Polypeptides that specifically bind nectin-4 and uses thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a polypeptide specifically combined with Nectin-4 and application thereof. 1 C1Z1C2Z2C3Z3C4X 2 The polypeptide can be specifically combined with Nectin-4, and can be used for treating and / or preventing diseases related to Nectin-4, in particular, cancers with high expression of Nectin-4.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a polypeptide that specifically binds to Nectin-4 and its uses. Background Technology

[0002] Cell adhesion molecules (CAMs) are a class of cell membrane surface glycoprotein molecules that mediate cell-cell and cell-extracellular matrix interactions. They play important roles in physiological processes such as cell polarization, differentiation, adhesion, proliferation, and migration, and are also involved in cell signal transduction. Therefore, abnormal CAM expression can reflect pathological changes in the body to some extent. CAMs can be broadly classified into the selectin family, integrin family, cadherin family, and immunoglobulin superfamily (IgSF). The expression of most CAMs in the body is nonspecific, and there are no significant differences in expression among different tissues and cells.

[0003] The term Nectin was first named and used by Japanese scientist Takahashi K in 1999, based on the Latin word "necto" (meaning connection). Nectin is a cell adhesion molecule belonging to the immunoglobulin (Ig) superfamily. It is a homolog of the poliovirus receptor (PVR / CD155) and is also known as the poliovirus receptor-related protein (PVRL). Nectin is an important transmembrane protein that constitutes adhesive junctions (AJs). The Nectin family consists of four members: Nectin-1, -2, -3, and -4, each with two to three splice variants. Except for nectin-1γ, which is a secreted protein lacking a transmembrane region, all members possess an extracellular domain with three Ig-like loops. Nectin is Ca2+-dependent. 2+ Nectin proteins, immunoglobulin-like intercellular adhesion molecules, play a role in cell-cell adhesion. They bind to the actin filament (F-actin)-binding protein afadin via their cytoplasmic tails and associate with the actin cytoskeleton, working in conjunction with other cell adhesion molecules and cell surface membrane receptors to regulate many other cellular activities.

[0004] Nectins play a crucial role in the formation of adhesion junctions between epithelial and endothelial cells. In addition to their pleiotropic function in intercellular adhesion, nectins are involved in a range of biological processes, including viral entry and immune regulation. Furthermore, some members of the nectin family can specifically interact with specific receptors expressed on immune cells (T cells and NK cells), mediating the transmission of immune regulatory signals.

[0005] Unlike other members of the Nectin family, which are widely expressed in various tissues of normal adults, Nectin-4 is expressed only in embryonic, placental, and mature epithelial tissues, and its expression is limited in healthy adult tissues. However, studies have found that Nectin-4 is abnormally overexpressed in various human cancers, especially in bladder cancer, breast cancer, and lung cancer, with positive rates exceeding 50% in each case. Existing research has confirmed that Nectin-4 protein is involved in mediating various cancer progression and metastasis processes, including cancer cell apoptosis resistance, angiogenesis, and lymphatic vessel development. Furthermore, its expression level in malignant tumors is closely related to the severity of the disease and patient survival. Therefore, Nectin-4 protein is an ideal biomarker for highly sensitive and specific cancer lesion localization imaging and targeted therapy. Simultaneously, as a receptor mediating measles virus invasion of cells, Nectin-4 itself has significant endocytosis-mediating capabilities.

[0006] Nectin-4 (Nectin cell adhesion molecule 4) is a cell adhesion molecule belonging to the Nectin and Nectin-like families. It is highly abundant in normal embryonic and fetal tissues, but its expression is very low in healthy adult tissues. Nectin-4 is a type I membrane protein; the extracellular region of the dimer Nectin-4 consists of three Ig domains: IgV and IgC. The metalloproteinase TACE / ADAM-17 can degrade the extracellular domain, producing soluble Nectin-4. This secreted form has been found in patients with breast cancer.

[0007] Studies have found that Nectin-4 promotes tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K / Akt pathway, and is believed to play an important role in the occurrence and metastasis of cancer. Based on this research background, drug research targeting this target has emerged. Enfortumab vedotin, a "first-in-class" drug jointly developed by Astellas and Seagen, is an ADC drug composed of the Nectin-4-targeting monoclonal antibody enfortumab and the microtubule disruptor MMAE linked by a cleavable dipeptide linker. Its anti-cancer mechanism of action lies in the fact that after binding to Nectin-4 on the surface of tumor cells, it enters the cells via endocytosis. The dipeptide linker is cleaved by intracellular proteolytic enzymes, releasing the toxin MMAE, thereby achieving targeted killing of tumor cells. 9MW2821 is the first domestically developed drug targeting the same target to be approved for clinical trials, and the second globally approved Nectin-4 ADC to be approved for clinical trials. BicycleTherapeutics has developed two Nectin-4-targeting therapies based on Bicycle technology. One type is BicycleToxin Conjugate (BTC)—BT8009, which is a Nectin-4-targeting toxin conjugate; the other is a bispecific bicyclic peptide—BT7480.

[0008] Although the mechanism of action of Nectin-4 in tumor progression and metastasis has not been fully elucidated, its clinical potential as a novel tumor-associated antigen is undeniable, providing great possibilities for the development of novel therapeutic drugs (such as nucleic acid drugs, peptide drugs and monoclonal antibodies). Therefore, there is an urgent need to develop more drugs targeting Nectin-4. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a polypeptide that specifically binds to Nectin-4 and its uses.

[0010] To achieve the above objectives, in one aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is: X 1 C1Z1C2Z2C3Z3C4X 2 ,in,

[0011] X 1 Selected from none or G;

[0012] X 2 G;

[0013] Z1 is X 3 X 4 X5 X 6 X 7 X 8 X 9 X 10 ;

[0014] Z2 is X 11 X 12 X 13 X 14 (X 15 ) n (X 16 ) p ;

[0015] Z3 is (Z4C5Z5C6) m X 17 X 18 X 19 X 20 X 21 ;

[0016] Z4 is X 22 X 23 X 24 ;

[0017] Z5 is X 25 ;

[0018] m, n, and p are each independently selected from 0 or 1;

[0019] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0020] X 4 ~X 25 Each amino acid is independently selected from any amino acid or non-natural amino acid;

[0021] C1, C2, C3, C4, C5, and C6 all represent cysteine ​​(cys, C).

[0022] In some embodiments, the polypeptide of the present invention is a linear peptide.

[0023] In some embodiments, the polypeptide of the present invention is a cyclic peptide.

[0024] In some implementations, any two cysteine ​​residues from C1, C2, C3, C4, C5, and C6 can form disulfide bonds, and one, two, or three disulfide bonds can be formed simultaneously.

[0025] In some implementations, disulfide bonds can be formed between C1 and C4.

[0026] In some implementations, disulfide bonds can be formed between C2 and C3.

[0027] In some implementations, disulfide bonds can be formed between C2 and C5.

[0028] In some implementations, disulfide bonds can be formed between C3 and C6.

[0029] In some implementations, disulfide bonds can be formed between C1 and C4, and disulfide bonds can be formed between C2 and C3.

[0030] In some implementations, disulfide bonds can be formed between C1 and C4, between C2 and C5, and between C3 and C6.

[0031] In some embodiments, the sequence has four cysteine ​​residues: C1, C2, C3, and C4, wherein C1 and C4 can form a disulfide bond, and C2 and C3 can form a disulfide bond.

[0032] In some embodiments, the sequence has six cysteine ​​residues: C1, C2, C3, C4, C5, and C6, wherein disulfide bonds can be formed between C1 and C4, between C2 and C5, and between C3 and C6.

[0033] In some embodiments, Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;in,

[0034] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0035] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0036] X 5 Selected from F, K, Y;

[0037] X 6 Selected from K, F, Y, W, E, S, L;

[0038] X 7 Selected from P and W;

[0039] X 8 Selected from P, W, K, H, D, Q, N;

[0040] X 9 Selected from W, G, R;

[0041] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0042] In some embodiments, Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ; where X 3 Selected from F and W.

[0043] In some embodiments, Z2 is X 11 X 12 X 13 X 14 (X 15 ) n (X 16 ) p ;in,

[0044] X 11 Let K be the value of K.

[0045] X 12 For Q;

[0046] X 13 The answer is D;

[0047] X 14 S;

[0048] X 15 Selected from G, D, and E;

[0049] X 16 Selected from V and F;

[0050] n and p are each independently selected from 0 or 1.

[0051] In some implementations, Z3 is (Z4C5Z5C6). m X 17 X 18 X 19 X 20 X 21 When m is 0, Z3 is X. 17 X 18 X 19 X 20 X 21 When m is 1, Z3 is Z4C5Z5C6X. 17 X 18 X 19 X 20 X 21 ;in,

[0052] X 17 Selected from E, A, L, D, Q, K, S;

[0053] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0054] X 19 Selected from V, I, F;

[0055] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0056] X 21 Selected from V, I, K, M, D;

[0057] Z4 is X 22 X 23 X 24 The X 22 For L; X 23 For A; X 24 G;

[0058] Z5 is X 25 The X 25 V.

[0059] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0060] GC1Z1C2KQDS(X 15 ) n (X 16 ) p C3X 17 X 18 X 19 X 20 X 21 C4G, among which...

[0061] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;

[0062] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0063] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0064] X5 Selected from F, K, Y;

[0065] X 6 Selected from K, F, Y, W, E, S, L;

[0066] X 7 Selected from P and W;

[0067] X 8 Selected from P, W, K, H, D, Q, N;

[0068] X 9 Selected from W, G, R;

[0069] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0070] X 15 Selected from G, D, and E;

[0071] X 16 Selected from V and F;

[0072] X 17 Selected from E, A, L, D, Q, K, S;

[0073] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0074] X 19 Selected from V, I, F;

[0075] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0076] X 21 Selected from V, I, K, M, D;

[0077] n and p are each independently selected from 0 or 1.

[0078] In some embodiments, n=0, p=0, and the amino acid sequence of the polypeptide is: GC1Z1C2KQDSDC3X 1 7 X 18 X 19 X 20 X 21 C4G, among which...

[0079] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10;

[0080] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0081] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0082] X 5 Selected from F, K, Y;

[0083] X 6 Selected from K, F, Y, W, E, S, L;

[0084] X 7 Selected from P and W;

[0085] X 8 Selected from P, W, K, H, D, Q, N;

[0086] X 9 Selected from W, G, R;

[0087] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0088] X 17 Selected from E, A, L, D, Q, K, S;

[0089] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0090] X 19 Selected from V, I, F;

[0091] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0092] X 21 Selected from V, I, K, M, D.

[0093] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0094] GC1WX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G.

[0095] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0096] GC1WX 4 FX 6 X 7 X 8 X 9 X 10 C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G.

[0097] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0098] GC1WX 4 FX 6 PX 8 X 9 X 10 C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G.

[0099] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0100] GC1WX 4 FX 6 PX 8 WX 10 C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G.

[0101] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0102] GC1FX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G.

[0103] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0104] GC1WX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3X 17 X 18 X 19 X 20 VC4G.

[0105] In some embodiments, n=1, p=0, and the amino acid sequence of the polypeptide is:

[0106] GC1Z1C2KQDSDX 15 C3X 17 X 18 X 19 X 20 X 21 C4G, where X 15 Selected from G, D, and E.

[0107] In some embodiments, n=1, p=1, and the amino acid sequence of the polypeptide is:

[0108] GC1Z1C2KQDSDX 15 X 16 C3X 17 X 18 X 19 X 20 X 21 C4G, where X 15 Selected from G, D, E, X 16 Selected from V and F.

[0109] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0110] GC1Z1C2KQDSD(X 15 ) n (X 16 ) p C3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, among which...

[0111] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X9 X 10 ;

[0112] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0113] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0114] X 5 Selected from F, K, Y;

[0115] X 6 Selected from K, F, Y, W, E, S, L;

[0116] X 7 Selected from P and W;

[0117] X 8 Selected from P, W, K, H, D, Q, N;

[0118] X 9 Selected from W, G, R;

[0119] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0120] X 15 Selected from G, D, and E;

[0121] X 16 Selected from V and F;

[0122] X 17 Selected from E, A, L, D, Q, K, S;

[0123] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0124] X 19 Selected from V, I, F;

[0125] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0126] X 21 Selected from V, I, K, M, D;

[0127] n and p are each independently selected from 0 or 1.

[0128] In some embodiments, n=0, p=0, and the amino acid sequence of the polypeptide is:

[0129] GC1Z1C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, among which...

[0130] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;

[0131] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0132] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0133] X 5 Selected from F, K, Y;

[0134] X 6 Selected from K, F, Y, W, E, S, L;

[0135] X 7 Selected from P and W;

[0136] X 8 Selected from P, W, K, H, D, Q, N;

[0137] X 9 Selected from W, G, R;

[0138] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0139] X 17 Selected from E, A, L, D, Q, K, S;

[0140] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0141] X 19 Selected from V, I, F;

[0142] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0143] X 21 Selected from V, I, K, M, D.

[0144] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0145] GC1WX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0146] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0147] GC1WX 4 FX 6 X 7 X 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0148] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0149] GC1WX 4 FX 6 PX 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0150] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0151] GC1WX 4 FX 6 PX 8 WX 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0152] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0153] GC1FX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0154] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0155] GC1WX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 VC4G.

[0156] In some embodiments, n=1, p=0, and the amino acid sequence of the polypeptide is:

[0157] GC1Z1C2KQDSDX 15 C3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, where X 15 Selected from G, D, and E.

[0158] In some embodiments, n=1, p=1, and the amino acid sequence of the polypeptide is:

[0159] GC1Z1C2KQDSDX 15 X 16 C3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, where X 15 Selected from G, D, E, X 16 Selected from V and F.

[0160] The present invention also provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is: C1Z1C2Z2C3Z3C4G, wherein,

[0161] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;

[0162] Z2 is X 11 X 12 X 13 X 14 (X 15 ) n (X 16 ) p ;

[0163] Z3 is (Z4C5Z5C6) m X 17 X 18 X 19 X 20 X 21 ;

[0164] Z4 is X 22 X 23 X 24 ;

[0165] Z5 is X 25 ;

[0166] m, n, and p are each independently selected from 0 or 1;

[0167] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0168] X 4 ~X 25 Each amino acid is independently selected from any amino acid or non-natural amino acid.

[0169] In some embodiments, Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;in,

[0170] X 3Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0171] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0172] X 5 Selected from F, K, Y;

[0173] X 6 Selected from K, F, Y, W, E, S, L;

[0174] X 7 Selected from P and W;

[0175] X 8 Selected from P, W, K, H, D, Q, N;

[0176] X 9 Selected from W, G, R;

[0177] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E.

[0178] In some embodiments, Z2 is X 11 X 12 X 13 X 14 (X 15 ) n (X 16 ) p ;in,

[0179] X 11 Selected from K;

[0180] X 12 Selected from Q;

[0181] X 13 Selected from D;

[0182] X 14 Selected from S;

[0183] X 15 Selected from G, D, and E;

[0184] X 16 Selected from V and F;

[0185] n and p are each independently selected from 0 or 1.

[0186] In some implementations, when m is 0, Z3 is X. 17 X 18 X 19 X 20 X21 When m is 1, Z3 is Z4C5Z5C6X. 17 X 18 X 19 X 20 X 21 ;in,

[0187] X 17 Selected from E, A, L, D, Q, K, S;

[0188] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0189] X 19 Selected from V, I, F;

[0190] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0191] X 21 Selected from V, I, K, M, D;

[0192] Z4 is X 22 X 23 X 24 The X 22 For L; X 23 For A; X 24 G;

[0193] Z5 is X 25 The X 25 V.

[0194] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0195] C1Z1C2KQDSD(X 15 ) n (X 16 ) p C3X 17 X 18 X 19 X 20 X 21 C4G, among which...

[0196] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;

[0197] X 3Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0198] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0199] X 5 Selected from F, K, Y;

[0200] X 6 Selected from K, F, Y, W, E, S, L;

[0201] X 7 Selected from P and W;

[0202] X 8 Selected from P, W, K, H, D, Q, N;

[0203] X 9 Selected from W, G, R;

[0204] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0205] X 15 Selected from G, D, and E;

[0206] X 16 Selected from V and F;

[0207] X 17 Selected from E, A, L, D, Q, K, S;

[0208] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0209] X 19 Selected from V, I, F;

[0210] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0211] X 21 Selected from V, I, K, M, D;

[0212] n and p are each independently selected from 0 or 1.

[0213] In some embodiments, p = 0, n = 0, and the amino acid sequence of the polypeptide is: C1Z1C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G, among which...

[0214] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;

[0215] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0216] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0217] X 5 Selected from F, K, Y;

[0218] X 6 Selected from K, F, Y, W, E, S, L;

[0219] X 7 Selected from P and W;

[0220] X 8 Selected from P, W, K, H, D, Q, N;

[0221] X 9 Selected from W, G, R;

[0222] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0223] X 17 Selected from E, A, L, D, Q, K, S;

[0224] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0225] X 19 Selected from V, I, F;

[0226] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0227] X 21 Selected from V, I, K, M, D.

[0228] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0229] C1WX 4 X 5 X 6 X 7 X8 X 9 X 10 C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G.

[0230] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0231] C1FX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G.

[0232] In some embodiments, n=1, p=0, and the amino acid sequence of the polypeptide is:

[0233] C1Z1C2KQDSDX 15 C3X 17 X 18 X 19 X 20 X 21 C4G, X 15 Selected from G, D, and E.

[0234] In some embodiments, n=1, p=1, and the amino acid sequence of the polypeptide is:

[0235] C1Z1C2KQDSDX 15 X 16 C3X 17 X 18 X 19 X 20 X 21 C4G, X 15 Selected from G, D, E; X 16 Selected from V and F.

[0236] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0237] C1Z1C2KQDSD(X 15 ) n (X 16 ) p C3LAGC5VC6X 17 X18 X 19 X 20 X 21 C4G, among which...

[0238] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;

[0239] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0240] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0241] X 5 Selected from F, K, Y;

[0242] X 6 Selected from K, F, Y, W, E, S, L;

[0243] X 7 Selected from P and W;

[0244] X 8 Selected from P, W, K, H, D, Q, N;

[0245] X 9 Selected from W, G, R;

[0246] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0247] X 15 Selected from G, D, and E;

[0248] X 16 Selected from V and F;

[0249] X 17 Selected from E, A, L, D, Q, K, S;

[0250] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0251] X 19 Selected from V, I, F;

[0252] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0253] X 21 Selected from V, I, K, M, D;

[0254] n and p are each independently selected from 0 or 1.

[0255] In some embodiments, n=0, p=0, and the amino acid sequence of the polypeptide is C1Z1C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, among which...

[0256] Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ;

[0257] X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of the amino acid;

[0258] X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A;

[0259] X 5 Selected from F, K, Y;

[0260] X 6 Selected from K, F, Y, W, E, S, L;

[0261] X 7 Selected from P and W;

[0262] X 8 Selected from P, W, K, H, D, Q, N;

[0263] X 9 Selected from W, G, R;

[0264] X 10 Selected from A, T, L, H, Q, S, G, M, N, D, E;

[0265] X 17 Selected from E, A, L, D, Q, K, S;

[0266] X 18 Selected from T, A, Q, K, P, T, Y, I;

[0267] X 19 Selected from V, I, F;

[0268] X 20 Selected from E, A, R, E, I, Y, V, L, T, G;

[0269] X 21 Selected from V, I, K, M, D;

[0270] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0271] C1WX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0272] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0273] C1WX 4 X 5 X 6 WX 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0274] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0275] C1FX 4 X 5 X 6 X 7 X 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0276] In some embodiments, the amino acid sequence of the polypeptide is as follows:

[0277] C1FX 4 X 5 X6 WX 8 X 9 X 10 C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G.

[0278] In some embodiments, n=1, p=0, and the amino acid sequence of the polypeptide is:

[0279] C1Z1C2KQDSDX 15 C3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, X 15 Selected from G, D, and E.

[0280] In some embodiments, n=1, p=1, and the amino acid sequence of the polypeptide is:

[0281] C1Z1C2KQDSDX 15 X 16 C3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, X 15 Selected from G, D, E; X 16 Selected from V and F.

[0282] On the other hand, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide comprising any one of the following (i) to (ii):

[0283] (i) an amino acid sequence having at least 80% identity with the amino acid sequence described above and retaining the original function; (ii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or modified in the amino acid sequence shown above and retain the original function.

[0284] In some embodiments, the polypeptide comprises any one of the following (i) to (ii):

[0285] (i) An amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO.1 and retaining the function of the sequence shown in SEQ ID NO.1;

[0286] (ii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or modified in the amino acid sequence shown in SEQ ID NO.1 and the function of the sequence shown in SEQ ID NO.1 is retained.

[0287] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.1 and retaining the function of the sequence shown in SEQ ID NO.1.

[0288] In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO.1 and retaining the function of the sequence shown in SEQ ID NO.1.

[0289] In some embodiments, the polypeptide comprises an amino acid sequence in which any one amino acid residue in the amino acid sequence shown in SEQ ID NO.1 is replaced by any amino acid or non-natural amino acid and retains the function of the sequence shown in SEQ ID NO.1.

[0290] In some embodiments, the polypeptide comprises an amino acid sequence in which any two amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are replaced by any amino acid or non-natural amino acid and the function of the sequence shown in SEQ ID NO.1 is retained.

[0291] In some embodiments, the polypeptide comprises an amino acid sequence that is missing one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 but retains the function of the sequence shown in SEQ ID NO.1.

[0292] In some embodiments, any one or more amino acids in the polypeptide are replaced by any amino acid or non-natural amino acid while retaining the original functional amino acid sequence.

[0293] On the other hand, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide being selected from the amino acid sequences of SEQ ID NO:1 to SEQ ID NO:98:

[0294] GCWHFKPPWACKQDSGVCETVEVCG(SEQ ID NO:1),

[0295] GCWFFFPWWTCKQDSDCLAGCVCEAVVVCG (SEQ ID NO: 2),

[0296] GCWVFYPPWLCKQDSEFCEAVEVCG(SEQ ID NO:3)、

[0297] GCWMFYPPWACKQDSGCAAVAVCG(SEQ ID NO:4)、

[0298] GCWVFWPQWNCKQDSDCLAVEVCG(SEQ ID NO:5)、

[0299] GCWTFYPKWQCKQDSDCLAVRVCG(SEQ ID NO:6)、

[0300] GCWTFYPKWHCKQDSDCLAGCVCDPVVVCG(SEQ ID NO:7)、

[0301] GCWKFFPPWTCKQDSDCLAGCVCDPIVVCG(SEQ ID NO:8)、

[0302] GCWVFYPPWSCKQDSDCLAGCVCEAVTVCG(SEQ ID NO:9)、

[0303] GCWVFFPHWTCKQDSDCLAGCVCEAVYICG(SEQ ID NO:10)、

[0304] GCWYFWPPWGCKQDSDCLAGCVCEPIEVCG(SEQ ID NO:11)、

[0305] GCWTFWPDWNCKQDSDCLAGCVCEPVLVCG(SEQ ID NO:12)、

[0306] GCWRFWPPWHCKQDSDCLAGCVCQPIVVCG(SEQ ID NO:13)、

[0307] GCWVFFPPWGCKQDSDCLAGCVCDAVYVCG(SEQ ID NO:14)、

[0308] GCWTFYPGWSCKQDSDCLAGCVCEPIVVCG(SEQ ID NO:15)、

[0309] GCWVFWPQWNCKQDSDCLAVEVCG(SEQ ID NO:16)、

[0310] GCWVFWPQWACKQDSDCLAGCVCEPVIVCG(SEQ ID NO:17)、

[0311] GCWYFYPEWMCKQDSDCLAGCVCEPIVVCG(SEQ ID NO:18)、

[0312] GCWTFWPPWACKQDSDCLAGCVCEQVVVCG(SEQ ID NO:19)、

[0313] GCWIFYPHWTCKQDSDCLAGCVCEPVEVCG(SEQ ID NO:20)、

[0314] GCWYFYPPWMCKQDSDCLAGCVCEPVEVCG(SEQ ID NO:21)、

[0315] GCWVFEPPWQCKQDSGVCEQVIVCG(SEQ ID NO:22)、

[0316] GCWIFSPPWTCKQDSCDKVYVCG(SEQ ID NO:23)、

[0317] GCWYFWPPWTCKQDSDCLAGCVCQTVIICG(SEQ ID NO:24)、

[0318] GCWTFYPPWMCKQDSDCLAGCVCKPVLVCG(SEQ ID NO:25)、

[0319] CWQYWWDGQCKQDSDCLAGCVCEYYGVCG(SEQ ID NO:26)、

[0320] CFWKLWHRQCKQDSDCLAGCVCAIYGKCG(SEQ ID NO:27)、

[0321] CFWKLWHRDCKQDSDCLAGCVCAIYGMCG(SEQ ID NO:28)、

[0322] CFWKLWHGECKQDSDCLAGCVCSIYKVCG(SEQ ID NO:29)、

[0323] CWAYWWNGDCKQDSDCLAGCVCEYFGDCG(SEQ ID NO:30)、

[0324] GCWHFKPPWACKQDSGVCETVLVCG(SEQ ID NO:31)、GCWHFKPPWACKQDSGVCEPVEVCG(SEQID NO:32)、GCWHFKPPWACKQDSGVCDTVEVCG(SEQ ID NO:33)、GCWTFKPPWACKQDSGVCETVEVCG(SEQ ID NO:34)、GCWHFYPPWACKQDSGVCETVEVCG(SEQ ID NO:35)、GCWHFKPKWACKQDSGVCETVEVCG(SEQ ID NO:36)、GCWHFKPDWACKQDSGVCETVEVCG(SEQ ID NO:37)、GCWHFKPPWNCKQDSGVCETVEVCG(SEQ ID NO:38)、GCWHFKPPWLCKQDSGVCETVEVCG(SEQ IDNO:39)、GCWHFKPPWTCKQDSGVCETVEVCG(SEQ ID NO:40)、GC{1-Nal}HFKPPWACKQDSGVCETVEVCG(SEQ ID NO:41)、GCWAFKPPWACKQDSGVCETVEVCG(SEQ ID NO:42)、GCWKFKPPWACKQDSGVCETVEVCG(SEQ ID NO:43)、GCWVFKPPWACKQDSGVCETVEVCG(SEQ ID NO:44)、GCWRFKPPWACKQDSGVCETVEVCG(SEQ ID NO:45)、GCWHFRPPWACKQDSGVCETVEVCG(SEQ IDNO:46)、GCWHFWPPWACKQDSGVCETVEVCG(SEQ ID NO:47)、GCWHFFPPWACKQDSGVCETVEVCG(SEQID NO:48)

[0325] GCWHF{Dab}PPWACKQDSGVCETVEVCG(SEQ ID NO:49)、GCWHFKPRWACKQDSGVCETVEVCG(SEQ ID NO:50)、GCWHFKPAWACKQDSGVCETVEVCG(SEQ ID NO:51)、GCWHFKP{Hyp}WACKQDSGVCETVEVCG(SEQ ID NO:52)、GCWHFKP{Dab}WACKQDSGVCETVEVCG(SEQ ID NO:53)、GCWHFKPP{1-Nal}ACKQDSGVCETVEVCG(SEQ ID NO:54)、GCWHFKPPW{Aib}CKQDSGVCETVEVCG(SEQ ID NO:55)、GCWHFKPPW{Abu}CKQDSGVCETVEVCG(SEQ ID NO:56)、GCWHFKPPWVCKQDSGVCETVEVCG(SEQ ID NO:57)、GCWHFKPPWSCKQDSGVCETVEVCG(SEQ ID NO:58)、GCWHFKPPWMCKQDSGVCETVEVCG(SEQ ID NO:59)、GCWHFKPPWGCKQDSGVCETVEVCG(SEQ IDNO:60)、GCWHFKPPWACKQDSGVCESVEVCG(SEQ ID NO:61)、GCWHFKPPWACKQDSGVCETVDVCG(SEQID NO:62)、GCWHFKPPWACKQDSGVCETVVVCG(SEQ ID NO:63)、GCWHFKPPWACKGCETVEVCG(SEQID NO:64)、GCWHFKPPWACKGGCETVEVCG(SEQ ID NO:65)、GCWHFKPPWACKGPCETVEVCG(SEQ IDNO:66)、GCWHFKPPWA{D-Pro}PETVEVCG(SEQ ID NO:67)、GCWTFWPDWNCWLSEECDFMCPCEPVLVCG(SEQ ID NO:68)、GCWTFWPDWNCHRMETLVGVIVACEPVLVCG(SEQ ID NO:69)、GCWTFWPDWNCNVWHQCTNTFICEPVLVCG(SEQ ID NO:70)、GCWTFWPDWNCNVWHQSTNTFICEPVLVCG(SEQ ID NO:71)、GCWTFWPDWNCLTGTATVNRSVWVFVHCEPVLVCG(SEQ IDNO:72)、

[0326] GCWTFWPDWNCLTGTATWNGNVVISVFCEPVLVCG(SEQ ID NO:73)、

[0327] GCWFFHPIWNCKQDSDCLAGCVCPLVTVCG(SEQ ID NO:74)、

[0328] GCWHF{Dab}PPWMCKQDSGVCETVEVCG(SEQ ID NO:75)、

[0329] GCWHF{Dab}PPW{Nle}CKQDSGVCETVEVCG(SEQ ID NO:76)、

[0330] GCWHFKPPW{Nle}CKQDSGVCETVEVCG(SEQ ID NO:77)、

[0331] GCWHF{D-Dab}PPWACKQDSGVCETVEVCG(SEQ ID NO:78)、

[0332] GCWHF{D-Tyr}PPWACKQDSGVCETVEVCG(SEQ ID NO:79)、

[0333] GCWHFKPPW{D-Ala}CKQDSGVCETVEVCG(SEQ ID NO:80)、

[0334] GCWTFWPDWNCKQDSDGSCLAGCVCEPVLVCG(SEQ ID NO:81)、

[0335] GCWTFWPDWNCKQDSDGGSCLAGCVCEPVLVCG(SEQ ID NO:82)、

[0336] GCWTFYPKWNCKQDSDCLAGCVCEPVVVCG(SEQ ID NO:83)、

[0337] GCWMFWPDWHCKQDSDCLAGCVCKPVIVCG(SEQ ID NO:84)、

[0338] GCWFFKPWWTCKQDSDCLAGCVCDPIVVCG(SEQ ID NO:85)、

[0339] GCWTFWPPWACKQDSDCLAGCVCEQIIVCG(SEQ ID NO:86)、

[0340] GCWIFWPPWGCKQDSDCLAGCVCEPVLVCG(SEQ ID NO:87)、

[0341] GCWYFFPPWTCKQDSDCLAGCVCEPVRICG(SEQ ID NO:88)、

[0342] GCWYFYPKWTCKQDSDCLAGCVCDPVVICG(SEQ ID NO:89)、

[0343] GCWVFWPPWGCKQDSDCLAGCVCEAVVVCG(SEQ ID NO:90)、

[0344] GCWVFFPWWSCKQDSDCLAGCVCEAVYICG(SEQ ID NO:91)、

[0345] GCWYFYPPWACKQDSDCLAGCVCDAIVICG(SEQ ID NO:92)、

[0346] GCWTFWPPWHCKQDSDCLAGCVCEPVVICG(SEQ ID NO:93)、

[0347] GCWTFYPWWECKQDSDCLAGCVCDAVVVCG(SEQ ID NO:94)、

[0348] GCWFFYPPWGCKQDSDCLAGCVCEPVTVCG(SEQ ID NO:95)、

[0349] GCWVFFPKWNCKQDSDCLAGCVCEPVEVCG(SEQ ID NO:96)、

[0350] GCWKFEPWWQCKQDSDCLAGCVCQPVIICG(SEQ ID NO:97)、

[0351] GCWRFKPWWACKQDSDCLAGCVCEIIEVCG(SEQ ID NO:98)。

[0352] When used herein, site-directed mutagenesis was used to identify the amino acids that play a key role in the activity of the peptide of the present invention. The amino acid residues that play a key role in the activity of the polypeptide molecule were identified as C1, C2, C3, C4, C5, C6, K at position 11, Q at position 12, D at position 13, and S at position 14.

[0353] Site-directed mutagenesis techniques, such as alanine scanning, introduce mutations into every residue of the polypeptide molecule and examine the biological activity of the resulting polypeptide molecule to identify the amino acid residues that play a key role in the activity of the polypeptide molecule; PCR site-directed mutagenesis techniques, such as designing primers containing non-specific paired bases, then introducing the mutation site into the product through PCR.

[0354] In some embodiments, the peptide is further modified. Such modifications include N-terminal modification, C-terminal modification, side chain modification, amino acid modification, peptide backbone modification, and binding to other peptides or proteins.

[0355] On the other hand, the present invention provides a polynucleotide whose sequence comprises a nucleotide sequence encoding the above-described amino acid sequence.

[0356] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or its pharmaceutically acceptable salt or polynucleotide, in combination with one or more pharmaceutically acceptable excipients.

[0357] On the other hand, the present invention provides the use of the above-described polypeptides or pharmaceutically acceptable salts, polynucleotides or pharmaceutical compositions thereof in the preparation of medicaments for the treatment and / or prevention of diseases related to Nectin-4.

[0358] In some implementations, the Nectin-4-related disease is a cancer that highly expresses Nectin-4.

[0359] In some embodiments, the cancers that highly express Nectin-4 are selected from at least one of gastric cancer, liver cancer, hepatocellular carcinoma, bladder cancer, urothelial carcinoma, urethral cancer, renal pelvis cancer, ureteral cancer, lung cancer, non-small cell lung cancer, breast cancer, ductal carcinoma of the breast, triple-negative breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer, or esophageal cancer.

[0360] Pharmaceutically acceptable salts of the compounds of this invention include salts derived from suitable inorganic acids and bases, as well as salts derived from organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts formed by amino groups with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or salts formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, disaccharide, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucono-heptahydrate, glyceryl phosphate, gluconate, hemisulfate, heptahydrate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectinate, persulfate, 3-phenylpropionate, phosphate, neopentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N salts. + (C 1–4 Alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium salts. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed where appropriate using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0361] In some embodiments, the pharmaceutically acceptable salt is selected from free acids or at least one of sodium, potassium, calcium, and ammonium salts.

[0362] Compared with existing technologies, the peptides provided by this invention have high specificity and high affinity, which helps to reduce the dosage of drugs and potential side effects.

[0363] As used herein, the term "an" entity refers to one or more of the entities mentioned. Therefore, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein.

[0364] The term "peptide" is intended to include both the singular and plural forms of "peptide" and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "peptide" refers to any one or more chains of two or more amino acids, not a specific length of the product. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains are included within the definition of "peptide," and the term "peptide" can be used in place of or interchanged with any of these terms. The term "peptide" also means a product modified after peptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, protein hydrolysis, or modification by non-naturally occurring amino acids. Peptides can be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be produced in any manner, including through chemical synthesis.

[0365] The term "amino acid" refers to any organic compound containing an amino group (--NH2) and a carboxyl group (--COOH); it is preferably in free group form or as part of a peptide bond after condensation. It includes naturally occurring amino acids (e.g., α-amino acids), non-natural amino acids, modified amino acids, and non-natural amino acids. It includes D-amino acids and L-amino acids.

[0366] Natural amino acids include those found in nature, such as the 23 amino acids that combine to form peptide chains, the building blocks of a wide range of proteins. These are primarily L stereoisomers, although small amounts of D-amino acids are present in bacterial envelopes and some antibiotics. The "twenty α-amino acids that form naturally encoded polypeptides" are understood in the field and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0367] The “non-standard” natural amino acids are pyrrolidone (found in methanogens and other eukaryotes), selenocysteine ​​(found in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts).

[0368] “Non-natural” amino acids are non-protein-derived amino acids (i.e., those not naturally encoded or discovered in the genetic code), which are either naturally occurring or chemically synthesized. More than 140 non-natural amino acids are known, and there are likely thousands more combinations. Examples of “non-natural” amino acids include β-amino acids (β3 and β2), high-molecular-weight amino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, cyclic-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, α-methyl amino acids, and N-methyl amino acids. Non-natural amino acids also include modified amino acids. “Modified” amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include one or more groups or chemical moieties that are not naturally present on the amino acid.

[0369] The term "amino acid derivative" refers to a compound generated from an amino acid by modifying its N and / or C terminus. Non-limiting examples include the conversion of the carboxyl group to a salt, ester, acylhydrazine, isohydroxamic acid, or amide, and the conversion of the amino group to amides, ureas, thioureas, thioamides, sulfonamides, phosphoramides, borate amides, or alkylamines. A part or moiety of a compound, generated by modification of the amino acid at its C and / or N terminus, may also be referred to as an amino acid unit. Furthermore, the amino acids can be derivatized in their side chains. Such derivatization is typically specifically indicated herein if the derivatized amino acid is one or more amino acids whose side chains have been derivatized once or several times. Preferred derivatization of the side chains can be carried out, particularly where the side chains have functional groups. Preferred functional groups are, for example, amino, carboxyl, thiohydroxy, or alcohol groups.

[0370] The term "amino acid analogue" refers to compounds derived from amino acids by substituting the amino and / or carboxyl groups with other groups that can mimic them. Non-limiting examples include thioamides, ureas, thioureas, acylhydrazides, esters, alkylamines, sulfonamides, phosphoramides, ketones, alcohols, borate amides, benzodiazepines, and other combinations of aromatic or non-aromatic heterocycles (for a review see MAEstiarte, DHRich in Burgers Medicinal Chemistry, 6th edition, volume 1, part 4, John Wiley & Sons, New York, 2002).

[0371] Non-natural amino acids include, but are not limited to, 2-amino fatty acids (Aad), 3-amino fatty acids (βAad), β-alanine, β-aminopropionic acid (βAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid, piperidine carboxylic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (βAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), and desmokinin (…). Des), 2,2”-Diaminopimelic acid (Dpm), 2,3-Diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesin (Ide), iso-isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6- N-Methyllysine (MeLys), N-Methylvaline (MeVal), novovaline (Nva), novoleucine (Nle), ornithine (Orn), Dab, 1-Naphthylalanine (1-Nal), 2-Naphthylalanine (2-Nal), 2-Fluoro-phenylalanine, 3-Fluoro-phenylalanine, 4-Fluoro-phenylalanine, 2-Chlorophenylalanine, 3-Chlorophenylalanine, 4-Chlorophenylalanine, 2-Bromophenylalanine, 2-Methylphenylalanine, 2-Trifluoromethylphenylalanine 2-Cyanophenylalanine, 2-Phenylenealanine, 2,4-Dichlorophenylalanine, 3,4-Dichlorophenylalanine, 2,6-Dimethylphenylalanine, High-Typtophan, 2-Methyl-Typtophan, 7-Bromo-Typtophan, 6-Chloro-Typtophan, 5-Hydroxy-Typtophan, 7-Methyl-Typtophan, High-Tyrosine, Meta-Tyrosine, 3-Hydroxy-Tyrosine, 3-Chloro-Tyrosine, 3-Bromo-Tyrosine, 3-Nitro-Tyrosine, 3,5-Diiodotyrosine, O-Methyltyrosine, O-Ethyltyrosine, O-Benzyltyrosine.

[0372] The terms "homology," "identity," or "similarity" refer to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which may be aligned for the purpose of comparison. Molecules are homologous at a position when a position in the compared sequences is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of shared matching or homologous positions. An "irrelevant" or "non-homologous" sequence has less than 40% identity with one of the sequences disclosed in this invention, but preferably less than 25%.

[0373] The term "treatment" or "therapy" refers to medical treatment and preventative or preventive measures aimed at preventing or slowing (reducing) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stable disease state (i.e., no worsening), delayed or slowed disease progression, improved or reduced disease state, and remission (partial or complete), whether detectable or undetectable. "Treatment" can also mean extended survival compared to expected survival without treatment. People who need treatment include those who already have the disease or disorder, those who are susceptible to the disease or disorder, or those who need prevention of the disease or disorder.

[0374] The term "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085, refers to a useful method for identifying residues or regions of a peptide that can be targeted for mutagenesis. In this method, residues or target residue groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the peptide with, for example, its receptor is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the peptide-receptor complex may be used to identify the contact points between the peptide and its receptor. Such contact residues and adjacent residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they possess the desired properties.

[0375] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. Attached Figure Description

[0376] Figure 1 To test the affinity of peptide 1 in Example 2 for Nectin-4;

[0377] Figure 2 To test the affinity of peptide 1 in Example 2 for Nectin-1;

[0378] Figure 3 To test the affinity of peptide 1 for Nectin-2 in Example 2;

[0379] Figure 4 To test the affinity of peptide 1 in Example 2 for Nectin-3;

[0380] Figure 5 To test the affinity of peptide 15 for Nectin-4 in Example 2;

[0381] Figure 6 To test the affinity of peptide 15 in Example 2 for Nectin-1;

[0382] Figure 7 To test the affinity of peptide 15 for Nectin-2 in Example 2;

[0383] Figure 8 To test the affinity of peptide 15 for Nectin-3 in Example 2;

[0384] Figure 9 The fluorescence image for test example 3BT8009 is shown in the fluorescence imaging detection. From left to right, the peptide concentrations are 2 μM, 0.4 μM, and 0.08 μM, respectively.

[0385] Figure 10 The fluorescence image of peptide 1 in test example 3 is shown. From left to right, the peptide concentrations are 2 μM, 0.4 μM, and 0.08 μM.

[0386] Figure 11 The fluorescence image of peptide 15 in test example 3 is shown. From left to right, the peptide concentrations are 2 μM, 0.4 μM, and 0.08 μM.

[0387] Figure 12 The results of fluorescence imaging detection for test example 3BT8009, peptide 1, and peptide 15;

[0388] Figure 13 The results of flow cytometry detection of peptide 1 in test example 4;

[0389] Figure 14 The results of flow cytometry detection of peptide 15 in test example 4 are shown. Specific Implementation

[0390] To make the objectives, features, and advantages of this invention more apparent and understandable, the invention will be further described in detail below with reference to the accompanying drawings and embodiments, but this is not intended to limit the invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0391] The present invention provides a method for synthesizing the linear precursors of polypeptide compounds and their derivatives using solid-phase synthesis. The precursors are oxidized with DMSO to form two and three intramolecular disulfide bonds. The synthesis support is Fmoc-Gly-Wang Resin resin. During the synthesis process, the Fmoc-Gly-Wang Resin resin is first fully swollen in N,N-dimethylformamide (DMF). Then, the solid support is repeatedly condensed with the activated amino acid derivative → washing → deprotecting the Fmoc → washing → the next round of amino acid condensation to achieve the desired polypeptide chain length. Finally, the polypeptide is cleaved from the solid support by reacting the resin with a mixed solution of trifluoroacetic acid:water:triisopropylsilane:aniline sulfide (90:2.5:2.5:5, v:v:v:v). After precipitation with frozen methyl tert-butyl ether, a solid crude linear precursor is obtained. The cleaved crude linear precursor is then subjected to disulfide bond oxidation in a weakly alkaline solution to obtain the target crude polypeptide. The crude polypeptide was purified and separated by a C-18 reversed-phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure polypeptides and their derivatives.

[0392] Experimental reagents

[0393]

[0394]

[0395] Example 1: Phage library screening and Nectin-4 high-affinity and internalized peptides

[0396] (1) Selection of strains and vectors

[0397] Escherichia coli strain SS320 (Sidhu, SS, et al. Methods Enzymol 328 (1999): 333-363.) exhibits high electroporation efficiency, and the introduced F' fragment is essential for phage infection and amplification. E. coli strain SS320 is frequently used as a bacterial host for phage production and expression. M13K07 (New England Biolabs, Beverly, MA) is commonly used for packaging and amplifying single-stranded phages. Vector selection and phage particle construction methods were described in Tonikian, R., et al. Nature Protocol 2.6 (2007): 1368-1386. The pGEX4T-2 vector was used to display the phage library; the vector contains single-stranded (f1) fragments. The replicon replicates both ori and double-stranded (dsDNAori) DNA, and also includes a selective marker, such as the β-lactamase gene (Ampr), which simultaneously confers resistance to ampicillin and carbenicillin. After insertion, the polypeptide sequence forms a fusion protein with the mature phage main coat protein (P3), and is displayed at the N-terminus.

[0398] (2) Preparation of phage display library

[0399] The most critical step in phage display is the mutagenesis process, which generates a random library. This invention uses oligonucleotide directed mutagenesis and efficient bacterial transformation to generate a highly diverse library. The oligonucleotide directed mutagenesis technique, first described by Kunkel, is a reliable method for constructing libraries by introducing random mutations into ssDNA templates. The method described in Liu, B., S. Long, and J. Liu, "New Biotechnology 56" (2019), was used to generate the random library. Subsequently, the random CCC-dsDNA library was electroporated into E. coli host SS320 (M13KO7) containing the F' fragment. Electroporation competent cells were prepared using M13KO7 pre-infected SS320 strains. Finally, the phage library was constructed according to (Tonikian, R., et al. Nature Protocol 2.6 (2007):1368-1386). The phage titer generated by PEG / NaCl precipitation was approximately 10. 13 pfu / mlLibrary1.

[0400] (3) Phage library screening yielded polypeptide sequences with high affinity for the Nectin4 target.

[0401] Nectin-4 (Nectin cell adhesion molecule 4) is a type I membrane protein used as a target protein in phage library panning (Nectin-4(His / Avi): Kaika Bio NEC-HM404B). Phage selection, also known as biopanning, is an affinity selection process for separating target-binding molecules. Using the target protein as the stationary phase and the phage display library as the mobile phase, after a period of co-incubation, unbound free phages are washed away. Then, phages bound to the target molecule are eluted using competitive receptors or acid. The eluted phages infect host cells, multiply, and amplify, undergoing another round of elution. After 3 to 5 rounds of "adsorption-elution-amplification," antibodies with high affinity for the target protein can be obtained (Padmanaban, G., et al. Journal of Biotechnology 187(2014):43-50 reveals a method for bioscreening phage libraries). This invention describes a method for obtaining high-affinity polypeptide sequences targeting Nectin-4 (His / Avi) from a phage library through four rounds of panning using MyOne Streptavidin T1 Dynabeads (10 mg / mL, Invitrogen, cat#65602) magnetic beads and Nectin-4 (His / Avi) target protein. After resuspending and washing the Dynabeads according to the manufacturer's instructions, 60 μg of Nectin-4 (His / Avi) was immobilized in 105 μL of magnetic beads, and 60 μL of PBS was added, bringing the total volume to 180 μL. The mixture was then incubated at 4°C for 16–24 h. After incubation, the target protein-bound magnetic beads were precipitated using a magnetic rack, the supernatant was removed, and the beads were washed four times with 1 mL of 0.1% PBST. Subsequently, the magnetic beads were blocked with 0.5% BSA-PBS at room temperature for 30 min. In the first round, 5 μL of 1×10⁻⁶ solution was added to the sealed magnetic beads. 11 The M13 phage library of PFU (dissolved in PBS) was gently mixed at 4°C and incubated for 1 hour. After incubation, unbound phages were repeatedly washed with PBS containing 0.1% Tween (PBST). In the next round of screening, the Tween concentration was gradually increased from 0.1% to 0.4%. Bound phages were eluted by incubation in 0.1M citrate (pH 3.1) for 2 minutes, followed immediately by neutralization with 1M TrisHCl (pH 9.1). 20 μL of the phage titer was determined. The remaining phages were amplified using SS320 strain, purified, and used for the next round of screening. The phage input in each round was 1 × 10⁻⁶. 11 pfu.

[0402] (4) Phage library screening and ELISA identification

[0403] 4.1. Coating: Dilute SA to 5 μg / mL with 50 mM sodium bicarbonate at pH 8.5, and take 50 μL to coat the substrate at 37℃ for 2 h;

[0404] 4.2 Blocking: Block with TBST + 20 mg / mL BSA blocking solution overnight at 4°C;

[0405] 4.3 Washing: Wash 3 times with 200 μL of washing solution (25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, 0.05% Tween-20) for 3 min each time;

[0406] 4.4 Binding of target protein: 200 ng of Nectin-4 target protein was incubated with an SA-coated ELISA plate at 37°C for 1 hour to bind;

[0407] 4.5 Washing: Wash 3 times with 200 μL of washing solution (25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, 0.05% Tween-20) for 3 min each time;

[0408] 4.6 Phage binding: 10 9 / 10 10 The phage supernatant of PFU was bound to a Nectin-4 coated ELISA plate at 37°C for 1 hour.

[0409] 4.7 Washing: Wash 5 times with 200 μL of washing solution (25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, 0.05% Tween-20) for 3 min each time;

[0410] 4.8. Binding antibody: Dilute M13 phage Antibody to 0.1 μg / mL, take 100 μL and bind it to the phage in the ELISA plate at 37℃ for 1 h;

[0411] 4.9 Washing: Wash 5 times with 200 μL of washing solution (25 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% BSA, 0.05% Tween-20) for 3 min each time;

[0412] 4.10. Color development: Mix TMB color development solution, A and B solutions in equal proportions, add 100 μL, and develop color at room temperature for 5 min;

[0413] 4.11. Termination: Add 100 μL of 2M H2SO4;

[0414] 4.12. Scanning: ELISA reader OD 450 reading.

[0415] The sequences with high affinity obtained from the phage library by ELISA are shown in Table 1.

[0416] Table 1. Results of ELISA identification of sequence affinity in phage library

[0417]

[0418]

[0419] Example 2. Preparation of polypeptide 1

[0420]

[0421] Step 1: Synthesis of linear precursor peptide chains

[0422] Linear precursor peptide chain of SEQ ID NO:1

[0423] GCWHFKPPWACKQDSGVCETV-EVCG.

[0424] 179 mg (0.1 mmol) of Fmoc-Gly-Wang resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the second C at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0425] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0426] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0427] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0428] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0429] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0430] Step 2: Cleavage of the linear precursor peptide chain

[0431] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washings with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product three times with MTBE and dry it under vacuum.

[0432] Step 3: Intramolecular disulfide bond formation

[0433] The crude product obtained in step 2 was dissolved in DMSO (DMSO volume was 20% of the total reaction volume). Then, 2 mM GSH was added to 50 mM ammonium bicarbonate buffer (pH = 8.0, containing 30% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer to a final concentration of 1 mg / mL. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0434] Step 4: Preparation of Peptides

[0435] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient was 20-45% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the pure peptide was obtained.

[0436] Step 5: Detection and Characterization Methods

[0437] The purity of the polypeptide from step 4 was determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, and the formation of two pairs of disulfide bonds within the compound was also determined.

[0438] Example 3. Preparation of polypeptide 15

[0439]

[0440] Step 1: Synthesis of linear precursor peptide chains

[0441] Linear precursor peptide chain of SEQ ID NO:15

[0442] GCWTFWPDWNCKQDSDCLAGC-VCEPVLVCG

[0443] 179 mg (0.1 mmol) of Fmoc-Gly-Wang resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the second C at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0444] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0445] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0446] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0447] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0448] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0449] Step 2: Cleavage of the linear precursor peptide chain

[0450] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 1, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washings with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product three times with MTBE and dry it under vacuum.

[0451] Step 3: Intramolecular disulfide bond formation

[0452] The crude product obtained in step 2 was dissolved in DMSO (DMSO volume was 20% of the total reaction volume). Then, 2 mM GSH was added to 50 mM ammonium bicarbonate buffer (pH = 8.0, containing 30% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer to a final concentration of 1 mg / mL. The mixture was shaken at room temperature for 16 hours. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0453] Step 4: Preparation of Peptides

[0454] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR C-18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 25-45% acetonitrile in 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0455] Step 5: Detection and Characterization Methods

[0456] The purity of the polypeptide from step 4 was determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, and the formation of three intramolecular disulfide bonds was also determined.

[0457] The remaining polypeptides of this invention were prepared using the same method as in Examples 1 and 2. The amino acid sequences of polypeptides 1 to 98 are shown in SEQ ID NO:1 to SEQ ID NO:98, respectively.

[0458] Test Example 1: BLI test to determine the affinity of the peptide sample for Nectin-4 protein.

[0459] (1) Experimental materials:

[0460] name factory Item number Anti-hisprobes Gator 160009 MAXplate Gator 06-0099 96-hole plate Luxcell 902558 HumanNectin-1 / PVRL1 / CD111Protein,FcTag Acro PV1-H5253 HumanNectin-2 / CD112Protein,FcTag Acro PV2-H5253 HumanNectin-3 / CD113Protein,FcTag Acro PV3-H5255 HumanNectin-4,FcTag Acro NE4-H5255

[0461] (2) Experimental steps:

[0462] The affinity of peptides for Nectin-4, Nectin-1, Nectin-2, and Nectin-3 was detected using Gator BLI technology. Peptides diluted to 10 μg / mL with Kbuffer were immobilized onto anti-his probes (biodetectors with an anti-his molecular layer). The anti-his probes were equilibrated twice in Kbuffer (PBST) for a total of 200 s. Then, the anti-his probes were added to 0-100 nM human Nectin-4, Nectin-1, Nectin-2, and Nectin-3 protein solutions for a 120 s binding reaction. Finally, the proteins were transferred to fresh Kbuffer for a 120 s dissociation reaction. The affinity KD values ​​between the peptides and each protein were calculated based on Kon and Koff.

[0463] The calculated affinity KD values ​​of each peptide with Nectin-4 are shown in Table 2. Table 2 shows that the peptides of this invention exhibit high affinity for Nectin-4. Specifically, peptide 1 has the highest affinity KD value for Nectin-4. d=2.57 nM, affinity K for peptide 15 to Nectin-4 d =3.32 nM. The affinity detection results for peptides 1 and 15 with Nectin-4, Nectin-1, Nectin-2, and Nectin-3 are as follows: Figures 1-8 As shown in the figure, both peptide 1 and peptide 15 specifically bind to Nectin-4, but not to Nectin-1, Nectin-2, or Nectin-3.

[0464] Table 2. Affinity KD values ​​of peptide samples to Nectin-4 protein as tested by BLI

[0465]

[0466]

[0467] Test Example 2: ELISA test of the binding of peptide samples to human Nectin-4

[0468] (1) Experimental materials:

[0469]

[0470] (2) Experimental steps:

[0471] The binding signal of peptide samples to Nectin-4 was tested using ELISA (Enzyme-Linked Immunosorbent Assay). HisTag Antibody and Mouse Mab were coated at 0.5 μg / mL, 25 μL / well, onto 384-well plates (Greiner 781097). After blocking the HisTag Antibody-coated ELISA plates, the peptide samples were serially diluted to 0–100 nM (8 concentrations in a 3-fold gradient from 100 nM downwards). 25 μL / well was transferred to each well using a workstation and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the plates were washed 3–5 times with 80 μL of 1×TBST washing buffer (pH 7.4) for 3–5 minutes each time. Nectin-4 protein (0.1 μg / mL, 25 μL / well) was added to the controlled-drying assay plate, and after brief centrifugation to remove air bubbles, the plates were incubated at 37°C for 1 h. After washing and drying the plate again, add 25 μL of TMB chromogenic solution to each well and incubate at 37°C for 30 min. Finally, add 25 μL of stop solution (1M HCl) to each well to terminate the reaction. Read the absorbance at 450 nm using a Cytation 5 microplate reader. The experimental results are shown in Table 3. The results show that the peptide of this invention has a high binding capacity to Nectin-4.

[0472] Table 3. ELISA results of peptide samples binding to human Nectin-4.

[0473]

[0474]

[0475] Test Example 3: Fluorescence Imaging Detection of Nectin-4 Internalization Induced by the Peptide in H_NECTIN4 CHO-K1 Cells

[0476] (1) Experimental materials:

[0477] name factory Item number H_NECTIN4CHO-K1CellLine Jiman Bio GM-C09528 F-12K basal medium Sigma N3520 puromycin Invivogen ant-pr-1 FBS EXCELLBio FSP500 4% paraformaldehyde fixative Beyotime P0099 DAPI staining solution Beyotime C1006 96-hole plate Agilent 204626-100

[0478] (2) Experimental steps:

[0479] Cell preparation: H_Nectin4 cells were cultured and grown in medium (F12K, 10% FBS, 4 μg / mL puromycin). When the cell density reached 80-90% of the culture flask, the cells were digested with 0.25% trypsin (containing 0.5 mM EDTA), and the cell suspension was collected into centrifuge tubes. The cells were centrifuged at 1000 rpm for 3 min, and the supernatant was removed. The cells were resuspended in 6-8 mL of fresh growth medium and passaged at a ratio of 1:3 to 1:5. The cells were then incubated at 37°C in a 5% CO2 incubator. The medium was changed or the cells were passaged every 2-3 days after passage.

[0480] 16-24 hours before the experiment, H_Nectin4 cells were passaged and expanded to the required number of cells. The cells were digested, centrifuged, and the cell pellet was collected. The cells were resuspended in an appropriate amount of complete culture medium, and cell viability was tested and counted. The cell concentration was then adjusted to 4 × 10⁶ cells / year with complete culture medium. 4 cells / mL. Seed 100 μL / well in the center wells of a 96-well plate, and fill the edge wells with the same volume of 100 μL / well DPBS. Incubate overnight at 37°C in a 5% CO2 incubator.

[0481] FITC-labeled peptides were dissolved to 1 mM using DMSO. During the experiment, the peptides were diluted to five different concentrations (0–50 μM) using cell growth medium. After overnight inoculation, the old medium in each well was aspirated and replaced with 100 μL of the diluted peptide per well. The plates were then incubated at 37°C with 5% CO2 for 4 h and then 24 h. Following fixation with fixative and DAPI nuclear staining, the cells were analyzed using confocal microscopy with fluorescence imaging.

[0482] Experimental results are as follows Figures 9-12 As shown. Among them, Figure 9 The results shown are fluorescence imaging results of BT8009. Figure 10 , 11The images show the fluorescence imaging results for peptide 1 and peptide 15, respectively. Figures 9-11 The peptide concentrations from left to right are 2 μM, 0.4 μM, and 0.08 μM, respectively. The experimental results show that peptides 1 and 15 of the present invention have good endocytosis effects and are superior to BT8009.

[0483] Test Example 4: Flow cytometry detection of peptide sample binding to or endocytosis with Nectin4

[0484] (1) Experimental materials:

[0485] name factory Item number eBioscienceFlowCytometrystainingBuffer Thermo 00-4222-57 DPBS self made NA

[0486] (2) Experimental steps:

[0487] The binding of peptides to Nectin-4 was detected using Attune Nxt flow cytometry. On the day of the experiment, H_Nectin4 cell suspensions were digested with 5 mM EDTA and collected. Cell growth medium was used, and the cell density was adjusted to 4 × 10⁶ cells / year. 5 Cells / mL were collected, 1 mL of cell suspension was centrifuged at 1500 rpm for 4 min, the culture medium was removed, and DPBS was added to wash the cells. This process was repeated twice. Finally, the cells were resuspended and blocked with DPBS solution containing 2% BSA.

[0488] Peptide samples were dissolved in DMSO and diluted to 0–30 μM with flow cytometry buffer. After washing the cells again, 200 μL of the diluted peptide sample was resuspended in the cells per tube and incubated at 4°C in the dark for 2.5 h. After incubation, the cells were centrifuged using a refrigerated centrifuge, the supernatant was discarded, and the cells were washed with pre-chilled DPBS, repeated four times. Finally, the cells were resuspended in 300 μL of DPBS and analyzed by flow cytometry. The experimental results are as follows. Figure 13 , 14 As shown in the figure, the experimental results show that peptides 1 and 15 of the present invention have good binding ability with Nectin-4 and endocytosis effect.

[0489] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, said polypeptide having an amino acid sequence as shown in any of the following: GCWHFKPPWACKQDSGVCETVEVCG (SEQ ID NO: 1), GCWTFKPPWACKQDSGVCETVEVCG (SEQ ID NO: 34), GCWHFYPPWACKQDSGVCETVEVCG (SEQ ID NO: 35), GCWHFKPKWACKQDSGVCETVEVCG (SEQ ID NO: 36), GCWHFKPDWACKQDSGVCETVEVCG (SEQ ID NO: 37), GC{1-Nal}HFKPPWACKQDSGVCETVEVCG (SEQ ID NO: 41), GCWAFKPPWACKQDSGVCETVEVCG (SEQ ID NO: 42), GCWKFKPPWACKQDSGVCETVEVCG (SEQ ID NO: 43), GCWVFKPPWACKQDSGVCETVEVCG (SEQ ID NO: 44), GCWRFKPPWACKQDSGVCETVEVCG (SEQ ID NO: 45), GCWHFRPPWACKQDSGVCETVEVCG (SEQ ID NO: 46), GCWHFWPPWACKQDSGVCETVEVCG (SEQ ID NO: 47), GCWHFFPPWACKQDSGVCETVEVCG (SEQ ID NO: 48), GCWHF{Dab}PPWACKQDSGVCETVEVCG (SEQ ID NO: 49), GCWHFKPRWACKQDSGVCETVEVCG (SEQ ID NO: 50), GCWHFKPAWACKQDSGVCETVEVCG (SEQ ID NO: 51), GCWHFKP{Hyp}WACKQDSGVCETVEVCG (SEQ ID NO: 52), GCWHFKP{Dab}WACKQDSGVCETVEVCG (SEQ ID NO: 53).

2. A polynucleotide encoding the polypeptide of claim 1.

3. A pharmaceutical composition comprising the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof or the polynucleotide of claim 2, in combination with one or more pharmaceutically acceptable excipients.

4. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the polynucleotide of claim 2, or the pharmaceutical composition of claim 3 in the preparation of a medicament for treating and / or preventing Nectin-4-related diseases, wherein the Nectin-4-related diseases are selected from at least one of gastric cancer, hepatocellular carcinoma, bladder cancer, urothelial carcinoma, lung cancer, breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer, or esophageal cancer.

5. The use according to claim 4, wherein, The hepatocellular carcinoma is liver cancer; the urothelial carcinoma is selected from at least one of urethral carcinoma, renal pelvis carcinoma, and ureteral carcinoma; the lung cancer is non-small cell lung cancer; and the breast cancer is selected from at least one of ductal carcinoma of the breast and triple-negative breast cancer.

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