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KR-20260067386-A - Composition containing a GPCR-based chimeric antigen receptor and method of using the same

KR20260067386AKR 20260067386 AKR20260067386 AKR 20260067386AKR-20260067386-A

Abstract

The present invention relates to a chimeric aGPCR and its uses. By redirecting intracellular signaling responses to a desired target, the chimeric aGPCR can be used in a manner similar to a chimeric antigen receptor. The chimeric aGPCR can also be used with platelets or engineered non-thrombotic platelets for targeted drug delivery.

Inventors

  • 패터슨, 제임스
  • 제스퍼스, 로랑

Assignees

  • 엑세이피 테라피틱스 리미티드

Dates

Publication Date
20260512
Application Date
20240801
Priority Date
20230802

Claims (20)

  1. As a chimeric adhesion G-protein coupled receptor (aGPCR), a) Intracellular tail domain, selectively C-terminal tail; b) 7 transmembrane domains (7TM) containing 3 intracellular loops (ICL); and c) (i) an intracellular tail domain and a heterogeneous target binding domain; (ii) A GPCR autoprotein-inducing domain (GAIN domain) containing a selectively tethered agonist peptide; and (iii) A chimeric aGPCR comprising an extracellular domain containing a linker between a GAIN domain and a target binding domain.
  2. A chimeric aGPCR according to claim 1, wherein the GAIN domain comprises a GPCR-proteolytic site (GPS) site located at the N-terminus of an optionally tethered peptide agonist.
  3. In paragraph 2, the GPS region is a chimeric aGPCR that is cleaved by the GAIN domain.
  4. In paragraph 2, the chimeric aGPCR in which the GPS region is not cleaved and is optionally not cleaved by the GAIN domain.
  5. In paragraph 4, the extracellular domain further comprises a protease site, and Optionally A protease site that is a cysteine protease site, a metalloprotease site, an aspartate protease site, a serine protease site, or a threonine protease site; and/or It includes a protease site that is a protease associated with the tumor microenvironment, and Optionally i) The chimeric aGPCR is activated when the protease site is cleaved while the target binding domain is not bound to the target, or; ii) A chimeric aGPCR that is activated only when cleavage of the protease site and binding between the target binding domain and the target occur.
  6. In any one of paragraphs 1 through 5, Intracellular tail domain, selectively the C-terminal tail is autogenous with respect to the 7TM domain; Intracellular tail domain, optionally the C-terminal tail is autodermal to the GAIN domain; and/or Intracellular tail domain, selectively, C-terminal tail is autogenous to the GPS motif, chimeric aGPCR.
  7. In any one of paragraphs 1 to 6, the chimeric aGPCR a) When the chimeric aGPCR is localized to the plasma membrane of a chassis, selectively a cell, platelet, or engineered platelet; b) A chassis, selectively activating intracellular signaling when cells, platelets, or engineered platelets are incubated and/or stirred in the presence of a target, and Optionally; the chassis, optionally the cell, platelet, or engineered platelet comprises a reporter system, and The reporter system selectively comprises a reporter protein that is expressed when intracellular signal transduction is activated, the reporter protein is selectively expressed from a promoter containing a nuclear factor (NFAT) response element of an activated T cell, and the reporter protein is a chimeric aGPCR that is a luciferase enzyme.
  8. In any one of claims 1 to 7, the intracellular domain comprises one or more substitutions, insertions, or deletions when compared to an autologous intracellular domain or a wild-type intracellular domain, and optionally One or more substitutions, insertions, or deletions increase intracellular signaling responses compared to an autologous intracellular domain or a wild-type intracellular domain that does not contain the same one or more substitutions, insertions, or deletions; One or more substitutions, insertions, or deletions reduce intracellular signaling responses compared to an autologous intracellular domain or a wild-type intracellular domain that does not contain the same one or more substitutions, insertions, or deletions; and/or A chimeric aGPCR in which one or more substitutions, insertions, or deletions alter the specificity of intracellular signaling responses compared to an autogenous intracellular domain or a wild-type intracellular domain that does not contain the same one or more substitutions, insertions, or deletions, thereby resulting in altered signaling pathway activation.
  9. A chimeric aGPCR according to any one of claims 1 to 8, further comprising a signal peptide selected to achieve a desired level of chimeric aGPCR on the surface of a cell, platelet, or engineered platelet.
  10. In any one of paragraphs 1 through 9, i) a) Intracellular tail; b) 7 membrane-penetrating domains (7TM); and c) a GPCR autoprotein-inducing domain (GAIN domain) containing a selectively tethered agonist peptide and selectively cleaving a GPS motif; and d) The GPS motif selectively truncated by the GAIN domain is They are self-derived from each other; or ii) The GAIN domain is 7TM Domain; Intracellular tail; and/or Heterogeneity with respect to the target binding domain; or iii) a) Intracellular tail; b) 7 penetrating domains (7TM); and c) GPCR autoprotein-inducing domain (GAIN domain); and d) The GPS motif is Derived from the same naturally occurring aGPCR; or iv) a) Intracellular tail; b) 7 membrane-penetrating domains (7TM); and c) GPCR autoprotein-inducing domain (GAIN domain); and/or d) The GPS motif is Derived from the same naturally occurring aGPCR, and a) Intracellular tail; b) 7 penetrating domains (7TM); and c) GPCR autoprotein-inducing domain (GAIN domain); and/or d) The GPS motif is A chimeric aGPCR comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with the corresponding domain of a naturally occurring aGPCR.
  11. In any one of paragraphs 1 through 10, the GAIN domain Derived from ADGRG1 and having the amino acid sequence of [SEQ No. 35]; Derived from ADGRL1 and having the amino acid sequence of [SEQ No. 27]; Derived from ADGRL3 and having the amino acid sequence of [SEQ No. 28]; Derived from ADGRE2 and having the amino acid sequence of [SEQ No. 29]; Derived from ADGRG2 and having the amino acid sequence of [Sequence No. 30]; A GAIN domain having an amino acid sequence having at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with SEQ ID NOs 35, 27, 28, 29, or 30, and optionally The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with sequence number 35: The intracellular tail does not originate from ADGRG1; The target binding domain does not originate from ADGRG1; and/or The 7TM domain does not originate from ADGRG1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with sequence number 27: The intracellular tail does not originate from ADGRL1; The target binding domain does not originate from ADGRL1; and/or The 7TM domain does not originate from ADGRL1; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or 100% sequence identity with sequence number 28; The intracellular tail does not originate from ADGRL3; The target binding domain does not originate from ADGRL3; and/or The 7TM domain does not originate from ADGRL3; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98% or 100% sequence identity with sequence number 29; The intracellular tail does not originate from ADGRE2; The target binding domain does not originate from ADGRE2; and/or 7TM domains do not originate from ADGRE2 or; The GAIN domain has at least 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, or 100% sequence identity with sequence number 30; The intracellular tail does not originate from ADGRG2; The target binding domain does not originate from ADGRG2; and/or The 7TM domain is a chimeric aGPCR that does not originate from ADGRG2.
  12. In any one of claims 1 to 11, the target binding domain is a chimeric aGPCR capable of binding to the following: Cell surface; Platelets or engineered platelet surfaces; Physical structure; Inside the blood vessel; long time; Solid tumor; A target non-covalently or covalently bonded to a fixed protein, or a fixed target; Targets that move in the opposite direction to the chimeric aGPCR when the chimeric aGPCR is present on the plasma membrane of cells, platelets, or engineered platelets; and/or Target immobilized on a solid substrate.
  13. In any one of paragraphs 1 to 12, the target is a chimeric aGPCR present on a cell surface or tissue surface.
  14. In any one of paragraphs 1 to 13, the target is a chimeric aGPCR that is a tumor antigen, a neoantigen, or an autoantigen.
  15. In any one of claims 1 to 14, the target binding domain An antibody or antibody fragment that specifically binds to the above target; Variable heavy chain domain and/or variable light chain domain; 1, 2, or 3 CDRs of the heavy chain and/or 1, 2, or 3 CDRs of the heavy chain; scFV; Nanobody; Fab; kappa light chain or its targeted fragment; Synthetic binding scaffold, e.g., monobody, apibody, DARPin or notin; anti-CD19 scFv domain, e.g., FMC63 scFv domain, optionally e.g., SEQ ID NO. 15; Anti-CD276 scFv domain, e.g., enoblituzumab scFv domain, optionally SEQ ID NO. 16 or ombrutamab, optionally SEQ ID NO. 17; and/or Anti-MAdCAM1 scFv domain, e.g., ontamalimab scFv domain, optionally SEQ ID NO. 18; A chimeric aGPCR comprising a target-binding ligand or a fragment thereof that specifically binds to the above target.
  16. In any one of claims 1 to 15, where the chimeric aGPCR is present on the membrane of a platelet or a engineered platelet, if the target binding domain binds to the target a) causing degranulation of platelets or manipulated platelets; b) causing the release of contents from platelets or manipulated platelets; c) resulting in the presence of intraplatelet contents on the plasma membrane of a platelet or a manipulated platelet; d) induce the release of extracellular vesicles through vesicles from the plasma membrane; and/or e) Chimeric aGPCR in which the shape of the platelet or engineered platelet changes from a biconcave disk to a fully unfolded cell fragment.
  17. In any one of claims 1 to 16, the intracellular tail and/or one, two, or three ICLs It includes a domain that binds to the G αq subunit, and when the chimeric aGPCR is activated, it activates beta-type phospholipase C (PLCβ); It includes a domain that binds to the Gαs subunit, and when the chimeric aGPCR is activated, it activates adenylyl cyclase and stimulates a cAMP-dependent pathway by increasing intracellular cAMP levels; It includes a domain that binds to the G αi/o subunit, and when the cGPCR is activated, it inhibits adenylyl cyclase and reduces intracellular cAMP levels; It contains a domain that binds to the Gα 12/13 subunit, and when the chimeric aGPCR is activated, it activates the RhoA pathway; and/or A chimeric aGPCR containing a domain that binds to the α16 subunit, which activates the PLC-β/PI3K/Akt/MAPK/NF-κB pathway when the chimeric aGPCR is activated.
  18. In any one of paragraphs 1 to 17, when the chimeric aGPCR is activated a) causing degranulation of platelets or manipulated platelets; b) causing the release of contents from platelets or manipulated platelets; c) resulting in the presence of intraplatelet contents on the plasma membrane of a platelet or a manipulated platelet; d) induce the release of extracellular vesicles through vesicles from the plasma membrane; and/or e) The shape of the platelet or manipulated platelet is changed from a biconcave disc to a fully unfolded cell fragment, and A chimeric aGPCR in which the intracellular signaling domain selectively contains a Gαq subunit.
  19. In any one of paragraphs 1 to 17, when the chimeric aGPCR is activated a) prevent degranulation of platelets or manipulated platelets; b) prevent release of contents from platelets or manipulated platelets; c) prevent the presence of platelet contents on the plasma membrane of platelets or manipulated platelets; d) prevent the release of extracellular vesicles through vesicles from the plasma membrane; and/or e) prevent the shape of the platelet or manipulated platelet from changing from a biconcave disc to a fully unfolded cell fragment; Optionally, an intracellular tail and/or one, two, or three ICLs are chimeric aGPCRs containing gas subunits.
  20. In any one of claims 1 to 19, the linker between the GAIN domain and the target binding domain is a peptide linker between the GAIN domain and the target binding domain, and optionally the linker comprises SPPHTAAHNA [SEQ ID NO. 10], a chimeric aGPCR.

Description

Composition containing a GPCR-based chimeric antigen receptor and method of use The present invention relates to the field of treatment, particularly the field of targeted drug delivery. A chimeric antigen receptor (CAR) is a synthetic receptor that combines the high affinity binding ability of antibodies with the internal signaling ITAM domain of T cell receptors. It has the ability to redirect the specificity of immune cells, particularly T cells, to targets of interest found on cancer cells. CARs are designed to enhance the immune system's ability to recognize and eliminate cancer cells. The ITAM domain is a short sequence found in the cytoplasmic tails of various immune receptors, including T cell receptors and B cell receptors. In CARs, the ITAM domain is incorporated into an intracellular signaling domain to initiate T cell activation upon antigen recognition. When a CAR binds to a target antigen on a cancer cell, the ITAM domain is phosphorylated, triggering a series of signaling events and activating the T cell. This activation ultimately leads to cancer cell death by T cells. By incorporating the ITAM domain into CARs, it is possible to 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, which is the general antigen recognition mode of T cells. This feature enables CAR T cell therapy to be applied to various types of cancer. CAR T therapy has achieved remarkable success in the treatment of certain blood malignancies, such as acute lymphoblastic leukemia and certain types of lymphoma. Ongoing research is focused on improving the efficacy and safety of CAR T cell therapy and expanding its application to solid tumors. Another example of a chimeric receptor containing an ITAM signaling domain is described in WO 2022/263824, which presents various chimeric ITAM-based receptors that can be used to redirect the natural degranulation response of platelets. Degranulation generally occurs as a reaction in which a receptor binds to a marker of physical damage, such as collagen, inducing thrombus formation. WO 2022/263824 describes a method of diverting this reaction using a chimeric platelet receptor to deliver a therapeutic cargo to a target of interest, for example, an anticancer drug to a tumor. WO 2022/263824 also describes a method in which platelets are engineered to express a cargo of interest within precursor megakaryocytes, or in which an exogenous cargo can be directly loaded onto precursor megakaryocytes or platelets. WO 2022/263824 also describes platelets engineered to eliminate or reduce thrombogenicity, that is, to prevent platelets from degranulating in response to natural signals found in a subject, for example, a patient requiring treatment. In this manner, WO 2022/263824 describes a complete system that may include non-thrombotic platelets (or Synlets) and a complete system that also includes a chimeric receptor, which can be used to deliver various types of cargo to a target site. However, there are potential drawbacks to the ITAM-based approach of these systems. ITAM receptors provide on-off responses with little nuance in the response that can be obtained compared to the intensity of the target signal. Furthermore, ITAM receptors tend to initiate signaling through the activation of a single kinase, such as Syk or Zap70, resulting in little diversity in the downstream effects that can be achieved by target binding. Finally, for example, receptor overexpression can cause receptor aggregation at the cell surface, leading to stochastic ITAM signaling even in the absence of target binding, which results in reduced sensitivity of signaling, i.e., a high background signaling rate. The present invention provides a receptor that solves these problems by utilizing a known chimeric receptor. G protein-coupled receptors (GPCRs) are a large family of transmembrane receptors that play an important role in intracellular signaling pathways. When GPCRs are activated by their ligands (e.g., neurotransmitters, hormones, or other signaling molecules), they undergo a conformational change, leading to the activation of intracellular signaling pathways. When a GPCR is activated, the G protein undergoes a conformational change (i.e., from an inactive conformation to an active conformation) to exchange GDP instead of GTP, which activates downstream effectors. Therefore, as herein, "activation of aGCCR" implies the initiation of downstream signaling through the corresponding G protein(s). Adhesive GPCRs (aGPCRs) are a specific class of GPCRs that, in addition to the standard seven transmembrane domains (7TMs) common to all GPCRs, possess an extracellular domain ( GPCRA utoproteolysis -inducing (GAIN) domain) that induces constitutive autoproteolytic cleavage in a region called the GPS site, which is located near the first transmembrane helix in most cases. The GAIN do