Compositions comprising GPCR-based chimeric antigen receptors and methods of use

Abstract

The present invention relates to chimeric aGPCR and uses thereof. By redirecting the intracellular signal response to the desired target, the chimeric aGPCR may be used in a manner similar to a chimeric antigen receptor. The chimeric aGPCR may also bind to platelets or engineered non-thrombogenic platelets for targeted drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of therapeutics, specifically to the field of targeted drug delivery. Background Technology

[0002] Chimeric antigen receptors (CARs) are synthetic receptors that combine the high affinity binding ability of antibodies with the internal signaling domain (ITAM) of T cell receptors. They enable the specific redirection of immune cells (typically T cells) to targets of interest, which are often found on cancer cells. CARs are engineered to enhance the immune system's ability to recognize and eliminate cancer cells.

[0003] The ITAM domain is a short sequence found in the cytoplasmic tail of various immune receptors, including T-cell and B-cell receptors. In CARs, the ITAM domain binds to an intracellular signaling domain to initiate T-cell activation upon antigen recognition. When the CAR binds to a target antigen on a cancer cell, the ITAM domain becomes phosphorylated, triggering a cascade of signaling events that activate T cells. This activation ultimately leads to T cells killing cancer cells.

[0004] Incorporating the ITAM domain into CARs can bypass several steps involved in the normal immune response against cancer cells. CAR T cells can recognize and kill cancer cells independently of major histocompatibility complex (MHC) presentation, the usual mode of T cell antigen recognition. This feature makes CAR T cell therapy applicable to a wide range of cancer types.

[0005] CAR T-cell therapy has shown significant success in treating certain hematologic malignancies, such as acute lymphoblastic leukemia and certain types of lymphoma. Ongoing research focuses on improving the efficacy and safety of CAR T-cell therapy and expanding its application to solid tumors.

[0006] Other examples of chimeric receptors that bind to the ITAM signaling domain are described in WO 2022 / 263824, providing a range of ITAM-based chimeric receptors that can be used to redirect the natural degranulation response of platelets.

[0007] Degranulation typically occurs in response to receptor binding to physical damage markers, such as collagen, and triggers thrombosis. WO 2022 / 263824 describes the redirection of this response by chimeric platelet receptors to deliver therapeutic cargo to targets of interest—for example, the delivery of anticancer agents to tumors. WO 2022 / 263824 also describes how platelets can be engineered to express cargo of interest from precursor megakaryocytes, or how exogenous cargo can be directly loaded into precursor megakaryocytes or platelets. WO 2022 / 263824 also describes platelets that have been engineered to remove or reduce procoagulant activity—that is, to prevent platelets from degranulating in response to natural signals that would be found in subjects requiring treatment, such as patients. In this way, WO 2022 / 263824 describes a complete system that may contain nonthrombotic platelets (or synlets) and chimeric receptors, and can be used to deliver various types of cargo to target sites.

[0008] However, these ITAM-based approaches also have some potential drawbacks. ITAM receptors exhibit a pronounced on-off response to the intensity of target signals, with little variation in the resulting responses. Furthermore, ITAM receptors tend to initiate signaling by activating a single kinase (such as Syk or Zap70), thus the downstream effects achievable through target binding are not significantly different. Finally, due to receptor aggregation on the cell surface (e.g., through receptor overexpression), random ITAM signaling can occur even without target binding, leading to a lack of sensitivity in signal transduction, i.e., a high background rate.

[0009] This invention provides receptors that address these problems using known chimeric receptors. Summary of the Invention

[0010] G protein-coupled receptors (GPCRs) are a large family of transmembrane receptors that play a crucial role in intracellular signaling pathways. Upon activation by their ligands (such as neurotransmitters, hormones, or other signaling molecules), GPCRs undergo a conformational change, leading to the activation of intracellular signaling pathways. Following GPCR activation, the G protein undergoes a conformational change (i.e., from an inactive conformation to an active conformation), enabling it to exchange GDP for GTP, thereby activating downstream effectors. Therefore, in this paper, "activation of aGCPR" is intended to encompass the initiation of downstream signaling through the corresponding G protein.

[0011] Adhesion GPCRs (aGPCRs) are a special class of GPCRs that, in addition to the standard seven-transmembrane domain (7TM) common to all GPCRs, also possess inclusion domains. GThe extracellular domain of the PCR-induced autoproteolytic (GAIN) domain induces constitutive autoproteolytic cleavage at a site (called the GPS site) located proximal to the first transmembrane helix. The GAIN domain typically terminates 7–18 residues before the start of the first transmembrane segment of the 7TM bundle. Cleavage at this site results in the generation of an N-terminal fragment (NTF) and a C-terminal fragment (CTF—containing 7TM). A dense network of hydrogen bonds within the GAIN domain allows the NTF and CTF to remain non-covalently bound after autoproteolysis, which is thought to occur early in receptor biosynthesis within the intracellular compartment. Both fragments of the whole receptor are transported to their respective plasma membranes, ready for signal transduction.

[0012] The GAIN domain also contains an agonist peptide, referred to as the "tethered agonist" or "Stachel sequence." Autologous proteolytic cleavage occurs at the N-terminus of this sequence, so the tethered agonist remains bound to the CTF. After cleavage, the tethered agonist remains hidden due to the non-covalent association between the NTF and CTF. However, the mechanical force exerted on this interaction due to ligand binding physically pulls the NTF away from the CTF, exposing the tethered agonist peptide. The tethered agonist peptide then interacts with the 7TM domain, activating aGPCR.

[0013] Some aGCPRs cannot be cleaved by proteolysis, but can still be activated by binding to their respective targets.

[0014] Despite the complex structure and intermolecular interactions of aGPCR receptors, the inventors have unexpectedly prepared and demonstrated the function of chimeric aGPCRs in which the extracellular ligand-binding domain has been modified or replaced to target new ligands, yet still triggers appropriate intracellular signaling upon ligand binding. In this way, the sensing repertoire of chimeric aGPCRs is expanded.

[0015] One potential advantage of chimeric aGPCR signaling over ITAM-based chimeric receptor signaling is that it allows for a more sensitive and dynamic response to extracellular stimuli, as GPCRs can activate multiple downstream effectors, including G proteins, β-repressor proteins, and various kinases. For example, with the Gαq subunit (also known as G...) αq / 11 or G αq / 11 / 14 / 15 GPCRs interacting with subunits activate β-phospholipase C (PLCβ); and with G... αs Subunit-interacting GPCRs stimulate the cAMP-dependent pathway by activating adenylate cyclase and increasing intracellular cAMP levels; and interact with G... αi / o Subunit-interacting GPCRs inhibit adenylate cyclase and reduce intracellular cAMP.

[0016] In contrast, ITAM signaling typically involves the activation of a single kinase, such as Syk or Zap70, leading to a more limited range of downstream effects. Complex signaling pathways with multilayered regulation can be designed using GPCRs or combinations of GPCRs and ITAMs.

[0017] GPCRs can be regulated through a variety of mechanisms, including receptor desensitization, internalization, and degradation, as well as through feedback mechanisms involving downstream effectors. This allows cells to modulate the intensity and duration of GPCR signaling in response to changing conditions.

[0018] In contrast, the response strength of ITAM-based signal transduction depends primarily on the number of activated receptors and operates more in an "on or off" manner.

[0019] Because aGPCRs are primarily activated by mechanical forces, and because cells (such as those found in the circulatory system, such as platelets and engineered platelets) are typically in a state of constant motion, they are able to exert mechanical forces on any target they bind to, thus expanding the ligand spectrum that can activate aGPCRs. Expression of these chimeric aGPCRs in cells, platelets, and engineered platelets provides opportunities for increased control and refinement in the activation of intracellular (or intraplatelet / engineered platelet) signaling and downstream effects, such as the release of therapeutic cargo in certain circumstances. Non-limiting examples include T cells, B cells, NK cells, erythrocytes, macrophages, megakaryocytes, pluripotent cells, or stem cells.

[0020] This invention provides a chimeric aGCPR receptor, compositions using the chimeric aGPCR, and methods.

[0021] The chimeric aGPCR described herein can be used to replace or supplement the ITAM-based chimeric receptor described in this patent in the context of platelets or nonthrombotic platelets as described in WO 2022 / 263824. The chimeric aGPCR described herein can also be used in different environments, such as different cellular environments. For example, the chimeric aGPCR can be expressed from T cells and used in a manner similar to current CAR-T therapies.

[0022] Chimerism is intended to encompass the meaning that portions or domains of an aGCPR differ from those common to the same aGPCRs found in nature. In some embodiments, chimeric properties arise from one or more substitutions, deletions, or insertions in the amino acid sequence of the aGPCR relative to the wild-type receptor. In some embodiments, chimeric properties of an aGPCR arise from the substitution of an entire domain of the aGPCR by a different domain (e.g., a different wild-type domain found in another aGPCR, or a different wild-type domain containing one or more mutations corresponding to that wild-type domain), or by a domain not derived from any aGPCR. For example, a chimeric aGPCR may contain a target-binding domain different from the target-binding domain of any natural aGPCR. For example, the target-binding domain may be an antibody or an antibody fragment thereof, or a ligand capable of binding a specific receptor.

[0023] The chimeric aGPCR described in this article can also be referred to as a redirected aGPCR, because the reaction triggered by the aGPCR upon target binding is redirected to a new target through substitution or mutation of the target-binding domain of the aGPCR.

[0024] Chimeric aGPCRs can also be considered synthetically activated G protein adhesion receptors (or abbreviated as SAGA receptors) because, in a preferred embodiment, the aGPCRs are designed to be activated by non-natural targets and are therefore considered synthetically activated.

[0025] Those skilled in the art will understand that aGPCRs (such as standard GPCRs) comprise a 7-transmembrane domain (7TM) and an intracellular tail domain. The intracellular tail domain is typically located at the C-terminus of the protein and is therefore preferably referred to as the C-terminal tail. Intracellular signal transduction from the binding of aGPCR to a target is achieved through intracellular interactions of different G proteins with intracellular loops (ICLs) (where there are 3 intracellular loops in the 7TM) and / or the intracellular tail domain (or the C-terminal tail domain). G proteins (such as β-repressor 1 / 2) may interact only with a single C-terminal tail domain (i.e., not also with all three ICLs) to achieve signal transduction, or they may interact with one, two, or all three intracellular loops of the 7TM. See also Figure 10 It depicts the 7TM and intracellular loop of the GPCR (note that the extracellular domain of the protein depicted is the extracellular domain of the GPCR, not the extracellular domain of the aGPCR). Therefore, the C-terminal tail and the three ICLs can be collectively referred to as the "intracellular signal transduction domain," even though the residues constituting this domain are not sequentially discontinuous.

[0026] Therefore, in a first aspect, the present invention provides a chimeric adhesion G protein-coupled receptor (chimeric aGPCR) comprising: a) Intracellular tail domain, optionally C-terminal tail; b) A seven-transmembrane domain (7TM) containing three intracellular loops (ICLs); and c) Extracellular domains, which include: (i) a target-binding domain heterologous to the intracellular tail domain; (ii) The GPCR autologous protein hydrolysis inducible domain (GAIN domain) optionally contains a tethered agonist peptide.

[0027] Chimeric aGPCRs may also include: (iii) A connector, optionally wherein the connector is located in the extracellular domain, for example, between the GAIN domain and the target-binding domain.

[0028] As described above, some aGPCRs do not require cleavage of the GPCR-GPS site for activation. Therefore, in some embodiments of chimeric aGCPRs, the GAIN domain does not contain GPS. In some embodiments, the GAIN domain contains GPS, but the GPS is non-cleavable, for example, GPS cannot be self-proteased. In other, more common embodiments, the GPS is cleavable. Preferably, the GPS is cleaved by the GAIN domain. In some embodiments, the GPS is constitutively cleaved by the GAIN domain. In some embodiments, the GPS is cleaved in vitro. In some embodiments, the GPS is cleaved when expressed by cells, such as eukaryotic cells like mammalian cells like human cells, or prokaryotic cells like bacterial cells. In some embodiments, the cleavage is constitutive.

[0029] In some embodiments, the extracellular domain comprises a GAIN domain that cannot be cleaved by the protein's own protease, but also includes an additional protease site that has been introduced or engineered into the protein. The protease sites are arranged such that the aGPCR is activated upon proteolysis. In some embodiments, the chimeric aGPCR is activated upon cleavage of the protease site and in the absence of a target-binding domain binding to the target. In some embodiments, the chimeric aGPCR is activated only upon cleavage of the protease site and in the presence of a target-binding domain binding to the target. Such embodiments, in which the chimeric aGPCR includes an additional protease site that has been introduced or engineered into the protein, are particularly useful in the sensing and detection of specific proteases. In a specific embodiment, the protease site is a site that can be cleaved by a protease expressed, for example, in the tumor microenvironment. In this way, the chimeric aGPCR will be activated only in the presence of the corresponding protease. This can be used to a) determine the presence of a specific protease in a specific environment such as the tumor microenvironment; and b) in embodiments where the chimeric aGPCR is activated only when the protease site is cleaved by the appropriate protease and where a target-binding domain is simultaneously bound to the target, which can help reduce off-target effects. Tumors are known to express many different types of proteases, such as cysteine ​​proteases, including cathepsins, caspases, and calpains; metalloproteinases, aspartic proteases, serine proteases, and threonine proteases. One or more of the corresponding proteolytic cleavage sites may be present in the extracellular domains of some embodiments of the chimeric aGPCR.

[0030] The GPS site is typically located within the N-terminus of the GAIN domain of the tethering peptide agonist, allowing the tethering peptide agonist to be retained along with the C-terminal fragment after cleavage.

[0031] As mentioned above, for most aGPCRs, after GPS is cleaved, the non-covalent interaction between the C-terminal fragment (CTF) and the N-terminal fragment (NTF) keeps the two fragments tightly associated and prevents receptor activation in the absence of external forces that may arise when the receptor binds to the target and may interfere with the non-covalent interaction between the CTF and NTF.

[0032] Therefore, in some embodiments of chimeric aGCPR, cleavage of the GPS motif produces an N-terminal fragment (NTF) and a C-terminal fragment (CTF), and the NTF and CTF remain bound to each other through non-covalent interactions. In some embodiments, when present in a lipid membrane, such as when 7TM is present in a lipid membrane (e.g., the lipid membrane of a cell, platelet, or engineered platelet), the CTF and NTF remain bound through non-covalent interactions. For clarity, the target-binding domain heterologous to the intracellular tail domain is part of the NTF.

[0033] In some implementations, the association of NTF and CTF prevents the tethered peptide agonist from interacting with the 7TM domain, thereby preventing activation of intracellular signaling.

[0034] In all embodiments, the chimeric aGPCR is inactive in the absence of target binding. Inactivity is intended to include the following meaning: failure to initiate the corresponding intracellular signaling cascade associated with a specific intracellular signaling domain (as described elsewhere, which consists of the three ICLs of 7TM and / or the intracellular tail domain (e.g., the C-terminal tail)). Inactivity is also intended to include the following meaning: basal level of activation. GPCRs exhibit varying levels of basal activity, which depend on the individual characteristics of each receptor. See, for example, Vizurraga et al., 2020 J Biol Chem 295: 14065-14083. Basal activity is a state of a GPCR in the dynamic energy landscape of active and inactive conformations. Those skilled in the art will understand that some “leakage” is typically present in any biological system, and even in the absence of a relevant target, some low level of receptor activation and subsequent intracellular signaling may exist due to the low-level switching between the inactive and active conformations in the absence of a target. However, technicians will also recognize the difference between the level of inactive (but basal) signal transduction and the level of signal transduction obtained in the presence of the relevant target.

[0035] Inactive also implies that the chimeric aGPCR is in an inactive conformation. Therefore, when the chimeric aGPCR binds to its target, the intracellular signaling level is higher than when it is not bound to the target. Thus, in some embodiments, the chimeric aGPCR: a) It is inactive in the absence of target-binding domain binding to the target; b) In the absence of target-binding domain binding to the target, it primarily occupies the inactive conformation; and / or c) It remains in a basic active state in the absence of target-binding domain binding to the target.

[0036] Those skilled in the art can readily test this. For example, constitutively active receptors will exhibit high levels of intracellular signaling regardless of the presence of a target; and receptors that cannot be activated by target binding will exhibit the same absence or basal levels of intracellular signaling, regardless of the presence of a target.

[0037] Suitable methods for determining receptor activity are described in the examples, such as the functional determination in Example 2 and the agitation method in Example 3.

[0038] In the case where a chimeric aGPCR is a receptor containing a cleavable GPS site and GPS cleavage produces NTF and CTF that are bound together by non-covalent interactions, the aGPCR can be activated by target binding, resulting in the physical removal of NTF from CTF, thereby allowing activation of the intracellular domain (“disruption” mechanism); or by modulating the non-covalent interaction between NTF and CTF, allowing CTF to be activated—that is, NTF and CTF remain bound in this case, but the conformational change due to the applied force allows for receptor activation (“regulatory” mechanism).

[0039] In some implementations, the binding of the target-binding domain to the target induces a mechanical force applied to the non-covalent interaction of the NTF / CTF.

[0040] In some implementation schemes, mechanical force can: a) Regulating the non-covalent interaction between NTF and CTF and activating intracellular signaling, for example by allowing tethered peptide agonists to interact with 7TM and activate intracellular signaling; or b) Disrupt the non-covalent interaction between NTF and CTF and activate intracellular signaling, for example by exposing tethered peptide agonists to interact with 7TM and activate intracellular signaling.

[0041] In some implementations of chimeric aGPCRs that do not contain cleavable GPS sites and do not undergo GPS cleavage, the binding of the target-binding domain to the target disrupts the receptor conformation, thereby activating intracellular signal transduction.

[0042] As described elsewhere in this document, in some embodiments, it is important to apply mechanical forces to the receptor via target binding. For example, in some embodiments where the GPS has been cleaved, target binding should apply sufficient mechanical force to disrupt the non-covalent interaction between the NTF and CTF; or sufficiently modulate those interactions to allow activation of intracellular signaling. In some cases, these forces can be triggered by a target acting as a “fixed” target. In some embodiments, “fixed” target is intended to include a target fixed to a physical structure (e.g., in vivo), such as a blood vessel, epithelial layer, or tumor. In the same or different implementations, "fixed" target is intended to include the following meanings: the kinetics of the target relative to the chimeric aGPCR are fixed, for example, there may be some relative motion differences between the aGPCR (which is typically present in platelets, engineered platelets or the plasma membrane of cells) and the target, such that when target binding occurs, some force is applied to the receptor, such as a force that can disrupt or modulate NTF / CTF interactions to trigger intracellular signal transduction; or a force sufficient to trigger intracellular signal transduction when GPS is absent or uncut.

[0043] However, some argue that binding to any target would generate sufficient force to induce intracellular signaling. For example, the random Brownian motion of a receptor within the plasma membrane relative to a target is expected to generate enough force to trigger receptor activation and intracellular signaling.

[0044] The forces required to disrupt or interfere with the non-covalent interactions between CTF and NTF can be designed using specific GAIN domains and / or by engineering sequences of GAIN domains—see, for example, Dumas (Uncovering and engineering the mechanical properties of the adhesion GPCR ADGRG1 GAIN domain doi: https: / / doi.org / 10.1101 / 2023.04.05.535724), and other discussions elsewhere in this paper.

[0045] As will be apparent from the disclosure herein, in some cases, the chimeric aGPCR of the present invention has the use as a drug delivery carrier in the context of platelets or engineered platelets. Upon binding to a target, the platelet or engineered platelet (which may load or express cargo such as therapeutic cargo) undergoes degranulation, releasing the cargo to the vicinity of the target. Clearly, the chimeric aGPCR described herein has particular use in cancer therapy, and in some embodiments, the target-binding domain is capable of binding to a cancer-associated target and triggering a response to that target.

[0046] However, the chimeric aGPCRs described herein are considered to have broader applications, not limited to platelet, drug delivery, and cancer fields. For example, in some embodiments, chimeric aGPCRs are considered to be used in the field of redirected immunotherapy, in a manner similar to the current application of chimeric antigen receptors in the T-cell field.

[0047] Therefore, the target can be any target. For example, the target can be any target against which specific binding activity can be directed, such as an antigen target against which an antibody or its antigen-binding fragment can be generated, or a receptor / ligand directed. It will be apparent to those skilled in the art, and as discussed elsewhere herein, that the target-binding domain can be an antibody or its antigen-binding fragment.

[0048] In some implementations, the target is a target that causes the following upon binding to the target-binding domain of an aGPCR: a) Regulating the non-covalent interaction between NTF and CTF and activating intracellular signaling, for example by enabling tethering peptide agonists to interact with 7TM and activate intracellular signaling; or b) Disrupt the non-covalent interaction between NTF and CTF and activate intracellular signaling, for example by exposing tethered peptide agonists to interact with 7TM and activate intracellular signaling.

[0049] In some implementations of chimeric aGPCRs that do not contain cleavable GPS sites and do not undergo GPS cleavage, the target is a target that, upon binding to the target-binding domain, causes conformational disruption of the receptor, thereby activating intracellular signal transduction.

[0050] The target can be a protein or peptide, an antigenic protein or peptide, a carbohydrate or lipid. The target can be of any type, and specific antibodies or antigen-binding fragments can be prepared against that target.

[0051] In some implementations, the target-binding domain is capable of binding to targets that are endogenous targets found on or in tissues or subsets of tissues in the subject’s body, or on cells, or at a specific location in the subject, such as cancerous tissue or cancer cells; and / or present on cell surfaces, on physical structures, on the inner walls of blood vessels, on organs, on solid tumors, on anchored targets, on targets that have opposite relative mobility to the chimeric aGPCR when the chimeric aGPCR is present in the plasma membrane of cells, platelets, or engineered platelets, on cell or tissue surfaces, on cellular matrix components, connective tissue, carbohydrates, collagen, and / or immobilized on solid matrices.

[0052] For example, in some implementations, the target is an endogenous target found on or in a tissue or subset of tissues in the subject’s body, or on cells, or in a specific location in the subject, such as cancerous tissue or cancer cells; and / or present on cell surfaces, on physical structures, on the inner walls of blood vessels, on organs, on solid tumors, on anchored targets, on targets that have opposite relative movement to the chimeric aGPCR when the chimeric aGPCR is present in the plasma membrane of cells, platelets, or engineered platelets, on cell or tissue surfaces, on cellular matrix components, connective tissue, carbohydrates, collagen, and / or immobilized on a solid matrix.

[0053] In some embodiments, the target is present only during one or more disease states, for example, in some embodiments, the target is a neoantigen that appears in tumor cells; and / or is present only in significant amounts, such as at abnormal levels in tissues or cells that do not normally express the target; and / or is present only locally during one or more disease states; and / or is an antigen associated with a disease, condition, or symptom, such as a tumor neoantigen or tumor-specific antigen.

[0054] In some implementations, the target-binding domain contains peptides related to autoimmunity, such as: Peptides or portions of any one or more of the following proteins: MOG, GAD65, MAG, PMP22, TPO, VGKC, PLP, AChR, TRIB2, NMDA, GluR, GAD2, ARMC9, CYP21A2, CASR, NSP, insulin, TSHR, thyroid peroxidase, desialyl glycoprotein receptor, CYP2D6, LF, TTG, H / K ATPase, factor XIII, β2-GPI, ITGB2, G-CSF, GP IIb / IIa, COLII, FBGβα, MPO, CYO, PRTN3, TGM, COLVII, COIL, DSG1, DSG3, SOX10, 70SNRNP70, SAG, and a3(IV)NC1 collagen; or Peptides or portions having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with any one or more of the following proteins: MOG, GAD65, MAG, PMP22, TPO, VGKC, PLP, AChR, TRIB2, NMDA, GluR, GAD2, ARMC9, CYP21A2, CASR, NSP, insulin, TSHR, thyroid peroxidase, desialyl glycoprotein receptor, CYP2D6, LF, TTG, H / K ATPase, factor XIII, β2-GPI, ITGB2, G-CSF, GP IIb / IIa, COLII, FBGβα, MPO, CYO, PRTN3, TGM, COLVII, COIL, DSG1, DSG3, SOX10, 70SNRNP70, SAG, and a3(IV)NC1 collagen.

[0055] This invention is considered particularly useful in the context of engineered platelets, such as those described in WO 2022 / 263824. In some embodiments, the platelet has been engineered to have reduced procoagulant activity or to be non-thrombotic. In these cases, when the chimeric aGPCR is present in the membrane of the platelet or engineered platelet, the target-binding domain binds to the target: a) Causes the platelet or engineered platelet to degranulate; b) Causes the release of contents from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment.

[0056] Some exemplary targets are described below - for example, in some implementations, the target-binding domain combines with the following targets: Endogenous targets; Endogenous targets, such as cancerous tissue or cancer cells, found on tissues or subsets of tissues in the subject, or on cells, or in specific locations within the subject; They exist on the cell surface; It exists in the physical structure, such as inside the body or on the pore walls of a plate; It exists on the inner wall of blood vessels; It exists in the plasma or blood of the subject; Found on organs; Existing in solid tumors Anchoring the target; When chimeric aGPCRs are present in the plasma membrane of cells, platelets, or engineered platelets, they have targets that move in a relative manner opposite to those of chimeric aGPCRs. Cell matrix components; It is connective tissue or exists on connective tissue. carbohydrate; Collagen; and / or Fixed on a solid substrate It exists on cancerous tissue or cancer cells; It exists only during one or more disease states; for example, in some implementations, the target is a neoantigen that appears in tumor cells. It exists only in significant amounts, such as at abnormal levels in tissues or cells that abnormally express the target, and / or only in a localized manner during one or more disease states; Antigens associated with diseases, symptoms, or conditions, such as tumor neoantigens or tumor-specific antigens; Artificial or exogenous targets; CD19; CD276; IL2; KLK; Amyloid protein; Notch receptor; OLR1; MadCAM1; Cytokine receptors; Collagen; Non-collagen; Drug design; Drugs designed using DREADD; Proteins selected from Table 2 on pages 23-31 of PCT / GB2020 / 053247, which are incorporated herein by reference; and / or Autoimmune B cells.

[0057] D276 is an identified immunomodulatory protein that is overexpressed in cancerous tissues (see Zhao et al., 2022 Journal of Hematology and Oncology 15: article 153). https: / / jhoonline.biomedcentral.com / articles / 10.1186 / s13045-022-01364-7 Mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) is a cell adhesion molecule expressed on the mucosal endothelium. This protein guides lymphocytes to specifically home to the mucosal tissue. Antibodies targeting MAdCAM1 have been developed for the treatment of ulcerative colitis and Crohn's disease (see Wang et al., 2020 J Clin Pharmacol 60: 903-914).

[0058] The target-binding domain preferably binds to an endogenous target as described herein. In some embodiments, the target-binding domain binds to an artificial or exogenous target.

[0059] In some implementations, the target is not a soluble target.

[0060] While chimeric aGCPRs are typically designed to have a target-binding domain that directly binds to the final or desired target (i.e., the target of a specific response, such as a neoantigen present on cancer cells), in some embodiments, aGPCRs can be considered generic aGPCRs designed to bind intermediate adaptor proteins or peptides, such as those that themselves contain a peptide tag and a target-binding domain that can bind to the final or desired target (i.e., any target the aGPCR is intended to target). This is predicated on administering an intermediate adaptor protein or peptide to a subject that itself binds to the target as described herein (referred to herein as the final target-binding domain in the context of the intermediate adaptor protein or peptide), but also contains a portion, such as a peptide tag, that can be bound by the target-binding domain of the chimeric aGPCR. In this way, the target is essentially coated by the intermediate adaptor protein or peptide, thereby allowing the chimeric aGPCR described herein to bind to the cell or target (i.e., the final target) via the intermediate adaptor protein or peptide. Alternatively, prior to administration, a chimeric aGPCR with a tag-binding target-binding domain (i.e., tag-binding domain) is pre-formed between an intermediate linker protein or peptide containing the final target-binding domain, thereby administering the pre-formed complex to the subject. An advantage of this system is that the aGPCR does not necessarily have to be redesigned for each new target; instead, new intermediate target proteins or peptides can be designed for relevant targets and incorporated with the corresponding tag peptide. Janh Hwan Cho et al. proposed such a system in the context of a chimeric antibody receptor containing ITAM in 2018 Cell 173:1426-1438.

[0061] In these cases, the target-binding domain of the chimeric aGPCR can be considered an adaptor protein or peptide-binding domain. The adaptor protein or peptide includes a portion capable of binding the target-binding domain of the chimeric aGPCR, as well as a portion that serves as the target-binding domain. The preference of the target-binding domain of the adaptor protein or peptide is as described elsewhere in this document regarding the target-binding domain of chimeric aGPCRs.

[0062] Preferably, the intermediate linker protein or peptide includes a portion serving as a peptide tag, and the target-binding domain of the chimeric aGPCR binds to this peptide tag. It should be understood that the present invention also provides a complex comprising: a) The chassis of the present invention, which expresses one or more chimeric aGPCRs of the present invention; and b) Intermediate linker proteins or peptides. The intermediate linker protein or peptide includes a final target-binding domain and a tag, optionally a peptide tag. Furthermore, the target-binding domain of the chimeric aGPCR of the present invention is a tag-binding domain that can bind to the tag of the intermediate linker protein or peptide, and the final target-binding domain of the intermediate linker polypeptide or protein can bind to both the final target and the chimeric aGPCR of the present invention simultaneously.

[0063] The final target-binding domain preference is as described in this paper for the target-binding domain of chimeric aGPCRs.

[0064] Those skilled in the art will understand that when any framework as described herein is described as expressing one or more chimeric aGPCRs, it is intended to include the following meaning: the framework expresses one or more chimeric aGPCRs on the cell membrane (i.e., in the plasma membrane), as is common for aGPCRs.

[0065] Therefore, the contact between the framework expressing chimeric aGPCR and the intermediate adaptor protein occurs on the cell surface, platelet surface, or engineered platelet surface, between the chimeric aGPCR present on the cell or platelet surface and the intermediate adaptor protein or peptide.

[0066] Therefore, in some embodiments, the target of the target-binding domain of the chimeric aGPCR is a peptide tag. In a preferred embodiment, the peptide tag is expressed as part of and is a component of a larger target peptide; that is, in such embodiments, the tagged intermediate adaptor protein or peptide is a single protein or peptide containing both the tag and the final target-binding domain.

[0067] In other implementations, the target-binding domain of the chimeric aGPCR does not bind to the peptide tag.

[0068] As described in this article, peptide tags are typically short peptide sequences (i.e., sequences of amino acids) well-known in the field of molecular biology, where it is routine to express the peptide or polypeptide sequence of interest, where the sequence has been extended to include a relatively short additional sequence encoding the tag. Examples of suitable peptide tags include FLAG-tags, V5-tags, Myc-tags, HA-tags, Spot-tags, T7-tags, NE-tags, and leucine zippers (Hwan et al., 2018 Cell 173: 1426-1438).

[0069] The peptide tag is also intended to include the meaning of a protein domain known to interact with the same or different protein domains. For example, in some embodiments, the target-binding domain of the aGPCR may include a leucine zipper domain. In this embodiment, the intermediate adaptor protein or peptide may include a final target-binding domain, such as an scFv fused to a leucine zipper domain, thereby allowing association between the chimeric aGPCR of the present invention and the leucine zipper of the intermediate adaptor protein or peptide. Similarly, coiled-coil domains that associate with each other (forming homodimers or heterodimers) may be employed to allow association between the chimeric aGPCR and the final target-binding domain of the intermediate adaptor protein or peptide.

[0070] Although tags are typically peptide tags, in some embodiments, the tag can be any part that can serve as a binding partner for a chimeric aGPCR. Therefore, non-peptide tags can be any chemical entity with an affinity for the tag-binding domain. Tags can be selected from, for example, any organic molecule, small molecule, or hapten. Tags can take the form of nucleic acids, such as aptamers.

[0071] Therefore, those skilled in the art will appreciate that the present invention also provides a complex comprising the chimeric aGPCR of the present invention, which binds to or interacts with an intermediate adaptor protein or peptide comprising a final target-binding domain and a tag (such as a peptide tag). In any embodiment of the present invention, the target-binding domain (or the final target-binding domain in the context of an intermediate adaptor protein or peptide) can be any domain having the ability to bind a desired target.

[0072] Typically, a target-binding domain is a protein domain, such as a target-binding ligand or a fragment thereof, or an antibody or an antigen-binding fragment thereof that specifically binds to the target.

[0073] The target-binding domain may be identical to or a variant of an endogenous target-binding domain. Endogenous is intended to include meanings such as being endogenous to a specific cell, to a specific tissue, or to a specific host organism (such as a mammal, such as a human). For example, in some embodiments, the target-binding domain is a human target-binding domain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with a human target-binding domain sequence. However, while the target-binding domain may be endogenous to a specific host or species (such as a human), it may still be heterologous to intracellular tail domains (e.g., the C-terminal tail domain of a chimeric aGPCR).

[0074] The target-binding domain may contain non-human target-binding domain sequences, such as humanized sequences or sequences derived from mice.

[0075] In some preferred embodiments, the target binding domain includes: Antibodies or antibody fragments that specifically bind to the target; Variable heavy chain domains and / or variable light chain domains of antibodies; scFv; Nanobodies; Fab; Targeted κ light chain or fragment thereof; Synthesize binding scaffolds, such as monomers, affinity molecules, designed ankyrin repeat sequence proteins (DARPin), or knottin. Anti-CD19 scFv domains, such as the FMC63 scFv domain, see, for example, SEQ ID NO: 15; Anti-CD276 scFv domains, such as the enotozumab scFv domain (see, for example, SEQ ID NO: 16) or aviolimab (see, for example, SEQ ID NO: 17); and / or Anti-MAdCAM1 scFv domains, such as the ontalimab scFv domain (see, for example, SEQ ID NO: 18).

[0076] The target-binding domain can be any antibody or antibody-derived fragment, including those mentioned above. Antigen-binding fragments of antibodies can be obtained in various forms, and this invention covers all natural or engineered / synthetic antigen-binding fragments and derivatives.

[0077] Technicians will understand that antibodies or their antigen-binding fragments typically comprise heavy and light chain polypeptides, each contributing to the variable epitope binding region via its three CDR regions, which are in turn supported by a framework region. Single-chain antibodies capable of specifically binding to targets are known to require only three CDR regions.

[0078] Therefore, in some embodiments, the target-binding domain comprises one, two, three, four, five, or six CDR regions, such as one, two, three, four, five, or six CDR regions from the light or heavy chain of the antibody. In some embodiments, the target-binding domain comprises one, two, or three CDR regions from the light chain. In other embodiments, the target-binding domain comprises one, two, or three CDRs from the light chain and one, two, or three CDRs from the heavy chain. In a particular embodiment, the target-binding domain comprises three CDRs from the light chain and three CDRs from the heavy chain.

[0079] As described above, the premise of the chimeric aGPCR of the present invention is to direct a specific intracellular signaling response to a specific target. For example, the chimeric aGPCR may include a target-binding domain that binds to the desired target, and a suitable intracellular tail domain, such as the C-terminal tail of 7TM and / or ICL that associates with a specific G protein to elicit the desired intracellular signaling response.

[0080] In this way, aGPCRs can be considered to comprise multiple modular units or domains that are interchangeable with other known units or with rationally designed or synthesized units. In the context of chimeric aGPCRs, all combinations of domains are considered appropriate.

[0081] In some embodiments, some domains of the chimeric aGPCR are naturally present together in the natural or wild-type aGPCR. For example, in some embodiments, the intracellular tail domain (e.g., the C-terminal tail) and the 7TM domain are autologous, meaning that the intracellular tail domain (e.g., the C-terminal tail) and 7TM coexist in nature, and only the N-terminal domain is interchanged. In some embodiments, the intracellular tail domain, such as the C-terminal tail, and the GAIN domain are autologous; and / or Intracellular tail domains, such as the C-terminal tail and the GPS motif, are self-contained.

[0082] In some implementations, the following is stated: a) Intracellular tail domains, such as the C-terminal tail; b) Seven-transmembrane domains (7TM); and c) The GPCR autoproteolytic inducible domain (GAIN domain) contains a tethered agonist peptide and constitutively cleaves the GPS motif; and d) GPS motifs segmented by the GAIN domain They are each other's own.

[0083] In some implementations, the following is stated: The GAIN domain and the following heterogeneous sources: 7TM structural domain; Intracellular tail domains, such as the C-terminal tail; and / or Target binding domain.

[0084] In some implementations, the following is stated: a) Intracellular tail domains, such as the C-terminal tail; b) Seven-transmembrane domains (7TM); and c) The GPCR autoproteolytic inducible domain (GAIN domain) contains a tethered agonist peptide and constitutively cleaves the GPS motif; and d) GPS motifs cleaved by the GAIN domain originate from the same naturally occurring aGPCR.

[0085] In some implementations, the following is stated: a) Intracellular tail domains, such as the C-terminal tail; b) Seven-transmembrane domains (7TM); and c) The GPCR autoproteolytic inducible domain (GAIN domain) contains a tethered agonist peptide and constitutively cleaves the GPS motif; and / or d) GPS motifs segmented by the GAIN domain Derived from the same naturally occurring aGPCR, and wherein one or more of the following are present: a) Intracellular tail domains, such as the C-terminal tail; b) Seven-transmembrane domains (7TM); and c) The GPCR autoproteolytic inducible domain (GAIN domain) contains a tethered agonist peptide and constitutively cleaves the GPS motif; and / or d) GPS motifs segmented by the GAIN domain It contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the corresponding domain of a naturally occurring aGPCR.

[0086] In any implementation, one or more of the three ICLs of 7TM may contain one or more mutations, substitutions, insertions, or deletions. For example, such mutations, substitutions, insertions, or deletions may be introduced to alter the interaction between 7TM and a specific G protein, such as allowing one or more ICLs to interact with a different G protein than the one or more ICLs would normally interact with in the absence of those mutations, substitutions, insertions, or deletions.

[0087] As described above, the chimeric aGPCR of the present invention can be fully modularized and considers all combinations of naturally occurring, synthetic, or engineered domains. In some embodiments, the chimeric aGPCR comprises the following combinations of naturally occurring and engineered and / or synthetic domains:

[0088]

[0089]

[0090]

[0091] Wherein (a) is an intracellular tail domain, such as a C-terminal tail domain; (b) is a seven-transmembrane domain (7TM) containing three ICLs; (c) is a GPCR autoproteolytic inducible domain (GAIN domain), optionally containing a tethered agonist; and (d) is an optional GPS motif, and in some embodiments, it is cleaved by the GAIN domain. And the engineered structural domains are: a) A domain containing at least one substituted, inserted, or deleted amino acid sequence relative to a naturally occurring or naturally present domain; b) A domain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with a naturally occurring domain; and / or c) is a rationally designed or synthesized structural domain.

[0092] A rationally designed or synthesized domain is intended to include the following meaning: the domain is designed de novo, rather than a relatively minor engineering of a known domain. A rationally designed or synthesized domain is also intended to include the following meaning: the domain has an amino acid sequence with less than 75% sequence identity to a corresponding known naturally occurring domain. For example, it is known that domains may be functionally and conformally equivalent, exhibiting very little sequence identity.

[0093] The inventors have discovered that different GAIN domains have different sequence and / or structural complexities while retaining similar functions, and that using relatively simple GAIN domains in the context of chimeric aGPCRs has some advantages. For example, in some embodiments, the GAIN domain of ADGRG1 aGCPR is considered relatively simple compared to the GAIN domain from ADGRG5. Therefore, in some embodiments of chimeric aGPCRs, the GAIN domain (domain (c) in the table above) is either the GAIN domain from ADGRG1 [SEQ ID NO: 35] or a GAIN domain having an amino acid sequence with at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 35. For example, the GAIN domain may be derived from the GAIN domain of ADGRG1 or may be derived from the GAIN domain (i.e., with some sequence differences from the GAIN domain), but the target binding domain, intracellular tail (e.g., C-terminal tail) and 7TM domain may all be derived from proteins other than ADGRG1, or in fact one or more of them may be rationally designed, engineered or synthetic.

[0094] For example, in some embodiments of the chimeric aGPCR, the GAIN domain is a GAIN domain having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 35, and wherein: The intracellular tail does not originate from ADGRG1; The target-binding domain is not derived from ADGRG1; and / or The 7TM structure domain is not from ADGRG1.

[0095] In some implementations, the GAIN domain is: The GAIN domain from ADGRL1 has the sequence of SEQ ID NO: 27; The GAIN domain from ADGRL3 has the sequence of SEQ ID NO: 28; The GAIN domain from ADGRE2 has the sequence SEQ ID NO: 29; The GAIN domain from ADGRG2 has the sequence of SEQ ID NO: 30; Or it is a GAIN domain of an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with any of SEQ ID NO: 27, 28, 29, or 30.

[0096] For example, in some embodiments, the GAIN domain is derived from ADDRL1 and has the sequence of SEQ ID NO: 27, or has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 27, but the target-binding domain, intracellular tail (e.g., C-terminal tail), and 7TM domain may all be derived from proteins other than ADDRL1, or in fact one or more of them may be rationally designed, engineered, or synthetic.

[0097] For example, in some embodiments, the GAIN domain is derived from ADDRL3 and has the sequence of SEQ ID NO: 28, or has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 28, but the target-binding domain, intracellular tail (e.g., C-terminal tail), and 7TM domain may all be derived from proteins other than ADDRL3, or in fact one or more of them may be rationally designed, engineered, or synthetic.

[0098] For example, in some embodiments, the GAIN domain is derived from ADGRE2 and has the sequence of SEQ ID NO: 29, or has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 29, but the target-binding domain, intracellular tail (e.g., C-terminal tail), and 7TM domain may all be derived from proteins other than ADGRE2, or in fact one or more of them may be rationally designed, engineered, or synthetic.

[0099] For example, in some embodiments, the GAIN domain is derived from ADGRG2 and has the sequence of SEQ ID NO: 30, or has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 30, but the target-binding domain, intracellular tail (e.g., C-terminal tail), and 7TM domain may all be derived from proteins other than ADGRG2, or in fact one or more of them may be rationally designed, engineered, or synthetic.

[0100] The GAIN domain can be an engineered GAIN domain. For example, Dumas et al. (Uncovering and engineering the mechanical properties of the adhesion GPCR ADGRG1 GAIN domain doi: https: / / doi.org / 10.1101 / 2023.04.05.535724) have shown that the GPS-Stachel fracture force can be tuned by computationally designing GAIN variants to lock the α and β subdomains and redecorating mechanically induced structural deformations. Therefore, in some embodiments, the GAIN domain is an engineered GAIN domain having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the naturally occurring GAIN domain. In some embodiments, the GAIN domain is a rationally designed or synthetic GAIN domain having less than 75% sequence identity with the naturally occurring GAIN domain. In some embodiments, the GAIN domain has been engineered to increase the force required to disrupt association between the GPS and the tethering agonist or between the NTF and CTF. In other implementations, the GAIN domain has been engineered to reduce the forces required to disrupt association between the GPS and the tethered agonist, or between the NTF and CTF.

[0101] The chimeric aGPCR of the present invention is considered to be capable of triggering intracellular signaling in response to a suitable target. In a preferred embodiment, the chimeric aGPCR is capable of activating intracellular signaling. For example, when the chimeric aGPCR is localized to the plasma membrane of a cell, platelet, or engineered platelet, it is capable of activating intracellular signaling. Those skilled in the art can readily test the ability of a particular aGPCR to activate intracellular signaling. For example, in some embodiments, cells, platelets, or engineered platelets expressing aGPCR are incubated and agitated in the presence of a target, and the activation status of intracellular signaling is determined. However, as described elsewhere herein, it is considered that the general molecular motion of the target-bound aGPCR relative to the cell and the target is sufficient to trigger chimeric aGPCR activation and intracellular signaling.

[0102] When using agitation to determine chimeric aGPCR activation in an assay, agitation can be performed by oscillating at the following rates: At least 10 rpm, 20 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm or at least 200 rpm; less than 200 rpm, 190 rpm, 180 rpm, 170 rpm, 160 rpm, 150 rpm, 140 rpm, 130 rpm. rpm, 120rpm, 110rpm, 100rpm, 90rpm, 80rpm, 70rpm, 60rpm, 50rpm, 40rpm, 30rpm, 20rpm or less than 10rpm; and / or 10rpm to 200rpm, 20rpm to 190rpm, 30rpm to 180rpm, 40rpm to 170rpm, 50rpm to 160rpm, 60rpm to 150rpm, 70rpm to 140rpm, 80rpm to 130rpm.

[0103] Technicians will understand various methods for identifying chimeric aGPCR activation and subsequent intracellular signaling. For example, in some cases, cells, platelets, or engineered platelets expressing chimeric aGPCRs also contain a reporter system that includes a reporter protein expressed when intracellular signaling is activated. For instance, the reporter protein may be expressed by a promoter containing an element that activates a T-cell nuclear factor (NFAT) response; the reporter protein could be luciferase. A similar assay can be performed using calcium indicator dyes such as Fluo-4 to detect increases in intracellular calcium. Other methods can be used to detect different intracellular signaling responses induced by the interaction of C-terminal signaling domains (which, as described elsewhere, may contain an intracellular tail, such as a C-terminal tail and / or one or more of 7TM) with different G proteins.

[0104] By detecting the expression or activity of reporter proteins, such as luciferase, it can be determined whether a specific chimeric aGPCR has been activated.

[0105] In addition to engineering aGPCRs to redirect intracellular signaling responses to new targets, intracellular signaling domains (i.e., any one, two, or three ICLs of the intracellular tail and / or 7TM) can be engineered to alter intracellular signaling responses.

[0106] As described elsewhere in this document, those skilled in the art will understand that the C-terminal intracellular signaling domain encompasses one, two, or three ICLs and / or the C-terminal tail of the 7TM intracellular loop (ICL). G proteins (especially G...) α The G protein interacts with the GPCR via the ICL in the 7TM region. The G protein also interacts with the C-terminal tail. In this way, the intracellular signal transduction domain is actually composed of four discontinuous regions (three ICLs and the C-terminal tail). The C-terminal tail primarily interacts with signaling proteins such as repressor proteins, which prevent GPCR activation and recruit other signaling molecules such as ERK / JNK.

[0107] For example, in some embodiments, the intracellular tail domain (e.g., the C-terminal tail domain) contains one or more substitutions, insertions, or deletions compared to the autologous intracellular tail (e.g., the autologous C-terminal tail) or the wild-type intracellular tail (e.g., the autologous C-terminal tail). Similarly, and as described above, in some embodiments, the 7TM domain contains one or more substitutions, insertions, or deletions compared to the autologous 7TM or the wild-type 7TM, for example in some embodiments: Compared to autologous intracellular tails (e.g., C-terminal tails and / or 7TM) or wild-type intracellular tails (e.g., C-terminal tails and / or 7TM) that do not contain the same one or more substitutions, insertions, or deletions, one or more substitutions, insertions, or deletions in the intracellular tail increases intracellular signaling response; or Compared to autologous intracellular tails (e.g., C-terminal tails and / or 7TM i) or wild-type intracellular tails (e.g., C-terminal tails and / or 7TM i) that do not contain the same one or more substitutions, insertions, or deletions, one or more substitutions, insertions, or deletions in the intracellular tail reduces intracellular signaling responses. And / or Compared to autologous intracellular tails (e.g., C-terminal tails and / or 7TM) or wild-type intracellular tails (e.g., C-terminal tails and / or 7TM i) that do not contain the same one or more substitutions, insertions, or deletions, one or more substitutions, insertions, or deletions in an intracellular tail alters the specificity of intracellular signal responses, thereby leading to the activation of altered signal transduction pathways.

[0108] Altering the specificity of intracellular signaling responses aims to include modifications to intracellular tails (e.g., C-terminal tails) and / or 7TMs to allow interaction with different or additional G proteins. For example, C-terminal tails and / or 7TM ICLs that typically interact with G proteins Gαq / Gαs are engineered by replacing the original Gαq C-terminal GPCR binding sequence with a sequence derived from Gαs. Chimeric G proteins Gαq / Gαs enable Gαs-dependent aGPCRs (instead of the original Gαq-dependent aGPCRs) to activate downstream Gαq signaling pathways upon activation.

[0109] In some implementations, any one or more of the three cytoplasmic loops or C-terminal tails of 7TM may be modified, for example, by including one or more substitutions, insertions or deletions, or otherwise engineered or designed sequences to achieve desired intracellular signal transduction.

[0110] The sensitivity of a cell, platelet, or engineered platelet response to a specific signal is considered to depend, at least in part, on the number of relevant chimeric aGPCR molecules present on the surface of the cell, platelet, or engineered platelet. One way to influence the level or amount of receptors present on the cell surface is to use appropriate signal peptides. Signal peptides are short peptide sequences that are read from inside the cell as a signal to transport proteins to the cell surface.

[0111] In some embodiments, the chimeric aGPCR of the present invention comprises a signal peptide. In some embodiments, the signal peptide is selected to achieve a desired level of chimeric aGPCR on the surface of cells, platelets, or engineered platelets. Those skilled in the art will understand how to select an appropriate signal peptide for a given situation. See, for example, O'Neill et al., 2023 Protein-specific signal peptides for mammalian vector engineering https: / / www.biorxiv.org / content / 10.1101 / 2023.03.14.532380v1 Exemplary signal peptides include the following: ADGRG1 signal peptide MTPQSLLQTTLFLLSLLFLVQGAHG [SEQ ID NO: 1] ADGRF5 signal peptide MKSPRRTTLCLMFIVIYSSKA [SEQ ID NO: 2] FCERG signal peptide MIPAVVLLLLLLVEQAAA [SEQ ID NO: 3] CD28 signal peptide MLRLLLALNLFPSIQVTG [SEQ ID NO: 4] Platelet signal peptide – GPIIb (aIIb)MARALCPLQALWLLEWVLLLLGPCAAPPAWA [SEQ ID NO:5] Platelet signal peptide – GPIIIa (β3)MRARPRPRPLWATVLALGALAGVGVG [SEQ ID NO: 6] Platelet signaling peptide - GPIBaMPLLLLLLLLPSPLHP [SEQ ID NO: 7] Platelet signaling peptide - GPIXMPAWGALFLLWATAEA [SEQ ID NO: 8] Platelet signaling peptide - GPVMLRGTLLCAVLGLLRA [SEQ ID NO: 9] MAPFASLASGILLLLSLITSSKA [SEQ ID NO: 36] MLLGPGHTLSAPALALAVTLTLLVRSASP [SEQ ID NO: 37] MLLSVPLLLGLLGLAAA [SEQ ID NO: 38] MQELRGILLCLLLAAAVPTTP [SEQ ID NO: 39] MRYVASYLLAALGGNS [SEQ ID NO: 40] MGKSPEAWCIVLFSVLASFSA [SEQ ID NO: 41] MASSGSVQQPRLVLLMLVLAGAARA [SEQID NO: 42]MRWKIIQLQYCFLLVPCMLTALEA [SEQ ID NO: 43] MLSRSLLCLALAWVARVGA [SEQ ID NO: 44] MRFSCLALLPGVALLLASARLAAA [SEQ ID NO: 45] MRVLWVLGLCCVLLTFGFVRA [SEQ ID NO: 46] MKFPMVAAALLLLCAVRA [SEQ ID NO: 47] MRSLLLASFCLLAVALA [SEQ ID NO: 48] MKILLLCVGLLLTWDNGMVLG [SEQ ID NO: 49] MLRISGRNMKVLFAAALIVGSVVFLLLPGPSVA [SEQ ID NO: 50]MAATVRRQRPRRLLCWTLVAVLLADLLALS [SE [SEQ ID NO: 51]MKMGVRLAARAWPLCGLLLAALGGVCA [SEQ ID NO: 52] MWWRLWWLLLLLLLLWLALAAAA [SEQ ID NO: 53] MGWSLILLFLVAVATRVLS [SEQ IDNO: 54] MDFQVQIISFLLISASVIMSRG [SEQ ID NO: 55] MEFGLSWVFLVALFRGVQC [SEQ ID NO: 56] MKWVTFISLLFLFSSAYS [SEQ ID NO: 57] MKLPVRLLVLMFWIPAASA [SEQ ID NO: 58] MNLLLILTFVAAAVA [SEQ ID NO: 59] MGSAALLLWVLLLWVPSSRA [SEQ ID NO: 60] MTRLTVLALLAGLLASSRA [SEQ ID NO: 61] MWWRLWWLLLLLLLLWPMVWA / AA [SEQ ID NO: 62] MKLPVRLLLVLMFWIPASSS [SEQ ID NO: 63] MDMRVPAQLLGLLLLWLSGARC [SEQ ID NO: 64] MKYLLPTAAAGLLLLAAQPAMA [SEQ ID NO: 65] MGVKVLFALICIAVAEA [SEQ ID NO: 66] MPLLLLLPLLWAGALA [SEQ ID NO: 67] MRARALLAVLLLLLLVGIAAAA synthetic design [SEQ ID NO: 68] MATATLLAVLLLLLLVGSAGGA synthetic design [SEQ ID NO: 69] MRARALLVVLVLVVLLGVASSA synthetic design [SEQ ID NO: 70] MPGPGAALLLLLLVLLGLGSAA Synthesis Design [SEQ ID NO: 71] MTTTTVLLLLVLVVLAGLTSGA Synthesis Design [SEQ ID NO: 72] The signal peptide may have at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or 100% sequence identity with the above sequence.

[0112] In some implementations, the signal peptide can be a naturally occurring signal peptide.

[0113] In some implementations, the signal peptide is not a naturally occurring signal peptide.

[0114] In some embodiments, the signal is an engineered signal peptide. For example, in some embodiments, the signal peptide comprises a sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with a naturally occurring signal peptide.

[0115] In some embodiments, the signal peptide is a rationally designed signal peptide, and in some further embodiments, it has less than 75% sequence identity with a naturally occurring signal peptide. For example, the signal peptide may have less than 75% sequence identity with any of SEQ ID NO: 1-9, 36-72.

[0116] As described elsewhere in this document, intracellular tail domains (e.g., C-terminal tails) are heterologous to target-binding domains, but intracellular tail domains (e.g., C-terminal tails) can be autologous to the GAIN domain and / or 7TM domain.

[0117] Various intracellular signaling domains derived from GPCRs (i.e., combinations of one, two, or three ICLs from 7TM and / or the C-terminal tail) are known, and they interact with different G proteins, triggering different intracellular signaling responses. In some embodiments, the C-terminal signaling domain (i.e., a combination of one, two, or three ICLs from 7TM and / or the C-terminal tail) is selected to provide an appropriate or desired intracellular signaling response when the target binds to the target-binding domain.

[0118] For example, when the target-binding domain binds to the target, Intracellular domains containing domains that interact with the Gαq subunit activate β-phospholipase C (PLCβ). Intracellular structures containing domains that interact with the Gαs subunit stimulate the cAMP-dependent pathway by activating adenylate cyclase and increasing intracellular cAMP levels. Intracellular domains containing domains that interact with the Gαi / o subunit inhibit adenylate cyclase and reduce intracellular cAMP. Intracellular domains containing domains that interact with the Gα 12 / 13 subunits activate the RhoA pathway; or Intracellular domains containing domains that interact with the Gα16 subunit activate the PLC-β / PI3K / Akt / MAPK / NF-κB pathway.

[0119] In the context of PLC-β, once PLC-β is activated, it hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 acts as a second messenger to release stored calcium into the cytoplasm, while DAG acts as a second messenger to activate protein kinase C (PKC).

[0120] For example, in some embodiments, the intracellular tail, such as the C-terminal tail: It contains a domain that interacts with the Gαq subunit and activates β-phospholipase C (PLCβ) when aGPCR is activated. It contains a domain that interacts with the Gαs subunit and, when asGPCR is activated, stimulates the cAMP-dependent pathway by activating adenylate cyclase and increasing intracellular cAMP levels. It contains a domain that interacts with the Gαi / o subunit, and when aGPCR is activated, it inhibits adenylate cyclase and reduces intracellular cAMP. It contains Gα 12 / 13 subunits and activates the RhoA pathway when the chimeric aGPCR is activated; and / or It contains a domain that interacts with the Gα16 subunit and activates the PLC-β / PI3K / Akt / MAPK / NF-κB pathway when the chimeric aGPCR is activated.

[0121] In platelets, intracellular calcium plays a crucial role in the regulation of degranulation. Inositol triphosphate (IP3), produced by activating Gα-coupled receptors on the platelet surface, binds to IP3 receptors on the endoplasmic reticulum, leading to the release of Ca2+ into the cytosol. This increase in intracellular Ca2+ concentration activates several downstream effectors, including protein kinase C (PKC) and calmodulin-dependent protein kinase II (CaMKII).

[0122] Both PKC and CaMKII contribute to the regulation of platelet degranulation. PKC phosphorylates many targets involved in granule release, including the cytoskeletal protein myosin light chain and the vesicle transport protein synaptic fusion protein-4. These phosphorylation events promote interactions between the platelet membrane and the granule membrane, leading to granule fusion and release.

[0123] CaMKII also plays a role in platelet degranulation by promoting the fusion of granules with the platelet membrane. CaMKII phosphorylates vesicle transporter synaptic protein, which is involved in the docking and fusion of granules with the platelet membrane.

[0124] Since the intracellular signaling response that triggers platelet degranulation is an increase in Ca2+, in a preferred embodiment, wherein a chimeric aGPCR is used for platelets or engineered platelets, such as those described in WO 2022 / 263824, the intracellular signaling domain is capable of triggering an increase in intracellular Ca2+. 2+ The increased amount of the structural domain leads to the degranulation of platelets or engineered platelets. For example, in these embodiments, the intracellular signaling domain may contain the Gαq subunit.

[0125] For example, in some implementations, the chimeric aGPCR is present in the membrane of platelets or engineered platelets, and once activated, the chimeric aGPCR: a) Causes the platelet or engineered platelet to degranulate; b) Causes the release of contents from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment; In some implementations, one, two, or three ICLs at the 7TM and / or C-terminal tail contain the Gαq subunit.

[0126] In the event of platelet degranulation, activation of the Gs pathway increases cAMP and inhibits platelet activation. In some cases, inhibition of platelet activation is necessary. In some embodiments, the chimeric aGPCR is present in the membrane of platelets or engineered platelets, and once activated, the chimeric aGPCR: a) Prevent the platelet or engineered platelet from degranulating; b) To prevent the contents from being released from the platelet or engineered platelet; c) Prevent the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) Preventing the release of extracellular vesicles from the plasma membrane via bubbling; and / or e) To prevent the shape of the platelet or engineered platelet from changing from a biconcave disc shape to a fully expanded cell fragment.

[0127] In some implementations, one, two, or three ICLs at the 7TM and / or C-terminal tail contain the Gαs subunit.

[0128] It is generally believed that for protein domains to function correctly, there must be no steric hindrance between the functional domains. To achieve this, linkers such as peptide linkers are typically used, which provide a sufficiently short amino acid segment to separate the two domains. In the context of this invention, in some embodiments, it is deemed necessary to include a linker, such as some linker amino acid residues between the GAIN domain and the target-binding domain. In some embodiments, the chimeric aGPCR includes a linker. In some preferred embodiments, the chimeric aGCPR includes a linker between the GAIN domain and the target-binding domain. In some embodiments, the chimeric aGPCR includes a peptide linker between the GAIN domain and the target-binding domain. For example, in a preferred embodiment, when the target-binding domain contains an antibody or its antigen-binding fragment as described herein, the chimeric aGPCR includes a linker between the GAIN domain and the target-binding domain. An exemplary linker includes SPPHTAAHNA [SEQ ID NO:10].

[0129] When the linker is a peptide linker, the linker length can be any number of amino acids. Preferably, the linker length is at least 5 amino acids, for example, at least 6, 7, 8, 9, or at least 10 amino acids.

[0130] As described elsewhere herein, the chimeric aGPCR of the present invention can be considered a universal chimeric aGPCR and can bind to an intermediate adaptor protein or peptide that itself binds to the final target—for example, it can bind to a tag present on the intermediate adaptor protein or peptide.

[0131] Therefore, the present invention also provides a system comprising: The chimeric aGPCR of the present invention; and Intermediate linker polypeptides or proteins, for example, containing a tag (e.g., a peptide tag) and a final target-binding domain (i.e., a binding domain that can bind to a target as described herein). The target-binding domain of the aGPCR can bind to an intermediate linker protein or peptide, such as a tag present on the linker polypeptide or peptide, and the final target-binding domain of the linker polypeptide or protein can simultaneously bind to the desired target—that is, the target to which the chimeric aGPCR is intended. The preferences for all features in this regard are described elsewhere in this document; for example, the preference for the final target is described elsewhere in this document for the target bound by the chimeric aGPCR, such as a target that may be present on a tumor or cancer cell. The preference for the tag is also described elsewhere.

[0132] Clearly, in the absence of simultaneous target binding between the intermediate adaptor protein and the final target, the binding of the intermediate adaptor protein or peptide itself to the target-binding domain of the chimeric aGPCR should not be sufficient to trigger receptor activation and intracellular signal transduction. For example, in the presence of the chimeric aGPCR in the platelet membrane or engineered platelet membrane, the binding of the intermediate adaptor peptide or peptide to the target-binding domain of the chimeric aGPCR is insufficient to activate degranulation of platelets or engineered platelets in the absence of simultaneous binding between the target-binding domain and the intermediate adaptor peptide or peptide.

[0133] On the other hand, the simultaneous binding of the final target-binding domain of the intermediate adaptor protein or peptide to the final target, as well as the binding of the target-binding domain of the chimeric aGPCR to the intermediate adaptor protein or peptide (e.g., binding to a tag on the intermediate adaptor protein or peptide, such as a peptide tag), triggers chimeric aGPCR activation and intracellular signal transduction.

[0134] The present invention also provides one or more polynucleotides encoding any or more of the chimeric aGPCRs described herein. The present invention also provides one or more nucleic acids encoding the intermediate adaptor proteins or peptides described herein.

[0135] In some implementations, the polynucleotide is DNA.

[0136] In some implementations, the polynucleotide is RNA.

[0137] Technicians understand the concept of a promoter and that, in order for transcription to begin from DNA, the promoter must be operatively linked to an appropriate open reading frame. Therefore, in some embodiments, a polynucleotide is operatively linked to the promoter. In some embodiments, the promoter is a heterologous promoter. Nucleic acids may also contain appropriate enhancers.

[0138] Depending on the context in which chimeric aGPCR is to be used, the promoter can be a cell-specific promoter. For example, in some embodiments, the cell-specific promoter is a megakaryocyte-specific promoter, or a pluripotent cell-specific promoter, or a stem cell-specific promoter, optionally an inducible pluripotent stem cell (iPSC) cell-specific promoter, or a T cell-specific promoter, or an NK cell-specific promoter, or a B cell-specific promoter.

[0139] The promoter can be an inducible promoter, such as one that is inducible in a specific cellular environment or in the intended subject. The promoter can also be constitutive, such as one that is constitutive in a specific cellular environment or in the intended subject.

[0140] The present invention also provides vectors comprising the polynucleotides of the invention, such as polynucleotides of the invention operably linked to a promoter that encode a chimeric aGPCR as described herein. The vectors may be plasmids or circular nucleic acids. The vectors may also contain suitable enhancers.

[0141] The present invention also provides viral vectors or viral particles, such as AAV or lentiviruses, comprising the polynucleotides of the present invention or the vectors of the present invention.

[0142] The present invention also provides a cell or framework comprising: a) One or more chimeric aGPCRs of the present invention; b) One or more polynucleotides of the present invention; c) One or more carriers of the present invention; d) One or more viral vectors of the present invention; e) The system of the present invention.

[0143] In a preferred embodiment, the cell or the substrate expresses the chimeric aGPCR of the present invention.

[0144] Those skilled in the art will recognize the term "framework" and understand that it refers to entities such as mammalian or microbial cells, or entities containing genetic or protein compartments, such as liposomes. In the context of this invention, the framework is an entity capable of expressing or carrying one or more chimeric aGPCRs of the invention. In some embodiments, the framework is a cell, platelet, or engineered platelet. In some embodiments, the cell is a T cell, B cell, NK cell, macrophage, or iPSC cell.

[0145] In some implementations, the chassis has been engineered. In some implementations, the chassis is engineered platelets, such as those described in WO 2022 / 263824, which is specifically incorporated herein by reference in its entirety, such as the various chassis described on pages 43-122 of WO 2022 / 263824, the pages of which are specifically incorporated herein by reference. In some implementations, platelets have been engineered: To disrupt thrombosis pathways, and / or engineer to disrupt platelet inflammatory signaling pathways, and / or engineer to reduce the immunogenicity of engineered platelets; and / or To enhance or disrupt one or more fundamental functions of the chassis, optionally wherein said one or more fundamental functions are involved in innate and / or adaptive immune responses, inflammation, angiogenesis, atherosclerosis, lymphoid development, and tumor growth.

[0146] In some implementation schemes, the base frame is: a) Progenitor cell framework, such as myeloid stem cells; iPSCs; adipocytes; adipose-derived mesenchymal matrix / stem cell lines (ASCLs); or cancer cell lines capable of producing a producer framework; or other immortalized cells capable of producing a producer framework; b) Producer framework, such as megakaryocytes; megakaryocytes; megakaryocyte-like cells; cancer cell lines capable of forming platelets, platelet-like membrane-bound cell fragments, or anucleate cell fragments, such as the MEG01 or DAMI cancer cell lines; or other immortalized cells capable of forming platelets, platelet-like membrane-bound cell fragments, or anucleate cell fragments; or c) Effector framework, such as platelets, platelet-like membrane-bound cell debris, or anucleate cell debris.

[0147] Platelets engineered to have reduced procoagulant activity may be referred to as “Synlets”, and as used herein, such as in the embodiments, the term Synlet refers to engineered platelets having a disruption or deletion of genes involved in procoagulant activity, resulting in platelets with reduced procoagulant activity. Such engineered platelets or Synlets are described in WO 2022 / 263824 and are contemplated by this invention.

[0148] In some implementations, the framework has been modified to drive differentiation into a producer framework, for example, to drive differentiation into megakaryocytes or megakaryocyte-like cells, and has been forward programmed to differentiate into megakaryocytes or megakaryocyte-like cells.

[0149] In some implementations, the chassis has been engineered to suppress the expression of the β2 microglobulin gene, for example, in which the β2 microglobulin gene has been knocked out or deleted.

[0150] The frame can be a mammal frame, such as a human frame, a cow frame, a horse frame, or a rat frame.

[0151] In some preferred embodiments, the framework has been engineered to disrupt platelet thrombosis pathways. For example, the framework may have been engineered to have reduced procoagulant properties compared to an unengineered framework with reduced thrombosis potential, for example, wherein the engineered framework does not have thrombosis potential.

[0152] In some cases, the framework has been engineered to disrupt or eliminate at least two, three, four, five, six, seven, eight, nine, or at least ten genes involved in the thrombosis pathway, for example, where these genes are selected from groups encoding the following: Proteins involved in recognizing primary stimuli for thrombus formation; Proteins involved in recognizing secondary mediators of thrombus formation; and / or Proteins involved in the release of secondary mediators of thrombus formation.

[0153] The base frame may include damage or loss of at least the following: A gene encoding a protein involved in recognizing primary stimuli for thrombus formation; A gene encoding a protein involved in recognizing secondary mediators of thrombus formation; and A gene encoding a protein involved in the release of secondary mediators of thrombus formation; For example, damage involving at least the following: Two genes encoding proteins involved in recognizing primary stimuli for thrombus formation; Two genes encoding proteins involved in recognizing secondary mediators of thrombus formation; and Two genes encoding proteins involved in the release of secondary mediators of thrombus formation; For example, damage involving at least the following: Three genes encoding proteins involved in recognizing primary stimuli for thrombus formation; Three genes encoding proteins involved in recognizing secondary mediators of thrombus formation; and Three genes encoding proteins involved in the release of secondary mediators of thrombus formation.

[0154] In some exemplary implementations: The genes encoding at least one, two, or three proteins involved in recognizing primary stimuli of thrombus formation are selected from the group consisting of: GPIb / V / IX and GPVI (GP6), ITGA2B, CLEC2, and integrin a. IIb b3, a2b1, a5b1 and a6b1, or choose the group consisting of GPVI and ITGA2B; The proteins encoding at least one, two, or three proteins involved in recognizing secondary mediators of thrombus formation are selected from the group consisting of: Par1, Par4, P2Y12, GPIb / V / IX, thromboxane receptor (TBXA2R), P2Y1, P2X1, and integrin a. IIb b3 or choose a group consisting of Par1, Par4, and P2Y12; and / or The at least one, two, or three genes encoding proteins involved in the release of secondary mediators of thrombus formation are selected from the group consisting of Cox1, HPS, and thromboxane A synthase (TBXAS1), or from the group consisting of Cox1 and HPS.

[0155] In some implementations of the chassis, each of the following genes is disrupted or missing: ITGA2B, Par1, and HPS; ITGA2B, P2Y12, and HPS; or GPVI, ITGA2B, Par1, Par4, P2Y12, Cox1, and HPS.

[0156] In some implementations, in addition to or besides the genes that have been disrupted as described above, the chassis may contain disruptions to one or more of the following genes: TBXAS1, ITGB1, TMEM16F and / or B2m.

[0157] Disruption is defined to include the complete knockout of protein expression of a gene. The gene itself may be missing, or it may be disrupted by other modifications that reduce or knock out gene expression.

[0158] In some implementation schemes, the base frame: a) No response to endogenous stimuli that typically lead to clot formation; b) Not recruited by other activated platelets; and / or c) During activation, the patient's endogenous platelets cannot be recruited and activated.

[0159] In some implementations, the chassis has been engineered to have reduced immunogenicity compared to non-engineered chassis.

[0160] In some implementation schemes for the chassis: a) The function of endogenous MHC class 1 and / or MHC class 2 has been impaired; and / or b) Expression of the β2 microglobulin gene has been disrupted, for example, it has been knocked out.

[0161] In some implementations, the framework has been engineered to disrupt the expression of one or more HLA genes, for example, to disrupt the expression of any one or more of HLA-A, HLA-B, and / or HLA-C. Optionally, the expression of HLA-A and HLA-B has been completely disrupted, but the expression of HLA-C has been partially disrupted. Optionally, the expression of both alleles of HLA-A and HLA-B has been disrupted, but the expression of only one allele of HLA-C has been disrupted.

[0162] In some implementations, the chassis has been engineered to overexpress any or more of the HLA class Ib genes, optionally any or more of HLA-G, HLA-E, CD47, and PD-L1, and may also be engineered, for example, to suppress the expression of the β2 microglobulin gene.

[0163] In some implementations, the chassis has been engineered to overexpress one or more immunomodulatory genes, optionally wherein the one or more immunomodulatory genes are selected from the group containing CD47 and PD-L1.

[0164] In some implementations, the chassis has been engineered to eliminate one or more genes whose products could negatively impact cargo effectiveness.

[0165] In some implementations, the chassis has been engineered to adjust the inherent / adaptive response upwards or downwards.

[0166] In some implementations, the underframe has been engineered to reduce inflammation, angiogenesis, atherosclerosis, lymphatic development, and tumor growth.

[0167] In some implementations, the underframe has been engineered to disrupt adhesion proteins and / or one or more genes of the cargo entity that may indirectly counteract the biological effects of the engineered cargo.

[0168] In some implementations, the chassis has been engineered to downregulate or suppress the expression of TGFb and / or GARP and / or CD40L.

[0169] In some implementations, the chassis has been engineered to downregulate or suppress CD36, NOD2, SRB1, TLR1, TLR2, TLR3, TLR4, TLR6, TLR9, CD40L, CD93 (C1qRp), C3aR, CD88 (C5aR), CD89 (FcαR1), CD23 (FcεR1), CD32 (FcγRIIa), and MHC. Expression of one or more of the following: Class 1, CD191 (CCR1), CD193 (CCR3), CD194 (CCR4), CD184 (CXCR4), CX3CR1, CD102 (ICAM-2), JAM-C / JAM-3, CD62P (P-selectin), CD31 (PECAM-1), CD150 (SLAMF1), CCL2, CCL3, CCL5, CXCL1, CXCL12, CXCL4 / PF4, CXCL5, CXCL8, NAP2 (CXCL7), and IL-1β.

[0170] In some implementations, the chassis has been engineered to disrupt or suppress the expression of TGFb and / or GARP2.

[0171] In some implementations, the chassis has been engineered to disrupt or inhibit the expression of any or more of Siglec-7, Siglec-9, Siglec-11, and TGFβ.

[0172] In some implementations, the framework has been engineered to express one or more additional ITAM receptors to enhance T cell signaling and stimulate immune responses.

[0173] In some implementations, the chassis has been engineered to have reduced immunogenicity relative to a non-engineered chassis, wherein the chassis has been engineered as follows: a) The function of MHC class 1 genes or proteins has been disrupted; b) The expression of the β2 microglobulin gene has been disrupted, and the β2 microglobulin gene has been optionally knocked out; c) The expression of one or more HLA genes has been disrupted; d) The expression of any one or more of HLA-A, HLA-B and / or HLA-C has been disrupted, optionally the expression of HLA-A and HLA-B has been completely disrupted, but the expression of HLA-C has been partially disrupted, optionally the expression of both alleles of HLA-A and HLA-B has been disrupted, but the expression of only one allele of HLA-C has been disrupted; e) Overexpressing any one or more of the HLA class Ib genes, optionally any or more of HLA-G, HLA-E, CD47 and PD-L1; f) engineered to overexpress any one or more of HLA-G, HLA-E, CD47, and PD-L1, and optionally engineered to have disrupted the expression of said β2 microglobulin; and / or g) Overexpressing one or more immunomodulatory genes, optionally wherein the one or more immunomodulatory genes are selected from the group containing CD47 and PD-L1.

[0174] In some implementation schemes, the underframe has been engineered as follows: a) The function of MHC class 1 genes or proteins has been disrupted; b) The expression of the β2 microglobulin gene has been disrupted, and the β2 microglobulin gene has been optionally knocked out; c) The expression of one or more HLA genes has been disrupted; d) The expression of any one or more of HLA-A, HLA-B and / or HLA-C has been disrupted, optionally the expression of HLA-A and HLA-B has been completely disrupted, but the expression of HLA-C has been partially disrupted, optionally the expression of both alleles of HLA-A and HLA-B has been disrupted, but the expression of only one allele of HLA-C has been disrupted; e) Overexpressing any one or more of the HLA class Ib genes, optionally any or more of HLA-G, HLA-E, CD47 and PD-L1; f) Overexpression of any one or more of HLA-G, HLA-E, CD47, and PD-L1, and optionally engineered to disrupt the expression of the β2 microglobulin gene; and / or g) Overexpressing one or more immunomodulatory genes, optionally wherein the one or more immunomodulatory genes are selected from the group containing CD47 and PD-L1; h) Elimination of one or more genes or gene products that can negatively affect the effectiveness of the goods; i) Upregulate or downregulate innate / adaptive responses; j) Reduce inflammation, angiogenesis, atherosclerosis, lymphatic development, and tumor growth; k) The expression of one or more genes encoding adhesion proteins and / or cargo entities that may indirectly counteract the biological effects of engineered cargo has been disrupted, potentially leading to a greater net therapeutic effect. l) Downregulate or inhibit the expression of TGFb and / or GARP and / or CD40L; n) Downregulation or inhibition of CD36, NOD2, SRB1, TLR1, TLR2, TLR3, TLR4, TLR6, TLR9, CD40L, CD93 (C1qRp), C3aR, CD88 (C5aR), CD89 (FcαR1), CD23 (FcεR1), CD32 (FcγRIIa), MHC Expression of one or more of the following: class I, CD191 (CCR1), CD193 (CCR3), CD194 (CCR4), CD184 (CXCR4), CX3CR1, CD102 (ICAM-2), JAM-C / JAM-3, CD62P (P-selectin), CD31 (PECAM-1), CD150 (SLAMF1), CCL2, CCL3, CCL5, CXCL1, CXCL12, CXCL4 / PF4, CXCL5, CXCL8, NAP2 (CXCL7), and IL-1β. o) Disrupt or inhibit the expression of TGFb and / or GARP; q) Disrupt or inhibit the expression of any or more of Siglec-7, Siglec-9, Siglec-11, or TGFβ s) Disrupt or inhibit the expression of any or more of GPIb / V / IX and GPVI (GP6), ITGA2B, CLEC2, integrin aIIbb3, a2b1, a5b1 and a6b1, GPVI and ITGA2B; t) Disrupt or inhibit the expression of any or more of Par1, Par4, P2Y12, GPIb / V / IX, thromboxane receptor (TBXA2R), P2Y1, P2X1 and integrin aIIbb3 or a group of Par1, Par4 and P2Y12. u) Disrupt or inhibit the expression of any or more of Cox1, Cox2, HPS, prothrombin, PDGF, EGF, von Willebrand factor, and thromboxane A synthase (TBXAS1). v) Synthesize a protein or RNA of interest in response to activation of the platelet or platelet-like membrane-bound cellular debris, optionally wherein the protein or RNA of interest is expressed by the untranslated region of BCL-3 mRNA, optionally the 5'UTR. z) Express one or more cargo proteins or cargo RNAs, optionally said cargo protein or cargo RNA contains an α-particle targeting signal, optionally containing platelet factor 4 (PF4) or von Willebrand factor (vWf). aa) Express at least two chimeric aGPCRs, optionally expressing at least 3, 4, 5, 6, 7, 8, 9 or at least 10 different aGPCRs as described herein; bb) express at least two chimeric aGPCRs, wherein the target-binding domains of the at least two chimeric aGPCRs target different targets; cc) expresses at least two chimeric aGPCRs, which work together to form logic circuits; Dd) expresses at least one chimeric aGPCR of the present invention and at least one chimeric platelet receptor as defined in WO2022263824, namely, a chimeric platelet receptor comprising: a) an intracellular domain which is a platelet-stimulating domain and includes a domain derived from an immune receptor tyrosine-based activation motif (ITAM) receptor; and b) a heterologous targeting domain that recognizes and binds to a target. (ee) expresses one or more goods, optionally said goods are selected from the group consisting of: a) Protein or peptide – optionally, said protein or peptide is: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). vi) A fusion protein comprising an exogenous targeting domain, wherein optionally the fusion protein comprises: a) Cargo proteins or peptides; and b) An exogenous target domain, optionally wherein the exogenous target domain is selected from the group consisting of or including the following: i) Exosome-specific membrane proteins or their exosome membrane-targeting portions, for example: Four-transmembrane proteins, such as CD63; or Non-quadriplete transmembrane proteins, such as PTGFRN or BASP1 ii) Exogenous targeting sequences from soluble proteins, optionally the WW domain of the Nedd4 ubiquitin ligase; iii) ubiquitous tags; and / or iv) Tag-binding domain, optionally targeting a tag, optionally targeting a GFP nanobody; b) Nucleic acid, optionally wherein the nucleic acid is: i) RNA, such as mRNA, miRNA, shRNA, and clustered regularly spaced short palindromic repeats (CRISPR) sequences; and / or ii) RNA containing an exosome-targeting domain, optionally wherein the exosome-targeting domain is selected from the group consisting of or including the following: a) Foreign bodies targeting hairpins or linear motifs; b) Viral foreign bodies that target RNA or foreign bodies that target its fragments; iii) RNA containing an aptamer domain, optionally wherein the aptamer domain is selected from: a) MS2 binding to stem-ring; b) C / D box; and / or c) AU-rich elements, wherein the RNA is optionally mRNA encoding Cas9; ff) expresses a fusion protein, wherein the fusion protein comprises: i) a phage capsid protein MS2 fused to an exogenous membrane protein, optionally wherein the exogenous membrane protein is selected from the group consisting of Lamp2b, VSVG, CD63, or a combination thereof; and / or ii) an archaeal ribosomal protein L7Ae fused to an exogenous body membrane protein, optionally wherein the exogenous body membrane protein is selected from the group consisting of Lamp2b, VSVG, CD63, or combinations thereof; and / or iii) CD9-HuR fusion protein; Optionally, the fusion protein further comprises a photoactivated dimerized protein; gg) expresses one or more goods only when one or more CPRs, a universal CPR, a complex of a universal CPR and a tagged targeting peptide, a SAPR, or an ePAR binds to the target, optionally wherein said goods are selected from the group consisting of: a) Proteins or peptides, optionally: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – In some implementations, the nucleic acid is: i) RNA, such as mRNA, miRNA, shRNA, and clustered regularly spaced short palindromic repeats (CRISPR) sequences. Optionally, the cargo is expressed by the Bcl-3 mRNA untranslated region, optionally by the 5'UTR.

[0175] When the substrate is platelets or engineered platelets, and as described in WO 2022 / 263824, the platelets or engineered platelets can express or load cargo. After the chimeric GCPR binds to the target, the platelets or engineered platelets degranulate, releasing the cargo near the target, thus enabling targeted drug delivery, etc.

[0176] The goods can be any kind of goods, for example: Therapeutic agents; Imaging agent, Non-therapeutic agents; and / or Cosmetic agent.

[0177] Therefore, in some embodiments, the chassis of the present invention includes one or more goods. For example, the chassis has already: a) The cargo is carrying one or more types of goods; and / or b) Engineered to express one or more goods.

[0178] In some implementations, the goods may be selected from one or more of the following: a) Protein or peptide – In some embodiments, the protein or peptide is: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – In some implementations, the nucleic acid is: i) RNA, such as mRNA, miRNA, shRNA, and clustered regularly spaced short palindromic repeats (CRISPR) sequences; or ii) DNA vector; c) Toxins; d) Small molecule drugs, imaging agents, radionucleotide drugs, radionucleotide-labeled antibodies, or any conjugates thereof; e) Viral vectors, such as AAV; f) Viruses, such as oncolytic viruses; g) Agents used for CRISPR-mediated gene editing; h) Foreign bodies, such as foreign bodies preloaded with a second cargo; i) or one or more nanoparticles; And / or j) Lipid nanoparticles (LNPs) containing RNA or mRNA. Or any combination thereof.

[0179] In some cases, goods are goods expressed endogenously.

[0180] For example, the endogenously expressed goods can be any one or more of the following: a) Protein or peptide – In some embodiments, the protein or peptide is: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – In some implementations, the nucleic acid is: i) RNA, such as mRNA, miRNA, shRNA and clustered regularly spaced short palindromic repeats (CRISPR) sequences.

[0181] In other cases, the goods are externally loaded into the rack. For example, externally loaded cargo can be any one or more of the following: a) Protein or peptide – In some embodiments, the protein or peptide is: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid—In some implementations, the nucleic acid is RNA, such as mRNA, miRNA, shRNA and clustered regularly spaced short palindromic repeats (CRISPR) sequences; c) Lipid nanoparticles containing RNA nucleic acids such as mRNA, miRNA, or shRNA.

[0182] Technicians will understand the meaning of the term lipid nanoparticles in the context of RNA delivery. See, for example, Hou et al., 2021 Nature Reviews Materials 6: 1078-1094.

[0183] In some cases, the cargo contains foreign body target domains.

[0184] In some cases, the cargo is a fusion protein containing: a) Cargo proteins or peptides; and b) An exogenous target domain, optionally wherein the exogenous target domain is selected from the group consisting of or including the following: i) Exosome-specific membrane proteins or their exosome membrane-targeting portions, for example: Four-transmembrane proteins, such as CD63; or Non-quadriplete transmembrane proteins, such as PTGFRN or BASP1 ii) Exogenous targeting sequences from soluble proteins, optionally the WW domain of the Nedd4 ubiquitin ligase; iii) ubiquitous tags; and / or iv) a tag-binding domain, optionally targeting a tag, optionally targeting a GFP nanobody; and / or v) Proteins selected from those listed in Table A; The cargo is RNA, and the target domain of the exogenous organism can be: a) Foreign bodies targeting hairpins or linear motifs; b) Viral foreign bodies that target RNA or foreign bodies that target its fragments; c) Fit, optional: i) MS2 binding to stem-ring; ii) C / D box; and / or iii) AU-rich elements, wherein the RNA is optionally mRNA encoding Cas9.

[0185] When the framework has been engineered to express the fusion protein, the fusion protein may contain: a) a phage capsid protein MS2 fused to an exogenous membrane protein, optionally wherein the exogenous membrane protein is selected from any one of or the group consisting of proteins comprising Lamp2b, VSVG, CD63, or Table A; and / or b) A fusion protein comprising an archaeal ribosomal protein L7Ae fused to an exogenous body membrane protein, optionally wherein the exogenous body membrane protein is selected from the group consisting of or comprising any of the following: Lamp2b, VSVG, CD63, or proteins listed in Table A; and / or c) An aptamer-binding protein fused to an exosome membrane protein, optionally wherein the exosome membrane protein is selected from the group consisting of or comprising any of the following: Lamp2b, VSVG, CD63, or proteins in Table A of WO 2022 / 263824, which are specifically incorporated herein by reference.

[0186] Fusion proteins may also include light-activated dimerized proteins.

[0187] In some cases, the chassis contains cargo, which is RNA comprising an exosome-targeting domain as an MS2-binding stem-loop, the chassis having been engineered to express a fusion protein comprising a phage coat protein MS2 fused to an exosome membrane protein, optionally wherein the exosome membrane protein is selected from the group consisting of Lamp2b, VSVG, CD63, or any of the proteins in Table A of WO 2022 / 263824, which is specifically incorporated herein by reference.

[0188] In some cases, the chassis contains cargo, which is RNA containing an exosome-targeting domain as a C / D box, the chassis having been engineered to express a fusion protein, wherein the fusion protein contains an archaeal ribosomal protein L7Ae fused to an exosome membrane protein, optionally wherein the exosome membrane protein is selected from the group consisting of or including any of the following: Lamp2b, VSVG, CD63, or proteins in Table A of WO 2022 / 263824, which is specifically incorporated herein by reference.

[0189] In some cases, the chassis contains cargo, which is RNA containing aptamers, the chassis having been engineered to express a fusion protein, wherein the fusion protein contains a protein or fragment thereof capable of binding to an aptamer fused with an exosome membrane protein, optionally wherein the exosome membrane protein is selected from the group consisting of Lamp2b, VSVG, CD63, or any of the proteins in Table A of WO 2022 / 263824, which is specifically incorporated herein by reference.

[0190] In some embodiments, the chassis contains a cargo, which is RNA containing an exogenous targeting domain as an AU-rich element, and the producer chassis or effector chassis has been engineered to express a fusion protein, wherein the fusion protein is a CD9-HuR fusion protein.

[0191] In some embodiments, the cargo is RNA encoding a Cas protein, optionally a Cas9 protein. In the same or different embodiments, the substrate has been engineered to express one or more sgRNAs.

[0192] Obviously, the present invention also provides for various therapeutic uses of the chimeric aGPCR, nucleic acid, system and chassis, and targeted delivery system described herein.

[0193] The present invention also provides a targeted delivery system comprising a chassis of the present invention, wherein the chassis expresses one or more chimeric aGPCRs of the present invention and comprises cargo, for example, wherein the targeted delivery system is a therapeutic targeted delivery system or a non-therapeutic delivery system. In a preferred embodiment, the chassis will comprise cargo as a therapeutic agent.

[0194] The present invention also provides chimeric aGPCRs, nucleic acids, vectors, systems, complexes or frameworks according to the invention for use in pharmaceuticals.

[0195] The present invention also provides chimeric aGPCRs, nucleic acids, vectors, systems, complexes or chassis according to the invention for: delivering therapeutic or imaging cargo; or treating or preventing cancer, autoimmune diseases, genetic diseases, cardiovascular diseases and / or infections.

[0196] The present invention also provides a method for delivering cargo, the method comprising applying an effective amount of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex or chassis of the present invention, wherein the chassis contains or expresses cargo.

[0197] The present invention also provides a method for targeted delivery of cargo to target cells, tissues or sites in vivo, wherein the method comprises administering an effective amount of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex or chassis of the present invention, such as a chassis of the present invention containing cargo.

[0198] The present invention also provides a non-therapeutic method for delivering goods to subjects in need, the method comprising administering an effective amount of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex, or chassis of the present invention, such as a chassis of the present invention containing non-therapeutic goods.

[0199] The present invention also provides a treatment method comprising administering an effective amount of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex, or chassis of the present invention, for example, wherein the method is used to treat or prevent any or more of cancer, autoimmune diseases, genetic diseases, cardiovascular diseases, and / or infections, for example, a chassis of the present invention comprising therapeutic goods.

[0200] The present invention also provides the use of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex or substrate of the present invention in the preparation of a medicament for the treatment or prevention of diseases or infections, such as for the treatment or prevention of any or more of cancer, autoimmune diseases, genetic diseases, cardiovascular diseases and / or infections, for example wherein the substrate is an effector substrate or an engineered effector substrate.

[0201] The present invention also provides a method for delivering goods (e.g., therapeutic agents) using the chimeric aGPCR, nucleic acid, vector, system, complex, or chassis of the present invention, namely by applying the chimeric aGPCR, nucleic acid, vector, system, complex, or chassis of the present invention.

[0202] This invention also provides various kits and kit portions comprising any one or more of the reagents described herein. For example, this invention provides a kit comprising any two or more of the following: The base frame of this invention; The chimeric aGPCR of the present invention; The nucleic acid of this invention; The carrier of this invention; The system of the present invention; The compound of the present invention.

[0203] The present invention also provides a kit comprising the chimeric aGPCR of the present invention and a corresponding intermediate adaptor protein or peptide, wherein the chimeric aGPCR can bind the intermediate adaptor protein or peptide.

[0204] The present invention also provides the following embodiments: Implementation Plan 1. A chimeric adhesion G protein-coupled receptor (aGPCR), said chimeric aGPCR comprising: a) Intracellular tail domain, optionally C-terminal tail; b) A seven-transmembrane domain (7TM) containing three intracellular loops (ICLs); and c) Extracellular domains, which include: (i) a target-binding domain heterologous to the intracellular tail domain; (ii) The GPCR autologous protein hydrolysis inducible domain (GAIN domain) optionally contains a tethered agonist peptide; And optionally also includes (iii) A connector, optionally wherein the connector is located in the extracellular domain, optionally between the GAIN domain and the target-binding domain.

[0205] 2. The chimeric aGPCR according to embodiment 1, wherein the GAIN domain includes a GPCR-protein hydrolysis site (GPS) optionally located at the N-terminus of the tethering peptide agonist.

[0206] 3. The chimeric aGPCR according to embodiment 2, wherein the GPS site is cleaved by the GAIN domain, optionally constitutively cleaved by the GAIN domain.

[0207] 4. The chimeric aGPCR according to embodiment 2, wherein the GPS site is not cleaved, optionally not cleaved by the GAIN domain.

[0208] 5. The chimeric aGPCR according to embodiment 4, wherein the N-terminal domain further comprises a protease site, optionally comprising a cysteine ​​protease site, a metalloproteinase site, an aspartic protease site, a serine protease site, or a threonine protease site, wherein optionally the protease site is a protease site associated with the tumor microenvironment.

[0209] 6. The chimeric aGPCR according to embodiment 5, wherein the chimeric aGPCR is activated upon cleavage of the protease site and in the absence of the target-binding domain binding to the target.

[0210] 7. The chimeric aGPCR according to embodiment 5, wherein the chimeric aGPCR is activated only when the protease site is cleaved and the target-binding domain binds to the target.

[0211] 8. The chimeric aGPCR according to embodiment 2 or 3, wherein the cleavage of the GPS motif produces an N-terminal fragment (NTF) and a C-terminal fragment (CTF), and wherein the NTF and the CTF are associated with each other by non-covalent interactions, optionally when present in a lipid membrane, optionally in the lipid membrane of a cell, platelet or engineered platelet, the association is maintained by non-covalent interactions.

[0212] 9. The chimeric aGPCR according to embodiment 8, wherein the association of the NTF and the CTF prevents the tethering peptide agonist from interacting with the 7TM domain, thereby preventing activation of intracellular signal transduction.

[0213] 10. The chimeric aGPCR according to any one of embodiments 1 to 9, wherein the chimeric aGPCR: a) It is inactive in the absence of binding between the target-binding domain and the target; c) In the absence of binding between the target-binding domain and the target, it primarily occupies the inactive conformation; and / or c) Remains in a basic active state in the absence of binding between the target-binding domain and the target.

[0214] 11. The chimeric aGPCR according to any one of embodiments 1 to 3 or 5 to 10, wherein the binding of the target-binding domain to the target results in: a) Modulate the non-covalent interaction between the NTF and the CTF and activate the CTF, optionally by causing the tethering peptide agonist to interact with the 7TM and activate intracellular signaling; or b) Disrupt the non-covalent interaction between the NTF and the CTF and activate the CTF, optionally by exposing the tethering peptide agonist to interact with the 7TM and activate intracellular signaling.

[0215] 12. The chimeric aGPCR according to any one of embodiments 1 to 3 or 5 to 11, wherein the binding of the target-binding domain to the target causes a mechanical force to be applied to the NTF / CTF non-covalent interaction.

[0216] 13. The chimeric aGPCR according to any one of embodiments 1 to 12, wherein: The intracellular tail domain, optionally the C-terminal tail, and the 7TM domain are self-organizing; The intracellular tail domain, optionally the C-terminal tail, and the GAIN domain are egodivided; and / or The intracellular tail domain, optionally the C-terminal tail, and the GPS motif are self-contained.

[0217] 14. The chimeric aGPCR according to any one of embodiments 1 to 13, wherein the chimeric aGPCR activates intracellular signal transduction under the following conditions: a) The chimeric aGPCR is localized to the plasma membrane of a substrate, optionally cells, platelets, or engineered platelets; and optionally in the following cases: b) Incubate and agitate the substrate, optionally the cells, the platelets, or the engineered platelets in the presence of the target.

[0218] 15. The chimeric aGPCR according to embodiment 14, wherein the substrate, optionally the cells, the platelets, or the engineered platelets include a reporter system. Optionally, the reporter system comprises a reporter protein expressed when intracellular signaling is activated, optionally the reporter protein is expressed by a promoter comprising an activated T cell nuclear factor (NFAT) response element, optionally the reporter protein is a luciferase.

[0219] 16. The chimeric aGPCR according to embodiment 15, wherein activation of intracellular signal transduction is determined by detecting the expression or activity of the reporter protein, and optionally by detecting the expression or activity of the luciferase.

[0220] 17. The chimeric aGPCR according to embodiment 16, wherein activation of intracellular signal transduction is detected by using a calcium indicator dye, optionally Fluo-4.

[0221] 18. The chimeric aGPCR according to any one of embodiments 14 to 17, wherein the agitation is performed by oscillation at a rate of: At least 10 rpm, 20 rpm, 30 rpm, 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm or at least 200 rpm; Less than 200 rpm, 190 rpm, 180 rpm, 170 rpm, 160 rpm, 150 rpm, 140 rpm, 130 rpm, 120 rpm, 110 rpm, 100 rpm, 90 rpm, 80 rpm, 70 rpm, 60 rpm, 50 rpm, 40 rpm, 30 rpm, 20 rpm or less than 10 rpm; and / or 10rpm to 200rpm, 20rpm to 190rpm, 30rpm to 180rpm, 40rpm to 170rpm, 50rpm to 160rpm, 60rpm to 150rpm, 70rpm to 140rpm, 80rpm to 130rpm.

[0222] 19. A chimeric aGPCR according to any one of embodiments 1 to 18, wherein the intracellular domain comprises one or more substitutions, insertions, or deletions compared to the autologous intracellular domain or the wild-type intracellular domain, optionally wherein Compared to the autologous intracellular domain or a wild-type intracellular domain that does not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions increase the intracellular signaling response; or Compared to the autologous intracellular domain or wild-type intracellular domains that do not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions reduce the intracellular signaling response; And / or Compared to the autologous intracellular domain or wild-type intracellular domains that do not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions alter the specificity of the intracellular signaling response, thereby leading to the activation of altered signal transduction pathways.

[0223] 20. The chimeric aGPCR according to any one of embodiments 1 to 19, wherein the chimeric aGPCR further comprises a signal peptide, optionally selected to achieve a desired level of the chimeric aGPCR signal peptide on the surface of the cell, platelet or engineered platelet.

[0224] 21. The chimeric aGPCR according to the implementation scheme, wherein the signal peptide is an engineered signal peptide.

[0225] 22. The chimeric aGPCR according to embodiment 20 or 21, wherein the signal peptide is selected from the group consisting of or comprised of the following: ADGRG1 signal peptide MTPQSLLQTTLFLLSLLFLVQGAHG [SEQ ID NO: 1] ADGRF5 signal peptide MKSPRRTTLCLMFIVIYSSKA [SEQ ID NO: 2] FCERG signal peptide MIPAVVLLLLLLVEQAAA [SEQ ID NO: 3] CD28 signal peptide MLRLLLALNLFPSIQVTG [SEQ ID NO: 4] Platelet signaling peptide – GPIIb (aIIb) MARALCPLQALWLLEWVLLLLGPCAAPPAWA [SEQ ID NO: 5] Platelet signaling peptide – GPIIIa (β3) MRARPRPRPLWATVLALGALAGVGVG [SEQ ID NO: 6] Platelet signaling peptide-GPIBaMPLLLLLLLLPSPLHP [SEQ ID NO: 7] Platelet signaling peptide - GPIXMPAWGALFLLWATAEA [SEQ ID NO: 8] Platelet signal peptide - GPVMLRGTLLCAVLGLLRA [SEQ ID NO: 9] MAPFASLASGILLLLSLITSSKA [SEQ ID NO: 36] MLLGPGHTLSAPALALAVTLTLLVRSASP [SEQ ID NO: 376] MLLSVPLLLGLLGLAAA [SEQ ID NO: 386] MQELRGILLCLLLAAAVPTTP [SEQ ID NO: 369] MRYVASYLLAALGGNS [SEQ ID NO: 40] MGKSPEAWCIVLFSVLASFSA [SEQ ID NO: 41] MASSGSVQQPRLVLLMLVLAGAARA [SEQ ID NO: 42] MRWKIIQLQYCFLLVPCMLTALEA [SEQ ID NO: 43] MLSRSLLCLALAWVARVGA [SEQ ID NO: 44] MRFSCLALLPGVALLLASARLAAA [SEQ ID NO: 45] MRVLWVLGLCCVLLTFGFVRA [SEQ ID NO: 46] MKFPMVAAALLLLCAVRA [SEQ ID NO: 47] MRSLLLASFCLLAVALA [SEQ ID NO: 48] MKILLLCVGLLLTWDNGMVLG [SEQ ID NO: 49] MLRISGRNMKVLFAAALIVGSVVFLLLPGPSVA [SEQ ID NO: 50] MAATVRRQRPRRLLCWTLVAVLLADLLALS [SE [SEQ ID NO: 51] MKMGVRLAARAWPLCGLLLAALGGVCA [SEQ ID NO: 52] MWWRLWWLLLLLLLLWLALAAAA [SEQ ID NO: 53] MGWSLILLFLVAVATRVLS [SEQ ID NO: 54] MDFQVQIISFLLISASVIMSRG [SEQ ID NO: 55] MEFGLSWVFLVALFRGVQC [SEQ ID NO: 56] MKWVTFISLLFLFSSAYS [SEQ ID NO: 57] MKLPVRLLLVLMMFWIPAASA [SEQ ID NO: 58] MNLLLILTFVAAAVA [SEQ ID NO: 59] MGSAALLLWVLLLWVPSSRA [SEQ ID NO: 60] MTRLTVLALLAGLLASSRA [SEQ ID NO: 61] MWWRLWWLLLLLLLLWPMVWA / AA [SEQ ID NO: 62] MKLPVRLLLVLMFWIPASSS [SEQ ID NO: 63] MDMRVPAQLLGLLLLWLSGARC [SEQ ID NO: 64] MKYLLPTAAAGLLLLAAQPAMA [SEQ ID NO: 65] MGVKVLFALICIAVAEA [SEQ ID NO: 66] MPLLLLLPLLWAGALA [SEQ ID NO: 67] MRARALLAVLLLLLLVGIAAAA [SEQ ID NO: 68] MATATLLAVLLLLLLVGSAGGA [SEQ ID NO: 69] MRARALLVVLVLVVLLGVASSA [SEQ ID NO: 70] MPGPGAALLLLLLLVLLGLGSAA [SEQ ID NO: 71] MTTTTVLLLLLVLVVLAGLTSGA [SEQ ID NO: 72] Or a signal peptide sequence that has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the above sequence.

[0226] 23. The chimeric aGPCR according to any one of embodiments 1 to 22, wherein: a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) A GPCR autoproteolytic inducible domain (GAIN domain), optionally containing a tethered agonist peptide and optionally cleaving the GPS motif; and d) GPS motifs arbitrarily segmented by GAIN domains They are each other's own.

[0227] 24. The chimeric aGPCR according to any one of embodiments 1 to 23, wherein: The GAIN domain and the following heterogeneous sources: 7TM structural domain; The intracellular tail; and / or Target binding domain.

[0228] 25. The chimeric aGPCR according to any one of embodiments 1 to 23, wherein: a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and d) GPS sequence They are derived from the same naturally occurring aGPCR.

[0229] 26. The chimeric aGPCR according to any one of embodiments 1 to 23, wherein: a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and / or d) GPS sequence Derived from the same naturally occurring aGPCR, and wherein one or more of the following are present: a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and / or d) GPS sequence It contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the corresponding domain of a naturally occurring aGPCR.

[0230] 27. The chimeric aGPCR according to any one of embodiments 1 to 26, wherein the GAIN domain is derived from: ADGRG1 [SEQ ID NO: 35]; ADGRL1, and has the amino acid sequence [SEQ ID NO: 27]; ADGRL3, and has the amino acid sequence [SEQ ID NO: 28]; ADGRE2, and having the amino acid sequence of [SEQ ID NO: 29]; ADGRG2, and having the amino acid sequence of [SEQ ID NO: 30]; Or it is a GAIN domain of an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 35, 27, 28, 29, or 30.

[0231] 28. The chimeric aGPCR according to embodiment 27, wherein: The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 35, and: The intracellular tail and / or one, two or three ICLs are not from ADGRG1; The target-binding domain is not derived from ADGRG1; and / or The 7TM structural domain is not from ADGRG1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 27, and: The intracellular tail and / or one, two or three ICLs are not from ADGRL1; The target-binding domain is not derived from ADGRL1; and / or The 7TM domain is not from ADGRL1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 28, and: The intracellular tail and / or one, two or three ICLs are not from ADGRL3; The target-binding domain is not derived from ADDRL3; and / or The 7TM domain is not from ADDRL3; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 29, and: The intracellular tail and / or one, two or three ICLs are not from ADGRE2; The target-binding domain is not derived from ADGRE2; and / or The 7TM domain is not from ADGRE2; or The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 30, and: The intracellular tail and / or one, two or three ICLs are not from ADGRG2; The target-binding domain is not derived from ADGRG2; and / or The 7TM domain is not from ADGRG2.

[0232] 29. The chimeric aGPCR according to any one of embodiments 1 to 28, wherein the chimeric aGPCR comprises the following combination of naturally occurring domains and engineered domains:

[0233]

[0234]

[0235]

[0236] (a) is an intracellular tail domain, such as a C-terminal tail; (b) is a seven-transmembrane domain (7TM); (c) is a GPCR autoproteolytic inducible domain (GAIN domain), which contains a tethered agonist peptide and constitutively cleaves the GPS motif; and (d) is a GPS motif cleaved by the GAIN domain. And the engineered structural domains are: a) A domain containing at least one substituted, inserted, or deleted amino acid sequence relative to a naturally occurring or naturally present domain; b) A domain comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with a naturally occurring domain; and / or c) is a rationally designed structural domain.

[0237] 30. The chimeric aGPCR according to any one of embodiments 1 to 29, wherein the target-binding domain is capable of binding a target, wherein the target: Fixed; and / or Once bound to the chimeric aGPCR, mechanical forces can be applied to the chimeric aGPCR through the relative movement of the target and the chimeric aGPCR (optionally, when the chimeric aGPCR is present in the plasma membrane of a cell, platelet, or engineered platelet).

[0238] 31. The chimeric aGPCR according to any one of embodiments 1 to 30, wherein the target-binding domain is capable of binding a target, wherein the target: They exist on the cell surface; It exists in the physical structure; It exists on the inner wall of blood vessels; Found on organs; It exists on solid tumors; Anchoring the target; When the chimeric aGPCR is present in the plasma membrane of cells, platelets, or engineered platelets, it has a target with relative mobility opposite to that of the chimeric aGPCR; and / or It is fixed on a solid substrate.

[0239] 32. The chimeric aGPCR according to any one of embodiments 1 to 31, wherein the target-binding domain is capable of binding a target, said target being a peptide tag, optionally a peptide tag present on an antibody or a fragment thereof, optionally an scFv, a nanobody, or a Fab.

[0240] 33. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the target-binding domain binds to a target that is endogenous to the subject, optionally wherein the target is a human target.

[0241] 34. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the target is present on the cell surface or tissue surface.

[0242] 35. The chimeric aGCPR according to any one of the foregoing embodiments, wherein the target binding domain comprises a human target binding domain sequence or a sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with a human target binding domain sequence.

[0243] 36. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the target-binding domain comprises a non-human target-binding domain sequence, optionally: Humanized sequence; or Sequence from mice.

[0244] 37. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the target-binding domain comprises a target-binding ligand or a fragment thereof that specifically binds to the target.

[0245] 38. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the target-binding domain comprises: Antibodies or antibody fragments that specifically bind to the target; Variable heavy chain domains and / or variable light chain domains of antibodies; One, two, or three CDRs of the heavy chain and / or one, two, or three CDRs of the light chain; scFV; Nanobodies; Fab; Targeted κ light chain or fragment thereof; Synthesize binding scaffolds, such as monomers, affinity molecules, and design ankyrin repeat sequence proteins or knotting agents; Anti-CD19 scFv domains, such as FMC63 scFv domains, optionally have example SEQ ID NO: 15; Anti-CD276 scFV domain, such as enotozumab scFv domain optionally having SEQ ID NO: 16 or avolimumab optionally having SEQ ID NO: 17; and / or Anti-MAdCAM1 scFV domain, such as the ontalimab scFv domain optionally having SEQ ID NO: 18.

[0246] 39. The chimeric aGPCR according to any one of the preceding paragraphs, wherein the target is a tumor antigen, a neoantigen, or an autoantigen.

[0247] 40. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the target-binding domain binds to a target, the target being: Endogenous targets, such as cancerous tissue or cancer cells, found on tissues or subsets of tissues in the subject, or on cells, or in specific locations within the subject; It exists in the plasma or blood of the subject; It exists only during one or more disease states; for example, in some implementations, the target is a neoantigen that appears in tumor cells. It exists only in significant amounts, such as at abnormal levels in tissues or cells that abnormally express the target, and / or only in a localized manner during one or more disease states; Antigens associated with diseases, symptoms, or conditions, such as tumor neoantigens or tumor-specific antigens; Artificial or exogenous targets; CD19; CD276 IL2 KLK amyloid Notch receptor OLR1 MadCAM1 Cytokine receptors Collagen Non-collagen Drug design; Drugs designed using DREADD; Proteins selected from Table 2 on pages 23-31 of PCT / GB2020 / 053247, the reference of which is incorporated herein by reference; and / or Autoimmune B cells.

[0248] 41. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the target-binding domain comprises an autoimmune-related peptide, optionally: Peptides or portions of any one or more of the following proteins: MOG, GAD65, MAG, PMP22, TPO, VGKC, PLP, AChR, TRIB2, NMDA, GluR, GAD2, ARMC9, CYP21A2, CASR, NSP, insulin, TSHR, thyroid peroxidase, desialyl glycoprotein receptor, CYP2D6, LF, TTG, H / K ATPase, factor XIII, β2-GPI, ITGB2, G-CSF, GP IIb / IIa, COLII, FBGβα, MPO, CYO, PRTN3, TGM, COLVII, COIL, DSG1, DSG3, SOX10, 70SNRNP70, SAG, and a3(IV)NC1 collagen; or Peptides or portions having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with any one or more of the following proteins: MOG, GAD65, MAG, PMP22, TPO, VGKC, PLP, AChR, TRIB2, NMDA, GluR, GAD2, ARMC9, CYP21A2, CASR, NSP, insulin, TSHR, thyroid peroxidase, desialyl glycoprotein receptor, CYP2D6, LF, TTG, H / K ATPase, factor XIII, β2-GPI, ITGB2, G-CSF, GP IIb / IIa, COLII, FBGβα, MPO, CYO, PRTN3, TGM, COLVII, COIL, DSG1, DSG3, SOX10, 70SNRNP70, SAG, and a3(IV)NC1 collagen.

[0249] 42. The chimeric aGPCR according to any one of the foregoing embodiments, wherein when the chimeric aGPCR is present in the membrane of a platelet or engineered platelet, the target-binding domain binds to the target: a) Causes the platelets or engineered platelets to degranulate; b) Causes the contents to be released from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment.

[0250] 43. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the intracellular tail, for example, the C-terminal tail: Includes G αq The subunit interaction domain, and when the chimeric aGPCR is activated, it activates β-phospholipase C (PLCβ). Includes G αs The subunit interaction domain, and when the chimeric aGPCR is activated, it stimulates the cAMP-dependent pathway by activating adenylate cyclase and increasing intracellular cAMP levels; or Includes G αi / o The subunit interaction domain, when cGPCR is activated, inhibits adenylate cyclase and reduces intracellular cAMP; Contains a domain that interacts with the Gα 12 / 13 subunit, and activates the RhoA pathway when the chimeric aGPCR is activated; and / or It contains a domain that interacts with the Gα16 subunit and activates the PLC-β / PI3K / Akt / MAPK / NF-κB pathway when the chimeric aGPCR is activated.

[0251] 44. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the intracellular tail, for example the C-terminal tail, once activated: a) Causes the platelets or engineered platelets to degranulate; b) Causes the contents to be released from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment. Optionally, the intracellular tail and / or one, two or three ICLs contain the Gαq subunit.

[0252] 45. The chimeric aGPCR according to any one of the foregoing embodiments, wherein the intracellular tail, for example the C-terminal tail, once activated: a) Prevent the platelets or engineered platelets from degranulating; b) Prevent the release of contents from the platelets or engineered platelets; c) Prevent the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) Preventing the release of extracellular vesicles from the plasma membrane via bubbling; and / or e) Prevent the shape of the platelet or engineered platelet from changing from a biconcave disc shape to a fully expanded cell fragment.

[0253] Optionally, the intracellular tail and / or one, two or three ICLs contain the Gαs subunit.

[0254] 46. ​​The chimeric aGPCR according to any one of the foregoing embodiments, wherein the chimeric aGPCR includes a linker between the GAIN domain and the target-binding domain, optionally including a peptide linker between the GAIN domain and the target-binding domain.

[0255] 47. The chimeric aGPCR according to embodiment 46, wherein the adapter comprises SPPHTAAHNA [SEQ ID NO: 10].

[0256] 48. A system comprising: Chimeric aGPCR as described in any of the foregoing embodiments; and An intermediate linker protein or peptide, optionally containing a tag, optionally a peptide tag, and a final target-binding domain; The target-binding domain of the chimeric aGPCR is capable of binding the intermediate linker protein or peptide, optionally binding the tag when present on the linker polypeptide or peptide, and the final target-binding domain of the linker polypeptide or protein is capable of binding the final target.

[0257] 49. The system according to embodiment 45, wherein the target is selected from or the group consisting of: Endogenous targets, such as cancerous tissue or cancer cells, found on tissues or subsets of tissues in the subject, or on cells, or in specific locations within the subject; It exists in the plasma or blood of the subject; It exists only during one or more disease states; for example, in some implementations, the target is a neoantigen that appears in tumor cells. It exists only in significant amounts, such as at abnormal levels in tissues or cells that abnormally express the target, and / or only in a localized manner during one or more disease states; Antigens associated with diseases, symptoms, or conditions, such as tumor neoantigens or tumor-specific antigens; Artificial or exogenous targets; CD19; CD276 IL2 KLK amyloid Notch receptor OLR1 MadCAM1 Cytokine receptors Collagen Non-collagen Drug design; Drugs designed using DREADD; Proteins selected from Table 2 on pages 23-31 of PCT / GB2020 / 053247, the reference of which is incorporated herein by reference; and / or Autoimmune B cells.

[0258] 50. The system according to embodiment 48 or 49, wherein, when the chimeric aGPCR is present in the platelet membrane or engineered platelet membrane, the binding of the intermediate linker protein or peptide to the target binding domain of the chimeric aGPCR is insufficient to activate the degranulation of the platelet or engineered platelet in the absence of the target-binding domain of the intermediate linker peptide or protein simultaneously binding to the target.

[0259] 51. The system according to any one of embodiments 48 to 50, wherein, when the chimeric aGPCR is present in the platelet membrane or engineered platelet membrane, the binding of the intermediate linker protein or peptide to the target binding domain of the chimeric aGPCR activates the degranulation of the platelet or engineered platelet when the target binding domain of the intermediate linker peptide or protein is simultaneously bound to the target.

[0260] 52. A complex comprising: Chimeric aGPCR as described in any of the foregoing embodiments; and An intermediate linker protein or peptide, wherein the intermediate linker protein or peptide comprises a final target-binding domain and a tag, optionally a peptide tag. The target-binding domain of the chimeric aGPCR binds to the tag of the intermediate adaptor protein or peptide.

[0261] 53. The complex according to embodiment 53, wherein the final target-binding domain of the intermediate linker polypeptide or protein is capable of binding the final target, and the target-binding domain of the chimeric aGPCR simultaneously binds the tag of the intermediate linker protein or peptide, optionally a peptide tag. 54. A polynucleotide encoding any one or more of the chimeric aGPCRs described in any of the preceding embodiments.

[0262] 55. The polynucleotide according to embodiment 54, wherein the polynucleotide is DNA.

[0263] 56. The polynucleotide according to embodiment 54, wherein the polynucleotide is RNA.

[0264] 57. The polynucleotide according to embodiment 55, wherein the polynucleotide is operatively linked to a promoter, optionally a heterologous promoter.

[0265] 58. The polynucleotide according to any one of embodiments 54 to 57, wherein the polynucleotide further comprises a cell-specific promoter, optionally a megakaryocyte-specific promoter, a pluripotent cell-specific promoter or a stem cell-specific promoter, optionally an induced pluripotent stem cell (iPSC) cell-specific promoter, a T cell-specific promoter, an NK cell-specific promoter or a B cell-specific promoter.

[0266] 59. The polynucleotide according to any one of paragraphs 57 or 58, wherein the promoter is an inducible promoter, optionally an inducible promoter in the intended subject.

[0267] 60. The polynucleotide according to any one of embodiments 57 to 59, wherein the promoter is a constitutive promoter, optionally constitutive in the intended subject.

[0268] 61. A vector comprising a polynucleotide according to any one of embodiments 54 to 61, wherein the vector is optionally a plasmid or a circular nucleic acid.

[0269] 62. A viral vector or viral particle comprising any one of embodiments 54 to 60, or the vector of embodiment 61, wherein optionally the viral vector is AAV or lentivirus.

[0270] 63. A base frame, the base frame comprising: a) One or more chimeric aGPCRs as described in any of the foregoing embodiments; b) One or more polynucleotides as described in any of the foregoing embodiments; c) One or more carriers as described in any of the foregoing implementation schemes; d) One or more viral vectors as described in any of the foregoing implementation schemes; e) The system described in any of the foregoing implementation schemes.

[0271] 64. The chassis according to embodiment 63, wherein the chassis is a cell, platelet or engineered platelet, optionally wherein the cell is a T cell, NK cell, B cell, macrophage or stem cell, optionally iPSC cell.

[0272] 65. The chassis according to embodiment 63 or 64, wherein the chassis expresses the chimeric aGPCR described in any of the preceding embodiments.

[0273] 66. The base frame according to any one of embodiments 63 to 65, wherein the base frame has been engineered: To disrupt thrombosis pathways, and / or engineer to disrupt platelet inflammatory signaling pathways, and / or engineer to reduce the immunogenicity of said engineered platelets; and / or To enhance or disrupt one or more fundamental functions of the chassis, optionally wherein said one or more fundamental functions are involved in innate and / or adaptive immune responses, inflammation, angiogenesis, atherosclerosis, lymphoid development, and tumor growth.

[0274] 67. The base frame according to any one of embodiments 63 to 67, wherein the base frame is: a) A progenitor cell framework, optionally a myeloid stem cell; iPSC; adipocyte; adipose-derived mesenchymal matrix / stem cell line (ASCL); or a cancer cell line capable of producing a producer framework; or other immortalized cells capable of producing a producer framework; b) A producer chassis, optionally a megakaryocyte; a megakaryocyte; a megakaryocyte-like cell; a cancer cell line capable of forming platelets, platelet-like membrane-bound cell fragments, or anucleate cell fragments, such as the MEG01 or DAMI cancer cell line; or other immortalized cells capable of forming platelets, platelet-like membrane-bound cell fragments, or anucleate cell fragments; or c) The effector substrate, optionally consisting of platelets, platelet-like membrane-bound cell fragments, or anucleate cell fragments.

[0275] 68. The chassis according to any one of embodiments 63 to 67, wherein the chassis is a mammal chassis, optionally a human chassis, a cattle chassis, a horse chassis, or a rat chassis.

[0276] 69. The chassis according to any one of embodiments 63 to 68, wherein the chassis has been engineered to disrupt the platelet thrombosis pathway.

[0277] 70. A chassis according to any one of embodiments 63 to 69, wherein the chassis has been engineered to have reduced procoagulant properties relative to a chassis that has not been engineered to have reduced thrombotic potential, optionally wherein the engineered chassis does not have thrombotic potential.

[0278] 71. A chassis according to any one of embodiments 63 to 70, wherein the chassis comprises disruption or deletion of at least two, three, four, five, six, seven, eight, nine, or at least ten genes involved in the thrombosis pathway, optionally wherein said genes are selected from the group of genes encoding: Proteins involved in recognizing primary stimuli for thrombus formation; Proteins involved in recognizing secondary mediators of thrombus formation; and / or Proteins involved in the release of secondary mediators of thrombus formation.

[0279] 72. The base frame according to any one of embodiments 63 to 71, wherein the base frame includes damage or loss of at least the following: A gene encoding a protein involved in recognizing primary stimuli for thrombus formation; A gene encoding a protein involved in recognizing secondary mediators of thrombus formation; and A gene encoding a protein involved in the release of secondary mediators of thrombus formation; Optionally includes at least the following destructions: Two genes encoding proteins involved in recognizing primary stimuli for thrombus formation; Two genes encoding proteins involved in recognizing secondary mediators of thrombus formation; and Two genes encoding proteins involved in the release of secondary mediators of thrombus formation; Optionally includes at least the following destructions: Three genes encoding proteins involved in recognizing primary stimuli for thrombus formation; Three genes encoding proteins involved in recognizing secondary mediators of thrombus formation; and Three genes encoding proteins involved in the release of secondary mediators of thrombus formation.

[0280] 73. The engineered base frame according to any one of embodiments 63 to 72, wherein: The genes encoding at least one, two, or three proteins involved in recognizing primary stimuli of thrombus formation are selected from the group consisting of: GPIb / V / IX and GPVI (GP6), ITGA2B, CLEC2, and integrin a. IIb b3, a2b1, a5b1 and a6b1, or choose the group consisting of GPVI and ITGA2B; The proteins encoding at least one, two, or three proteins involved in recognizing secondary mediators of thrombus formation are selected from the group consisting of: Par1, Par4, P2Y12, GPIb / V / IX, thromboxane receptor (TBXA2R), P2Y1, P2X1, and integrin a. IIb b3 or choose a group consisting of Par1, Par4, and P2Y12; and / or The at least one, two, or three genes encoding proteins involved in the release of secondary mediators of thrombus formation are selected from the group consisting of Cox1, HPS, and thromboxane A synthase (TBXAS1), or from the group consisting of Cox1 and HPS.

[0281] 74. The chassis according to any one of embodiments 63 to 73, wherein each of the following genes is destroyed or deleted: ITGA2B, Par1, and HPS; ITGA2B, P2Y12, and HPS; or GPVI, ITGA2B, Par1, Par4, P2Y12, Cox1, and HPS.

[0282] 75. The chassis according to any one of claims 63 to 74, wherein the chassis comprises the disruption of any one or more of the following genes: TBXAS1, ITGB1, TMEM16F and / or B2m.

[0283] 76. The chassis according to any one of the foregoing embodiments, wherein the chassis has been engineered to have reduced immunogenicity relative to a non-engineered chassis, wherein the chassis has been engineered as follows: a) The function of MHC class 1 genes or proteins has been disrupted; b) The expression of the β2 microglobulin gene has been disrupted, and the β2 microglobulin gene has been optionally knocked out; c) The expression of one or more HLA genes has been disrupted; d) The expression of any one or more of HLA-A, HLA-B and / or HLA-C has been disrupted, optionally the expression of HLA-A and HLA-B has been completely disrupted, but the expression of HLA-C has been partially disrupted, optionally the expression of both alleles of HLA-A and HLA-B has been disrupted, but the expression of only one allele of HLA-C has been disrupted; e) Overexpressing any one or more of the HLA class Ib genes, optionally any or more of HLA-G, HLA-E, CD47 and PD-L1; f) engineered to overexpress any one or more of HLA-G, HLA-E, CD47, and PD-L1, and optionally engineered to have disrupted the expression of said β2 microglobulin; and / or g) Overexpressing one or more immunomodulatory genes, optionally wherein the one or more immunomodulatory genes are selected from the group containing CD47 and PD-L1.

[0284] 77. The chassis according to any one of the foregoing embodiments, wherein the chassis comprises one or more goods, optionally wherein the chassis has: a) The cargo is carrying one or more types of goods; and / or b) Engineered to express one or more goods.

[0285] 78. The chassis according to any one of the foregoing embodiments, wherein the goods are selected from any one or more of the following: a) Protein or peptide – In some embodiments, the protein or peptide is: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – In some implementations, the nucleic acid is: i) RNA, such as mRNA, miRNA, shRNA, and clustered regularly spaced short palindromic repeats (CRISPR) sequences; or ii) DNA vector; c) Toxins; d) Small molecule drugs, imaging agents, radionucleotide drugs, radionucleotide-labeled antibodies, or any conjugates thereof; e) Viral vectors, such as AAV; f) Viruses, such as oncolytic viruses; g) Agents used for CRISPR-mediated gene editing; h) Foreign bodies, such as foreign bodies preloaded with a second cargo; i) one or more nanoparticles; and / or j) Lipid nanoparticles (LNPs) containing RNA or mRNA. or any combination thereof Or any combination thereof.

[0286] 79. The chassis according to any one of the foregoing embodiments, wherein the cargo is endogenously expressed cargo, optionally wherein the endogenously expressed cargo is any one or more of the following: a) Protein or peptide – In some embodiments, the protein or peptide is: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – In some implementations, the nucleic acid is: i) RNA, such as mRNA, miRNA, shRNA and clustered regularly spaced short palindromic repeats (CRISPR) sequences.

[0287] 80. The chassis according to any one of the foregoing embodiments, wherein goods are externally loaded onto the chassis, optionally wherein the externally loaded goods are any one or more of the following: a) Protein or peptide – In some embodiments, the protein or peptide is: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – In some implementations, the nucleic acid is: i) RNA, such as mRNA, miRNA, shRNA and clustered regularly spaced short palindromic repeats (CRISPR) sequences; j) Lipid nanoparticles (LNPs) containing RNA or mRNA. Or any combination thereof.

[0288] 81. The chassis according to any one of the foregoing embodiments, wherein the chassis contains cargo, and wherein the cargo has been exogenously loaded into or onto the chassis, optionally loaded into the cytoplasm, loaded into the plasma membrane, or loaded onto the extracellular surface.

[0289] 82. The chassis according to any one of the preceding paragraphs, wherein the chassis contains cargo, wherein the cargo is RNA containing an exogenous targeting domain as an AU-rich element, and the producer chassis or effector chassis has been engineered to express a fusion protein, wherein the fusion protein is a CD9-HuR fusion protein.

[0290] 83. The chassis according to any one of the preceding paragraphs, wherein the cargo is RNA encoding a Cas protein, optionally a Cas9 protein.

[0291] 84. The chassis according to any one of the preceding paragraphs, wherein the progenitor chassis, producer chassis or effector chassis has been engineered to express one or more sgRNAs.

[0292] 85. The chassis according to any one of the preceding paragraphs, wherein the chassis contains cargo, and the cargo is: Therapeutic agents; Imaging agent, Non-therapeutic agents; and / or Cosmetic agent.

[0293] 86. A targeted delivery system comprising a chassis according to any one of the foregoing embodiments, wherein the chassis expresses one or more chimeric aGPCRs as described in the foregoing embodiments, optionally wherein the targeted delivery system is a therapeutic targeted delivery system or a non-therapeutic delivery system.

[0294] 87. A nonthrombotic targeted delivery system comprising a chassis according to any one of the preceding embodiments, wherein the chassis expresses one or more chimeric aGPCRs according to any one of the preceding embodiments, and wherein the chassis has been engineered to disrupt the thrombotic pathway targeted delivery system.

[0295] 88. The targeted delivery system or non-thrombotic targeted delivery system according to the foregoing embodiments, wherein the system further comprises one or more cargoes, optionally wherein the cargoes comprise one or more target domains, optionally including exogenous target domains.

[0296] 89. The chimeric aGPCR, nucleic acid, vector, system, complex or framework according to any one of the foregoing embodiments, for use in a pharmaceutical.

[0297] 90. A chimeric aGPCR, nucleic acid, vector, system, complex, or chassis according to any one of the foregoing embodiments, used for: delivering therapeutic or imaging cargo; or treating or preventing cancer, autoimmune diseases, genetic diseases, cardiovascular diseases, and / or infections, optionally wherein the chassis is an effector chassis or an engineered effector chassis.

[0298] 91. A method of delivering goods, the method comprising applying an effective amount of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex or chassis described in any of the foregoing embodiments.

[0299] 92. A method for targeted delivery of cargo to target cells, tissues or sites in vivo, wherein the method comprises administering an effective amount of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex or chassis described in any of the preceding embodiments.

[0300] 93. A non-therapeutic method for delivering cargo to a subject in need, the method comprising administering an effective amount of any or more of the chimeric aGPCR, nucleic acid, vector, system, complex, or chassis described in any of the foregoing embodiments, wherein the chassis contains the non-therapeutic cargo. 94. A treatment method comprising administering an effective amount of any one or more of the chimeric aGPCR, nucleic acid, vector, system, complex, or substrate described in any of the preceding embodiments, optionally wherein the method is for the treatment or prevention of any one or more of cancer, autoimmune disease, genetic disease, cardiovascular disease, and / or infection, optionally wherein the substrate is an effector substrate or an engineered effector substrate.

[0301] 95. Use of any one or more of the chimeric aGPCR, nucleic acid, vector, system, complex or substrate according to any of the foregoing embodiments in the preparation of a medicament for the treatment or prevention of a disease or infection, optionally for the treatment or prevention of any one or more of cancer, autoimmune diseases, genetic diseases, cardiovascular diseases and / or infections, optionally wherein the substrate is an effector substrate or an engineered effector substrate.

[0302] 96. A method for delivering goods, optionally therapeutic agents, using a chimeric aGPCR, nucleic acid, vector, system, complex, or chassis as described in any of the foregoing embodiments, said method being carried out by applying a chimeric aGPCR, nucleic acid, vector, system, complex, or chassis as described in any of the foregoing embodiments.

[0303] 97. A kit comprising any two or more of the following: The base frame as described in any of the aforementioned implementation schemes; Chimeric aGPCR as described in any of the aforementioned implementation schemes; Nucleic acid as described in any of the aforementioned implementation schemes; The carrier described in any of the aforementioned implementation schemes; The system described in any of the foregoing implementation schemes; The complex as described in any of the foregoing embodiments.

[0304] 98. A kit comprising a chimeric aGPCR as described in any of the preceding embodiments and a corresponding intermediate adaptor protein or peptide as described in any of the preceding embodiments, wherein the chimeric aGPCR is capable of binding the intermediate adaptor protein or peptide.

[0305] 99. A complex comprising: a) A chassis as described in any of the foregoing embodiments, wherein the chassis expresses one or more chimeric aGPCRs of the present invention; and b) Intermediate linker proteins or peptides. The intermediate linker protein or peptide described herein includes a final target-binding domain and a tag, optionally a peptide tag. Furthermore, the target-binding domain of the chimeric aGPCR of the present invention is a tag-binding domain that can bind to the tag of the intermediate linker protein or peptide, and the final target-binding domain of the intermediate linker polypeptide or protein can bind to both the final target and the chimeric aGPCR of the present invention simultaneously.

[0306] The enumeration or discussion of previously disclosed documents in this specification should not be construed as an admission that such documents are part of the prior art or common general knowledge.

[0307] Unless the context otherwise indicates, preferences and options for a given aspect, feature, or parameter of the invention should be considered as having been disclosed in combination with any and all preferences and options for all other aspects, features, and parameters of the invention. For example, the invention provides: A chimeric aGPCR, the chimeric aGPCR comprising: Extracellular domains, the extracellular domains comprising: A target-binding domain, wherein the target-binding domain is an antigen-binding domain derived from an antibody capable of binding to a cancer neoantigen; and GAIN domains, which have been engineered to increase the force required to disrupt association between GPS and tethered agonists or between NTF and CTF. and The intracellular tail can trigger platelet or engineered platelet degranulation when the target-binding domain binds to the cancer neoantigen and the association between GPS and tethering peptide or between CTF and NTF is disrupted.

[0308] A nonthrombotic platelet or platelet with reduced procoagulability, comprising at least one chimeric aGPCR of the present invention, wherein the aGPCR is capable of triggering degranulation of the nonthrombotic platelet or platelet with reduced procoagulability upon binding of the target-binding domain to the target, and wherein the nonthrombotic platelet or platelet with reduced procoagulability comprises a therapeutic cargo released upon degranulation of the nonthrombotic platelet or platelet with reduced procoagulability.

[0309] Table 1. Sequence information of chimeric aGPCRs

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324] The sequence of the portion of the aGPCR involved in intracellular signal transduction (i.e., the three intracellular loops and the C-terminal tail):

[0325]

[0326] A series of GPS cleavage sites and CTF stem sequences for aGPCR.

[0327] TA refers to tethering agonists.

[0328] Attached Figure Description

[0329] Figure 1 - Design and engineering of chimeric aGPCRs. A) Schematic diagram of aGPCRs and their activation mechanism. A unique feature of the aGPCR family members is the highly conserved GAIN domain ( G PCR since Somatic protein hydrolysis Temptation The process involves the hydrolysis of the ligand-binding domain (GAIN). This leads to nonvalent association between the N-terminal extracellular domain (ECD) and the tethering agonist-7 transmembrane domain / cytoplasmic C-terminal fragment. Upon binding to the target ligand, mechanical forces remove the N-terminal fragment, exposing the tethering agonist peptide to activate the aGPCR. B) The concept of chimeric aGPCR design. To expand the sensor library of synthetic or chimeric aGPCRs, the original ligand-binding domain of the aGPCR is replaced with a different heterologous ligand-binding scaffold (e.g., scFv). Mechanical forces induced by antibody-antigen interactions remove the GAIN domain and expose the tethering agonist peptide to activate the GPCR signaling pathway. C) General strategies for the design and engineering of chimeric aGPCRs. The chimeric aGPCR scaffold consists of: 1. different aGPCR signal peptides for regulating receptor expression levels; 2. different synthetic sensing scaffolds for different targets of interest; 3. adapter sequences crucial for receptor performance; 4. modular aGPCR variants consisting of a GAIN domain, a corresponding tethering agonist, and seven transmembrane and cytoplasmic domains; 5. gene circuit design for signal processing engineering; and 6. N-terminal c-Myc tags and C-terminal HiBIT tags for validating receptor cell surface expression levels and autoproteolytic states of the chimeric aGPCR.

[0330] Figure 2 – Validation of the expression levels of chimeric aGPCR on different cell surfaces.

[0331] A) Expression of chimeric aGPCRs in different cell types. Chimeric aGPCRs were introduced into: 1) the Jurkat NFAT-luciferase system (Promega) transfected with mRNA; 2) MK cells transfected with mRNA or infected with lentivirus; and 3) iPSC cells knocked in via CRISPR and further converted into MK cells and Synlet cells through directed differentiation and plate shaking. B) Analysis of chimeric aGPCR expression levels by flow cytometry and Western blotting. C) Jurkat cell surface expression levels of four different chimeric aGPCRs. Four chimeric aGPCRs were designed based on ADGRG1 and ADGRF5, fused with the FMC63 single-stranded variable fragment (scFv) that recognizes the CD19 antigen (see Supplementary Information for sequence and design details). Their cell surface expression levels were analyzed by anti-FMC63-PE conjugate antibody and flow cytometry.

[0332] Figure 3 – Functional analysis of chimeric aGPCRs in the Jurkat NFAT-luciferase system. A) Functional analysis of FMC63 chimeric aGPCRs in the Jurkat NFAT-luciferase system (Promega). Jurkat cells transfected with IVT mRNA from different chimeric aGPCR constructs were incubated in 96-well streptavidin plates coated with biotinylated antigen CD19 at 37°C with gentle shaking (120 rpm). The N-terminal scFv FMC63-GAIN domain fusion protein was separated from the C-terminal aGPCR fragment by the mechanical force generated by FMC63-CD19 interaction and plate shaking, and then the expression of downstream luciferase reporter genes was further activated through the NFAT signaling pathway. B) Functional analysis results of four different chimeric aGPCR constructs in the Jurkat NFAT-luciferase system.

[0333] Figure 4 - Detection of N-terminal scFv FMC63-GAIN domain fusion protein release. A) Schematic diagram of N-terminal scFv FMC63-GAIN domain fusion protein release. Release of the N-terminal scFv FMC63-GAIN domain fusion protein from the surface of Jurkat cells was analyzed under four different incubation conditions: i. not binding to antigen CD19 and not undergoing plate shaking; ii. not binding to antigen CD19 but undergoing plate shaking; iii. binding to antigen CD19 but not undergoing plate shaking; and iv. binding to antigen CD19 and undergoing plate shaking. B) Flow cytometry analysis of residual scFv FMC63-GAIN domain fusion protein on the surface under the four different incubation conditions. C) Functional analysis of ADGRG1.2 cells under the four different incubation conditions. Analysis was performed using three different cell amounts.

[0334] Figure 5 – The engineered FMC63-ADGRG1.2wt and FMC63-ADGRG1.2_H381S exhibit different responses to ligand binding and plate oscillation.

[0335] The H381S mutation is located at the GPCR proteolytic site (GPS) and inhibits aGPCR autoproteolytic activity. Mechanical forces generated by CD19-FMC63 scFv interactions and plate oscillations should not be able to dissociate the GAIN domain of the mutated ADGRG1.2_H381S from the tethering peptide agonist and should not trigger downstream aGPCR signaling responses. Transfection of 10.5 μg and 15 μg of ADGRG1.2wt and ADGRG1.2_H381S mRNA into Jurkat cells for functional assays. Compared to ADGRG1.2_H381S, ADGRG1.2wt showed a more significant response to ligand binding and plate oscillations.

[0336] Figure 6 - Functional analysis of chimeric aGPCRs in MK cells or synlets. Since activation of chimeric aGPCRs triggers downstream α-granule release via the PLCβ pathway, the function of chimeric aGPCRs in MK cells or synlets can be analyzed through: exposure to P-selectin / CD62p on the cell surface; release of platelet factor 4 (PF4); and release of pre-loaded therapeutic cargo. Other molecules involved in related signaling pathways will also be considered for further engineering.

[0337] Figure 7 – In vitro and in vivo mouse models for chimeric aGPCR functional analysis. A) Establishment of endothelial cell lines (e.g., MS1) with tunable expression levels of cell surface antigens (e.g., CD19 / CD276 / MadCam1). B) Multistage in vitro screening of chimeric aGPCRs. To efficiently select functional constructs, chimeric aGPCR variants will be selected stepwise through the following steps: I.) 2D cultured cell surface binding assay; II.) Transwell therapeutic cargo release assay; and III.) 3D microfluidic co-culture system for mimicking the cancer microenvironment. C) Establishment of in vivo mouse models. MS1 cells expressing the target antigen form hemangiomas in NGS mice and will be used to evaluate the therapeutic efficacy of chimeric aGPCRs (left). Co-injection of human cancer cell lines and MS1 cells expressing or not expressing the target antigen will form xenograft tumor vessels as positive and negative controls for chimeric aGPCR functional analysis (right).

[0338] Figure 8Rewire chimeric aGPCR signaling pathways via gene circuit design. a) Reconnecting downstream aGPCR signaling pathways via chimeric G proteins (left) or modified aGPCR G protein binding motifs. Chimeric G proteins can be engineered with alternating C-terminal GPCR binding sequences. For example, the chimeric G protein Gαq / Gαs is engineered by replacing the original Gαq C-terminal GPCR binding sequence with a sequence from Gαs. The chimeric G protein Gαq / Gαs enables Gαs-dependent aGPCR (instead of the original Gαq-dependent aGPCR) to activate downstream Gαq signaling pathways upon activation. b) Synthetic gene circuit design based on close proximity induced by aGPCR activation. Nter: N-terminus. Cter: C-terminus.

[0339] Figure 9 A series of GPS cleavage sites and CTF stem sequences for aGPCR.

[0340] TA refers to tethering agonists.

[0341] Figure 10 A schematic diagram depicting the location of a GPCR protein with a C-terminal tail and three intracellular loops involved in intracellular signal transduction.

[0342] Figure 11 A – A schematic diagram depicting the extracellular domain and intracellular tail of an embodiment of the chimeric aGPCR of the present invention. B – A schematic diagram depicting the arrangement of intermediate adaptor proteins or peptides, and corresponding tag and final target-binding domains.

[0343] Example Overview aGPCRs sense and respond to environmental stimuli through the mechanical forces generated by the interaction between the target ligand and its own ligand-binding motif (Figure 1a). To expand the aGPCR sensor library, we designed and engineered chimeric aGPCRs by exchanging their native ligand-binding motifs with synthetic (or non-natural) sensing scaffolds (Figure 1b). General strategies for engineering chimeric aGPCRs include: a) fine-tuning receptor expression levels by screening for different aGPCR signal peptides; b) validating receptor cell surface expression levels by adding an N-terminal c-myc tag; c) exchanging native sensing domains for different sensing domains, such as nanobodies, scFvs, or Fabs targeting different targets of interest; d) modulating signal thresholds, signal output intensity, and signal-to-noise ratio (S / N ratio) by modularizing different aGPCR GAIN domains, 7-transmembrane regions, and cytoplasmic domains; e) rewiring downstream signal processing through gene circuit design and engineering; and f) validating autologous protein hydrolysis status using C-terminal HiBiT tags (Figure 1c). Further details are described below.

[0344] Example 1. Expression of chimeric aGPCR on cell surface Based on their intended use and purpose, chimeric aGPCRs were expressed in different cell types using various methods, including: 1) transfecting the nuclei of in vitro transcribed (IVT) mRNA from different chimeric aGPCR constructs into the Jurkat NFAT-luciferase system for high-throughput (HTP) screening of functional chimeric aGPCR variants; 2) infecting iPSCs or directly differentiated human stem cell (hSC) MK cells with lentiviral constructs of selected functional chimeric aGPCR variants for further validation; and 3) knocking selected candidates of functional chimeric aGPCR constructs into iPSC cell lines and further directing their differentiation into MK cells for functional analysis. These MK cells will then be used to generate synlets via plate shaking (Figure 2a). The surface expression level of the chimeric aGPCR will be analyzed using the following materials: 1) a fluorophore-conjugated anti-c-Myc tag antibody to detect the N-terminal c-Myc tag of the chimeric aGPCR by flow cytometry; 2) a fluorophore-conjugated target ligand to detect ligand-sensor scaffold interactions by flow cytometry; and 3) a fluorophore-conjugated anti-sensor scaffold antibody to detect scaffold folding / expression. Furthermore, Western blotting will be used to analyze the autologous proteolytic state of the chimeric aGPCR construct via the C-terminal HiBIT tag (Figure 2b).

[0345] Expression levels of four FMC63 chimeric aGPCRs were analyzed on the surface of Jurkat cells. FMC63 is a CD19-specific IgG1 mouse monoclonal antibody. These chimeric aGPCRs were designed based on ADGRG1 and ADGRF5. Their ligand-binding domains were exchanged with FMC63 scFvs that recognize the CD19 antigen (design and sequence details are shown in Table 1). Cell surface expression levels were analyzed using an anti-FMC63-PE conjugate antibody (Acrobiosystems) and flow cytometry (Figure 2c). All four designed constructs were able to express on the surface of Jurkat cells. The two constructs based on ADGRG1 (ADGRG1.1 and ADGRG1.2) showed significantly higher expression levels than the other two constructs based on ADGRF5 (ADGRF5.1 and ADGRF5.2). The differences between ADGRG1.1 / ADGRG1.2 (with or without additional connectors) or ADGRF5.1 / ADGRF5.2 (with or without the HormR domain) appear to have little or no effect on their surface expression levels.

[0346] >In vitro transcription of mRNA IVT mRNA production was performed according to the instructions of the HiScribe T7 mRNA kit with CleanCap Reagent AG. In short: the T7 promoter, eiF4G aptamer, kozak sequence, and 3'UTR were added to the target gene sequence by PCR amplification. The purified DNA fragment was further transcribed into mRNA in vitro using a CleanCap reaction containing a mixture of pseudoUTPs and other NTPs, reaction buffer, 5'Cap analogue, and T7 RNA polymerase. After removing the DNA template by DNase I, a polyA tail was added using E. coli Poly(A) polymerase.

[0347] >Transfect mRNA nucleus into Jurkat cells After washing with PBS to remove cell culture medium, 1 × 10^6 Jurkat cells were resuspended in 20 μL SE buffer (Lonza). 1 μg–6 μg IVT mRNA in 2 μL of nuclease-free water was added to the Jurkat cells and transferred to a nuclear transfection cuvette. Cells were nuclear transfected using the CM-120 program in a 4D nuclear transfection instrument. After rescue with 100 μL of Jurkat cell culture medium, the cells were further transferred to 2 mL of medium in 6-well plates and incubated overnight at 37°C for further analysis.

[0348] > Surface expression level measurement One × 10^5 Jurkat cells transfected with chimeric aGPCR mRNA were labeled in the dark for 20–30 minutes with either CD19-PE (Bepsys, 1:100) or anti-FMC63 antibody-PE (Bepsys, 1:100). Cells were washed with MACSQuant run buffer to remove unbound CD19-PE or antibody-PE. The precipitated cells were resuspended in MACSQuant run buffer (1:200) containing DAPI and then analyzed by MACSQuant flow cytometry.

[0349] Example 2. Functional assay of chimeric aGPCR in Jurkat NFAT-luciferase system The function of chimeric aGPCRs was tested in the Jurkat NFAT-luciferase system (Promega). Activation of aGPCRs increases cytoplasmic calcium concentration via the phospholipase C (PLC) pathway, which further activates downstream NFAT-luciferase reporter gene expression.

[0350] Jurkat NFAT-luciferase reporter cells nuclear-transfected with IVT mRNA from different FMC63 chimeric aGPCR constructs were incubated in 96-well streptavidin plates coated with biotinylated antigen CD19. The plates were incubated at 37°C and gently agitated to generate mechanical force to activate the chimeric aGPCR and downstream reporter gene expression. Figure 3 a). Functional assays of four FMC63 chimeric aGPCRs showed that two chimeric aGPCRs (ADGRG1.1 and ADGRG1.2) designed based on ADGRG1 were responsive to the target antigen CD19. Figure 3 b). The difference in functional performance between ADGRG1.1 and ADGRG1.2 should be attributed to the presence of an additional adapter sequence between the FMC63 scFv and GAIN domains, highlighting the importance of the adapter sequence for the function of ADGRG1-based chimeric aGPCR designs (see Supplementary Information for sequence details). The functional loss of the FMC63 chimeric aGPCR design based on two ADGRF5s may be due to lower expression levels compared to the ADGRG1-based design. Figure 3 c). Further adjusting the surface expression level of ADGRF5-based chimeric designs through signal peptide engineering and verifying the folding and binding ability of the surface-expressed FMC63 scFv will be the main tasks for improving ADGRF5-based chimeric aGPCR designs.

[0351] Example 3. Effects of agitation and the cutability of GPS sites The effects of release of the N-terminal scFv FMC63-GAIN domain fusion protein and plate oscillation on aGPCR activation were verified using the chimeric aGPCR construct ADGRG1.2. Jurkat NFAT-luciferase reporter cells nuclearly transfected with ADGRG1.2 mRNA were incubated under four different conditions (Fig. 4a). Due to ligand binding and plate oscillation, the surface expression level of the scFv FMC63-GAIN domain was significantly reduced (Fig. 4b). Jurkat NFAT-luciferase reporter cells nuclearly transfected with ADGRG1.2 were activated only under the conditions of ligand binding and plate oscillation (Fig. 4c).

[0352] To verify whether exposure to tethering peptide agonists is necessary for the activation of chimeric aGPCRs, the ADGRG1 autoproteolytic resistant variant H381S was used as a negative control. Figure 5 The significantly lower ligand binding and plate oscillation responses of ADGRG1.2_H381S compared to wild-type ADGRG1.2 suggest that proteolysis and exposure to tethered peptide agonists are important for aGPCR function.

[0353] Functional assay of the Jurkat NFAT-luciferase system One day prior to the Jurkat NFAT-luciferase assay, 96-well white streptavidin plates (Pierce) were coated with 0.4 μg CD19-Fc-Avitag (bio-techne) per well and stored overnight at 4°C. Five × 10^4 Jurkat cells transfected with chimeric aGPCR mRNA were resuspended in Jurkat cell culture medium and added to each well. The plates were incubated at 37°C with gentle shaking (120 rpm) for 6 hours. After incubation, 50 μL of Bio-Glo luciferase assay buffer (Promega) was added to each well. Luciferase signals were further analyzed using a FLUOstar Omega microplate reader.

[0354] Example 4. Functional assay of chimeric aGPCR in MK cells Activation of chimeric aGPCRs will lead to activation of PLCs, which in turn will further promote the release of α-granules of cargo, such as platelet factor 4 (PF4) or pre-loaded therapeutic cargo (e.g., scFv or cytokines), or exposure of platelets to P-selectin / CD62p. Figure 6 Exposure to P-selectin can be assessed by labeling with fluorophore-conjugated anti-P-selectin antibodies and analyzing the results by flow cytometry. Release of PF4 or pre-loaded therapeutic cargo can also be analyzed by ELISA. These assays provide us with a variety of methods to test the functional performance of chimeric aGPCRs in MK cells.

[0355] Example 5. Functional determination of chimeric aGPCR in synlet Synlet was generated overnight by gently agitating MK cell culture plates. The function of the chimeric aGPCR was tested by: 1) determining the exposure of P-selectin on the surface of the Synlet by flow cytometry; 2) releasing PF4 or pre-loaded therapeutic cargo by ELISA.

[0356] Example 6. Expanding the sensor library of chimeric aGPCRs To expand the sensor library for chimeric aGPCRs, further chimeric aGPCR designs will be conducted using various sensor scaffolds, including nanobodies, scFv, and Fab. Different targets of interest (such as CD19, CD276, and MadCAM1) and their corresponding sensor scaffolds will also be incorporated into the chimeric aGPCR designs.

[0357] Example 7. Functional chimeric aGPCR in in vitro screening cell models In vitro screening of functional chimeric aGPCRs established in cell models will be conducted to further evaluate their ability to be activated by target antigens expressed on the cell surface.

[0358] Immortalized mouse endothelial cell models (e.g., the MS1 cell line) have been shown to support in vitro and in vivo targeting of endothelial cell markers. The expression levels of target proteins in endothelial cell models can also be fine-tuned by selecting different promoters or viral MOIs. Figure 7 a).

[0359] Functional chimeric aGPCRs will be selected using three in vitro models. Figure 7 b): a) Select aGPCR design variant that can bind / interact with MS1 target-presenting cells in 2D cell culture plates ( Figure 7 b, left); b) Selected variants will be further analyzed in a transwell assay to test their ability to release functional therapeutic cargo upon interaction with MS1 target-presenting cells ( Figure 7 b, middle); c) The variants selected after the two-stage screening will be applied to 3D microfluidics assays to simulate the environment in blood flow (b, middle); Figure 7 b, right).

[0360] Example 8. Functional assay of chimeric aGPCR in MS1 ​​mouse model .

[0361] MS1 cell lines expressing the target antigen were able to form hemangiomas in NGS mice. These hemangiomas have been shown to be removed by anti-target antigen CAR-T cells. Therefore, using an NGS mouse model of hemangiomas formed by MS1 cells expressing the target antigen was used as a first step in evaluating the therapeutic efficacy of chimeric aGPCR. Figure 7 c, left).

[0362] Furthermore, it has been demonstrated that co-injection of human cancer cell lines and MS1 cells into mice results in the formation of xenograft tumor angiogenesis derived from MS1 cells. Co-injection of human cancer cell lines and MS1 cells expressing or not expressing the target antigen will serve as positive and negative controls to evaluate the therapeutic effect of chimeric aGPCR. Figure 7 c, right).

[0363] Example 9. Rewiring chimeric aGPCR signal transduction via gene circuit design .

[0364] Modularizing and adapting various aGPCRs to the Synlet platform is a key step in optimizing the signal transduction performance of chimeric aGPCRs. To achieve this, a primary task is to rewire various downstream signaling pathways with the Gαq protein pathway, the main pathway for α-granule release from platelets via GPCRs. Strategies such as introducing chimeric G proteins onto GPCRs based on structural information or modifying G protein interaction motifs will be applied in this project. Figure 8 a).

[0365] Implantation of different Boolean logic gates has shown great potential in improving the precision of therapeutic cells. However, the possibility of synthesizing ITAM and designing aGPCR receptor-based logic gates is greatly limited due to the sharing of the same downstream calcium signaling pathway. Therefore, there is an urgent need for orthogonal gene circuit designs for rewiring downstream aGPCR signaling. In this project, we will recruit close proximity induced by aGPCR activation-based gene circuit design. Figure 7 b): The C-terminus of the aGPCR fuses with the V2 tail sequence from arginine vasopressin receptor 2 (AVPR2) and a portion of the downstream cleavage reporter (e.g., the C-terminal fragment of cleavage PLCβ); and the adaptor protein, β-repressor-2 (ARRB2), fuses with another portion of the downstream cleavage reporter (e.g., the N-terminal fragment of cleavage PLCβ). The interaction induced by aGPCR activation between the V2 tail sequence and ARRB2 will bring the two cleavage reporter fragments close together, thereby restoring PLCβ function. Dimeric PLCβ can be further activated via an ITAM-based signaling pathway to release therapeutic cargo in the α-particle and form an AND Boolean logic gate to improve the precision of the synlet.

[0366] The present invention also provides the following embodiments with the following numbers. 1. A chimeric adhesion G protein-coupled receptor (aGPCR), said chimeric aGPCR comprising: a) Intracellular tail domain, optionally C-terminal tail; b) A seven-transmembrane domain (7TM) containing three intracellular loops (ICLs); and c) Extracellular domains, which include: (i) a target-binding domain heterologous to the intracellular tail domain; (ii) The GPCR autologous protein hydrolysis inducible domain (GAIN domain) optionally contains a tethered agonist peptide; And optionally also includes (iii) A connector, optionally wherein the connector is located in the extracellular domain, optionally between the GAIN domain and the target-binding domain.

[0367] 2. The chimeric aGPCR according to embodiment 1, wherein the GAIN domain includes a GPCR-protein hydrolysis site (GPS) optionally located at the N-terminus of the tethering peptide agonist.

[0368] 3. The chimeric aGPCR according to embodiment 2, wherein the GPS site is cleaved by the GAIN domain.

[0369] 4. The chimeric aGPCR according to embodiment 2, wherein the GPS site is not cleaved, optionally not cleaved by the GAIN domain.

[0370] 5. The chimeric aGPCR according to embodiment 4, wherein the extracellular domain further comprises a protease site. Optional to include Protease sites that are cysteine ​​protease sites, metalloproteinase sites, aspartic protease sites, serine protease sites, or threonine protease sites; and / or For protease sites of proteases associated with the tumor microenvironment, Choose one of them i) The chimeric aGPCR is activated upon cleavage of the protease site and in the absence of the target-binding domain binding to the target; or ii) The chimeric aGPCR is activated by cleaving only the protease site and binding the target to the target-binding domain.

[0371] 6. The chimeric aGPCR according to any one of embodiments 1 to 5, wherein: The intracellular tail domain, optionally the C-terminal tail, and the 7TM domain are self-organizing; The intracellular tail domain, optionally the C-terminal tail, and the GAIN domain are egodivided; and / or The intracellular tail domain, optionally the C-terminal tail, and the GPS motif are self-contained.

[0372] 7. The chimeric aGPCR according to any one of embodiments 1 to 6, wherein the chimeric aGPCR activates intracellular signal transduction under the following conditions: a) The chimeric aGPCR is localized to the plasma membrane of a substrate, optionally cells, platelets, or engineered platelets; and when b) Incubate and / or agitate the chassis, optionally the cells, the platelets, or the engineered platelets in the presence of the target. Optionally, the chassis, the cells, the platelets, or the engineered platelets include a reporting system. Optionally, the reporter system comprises a reporter protein expressed when intracellular signaling is activated, optionally the reporter protein is expressed by a promoter comprising an activated T cell nuclear factor (NFAT) response element, optionally the reporter protein is a luciferase.

[0373] 8. A chimeric aGPCR according to any one of embodiments 1 to 7, wherein the intracellular domain comprises one or more substitutions, insertions, or deletions compared to the autologous intracellular domain or the wild-type intracellular domain, optionally wherein Compared to the autologous intracellular domain or a wild-type intracellular domain that does not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions increase the intracellular signaling response; or Compared to the autologous intracellular domain or wild-type intracellular domains that do not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions reduce the intracellular signaling response; And / or Compared to the autologous intracellular domain or wild-type intracellular domains that do not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions alter the specificity of the intracellular signaling response, thereby leading to the activation of altered signal transduction pathways.

[0374] 9. The chimeric aGPCR according to any one of embodiments 1 to 8, wherein the chimeric aGPCR further comprises a signal peptide, optionally selected to achieve a desired level of the chimeric aGPCR signal peptide on the surface of the cell, platelet or engineered platelet.

[0375] 10. The chimeric aGPCR according to any one of embodiments 1 to 9, wherein: i) a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) A GPCR autoproteolytic inducible domain (GAIN domain), optionally containing a tethered agonist peptide and optionally cleaving the GPS motif; and d) GPS motifs arbitrarily segmented by GAIN domains They are each other's own; or ii) The GAIN domain and the following heterogeneous sources: 7TM structural domain; The intracellular tail; and / or The target binding structural domain; or iii) a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and d) GPS sequence Derived from the same naturally occurring aGPCR; or iv) a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and / or d) GPS sequence Derived from the same naturally occurring aGPCR, and wherein one or more of the following are present: a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and / or d) GPS sequence It contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the corresponding domain of a naturally occurring aGPCR.

[0376] 11. The chimeric aGPCR according to any one of embodiments 1 to 10, wherein the GAIN domain is derived from: Derived from ADGRG1 and having the amino acid sequence [SEQ ID NO: 35]; Derived from ADGRL1 and having the amino acid sequence [SEQ ID NO: 27]; It is derived from ADGRL3 and has the amino acid sequence [SEQ ID NO: 28]; It is derived from ADGRE2 and has the amino acid sequence [SEQ ID NO: 29]; Derived from ADGRG2 and having the amino acid sequence [SEQ ID NO: 30]; Or a GAIN domain having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with an amino acid sequence of SEQ ID NO: 35, 27, 28, 29, or 30, optionally wherein: The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 35, and: The intracellular tail is not from ADGRG1; The target-binding domain is not derived from ADGRG1; and / or The 7TM structural domain is not from ADGRG1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 27, and: The intracellular tail is not from ADGRL1; The target-binding domain is not derived from ADGRL1; and / or The 7TM domain is not from ADGRL1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 28, and: The intracellular tail is not from ADGRL3; The target-binding domain is not derived from ADDRL3; and / or The 7TM domain is not from ADDRL3; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 29, and: The intracellular tail is not from ADGRE2; The target-binding domain is not derived from ADGRE2; and / or The 7TM domain is not from ADGRE2; or The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 30, and: The intracellular tail is not from ADGRG2; The target-binding domain is not derived from ADGRG2; and / or The 7TM domain is not from ADGRG2.

[0377] 12. The chimeric aGPCR according to any one of embodiments 1 to 11, wherein the target-binding domain is capable of binding a target, the target being non-covalently bound, covalently bound, or anchored in one of the following ways: Cell surface; The surface of platelets or engineered platelets; Physical structure; Inner wall of blood vessels; organ; Solid tumors; Anchored proteins; When the chimeric aGPCR is present in the plasma membrane of cells, platelets, or engineered platelets, it has a target with relative mobility opposite to that of the chimeric aGPCR; and / or It is fixed on a solid substrate.

[0378] 13. The chimeric aGPCR according to any one of embodiments 1 to 12, wherein the target is present on the cell surface or tissue surface.

[0379] 14. A chimeric aGPCR according to any one of embodiments 1 to 13, wherein the target is a tumor antigen, a neoantigen, or an autoantigen.

[0380] 15. The chimeric aGPCR according to any one of embodiments 1 to 14, wherein the target-binding domain comprises: Antibodies or antibody fragments that specifically bind to the target; Variable heavy chain domains and / or variable light chain domains of antibodies; One, two, or three CDRs of the heavy chain and / or one, two, or three CDRs of the light chain; scFV; Nanobodies; Fab; Targeted κ light chain or fragment thereof; Synthetic binding scaffolds, such as monomers, affinity molecules, designed ankyrin repeat sequence proteins, or knotting agents; anti-CD19scFv domains, such as FMC63 scFv domains, optionally having example SEQ ID NO: 15; Anti-CD276 scFV domain, such as enotozumab scFv domain optionally having SEQ ID NO: 16 or avolimumab optionally having SEQ ID NO: 17; and / or Anti-MAdCAM1 scFV domain, such as ontalitumab scFv domain optionally having SEQ ID NO: 18; Target-binding ligands or fragments thereof that specifically bind to the target.

[0381] 16. The chimeric aGPCR according to any one of embodiments 1 to 15, wherein when the chimeric aGPCR is present in the membrane of a platelet or engineered platelet, the target-binding domain binds to the target: a) Causes the platelets or engineered platelets to degranulate; b) Causes the contents to be released from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment.

[0382] 17. A chimeric aGPCR according to any one of embodiments 1 to 16, wherein the intracellular tail and / or one, two, or three ICLs: Includes G αq The subunit-binding domain, and when the chimeric aGPCR is activated, it activates β-phospholipase C (PLCβ). It contains a domain that binds to the Gαs subunit, and when the chimeric aGPCR is activated, it stimulates the cAMP-dependent pathway by activating adenylate cyclase and increasing intracellular cAMP levels. Includes G αi / o The subunit-binding domain, when cGPCR is activated, inhibits adenylate cyclase and reduces intracellular cAMP; Contains a domain that binds to the Gα 12 / 13 subunit, and activates the RhoA pathway when the chimeric aGPCR is activated; and / or It contains a G domain that binds to the α16 subunit, and when the chimeric aGPCR is activated, it activates the PLC-β / PI3K / Akt / MAPK / NF-κB pathway.

[0383] 18. The chimeric aGPCR according to any one of embodiments 1 to 17, wherein once activated: a) Causes the platelets or engineered platelets to degranulate; b) Causes the contents to be released from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment. Optionally, the intracellular signal transduction domain includes the Gαq subunit.

[0384] 19. The chimeric aGPCR according to any one of embodiments 1 to 17, wherein once activated: a) Prevent the platelets or engineered platelets from degranulating; b) Prevent the release of contents from the platelets or engineered platelets; c) Prevent the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) Preventing the release of extracellular vesicles from the plasma membrane via bubbling; and / or e) Prevent the shape of the platelets or engineered platelets from changing from a biconcave disc shape to a fully expanded cell fragment; Optionally, the intracellular tail and / or one, two or three ICLs contain the Gαs subunit.

[0385] 20. A chimeric aGPCR according to any one of embodiments 1 to 19, wherein the chimeric aGPCR includes a linker between the GAIN domain and the target-binding domain, optionally including a peptide linker between the GAIN domain and the target-binding domain, optionally wherein the linker includes... SPPHTAAHNA [SEQ ID NO: 10].

[0386] 21. A system comprising: Chimeric aGPCR according to any one of embodiments 1 to 20; and An intermediate adaptor protein or peptide containing a final target-binding domain and an optional peptide tag. The target-binding domain of the chimeric aGPCR is capable of binding the intermediate adaptor protein or peptide, optionally binding the tag when present on the adaptor polypeptide or peptide, and the final target-binding domain of the adaptor polypeptide or protein is capable of binding the final target. Optional location: When the chimeric aGPCR is present in the platelet membrane or engineered platelet membrane, and in the absence of simultaneous binding of the final target-binding domain of the intermediate linker peptide or protein to the final target, the binding of the intermediate linker protein or peptide to the target-binding domain of the chimeric aGPCR is insufficient to activate degranulation of the platelet or engineered platelet; and / or When the chimeric aGPCR is present in the platelet membrane or engineered platelet membrane, and when the final target-binding domain of the intermediate linker polypeptide or protein binds to the final target simultaneously, the binding of the intermediate linker protein or peptide to the target-binding domain of the chimeric aGPCR activates the degranulation of the platelet or engineered platelet.

[0387] 22. A polynucleotide encoding any one or more of the chimeric aGPCRs described in any of the preceding embodiments.

[0388] 23. A vector comprising the polynucleotide of embodiment 22 operably linked to a promoter and / or enhancer, optionally wherein the vector is a plasmid.

[0389] 24. A viral vector comprising the polynucleotide of embodiment 22, optionally wherein the viral vector is AAV or lentivirus.

[0390] 25. A cell or platelet or engineered platelet comprising any one or more of the chimeric aGPCRs described in any of the preceding embodiments, wherein the cell is optionally a T cell, B cell, NK cell, erythrocyte, macrophage, megakaryocyte, pluripotent stem cell or stem cell, optionally an induced pluripotent stem cell (iPSC).

[0391] 26. A base frame, the base frame comprising: a) One or more chimeric aGPCRs as described in any of the foregoing embodiments; b) One or more polynucleotides as described in any of the foregoing embodiments; c) The system described in any of the foregoing implementation schemes.

[0392] 27. The chassis according to embodiment 26, wherein the chassis is a cell, platelet or engineered platelet, optionally wherein the cell is a T cell, NK cell, macrophage, B cell, erythrocyte, megakaryocyte, pluripotent stem cell or stem cell, optionally induced pluripotent stem cell (iPSC cell).

[0393] 28. The base frame according to any one of embodiments 26 or 27, wherein the base frame has been engineered: To disrupt the pathway of thrombus formation; To disrupt the platelet inflammatory signal transduction pathway; Engineering to reduce the immunogenicity of the engineered platelets; and / or To enhance or disrupt one or more fundamental functions of the chassis. Optionally, one or more of the aforementioned basic functions are involved in innate and / or adaptive immune responses, inflammation, angiogenesis, atherosclerosis, lymphoid development, and tumor growth.

[0394] 29. A chassis according to any one of embodiments 26 to 28, wherein the chassis has been engineered to have reduced procoagulant properties relative to a chassis that has not been engineered to have reduced thrombotic potential, optionally wherein the engineered chassis has no thrombotic potential.

[0395] 30. The chassis according to any one of the foregoing embodiments, wherein the chassis comprises one or more goods, wherein the chassis has: a) The cargo is carrying one or more types of goods; and / or b) Engineered to enable the endogenous expression of one or more goods. Optionally, the goods mentioned herein are selected from one or more of the following: a) Proteins or peptides – optionally: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – optionally: i) RNA, such as mRNA, miRNA, shRNA, and clustered regularly spaced short palindromic repeats (CRISPR) sequences; or ii) DNA vector; c) Toxins; d) Small molecule drugs, imaging agents, radionucleotide drugs, radionucleotide-labeled antibodies, or any conjugates thereof; e) Viral vectors, such as AAV; f) Viruses, such as oncolytic viruses; g) Agents used for CRISPR-mediated gene editing; h) an exogenous body, such as an exogenous body preloaded with a second cargo; and / or i) or one or more nanoparticles; And / or j) Lipid nanoparticles (LNPs) containing RNA or mRNA.

[0396] Or any combination thereof.

[0397] 31. A complex comprising: a) A chassis as described in any of the preceding embodiments, the chassis expressing one or more chimeric aGPCRs as described in any of the preceding embodiments; and b) Intermediate linker proteins or peptides. The intermediate linker protein or peptide described herein includes a final target-binding domain and a tag, and optionally a peptide tag. Furthermore, the final target-binding domain of the intermediate linker polypeptide or protein can simultaneously bind to the final target.

[0398] 32. A targeted delivery system comprising a chassis according to any one of the foregoing embodiments, wherein the chassis expresses one or more chimeric aGPCRs as described in the foregoing embodiments, and wherein the chassis contains one or more cargoes.

[0399] 33. The framework, system, or complex according to any one of the foregoing embodiments, for use in a pharmaceutical product, Optional for use Treatment or prevention of cancer, autoimmune diseases, genetic diseases, cardiovascular diseases and / or infections; or Used for delivering medical or imaging goods.

[0400] 34. A non-therapeutic method for delivering goods to a subject in need, the method comprising administering an effective amount of a chassis according to any one of the preceding embodiments, wherein the chassis comprises one or more of a chimeric aGPCR according to any one of the preceding embodiments and one or more non-therapeutic goods.

[0401] 35. A kit comprising any two or more of the following: The base frame as described in any of the aforementioned implementation schemes; Chimeric aGPCR as described in any of the aforementioned implementation schemes; Nucleic acid as described in any of the aforementioned implementation schemes; The carrier described in any of the aforementioned implementation schemes; The system described in any of the foregoing implementation schemes; The complex as described in any of the foregoing embodiments.

[0402] 36. A kit comprising a chimeric aGPCR as described in any of the preceding embodiments and a corresponding intermediate adaptor protein or peptide as described in any of the preceding embodiments, wherein the target-binding domain of the chimeric aGPCR is capable of binding the intermediate adaptor protein or peptide.

Claims

1. A chimeric adhesion G protein-coupled receptor (aGPCR), said chimeric aGPCR comprising: a) Intracellular tail domain, optionally C-terminal tail; b) A seven-transmembrane domain (7TM) containing three intracellular loops (ICLs); and c) Extracellular domains, which include: (i) a target-binding domain heterologous to the intracellular tail domain; (ii) The GPCR autologous protein hydrolysis inducible domain (GAIN domain) optionally contains a tethered agonist peptide; and (iii) The junction between the GAIN domain and the target binding domain.

2. The chimeric aGPCR of claim 1, wherein the GAIN domain comprises a GPCR-protein hydrolysis site (GPS) optionally located at the N-terminus of the tethering peptide agonist.

3. The chimeric aGPCR according to claim 2, wherein the GPS site is cleaved by the GAIN domain.

4. The chimeric aGPCR of claim 2, wherein the GPS site is not cleaved, optionally not cleaved by the GAIN domain.

5. The chimeric aGPCR according to claim 4, wherein the extracellular domain further comprises a protease site. Optional to include Protease sites that are cysteine ​​protease sites, metalloproteinase sites, aspartic protease sites, serine protease sites, or threonine protease sites; and / or For protease sites of proteases associated with the tumor microenvironment, Choose one of them i) The chimeric aGPCR is activated upon cleavage of the protease site and in the absence of the target-binding domain binding to the target; or ii) The chimeric aGPCR is activated by cleaving only the protease site and binding the target to the target-binding domain.

6. The chimeric aGPCR according to any one of claims 1 to 5, wherein: The intracellular tail domain, optionally the C-terminal tail, and the 7TM domain are self-organizing; The intracellular tail domain, optionally the C-terminal tail, and the GAIN domain are egodivided; and / or The intracellular tail domain, optionally the C-terminal tail, and the GPS motif are self-contained.

7. The chimeric aGPCR according to any one of claims 1 to 6, wherein the chimeric aGPCR activates intracellular signal transduction under the following conditions: a) The chimeric aGPCR is localized to the substrate, optionally to the plasma membrane of cells, platelets, or engineered platelets; and when b) Incubate and / or agitate the chassis, optionally the cells, the platelets, or the engineered platelets in the presence of the target. Optionally, the chassis, the cells, the platelets, or the engineered platelets include a reporting system. Optionally, the reporter system comprises a reporter protein expressed when intracellular signaling is activated, optionally the reporter protein is expressed by a promoter comprising an activated T cell nuclear factor (NFAT) response element, optionally the reporter protein is a luciferase.

8. The chimeric aGPCR according to any one of claims 1 to 7, wherein the intracellular domain comprises one or more substitutions, insertions, or deletions compared to the autologous intracellular domain or the wild-type intracellular domain, optionally wherein Compared to the autologous intracellular domain or a wild-type intracellular domain that does not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions increase the intracellular signaling response; or Compared to the autologous intracellular domain or wild-type intracellular domains that do not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions reduce the intracellular signaling response; And / or Compared to the autologous intracellular domain or wild-type intracellular domains that do not contain the same one or more substitutions, insertions, or deletions, the one or more substitutions, insertions, or deletions alter the specificity of the intracellular signaling response, thereby leading to the activation of altered signal transduction pathways.

9. The chimeric aGPCR according to any one of claims 1 to 8, wherein the chimeric aGPCR further comprises a signal peptide, optionally selected to achieve a desired level of chimeric aGPCR signal peptide on the surface of the cell, platelet or engineered platelet.

10. The chimeric aGPCR according to any one of claims 1 to 9, wherein: i) a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) A GPCR autoproteolytic inducible domain (GAIN domain), optionally containing a tethered agonist peptide and optionally cleaving the GPS motif; and d) GPS motifs arbitrarily segmented by GAIN domains They are each other's own; or ii) The GAIN domain and the following heterogeneous sources: 7TM structural domain; The intracellular tail; and / or The target binding structural domain; or iii) a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and d) GPS sequence Derived from the same naturally occurring aGPCR; or iv) a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and / or d) GPS sequence Derived from the same naturally occurring aGPCR, and wherein one or more of the following are present: a) Intracellular tail; b) Seven-transmembrane domains (7TM); and c) GPCR autologous protein hydrolysis-inducible domain (GAIN domain); and / or d) GPS sequence It contains an amino acid sequence that has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the corresponding domain of a naturally occurring aGPCR.

11. The chimeric aGPCR according to any one of claims 1 to 10, wherein the GAIN domain: Derived from ADGRG1 and having the amino acid sequence [SEQ ID NO: 35]; Derived from ADGRL1 and having the amino acid sequence [SEQ ID NO: 27]; It is derived from ADGRL3 and has the amino acid sequence [SEQ ID NO: 28]; It is derived from ADGRE2 and has the amino acid sequence [SEQ ID NO: 29]; Derived from ADGRG2 and having the amino acid sequence [SEQ ID NO: 30]; Or a GAIN domain having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with an amino acid sequence of SEQ ID NO: 35, 27, 28, 29, or 30, optionally wherein: The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 35, and: The intracellular tail is not from ADGRG1; The target-binding domain is not derived from ADGRG1; and / or The 7TM structural domain is not from ADGRG1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 27, and: The intracellular tail is not from ADGRL1; The target-binding domain is not derived from ADGRL1; and / or The 7TM domain is not from ADGRL1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 28, and: The intracellular tail is not from ADGRL3; The target-binding domain is not derived from ADDRL3; and / or The 7TM domain is not from ADDRL3; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 29, and: The intracellular tail is not from ADGRE2; The target-binding domain is not derived from ADGRE2; and / or The 7TM domain is not from ADGRE2; or The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NO: 30, and: The intracellular tail is not from ADGRG2; The target-binding domain is not derived from ADGRG2; and / or The 7TM domain is not from ADGRG2.

12. The chimeric aGPCR according to any one of claims 1 to 11, wherein the target-binding domain is capable of binding a target, wherein the target is non-covalently bound, covalently bound, or anchored in one of the following ways: Cell surface; The surface of platelets or engineered platelets; Physical structure; Inner wall of blood vessels; organ; Solid tumors; Anchored proteins; When the chimeric aGPCR is present in the plasma membrane of cells, platelets, or engineered platelets, it has a target with relative mobility opposite to that of the chimeric aGPCR; and / or It is fixed on a solid substrate.

13. The chimeric aGPCR according to any one of claims 1 to 12, wherein the target is present on the cell surface or tissue surface.

14. The chimeric aGPCR according to any one of claims 1 to 13, wherein the target is a tumor antigen, a neoantigen, or an autoantigen.

15. The chimeric aGPCR according to any one of claims 1 to 14, wherein the target-binding domain comprises: Antibodies or antibody fragments that specifically bind to the target; Variable heavy chain domains and / or variable light chain domains of antibodies; One, two, or three CDRs of the heavy chain and / or one, two, or three CDRs of the light chain; scFV; Nanobodies; Fab; Targeted κ light chain or fragment thereof; Synthesize binding scaffolds, such as monomers, affinity molecules, and design ankyrin repeat sequence proteins or knotting agents; Anti-CD19 scFv domains, such as FMC63 scFv domains, optionally have example SEQ ID NO: 15; Anti-CD276 scFV domain, such as enotozumab scFv domain optionally having SEQ ID NO: 16 or avolimumab optionally having SEQ ID NO: 17; and / or Anti-MAdCAM1 scFV domain, such as ontalitumab scFv domain optionally having SEQ ID NO: 18; Target-binding ligands or fragments thereof that specifically bind to the target.

16. The chimeric aGPCR according to any one of claims 1 to 15, wherein when the chimeric aGPCR is present in the membrane of a platelet or engineered platelet, the target-binding domain binds to the target: a) Causes the platelets or engineered platelets to degranulate; b) Causes the contents to be released from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment.

17. The chimeric aGPCR according to any one of claims 1 to 16, wherein the intracellular tail and / or one, two, or three ICLs: Includes G αq The subunit-binding domain, and when the chimeric aGPCR is activated, it activates β-phospholipase C (PLCβ). Includes G αs The subunit-binding domain, and when the chimeric aGPCR is activated, it stimulates the cAMP-dependent pathway by activating adenylate cyclase and increasing intracellular cAMP levels. Includes G αi / o The subunit-binding domain, when cGPCR is activated, inhibits adenylate cyclase and reduces intracellular cAMP; Contains a domain that binds to the Gα 12 / 13 subunit, and activates the RhoA pathway when the chimeric aGPCR is activated; and / or It contains a G domain that binds to the α16 subunit, and when the chimeric aGPCR is activated, it activates the PLC-β / PI3K / Akt / MAPK / NF-κB pathway.

18. The chimeric aGPCR according to any one of claims 1 to 17, wherein once activated: a) Causes the platelets or engineered platelets to degranulate; b) Causes the contents to be released from the platelet or engineered platelet; c) This results in the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) causing extracellular vesicles to be released from the plasma membrane via bubbling; and / or e) This causes the shape of the platelet or engineered platelet to change from a biconcave disc shape to a fully expanded cell fragment. Optionally, the intracellular signal transduction domain includes the Gαq subunit.

19. The chimeric aGPCR according to any one of claims 1 to 17, wherein once activated: a) Prevent the platelets or engineered platelets from degranulating; b) Prevent the release of contents from the platelets or engineered platelets; c) Prevent the presence of platelet contents on the plasma membrane of the platelet or engineered platelet; d) Preventing the release of extracellular vesicles from the plasma membrane via bubbling; and / or e) Prevent the shape of the platelets or engineered platelets from changing from a biconcave disc shape to a fully expanded cell fragment; Optionally, the intracellular tail and / or one, two or three ICLs contain the Gαs subunit.

20. The chimeric aGPCR according to any one of claims 1 to 19, wherein the linker between the GAIN domain and the target-binding domain is a peptide linker between the GAIN domain and the target-binding domain, optionally wherein the linker comprises SPPHTAAHNA [SEQ ID NO: 10].

21. A system comprising: The chimeric aGPCR according to any one of claims 1 to 20; and An intermediate adaptor protein or peptide containing a final target-binding domain and an optional peptide tag. The target-binding domain of the chimeric aGPCR is capable of binding the intermediate adaptor protein or peptide, optionally binding the tag when present on the adaptor polypeptide or peptide, and the final target-binding domain of the adaptor polypeptide or protein is capable of binding the final target. Optional location: When the chimeric aGPCR is present in the platelet membrane or engineered platelet membrane, and in the absence of simultaneous binding of the final target-binding domain of the intermediate linker peptide or protein to the final target, the binding of the intermediate linker protein or peptide to the target-binding domain of the chimeric aGPCR is insufficient to activate degranulation of the platelet or engineered platelet; and / or When the chimeric aGPCR is present in the platelet membrane or engineered platelet membrane, and when the final target-binding domain of the intermediate linker polypeptide or protein binds to the final target simultaneously, the binding of the intermediate linker protein or peptide to the target-binding domain of the chimeric aGPCR activates the degranulation of the platelet or engineered platelet.

22. A polynucleotide encoding any one or more of the chimeric aGPCRs described in any of the preceding claims.

23. A vector comprising the polynucleotide of claim 22 operably linked to a promoter and / or enhancer, optionally wherein the vector is a plasmid.

24. A viral vector comprising the polynucleotide of claim 22, optionally wherein the viral vector is AAV or lentivirus.

25. A cell or platelet or engineered platelet comprising any one or more of the chimeric aGPCRs of any of the preceding claims, wherein the cell is optionally a T cell, B cell, NK cell, erythrocyte, macrophage, megakaryocyte, pluripotent stem cell or stem cell, optionally an induced pluripotent stem cell (iPSC).

26. A base frame, the base frame comprising: a) One or more chimeric aGPCRs as described in any of the preceding claims; b) One or more polynucleotides as described in any of the preceding claims; c) The system according to any one of the preceding claims.

27. The chassis of claim 26, wherein the chassis is a cell, platelet or engineered platelet, optionally wherein the cell is a T cell, NK cell, macrophage, B cell, erythrocyte, megakaryocyte, pluripotent stem cell or stem cell, optionally induced pluripotent stem cell (iPSC cell).

28. The chassis according to any one of claims 26 or 27, wherein the chassis has been engineered: To disrupt the pathway of thrombus formation; To disrupt the platelet inflammatory signal transduction pathway; Engineering to reduce the immunogenicity of the engineered platelets; and / or To enhance or disrupt one or more fundamental functions of the chassis. Optionally, one or more of the aforementioned basic functions are involved in innate and / or adaptive immune responses, inflammation, angiogenesis, atherosclerosis, lymphoid development, and tumor growth.

29. The chassis according to any one of claims 26 to 28, wherein the chassis has been engineered to have reduced procoagulant properties relative to a chassis that has not been engineered to have reduced thrombotic potential, optionally wherein the engineered chassis does not have thrombotic potential.

30. The chassis according to any one of the foregoing embodiments, wherein the chassis comprises one or more goods, wherein the chassis has: a) The cargo is carrying one or more types of goods; and / or b) Engineered to enable the endogenous expression of one or more goods. Optionally, the goods mentioned herein are selected from one or more of the following: a) Proteins or peptides – optionally: i) An antibody or its antigen-binding fragment, for example, an antibody or its antigen-binding fragment binds to a tumor antigen or a neoantigen; ii) Enzymes, such as nucleases, for example, TALEN; iii) Cytokines, such as IL-10; or iv) CRISPR-related proteins, such as Cas9; v) Bispecific proteins, such as bispecific antibodies or optional T-cell conjugates (BiTEs). b) Nucleic acid – optionally: i) RNA, such as mRNA, miRNA, shRNA, and clustered regularly spaced short palindromic repeats (CRISPR) sequences; or ii) DNA vector; c) Toxins; d) Small molecule drugs, imaging agents, radionucleotide drugs, radionucleotide-labeled antibodies, or any conjugates thereof; e) Viral vectors, such as AAV; f) Viruses, such as oncolytic viruses; g) Agents used for CRISPR-mediated gene editing; h) an exogenous body, such as an exogenous body preloaded with a second cargo; and / or i) or one or more nanoparticles; and / or j) Lipid nanoparticles (LNPs) containing RNA or mRNA. Or any combination thereof.

31. A complex comprising: a) A chassis according to any one of the preceding claims, the chassis expressing one or more chimeric aGPCRs according to any one of the preceding claims; and b) Intermediate linker proteins or peptides. The intermediate linker protein or peptide described herein includes a final target-binding domain and a tag, and optionally a peptide tag. Furthermore, the final target-binding domain of the intermediate linker polypeptide or protein can simultaneously bind to the final target.

32. A targeted delivery system comprising a chassis according to any one of the preceding claims, wherein the chassis expresses one or more chimeric aGPCRs as described in the preceding claims, and wherein the chassis contains one or more cargoes.

33. The framework, system, or complex according to any one of the preceding claims, for use in a pharmaceutical medicament. Optional for use Treatment or prevention of cancer, autoimmune diseases, genetic diseases, cardiovascular diseases and / or infections; or Used for delivering medical or imaging goods.

34. A non-therapeutic method for delivering cargo to a subject in need, the method comprising administering an effective amount of a chassis according to any one of the preceding claims, wherein the chassis comprises one or more of a chimeric aGPCR according to any one of the preceding claims and one or more non-therapeutic cargoes.

35. A kit comprising any two or more of the following: The base frame as described in any of the preceding claims; Chimeric aGPCR as described in any of the preceding claims; Nucleic acid as described in any of the preceding claims; The carrier as described in any of the preceding claims; The system according to any one of the preceding claims; The complex of any one of the preceding claims.

36. A kit comprising a chimeric aGPCR as described in any of the preceding claims and a corresponding intermediate adaptor protein or peptide as described in any of the preceding claims, wherein the target-binding domain of the chimeric aGPCR is capable of binding the intermediate adaptor protein or peptide.

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