Tumor drug marker protein kinase HUNK and application thereof

Abstract

This invention discloses the application of a tumor drug marker, the protein kinase HUNK, in the preparation of drugs for treating tumors and metastatic diseases. These drugs include components for inhibiting HUNK expression and nanomedicines. This invention inhibits cellular endocytosis mediated by caveolin, a major pathway for nanomedicines such as albumin-bound paclitaxel to enter cells. The sirna of this protein and its inhibitors can serve as nanomedicine therapies for cancers with high HUNK gene expression. In experimental applications, tumor cells with high HUNK gene expression are insensitive to albumin-bound paclitaxel, while cells treated with HUNK knockout or its inhibitor are more sensitive. Experiments showed that knockout or inhibition of HUNK promotes albumin endocytosis and its entry into lysosomes for degradation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a tumor drug marker protein kinase HUNK and its applications. Background Technology

[0002] A tumor is a new growth formed by the proliferation and abnormal differentiation of cells under the influence of various initiating and promoting factors. Tumor growth is not regulated by normal bodily physiology; instead, it destroys normal tissues and organs. Based on their biological behavior, tumors can be classified as benign tumors, malignant tumors, and borderline tumors between benign and malignant. Tumors with a clearly defined mass are solid tumors, while those without a clearly defined mass are non-solid tumors, most of which are hematologic malignancies. Antibody drugs and nanomedicines are commonly used to treat tumors. Nanomedicines are broad-spectrum tumor drugs, but their limited internalization efficiency restricts their effectiveness in clinical tumor treatment. Summary of the Invention

[0003] The purpose of this invention is to apply the protein kinase HUNK marker in the preparation of drugs for treating tumors and diseases related to tumor metastasis. By inhibiting cellular endocytosis mediated by caveolin through HUNK, this endocytosis pathway is a major route for nanomedicines such as albumin-bound paclitaxel to enter cells. The sirna of this protein and its inhibitors can be used as nanomedicines for the treatment of cancers with high expression of this gene.

[0004] The objective of this invention can be achieved through the following technical solutions:

[0005] A pharmaceutical composition comprising a) a nanomedicine, and at least one of b) and c) below:

[0006] b) Reagents that reduce or eliminate HUNK gene expression.

[0007] c) Reagents that reduce or eliminate the kinase activity of HUNK proteins.

[0008] Furthermore, the reagent for reducing or eliminating HUNK gene expression is selected from at least one of the following:

[0009] b1) A polynucleotide comprising a nucleotide sequence that reduces or eliminates the expression level of the HUNK gene.

[0010] b2) A recombinant vector comprising the polynucleotide sequence shown in b1).

[0011] Preferably, the polynucleotide shown in b1) comprises shRNA, siRNA, and / or sgRNA targeting the HUNK gene.

[0012] Furthermore, the polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO:XX, or a nucleotide sequence having at least 90% sequence identity with it.

[0013] Furthermore, the reagents for reducing or eliminating the kinase activity of HUNK protein are selected from:

[0014] Serine / threonine protein kinase inhibitors and tyrosine protein kinase inhibitors.

[0015] Furthermore, the nanomedicine is selected from taxanes; preferably paclitaxel or albumin-bound paclitaxel; more preferably, the concentration of albumin-bound paclitaxel in the pharmaceutical composition is 0.1-1000 nM, more preferably 10-1000 nM.

[0016] Furthermore, the use of the pharmaceutical composition in the preparation of medicaments for treating cancer or tumors;

[0017] Preferably, the cancer or tumor is selected from colon cancer, rectal cancer, breast cancer, endometrial cancer, squamous cell carcinoma, follicular lymphoma, renal cell carcinoma, uveal melanoma, cervical cancer, head and neck cancer, Hodgkin's disease, astrocytoma, lung adenocarcinoma, mesothelioma, choriocarcinoma, melanoma, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, brain cancer, stomach cancer, bladder cancer, esophageal cancer, cervical cancer, multiple myeloma, leukemia, lymphoma, or glioblastoma.

[0018] Furthermore, the use of at least one reagent selected from b) and c) below in the preparation of a medicament for promoting endocytosis:

[0019] b) Reagents to reduce or eliminate HUNK gene expression.

[0020] c) Reagents that reduce or eliminate the kinase activity of HUNK proteins.

[0021] Preferably, the drug for promoting cellular endocytosis is a drug for promoting the endocytosis of nanomedicines.

[0022] Preferably, the nanomedicine is selected from chemotherapeutic agents, immune checkpoint inhibitors, or antibody-drug conjugates.

[0023] Furthermore, the use of at least one reagent selected from b) and c) below in the preparation of a medicament for inhibiting AGAP3 protein phosphorylation:

[0024] b) Reagents to reduce or eliminate HUNK gene expression.

[0025] c) Reagents that reduce or eliminate the kinase activity of HUNK proteins.

[0026] Furthermore, a method for promoting cell endocytosis includes the step of administering at least one of the following reagents to the cells:

[0027] b) Reagents to reduce or eliminate HUNK gene expression.

[0028] c) Reagents that reduce or eliminate the kinase activity of HUNK proteins.

[0029] Preferably, the cells are tumor cells.

[0030] Optionally, the cells are cells cultured in vitro or cells contained within the subject's body.

[0031] Furthermore, a method for inhibiting AGAP3 protein phosphorylation includes the step of administering at least one of the following reagents to cells:

[0032] b) Reagents to reduce or eliminate HUNK gene expression.

[0033] c) Reagents that reduce or eliminate the kinase activity of HUNK proteins.

[0034] Preferably, the cells are tumor cells.

[0035] Optionally, the cells are cells cultured in vitro or cells contained within the subject's body.

[0036] The beneficial effects of this invention are:

[0037] 1. In experimental applications, tumor cells that highly express the HUNK gene are not sensitive to albumin-bound paclitaxel, while cells treated with HUNK knockout or inhibitors are more sensitive to albumin-bound paclitaxel. The experiment found that knocking out or inhibiting HUNK can promote the endocytosis of bovine serum albumin and promote its entry into lysosomes for degradation.

[0038] 2. The biomarker of this invention promotes the treatment of tumors with nanomedicines by targeting the HUNK gene with sirna or drugs, thereby overcoming the drug resistance of nanomedicines and improving the treatment effect.

[0039] 3. This invention proposes a new biomarker that can be used in combination with nanomedicines such as albumin-bound paclitaxel, which can reduce the toxicity and dosage of nanomedicines, thereby improving drug efficacy and reducing side effects. Attached Figure Description

[0040] The invention will now be further described with reference to the accompanying drawings.

[0041] Figure 1 This invention enriches pathways in HUNK knockout cell lines.

[0042] Figure 2This invention relates to the HUNK knockout KO cell line containing endocytosed material.

[0043] Figure 3 This is the result of MOCK simulation treatment of the cells in this invention;

[0044] Figure 4 This invention is for detecting the amount of protein endocytosed by BSA;

[0045] Figure 5 This invention detects the fluorescence pattern of BSA;

[0046] Figure 6 This invention detects cell fluorescence at different time points;

[0047] Figure 7 This is a lysosomal ratio diagram of the BSA located in this invention and the whole BSA;

[0048] Figure 8 The present invention uses ab7 and lamp1 to co-locate and measure late-stage endocrine status;

[0049] Figure 9 It involves adding BSA at different time points to detect BSA protein levels;

[0050] Figure 10 This refers to the total amount of late-stage endosome lysosomes co-located by ab7 and lamp1 in this invention;

[0051] Figure 11 This describes the size of late-stage endosome lysosomes after the invention.

[0052] Figure 12 This invention relates to a detector based on lysotracker-stained lysosome extraction.

[0053] Figure 13 This describes the number and size of late-stage endosome lysosomes in the Iko cells of this invention;

[0054] Figure 14 These are the neo control plasmid overexpressing neo in ko cells, the hunk gene plasmid, and the hunk gene kinase activity mutant plasmids dk1 and dk2;

[0055] Figure 15 The method involves adding BSA to cells to detect protein levels.

[0056] Figure 16 This is the condition after treatment with the hunk kinase inhibitor STS;

[0057] Figure 17 After STS processing, rab7 and lamp1 are co-located;

[0058] Figure 18The late-stage endosomal lysosomes increased, and after STS treatment, rab7 and lamp1 co-localized and increased.

[0059] Figure 19 The phosphorylation status of rab7 and lamp1 in AWT and ko cell lines by hunk;

[0060] Figure 20 The endocytosis of BSA was detected by Western blotting, and S395D inhibited BSA endocytosis.

[0061] Figure 21 GST precipitation protein phosphorylation detection revealed HUNK phosphorylation at s395;

[0062] Figure 22 This describes the downregulation of S395 phosphorylation after hunk knockout;

[0063] Figure 23 This describes the protein extraction results for expressing hunk and mutant proteins;

[0064] Figure 24 This refers to the drug sensitivity of knockout cells and control cells after treatment with albumin-bound paclitaxel.

[0065] Figure 25 This refers to the drug sensitivity of patients after adding albumin-bound paclitaxel following STS and mock treatment.

[0066] Figure 26 This is a comparison of the drug toxicity of albumin-bound paclitaxel to 3D cultured WT, KO, and STS cells.

[0067] Figure 27 These are the gray values ​​of cell spheres from WT, KO, and STS cells cultured in three dimensions under different culture conditions.

[0068] Figure 28 This invention relates to the gene expression of WT and KO. Detailed Implementation

[0069] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Example 1

[0071] This embodiment provides a tumor drug biomarker that targets the protein kinase HUNK. The HUNK gene is knocked down using sirna. A HUNK knockout cell line KO is constructed in SW480 cells, where WT represents normal SW480 cells and KO represents HUNK knockout cells. The biomarker is applied in experiments using nanomedicine therapy in combination with HUNK sirna or its inhibitors. Specific experiments are as follows:

[0072] RNA was extracted from WT and KO cell lines in the HUNK knockout cell line, and cDNA libraries were constructed by reverse transcription and transcriptome sequencing was performed. Differentially expressed genes were then compared, and KEGG pathway enrichment was performed using GSEA software. The top 20 enriched pathways were plotted as a bubble diagram, such as... Figure 1 As shown.

[0073] Bioinformatics pathway enrichment revealed enrichment of the lysosomal and endocytosis endocytosis pathways. WT and KO cell lines were subjected to 24-hour serum-free culture starvation. In the HUNK knockout KO cell line, four different endocytokines were added: Alexa-488-conjugated bovine serum albumin (BSA), Alexa-555-conjugated sphingomyelin (CTB), Alexa-488-conjugated transferrin (Tfn), and Alexa-555-conjugated dextran. One hour after addition, Hoechst nuclear dye was added for 5 minutes, followed by confocal microscopy. BSA and CTB were identified as endocytokines of the pitot pathway, while Tfn and dextran were identified as endocytokines of the lattice pathway. Results are as follows: Figure 2 As shown in the figure, by quantifying the fluorescence intensity of endocytokines, BSA and CTB were found to be significantly increased in KO cells;

[0074] Using MOCK simulations in cells, such as Figure 3 As shown in the figure, dimethyl sulfoxide solvent was added as a control, and WT and KO were each treated with MOCK. In the KO group, beta-cyclodextrin, which inhibits cavernin endocytosis, and CPZ, which inhibits clathrin, were added to quantify BSA endocytosis bound to green fluorescent protein. The specific experimental procedure was as follows: after treatment with serum-free medium for 24 hours, inhibitors were added for pretreatment for 30 minutes, followed by treatment with fluorescently coupled BSA for 1 hour. The cavernin inhibitor beta-cyclodextrin significantly inhibited BSA endocytosis.

[0075] Detection of BSA endocytosis by Western blotting, such as Figure 4As shown in the figure, WT and KO cell lines were subjected to 24 hours of serum-free starvation, followed by incubation with 10 μm BSA in the culture medium. After 1 hour, the cells were washed with PBS, and after removing the PBS, lysis buffer was added to extract proteins. Western blot analysis of the proteins was performed, and the protein expression level was detected by antibodies against HUNK, BSA and GAPDH, with GAPDH serving as an internal control.

[0076] In the KO cell line, siRNA knockdown of caverin CAV1 and clathrin was performed, with siNC serving as the knockdown control. Cells were transfected with siRNA for 24 hours, followed by a 24-hour serum-free treatment, then fluorescently conjugated BSA was added. One hour after BSA addition, Hoechst was added for nuclear staining, followed by confocal microscopy imaging. Figure 5 As shown, knocking down the fossa significantly inhibited BSA endocytosis.

[0077] Example 2

[0078] This embodiment detects the co-localization of rabbit5 and rabbit7, and performs dual-color fluorescent staining of early endosomal marker RAB5 and late endosomal marker RAB7 in WT and KO cell lines to assess the expression of early and late endosome lysosomes. The specific experimental procedures are as follows:

[0079] WT and KO cell lines were subjected to 24 hours of serum-free starvation, followed by treatment with lysotracker (a red dye for lysosomes) for 30 minutes. Then, fluorescently coupled BSA was added at different time points. Confocal microscopy was performed after adding BSA at different time gradients. Hoechst was added 5 minutes before imaging for nuclear staining. Figure 6 As shown, bsa was significantly enriched in lysosomes in the KO cell line, and Western blot results showed the same.

[0080] Lysosomes were detected using Lysotracker. After fluorescence intensity quantification, the percentage of green BSA fluorescence values ​​overlapping with the red lysosomes was statistically compared to the total BSA fluorescence value. This allowed for the calculation of the proportion of BSA in lysosomes relative to all internalized BSA after BSA addition at different time points. The proportion of BSA entering lysosomes was determined by comparing the localization of BSA within lysosomes with that of overall BSA, and fluorescence intensity was then quantified and statistically analyzed. Figure 7 As shown, Figure 7 for Figure 6 Quantitative statistics of fluorescence intensity;

[0081] After 24 hours of serum-free culture and pretreatment, BSA protein levels were measured at different time points. Protein was then extracted using lysis buffer, and the total amount of endocytosed BSA protein was determined by Western blotting. GAPDH was used as an internal control. Results are as follows: Figure 8 As shown;

[0082] Through such Figure 9 and Figure 10 The detection of colocalization of rabbit5 and rabbit7 was shown. WT and KO cell lines were subjected to dual-color fluorescent staining for the early endosomal marker RAB5 and the late endosomal marker RAB7. The colocalization of the two markers represents the late endosomal formation. WT and KO cell lines were also subjected to dual-color fluorescent staining for the late endosomal marker RAB7 and lysosomal LAMP1. The colocalization of the two markers represents the late endosomal lysosomal formation. Gene knockout significantly increased the formation of late endosomal lysosomes.

[0083] like Figure 11 The image shows the super-resolution results of LAMP1 staining. Using sim super-resolution analysis, late endosomal lysosomes were detected. In the KO cell line, LAMP1-labeled late endosomal lysosomes were significantly larger than those in WT cells. Figure 12 As shown, the cultured WT and KO cell lines were treated with lysotracker for 30 minutes, and then the cells were repeatedly broken with a syringe. Lysosomes were extracted from the cells by density gradient centrifugation. The size and number of lysosomes with green fluorescence were detected by the nanoparticle analysis system NTA. The late endosomes in KO cells were larger, and the late endosomes in H-Iko cells were larger and more numerous.

[0084] Detected by a nanoparticle detector (NTA), such as Figure 13 and Figure 14 As shown, this suggests an increase in early and late endosome lysosomes, and the increased volume of lysosomes suggests an increase in endocytosis.

[0085] Example 3

[0086] This embodiment uses lentiviral mutants that overexpress the full-length HUNK gene and lack kinase activity to detect the expression of the HUNK inhibitor STS in late endosomal lysosomes, including the following steps:

[0087] In WT and KO cell lines, lentiviruses were used to transfect the full-length HUNK gene and kinase-deficient mutants. DK#1 and DK#2 were two mutations at amino acid 91. Neo was used as an empty vector as a blank control. After cells were stably transfected with the full-length HUNK gene, the kinase-deficient mutant, and the blank vector, they were starved of serum for 24 hours, treated with fluorescent BSA for 1 hour, and Hoechst was added to indicate the cell nuclei before imaging. The fluorescence intensity was then quantified and statistically analyzed. Figure 15 As shown, cells were treated with BSA for 1 hour, followed by washing with PBS, and lysis buffer was added to extract cell proteins. Western blotting was used to detect protein expression in the cells. The results are shown below. Figure 16 As shown.

[0088] Sw480 cell lines were subjected to 24 hours of serum starvation, followed by treatment with the HUNK inhibitor STS for 30 minutes, and then treatment with fluorescently conjugated BSA for 1 hour. MOCK was used as a control treatment with dimethyl sulfoxide as the solvent. Hoechst was added before imaging to indicate the location of cell nuclei. Quantitative analysis of fluorescent BSA was performed, as shown below. Figure 17 As shown.

[0089] Sw480 cell lines were subjected to 24 hours of serum starvation, followed by treatment with the HUNK inhibitor STS for 30 minutes. Immunofluorescence staining for late endosomal marker RAB7 and lysosomal marker LAMP1 was performed. Figure 18 As shown, the fluorescence intensity quantification of Rab7 and LAMP1 indicates that the production of late endosomal lysosomes increased after HUNK gene knockout; as Figure 19 As shown Figure 18 The colocation coefficient quantification also showed that the production of late endosomal lysosomes increased after HUNK gene knockout.

[0090] In summary, adding BSA to WT and KO cells, with KO cells overexpressing the neo control plasmid, hunk gene plasmid, and hunk gene kinase activity mutant plasmids dk1 and dk2, revealed that hunk overexpression inhibited BSA endosomes, while dk1 and dk2 did not. Protein levels were also measured by adding BSA to the cells; similarly, dk1 and dk2 did not inhibit BSA endocytosis. Treatment with the hunk kinase inhibitor STS showed increased BSA endocytosis. STS treatment increased the colocalization of rab7 and lamp1, indicating an increase in late-stage lysosomes.

[0091] Example 4

[0092] This study demonstrated that the phosphorylation mutation S395D at the S395 site of AGAP3 can inhibit BSA endocytosis in HUNK knockout cell lines. The specific procedure is as follows:

[0093] First, the control Neo vector, HUNK gene, full-length AGAP3 gene, and two simulated phosphorylation mutations at two sites in the AGAP3 gene were overexpressed in WT and KO cell lines. RAB7 and LAMP1 fluorescence staining was then performed, followed by fluorescence intensity quantification. Figure 20The diagram shows that the phosphorylation-mimicking mutation at the S395 site of AGAP3 significantly inhibits the production of late endosomal lysosomes in HUNK knockout cell lines, while wild-type AGAP3 does not, suggesting that HUNK phosphorylation of AGAP3 is involved in this function. The serine residue at S395 of AGAP3 can be phosphorylated by HUNK, and the S395 mutation inhibits the formation of late endosomal lysosomes represented by rabbit7 and lamp1.

[0094] Then, in WT and KO cell lines, the control Neo vector, the full-length HUNK gene, and two simulated phosphorylation mutations at two sites in the AGAP3 gene were overexpressed. BSA endocytosis experiments were performed. Cells were starved of serum for 24 hours, followed by BSA treatment for 1 hour, and then Western blotting was performed. Figure 21 The results show that the phosphorylation-mimicking mutation S395D at the S395 site of AGAP3 significantly inhibits BSA endocytosis in HUNK knockout cell lines, while wild-type AGAP3 does not, suggesting that HUNK phosphorylation of AGAP3 is involved in this function. Western blot analysis showed that S395 inhibited BSA endocytosis.

[0095] A half-length fragment (M2) of AGAP3 (amino acids 1-411) was constructed and expressed in bacteria using GST-M2. The fusion protein was then purified using GST pulldown technology. HUNK protein was translated in vitro using an in vitro translation kit. The translated full-length HUNK-FLAG protein (500 ng) and GST-M2 protein (1 μg) were subjected to in vitro phosphorylation experiments. The phosphorylation level of AGAP3 was detected using a phosphorylated AGAP3 antibody (p-AGAP3). Figure 22 As shown, the protein phosphorylation detection of GST precipitate revealed that HUNK can phosphorylate s395, but cannot phosphorylate the s395a mutation.

[0096] Phosphorylation levels of AGAP3 were examined in WT and KO cell lines. It was found that KO significantly downregulated phosphorylation of AGAP3 at the S395 site. Figure 23 The results showed that S395 phosphorylation was downregulated after hunk knockout. In WT and KO cell lines, lentiviral overexpression of the full-length hunk gene and kinase-deficient mutants (DK#1 and DK#2, two mutations at amino acid 91) was performed. Cellular proteins were extracted, and AGAP3 phosphorylation levels were detected by Western blotting. AGAP3 phosphorylation was significantly downregulated after KO, while overexpression of hunk could compensate for this downregulation. However, hunk with kinase-activation mutations could not compensate for this downregulation. Figure 24 As shown.

[0097] Example 5

[0098] This embodiment demonstrates that by adding different concentrations of albumin-bound paclitaxel, the activity of KO and STS cell lines was significantly lower than that of WT cells. Specific experiments are as follows:

[0099] First, albumin-bound paclitaxel (Nab-paclitaxel) was added to WT and KO cell lines, and cell viability was measured. Different concentrations of albumin-bound paclitaxel were added and tested 48 hours later. The KO cell line showed greater sensitivity to the drug. Figure 25 As shown, the viability of the KO cell line was significantly lower than that of the WT cell line;

[0100] Then, albumin-bound paclitaxel (Nab-paclitaxel) was added to both MOCK and STS cell lines. After adding different concentrations of albumin-bound paclitaxel, the activity of the STS cell line was significantly lower than that of the MOCK cell line. Figure 26 As shown;

[0101] The drug toxicity of WT, KO, and STS cells cultured in three dimensions was assessed. Cells were seeded in contactless culture dishes and formed three-dimensional spheres after 72 hours. Nab-paclitaxel (Nab-paclitaxel) was added to the WT, KO, and STS-treated cell lines. Cell images were taken after 7 days of Nab-paclitaxel treatment, and the grayscale of the cell spheres under different culture conditions was compared to assess cell viability. Figure 27 , Figure 28 The results showed that after adding different concentrations of albumin-bound paclitaxel, the viability of KO and STS cell lines was significantly lower than that of WT cells. Figure 27 yes Figure 28 Quantification, Figure 28 These are three-dimensionally cultured cells. After 48 hours of culture, wt and KO cell lines formed spheres and were treated with white and purple for 7 days. The size of the three-dimensional spheres was then measured. A low concentration of STS was used for treatment without affecting the size of the spheres, while different concentrations of white and purple were added. The STS group used a concentration that did not affect the size of the cell spheres.

[0102] The targeting siRNA sequence for HUNK is as follows, where si-NC is the control:

[0103] Table 2 Sequences of siRNA for KD.

[0104]

[0105] According to the hunk gene sequence listing, the HUNK mutant DK#1 has a mutation of K to M at amino acid position 91, and DK#2 has a mutation of K to R at amino acid position 91; in the AGAP3 protein sequence listing, S395A has a mutation of S to A at position 395, and S395D has a mutation of S to D at position 395.

[0106] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," 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 invention. In this specification, 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.

[0107] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the following components: a) Nanomedicines, and b) A reagent that reduces or eliminates HUNK gene expression, wherein the reagent is a polynucleotide selected from any of the nucleotide sequences shown in SEQ ID NO:3-6; The pharmaceutical composition promotes the internalization of the nanomedicine by tumor cells through the reagent in b), thereby enhancing the therapeutic effect of the nanomedicine on tumors. The nanomedicine is albumin-bound paclitaxel; The tumor is colon cancer or rectal cancer.

2. The pharmaceutical composition of claim 1, wherein, The concentration of the albumin-bound paclitaxel is 0.1-1000 nM.

Images