Polypeptide targeting GPC3, and drug conjugate thereof and use thereof

Abstract

The present invention relates to the technical field of pharmaceuticals, and specifically relates to a polypeptide targeting GPC3, and a drug conjugate thereof and the use thereof. The polypeptide has an amino acid sequence as shown in general formula (I), and the structure of the polypeptide-drug conjugate is as shown in general formula (1). The polypeptide and polypeptide-drug conjugate of the present invention are used in the development or screening of diagnostic or therapeutic drugs against GPC3 expressing cells or tumors, and the obtained drug has a good specificity and affinity and a significant tumor inhibitory effect.

Description

A GPC3-targeting peptide and its drug conjugate and applications Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a polypeptide targeting GPC3, its drug conjugate, and its applications. Background Technology

[0002] Liver cancer is one of the most common malignant tumors worldwide, with its incidence and mortality rates increasing year by year, seriously threatening human health. Treatment methods for liver cancer mainly include liver transplantation, tumor resection, and non-resectable local therapies such as hepatic artery chemoembolization. However, due to the high rate of early metastasis and recurrence after treatment in liver cancer, especially hepatocellular carcinoma (HCC), the above treatment methods are not very effective. Therefore, new immunotherapies are urgently needed, and the identification of immunotherapy targets has become a hot topic in HCC treatment research in recent years.

[0003] Glypicans-3 (GPC3) is a tumor marker for primary liver cancer that has been extensively studied in recent years. Targeted therapy targeting the GPC3 receptor holds promise for changing the current passive approach to treating advanced hepatocellular carcinoma. GPC3 is a proteoglycan belonging to the heparan sulfate proteoglycan (HSPG) family, with a molecular weight of approximately 70,000. It is anchored to the cell membrane surface via glycosylphosphatidyl inositol (GPI). Studies have found that GPC3 is highly expressed in HCC tissues, but not expressed or expressed at low levels in normal adult tissues, making it a specific tissue marker closely related to prognosis. GPC3 participates in the development and progression of HCC through multiple signaling pathways, such as stimulating the Wnt signaling pathway, interacting with growth factors, stimulating macrophage recruitment, and promoting epithelial-mesenchymal transition. Targeted immunotherapy based on GPC3 has become a research hotspot in HCC treatment.

[0004] GPC3 has clinical application value as a tumor target. For example, engineered immune cells such as TCR-T and CAR-T targeting GPC3, as well as therapeutic antibodies against GPC3, have shown some efficacy in clinical trials. Roche's Cdrituzumab (GC33) is the first GPC3-targeting antibody to enter the clinical stage. Radiolabeled probes targeting GPC3 mainly focus on antibody probes and peptide probes. GPC3 monoclonal antibodies have good affinity and specificity for HCC. 89Zr-labeled GPC3 monoclonal antibodies can visualize HCC tumor tissue, but these antibodies suffer from drawbacks such as large molecular weight, high cost, difficulty in labeling, slow in vivo clearance, and potential immunogenicity. In contrast, peptides offer advantages such as low molecular weight, low immunogenicity, ease of synthesis, modification, and radiolabeling, and good tumor penetration. Reported GPC3-targeting peptide probes include… 99m Tc-HPG, [ 18 F]AlF-NOTA-TJ12P2 and [ 68 Ga]DOTA-F3, [ 18 [F]AlF-GP2633, etc., these GPC3-targeting peptide probes can specifically target GPC3-positive HCC tumors, but they have shortcomings such as low tumor uptake, short tumor retention time, and poor pharmacokinetic characteristics (e.g., non-specific uptake in the abdomen). RayzeBio's RAYZ-8009 consists of a macrocyclic peptide that binds to GPC3 and a chelating agent that binds to a radioactive metal isotope. The GPC3 macrocyclic peptide was discovered using PeptiDream Inc.'s proprietary screening system and further chemically optimized before being fitted with a metal chelating agent. CN118909052A discloses a GPC3-targeting peptide and its application.

[0005] The development of GPC3-targeted new drugs has brought new hope to the treatment of liver cancer. Therefore, the development of GPC3-targeting peptides has a very high application prospect for the diagnosis and treatment of malignant tumors with high GPC3 specific expression, especially HCC.

[0006] Currently, screening methods for cyclic peptide drugs can be broadly categorized into in vitro screening and in vivo screening. In vitro screening primarily relies on the binding affinity between the cyclic peptide and the target protein, selecting peptides with higher affinity through a series of screening steps. A widely used screening technique is phage display. Phage display involves inserting different exogenous genes into phage vectors. These exogenous genes are expressed along with the capsid protein, resulting in the peptide or protein appearing as a fusion protein on the phage surface, thus maintaining relative spatial structure and biological activity. The linear library can then cyclize by linking to reactants via its cysteine ​​residue (Cys). Subsequently, screening is performed using specific target proteins. Summary of the Invention

[0007] Current drug development still falls short of actual clinical needs, and there is still room for development in drugs targeting GPC3. Therefore, one objective of this invention is to provide a GPC3-targeting peptide developed using phage display technology. This peptide binds to GPC3 overexpressed in tumor cells, possessing significant application value in tumor molecular diagnostics and targeted therapy. A second objective is to provide a peptide-drug conjugate based on the aforementioned GPC3-targeting peptide. A third objective is to provide an application of the GPC3-targeting peptide or its drug conjugate.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] On one hand, the present invention provides a polypeptide having the amino acid sequence shown in general formula (I), X1CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CX 19 (I)

[0010] in,

[0011] X1 either does not exist or is G;

[0012] X 19 It does not exist or is G;

[0013] X4, X8, X9, X 10 X 11 X 12 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids;

[0014] Each amino acid in the general formula is independently selected from either the D- or L-isomer.

[0015] In some embodiments, the polypeptide has the amino acid sequence shown in general formula (II), GCPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CG (II).

[0016] In some embodiments, the polypeptide has the amino acid sequence shown in general formula (III), GCPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 C (III).

[0017] In some embodiments, the polypeptide has the amino acid sequence shown in general formula (IV), CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CG (IV).

[0018] In some embodiments, the polypeptide has the amino acid sequence shown in general formula (V), CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 C (V).

[0019] In some embodiments, in the general formula (I),

[0020] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R;

[0021] X8 is selected from I, T, H, F, V, N, L, R, Y, S, Q;

[0022] X9 is selected from F and M;

[0023] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0024] X 11 Selected from E, D, Y, H, Q, M;

[0025] X 12 Selected from R and W;

[0026] X 16 Selected from I, H, Y, W, F, E, D, A;

[0027] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0028] In some embodiments, in the general formula (I),

[0029] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I;

[0030] X8 is selected from I, T, H, F, V, N, L, R;

[0031] X9 is selected from F and M;

[0032] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0033] X 11 Selected from E, D, Y, H, Q;

[0034] X 12 Selected from R and W;

[0035] X 16 Selected from I, H, Y, W, F, E, D, A;

[0036] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0037] In some embodiments, in the general formula (I),

[0038] X4 is selected from S, N, D, G, K, A, H;

[0039] X8 is selected from I, T, H, F, V, N;

[0040] X9 is selected from F;

[0041] X 10 Selected from H, V, T, K, P, M, G, Q;

[0042] X 11 Selected from E, D, and Y;

[0043] X 12 Selected from R;

[0044] X 16 Selected from I, H, Y, W, F, E, D;

[0045] X 17 Selected from K, I, Y, E, Q, M, H.

[0046] In some embodiments, in the general formula (I),

[0047] X4 is selected from S, N, D, and G;

[0048] X8 is selected from I, T, H, F, V;

[0049] X9 is selected from F;

[0050] X 10 Selected from H, V, T, K, P, M, G;

[0051] X 11 Selected from E and D;

[0052] X 12 Selected from R;

[0053] X 16 Selected from I, H, Y, W, F;

[0054] X 17 Selected from K, I, Y, E, Q, M.

[0055] In some embodiments, in the general formula (I),

[0056] X4 is selected from S and N;

[0057] X8 is selected from I, T, H, and V;

[0058] X9 is selected from F;

[0059] X 10 Selected from H, V, T, K, G;

[0060] X 11 Selected from E and D;

[0061] X 12 Selected from R;

[0062] X 16 Selected from I, H, Y, W;

[0063] X 17 Selected from K, I, Y, E.

[0064] In some embodiments, in the general formula (I),

[0065] X4 is selected from N and E;

[0066] X8 is selected from V and Q;

[0067] X9 is selected from F;

[0068] X 10 Selected from G and Q;

[0069] X 11 Selected from E and Q;

[0070] X 12 Selected from R;

[0071] X 16 Selected from W;

[0072] X 17 Selected from I and Q.

[0073] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (II), (III), (IV) or (V), GCPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CG (II), GCPX4YCTX8X9X 10 X11 X 12 YECX 16 X 17 C (III), CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CG (IV), CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 C (V),

[0074] in,

[0075] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R;

[0076] X8 is selected from I, T, H, F, V, N, L, R, Y, S, Q;

[0077] X9 is selected from F and M;

[0078] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0079] X 11 Selected from E, D, Y, H, Q, M;

[0080] X 12 Selected from R and W;

[0081] X 16 Selected from I, H, Y, W, F, E, D, A;

[0082] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0083] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (II), (III), (IV) or (V), wherein,

[0084] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I;

[0085] X8 is selected from I, T, H, F, V, N, L, R;

[0086] X9 is selected from F and M;

[0087] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0088] X 11 Selected from E, D, Y, H, Q;

[0089] X 12 Selected from R and W;

[0090] X 16 Selected from I, H, Y, W, F, E, D, A;

[0091] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0092] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (II), (III), (IV) or (V), wherein,

[0093] X4 is selected from S, N, D, G, K, A, H;

[0094] X8 is selected from I, T, H, F, V, N;

[0095] X9 is selected from F;

[0096] X 10 Selected from H, V, T, K, P, M, G, Q;

[0097] X 11 Selected from E, D, and Y;

[0098] X 12 Selected from R;

[0099] X 16 Selected from I, H, Y, W, F, E, D;

[0100] X 17 Selected from K, I, Y, E, Q, M, H.

[0101] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (II), (III), (IV) or (V), wherein,

[0102] X4 is selected from S, N, D, and G;

[0103] X8 is selected from I, T, H, F, V;

[0104] X9 is selected from F;

[0105] X 10 Selected from H, V, T, K, P, M, G;

[0106] X 11 Selected from E and D;

[0107] X 12 Selected from R;

[0108] X16 Selected from I, H, Y, W, F;

[0109] X 17 Selected from K, I, Y, E, Q, M.

[0110] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (II), (III), (IV) or (V), wherein,

[0111] X4 is selected from S and N;

[0112] X8 is selected from I, T, H, and V;

[0113] X9 is selected from F;

[0114] X 10 Selected from H, V, T, K, G;

[0115] X 11 Selected from E and D;

[0116] X 12 Selected from R;

[0117] X 16 Selected from I, H, Y, W;

[0118] X 17 Selected from K, I, Y, E.

[0119] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (II), (III), (IV) or (V), wherein,

[0120] X4 is selected from N and E;

[0121] X8 is selected from V and Q;

[0122] X9 is selected from F;

[0123] X 10 Selected from G and Q;

[0124] X 11 Selected from E and Q;

[0125] X 12 Selected from R;

[0126] X 16 Selected from W;

[0127] X 17 Selected from I and Q.

[0128] In some embodiments, the polypeptide has the amino acid sequence shown in general formula (VI), GCPX4YCTX8FX 10 X 11 RYECX16 X 17 CG (VI)

[0129] X4, X8, X 10 X 11 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids;

[0130] Each amino acid in the general formula is independently selected from either the D- or L-isomer.

[0131] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (VIII), (IX), or (X), CPX4YCTX8FX 10 X 11 RYECX 16 X 17 CG (VIII), GCPX4YCTX8FX 10 X 11 RYECX 16 X 17 C (IX), CPX4YCTX8FX 10 X 11 RYECX 16 X 17 C (X),

[0132] X4, X8, X 10 X 11 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids;

[0133] Each amino acid in the general formula is independently selected from either the D- or L-isomer.

[0134] In some embodiments, in formulas (VI), (VIII), (IX), or (X),

[0135] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R;

[0136] X8 is selected from I, T, H, F, V, N, L, R, Y, S, Q;

[0137] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0138] X 11 Selected from E, D, Y, H, Q, M;

[0139] X 16Selected from I, H, Y, W, F, E, D, A;

[0140] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0141] In some embodiments, in formulas (VI), (VIII), (IX), or (X),

[0142] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I;

[0143] X8 is selected from I, T, H, F, V, N, L, R;

[0144] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0145] X 11 Selected from E, D, Y, H, Q;

[0146] X 16 Selected from I, H, Y, W, F, E, D, A;

[0147] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0148] In some embodiments, in formulas (VI), (VIII), (IX), or (X),

[0149] X4 is selected from S, N, D, G, K, A, H;

[0150] X8 is selected from I, T, H, F, V, N;

[0151] X 10 Selected from H, V, T, K, P, M, G, Q;

[0152] X 11 Selected from E, D, and Y;

[0153] X 16 Selected from I, H, Y, W, F, E, D;

[0154] X 17 Selected from K, I, Y, E, Q, M, H.

[0155] In some embodiments, in formulas (VI), (VIII), (IX), or (X),

[0156] X4 is selected from S, N, D, and G;

[0157] X8 is selected from I, T, H, F, V;

[0158] X 10 Selected from H, V, T, K, P, M, G;

[0159] X 11 Selected from E and D;

[0160] X 16 Selected from I, H, Y, W, F;

[0161] X 17 Selected from K, I, Y, E, Q, M.

[0162] In some embodiments, in formulas (VI), (VIII), (IX), or (X),

[0163] X4 is selected from S and N;

[0164] X8 is selected from I, T, H, and V;

[0165] X 10 Selected from H, V, T, K, G;

[0166] X 11 Selected from E and D;

[0167] X 16 Selected from I, H, Y, W;

[0168] X 17 Selected from K, I, Y, E.

[0169] In some embodiments, in formulas (VI), (VIII), (IX), or (X),

[0170] X4 is selected from N and E;

[0171] X8 is selected from V and Q;

[0172] X 10 Selected from G and Q;

[0173] X 11 Selected from E and Q;

[0174] X 16 Selected from W;

[0175] X 17 Selected from I and Q.

[0176] In some implementations, X8 is selected from V;

[0177] In some embodiments, the polypeptide has the amino acid sequence shown in general formula (VII), GCPX4YCTVFX 10 X 11 RYECWX 17 CG (VII)

[0178] in,

[0179] X4, X 10 X 11 X 17 Each amino acid is independently selected from natural or non-natural amino acids;

[0180] Each amino acid in the general formula is independently selected from either the D- or L-isomer.

[0181] In some embodiments, the polypeptide has an amino acid sequence represented by general formula (XI), (XII), or (XIII), CPX4YCTVFX 10 X 11 RYECWX 17 CG (XI), GCPX4YCTVFX 10 X 11 RYECWX 17 C (XII), CPX4YCTVFX 10 X 11 RYECWX 17 C (XIII),

[0182] in,

[0183] X4, X 10 X 11 X 17 Each amino acid is independently selected from natural or non-natural amino acids;

[0184] Each amino acid in the general formula is independently selected from either the D- or L-isomer.

[0185] In some embodiments, in general formulas (VII), (XI), (XII) or (XIII),

[0186] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R;

[0187] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0188] X 11 Selected from E, D, Y, H, Q, M;

[0189] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0190] In some embodiments, in general formulas (VII), (XI), (XII) or (XIII),

[0191] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I;

[0192] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0193] X 11 Selected from E, D, Y, H, Q;

[0194] X 17 Selected from K, I, Y, E, Q, M, H, V.

[0195] In some embodiments, in general formulas (VII), (XI), (XII) or (XIII),

[0196] X4 is selected from S, N, D, G, K, A, H;

[0197] X 10 Selected from H, V, T, K, P, M, G, Q;

[0198] X 11 Selected from E, D, and Y;

[0199] X 17 Selected from K, I, Y, E, Q, M, H.

[0200] In some embodiments, in general formulas (VII), (XI), (XII) or (XIII),

[0201] X4 is selected from S, N, D, and G;

[0202] X 10 Selected from H, V, T, K, P, M, G;

[0203] X 11 Selected from E and D;

[0204] X 17 Selected from K, I, Y, E, Q, M.

[0205] In some embodiments, in general formulas (VII), (XI), (XII) or (XIII),

[0206] X4 is selected from S and N;

[0207] X 10 Selected from H, V, T, K, G;

[0208] X 11 Selected from E and D;

[0209] X 17 Selected from K, I, Y, E.

[0210] In some embodiments, in general formulas (VII), (XI), (XII) or (XIII),

[0211] X4 is selected from N and E;

[0212] X 10 Selected from G and Q;

[0213] X 11 Selected from E and Q;

[0214] X 17 Selected from I and Q.

[0215] On one hand, the present invention provides a polypeptide having the amino acid sequence shown in general formula (XIV), X1CPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 CX 19 (XIV)

[0216] in,

[0217] X1 either does not exist or is G;

[0218] X 19 It does not exist or is G;

[0219] X4, X8, X9, X 10 X 11 X 12 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids;

[0220] Each amino acid in the general formula is independently selected from either the D- or L-isomer.

[0221] In some embodiments, the polypeptide has an amino acid sequence represented by the general formula (XV), (XVI), (XVII), (XVIII), GCPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 CG (XV), GCPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 C (XVI), CPX4YCQX8X9X 10 X 11 X 12YECX 16 X 17 CG (XVII), CPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 C (XVIII),

[0222] in,

[0223] X1 either does not exist or is G;

[0224] X 19 It does not exist or is G;

[0225] X4, X8, X9, X 10 X 11 X 12 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids;

[0226] Each amino acid in the general formula is independently selected from either the D- or L-isomer.

[0227] In some embodiments, the polypeptide has an amino acid sequence represented by the general formula (XV), (XVI), (XVII), (XVIII), wherein,

[0228] X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R;

[0229] X8 is selected from I, T, H, F, V, N, L, R, Y, S, Q;

[0230] X9 is selected from F and M;

[0231] X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E;

[0232] X 11 Selected from E, D, Y, H, Q, M;

[0233] X 12 Selected from R and W;

[0234] X 16 Selected from I, H, Y, W, F, E, D, A, N;

[0235] X 17 Selected from K, I, Y, E, Q, M, H, V, A.

[0236] In some embodiments, the polypeptide has an amino acid sequence represented by the general formula (XV), (XVI), (XVII), (XVIII), wherein,

[0237] X4 is selected from S, N, D, G, and E;

[0238] X8 is selected from I, T, H, F, V;

[0239] X9 is selected from F;

[0240] X 10 Selected from H, V, T, K, P, M, G;

[0241] X 11 Selected from E and D;

[0242] X 12 Selected from R;

[0243] X 16 Selected from I, H, Y, W, F, N;

[0244] X 17 Selected from K, I, Y, E, Q, M, A.

[0245] In some embodiments, the polypeptide has an amino acid sequence represented by the general formula (XV), (XVI), (XVII), (XVIII), wherein,

[0246] X4 is selected from N and E;

[0247] X8 is selected from V and Q;

[0248] X9 is selected from F;

[0249] X 10 Selected from G, Q, K;

[0250] X 11 Selected from E and Q;

[0251] X 12 Selected from R;

[0252] X 16 Selected from W and N;

[0253] X 17 Selected from I and A.

[0254] In some embodiments, the polypeptide has an amino acid sequence represented by the general formula (XV), (XVI), (XVII), (XVIII), wherein,

[0255] X4 is selected from N;

[0256] X8 is selected from V;

[0257] X9 is selected from F;

[0258] X 10 Selected from G, Q, K;

[0259] X 11 Selected from E and Q;

[0260] X 12 Selected from R;

[0261] X 16 Selected from W and N;

[0262] X 17 Selected from I and A.

[0263] In some embodiments, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID No. 1 to SEQ ID No. 244.

[0264] SEQ ID No.1: GCPNYCTVFGERYECWICG,

[0265] SEQ ID No.2: GCPSYCTIFHERYECIKCG,

[0266] SEQ ID No.3: GCPSYCTTFVDRYECHICG,

[0267] SEQ ID No.4: GCPNYCTTFTERYECYYCG,

[0268] SEQ ID No.5: GCPSYCTHFKDRYECWECG,

[0269] SEQ ID No.6: GCPDYCTFFPERYECWQCG,

[0270] SEQ ID No.7: GCPGYCTHFMERYECYMCG,

[0271] SEQ ID No.8: GCPNYCTHFKERYECFQCG,

[0272] SEQ ID No.9: GCPKYCTIFHERYECEKCG,

[0273] SEQ ID No.10: GCPAYCTHFQERYECHYCG,

[0274] SEQ ID No.11:GCPDYCTNFQYRYECFICG、

[0275] SEQ ID No.12:GCPSYCTVFGERYECIHCG、

[0276] SEQ ID No.13:GCPHYCTVFKERYECDQCG、

[0277] SEQ ID No.14:GCPGYCTHFTERYECHICG、

[0278] SEQ ID No.15:GCPGYCTIFQERYECEHCG、

[0279] SEQ ID No.16:GCPEYCTIFSHRYECYICG、

[0280] SEQ ID No.17:GCPGYCTIFRDRYECAMCG、

[0281] SEQ ID No.18:GCPEYCTIFKERYECHHCG、

[0282] SEQ ID No.19:GCPSYCTLFTERYECYMCG、

[0283] SEQ ID No.20:GCPTYCTHFRERYECWQCG、

[0284] SEQ ID No.21:GCPQYCTTFKDRYECYICG、

[0285] SEQ ID No.22:GCPDYCTRMLHWYECHVCG、

[0286] SEQ ID No.23:GCPTYCTHFTERYECFTCG、

[0287] SEQ ID No.24:GCPTYCTNFKERYECFHCG、

[0288] SEQ ID No.25:GCPSYCTLFHERYECYICG、

[0289] SEQ ID No.26:GCPIYCTHFKERYECFMCG、

[0290] SEQ ID No.27:GCPTYCTIFNHRYECFHCG、

[0291] SEQ ID No.28:GCPKYCTIFMERYECEKCG、

[0292] SEQ ID No.29:GCPQYCTHFEQRYECWKCG、

[0293] SEQ ID No.30:GCPHYCTNFKERYECFMCG、

[0294] SEQ ID No.31:GCPSYCTYFEHRYECYTCG、

[0295] SEQ ID No.32:GCPKYCTTFQERYECHYCG、

[0296] SEQ ID No.33:GCPHYCTVFSERYECHMCG、

[0297] SEQ ID No.34:GCPGYCTTFHERYECFTCG、

[0298] SEQ ID No.35:GCPKYCTTFRDRYECWECG、

[0299] SEQ ID No.36:GCPAYCTIFEHRYECHHCG、

[0300] SEQ ID No.37:GCPHYCTSFEQRYECYICG、

[0301] SEQ ID No.38:GCPGYCTIFMERYECHHCG、

[0302] SEQ ID No.39:GCPAYCTHFVERYECYTCG、

[0303] SEQ ID No.40:GCPPYCTVFEQRYECIHCG、

[0304] SEQ ID No.41:GCPHYCTTFRERYECFVCG、

[0305] SEQ ID No.42:GCPHYCTHFQERYECFECG、

[0306] SEQ ID No.43:GCPKYCTQMPERYECFYCG、

[0307] SEQ ID No.44:GCPRYCTIFSERYECETCG、

[0308] SEQ ID No.45:GCPGYCTVFEMRYECAVCG、

[0309] SEQ ID No.46:GCPHYCTYFQERYECEYCG、

[0310] SEQ ID No.47:GCPGYCTYFNDRYECYTCG、

[0311] SEQ ID No.48:GCPPYCTYFSERYECYICG、

[0312] SEQ ID No.49:GCPHYCTFFMERYECHICG、

[0313] SEQ ID No.50:GCPDYCTIFMMRYECIECG、

[0314] SEQ ID No.51:GCPSYCTIFHERYECIKC、

[0315] SEQ ID No.52:GCPSYCTTFVDRYECHIC、

[0316] SEQ ID No.53:GCPNYCTTFTERYECYYC、

[0317] SEQ ID No.54:GCPSYCTHFKDRYECWEC、

[0318] SEQ ID No.55:GCPDYCTFFPERYECWQC、

[0319] SEQ ID No.56:GCPGYCTHFMERYECYMC、

[0320] SEQ ID No.57:GCPNYCTHFKERYECFQC、

[0321] SEQ ID No.58:GCPNYCTVFGERYECWIC、

[0322] SEQ ID No.59:GCPKYCTIFHERYECEKC、

[0323] SEQ ID No.60:GCPAYCTHFQERYECHYC、

[0324] SEQ ID No.61:GCPDYCTNFQYRYECFIC、

[0325] SEQ ID No.62:GCPSYCTVFGERYECIHC、

[0326] SEQ ID No.63:GCPHYCTVFKERYECDQC、

[0327] SEQ ID No.64:GCPGYCTHFTERYECHIC、

[0328] SEQ ID No.65:GCPGYCTIFQERYECEHC、

[0329] SEQ ID No.66:GCPEYCTIFSHRYECYIC、

[0330] SEQ ID No.67:GCPGYCTIFRDRYECAMC、

[0331] SEQ ID No.68:GCPEYCTIFKERYECHHC、

[0332] SEQ ID No.69:GCPSYCTLFTERYECYMC、

[0333] SEQ ID No.70:GCPTYCTHFRERYECWQC、

[0334] SEQ ID No.71:GCPQYCTTFKDRYECYIC、

[0335] SEQ ID No.72:GCPDYCTRMLHWYECHVC、

[0336] SEQ ID No.73:GCPTYCTHFTERYECFTC、

[0337] SEQ ID No.74:GCPTYCTNFKERYECFHC、

[0338] SEQ ID No.75:GCPSYCTLFHERYECYIC、

[0339] SEQ ID No.76:GCPIYCTHFKERYECFMC、

[0340] SEQ ID No.77:GCPTYCTIFNHRYECFHC、

[0341] SEQ ID No.78:GCPKYCTIFMERYECEKC、

[0342] SEQ ID No.79:GCPQYCTHFEQRYECWKC、

[0343] SEQ ID No.80:GCPHYCTNFKERYECFMC、

[0344] SEQ ID No.81:GCPSYCTYFEHRYECYTC、

[0345] SEQ ID No.82:GCPKYCTTFQERYECHYC、

[0346] SEQ ID No.83:GCPHYCTVFSERYECHMC、

[0347] SEQ ID No.84:GCPGYCTTFHERYECFTC、

[0348] SEQ ID No.85:GCPKYCTTFRDRYECWEC、

[0349] SEQ ID No.86:GCPAYCTIFEHRYECHHC、

[0350] SEQ ID No.87:GCPHYCTSFEQRYECYIC、

[0351] SEQ ID No.88:GCPGYCTIFMERYECHHC、

[0352] SEQ ID No.89:GCPAYCTHFVERYECYTC、

[0353] SEQ ID No.90:GCPPYCTVFEQRYECIHC、

[0354] SEQ ID No.91:GCPHYCTTFRERYECFVC、

[0355] SEQ ID No.92:GCPHYCTHFQERYECFEC、

[0356] SEQ ID No.93:GCPKYCTQMPERYECFYC、

[0357] SEQ ID No.94:GCPRYCTIFSERYECETC、

[0358] SEQ ID No.95:GCPGYCTVFEMRYECAVC、

[0359] SEQ ID No.96:GCPHYCTYFQERYECEYC、

[0360] SEQ ID No.97:GCPGYCTYFNDRYECYTC、

[0361] SEQ ID No.98:GCPPYCTYFSERYECYIC、

[0362] SEQ ID No.99:GCPHYCTFFMERYECHIC、

[0363] SEQ ID No.100:GCPDYCTIFMMRYECIEC、

[0364] SEQ ID No.101:CPSYCTIFHERYECIKCG、

[0365] SEQ ID No.102:CPSYCTTFVDRYECHICG、

[0366] SEQ ID No.103:CPNYCTTFTERYECYYCG、

[0367] SEQ ID No.104:CPSYCTHFKDRYECWECG、

[0368] SEQ ID No.105:CPDYCTFFPERYECWQCG、

[0369] SEQ ID No.106:CPGYCTHFMERYECYMCG、

[0370] SEQ ID No.107:CPNYCTHFKERYECFQCG、

[0371] SEQ ID No.108:CPNYCTVFGERYECWICG、

[0372] SEQ ID No.109:CPKYCTIFHERYECEKCG、

[0373] SEQ ID No.110:CPAYCTHFQERYECHYCG、

[0374] SEQ ID No.111:CPDYCTNFQYRYECFICG、

[0375] SEQ ID No.112:CPSYCTVFGERYECIHCG、

[0376] SEQ ID No.113:CPHYCTVFKERYECDQCG、

[0377] SEQ ID No.114:CPGYCTHFTERYECHICG、

[0378] SEQ ID No.115:CPGYCTIFQERYECEHCG、

[0379] SEQ ID No.116:CPEYCTIFSHRYECYICG、

[0380] SEQ ID No.117:CPGYCTIFRDRYECAMCG、

[0381] SEQ ID No.118:CPEYCTIFKERYECHHCG、

[0382] SEQ ID No.119:CPSYCTLFTERYECYMCG、

[0383] SEQ ID No.120:CPTYCTHFRERYECWQCG、

[0384] SEQ ID No.121:CPQYCTTFKDRYECYICG、

[0385] SEQ ID No.122:CPDYCTRMLHWYECHVCG、

[0386] SEQ ID No.123:CPTYCTHFTERYECFTCG、

[0387] SEQ ID No.124:CPTYCTNFKERYECFHCG、

[0388] SEQ ID No.125:CPSYCTLFHERYECYICG、

[0389] SEQ ID No.126:CPIYCTHFKERYECFMCG、

[0390] SEQ ID No.127:CPTYCTIFNHRYECFHCG、

[0391] SEQ ID No.128:CPKYCTIFMERYECEKCG、

[0392] SEQ ID No.129:CPQYCTHFEQRYECWKCG、

[0393] SEQ ID No.130:CPHYCTNFKERYECFMCG、

[0394] SEQ ID No.131:CPSYCTYFEHRYECYTCG、

[0395] SEQ ID No.132:CPKYCTTFQERYECHYCG、

[0396] SEQ ID No.133:CPHYCTVFSERYECHMCG、

[0397] SEQ ID No.134:CPGYCTTFHERYECFTCG、

[0398] SEQ ID No.135:CPKYCTTFRDRYECWECG、

[0399] SEQ ID No.136:CPAYCTIFEHRYECHHCG、

[0400] SEQ ID No.137:CPHYCTSFEQRYECYICG、

[0401] SEQ ID No.138:CPGYCTIFMERYECHHCG、

[0402] SEQ ID No.139:CPAYCTHFVERYECYTCG、

[0403] SEQ ID No.140:CPPYCTVFEQRYECIHCG、

[0404] SEQ ID No.141:CPHYCTTFRERYECFVCG、

[0405] SEQ ID No.142:CPHYCTHFQERYECFECG、

[0406] SEQ ID No.143:CPKYCTQMPERYECFYCG、

[0407] SEQ ID No.144:CPRYCTIFSERYECETCG、

[0408] SEQ ID No.145:CPGYCTVFEMRYECAVCG、

[0409] SEQ ID No.146:CPHYCTYFQERYECEYCG、

[0410] SEQ ID No.147:CPGYCTYFNDRYECYTCG、

[0411] SEQ ID No.148:CPPYCTYFSERYECYICG、

[0412] SEQ ID No.149:CPHYCTFFMERYECHICG、

[0413] SEQ ID No.150:CPDYCTIFMMRYECIECG、

[0414] SEQ ID No.151:CPSYCTIFHERYECIKC、

[0415] SEQ ID No.152:CPSYCTTFVDRYECHIC、

[0416] SEQ ID No.153:CPNYCTTFTERYECYYC、

[0417] SEQ ID No.154:CPSYCTHFKDRYECWEC、

[0418] SEQ ID No.155:CPDYCTFFPERYECWQC、

[0419] SEQ ID No.156:CPGYCTHFMERYECYMC、

[0420] SEQ ID No.157:CPNYCTHFKERYECFQC、

[0421] SEQ ID No.158:CPNYCTVFGERYECWIC、

[0422] SEQ ID No.159:CPKYCTIFHERYECEKC、

[0423] SEQ ID No.160:CPAYCTHFQERYECHYC、

[0424] SEQ ID No.161:CPDYCTNFQYRYECFIC、

[0425] SEQ ID No.162:CPSYCTVFGERYECIHC、

[0426] SEQ ID No.163:CPHYCTVFKERYECDQC、

[0427] SEQ ID No.164:CPGYCTHFTERYECHIC、

[0428] SEQ ID No.165:CPGYCTIFQERYECEHC、

[0429] SEQ ID No.166: CPEYCTIFSHRYECYIC

[0430] SEQ ID No.167:CPGYCTIFRDRYECAMC、

[0431] SEQ ID No.168:CPEYCTIFKERYECHHC、

[0432] SEQ ID No.169:CPSYCTLFTERYECYMC、

[0433] SEQ ID No. 170: CPTYCTHFRERYECWQC

[0434] SEQ ID No.171:CPQYCTTFKDRYECYIC、

[0435] SEQ ID No.172:CPDYCTRMLHWYECHVC、

[0436] SEQ ID No.173:CPTYCTHFTERYECFTC、

[0437] SEQ ID No.174:CPTYCTNFKERYECFHC、

[0438] SEQ ID No.175:CPSYCTLFHERYECYIC、

[0439] SEQ ID No.176:CPIYCTHFKERYECFMC、

[0440] SEQ ID No.177: CPTYCTIFNHRYECFHC

[0441] SEQ ID No.178:CPKYCTIFMERYECEKC、

[0442] SEQ ID No.179:CPQYCTHFEQRYECWKC、

[0443] SEQ ID No.180:CPHYCTNFKERYECFMC、

[0444] SEQ ID No.181:CPSYCTYFEHRYECYTC、

[0445] SEQ ID No.182:CPKYCTTFQERYECHYC、

[0446] SEQ ID No.183:CPHYCTVFSERYECHMC、

[0447] SEQ ID No.184:CPGYCTTFHERYECFTC、

[0448] SEQ ID No.185:CPKYCTTFRDRYECWEC、

[0449] SEQ ID No.186:CPAYCTIFEHRYECHHC、

[0450] SEQ ID No.187:CPHYCTSFEQRYECYIC、

[0451] SEQ ID No.188:CPGYCTIFMERYECHHC、

[0452] SEQ ID No.189:CPAYCTHFVERYECYTC、

[0453] SEQ ID No.190:CPPYCTVFEQRYECIHC、

[0454] SEQ ID No.191:CPHYCTTFRERYECFVC、

[0455] SEQ ID No.192:CPHYCTHFQERYECFEC、

[0456] SEQ ID No.193:CPKYCTQMPERYECFYC、

[0457] SEQ ID No.194:CPRYCTIFSERYECETC、

[0458] SEQ ID No.195:CPGYCTVFEMRYECAVC、

[0459] SEQ ID No.196:CPHYCTYFQERYECEYC、

[0460] SEQ ID No.197:CPGYCTYFNDRYECYTC、

[0461] SEQ ID No.198:CPPYCTYFSERYECYIC、

[0462] SEQ ID No.199:CPHYCTFFMERYECHIC、

[0463] SEQ ID No.200:CPDYCTIFMMRYECIEC、

[0464] SEQ ID No.228:GCPNYCTQFGERYECWICG、

[0465] SEQ ID No.229:GCPNYCTVFQERYECWICG、

[0466] SEQ ID No.230:GCPNYCTVFGQRYECWICG、

[0467] SEQ ID No.231:GCPNYCTVFGERYECWQCG、

[0468] SEQ ID No.232:GCPNYCTQFGERYECWQCG、

[0469] SEQ ID No.233:GCPNYCTVFGQRYECWQCG、

[0470] SEQ ID No.234:GCPNYCTVFQERYECWQCG、

[0471] SEQ ID No.235:GCPNYCTQFGQRYECWQCG、

[0472] SEQ ID No.236:GCPEYCTVFGERYECWICG、

[0473] SEQ ID No.237:GCPNYCQVFGERYECWICG、

[0474] SEQ ID No.238:GCPEYCQVFGERYECWICG、

[0475] SEQ ID No.239:GCPNYCTIFGERYECWICG、

[0476] SEQ ID No.240:GCPNYCTVFKERYECWICG、

[0477] SEQ ID No.241:GCPNYCTVFGERYECNICG、

[0478] SEQ ID No.242:GCPNYCQVFKERYECWICG、

[0479] SEQ ID No.243: GCPNYCQVFGERYECNICG,

[0480] SEQ ID No. 244: GCPNYCQVFKERYECNICG.

[0481] In some embodiments, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID No. 1 to SEQ ID No. 50.

[0482] In some embodiments, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID No. 51 to SEQ ID No. 100.

[0483] In some embodiments, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID No. 101 to SEQ ID No. 150.

[0484] In some embodiments, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID No. 151 to SEQ ID No. 200.

[0485] In some embodiments, the amino acid sequence of the polypeptide is as shown in any one of SEQ ID No. 1 to SEQ ID No. 50 and SEQ ID No. 228 to SEQ ID No. 244.

[0486] In some embodiments, the polypeptide differs from SEQ ID No. 1 by 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0487] In some embodiments, the polypeptide differs from SEQ ID No. 1 by 5, 4, 3, 2, or 1 amino acid.

[0488] In some embodiments, the polypeptide differs from SEQ ID No. 1 by 3, 2, or 1 amino acids.

[0489] In some embodiments, the polypeptide differs from SEQ ID No. 1 by one amino acid.

[0490] On the other hand, the present invention provides a polypeptide selected from the following polypeptides or combinations thereof:

[0491] (1) N-terminal and / or C-terminal truncated peptides of the polypeptides represented by general formula (I);

[0492] (2) Derivative peptides obtained by amino acid scanning mutation of the polypeptide shown in general formula (I);

[0493] (3) The polypeptide of formula (I), or the modified product of the polypeptide of formula (1) or (2).

[0494] In some embodiments, the truncated peptide is obtained by removing one or more amino acids from the N-terminus and / or C-terminus of a polypeptide.

[0495] In some embodiments, the truncated peptide is obtained by removing one amino acid from the N-terminus and / or C-terminus of a polypeptide.

[0496] In some embodiments, the polypeptide is an N-terminal and / or C-terminal truncated peptide of the polypeptide represented by general formula (I).

[0497] In some embodiments, the polypeptide is a truncated peptide obtained by removing one amino acid from the N-terminus and / or C-terminus of the polypeptide of general formula (I).

[0498] In some embodiments, the polypeptide is an N-terminal and / or C-terminal truncated peptide of any of the polypeptides shown in formulas (II) to (XIII).

[0499] In some embodiments, the polypeptide is an N-terminal and / or C-terminal truncated peptide of any of the polypeptides shown in formulas (II) to (XVIII).

[0500] In some embodiments, the derived peptide is selected from any of the following:

[0501] (a) The derived peptide is obtained by truncating one amino acid from the N-terminus of the above polypeptide;

[0502] (b) The derived peptide is obtained by truncating one amino acid from the C-terminus of the above polypeptide;

[0503] (c) The derived peptide is obtained by shortening one amino acid at the N-terminus and one amino acid at the C-terminus of the above polypeptide.

[0504] In some embodiments, the derived peptide is obtained by performing neutral amino acid scanning mutations or D-type amino acid substitution mutations on a polypeptide.

[0505] In some embodiments, the derived peptide is obtained by performing alanine scanning mutation and D-type amino acid substitution mutation on the polypeptide.

[0506] In some embodiments, the polypeptide is a derivative peptide obtained by scanning amino acid mutations of the polypeptide represented by general formula (I).

[0507] In some embodiments, the polypeptide is a derivative peptide obtained by scanning amino acid mutation of any of the polypeptides shown in formulas (II) to (XIII).

[0508] In some embodiments, the polypeptide is a derivative peptide obtained by scanning amino acid mutation of any of the polypeptides represented by formulas (II) to (XVIII).

[0509] In some embodiments, the polypeptide is a derivative peptide obtained by scanning amino acid mutation of any of the polypeptides shown in formulas (II) to (XIII).

[0510] In some embodiments, the polypeptide is a derivative peptide obtained by scanning amino acid mutation of any of the polypeptides represented by formulas (II) to (XVIII).

[0511] In some embodiments, the polypeptide is selected from the following polypeptides or combinations thereof:

[0512] (1) N-terminal and / or C-terminal truncated peptides of the polypeptides shown in SEQ ID No. 1 to SEQ ID No. 50, SEQ ID No. 228 to SEQ ID No. 244;

[0513] (2) Derivative peptides obtained by amino acid scanning mutation of the peptides shown in SEQ ID No.1~SEQ ID No.50 and SEQ ID No.228~SEQ ID No.244.

[0514] In some embodiments, the polypeptide is a truncated peptide obtained by removing one amino acid from the N-terminus and / or C-terminus of the polypeptide shown in SEQ ID No. 1, wherein the amino acid sequence of the polypeptide is shown in any one of SEQ ID No. 58, SEQ ID No. 108 or SEQ ID No. 158.

[0515] In some embodiments, the polypeptide is a truncated peptide obtained by removing one amino acid from the N-terminus and / or C-terminus of the polypeptide shown in SEQ ID No. 2 to 50, wherein the amino acid sequence of the polypeptide is shown in any one of SEQ ID No. 51 to 57, SEQ ID No. 59 to 100, SEQ ID No. 101 to 107, SEQ ID No. 109 to 150, SEQ ID No. 151 to 157 or SEQ ID No. 159 to 200.

[0516] In some embodiments, the polypeptide is selected from the following polypeptides or combinations thereof:

[0517] (1) N-terminal and / or C-terminal truncated peptides of the polypeptides shown in SEQ ID No. 1, SEQ ID No. 58, SEQ ID No. 108 or SEQ ID No. 158;

[0518] (2) Derivative peptides obtained by amino acid scanning mutation of the polypeptides shown in SEQ ID No.1, SEQ ID No.58, SEQ ID No.108 or SEQ ID No.158.

[0519] In some embodiments, the polypeptide is selected from the following polypeptides or combinations thereof:

[0520] (1) The N-terminal and / or C-terminal truncated peptide of the polypeptide shown in SEQ ID No. 1;

[0521] (2) Derivative peptides obtained by amino acid scanning mutation of the polypeptide shown in SEQ ID No.1.

[0522] In some embodiments, the polypeptide is obtained by scanning amino acid mutations on the N-terminus and / or C-terminus of the polypeptide shown in SEQ ID No. 1 or the polypeptide shown in SEQ ID No. 1.

[0523] In some embodiments, the polypeptide is obtained by performing neutral amino acid scanning mutations or D-type amino acid substitution mutations on the N-terminus and / or C-terminus truncated peptide of the polypeptide shown in SEQ ID No. 1 or the polypeptide shown in SEQ ID No. 1.

[0524] In some embodiments, the polypeptide is obtained by performing alanine scanning mutation or D-type amino acid substitution mutation on the N-terminus truncated peptide of the polypeptide shown in SEQ ID No. 1 or the truncated peptide of the polypeptide shown in SEQ ID No. 1.

[0525] In some embodiments, the polypeptide is obtained by performing an alanine scan mutation on the polypeptide shown in SEQ ID No. 1, and the amino acid sequence of the alanine scan-derived peptide obtained by performing an alanine scan mutation on the polypeptide shown in SEQ ID No. 1 is shown in any one of SEQ ID No. 201 to SEQ ID No. 215.

[0526] SEQ ID No.201: ACPNYCTVFGERYECWICG,

[0527] SEQ ID No.202: GCANYCTVFGERYECWICG,

[0528] SEQ ID No.203: GCPAYCTVFGERYECWICG,

[0529] SEQ ID No.204: GCPNACTVFGERYECWICG,

[0530] SEQ ID No.205: GCPNYCAVFGERYECWICG,

[0531] SEQ ID No.206: GCPNYCTAFGERYECWICG,

[0532] SEQ ID No.207: GCPNYCTVAGERYECWICG,

[0533] SEQ ID No.208: GCPNYCTVFAERYECWICG,

[0534] SEQ ID No.209: GCPNYCTVFGARYECWICG,

[0535] SEQ ID No.210: GCPNYCTVFGEAYECWICG,

[0536] SEQ ID No.211: GCPNYCTVFGERAECWICG,

[0537] SEQ ID No.212: GCPNYCTVFGERYACWICG,

[0538] SEQ ID No.213: GCPNYCTVFGERYECAICG,

[0539] SEQ ID No.214: GCPNYCTVFGERYECWACG,

[0540] SEQ ID No. 215: GCPNYCTVFGERYECWICA.

[0541] In some embodiments, the derived peptide is obtained by performing a D-type amino acid substitution mutation on the polypeptide shown in SEQ ID No. 1; the amino acid sequence of the D-type amino acid substitution derived peptide obtained by performing a D-type amino acid substitution mutation on the polypeptide shown in SEQ ID No. 1 is shown in any one of SEQ ID No. 216 to SEQ ID No. 227, SEQ ID No. 216: GC{D-Pro}NYCTVFGERYECWICG,

[0542] SEQ ID No.217:GCP{D-Asn}YCTVFGERYECWICG、

[0543] SEQ ID No.218: GCPN{D-Tyr}CTVFGERYECWICG,

[0544] SEQ ID No.219:GCPNYC{D-Thr}VFGERYECWICG、

[0545] SEQ ID No.220:GCPNYCT{D-Val}FGERYECWICG、

[0546] SEQ ID No.221:GCPNYCTV{D-Phe}GERYECWICG、

[0547] SEQ ID No.222: GCPNYCTVFG{D-Glu}RYECWICG,

[0548] SEQ ID No.223:GCPNYCTVFGE{D-Arg}YECWICG、

[0549] SEQ ID No.224:GCPNYCTVFGER{D-Tyr}ECWICG、

[0550] SEQ ID No.225: GCPNYCTVFGERY{D-Glu}CWICG,

[0551] SEQ ID No.226: GCPNYCTVFGERYEC{D-Trp}ICG,

[0552] SEQ ID No. 227: GCPNYCTVFGERYECW{D-Ile}CG.

[0553] In some embodiments, the derived peptides of the present invention include polypeptide sequences resulting from a combination of the above-described modification strategies. Examples include analogs obtained by simultaneously employing both arginine scanning mutation and D-type amino acid substitution mutation strategies; analogs obtained by simultaneously employing both alanine scanning mutation and D-type amino acid substitution mutation strategies, etc.

[0554] In some embodiments, the modifications to the modified product include, but are not limited to, N-terminal modification, C-terminal modification, side chain modification, and amino acid modification.

[0555] In some embodiments, the modification is selected from the following forms or combinations thereof:

[0556] Modification of N-terminal or side-chain amino acids: acetylation, formylation, trifluoroacetylation, benzoylation, 2-aminobenzoylation;

[0557] C-terminal modifications: amidation, esterification, aldehydeation, alcoholation;

[0558] Alkylation modifications: N-methylation, side-chain methylation, N-ethylation, N-phenylpropylation, N-allylation, etc.

[0559] In some embodiments, the modification is selected from the following forms or combinations thereof:

[0560] Modification of N-terminal or side-chain amino acids: acetylation, formylation;

[0561] C-terminal modification: amidation;

[0562] Alkylation modifications: N-methylation, side-chain methylation, N-ethylation.

[0563] In some embodiments, the polypeptide of the present invention is a polymer, which is a dimer, trimer, or tetramer. A dimer, trimer, tetramer, or polymer refers to two, three, four, or more monomers forming one polypeptide molecule. The dimer, trimer, tetramer, or polymer can be a homodimer, homotrimer, homotetramer, or homopolymer, wherein each monomer forming the dimer, trimer, tetramer, or polymer contains the same amino acid sequence. The dimer, trimer, tetramer, or polymer can be a heterodimer, heterotrimer, heterotetramer, or heteropolymer, wherein each monomer forming the dimer, trimer, tetramer, or polymer contains a different amino acid sequence.

[0564] In some embodiments, the polypeptide of the present invention contains four cysteine ​​residues, which may or may not form intramolecular disulfide bonds.

[0565] In some embodiments, the polypeptide of the present invention has a linear spatial structure and does not form intramolecular disulfide bonds.

[0566] In some embodiments, the polypeptide of the present invention has a cyclic peptide spatial structure containing a pair of disulfide bonds formed from the first and second cysteine ​​residues at the N-terminus; or from the first and third cysteine ​​residues at the N-terminus; or from the first and fourth cysteine ​​residues at the N-terminus; or from the second and third cysteine ​​residues at the N-terminus; or from the second and fourth cysteine ​​residues at the N-terminus; or from the third and fourth cysteine ​​residues at the N-terminus.

[0567] In some embodiments, the polypeptide of the present invention has a cyclic peptide spatial structure containing two pairs of disulfide bonds, with disulfide bonds formed from the first and second cysteine ​​residues at the N-terminus, and from the third and fourth cysteine ​​residues; or with disulfide bonds formed from the first and fourth cysteine ​​residues at the N-terminus, and from the second and third cysteine ​​residues; or with disulfide bonds formed from the first and third cysteine ​​residues at the N-terminus, and from the second and fourth cysteine ​​residues.

[0568] In some embodiments, the polypeptide of the present invention has a cyclic peptide spatial structure containing two pairs of disulfide bonds, with the first and fourth cysteine ​​residues at the N-terminus forming a disulfide bond, and the second and third cysteine ​​residues forming a disulfide bond.

[0569] In some embodiments, the polypeptide spatial structures shown in SEQ ID No. 1 to SEQ ID No. 244 of the present invention are cyclic peptides containing two pairs of disulfide bonds, with disulfide bonds formed from the first and fourth cysteine ​​residues at the N-terminus, and from the second and third cysteine ​​residues. SEQ ID No. 1 to SEQ ID No. 244 containing the two pairs of disulfide bonds are respectively named polypeptide 1 to polypeptide 244. For example, the structure of polypeptide 1 is:

[0570] In some embodiments, the polypeptides shown in SEQ ID No. 1 to SEQ ID No. 244 of the present invention have a cyclic peptide spatial structure containing two pairs of disulfide bonds, with disulfide bonds formed from the first and fourth cysteine ​​residues at the N-terminus, and from the second and third cysteine ​​residues. SEQ ID No. 1 to SEQ ID No. 244 containing these two pairs of disulfide bonds are respectively named polypeptide 1 to polypeptide 244. N-terminal acetylation, C-terminal amidation, and methylation of the side chain are respectively named -1, -2, and -3, as follows:

[0571] On the other hand, the present invention provides a peptide drug conjugate comprising the above-mentioned peptide, and the general structural formula (1) of the peptide drug conjugate is shown below: Peptide-Linker-Payload (1)

[0572] in,

[0573] The linker is absent or is a linking group;

[0574] Payload is a payload group, which includes cytotoxic drugs, radionuclide complex groups, or fluorescent groups.

[0575] Peptide is a polypeptide, and the polypeptide can be any of the above-mentioned polypeptides.

[0576] In some embodiments, the Payload in the general formula (1) of the polypeptide drug conjugate is a fluorescent group, which includes, but is not limited to, infrared fluorescent dyes, compounds containing organic chromophores, compounds containing organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, and bioluminescent molecules.

[0577] In some embodiments, the fluorescent groups include, but are not limited to, near-infrared fluorescent dyes MPA, IRDye800CW, Cy7, Cy7.5, Cy3, Cy5, Cy5.5, ICG, FIGT, FAM, MCA, TAMRA, Biotin, HEX, AMC, and Rhodamine B.

[0578] In some embodiments, the fluorescent group is selected from one or more of Cy5, Cy5.5, ICG, FIGT, FAM, TAMRA, and Biotin.

[0579] In some embodiments, the fluorescent group is selected from one or more of Cy5, Cy5.5, FITC, and TAMRA.

[0580] In some embodiments, the payload is a radionuclide complexing group, which includes a radionuclide and a bifunctional chelating agent for radionuclide labeling; the bifunctional chelating agent for radionuclide labeling includes, but is not limited to, NOTA, DOTA, DOTAM, DOTAGA, NODAGA, DTPA, CHX-DTPA, HYNIC, DFO, p-SCN-Bn-DFO, NODAGA, NO2A, DO3A, and MAG3; the radionuclide includes, but is not limited to, […]. 18 F, 125 I, 131 I, 64 Cu、 67 Ga、 68 Ga、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 153 Sm、 177 Lu、 186 Re、 188 Re、 211 At、 212 Pb, 223 Ra、 225 Ac.

[0581] In some embodiments, the bifunctional chelating agent for radionuclide labeling is selected from NOTA, DOTA, DOTAGA, or NODAGA.

[0582] In some embodiments, the radionuclide is selected from... 18 F, 64 Cu、 68 Ga、 99m Tc, 177Lu or 225 Ac.

[0583] In some embodiments, the payload is a cytotoxic drug selected from: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0584] In some embodiments, the Payload is selected from auristatins and their derivatives (such as MMAE, MMAF, MMAD), maytansin and its derivatives (such as DM1, DM2, DM3, DM4), tubulysins, cryptomycin, spindle kinesin, gemcitabine, pyrrolo[2,1-c][1,4]benzodiazepines, ducamycin, camptothecin and its derivatives (such as SN38, eczetidine, Dxd, topotecan, irinotecan), cazithromycin, amatoxins, paclitaxel, taxane, vincristine, vinblastine, etoposide, doxorubicin, cyclophosphamide, docetaxel, methotrexate, cisplatin, cytarabine, phenylalanine mustard, and chlorambucil, or combinations thereof.

[0585] In some embodiments, the payload is selected from auristatins and their derivatives (such as MMAE, MMAF, MMAD), maytansin and its derivatives (such as DM1, DM2, DM3, DM4), tubulysins, gemcitabine, camptothecin and its derivatives (such as SN38, Exatecan, Dxd, topotecan, irinotecan), paclitaxel, and taxanes or combinations thereof.

[0586] In some embodiments, the Payload is selected from auristatins and their derivatives (such as MMAE, MMAF, MMAD), maytansin and its derivatives (such as DM1, DM2, DM3, DM4), camptothecin and its derivatives (such as SN38, Exatecan, Dxd, topotecan, irinotecan) or combinations thereof.

[0587] In some implementations, the payload is selected from MMAE, MMAF, DM1, DM4, Exatecan, and Dxd, or combinations thereof; that is, the payload is selected from the following structures or combinations thereof.

[0588] In some embodiments, the Linker is absent in the general formula (1) of the peptide drug conjugate, the Payload is directly linked to the Peptide, the structure of the peptide drug conjugate is Peptide-Payload, the Payload is the effective payload group, and the effective payload group includes cytotoxic drugs, radionuclide complexing groups or fluorescent groups.

[0589] In some embodiments, the Linker in the general structural formula (1) of the polypeptide drug conjugate is a linking group. The Linker includes a non-cleavable linker or a cleavable linker. The non-cleavable linker is selected from PEG linkers, linkers with thioether groups, linkers with oxime groups, or combinations thereof. The cleavable linker is selected from linkers with disulfide bonds, dipeptide linkers, tripeptide linkers, tetrapeptide linkers, peptide-like linkers, β-glucuronidase-cleavable linkers, β-galactosidase-cleavable linkers, phosphatase-cleavable linkers, pH-sensitive linkers, sulfatase-cleavable linkers, or combinations thereof.

[0590] In some embodiments, the flexible polypeptide linker is composed of simple small amino acids, such as glycine (Gly), serine (Ser), or alanine (Ala), and the flexible linker includes, but is not limited to, Gly. n (Glu) n (γGlu) n (GS) n 、(GGGS)n.

[0591] In some embodiments, the rigid polypeptide linker is a linker containing proline (Pro) or other cyclic amino acids, including but not limited to (AP)n, (A{D-Pro})n, (PPP)n, (EAAAK)n, and (GP). n (Gp) n (2-Nal-Y1)n, (PX) n Y1 is selected from none or G; X is selected from any amino acid; m is an integer from 0 to 24; n is an integer from 1 to 10.

[0592] In some embodiments, the dipeptide linker is selected from valine-citrulline (Val-Cit) dipeptide linkers, phenylalanine-lysine (Phe-Lys) dipeptide linkers, and valine-alanine (Val-Ala) dipeptide linkers.

[0593] In some embodiments, the tripeptide linker is selected from glutamic acid-valine-citrulline (Glu-Val-Cit) tripeptide linker, alanine-valine-citrulline (Ala-Val-Cit) tripeptide linker, methionine-valine-lysine (Met-Val-Lys) tripeptide linker, and glycine-phenylalanine-lysine (Gly-Phe-Lys) tripeptide linker.

[0594] In some embodiments, the tetrapeptide linker is selected from the glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) tetrapeptide linker and the aspartic acid-glutamic acid-valine-aspartic acid (Asp-Glu-Val-Asp) tetrapeptide linker.

[0595] In some embodiments, the linker in the general structural formula (1) of the peptide drug conjugate is a linking group, said linker being connected to the N-terminus and / or C-terminus of the peptide. The linker comprises the following structures or combinations thereof: (Gly) n (Glu) n (γGlu) n (GS) n GGSG (D-Gly) n (D-Glu) n (Gln) n (D-Gln) n , (GP)n, (Gp)n, (pGp)n, (AP)n, (2-Nal-Y1)n, (GGGS) n ,

[0596] R is selected from -OH, -NH2, -NH-Glu, -NH-Gln, methylamino, ethylamino, propanamino, butylamino, methoxy, ethoxy, propoxy, and butoxy; m is an integer from 0 to 24; and n is an integer from 1 to 10.

[0597] Preferably, m is an integer from 0 to 10;

[0598] More preferably, m is 2, 4 or 6.

[0599] Preferably, n is 3, 4, 5 or 6.

[0600] In some embodiments, the Linker structure further includes a PABC spacer base, wherein the PABC spacer base structure is as follows:

[0601] In some embodiments, the connector substructure includes a 4-AMC spacer base, the 4-AMC spacer base structure being...

[0602] In some embodiments, the Linker structure further includes an AMBA spacer base, wherein the AMBA spacer base structure is as follows:

[0603] In some embodiments, the Linker structure also includes a β-Ala spacer base.

[0604] In some implementations, the Linker structure also includes [Sar]. n The interval basis, n, is selected from integers from 1 to 10.

[0605] In some embodiments, the Linker structure further includes a β-Ala-[Sar]n spacer group, where n is an integer selected from 1 to 10, and the carboxyl group in [Sar]n is linked to the Peptide moiety via an amide bond.

[0606] In some implementations, n is selected from 4 or 6.

[0607] In some implementations, the Linker includes the following structures or combinations thereof. m is an integer between 0 and 24.

[0608] In some implementations, the Linker includes the following structures or combinations thereof. m is an integer between 0 and 24.

[0609] In some implementations, the Linker comprises the following structures or combinations thereof, (Glu) n (γGlu) n , m is an integer between 0 and 24.

[0610] In some embodiments, the connector comprises the following structure or a combination thereof: (D-Glu) n AEEA, (Gln) n (Gp)n

[0611] In some embodiments, the Linker structure also includes a β-Ala spacer base.

[0612] In some implementations, the Linker structure also includes [Sar]. n The interval basis, n, is selected from integers from 1 to 10.

[0613] In some embodiments, the Linker structure further includes a β-Ala-[Sar]n spacer group, where n is an integer selected from 1 to 10, and the carboxyl group in [Sar]n is linked to the Peptide moiety via an amide bond.

[0614] In some implementations, n is selected from 4 or 6.

[0615] In some implementations, the Linker comprises the following structures or combinations thereof: G{D-Pro}, Gln2, AEEA, 2-Nal,

[0616] In some embodiments, the structure of the polypeptide drug conjugate is shown as any one of GPC3-1 to GPC3-85 in Table 1.

[0617] Table 1. Structure of the polypeptide drug conjugate of the present invention

[0618] On the other hand, the present invention provides a nucleotide that encodes a polypeptide targeting GPC3.

[0619] In some embodiments, the nucleotide encodes a polypeptide targeting GPC3 as shown in at least one of SEQ ID No. 1 to SEQ ID No. 244.

[0620] On the other hand, the present invention provides a composition comprising the polypeptide or polypeptide drug conjugate described in the present invention.

[0621] On the other hand, the present invention provides the use of the polypeptides, polypeptide drug conjugates, nucleotides or compositions described herein in the preparation of tumor PET imaging agents, tumor SPECT imaging agents, or in the preparation of tumor peptide targeted therapy drugs.

[0622] In some embodiments, the tumor peptide-targeted therapeutic agent is used for tumor treatment.

[0623] In some embodiments, the tumor includes a tumor that expresses GPC3 positively.

[0624] In some embodiments, the GPC3-positive tumors include, but are not limited to, liver cancer, ovarian cancer, lung cancer, melanoma, gastric cancer, thyroid cancer, colon cancer, pancreatic cancer, bladder cancer, and myeloma.

[0625] In some embodiments, the GPC3-positive tumor is liver cancer.

[0626] In some embodiments, the GPC3-positive tumor is HCC.

[0627] Terminology Explanation

[0628] Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0629] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0630] In the description of this specification, natural amino acids refer to 20 common naturally occurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V).

[0631] In the description of this specification, examples of non-natural amino acids include, but are not limited to: hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azacyclobutanecarboxylic acid, 2-aminohexanoic acid, 3-aminohexanoic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid, desmodium, 2,2-diaminopimelic acid. Acids, 2,3-diaminopropionic acid, N-ethylglycine, N-methylglycine, N-ethylasparagine, homoproline, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesin, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, valine, leucine, ornithine, para-aminophenylalanine, pentylglycine, piperidine acid, and thioproline.

[0632] The term "amino acid scan" refers to a site-directed mutagenesis technique used to determine the contribution of a specific wild-type residue to the stability or function (e.g., binding affinity) of a given protein or peptide. This technique involves replacing a wild-type residue in a peptide with a specific amino acid, such as alanine, and then assessing the stability or function (e.g., binding affinity) of the alanine-substituted derivative or mutant peptide and comparing it to the wild-type peptide. Techniques for replacing wild-type residues in peptides with alanine are known in the art.

[0633] The term "derivative" refers to a variation of the parent molecule. For example, a derivative of a polypeptide can include a variation in which one or more amino acids are substituted relative to the polypeptide, preferably a conserved variant sequence. It can also include modifications to the polypeptide, including, but not limited to, non-naturally occurring amino acids, D-type amino acids, amino acids with N- or C-terminal (N- or C-terminal) modifications, particularly modifications to the N-terminal amino and / or C-terminal carboxyl groups, fatty acid modifications, peptide mimics, and pseudopeptides, etc. For example, a derivative of a cytotoxic drug can include compounds with the same or similar biological activities (e.g., inhibition of topoisomerase I, antitumor activity) obtained through chemical modification based on the parent nucleus structure.

[0634] The term "radionoid," also known as an unstable nuclide, is used in contrast to stable nuclides. It refers to an unstable atomic nucleus that spontaneously emits radiation (such as alpha rays, beta rays, etc.) and decays to form a stable nuclide. "Radionoid" includes, but is not limited to, diagnostic radionuclides and therapeutic radionuclides, such as... 68 Ga、 64 Cu、 18 F, 86 Y、90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I, 124 Class I, the therapeutic radionuclide such as 177 Lu、 125 I, 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bi、 212 Pb, etc.

[0635] The term "Linker" refers to a linking group or bond that is connected to a polypeptide at one end and to a payload at the other end. When the linker is a linking group, it may contain one or more links selected from non-cleavable linkers or cleavable linkers. Non-cleavable linkers are selected from PEG linkers, linkers with thioether groups, linkers with oxime groups, or combinations thereof. Cleavable linkers are selected from linkers with disulfide bonds, dipeptide linkers, tripeptide linkers, tetrapeptide linkers, peptide-like linkers, linkers cleaved by β-glucuronidase, linkers cleaved by β-galactosidase, linkers based on phosphatase cleavage, pH-sensitive linkers, and linkers cleaved by sulfatase.

[0636] The term "payload" refers to the active portion of the polypeptide of the present invention, which is linked to the polypeptide of the present invention via a linker, that is, linked to the polypeptide of the present invention via a linker bond or a linker group. In some embodiments, the payload group is a cytotoxic drug, a radionuclide complex group, or a fluorescent group.

[0637] The term "peptide drug conjugate" refers to a structure / molecule obtained by linking a peptide to a payload via a linker bond or linker group.

[0638] The term "phage display" refers to the genetic engineering technique of inserting a foreign gene encoding a polypeptide or protein into an appropriate position on the structural gene of a bacteriophage capsid protein. This causes the foreign polypeptide or protein to form a fusion protein on the phage capsid protein, which is then presented on the phage surface during the reassembly of progeny phages, thus forming a phage display library. Phage display technology can maintain the relative spatial structure and biological activity of the foreign polypeptide or protein. Then, using a target molecule and appropriate washing methods, non-specifically bound phages can be washed away, and phages bound to the target molecule can be eluted with acids, bases, or competing molecules. The neutralized phages are then used to infect *E. coli* for amplification. After 3-5 rounds of screening and enrichment, the polypeptide or protein that recognizes the target molecule can finally be obtained. Phage display technology is an operating system for expressing foreign genes, with advantages such as simplicity, effectiveness, and ease of control. It can achieve phenotypic and genotypic uniformity and has broad application prospects. Attached Figure Description

[0639] Figure 1 shows the liquid phase detection results of peptide 1;

[0640] Figure 2 shows the LC-MS detection results of peptide 1;

[0641] Figure 3 shows the liquid phase detection results of GPC3-1;

[0642] Figure 4 shows the LC-MS detection results of GPC3-1;

[0643] Figure 5 shows the liquid phase detection results of GPC3-2;

[0644] Figure 6 shows the LC-MS detection results of GPC3-2;

[0645] Figure 7 shows the liquid phase detection results of GPC3-3;

[0646] Figure 8 shows the LC-MS detection results of GPC3-3;

[0647] Figure 9 shows the liquid phase detection results of GPC3-4;

[0648] Figure 10 shows the LC-MS detection results of GPC3-4;

[0649] Figure 11 shows the liquid phase detection results of GPC3-5;

[0650] Figure 12 shows the LC-MS detection results of GPC3-5;

[0651] Figure 13 shows the liquid phase detection results of GPC3-74;

[0652] Figure 14 shows the LC-MS detection results of GPC3-74;

[0653] Figure 15 shows the liquid phase detection results of GPC3-77;

[0654] Figure 16 shows the LC-MS detection results of GPC3-77;

[0655] Figure 17 shows the SPR affinity test results of peptide 1 and GPC3;

[0656] Figure 18 shows the affinity test results of GPC3-1 and GPC3 SPR.

[0657] Figure 19 shows the affinity test results of GPC3-4 and GPC3 SPR.

[0658] Figure 20 shows the affinity test results of GPC3-5 and GPC3 SPR.

[0659] Figure 21 shows the results of the GPC3-2 cell surface binding test. Figure 21(a) is a confocal image of HEK293 cells at 4℃ for 1 hour of surface binding, and Figure 21(b) and Figure 21(c) are confocal images of HEK293 / GPC3 cells at 4℃ for 1 hour of surface binding.

[0660] Figure 22 shows the results of the GPC3-3 cell surface binding test. Figure 22(a) is a confocal image of HEK293 cells at 4℃ for 1 hour of surface binding, and Figures 22(b) and 22(c) are confocal images of HEK293 / GPC3 cells at 4℃ for 1 hour of surface binding.

[0661] Figure 23 shows the results of the GPC3-2 endocytosis test. Figure 23(a) is a confocal image of HEK293 cells endocytosis at 37°C for 2 hours, and Figures 23(b) and 23(c) are confocal images of HEK293 / GPC3 cells endocytosis at 37°C for 2 hours.

[0662] Figure 24 shows the results of the GPC3-3 endocytosis test. Figure 24(a) is a confocal image of HEK293 cells endocytosis at 37°C for 2 hours, and Figures 24(b) and 24(c) are confocal images of HEK293 / GPC3 cells endocytosis at 37°C for 2 hours.

[0663] Figure 25 shows the selectivity results of GPC3 and GPC6 detected by the ELISA method;

[0664] Figure 26 shows the results of the plasma metabolic stability test;

[0665] Figure 27 is 68 In vivo distribution of Ga-GPC3-4 in the Huh7 model;

[0666] Figure 28 is 68 In vivo distribution results of Ga-GPC3-5 in the Huh7 model. Detailed Implementation

[0667] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0668] Example 1: Screening of peptides

[0669] 1. Phage library screening to obtain peptides with high affinity for the GPC3 target.

[0670] The selected protein was hGPC3 (Bepsys GP3-H82E5). Using the target protein as the stationary phase and a phage display library (self-made) as the mobile phase, after a period of co-incubation, unbound free phages were washed away. Then, phages bound to the target molecule were eluted using a competitive receptor or acid. The eluted phages infected host cells, multiplied, and were then eluted again. After four rounds of "adsorption-elution-amplification," a peptide with high affinity for the target protein was obtained.

[0671] 2. Phage ELISA identification

[0672] 2.1. Coating: Dilute SA to 5 μg / mL with 50 mM sodium bicarbonate at pH 8.5, and coat 50 μL at 37℃ for 2 h;

[0673] 2.2. Blocking: Block with TBST + 20 mg / mL BSA blocking solution overnight at 4°C;

[0674] 2.3. 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;

[0675] 2.4. Binding of target protein: 200 ng of GPC3 target protein was incubated with an SA-coated ELISA plate at 37°C for 1 hour to bind;

[0676] 2.5. 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;

[0677] 2.6. Phage binding: 10 9 / 10 10The phage supernatant of PFU was bound to the GPC3 ELISA plate at 37°C for 1 hour;

[0678] 2.7. 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;

[0679] 2.8. Binding antibody: M13 phage Antibody was diluted to 0.1ug / mL, and 100μL was used to bind the phage in the ELISA plate at 37℃ for 1h;

[0680] 2.9. 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;

[0681] 2.10. 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;

[0682] 2.11. Termination: Add 100 μL of 2M H2SO4;

[0683] 2.12. Scan: ELISA reader OD 450 reading.

[0684] 2.13. Select samples with better signals from the above samples for sequencing to obtain polypeptide sequences.

[0685] 3. Experimental Results

[0686] As the number of phage library screening rounds increased, the affinity of the eluted peptides for GPC3 gradually increased. A series of peptides with good affinity for GPC3 were obtained from the phage library screening. Further ELISA experiments were conducted to verify their binding ability to GPC3. BSA was used as a blank control. The experimental groups showed significant differences. The peptides with better verification results were selected for sequencing to obtain the amino acid sequences of peptides 1 to 50. The detection results of peptides 1 to 50 are shown in Table 2.

[0687] Table 2. Identification results of the polypeptides of this invention by ELISA

[0688] Example 2 Synthesis of polypeptides and polypeptide drug conjugates

[0689] The peptides and peptide-drug conjugates provided in this study employ a solid-phase synthesis method to synthesize their linear precursors, which are then oxidized with DMSO to form two pairs of intramolecular disulfide bonds. The synthetic support is Fmoc-Gly-Wang Resin or Rink Amide resin. During the synthesis process, the Fmoc-Gly-Wang Resin or Rink Amide 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 peptide chain length. Finally, the peptide 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 peptide. 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.

[0690] Experimental reagents

[0691] (1) Synthesis of polypeptide 1

[0692] Step 1: Couple the first amino acid Fmoc-Gly-OH

[0693] 90 mg (0.1 mmol) of Wang resin was fully swollen in DCM for 1 h. An amino acid solution was prepared by dissolving 0.25 mmol of Fmoc-Gly-OH and 0.25 mmol of 1-hydroxybenzotriazole (HOBT) in 5 mL of DCM, and adding N,N'-diisopropylcarbodiimide (DIC, 0.25 mmol). After the resin swelled, the DCM was drained, the prepared amino acid solution was added, and 0.01 mmol of 4-dimethylaminopyridine (DMAP) was added. The mixture was shaken at room temperature for 15 h. After the reaction was complete, the resin was washed 5 times with DCM, and then blocked with blocking buffer (10 mL) DCM:methanol:DIEA (85:10:5, v:v:v) for 10 min at room temperature. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0694] Step 2: Synthesis of linear precursor peptide chains

[0695] The linear precursor peptide chain of peptide 1: GCPNYCTVFGERYECWICG.

[0696] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the straight-chain precursor sequence from the second C at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0697] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0698] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0699] 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;

[0700] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0701] 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.

[0702] Step 3: Cleavage of the linear precursor peptide chain

[0703] 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 2, 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.

[0704] Step 4: Intramolecular disulfide bond formation

[0705] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0706] Step 5: Preparation of Peptides

[0707] 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-C18 (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 25-40% 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.

[0708] Step 6: Detection and Characterization Methods

[0709] The purity of the polypeptide obtained in step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the formation of intramolecular disulfide bonds in the compound was also determined. The results of liquid chromatography and LC-MS detection are shown in Figure 1 and Figure 2.

[0710] (2) Synthesis of GPC3-1

[0711] Step 1: Couple the first amino acid Fmoc-Lys(Mtt)-OH

[0712] 90 mg (0.1 mmol) of Wang resin was fully swollen in DCM for 1 h. An amino acid solution was prepared by dissolving 0.25 mmol of Fmoc-Lys(Mtt)-OH and 0.25 mmol of 1-hydroxybenzotriazole (HOBT, 0.25 mmol) in 5 mL of DCM, and adding N,N'-diisopropylcarbodiimide (DIC, 0.25 mmol). After the resin swelled, the DCM was drained, the prepared amino acid solution was added, and 0.01 mmol of 4-dimethylaminopyridine (DMAP, 0.01 mmol) was added. The mixture was shaken at room temperature for 15 h. After the reaction was complete, the resin was washed 5 times with DCM, and then blocked with blocking buffer (10 mL) DCM:methanol:DIEA (85:10:5, v:v:v) for 10 min at room temperature. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0713] Step 2: Synthesis of linear precursor peptide chains

[0714] GPC3-1 linear precursor peptide chain:

[0715] {Boc-Gly-OH}-CPNYCTVFGERYECWICG-PEG6-K(Mtt).

[0716] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the linear precursor sequence from the second carboxyl-terminal PEG6 to the amino-terminal. Each coupling cycle was performed as follows:

[0717] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0718] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0719] 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;

[0720] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0721] After the linear polypeptide was synthesized, the resin was rinsed 5 times with DMF and 5 times with DCM.

[0722] Step 3: C-terminal lysine side chain coupling with biotin

[0723] Removal of the Mtt protecting group from the lysine side chain: After swelling the resin with DCM for 1 hour, add a mixed solution of hexafluoroisopropanol / dichloromethane (30% v / v, 10 mL) to the resin, shake and react at room temperature for 45 minutes, then remove the Mtt protecting group. Repeat the operation once. After the reaction is complete, wash the resin with DCM 5 times and DMF 6 times.

[0724] Lysine side chain coupling with Biotin: Weigh 0.5 mmol Biotin, 0.5 mmol HCTU and 1 mmol NMM and dissolve them in 8 mL DMF. Then add the mixed solution to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution, wash with DMF until the discharged liquid is colorless, wash with DCM, and dry the resin under vacuum.

[0725] Step 4: Cleavage of the linear precursor peptide chain

[0726] 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 3, 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 washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0727] Step 5: Intramolecular disulfide bond formation

[0728] The crude product obtained in step 4 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0729] Step 6: Preparation of Peptides

[0730] 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-C18 (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 25-40% 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.

[0731] Step 7: Detection and Characterization Methods

[0732] The purity of the polypeptide obtained in step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the C-terminal lysine side chain of the compound was coupled with biotin and intramolecular disulfide bond was formed. The liquid chromatography and LC-MS detection results are shown in Figures 3 and 4.

[0733] (3) Synthesis of GPC3-2

[0734] Step 1: Couple the first amino acid Fmoc-Lys(Mtt)-OH

[0735] 90 mg (0.1 mmol) of Wang resin was fully swollen in DCM for 1 h. An amino acid solution was prepared by dissolving 0.25 mmol of Fmoc-Lys(Mtt)-OH and 0.25 mmol of 1-hydroxybenzotriazole (HOBT, 0.25 mmol) in 5 mL of DCM, and adding N,N'-diisopropylcarbodiimide (DIC, 0.25 mmol). After the resin swelled, the DCM was drained, the prepared amino acid solution was added, and 0.01 mmol of 4-dimethylaminopyridine (DMAP, 0.01 mmol) was added. The mixture was shaken at room temperature for 15 h. After the reaction was complete, the resin was washed 5 times with DCM, and then blocked with blocking buffer (10 mL) DCM:methanol:DIEA (85:10:5, v:v:v) for 10 min at room temperature. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0736] Step 2: Synthesis of linear precursor peptide chains

[0737] The linear precursor peptide chain of GPC3-2 is: {Boc-Gly-OH}-CPNYCTVFGERYECWICG-PEG6-K(Mtt).

[0738] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the linear precursor sequence from the second carboxyl-terminal PEG6 to the amino-terminal. Each coupling cycle was performed as follows:

[0739] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0740] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0741] 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;

[0742] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0743] After the linear polypeptide was synthesized, the resin was rinsed 5 times with DMF and 5 times with DCM.

[0744] Step 3: C-terminal lysine side chain coupling with 5-TAMRA

[0745] Removal of the Mtt protecting group from the lysine side chain: After swelling the resin with DCM for 1 hour, add a mixed solution of hexafluoroisopropanol / dichloromethane (30% v / v, 10 mL) to the resin, shake and react at room temperature for 45 minutes, then remove the Mtt protecting group. Repeat the operation once. After the reaction is complete, wash the resin with DCM 5 times and DMF 6 times.

[0746] Lysine side chain coupling with 5-TAMRA: Weigh 0.2 mmol 5-TAMRA, 0.5 mmol HCTU and 1 mmol NMM and dissolve them in 8 mL DMF. Then add the mixed solution to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution, wash with DMF until the drained liquid is colorless, wash with DCM, and dry the resin under vacuum.

[0747] Step 4: Cleavage of the linear precursor peptide chain

[0748] 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 3, 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 washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0749] Step 5: Intramolecular disulfide bond formation

[0750] The crude product obtained in step 4 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0751] Step 6: Preparation of Peptides

[0752] 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-C18 (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 25-45% 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.

[0753] Step 7: Detection and Characterization Methods

[0754] The purity of the polypeptide obtained in step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry. The compound was coupled with 5-TAMRA to the C-terminal lysine side chain and formed an intramolecular disulfide bond. The liquid chromatography and LC-MS detection results are shown in Figures 5 and 6.

[0755] (4) Synthesis of GPC3-3

[0756] Step 1: Synthesis of linear precursor peptide chains

[0757] The linear precursor peptide of GPC3-3: PEG6-GCPNYCTVFGERYECWICG.

[0758] 147 mg (0.1 mmol) of RinkAmide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0759] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0760] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0761] 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;

[0762] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0763] After the linear polypeptide was synthesized, the resin was rinsed five times with DMF.

[0764] Step 2: N-terminal coupling of 5-TAMRA

[0765] Weigh 0.2 mmol 5-TAMRA, 0.5 mmol HCTU, and 1 mmol NMM and dissolve them in 8 mL DMF. Then add the mixed solution to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution, wash with DMF until the discharged liquid is colorless, wash with DCM, and dry the resin under vacuum.

[0766] Step 3: Cleavage of the linear precursor peptide chain

[0767] 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 2, 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.

[0768] Step 4: Intramolecular disulfide bond formation

[0769] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0770] Step 5: Preparation of Peptides

[0771] 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-C18 (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 30-50% 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.

[0772] Step 6: Detection and Characterization Methods

[0773] The purity of the polypeptide obtained in step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the N-terminal coupling of the compound with 5-TAMRA and the formation of intramolecular disulfide bonds were completed. The liquid chromatography and LC-MS detection results are shown in Figures 7 and 8.

[0774] (5) Synthesis of GPC3-4

[0775] Step 1: Couple the first amino acid Fmoc-Lys(Mtt)-OH

[0776] 90 mg (0.1 mmol) of Wang resin was fully swollen in DCM for 1 h. An amino acid solution was prepared by dissolving 0.25 mmol of Fmoc-Lys(Mtt)-OH and 0.25 mmol of 1-hydroxybenzotriazole (HOBT, 0.25 mmol) in 5 mL of DCM, and adding N,N'-diisopropylcarbodiimide (DIC, 0.25 mmol). After the resin swelled, the DCM was drained, the prepared amino acid solution was added, and 0.01 mmol of 4-dimethylaminopyridine (DMAP, 0.01 mmol) was added. The mixture was shaken at room temperature for 15 h. After the reaction was complete, the resin was washed 5 times with DCM, and then blocked with blocking buffer (10 mL) DCM:methanol:DIEA (85:10:5, v:v:v) for 10 min at room temperature. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0777] Step 2: Synthesis of linear precursor peptide chains

[0778] The linear precursor peptide chain of GPC3-4 is: {Boc-Gly-OH}-CPNYCTVFGERYECWICG-PEG6-K(Mtt)

[0779] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the linear precursor sequence from the second carboxyl-terminal PEG6 to the amino-terminal. Each coupling cycle was performed as follows:

[0780] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0781] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0782] 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;

[0783] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0784] After the linear polypeptide was synthesized, the resin was rinsed 5 times with DMF and 5 times with DCM.

[0785] Step 3: C-terminal lysine side chain coupling with DOTA

[0786] Removal of the Mtt protecting group from the lysine side chain: After swelling the resin with DCM for 1 hour, add a mixed solution of hexafluoroisopropanol / dichloromethane (30% v / v, 10 mL) to the resin, shake and react at room temperature for 45 minutes, then remove the Mtt protecting group. Repeat the operation once. After the reaction is complete, wash the resin with DCM 5 times and DMF 6 times.

[0787] Lysine side-chain coupling with DOTA: Weigh 0.5 mmol of tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 0.5 mmol of ethyl 2-oxime cyanoacetate, dissolve them in 8 mL of DMF, then add 80 μL of DIC for pre-activation for 3 min. Add the mixed solution to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution, wash 4-5 times with DMF, wash 4-5 times with DCM, and dry the resin under vacuum.

[0788] Step 4: Cleavage of the linear precursor peptide chain

[0789] 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 3, 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 washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0790] Step 5: Intramolecular disulfide bond formation

[0791] The crude product obtained in step 4 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0792] Step 6: Preparation of Peptides

[0793] 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-C18 (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 20-40% 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.

[0794] Step 7: Detection and Characterization Methods

[0795] The purity of the polypeptide obtained in step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the C-terminal lysine side chain of the compound was coupled with DOTA and intramolecular disulfide bond was formed. The liquid chromatography and LC-MS detection results are shown in Figures 9 and 10.

[0796] (6) Synthesis of GPC3-5

[0797] Step 1: Couple the first amino acid Fmoc-Lys(Mtt)-OH

[0798] 90 mg (0.1 mmol) of Wang resin was fully swollen in DCM for 1 h. An amino acid solution was prepared by dissolving 0.25 mmol of Fmoc-Lys(Mtt)-OH and 0.25 mmol of 1-hydroxybenzotriazole (HOBT, 0.25 mmol) in 5 mL of DCM, and adding N,N'-diisopropylcarbodiimide (DIC, 0.25 mmol). After the resin swelled, the DCM was drained, the prepared amino acid solution was added, and 0.01 mmol of 4-dimethylaminopyridine (DMAP, 0.01 mmol) was added. The mixture was shaken at room temperature for 15 h. After the reaction was complete, the resin was washed 5 times with DCM, and then blocked with blocking buffer (10 mL) DCM:methanol:DIEA (85:10:5, v:v:v) for 10 min at room temperature. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0799] Step 2: Synthesis of linear precursor peptide chains

[0800] The linear precursor peptide chain of GPC3-5 is: {Boc-Gly-OH}-CPNYCTVFGERYECWICGEEE-EEK(Mtt).

[0801] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the linear precursor sequence from the second carboxyl-terminal PEG6 to the amino-terminal. Each coupling cycle was performed as follows:

[0802] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0803] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0804] 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;

[0805] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0806] After the linear polypeptide was synthesized, the resin was rinsed 5 times with DMF and 5 times with DCM.

[0807] Step 3: C-terminal lysine side chain coupling with DOTA

[0808] Removal of the Mtt protecting group from the lysine side chain: After swelling the resin with DCM for 1 hour, add a mixed solution of hexafluoroisopropanol / dichloromethane (30% v / v, 10 mL) to the resin, shake and react at room temperature for 45 minutes, then remove the Mtt protecting group. Repeat the operation once. After the reaction is complete, wash the resin with DCM 5 times and DMF 6 times.

[0809] Lysine side-chain coupling with DOTA: Weigh 0.5 mmol of tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 0.5 mmol of ethyl 2-oxime cyanoacetate, dissolve them in 8 mL of DMF, then add 80 μL of DIC for pre-activation for 3 min. Add the mixed solution to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution, wash 4-5 times with DMF, wash 4-5 times with DCM, and dry the resin under vacuum.

[0810] Step 4: Cleavage of the linear precursor peptide chain

[0811] 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 3, 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 washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0812] Step 5: Intramolecular disulfide bond formation

[0813] The crude product obtained in step 4 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0814] Step 6: Preparation of Peptides

[0815] 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-C18 (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 25-40% 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.

[0816] Step 7: Detection and Characterization Methods

[0817] The purity of the polypeptide from step 6 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry, and the C-terminal lysine side chain of the compound was coupled with DOTA and intramolecular disulfide bond was formed. The liquid chromatography and LC-MS detection results are shown in Figures 11 and 12.

[0818] (7) Synthesis of polypeptide 158

[0819] Step 1: Couple the first amino acid Fmoc-Cys(Trt)-OH

[0820] 90 mg (0.1 mmol) of Wang resin was fully swollen in DCM for 1 h. An amino acid solution was prepared by dissolving 0.25 mmol of Fmoc-Cys(Trt)-OH and 0.25 mmol of 1-hydroxybenzotriazole (HOBT, 0.25 mmol) in 5 mL of DCM, and adding N,N'-diisopropylcarbodiimide (DIC, 0.25 mmol). After the resin swelled, the DCM was drained, the prepared amino acid solution was added, and 0.01 mmol of 4-dimethylaminopyridine (DMAP, 0.01 mmol) was added. The mixture was shaken at room temperature for 15 h. After the reaction was complete, the resin was washed 5 times with DCM, and then blocked with blocking buffer (10 mL) DCM:methanol:DIEA (85:10:5, v:v:v) for 10 min at room temperature. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0821] Step 2: Synthesis of linear precursor peptide chains

[0822] The linear precursor peptide chain of polypeptide 158: CPNYCTVFGERYECWIC.

[0823] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the straight-chain precursor sequence from the second C at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0824] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0825] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0826] 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;

[0827] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0828] 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.

[0829] Step 3: Cleavage of the linear precursor peptide chain

[0830] 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 2, 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.

[0831] Step 4: Intramolecular disulfide bond formation

[0832] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0833] Step 5: Preparation of Peptides

[0834] 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-C18 (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 25-40% 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.

[0835] Step 6: Detection and Characterization Methods

[0836] The purity of the polypeptide from step 5 was determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, and the formation of intramolecular disulfide bonds in the compound was also determined.

[0837] (8) Synthesis of GPC3-55

[0838] Step 1: Synthesis of linear precursor peptide chains

[0839] The linear precursor peptide of GPC3-55: PEG6-CPNYCTVFGERYECWIC-PEG6.

[0840] 147 mg (0.1 mmol) of RinkAmide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0841] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0842] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0843] 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;

[0844] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0845] After the linear polypeptide was synthesized, the resin was rinsed five times with DMF.

[0846] Step 2: Cleavage of the linear precursor peptide chain

[0847] 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 2, 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.

[0848] Step 3: Intramolecular disulfide bond formation

[0849] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0850] Step 4: Preparation of Peptides

[0851] 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-C18 (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 30-50% 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.

[0852] Step 5: Detection and Characterization Methods

[0853] The purity of the polypeptide from step 4 was determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, as well as the formation of intramolecular disulfide bonds.

[0854] (9) Synthesis of polypeptide 108

[0855] Step 1: Couple the first amino acid Fmoc-Gly-OH

[0856] 90 mg (0.1 mmol) of Wang resin was fully swollen in DCM for 1 h. An amino acid solution was prepared by dissolving 0.25 mmol of Fmoc-Gly-OH and 0.25 mmol of 1-hydroxybenzotriazole (HOBT) in 5 mL of DCM, and adding N,N'-diisopropylcarbodiimide (DIC, 0.25 mmol). After the resin swelled, the DCM was drained, the prepared amino acid solution was added, and 0.01 mmol of 4-dimethylaminopyridine (DMAP) was added. The mixture was shaken at room temperature for 15 h. After the reaction was complete, the resin was washed 5 times with DCM, and then blocked with blocking buffer (10 mL) DCM:methanol:DIEA (85:10:5, v:v:v) for 10 min at room temperature. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0857] Step 2: Synthesis of linear precursor peptide chains

[0858] The linear precursor peptide chain of polypeptide 108: CPNYCTVFGERYECWICG.

[0859] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the straight-chain precursor sequence from the second C at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0860] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0861] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0862] 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;

[0863] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0864] 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.

[0865] Step 3: Cleavage of the linear precursor peptide chain

[0866] 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 2, 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.

[0867] Step 4: Intramolecular disulfide bond formation

[0868] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0869] Step 5: Preparation of Peptides

[0870] 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-C18 (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 25-40% 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.

[0871] Step 6: Detection and Characterization Methods

[0872] The purity of the polypeptide from step 6 was determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, and the formation of intramolecular disulfide bonds in the compound was also determined.

[0873] (8) Synthesis of GPC3-52

[0874] Step 1: Synthesis of linear precursor peptide chains

[0875] The linear precursor peptide of GPC3-52: PEG6-CPNYCTVFGERYECWICG.

[0876] 147 mg (0.1 mmol) of RinkAmide-AM Resin resin was fully swollen in DMF for 1 h. Then, the linear precursor was synthesized sequentially from the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0877] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0878] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0879] 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;

[0880] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0881] After the linear polypeptide was synthesized, the resin was rinsed five times with DMF.

[0882] Step 2: Cleavage of the linear precursor peptide chain

[0883] 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 2, 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.

[0884] Step 3: Intramolecular disulfide bond formation

[0885] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0886] Step 4: Preparation of Peptides

[0887] 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-C18 (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 30-50% 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.

[0888] Step 5: Detection and Characterization Methods

[0889] The purity of the polypeptide from step 4 was determined by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry, as well as the formation of intramolecular disulfide bonds.

[0890] (9) Synthesis of GPC3-74

[0891] Step 1: Synthesis of linear precursor peptide chains

[0892] The linear precursor peptide chain of GPC3-74 is {D-Pro}-G-{D-Pro}-GGCPEYCTVFGERYECWICG.

[0893] 306 mg (0.1 mmol) of Fmoc-Gly-Wang Resin 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:

[0894] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0895] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0896] 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;

[0897] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0898] 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.

[0899] Step 2: Connecting DOTA to the N-end

[0900] Weigh out 0.5 mmol of tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 0.5 mmol of ethyl 2-oxime cyanoacetate, dissolve them in 8 mL of DMF, then add 80 μL of DIC for pre-activation for 3 min. Add the mixture to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution, wash 4-5 times with DMF, wash 4-5 times with DCM, and dry the resin under vacuum.

[0901] Step 3: Cleavage of the linear precursor peptide chain

[0902] 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 2, 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.

[0903] Step 4: Intramolecular disulfide bond formation

[0904] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0905] Step 5: Preparation of Peptides

[0906] 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-C18 (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 27-45% 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.

[0907] Step 6: Detection and Characterization Methods

[0908] The purity of the polypeptide obtained in step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry (LC / MS), and the formation of intramolecular disulfide bonds in the compound was confirmed. The results of LC and MS detection are shown in Figures 13 and 14.

[0909] (10) Synthesis of GPC3-77

[0910] Step 1: Synthesis of linear precursor peptide chains

[0911] The linear precursor peptide chain of GPC3-77 is {4-Amb}-MV-{K(Me)2}-PEG6-GCPNYCTVFGERYECWICG.

[0912] 306 mg (0.1 mmol) of Fmoc-Gly-Wang Resin 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:

[0913] Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time;

[0914] Rinse the resin with DMF 6-8 times until neutral pH is reached;

[0915] 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;

[0916] Rinse the resin with DMF 4-6 times before the next amino acid coupling.

[0917] 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.

[0918] Step 2: Connecting DOTA to the N-end

[0919] Weigh out 0.5 mmol of tri-tert-butyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 0.5 mmol of ethyl 2-oxime cyanoacetate, dissolve them in 8 mL of DMF, then add 80 μL of DIC for pre-activation for 3 min. Add the mixture to the resin obtained in the previous step and shake to react for 3 h. After the reaction, drain the reaction solution, wash 4-5 times with DMF, wash 4-5 times with DCM, and dry the resin under vacuum.

[0920] Step 3: Cleavage of the linear precursor peptide chain

[0921] 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 2, 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.

[0922] Step 4: Intramolecular disulfide bond formation

[0923] The crude product obtained in step 3 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 50% acetonitrile). The dissolved peptide solution was slowly added dropwise to the buffer solution to a final peptide 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.

[0924] Step 5: Preparation of Peptides

[0925] 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-C18 (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 20-40% 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.

[0926] Step 6: Detection and Characterization Methods

[0927] The purity of the polypeptide obtained in step 5 was determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry (LC / MS), and the formation of intramolecular disulfide bonds in the compound was confirmed. The results of LC and MS detection are shown in Figures 15 and 16.

[0928] Example 3: SPR test for affinity

[0929] 1. Experimental materials:

[0930] 2. Experimental steps:

[0931] The affinity of peptides for GPC3 was tested using a Biacore T200 microarray. Approximately 1000-3000 RU of GPC3 protein was captured at 25°C using a ProteinA chip. Binding experiments were performed at 25°C with 1×PBS-P, pH 7.4 as the running buffer. The peptide analysis flow rate was 30 μL / min, with association time of 120 s and dissociation time of 600 s. A single-cycle Kinetics / Affinity assay was used to detect the binding of peptide samples to the protein. Gly-HCl at pH 1.5 was used at a flow rate of 30 μL / min, with chip regeneration performed after 30 s. Data were fitted using a 1:1 binding model.

[0932] 3. Experimental Results

[0933] The experimental results of some peptides and peptide drug conjugates of this invention are shown in Table 2 and Figures 17 to 20.

[0934] The control group has the following structure, and the control group used in subsequent embodiments also has this structure.

[0935] The peptides and peptide drug conjugates of the present invention show significant binding to GPC3, and the peptides and peptide drug conjugates of the present invention have a very good binding ability to GPC3.

[0936] The results of peptides with alanine substitution and D-type amino acid substitution also demonstrate that the Y at position 5, F at position 9, R at position 12, Y at position 13, and E at position 14 have a significant impact on peptide activity. Furthermore, the L configuration (V at position 8, F at position 9, R at position 12, Y at position 13, and E at position 14) also significantly affects peptide activity.

[0937] The peptides of this invention retain good activity even after the G terminus is deleted from the N-terminus and / or C-terminus.

[0938] Furthermore, the peptides and peptide drug conjugates of the present invention show significant binding to GPC3 in monkeys (e.g., cynomolgus monkeys).

[0939] Example 4 Cell Surface Binding Data

[0940] 1. Experimental materials:

[0941] 2. Experimental steps:

[0942] Cell preparation: H_GPC3 cells were cultured and grown in DMEM, 10% FBS, and 0.75 μg / mL puromycin medium; Huh-7 cells were cultured and grown in DMEM + 10% FBS + 1% P / S medium; and HEK293 cells were cultured and grown in DMEM, 10% FBS medium. 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.

[0943] 16-24 hours before the experiment, H_GPC3, Huh-7, and HEK293 cells were passaged and expanded to the required cell numbers. The cells were digested, centrifuged to collect the cell pellet, resuspended in an appropriate amount of complete culture medium, and cell viability was assessed and counted. The cell concentration was then adjusted to 6 x 10⁶ cells / year using 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 of DPBS. Incubate overnight at 37°C in a 5% CO2 incubator.

[0944] The peptides were dissolved in DMSO to a concentration of 2 mM. During the experiment, the peptides were diluted to the desired concentration using cell basal medium. After overnight inoculation, the old medium in the wells was aspirated and replaced with 100 μl of diluted peptide per well. The cells were then incubated at 4°C for 1 h. After washing the cells twice with pre-cooled DPBS, the cells were analyzed using confocal microscopy with fluorescence imaging.

[0945] 3. Experimental Results

[0946] The experimental results are shown in Figures 21 and 22. The polypeptides and polypeptide drug conjugates of the present invention can bind to GPC3 on the cell surface.

[0947] Example 5: Cell Endocytosis

[0948] 1. Experimental materials:

[0949] 2. Experimental steps:

[0950] Cell preparation: H_GPC3 cells were cultured in DMEM, 10% FBS, and 0.75 μg / mL puromycin medium; Huh-7 cells were cultured in DMEM, 10% FBS, and 1% P / S medium; and HEK293 cells were cultured in DMEM, 10% FBS medium. 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.

[0951] 16-24 hours before the experiment, H_GPC3, Huh-7, and HEK293 cells were passaged and expanded to the required cell numbers. The cells were digested, centrifuged to collect the cell pellet, resuspended in an appropriate amount of complete culture medium, and cell viability was assessed and counted. The cell concentration was then adjusted to 6 x 10⁶ cells / year using 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 of DPBS. Incubate overnight at 37°C in a 5% CO2 incubator.

[0952] The peptides were dissolved in DMSO to a concentration of 2 mM. During the experiment, the peptides were diluted to the desired concentration using cell basal medium. After overnight inoculation, the cell plates were removed, the old medium in the wells was aspirated, and 100 μl of diluted peptide per well was replaced. The plates were then incubated at 37°C in a 5% CO2 incubator for 2 hours. After fixation with fixative and nuclear staining with DAPI, the cells were analyzed using confocal microscopy fluorescence imaging.

[0953] 3. Experimental Results

[0954] The experimental results are shown in Figures 23 and 24. The polypeptides and polypeptide drug conjugates of the present invention can be internalized into cells and have a high internalization efficiency.

[0955] Example 6 Specific Identification Analysis

[0956] (1) Surface plasmon resonance (SPR) detection of selectivity between peptides and GPC family proteins

[0957] 1. Experimental materials:

[0958] 2. Experimental steps:

[0959] 2.1 Install the CM5 / ProteinA chip (the ProteinA chip is used to test GPC1, GPC2, and GPC3; the CM5 chip is used to test GPC3, GPC4, and GPC5).

[0960] 2.2 Immobilization of ligand proteins: The protein immobilization signal RU value was calculated. Using a flow rate of 10 μL / min, 2200 RU GPC1 protein was immobilized using a ProteinA chip with 2 channels, 2200 RU GPC2 protein was immobilized using a 3-channel chip, 3000 RU GPC3 protein was immobilized using a 4-channel chip, and 1 channel was used as a reference channel. 2200 RU GPC3 protein was immobilized using a CM5 chip with 2 channels, 2000 RU GPC4 protein was immobilized using a 3-channel chip, 2000 RU GPC5 protein was immobilized using a 4-channel chip, and 1 channel was used as a reference channel.

[0961] 2.3 Single concentration test: Test whether the peptide and peptide drug conjugate bind to GPC1-GPC5 at concentrations of 50-100 nM, 1 μM and 3 μM respectively.

[0962] Detection conditions (parameters): flow rate 30 μL / min, binding time 120 s.

[0963] Buffer: 1×PBS-P+.

[0964] Regeneration conditions: 10 mM Glycine-HCl, 30 s.

[0965] (2) ELISA assay to detect the binding of peptides to GPC6 protein

[0966] 1. Experimental materials:

[0967] 2. Experimental steps:

[0968] 2.1 Coating: Add 25 μL of 4 μg / mL GPC3 (Fc tag) and 4 μg / mL LGPC6 (His tag) to different positions on the 384-well detection plate, and set up an uncoated group (add only 25 μL of empty coating solution); incubate at 4°C for 24 h.

[0969] 2.2 Sealing: After coating, remove the liquid and add 80 μL of washing buffer per well. Shake on a shaker for 1 minute, then remove the washing buffer and add fresh washing buffer. Repeat this process 3 times. Add 80 μL of sealing buffer per well, centrifuge, and incubate at 37°C for 2 hours.

[0970] 2.3 Reagent Preparation:

[0971] a. The polypeptide drug conjugate of the present invention: prepared into 50 nM using diluent, serially diluted 5 times to obtain 4 concentrations, and then set up a 0 concentration control.

[0972] b.SA-HRP: Mixed at a ratio of 1:10000.

[0973] 2.4 Operating Steps:

[0974] ① After the 384 coated plate is blocked and cleaned, add 25uL of different concentrations of peptides and 0 concentration control according to the plate layout. Incubate at 37 degrees for 60 min, remove the liquid and add 80uL of washing buffer / well. Shake on a shaker for 1 minute, remove the washing buffer, add new washing buffer, and repeat 3 times.

[0975] ② Add 25 uL LSA-HRP, centrifuge, incubate at 37°C for 1 hour, wash the plate with 80 uL washing solution per well, shake on a shaker for 1 minute, remove the washing solution, add new washing solution, and repeat 3 times.

[0976] ③ Add 25 μL / well of TMB colorimetric solution (a 1:1 mixture of solution A and solution B) and incubate at 37°C for 12 min.

[0977] ④ Add 25 μL / well of 1 M HCl to terminate the reaction and measure the OD. 450 .

[0978] (3) Experimental Results

[0979] The experimental results are shown in Table 3 and Figure 25. Only the sample showed affinity for GPC3 protein, while exhibiting no affinity for the other five proteins in the same family, demonstrating excellent selectivity. These results indicate that the peptides and peptide-drug conjugates of this invention can selectively target GPC3.

[0980] Table 3. SPR test sample selectivity results

[0981] Example 7 Metabolic stability in mouse plasma

[0982] 1. Experimental materials:

[0983] Mouse plasma (heparin sodium) was collected by Slack himself.

[0984] 2. Experimental Procedure

[0985] (1) Preparation of mixing buffer: Dilute the sample to 0.1 mM using DMSO. Add (50 μL × (6 time points) + 1) 350 μL of plasma (heparin sodium) to a 1.5 mL EP tube. Prepare one tube of mixing buffer for each time point with at least 3 replicates. Incubate on ice for 5 min. Add 3.5 μL of the sample to be tested to each tube to achieve a final concentration of 1 μM. Vortex to mix. Aliquot 50 μL into EP tubes according to the time gradient and incubate.

[0986] (2) Incubation: Incubate in a 37℃ water bath at six time points: 0 min, 15 min, 30 min, 60 min, 120 min, and 240 min.

[0987] (3) Termination of reaction: After incubation, different pretreatments are performed according to the pretreatment methods required for bioanalysis.

[0988] (4) Mixing: Mix by oscillating on a vortex oscillator.

[0989] (5) Centrifugation: Centrifuge at 4°C and 13,000 rpm for 10 min in a low-temperature high-speed centrifuge.

[0990] (6) Take 80 μL of the supernatant, transfer it to a sample vial, and analyze it using LC-MS / MS.

[0991] (7) The peak area of ​​the peptide at different time points was detected by LC-MS / MS, and the results were expressed as the percentage of original drug remaining.

[0992] 3. Experimental Results

[0993] The polypeptides and polypeptide drug conjugates of the present invention have good plasma metabolic stability. The experimental results of some polypeptides and polypeptide drug conjugates are shown in Table 4 and Figure 26. The structure of the control is the same as that in Example 3.

[0994] Table 4 Results of Plasma Metabolic Stability Test

[0995] Example 8: Metabolic stability in mouse liver and kidney homogenates

[0996] 1. Experimental materials:

[0997] 2. Experimental steps:

[0998] (1) Liver and kidney homogenate: The tissue was taken out from the tissue preservation solution, and the connective tissue on the liver and kidney was removed. The liver and kidney were cut into pieces of about 0.4g each, rinsed twice with PBS solution, and the liver and kidney were placed on clean sterile filter paper (with the blood vessels facing down) to absorb excess blood and PBS. The weight was recorded, and the tissue pieces were then slightly cut into smaller pieces for easy grinding.

[0999] (2) Homogenization of liver and kidney: The minced tissue was placed in 2mL centrifuge tubes supplied with the homogenizer, and the corresponding amount of 50mM Tris-HCl (pH 7.4) solution was added to achieve a homogenate concentration of 0.25g / mL. Two steel balls were added to each centrifuge tube, and homogenization was performed at 65Hz for 60s. After homogenization, the mixture was centrifuged at 2000g for 10 minutes at 4℃. The supernatant was collected after centrifugation and stored at -80℃. It should be used within three days.

[1000] (3) The protein concentration of the homogenate was detected using a protein detection kit: kidney: 13 mg / mL, liver: 23 mg / mL.

[1001] (4) Dilute the above homogenate with PBS to a concentration of 0.5 mg / mL before use.

[1002] (5) Preparation of homogenate: Add 350 μL of mouse liver and kidney homogenate to a 1.5 mL EP tube. Prepare one homogenate for at least three parallel samples at each time point. Add 3.5 μL of the test sample to each tube to make a final concentration of 1 μM. Vortex for 30 s. Repeat the process by aliquoting 50 μL into EP tubes at different time points and incubate.

[1003] (6) Incubation: Incubate in a 37℃ water bath at six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min.

[1004] (7) Termination of reaction: After incubation, different pretreatments are performed according to the pretreatment methods required for bioanalysis.

[1005] (8) Mixing: Mix by oscillating on a vortex oscillator.

[1006] (9) Centrifugation: Centrifuge at 4°C and 13000g for 10 min in a low-temperature high-speed centrifuge.

[1007] (10) Take 80 μL of the supernatant and transfer it to a vial for analysis by LC-MS / MS.

[1008] (11) The peak area of ​​the peptide at different time points was detected by LC-MS / MS, and the results were expressed as the percentage of original drug remaining.

[1009] 3. Experimental Results

[1010] The stability of some peptides and peptide drug conjugates in liver and kidney homogenates of this invention is shown in Tables 5 and 6.

[1011] Table 5 Stability of mouse liver homogenate

[1012] Table 6 Stability of mouse kidney homogenate

[1013] The polypeptides and polypeptide drug conjugates of this invention exhibit good stability in liver and kidney homogenates.

[1014] Example 9: Plasma Protein Binding

[1015] (I) Mouse plasma protein binding

[1016] 1. Experimental materials:

[1017] 2. Experimental steps:

[1018] (1) Plasma thawing: Take out the plasma (1 mL / sample number) from the -80℃ freezer and thaw it quickly in a 37℃ water bath.

[1019] (2) Installation of the equilibration dialysis apparatus: Install the equilibration dialysis apparatus according to the standard operating procedure (SOP). Quickly add 100 μL of PBS solution to the sample receiving end to prevent the dialysis membrane from drying out.

[1020] (3) Plasma pretreatment: Add an appropriate amount of plasma to a 1.5 mL EP tube, and add an appropriate amount of protease inhibitor mixture and 0.5 M EDTA at a ratio of inhibitor: plasma = 1:100. Vortex mix and place on ice.

[1021] (4) Prepare EP tubes for sampling: Prepare 6 blank EP tubes (1 for receiving end + 1 for drug delivery end) × 3 duplicates, and label them. At time 0, add 50 μL of blank plasma to the sample tube at the receiving end and add an appropriate amount of precipitant (refer to the sample pretreatment procedure for bioanalysis).

[1022] (5) Add the test sample: Add an appropriate amount (1% of the total volume) of the test sample to the pretreated plasma to achieve a final concentration of 1 μM. Perform three replicates per sample at two time points. Vortex thoroughly on a vortex mixer.

[1023] (6) Take the 0-hour sample: Add 100 μL of the above 7.5 solution to the sample delivery end and 100 μL of PBS to the receiving end. After adding the equilibration dialysis device, take 50 μL of the above 7.5 solution as the 0-hour delivery end sample and add it to the EP tube prepared in 7.4. After adding the precipitant, add 50 μL of PBS. Take 50 μL of blank PBS as the 0-hour receiving end sample and add it to the EP tube prepared in 7.4. Vortex thoroughly to mix. Seal the equilibration dialysis device with sealing film.

[1024] (7) Incubation: Secure the equilibration dialysis apparatus and place ice packs. Incubate at 4°C for 6 hours in a benchtop constant temperature shaker until equilibrium is reached. If the sample is stable in plasma stability testing, incubation at 37°C can be used.

[1025] (8) Sample taking after incubation: Take 50 μL of the receiving end sample and 50 μL of the dosing end sample into the EP tube prepared in 7.4 above.

[1026] (9) Mixing: Mix thoroughly in a vortex oscillator.

[1027] (10) Centrifugation: 4℃, 13000rpm, 10min.

[1028] (11) Take 70 μL of the supernatant, transfer it to the injection tube, and send it to LC-MS / MS for analysis.

[1029] (ii) Human plasma protein binding

[1030] 1. Experimental Materials

[1031] Experimental reagents and consumables: methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); human plasma (Jiangsu Kewei); warfarin (purchased from Aladdin); 1×DPBS (self-made); protease inhibitor (Beyotime);

[1032] Experimental apparatus: Equilibrium dialysis apparatus HTD (Shanghai Meixing Gaode); LC-MS / MS (AB Sciex 6500+ and Waters I-Class UPLC).

[1033] 2. Experimental Methods

[1034] (1) Plasma thawing: Take out the plasma (1 ml / sample number) from the -80℃ freezer and thaw it quickly in a 37℃ water bath.

[1035] (2) Installation of the equilibration dialysis apparatus: Install the equilibration dialysis apparatus according to the standard operating procedure (SOP). Quickly add 100 μL of PBS solution to the sample receiving end to prevent the dialysis membrane from drying out.

[1036] (3) Plasma treatment: Take an appropriate amount of plasma and add it to a 1.5 mL EP tube, along with 1% protease inhibitor mixture and 1% 0.5 M EDTA.

[1037] (4) Matrix leveling: Prepare 6 blank EP tubes ((receiving end 1 + dosing end 1) × 3 replicates) for each time point of each sample and mark them.

[1038] (5) Add 50 μL of blank plasma to the receiving end sample tube, add 50 μL of blank PBS to the dosing end sample tube, and add 4 times the volume of 0.1% formic acid methanol precipitant.

[1039] (6) Add an appropriate amount of the test sample: Add 1% of the test sample to the pretreated plasma to make a final concentration of 10 μM. Each sample is repeated 3 times at 2 time points. Vortex the sample to mix.

[1040] (7) Sample transfer to the equilibrium dialysis apparatus: Add 100 μL of the sample solution to be tested to the sample delivery end, and seal the equilibrium dialysis apparatus with sealing film. Incubation temperature: Fix the equilibrium dialysis apparatus and place ice packs, incubate at 4°C for 6 hours in a benchtop constant temperature shaker.

[1041] (8) Samples after incubation: Take 50 μL of the receiving end sample and 50 μL of the dosing end sample and put them into the EP tubes that have been filled with the matrix.

[1042] (9) Mixing: Vortex on a vortex mixer. Centrifugation: 4℃, 13000-15000 rpm, 10 min. Take 80 μL of the supernatant, transfer it to a sample injection tube, and analyze it by LC-MS / MS. 3. Experimental Results

[1043] Most peptide samples are unstable in plasma. To ensure the results of plasma protein binding experiments, this experiment set up conditions of low temperature and inhibitors to test their plasma protein binding rate (%).

[1044] 3. Experimental Results

[1045] The polypeptides and polypeptide drug conjugates of this invention have a low plasma protein binding rate, which is lower than that of warfarin, and the concentration of usable free components in plasma is high.

[1046] Example 10: Study on tissue distribution in HuH-7 tumor-bearing mice

[1047] 1. Materials and Methods

[1048] 1.1 The polypeptide drug conjugate of the present invention.

[1049] 1.2 Reagents and consumables: 1mL insulin injection, DMSO, high-purity hydrochloric acid, sodium acetate.

[1050] 1.3 Experimental animals: Six NOD Scid mice, female, 6-8 weeks old, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.

[1051] 2. Experimental Methods

[1052] 2.1 Female NOD Scid mice were housed for 6-8 weeks under standard conditions. HuH-7 cells were suspended in a suspension of PBS and matrix gel, and each mouse was inoculated with 1×10⁻⁶ cells. 7 One tumor cell. When the tumor volume reaches 300-500 mm. 3 At that time, the mice were evenly divided into 3 groups according to their weight, with 2 mice in each group.

[1053] 2.2 Dissolve the peptide drug conjugate to 1 mg / mL using DMSO.

[1054] 2.3 The germanium-gallium generator was rinsed in fractions with 5 mL of 0.1 M HCl, and 500 μL of the fraction with the highest activity was taken for peptide labeling. The amount of peptide molecules added was calculated based on the activity. The reaction system was adjusted to pH 3-4 using 1 M metal-free sodium acetate buffer with pH=7, and the reaction was carried out at 95℃ for 10 min to obtain the final product.

[1055] Tail vein injection 68 The Ga-labeled product (120 μCi / animal) was administered in a volume of 120 μL. Dynamic PET / CT scans were performed 30 min, static scans 2 h, and static scans 4 h post-injection. Animals were euthanized 4.5 h after cervical vertebrae fracture following the scans. Data processing using PMOD software yielded PET / CT images and indicators such as the standard uptake value (SUVmax).

[1056] 3. Experimental Results

[1057] The test results are shown in Figures 27 and 28. In HuH-7 tumor-bearing mice, the tumor... 68 Ga-GPC3-4 68 Significant Ga-GPC3-5 uptake indicates 68 Ga-GPC3-4 68 Ga-GPC3-5 exhibits specific uptake in HuH-7 tumors. In the livers of HuH-7 tumor-bearing mice, 68 Ga-GPC3-4 68 Ga-GPC3-5 were essentially not taken up, indicating that neither molecule is hepatotoxic.

[1058] Example 11 68 Study on tissue distribution of Ga-labeled peptide drug conjugate in HepG2 tumor-bearing mice

[1059] 1. Materials and Methods

[1060] 1.1 68 Ga-labeled polypeptide drug conjugates of the present invention.

[1061] 1.2 Reagents and consumables: 1mL insulin injection, DMSO, high-purity hydrochloric acid, sodium acetate.

[1062] 1.3 Experimental animals: Six male HepG2 tumor-bearing mice (Balb / c nu), 5-6 weeks old, were purchased from Pengli Biomedical Technology (Shanghai) Co., Ltd.

[1063] 2. Experimental Methods

[1064] 2.1 When the tumor volume reaches 300-500 mm 3 At that time, the mice were evenly divided into 3 groups according to their weight, with 2 mice in each group.

[1065] 2.2 Dissolve the peptide drug conjugate to 1 mg / mL using DMSO.

[1066] 2.3 The germanium-gallium generator was rinsed in fractions with 5 mL of 0.1 M HCl, and 500 μL of the fraction with the highest activity was taken for peptide labeling. The amount of peptide molecules added was calculated based on the activity. The reaction system was adjusted to pH 3-4 using 1 M metal-free sodium acetate buffer with pH=7, and the reaction was carried out at 95℃ for 10 min to obtain the final product.

[1067] Tail vein injection 68 The Ga-labeled product (120 μCi / animal) was administered in 120 μL volumes. Dynamic PET / CT scans were performed 30 min, static PET / CT scans 2 h, and static PET / CT scans 4 h post-injection. Animals were euthanized 4.5 h after cervical vertebrae were broken. Dissection was performed on tumors, liver, kidneys, blood, tail, spleen, brain, and bladder. Radioactivity measurements were taken from these tissues, and the %ID / g per gram of tissue was calculated. Data processing using PMOD software yielded PET / CT images and SUVmax parameters.

[1068] 3. Experimental Results

[1069] In HepG2 tumor-bearing mice, tumors affect... 68 Ga-GPC3-4 68 Significant Ga-GPC3-5 uptake indicates 68 Ga-GPC3-4 68 Ga-GPC3-5 exhibits specific uptake in HepG2 tumors. In the livers of HepG2 tumor-bearing mice, 68 Ga-GPC3-4 68 Ga-GPC3-5 were essentially not taken up, indicating that neither molecule is hepatotoxic.

[1070] Example 12 Human plasma stability

[1071] 1. Experimental Materials

[1072] Formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); methanol (purchased from Sigma); acetonitrile (purchased from Sigma)

[1073] 2. Experimental Procedure

[1074] Sample preparation: Dissolve the peptide to be tested in 50% methanol, water and 1% formic acid to 20 μM, and store at -4℃ for later use.

[1075] Plasma thawing: Remove the plasma from the -80℃ freezer and thaw it rapidly in a 37℃ water bath.

[1076] Preparation of reaction solution: Take 47.5 μL of plasma (heparin sodium) from each tube and add plasma from all time points to a 1.5 mL EP tube, with 3 replicates per time point. At 240 min, add 2.5 μL of the test sample to achieve a final concentration of 1 μM. Incubate all plasma at 37°C. At 120 min, add another 2.5 μL of the test sample to achieve a final concentration of 1 μM and incubate at 37°C for another 120 min. Prepare samples sequentially according to the following time points.

[1077] Incubation: Incubate in a 37℃ water bath at seven time points: 0 min, 30 min, 60 min, 120 min, 240 min, 480 min, and 1440 min.

[1078] Termination of reaction: After incubation, add 4 volumes of 0.1% formic acid and 75% acetonitrile aqueous solution as precipitant to all samples.

[1079] Mix well: Vortex until homogenized. Centrifuge: Centrifuge at 15000 rpm for 10 min at 4 °C. Take 80 μL of the supernatant, transfer it to a sample injection tube, and send it to LC-MS / MS for analysis.

[1080] A line graph with the ordinate representing the residual percentage of the original drug (%) and the abscissa representing time shows the trend of sample degradation in in vitro plasma over time, thus providing the results of sample stability.

[1081] Calculate the half-life T of the drug in plasma 1 / 2

[1082] The elimination rate constant (Ke) was calculated using first-order kinetics. Furthermore, the Ti of the compound in plasma was determined using the same formula. 1 / 2 (min). T 1 / 2 =-0.693 / Ke

[1083] 3. Experimental Results:

[1084] The polypeptides and polypeptide drug conjugates of this invention have good stability in human plasma.

[1085] Example 13: Study on the distribution of Blocking tissue in HepG2 tumor-bearing mice

[1086] 1. Materials and Equipment:

[1087] 68 Ge / 68 Ga generator: Chengdu New Nucleotide Pharmaceutical Technology Co., Ltd. (Xiangya No. 2 Hospital of Central South University);

[1088] High-performance liquid chromatograph (HPLC) for quality control: Shimadzu / Thermo Scientific Vanquish series;

[1089] Small Animal Positron Emission Tomography / Computed Tomography System (Micro-PET / CT): PINGSENG, China;

[1090] γ counter: CAPRAC-t type;

[1091] Reagents: Dimethyl sulfoxide (DMSO, Aladdin), ultrapure water (Merck), sterile water for injection (Hunan Kelun Pharmaceutical);

[1092] Sample grouping: 68 Ga-labeled peptide drug conjugates of the present invention (1 mCi / nmol, 0.1 mCi / nmol), Blocking group (500 μg).

[1093] 2. Experimental Procedure

[1094] 2.1 68 Ga-labeled synthesis process:

[1095] The tracer was dissolved in dimethyl sulfoxide (DMSO) to prepare a solution with a concentration of 1 mg / mL;

[1096] in accordance with 68 Ge / 68 The instruction manual for the Ga generator states that the generator should be rinsed with 5 mL of 0.1 M HCl and the eluted fraction with the highest radioactivity should be collected for tracer labeling.

[1097] The pH of the reaction system was adjusted to 3-4 using 1M sodium acetate buffer solution free of metal contamination. After adding the tracer, the reaction was carried out at 95°C for 10 minutes to obtain the final product.

[1098] Add 0.5 mg of ascorbic acid and dilute with physiological saline. 68 The Ga-labeled tracer was diluted to 1 mCi / ml;

[1099] The solution was filtered into a sterile bottle through a 0.22 μm filter membrane.

[1100] Radiochemical yield and purity (requirement >95%) were verified using radio-high performance liquid chromatography (radio-HPLC).

[1101] HPLC conditions: Mobile phase: Phase A (0.1% TFA), Phase B (0.1% TFA-ACN); Gradient elution program: 5% B to 95% B, elution time 20 min.

[1102] 2.2 Mouse PET Imaging Experiment:

[1103] Will 68 The Ga-labeled tracer was diluted to 1 μCi / μl, and 100-120 μCi was injected intravenously into each mouse. Static PET / CT scans were performed at 0.5, 2, and 4 hours after injection. After the scans, the mice were euthanized 4.5 hours after injection, and samples of tumors, liver, kidneys, blood, tail, and brain tissue were collected for radioactivity testing.

[1104] 3. Experimental Results

[1105] Experimental results show 68 In both groups of mice, the Ga-labeled peptide drug conjugate of this invention showed significant radioactive accumulation at the tumor site. However, in the Blocking group, after prior administration of an excessive amount of unlabeled protein to block the GPC3 receptor, no radioactive accumulation was observed at the tumor site, and the absorbance was far lower than that of the Ga-labeled group. 68 The absorbance values ​​of the two groups of peptide drug conjugates of the present invention labeled with Ga indicate that the peptide drug conjugates of the present invention can specifically bind to GPC3-positive tumors.

[1106] Example 14 177 Tissue distribution of Lu-labeled peptide drug conjugates in HepG2 mice

[1107] 1. Experimental Materials

[1108] 1.1 177 Lu-labeled polypeptide drug conjugates of the present invention.

[1109] 1.2 Reagents and consumables: 1mL insulin injection, DMSO, high-purity hydrochloric acid, sodium acetate.

[1110] 1.3 Experimental animals: 12 HepG2 tumor-bearing rats (Balb / c nu), female, 18.4-22.2g, were purchased from the Miluo branch of Hunan Slack Jingda Experimental Animal Co., Ltd.

[1111] 2. Experimental Methods

[1112] 2.1 177 Lu mark

[1113] 1) The precursor was dissolved in DMSO to prepare a 10 mg / mL stock solution. A certain amount of the stock solution was then dissolved in 0.45 M pH 4.5 labeling buffer to prepare 0.1 mg / mL and 1 mg / mL solutions.

[1114] 2) Labeling according to the ratio of nuclide (nmol): precursor (nmol) = 1:15, the ratio of nuclide (mCi): precursor (nmol) = 1mCi: 1 nmol, and the ratio of 0.1mCi: 1 nmol: 177 Lu COA calculates precursor volume and 177 The volume of Lu solution, and the volume of buffer solution are 150 μL minus the volume of the precursor. 177 The volume of Lu.

[1115] 3) Add the labeling buffer and precursor solution sequentially. 177 Lu solution was vortexed and mixed, and the pH and activity were measured. The final reaction volume was 150 μL.

[1116] 4) Seal the centrifuge tubes with sealing film and react at 100°C for 20 minutes on a constant temperature heater until the reaction is complete.

[1117] 5) Perform Radio-HPLC analysis on the reaction solution and record the results. The analytical method is as follows:

[1118] 2.2 Experimental Design

[1119] Dosage group design for imaging trials.

[1120] 2.3 Dosage Design

[1121] Based on previous studies on tissue distribution in HepG2 mice, and combined with the sensitivity of Micro-SPECT / CT, it was found that at this dose (100 μCi / mouse), the major organs had already responded to the isotope ( 177 Lu) has significant uptake, therefore this dosage was chosen.

[1122] Route of administration: Intravenous injection.

[1123] 2.4. Animal drug administration and test substance preparation

[1124] The activity of the prepared test sample was measured by an activity meter. According to the experimental requirements, the corresponding stock solution was extracted and diluted with physiological saline to the appropriate volume before administration.

[1125] 2.5 Micro-SPECT / CT scan of experimental animals

[1126] 1) Power on the machine according to the operating procedures, open the scanning software, and allow the CT scanner to warm up;

[1127] 2) Create a new research folder according to the needs of the experiment;

[1128] 3) Create a new CT acquisition program and complete all necessary calibrations for CT acquisition;

[1129] 4) Create a new SPECT acquisition program and set the experimental parameters (acquisition method, use of isotopes, time window);

[1130] 5) Save this data collection program;

[1131] 6) Turn on the anesthesia machine, fix the experimental animal in a prone position on the bed board, and adjust the animal's position;

[1132] 7) Adjust the scanning range to cover the region of interest;

[1133] 8) Close the rack cavity cover;

[1134] 9) Input animal information into the scanning software, adjust the field of view of SPECT and CT, and then start the scan;

[1135] 10) When reconstructing data, input the pre-drug activity, residual activity, injection time, and activity measurement time into the software;

[1136] 11) After the procedure, remove the animal from the bed board, put it in the rat cage, and turn off the anesthetic.

[1137] 2.6 Drug administration to experimental animals

[1138] Anesthesia time: from the start of the scan to the end of the scan

[1139] 1) Restrain the animal and inject the test substance via the tail vein;

[1140] 2) Whole-body CT scans and static SPECT scans were performed at 1h, 4h, 24h, 48h and 96h after injection to collect raw imaging data of the animals.

[1141] 2.7 Animal disposal

[1142] The mice were euthanized after the experiment.

[1143] 3. Data processing and statistical analysis

[1144] Process the scanned images using PMOD software:

[1145] 1) Import the obtained CT and SPECT images into the software;

[1146] 2) Rigidly fuse CT and SPECT images;

[1147] 3) Adjust the window width and window level of the fused image;

[1148] 4) Save animal images;

[1149] 5) Use the tools in the software to outline the heart, liver, kidneys, tibia, blood, intestines, muscles, and tumors;

[1150] 6) Read the %ID / cc value of the outlined area and save it as an Excel file.

[1151] 3. Experimental Results

[1152] Experimental results show that the polypeptide drug conjugate of this invention, after... 177 After Lu labeling, the drug rapidly accumulates in the tumor after administration. The tumor has a higher uptake value than other organs in the body. The uptake values ​​of other organs or tissues such as the heart, liver, intestine, muscle, and bone are lower. At the same time, the kidney, as the main metabolic pathway, also has a high uptake value.

[1153] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide has the amino acid sequence shown in general formula (I). X1CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CX 19 (I) in, X1 either does not exist or is G; X 19 It does not exist or is G; X4, X8, X9, X 10 X 11 X 12 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids.

2. The polypeptide according to claim 1, characterized in that, The polypeptide has an amino acid sequence represented by general formula (II), (III), (IV) or (V). GCPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CG (II) GCPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 C (III) CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 CG (IV) CPX4YCTX8X9X 10 X 11 X 12 YECX 16 X 17 C (V)。 3. The polypeptide according to claim 1 or 2, characterized in that, in, X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R; X8 is selected from I, T, H, F, V, N, L, R, Y, S, Q; X9 is selected from F and M; X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E; X 11 Selected from E, D, Y, H, Q, M; X 12 Selected from R and W; X 16 Selected from I, H, Y, W, F, E, D, A; X 17 Selected from K, I, Y, E, Q, M, H, V.

4. The polypeptide according to claim 1 or 2, characterized in that, in, X4 is selected from S, N, D, G, K, A, H, E, T, Q, I; X8 is selected from I, T, H, F, V, N, L, R; X9 is selected from F and M; X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E; X 11 Selected from E, D, Y, H, Q; X 12 Selected from R and W; X 16 Selected from I, H, Y, W, F, E, D, A; X 17 Selected from K, I, Y, E, Q, M, H, V.

5. The polypeptide according to claim 1 or 2, characterized in that, in, X4 is selected from S, N, D, and G; X8 is selected from I, T, H, F, V; X9 is selected from F; X 10 Selected from H, V, T, K, P, M, G; X 11 Selected from E and D; X 12 Selected from R; X 16 Selected from I, H, Y, W, F; X 17 Selected from K, I, Y, E, Q, M.

6. The polypeptide according to claim 1 or 2, characterized in that, in, X4 is selected from S and N; X8 is selected from I, T, H, and V; X9 is selected from F; X 10 Selected from H, V, T, K, G; X 11 Selected from E and D; X 12 Selected from R; X 16 Selected from I, H, Y, W; X 17 Selected from K, I, Y, E.

7. The polypeptide according to claim 1 or 2, characterized in that, in, X4 is selected from N and E; X8 is selected from V and Q; X9 is selected from F; X 10 Selected from G and Q; X 11 Selected from E and Q; X 12 Selected from R; X 16 Selected from W; X 17 Selected from I and Q.

8. A polypeptide, characterized in that, The polypeptide has an amino acid sequence represented by general formula (VI), (VIII), (IX) or (X). GCPX4YCTX8FX 10 X 11 RYECX 16 X 17 CG (VI) CPX4YCTX8FX 10 X 11 RYECX 16 X 17 CG (VIII) GCPX4YCTX8FX 10 X 11 RYECX 16 X 17 C (IX) CPX4YCTX8FX 10 X 11 RYECX 16 X 17 C (X)、 X4, X8, X 10 X 11 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids; Each amino acid in the general formula is independently selected from either the D- or L-isomer.

9. The polypeptide according to claim 8, characterized in that, in, X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R; X8 is selected from I, T, H, F, V, N, L, R, Y, S, Q; X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E; X 11 Selected from E, D, Y, H, Q, M; X 16 Selected from I, H, Y, W, F, E, D, A; X 17 Selected from K, I, Y, E, Q, M, H, V.

10. The polypeptide according to claim 8, characterized in that, in, X4 is selected from S, N, D, G, K, A, H, E, T, Q, I; X8 is selected from I, T, H, F, V, N, L, R; X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E; X 11 Selected from E, D, Y, H, Q; X 16 Selected from I, H, Y, W, F, E, D, A; X 17 Selected from K, I, Y, E, Q, M, H, V.

11. The polypeptide according to claim 8, characterized in that, in, X4 is selected from S, N, D, G, K, A, H; X8 is selected from I, T, H, F, V, N; X 10 Selected from H, V, T, K, P, M, G, Q; X 11 Selected from E, D, and Y; X 16 Selected from I, H, Y, W, F, E, D; X 17 Selected from K, I, Y, E, Q, M, H.

12. The polypeptide according to claim 8, characterized in that, in, X4 is selected from S, N, D, and G; X8 is selected from I, T, H, F, V; X 10 Selected from H, V, T, K, P, M, G; X 11 Selected from E and D; X 16 Selected from I, H, Y, W, F; X 17 Selected from K, I, Y, E, Q, M.

13. The polypeptide according to claim 8, characterized in that, in, X4 is selected from S and N; X8 is selected from I, T, H, and V; X 10 Selected from H, V, T, K, G; X 11 Selected from E and D; X 16 Selected from I, H, Y, W; X 17 Selected from K, I, Y, E.

14. The polypeptide according to claim 8, characterized in that, in, X4 is selected from N and E; X8 is selected from V and Q; X 10 Selected from G and Q; X 11 Selected from E and Q; X 16 Selected from W; X 17 Selected from I and Q.

15. A polypeptide, characterized in that, The polypeptide has an amino acid sequence represented by general formula (VII), (XI), (XII) or (XIII). GCPX4YCTVFX 10 X 11 RYECWX 17 CG (VII) CPX4YCTVFX 10 X 11 RYECWX 17 CG (XI) GCPX4YCTVFX 10 X 11 RYECWX 17 C (XII) CPX4YCTVFX 10 X 11 RYECWX 17 C (XIII) in, X4, X 10 X 11 X 17 Each amino acid is independently selected from natural or non-natural amino acids; Each amino acid in the general formula is independently selected from either the D- or L-isomer.

16. The polypeptide according to claim 15, characterized in that, in, X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R; X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E; X 11 Selected from E, D, Y, H, Q, M; X 17 Selected from K, I, Y, E, Q, M, H, V.

17. The polypeptide according to claim 15, characterized in that, in, X4 is selected from S, N, D, G, K, A, H, E, T, Q, I; X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E; X 11 Selected from E, D, Y, H, Q; X 17 Selected from K, I, Y, E, Q, M, H, V.

18. The polypeptide according to claim 15, characterized in that, in, X4 is selected from S, N, D, G, K, A, H; X 10 Selected from H, V, T, K, P, M, G, Q; X 11 Selected from E, D, and Y; X 17 Selected from K, I, Y, E, Q, M, H.

19. The polypeptide according to claim 15, characterized in that, in, X4 is selected from S, N, D, and G; X 10 Selected from H, V, T, K, P, M, G; X 11 Selected from E and D; X 17 Selected from K, I, Y, E, Q, M.

20. The polypeptide according to claim 15, characterized in that, in, X4 is selected from S and N; X 10 Selected from H, V, T, K, G; X 11 Selected from E and D; X 17 Selected from K, I, Y, E.

21. The polypeptide according to claim 15, characterized in that, in, X4 is selected from N and E; X 10 Selected from G and Q; X 11 Selected from E and Q; X 17 Selected from I and Q.

22. A polypeptide, characterized in that, The polypeptide has amino acid sequences represented by the general formulas (XIV), (XV), (XVI), (XVII), and (XXIII). X1CPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 CX 19 (XIV) GCPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 CG (XV) GCPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 C (XVI) CPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 CG (XVII) CPX4YCQX8X9X 10 X 11 X 12 YECX 16 X 17 C (XVIII) in, X1 either does not exist or is G; X 19 It does not exist or is G; X4, X8, X9, X 10 X 11 X 12 X 16 X 17 Each amino acid is independently selected from natural or non-natural amino acids; Each amino acid in the general formula is independently selected from either the D- or L-isomer.

23. The polypeptide according to claim 22, characterized in that, in, X4 is selected from S, N, D, G, K, A, H, E, T, Q, I, P, R; X8 is selected from I, T, H, F, V, N, L, R, Y, S, Q; X9 is selected from F and M; X 10 Selected from H, V, T, K, P, M, G, Q, S, R, L, N, E; X 11 Selected from E, D, Y, H, Q, M; X 12 Selected from R and W; X 16 Selected from I, H, Y, W, F, E, D, A, N; X 17 Selected from K, I, Y, E, Q, M, H, V, A.

24. The polypeptide according to claim 22, characterized in that, in, X4 is selected from S, N, D, G, and E; X8 is selected from I, T, H, F, V; X9 is selected from F; X 10 Selected from H, V, T, K, P, M, G; X 11 Selected from E and D; X 12 Selected from R; X 16 Selected from I, H, Y, W, F, N; X 17 Selected from K, I, Y, E, Q, M, A.

25. The polypeptide according to claim 22, characterized in that, in, X4 is selected from N and E; X8 is selected from V and Q; X9 is selected from F; X 10 Selected from G, Q, K; X 11 Selected from E and Q; X 12 Selected from R; X 16 Selected from W and N; X 17 Selected from I and A.

26. The polypeptide according to claim 22, characterized in that, in, X4 is selected from N; X8 is selected from V; X9 is selected from F; X 10 Selected from G, Q, K; X 11 Selected from E and Q; X 12 Selected from R; X 16 Selected from W and N; X 17 Selected from I and A.

27. A polypeptide, characterized in that, The polypeptide is selected from the following polypeptides or combinations thereof: (1) N-terminal and / or C-terminal truncated peptides of the polypeptides represented by general formula (I); (2) Derivative peptides obtained by amino acid scanning mutation of the polypeptide shown in general formula (I); (3) The polypeptide of formula (I), or the modified product of the polypeptide of formula (1) or (2).

28. The polypeptide according to claim 27, characterized in that, The polypeptide is selected from the following polypeptides or combinations thereof: (1) N-terminal and / or C-terminal truncated peptides of any of the polypeptides shown in general formulas (II) to (XVIII); (2) Derivative peptides obtained by amino acid scanning mutation of any of the polypeptides shown in general formulas (II) to (XVIII); (3) Any polypeptide of formula (II) to (XVIII), or a modified product of the polypeptide of (1) or (2).

29. The polypeptide according to claim 27, characterized in that, The polypeptide is selected from the following polypeptides or combinations thereof: (1) N-terminal and / or C-terminal truncated peptides of the polypeptides shown in SEQ ID No. 1, SEQ ID No. 58, SEQ ID No. 108 or SEQ ID No. 158; (2) Derivative peptides obtained by amino acid scanning mutation of the polypeptides shown in SEQ ID No. 1, SEQ ID No. 58, SEQ ID No. 108 or SEQ ID No. 158; (3)(1) or (2) modified products of the polypeptides.

30. The polypeptide according to claim 27, characterized in that, The polypeptide is selected from the following polypeptides or combinations thereof: (1) The N-terminal and / or C-terminal truncated peptide of the polypeptide shown in SEQ ID No. 1; (2) Derivative peptides obtained by amino acid scanning mutation of the polypeptide shown in SEQ ID No. 1; (3)(1) or (2) modified products of the polypeptides.

31. The polypeptide according to claim 27, characterized in that, The modification is selected from the following forms or combinations thereof: modification of N-terminal or side chain amino acids: acetylation, formylation, trifluoroacetylation, benzoylation, 2-aminobenzoylation; C-terminal modifications: amidation, esterification, aldehydeation, alcoholation; Alkylation modifications: N-methylation, side-chain methylation, N-ethylation, N-phenylpropylation, N-allylation, etc.

32. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in any one of SEQ ID No. 1 to SEQ ID No.

244.

33. The polypeptide according to any one of claims 1-32, characterized in that, The polypeptide has a cyclic peptide structure, which is formed by intramolecular cysteine ​​residues through disulfide bonds.

34. The polypeptide according to claim 33, characterized in that, The cyclic peptide contains a pair of disulfide bonds formed from the first and second cysteine ​​residues at the N-terminus; or from the first and third cysteine ​​residues at the N-terminus; or from the first and fourth cysteine ​​residues at the N-terminus; or from the second and third cysteine ​​residues at the N-terminus; or from the second and fourth cysteine ​​residues at the N-terminus; or from the third and fourth cysteine ​​residues at the N-terminus.

35. The polypeptide according to claim 33, characterized in that, The cyclic peptide contains two pairs of disulfide bonds, formed from the first and second cysteine ​​residues at the N-terminus, and from the third and fourth cysteine ​​residues; or formed from the first and fourth cysteine ​​residues at the N-terminus, and from the second and third cysteine ​​residues; or formed from the first and third cysteine ​​residues at the N-terminus, and from the second and fourth cysteine ​​residues.

36. The polypeptide according to claim 33, characterized in that, The cyclic peptide contains two pairs of disulfide bonds, formed from the first and fourth cysteine ​​residues at the N-terminus, and from the second and third cysteine ​​residues.

37. A polypeptide drug conjugate, characterized in that, The general structural formula (1) of the polypeptide drug conjugate is shown below: Peptide-Linker-Payload(1) in, The linker is absent or is a linking group; Payload is a payload group, which includes cytotoxic drugs, radionuclide complex groups, or fluorescent groups. Peptide is a polypeptide, and the polypeptide is the polypeptide described in any one of claims 1 to 36.

38. The polypeptide drug conjugate according to claim 37, characterized in that, The payload is a fluorescent group, which is selected from at least one of infrared fluorescent dyes, compounds containing organic chromophores, compounds containing organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, or bioluminescent molecules.

39. The polypeptide drug conjugate according to claim 37, characterized in that, The fluorescent group is selected from at least one of the following near-infrared fluorescent dyes: MPA, IRDye800CW, Cy7, Cy7.5, Cy3, Cy5, Cy5.5, ICG, FIGT, FAM, MCA, TAMRA, Biotin, HEX, AMC, or Rhodamine B.

40. The polypeptide drug conjugate according to claim 37, characterized in that, The payload is a radionuclide complexing group, which includes a radionuclide and a bifunctional chelating agent for radionuclide labeling; the bifunctional chelating agent for radionuclide labeling is selected from at least one of NOTA, DOTA, DOTAM, DOTAGA, NODAGA, DTPA, CHX-DTPA, HYNIC, DFO, p-SCN-Bn-DFO, NODAGA, NO2A, DO3A, and MAG3; the radionuclide is selected from... 18 F, 125 I, 131 I, 64 Cu、 67 Ga、 68 Ga、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 153 Sm、 177 Lu、 186 Re、 188 Re、 211 At、 212 Pb, 223 Ra、 225 At least one of Ac.

41. The polypeptide drug conjugate according to claim 37, characterized in that, The radionuclide complexing group includes a radionuclide and a bifunctional chelating agent for radionuclide labeling; the bifunctional chelating agent for radionuclide labeling is selected from at least one of NOTA, DOTA, DOTAGA, and NODAGA; the radionuclide is selected from... 18 F, 64 Cu、 68 Ga、 99m Tc, 177 Lu、 225 At least one of Ac.

42. The polypeptide drug conjugate according to claim 37, characterized in that, The payload is a cytotoxic drug selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

43. The polypeptide drug conjugate according to claim 37, characterized in that, The payload is selected from auristatins and their derivatives (such as MMAE, MMAF, MMAD), maytansin and its derivatives (such as DM1, DM2, DM3, DM4), tubulysins, cryptomycin, spindle kinesin, gemcitabine, pyrrolo[2,1-c][1,4]benzodiazepines, ducamycin, camptothecin and its derivatives (such as SN38, eczetidine, Dxd, topotecan, irinotecan), cazithromycin, amatoxins, paclitaxel, taxane, vincristine, vinblastine, etoposide, doxorubicin, cyclophosphamide, docetaxel, methotrexate, cisplatin, cytarabine, phenylalanine mustard, and chlorambucil mustard, or combinations thereof.

44. The polypeptide drug conjugate according to claim 37, characterized in that, The Linker is absent or is a linking group; the Linker contains non-cleavable linkers or cleavable linkers. Non-cleavable linkers are selected from PEG linkers, linkers with thioether groups, linkers with oxime groups, or combinations thereof; cleavable linkers are selected from linkers with disulfide bonds, dipeptide linkers, tripeptide linkers, tetrapeptide linkers, peptide-like linkers, β-glucuronidase-cleavable linkers, β-galactosidase-cleavable linkers, phosphatase-based linkers, pH-sensitive linkers, sulfatase-cleavable linkers, or combinations thereof.

45. The polypeptide drug conjugate according to claim 37, characterized in that, Linker includes the following structures or combinations thereof: (Gly) n (Glu) n (γGlu) n (GS) n GGSG (D-Gly) n (D-Glu) n (Gln) n (D-Gln) n , (GP)n, (Gp)n, (pGp)n, (AP)n, (2-Nal-Y1)n, (GGGS) n , R is selected from -OH, -NH2, -NH-Glu, -NH-Gln, methylamino, ethylamino, propanamino, butylamino, methoxy, ethoxy, propoxy, and butoxy; m is an integer from 0 to 24; and n is an integer from 1 to 10.

46. ​​The polypeptide drug conjugate according to claim 37, characterized in that, The linker structure also includes PABC spacer groups, 4-AMC spacer groups, and / or AMBA spacer groups; the PABC spacer group structure is as follows: The AMBA spacer structure is The 4-AMC spacer structure is 47. The polypeptide drug conjugate according to claim 37, characterized in that, The linker structure also contains β-Ala spacer groups and / or [Sar] groups. n The interval basis, n, is selected from integers from 1 to 10.

48. The polypeptide drug conjugate according to claim 37, characterized in that, Linker includes the following structures or combinations thereof: G{D-Pro}, Gln2, AEEA, 2-Nal, 49. The polypeptide drug conjugate according to claim 37, characterized in that, The structure of the polypeptide drug conjugate is shown in any one of GPC3-1 to GPC3-85.

50. A nucleotide, characterized in that, The nucleotide encodes the polypeptide according to any one of claims 1 to 36.

51. A composition, characterized in that, The composition comprises a polypeptide according to any one of claims 1 to 36, a polypeptide drug conjugate according to any one of claims 37 to 49, or a nucleotide according to claim 50.

52. The use of the polypeptide of any one of claims 1 to 36, the polypeptide drug conjugate of any one of claims 37 to 49, the nucleotide of claim 50, or the composition of claim 51 in the preparation of tumor PET imaging agents, tumor SPECT imaging agents, or in the preparation of tumor peptide targeted therapy drugs, wherein the tumor is a GPC3-positive tumor.

53. The application according to claim 52, characterized in that, The tumors expressing GPC3 positively are selected from at least one of liver cancer, ovarian cancer, lung cancer, melanoma, gastric cancer, thyroid cancer, colon cancer, pancreatic cancer, bladder cancer, and myeloma.

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