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WO2025062139A1 - Conjugué du domaine c-terminal de pcsk9 et d'un anticorps anti-récepteur ou d'un fragment de liaison à l'antigène de celui-ci - Google Patents

Conjugué du domaine c-terminal de pcsk9 et d'un anticorps anti-récepteur ou d'un fragment de liaison à l'antigène de celui-ci Download PDF

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WO2025062139A1
WO2025062139A1 PCT/GB2024/052428 GB2024052428W WO2025062139A1 WO 2025062139 A1 WO2025062139 A1 WO 2025062139A1 GB 2024052428 W GB2024052428 W GB 2024052428W WO 2025062139 A1 WO2025062139 A1 WO 2025062139A1
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seq
receptor
antigen
conjugate
frizzled
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Bernard Kelly
David Owen
Stephen Graham
John Sinclair
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Cambridge Enterprise Ltd
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Cambridge Enterprise Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
    • C07K16/089—Cytomegalovirus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6454—Dibasic site splicing serine proteases, e.g. kexin (3.4.21.61); furin (3.4.21.75) and other proprotein convertases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21061—Kexin (3.4.21.61), i.e. proprotein convertase subtilisin/kexin type 9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the present invention relates to a conjugate, in particular to a conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9) or a homolog thereof, wherein the antigen-binding moiety is operable to bind to a receptor on the surface of a cell, and wherein the receptor is constitutively internalised and recycled.
  • the conjugates may be used in methods for degrading a cell surface receptor and/or inhibiting the growth of a cell.
  • Antibodies are well-known for their target specificity and are used as therapeutics to treat a wide variety of human diseases. Because of their large size ( ⁇ 150kDa) and inability to cross the plasma membrane (PM), antibodies are generally not able to engage cytoplasmic targets in vivo, being instead well-suited to targeting secreted proteins. Typically, antibody therapeutics work by blockade of a functional binding surface, by recruitment of immune effector functions leading to target cell killing (ADCC, antibody-dependent cellular cytotoxicity), or by mediating endolysosomal delivery of cytotoxic drug payloads (ADCs, antibody-drug conjugates) (Drago et al, Nat Rev Clin Oncol 18: 327-344, 2021).
  • ADCC target cell killing
  • ADCs cytotoxic drug payloads
  • PROTACs Proteolysis Targeting Chimeras
  • PROTACs which are heterobifunctional small molecule compounds consisting of a ligand for the target protein, a linker, and a ligand to recruit E3 ligase, have been utilised to induce degradation of the target proteins.
  • PROTACs forcibly recruit cytoplasmic E3 ubiquitin ligases to a target to stimulate its ubiquitination and subsequent proteasomal degradation. It has thus emerged as an alternative approach to target 'undruggable' cytoplasmic proteins.
  • LYTACs lysosome-targeting chimaeras
  • CIMPR cationindependent mannose-6-phosphate receptor
  • TM proteins transmembrane proteins
  • PM plasma membrane
  • TM proteins Internalisation from the PM is most commonly effected by clathrin-mediated endocytosis.
  • TM proteins are packaged into clathrin-coated vesicles that bud from the PM and migrate to the cell interior for onward processing.
  • TM proteins are marked as 'cargo' for clathrin-mediated endocytosis by the presence of short, linear amino acid motifs present in the unstructured parts of their cytosolic regions. These motifs are recognised and bound by 'clathrin adaptors', which simultaneously bind clathrin in order to draw the cargo into clathrin-coated vesicles as they form.
  • TM proteins have been internalised into clathrin-coated vesicles, the vesicles fuse to form early endosomes. Early endosomes undergo a complex process of maturation to become 'sorting endosomes'. A subset of TM proteins that have been internalised are trafficked back to the Golgi I trans-Golgi via the clathrin adaptor complex AP1 . Other TM proteins are recycled to the PM, and this is an important but relatively poorly-understood process. In it, TM proteins are selected as cargo to be packaged into 'tubules' that bud off from sorting endosomes and are transported back to the PM, where they fuse. Several different protein complexes are known to be involved in this process, including the retromer and retriever complexes. It is likely that these complexes recognise motifs in the cytosolic portions of the TM proteins.
  • a conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9) or a homolog thereof; wherein the antigen-binding moiety is operable to bind to a receptor on the surface of a cell; and wherein the receptor is constitutively internalised and recycled.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • a method of degrading a cell surface receptor that is constitutively internalised and recycled, the method comprising contacting the receptor with a conjugate according to the first aspect of the present invention; wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.
  • a method of inhibiting the growth of a cell having a receptor on the surface thereof comprising contacting the cell with a conjugate according to the first aspect of the present invention; wherein the receptor is constitutively internalised and recycled; wherein the antigen-binding moiety is operable to bind to the receptor; and wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.
  • a conjugate according to the first aspect of the present invention to degrade a cell surface receptor that is constitutively internalised and recycled.
  • a conjugate according to the first aspect of the present invention to inhibit the growth of a cell having receptor on the surface thereof; wherein the receptor is constitutively internalised and recycled; and wherein the antigen-binding moiety is operable to bind to the receptor.
  • a conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9), wherein the C-terminal domain of PCSK9 consists of an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36 or SEQ ID NO. 39.
  • PCSK9 proprotein convertase subtilisin/kexin type 9
  • Figure 1 shows (a) a GFP immunoblot detecting the presence of TfR-GFP in HeLa cells treated with PCTD-aGFP and aGFP alone; (b) shows TfR-GFP degradation assessed by densitometry. Data shown as Superplot: biological replicates differ by shape; large symbols indicate means.
  • receptors that are constitutively internalised and recycled will be known to a person skilled in the art.
  • the ability of a receptor to undergo constitutive internalisation and recycling may be predicted based on the presence of one or more of the internalisation and/or recycling motif defined herein, suitably based on the presence of one or more of the internalisation and/or recycling motif defined herein in a cytosolic portion of the receptor.
  • the internalisation and/or recycling motif(s) provided herein should be present in a cytosolic portion of a suitable receptor in order for said motif(s) to drive internalisation and/or recycling. Further, said motif(s) should suitably be accessible.
  • the presence of one or more of the internalisation and/or recycling motif(s) defined herein may not be indicative of internalisation and/or recycling alone, rather said motif(s) should also be present in a cytosolic portion of the receptor and, preferably, be accessible (to the endosomal trafficking system, for example).
  • the internalisation and/or recycling motif(s) may be accessible by virtue of being in an unstructured part of the cytosolic portion of the receptor, for example.
  • the antigen-binding moiety may bind to one or more receptor(s) selected from the group consisting of: HCMV US28; Erbb2; CAIX; p-glycoprotein; ADAMs, such as ADAM17; integrins; CDs, such as CD22; CD33, CD56, CD70 and CD71 ; GLUT1 ; GPCRs; LDLR; PD-1 ; PD-L1 ; voltage-gated sodium channels, such as Nav1.1 , Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Na v 1.7, Na v 1.8 and Na v 1.9; TRKA/NTRK1 channels; R-SNAREs, such as VAMP2, VAMP3, VAMP4, VAMP7 and VAMP8; LAMP1 ; LAMP2; HER2; HER3; CTLA-4, Frizzled receptors, such as Frizzled-1 , Frizzled-2, Frizzled-3, Frizzled-4, Frizzled-5
  • each of these receptors are constitutively internalised and/or recycled. It will also be appreciated by a person skilled in the art that each of these receptors i) comprise one or more of the internalisation and/or recycling motifs defined herein and/or ii) have been shown experimentally to be internalising and/or recycling.
  • the antigen-binding moiety may bind to US28.
  • US28 refers to a G-protein coupled receptor that is encoded by herpesviruses, especially CMV.
  • US28 is a rare multi-chemokine family binding receptor with the ability to bind ligands such as CCL2/MCP-1 , CCL5/RANTES, and CX3CLI/fraktalkine as ligands.
  • Ligand binding to US28 activates cell-type and ligand-specific signalling pathways leading to cellular migration, which is an important example of receptor functional selectivity. Additionally, US28 has been demonstrated to constitutively activate Gaq, phospholipase C (PLC) and NF-kB signaling pathways, amongst others.
  • PLC phospholipase C
  • NF-kB NF-kB signaling pathways
  • the antigen-binding moiety may comprise an anti-US28 antibody or fragment thereof, such as an antibody or fragment thereof comprising the amino acid sequence as set forth in SEQ ID NO. 6.
  • the antigen-binding moiety may comprise two or more copies of the amino acid sequence as set forth in SEQ ID NO. 6, for example linked by a suitable peptide linker (such as the Gly-Ser linkers described hereinbelow).
  • the antigen-binding moiety may comprise an anti-US28 antibody as set forth in SEQ ID NO. 7.
  • the antigen-binding moiety may comprise an anti-CD71 antibody or fragment thereof, such as an antibody or fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO. 8 and/or SEQ ID NO. 9, preferably as set forth in SEQ ID NO. 8.
  • the antigenbinding moiety may comprise OKT9.
  • the antigen-binding moiety may comprise a humanized version of the amino acid sequences as set forth in SEQ ID NO. 8 and/or SEQ ID NO. 9, preferably a humanized version of the amino acid sequence as set forth in SEQ ID NO. 8.
  • the antigen-binding moiety may comprise an anti-HER2 antibody or fragment thereof, such as an antibody or fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO. 10 and/or SEQ ID NO. 11 , preferably as set forth in SEQ ID NO. 10.
  • the antigenbinding moiety may comprise Trastuzumab.
  • the antigen-binding moiety may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11 .
  • the conjugate comprises the C-terminal domain of PCSK9 or a homolog thereof.
  • Human PCSK9 (SEQ ID NO. 12) has 692 amino acids and contains a pro-domain (residues 31-152); a catalytic domain (residues 153-451); and a C-terminal domain (residues 452-692), which is further divided into three modules (Du F, Hui Y, Zhang M, Linton MF, Fazio S, Fan D (December 2011). "Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein". Journal of Biological Chemistry. 286 (50): 43054-43061).
  • the N-terminal pro-domain has a flexible crystal structure and is responsible for regulating PCSK9 function by interacting with and blocking the catalytic domain, which otherwise binds the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR (Du F, Hui Y, Zhang M, Linton MF, Fazio S, Fan D (December 2011). "Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein". Journal of Biological Chemistry. 286 (50): 43054-43061 , Lo Surdo P, Bottomley MJ, Calzetta A, Settieri EC, Cirillo A, Pandit S, et al. (December 2011).
  • the C-terminal domain of PCSK9 may comprise or consist of amino acid residues 450 to 692 of human PCSK9 as set forth in SEQ ID NO. 1 .
  • the C-terminal domain of PCSK9 may comprise or consist of amino acid residues 452 to 692 of human PCSK9 as set forth in SEQ ID NO. 2.
  • the C-terminal domain of PCSK9 may comprise or consist of amino acid residues 450 to 529 of human PCSK9 as set forth in SEQ ID NO. 3.
  • a homolog of a nucleic acid sequence or amino acid sequence has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%,
  • the conjugate may further comprise a payload.
  • the cytotoxic or cytostatic agent may be a tubulin inhibitor; or a DNA interacting agent.
  • Tubulin inhibitors modulate tubulin polymerisation.
  • DNA interacting agents target cellular DNA.
  • the cytotoxic or cytostatic agent is a tubulin inhibitor.
  • the tubulin inhibitor is selected from the group consisting of: (a) an auristatin; and (b) a maytansine derivative.
  • the cytotoxic or cytostatic agent is an auristatin.
  • Auristatins include synthetic derivatives of the naturally occurring compound Dolastatin-10.
  • Auristatins are a family of antineoplastic I cytostatic pseudopeptides. Dolastatins are structurally unique due to the incorporation of 4 unusual amino acids (Dolavaine, Dolaisoleuine, Dolaproine and Dolaphenine) identified in the natural biosynthetic product.
  • the auristatin is selected from the group consisting of: Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF); vcMMAE; vcMMAF; mcMMAE and mcMMAF.
  • the cytotoxic or cytostatic agent is a maytansine or a structural analogue of maytansine.
  • the cytotoxic or cytostatic agent is a maytansine.
  • Maytansines include structurally complex antimitotic polypeptides. Maytansines are potent inhibitors of microtubulin assembly which leads towards apoptosis of tumour cells.
  • the maytansine is selected from the group consisting of: Mertansine (DM1); and a structural analogue of maytansine such as DM3 or DM4.
  • the drug is MMAE, MMAF or auristatin MMAF.
  • the cytotoxic or cytostatic agent is DNA interacting agent.
  • the DNA interacting agent is selected from the group consisting of: (a) calicheamicins, (b) duocarmycins and (c) pyrrolobenzodiazepines (PBDs).
  • the cytotoxic or cytostatic agent is a calicheamicin.
  • Calicheamicin is a potent cytotoxic agent that causes double-strand DNA breaks, resulting in cell death.
  • Calicheamicin is a naturally occurring enediyne antibiotic (A. L. Smith et al, J. Med. Chem., 1996, 39,11 , 2103-21 17).
  • Calicheamicin was found in the soil microorganism Micromonosporaechinospora.
  • the calicheamicin is calicheamicin gamma 1.
  • the drug is a duocarmycin.
  • Duocarmycins are potent anti-tumour antibiotics that exert their biological effects through binding sequence-selectively in the minor groove of DNA duplex and alkylating the N3 of adenine (D. Boger, Pure & Appl. Chem., 1994, 66, 4, 837-844).
  • PBDs exert their anti-tumour activity by covalently binding to the DNA in the minor groove specifically at purine-guanine-purine units. They insert on to the N2 of guanine via an aminal linkage and, due to their shape, they cause minimal disruption to the DNA helix. It is believed that the formation of the DNA-PBD adduct inhibits nucleic acid synthesis and causes excision-dependent single and double stranded breaks in the DNA helix. As synthetic derivatives the joining of two PBD units together via a flexible polymethylene tether allows the PBD dimers to cross-link opposing DNA strands producing highly lethal lesions.
  • the cytotoxic or cytostatic agent is a synthetic derivative of two pyrrolobenzodiazepines units joined together via a flexible polymethylene tether.
  • the pyrrolobenzodiazepine is selected from the group consisting of: Anthramycin (and dimers thereof); Mazethramycin (and dimers thereof); Tomaymycin (and dimers thereof); Prothracarcin (and dimers thereof); Chicamycin (and dimers thereof); Neothramycin A (and dimers thereof); Neothramycin B (and dimers thereof); DC-81 (and dimers thereof); Sibiromycin (and dimers thereof); Porothramycin A (and dimers thereof); Porothramycin B (and dimers thereof); Sibanomycin (and dimers thereof); Abbeymycin (and dimers thereof); SG2000; and SG2285.
  • the cytotoxic or cytostatic agent is a drug that targets DNA interstrand crosslinks through alkylation.
  • a drug that targets DNA interstrand crosslinks through alkylation is selected from: a DNA targeted mustard; a guanine-specific alkylating agent; and an adeninespecific alkylating agent.
  • the cytotoxic or cytostatic agent is a DNA targeted mustard.
  • the DNA targeted mustard may be selected from the group consisting of: an oligopyrrole; an oligoimidazole; a Bis-(benzimidazole) carrier; a Polybenzamide Carrier; and a 9-Anilinoacridine-4-carboxamide carrier.
  • the cytotoxic or cytostatic agent is selected from the group consisting of: Netropsin; Distamycin; Lexitropsin; Tallimustine; Dibromotallimustine; PNU 157977; and MEN 10710.
  • the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier.
  • the drug is Hoechst 33258.
  • a guanine-specific alkylating agent is a highly regiospecific alkylating agents that reacts at specific nucleoside positions.
  • the cytotoxic or cytostatic agent is a guaninespecific alkylating agent selected from the group consisting of: a G-N2 alkylators; a A-N3 alkylator; a mitomycin; a carmethizole analogue; a ecteinascidin analogue.
  • the mitomycin is selected from: Mitomycin A; Mitomycin C; Porfiromycin; and KW-2149.
  • the a carmeth izole analogue is selected from: Bis-(Hydroxymethyl)pyrrolizidine; and NSC 602668.
  • the ecteinascidin analogue is Ecteinascidin 743.
  • Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.
  • Cyclopropaindolones and duocamycins may be defined as adenine-specific alkylators.
  • the cytotoxic or cytostatic agent is a cyclopropaindolone analogue.
  • the drug is selected from: adozelesin; and carzelesin.
  • the cytotoxic or cytostatic agent is bizelesin.
  • the cytotoxic or cytostatic agent is a Marine Antitumour Drug. Marine Antitumour Drugs has been a developing field in the antitumour drug development arena (I. Bhatnagaret al, Mar. Drugs 2010, 8, P2702-2720 and T. L. Simmons et al, Mol. Cancer Ther. 2005, 4(2), P333-342). Marine organisms including sponges, sponge-microbe symbiotic association, gorgonian, actinomycetes, and soft coral have been widely explored for potential anticancer agents.
  • the cytotoxic or cytostatic agent is selected from: Cytarabine, Ara-C; Trabectedin (ET-743); and EribulinMesylate.
  • the EribulinMesylate is selected from: (E7389); Soblidotin (TZT 1027); Squalamine lactate; CemadotinPlinabulin (NPI-2358); Plitidepsin; Elisidepsin; Zalypsis; Tasidotin, Synthadotin; (ILX-651); Discodermolide; HT1286; LAF389; Kahalalide F; KRN7000; Bryostatin 1 ; Hemiasterlin (E7974); Marizomib; Salinosporamide A; NPI-0052); LY355703; CRYPTO 52; Depsipeptide (NSC630176); Ecteinascidin 743; Synthadotin; Kahalalide
  • the payload may be conjugated to the antigen-binding moiety and/or the C-terminal domain of PCSK9.
  • the payload may be conjugated to the antigen-binding moiety.
  • Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent.
  • Methods for conjugating the payload to the antigen-binding moiety or C-terminal domain of PCSK9 may utilise the presence of one or more non-naturally occurring amino acids.
  • the payload and/or linker may be attached via a non-naturally occurring amino acid residue present in the antigen-binding moiety or C-terminal domain of PCSK9. Methods to introduce non- naturally occurring amino acid residues are known in the art.
  • site directed mutagenesis may be used to introduce a suitable amino acid residue at a suitable position within the binding molecule.
  • a non-canonical amino acid is used to attach the payload or linker.
  • a “non-canonical” amino acid refers to one of the non-proteinogenic (unnatural) amino acids i.e., an amino acid which is not introduced via the cell’s natural translation machinery.
  • non-canonical amino acids there are many examples of non-canonical amino acids in the art many of which provide a bio- orthogonal handle on which to attach a payload.
  • non-canonical amino acid is used to attach the payload linker
  • suitable techniques are known in the art to introduce such non-canonical amino acid residues such as chemical modification, tRNA suppressor technology, engineered tRNA,/tRNA synthetase pairs.
  • the payload may be attached at various positions within the conjugate.
  • One or more copies of the payload may be attached to the conjugate, for example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 copies of the payload may be attached.
  • a method of degrading a cell surface receptor that is constitutively internalised and recycled, the method comprising contacting the receptor with a conjugate according to the first aspect of the present invention; wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.
  • the cell may be a tumour cell.
  • the conjugate may be operable to bind to a cancer cell.
  • the cancer may be selected from lung cancer, breast cancer, ovarian cancer, bowel cancer, prostate cancer, bladder cancer, colorectal cancer, pancreas carcinoma, kidney cancer, renal cancer, leukaemias, multiple myeloma, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's Lymphoma), brain cancer and other CNS and intracranial tumours cancer, head and neck cancer, oesophageal cancers, solid tumours such as sarcoma and carcinomas, mesothelioma, osteosarcoma, endometrial cancer or melanoma.
  • the cancer may be brain cancer.
  • the tumour cell may be a glioblastoma.
  • the method may comprise administering a conjugate and/or composition of the invention to a subject.
  • a subject refers to an animal which is the object of diagnosis, treatment, observation, or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.
  • the subject is preferably a human.
  • the subject may be male or female.
  • the subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric.
  • the subject may be a smoker, a former smoker or a non- smoker.
  • the subject may have a personal or family history of cancer.
  • the subject may have a cancer-free personal or family history.
  • the subject may exhibit one or more symptoms of a disease, such as a cancer.
  • Administration may by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.
  • Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration.
  • the compositions are administered orally, for example as a liquid, capsule or tablet, such as a slow release formulation.
  • the route of administration depends on the disease of interest and the target antigen. For instance, if the target antigen is present in the gastrointestinal tract, oral administration is preferable, while in case of hepatic expression either oral or intravenous administration could constitute viable options. In the case of a lung disease, such as lung cancer oral administration or inhalation may be used.
  • a conjugate according to the first aspect of the present invention to inhibit the growth of a cell having receptor on the surface thereof; wherein the receptor is constitutively internalised and recycled; and wherein the antigen-binding moiety is operable to bind to the receptor.
  • the composition may comprise one or more conjugate of the invention.
  • the composition may comprise only one conjugate of the invention.
  • the composition may comprise two or more conjugates of the invention.
  • the pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form.
  • carrier refers to a diluent, adjuvant or excipient, with which conjugate of the present invention is administered.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • auxiliary, stabilizing, thickening, lubricating and colouring agents can be used.
  • the conjugates, compositions and/or pharmaceutically acceptable carriers may suitably be sterile.
  • Water is a preferred carrier when the compound of the present invention is to be administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the present compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be in the form of a liquid, e.g., a solution, emulsion or suspension.
  • the liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously.
  • composition When intended for oral administration, the composition may be in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.
  • the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • a solid composition typically contains one or more inert diluents.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent.
  • a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.
  • the composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension.
  • the liquid can be useful for oral administration or for delivery by injection.
  • a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
  • compositions can take the form of one or more dosage units.
  • the term “effective amount” means an amount of conjugate that, when administered to a cell, tissue, or subject, is effective to achieve the desired diagnostic and/or therapeutic effect under the conditions of administration.
  • the term “effective amount” is intended to denote a non- lethal but sufficient amount to allow diagnosis of the disorder.
  • the term “effective amount” is intended to denote one which eliminates or diminishes the symptoms associated with the disorder (e.g., by eliminating or reducing the size of a tumour, for example).
  • An effective amount may be determined by one of ordinary skill in the art, using routine experimentation.
  • [250] piggyBac construction of BC2-CD8a reporter cell lines Cargo vectors encoding the NPXY, AMOTIF and YQRL reporter constructs under the control of a constitutive chicken b-actin promoter (with CMV enhancer and chicken p-actin/rabbit p-globin chimeric intron) flanked by PiggyBac Transposase recognition sequences was mixed with Super PiggyBac transposase vector (System Biosciences) in a 5:1 mass ratio (cargo:transposase) and transfected into HeLa cells (grown in a 6-well dish) using a TransIT-HeLaMONSTER transfection kit according to the manufacturer's protocol.
  • the cells were dissociated with Accutase and plated onto a T75 tissue culture flask. Once near confluence the cells were dissociated with Accutase, pelleted by centrifugation at 300g, the pellet washed with PBS and gently resuspended in PBS supplemented with 1% FBS, and the cells sorted for GFP fluorescence on a Becton Dickinson FACSMelody sorter.
  • TfR-GFP reporter cell line by retroviral transduction Transient DNA transfections were carried out using TransIT-HeLaMONSTER® kit (Mirus, Cambridge Bioscience Ltd, UK) following the manufacturer’s instructions.
  • HEK 293ET cells were co-transfected with the appropriate retroviral vector and the packaging plasmids pMD.GagPol and pMD.VSVG (University of Cambridge) in the ratio of 50:30:15.
  • Virus-containing supernatant was filtered through a 0.45 pm filter and applied directly to the target cells. The cells were subsequently sorted for GFP fluorescence on a Becton Dickinson FACSMelody sorter.
  • HEK F expression of proteins by transient transfection pHLSec vectors containing PCTD-nanobody fusions or OKT9 fab fragments were transfected into 30ml HEK F cells (Thermo Fisher) at a density of 1x10 6 cells/ml with 30pg DNA using 293Fectin (Thermo Fisher), following the manufacturer's instructions.
  • OKT9 fab fragments 15pg of each vector were mixed and transfected in the same way.
  • E. coli expression of nanobodies pMW vectors encoding the aBC2 and aGFP nanobodies were expressed in E. coli strain BL21 (DE3)pLysS and purified by NiNTA affinity chromatography as for the HEK F-expressed proteins.
  • Antibodies The following antibodies were used:
  • Anti-GFP (Abeam, ab6556, rabbit polyclonal);
  • Anti-actin (Sigma, A2066, rabbit polyclonal);
  • NiNTA beads were collected in a glass column, washed with 50 ml of 20mM Tris pH 8, 300 mM NaCI, and bound protein eluted with 20mM Tris pH 8, 300 mM NaCI, 300 mM imidazole (pH 8), concentrated in Amicon Ultra-15 concentrators and dialyzed against Dulbecco's PBS.
  • Total protein concentration was measured using a BCA assay (Pierce) and typically 40pg of protein was loaded on a Bio-Rad TGX 4-20% SDS-PAGE gel and blotted using a Bio-Rad wet transfer system onto Amersham Protran nitrocellulose membranes for 2 hours at 70V constant. The membranes were blocked for 1 hour at 21 °C with PBS + 5% skimmed milk powder (Marvel). Anti-GFP, anti-actin and anti-TfR were used at 1 :1000 dilutions into PBS + 5% skimmed milk powder + 0.1 % Tween-20, and incubated with the blot overnight at 4°C.
  • Blots were washed three times with PBS + 0.1 % Tween- 20 (5 minutes per wash) before addition of goat Anti-Rabbit IgG H&L (Alexa Fluor® 680) diluted 1 :1000 in PBS + 5% skimmed milk powder + 0.1 % Tween-20 and incubation for 1 hour at 21 °C. Blots were washed three times as above and imaged using a Li-Cor Odyssey near-infrared fluorescence imaging system. Blot bands were quantitated by densitometry using Fiji.
  • PCTD- nanobody fusions 5pg/ml concentrations of PCTD- nanobody fusions were used unless otherwise indicated, or a molar equivalent (120 nM) of the nanobody. 'Overnight' treatments were typically done for 16 hours before cells were lysed. Where used, Bafilomycin A1 (or an equal volume of DMSO) was added at 100nM 2 hours prior to treatment with PCTD fusions or control antibodies.
  • the anti- TfR antibody OKT9 was fused to PCTD. As described in detail above, this was achieved by expressing and purifying a Fab fragment of OKT9 with PCTD fused to the C terminus of the truncated heavy chain (OKT9FabPCTD) via co-transfection of HEK-293F cells with vectors encoding the light chain (VL-CL) and the heavy chain-PCTD fusion (VH-CH1-PCTD). As a control, a modified version of this construct with the PCTD domain replaced by a dual HA (hemagglutinin) tag (OKT9FabHA), was expressed and purified.
  • NPXY an NPXY-type motif that drives both internalisation by clathrin-mediated endocytosis and PM recycling (Chen, W. J., Goldstein, J. L. & Brown, M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J. Biol. Chem. 265, 3116-3123 (1990) and Steinberg, F., Heesom, K. J., Bass, M. D. & Cullen, P. J. SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways. J. Cell Biol. 197, 219-230 (2012));
  • AMOTIF lacks known internalisation motifs
  • YQRL a motif known to direct efficient internalisation from the PM and rapid rerouting from early endosomal compartments to the trans-Golgi network (Bos, K., Wraight, C. & Stanley, K. K. TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain. EMBO J. 12, 2219-2228 (1993)).
  • a virus-specific nanobody that functions as a partial inverse agonist of US28 and induces the reactivation of HCMV from latency, has recently been developed (De Groof, T. W. M. et al. Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies. Nat. Common. 12, 4436 (2021)).
  • the induction of viral IE gene expression offers therapeutic potential, as reactivation allows infected cells to be recognized by HCMV-specific cytotoxic T-cells.
  • Vun100bv (SEQ ID NO. 7) was fused to the C terminus of PCTD to generate a virus-specific nanobody conjugate (PCTD-Vun100bv; SEQ ID NOs 34 and 35, wherein SEQ ID NO. 35 is the amino acid sequence as expressed).
  • PCTD-Vun100bv was fused to the C terminus of PCTD to generate a virus-specific nanobody conjugate (PCTD-Vun100bv; SEQ ID NOs 34 and 35, wherein SEQ ID NO. 35 is the amino acid sequence as expressed).
  • PCTD was fused to the C-terminus of the light chain (SEQ ID NO. 30) and co-expressed with the heavy chain (SEQ ID 10); in the other, PCTD was fused to the C-terminus of the heavy chain (SEQ ID NO. 31) and co-expressed with the light chain (SEQ ID 11).
  • the PCTD fused to the C-terminus of the heavy chain as expressed in HeLa cells is provided in SEQ ID NO. 32.
  • SKBR3 cells were seeded at a density of 1x10 6 cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium containing trastuzumab at 5pg/ml, a molar equivalent of trastuzumab-PCTD fusion, or PBS.
  • PCTD comprises subdomains defined here as M1 (residues 450-529), M2 (residues 530- 605) and M3 (residues 606-692).
  • M1 residues 450-529
  • M2 residues 530- 605
  • M3 residues 606-692
  • a fusion of the M1 subdomain with the aBC2 nanobody (M1- aBC2) was expressed in HEK F cells (SEQ ID NO.33), and tested its ability to cause degradation of a BC2-tagged NPXY reporter construct as follows.
  • HeLa cells stably expressing a chimeric NPXY reporter comprising an extracellular CD8 a-chain tagged with the p-catenin-derived BC2 peptide tag, a single transmembrane helix, a cytoplasmic domain comprising an 'NPXY' sorting motif embedded within an unstructured sequence and an intracellular EGFP for fluorescence detection, were seeded at a density of 1x10 6 cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium supplemented with M1-aBC2 or PCTD-aBC2 at 5pg/ml.
  • HeLa cells stably expressing a chimeric NPXY reporter comprising an extracellular CD8 a-chain tagged with the p-catenin-derived BC2 peptide tag, a single transmembrane helix, a cytoplasmic domain comprising an 'NPXY' sorting motif embedded within an unstructured sequence and an intracellular EGFP for fluorescence detection, were seeded at a density of 1x10 6 cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium supplemented with M1-aBC2 or PCTD-aBC2 at 5pg/ml.
  • M1 SOL1 When expressed in isolation, the M1 subdomain displays limited solubility, beginning to precipitate above concentrations of ⁇ 1 mg/ml in PBS.
  • Trp453, Leu455 and Leu529 which were identified as key hydrophobic residues by removal of M2/M3, were each mutated to serine (M1 SOL1 ; SEQ ID NO. 36).
  • M1 SOL1-ocBC2 A fusion of M1 SOL1 with the ocBC2 nanobody was expressed in HEK F cells (M1 SOL1-ocBC2; SEQ ID NO. 37, wherein SEQ ID NO. 38 is the amino acid sequence as expressed).
  • This construct displayed improved solubility, not beginning to precipitate until concentrations above ⁇ 4mg/ml in PBS.
  • the ability of M1 SOL1-aBC2 to cause degradation of the BC2-tagged NPxY reporter construct was tested as follows.
  • HeLa cells stably expressing the chimeric NPxY reporter were seeded at a density of 1x10 6 cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium supplemented with M1 SOL1-aBC2 or PCTD-aBC2 at 5pg/ml, or left untreated. The following morning the cells were washed in PBS, dissociated with Accutase, washed in complete medium, resuspended in PBS, and analysed by flow cytometry. EGFP fluorescence was measured on a Becton Dickinson LSR Fortessa and the data analysed with FlowJo software. The results are shown in Figure 13.
  • NPXY reporter remaining after treatment is shown as a % of untreated. Means and standard deviation from 2 experiments is shown.
  • One-way ANOVA analysis of mean fluorescence indicates that M1 SOL drives substantial degradation, indicated by significant (* denotes p ⁇ 0.01) reduction of mean reporter fluorescence, similar to PCTD (ns indicates no significant difference between PCTD and M1 SOL).
  • NPXY reporter remaining after treatment is shown as a % of untreated. Means and standard deviation from 2 or 3 (for M1-M2-ocBC2 and M1-M3-ocBC2) experiments shown.
  • Tukey Tukey
  • One-way ANOVA analysis of mean fluorescence indicates that both M1-M2 and M1-M3 drove degradation, indicated by significant (*** denotes p ⁇ 0.0001) reduction of mean reporter fluorescence, with the M1-M3 fusion being the most effective.

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Abstract

La présente invention concerne un conjugué, en particulier un conjugué comprenant une fraction de liaison à l'antigène et le domaine C-terminal de la proprotéine convertase subtilisine/kexine de type 9 (PCSK9) ou un homologue de celui-ci, la fraction de liaison à l'antigène étant utilisable pour se lier à un récepteur sur la surface d'une cellule, et le récepteur étant constitutivement internalisé et recyclé. Les conjugués peuvent être utilisés dans des procédés de dégradation d'un récepteur de surface cellulaire et/ou d'inhibition de la croissance d'une cellule.
PCT/GB2024/052428 2023-09-20 2024-09-20 Conjugué du domaine c-terminal de pcsk9 et d'un anticorps anti-récepteur ou d'un fragment de liaison à l'antigène de celui-ci Pending WO2025062139A1 (fr)

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