Polypeptide specifically binding to Nectin-4 and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to a polypeptide specifically combined with Nectin-4 and application of the polypeptide. The polypeptide provided by the invention comprises an amino acid sequence: X1C1Z1C2Z2C3Z3C4X2, can be specifically combined with Nectin-4, and can be used for treating and / or preventing diseases related to Nectin-4, in particular cancers highly expressing Nectin-4.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a polypeptide specifically binding to nectin-4 and a use thereof. Background Art

[0002] Cell Adhesion Molecular (CAM) is a type of cell membrane surface glycoprotein molecule that mediates cell-cell and cell-extracellular matrix interactions. It plays an important role in mediating physiological processes such as cell polarization, differentiation, adhesion, proliferation, and migration, and is also involved in cell signal transduction. Therefore, abnormal expression of CAM can reflect pathological changes in the body to a certain extent. CAM can be roughly divided into the selectin family, integrin family, cadherin family, and immunoglobulin superfamily (Immunoglobulinsuperfamily, IgSF). The expression of most CAMs in the body is not specific, and there is no significant difference in the expression of various tissue cells.

[0003] The term nectin was first named and used by Japanese scientist Takahashi K in 1999 based on the Latin word "necto" (which means connection in Chinese). 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 adherens junctions (AJs). The nectin family consists of four members, Nectin-1, -2, -3 and -4, each of which has two to three splice variants. Except for nectin-1γ, which is a secreted protein that lacks a transmembrane region, all members have an extracellular domain with three Ig-like loops. Nectin is Ca-independent. 2+ Nectin proteins are immunoglobulin-like intercellular adhesion molecules that play a role in cell-cell adhesion. They bind to the actin filament (F-actin) binding protein afadin through their cytoplasmic tails and associate with the actin cytoskeleton, cooperating with other cell adhesion molecules and cell surface membrane receptors to regulate many other cellular activities.

[0004] Nectin plays an important role in the formation of adhesion junctions between epithelial and endothelial cells. In addition to its pleiotropic functions in cell-to-cell adhesion, nectin is also involved in a series of biological processes, including virus entry and immune regulation. At the same time, some nectin family members can also specifically interact with specific receptors expressed on immune cells (T cells and NK cells) to mediate 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 only expressed in embryos, placentas, and mature epithelial tissues, and its expression is limited in healthy adult tissues. However, studies have found that Nectin-4 is abnormally overexpressed in a variety of human cancers, especially in bladder cancer, breast cancer, and lung cancer, with the proportion of positive patients exceeding 50%. Studies have confirmed that Nectin-4 protein is involved in mediating the progression and metastasis of various cancers such as cancer cell apoptosis resistance, angiogenesis, and lymphatic vessels, and its expression level in malignant tumors is closely related to the malignancy of the disease and the survival of patients. Therefore, Nectin-4 protein is an ideal biomarker for highly sensitive and specific cancer lesion localization imaging and targeted therapy. At the same time, as a receptor that mediates measles virus invasion of cells, Nectin-4 itself has a significant ability to mediate endocytosis.

[0006] Nectin-4 (Nectin cell adhesion molecule 4) is a cell adhesion molecule of the nectin and nectin-like families. It is highly expressed in normal embryonic and fetal tissues, but very low in healthy adult tissues. Nectin-4 is a type I membrane protein. The extracellular region of the dimeric nectin-4 consists of three Ig domains, Ig V type and IgC type. The metalloproteinase TACE / ADAM-17 can degrade the extracellular domain to produce soluble nectin-4. This secreted form has been found in patients with breast tumors.

[0007] Studies have found that Nectin-4 promotes tumor cell proliferation, differentiation, migration, invasion, etc. by activating the PI3K / Akt pathway, and is believed to play an important role in the occurrence and metastasis of cancer. It is based on these research backgrounds that drug research targeting this target has also emerged. Enfortumab vedotin is a "first-in-class" new drug jointly developed by Astellas and Seagen. It is an ADC drug formed by connecting the monoclonal antibody enfortumab targeting Nectin-4 and the microtubule disruptor MMAE through a cleavable dipeptide linker. Its anti-cancer mechanism is that when it binds to Nectin-4 on the surface of tumor cells, it enters the cell through endocytosis of tumor cells. After the dipeptide linker is cleaved by the proteolytic enzyme in the cell, the toxin MMAE can be released, thereby achieving targeted killing of tumors. 9MW2821 is the first variety of drugs with the same target approved for clinical research by domestic companies, and it is also the second Nectin-4 ADC variety approved for clinical research in the world. Bicycle Therapeutics has developed two Nectin-4-targeted therapies based on Bicycle technology. One type is BicycleToxin Conjugate (BTC)—BT8009, which is a Nectin-4-targeted 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 new type of tumor-associated antigen is unquestionable, providing great possibilities for the development of new 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 the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a polypeptide specifically binding to nectin-4 and its use.

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

[0011] X 1 Select None or G;

[0012] X 2 is 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 for X 22 X 23 X 24 ;

[0017] Z5 to 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 said amino acids;

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

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

[0022] In some embodiments, the polypeptides of the invention are linear peptides.

[0023] In some embodiments, the polypeptides of the invention are cyclic peptides.

[0024] In some embodiments, any two cysteines among C1, C2, C3, C4, C5, and C6 can form a disulfide bond, and 1, 2, or 3 disulfide bonds can be formed simultaneously.

[0025] In some embodiments, a disulfide bond can be formed between C1 and C4.

[0026] In some embodiments, a disulfide bond can be formed between C2 and C3.

[0027] In some embodiments, a disulfide bond can be formed between C2 and C5.

[0028] In some embodiments, a disulfide bond can be formed between C3 and C6.

[0029] In some embodiments, a disulfide bond can be formed between C1 and C4, and a disulfide bond can be formed between C2 and C3.

[0030] In some embodiments, a disulfide bond can be formed between C1 and C4, a disulfide bond can be formed between C2 and C5, and a disulfide bond can be formed between C3 and C6.

[0031] In some embodiments, the sequence has four cysteines: C1, C2, C3, and C4, wherein a disulfide bond can be formed between C1 and C4, and a disulfide bond can be formed between C2 and C3.

[0032] In some embodiments, the sequence has 6 cysteines: C1, C2, C3, C4, C5, and C6, wherein a disulfide bond can be formed between C1 and C4, a disulfide bond can be formed between C2 and C5, and a disulfide bond can be formed 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 said amino acids;

[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, W;

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

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

[0041] X 10 Select 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 ; Among them, X 3 Selected from F, 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 is K;

[0045] X 12 is Q;

[0046] X 13 is D;

[0047] X 14 is S;

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

[0049] X 16 Selected from V, F;

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

[0051] In some embodiments, Z3 is (Z4C5Z5C6) m X 17 X 18 X 19 X 20 X 21 , when m is 0, the Z3 is X 17 X 18 X 19 X 20 X 21 ; When m is 1, the 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 for X 22 X 23 X 24 ; said X 22 L; X 23 A; X 24 is G;

[0058] Z5 to X 25 ; said X 25 is V.

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

[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 said amino acids;

[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, W;

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

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

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

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

[0071] X 16 Selected from V, 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 said amino acids;

[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, W;

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

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

[0087] X 10 Select 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:

[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:

[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:

[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:

[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:

[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:

[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, 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, F.

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

[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 said amino acids;

[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, W;

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

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

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

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

[0121] X 16 Selected from V, 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 said amino acids;

[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, W;

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

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

[0138] X 10 Select 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:

[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:

[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:

[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:

[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:

[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:

[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, 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, 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 for X 22 X 23 X 24 ;

[0165] Z5 to 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 said amino acids;

[0168] X 4 ~X 25 Each is independently selected from any amino acid or unnatural 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 said amino acids;

[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, 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, E;

[0184] X 16 Selected from V, F;

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

[0186] In some embodiments, when m is 0, Z3 is X 17 X 18 X 19 X 20 X21 ; When m is 1, the 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 for X 22 X 23 X 24 ; said X 22 L; X 23 A; X 24 is G;

[0193] Z5 to X 25 ; said X 25 is V.

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

[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 said amino acids;

[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, W;

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

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

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

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

[0206] X 16 Selected from V, 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 said amino acids;

[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, W;

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

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

[0222] X 10 Select 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:

[0229] C1W 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:

[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, 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, F.

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

[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 said amino acids;

[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, W;

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

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

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

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

[0248] X 16 Selected from V, 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 said amino acids;

[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, W;

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

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

[0264] X 10 Select 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:

[0271] C1W 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:

[0273] C1W 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:

[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:

[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, 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, F.

[0282] In another aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises any one of the following (i) to (ii):

[0283] (i) an amino acid sequence that has at least 80% identity with the above-mentioned amino acid sequence and retains the original function; (ii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or modified in the above-mentioned amino acid sequence and retains 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 as shown in SEQ ID NO.1 while retaining the function of the sequence as shown in SEQ ID NO.1.

[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 retains 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 to the amino acid sequence shown in SEQ ID NO.1 and retains 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 the function of the sequence shown in SEQ ID NO.1 is retained.

[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 in which one or more amino acid residues are deleted from the amino acid sequence shown in SEQ ID NO.1 and the function of the sequence shown in SEQ ID NO.1 is retained.

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

[0293] In another aspect, the present invention provides a polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide is selected from the following 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 in this article, site-directed mutagenesis technology is used to identify amino acids that play a key role in the activity of the peptide of the present invention, and the amino acid residues that play a key role in the activity of the polypeptide molecule are determined to be 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 test the biological activity of the resulting polypeptide molecule to determine the amino acid residues that play a key role in the activity of the polypeptide molecule; such as PCR site-directed mutagenesis techniques, by designing primers containing non-specific paired bases, and then introducing mutation sites into the product through PCR.

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

[0355] In another aspect, the present invention provides a polynucleotide, the sequence of which comprises a nucleotide sequence encoding the above-mentioned amino acid sequence.

[0356] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide or a pharmaceutically acceptable salt or polynucleotide thereof, in combination with one or more pharmaceutically acceptable excipients.

[0357] In another aspect, the present invention provides use of the above polypeptide or a pharmaceutically acceptable salt thereof, polynucleotide or pharmaceutical composition in the preparation of a drug for treating and / or preventing a disease associated with nectin-4.

[0358] In some embodiments, the disease associated with Nectin-4 is a cancer with high expression of Nectin-4.

[0359] In some embodiments, the cancer with high expression of nectin-4 is 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, breast ductal carcinoma, 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 the invention include salts derived from suitable inorganic acids and bases and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of an amino group 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 by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N + (C 1–4 Representative alkali metal or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed when 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 at least one of a free acid or a sodium, potassium, calcium, or ammonium salt.

[0362] Compared with the prior art, the polypeptide provided by the present invention has high specificity and high affinity, which helps to reduce the dosage of the drug and potential side effects.

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

[0364] The term "polypeptide" is intended to include the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, rather than the specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to two or more amino acid chains are included in the definition of "polypeptide", and the term "polypeptide" can be used to replace or interchange with any of these terms. The term "polypeptide" also means a product modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be produced in any way, including by chemical synthesis.

[0365] The term "amino acid" refers to any organic compound containing an amino group (--NH2) and a carboxyl group (--COOH); preferably in the form of a free group 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, for example, the 23 amino acids that combine into peptide chains to form the building blocks of a large number of proteins. These are primarily L stereoisomers, although small amounts of D-amino acids are present in bacterial envelopes and some antibiotics. The "twenty alpha amino acids that form naturally encoded polypeptides" are understood in the art 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 pyrrolysine (found in methanogens and other eukaryotic organisms), selenocysteine ​​(present in many non-eukaryotic organisms as well as most eukaryotic organisms), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts).

[0368] "Non-natural" amino acids are non-proteinogenic amino acids (i.e., those not naturally encoded or found in the genetic code), which are naturally occurring or chemically synthesized. More than 140 non-natural amino acids are known and thousands of more combinations are possible. Examples of "non-natural" amino acids include β-amino acids (β3 and β2), homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-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" is a compound produced by modifying the N and / or C terminus of an amino acid. Non-limiting examples are the conversion of a carboxyl group to a salt, ester, acyl hydrazide, hydroxamic acid or amide, and the conversion of an amino group to amides, ureas, thioureas, thioamides, sulfonamides, phosphoric acid amides, boric acid amides or alkylamines. Parts or moieties of a compound, produced by the modification of an amino acid at the C and / or N terminus, may also be referred to as amino acid units. Moreover, the amino acids may also be derivatized at their side chains. If the derivatized amino acid is an amino acid whose side chain has been derivatized once or several times, the derivatization of this type is usually specifically indicated here. Preferred derivatization of the side chain may be performed, in particular where the side chain has a functional group. Preferred functional groups are, for example, amino, carboxyl, thiol or alcohol groups.

[0370] The term "amino acid analogues" is a compound produced by amino acids by replacing amino and / or carboxyl groups with other groups that can mimic them. Non-limiting examples are thioamides, ureas, thioureas, acyl hydrazides, esters, alkylamines, sulfonamides, phosphoric acid amides, ketones, alcohols, boric acid amides, benzodiazepines and other aromatic or non-aromatic heterocyclic combinations (for a review see MA Estiarte, DH Rich in Burgers Medicinal Chemistry, 6th edition, volume 1, part 4, John Wiley & Sons, New York, 2002).

[0371] Unnatural amino acids include, but are not limited to, 2-amino fatty acid (Aad), 3-amino fatty acid (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), desmosine ( Des), 2,2"-diaminopimelane (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6- N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle) and 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-phenylphenylalanine, 2,4-dichlorophenylalanine, 3,4-dichlorophenylalanine, 2,6-dimethylphenylalanine, homotryptophan, 2-methyl-tryptophan, 7-bromo-tryptophan, 6-chloro-tryptophan, 5-hydroxytryptophan, 7-methyl-tryptophan, homotyrosine, meta-tyrosine, 3-hydroxytyrosine, 3-chlorotyrosine, 3-bromotyrosine, 3-nitrotyrosine, 3,5-diiodotyrosine, O-methyltyrosine, O-ethyltyrosine, O-benzyltyrosine.

[0372] The terms "homology" or "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 positions may be aligned for the purpose of comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence has less than 40% identity, but preferably less than 25% identity to one of the sequences disclosed in the present invention.

[0373] The term "treatment" or "therapy" refers to both medical treatment and prophylactic or preventative measures, wherein the purpose is to prevent or slow down (mitigate) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival without treatment. People in need of treatment include people who already have the disease or disorder, as well as people who are susceptible to the disease or disorder, or people in need of prevention of the disease or disorder.

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

[0375] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article. BRIEF DESCRIPTION OF THE DRAWINGS

[0376] Figure 1 The affinity between peptide 1 and nectin-4 in test example 2;

[0377] Figure 2 The affinity between peptide 1 and Nectin-1 in test example 2;

[0378] Figure 3 The affinity between peptide 1 and Nectin-2 in test example 2;

[0379] Figure 4 The affinity between peptide 1 and nectin-3 in test example 2;

[0380] Figure 5 The affinity of peptide 15 and nectin-4 in test example 2;

[0381] Figure 6 The affinity of peptide 15 and nectin-1 in test example 2 is tested;

[0382] Figure 7 The affinity of peptide 15 and Nectin-2 in test example 2 is tested;

[0383] Figure 8 The affinity between peptide 15 and nectin-3 in test example 2;

[0384] Fig. 9 This is the fluorescence image of BT8009 fluorescence imaging detection in test example 3. From left to right, the peptide concentrations are 2μM, 0.4μM, and 0.08μM;

[0385] Fig.10 This is a fluorescence image of the peptide 1 in Test Example 3. The peptide concentrations from left to right are 2 μM, 0.4 μM, and 0.08 μM.

[0386] Fig.11 This is a fluorescence image of the peptide 15 fluorescence imaging detection in Test Example 3. From left to right, the peptide concentrations are 2 μM, 0.4 μM, and 0.08 μM;

[0387] Fig.12 Fluorescence imaging test results of test case 3 BT8009, peptide 1, and peptide 15;

[0388] Fig.13 This is the flow cytometry result of peptide 1 in test example 4;

[0389] Fig.14 This is the flow cytometry detection result of peptide 15 in test example 4. Specific embodiments

[0390] In order to make the objects, features and advantages of the present invention more obvious and understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited thereto. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0391] The polypeptide compound and its derivative provided by the present invention adopt a solid phase synthesis method to synthesize its linear precursor, and DMSO is used for oxidation to form two pairs of disulfide bonds and three pairs of disulfide bonds in the molecule. The synthetic carrier is Fmoc-Gly-Wang Resin resin. In the synthesis process, the Fmoc-Gly-Wang Resin resin is first fully swollen in N,N-dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90:2.5:2.5:5, v:v:v:v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The cut linear precursor crude product is subjected to disulfide bond oxidation in a weakly alkaline solution to obtain the target polypeptide crude product. The crude polypeptide was purified and separated by C-18 reverse phase preparative chromatography in 0.1% trifluoroacetic acid acetonitrile / water system to obtain pure polypeptide and its derivatives.

[0392] Experimental reagents

[0393]

[0394]

[0395] Example 1 Phage Library Screening and Nectin-4 High Affinity Internalized Polypeptide

[0396] (1) Selection of strains and vectors

[0397] Escherichia coli strain SS320 (Sidhu, SS, et al. "Methods Enzymol 328 (1999): 333-363.) has a high electroporation efficiency, and the introduced F' fragment is necessary for phage infection and amplification. Escherichia coli strain SS320 is often used as a bacterial host for phage production and expression. M13K07 (New England Biolabs, Beverly, MA) is often used for packaging to obtain single-chain and amplified phage. The vector selection and phagemid construction method are described by Tonikian, R., et al. Nature Protocol 2.6 (2007): 1368-1386. The pGEX4T-2 vector is used to display the phage library. The vector contains a single-chain (f1 ori) and double-stranded (dsDNAori) DNA replication replicons, in addition to a selectable marker, such as the β-lactamase gene (Ampr), which also makes it resistant to ampicillin and carbenicillin. After the polypeptide sequence is inserted, it forms a fusion protein with the mature major coat protein (P3) of the phage for N-terminal display.

[0398] (2) Preparation of phage display library

[0399] The most critical step in the phage display method is the mutation process, which produces a random library. The present invention uses oligonucleotide-directed mutagenesis and efficient bacterial transformation to generate a highly diverse library. The oligonucleotide-directed mutagenesis technology first described by Kunkel is a reliable method for constructing a library by introducing random mutations into an ssDNA template. The random library was generated using the method of Liu, B., S. Long, and J. Liu. "New Biotechnology 56 (2019). Subsequently, the random CCC-dsDNA library was electroporated into the Escherichia coli host SS320 (M13KO7) containing the F' fragment, and the SS320 strain pre-infected with M13KO7 was used to prepare the electroporation competent state, and finally the phage library was packaged according to (Tonikian, R., et al. Nature Protocol 2.6 (2007): 1368-1386) to complete the construction of the phage library. The phage titer produced by PEG / NaCl precipitation is approximately 10 13 pfu / mlLibrary1.

[0400] (3) Phage library selection to obtain peptide sequences with high affinity to the Nectin4 target

[0401] Nectin-4 (Nectin cell adhesion molecule 4) is a type I membrane protein used as a target protein for phage library panning (Nectin-4 (His / Avi): Kaixia Bio NEC-HM404B). The phage selection method, called bio-panning, is an affinity selection process for separating target binding molecules. With the target protein as the stationary phase and the phage display library as the mobile phase, after a period of co-incubation, the unbound free phage is washed away, and then the phage bound to the target molecule is eluted with a competitive receptor or acid. After the eluted phage infects the host cell, it is propagated and amplified, and the next round of elution is carried out. After 3 to 5 rounds of "adsorption-elution-amplification", antibodies with high affinity to the target protein can be obtained (Padmanaban, G., et al. Journal of Biotechnology 187 (2014): 43-50 discloses the method of biological screening of phage libraries). The present invention uses MyOne Streptavidin T1 Dynabeads (10 mg / mL, Invitrogen, cat#65602) magnetic beads and Nectin-4 (His / Avi) target protein to perform 4 rounds of panning according to the method. A polypeptide sequence with high affinity to the Nectin-4 target protein is obtained from the biological screening of the phage library. After the Dynabeads are resuspended and washed according to the instructions, 60 μg of Nectin-4 (His / Avi) is fixed to 105 μL of magnetic beads, and 60 μL of PBS is added, and the final total volume is 180 μL. Place on a rotating mixer and mix at 4°C for 16-24 hours. After the incubation is completed, the magnetic beads bound to the target protein are precipitated with a magnetic stand, the supernatant is removed, and the magnetic beads are washed 4 times with 1 mL of 0.1% PBST. Subsequently, the magnetic beads are blocked and the magnetic beads are blocked with 0.5% BSA-PBS for 30 minutes at room temperature. In the first round, 5 μL of 1×10 11 pfu of M13 phage library (dissolved in PBS), gently mix at 4°C, and incubate for 1 hour. After incubation, unbound phages were repeatedly washed with PBS containing 0.1% Tween (PBST). In the next round of panning, the concentration of Tween was gradually increased from 0.1% to 0.4%. The elution method used 0.1M citrate (pH 3.1) to incubate for 2 minutes to elute the bound phages, and immediately neutralized with 1M TrisHCl (pH 9.1). Take 20μL for phage titer determination, and the remaining phages are amplified using SS320 strains. The phages are purified and used for the next round of screening. The amount of phage input in each round is 1×10 11 pfu.

[0402] (4) Phage library selection ELISA identification

[0403] 4.1. Plate coating: dilute SA to 5 μg / mL with 50 mM sodium bicarbonate pH 8.5, take 50 μL and coat at 37°C for 2 h;

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

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

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

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

[0408] 4.6. Phage binding: 10 9 / 10 10 pfu of phage supernatant was bound to the coated Nectin-4 ELISA plate at 37°C for 1 h;

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

[0410] 4.8. Binding antibody: dilute M13 phageAntibody to 0.1 μg / mL, take 100 μL and bind to the phage in the ELISA plate at 37°C for 1 hour;

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

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

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

[0414] 4.12 Scanning: OD reader 450 reading.

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

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

[0417]

[0418]

[0419] Example 2. Preparation of polypeptide 1

[0420]

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

[0422] The 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 sequence was synthesized from the second C of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

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

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

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

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

[0429] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

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

[0431] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90: 2.5: 2.5: 5:, v: v: v: v) was added to the resin obtained in step 1 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0432] Step 3: Intramolecular disulfide bond formation

[0433] Add DMSO to the crude product obtained in step 2 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), then add 2mM GSH to 50mM ammonium bicarbonate buffer (pH=8.0, containing 30% acetonitrile), slowly add the dissolved polypeptide solution dropwise to the above buffer, the final concentration is 1mg / mL, and shake at room temperature for 16 hours. LC-MS monitors the reaction results, and purification preparation is directly carried out after the reaction is completed.

[0434] Step 4: Peptide purification

[0435] After filtering through a 0.45um membrane, the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR C-18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230nm, the flow rate was 15mL / min, and the gradient was 20-45% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0436] Step 5: Detection and characterization methods

[0437] The purity of the purified polypeptide in step 4 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and the formation of two pairs of disulfide bonds within the molecule.

[0438] Example 3. Preparation of polypeptide 15

[0439]

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

[0441] The 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 sequence was synthesized from the second C of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

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

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

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

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

[0448] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

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

[0450] Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90: 2.5: 2.5: 5:, v: v: v: v) was added to the resin obtained in step 1 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 volumes of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

[0451] Step 3: Intramolecular disulfide bond formation

[0452] Add DMSO to the crude product obtained in step 2 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), then add 2mM GSH to 50mM ammonium bicarbonate buffer (pH=8.0, containing 30% acetonitrile), slowly add the dissolved polypeptide solution dropwise to the above buffer, the final concentration is 1mg / mL, and shake at room temperature for 16 hours. LC-MS monitors the reaction results, and purification preparation is directly carried out after the reaction is completed.

[0453] Step 4: Peptide purification

[0454] After filtering through a 0.45um membrane, the product was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR C-18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 25-45% acetonitrile in 40 min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide.

[0455] Step 5: Detection and characterization methods

[0456] The purity of the purified polypeptide in step 4 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and the formation of three pairs of disulfide bonds within the molecule.

[0457] The remaining polypeptides of the present invention were prepared by 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 of the affinity of peptide samples to Nectin-4 protein

[0459] (1) Experimental materials:

[0460] name factory Part Number Anti-hisprobes Gator 160009 MAXplate Gator 06-0099 96-well 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] Gator BLI technology was used to detect the affinity of peptides with Nectin-4, Nectin-1, Nectin-2, and Nectin-3. Peptides diluted to 10 μg / mL using Kbuffer were fixed on Anti-his probes (biodetectors with anti-his molecule layers), and Anti-his probes were placed in Kbuffer (PBST) for two equilibrations for a total of 200 s. Anti-hisprobes were then placed in 0-100 nM human Nectin-4, Nectin-1, Nectin-2, and Nectin-3 protein solutions for 120 s binding reaction, and finally placed in new KBuffer for 120 s dissociation reaction. The affinity KD value of the peptide with each protein was calculated based on Kon and Koff.

[0463] The calculated affinity KD values ​​of each polypeptide and nectin-4 are shown in Table 2. From Table 2, it can be seen that the polypeptide of the present invention has a high affinity for binding to nectin-4. Among them, the affinity KD value of polypeptide 1 and nectin-4 is d=2.57 nM, affinity K of peptide 15 for nectin-4 d =3.32 nM. The results of the affinity test of peptide 1 and peptide 15 with nectin-4, nectin-1, nectin-2, and nectin-3 are shown in Figures 1 to 8 As shown in the figure, it can be seen that polypeptide 1 and polypeptide 15 only specifically bind to nectin-4, but not to nectin-1, nectin-2, and nectin-3.

[0464] Table 2 Affinity KD values ​​of BLI tested peptide samples and Nectin-4 protein

[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 ELISA (enzyme-linked immunosorbent assay) method was used to test the binding signal of peptide samples with Nectin-4. HisTag Antibody, Mouse Mab were coated on a 384-well plate (greiner 781097) at 0.5μg / mL, 25μL / well. After blocking the ELSIA plate coated with HisTag Antibody, the peptide sample was diluted to 0-100nM (from 100nM down 3-fold gradient dilution for a total of 8 concentrations). Use the workstation to transfer 25μL / well to the 384-well plate and incubate at 37℃ for 1h. Discard the liquid in the well, add 80μL of 1×TBST washing solution with pH=7.4 to wash the plate 3-5 times, each time for 3min~5min. Control the dryness of the test plate and add 0.1μg / mL of Nectin-4 protein, 25μL / well. After instant centrifugation to remove bubbles, put it in a 37℃ incubator and incubate for 1h. After washing the plate again and drying the test plate, add 25 μL of TMB colorimetric solution to each well and continue to incubate in an incubator at 37°C for 30 min. Finally, add 25 μL of stop solution (1M HCL) to each well to terminate the reaction. Use an ELISA reader cytation5 to read the absorbance at 450 nm. The experimental results are shown in Table 3. The experimental results show that the polypeptide of the present invention has a high ability to bind to Nectin-4.

[0472] Table 3 ELISA test results of peptide samples binding to human nectin-4

[0473]

[0474]

[0475] Test Example 3 Fluorescence imaging detection of peptides causing nectin-4 endocytosis in H_NECTIN4 CHO-K1 cells

[0476] (1) Experimental materials:

[0477] name factory Part Number H_NECTIN4CHO-K1CellLine Yoshiman Biology 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-well plate Agilent 204626-100

[0478] (2) Experimental steps:

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

[0480] 16-24 h before the experiment, H_Nectin4 cells were subcultured and expanded to the required cell number, the cells were digested and centrifuged to collect the cell pellet, the cells were resuspended in an appropriate amount of complete medium, the cell viability was detected and counted, and the cell concentration was adjusted to 4×10 4 The middle well of a 96-well plate was inoculated with 100 μL / well, and the edge wells were filled with the same volume of 100 μL / well DPBS, and incubated in a 37°C, 5% CO2 incubator overnight.

[0481] The FITC-labeled peptide was dissolved in DMSO to 1mM, and the peptide was diluted to 5 concentrations of 0-50μM using cell growth medium during the experiment. The cell plate inoculated overnight was removed, the old medium in the well was discarded, and replaced with 100μL / well of the diluted peptide, and then placed in a 37°C, 5% CO2 incubator for 4h / 24h, and then fixed with fixative, nuclear stained with DAPI, and analyzed by confocal microscopy fluorescence imaging.

[0482] The experimental results are as follows Figures 9 to 12 As shown. Among them, Fig. 9 The results of BT8009 fluorescence imaging are shown. Fig.10 , 11These are the fluorescence imaging results of peptide 1 and peptide 15, 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 the peptide 1 and peptide 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 Nectin4 or internalization

[0484] (1) Experimental materials:

[0485] name factory Part Number eBioscienceFlowCytometrystainingBuffer Thermo 00-4222-57 DPBS self made NA

[0486] (2) Experimental steps:

[0487] Attune Nxt flow cytometer was used to detect the binding of peptides to Nectin-4. On the day of the experiment, 5 mM EDTA was used to digest and collect H_Nectin4 cell suspension, and cell growth medium was used and the cell density was adjusted to 4 × 10 5 1 mL of cell suspension was taken, centrifuged at 1500 rpm for 4 min, the culture medium was removed, and DPBS was added to wash the cells, and the process was repeated twice. Finally, the cells were resuspended and blocked with DPBS solution containing 2% BSA.

[0488] The peptide sample was dissolved in DMSO and diluted to 0-30 μM using flow cytometry buffer. After washing the cells again, the diluted peptide sample was resuspended in 200 μL / tube and incubated at 4°C in a dark environment for 2.5 hours. After incubation, a low-temperature refrigerated centrifuge was used to centrifuge and remove the supernatant. The cells were washed with pre-cooled DPBS and repeated 4 times. Finally, 300 μL DPBS was resuspended and the cells were tested on a flow cytometer. The experimental results are shown in Figure 2. Fig.13 , 14 As shown, the experimental results show that the polypeptide 1 and polypeptide 15 of the present invention have good binding ability with nectin-4 and internalization effect.

[0489] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is: X 1 C1Z1C2Z2C3Z3C4X 2 ,in, X 1 Select from None or G; X 2 is G; Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ; Z2 is X 11 X 12 X 13 X 14 (X 15 ) n (X 16 ) p ; Z3 is (Z4C5Z5C6) m X 17 X 18 X 19 X 20 X 21 ; Z4 for X 22 X 23 X 24 ; Z5 to X 25 ; m, n, and p are each independently selected from 0 or 1; X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of said amino acids; X 4 ~X 25 Each is independently selected from any amino acid or unnatural amino acid.

2. The polypeptide or pharmaceutically acceptable salt thereof according to claim 1, wherein the amino acid sequence of the polypeptide is: GC1Z1C2KQDS(X 15 ) n (X 16 ) p C3X 17 X 18 X 19 X 20 X 21 C4G or C1Z1C2KQDSD(X 15 ) n (X 16 ) p C3X 17 X 18 X 19 X 20 X 21 C4G, among which Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ; X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of said amino acids; X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A; X 5 Selected from F, K, Y; X 6 Selected from K, F, Y, W, E, S, L; X 7 Selected from P, W; X 8 Selected from P, W, K, H, D, Q, N; X 9 Selected from W, G, R; X 10 Select from A, T, L, H, Q, S, G, M, N, D, E; X 15 Selected from G, D, E; X 16 Selected from V, F; X 17 Selected from E, A, L, D, Q, K, S; X 18 Selected from T, A, Q, K, P, T, Y, I; X 19 Selected from V, I, F; X 20 Selected from E, A, R, E, I, Y, V, L, T, G; X 21 Selected from V, I, K, M, D; n and p are each independently selected from 0 or 1.

3. The polypeptide or pharmaceutically acceptable salt thereof according to claim 2, wherein the amino acid sequence of the polypeptide is: GC1Z1C2KQDSDC3X 17 X 18 X 19 X 20 X 21 C4G, among which Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ; X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of said amino acids; X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A; X 5 Selected from F, K, Y; X 6 Selected from K, F, Y, W, E, S, L; X 7 Selected from P, W; X 8 Selected from P, W, K, H, D, Q, N; X 9 Selected from W, G, R; X 10 Select from A, T, L, H, Q, S, G, M, N, D, E; X 17 Selected from E, A, L, D, Q, K, S; X 18 Selected from T, A, Q, K, P, T, Y, I; X 19 Selected from V, I, F; X 20 Selected from E, A, R, E, I, Y, V, L, T, G; X 21 Selected from V, I, K, M, D.

4. The polypeptide according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is GC1Z1C2KQDSD(X 15 ) n (X 16 ) p C3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G or C1Z1C2KQDSD(X 15 ) n (X 16 ) p C3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, among which Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ; X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of said amino acids; X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A; X 5 Selected from F, K, Y; X 6 Selected from K, F, Y, W, E, S, L; X 7 Selected from P, W; X 8 Selected from P, W, K, H, D, Q, N; X 9 Selected from W, G, R; X 10 Select from A, T, L, H, Q, S, G, M, N, D, E; X 15 Selected from G, D, E; X 16 Selected from V, F; X 17 Selected from E, A, L, D, Q, K, S; X 18 Selected from T, A, Q, K, P, T, Y, I; X 19 Selected from V, I, F; X 20 Selected from E, A, R, E, I, Y, V, L, T, G; X 21 Selected from V, I, K, M, D; n and p are each independently selected from 0 or 1.

5. The polypeptide or pharmaceutically acceptable salt thereof according to claim 4, wherein the amino acid sequence of the polypeptide is: GC1Z1C2KQDSDC3LAGC5VC6X 17 X 18 X 19 X 20 X 21 C4G, among which Z1 is X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 ; X 3 Selected from F, D-Phe, W, D-Trp, Y, D-Tyr or analogs or derivatives of said amino acids; X 4 Selected from H, F, V, M, K, T, R, Y, I, Q, W, A; X 5 Selected from F, K, Y; X 6 Selected from K, F, Y, W, E, S, L; X 7 Selected from P, W; X 8 Selected from P, W, K, H, D, Q, N; X 9 Selected from W, G, R; X 10 Select from A, T, L, H, Q, S, G, M, N, D, E; X 17 Selected from E, A, L, D, Q, K, S; X 18 Selected from T, A, Q, K, P, T, Y, I; X 19 Selected from V, I, F; X 20 Selected from E, A, R, E, I, Y, V, L, T, G; X 21 Selected from V, I, K, M, D.

6. A polypeptide or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO: 1 to SEQ ID NO: 98: GCWHFKPPWACKQDSGVCETVEVCG(SEQ ID NO:1), GCWFFFPWWTCKQDSDCLAGCVCEAVVVCG (SEQ ID NO: 2), GCWVFYPPWLCKQDSEFCEAVEVCG(SEQ ID NO:3)、 GCWMFYPPWACKQDSGCAAVAVCG(SEQ ID NO:4)、 GCWVFWPQWNCKQDSDCLAVEVCG(SEQ ID NO:5)、 GCWTFYPKWQCKQDSDCLAVRVCG(SEQ ID NO:6), GCWTFYPKWHCKQDSDCLAGCVCDPVVVCG (SEQ ID NO:7), GCWKFFPPWTCKQDSDCLAGCVCDPIVVCG (SEQ ID NO:8), GCWVFYPPWSCKQDSDCLAGCVCEAVTVCG(SEQ ID NO:9), GCWVFFPHWTCKQDSDCLAGCVCEAVYICG (SEQ ID NO: 10), GCWYFWPPWGCKQDSDCLAGCVCCEPIEVCG(SEQ ID NO:11), GCWTFWPDWNCKQDSDCLAGCVCEPVLVCG(SEQ ID NO:12), GCWRFWPPWHCKQDSDCLAGCVCQPIVVCG(SEQ ID NO:13), GCWVFFPPWGCKQDSDCLAGCVCDAVYVCG (SEQ ID NO: 14), GCWTFYPGWSCKQDSDCLAGCVCEPIVVCG(SEQ ID NO:15), GCWVFWPQWNCKQDSDCLAVEVCG(SEQ ID NO:16)、 GCWVFWPQWACKQDSDCLAGCCEPVIVCG (SEQ ID NO:17), GCWYFYPEWMCKQDSDCLAGCVCEPIVVCG(SEQ ID NO:18), GCWTFWPPWACKQDSDCLAGCVCEQVVVCG(SEQ ID NO:19), GCWIFYPHWTCKQDSDCLAGCVCEPVEVCG(SEQ ID NO:20)、 GCWYFYPPWMCKQDSDCLAGCVCEPVEVCG(SEQ ID NO:21)、 GCWVFEPPWQCKQDSGVCEQVIVCG(SEQ ID NO:22)、 GCWIFSPPWTCKQDSCDKVYVCG(SEQ ID NO:23)、 GCWYFWPPWTCKQDSDCLAGCVCQTVIICG(SEQ ID NO:24)、 GCWTFYPPWMCKQDSDCLAGCVCKPVLVCG(SEQ ID NO:25)、 CWQYWWDGQCKQDSDCLAGCVCEYYGVCG(SEQ ID NO:26)、 CFWKLWHRQCKQDSDCLAGCVCAIYGKCG(SEQ ID NO:27)、 CFWKLWHRDCKQDSDCLAGCVCAIYGMCG(SEQ ID NO:28)、 CFWKLWHGECKQDSDCLAGCVCSIYKVCG(SEQ ID NO:29)、 CWAYWWNGDCKQDSDCLAGCVCEYFGDCG(SEQ ID NO:30)、 GCWHFKPPWACKQDSGVCETVLVCG(SEQ ID NO:31)、 GCWHFKPPWACKQDSGVCEPVEVCG(SEQ ID 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 ID NO: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 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)、 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 ID NO:60)、 GCWHFKPPWACKQDSGVCESVEVCG(SEQ ID NO:61)、 GCWHFKPPWACKQDSGVCETVDVCG(SEQ ID NO:62)、 GCWHFKPPWACKQDSGVCETVVVCG(SEQ ID NO:63)、 GCWHFKPPWACKGCETVEVCG(SEQ ID NO:64)、 GCWHFKPPWACKGGCETVEVCG(SEQ ID NO:65)、 GCWHFKPPWACKGPCETVEVCG(SEQ ID NO: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 ID NO:72)、GCWTFWPDWNCLTGTATWNGNVVISVFCEPVLVCG(SEQ ID NO:73)、GCWFFHPIWNCKQDSDCLAGCVCPLVTVCG(SEQ ID NO:74)、 GCWHF{Dab}PPWMCKQDSGVCETVEVCG(SEQ ID NO:75)、 GCWHF{Dab}PPW{Nle}CKQDSGVCETVEVCG(SEQ ID NO:76)、 GCWHFKPPW{Nle}CKQDSGVCETVEVCG(SEQ ID NO:77)、 GCWHF{D-Dab}PPWACKQDSGVCETVEVCG(SEQ ID NO:78)、 GCWHF{D-Tyr}PPWACKQDSGVCETVEVCG(SEQ ID NO:79)、 GCWHFKPPW{D-Ala}CKQDSGVCETVEVCG(SEQ ID NO:80)、 GCWTFWPDWNCKQDSDGSCLAGCVCEPVLVCG(SEQ ID NO:81)、 GCWTFWPDWNCKQDSDGGSCLAGCVCEPVLVCG(SEQ ID NO:82)、 GCWTFYPKWNCKQDSDCLAGCVCEPVVVCG(SEQ ID NO:83)、 GCWMFWPDWHCKQDSDCLAGCVCKPVIVCG(SEQ ID NO:84)、 GCWFFKPWWTCKQDSDCLAGCVCDPIVVCG(SEQ ID NO:85)、 GCWTFWPPWACKQDSDCLAGCVCEQIIVCG(SEQ ID NO:86)、 GCWIFWPPWGCKQDSDCLAGCVCEPVLVCG(SEQ ID NO:87)、 GCWYFFPPWTCKQDSDCLAGCVCEPVRICG (SEQ ID NO: 88), GCWYFYPKWTCKQDSDCLAGCVCDPVVICG (SEQ ID NO: 89), GCWVFWPPWGCKQDSDCLAGCVCEAVVVCG (SEQ ID NO: 90), GCWVFFPWWSCKQDSDCLAGCVCEAVYICG (SEQ ID NO: 91), GCWYFYPPWACKQDSDCLAGCVCDAIVICG (SEQ ID NO: 92), GCWTFWPPWHCKQDSDCLAGCVCEPVVICG (SEQ ID NO: 93), GCWTFYPWWECKQDSDCLAGCCDAVVVCG (SEQ ID NO: 94), GCWFFYPPWGCKQDSDCLAGCVCEPVTVCG (SEQ ID NO: 95), GCWVFFPKWNCKQDSDCLAGCVCEPVEVCG (SEQ ID NO: 96), GCWKFEPWWQCKQDSDCLAGCVCQPVIICG (SEQ ID NO: 97), GCWRFKPWWACKQDSDCLAGCVCCEIIEVCG (SEQ ID NO: 98).

7. A polypeptide or a pharmaceutically acceptable salt thereof, wherein the polypeptide comprises any one of the following (i) to (ii): (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; (ii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or modified in the amino acid sequence as shown in SEQ ID NO.1 while retaining the function of the sequence as shown in SEQ ID NO.

1.

8. A polynucleotide, the sequence of which comprises a nucleotide sequence encoding the amino acid sequence according to any one of claims 1 to 7.

9. A pharmaceutical composition comprising the polypeptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7 or the polynucleotide according to claim 8, in combination with one or more pharmaceutically acceptable excipients.

10. Use of the polypeptide or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, the polynucleotide according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of a drug for treating and / or preventing a disease associated with Nectin-4, wherein the disease associated with Nectin-4 is a cancer with high expression of Nectin-4. Preferably, the cancer with high expression of nectin-4 is 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, breast ductal carcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, head and neck cancer, colon cancer, rectal cancer or esophageal cancer.

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