WO2025072425A2 - Chimères ciblant les sheddases pour la modulation de la protéostase - Google Patents

Chimères ciblant les sheddases pour la modulation de la protéostase Download PDF

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WO2025072425A2
WO2025072425A2 PCT/US2024/048537 US2024048537W WO2025072425A2 WO 2025072425 A2 WO2025072425 A2 WO 2025072425A2 US 2024048537 W US2024048537 W US 2024048537W WO 2025072425 A2 WO2025072425 A2 WO 2025072425A2
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protease
targeting domain
fusion molecule
protein
substrate
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WO2025072425A3 (fr
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Brett Garabedian
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Scripps Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody

Definitions

  • proteases that are delivered to the cell surface where they proteolyze broad collections of membrane proteins, as described in Pedram et al., bioRxiv, 2022.05.20.492748 (2022), Romei et al., J. Biolo 299(5), 104685 (2023), and in US20220363778A1. Notably, all these methods are limited by the need for ubiquitylation, lysosomal trafficking, or the requirement for delivery of exogeneous proteases.
  • the invention provides fusion molecules that contain a protease-targeting domain and a covalently linked substrate-targeting domain.
  • fusion molecules also termed SHEDTACs (Sheddase-Targeting Chimeras) herein
  • the protease-targeting domain can specifically bind to an integral membrane protease or to an auxiliary membrane protein in a multimeric integral membrane protease complex
  • the substrate-targeting domain can specifically bind to a transmembrane substrate protein (also termed herein a target integral membrane protein) that is bound to the same phospholipid membrane
  • the membrane bound protease can cleave the extracellular domain of the target transmembrane substrate protein.
  • the integral membrane protease to be targeted is a sheddase.
  • the sheddase to be targeted is a member of the A Disintegrin And Metalloprotease (ADAM) family of proteases, e.g., ADAM 10.
  • ADAM A Disintegrin And Metalloprotease
  • Some of the fusion molecules of the invention are intended to target a transmembrane substrate protein that is a cellular signaling receptor or a receptor ligand.
  • Some transmembrane substrate proteins targeted by the fusion molecules of the invention are tumor-associated antigens.
  • the protease-targeting domain and the substrate-targeting domain are each an antibody or an antigen-binding fragment.
  • the protease-targeting domain and the substrate-targeting domain are each a single-domain molecule or a single chain variable fragment (scFv).
  • the protease-targeting domain and the substrate-targeting domain in some SHEDTACs of the invention are each a single domain antibody (sdAb).
  • the protease-targeting domain and the substrate-targeting domain are linked via a linker motif.
  • some SHEDTACs of the invention contain a protease-targeting domain that recognizes ADAMI 0 and a substratetargeting domain that recognizes Lymphocyte Activation Gene 3 (LAG3, CD233) or programmed cell death protein 1 (PD-1; CD279).
  • the protease-targeting domain contains the amino acid sequence shown in any one of SEQ ID NOs: 12-16
  • the substrate-targeting domain contains the amino acid sequence shown in any one of SEQ ID NOs: 17-21.
  • some SHEDTACS of the invention can additionally contain a covalently or non-covalently linked carrier moiety.
  • the carrier moiety extends serum half-life in vivo and is a carrier protein, a Fc domain, or a PEG molecule that is covalently linked to the protease-targeting domain or the substrate-targeting domain.
  • the invention provides polynucleotides that encode the novel fusion molecules described herein.
  • the invention provides vectors that contain one of such polynucleotides, as well as cells that harbor one or more of these vectors or the encoded polynucleotide molecules.
  • the invention provides pharmaceutical compositions that contain a SHEDTAC molecule disclosed herein or a polynucleotide encoding the SHEDTAC, and a pharmaceutically acceptable carrier.
  • the invention provides methods for cleaving the ectodomain of a membrane-bound protein on the surface of a cell.
  • These methods entail first constructing a fusion molecule that contains a protease-targeting domain and a covalently linked substratetargeting domain.
  • the substrate-targeting domain and the protease-targeting domain specifically bind to the extracellular portion of the membrane-bound protein and a protease that is bound to the same membrane surface, respectively.
  • the fusion molecule is generated, it is applied to contact with the cell that expresses the membrane-bound protein and the membrane-bound protease, leading to cleaving of the ectodomain of the membranebound protein.
  • the targeted membrane-bound protein is a cellular receptor or a receptor ligand
  • the targeted protease is a sheddase.
  • the targeted cellular receptor is LAG3 or PD-1
  • the targeted sheddase is ADAM 10.
  • the substrate-targeting domain and the protease-targeting domain are antibodies or antigen-binding fragments (e.g., VHH sdAbs) that specifically binds to the membrane-bound protein and the protease, respectively.
  • the fusion molecule can be contacted with the cell in vitro or ex vivo.
  • the fusion molecule or a polynucleotide encoding the fusion protein can be administered to a subject, allowing in vivo contact between the fusion molecule and the cellular targets.
  • FIG 1 shows a scheme of the function of Sheddase-Targeting Chimeras (SHEDTACs).
  • SHEDTACs recruit an integral membrane protease (sheddase) to a target substrate on the same-cell membrane (in cis).
  • Dadase integral membrane protease
  • C decoupling associated signaling events
  • D-F A similar process is indicated for SHEDTACs that catalyze proteolysis of target substrate complexes.
  • FIG. 1 illustrates cleavage of target membrane proteins by SHEDTACs.
  • Substrate proteolysis catalyzed by SHEDTACs produces substrate fragments that include extracellular domains (ECD) and intracellular domains (ICD).
  • ECD extracellular domains
  • ICD intracellular domains
  • B) SHED T AC -generated ICDs may translocate to the nucleus to initiate gene transcription.
  • C SHEDTAC-generated ECDs may engage soluble ligands to affect downstream signaling processes.
  • D SHEDTAC-generated ECDs may ligate same-cell (cis) receptors to initiate signaling.
  • E SHEDTAC-generated ECDs may ligate (trans) receptors on neighboring cells to initiate signaling.
  • FIG. 3 shows general configurations for protein SHEDTACs.
  • A Generalized SHEDTAC format, consisting of a sheddase-targeting domain and a substrate-targeting domain. Modular targeting domains may be reconfigured as needed.
  • B Reducing SDS- PAGE analysis of a representative SHEDTAC.
  • FIG. 4 shows LAG3-SHEDTAC library construction and validation.
  • A (top) Design of a focused LAG3-SHEDTAC library, (bottom) Reducing SDS-PAGE analysis indicating purified LAG3 -SHEDTACs (used to treat cells in (B-D)).
  • B Western blot analysis of cell pellets indicating levels of full-length LAG3 on cells following 24h treatment with 500nM LAG3-SHEDTACs.
  • Band intensity is expressed as percent control of cells treated with vehicle in the absence of PMA/ionomycin, indicated by (C) Western blot analysis of cell pellets and corresponding cell supernatants treated with IpM BMGX0016, sampled every 10 minutes for 60 minutes, indicating time-dependent decreases in full-length ( ⁇ 70kDa) LAG3 and concomitant increases in soluble LAG3 (sLAG3, ⁇ 60kDa) ectodomain released into the growth medium by ADAM10.
  • FIG. 5 shows that LAG3-SHEDTACs accelerate LAG3 ectodomain cleavage by ADAMI 0.
  • A Flow cytometry contour plots indicating PBMC gating strategy for CD3+ADAM10+ cells.
  • B Reducing SDS-PAGE analysis indicating LAG3-SHEDTAC (left) and TEV-proteolyzed LAG3-SHEDTAC (right) used to treat cells in (C-E).
  • C-E Flow cytometry contour plots showing cell surface LAG3 abundance on CD3+ADAM10+ cells following Ih treatment with (C) vehicle, (D) LAG3-SHEDTACs, (E) TEV-proteolyzed LAG3-SHEDTACs.
  • (F) Flow cytometry contour plots indicating dose-dependent reductions in detectable cell surface LAG3 levels following treatment with LAG3-SHEDTACs at various concentrations.
  • G Western blot analysis of cell pellets indicating levels of full- length LAG3 ( ⁇ 70kDa) on cells following 24h treatment with vehicle or PMA ⁇ LAG3- SHEDTACs.
  • H, I Western blot analysis of cell supernatants indicating levels of soluble LAG3 (sLAG3, ⁇ 60kDa) ectodomain released into the growth medium by ADAM 10, following 24h treatment with LAG3-SHEDTACs at various concentrations.
  • FIG. 6 shows that protein depletion by SHEDTACs does not require proteasomes or lysosomes.
  • Flow cytometry contour plots showing cell surface LAG3 abundance on CD3+ADAM10+ cells treated with LAG3 -SHEDTACs (top) or TEV-digested LAG3-SHEDTAC (monospecific controls, bottom). Prior to treatment, cells were incubated for 2h at 37°C with (A) vehicle, (B) proteasome inhibitor (MG132, lOpM), or (C) lysosome inhibitor (Dynasore, 50pM).
  • FIG. 7 shows that SHEDTAC -mediated LAG3 shedding from T cells enhances
  • LAG3 suppresses T cell signaling through interactions with the TCR on T cells and MHCII on antigen presenting cells (APCs) (left). LAG3-SHEDTACs catalyze LAG3 ectodomain cleavage by the integral membrane protease ADAMI 0 to restore TCR signaling and induce a luciferase reporter (right).
  • B Flow cytometry contour plots indicating LAG3 abundance on ADAMI 0(+) Jurkat reporter cells treated with isotype- SHEDTACs or LAG3-SHEDTACS.
  • C dose-dependent luminescence increases following treatment with LAG3-SHEDTACs at various concentrations, indicating enhanced TCR signaling through LAG3 shedding.
  • RLU relative luminescence units
  • FIG. 8 shows that SHEDTACs catalyze depletion of the immune checkpoint PD-1 from T cells.
  • T cell activation proceeds through T cell receptor (TCR) recognition of antigen-loaded major histocompatibility complex (MHC), followed by ligation of costimulatory CD28 to its B7 ligands CD80/86 on antigen presenting cells (APCs).
  • MHC major histocompatibility complex
  • APCs antigen presenting cells
  • the immune checkpoint PD1 suppresses T cell signaling by binding its B7 ligands on APCs.
  • B PD1 -SHEDTACs catalyze PD1 ectodomain shedding by the integral membrane protease ADAMIO to restore T cell function.
  • the present invention provides novel compositions and related methods to proteolyze integral membrane proteins on demand by using endogenous proteases.
  • this is accomplished by using a multi-specific reagent that binds and induces proximity between an integral membrane protease (sheddase) and a target membrane protein.
  • This multi-specific chimera recruits a sheddase to its target and has therefore been termed a Sheddase-T argeting Chimera (SHEDTAC).
  • SHEDTACs catalyze ectodomain cleavage of a designated target protein ( Figure 1).
  • SHEDTAC-derived cleavage products include a soluble extracellular domain (ECD) and a transmembrane domain which may be further processed into a soluble, intracellular domains (ICD). These ECD/ICD cleavage products may affect downstream processes, including gene transcription, soluble ligand complexation, or receptor ligation on the same cell (in cis) or on a distant cell (in trans) (Fig. 2, Panels B-E).
  • LAG3 -targeting SHEDTACS were able to promote robust LAG3 shedding through induced proximity, and reverse LAG3 suppression of T cell function.
  • SHEDTACs described herein apparently are also able to cleave ECDs of membrane proteins that are not known to be sheddase substrates. This is demonstrated herein with SHEDTACs that target Programmed Cell Death-1 (PD-1) on T lymphocytes.
  • PD-1 Programmed Cell Death-1
  • the fusion molecules can be readily employed as pharmaceuticals for their ability to therapeutically modulate membrane proteostasis and alleviate disease phenotypes.
  • methods utilizing SHEDTACs for targeted proteolysis operate independent of the ubiquitylation or lysosomal trafficking, and do not require delivery of exogeneous proteases. Instead, these fusion proteins catalyze target protein ectodomain cleavage by local, endogenous proteases (sheddases) ( Figure 1).
  • SHEDTAC- derived cleavage products may affect downstream processes on a same cell (in cis) or distant cell (in trans) ( Figure 2). This novel mechanism of action cannot be achieved by existing technologies and is entirely unique to SHEDTACs.
  • amino acid of a peptide refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.
  • the term "consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term “contacting” has its normal meaning and refers to combining two or more agents (e.g., polypeptides or small organic molecules), combining agents and cells, or combining two populations of different cells. Contacting can occur in vitro, e.g., mixing two polypeptides or mixing a protein with a population of cells in a test tube or growth medium. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by coexpression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate.
  • agents e.g., polypeptides or small organic molecules
  • “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • betabranched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • engineered cell or “recombinant host cell” (or simply “host cell”) refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • a “fusion” protein or polypeptide refers to a polypeptide comprised of at least two polypeptides and a linking sequence or a linkage to operatively link the two polypeptides into one continuous polypeptide.
  • the two polypeptides linked in a fusion polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature.
  • Heterologous when used with reference to two polypeptides, indicates that the two are not found in the same cell or microorganism in nature. Allelic variations or naturally occurring mutational events do not give rise to a heterologous biomolecule or sequence as defined herein.
  • a "heterologous" region of a vector construct is an identifiable segment of polynucleotide within a larger polynucleotide molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by polynucleotide that does not flank the mammalian genomic polynucleotide in the genome of the source organism.
  • isolated means a polypeptide or protein is removed from its natural surroundings. However, some of the components found with it may continue to be with an “isolated” protein. Thus, an “isolated polypeptide” is not as it appears in nature but may be substantially less than 100% pure protein.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • Lymphocyte Activation Gene 3 belongs to the immunoglobulin (Ig) superfamily and comprises a 503-amino acid type I transmembrane protein with four extracellular Ig-like domains, designated DI to D4.
  • LAG3 is expressed on activated T cells, natural killer cells, B cells and plasmacytoid dendritic cells.
  • LAG3 plays a regulatory role in the immune system comparable to PD-1 and CTLA-4, generally consisting of inhibition of cell proliferation, immune function, cytokine secretion, and homeostasis. It functions by delivering inhibitory signals that regulate immune cell homeostasis, T cell activation, proliferation, cytokine production, cytolytic activity and other functions.
  • LAG3 can be upregulated under various antigen stimulation conditions.
  • LAG3 inhibitors can directly bind LAG3 molecules or their ligands, blocking the interaction between ligands and LAG3, and downregulating the inhibitory efficacy of LAG3 toward the immune system.
  • nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
  • Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.
  • a "ligand” is a molecule that is recognized by a particular antigen, receptor or target molecule.
  • ligands that can be employed in the practice of the present invention may include, but are not restricted to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones, hormone receptors, polypeptides, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g., opiates, steroids, etc.), lectins, sugars, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.
  • Linkage refers to means of operably or functionally connecting two biomolecules (e.g., polypeptides or polynucleotides encoding two polypeptides), including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding. "Fused” refers to linkage by covalent bonding.
  • a “linker” or “spacer” refers to a molecule or group of molecules that connects two biomolecules, and serves to place the two molecules in a preferred configuration with minimal steric hindrance.
  • operably linked when referring to a nucleic acid, refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • PD-1 Programmed cell death protein 1
  • CD279 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro-B cells.
  • PD- 1 binds two ligands, PD-L1 and PD-L2, and plays an important role in regulating the immune system's response. It is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigenspecific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti- inflammatory, suppressive T cells). See, e.g., Francisco et al., Immunol. Rev.
  • polypeptide and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues. They encompass both short oligopeptides (e.g., peptides with less than about 25 residues) and longer polypeptide molecules (e.g., polymers of more than about 25 or 30 amino acid residues).
  • the candidate peptide or polypeptide ligands used in the invention can comprise from about 4 amino acid residues to about 350 or more amino acid residues in length.
  • the peptides or polypeptides comprise from about 6 amino acid residues to about 60 amino acid residues in length. In some other embodiments, they can comprise from about 8 amino acid residues to about 40 amino acid residues in length.
  • the peptides or polypeptides can include naturally occurring amino acid polymers and non-naturally occurring amino acid polymer, as well as amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
  • peptide mimetic or “peptidomimetic” refers to a derivative compound of a reference peptide that biologically mimics the peptide’s functions.
  • the peptidomimetic derivative has at least 50%, at least 75% or at least 90% of the biological function of the reference polypeptide.
  • receptor broadly refers to a molecule that has an affinity for a given ligand. Receptors may -be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species. Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance.
  • a typical example of receptors which can be employed in the practice of the invention is cell surface signaling receptor.
  • signal transduction pathway or “signaling activities” refers to at least one biochemical reaction, but more commonly a series of biochemical reactions, which result from interaction of a cell with a stimulatory compound or agent.
  • signal transduction pathway refers to at least one biochemical reaction, but more commonly a series of biochemical reactions, which result from interaction of a cell with a stimulatory compound or agent.
  • the interaction of a stimulatory compound with a cell generates a “signal” that is transmitted through the signal transduction pathway, ultimately resulting in a cellular response.
  • the term “variant” refers to a molecule (e.g., a polypeptide or an antibody fragment) that contains a sequence that is substantially identical to the sequence of a reference molecule.
  • the reference molecule can be an enzymatic polypeptide disclosed herein or a fusion thereof.
  • the reference molecule can also be a polynucleotide encoding the polypeptide.
  • the variant can share at least 50%, at least 70%, at least 80%, at least 90%, at least 95% or more sequence identity with the reference molecule.
  • the variant differs from the reference molecule by having one or more conservative amino acid substitutions.
  • a variant of a reference molecule is a conservatively modified variant, e.g., a variant which has altered amino acid sequences (e.g., with one or more conservative amino acid substitutions) but substantially retains the biological activity of the reference molecule. Conservative amino acid substitutions are well known to one skilled in the art.
  • vector is intended to refer to a polynucleotide molecule capable of transporting another polynucleotide to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non- episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • integral membrane protein refers to proteins, polypeptides, peptidomimetics or conjugates thereof, characterized by their ability to span or traverse the lipid bilayer of biological membranes, which can be found in cells or organelles like the cell membrane, endoplasmic reticulum, Golgi apparatus, mitochondria, and others.
  • integral membrane proteins include transporters, receptors, cytokines and enzymes.
  • Integral membrane protease is a type of integral membrane protein that is also a protease.
  • Proteases in general, are enzymes that catalyze the cleavage of peptide bonds in proteins, leading to the breakdown of proteins into smaller peptide fragments or individual amino acids.
  • Integral membrane proteases hydrolyze polypeptides located near, or embedded within, biological lipid membranes, such as those constituting cells and organelles, e.g. cell membrane, endoplasmic reticulum, Golgi apparatus, and mitochondria.
  • SHEDTACs for inducing proximity between sheddases and target proteins
  • the invention provides Sheddase-Targ eting Chimeras (SHEDTACs), which by induced proximity between an integral membrane protein (substrate or target) and an integral membrane protease (sheddase), can catalyze ectodomain cleavage of designated target proteins.
  • SHEDTACs contain a protease-targeting domain and a substratetargeting domain.
  • the protease-targeting domain e.g., an antibody or a ligand, is able to specifically bind to an endogenous or local protease.
  • the substrate-targeting domain e.g., an antibody or a ligand, specifically binds to a target membrane protein.
  • Any molecules or compounds that can specifically bind to a target protein of interest and a corresponding sheddase can be used as the substrate-targeting domain and the protease-targeting domain, respectively. They can contain small molecules, amino acids, nucleotides, lipids, carbohydrates, antibody or antibody fragments, oligopeptides, oligonucleotides, polypeptides, polynucleotides, cytokines, chemokines, DNA/RNA aptamers, or combinations thereof.
  • the protease-targeting domain and the substrate-targeting domain are covalently linked in the SHEDTACs.
  • a liker motif is used to fuse the protease-targeting domain to the substratetargeting domain.
  • SHEDTACs may be constructed in diverse formats, as monomeric and multimeric fusions, using targeting molecules or moieties for multi-specific targeting to an integral membrane protease (sheddase) and a target substrate.
  • SHEDTACs General configurations for protein SHEDTACs are shown in Figure 3. Exemplifications are provided herein for SHEDTACs that direct the LAG3 or PD-1 membrane protein for proteolysis by an integral membrane sheddase, such as A Disintegrin And Metalloprotease (ADAM10).
  • LAG3 is an immune checkpoint, which is a co-inhibitory receptor to suppress T cells activation and cytokines secretion, thereby ensuring a state of immune homeostasis. LAG3 exerts differential inhibitory impacts on various types of lymphocytes.
  • a library of LAG3-ADAM10 engaging SHEDTACs was constructed and recombinantly expressed in Escherichia coli.
  • This SHEDTAC contains a LAG3-binding sdAb and a ADAMI O-binding sdAb.
  • the two targeting domains can be linked a linker motif, e.g., a GGGGS (SEQ ID NO: 11) tandem repeat linker as exemplified herein.
  • this exemplary SHEDTAC has a molecular weight of ⁇ 34kDa.
  • the substrate-targeting domain can be fused at its C-terminus to the N-terminus of the proteasetargeting domain.
  • the protease-targeting domain can be fused at its C-terminus to the N-terminus of the substrate-targeting domain.
  • substrate-targeting domains or protease-targeting domains can be fused at their C-terminus or N-terminus with molecules that extend serum half-life in vivo, e.g., carrier proteins, albumin binding polypeptides, Fc domains, antibody fragments, polyethylene glycol (PEG) chains, nucleic acids, lipids, or carbohydrates.
  • carrier proteins e.g., albumin binding polypeptides, Fc domains, antibody fragments, polyethylene glycol (PEG) chains, nucleic acids, lipids, or carbohydrates.
  • PEG polyethylene glycol
  • the protease-targeting domain and the substratetargeting domain of the SHEDTACs are antibodies or antigen-binding fragments thereof. Any antibody sequences can be employed in the invention.
  • each of the protease-targeting domain and the substrate-targeting domain is an antibody fragment or antigen-binding fragment.
  • Antibody fragments or antigen-binding fragments contain the antigen-binding portions of an intact antibody that retain capacity to bind the cognate antigen.
  • antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (sdAb) or sdAb which consists of a VH domain (see, e.g., Ward et al., Nature 341 :544-546, 1989); and (vii) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the V
  • antibodies employed for practicing the present invention are single chain antibodies.
  • the term "single chain antibody” refers to a polypeptide comprising a VH domain and/or a VL domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and/or carboxyl-termini.
  • a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide.
  • a single chain variable region fragment (scFv) is a single-chain antibody.
  • a scFv Compared to the VL and VH domains of the Fv fragment which are coded for by separate genes, a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.
  • the protease-targeting domain and the substrate-targeting domain of the SHEDTACs are single domain antibodies such as VHH sdAb domains as exemplified herein.
  • the protease-targeting domain can be a ADAMlO-binding sdAb that contains the amino acid sequence shown in any one of SEQ ID NOs: 12-16, a substantially identical sequence, or a conservatively modified variant thereof
  • the substrate-targeting domain can be a LAG3-binding sdAb or PD-1 binding sdAb that contains the amino acid sequence shown in any one of SEQ ID NOs: 17-21, a substantially identical sequence, or a conservatively modified variant thereof.
  • Antibodies or antigen-binding fragments for practicing the invention can be produced by enzymatic or chemical modifications of the intact antibodies, or synthesized de novo using recombinant DNA methodologies, or identified using phage display libraries. Methods for generating these antibodies or antigen-binding molecules are all well known in the art. For example, single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; and U.S. Pat. No. 4,946,778).
  • scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988.
  • Fv antibody fragments can be generated as described in Skerra and Pliickthun, Science 240: 1038-41, 1988.
  • Disulfide- stabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67: 113-23, 1996.
  • single domain antibodies can be produced by recombinant methods as exemplified herein and/or methods known in the art. See, e.g., Ward et al., Nature 341 :544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996.
  • Camelid single domain antibodies can be produced using methods well known in the art, e.g., Dumoulin et al., Nature Struct. Biol. 11 :500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J Mol Biol.
  • sdAb fragments used in the SHEDTACs of the invention can be produced via recombinant expression.
  • the SHEDTACs of the invention can be further conjugated to a carrier moiety to enhance their stability and serum half-lives.
  • the conjugation can be either covalent or non-covalent via any conventional methods.
  • the “carrier moiety” (“carrier” or “carrier molecule”) is typically a large, slowly metabolized macromolecule such as proteins; polysaccharides (such as latex functionalized SEPHAROSETM, agarose, cellulose, cellulose beads and the like); polymeric amino acids (such as polyglutamic acid, polylysine, and the like); amino acid copolymers; and inactive virus particles or attenuated bacteria, such as Salmonella.
  • the carrier moiety can be a carrier protein, an immunoglobulin, a Fc domain, a PEG molecule or other polymer.
  • the employed carrier moiety is the Fc domain of an IgG. Fusion with an Fc-domain provides a number of beneficial biological and pharmacological properties. For example, the presence of the Fc domain can markedly increase the plasma half-life of the hybrid molecule, which prolongs therapeutic activity, owing to its interaction with the salvage neonatal Fc-receptor, as well as to the slower renal clearance for larger sized molecules.
  • the carrier moiety is a protein. Integral membrane proteins from, e.g., E. coli and other bacteria are useful conjugation partners. Especially useful carrier proteins are serum albumins, keyhole limpet hemocyanin (KLH), certain immunoglobulin molecules, thyroglobulin, ovalbumin, bovine serum albumin (BSA), tetanus toxoid (TT), and diphtheria toxoid (CRM).
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • TT tetanus toxoid
  • CCM diphtheria toxoid
  • the employed carrier moiety is a protein that can bind serum molecules, e.g., albumin.
  • the carrier moiety can be a polymer other than a protein or polypeptide. Examples of such polymers include, e.g., carbohydrates such as dextran, mannose or mannan.
  • the various SHEDTACs of the invention can be generated in accordance with routinely practiced biological and/or chemical methods (e.g., recombination technology as exemplified herein).
  • the method for generating the SHEDTAC fusion proteins is not subject to any particular limitation.
  • the fusion proteins may be a fusion protein synthesized by chemical synthesis, or a recombinant fusion protein produced by a genetic engineering technique as exemplified herein.
  • fusion protein of the invention is to be chemically synthesized, synthesis may be carried out by, for example, the Fmoc (fluorenylmethyloxy carbonyl) process or the tBoc (t-butyloxy carbonyl) process.
  • peptide synthesizers available from, for example, Advanced ChemTech, PerkinElmer, Pharmacia, Protein Technology Instrument, Syntheceh-Vega, PerSeptive and Shimadzu Corporation may be used for chemical synthesis.
  • SHEDTAC fusion protein is to be produced by a genetic engineering technique, production may be carried out using the conventional recombination techniques routinely practiced in the art.
  • the SHEDTAC fusion protein can be produced by inserting a polynucleotide (e.g., DNA) encoding the fusion protein into a suitable vector, followed by introducing the vector into an appropriate expression system, e.g., a pET23a vector for expression in E. coli as exemplified herein.
  • a polynucleotide e.g., DNA
  • an appropriate expression system e.g., a pET23a vector for expression in E. coli as exemplified herein.
  • the SHEDTACs of the invention can be readily employed to specifically target a cellular membrane protein (e.g., a regulatory protein such as LAG3 or PD-1 as exemplified herein) for proteolytic cleavage in vivo by a corresponding endogenous sheddase (e.g., ADAM sheddase as exemplified herein).
  • a cellular membrane protein e.g., a regulatory protein such as LAG3 or PD-1 as exemplified herein
  • ADAM sheddase e.g., ADAM sheddase as exemplified herein
  • these applications involve contacting in vivo (e.g., by administration to a subject) or ex vivo a SHEDTAC described herein that specifically target a membrane protein for proteolysis and a sheddase enzyme that specifically proteolyze the target protein of interest.
  • the employed SHEDTAC is a protein.
  • the membrane protein to be targeted for proteolysis is specifically or differentially expressed in a given cell or tissue type (e.g., LAG3 in CD4 + and CD8 + T cells, and PD-1 on T cells and B cells).
  • the target protein can be a regulatory receptor predominantly expressed on the surface of immune cells such as T cells.
  • the target protein can be a membrane protein that is specifically or differentially expressed on a diseased tissue or cell such as a tumor cell.
  • proteases for degrading target proteins via induced proximity with SHEDTACs can be any protease that is capable of proteolyzing a membrane bound protein of interest on the surface of a cell (e.g., a leukocyte).
  • a cell e.g., a leukocyte
  • the protease is expressed by and bound to the membrane of the same cell.
  • the membrane-bound protease is a sheddase as described herein.
  • Sheddases are membrane-bound enzymes that cleave extracellular portions of transmembrane proteins, releasing the soluble ectodomains from the cell surface.
  • Many sheddases are members of the ADAM or aspartic protease (BACE) protein families. These enzymes can activate a transmembrane protein if it is a receptor (e.g., HER2), or cut off the part of the transmembrane protein which has already bound a ligand (e.g., in the case of EGFR), allowing this ligand to engage a receptor on another cell. Due to the nature of the mechanisms and functions of sheddase enzymes, they have been studied on the basis of discovering possible uses in medicine.
  • Therapeutic uses of sheddases can encompass various diseases and disorders, e.g., brain disorders, e.g. Alzheimer’s disease, Prion disease, Fragile X-syndrome and Huntington’s disease. Additional medical uses can be applied to address immune disorders, e.g. psoriasis, systemic lupus erythematosus (SLE), rheumatoid arthritis, bacterial or viral infection, chronic tissue and organ inflammation and atherosclerosis. Additional medical uses can be found in areas of cancer, e.g.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • cHL classical Hodgkin lymphoma
  • PMBCL primary mediastinal large B-cell lymphoma
  • urothelial carcinoma microsatellite instability-high cancer
  • gastric cancer esophageal cancer
  • cervical cancer hepatocellular carcinoma
  • merkel cell carcinoma renal cell carcinoma and endometrial carcinoma.
  • Possible therapeutic substrates contained within these examples could include membranebound and soluble forms of proteins or their ligands that include amyloid precursor protein (APP), prion protein PrPc, PrPsc, Fragile X Mental Retardation Protein (FMRP), htt, N- cadherin, VE-cadherin, IL6, IL6R, CXCL16, CXCL1, EGF, EGFR, LAG3, Notch 1, Delta, HB-EFG, TGFa, Axl, TACI, CX3CLl/fractalkine, GM-CSF, CD23, JAM-A, JAM-C, IL8, TNFa, c-Met, IL10, including others listed in Table 1.
  • APP amyloid precursor protein
  • PrPc PrPsc
  • FMRP Fragile X Mental Retardation Protein
  • htt N- cadherin
  • VE-cadherin IL6, IL6R, CXCL16, CXCL1,
  • the SHEDTAC fusion constructs of the invention are intended to direct a target membrane protein of interest (e.g., a signaling receptor) to an endogenous sheddase for proteolysis, thereby eliciting desired cellular responses and/or therapeutic activities.
  • a target membrane protein of interest e.g., a signaling receptor
  • Many sheddases known in the art can be targeted by the SHEDTACs of the invention.
  • the targeted sheddases are A Disintegrin And Metalloprotease (ADAM) proteases such as ADAM 10 as exemplified herein.
  • ADAM Disintegrin And Metalloprotease
  • SHEDTACs of the invention are intended to target BACE proteases (aspartyl proteases). Examples of BACE proteases to be targeted include BACE1, BACE2 and etc. Some SHEDTACs of the invention are intended to target the Site-1 protease (serine protease), also known as SKI-1 or SIP. Some SHEDTACs of the invention can target the meprin P metalloprotease. In some other embodiments, the sheddase to be targeted by the SHEDTACs of the invention is a membrane-type matrix metalloproteinase (MT-MMP).
  • MT-MMP membrane-type matrix metalloproteinase
  • the sheddase to be targeted by the SHEDTACs of the invention is a pro-protein convertase (serine protease).
  • pro-protein convertases include PCSK1/3, PCSK2, furin, PCSK4, PCSK5/6, PACE4, PCSK7 and PCSK9.
  • the sheddase to be targeted is a transmembrane serine protease.
  • transmembrane serine proteases that can be targeted by the SHEDTACs of the invention include
  • the sheddase to be targeted is a matrix metalloprotease (MMP).
  • MMPs matrix metalloproteases
  • Specific examples of MMPs that can be targeted include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP 10, MMP11, MMP 12, MMP 13, MMP 19, MMP20, MMP21, MMP23, MMP26, MMP27, and MMP28.
  • the SHEDTACs of the invention are intended to direct target proteins for proteolysis by additional sheddases such as Legumain ( 6 -secretase), cysteine proteases, Cathepsin S and L, Rhomboid proteases (e g., RHBDL1, RHBDL2, RHBDL3, and RHBDL4), the SPP/SPPL family of aspartyl proteases (e.g., SPPL3, SPP, SPPL2a, SPPL2b, and SPPL2c), and the Presenilin/ y -secretase aspartyl protease (e.g., Presenilin-1 and Presenilin-2).
  • Legumain 6 -secretase
  • cysteine proteases e.g., Cathepsin S and L
  • Rhomboid proteases e g., RHBDL1, RHBDL2, RHBDL3, and RHBDL4
  • SPP/SPPL family of aspartyl proteases e.
  • the SHEDTACs of the invention can also target the protease (e.g., sheddase) indirectly by binding to a molecule that interacts with the protease.
  • Integral membrane proteases such as sheddases typically co-localize with auxiliary membrane proteins to form integral membrane protease complexes capable of proteolyzing membrane protein substrates.
  • SHEDTACs of the invention are intended to induce proximity between a target membrane protein and integral membrane protease complex.
  • Target proteins for proteolysis by sheddases brought in proximity via SHEDTACs Many proteins that are “shed” from cellular phospholipid membranes represent sheddase targets. By association, these proteins are target proteins or substrates for proteolysis by sheddases that can be brought in proximity via SHEDTACs of the invention. In general, any known protein attached to a phospholipid membrane (e.g., transmembrane proteins) can be targeted, provided that a membrane-bound protease expressed on the same membrane recognizes the protein for proteolysis.
  • Such candidate target proteins include all computationally predicted and/or experimentally validated to localize to the cellular plasma membrane, which can be retrieved from the EMBL-EBI database (www.ebi.ac.uk/QuickGO/term/GO:0005886).
  • these membrane proteins can all be targeted by a corresponding SHEDTAC molecule that can be generated in accordance with the invention.
  • the transmembrane substrate protein to be targeted by the SHEDTACs of the invention is a cellular signaling receptor or a receptor ligand.
  • the target transmembrane substrate protein is a tumor-associated antigen.
  • the target protein is a known or suggested substrate of a sheddase.
  • a non-exhaustive list of annotated sheddase targets has been compiled on the SheddomeDB. See, e.g., Tien et al., BMC Bioinformatics 18, 42 (2017).
  • the substratetargeting domain in the SHEDTACs of the invention can be a binding moiety (e.g., a single domain VHH sdAb or a single chain scFv fragment) that specifically recognizes any of these target proteins. Examples of known and potential sheddase substrates in humans are summarized in Table 1.
  • the target substrate to be engaged by the SHEDTACs for proteolysis is a protein whose depletion at the plasma membrane provides therapeutic benefit.
  • Some specific examples of such target proteins include immune checkpoint receptors such as PD-1, CTLA4, TIM3 and TIGIT.
  • the substrate to be targeted for proteolysis is a membrane protein whose soluble extracellular domain provides therapeutic benefit once released from the membrane.
  • Some specific examples of such target proteins include immune checkpoint receptors such as PD-1, CTLA4, TIM3, TIGIT, CD33-related Siglecs, HER2, EGFR, TNFRVII, Notchl, Delta, APP. Additional sheddase substrates whose depletion at the membrane directly confers therapeutic benefit, or whose soluble extracellular or intracellular domain provides therapeutic benefit, are listed in Table 1.
  • the invention also provides polynucleotide sequences that encode such fusions, expression constructs for expressing the fusion proteins, multimeric fusion protein combinations, and host cells that harbor the polynucleotides or expression constructs.
  • the polynucleotide sequences of the invention can be any polynucleotide having a nucleotide sequence that encodes the fusion protein of the invention, although DNA is preferred.
  • the recombinant constructs or expression vectors of the invention harbor a polynucleotide sequence of the invention that encodes a SHEDTAC fusion polypeptide.
  • the recombinant constructs of the invention may be obtained by ligating (inserting) the polynucleotide (DNA) of the invention into a suitable vector. More specifically, the recombinant vector may be obtained by cleaving purified polynucleotide (DNA) with a suitable restriction enzyme, then inserting the cleaved polynucleotide to a restriction enzyme site or multicloning site on a suitable vector, and ligating the polynucleotide to the vector.
  • the vector for inserting the polynucleotide sequence is not subject to any particular limitation, provided it is capable of replication in an appropriate host.
  • the expression vectors of the invention are not subject to any particular limitation, and may be, for example, bacteriophages, plasmids, cosmids or phagemids.
  • examples of recombinant bacteriophage or phagemid vectors include that based on a filamentous phage such as M13.
  • Plasmid vectors include those based on plasmids from, e.g., E.
  • the vector for expressing the SHEDTAC fusion proteins of the invention is a viral vector.
  • Various viral expression vectors can be used in the invention.
  • lentiviral vectors are suitable for introducing into and expressing in the host cell the combinatorial library of candidate binding partners.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect both dividing and non-dividing cells and typically produce high viral titers.
  • Examples of lentiviral based vectors that may be employed and modified for the invention include, e.g., pLV2, pLVX-Puro, pLVX-IRES- Neo, pLVX-IRES-Hyg, and pLVX-IRES-Puro.
  • the various lentiviral vectors with cloned candidate ligand sequences can be introduced into an appropriate host cell for expressing the candidate ligand library.
  • the HEK293 cell line, the HEK293T cell line, and the TF-1 cell line are all suitable for the invention.
  • Many other packaging cell lines well known in the art e.g., Lenti-X 293T cell line
  • Lenti-X 293T cell line may also be employed for expressing the combinatorial library in the invention.
  • retroviral based vectors and expression systems may also be employed in the practice of the methods of the invention.
  • MMLV based vectors pQCXIN, pQCXIQ and pQCXIH include MMLV based vectors pQCXIN, pQCXIQ and pQCXIH, and compatible producer cell lines such as HEK 293 based packaging cell lines GP2-293, EcoPack 2-293 and AmphoPack 293, as well as NIH/3T3-based packaging cell line RetroPack PT67.
  • the polynucleotide encoding the SHEDTAC fusion is generally ligated downstream from the promoter in a suitable vector in such a way as to be expressible.
  • preferred promoters include promoters from SV40, retrovirus promoters, metallothionein promoters, heat shock promoters, cytomegalovirus promoters and the SRa promoter.
  • preferred promoters include the tetracycline promoter, the Trp promoter, the T7 promoter, the lac promoter, the recA promoter, the 1 promoter and the Ipp promoter.
  • preferred promoters include the SPO1 promoter, the SPO2 promoter and the penP promoter. If the host is a yeast, preferred promoters include the PHO5 promoter, the PGK promoter, the GAP promoter, the ADH1 promoter and the GAL promoter. If the host is an insect cell, preferred promoters include the polyhedrin promoter and the PIO promoter.
  • the recombinant vector used in the invention may contain, if desired, an enhancer, a splicing signal, a poly(A) addition signal, a ribosome binding sequence (SD sequence), a selective marker and the like.
  • selective markers include the tetracycline resistance gene, the carbencillin resistance gene, the dihydrofolate reductase gene, the ampicillin resistance gene and the neomycin resistance gene.
  • the recombinant vector of the invention may additionally include a polynucleotide having a nucleotide sequence encoding an amino acid sequence for enhancing translation and/or a polynucleotide having a nucleotide sequence encoding a peptide sequence for purification.
  • the vectors can employ a translational enhancer element (TEE) sequence (see, e.g., Batten et al., FEBS Lett. 580:2591-7, 2006).
  • TEE translational enhancer element
  • the invention further provides host cells that express the SHEDTAC fusion polypeptides described herein.
  • the host cells are genetically engineered (transduced, transformed or transfected) with the recombinant constructs or expression vectors disclosed herein for production of the fusion protein or examination of its activity.
  • a recombinant construct which harbors and expresses a SHEDTAC fusion sequence can be introduced into a suitable host.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying particular genes such as the fusion gene encoding a SHEDTAC fusion polypeptide.
  • the SHEDTAC fusion sequence is stably integrated into the chromosome of the host cells. With such host cells, the SHEDTAC sequence and its expression are substantially maintained in successive generations of cells. They are distinguished from host cells which transiently express the fusion polypeptide as detailed herein.
  • the host cell for production or expression of a SHEDTAC construct of the invention can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • a higher eukaryotic cell such as a mammalian cell
  • a lower eukaryotic cell such as a yeast cell
  • the host cell can be a prokaryotic cell, such as a bacterial cell.
  • the host cell employed is suitable for expression of the SHEDTAC fusion protein.
  • Representative examples of appropriate host cells suitable for practicing the present invention include, but need not be limited to, bacterial cells, such as E.
  • coli Streptomyces, Salmonella tvphimurium
  • fungal cells such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or 293 cells
  • adenoviruses plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo.
  • Introduction of the vector or expression construct into the host cell can be affected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)).
  • SHEDTAC fusion polypeptide in a transfected or transformed host cell can be carried out in accordance with any of the routinely practiced methods in the art, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., (3 rd ed., 2000); and Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).
  • the host cell harboring the expression vector is cultured under appropriate conditions that allow the polynucleotide (DNA) encoding the fusion protein to be expressed, thereby inducing formation and accumulation of the fusion polypeptide, then isolating and purifying the fusion polypeptide.
  • the fusion protein expressed in the host cell can be readily isolated and purified.
  • an extract of the fusion protein of the invention may be obtained by a conventional method such as centrifugation or filtration after using a conventional technique (e.g., detergents, ultrasound, lysozymes, freezing and thawing) to disrupt the bacteria or cells.
  • a conventional technique e.g., detergents, ultrasound, lysozymes, freezing and thawing
  • an extract containing the target protein may be obtained by a conventional method such as osmotic shock.
  • a culture supernatant containing the inventive fusion protein may be obtained by using a conventional method such as centrifugation or filtration to separate the culture supernatant from the bacteria or cells.
  • Sheddase-Targ eting Chimeras can find many therapeutic or prophylactic applications. These fusion molecules can be employed for targeted proteolysis of many membrane-bound proteins for modulating cellular signaling events and/or for treating diseases. In some embodiments, the compositions and methods described herein are employed to proteolyze a surface antigen that is associated with progression of cancers or tumors.
  • tumor antigens associated with cardiac cancer e.g., sarcoma, myxoma, rhabdomyoma, fibroma, lipoma and teratoma
  • lung cancer e.g., bronchogenic carcinoma, alveolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma
  • various gastrointestinal cancer e.g., cancers of esophagus, stomach, pancreas, colon, small bowel, and large bowel
  • genitourinary tract cancer e.g., kidney, bladder and urethra, prostate, testis
  • liver cancer e.g., hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma
  • bone cancer e.g., osteogenic sarcoma
  • the SHEDTAC fusion molecules described herein can be used in either prophylactic or therapeutic applications (e.g., cancer immunotherapies).
  • prophylactic applications pharmaceutical compositions or medicaments are administered to a subject susceptible to, or otherwise at risk of, developing a disease or condition (/. ⁇ ., melanoma) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • compositions or drugs are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. Often, the therapeutic benefit is monitored and repeated dosages are given if the benefit starts to wane.
  • the SHEDTAC fusion molecules for use in the methods of the invention should be administered to a subject in an amount that is sufficient to achieve the desired therapeutic effect (e.g., eliminating or ameliorating symptoms associated with a relevant condition).
  • An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose.
  • a therapeutically- or prophylactically-effective dose or efficacious dose of the functional derivative effector polypeptide is employed in the pharmaceutical compositions of the invention.
  • agents are usually administered in several dosages until a sufficient benefit has been achieved.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, and the rate of excretion of the particular compound being employed.
  • a pharmaceutically effective dosage would be between about 0.001 and 100 mg/kg body weight of the subject to be treated.
  • the SHEDTACs of the invention are formulated in pharmaceutical compositions for the therapeutic or prophylactic applications disclosed herein.
  • the pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • the pharmaceutical compositions typically contain a therapeutically effective amount of the active effector molecule (e.g., LAG3- or PD-1 -targeting SHEDTAC of the invention).
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100% by weight.
  • the compositions may contain pharmaceutically acceptable carriers and other ingredients known to facilitate administration and/or enhance uptake.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxy ethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, e.g., polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • Therapeutic formulations are prepared by any methods well known in the art of pharmacy. The therapeutic formulations can be delivered by any effective means which could be used for treatment.
  • compositions are preferably manufactured under GMP conditions.
  • the pharmaceutical compositions are usually administered to the subjects on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the derivative effector polypeptide and the other therapeutic agents used in the subject. In some methods, dosage is adjusted to achieve a plasma concentration of 1-1000 pg/ml, and in some methods 25-300 pg/ml or 10-100 pg/ml. Alternatively, the therapeutic agents can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the effector polypeptide and the other drugs in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the subject can be administered a prophylactic regime.
  • kits for carrying out the therapeutic applications disclosed herein are provided herein.
  • the invention provides therapeutic kits for immunotherapies in the treatment of subjects afflicted with tumors, e.g., melanoma.
  • the therapeutic kits of the invention typically comprise as active agent one or more of the described SHEDTACs.
  • the kits can optionally contain suitable pharmaceutically acceptable carriers or excipients for administering the active agents.
  • the pharmaceutically acceptable carrier or excipient suitable for the kits can be coatings, isotonic and absorption delaying agents, binders, adhesives, lubricants, di sintergrants, coloring agents, flavoring agents, sweetening agents, absorbants, detergents, and emulsifying agents.
  • kits include antioxidants, vitamins, minerals, proteins, fats, and carbohydrates.
  • the therapeutic kits can further include packaging material for packaging the reagents and a notification in or on the packaging material.
  • the kits can additionally include appropriate instructions for use and labels indicating the intended use of the contents of the kit. The instructions can be present on any written material or recorded material supplied on or with the kit or which otherwise accompanies the kit.
  • LAG3 -targeting SHEDTACs Construction and validation of LAG3 -targeting SHEDTACs are shown in Figure 4.
  • a total of 30 SHEDTACs that target membrane protein LAG3 and ADAM protease ADAM10 were generated.
  • the LAG3 -targeting domain and the ADAM10- targeting domain in these SHEDTAC constructs are both VHH sdAb domains (variable domains of heavy chain of heavy chain-only antibody).
  • 3 sdAb domains targeting LAG3 and 5 sdAb domains targeting ADAM 10 are used. Each sdAb has a different sequence and targets a different epitope on LAG3 or ADAM 10.
  • 15 have the ADAM 10-targ eting domain at the N-terminus and the LAG3 -targeting domain at the C-terminus, with the other 15 having the ADAM 10-targ eting domain at the C-terminus and the LAG3 -targeting domain at the N-terminus.
  • a linker sequence is used to connect the 2 targeting domains in the SHEDTACs.
  • PD-1 targeting SHEDTACs detailed in Example 4 were similarly produced using the protocols as described herein for LAG3 -targeting SHEDTACs.
  • SEQ ID NO: 12 ADAM10 targeting sdAb 1 (32-G6)
  • SEQ ID NO: 13 AD AM10 targeting sdAb 2 (33 -C6)
  • SEQ ID NO: 14 ADAM10 targeting sdAb 3 (39-B1)
  • SEQ ID NO : 15 ADAM 10 targeting sdAb 4 (39-C 1 )
  • SEQ ID NO : 16 ADAM 10 targeting sd Ab 5 (39-D 1 )
  • SEQ ID NO: 17 LAG3 targeting sdAb 1 (W3396-R2-13)
  • SEQ ID NO: 18 LAG3 targeting sdAb 2 (AS20592VH10)
  • SEQ ID NO: 19 LAG3 targeting sdAb 3 (F023700842)
  • SEQ ID NO:20 PD-1 targeting sdAb 1 (AS15193)
  • SEQ ID NO:21 PD-1 targeting sdAb 2 (NB52)
  • LAG3/ADAM10-targeting or PD-l/ADAM10-targeting SHEDTACs are all known in the art. Specifically, sequences and activities of the 5 ADAMlO-targeting sdAb domains are described in European patent application EP2514767A1 (Clones 32-G6, 33-C6, 39-B1, 39- C1 and 39-D1). The 3 LAG3 -targeting sdAb domains are respectively described in WO2019179365 (Clone W3396-R2-13), W02019185040 (Clone AS20592VH10) and KR20190116546 A (Clone F023700842).
  • Genes corresponding to each sdAb were synthesized as gene fragments (Twist Biosciences), each containing a 5’ adapter nucleotide sequence (atggttaaattttctgaagtgcagttagttgaatct) (SEQ ID NO: 1) and a 3’ adapter nucleotide sequence (acgcaggtgactgtgagctca) (SEQ ID NO:2).
  • Synthetic genes are amplified using primers that anneal to these 5’ or 3’ adapter regions using Q5® Hot Start High-Fidelity DNA Polymerase (New England Biolabs, M0493S) according to the manufacturer’s protocol.
  • N-terminal LAG3-SHEDTAC domains are amplified as “A” fragments using A.fwd and A.rev, while genes encoding C-terminal LAG3-SHEDTAC domains are amplified as “B” fragments using B.fwd and B.rev.
  • N-terminal and C-terminal domains are separated by a linker region with amino acid sequence
  • GGGGSGGGGSGGGGS/A7.F/ O GGGGGSGGGGSGGGGS (SEQ ID NO:3), containing a TEV-cleavage site (ENLYFQG; SEQ ID NO:4) indicated in italicized where the cut site is denoted by “
  • a pET23a vector backbone (Novagen) was amplified as a “C” fragment using primers C.fwd and C.rev. A, B, and C DNA fragments are assembled using NEBuilder® HiFi DNA Assembly (New England Biolabs, E2621S) according to the manufacturer’s protocol.
  • A.fwd atggttaaattttctgaagtgcagttag (SEQ ID NO: 5)
  • C.fwd acgcaggtgactgtgagctcacatcatcaccatcaccattaa-tgagatccg (SEQ ID NO:9)
  • C.rev ctaactgcacttcagaaaatttaaccatatgacctcctcttaaa-gttaacaaaattatttctag (SEQ ID NO: 10)
  • E. coli strain BL21(DE3) Invitrogen
  • E. coli harboring the pET23-LAG3-SHEDTAC plasmid are cultured overnight in sterile 15mm glass culture tubes containing 5ml of Luria Broth supplemented with lOOpg/ml of carbenicillin. The following day, cells are diluted 1 : 100 into a sterile 500ml baffled glass flask containing 100ml of Terrific Broth supplemented with lOOpg/ml carbenicillin, and are grown at 37°C with shaking at 200RPM.
  • Cells are lysed by stirring at room temperature for 30min, cooled on ice, and further lysed using a sonicator (Fisher model FB50, amplitude 90, 3min continuous sonication). Following sonication, the 50ml cell lysate was centrifuged for 30min at 10,000 RCF, 4°C. The resulting supernatant was applied to nickel -nitrilotriacetic acid (Ni-NTA) resin and soluble LAG3- SHEDTAC (containing a C-terminal-hexahistidine affinity tag) was eluted with DPBS containing 500mM imidazole. Purified protein was buffer-exchanged into DPBS, boiled in reducing sodium dodecyl sulfate and analyzed by polyacrylamide gel electrophoresis.
  • Ni-NTA nickel -nitrilotriacetic acid
  • LAG3- SHEDTAC containing a C-terminal-hexahistidine affinity tag
  • LAG3-SHEDTACs BMGX0008, BMGX0009, BMGX0010, BMGX0012, BMGX0013, BMGX0016, BMGX0021, BMGX0023, BMGX0026
  • LAG3-SHEDTAC BMGX0016 was used to demonstrate time-dependent effects on LAG3 abundance resulting from treatment with LAG3-SHEDTACs ( Figure 4, Panels C- D).
  • TEV proteolysis additionally controls for the critical requirement of induced proximity between ADAMIO and LAG3 by intact LAG3-SHEDTACs. Indeed, cells treated with TEV-proteolyzed LAG3-SHEDTAC displayed cell surface LAG3 levels comparable to the vehicle control ( Figure 5, Panels B, E), demonstrating the requirement for simultaneous and bispecific engagement of LAG3 and ADAMIO. LAG3 cleavage by ADAMIO was accelerated by LAG3-SHEDTAC in a dose dependent manner following Ih of treatment as determined by flow cytometry (Figure 5, Panel F).
  • Example 3 Additional studies on the activities of LAG3 -targeting SHEDTACs [0098] Induced proximity between an integral membrane protease and a membrane protein substrate represents a novel modality to modulate proteostasis that is distinct from technologies that rely on proteosomes and lysosomes (e.g. PROTACs, LYTACs, and others). To demonstrate this point, PMBCs were pre-treated for 2h with MG132 (www.cell.com/cell-chemical-biology/fulltext/S1074-5521(15)00193-3 ) or Dynasore (pubmed.
  • MG132 www.cell.com/cell-chemical-biology/fulltext/S1074-5521(15)00193-3
  • Dynasore pubmed.
  • SHEDTACs represent a promising platform technology that can be reconfigured to induce shedding of broad membrane protein targets.
  • SHEDTACs are capable of inducing ectodomain shedding of membrane proteins that are otherwise not shed (not known to be substrates of sheddases or do not shed naturally.
  • One such protein is the immune checkpoint receptor Programmed Cell Death-1 (PD-1) on T lymphocytes. Soluble forms of PD-1 have been measured in serum and are attributed to alternative gene splicing (www.ncbi.nlm.nih.gov/pmc/articles/PMC7710690/) (pubmed. ncbi.nlm.nih.gov/16171790/) (pubmed.ncbi.nlm.nih.gov/16171790/).
  • PD1 -SHEDTACs 8 SHEDTACs (BMGX221-228) targeting PD-1 or ADAM10, termed “PD1 -SHEDTACs” were generated. These reagents were composed of two separate sdAb domains targeting PD-1 (SEQ IDs:20- 21), and five separate sdAb domains targeting ADAM10 (SEQ IDs: 12-16) separated by a linker (SEQ ID:3).
  • PDl-SHEDTACs BMGX221-225 respectively contain at the N-terminus an ADAM10 binding sdAb shown in SEQ ID NOs: 12-16, plus at the C- terminus a PD-1 binding sdAb shown in SEQ ID NO:20.
  • PDl-SHEDTACs BMGX226-228 respectively contain at the N-terminus an ADAM 10 binding sdAb shown in SEQ ID NOs: 12-14, plus at the C-terminus a PD-1 binding sdAb shown in SEQ ID NO:21.
  • PDl-SHEDTACs Upon expression and purification of the PDl-SHEDTACs, they were examined for effects on cell surface PD-1 levels. Specification, PBMCs were stimulated with PMA/ionomycin to induce ADAM10/17 activity. PDl-SHEDTACs were applied to cells for 24h at 37°C in cell culture medium before analysis by western blot. Western blot analysis revealed increased PD1 expression by PMA/ionomycin that was robustly diminished by the addition PDl-SHEDTACs spanning multiple epitopes on PD-1 and ADAM10.
  • SHEDTACs described in the present disclosure represent a generalized approach that (i) can be applied to proteins that are not obviously known as sheddase substrates and (ii) maintains high efficiency despite targeting diverse epitopes on the protease and/or target.
  • PBMCs peripheral blood mononuclear cells
  • T cells are activated by the addition of 4pg sterile anti-human CD28 antibody (Biolegend, 302934) per well (2pg/ml final) and cells are cultured for 72h at 37°C in 5% CO2 atmosphere and 95% relative humidity.
  • Flow cytometry Following 72h activation, cells are pooled into a sterile 50ml centrifuge tube (Genesee Scientific, 21-108) and pelleted by centrifugation at 350g for 5min at room temperature. Pelleted cells are resuspended in T complete growth medium to a density of 5xl0 6 cells per ml and seeded into a 96-well U-bottom tissue culture plate (Falcon, 353077) at 0.5xl0 6 cells per well in a lOOpl final volume.
  • Cells are treated by directly adding lOpl of a I M stock of SHEDTAC in PBS, or Tobacco etch virus (TEV)- proteolyzed SHEDTAC for a final SHEDTAC concentration of lOOnM. Cells are incubated with SHEDTACs for Ih at 37°C in 5% CO2 atmosphere and 95% relative humidity without shaking.
  • TSV Tobacco etch virus
  • cells are pelleted by centrifugation at 350g for 5min at room temperature and resuspended in 200pl of sort buffer, consisting of 0.22pm sterile-filtered PBS containing 1% w/v bovine serum albumin (Prometheus, 25-529C) and ImM ethylenediaminetetraacetic acid (EDTA, Invitrogen, AM9260G). Cells are similarly pelleted and resuspended in 200pl sort buffer for two total washes.
  • sort buffer consisting of 0.22pm sterile-filtered PBS containing 1% w/v bovine serum albumin (Prometheus, 25-529C) and ImM ethylenediaminetetraacetic acid (EDTA, Invitrogen, AM9260G).
  • Washed PBMCs are suspended in 50pl of sort buffer containing 1 :400 dilutions of APC anti-human CD156c (ADAM10) antibody (Biolegend, 352706); Brilliant Violet 421TM anti-human CD223 (LAG-3) antibody (Biolegend, 369314); and BD PharmingenTM FITC Mouse anti-human CD3 antibody (BD Biosciences, 555332) and incubated for Ih at 4°C. Stained cells are washed twice with sort buffer and analyzed on a ZE5 Cell Analyzer (Bio-Rad) for mean fluorescence intensities corresponding to cell surface CD3, ADAM 10 and LAG3.
  • ADAM10 APC anti-human CD156c
  • LAG-3 Brilliant Violet 421TM anti-human CD223
  • BD PharmingenTM FITC Mouse anti-human CD3 antibody BD Biosciences, 555332
  • the entire cell pellet or 80pl of cell culture medium was boiled in reducing SDS buffer, resolved by SDS-PAGE (Bio-Rad 4568096) and transferred to a nitrocellulose membrane (Bio-Rad 1620233) in 20mM Tris, 2mM glycine and 20% methanol using an XCell II Blot Module (Thermo Fisher) powered at 35V for 120min at room temperature.
  • the membrane was blocked at 4°C overnight in 5% w/v BSA in PBS + 0.1% v/v Tween-20 (PBST).
  • the membrane was incubated for Ih with a 5ml volume of polyclonal goat IgG anti -human LAG3 (R&D Systems, AF2319) and rat antihuman GAPDH (Biolegend, 607902) each diluted 1 : 1000 in PBS containing 0.1% w/v BSA in PBST.
  • the membrane was washed in PBST 3xl5min and subsequently incubated for Ih with a 5ml volume of donkey anti-goat IgG-HRP (Santa Cruz, SC-2020) diluted 1 :2000 in PBST, and mouse-anti rat IgG2a-AF647 (Southern Biotech, 3065-31) diluted 1 : 1000 in PBST.
  • the membrane was washed 3xl5min in PBST and stained with 3ml of SuperSignal Plus PICO West (Thermo Fisher). Chemiluminescence was immediately measured using a ChemiDoc MP Gel imaging system (Bio-Rad) at 60s exposure. Fluorescence at 647nm was similarly measured using the factory filter set and a 30s exposure.

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Abstract

L'invention concerne des molécules de fusion pour induire la proximité entre des protéines membranaires intégrales et des protéases membranaires intégrales (sheddases), favorisant ainsi la protéolyse ciblée (changement) de l'ectodomaine de protéine membranaire cible. Ces molécules de fusion, chimères ciblant les sheddases (SHEDTAC), contiennent un domaine de ciblage de protéase et un domaine de ciblage de substrat qui se lient respectivement à une sheddase et à une protéine cible d'intérêt (par exemple, un récepteur de surface cellulaire). L'invention concerne également des polynucléotides codant pour les molécules de fusion, des vecteurs d'expression associés et des cellules hôtes, ainsi que des procédés utilisant les molécules de fusion pour accélérer la protéolyse de membrane intégrale par des sheddases locales.
PCT/US2024/048537 2023-09-26 2024-09-26 Chimères ciblant les sheddases pour la modulation de la protéostase Pending WO2025072425A2 (fr)

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