Polypeptide targeting KRAS mutant protein and use thereof, drug, and method

Abstract

Provided are a polypeptide targeting a KRAS mutant protein and a use thereof, a drug, and a method. The polypeptide can specifically bind to a KRAS G12 mutant protein binding domain, can exhibit high affinity to a KRAS G12X mutant protein (X=D, V, R, or C), with a picomolar-level equilibrium dissociation constant KD, and can specifically regulate and control a secondary structure of the KRAS G12X mutant protein (X=D, V, R, or C). The polypeptide conforming to specific amino acid composition and sequence characteristics and including SEQ ID NOs. 1-9 has the ability to induce death of KRAS G12X mutant (X=D, V, R, or C) tumor cells and, on this basis, enhances the therapeutic effect of existing chemotherapy and tumor immunotherapy. The polypeptide can be used as a therapeutic or auxiliary therapeutic drug for diseases, such as colon cancer, lung cancer, pancreatic cancer, breast cancer, osteogenic sarcoma and / or ovarian cancer, and has clinical application potential.

Description

Polypeptides targeting KRAS mutant proteins, uses thereof, medicaments and methods

[0001] Cross-reference to Related Applications

[0002] This application claims priority to Chinese Patent Application No. CN 202410474321.9, filed April 19, 2024, the contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of biological medicine, and relates to a polypeptide targeting KRAS mutant proteins, uses thereof, medicaments and methods. BACKGROUND

[0004] The global incidence of cancer is on the rise and is currently the leading cause of morbidity and mortality worldwide. It is estimated that the number of global cancer cases will reach 28.4 million cases in 2040, an increase of 47% compared to 2020. According to statistics, lung cancer, colorectal cancer and pancreatic cancer rank first, third and fourth, respectively, in terms of tumor-related causes of death in men and women. The most commonly used treatment for cancers such as pancreatic cancer, colorectal cancer and lung cancer is surgery combined with radiotherapy, targeted therapy and immunotherapy. However, overall, the prognosis of tumor patients is poor, and postoperative recurrence is common. Late-stage cancer patients and elderly patients have poor tolerance to chemotherapy, resulting in low survival rates for cancer patients. For example, the 5-year survival rate for pancreatic cancer patients is about 5%, and there is an urgent need to develop more effective treatment strategies for clinical treatment.

[0005] KRAS is one of the most common mutated oncogenes in human malignancies, and plays an important role in regulating cell growth, differentiation, proliferation and survival signaling pathways. In healthy cells, KRAS is a "switch" that regulates cell growth. However, when the KRAS gene is mutated, the KRAS protein is continuously activated, abnormally activates downstream signaling pathways, leading to uncontrolled cell growth and tumor production. KRAS gene mutations are most common in pancreatic cancer, colorectal cancer and non-small cell lung cancer, accounting for 67.61%, 35.77% and 20.42%, respectively. KRAS gene mutations are mainly single base missense mutations, and 98% of mutations occur at codon 12 (G12), codon 13 (G13) or codon 61 (Q61), with codon 12 mutations being the most common. According to the different amino acids expressed after mutation, KRAS G12 is divided into G12A, G12C, G12D, G12V, G12V, G12R and other subtypes. For example, KRAS G12C represents a mutation of the 12th glycine (Glycine) codon in the KRAS gene to a cysteine (Cysteine) codon. KRAS mutation profiles also differ significantly among different cancer types. KRAS G12C is the most common in non-small cell lung cancer, accounting for about 14%; in pancreatic cancer and colorectal cancer, KRAS G12D is the most common, accounting for 12% and 36%, respectively, followed by KRAS G12V, accounting for 9% and 21%, respectively. Therefore, targeting the function of KRAS G12 mutant proteins can effectively inhibit the occurrence and progression of KRAS G12 site mutation-driven cancers.

[0006] The important role of KRAS mutations in tumors has been widely recognized, but for a long time, KRAS mutants have been considered a difficult target. On the one hand, KRAS has a very strong binding force with substrate GTP, with an affinity coefficient reaching picomolar concentration, making it difficult to develop competitive inhibitors targeting the GTP pocket directly. On the other hand, the surface of the KRAS protein lacks an ideal small molecule binding pocket, making it difficult to design a high-affinity allosteric inhibitor, resulting in a lack of KRAS-targeting drugs. Existing small molecule drugs targeting KRAS G12C (AMG510 and MRTX849) have successfully entered clinical trials for the treatment of non-small cell lung cancer, but due to potential liver toxicity and drug resistance caused by new mutations at the KRAS G12 site, their clinical application is limited, and there is an urgent need to develop more efficient and less toxic new technologies and treatment methods. Developing antagonists targeting KRAS G12X mutant proteins is crucial for controlling cancer progression and improving cancer cure rates.

[0007] Polypeptide molecules have the advantages of low molecular weight, small molecular size, simple synthesis, easy storage, high affinity and high selectivity comparable to antibodies, and easy metabolism in the human body, and therefore have high drug potential. Therefore, designing and screening a kind of polypeptide which can bind to KRAS G12X mutant protein (X=D, V, R, C) binding domain with high efficiency and selectively kill tumor cells can provide potential new technologies and methods for the treatment of refractory cancers including lung cancer, colorectal cancer and pancreatic cancer.

[0008] SUMMARY

[0009] The present application aims to overcome the defects in the prior art and provide a polypeptide targeting KRAS mutant protein, its application, a drug and a method. The polypeptide of the present application can have high affinity with KRAS G12X mutant protein (X=D, V, R, C), and the equilibrium dissociation constant KD is in the picomolar level. The polypeptide comprising SEQ ID NO. 1-9 according to the present application has the ability to induce the death of KRAS G12X mutant (X=D, V, R, C) tumor cells, and on this basis, enhances the therapeutic effect of existing chemotherapy and tumor immunotherapy, and can be used as a therapeutic or adjuvant therapy drug for diseases such as colon cancer, lung cancer, pancreatic cancer, breast cancer, osteosarcoma and / or ovarian cancer.

[0010] Before setting forth the application, definitions of terms used in the specification are set forth below:

[0011] The term "KRAS" refers to: Kirsten rat sarcoma viral oncogene homolog protein (Kirsten rat sarcoma viral oncogene homolog protein).

[0012] The term "KRAS G12D" refers to: the amino acid at position 12 of the KRAS protein is mutated from glycine to aspartic acid.

[0013] The term "KRAS G12V" refers to: the amino acid at position 12 of the KRAS protein is mutated from glycine to valine.

[0014] The term "KRAS G12R" refers to: the amino acid at position 12 of the KRAS protein is mutated from glycine to arginine.

[0015] The term "KRAS G12C" refers to: the amino acid at position 12 of the KRAS protein is mutated from glycine to cysteine.

[0016] The term "KRAS G12A" refers to: the amino acid at position 12 of the KRAS protein is mutated from glycine to alanine.

[0017] The term "KRAS G12S" refers to: the amino acid at position 12 of the KRAS protein is mutated from glycine to serine.

[0018] The term "D" refers to: the abbreviation of aspartic acid, Asp.

[0019] The term "V" refers to: the abbreviation of valine, Val.

[0020] The term "R" refers to: the abbreviation of arginine, Arg.

[0021] The term "C" refers to: the abbreviation of cysteine, Cys.

[0022] The term "A" refers to: the abbreviation of alanine, Ala.

[0023] The term "S" refers to: the abbreviation of serine, Ser.

[0024] To achieve the above object, the first aspect of the present application provides a polypeptide targeting KRAS mutant protein, which can specifically bind to KRAS G12X mutant protein, wherein X is selected from one or more of the following amino acids: D, V, R, C, A, S, preferably selected from one or more of the following amino acids: D, V, R, C; wherein,

[0025] The amino acid sequence of the C-terminal of the polypeptide comprises: YGRKKRRQRRR; and,

[0026] The polypeptide consists of 22-30 amino acids, preferably 24-30 amino acids, and more preferably 26-30 amino acids.

[0027] The polypeptide according to the first aspect of the present application, wherein the amino acid sequence of the polypeptide from N-terminal to C-terminal is B-YGRKKRRQRRR, the amino acid sequence of B is B1VVB2RB3; wherein,

[0028] The amino acid in the amino acid sequence of B1 is selected from one or more of the following: D, R, Q, Y, L, V;

[0029] The amino acid in the amino acid sequence of B2 is selected from one or more of the following: I, R, G, W, V, A;

[0030] The amino acid in the amino acid sequence of B3 is selected from one or more of the following: V, G, D, S, T, C, I, L, R.

[0031] The polypeptide according to the first aspect of the present application, wherein the amino acid in the amino acid sequence of B2 is selected from one or more of the following: I, R, G, W, V.

[0032] The polypeptide according to the first aspect of the present application, wherein,

[0033] the number of amino acids in the amino acid sequence of B1 is 2-10, preferably 3-8, more preferably 3-5;

[0034] the number of amino acids in the amino acid sequence of B2 is 2-6, preferably 2-4, most preferably 3; and / or

[0035] the number of amino acids in the amino acid sequence of B3 is 3-10, preferably 3-9, more preferably 4-8.

[0036] the polypeptide according to the first aspect of the present application, wherein,

[0037] the amino acid sequence of the polypeptide is selected from one or more of the following: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9;

[0038] preferably selected from one or more of the following: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8;

[0039] more preferably selected from one or more of the following: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 7.

[0040] the polypeptide according to the first aspect of the present application, wherein the amino acid sequence of the polypeptide is N-terminally modified by one or more of the following modification groups: biotin, fatty acid chain, polyethylene glycol, preferably by biotin or fatty acid chain, most preferably by biotin.

[0041] The second aspect of the present application provides use of the polypeptide according to the first aspect of the present application in the manufacture of a medicament for treating or adjuvant treatment of cancer.

[0042] The third aspect of the present application provides a medicament for treating or adjuvant treatment of cancer, the medicament comprising: the polypeptide according to the first aspect of the present application.

[0043] The fourth aspect of the present application provides a pharmaceutical composition, the pharmaceutical composition comprising:

[0044] 1) the polypeptide according to the first aspect of the present application or the medicament for treating or adjuvant treatment of cancer according to the third aspect of the present application; and

[0045] 2) one or more pharmaceutically acceptable carriers.

[0046] A fifth aspect of the present application provides a method for treating or adjuvant treatment of cancer, the method comprising: administering to a subject in need thereof the polypeptide of the first aspect, the medicament for treating or adjuvant treatment of cancer of the third aspect or the pharmaceutical composition of the fourth aspect.

[0047] A sixth aspect of the present application provides a targeted intervention method, the targeted intervention method comprising: administering to a subject in need thereof the polypeptide of the first aspect, the medicament for treating or adjuvant treatment of cancer of the third aspect or the pharmaceutical composition of the fourth aspect.

[0048] A seventh aspect of the present application provides a drug-mediated method, the drug-mediated method comprising: administering to a subject in need thereof the polypeptide of the first aspect, the medicament for treating or adjuvant treatment of cancer of the third aspect or the pharmaceutical composition of the fourth aspect.

[0049] The use according to the second aspect, the medicament for treating or adjuvant treatment of cancer according to the third aspect, the pharmaceutical composition according to the fourth aspect, the method for treating or adjuvant treatment of cancer according to the fifth aspect, the targeted intervention method according to the sixth aspect or the drug-mediated method according to the seventh aspect, wherein the cancer is selected from one or more of: colon cancer, lung cancer, pancreatic cancer, breast cancer, osteosarcoma and / or ovarian cancer, preferably selected from one or more of: colon cancer, lung cancer, pancreatic cancer, breast cancer and / or ovarian cancer, more preferably selected from one or more of: colorectal cancer, lung cancer, pancreatic cancer.

[0050] The use according to the second aspect, the medicament for treating or adjuvant treatment of cancer according to the third aspect, the pharmaceutical composition according to the fourth aspect, the method for treating or adjuvant treatment of cancer according to the fifth aspect, the targeted intervention method according to the sixth aspect or the drug-mediated method according to the seventh aspect, wherein,

[0051] The polypeptide is capable of specifically modulating the secondary structure of a KRAS G12X mutant protein, wherein X is selected from one or more of the following amino acids: D, V, R, C, A, S, preferably selected from one or more of the following amino acids: D, V, R, C; and / or

[0052] The polypeptide has the ability to induce death of KRAS G12X mutant tumor cells.

[0053] According to the second aspect, the drug for treating or adjuvant treatment of cancer of the third aspect, the pharmaceutical composition of the fourth aspect, the method for treating or adjuvant treatment of cancer of the fifth aspect, the targeted intervention method of the sixth aspect or the drug-mediated method of the seventh aspect, wherein the KRAS G12 is selected from one or more subtypes of KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, KRAS G12A, KRAS G12S, preferably selected from one or more subtypes of KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C.

[0054] According to a specific embodiment of the present application, the first aspect of the present application provides a polypeptide specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C) cytotoxicity on KRAS G12D mutant pancreatic cancer cells, KRAS WT / G12D mutant human pancreatic cancer organoids and KRAS WT normal pancreatic ductal epithelial cells and human embryonic kidney cells;

[0055] The second aspect of the present application provides a polypeptide specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C) cytotoxicity on KRAS G12D mutant lung cancer cells and colorectal cancer cells and the effect on apoptosis of KRAS G12D mutant pancreatic cancer cells;

[0056] The third aspect of the present application provides the affinity of a polypeptide specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C) of the first aspect and the specific regulation of the secondary structure change of KRAS G12X protein.

[0057] According to another specific embodiment of the present application, the first aspect of the present application provides the cytotoxicity of a polypeptide specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C) on KRAS G12D mutant pancreatic cancer cells, KRAS WT / G12D mutant human pancreatic cancer organoids and KRAS WT normal pancreatic ductal epithelial cells and human embryonic kidney cells. The polypeptide can specifically inhibit the proliferation of KRAS G12X (X=D, V, R, C) mutant pancreatic cancer cells, and the cytotoxicity has a safety window of 3-5 times compared to the cytotoxicity of the polypeptide on normal pancreatic ductal epithelial cells and human embryonic kidney cells. The polypeptide can specifically inhibit the progression of KRAS G12D mutant pancreatic cancer organoids, which is significantly better than the toxicity on KRAS WT pancreatic cancer organoids, indicating the selectivity of the polypeptide to KRAS G12D mutant. Therefore, the first aspect clearly shows the toxicity of the polypeptide to KRAS G12X mutant pancreatic cancer and the toxicity of the polypeptide to KRAS G12D mutant pancreatic cancer organoids, and the safety of the polypeptide to KRAS WT pancreatic ductal epithelial cells and human embryonic kidney cells.

[0058] The second aspect of the present application provides the cytotoxicity of a polypeptide specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C) on KRAS G12D mutant lung cancer cells and colorectal cancer cells and the effect of the polypeptide on the apoptosis of KRAS G12D mutant pancreatic cancer cells, further clarifying the toxicity of the polypeptide to KRAS G12D mutant pancreatic cancer, colorectal cancer and lung cancer cells

[0059] The third aspect of the present application provides that the affinity of a polypeptide specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C) to KRAS G12X protein reaches the picomolar level, and the polypeptide can specifically regulate the secondary structure change of KRAS G12X protein, which preliminarily clarifies the molecular level mechanism of the selectivity of the polypeptide to the toxicity of KRAS G12X (X=D, V, R, C) mutant pancreatic cancer, lung cancer and colorectal cells.

[0060] The present application provides amino acid components and sequence characteristics of a polypeptide capable of specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C). The amino acid sequence of the polypeptide is B-YGRKKRRQRRR (N-terminal-C-terminal), and the B amino acid sequence is a B1VVB2RB3YGRKKRRORRR sequence fragment, wherein the B1 amino acid sequence is a sequence of 3-5 amino acids selected from one or more of the following: D, R, Q, Y, L, V; the B2 amino acid sequence is a sequence of 3 amino acids selected from one or more of the following: I, R, G, W, V, A; and the B3 amino acid sequence is a sequence of 4-8 amino acids selected from one or more of the following: V, G, D, S, T, C, I, L, R. The N-terminal of the polypeptide can be modified or unmodified biotin.

[0061] The present application provides a polypeptide capable of specifically binding to the binding domain of KRAS G12X mutant protein (X=D, V, R, C), and the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 1-9:

[0062] SEQ ID NO. 1: Biotin-RYDLVVVGARGVGDSYGRKKRRQRRR (Due to the standard format of the sequence list attached to the present application, Biotin- cannot be reflected, therefore, SEQ ID NO. 1 is based on the sequence information recorded herein in the specification).

[0063] SEQ ID NO. 2: Biotin-RYDLVVVWRRRVGDSYGRKKRRQRRR (Due to the standard format of the sequence list attached to the present application, Biotin- cannot be reflected, therefore, SEQ ID NO. 2 is based on the sequence information recorded herein in the specification).

[0064] SEQ ID NO. 3: Biotin-DDQVVIRGRTCLLRILRYGRKKRRQRRR (Due to the standard format of the sequence list attached to the present application, Biotin- cannot be reflected, therefore, SEQ ID NO. 3 is based on the sequence information recorded herein in the specification).

[0065] SEQ ID NO. 4: Biotin-VDDQVVWRGRTCLLRRLRYGRKKRRQRRR (Due to the standard format of the sequence list attached to the present application, Biotin- cannot be reflected, therefore, SEQ ID NO. 4 is based on the sequence information recorded herein in the specification).

[0066] SEQ ID NO. 5: Biotin-DRQVVRRGRLLRVGDSYGRKKRRQRRR (Due to the standard format of the sequence listing attached to the present application, Biotin- cannot be embodied, therefore, SEQ ID NO. 5 is subject to the sequence information recorded herein in the specification).

[0067] SEQ ID NO. 6: Biotin-DDQVVWIGRLLRILRVGYGRKKRRQRRR (Due to the standard format of the sequence listing attached to the present application, Biotin- cannot be embodied, therefore, SEQ ID NO. 6 is subject to the sequence information recorded herein in the specification).

[0068] SEQ ID NO. 7: Biotin-RYDLVVVIRRRVGDSYGRKKRRQRRR (Due to the standard format of the sequence listing attached to the present application, Biotin- cannot be embodied, therefore, SEQ ID NO. 7 is subject to the sequence information recorded herein in the specification).

[0069] SEQ ID NO. 8: Biotin-RYDDQVVIRGRLLRILRYGRKKRRQRRR (Due to the standard format of the sequence listing attached to the present application, Biotin- cannot be embodied, therefore, SEQ ID NO. 8 is subject to the sequence information recorded herein in the specification).

[0070] SEQ ID NO. 9: Biotin-DRQYLVVWRRRTCLLRILRYGRKKRRQRRR (Due to the standard format of the sequence listing attached to the present application, Biotin- cannot be embodied, therefore, SEQ ID NO. 9 is subject to the sequence information recorded herein in the specification).

[0071] The polypeptide targeting KRAS mutant protein of the present application can have, but is not limited to, the following beneficial effects:

[0072] 1. The present application provides a polypeptide sequence capable of having high affinity with KRAS G12X mutant protein (X = D, V, R, C), with a balance dissociation constant KD of picomolar level, which can specifically regulate the secondary structure of KRAS G12X mutant (X = D, V, R, C) protein.

[0073] 2. The polypeptide containing SEQ ID NO. 1-9 according to the present application has the ability to induce the death of KRAS G12X mutant (X = D, V, R, C) tumor cells, and on this basis, enhances the therapeutic effect of existing chemotherapy and tumor immunotherapy, and can be used as a therapeutic or adjuvant therapy drug for diseases such as colon cancer, lung cancer, pancreatic cancer, breast cancer, osteosarcoma and / or ovarian cancer.

[0074] BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0076] Figure 1 shows the cytotoxicity of SEQ ID NO.1 against KRAS G12X (X = D, V, R, C) pancreatic cancer cells; Figure 1A shows the IC values ​​of SEQ ID NO.1 in AsPc-1 cells (KRAS G12D mutation) at 24h and 48h. 50 ; Figure 1B shows the IC of SEQ ID NO.1 in Panc-1 cells (KRAS G12D mutation) for 48h 50 ; Figure 1C shows the IC of SEQ ID NO.1 in Patu8988 (KRAS G12V mutation) at 48h 50 ; Figure 1D shows the IC of SEQ ID NO.1 in Mia-Paca2 cells (KRAS G12C mutation) at 48h 50 ; Figure 1E shows the IC of SEQ ID NO.1 in PSN1 cells (KRAS G12R mutation) at 48h 50 .

[0077] Figure 2 shows the cytotoxicity of SEQ ID NO.2 against KRAS G12X (X = D, V, R, C) pancreatic cancer cells; Figure 2A shows the IC values ​​of SEQ ID NO.2 in AsPc-1 cells (KRAS G12D mutation) at 24h and 48h. 50 ; Figure 2B shows the IC of SEQ ID NO.2 in Panc-1 cells (KRAS G12D mutation) for 48h 50 ; Figure 2C shows the IC of SEQ ID NO.2 in Patu8988 (KRAS G12V mutation) at 48h 50 ; Figure 2D shows the IC of SEQ ID NO.2 in Mia-Paca2 cells (KRAS G12C mutation) at 48h 50 ; Figure 2E shows the IC of SEQ ID NO.2 in PSN1 cells (KRAS G12R mutation) at 48h 50 .

[0078] Figure 3 shows the cytotoxicity of SEQ ID NO.3 against KRAS G12X (X = D, V, R, C) pancreatic cancer cells; Figure 3A shows the IC values ​​of SEQ ID NO.3 in AsPc-1 cells (KRAS G12D mutation) at 24h and 48h. 50Figure 3A shows IC of SEQ ID NO. 3 at 24h and 48h in Panc-1 cells (KRAS G12D mutant) 50 Figure 3C shows IC of SEQ ID NO. 3 at 24h and 48h in Patu8988 (KRAS G12V mutant) 50 Figure 3D shows IC of SEQ ID NO. 3 at 24h and 48h in Mia-Paca2 cells (KRAS G12C mutant) 50 Figure 3E shows IC of SEQ ID NO. 3 at 24h and 48h in PSN1 cells (KRAS G12R mutant) 50 .

[0079] Figure 4 shows cytotoxicity of SEQ ID NO. 1-3 on KRAS WT normal pancreatic ductal epithelial cells and human embryonic kidney cells; wherein Figure 4A shows IC of SEQ ID NO. 1 at 48h in HPNE cells (KRAS WT) 50 Figure 4B shows IC of SEQ ID NO. 1 at 48h in 293T cells (KRAS WT) 50 Figure 4C shows IC of SEQ ID NO. 2 at 48h in HPNE cells (KRAS WT) 50 Figure 4D shows IC of SEQ ID NO. 2 at 48h in 293T cells (KRAS WT) 50 Figure 4E shows IC of SEQ ID NO. 3 at 24h and 48h in HPNE cells (KRAS WT) 50 Figure 4F shows IC of SEQ ID NO. 3 at 24h and 48h in 293T cells (KRAS WT) 50 .

[0080] Figure 5 shows toxicity of SEQ ID NO. 3 on KRAS WT / G12D mutant human pancreatic cancer organoids; wherein Figure 5A shows that no obvious toxicity of SEQ ID NO. 3 on KRAS WT organoids was observed within 4h, while concentration-dependent inhibition of organoid proliferation was observed after 24h and 48h of treatment. While SEQ ID NO. 3 was used to treat KRAS G12D mutant organoids, obvious concentration-dependent inhibition of organoid proliferation was observed within 4h, and concentration-dependent inhibition of organoid proliferation was observed after 24h and 48h of treatment, which was significantly higher than that of KRAS WT pancreatic cancer organoids. Figure 5B shows IC of SEQ ID NO. 3 at 48h in KRAS G12D mutant organoids 50IC of SEQ ID NO. 3 at 48h on KRAS WT mutant organoids 50 .

[0081] Figure 6 shows the toxicity of SEQ ID NO. 1-3 on KRAS G12D mutant colorectal and lung cancer cell lines; wherein Figure 6A shows IC of SEQ ID NO. 1 at 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 ; Figure 6B shows IC of SEQ ID NO. 1 at 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 ; Figure 6C shows IC of SEQ ID NO. 2 at 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 ; Figure 6D shows IC of SEQ ID NO. 2 at 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 ; Figure 6E shows IC of SEQ ID NO. 3 at 24h and 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 ; Figure 6F shows IC of SEQ ID NO. 3 at 24h and 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 .

[0082] Figure 7 shows the toxicity of SEQ ID NO. 4 on KRAS G12X (X=D, V, R, C) mutant pancreatic, KRAS G12D mutant lung and KRAS G12D mutant colorectal cancer cells; wherein Figure 7A shows IC of SEQ ID NO. 4 at 24h and 48h on AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 7B shows IC of SEQ ID NO. 4 at 48h on Panc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 7C shows IC of SEQ ID NO. 4 at 48h on Patu8988 (KRAS G12V mutant) 50 ; Figure 7D shows IC of SEQ ID NO. 4 at 48h on PSN1 cells (KRAS G12R mutant) 50 Figure 7E shows IC of SEQ ID NO. 4 at 48h on Mia-Paca 2 cells (KRAS G12C mutant) 50 Figure 7F shows IC of SEQ ID NO. 4 at 24h and 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50; Figure 7G shows the IC50of SEQ ID NO. 4 at 24h and 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 .

[0083] Figure 8 shows the toxicity of SEQ ID NO. 5 on KRAS G12X (X=D, V, R, C) mutant pancreatic cancer, KRAS G12D mutant lung cancer and KRAS G12D mutant colorectal cancer cells; wherein Figure 8A shows the IC50of SEQ ID NO. 5 at 24h and 48h on AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 8B shows the IC50of SEQ ID NO. 5 at 48h on Panc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 8C shows the IC50of SEQ ID NO. 5 at 48h on Patu8988 (KRAS G12V mutant) 50 ; Figure 8D shows the IC50of SEQ ID NO. 5 at 48h on PSN1 cells (KRAS G12R mutant) 50 ; Figure 8E shows the IC50of SEQ ID NO. 5 at 48h on Mia-Paca 2 cells (KRAS G12C mutant) 50 ; Figure 8F shows the IC50of SEQ ID NO. 5 at 24h and 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 ; Figure 8G shows the IC50of SEQ ID NO. 5 at 24h and 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 .

[0084] Figure 9 shows the toxicity of SEQ ID NO. 6 on KRAS G12X (X=D, V, R, C) mutant pancreatic cancer, KRAS G12D mutant lung cancer and KRAS G12D mutant colorectal cancer cells; wherein Figure 9A shows the IC50of SEQ ID NO. 6 at 24h and 48h on AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 9B shows the IC50of SEQ ID NO. 6 at 48h on Panc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 9C shows the IC50of SEQ ID NO. 6 at 48h on Patu8988 (KRAS G12V mutant) 50 ; Figure 9D shows the IC50of SEQ ID NO. 6 at 48h on PSN1 cells (KRAS G12R mutant) 50; Figure 9E shows IC of SEQ ID NO. 6 at 48h on Mia-Paca 2 cells (KRAS G12C mutant) 50 ; Figure 9F shows IC of SEQ ID NO. 6 at 24h and 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 ; Figure 9G shows IC of SEQ ID NO. 6 at 24h and 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 .

[0085] Figure 10 shows toxicity of SEQ ID NO. 7 on KRAS G12X (X=D, V, R, C) mutant pancreatic cancer, KRAS G12D mutant lung cancer and KRAS G12D mutant colorectal cancer cells; wherein Figure 10A shows IC of SEQ ID NO. 7 at 24h and 48h on AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 10A shows IC of SEQ ID NO. 7 at 48h on Panc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 10C shows IC of SEQ ID NO. 7 at 48h on Patu8988 (KRAS G12V mutant) 50 ; Figure 10D shows IC of SEQ ID NO. 7 at 48h on PSN1 cells (KRAS G12R mutant) 50 ; Figure 10E shows IC of SEQ ID NO. 7 at 48h on Mia-Paca 2 cells (KRAS G12C mutant) 50 ; Figure 10F shows IC of SEQ ID NO. 7 at 24h and 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 ; Figure 10G shows IC of SEQ ID NO. 7 at 24h and 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 .

[0086] Figure 11 shows toxicity of SEQ ID NO. 8 on KRAS G12X (X=D, V, R, C) mutant pancreatic cancer, KRAS G12D mutant lung cancer and KRAS G12D mutant colorectal cancer cells; wherein Figure 11A shows IC of SEQ ID NO. 8 at 24h and 48h on AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 ; Figure 11B shows IC of SEQ ID NO. 8 at 48h on Panc-1 pancreatic cancer cell line (KRAS G12D mutant)50 Figure 11C shows IC of SEQ ID NO. 8 at 48h on Patu8988 (KRAS G12V mutant) 50 Figure 11D shows IC of SEQ ID NO. 8 at 48h on PSN1 cells (KRAS G12R mutant) 50 Figure 11E shows IC of SEQ ID NO. 8 at 48h on Mia-Paca 2 cells (KRAS G12C mutant) 50 Figure 11F shows IC of SEQ ID NO. 8 at 24h and 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 Figure 11G shows IC of SEQ ID NO. 8 at 24h and 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 .

[0087] Figure 12 shows toxicity of SEQ ID NO. 9 on KRAS G12X (X=D, V, R, C) mutant pancreatic cancer, KRAS G12D mutant lung cancer and KRAS G12D mutant colorectal cancer cells; wherein Figure 12A shows IC of SEQ ID NO. 9 at 24h and 48h on AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 Figure 12B shows IC of SEQ ID NO. 9 at 48h on Panc-1 pancreatic cancer cell line (KRAS G12D mutant) 50 Figure 12C shows IC of SEQ ID NO. 9 at 48h on Patu8988 (KRAS G12V mutant) 50 Figure 12D shows IC of SEQ ID NO. 9 at 48h on PSN1 cells (KRAS G12R mutant) 50 Figure 12E shows IC of SEQ ID NO. 9 at 48h on Mia-Paca 2 cells (KRAS G12C mutant) 50 Figure 12F shows IC of SEQ ID NO. 9 at 24h and 48h on LS513 colorectal cancer cell line (KRAS G12D mutant) 50 Figure 12G shows IC of SEQ ID NO. 9 at 24h and 48h on SK-LU-1 lung cancer cell line (KRAS G12D mutant) 50 .

[0088] Figure 13 shows the cytotoxicity of SEQ ID NO. 4 to 6 on KRAS WT normal pancreatic ductal epithelial cells and human embryonic kidney cells; Figure 13A shows the IC of SEQ ID NO. 4 in HPNE cells (KRAS WT) at 48h. 50 ; Figure 13B shows the IC of SEQ ID NO.4 in 293T cells (KRAS WT) 48h 50 ; Figure 13C shows the IC of SEQ ID NO.5 in HPNE cells (KRAS WT) 48h 50 ; Figure 13D shows the IC of SEQ ID NO.5 in 293T cells (KRAS WT) 48h 50 ; Figure 13E shows the IC of SEQ ID NO.6 in HPNE cells (KRAS WT) 48h 50 ; Figure 13F shows the IC of SEQ ID NO.6 in 293T cells (KRAS WT) 48h 50 .

[0089] Figure 14 shows the cytotoxicity of SEQ ID NO.7 to 9 on KRAS WT normal pancreatic ductal epithelial cells and human embryonic kidney cells; Figure 14A shows the IC of SEQ ID NO.7 in HPNE cells (KRAS WT) at 48h. 50 ; Figure 14B shows the IC of SEQ ID NO.7 in 293T cells (KRAS WT) 48h 50 ; Figure 14C shows the IC of SEQ ID NO.8 in HPNE cells (KRAS WT) 48h 50 ; Figure 14D shows the IC of SEQ ID NO.8 in 293T cells (KRAS WT) 48h 50 ; Figure 14E shows the IC of SEQ ID NO.9 in HPNE cells (KRAS WT) 48h 50 ; Figure 14F shows the IC of SEQ ID NO.9 in 293T cells (KRAS WT) 48h 50 .

[0090] Figure 15 shows the effect of SEQ ID NO.3 on apoptosis of KRAS G12D mutant pancreatic cancer cells AsPc-1; Figure 15A shows representative data of flow cytometry detection of cell apoptosis caused by polypeptide SEQ ID NO.3 after treatment of KRAS G12D mutant pancreatic cancer cells AsPc-1; Figure 15B shows a statistical analysis of the proportion of cells causing late apoptosis after polypeptide treatment of AsPc-1.

[0091] Figure 16 shows the effect of SEQ ID NO. 3 on the apoptosis of KRAS G12D mutant human pancreatic cancer cell line Panc-1; wherein, Figure 16A shows the representative data of flow cytometry detection of the apoptosis of polypeptide SEQ ID NO. 3 treated KRAS G12D mutant pancreatic cancer cell line Panc-1; Figure 16B shows the statistical analysis of the proportion of cells in late apoptosis after polypeptide treatment of Panc-1.

[0092] Figure 17 shows the results of circular dichroism experiments of SEQ ID NO. 3 on KRAS G12X (X = D, V, R, C); wherein, Figure 17A shows the secondary structure change of KRAS G12D protein caused by SEQ ID NO. 3; Figure 17B shows the secondary structure change of KRAS G12V protein caused by SEQ ID NO. 3; Figure 17C shows the secondary structure change of KRAS G12R protein caused by SEQ ID NO. 3; Figure 17D shows the secondary structure change of KRAS G12C protein caused by SEQ ID NO. 3.

[0093] Best mode for carrying out the invention

[0094] The present application is further described in detail by the following specific examples. However, it is to be understood that these examples are merely illustrative of the present application and are not to be construed as limiting the present application in any way.

[0095] This section describes the materials and methods used in the experiments of the present application in general. Although many of the materials and methods used to achieve the objectives of the present application are well known in the art, the present application is described in as much detail as possible. It is clear to those skilled in the art that, in the context, if not specifically stated, the materials and methods used in the present application are well known in the art.

[0096] The following examples will help to further understand the present application, but do not limit the content of the present application.

[0097] As not specifically pointed out, NA in the following test examples means not applicable. When some data in the following table are not applicable or do not exist in specific cases, they are marked as NA.

[0098] The experimental methods used in the following examples are routine methods unless otherwise specified.

[0099] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0100] The AsPc-1 cell line, the Panc-1 cell line, the Patu8988 cell line, the Mia-Paca2 cell line, the PSN1 cell line, the HPNE cell line, the 293T cell line, the LS513 cell line and the SK-LU-1 cell line used in the following examples are purchased from the Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, unless otherwise specified.

[0101] The polypeptides used in the following examples are synthesized by Anhui Guoping Pharmaceutical Co., Ltd. according to the given sequences, and the purity is 98%, unless otherwise specified. The polypeptides used are prepared into a mother liquor of a suitable concentration with sterile ultrapure water and cell culture medium before the experiment.

[0102] Unless otherwise specified, the reagents and instrument information used in the following examples are as follows:

[0103] The solvent of the aqueous solution used is sterile ultrapure water solution, and the water quality parameter is resistivity 18.2 MΩ·cm @ 25℃.

[0104] The cell culture medium (DMEM medium and RPMI 1640 medium), trypsin, fetal bovine serum, PBS buffer, and ampicillin and streptomycin double-antibiotic solution are purchased from Thermo Fisher Scientific Company.

[0105] Corning 96-well plates and Corning 6-well plates are purchased from Beijing Huayide Company.

[0106] The CCK8 kit is purchased from Beijing Yihong Guangjie Biological Company.

[0107] The 10% fetal bovine serum and 1% ampicillin and streptomycin contained in the complete culture medium for cell culture refer to the volume fraction percentage.

[0108] The surface plasmon resonance instrument (SPR) is BIACORE3000.

[0109] The instrument for measuring circular dichroism (CD) spectrum is a polarimeter instrument, model J-1500, JASCO.

[0110] The continuous spectrum multifunctional enzyme label instrument is purchased from Tecan Company, model Infinite M200.

[0111] The centrifuge is purchased from Beijing Leboer Centrifuge Co., Ltd., model LD5-2A.

[0112] Example 1

[0113] This example is used to illustrate the polypeptide targeting KRAS mutant protein of the present application.

[0114] The polypeptides used in the following examples were all synthesized by Anhui Guoping Pharmaceutical Co., Ltd. according to the given sequences.

[0115] SEQ ID NO. 1: Biotin-RYDLVVVGARGVGDSYGRKKRRQRRR.

[0116] SEQ ID NO. 2: Biotin-RYDLVVVWRRRVGDSYGRKKRRQRRR.

[0117] SEQ ID NO. 3: Biotin-DDQVVIRGRTCLLRILRYGRKKRRQRRR.

[0118] SEQ ID NO. 4: Biotin-VDDQVVWRGRTCLLRRLRYGRKKRRQRRR.

[0119] SEQ ID NO. 5: Biotin-DRQVVRRGRLLRVGDSYGRKKRRQRRR.

[0120] SEQ ID NO. 6: Biotin-DDQVVWIGRLLRILRVGYGRKKRRQRRR.

[0121] SEQ ID NO. 7: Biotin-RYDLVVVIRRRVGDSYGRKKRRQRRR.

[0122] SEQ ID NO. 8: Biotin-RYDDQVVIRGRLLRILRYGRKKRRQRRR.

[0123] SEQ ID NO. 9: Biotin-DRQYLVVWRRRTCLLRILRYGRKKRRQRRR.

[0124] Test Example 1

[0125] Cytotoxicity of SEQ ID NO. 1-3 against KRAS G12X (X = D, V, R, C) pancreatic cancer cells

[0126] The KRAS G12X (X = D, V, R, C) human pancreatic cancer cell lines were used as a model system to study the cytotoxicity of SEQ ID NO. 1-3 (AsPc-1 cells (KRAS G12D mutation, 1640 culture medium), Panc-1 cells (KRAS G12D mutation, DMEM culture medium), Patu8988 (KRAS G12V mutation, DMEM culture medium), Mia-Paca2 cells (KRAS G12C mutation, DMEM culture medium) and PSN1 cells (KRAS G12R mutation, 1640 culture medium)). In a Corning 96-well plate, 5 x 10 3 cells were cultured in each well using 100 μL of complete culture medium (containing 10% fetal bovine serum FBS and 1% penicillin-streptomycin), and the 96-well plate was pre-cultured for 24 h at 37°C in a 5% carbon dioxide incubator to allow the cells to adhere. 10 μL of different concentrations of SEQ ID NO. 1-3 polypeptide PBS solution was added to each well of the Corning 96-well plate, so that the final concentration of SEQ ID NO. 1-3 polypeptide was 2 μM, 5 μM, 10 μM, 15 μM, 30 μM and 50 μM (or 100 μM), and the blank control group only added 10 μL of PBS solution. The 96-well cell culture plate was incubated in the incubator for 24 h and 48 h. After 24 h and 48 h of incubation, the culture medium was discarded, and the basal culture medium was mixed with the CCK8 reagent at a ratio of 10:1, and 100 μL of the mixture was added to the 96-well plate. After 1-2 h of incubation, the fluorescence intensity at an emission wavelength of 450 nm was detected using a microplate reader, and the IC 50 value was calculated based on the fluorescence intensity value.

[0127] SEQ ID NO. 1 can significantly inhibit the activity of KRAS G12X mutant human pancreatic cancer cells (AsPc-1 cells (KRAS G12D mutation) and Panc-1 cells (KRAS G12D mutation)).

[0128] SEQ ID NO. 2 can significantly inhibit the activity of KRAS G12X mutant human pancreatic cancer cells (AsPc-1 cells (KRAS G12D mutation) and Panc-1 cells (KRAS G12D mutation), Patu8988 (KRAS G12V mutation) and PSN1 cells (KRAS G12R mutation)).

[0129] SEQ ID NO. 3 significantly inhibited the activity of KRAS G12X mutant human pancreatic cancer cells (AsPc-1 cells (KRAS G12D mutation), Panc-1 cells (KRAS G12D mutation), Patu8988 (KRAS G12V mutation), Mia-Paca2 cells (KRAS G12C mutation) and PSN1 cells (KRAS G12R mutation).

[0130] As shown in FIG. 1A and Table 1, the IC50 of SEQ ID NO. 1 in AsPc-1 cells (KRAS G12D mutation) at 24h and 48h was 100.3 μM and 223.4 μM, respectively. 50 As shown in FIG. 1B and Table 1, the IC50 of SEQ ID NO. 1 in Panc-1 cells (KRAS G12D mutation) at 48h was 42.33 μM. 50 As shown in FIG. 1C and Table 1, the IC50 of SEQ ID NO. 1 in Patu8988 (KRAS G12V mutation) at 48h was NA. 50 As shown in FIG. 1D and Table 1, the IC50 of SEQ ID NO. 1 in Mia-Paca2 cells (KRAS G12C mutation) at 48h was NA. 50 As shown in FIG. 1E and Table 1, the IC50 of SEQ ID NO. 1 in PSN1 cells (KRAS G12R mutation) at 48h was NA. 50 As shown in FIG. 1E and Table 1, the IC50 of SEQ ID NO. 1 in PSN1 cells (KRAS G12R mutation) at 48h was NA.

[0131] As shown in FIG. 2A and Table 1, the IC50 of SEQ ID NO. 2 in AsPc-1 cells (KRAS G12D mutation) at 24h and 48h was 7.58 μM and 8.06 μM, respectively. 50 As shown in FIG. 2B and Table 1, the IC50 of SEQ ID NO. 2 in Panc-1 cells (KRAS G12D mutation) at 48h was 70.01 μM. 50 As shown in FIG. 2C and Table 1, the IC50 of SEQ ID NO. 2 in Patu8988 (KRAS G12V mutation) at 48h was 99.17 μM. 50 As shown in FIG. 2D and Table 1, the IC50 of SEQ ID NO. 2 in Mia-Paca2 cells (KRAS G12C mutation) at 48h was NA. 50 As shown in FIG. 2E and Table 1, the IC50 of SEQ ID NO. 2 in PSN1 cells (KRAS G12R mutation) at 48h was 64.17 μM. 50 As shown in FIG. 2E and Table 1, the IC50 of SEQ ID NO. 2 in PSN1 cells (KRAS G12R mutation) at 48h was 64.17 μM.

[0132] As shown in FIG. 3A and Table 1, the IC50 of SEQ ID NO. 3 in AsPc-1 cells (KRAS G12D mutation) at 24h and 48h was50 were 33.41 μΜ and 28.23 μΜ. As shown in Figure 3B and Table 1, the IC50of SEQ ID NO. 3 against Panc-1 cells (KRAS G12D mutant) at 24h and 48h were 50 were 33.19 μΜ and 22.00 μΜ. As shown in Figure 3C and Table 1, the IC50of SEQ ID NO. 3 against Patu8988 (KRAS G12V mutant) at 24h and 48h were 50 were 19.00 μΜ and 27.61 μΜ. As shown in Figure 3D and Table 1, the IC50of SEQ ID NO. 3 against Mia-Paca2 cells (KRAS G12C mutant) at 24h and 48h were 50 were 48.39 μΜ and 29.62 μΜ. As shown in Figure 3E and Table 1, the IC50of SEQ ID NO. 3 against PSN1 cells (KRAS G12R mutant) at 24h and 48h were 50 were 9.35 μΜ and 10.48 μΜ.

[0133] Table 1. Cytotoxicity of SEQ ID NO. 1-3 against KRAS G12X (X=D, V, R, C) pancreatic cancer cells (48 hours)

[0134] Test Example 2

[0135] Cytotoxicity of SEQ ID NO. 1-3 against KRAS WT normal pancreatic ductal epithelial cells and human embryonic kidney cells

[0136] To determine whether SEQ ID NO. 1-3 have selectivity at KRAS G12 mutant sites, the present inventors selected HPNE cells (human normal pancreatic ductal epithelial cells, DMEM medium) and 293T (human normal embryonic kidney cells, DMEM medium) with KRAS WT as controls. In Corning 96-well plates, 5 x 10 3The 96-well plate was pre-cultured at 37°C, 5% carbon dioxide condition in the incubator for 24h to make the cells adhere to the wall. 10μL of different concentrations of SEQ ID NO. 1-3 polypeptide PBS solution was added to each well of the Corning 96-well plate, so that the final concentration of SEQ ID NO. 1-3 polypeptide was 2μM, 5μM, 10μM, 15μM, 30μM and 50μM (or 100μM), and the blank control group only added 10μL of PBS solution. The 96-well cell culture plate was incubated in the incubator for 24h and 48h. After 24h and 48h of incubation, the culture medium was discarded, and the basal medium was mixed with the CCK8 reagent at a ratio of 10:1, and 100μL of the mixed solution was added to the 96-well plate. After 1-2h of incubation, the fluorescence intensity at an emission wavelength of 450nm was detected by a labeling instrument, and the IC 50 value was calculated according to the fluorescence intensity value.

[0137] As shown in FIG. 4A and Table 2, the IC 50 of SEQ ID NO. 1 in HPNE cells (KRAS WT) for 48h was 30.63μM. As shown in FIG. 4B and Table 2, the IC 50 of SEQ ID NO. 1 in 293T cells (KRAS WT) for 48h was NA.

[0138] As shown in FIG. 4C and Table 2, the IC 50 of SEQ ID NO. 2 in HPNE cells (KRAS WT) for 48h was 34.4μM. As shown in FIG. 4D and Table 2, the IC 50 of SEQ ID NO. 2 in 293T cells (KRAS WT) for 48h was 87.04μM.

[0139] As shown in FIG. 4E and Table 2, the IC 50 of SEQ ID NO. 3 in HPNE cells (KRAS WT) for 24h and 48h was 151.3μM and 70.61μM. As shown in FIG. 4F and Table 2, the IC 50 of SEQ ID NO. 3 in 293T cells (KRAS WT) for 24h and 48h was 69.60μM and 47.58μM. Thus, SEQ ID NO. 3 has a safety window of 3-5 times in KRAS G12X cells in the pancreas and pancreatic duct epithelial cells, and a safety window of 2-4 times in KRAS G12X cells in the pancreas and human normal embryonic kidney cells.

[0140] Table 2. Cytotoxicity of SEQ ID NO. 1-3 to KRAS WT normal pancreatic duct epithelial cells and human embryonic kidney cells (48 hours)

[0141] Test Example 3

[0142] Toxicity of SEQ ID NO. 3 to KRAS WT / G12D mutant human pancreatic cancer organoids

[0143] To further clarify whether there is selectivity of KRAS site for the toxicity of SEQ ID NO. 3 in human pancreatic cancer samples, the inventors constructed human pancreatic cancer organoid samples, plated 2000 organoids per well using organoid medium mixed with Matrigel, added 10 μL of different concentrations of SEQ ID NO. 3 polypeptide PBS solution to each well of the Corning 96-well plate, so that the final concentration of SEQ ID NO. 3 polypeptide was 10 μM, 15 μM, 30 μM and 50 μM, and the blank control group only added 10 μL of PBS solution. The 96-well cell culture plate was incubated in an incubator for 4 h, 24 h and 48 h, and the morphology of the organoids was observed and photographed. After the organoids were digested and resuspended, they were inoculated in a 96-well plate, and the organoids were counted as 6000 cells / ml. Then 10 μL of different concentrations of SEQ ID NO. 3 polypeptide PBS solution was added, so that the final concentration of SEQ ID NO. 3 polypeptide was 2 μM, 5 μM, 10 μM, 15 μM, 30 μM and 50 μM, and the blank control group only added 10 μL of PBS solution. After 24 h and 48 h, 100 μL of 2.0 Reagent. Place the 96-well plate on a 37°C shaker for 2 min to lyse the cells. After lysis is complete, place the well plate at room temperature for 10 min. Load the machine, record the luminescence value, and calculate the IC 50 value according to the fluorescence intensity value.

[0144] As shown in FIG. 5A and Table 3, after treating KRAS WT organoids with SEQ ID NO. 3, no obvious toxicity was observed in the organoids within 4 h, but concentration-dependent inhibition of organoid proliferation was observed after 24 h and 48 h of treatment. When SEQ ID NO. 3 was used to treat KRAS G12D mutant organoids, concentration-dependent inhibition of organoid proliferation was observed within 4 h, and concentration-dependent inhibition of organoid proliferation was observed after 24 h and 48 h of treatment, with an inhibition efficiency significantly higher than that of KRAS WT pancreatic cancer organoids. As shown in FIG. 5B and Table 3, the IC 50 of SEQ ID NO. 3 in KRAS G12D mutant organoids for 48 h was 24.48 μM. The IC 50 of SEQ ID NO. 3 in KRAS WT mutant organoids for 48 h was 7.93 μM. Thus, there is about 3-fold difference between KRAS G12D mutant organoids and KRAS WT organoids in the pancreas for SEQ ID NO. 3, and therefore there is selectivity of KRAS G12 mutation site for SEQ ID NO. 3.

[0145] Table 3 Toxicity of SEQ ID NO. 3 against KRAS WT human pancreatic cancer organoids and KRAS G12D mutant human pancreatic cancer organoids for 48 hours

[0146] Test Example 4

[0147] Toxicity of SEQ ID NO. 1-3 against KRAS G12D mutant colorectal and lung cancer cell lines

[0148] KRAS is also one of the initiating mutations in colorectal and lung cancer. To expand the indications of SEQ ID NO. 1-3, the inventors further determined the cytotoxicity of SEQ ID NO. 1-3 in KRAS G12D mutant colorectal (LS513, 1640 medium) and lung cancer cell lines (SK-LU-1, MEM medium). In Corning 96-well plates, 5x10 3 cells were cultured in each well using 100 μL of complete medium (containing 10% fetal bovine serum FBS and 1% penicillin-streptomycin), and the 96-well plates were pre-cultured for 24 h in an incubator at 37°C, 5% carbon dioxide to allow the cells to adhere. 10 μL of different concentrations of SEQ ID NO. 1-3 polypeptide PBS solution was added to each well of the Corning 96-well plate, so that the final concentration of SEQ ID NO. 1-3 polypeptide was 2 μM, 5 μM, 10 μM, 15 μM, 30 μM, 50 μM and 100 μM, and the blank control group only added 10 μL of PBS solution, and the 96-well cell culture plate was incubated in the incubator for 24 h and 48 h. After 24 h and 48 h of incubation, the culture medium was discarded, and the basal medium was mixed with CCK8 reagent at a ratio of 10:1, and 100 μL of the mixture was added to the 96-well plate. After 1-2 h of incubation, the fluorescence intensity at an emission wavelength of 450 nm was detected with a labeling instrument, and the IC 50 value was calculated according to the fluorescence intensity value.

[0149] As shown in FIG. 6A and Table 4, the IC 50 of SEQ ID NO. 1 in LS513 colorectal cancer cell lines (KRAS G12D mutant) for 48 h was 103.9 μM. As shown in FIG. 6B and Table 4, the IC 50 of SEQ ID NO. 1 in SK-LU-1 lung cancer cell lines (KRAS G12D mutant) for 48 h was 70.85 μM.

[0150] As shown in FIG. 6C and Table 4, the IC 50was 99.16 μΜ. As shown in Figure 6D and Table 4, the IC50of SEQ ID NO. 2 in SK-LU-1 lung cancer cell line (KRAS G12D mutant) at 48h was 50 was 45.71 μΜ.

[0151] As shown in Figure 6E and Table 4, the IC50of SEQ ID NO. 3 in LS513 colorectal cancer cell line (KRAS G12D mutant) at 24h and 48h was 50 were 38.37 μΜ and 13.03 μΜ. As shown in Figure 6F and Table 4, the IC50of SEQ ID NO. 3 in SK-LU-1 lung cancer cell line (KRAS G12D mutant) at 24h and 48h was 50 were 13.41 μΜ and 27.70 μΜ.

[0152] Table 4. Toxicity of SEQ ID NO. 1-3 against KRAS G12D mutant colorectal and lung cancer cell lines

[0153] Test Example 5

[0154] Toxicity of SEQ ID NO. 4-NO. 9 against KRAS G12X (X=D, V, R, C) mutant pancreatic cancer, KRAS G12D mutant lung cancer and KRAS G12D mutant colorectal cancer cells

[0155] KRAS G12X (X=D, V, R, C) human pancreatic cancer cell lines and KRAS G12D mutant colorectal and lung cancer cell lines were used as model systems to study the cytotoxicity of SEQ ID NO. 4-NO. 9 ((AsPc-1 cells (KRAS G12D mutant, 1640 medium), Panc-1 cells (KRAS G12D mutant, DMEM medium), Patu8988 (KRAS G12V mutant, DMEM medium), Mia-Paca2 cells (KRAS G12C mutant, DMEM medium) and PSN1 cells (KRAS G12R mutant, 1640 medium), KRAS G12D mutant colorectal cancer (LS513, 1640 medium) and KRAS G12D mutant lung cancer cell line (SK-LU-1, MEM medium). 5 x 10 3cells, and pre-cultured the 96-well plate in an incubator at 37°C and 5% carbon dioxide for 24 hours to allow the cells to adhere. 10 μL of PBS solution of SEQ ID NO.4-NO.9 polypeptides of different concentrations was added to each well of the Corning 96-well plate to make the final concentration of SEQ ID NO.4-NO.9 polypeptides 2μM, 5μM, 10μM, 15μM, 30μM and 50μM (or 100μM). Only 10μL of PBS solution was added to the blank control group, and the 96-well cell culture plate was incubated in the incubator for 24 hours and 48 hours. After incubation for 24 hours and 48 hours, the culture medium was discarded, and the basal culture medium and CCK8 reagent were mixed at a ratio of 10:1. 100 μL of the mixture was added to the 96-well plate. After incubation for 1-2 hours, the fluorescence intensity at an emission wavelength of 450nm was detected using a calibration instrument, and the IC was calculated based on the fluorescence intensity value. 50 Numeric value.

[0156] As shown in Figure 7A, Table 5 and Table 6, the IC values ​​of SEQ ID NO. 4 at 24h and 48h in the AsPc-1 pancreatic cancer cell line (KRAS G12D mutation) were 50 As shown in Figure 7B, Table 5 and Table 6, the IC values ​​of SEQ ID NO.4 in Panc-1 pancreatic cancer cell line (KRAS G12D mutation) for 48 h were 52.82 μM and 35.22 μM. 50 As shown in Figure 7C, Table 5 and Table 6, the IC value of SEQ ID NO.4 in Patu8988 (KRAS G12V mutation) for 48h was 24.23μM. 50 As shown in Figure 7D, Table 5 and Table 6, the IC value of SEQ ID NO.4 in PSN1 cells (KRAS G12R mutation) for 48 h was 22.78 μM. 50 As shown in Figure 7E, Table 5 and Table 6, the IC value of SEQ ID NO.4 in Mia-Paca2 cells (KRAS G12C mutation) for 48 h was 25.34 μM. 50 As shown in Figure 7F, Table 5 and Table 6, the IC values ​​of SEQ ID NO. 4 in the LS513 colorectal cancer cell line (KRAS G12D mutation) at 24h and 48h were 30.03μM. 50 As shown in Figure 7G, Table 5 and Table 6, the IC values ​​of SEQ ID NO.4 in SK-LU-1 lung cancer cell line (KRAS G12D mutation) at 24h and 48h were 59.56μM and 32.63μM, respectively. 50 65.97 μM and 42.97 μM.

[0157] As shown in Figure 8A, Table 5 and Table 6, the IC values ​​of SEQ ID NO.5 at 24h and 48h in the AsPc-1 pancreatic cancer cell line (KRAS G12D mutation) were50 NA and 2.15 mM. As shown in Figure 8B, Table 5 and Table 6, the IC50of SEQ ID NO. 5 at 48h in Panc-1 pancreatic cancer cell line (KRAS G12D mutation) was 50 NA. As shown in Figure 8C, Table 5 and Table 6, the IC50of SEQ ID NO. 5 at 48h in Patu8988 (KRAS G12V mutation) was 50 NA. As shown in Figure 8D, Table 5 and Table 6, the IC50of SEQ ID NO. 5 at 48h in PSN1 cells (KRAS G12R mutation) was 50 NA. As shown in Figure 8E, Table 5 and Table 6, the IC50of SEQ ID NO. 5 at 48h in Mia-Paca 2 cells (KRAS G12C mutation) was 50 NA. As shown in Figure 8F, Table 5 and Table 6, the IC50of SEQ ID NO. 5 at 24h and 48h in LS513 colorectal cancer cell line (KRAS G12D mutation) was 50 NA. As shown in Figure 8G, Table 5 and Table 6, the IC50of SEQ ID NO. 5 at 24h and 48h in SK-LU-1 lung cancer cell line (KRAS G12D mutation) was 50 NA and 296.2 mM.

[0158] As shown in Figure 9A, Table 5 and Table 6, the IC50of SEQ ID NO. 6 at 24h and 48h in AsPc-1 pancreatic cancer cell line (KRAS G12D mutation) was 50 22.00 mM and 15.37 mM. As shown in Figure 9B, Table 5 and Table 6, the IC50of SEQ ID NO. 6 at 48h in Panc-1 pancreatic cancer cell line (KRAS G12D mutation) was 50 35.23 mM. As shown in Figure 9C, Table 5 and Table 6, the IC50of SEQ ID NO. 6 at 48h in Patu8988 (KRAS G12V mutation) was 50 17.80 mM. As shown in Figure 9D, Table 5 and Table 6, the IC50of SEQ ID NO. 6 at 48h in PSN1 cells (KRAS G12R mutation) was 50 32.04 mM. As shown in Figure 9E, Table 5 and Table 6, the IC50of SEQ ID NO. 6 at 48h in Mia-Paca2 cells (KRAS G12C mutation) was 50 20.35 mM. As shown in Figure 9F, Table 5 and Table 6, the IC50of SEQ ID NO. 6 at 24h and 48h in LS513 colorectal cancer cell line (KRAS G12D mutation) was 50were 30.30 μΜ and 21.74 μΜ. As shown in Figure 9G, Table 5 and Table 6, the IC50of SEQ ID NO. 6 at 24h and 48h in the SK-LU-1 lung cancer cell line (KRAS G12D mutant) were 50 were 59.56 μΜ and 32.63 μΜ.

[0159] As shown in Figure 10A, Table 5 and Table 6, the IC50of SEQ ID NO. 7 at 24h and 48h in the AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) were 50 were 27.52 μΜ and 22.94 μΜ. As shown in Figure 10B, Table 5 and Table 6, the IC50of SEQ ID NO. 7 at 48h in the Panc-1 pancreatic cancer cell line (KRAS G12D mutant) was 50 was 27.20 μΜ. As shown in Figure 10C, Table 5 and Table 6, the IC50of SEQ ID NO. 7 at 48h in Patu8988 (KRAS G12V mutant) was 50 was 28.30 μΜ. As shown in Figure 10D, Table 5 and Table 6, the IC50of SEQ ID NO. 7 at 48h in PSN1 cells (KRAS G12R mutant) was 50 was 35.23 μΜ. As shown in Figure 10E, Table 5 and Table 6, the IC50of SEQ ID NO. 7 at 48h in Mia-Paca2 cells (KRAS G12C mutant) was 50 was 30.51 μΜ. As shown in Figure 10F, Table 5 and Table 6, the IC50of SEQ ID NO. 7 at 24h and 48h in the LS513 colorectal cancer cell line (KRAS G12D mutant) were 50 were 34.99 μΜ and 20.51 μΜ. As shown in Figure 10G, Table 5 and Table 6, the IC50of SEQ ID NO. 7 at 24h and 48h in the SK-LU-1 lung cancer cell line (KRAS G12D mutant) were 50 were 24.35 μΜ and 15.71 μΜ.

[0160] As shown in Figure 11A, Table 5 and Table 6, the IC50of SEQ ID NO. 8 at 24h and 48h in the AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) were 50 were 34.65 μΜ and 39.89 μΜ. As shown in Figure 11B, Table 5 and Table 6, the IC50of SEQ ID NO. 8 at 48h in the Panc-1 pancreatic cancer cell line (KRAS G12D mutant) was 50 was 344.3 μΜ. As shown in Figure 11C, Table 5 and Table 6, the IC50of SEQ ID NO. 8 at 48h in Patu8988 (KRAS G12V mutant) was 50was 105.3 μΜ. As shown in Figure 11D, Table 5 and Table 6, the IC50of SEQ ID NO. 8 at 48h in PSN1 cells (KRAS G12R mutant) was 50 was 20.67 μΜ. As shown in Figure 11E, Table 5 and Table 6, the IC50of SEQ ID NO. 8 at 48h in Mia-Paca2 cells (KRAS G12C mutant) was 50 was 720.2 μΜ. As shown in Figure 11F, Table 5 and Table 6, the IC50of SEQ ID NO. 8 at 24h and 48h in LS513 colorectal cancer cell line (KRAS G12D mutant) was 50 was 53.39 μΜ and 33.08 μΜ. As shown in Figure 11G, Table 5 and Table 6, the IC50of SEQ ID NO. 8 at 24h and 48h in SK-LU-1 lung cancer cell line (KRAS G12D mutant) was 50 was 40.00 μΜ and 14.90 μΜ.

[0161] As shown in Figure 12A, Table 5 and Table 6, the IC50of SEQ ID NO. 9 at 24h and 48h in AsPc-1 pancreatic cancer cell line (KRAS G12D mutant) was 50 was 47.76 μΜ and 21.57 μΜ. As shown in Figure 12B, Table 5 and Table 6, the IC50of SEQ ID NO. 9 at 48h in Panc-1 pancreatic cancer cell line (KRAS G12D mutant) was 50 was NA. As shown in Figure 12C, Table 5 and Table 6, the IC50of SEQ ID NO. 9 at 48h in Patu8988 (KRAS G12V mutant) was 50 was NA. As shown in Figure 12D, Table 5 and Table 6, the IC50of SEQ ID NO. 9 at 48h in PSN1 cells (KRAS G12R mutant) was 50 was NA. As shown in Figure 12E, Table 5 and Table 6, the IC50of SEQ ID NO. 9 at 48h in Mia-Paca 2 cells (KRAS G12C mutant) was 50 was 116.3 μΜ. As shown in Figure 12F, Table 5 and Table 6, the IC50of SEQ ID NO. 9 at 24h and 48h in LS513 colorectal cancer cell line (KRAS G12D mutant) was 50 was 33.64 μΜ and 27.07 μΜ. As shown in Figure 12G, Table 5 and Table 6, the IC50of SEQ ID NO. 9 at 24h and 48h in SK-LU-1 lung cancer cell line (KRAS G12D mutant) was 50 was 111.5 μΜ and 93.35 μΜ.

[0162] Table 5. Toxicity of SEQ ID NO. 4-9 against KRAS G12X (X = D, V, R, C) mutant colorectal and lung cancer cell lines (48 hours)

[0163] Table 6. Toxicity of SEQ ID NO. 1-3 against KRAS G12D mutant colorectal and lung cancer cell lines

[0164] Test Example 6

[0165] Cytotoxicity of SEQ ID NO. 4-9 on KRAS WT normal pancreatic duct epithelial cells and human embryonic kidney cells

[0166] To determine whether SEQ ID NO. 4-9 is selective at the KRAS G12 mutant site, the inventors selected KRAS WT HPNE cells (human normal pancreatic duct epithelial cells, DMEM medium) and 293T (human normal embryonic kidney cells, DMEM medium) as controls. In a Corning 96-well plate, 5 x 10 3 cells were cultured in each well using 100 μL of complete medium (containing 10% fetal bovine serum FBS and 1% penicillin-streptomycin), and the 96-well plate was pre-cultured for 24 h in an incubator at 37°C, 5% carbon dioxide to allow the cells to adhere. 10 μL of different concentrations of SEQ ID NO. 4-9 polypeptide PBS solution was added to each well of the Corning 96-well plate, so that the final concentration of SEQ ID NO. 4-9 polypeptide was 2 μM, 5 μM, 10 μM, 15 μM, 30 μM and 50 μM (or 100 μM), and the blank control group only added 10 μL of PBS solution. The 96-well cell culture plate was incubated in the incubator for 24 h and 48 h. After 24 h and 48 h of incubation, the culture medium was discarded, and the basal medium was mixed with CCK8 reagent at a ratio of 10:1, and 100 μL of the mixture was added to the 96-well plate. After 1-2 h of incubation, the fluorescence intensity at an emission wavelength of 450 nm was detected using a microplate reader, and the IC 50 value was calculated according to the fluorescence intensity value.

[0167] As shown in FIG. 13A and Table 7, the IC 50 of SEQ ID NO. 4 in HPNE cells (KRAS WT) for 48 h was 48.50 μM. As shown in FIG. 13B and Table 7, the IC 50 of SEQ ID NO. 4 in 293T cells (KRAS WT) for 48 h was 13.58 μM.

[0168] As shown in FIG. 13C and Table 7, the IC 50NA. As shown in FIG. 13D and Table 7, the IC50 of SEQ ID NO. 5 on HPNE cells (KRAS WT) for 48h was 50 NA.

[0169] As shown in FIG. 13E and Table 7, the IC50 of SEQ ID NO. 6 on HPNE cells (KRAS WT) for 48h was 50 9.88 μΜ. As shown in FIG. 13F and Table 7, the IC50 of SEQ ID NO. 6 on 293T cells (KRAS WT) for 48h was 50 52.22 μΜ.

[0170] As shown in FIG. 14A and Table 7, the IC50 of SEQ ID NO. 7 on HPNE cells (KRAS WT) for 48h was 50 33.12 μΜ. As shown in FIG. 14B and Table 7, the IC50 of SEQ ID NO. 7 on 293T cells (KRAS WT) for 48h was 50 75.81 μΜ.

[0171] As shown in FIG. 14C and Table 7, the IC50 of SEQ ID NO. 8 on HPNE cells (KRAS WT) for 48h was 50 29.49 μΜ. As shown in FIG. 14D and Table 7, the IC50 of SEQ ID NO. 8 on 293T cells (KRAS WT) for 48h was 50 114.4 μΜ.

[0172] As shown in FIG. 14E and Table 7, the IC50 of SEQ ID NO. 9 on HPNE cells (KRAS WT) for 48h was 50 85.10 μΜ. As shown in FIG. 14F and Table 7, the IC50 of SEQ ID NO. 9 on 293T cells (KRAS WT) for 48h was 50 NA.

[0173] Table 7. Cytotoxicity (48 hours) of SEQ ID NO. 4-9 against KRAS WT normal pancreatic ductal epithelial cells and human embryonic kidney cells

[0174] Test Example 7

[0175] Effect of SEQ ID NO. 3 on apoptosis of KRAS G12D mutant pancreatic cancer cell AsPc-1

[0176] The KRAS G12D human pancreatic cancer cell line was used as a model system to study the cytotoxicity of SEQ ID NO. 3 (AsPc-1 cells (KRAS G12D mutation, 1640 culture medium). 3×10 cells were cultured in each well of a Corning 6-well plate using 2 mL of complete culture medium (containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin). 6 Cells were pre-incubated in a 6-well plate at 37°C, 5% CO2 incubator for 24 hours to allow attachment. 10 μL of PBS solution containing different concentrations of the SEQ ID NO. 3 peptide was added to each well of a Corning 6-well plate, resulting in final concentrations of 10 μM and 30 μM. A blank control group was treated with 10 μL of PBS solution alone. The 6-well plate was incubated in an incubator for 48 hours. After 48 hours of incubation, the cells were trypsinized with EDTA-free trypsin, washed with PBS (300g, 5 minutes), and resuspended in 1 mL of 1x Binding Buffer to a cell density of 1x106 cells / mL. 100 μL of cells were added to each tube. 10 μL of Annexin V and PI reagent (1:1) were added to the tube and incubated at room temperature in the dark for 5 minutes. PBS was then added to 500 μL, mixed, and analyzed by flow cytometry within 1 hour.

[0177] Utilize flow cytometer (FCM, Applied Biosystems, Life Technologies, Carlsbad, CA), set emission wavelength 488nm, detection wavelength 535nm (1 channel) and emission wavelength 617nm, detection wavelength and 647nm (3 channels). Negative control group cell sample is placed on instrument sample holder and starts detection, according to cell size, in the scatter plot of forward angle signal (FSC), lateral angle signal (SSC), set gate, and set threshold value before recording detection result, make the quantitative statistical peak graph of fluorescence intensity, the cell fluorescence intensity in the gate is higher than the number of above threshold fluorescence intensity less than 1%, after setting, count 10,000 cells. Under the above-mentioned gate setting and counting conditions, detect blank control group and experimental group samples in turn, record corresponding detection value, i.e., the number percentage and statistical analysis result that are higher than Annexin-V threshold fluorescence intensity.

[0178] As shown in FIG15A , SEQ ID NO. 3 can promote the apoptosis of AsPc-1 in a concentration-dependent manner, and there are significant statistical differences in the apoptosis rates among the groups ( FIG15B ).

[0179] Test Example 8

[0180] Effect of SEQ ID NO.3 on apoptosis of KRAS G12D mutant human pancreatic cancer cells Panc-1

[0181] The KRAS G12D human pancreatic cancer cell line was used as a model system to study the cytotoxicity of SEQ ID NO. 3 (Panc-1 cells (KRAS G12D mutant, DMEM medium)). 3 x 10 6 The cells were pre-cultured in a Corning 6-well plate for 24 h at 37°C in a 5% CO2 incubator to allow the cells to adhere. 10 μL of a PBS solution of SEQ ID NO. 3 polypeptide at different concentrations was added to each well of the Corning 6-well plate to give a final concentration of 10 μM and 30 μM of SEQ ID NO. 3 polypeptide, and 10 μL of a PBS solution was added to the blank control group. The 6-well cell culture plate was incubated in the incubator for 48 h. After 48 h of incubation, the cells were trypsinized without EDTA, washed with PBS (300 g, 5 min), resuspended with 1 mL of 1x Binding Buffer, and the density of the cells was adjusted to 1 x 106 cells / mL. 100 μL of the cells was added to each tube, 10 μL of Annexin V and PI reagent (1:1) was added to the tube, and the tube was incubated at room temperature in the dark for 5 min. PBS was added to 500 μL, mixed, and then detected by flow cytometry within 1 h. The flow cytometer (FCM, Applied Biosystems, Life Technologies, Carlsbad, CA) was set to an emission wavelength of 488 nm, a detection wavelength of 535 nm (channel 1), and an emission wavelength of 617 nm, a detection wavelength of 647 nm (channel 3). The negative control group of cell samples was placed in the instrument sample holder and detection was started. According to the cell size, the forward angle signal (FSC) and the lateral angle signal (SSC) were gated in the scatter plot, and the threshold value was set before recording the detection results, so that the number of cells in the gate with a fluorescence intensity higher than the threshold fluorescence intensity was less than 1% in the number statistics peak graph of fluorescence intensity. After the settings were completed, 10,000 cells were counted. Under the above gate setting and counting conditions, the blank control group and the experimental group samples were detected in turn, and the corresponding detection values, i.e., the percentage of the number of cells with a fluorescence intensity higher than the threshold fluorescence intensity of Annexin-V and the statistical analysis results were recorded.

[0182] As shown in FIG. 16A, SEQ ID NO. 3 can promote apoptosis of AsPc-1 in a concentration-dependent manner, and there is a significant statistical difference in the apoptosis rate of each group (FIG. 16B).

[0183] Test Example 9

[0184] Results of surface plasmon resonance experiments on SEQ ID NO. 1-9 on KRAS G12X (X = D, V, R, C)

[0185] The binding affinity of SEQ ID NO. 3 to KRAS G12X (X = D, V, R, C) mutant proteins was determined by BIACORE model 3000 surface plasmon resonance (SPR). The polypeptide SEQ ID NO. 3 was immobilized on a chip. Proteins were diluted in assay running buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.5 mM TCEP, 0.03% Triton X-100 and 5 mM GDP). The inlet was cleaned between cycles with a solution containing 50 mM NaOH and 1 M NaCl. Experimental data were subtracted from the reference cell, and a third equilibrium cycle was used to subtract baseline drift. Data were processed using T200 Bia evaluation software and a binary fit model. The KD value for each interaction is reported as shown in Table 8. The binding affinity of SEQ ID NO. 3 to KRAS G12D mutant protein was 0.329 pM, and the binding affinity of SEQ ID NO. 3 to KRAS G12C, KRAS G12V and KRAS G12R mutant proteins were all < 0.01 pM, even postulated to not dissociate after binding. Thus, the binding affinity of SEQ ID NO. 3 to KRAS G12X (X = D, V, R, C) mutant proteins was very strong.

[0186] Table 8. Affinity (KD) of SEQ ID NO. 3 to KRAS G12X (X = D, V, R, C) mutant proteins

[0187] Test Example 10

[0188] Circular dichroism experimental results for SEQ ID NO. 1-9 to KRAS G12X (X = D, V, R, C)

[0189] The effect of polypeptide SEQ ID NO. 3 on the secondary structure of KRAS G12X (X = D, V, R, C) mutant protein was determined by circular dichroism (CD) spectroscopy. The experiment used a 0.1 cm path length quartz cuvette and a polarimeter instrument (J-1500, JASCO, Japan). Polypeptide SEQ ID NO. 3 was dissolved in ultrapure water at room temperature, and KRAS G12X (X = D, V, R, C) mutant protein was dissolved in buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.5 mM TCEP, 0.03% Triton X-100, and 5 mM GDP). The mixed solution of KRAS G12X (X = D, V, R, C) mutant protein (0.1 mM) and polypeptide SEQ ID NO. 3 (0.01 mM, 0.1 mM, 1 mM, and 10 mM) was measured and recorded on the polarimeter instrument at 190-240 nm. The CD spectroscopy data used in this paper were blank corrected, and the secondary structure of the protein was fitted using a fitting model. The data of polypeptide SEQ ID NO. 3 on the secondary structure of KRAS G12X (X = D, V, R, C) mutant protein are shown in FIG. 17 and Table 9. The change in the secondary structure of KRAS G12X (X = D, V, R, C) mutant protein by polypeptide SEQ ID NO. 3 affects the function of the protein.

[0190] As shown in FIG. 17A, SEQ ID NO. 3 can cause the KRAS G12D protein to decrease in alpha helix, increase in beta sheet, increase in beta turn, and decrease in random coil, and the alpha helix to shift to beta sheet.

[0191] As shown in FIG. 17B, SEQ ID NO. 3 can cause the KRAS G12V protein to decrease in alpha helix, increase in beta sheet, decrease in beta turn, and decrease then increase in random coil, and the alpha helix to shift to beta sheet.

[0192] As shown in FIG. 17C, SEQ ID NO. 3 can cause the KRAS G12R protein to increase in alpha helix, decrease then increase in beta sheet, increase then decrease in beta turn, and decrease then increase in random coil.

[0193] As shown in FIG. 17D, SEQ ID NO. 3 can cause the KRAS G12C protein to decrease in alpha helix, increase in beta sheet, decrease then increase in beta turn, and decrease then increase in random coil, and the alpha helix to shift to beta sheet.

[0194] Table 9. Effect of SEQ ID NO. 3 on the secondary structure of KRAS G12X (X = D, V, R, C) mutant protein

[0195] Although the above test examples 1-10 show the effects of some embodiments, those skilled in the art should understand that according to the concept of the present application, the foregoing other embodiments not specifically showing the effects or other technical solutions of the present application not shown in the embodiments or test examples can also achieve the following technical effects declared in the part of the invention content as comparable to test examples 1-10:

[0196] 1. The prior art switches and combines the operation by multiple gas circuits circulation and refrigeration mode, the process of temperature rising and re-cooling needs partial gas collection and re-liquefaction, consumes time, and is inconvenient to operate; the scheme proposed in the present application can realize large temperature range temperature variation by only switching the thermal switch, does not affect the operation of the dilution refrigeration cycle, and does not need gas collection and re-liquefaction, and is simple and convenient to operate.

[0197] 2. The prior art performs temperature variation (convective heat exchange) in the high temperature zone by helium circulation; the scheme proposed in the present application establishes thermal connection (thermal conduction) between the variable temperature cold plate and the cold plates of each stage of the dilution refrigerator by the thermal switch, and is expected to realize higher temperature control precision.

[0198] Although the present application has been described to a certain extent, obviously, appropriate changes can be made to each condition without departing from the spirit and scope of the present application. It can be understood that the present application is not limited to the described embodiments, but is subject to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A polypeptide targeting a KRAS mutant protein, characterized in that, the polypeptide is capable of specifically binding to a KRAS G12X mutant protein, wherein X is selected from one or more of the following amino acids: D, V, R, C, A, S, preferably selected from one or more of the following amino acids: D, V, R, C; wherein, the amino acid sequence of the C-terminus of the polypeptide comprises: YGRKKRRQRRR; and, the polypeptide consists of 22-30 amino acids, preferably 24-30 amino acids, more preferably 26-30 amino acids.

2. The polypeptide of claim 1, wherein, the amino acid sequence of the polypeptide from N-terminus to C-terminus is B-YGRKKRRQRRR, the amino acid sequence of B is B1VVB2RB3; wherein, the amino acid in the amino acid sequence of B1 is selected from one or more of the following: D, R, Q, Y, L, V; the amino acid in the amino acid sequence of B2 is selected from one or more of the following: I, R, G, W, V, A; the amino acid in the amino acid sequence of B3 is selected from one or more of the following: V, G, D, S, T, C, I, L, R.

3. The polypeptide of claim 2, wherein, the amino acid in the amino acid sequence of B2 is selected from one or more of the following: I, R, G, W, V.

4. The polypeptide of claim 2 or 3, wherein: the number of amino acids in the amino acid sequence of B1 is 2-10, preferably 3-8, more preferably 3-5; the number of amino acids in the amino acid sequence of B2 is 2-6, preferably 2-4, most preferably 3; and / or the number of amino acids in the amino acid sequence of B3 is 3-10, preferably 3-9, more preferably 4-8.

5. The polypeptide of any one of claims 1-4, wherein: the amino acid sequence of the polypeptide is selected from one or more of the following: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9; preferably selected from one or more of the following: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8; more preferably selected from one or more of the following: SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.

7. the N-terminus of the amino acid sequence of the polypeptide can be modified by one or more of the following modification groups: biotin, fatty acid chain, polyethylene glycol, preferably modified by biotin or fatty acid chain, most preferably modified by biotin.

6. The polypeptide according to any one of claims 1 to 5, characterized in that, 7. Use of the polypeptide of any one of claims 1-6 in the preparation of a medicament for the treatment or adjuvant treatment of cancer. The medicament comprises: the polypeptide of any one of claims 1-6.

8. A medicament for the treatment or adjuvant treatment of cancer, characterized in that, The pharmaceutical composition comprises:

9. A pharmaceutical composition, characterized by, ​ 1) the polypeptide of any one of claims 1 to 6 or the medicament for treating or adjuvant treatment of cancer of claim 8; and 2) one or more pharmaceutically acceptable carriers.

10. A method for the treatment or adjuvant treatment of cancer, characterized in that, The method comprises administering to a subject in need thereof the polypeptide of any one of claims 1 to 6, the medicament for treating or adjuvant treatment of cancer of claim 8 or the pharmaceutical composition of claim 9.

11. A targeted intervention method, characterized in that, The method of targeted intervention comprises administering to a subject in need thereof the polypeptide of any one of claims 1 to 6, the medicament for treating or adjuvant treatment of cancer of claim 8 or the pharmaceutical composition of claim 9.

12. A method of drug delivery, comprising: The method of drug-mediated comprises administering to a subject in need thereof the polypeptide of any one of claims 1 to 6, the medicament for treating or adjuvant treatment of cancer of claim 8 or the pharmaceutical composition of claim 9.

13. Use according to claim 7, medicament for the treatment or adjuvant treatment of cancer according to claim 8, pharmaceutical composition according to claim 9, method for the treatment or adjuvant treatment of cancer according to claim 10, method of targeted intervention according to claim 11 or method of drug mediation according to claim 12, characterized in that, The cancer is selected from one or more of the following: colon cancer, lung cancer, pancreatic cancer, breast cancer, osteosarcoma and / or ovarian cancer, preferably from one or more of the following: colon cancer, lung cancer, pancreatic cancer, breast cancer and / or ovarian cancer, more preferably from one or more of the following: colorectal cancer, lung cancer, pancreatic cancer.

14. The use of claim 7, the medicament for treating or adjuvant treatment of cancer of claim 8, the pharmaceutical composition of claim 9, the method for treating or adjuvant treatment of cancer of claim 10, the method of targeted intervention of claim 11 or the method of drug-mediated of claim 12, characterized in that: The polypeptide is capable of specifically regulating the secondary structure of KRAS G12X mutant protein, wherein X is selected from one or more of the following amino acids: D, V, R, C, A, S, preferably from one or more of the following amino acids: D, V, R, C; and / or The polypeptide has the ability to induce the death of KRAS G12X mutant tumor cells.

15. Use according to claim 7, medicament for the treatment or adjuvant treatment of cancer according to claim 8, pharmaceutical composition according to claim 9, method for the treatment or adjuvant treatment of cancer according to claim 10, method of targeted intervention according to claim 11 or method of drug mediation according to claim 12, characterized in that, The KRAS G12X is selected from one or more of the following subtypes: KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, KRAS G12A, KRAS G12S, preferably from one or more of the following subtypes: KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C.

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