).

[0011] Intracellular activity may also include at least one secreted fusion protein consisting of a human CTLA4 extracellular domain fused to a wild-type or modified Fc region of human immunoglobulin G (IgG). In some embodiments and by way of illustration, human IgG may be at least one of IgG1, IgG2, or IgG4.

[0012] Intracellular domain activity may also include transgenes of at least one human interleukin.

[0013] For example, in some embodiments, intracellular activity includes at least one transgene encoding human interleukin 2. In yet another embodiment, at least one transgene may encode human interleukin 12. In some embodiments, at least one transgene may encode 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.

[0014] In some aspects 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, worms, pigs, or mice.

[0015] The receptor may comprise a human CD3-specific single-chain Fv molecule fused to the Fc region of human IgG1. In these embodiments, 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 SEQ ID NO:1 or SEQ ID NO:3. The receptor may also comprise a mouse CD3-specific single-chain Fv 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:3.

[0016] In some embodiments, the synthetic receptor may further comprise a single-chain Fv molecule derived from dartuximab fused to the Fc region of human IgG1. In these embodiments, the receptor comprises a polypeptide sequence having at least 85% amino acid sequence identity with SEQ ID NO:2.

[0017] In some implementations, the receptor further comprises a transgene encoding a fusion protein formed by the fusion of the extracellular domain of human CTLA4 with the Fc region of human IgG1.

[0018] In some 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.

[0019] In a further embodiment, the receptor comprises a transgene encoding an engineered single-chain human interleukin-12 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:5.

[0020] This document also provides a method for modulating target cell activity using a synthetic receptor. The method may include contacting the synthetic receptor with a receptor on a target cell, allowing the synthetic receptor to be cleaved while the extracellular binding domain is still bound to the target cell, releasing the intracellular domain into the cell nucleus, and inducing gene expression in the cell nucleus. In some aspects of this disclosure, the extracellular domain may be an antibody or a fragment thereof. In some embodiments, the extracellular domain may also be a single-chain Fv molecule of an antibody fused to the Fc region of human IgG1 and binding to glycolipid disialic acid ganglioside.

[0021] The intracellular activity of this method may include at least one fusion protein consisting of a human CTLA4 extracellular domain fused to a wild-type or modified Fc region of human IgG. In some embodiments, IgG may include IgG1, IgG2, or IgG4. Intracellular activity may also include at least one transgene encoding human interleukin. In some embodiments, human interleukin is human IL2. In some other embodiments, intracellular activity includes at least one transgene encoding human IL12. In some embodiments, at least one transgene can encode 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. Any of 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.

[0022] Contacting a synthetic receptor with a receptor on a target cell can include administering the synthetic receptor to a subject with cancer. Further contact can include administering the synthetic receptor to a subject with an autoimmune disease. Further contact can include administering the synthetic receptor to a subject following allogeneic or xenotransplantation. Attached Figure Description

[0023] Figure 1 A schematic diagram of the synthetic receptor of this disclosure is shown.

[0024] Figures 2A-2E The results of polyacrylamide gel electrophoresis are shown, demonstrating that the correctly sized product was produced from the synthetic construct. Figure 2A This demonstrates a synthetic receptor formed by the fusion of a human CD3-specific single-chain Fv molecule with the Fc region of human IgG1. Figure 2B This shows the product derived from dartuximab (a trademark of United Therapeutics Corp.). A synthetic receptor formed by fusing a single-chain Fv molecule of dinutuximab with the Fc region of human IgG1. Figure 2C The transgene encoding Belatacept is shown. Belatacept is a fusion protein formed by fusing the extracellular domain of human CTLA4 with the Fc region of human IgG1. Figure 2D Transgenic models encoding human interleukin-2 molecules fused to the Fc region of human IgG1 were depicted. Figure 2ETransgenic single-stranded human interleukin-12 molecules encoded by fusion with the Fc region of human IgG1 are shown.

[0025] Figure 3 The illustration shows flow cytometry analysis demonstrating that the transgenic berazepam, which is formed by the fusion of the extracellular domain of human CTLA4 and the Fc region of human IgG1, binds to the porcine CD80 / CD86 molecule.

[0026] Figure 4 This illustration demonstrates the function of a synthetic receptor with an extracellular domain, formed by the fusion of a human CD3-specific single-chain Fv molecule with the Fc region of human IgG1. This synthetic receptor recognizes CD3 and responds to the expression and secretion of beraccept. Porcine aortic endothelial cells engineered with reactive transgenes containing anti-hCD3 synthetic receptors and beraccept were exposed to natively CD3-expressing human Jurkat T cells for 48 hours, and beraccept expression was analyzed. The results showed that only cells expressing the synthetic receptor induced beraccept expression in the presence of human T cells, which would have the dual benefit of blocking both CD3 and CD80 / 86.

[0027] Figures 5A-5B . Figure 5A The illustration shows the expression of dalutuximab (trademarked). (For sale) Production of IL-2Fc when porcine aortic endothelial cells that synthesize the receptor are co-cultured with CHP-134 cells that naturally express the target glycolipid disialic acid ganglioside (GD2) for 48 hours. Figure 5B The figure illustrates the scIL-12Fc production level when porcine aortic endothelial cells expressing the unituxin synthesis receptor were co-cultured with CHP-134 cells naturally expressing the target glycolipid disialothorax (GD2) for 48 hours. IL-2Fc and scIL-12-Fc protein levels were quantified by ELISA.

[0028] Figures 6A-6B . Figure 6A The illustration shows that the engineered human IL2-Fc protein encoded by the transgene functions at a level similar to that of natural human IL2 in the CTLL-2 proliferation assay (relative fluorescence units). Figure 6B The results of a CTLL-2 proliferation assay are shown, in which porcine aortic endothelial cells engineered with reactive transgenes resistant to the GD2 synthetic receptor and encoding human IL2-Fc were cultured for 48 hours with or without human CHP-134 cells naturally expressing the target glycolipid disialotetine ganglioside (GD2), and the supernatant was analyzed. “Negative” refers to CTLL-2 cells alone, and “positive” refers to CTLL-2 cells cultured with purified human IL2-Fc protein.

[0029] Figure 7The image shows an alignment of the transmembrane domain and flanking first and second cleavage recognition sequences of the Notch receptor from flies (dNotch), worms (GLP-1), pigs (pNotch1), mice (mNotch1), and humans (hNotch1-4). Detailed Implementation

[0030] definition:

[0031] As used herein and in the appended claims, in the context of describing an element, singular articles such as “a,” “an,” “the,” and similar designations are interpreted to cover both singular and plural, unless otherwise stated herein or clearly contradicted by the context.

[0032] As used herein, “about” is as understood by one of ordinary skill in the art and may vary to some extent depending on the context in which it is used. If one of ordinary skill in the art is unaware of the usage of the term “about” in the context of its use, “about” will refer to no more than plus or minus 10% of that particular term.

[0033] As will be understood by those skilled in the art, for any and all purposes, all scopes disclosed herein also cover any and all possible subscopes and combinations thereof. Furthermore, as will be understood by those skilled in the art, the scope includes each individual member.

[0034] As used herein, the term “exemplary” means “used as an example, instance, or illustration”, and not “preferred” or “superior to other implementations”.

[0035] As used in this article, "antibody-dependent cytotoxicity" (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, can refer to a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells, whose membrane surface antigens have been bound by specific antibodies. It is one of the mechanisms by which antibodies, as part of the humoral immune response, can limit and suppress infection.

[0036] As used herein, "beraccept" can refer to a soluble fusion protein that links the extracellular domain of human cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) to the Fc (hinge, CH2, and CH3 domains) portion of modified 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 357 amino acids long. It is produced in mammalian CHO cells using recombinant DNA technology. This drug exhibits activity as a selective co-stimulatory modulator and has inhibitory activity against T lymphocytes.

[0037] As used in this article, “complement-dependent cytotoxicity” (CDC) can refer to the effector function of immunoglobulins, typically IgG and IgM antibodies. When they bind to surface antigens on target cells (e.g., cells infected by bacteria or viruses), the classical complement pathway is triggered by the binding of protein C1q to these antibodies, leading to the formation of the membrane attack complex (MAC) and the lysis of the target cell.

[0038] As used in this article, "dataximab" (trademarked by United Therapeutics Corp.) Dinutuximab is a GD2-binding monoclonal antibody that is used in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid (RA) to treat pediatric patients with high-risk neuroblastoma who have achieved at least partial remission with prior first-line multi-drug, multimodal therapy.

[0039] As used herein, unless otherwise stated, "human interleukin" can 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 important role in the activation and differentiation of immune cells, as well as their proliferation, maturation, migration, and adhesion. They also possess pro-inflammatory and anti-inflammatory properties.

[0040] Synthetic receptors:

[0041] The synthetic receptor includes a) at least one domain comprising an extracellular domain configured to specifically bind to one or more ligands and selectively release from the synthetic receptor upon binding to the ligands, b) at least one domain comprising a transmembrane domain derived from the Notch receptor, and c) at least one domain comprising an intracellular domain configured to selectively initiate one or more functional activities upon release from the synthetic receptor.

[0042] In some embodiments, after the extracellular domain binds to a specific ligand, the synthetic receptor can undergo proteolytic cleavage to release one or both of the extracellular and intracellular domains. Despite release, the extracellular binding domain can continue to bind to homologous ligands and exercise one or more functional activities. These functional activities can include at least one of antibody-dependent cytotoxicity, complement-dependent cytotoxicity, or enzyme function. The activity can be at least one of blocking or inducing protein-protein interactions or secreting extracellular functional molecules. Antibody-dependent cytotoxicity (ADCC), also known as antibody-dependent cell-mediated cytotoxicity, is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies. It is one of the mechanisms by which antibodies, as part of a humoral immune response, can limit and suppress infection. In some embodiments, the extracellular functional molecule can be at least one of cytokines or chemokines.

[0043] If the intracellular domain of a synthetic receptor is released, it can stimulate or inhibit one or more intracellular or extracellular activities.

[0044] In certain aspects of this disclosure, intracellular domains may be secreted proteins that stimulate or inhibit extracellular activity. These extracellular activities may include, but are not limited to, at least one of signal transduction, transport, adhesion, blocking 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.

[0045] The extracellular domain may include an antibody or a fragment thereof. For example, in some embodiments, the extracellular domain may be a single-chain Fv molecule of an antibody fused to the Fc region of human IgG1 and binding glycolipid disialic acid ganglioside (GD2).

[0046] Intracellular activity may also include a secreted fusion protein fused with at least one wild-type or modified Fc region of a human CTLA4 extracellular domain and human immunoglobulin G (IgG). In some embodiments and by way of illustration, human IgG may be at least one of IgG1, IgG2, or IgG4. Reference Figure 3 The illustration shows flow cytometry analysis demonstrating that the transgenic berazepam, which is formed by the fusion of the extracellular domain of human CTLA4 and the Fc region of human IgG1, binds to the porcine CD80 / CD86 molecule.

[0047] Intracellular domain activity may also include transgenes of at least one human interleukin.

[0048] For example, in some embodiments, intracellular activity includes at least one transgene encoding human interleukin 2. In yet another embodiment, at least one transgene may encode human interleukin 12. In some embodiments, at least one transgene may encode 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.

[0049] In some aspects 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, worms, pigs, mice, or humans.

[0050] The receptor may comprise a human CD3-specific single-chain Fv molecule fused to the Fc region of human IgG1. In some embodiments, 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 SEQ ID NO:1, as disclosed in Table 1. The receptor may also comprise a mouse CD3-specific single-chain Fv molecule fused to the Fc region of human IgG1. In some embodiments, 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 SEQ ID NO:3, as disclosed in Table 3. The Notch receptor may be derived from other members of the human Notch receptor family (e.g., Figure 7 (as shown), or members of the Notch receptor family from other species, which have different sequences but retain protein hydrolysis and cleavage functions. Figure 7 Partial alignments of the first and second cleavage regions after the extracellular domain binds to a specific ligand are shown. Sequences of Notch receptors from flies (dNotch), worms (GLP-1), pigs (pNotch1), mice (mNotch1), and humans (hNotch1-4) are shown.

[0051] In some embodiments, the synthetic receptor may further comprise a single-chain Fv molecule derived from dartuximab fused to the Fc region of human IgG1. In these embodiments, 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 SEQ ID NO:2, as disclosed in Table 2.

[0052] In some implementations, the receptor further comprises a transgene encoding a fusion protein that fuses the extracellular domain of human CTLA4 with the Fc region of human IgG1.

[0053] In some 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%, at least 90%, at least 95%, or at least 98% amino acid sequence identity with SEQ ID NO:4, as disclosed in Table 4.

[0054] In a further embodiment, the receptor comprises a transgene encoding an engineered single-chain human interleukin-12 molecule fused to the Fc region of human IgG1. In these embodiments, 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 SEQ ID NO:5, as disclosed in Table 5.

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] Usage and treatment methods:

[0063] This article also provides a method for modulating target cell activity using a synthetic receptor. This method may include contacting the synthetic receptor with a receptor on a target cell, allowing the synthetic receptor to be cleaved while its extracellular binding domain remains bound to the target cell, releasing the intracellular domain into the nucleus, and inducing gene expression in the nucleus.

[0064] refer to Figure 1This document provides a schematic diagram of the synthetic receptor of this disclosure, referencing the possibilities of first and second protein hydrolysis cleavage following binding of a homologous ligand to the extracellular binding domain. It is shown here that, in some embodiments, the extracellular domain of the synthetic receptor binds to the receptor on a target cell. This binding can induce cleavage of the synthetic receptor. The extracellular binding domain can maintain its binding to the target cell. The intracellular transcription activator domain can be released into the cytoplasm of the cell. The intracellular domain can then translocate to the nucleus, where it can induce gene expression, after which the gene product is secreted from the cell.

[0065] In some aspects of this disclosure, the extracellular domain may be an antibody or a fragment thereof. In some embodiments, the extracellular domain may also be a single-chain Fv molecule of an antibody fused to the Fc region of human IgG1 and binding glycolipid disialic acid ganglioside (GD2).

[0066] The intracellular activity of this method may include at least one fusion protein consisting of a human CTLA4 extracellular domain fused to a wild-type or modified Fc region of human IgG. In some embodiments, the IgG may include IgG1, IgG2, or IgG4. The intracellular activity may also include at least one transgene encoding human interleukin. In some embodiments, human interleukin is human IL2. In some other embodiments, the intracellular activity includes at least one transgene encoding human IL12. In some embodiments, at least one transgene can encode 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. Any of 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.

[0067] Contacting a synthetic receptor with a receptor on a target cell can include administering the synthetic receptor to a subject with cancer. Further contact can include administering the synthetic receptor to a subject with an autoimmune disease. Further contact can include administering the synthetic receptor to a subject following allogeneic or xenotransplantation.

[0068] Potential applications:

[0069] The synthetic receptors of this application encompass a wide range of potential applications. By way of illustration and not limitation, the synthetic receptors can function in allogeneic and xenograft patients by recognizing allogeneic and xenoantigens and inducing a tolerable response. The receptors disclosed herein can also be used as therapeutic agents in oncology, recognizing tumor antigens and inducing an immunogenic response (i.e., immune activation). The receptors disclosed herein can also be used to modulate autoimmunity by recognizing pro-inflammatory or immune mediators and inducing an anti-inflammatory response (i.e., immunosuppression).

[0070] Example

[0071] Example 1: Engineering Concept Proof

[0072] Figures 2A-2E The results of polyacrylamide gel electrophoresis are shown, demonstrating that the correctly sized product was produced from the synthetic construct. Figure 2A This demonstrates a synthetic receptor formed by the fusion of a human CD3-specific single-chain Fv molecule with the Fc region of human IgG1. Figure 2B This shows the product derived from dartuximab (trademarked). A synthetic receptor formed by fusing a single-chain Fv molecule (for sale) with the Fc region of human IgG1. Figure 2C Berazip was shown, which is a fusion protein formed by the fusion of the extracellular domain of human CTLA4 and the Fc region of human IgG1. Figure 2D Human interleukin-2, which fuses with the Fc region of human IgG1, was depicted. Figure 2E The engineered single-chain human interleukin-12 molecule fused to the Fc region of human IgG1 is shown.

[0073] Example 2: Anti-hCD3 Synthesis Receptor

[0074] See now Figure 4 This study demonstrates the ability of a synthetic receptor with an extracellular domain to bind to human CD3 and respond to beraccept expression and secretion. Here, porcine aortic endothelial cells engineered with a reactive transgene encoding the anti-hCD3 synthetic receptor and beraccept were exposed to human Jurkat T cells for 48 hours. Cell supernatants were collected and beraccept expression was analyzed. The synthetic receptor induces beraccept expression only in the presence of human T cells, which would have the dual benefit of blocking both CD3 and CD80 / CD86.

[0075] Example 3: scFV dartuximab fused with Fc-IgG1 (brand name: [brand name not provided]) sell)

[0076] A synthetic receptor was constructed by fusing a single-chain Fv derived from dartuximab with the Fc moiety of an IgG1 antibody. This synthetic receptor binds to GD2 on tumor cells and responds to the expression and secretion of human IL-2Fc or human scIL-12Fc. Porcine aortic endothelial cells engineered with reactive transgenes encoding the anti-GD2 synthetic receptor and human IL-2Fc or human scIL-12Fc were exposed to human CHP134 cells for 48 hours. Cell supernatants were collected and the expression of human IL-2Fc or human scIL-12Fc was analyzed. Figure 5A and Figure 5B The results of this scheme are shown. Figure 5A The result is from IL-2Fc. Figure 5B The result is scIL-12Fc. The synthetic receptor induces the expression of human IL2-Fc or human scIL12-Fc only in the presence of GD2-expressing CHP-134 cells, which will have the dual benefits of blocking GD2 and producing anti-tumor cytokines.

[0077] Example 4: CTLL-2 proliferation assay

[0078] CTLL is a subclone of T cells derived from C57BL / 6 mice. The growth of these cells requires IL-2, and the presence of IL-2 in conditioned medium is used to detect this, thus allowing for the determination of T cell cytokine presence by measuring CTLL-2 cell proliferation. In this example, porcine aortic endothelial cells engineered with reactive transgenes encoding the GD2 synthetic receptor and human IL-2 Fc were co-cultured with or without human CHP-134 cells for 48 hours, and CTLL-2 proliferation was measured in the supernatant. The results showed... Figure 6A and Figure 6B middle. Figure 6A The illustration shows that purified engineered human IL2-Fc protein encoded by the transgene functions at a level similar to that of natural human IL2 in the CTLL-2 proliferation assay (RFU refers to relative fluorescence units). Figure 6B The activity of IL-2-Fc in CTLL-2 proliferation assay was demonstrated. Figure 6B The illustration shows the results of porcine aortic endothelial cells with reactive transgenes encoding the GD2 synthetic receptor and human IL2-Fc cultured for 48 hours with or without GD2-expressing CHP134 cells. Figure 6B In this context, CHP-134(-) means not cultured with CHP-134 cells, and CHP-134(+) means cultured with CHP-134 cells. "Negative" refers to CTLL-2 cells alone, and "positive" refers to CTLL-2 cells with purified human IL2-Fc protein. For example... Figure 6BAs shown, the synthetic receptor induces IL2-Fc expression only when co-cultured with human CHP-134 cells expressing GD2, which is reflected in the ability of CHP-134 supernatant to stimulate CTLL-2 proliferation.

[0079] Equivalent solution:

[0080] The technology has been described broadly and generally in this document. Each narrower group of species and subgenus falling within the general disclosure also constitutes part of this technology. This includes a general description of the technology, including provisos or negative qualifiers that exclude any subject from that genus, regardless of whether the removed material is specifically described herein.

[0081] Furthermore, where features or aspects of this technology are described in the Markush group, those skilled in the art will recognize that this technology is therefore also described in any single member or subgroup of the Markush group.

[0082] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated in their entirety by reference, to the same extent as they would be individually incorporated by reference. In case of conflict, this specification (including definitions) shall prevail. The appendices are incorporated herein by reference.

Claims

1. Modular synthetic receptors, including: a) An extracellular domain configured to specifically bind to one or more ligands and selectively release from the synthetic receptor upon binding to the ligand. b) A transmembrane domain derived from the Notch receptor, and c) An intracellular domain configured to selectively activate functionally active intracellular domains upon release from the synthetic receptor. The extracellular domain comprises: (i) a human CD3-specific single-chain Fv molecule fused to the Fc region of human IgG1 or (ii) a single-chain Fv molecule derived from dartuximab fused to the Fc region of human IgG1.

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

3. The synthetic receptor according to claim 1, wherein the extracellular domain continues to bind to homologous ligands and perform one or more functional activities after being released.

4. The synthetic receptor according to claim 1, wherein the functional activity includes at least one of antibody-dependent cytotoxicity, complement-dependent cytotoxicity, or enzyme function.

5. The synthetic receptor according to claim 1, wherein the intracellular domain, upon release, stimulates or inhibits one or more intracellular activities.

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

7. The synthetic receptor according to claim 6, wherein the human IgG comprises IgG1, IgG2 or IgG4.

8. The synthetic receptor according to claim 5, wherein the intracellular activity comprises a transgene of at least one human interleukin.

9. The synthetic receptor of claim 5, wherein the intracellular activity comprises at least one transgene encoding human interleukin-2.

10. The synthetic receptor of claim 5, wherein the intracellular activity comprises at least one transgene encoding human interleukin-12.

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

12. The synthetic receptor of claim 1, wherein the Notch receptor is a member of the Notch receptor family from at least one of flies, worms, pigs, mice or humans.

13. The synthetic receptor of claim 1, wherein the receptor further comprises a fusion protein formed by fusing the extracellular domain of human CTLA4 with the Fc region of human IgG1.

14. The synthetic receptor of claim 1, wherein the receptor further comprises a human interleukin-2 molecule fused to the Fc region of human IgG1.

15. The synthetic receptor of claim 1, wherein the extracellular domain comprises a human CD3-specific single-chain Fv molecule fused to the Fc region of human IgG1.

16. The synthetic receptor of claim 1, wherein the extracellular domain comprises a single-chain Fv molecule derived from dartuximab fused to the Fc region of human IgG1.

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