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WO2025206033A1 - PEPTIDE HLH LIANT uPAR, CONJUGUÉ PEPTIDE−MÉDICAMENT ET COMPOSITION - Google Patents

PEPTIDE HLH LIANT uPAR, CONJUGUÉ PEPTIDE−MÉDICAMENT ET COMPOSITION

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Publication number
WO2025206033A1
WO2025206033A1 PCT/JP2025/012193 JP2025012193W WO2025206033A1 WO 2025206033 A1 WO2025206033 A1 WO 2025206033A1 JP 2025012193 W JP2025012193 W JP 2025012193W WO 2025206033 A1 WO2025206033 A1 WO 2025206033A1
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WO
WIPO (PCT)
Prior art keywords
upar
peptide
binding
amino acid
helix
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/012193
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English (en)
Japanese (ja)
Inventor
郁雄 藤井
雅孝 道上
勇登 中谷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University Public Corporation Osaka
Original Assignee
University Public Corporation Osaka
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Filing date
Publication date
Application filed by University Public Corporation Osaka filed Critical University Public Corporation Osaka
Publication of WO2025206033A1 publication Critical patent/WO2025206033A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

Definitions

  • Urokinase-type plasminogen activator receptor is a GPI-anchored membrane receptor expressed on the cell surface and is generally known to be involved in all physiological and pathological processes involving extracellular matrix (ECM) remodeling, such as embryogenesis, inflammation, tissue repair, tumor invasion, and metastasis.
  • ECM extracellular matrix
  • uPAR binds to its ligand, urokinase-type plasminogen activator (uPA), promoting pericellular activation of the plasmin system by uPA and subsequent degradation of the extracellular matrix (ECM), which is thought to contribute to cell migration and other processes.
  • ECM extracellular matrix
  • Peptides with a helix-loop-helix (HLH) structure are disclosed in Patent Documents 1 and 2, among others, as peptide-based molecules that bind to targets.
  • Peptides with a helix-loop-helix structure include an N-terminal helix region, a C-terminal helix region, and a loop region connecting them.
  • the N-terminal and C-terminal helices each form an ⁇ -helical coiled-coil structure.
  • these peptides adopt stable secondary structures in solution, making it easy to introduce functional groups with different chemical properties into the solvent-exposed portions of the molecule (Non-Patent Document 1). Utilizing these properties, various peptides with helix-loop-helix structures that possess physiological activity have been proposed.
  • the present disclosure aims to provide uPAR-binding HLH peptides, peptide-drug conjugates (PDCs), and compositions containing the uPAR-binding HLH peptides or the PDCs.
  • PDCs peptide-drug conjugates
  • the inventors constructed a peptide library that retained its three-dimensional structure using phage surface display, performed screening, and obtained peptides that bind to uPAR. Furthermore, they obtained PDCs in which drugs were linked to the obtained peptides.
  • the present disclosure relates to a peptide (“uPAR-binding peptide”) that binds to the urokinase-type plasminogen activator receptor (uPAR), comprising a (helix 1)-(loop)-(helix 2) structure from the N-terminus to the C-terminus.
  • uPAR-binding peptide that binds to the urokinase-type plasminogen activator receptor (uPAR), comprising a (helix 1)-(loop)-(helix 2) structure from the N-terminus to the C-terminus.
  • the present disclosure relates to a uPAR-binding peptide-drug conjugate (PDC) comprising the uPAR-binding peptide and a drug linked to the peptide.
  • PDC uPAR-binding peptide-drug conjugate
  • the present disclosure relates to a composition comprising the uPAR-binding peptide or the PDC.
  • the present disclosure provides a uPAR-binding HLH peptide, a peptide-drug conjugate (PDC) in which a drug is linked to the uPAR-binding HLH peptide, and a composition comprising the peptide or the PDC.
  • PDC peptide-drug conjugate
  • the present disclosure relates to peptides ("uPAR-binding peptides”) that bind to the urokinase-type plasminogen activator receptor (uPAR), including a helix-loop-helix (HLH) structure, peptide-drug conjugates (PDCs) in which a drug is linked to the uPAR-binding peptide, and compositions containing the uPAR-binding peptide or PDC, as well as uses thereof.
  • uPAR-binding peptides that bind to the urokinase-type plasminogen activator receptor (uPAR), including a helix-loop-helix (HLH) structure, peptide-drug conjugates (PDCs) in which a drug is linked to the uPAR-binding peptide, and compositions containing the uPAR-binding peptide or PDC, as well as uses thereof.
  • the present disclosure relates to a uPAR-binding peptide (also referred to herein as a uPAR-binding HLH peptide or simply as a uPAR-binding peptide) that comprises a (helix 1)-(loop)-(helix 2) structure from the N-terminus to the C-terminus.
  • a uPAR-binding peptide also referred to herein as a uPAR-binding HLH peptide or simply as a uPAR-binding peptide
  • helix 1 and helix 2 are each an ⁇ -helix.
  • the uPAR-binding HLH peptide can form a stable structure by bringing the two helices closer together through hydrophobic interactions between the leucine residues contained in helix 1 and helix 2 and electrostatic interactions between glutamic acid and lysine residues.
  • the amino acid residues located in the solvent-exposed regions of the two helices that are closer together contribute little to the three-dimensional structure of the HLH peptide; rather, by constituting the regions that interact with the target, they can independently contribute to the binding activity of the HLH peptide to its target.
  • Helix 1 and helix 2 may have any sequence length that maintains a stable helix structure, for example, approximately 4 to 40 residues.
  • the sequence length of the helix is determined appropriately depending on factors such as target specificity, stability, and intracellular internalization.
  • the lengths of helix 1 and helix 2 may be equal or may differ by no more than two amino acid residues.
  • helix 1 and helix 2 may be 10 to 20 amino acid residues.
  • helix 1 consists of 14 amino acid residues and helix 2 consists of 14 or 15 amino acid residues.
  • helix 1 and helix 2 consist of 14 amino acid residues.
  • X9 , X10 , X12 , and X14 may be any amino acid.
  • X9 , X10 , X12 , and X14 are amino acids suitable for maintaining a helix structure, such as amino acids other than C, P, G, or S, and more preferably amino acids selected from the group consisting of A, R, N, E, H, L, K, M, F, V, and Y.
  • X9 is A.
  • X10 is A.
  • X12 is A.
  • X14 is A.
  • Helix 2 of the uPAR-binding HLH peptide can be helix 2-2 comprising or consisting of the amino acid sequence KLX17X18LX19X20KLX21X22LKX23X24 (SEQ ID NO : 3).
  • X20 is F, Y , or W
  • X22 is R or K
  • X23 is L , I , or V
  • X24 is L, I, or V.
  • X20 is F, Y , or W
  • X22 is R
  • X23 is L, I, or V
  • X24 is L , I, or V.
  • X17 , X18 , X19 , and X21 may be any amino acid.
  • X17 , X18 , X19 , and X21 are amino acids suitable for maintaining a helix structure, such as amino acids other than C, P, G, or S, and more preferably amino acids selected from the group consisting of A, R, N, E, H, L, K, M, F, V, and Y.
  • X17 is A.
  • X18 is A.
  • X19 is K.
  • X21 is A.
  • helix 2-1 comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 13-18.
  • helix 2-2 comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 20-25.
  • helix 2-1 comprises the amino acid sequence set forth in any of SEQ ID NOs: 13-18.
  • helix 2-2 comprises the amino acid sequence set forth in any of SEQ ID NOs: 20-25.
  • helix 2 may not have specific amino acid residues that contribute to maintaining binding to uPAR.
  • X9 to X24 may be any amino acid, preferably an amino acid that is suitable for maintaining a helix structure, for example, an amino acid other than C, P, G, or S, and more preferably an amino acid selected from the group consisting of A, R, N, E, H, L, K, M, F, V, and Y.
  • helix 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 26.
  • helix 2 consists of the amino acid sequence set forth in SEQ ID NO: 19 or 26.
  • the uPAR-binding HLH peptide comprises helix 1 comprising or consisting of the amino acid sequence of X 1 ELX 2 X 3 LX 4 X 5 ELX 6 X 7 LX 8 (SEQ ID NO: 1), and helix 2, which is helix 2-1 comprising or consisting of the amino acid sequence of KLX 9 X 10 LX 11 X 12 KLX 13 X 14 LX 15 X 16 (SEQ ID NO: 2) or helix 2-2 comprising or consisting of the amino acid sequence of KLX 17 X 18 LX 19 X 20 KLX 21 X 22 LKX 23 X 24 (SEQ ID NO: 3), wherein X 1 to X 24 represent amino acid residues;
  • These amino acid sequences satisfy the following conditions (a) to (e): (a) X2 is F, Y or W, X5 is R or K, and X6 and X7 are each independently L, I or V; (b) X 11 is
  • the uPAR-binding HLH peptide comprises a helix 1 comprising or consisting of the amino acid sequence X 1 ELX 2 X 3 LX 4 X 5 ELX 6 X 7 LX 8 (SEQ ID NO: 1), and a helix 2-1 comprising or consisting of the amino acid sequence KLX 9 X 10 LX 11 X 12 KLX 13 X 14 LX 15 X 16 (SEQ ID NO: 2), wherein X 1 to X 16 represent amino acid residues; X2 is F, Y or W, X5 is R or K, X6 is L, I or V, X7 is L, I or V, and optionally, X1 , X3 , X4 and X8 to X16 are each independently any amino acid, preferably an amino acid suitable for maintaining a helical structure, for example, an amino acid other than C, P, G or S, more preferably an amino acid selected from the group consisting of A, R
  • the uPAR-binding HLH peptide comprises a helix 1 comprising or consisting of the amino acid sequence X 1 ELX 2 X 3 LX 4 X 5 ELX 6 X 7 LX 8 (SEQ ID NO: 1), and a helix 2-1 comprising or consisting of the amino acid sequence KLX 9 X 10 LX 11 X 12 KLX 13 X 14 LX 15 X 16 (SEQ ID NO: 2), wherein X 1 to X 16 represent amino acid residues; X11 is F, Y or W, X13 is R or K, X15 and X16 are each independently L, I or V, and optionally, X1 to X8 , X9 , X10 , X12 and X14 are each independently any amino acid, preferably an amino acid suitable for maintaining a helix structure, for example, an amino acid other than C, P, G or S, more preferably an amino acid selected from the group consisting of A,
  • the uPAR-binding HLH peptide comprises a helix 1 comprising or consisting of the amino acid sequence X 1 ELX 2 X 3 LX 4 X 5 ELX 6 X 7 LX 8 (SEQ ID NO: 1), and a helix 2-2 comprising or consisting of the amino acid sequence KLX 17 X 18 LX 19 X 20 KLX 21 X 22 LKX 23 X 24 (SEQ ID NO: 3), wherein X 1 to X 8 and X 17 to X 24 represent amino acid residues;
  • These amino acid sequences satisfy the following conditions (a) and (b): (a) X2 is F, Y or W, X5 is R or K, and X6 and X7 are each independently L, I or V; (b) X20 is F, Y or W, X22 is R or K, and X23 and X24 are each independently L, I or V; and optionally, X1 , X3 ,
  • the loop sequence may be any amino acid sequence that does not prevent the two helices from interacting with each other to maintain a stable configuration.
  • the loop sequence may or may not contain a 3D structure such as a helix or sheet, but preferably does not contain a 3D structure.
  • G or P may be selected as an amino acid suitable for the loop sequence because it is less likely to form a 3D structure, and G is preferably selected from the perspective of simple structure.
  • the loop sequence contains at least one G or P, for example, 1 to 20 G or P. The length of the loop sequence is determined appropriately depending on the stability of the peptide, its intracellular transport, the activity of the peptide-drug conjugate, and the like.
  • the loop sequence has a length of 3 to 20, 5 to 15, or 7 to 11 amino acids. In preferred embodiments, the loop sequence is 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or 100%, of its sequence is G or P, preferably G. In some embodiments, the loop sequence is a sequence of 3 to 20 consecutive Gs, or a sequence with 1, 2, 3, 4, or 5 amino acid substitutions therein. In some embodiments, the loop is a sequence of 7 consecutive Gs, or a sequence with 1 or 2 amino acid substitutions therein. In some embodiments, the loop is a sequence of 7 consecutive Gs, or a sequence with 1 or 2 amino acid substitutions at the second and/or seventh positions.
  • the amino acid substitutions may be with any amino acid, but are preferably with K.
  • the loop sequence consists of G and K.
  • the loop sequence comprises or consists of a sequence set forth in any of SEQ ID NOs: 27-30.
  • the uPAR-binding HLH peptide may further comprise 1 to 3 amino acids at the N-terminus of helix 1 and/or the C-terminus of helix 2. These amino acids make a small contribution to maintaining the HLH structure and the uPAR-binding activity of the HLH peptide, and may be any amino acid sequence. In some embodiments, the uPAR-binding HLH peptide further comprises one or two amino acids at the N-terminus of helix 1 and/or the C-terminus of helix 2.
  • the uPAR-binding HLH peptide further comprises two amino acids at the N-terminus of helix 1 and two amino acids at the C-terminus of helix 2-1 or one amino acid at the C-terminus of helix 2-2. In some embodiments, the uPAR-binding HLH peptide further comprises CA at the N-terminus of helix 1 and CA at the C-terminus of helix 2-1 or C at the C-terminus of helix 2-2.
  • the linker is any linker that does not affect the helix structure and higher-order structure of the uPAR-binding HLH peptide. Therefore, a linker with a high degree of structural flexibility is preferred; for example, the linker does not include a specific three-dimensional structure such as a helix or sheet.
  • linkers include linkers in which carbon atoms are linked in a linear chain and linkers in which amino acids are peptide-bonded. The length of the linker is determined appropriately depending on the stability of the peptide, its intracellular transport, the activity of the peptide-drug conjugate, and other factors.
  • the uPAR-binding HLH peptide comprises a cyclic structure in which the N-terminal and C-terminal cysteine residues are linked by a disulfide bond. In some embodiments, the uPAR-binding HLH peptide comprises a cyclic structure in which a chloroacetylated amino acid at either the N-terminus or the C-terminus is linked to a cysteine residue at the other terminus by forming a thioether bond.
  • the uPAR-binding HLH peptide may have a sequence length of at least 35 amino acids.
  • the sequence length of the uPAR-binding HLH peptide may be 35 to 120 amino acids.
  • the uPAR-binding HLH peptide is 120 amino acids or less, 110 amino acids or less, 100 amino acids or less, 90 amino acids or less, 80 amino acids or less, 70 amino acids or less, 60 amino acids or less, 55 amino acids or less, or 50 amino acids or less.
  • sequence length of the uPAR-binding HLH peptide is 35 to 45 amino acids, for example, 45 amino acids, 44 amino acids, 43 amino acids, 42 amino acids, 41 amino acids, 40 amino acids, 39 amino acids, 38 amino acids, 37 amino acids, 36 amino acids, or 35 amino acids. In some embodiments, the sequence length of the uPAR-binding HLH peptide is 39 amino acids.
  • C represents cysteine
  • A represents alanine
  • Y represents tyrosine
  • H represents histidine
  • R represents arginine
  • G represents glycine
  • E represents glutamic acid
  • L represents leucine
  • V valine
  • W represents tryptophan
  • T represents threonine
  • S represents serine
  • I represents isoleucine
  • F represents phenylalanine
  • D represents aspartic acid
  • N represents asparagine
  • Q represents glutamine
  • M represents methionine
  • K represents lysine
  • P proline.
  • amino acid sequence of a peptide is written from left to right from the amino terminus (N-terminus) to the carboxy terminus (C-terminus).
  • amino acid mutations include amino acid deletions, substitutions, insertions, and additions.
  • An amino acid mutation refers to an amino acid residue that differs from that of a reference sequence when a sequence is aligned to the reference sequence according to standard methods in the art. Because amino acid deletions, insertions, and additions change the length of the sequence, when referring to amino acids within a helical region, the amino acid mutation is preferably an amino acid substitution.
  • six or fewer amino acid mutations may be allowed when the full length of the uPAR-binding HLH peptide is used as the reference sequence.
  • two or fewer amino acid mutations may be allowed when the sequence of one helical region is used as the reference sequence.
  • two or fewer amino acid mutations may be allowed when a sequence other than the helical region is used as the reference sequence.
  • the uPAR-binding HLH peptide exhibits binding affinity to human urokinase-type plasminogen activator receptor (uPAR).
  • binding affinity is represented by a dissociation constant ( KD ) measured by the method described in the Examples.
  • KD dissociation constant
  • a peptide is said to bind to a target if it exhibits a KD of at least about 10,000 nM or less for the target.
  • the uPAR-binding HLH peptide exhibits a KD of at least about 10,000 nM or less, preferably 1000 nM or less, 900 nM or less, 800 nM or less, 700 nM or less, 600 nM or less, 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 5 ...500 nM or less, 600 nM or less, 700 nM or less, 800 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900 nM or less, 900
  • the uPAR-binding HLH peptides exhibit a KD of 600 nM or less. In preferred embodiments, the uPAR-binding HLH peptides exhibit a KD of 5 nM or less.
  • the uPAR-binding HLH peptide inhibits the interaction between uPAR and uPA.
  • the inhibitory activity of the uPAR-binding HLH peptide against uPAR-uPA interaction is measured by the method described in the Examples.
  • "a peptide inhibits interaction” means that addition of the peptide reduces uPAR-uPA interaction by at least 30%, relative to uPAR-uPA interaction under conditions in which the peptide is not added.
  • the uPAR-binding HLH peptide reduces uPAR-uPA interaction by at least 30%, preferably 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%, relative to uPAR-uPA interaction under conditions in which the uPAR-binding HLH peptide is not added.
  • the uPAR-binding HLH peptide exhibits an IC50 of at least about 1000 nM or less, preferably 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 0.1 nM or less, or 0.01 nM or less, in inhibiting the uPA- uPAR interaction as measured according to the methods described in the Examples.
  • the uPAR-binding HLH peptides of the present disclosure inhibit the uPAR-uPA interaction, thereby reducing the degradation of the extracellular matrix by plasmin and functioning to suppress cell migration, i.e., metastasis, of cells, particularly cancer cells.
  • the uPAR-binding HLH peptides of the present disclosure can be translocated into cells expressing uPAR. It has been reported in the art that uPAR is translocated into cells by endocytosis. Therefore, since ligands that bind to uPAR are translocated into cells along with the translocation of uPAR, it can be considered that ligands with high binding affinity for uPAR are advantageous for translocation into cells. While not wishing to be bound by any particular theory, uPAR-binding HLH peptides have an advantage in translocation into cells by endocytosis because they have a smaller molecular weight than, for example, polymeric ligands such as anti-uPAR antibodies.
  • uPAR-binding HLH peptides can be modified as needed to adjust any of their properties, including peptide stability, solubility, intracellular transport, bioavailability, and peptide-drug conjugate activity, as desired. Examples of modifications include halogenation, polyethylene glycol (PEG)ation, acetamidomethyl (Acm)ation, phosphorylation, glycosylation, biotinylation, glutathione-S-transferase (GST) fusion, and protein A/G fusion.
  • the uPAR-binding HLH peptide is PEGylated.
  • the uPAR-binding HLH peptide is chlorinated for cyclization.
  • the uPAR-binding HLH peptide is Acmylated to protect cysteine residues.
  • the uPAR-binding HLH peptides of the present disclosure may be produced by any method known to those of skill in the art.
  • the uPAR-binding HLH peptides may be produced chemically synthetically using solid-phase synthesis.
  • the uPAR-binding HLH peptides may be produced by transient or stable expression in host cells. Examples of host cells include E. coli, yeast, plant cells, insect cells, and mammalian cells.
  • uPAR-binding HLH peptides can be purified by any method known to those of skill in the art. Examples of purification methods include, for example, chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion chromatography), centrifugation, differential solubility, or other standard techniques for protein purification. In some embodiments, uPAR-binding HLH peptides are purified by reverse-phase high-performance liquid chromatography (RP-HPLC).
  • RP-HPLC reverse-phase high-performance liquid chromatography
  • the present disclosure relates to a nucleic acid sequence encoding the uPAR-binding HLH peptide of the present disclosure.
  • the nucleic acid sequence encoding the uPAR-binding HLH peptide of the present disclosure can be codon-optimized as appropriate by one of skill in the art depending on its intended use.
  • the nucleic acid sequence encodes the amino acid sequence set forth in any of SEQ ID NOs: 32-43.
  • the nucleic acid sequence is the sequence set forth in any of SEQ ID NOs: 47-58.
  • the present disclosure relates to a vector comprising a nucleic acid sequence encoding a uPAR-binding HLH peptide of the present disclosure.
  • a vector is any molecule used to transfer protein-coding information into a host cell, and includes, for example, a nucleic acid, a plasmid, a bacteriophage, or a viral vector.
  • the vector may further comprise any regulatory sequence, such as a promoter, for regulating expression of the HLH peptide of the present disclosure.
  • the present disclosure relates to a peptide-drug conjugate (PDC) comprising a uPAR-binding HLH peptide of the present disclosure and a drug linked to the peptide.
  • the drug is any molecule that has an independent action function
  • the uPAR-binding HLH peptide contributes to delivering such a drug to its site of action, particularly to cells expressing uPAR or tissues containing such cells, or to lesions.
  • the drug is a detectable label, including enzyme labels, fluorescent labels, radiolabels, etc. Because the detectable label does not require endocytosis by uPAR-expressing cells, labels of any molecular weight can be used.
  • exemplary labels include enzyme labels such as horseradish peroxidase (HRP), alkaline phosphatase (AP), or glucose oxidase.
  • example labels include Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor® 750, BODIPY® FL, coumarin, Cy® 3, Cy® 5, fluorescein (FITC), Oregon Green®, Pacific Examples of labels include fluorescent labels such as BlueTM, Pacific GreenTM, Pacific OrangeTM, tetramethylrhodamine (TRITC), Texas Red®, or other fluorescent labels.
  • Alexa Fluor® 350 Alexa Fluor® 405, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor® 750, BODIPY® FL
  • the drug is a therapeutic agent, such as a cancer therapeutic agent or an immunosuppressant.
  • the therapeutic agent is a cancer therapeutic agent.
  • the cancer therapeutic agent includes a cytotoxic agent. The cytotoxic agent exerts cytotoxicity by targeting, for example, DNA or microtubules.
  • the cytotoxic agent is an auristatin (such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), Aur0101, PF06380101, auristatin W, or auristatin F) or a derivative thereof, maytansinoid (such as DM1 or DM4), pyrrolobenzodiazepine (PBD) (such as SGD1882 or SG3199), indolinobenzodiazepine (such as DGN462 or DGN549), calicheamicin (ozogamicin) (such as CM1), camptothecin analogs (SN38, DX-8951f, or is a DX-8951f derivative), duocarmycin (such as seco-duocarmycin-hydroxy-benzamide-azaindole (seco-DUBA), minor groove binding alkylating agent (MGBA), or MED-2460), tubulin inhibitor (such as cryptophycin), tubulys
  • the PDC of the present disclosure may include at least one drug, e.g., one, two, three, or more drugs.
  • the PDC of the present disclosure is linked to a drug at a natural or unnatural amino acid at any position within the peptide that is suitable for linking a drug. Any method known in the art may be used to link a drug to a peptide.
  • the drug is linked via a covalent bond to the side chain of an amino acid residue at the terminus of the peptide or at another position.
  • the drug may be linked via a carboxyl group at the peptide terminus, an amino group at K, or a thiol group at C.
  • the drug is linked via a reaction of a thiol at C introduced at any position with a maleimide.
  • the drug is linked to the peptide via a C residue at the C terminus.
  • the drug is linked to the peptide via a C residue at the N terminus.
  • the PDC of the present disclosure includes a linker that connects the uPAR-binding peptide and the drug.
  • linkers include linkers in which carbon atoms are bonded in a linear chain, and linkers in which amino acids are bonded by peptide bonds. The length of the linker is determined appropriately depending on the stability of the peptide, its intracellular transport, the activity of the peptide-drug conjugate, and the like.
  • the linker includes a linker in which one or more methylene groups are bonded in a linear chain.
  • the linker includes a peptide chain in which one or more amino acids are bonded by amide bonds.
  • the linker is a cleavable linker or a non-cleavable linker.
  • the linker is preferably a non-cleavable linker.
  • the linker is preferably a cleavable linker.
  • the linker comprises one or more PEGs.
  • the cleavable linker may be cleavable under intracellular conditions (e.g., intracellular enzymes, pH, redox, temperature).
  • intracellular conditions e.g., intracellular enzymes, pH, redox, temperature.
  • Exemplary cleavable linker components include valine-citrulline (Val-Cit), valine-alanine (VA), phenylalanine-lysine (FK), maleimidocaproic acid (MC), p-aminobenzyl alcohol (PAB), hydrazone, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), N-hydroxysuccinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB), N-hydroxysuccinimidyl 4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), and CL2A (MedChemExpress).
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carbox
  • the PDCs of the present disclosure comprise a linker that is cleavable by an enzyme present in uPAR-expressing cells (e.g., cathepsin B, cathepsin D, plasmin, etc.).
  • the PDCs of the present disclosure comprise a linker that is cleavable by cathepsin B, for example, MC-Val-Cit-PAB.
  • the PDCs of the present disclosure comprise (PEG) 2 -MC-Val-Cit-PAB.
  • the present disclosure relates to a composition comprising a uPAR-binding peptide of the present disclosure or a uPAR-binding peptide-drug conjugate of the present disclosure.
  • compositions of the present disclosure may further comprise a pharmaceutically acceptable carrier in addition to the uPAR-binding peptide or uPAR-binding peptide-drug conjugate of the present disclosure.
  • a pharmaceutically acceptable carrier is any carrier, excipient, or other ingredient commonly used in the art that has no adverse effects on the subject and is understood by those skilled in the art. Examples of carriers or excipients and other ingredients include water, saline, buffer solutions, etc. as carriers; lactose, starch, sorbitol, D-mannitol, sucrose, etc. as excipients; other agents include starch, carboxymethylcellulose, calcium carbonate, etc. as disintegrants; phosphates, citrates, acetates, etc.
  • compositions disclosed herein can be prepared in a variety of dosage forms and administered to a subject via an appropriate route, frequency, and interval.
  • the term "subject" refers to an animal to which the uPAR-binding peptide, uPAR-binding peptide-drug conjugate, or composition of the present disclosure is administered.
  • the subject includes a mammalian, avian, or reptilian subject.
  • the subject includes a mammal, such as a primate, with or without a human.
  • the subject includes pets, such as dogs and cats, and livestock, such as cows and horses.
  • the subject is a human.
  • the subject is a subject in need of treatment for a disease (e.g., cancer) involving cells that express uPAR, such as a cancer patient.
  • a disease e.g., cancer
  • compositions of the present disclosure may be prepared in any dosage form appropriate for their intended use.
  • the compositions of the present disclosure may be solid, such as powder, fine granules, or granules; liquid, such as a suspension or emulsion; or semi-solid, such as a gel.
  • the compositions of the present disclosure may be stored in a solid, liquid, or semi-solid form that has been preserved (e.g., concentrated, frozen, dried, or lyophilized), and may be used after undergoing a preparation process (e.g., melting, diluting, gelling, or mixing with a carrier or solvent) prior to administration to a subject.
  • a preparation process e.g., melting, diluting, gelling, or mixing with a carrier or solvent
  • compositions of the present disclosure may be pharmaceutical compositions used in the treatment of diseases involving cells expressing uPAR.
  • uPAR is known to be expressed particularly in cancer cells
  • the disease is cancer.
  • cancer include melanoma (e.g., advanced melanoma or metastatic melanoma), non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, glioblastoma, glioma, lung squamous cell carcinoma, small cell lung cancer, hepatocellular carcinoma, bladder cancer, upper tract urothelial carcinoma, esophageal cancer, gastroesophageal junction cancer, stomach cancer, liver cancer, colon cancer, colorectal cancer, multiple myeloma, sarcoma, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome, nasopharyngeal
  • ductal carcinoma include ductal carcinoma, urothelial carcinoma, HTLV-associated T-cell leukemia/lymphoma, prostate cancer, genitourinary cancer, meningioma, adrenocortical carcinoma, gliosarcoma, fibrosarcoma, kidney cancer, breast cancer, pancreatic cancer, endometrial cancer, basal cell carcinoma of the skin, appendix cancer, bile duct cancer, salivary gland cancer, advanced Merkel cell carcinoma, diffuse large B-cell lymphoma, follicular lymphoma, mesothelioma, neuroendocrine tumors, urinary tract cancer, bone cancer, breast cancer, respiratory tract cancer, adenoid cystic carcinoma, cervical cancer, astrocytoma, chordoma, neuroblastoma, oral cancer, squamous cell carcinoma of the skin, thyroid cancer, Kaposi's sarcoma, anal cancer, gallbladder cancer, thymic carcinoma, uterine cancer, and solid
  • a urokinase-type plasminogen activator receptor (uPAR)-binding peptide which comprises a (helix 1)-(loop)-(helix 2) structure from the N-terminus to the C-terminus.
  • Helix 1 consists of the amino acid sequence: X1ELX2X3LX4X5ELX6X7LX8 ( SEQ ID NO : 1), and helix 2 is helix 2-1 consisting of the amino acid sequence : KLX9X10LX11X12KLX13X14LX15X16 ( SEQ ID NO: 2 ) , or helix 2-2 consisting of the amino acid sequence : KLX17X18LX19X20KLX21X22LKX23X24 (SEQ ID NO: 3 ) , where X 1 to X 24 represent amino acid residues, These amino acid sequences satisfy the following conditions (a) to (e): (a) X2 is F, Y or W, X5 is R or K, and X6 and X7 are each independently L, I or V; (b) X 11 is F, Y or W, X 13 is R or K, and X 15 and X 16 are each independently L, I or V; (b)
  • Item 2. The uPAR-binding peptide according to Item 1.
  • Item 3. The uPAR-binding peptide according to Item 2, wherein X n (n is an integer of 1 to 24) not specified in each of the conditions (a) to (e) is independently selected from amino acids other than C, P, G, or S.
  • Item 4. The uPAR-binding peptide according to Item 3, wherein X n (n is an integer of 1 to 24) not specified in each of conditions (a) to (e) is independently selected from the group consisting of A, R, N, E, H, L, K, M, F, V and Y.
  • Item 6 The uPAR-binding peptide according to any one of Items 2 to 5, wherein X3 is L, V, R, K, or Y.
  • Item 7 The uPAR-binding peptide according to any one of Items 2 to 6, wherein X4 is E or N.
  • Item 8 The uPAR-binding peptide according to any one of Items 2 to 7, wherein X4 is N.
  • Item 16 The uPAR-binding peptide according to any one of Items 2 to 9, 14 and 15, wherein X 19 is K.
  • Item 17 The uPAR-binding peptide according to any one of Items 2 to 9 and 14 to 16, wherein X 21 is A.
  • X1 is Y, L, R, or H
  • X3 is L, V, R, K, or Y
  • X4 is E or N
  • X8 is E
  • X9 is A and X10 is A
  • X 12 is A
  • X 14 is A; Item 14.
  • X1 is Y, L, R, or H
  • X3 is L, V, R, K, or Y
  • X4 is E or N
  • X8 is E
  • X 17 is A and X 18 is A
  • X 19 is K and X 21 is A
  • Item 14 The uPAR-binding peptide according to any one of Items 2 to 13.
  • Item 19 The uPAR-binding peptide according to any one of Items 2 to 13 and 18, wherein helix 2 consists of the amino acid sequence KLAALKAKLAALKA (SEQ ID NO: 19). [21] 20.
  • X 2 , X 5 , X 6 and X 7 are, respectively, in order: (i) F, R, I and L; (ii) F, R, L and I; (iii) W, R, L and V; (iv) Y, R, L and I, (v) F, R, L and V, or (vi) F, R, L and L Item 22.
  • Helix 1 is YELFLERELILLE (SEQ ID NO: 4), LELFVLERELLILE (SEQ ID NO: 5), RELWRLERELLVLE (SEQ ID NO: 6), HELYKLERELLILE (SEQ ID NO: 7), LELFLLERELLILE (SEQ ID NO: 8), RELFYLERELLVLE (SEQ ID NO: 9), RELFYLNRELLVLE (SEQ ID NO: 10), LELFLLERELLLLE (SEQ ID NO: 11) or LELFLLERELLVLE (SEQ ID NO: 12), Item 23.
  • the uPAR-binding peptide according to any one of Items 1 to 25 wherein the loop has a sequence length of 7 to 11 amino acids.
  • Item 27 Item 27.
  • Item 28. The uPAR-binding peptide according to any one of Items 1 to 27, wherein the loop is a sequence consisting of seven consecutive Gs or a sequence having one or two amino acid substitutions therein.
  • 29. The uPAR-binding peptide of claim 28, wherein the loop comprises an amino acid substitution at the second and/or seventh position.
  • the loop GGGGGGG (SEQ ID NO: 27), GKGGGGGG (SEQ ID NO: 28), GGGGGGK (SEQ ID NO: 29), or GKGGGGK (SEQ ID NO: 30) Item 30.
  • uPAR-binding peptides in which some amino acids of R4I-7 were substituted, R4I-7 E9N, R4I-7 G18K, R4I-7 G23K, and R4I-7 E9N/G18K/G23K mutants were identified.
  • the amino acid sequences of the identified uPAR-binding peptides are shown in Table 1 below.
  • the purified fractions were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) on an Autoflex II (Bruker Daltonics) instrument.
  • the purified peptide was dissolved in 50 mL of 20 mM NH 4 HCO 3 (pH 8) and stirred for 12 hours to form a disulfide bridge between the thiol group of the N-terminal Cys and the thiol group of the C-terminal Cys, thereby cyclizing the peptide.
  • the cyclized peptide was purified using RP-HPLC. The purity was 95% or higher.
  • R4I-7 E9N, R4I-7 G18K, R4I-7 G23K, and R4I-7 E9N/G18K/G23K mutants all showed superior uPAR binding activity compared to wild-type R4I-7.
  • R4I-7 exhibited a K D of 4.39 nM, R4I-7 E9N a K D of 0.44 nM, R4I-7 G18K a K D of 3.61 nM, R4I-7 G23K a K D of 3.36 nM, and R4I-7 E9N/G18K/G23K a K D of 0.17 nM.
  • R4I-7-linker (ClCH 2 CO-GAREFYLERELLVLEGGGGGGGGKLAALKAKLAALKAAAC(Peg)(Peg)C-NH 2 ) (SEQ ID NO: 45) was synthesized by Fmoc solid-phase synthesis, in which 2-[2-( 2 -aminoethoxy)ethoxy]acetic acid (amino-PEG2-acetic acid) was linked to R4I-7 as a linker.
  • YT1-S-linker (ClCH 2 CO-GAAELAALEAELAALEGGGGGGGGKLAALKAKLAALKAYC(Peg)(Peg)C-NH 2 ) (SEQ ID NO: 46) was synthesized.
  • a cocktail of TFA/HO/triisopropylsilane (95/2.5/2.5) was used for deprotection and deprotection, and the C-terminal Cys was protected with an acetamidomethyl group (Acm).
  • the peptide was cyclized by reacting the chloro group of the N-terminal Chloroacetyl-Gly with the thiol group of the 39th Cys residue to form a thioether bridge.
  • the C-terminal Cys (Acm) was deprotected, and the thiol group of the C-terminal Cys of the peptide R4I-7-linker was reacted with the maleimide group of MC-Val-Cit-PAB-MMAE (MedChemExpress) to synthesize the conjugate.
  • the crude product was purified by RP-HPLC using a C18 column (AM12S05-2510WT, YMC), and the purified fractions were analyzed by MALDI-TOF-MS. The purity of the purified R4I-7-MMAE was 95% or higher.
  • the results of evaluating the binding activity of uPAR-binding PDC are shown in Figure 6.
  • the R4I-7-MMAE complex showed a K D of 8 nM for uPAR, confirming its binding activity.
  • the YT1-S-MMAE complex showed no binding activity to uPAR.
  • the cell proliferation inhibitory effect of the R4I-7-MMAE conjugate on cancer cells was evaluated.
  • the R4I-7-MMAE conjugate, YT1-S-MMAE conjugate, R4I-7 (thioether), and YT1-S (thioether) were added to MDA-MB-231 cells (uPAR-positive cells) and MCF-7 cells (uPAR-negative cells).
  • the cells were cultured at 37°C for 3 days, after which Cell Proliferation Reagent WST-1 (Roche) was added.
  • the absorbance at 450 nm of each well was measured using a plate reader.
  • the cell viability was normalized by setting the cell viability in the presence of only the medium to the cells at 100% and the cell viability in the absence of cells at 0%.
  • the normalized relative values were used as the cell growth inhibitory activity of the compound.
  • Figure 7 shows the results of evaluating the cancer cell growth inhibitory ability of uPAR-binding PDC.
  • the R4I-7-MMAE conjugate exhibited a significant concentration-dependent inhibitory effect on cell growth in MDA-MB-231 cells, but not in MCF-7 cells. In both cell types, a slight decrease in cell viability was observed at high concentrations of the YT1-S-MMAE conjugate, but no significant concentration-dependent inhibitory effect on cell growth was observed. Furthermore, R4I-7 (thioether) and YT1-S (thioether), which do not contain MMAE, did not exhibit any inhibitory effect on cell growth in either cell type or at any concentration. These results indicate that after binding to uPAR, the R4I-7-MMAE conjugate was transported into the cell via uPAR endocytosis and released MMAE, thereby inhibiting cell growth.
  • the expression level of uPAR in human cancer cell lines was quantified by immunostaining using a fluorescently labeled anti-uPAR antibody.
  • Cultured human cancer cell lines MCF-7, U-87MG, MAD-MB-231, and HeLa were collected and suspended in 1 mL of PBS (+1% FCS) to obtain a cell suspension of 1.0 x 10 6 /mL.
  • 5 ⁇ L of fluorescent dye (APC)-labeled anti-uPAR antibody or isotype control antibody was added to 100 ⁇ L of each cell suspension and allowed to stand for 1 hour for antibody staining. After washing the cells three times with PBS (+1% FCS), fluorescence intensity was measured using a flow cytometer (Becton Dickinson and Company, FACS Aria TM IIIu Cell Sorter).
  • R4I-7 E9N/G18K/G23K Mutant Peptide 4-1 Preparation of Fluorescently Labeled Peptide
  • R4I-7(3M) peptide The intracellular transport of the fluorescently labeled R4I-7(3M) peptide was evaluated.
  • Human cancer cell lines U-87MG and HeLa (uPAR-positive cells) and MCF-7 cells (uPAR-negative cells) were immunostained and observed using a confocal microscope.
  • R4I-7(3M)-linker (CGCARELFYLNRELLVLEGKGGGGKKLAALKAKLAALKAAC) (SEQ ID NO: 59) was synthesized by Fmoc solid-phase synthesis, in which Cys and Gly were linked as a linker to the N-terminus of R4I-7(3M).
  • the peptide R4I-7(3M) was cyclized by reacting the thiol groups of the third residue from the N-terminus and the C-terminal Cys residue of the linker to form disulfide bonds.
  • Fluorescently labeled peptide R4I-7(3M) was synthesized by reacting the thiol group of the N-terminal Cys residue of the linker with the maleimide group of a maleimide-derivatized fluorescent dye (Alexa Fluor® 488C 5 Maleimide) (Thermo Fisher Scientific).
  • the crude product was purified by RP-HPLC using a C18 column (AM12S05-2510WT, YMC), and the purified fraction was analyzed by MALDI-TOF-MS to confirm that the fluorescently labeled peptide R4I-7(3M) had been synthesized.
  • the structure of the R4I-7(3M)-linker is shown below. (SEQ ID NO: 59)
  • Each cancer cell line was seeded at a seeding density of 2.4 x 104 cells/well on a glass-based dish ( ⁇ 27 mm) (IWAKI) and cultured overnight at 37°C, 5% CO2 .
  • Fluorescently labeled R4I-7 (3M) 1 ⁇ M was added at 100 ⁇ L/well, and after 24 hours, the cells were observed using a confocal laser microscope (FV1200, Olympus). Prior to observation, cell nuclei were stained with Hoechst 33342 (Nacalai Tesque).
  • uPAR-binding PDC was prepared by conjugating the peptide R4I-7(3M) with monomethyl auristatin F (MMAF) as an anticancer agent.
  • MMAF is a known tubulin polymerization inhibitor.
  • R4I-7(3M)-linker (CGCARELFYLNRELLVLEGKGGGGKKLAALKAKLAALKAAC) (SEQ ID NO: 59) was synthesized by Fmoc solid-phase synthesis, in which Cys and Gly were linked to the N-terminus of R4I-7(3M).
  • YT1-S-linker (CGCAAELAALEAELAALEGGGGGGGGKLAALKAKLAALKAYC) (SEQ ID NO: 60) was synthesized.
  • the peptide R4I-7(3M) was cyclized by reacting the thiol groups of the third residue from the N-terminus and the C-terminal Cys residue of the linker to form disulfide bonds.
  • a peptide-drug conjugate was synthesized by reacting the thiol group of the N-terminal Cys residue of the linker with the maleimide group of MC-Val-Cit-PAB-MMAF (MedChemExpress).
  • the crude product was purified by RP-HPLC using a C18 column (AM12S05-2510WT, YMC), and the purified fraction was analyzed by MALDI-TOF-MS to confirm the synthesis of the R4I-7(3M)-MMAF conjugate.
  • YT1-S-MMAF was also synthesized in the same manner as a control.

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Abstract

La présente divulgation concerne un peptide HLH de liaison à l'uPAR et un conjugué peptide−médicament (PDC) dans lequel un médicament est lié au peptide HLH de liaison à l'uPAR.
PCT/JP2025/012193 2024-03-26 2025-03-26 PEPTIDE HLH LIANT uPAR, CONJUGUÉ PEPTIDE−MÉDICAMENT ET COMPOSITION Pending WO2025206033A1 (fr)

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