WO2025206033A1 - uPAR-BINDING HLH PEPTIDE, PEPTIDE−DRUG CONJUGATE, AND COMPOSITION - Google Patents

Abstract

The present disclosure relates to a uPAR-binding HLH peptide and a peptide−drug conjugate (PDC) in which a drug is linked to the uPAR-binding HLH peptide.

Description

uPAR-binding HLH peptides, peptide-drug conjugates and compositions

This application claims priority to Japanese Patent Application No. 2024-049559, the entire contents of which are incorporated herein by reference.
The present disclosure relates to uPAR-binding HLH peptides, and peptide-drug conjugates (PDCs) in which a drug is linked to the uPAR-binding HLH peptides.

Urokinase-type plasminogen activator receptor (uPAR) 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. 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.

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 α-helical coiled-coil structure. Despite their low molecular weight, 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.

JP 2014-047156 A International Publication No. 2016/208761

Ikuo Fujii, Daisuke Fujiwara, Masataka Michigami, "New drug discovery modality based on molecularly targeted HLH peptides," Drug Delivery System, 2020, Vol. 35, No. 3, pp. 212-221, published October 25, 2020

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.

In order to solve the above problems, 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.

In one aspect, 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.

In one aspect, the present disclosure relates to a uPAR-binding peptide-drug conjugate (PDC) comprising the uPAR-binding peptide and a drug linked to the peptide.

In one aspect, 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.

Spectral evaluation of peptides (R4G-6, R4G-8, R4G-10, R4I-7, and R4I-12) Evaluation of uPAR binding activity of peptide (R4G-6) Evaluation of uPAR binding activity of peptide (R4G-8) Evaluation of uPAR binding activity of peptide (R4G-10) Evaluation of uPAR binding activity of peptide (R4I-12) Evaluation of uPAR binding activity of peptide (R4I-7) Evaluation of uPAR binding activity of peptide (R4I-7 E9N) Evaluation of uPAR binding activity of peptide (R4I-7 G18K) Evaluation of uPAR binding activity of peptide (R4I-7 G23K) Evaluation of uPAR binding activity of peptide (R4I-7 E9N/G18K/G23K) Evaluation of peptide (R4I-7) for inhibition of uPA-uPAR interaction Assessment of intracellular internalization of anti-uPAR antibodies by confocal microscopy Evaluation of intracellular internalization of peptide (R4I-7) by confocal microscopy PDC (R4I-7-MMAE) production scheme Evaluation of uPAR binding activity of PDC (R4I-7-MMAE) Evaluation of the cytotoxic activity of PDC (R4I-7-MMAE) in MDA-MB-231 and MCF-7 cells Flow cytometry analysis of uPAR expression Evaluation of intracellular internalization of peptide R4I-7 (3M) by confocal microscopy Evaluation of uPAR binding activity of PDC (R4I-7(3M)-MMAF) Evaluation of cytotoxic activity of PDC (R4I-7(3M)-MMAF) in MCF-7 cells Evaluation of cytotoxic activity of PDC (R4I-7(3M)-MMAF) in U-87MG cells Evaluation of cytotoxic activity of PDC (R4I-7(3M)-MMAF) in HeLa cells

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.

In one aspect, 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.

In the uPAR-binding HLH peptide of the present disclosure, helix 1 and helix 2 are each an α-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. In some embodiments, the lengths of helix 1 and helix 2 may be equal or may differ by no more than two amino acid residues. In some embodiments, helix 1 and helix 2 may be 10 to 20 amino acid residues. In some embodiments, helix 1 consists of 14 amino acid residues and helix 2 consists of 14 or 15 amino acid residues. In some embodiments, helix 1 and helix 2 consist of 14 amino acid residues.

The present inventors used phage surface display to screen a library of randomized HLH peptides and obtained the amino acid sequences of several HLH peptides that are thought to have high binding affinity to uPAR. The types of amino acids at some outer positions of the HLH peptides were limited. The amino acids at these positions include ^X2 , ^{X5 , X6 , and X7} when helix ¹ has the amino ^acid sequence ^{X1ELX2X3LX4X5ELX6X7LX8} (SEQ ID NO: 1), which structurally corresponds to ^X11 , ^X13 , ^{X15 , and X16} when helix ² has the amino acid sequence ^{KLX9X10LX11X12KLX13X14LX15X16} (helix ^2-1 ; SEQ ID ^NO : ^{2 ), or X20 and X22 when helix 2} has ^the amino acid sequence ^{KLX17X18LX19X20KLX21X22LKX23X24 ( helix 2-2} ; SEQ ID NO ^: 3 ^{) .} , ^X23 , and ^X24 , respectively. Without wishing to be bound by a particular theory, it is believed that the amino acid residues at these positions are important for the interaction with uPAR, and that the specific amino acid residues strongly interact with the epitope of uPAR, thereby contributing to maintaining the binding ability of the uPAR-binding HLH peptide to uPAR.

Helix 1 of the uPAR-binding HLH peptide may comprise or consist of the amino acid sequence ^{X1ELX2X3LX4X5ELX6X7LX8} (SEQ ID NO: 1). In some embodiments, ^{X2 is} F, ^Y , ^{or W} , ^X5 is R ^or K, ^X6 is ^L , I, or V, and ^X7 is L, I, or V. In some embodiments, ^X2 is F, Y, or ^W , ^X5 is R, ^X6 is L, I, or V, and ^X7 is L, I, or V. In some embodiments, ^X2 , ^X5 , ^X6 , and ^X7 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.

In helix 1, ^X1 , ^X3 , ^X4 , and ^X8 may be any amino acid. Preferably, ^X1 , ^X3 , ^X4 , and ^X8 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. In some embodiments, ^X1 is Y, L, R, or H. In some embodiments, ^X3 is L, V, R, K, or Y. In some embodiments, ^X4 is E or N. In some embodiments, ^X8 is E.

In some embodiments, Helix 1 comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 4-12. In some embodiments, Helix 1 consists of the amino acid sequence set forth in any of SEQ ID NOs: 4-12.

In some embodiments, Helix 1 may not have specific amino acid residues that contribute to maintaining the binding to uPAR. In such embodiments, X ¹ to X ⁸ in Helix 1 may be any amino acid, preferably an amino acid that is suitable for maintaining the helix structure, such as 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 of the uPAR-binding HLH peptide can be helix 2-1 comprising or consisting of the amino acid sequence ^{KLX9X10LX11X12KLX13X14LX15X16} (SEQ ID ^NO : 2). In some embodiments, ^X11 is ^F , ^{Y , or W, X13 is R or K, X15 is L ,} I, or V, and ^X16 is L, I, or V. In some embodiments, ^X11 is F, Y ^{, or W, X13 is R, X15 is L, I, or V, and X16 is L ,} I, ^or V. In some embodiments, X ¹¹ , X ¹³ , X ¹⁵ and X ¹⁶ 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.

In helix 2-1, ^X9 , ^X10 , ^X12 , and ^X14 may be any amino acid. Preferably, ^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. In some embodiments, ^X9 is A. In some embodiments, ^X10 is A. In some embodiments, ^X12 is A. In some embodiments, ^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). In some embodiments, ^{X20 is} F, Y ^{, or W, X22 is R or K, X23 is L , I} , or V, and ^X24 is L, I, or V. In some embodiments, ^X20 is F, Y ^{, or W, X22 is R, X23 is L, I, or V, and X24 is L ,} I, or V. In some embodiments, ^X20 , ^X22 , ^X23 and ^X24 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.

In helix 2-2, ^X17 , ^X18 , ^X19 , and ^X21 may be any amino acid. Preferably, ^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. In some embodiments, ^X17 is A. In some embodiments, ^X18 is A. In some embodiments, ^X19 is K. In some embodiments, ^X21 is A.

In some embodiments, helix 2-1 comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 13-18. In some embodiments, helix 2-2 comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 20-25. In some embodiments, helix 2-1 comprises the amino acid sequence set forth in any of SEQ ID NOs: 13-18. In some embodiments, helix 2-2 comprises the amino acid sequence set forth in any of SEQ ID NOs: 20-25.

In some embodiments, helix 2 may not have specific amino acid residues that contribute to maintaining binding to uPAR. In such embodiments, in helix 2-1 or helix 2-2, ^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. In some embodiments, helix 2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or 26. In some embodiments, helix 2 consists of the amino acid sequence set forth in SEQ ID NO: 19 or 26.

In some embodiments, 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 F, Y or W, X ¹³ is R or K, and X ¹⁵ and X ¹⁶ are each independently L, I or V;
(c) X ²⁰ is F, Y or W, X ²² is R or K, and X ²³ and X ²⁴ are each independently L, I or V;
(d) Both conditions (a) and (b) above are satisfied; or (e) Both conditions (a) and (c) above are satisfied.
Optionally, each unspecified X ⁿ (n is an integer from 1 to 24) in each of (a) to (e) is 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, N, E, H, L, K, M, F, V and Y.

In some embodiments, 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, N, E, H, L, K, M, F, V and Y.

In some embodiments, 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, R, N, E, H, L, K, M, F, V and Y.

In some embodiments, 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;
^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 , ^X8 and ^X17 to ^X24 each independently represent 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, N, E, H, L, K, M, F, V and Y.

In some embodiments, 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;
^X20 is F, Y or W, ^X22 is R or K, ^X23 is L, I or V, ^X24 is L, I or V, and optionally, ^X1 to ^X8 , ^X17 , ^X18 , ^X19 and ^X21 each independently represent 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, R, N, E, H, L, K, M, F, V and Y.

In some embodiments, 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;
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) ^X11 is F, Y or W, ^X13 is R or K, and ^X15 and ^X16 are each independently L, I or V; and optionally, ^X1 , ^X3 , ^X4 , ^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, R, N, E, H, L, K ^, M, F, V and Y.

In some embodiments, 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 , ^X4 , X8, ^X17 , ^X18 , ^X19 and ^X21 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, N, E, H, L, K ^, M, F, V and Y.

In a uPAR-binding HLH peptide, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the loop sequence consists of G and K. In some embodiments, 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. In some embodiments, 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 uPAR-binding HLH peptide may be either a non-cyclic peptide or a cyclic peptide, but is preferably a cyclic peptide. Even if it does not form a cyclic structure, the uPAR-binding HLH peptide of the present disclosure can maintain a stable structure due to the interaction between the two helices, but forming a cyclic structure is more stable and has advantages in terms of resistance to physicochemical degradation, half-life, bioavailability, etc.

In some embodiments, the uPAR-binding HLH peptide is a cyclic peptide in which the N-terminal amino acid and the C-terminal amino acid are directly or indirectly linked. Here, "directly linked" means that the N-terminal amino acid and the C-terminal amino acid are linked without a linker, including, for example, embodiments in which the N-terminal and C-terminal amino acids are linked via an amide bond (peptide bond) and embodiments in which the N-terminal and C-terminal cysteine residues are linked via a disulfide bond. Here, "indirectly linked" means that the N-terminal amino acid and the C-terminal amino acid are linked via a linker. 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. Examples of 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. In some embodiments, the linker includes a linker in which one or more methylene groups are linked in a linear chain, or a linker to which one or more polyethylene glycols (PEGs) are linked. In some embodiments, the linker includes a peptide chain in which 1 to 10 amino acids are linked via amide bonds. In some embodiments, the linker includes a peptide chain in which 1 to 9, for example 1 to 5, consecutive glycines are linked.

Cyclization of peptides may be carried out by any method known in the art. The N-terminal and/or C-terminal amino acids of the peptide to be cyclized may be natural or unnatural amino acids suitable for cyclization reactions. In some embodiments, the uPAR-binding HLH peptide comprises a cyclic structure formed by linking two amino acid residues at the N-terminus and C-terminus via a disulfide bond, peptide bond, alkyl bond, alkenyl bond, ester bond, thioester bond, ether bond, thioether bond, phosphonate ether bond, azo bond, C-S-C bond, C-N-C bond, C=N-C bond, amide bond, lactam bridge, carbamoyl bond, urea bond, thiourea bond, amine bond, thioamide bond, or triazole bond. In some embodiments, 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.

In some embodiments, the uPAR-binding HLH peptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 32-43, or comprises or consists of an amino acid sequence containing 1, 2, 3, 4, 5, or 6 amino acid mutations relative to the amino acid sequence set forth in any one of SEQ ID NOs: 32-43. Here, the mutations are preferably substitutions, particularly in the helices, and more preferably substitutions with amino acids other than C, P, G, or S. To introduce multiple mutations to maintain or improve the activity of the uPAR-binding HLH peptide while maintaining its structure, slight mutations (e.g., no more than two substitutions) may be tolerated at positions contributing to maintaining the HLH structure, positions contributing to uPAR binding, and other positions, including the loop regions and each terminus. In some embodiments, no more than two mutations may be tolerated at positions contributing to maintaining the HLH structure and positions contributing to uPAR binding in the two helices, as well as other positions. In some embodiments, no more than one mutation may be tolerated at each of the positions in the two helices that contribute to maintaining the HLH structure and contributing to uPAR binding, as well as at each of the other positions. In some embodiments, no more than one mutation may be tolerated at each of the positions in one helix that contribute to maintaining the HLH structure and contributing to uPAR binding.

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. In some embodiments, 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. In some embodiments, the 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.

The uPAR-binding HLH peptide may have a molecular mass of 3 to 15 kDa. In some embodiments, the uPAR-binding HLH peptide has a molecular mass of 15 kDa or less, 14 kDa or less, 13 kDa or less, 12 kDa or less, 11 kDa or less, 10 kDa or less, 9 kDa or less, 8 kDa or less, 7 kDa or less, 6 kDa or less, 5 kDa or less, or 4 kDa or less. In some embodiments, the uPAR-binding HLH peptide has a molecular mass of 3.5 to 4.5 kDa.

In this specification, various amino acid residues are represented by single-letter symbols according to the standard practice in the art. For example, 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 represents 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, and P represents proline. Furthermore, in this specification, X ⁿ (n is a natural number) represents an amino acid, and each of these may represent one type of amino acid selected from a group consisting of multiple defined amino acids. For example, in X ¹ ELX ² X ³ LX ⁴ X ⁵ ELX ⁶ X ⁷ LX ⁸ (SEQ ID NO: 1), "X ² is F, Y, or W" means that the amino acid represented by X ² is any one of phenylalanine, tyrosine, and tryptophan.

In this specification, the 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 acids may be natural or unnatural amino acids. As used herein, unnatural amino acids are amino acids that are not naturally encoded and are generated, for example, by modification (e.g., post-translational modification) of the 20 common amino acids or are chemically synthesized. Unnatural amino acids may be introduced into uPAR-binding peptides for linking to drugs, linkers, etc. Unnatural amino acids may be derivatives of the 20 common amino acids, including halogen groups, carbonyl groups, acetyl groups, aminooxy groups, hydrazine groups, hydrazide groups, semicarbazide groups, azide groups, or alkyne groups.

As used herein, 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. In some embodiments, 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. In some embodiments, two or fewer amino acid mutations may be allowed when the sequence of one helical region is used as the reference sequence. In some embodiments, 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). Here, binding affinity is represented by a dissociation constant ( _KD ) measured by the method described in the Examples. As used herein, 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. In some embodiments, 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 nM or less, 900 nM or less, 900 nM or less The uPAR-binding HLH peptides exhibit a KD of 0 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 _nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, or 0.05 nM or less. In some embodiments, 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.

In some embodiments, 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. As used herein, "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. In some embodiments, 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.

In some embodiments, 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 can exert their physiological activity independently through inhibition of the uPAR-uPA interaction. It is known in the art that uPA binds to uPAR on the cell surface and converts plasminogen into active plasmin, that plasmin contributes to the degradation of structural proteins in the extracellular matrix, and that uPAR is more highly expressed in cancer cells than in normal cells. Therefore, without wishing to be bound by any particular theory, it is believed that 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.

In some embodiments, 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. In some embodiments, the uPAR-binding HLH peptide is PEGylated. In some embodiments, the uPAR-binding HLH peptide is chlorinated for cyclization. In some embodiments, 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. In some embodiments, the uPAR-binding HLH peptides may be produced chemically synthetically using solid-phase synthesis. In some embodiments, 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).

In one aspect, 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. In some embodiments, the nucleic acid sequence encodes the amino acid sequence set forth in any of SEQ ID NOs: 32-43. In some embodiments, the nucleic acid sequence is the sequence set forth in any of SEQ ID NOs: 47-58.

In one aspect, the present disclosure relates to a vector comprising a nucleic acid sequence encoding a uPAR-binding HLH peptide of the present disclosure. Here, 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.

In one aspect, the present disclosure relates to a host cell comprising a vector including a nucleic acid sequence encoding a uPAR-binding HLH peptide of the present disclosure. In one aspect, the present disclosure relates to a host cell that expresses a uPAR-binding HLH peptide of the present disclosure. Examples of host cells include E. coli, phage, yeast, plant cells, insect cells, and mammalian cells.

In one aspect, 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. Here, the drug is any molecule that has an independent action function, and 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.

In some embodiments, 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. In some embodiments, exemplary labels include enzyme labels such as horseradish peroxidase (HRP), alkaline phosphatase (AP), or glucose oxidase. In some embodiments, 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 Blue™, Pacific Green™, Pacific Orange™, tetramethylrhodamine (TRITC), Texas Red®, or other fluorescent labels. In some embodiments, example labels include chelators such as Cyclen, Cyclam, DO2A, DOTP, DOTMA, TETA, DOTAM, CB-T2A, DOTA, or NOTA. In some embodiments, exemplary labels include radioisotopes such as ³² P, ³³ P, ³ H, ¹⁴ C, ¹²⁵ I, ¹⁸ F, ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁹ Zr, ¹¹ C, ¹³ N, ¹⁵ O, ⁶² Cu, ¹²⁴ I, ⁷⁶ Br, ⁸² Rb, or other radioisotopes. In certain embodiments, the label is fluorescein (FITC).

In some embodiments, the drug is a therapeutic agent, such as a cancer therapeutic agent or an immunosuppressant. In some embodiments, 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. In some embodiments, 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), tubulysin or tubulysin analog (such as AZ13599185), amberstatin 269, doxorubicin, antibiotic (such as rifalog), anthracycline (such as PNU-159682), microtubule inhibitor (such as rhizoxin), spliceostatin, or tylanstatin. In certain embodiments, the cytotoxic drug is monomethyl auristatin E (MMAE).

In some embodiments, the PDC of the present disclosure may include at least one drug, e.g., one, two, three, or more drugs. In some embodiments, 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. In some embodiments, 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. For example, the drug may be linked via a carboxyl group at the peptide terminus, an amino group at K, or a thiol group at C. In some embodiments, the drug is linked via a reaction of a thiol at C introduced at any position with a maleimide. In certain embodiments, the drug is linked to the peptide via a C residue at the C terminus. In certain embodiments, the drug is linked to the peptide via a C residue at the N terminus.

In some embodiments, the PDC of the present disclosure includes a linker that connects the uPAR-binding peptide and the drug. Examples of 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. In some embodiments, the linker includes a linker in which one or more methylene groups are bonded in a linear chain. In some embodiments, the linker includes a peptide chain in which one or more amino acids are bonded by amide bonds.

In some embodiments, the linker is a cleavable linker or a non-cleavable linker. In embodiments in which the drug is a detectable label, the linker is preferably a non-cleavable linker. In embodiments in which the drug is a therapeutic agent, the linker is preferably a cleavable linker. In some embodiments, the linker comprises one or more PEGs.

In some embodiments, the cleavable linker may be cleavable under intracellular conditions (e.g., intracellular enzymes, pH, redox, temperature). By linking a drug using a linker that is cleavable under intracellular conditions, the PDC of the present disclosure is stable in uPAR-expressing cells, for example in the blood, until it is translocated into the cells, and the drug exerts its physiological activity upon cleavage within the cells. 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).

In some embodiments, 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.). In some embodiments, the PDCs of the present disclosure comprise a linker that is cleavable by cathepsin B, for example, MC-Val-Cit-PAB. In some embodiments, the PDCs of the present disclosure comprise (PEG) ₂ -MC-Val-Cit-PAB. In some embodiments, the PDCs of the present disclosure comprise MC-Val-Cit-PAB-MMAE (MedChemExpress), for example, peptide-MC-Val-Cit-PAB-MMAE, represented by the following formula:
In some embodiments, the PDC of the present invention comprises MC-Val-Cit-PAB-MMAF (MedChemExpress), for example, peptide-MC-Val-Cit-PAB-MMAF, represented by the following formula:

After conjugation of the peptide and drug, the PDCs of the present disclosure 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 certain embodiments, the uPAR-binding HLH peptides of the present disclosure are purified by reverse-phase high-performance liquid chromatography (RP-HPLC).

In one aspect, 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.

The 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. as buffers; gum arabic, sodium alginate, tragacanth, etc. as emulsifiers; glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. as suspending agents; benzyl alcohol, chlorobutanol, sorbitol, etc. as soothing agents; propylene glycol, ascorbic acid, etc. as stabilizers; phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. as preservatives; benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. as antiseptics; hyaluronic acid, collagen, etc. as gelling agents.

The compositions disclosed herein can be prepared in a variety of dosage forms and administered to a subject via an appropriate route, frequency, and interval.

As used herein, 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. In some embodiments, the subject includes a mammalian, avian, or reptilian subject. In some embodiments, the subject includes a mammal, such as a primate, with or without a human. In some embodiments, the subject includes pets, such as dogs and cats, and livestock, such as cows and horses. In some embodiments, the subject is a human. In some embodiments, 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.

The compositions of the present disclosure may be prepared in any dosage form appropriate for their intended use. In some embodiments, 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. In some embodiments, 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.

The compositions of the present disclosure may be prepared for systemic or local administration. Examples of routes of administration of the compositions of the present disclosure include oral, rectal, nasal, intravenous, intra-articular, intraconjunctival, intracranial, intraperitoneal, intrapleural, intramuscular, intrathecal, transdermal, or subcutaneous routes of administration. In some embodiments, the compositions of the present disclosure are administered intravenously.

The compositions of the present disclosure may be pharmaceutical compositions used in the treatment of diseases involving cells expressing uPAR. Because uPAR is known to be expressed particularly in cancer cells, in some embodiments, the disease is cancer. Examples of 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 carcinoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, small lymphocytic lymphoma, ovarian cancer, gastrointestinal cancer, primary peritoneal carcinoma, fallopian tube cancer, and others. These 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 tumors. The cancer may be, for example, in an early stage, advanced stage, or metastatic stage.

The present disclosure relates, for example, to the following:
[1]
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.
[2]
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;
(c) X ²⁰ is F, Y or W, X ²² is R or K, and X ²³ and X ²⁴ are each independently L, I or V;
(d) Both conditions (a) and (b) above are satisfied; or (e) Both conditions (a) and (c) above are satisfied.
Item 2. The uPAR-binding peptide according to Item 1.
[3]
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.
[4]
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.
[5]
Item 5. The uPAR-binding peptide according to any one of Items 2 to 4, wherein X ¹ is Y, L, R, or H.
[6]
Item 6. The uPAR-binding peptide according to any one of Items 2 to 5, wherein ^X3 is L, V, R, K, or Y.
[7]
Item 7. The uPAR-binding peptide according to any one of Items 2 to 6, wherein ^X4 is E or N.
[8]
Item 8. The uPAR-binding peptide according to any one of Items 2 to 7, wherein ^X4 is N.
[9]
Item 9. The uPAR-binding peptide according to any one of Items 2 to 8, wherein ^X8 is E.
[10]
Item 10. The uPAR-binding peptide according to any one of Items 2 to 9, wherein X ⁹ is A.
[11]
Item 11. The uPAR-binding peptide according to any one of Items 2 to 10, wherein X ¹⁰ is A.
[12]
Item 12. The uPAR-binding peptide according to any one of Items 2 to 11, wherein X ¹² is A.
[13]
Item 13. The uPAR-binding peptide according to any one of Items 2 to 12, wherein X ¹⁴ is A.
[14]
Item 10. The uPAR-binding peptide according to any one of Items 2 to 9, wherein X ¹⁷ is A.
[15]
Item 15. The uPAR-binding peptide according to any one of Items 2 to 9 and 14, wherein X ¹⁸ is A.
[16]
Item 16. The uPAR-binding peptide according to any one of Items 2 to 9, 14 and 15, wherein X ¹⁹ is K.
[17]
Item 17. The uPAR-binding peptide according to any one of Items 2 to 9 and 14 to 16, wherein X ²¹ is A.
[18]
^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, and X ¹⁴ is A;
Item 14. The uPAR-binding peptide according to any one of Items 2 to 13.
[19]
^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.
[20]
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. The uPAR-binding peptide according to any one of Items 2 to 9, 14 to 17 and 19, wherein helix 2 consists of the amino acid sequence KLAALKAKLAALKAA (SEQ ID NO: 26).
[22]
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. The uPAR-binding peptide according to any one of Items 2 to 21, wherein:
[23]
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 2 to 22, which consists of any one of the amino acid sequences:
[24]
Item 24. The uPAR-binding peptide according to any one of Items 1 to 23, wherein the loop has a sequence length of 3 to 20 amino acids.
[25]
Item 25. The uPAR-binding peptide according to any one of Items 1 to 24, wherein the loop has a sequence length of 5 to 15 amino acids.
[26]
Item 26. 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.
[27]
Item 27. The uPAR-binding peptide according to any one of Items 1 to 26, wherein the loop is a sequence consisting of consecutive Gs or a sequence having 1, 2, 3, 4, or 5 amino acid substitutions therein.
[28]
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]
29. The uPAR-binding peptide of claim 28, wherein the loop comprises an amino acid substitution at the second and/or seventh position.
[30]
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. The uPAR-binding peptide according to any one of Items 1 to 29, which consists of any one of the amino acid sequences:
[31]
Item 31. The uPAR-binding peptide according to any one of Items 1 to 30, wherein the peptide further comprises 1 to 3 amino acids at the N-terminus of helix 1 and/or the C-terminus of helix 2.
[32]
Item 32. The uPAR-binding peptide according to Item 31, wherein the peptide further comprises 1 to 3 amino acids at the N-terminus of helix 1 and/or the C-terminus of helix 2, respectively, and the amino acid at the N-terminus and/or the C-terminus of the peptide is C.
[33]
Item 33. The uPAR-binding peptide according to Item 32, wherein the peptide further comprises CA at the N-terminus of helix 1 and further comprises AC at the C-terminus of helix 2-1, or further comprises CA at the N-terminus of helix 1 and further comprises C at the C-terminus of helix 2-2.
[34]
Item 34. The uPAR-binding peptide according to any one of Items 1 to 33, wherein the peptide has a sequence length of 60 amino acids or less.
[35]
Item 35. The uPAR-binding peptide according to Item 34, wherein the peptide has a sequence length of 35 to 45 amino acids.
[36]
Item 36. The uPAR-binding peptide according to Item 35, wherein the peptide has a sequence length of 39 amino acids.
[37]
Item 37. The uPAR-binding peptide according to any one of Items 1 to 36, which consists of the amino acid sequence set forth in any one of SEQ ID NOs: 32 to 43, or a sequence containing 1, 2, 3, 4, 5, or 6 amino acid mutations in the amino acid sequence.
[38]
Item 38. The uPAR-binding peptide according to Item 37, wherein the amino acid mutation is an amino acid substitution.
[39]
Item 38. The uPAR-binding peptide according to Item 37, consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 32 to 43.
[40]
Item 41. The uPAR-binding peptide according to any one of Items 1 to 39, wherein the N-terminus and C-terminus of the peptide are directly or indirectly linked.
Item 41. A uPAR-binding peptide according to any one of Items 1 to 40, wherein the N-terminus and C-terminus of the peptide are bonded via a disulfide bond or a thioether bond, or linked via a linker.
[42]
Item 42. The uPAR-binding peptide according to Item 41, wherein the N-terminus and C-terminus of the peptide are linked by a disulfide bond.
[43]
Item 42. The uPAR-binding peptide according to Item 41, wherein the N-terminus and C-terminus of the peptide are linked by a thioether bond.
[44]
Item 44. A uPAR-binding peptide-drug conjugate comprising the uPAR-binding peptide according to any one of Items 1 to 43 and a drug linked to the peptide.
[45]
Item 45. The uPAR-binding peptide-drug conjugate according to Item 44, wherein the drug is a cancer therapeutic agent.
[46]
Item 46. The uPAR-binding peptide-drug conjugate according to Item 45, wherein the cancer therapeutic agent is monomethyl auristatin E (MMAE).
[47]
Item 47. The uPAR-binding peptide-drug conjugate according to any one of Items 44 to 46, further comprising a linker connecting the peptide and the drug.
[48]
Item 48. The uPAR-binding peptide-drug conjugate according to Item 47, wherein the linker comprises PEG.
[49]
Item 49. The uPAR-binding peptide-drug conjugate according to Item 47 or 48, wherein the linker is cleavable by an intracellular enzyme.
[50]
Item 50. The uPAR-binding peptide-drug conjugate according to Item 49, wherein the linker cleavable by an intracellular enzyme comprises valine-citrulline (Val-Cit).
[51]
51. The uPAR-binding peptide-drug conjugate according to any one of Items 47 to 50, wherein the linker comprises (PEG) ₂ -MC-Val-Cit-PAB.
[52]
A composition comprising the uPAR-binding peptide according to any one of Items 1 to 43 or the uPAR-binding peptide-drug conjugate according to any one of Items 44 to 51.
[53]
53. The composition of claim 52 for use in the treatment of cancer.
[54]
A method for treating cancer, comprising administering to a subject in need of treatment the uPAR-binding peptide according to any one of Items 1 to 43, or the uPAR-binding peptide-drug conjugate according to any one of Items 44 to 51.
[55]
A uPAR-binding peptide according to any one of Items 1 to 43 or a uPAR-binding peptide-drug conjugate according to any one of Items 44 to 51 for treating cancer.
[56]
Use of the uPAR-binding peptide according to any one of paragraphs 1 to 43 or the uPAR-binding peptide-drug conjugate according to any one of paragraphs 44 to 51 for the treatment of cancer.
[57]
Use of the uPAR-binding peptide according to any one of Items 1 to 43 or the uPAR-binding peptide-drug conjugate according to any one of Items 44 to 51 for the manufacture of a medicament for treating cancer.

1. Preparation and Evaluation of uPAR-Binding HLH Peptides 1-1. Library Construction A phage surface-displayed peptide library was constructed by fusing HLH peptides to the phage surface protein pIII. In this study, a library in which the N-terminal helix portion of HLH peptides was randomized was used: CAXELXXLEXELXXLEGGGGGGGGKLAALKAKLAALKAAC (SEQ ID NO: 31) (where X represents an amino acid selected from 17 natural amino acids excluding P, T, and A). Specifically, the nucleic acid sequence encoding the HLH peptide was amplified by polymerase chain reaction (PCR) and introduced by direct ligation into the phage surface display vector pComb3d. The constructed vector was introduced into Escherichia coli TG1 by electroporation and cultured at 37°C until logarithmic growth phase in SB medium supplemented with 100 mg/mL ampicillin. The helper phage VCM13 solution was then added to the E. coli culture and incubated at 37°C for 30 minutes. The cells were collected by centrifugation at 6000g for 5 minutes, and the collected E. coli was cultured overnight at 30°C in SB medium supplemented with 100 mg/mL ampicillin and 50 mg/mL kanamycin. The phages grown in the culture were collected by PEG precipitation.

1-2. Screening The peptide-displaying phage library was reacted with uPAR-Fc (a fusion protein of uPAR and immunoglobulin G Fc fragment). Unbound phages were removed by capturing the phage and uPAR-Fc complex with protein A magnetic beads. The captured phages were recovered by adding 0.2 M Gly-HCl (pH 2.2) and amplified by infecting Escherichia coli. This series of procedures constitutes one round, and was repeated up to four rounds. uPAR-binding peptides were identified by DNA sequence analysis of the finally selected phage clones.

Furthermore, as 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.

1-3. Synthesis of uPAR-binding peptides Based on the amino acid sequences of the identified uPAR-binding peptides, each uPAR-binding peptide was chemically synthesized by Fmoc solid-phase peptide synthesis using an automated peptide synthesizer (PSSM-8, Shimadzu). Specifically, solid-phase synthesis was performed using Fmoc-NH-SAL-PEG resin (substitution ratio: 0.22 mmol/g). A cocktail of 2,2,2-trifluoroacetic acid (TFA)/H _O /1,2-ethanedithiol/triisopropylsilane (94/2.5/2.5/1) was added to the resin and reacted for 3 hours to remove the resin and deprotect the peptide. Ice-cold diethyl ether was added to the recovered filtrate to precipitate the peptide. After washing three times with ice-cold diethyl ether, the crude peptide was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (SP-120-5-ODS-RPS, DAISOPAK). 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. As a negative control, the HLH scaffold peptide YT1-S (CAAELAALEAELAALEGGGGGGGGKLAALKAKLAALKAYC) (SEQ ID NO: 44), which does not bind to uPAR, was also synthesized in the same manner.

1-4. Evaluation of the Structure of uPAR-Binding Peptides The secondary structure of each peptide sample, R4G-6, R4G-8, R4G-10, R4I-7, and R4I-12, was evaluated by circular dichroism spectroscopy using a J-820 (Jasco). The spectrum of each peptide is shown in Figure 1. Each peptide had a positive maximum at around 191 nm and negative maxima at around 208 nm and 222 nm, confirming that it had an α-helical structure.

1-5. Measurement of uPAR-binding activity of uPAR-binding peptides. The binding activity of peptides R4G-6, R4G-8, R4G-10, R4I-7, and R4I-12 to uPAR was evaluated by measuring the dissociation constant using surface plasmon resonance (SPR). Each concentration of peptide was added to human uPAR (Acro biosystems) immobilized on a sensor chip, and the binding response was measured using a Biacore T200 (Cytiva). Measurement conditions were a running buffer of HBS-EP+ and a temperature of 25°C. Measurement data were fitted with a 1:1 binding model using Biacore T200 Evaluation Software. Furthermore, the uPAR binding activity of peptides R4I-7E9N, G18K, G23K, and E9N/G18K/G23K mutants was similarly measured.

The results of measuring the uPAR binding activity of the uPAR-binding peptides are shown in Figures 2A to 2I. Binding to uPAR was observed for all five peptides (R4G-6: K _D = 507 nM, R4G-8: K _D = 595 nM, R4G-10: K _D = 167 nM, R4I-12: K _D = 311 nM, and R4I-7: K _D = 4 nM). R4I-7 showed the lowest K _D for uPAR binding activity. Furthermore, the 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. Specifically, 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.

1-6. Inhibition of uPA Activity by uPAR-Binding Peptides The R4I-7 peptide was evaluated for its inhibitory activity against uPA-uPAR interactions by measuring the dissociation constant using surface plasmon resonance (SPR). A mixed solution of various concentrations of peptide and uPAR (3 nM) was added to human uPA (Sino Biological) immobilized on a sensor chip, and the binding response was measured using a Biacore T200 (Cytiva). Measurement conditions were HBS-EP+ running buffer and 25°C temperature. The response value obtained when only uPAR was added (uPAR: 3 nM, measurement sample: 0 nM) was converted to 100% uPA-uPAR interaction, and the measurement data were normalized. As a negative control, the inhibitory activity of YT1-S was similarly evaluated.

The results of the inhibition of uPA activity by the R4I-7 peptide are shown in Figure 3. As a result of the measurement, R4I-7 completely inhibited the uPA-uPAR interaction, showing an IC ₅₀ of 5 nM. No inhibitory effect was observed with the negative control, YT1-S.

1-7. Evaluation of Intracellular Internalization of uPAR-Binding Peptides. The intracellular internalization of fluorescently labeled peptide R4I-7 and anti-uPAR antibodies was examined using a confocal microscope by immunostaining MDA-MB-231 cells (uPAR-positive cells) and MCF-7 cells (uPAR-negative cells). Specifically, MDA-MB-231 cells were seeded at a density of ⁴ x 104 cells/well in multiwell glass-bottom dishes (MATSUNAMI), or MCF-7 cells were seeded at a density of 6 x ¹⁰⁴ cells/well, and cultured at 37°C, 5% CO2 for ₂ days. To one well of plated cells, biotinylated anti-uPAR goat antibody (R&D Systems) was added at 2 μg/mL (100 μL/well), followed by streptavidin-APC (Thermo Fisher Scientific) at 3 μg/mL (100 μL/well). To another well, fluorescein (FITC)-labeled peptide R4I-7 was added at 1 μM (100 μL/well). The remaining wells received DMEM medium. After incubation at 37°C for 1 hour, Hoechst 33342 (Nacalai Tesque) was added at 20 μg/mL (100 μL/well) for nuclear staining and incubated at 37°C for 15 minutes. After washing three times with DMEM medium, 100 μL of DMEM medium was added again, and the stained cells were observed using a confocal microscope (FV1200 (OLYMPUS)).

The results of observation using a confocal microscope are shown in Figures 4A and 4B. The biotinylated uPAR antibody was observed to be localized on the cell surface of MDA-MB-231 cells, but not in MCF-7 cells (Figure 4A). This suggests that uPAR is expressed on the cell surface of MDA-MB-231 cells, but not in MCF-7 cells. The fluorescently labeled peptide R4I-7 was observed to be localized in the cytoplasm of MDA-MB-231 cells, but not in MCF-7 cells (Figure 4B). These results indicate that the uPAR-binding HLH peptide of the present disclosure is capable of selective intracellular transport into uPAR-positive cancer cells.

2. Preparation and Evaluation of uPAR-Binding HLH Peptide-Drug Conjugates (PDC) 2-1. Preparation of uPAR-Binding PDC uPAR-binding PDC was prepared by conjugating the peptide R4I-7 with monomethyl auristatin E (MMAE) as an anticancer agent. MMAE is a known tubulin polymerization inhibitor. A schematic diagram of the synthesis scheme of R4I-7-MMAE is shown in Figure 5. 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. Similarly, 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. As controls, the R4I-7 peptide cyclized by a thioether bond (R4I-7(thioether)) and the YT1-S peptide (YT1-S(thioether)), as well as YT1-S-MMAE, were also synthesized in the same manner.

2-2. Evaluation of uPAR-binding PDC binding activity The R4I-7-MMAE complex and YT1-S-MMAE complex were evaluated for their binding activity to uPAR by SPR. Various concentrations of PDC were added to human uPAR (Acro Biosystems) immobilized on a sensor chip, and the binding response was measured using a Biacore T200 (Cytiva). The measurement data was fitted with a 1:1 binding model using Biacore T200 Evaluation Software.

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.

2-3. Evaluation of the ability of uPAR-binding PDC to inhibit cancer cell proliferation. 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.

3. Analysis of uPAR Expression in Human Cancer Cell Lines 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 μL of fluorescent dye (APC)-labeled anti-uPAR antibody or isotype control antibody was added to 100 μL 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 ^™ IIIu Cell Sorter).

The results of uPAR expression analysis in human cancer cell lines are shown in Figure 8. uPAR expression was confirmed in U-87MG, MAD-MB-231, and HeLa cells. However, uPAR expression was minimal in MCF cells.

4. Tests Using the R4I-7 E9N/G18K/G23K Mutant Peptide 4-1. Preparation of Fluorescently Labeled Peptide Hereinafter, the R4I-7 E9N/G18K/G23K mutant peptide will be referred to as the 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)

4-2. Evaluation of intracellular internalization of fluorescently labeled peptides. Each cancer cell line was seeded at a seeding density of 2.4 x ¹⁰⁴ cells/well on a glass-based dish (φ27 mm) (IWAKI) and cultured overnight at 37°C, 5% _CO2 . Fluorescently labeled R4I-7 (3M) 1 μM was added at 100 μL/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).

The results of observation using a confocal microscope are shown in Figure 9. In U-87MG cells and HeLa cells, many dot-shaped fluorescent puncta were observed localized in the cytoplasm compared to MCF-7 cells. The fact that more fluorescent puncta were observed in U-87MG and HeLa cells, which have high uPAR expression, indicates that the uptake of the uPAR-binding peptide into the cells is uPAR-specific. Furthermore, the observation of dot-shaped fluorescent puncta suggests that the uPAR-binding peptide is taken up into the cells via the endocytic pathway of the cells.

4-3. Preparation of uPAR-binding PDC A 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). Similarly, 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.

4-4. Evaluation of Binding Activity of uPAR-Binding PDC The binding activity of the R4I-7(3M)-MMAF complex and the YT1-S-MMAF complex to uPAR was evaluated by the SPR method. Various concentrations of PDC were added as an analyte to recombinant human uPAR (Acro biosystems) immobilized on a CM sensor chip by the amine coupling method, and the binding response was measured using a Biacore T200 (Cytiva). Measurements were performed in HBS-EP+ buffer at 25°C and a flow rate of 30 μL/min. The measurement data was fitted with a 1:1 binding model using Biacore T200 Evaluation Software.

The results of the binding activity assessment of the R4I-7(3M)-MMAF conjugate are shown in Figure 10A. The R4I-7(3M)-MMAF conjugate exhibited a K _D of 0.47 nM for uPAR, confirming its binding activity. No binding activity for uPAR was observed for the YT1-S-MMAF conjugate. Therefore, these results indicate that the HLH peptide moiety of the present disclosure in PDC is important for molecular recognition of uPAR.

4-5. Evaluation of the Cancer Cell Growth Inhibitory Ability of uPAR-Bound PDCs. The cell growth inhibitory effects of the R4I-7(3M)-MMAF conjugate and YT1-S-MMAF conjugate on cancer cells were evaluated. Human cancer cell lines U-87MG cells and HeLa cells (uPAR-positive cells), and MCF-7 cells (uPAR-negative cells) were seeded at 3,000 cells/well/100 μL in a 96-well plate and cultured overnight at 37°C and 5% _CO2 . The R4I-7(3M)-MMAF conjugate or YT1-S-MMAF conjugate was added to each well. After 96 hours of culture, Cell Proliferation Reagent WST-1 (Roche) was added. The absorbance at 450 nm of each well was measured using a plate reader. Here, the cell viability was normalized by setting the cell viability under the condition where only medium was added to the cells to 100% and the cell viability under the condition where only medium was added without cells to 0%, and the relative value after normalization was used as the cell proliferation inhibitory activity of the compound.

The results of evaluating the cancer cell growth inhibitory activity of uPAR-binding PDC are shown in Figures 10B-C. The R4I-7(3M)-MMAF conjugate exhibited superior cell growth inhibitory activity compared to the YT1-S-MMAF conjugate in U-87MG and HeLa cells. However, no significant difference was observed in the cell growth inhibitory activity of the R4I-7(3M)-MMAF conjugate and YT1-S-MMAF in MCF-7 cells. Furthermore, cell growth inhibitory activity was observed even with the YT1-S-MMAF conjugate, which does not exhibit uPAR binding activity. This is thought to be due to cleavage of the linker between the peptide and MMAF during culture, resulting in the release of MMAF, which becomes toxic. Since the R4I-7(3M)-MMAF conjugate showed superior cell growth inhibitory effects compared to the YT1-S-MMAF conjugate only in uPAR-positive cells, these results indicate that the R4I-7(3M)-MMAF conjugate is transported into cells via binding to uPAR expressed in the cells, and inhibits cell growth by releasing MMAF.

Sequence Listing

Claims (19)

A urokinase-type plasminogen activator receptor (uPAR)-binding peptide that contains 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;
(c) X ²⁰ is F, Y or W, X ²² is R or K, and X ²³ and X ²⁴ are each independently L, I or V;
(d) Both conditions (a) and (b) above are satisfied; or (e) Both conditions (a) and (c) above are satisfied.
The uPAR-binding peptide according to claim 1. The uPAR-binding peptide according to claim 2, wherein in each of conditions (a) to (e), unspecified X ⁿ (n is an integer of 1 to 24) is independently selected from amino acids other than C, P, G, or S. 4. The uPAR-binding peptide according to claim 3, wherein in each of conditions (a) to (e), unspecified X ⁿ (n is an integer of 1 to 24) is independently selected from the group consisting of A, R, N, E, H, L, K, M, F, V and Y. ^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, and X ¹⁴ is A;
The uPAR-binding peptide according to claim 4. ^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;
The uPAR-binding peptide according to claim 4. The uPAR-binding peptide of claim 2, wherein helix 2 consists of the amino acid sequence KLAALKAKLAALKAA (SEQ ID NO: 19) or KLAALKAKLAALKAA (SEQ ID NO: 26). 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
The uPAR-binding peptide of claim 2, wherein 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)
3. The uPAR-binding peptide according to claim 2, which consists of any one of the amino acid sequences: The uPAR-binding peptide of claim 1, wherein the loop has a sequence length of 3 to 20 amino acids. The loop
GGGGGGG (SEQ ID NO: 27),
GKGGGGGG (SEQ ID NO: 28),
GGGGGGK (SEQ ID NO: 29) or GKGGGGK (SEQ ID NO: 30)
2. The uPAR-binding peptide according to claim 1, which consists of any one of the amino acid sequences: The uPAR-binding peptide of claim 2, wherein the peptide further comprises 1 to 3 amino acids at the N-terminus of helix 1 and/or the C-terminus of helix 2. The uPAR-binding peptide of claim 12, wherein the peptide further comprises CA at the N-terminus of helix 1 and AC at the C-terminus of helix 2-1, or CA at the N-terminus of helix 1 and C at the C-terminus of helix 2-2. The uPAR-binding peptide of claim 1, consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 32 to 43, or a sequence containing 1, 2, 3, 4, 5, or 6 amino acid mutations in said amino acid sequence. The uPAR-binding peptide of claim 1, wherein the N-terminus and C-terminus of the peptide are directly or indirectly linked. The uPAR-binding peptide of claim 15, wherein the N-terminus and C-terminus of the peptide are linked by a disulfide bond or a thioether bond, or are linked via a linker. A uPAR-binding peptide-drug conjugate comprising the uPAR-binding peptide of any one of claims 1 to 16 and a drug linked to the peptide. A composition comprising a uPAR-binding peptide described in any one of claims 1 to 16, or a uPAR-binding peptide-drug conjugate described in claim 17. The composition of claim 18 for use in the treatment of cancer.