CN117731772A

CN117731772A - Use of pharmaceutical combinations

Info

Publication number: CN117731772A
Application number: CN202311744204.1A
Authority: CN
Inventors: 徐晓燕; 夏宏光; 谢庭雪; 周梦欣; 谢彦奇; 陈子彦
Original assignee: Liangzhu Laboratory
Current assignee: Liangzhu Laboratory
Priority date: 2023-12-18
Filing date: 2023-12-18
Publication date: 2024-03-22

Abstract

The application discloses a use of a pharmaceutical combination. The medicine combination provided by the application can be used for activating breast cancer cell endosomal micro autophagy so as to promote immune checkpoint protein degradation, remarkably overcome breast cancer immunotherapy drug resistance and enhance curative effect.

Description

Use of pharmaceutical combinations

Technical Field

The invention relates to the field of biological medicine, in particular to application of a pharmaceutical composition.

Background

Cancer is the major disease affecting human health and has become the second leading cause of death worldwide. Immunosuppression and dysfunction are one of the hallmarks of cancer, while restoring an anti-tumor immune response is a rapidly evolving area of cancer treatment. PD-1/PD-L1 Immune Checkpoint Inhibition (ICI) is a promising clinical treatment for certain types of cancer, and T cell mediated anti-tumor immunity can be reactivated by blocking PD-L1 binding on tumor cell membranes to PD-1 on activated T cells, but a significant fraction of patients develop resistance. In recent years, immune checkpoint blocking therapy targeting PD-1/PD-L1 achieves good effects in various malignant tumors, is a main treatment scheme of various blood and solid tumors, but has low overall response rate on tumors, high primary or acquired drug resistance rate, and finally leads to failure of the PD-1/PD-L1 blocking therapy to achieve the expected curative effect. PD-L1 molecules have a broad distribution inside and outside cells, which can be located outside cells, inside cells and on cell membranes, and redistribution of PD-L1 stored and endocytosed inside cells to cell membranes can restore the ability of tumors to immune escape. Blocking the interaction of PD-1 with PD-L1 with monoclonal antibodies can block immune escape. However, the inventors have found in the study that PD-L1 is prone to recycle back to the cell membrane after internalization in combination with monoclonal antibodies, which prevents PD-L1 lysosome mediated degradation. A large amount of PD-L1 is stored in circulating endosomes and repopulates to the cell membrane, mediating resistance to ICI treatments targeting PD-L1. If an effective method for promoting the degradation of the PD-L1 lysosome and inhibiting the PD-L1 circulation is found based on the path, the method has a great promoting effect on overcoming the drug resistance of ICI treatment and enhancing the immunotherapy.

Endosomal microalbumin (endosomal microautophagy, eMI) is one of the important selectively-active lysosomal autophagy-related protein degradation pathways, and the molecular mechanism of endosomal microalbumin and its role in the development of diseases such as tumors have attracted increasing attention. Therefore, screening for small molecules that can enhance anti-tumor immunity by degrading PD-L1 by endosomal microalbumina is an important therapeutic strategy to suppress tumors and alleviate disease progression. However, there is no effective anticancer drug developed for the related pathway of degradation of PD-L1 by endosomal microautophagy in the prior art, and there is a need in the art to find drugs for enhancing immunotherapy targeting the endosomal microautophagy pathway.

Disclosure of Invention

The invention aims to provide a use of a pharmaceutical combination.

It is another object of the present invention to provide a method of treating breast cancer.

It is another object of the present invention to provide a method for inducing in vitro endosomal microalbumina of tumor cells.

To solve the above technical problems, the first aspect of the present invention provides a use of a pharmaceutical composition,

(i) Up-regulating Hsc70 expression levels (preferably up-regulating Hsc70 expression levels in tumor cells);

(ii) Promoting endosomal microalbuminiscence (preferably promoting endosomal microalbuminiscence in tumor cells);

(iii) Promoting PD-L1 degradation or down-regulating PD-L1 levels (preferably down-regulating PD-L1 levels on tumor cell membrane surfaces);

(iv) Enhancing the interaction of PD-L1 and Hsc 70;

(v) Increasing CD8 in immune cells ⁺ And/or GzmB ⁺ Proportion of cells;

(vi) Treating breast cancer; and/or

(vii) Preparing a medicament for treating breast cancer;

wherein the pharmaceutical combination comprises: a first drug which is a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof; and a second agent that is an immune checkpoint inhibitor;

in some preferred embodiments, the amount of the first drug in the pharmaceutical combination is 10-1000mg;

and the amount of the second drug is 10-5000mg.

In some preferred embodiments, the first and second drugs are separate from each other.

In some preferred embodiments, the immune checkpoint inhibitor comprises at least one of a PD-1/PD-L1 inhibitor and a CTLA-4 inhibitor. Preferably, the PD-1/PD-L1 inhibitor is a PD-1/PD-L1 antibody, CTLA-4 antibody or antigen-binding fragment thereof.

In a second aspect of the invention, there is provided a method of treating breast cancer, the method comprising the steps of:

Administering to a patient a therapeutically effective amount of a pharmaceutical combination according to the first aspect of the invention.

In some preferred embodiments, the first agent is administered at a dose of 10-30mg/kg in the pharmaceutical combination; the dosage of the second medicine is 1-10mg/kg.

In some preferred embodiments, a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt, stereoisomer, solvate, or prodrug thereof, is administered to the patient every 1 week, and a therapeutically effective amount of an immune checkpoint inhibitor is administered to the patient every 2-4 weeks.

In some preferred embodiments, the patient is a human, and a fixed dose of 200-2000mg of immune checkpoint inhibitor is administered to the patient each time.

In some preferred embodiments, the patient is a human, at a rate of 60-80mg/m each time ² Is administered to the patient a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.

In a third aspect of the invention, there is provided a method of inducing in vitro tumor cell endosomal microalbumina comprising the steps of:

culturing the tumor cells in a culture system comprising a pharmaceutical combination according to the first aspect of the invention.

In some preferred embodiments, endosomal microalbumination of tumor cells is induced by up-regulating the level of Hsc70 expression in the tumor cells.

In some preferred embodiments, tumor cell endosomal microalbumination is induced by enhancing the interaction of PD-L1 and Hsc 70.

In a fourth aspect of the present invention, there is provided a method of up-regulating in vitro the expression level of Hsc70 in a tumour cell, said method comprising the steps of:

The invention has at least the following advantages over the prior art:

the application of the medicine combination provided by the invention comprises the steps of activating tumor cell endosomal micro autophagy, thereby promoting immune checkpoint protein degradation, obviously overcoming tumor immunotherapy drug resistance and enhancing curative effect.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.

FIG. 1 is a graph showing measurement of the expression level of PD-L1 in cell membranes by flow cytometry when the PD-L1 mab is treated in 4T1 cells for various periods of time in accordance with an embodiment of the present invention;

FIG. 2 is a graph of flow analysis data for PD-L1 after various times of treatment with PD-L1 mab in 4T1 cells and AUY-922 in accordance with an embodiment of the present invention;

FIG. 3 is a Western blotting graph of 1. Mu.M AUY-922 treated MCF-7, PANC1, U937 and A375 cells according to an embodiment of the invention;

FIG. 4 is a graph showing the statistics of q-PCR of 1. Mu.M AUY-922 treated MCF-7 cells according to an embodiment of the present invention;

FIG. 5 is a schematic illustration of 1. Mu.M AUY-922 and MG132, E-64D, NH in accordance with an embodiment of the invention ₄ Western blotting of MCF-7 cells is jointly processed by Cl+Leup and 3-MA;

FIG. 6 is a statistical plot of Western blotting and flow analysis of cell membrane PD-L1 with MCF-7 cell knockdown expressing Hsc70 and treatment with 1. Mu.M AUY-922 according to an embodiment of the invention;

FIG. 7 is a statistical plot of Western blotting and cell membrane PD-L1 with 1. Mu.M AUY-922 treatment using MCF-7 cell knockdown expressing Lamp2a according to an embodiment of the invention;

FIG. 8 is a Western blotting graph of MCF-7 and a statistical graph of flow analysis of cell membrane PD-L1 treated by U18666A and AUY-922 according to an embodiment of the invention;

FIG. 9 is a statistical plot of Western blotting and flow analyses of cell membrane PD-L1 after MCF-7 cell knockdown expressing TSG101 and VPS4 and treated with 1. Mu.M AUY-922 according to an embodiment of the present invention;

FIG. 10 is a Western blotting graph of MCF-7 cells transfected with PD-L1 and treated with AUY-922, wherein AUY-922 enhances interaction of Hsc70 with PD-L1 according to an embodiment of the invention;

FIG. 11 is a co-localized immunofluorescence of Hsc70 with late endosomes (RAB 7) following treatment of MCF-7 cells AUY-922 in accordance with an embodiment of the present invention;

FIG. 12 is a co-localized immunofluorescence of Hsc70 with lysosomes (LAMP 1) after treatment of MCF-7 cells AUY-922 according to an embodiment of the invention;

FIG. 13 is a co-localized immunofluorescence of Hsc70 with circulating endosomes (RAB 11) following treatment of MCF-7 cells AUY-922 in accordance with an embodiment of the present invention;

FIG. 14 is a statistical graph of Western blotting and flow analysis of cell membrane PD-L1 with HSP90 expressed by MCF-7 cell knockdown according to an embodiment of the present invention;

FIG. 15 is a statistical graph of flow analysis of Western blotting of MCF-7 cells simultaneously knocked down expressing HSP90 and Hsc70 and cell membrane PD-L1 according to an embodiment of the present invention;

FIG. 16 is a Western blotting graph of down-expression of HSP90 by MCF-7 cells according to an embodiment of the present invention;

FIG. 17 is a Western blotting graph of AUY-922 treatment of MCF-7 according to an embodiment of the invention;

FIG. 18 is a Western blotting graph of MCF-7 cell knockdown expressing CMTM6 according to an embodiment of the invention;

FIG. 19 is a statistical graph of flow analysis of Western blotting of MCF-7 cells simultaneously knocked down to express CMTM6 and Hsc70 and cell membrane PD-L1 according to an embodiment of the invention;

FIG. 20 is a Western blotting graph of MCF-7 cell knockdown expressing CMTM6 enhancing interaction of Hsc70 with PD-L1 according to an embodiment of the invention;

FIG. 21 is a Western blotting graph of 17-AAG-treated MCF-7 cells according to an embodiment of the invention;

FIG. 22 is a Western blotting graph of 17-DMAG treated MCF-7 cells according to the present invention;

FIG. 23 shows the overexpression of Hsc70 by MCF-7 and PANC1 cells and the use of MG132, E-64D, NH according to the examples of the invention ₄ Western blotting graph jointly treated by Cl+Leup and 3-MA;

FIG. 24 is a Western blotting graph of a stable 4T1 cell line over-expressing Hsc70-WT and Hsc70-3KA according to an example of the present invention;

FIG. 25 is a schematic representation of a breast cancer tumor model of a 4T1 cell-vaccinated BALB/c mouse overexpressing Hsc70-WT and Hsc70-3KA according to an embodiment of the present invention;

FIG. 26 is a statistical plot of breast cancer tumor growth seeded with 4T1 cells overexpressing Hsc70-WT and Hsc70-3KA in accordance with an embodiment of the present invention;

FIG. 27 is a weight graph of breast cancer tumor lysis following seeding of 4T1 cells overexpressing Hsc70-WT and Hsc70-3KA in accordance with an embodiment of the present invention;

FIG. 28 is a schematic representation of breast cancer tumor dissection following seeding of 4T1 cells overexpressing Hsc70-WT and Hsc70-3KA in accordance with an embodiment of the present invention;

FIG. 29 is a graph showing the detection of CD8 in tumor cells after breast cancer tumor ablation inoculated with 4T1 cells overexpressing Hsc70-WT and Hsc70-3KA in accordance with an embodiment of the present invention ⁺ GzmB ⁺ Flow chart of immune cell ratios;

FIG. 30 is a diagram of immunohistochemical analysis and analysis of CD8 and GzmB after breast cancer tumor lysis with 4T1 cells over-expressing Hsc70-WT and Hsc70-3KA inoculated in accordance with the present invention;

FIG. 31 is a Western blotting graph of tumor tissue after breast cancer tumor dissection inoculated with 4T1 cells overexpressing Hsc70-WT and Hsc70-3KA in accordance with an embodiment of the present invention;

FIG. 32 is a schematic representation of a breast cancer tumor model vaccinated with 4T1 cells according to an embodiment of the present invention with AUY-922 and Ganetespib dosing treatments;

FIG. 33 is a graph showing the growth statistics of AUY-922 and Ganetespib treatments on breast cancer tumors vaccinated with 4T1 cells according to an embodiment of the invention;

FIG. 34 is a weight graph of AUY-922 and Ganetespib treatment vs. breast cancer tumor dissection seeded with 4T1 cells according to an embodiment of the present invention;

FIG. 35 is a graph showing AUY-922 and Ganetespib treatment versus CD8 in tumor tissue according to an embodiment of the present invention ⁺ T cells and CD8 ⁺ GzmB ⁺ Flow cytometry analysis of T cells;

FIG. 36 is a graph showing the growth statistics of AUY-922, ganetespib and PD-L1 mab treatment on breast cancer tumors vaccinated with 4T1 cells according to an embodiment of the invention;

FIG. 37 is a graph of the growth of anti-PD-L1 (100 μg), ganetespib (15 mg/kg) co-treated with anti-PD-L1 and AUY-922 (15 mg/kg) and anti-PD-L1 co-treated with 4T1 breast cancer tumors according to an embodiment of the invention;

FIG. 38 is a statistical plot of anti-PD-L1 (100 μg), ganetespib (15 mg/kg) co-treatment with anti-PD-L1 and AUY-922 (15 mg/kg) and anti-PD-L1 co-treatment versus 4T1 breast cancer tumor weight in an embodiment according to the invention;

FIG. 39 is a graph of CD8 in tumor tissue co-treatment of anti-PD-L1 (100. Mu.g), ganetespib (15 mg/kg) with anti-PD-L1 and AUY-922 (15 mg/kg) and anti-PD-L1 in accordance with an embodiment of the invention ⁺ T cells and GzmB ⁺ Flow cytometry analysis of T cells.

FIG. 40 is a flow chart of tumor cell membrane PD-L1 co-treatment with anti-PD-L1 (100 μg), ganetespib (15 mg/kg) and AUY-922 (15 mg/kg) and anti-PD-L1 co-treatment according to an embodiment of the invention.

FIG. 41 is a graph of anti-CTLA4 (100 μg), ganetespib (15 mg/kg) co-treatment with anti-CTLA4 and AUY-922 (15 mg/kg) and anti-CTLA4 co-treatment versus 4T1 breast cancer tumor growth in accordance with an embodiment of the invention;

FIG. 42 is a graph of anti-CTLA4 (100 μg), ganetespib (15 mg/kg) co-treatment with anti-CTLA4 and AUY-922 (15 mg/kg) and anti-CTLA4 co-treatment versus 4T1 breast cancer tumor weight statistics in accordance with an embodiment of the invention;

FIG. 43 is a graph of anti-CTLA4 (100 μg), ganetespib (15 mg/kg) co-processed with anti-CTLA4 and AUY-922 (15 mg/kg) and anti-CT according to an embodiment of the inventionCo-treatment of LA4 with CD8 in tumor tissue ⁺ T cells and GzmB ⁺ Flow cytometry analysis of T cells;

FIG. 44 is a flow chart of anti-CTLA4 (100. Mu.g), ganetespib (15 mg/kg) co-treatment with anti-CTLA4 and AUY-922 (15 mg/kg) and anti-CTLA4 co-treatment with tumor cell membrane PD-L1 according to an embodiment of the invention.

Detailed Description

The present inventors have conducted extensive and intensive studies and have found that HSP90 inhibitors are capable of enhancing the therapeutic efficacy of antibodies against immune checkpoint proteins by activating or promoting the endosomal microautoautophagy pathway to enhance Hsc70 levels and promote immune checkpoint protein degradation. On this basis, a combination of drugs comprising an HSP90 inhibitor and an immune checkpoint inhibitor has been developed, which has been shown to improve tumor immunotherapy resistance, thereby enhancing the immune efficacy.

Pharmaceutical combination

The present invention relates to a pharmaceutical combination comprising: a first drug and a second drug, wherein the first drug is a compound of formula I or a derivative thereof; the second agent is an immune checkpoint inhibitor.

"AUY-922" as used in the present invention has the formula: c (C) ₂₆ H ₃₁ N ₃ O ₅ The CAS number is: 747412-49-3, and has a structural formula shown in formula I below. It will be appreciated that various forms of derivatives of AUY-922, such as pharmaceutically acceptable salts, isomers, solvates or prodrugs thereof, should also be capable of performing a similar function as AUY-922 in the present invention.

As used herein, the term "Immune Checkpoint Inhibitor (ICI)" refers to a series of monoclonal antibodies developed against immune checkpoint molecules of the body, which can block the interaction between tumor cells expressing immune checkpoints and immune cells, and block the inhibition of tumor cells and immune cells, thereby realizing immunotherapy. Preferred immune checkpoints in the present invention include PD-1, PD-L1, CTLA4, and the like. In a preferred embodiment of the invention, the immune checkpoint inhibitor is a PD-1/PD-L1 antibody, a CTLA-4 antibody, a PD-1/CTLA-4 diabody or an antigen-binding fragment thereof. Immune checkpoint inhibitors in the present invention also include diabodies, such as PD-1/CTLA-4 diabodies, that target two immune checkpoints simultaneously.

As used herein, the term "PD-L1" refers to apoptosis-ligand 1. The term "PD-L1 antibody" refers to any antibody that targets PD-L1, preferably the PD-L1 antibody is a PD-L1 humanized IgG monoclonal antibody. For example: human PD-L1 monoclonal antibody atezolizumab (trade name Tecentriq), human PD-L1 monoclonal antibody avelumab (trade name Bavendio)), human PD-L1 monoclonal antibody durvalumab (trade name Imfinzi), and the like.

As used herein, the term "CTLA4" refers to cytotoxic T lymphocyte-associated antigen 4 (cytotoxic Tlymphocyte-associated antigen-4, CTLA-4), also known as CD152, which is a membrane protein that activates T cell expression, down regulates T cell proliferation, and belongs to an immunoglobulin superfamily member. CTLA-4 is an important costimulatory molecule involved in down-regulating T cell function, similar in structure to CD28, and competes for binding to the same ligand B7. The term "CTLA4" antibody refers to any antibody that targets CTLA 4. Such as the commercially available recombinant humanized monoclonal antibodies Yervoy, the CTLA-4 antibodies Zalifrelimab, BMS-986218 to fully human IgG1, BMS-986288, BMS-986249, AGEN1181, ADG-116, ADG-126, quavonlimab, HBM-4003, ONC-392, YH-001, XTX101, JS007, BA3071, and the like.

In the present invention, the term "PD-1/CTLA-4 diabodies" refers to monovalent bispecific antibodies that target both PD-1 and CTLA-4. Examples of PD-1/CTLA-4 diabodies are the commercially available drug MEDI5752.

In the pharmaceutical combination of the present invention, the first and second drugs may or may not be present in separate form, and in a preferred embodiment the first and second drugs are separated from each other. In a preferred embodiment of the invention, the amount of the first drug in the pharmaceutical combination is 10-1000mg and the amount of the second drug is 10-5000mg.

Combined medicine box

The invention also relates to a combination kit comprising a first kit and a second kit, which are independently packaged, wherein the first kit comprises a compound of formula I or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof and a pharmaceutically acceptable carrier, and the second kit comprises an immune checkpoint inhibitor and a pharmaceutically acceptable carrier.

In the present invention, the first kit and the second kit may be prepared into any one of the following dosage forms, respectively, according to the administration conditions: powders, granules, tablets, suppositories and injections, and depending on the dosage form, different pharmaceutically acceptable carriers may be selected, such as liquid or solid fillers, diluents, emulsifiers, solubilizers, dispersants, solvents or encapsulating materials, etc., each of which is "pharmaceutically acceptable" in one embodiment, meaning that it is compatible with the other ingredients in the pharmaceutical formulation and suitable for contact with tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Use of medicine combination or combined medicine box and indication

The invention also relates to application of the medicine combination or the combined medicine box in preparing medicines for treating diseases, wherein the diseases are various diseases suitable for immunotherapy treatment, in particular to various diseases which can be treated by PD-L1 antibody therapy, CTLA-4 antibody therapy and PD-1/CTLA-4 double antibody combination therapy. Exemplary diseases that can be treated with PD-L1 antibody therapy are malignant tumors, cardiovascular diseases or autoimmune diseases. In a preferred embodiment of the invention, the above pharmaceutical combination or kit is used for the treatment of malignant tumors or for the preparation of a medicament for the treatment of malignant tumors. Malignant tumors in the present invention include hematological malignant tumors or solid malignant tumors, more preferably malignant tumors such as, for example, melanoma, lung cancer (e.g., small cell lung cancer or non-small cell lung cancer), kidney cancer, bladder cancer, stomach cancer, intestinal cancer, ovarian cancer, liver cancer, breast cancer, pancreatic cancer, bladder cancer, prostate cancer, glioma, mesothelioma, lymphoma (e.g., hodgkin lymphoma), urothelial cancer, etc., most preferably pancreatic cancer. Examples of diseases treatable by PD-L1 antibody therapy include melanoma, skin cancer, renal cell carcinoma, carcinoma of large intestine, hepatocellular carcinoma, etc. Diseases which can be treated by the PD-1/CTLA-4 dual antibody combination therapy are exemplified by gastric cancer, colorectal cancer, liver cancer and the like.

In other embodiments of the invention, the above pharmaceutical combination or co-kit is used for the treatment of cardiovascular diseases or for the preparation of a medicament for the treatment of cardiovascular diseases, such as for example coronary heart disease, dilated cardiomyopathy, myocarditis, etc.

In other embodiments of the invention, the above pharmaceutical combination or kit is used for treating an autoimmune disease or for preparing a medicament for treating an autoimmune disease. Such autoimmune diseases are exemplified by autoimmune diabetes, autoimmune hepatitis, and the like.

In a most preferred embodiment of the invention, the pharmaceutical combination or co-kit is used for the treatment of breast cancer or for the preparation of a medicament for the treatment of breast cancer. Compared with other cancers, the pharmaceutical combination or the combined kit provided by the invention has better curative effect enhancing capability in the aspect of treating breast cancer.

The pharmaceutical combination of the invention can also be used for: up-regulating Hsc70 expression levels (preferably up-regulating Hsc70 expression levels in tumor cells); promoting endosomal microalbuminiscence (preferably promoting endosomal microalbuminiscence in tumor cells); promoting PD-L1 degradation or down-regulating PD-L1 levels (preferably down-regulating PD-L1 levels on tumor cell membrane surfaces); enhancing the interaction of PD-L1 and Hsc 70; increasing CD8 in immune cells ⁺ And/or GzmB ⁺ Proportion of cells. Preferably these uses are in vitro uses. In some embodiments, these uses are non-therapeutic uses. In some embodiments, the pharmaceutical combination is used to up-regulate Hsc70 expression levels in vitro; and/or promote endosomal microalbuminiscence; and/or promote PD-L1 degradation or down-regulate PD-L1 expression levels; and/or enhance the interaction of PD-L1 and Hsc 70.

Therapeutic method

The invention also relates to a method of treating a malignancy, comprising the steps of: a therapeutically effective amount of a pharmaceutical combination or combination kit of the invention is administered to a subject.

As used herein, the term "subject" is defined herein to include animals, such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, and the like. In a particular embodiment, the subject is a human.

As used herein, a "therapeutically effective amount" refers to an amount of a compound that is sufficient to provide a therapeutic effect in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder. A therapeutically effective amount of a compound refers to the amount of a therapeutic agent that, when used alone or in combination with other therapies, provides a therapeutic effect in the treatment or management of a disease or disorder. The term "therapeutically effective amount" may include an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. The appropriate amount of the pharmaceutical combination of the present invention to be used depends on the type of the first drug and the second drug to be used in combination, the dosage, the symptoms of the patient, the age, the administration method, etc. For example, in the case of oral administration, the lower limit is 0.01mg/kg body weight (preferably 0.1mg/kg body weight), the upper limit is 1000mg/kg body weight (preferably 100mg/kg body weight), the lower limit is 0.001mg/kg body weight (preferably 0.01mg/kg body weight), and the upper limit is 1000mg/kg body weight (preferably 100mg/kg body weight). Depending on the symptoms, one to several times per day may be taken. However, this numerical range is merely an example and is not limiting the scope of the present invention. In a preferred embodiment of the invention, the first agent is AUY-922 and the second agent is a human PD-L1 monoclonal antibody or CTLA-4 antibody, and the first agent is administered at a dose of 10-30mg/kg, for example 12mg/kg,25mg/kg. The second drug is administered at a dose of 1-10mg/kg, for example 2.5mg/kg (100 ug/mouse per 25 g).

Method for in vitro induction of tumor cell endosomal microalbumina

The invention also relates to a method for inducing in vitro endosomal microalbumina of tumor cells (preferably breast cancer cells), comprising the steps of: tumor cells are cultured in a culture system containing the pharmaceutical combination.

Kit for detecting a substance in a sample

The invention also relates to a kit for use according to any one of (I) - (iv), comprising a compound of formula I or a derivative thereof; and immune checkpoint inhibitors;

(i) Up-regulating the expression level of Hsc70 in tumor cells;

(ii) Promoting endosomal microalbuminiscence in tumor cells;

(iii) Promoting the degradation of PD-L1 or down regulating the expression level of PD-L1 on the surface of tumor cell membrane;

(iv) Enhancing the interaction of PD-L1 and Hsc 70.

In some embodiments, the use described in (i) - (iv) above is an in vitro use. In some embodiments, the use described in (i) - (iv) above is a non-therapeutic use.

The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit the exemplary embodiments of this application.

The term "or" means and is used interchangeably with the term "and/or" unless otherwise indicated.

As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

EXAMPLE 1, recirculation of PD-L1 back to the cell membrane after internalization in combination with monoclonal antibody

4T1 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody and passaged at 4X 10 ⁵ Inoculating the cells to a 6-well plate, simultaneously adding IFN-gamma to stimulate the expression of PD-L1, adding a monoclonal antibody of PD-L1 after 24h stimulation, collecting the cells at 24h, 48h and 96h respectively, and analyzing the expression condition of PD-L1 on the cell surface by using a flow experiment.

The flow detection method comprises the following steps: the cells were removed from the medium, washed 2 times with PBS, 0.5ml of pancreatin was added to each well, digested for 2-3min, neutralized with medium, collected by centrifugation at 1000g for 5min, incubated with PBS containing 0.5% BSA at room temperature for 10min, incubated with PE-conjugated PD-L1 antibody and matched isotype control for 30min at 4℃in dark. After three washes with PBS, the cells were analyzed by flow cytometry (Beckman-Colter-Cytofelx) and the data were processed using CytExpert2.3 and Flowjo software analysis.

As shown in fig. 1, the experimental result shows that the IFN-gamma stimulation can significantly increase the expression of the cell membrane PD-L1, the PD-L1 monoclonal antibody is treated for 24 hours to significantly reduce the amount of the cell membrane PD-L1, but the action effect of the PD-L1 monoclonal antibody is less and less obvious along with the extension of time, and the level of the PD-L1 on the surface of the cell membrane is significantly increased for 96 hours. It was shown that PD-L1 mab may be re-circulated back to the cell membrane after internalization upon binding to the cell membrane PD-L1.

Example 2, AUY-922 in combination with PD-L1 mab drug can reduce recovery of Membrane PD-L1

4T1 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody and passaged at 4X 10 ⁵ Inoculating the cell density of (2) in a 6-hole plate, simultaneously adding IFN-gamma to stimulate the expression of PD-L1, adding a monoclonal antibody of PD-L1 after 24h stimulation, adding 1 mu M AUY-922 when the PD-L1 monoclonal antibody is treated for 72h, treating for 24h, finally collecting cells when the PD-L1 monoclonal antibody is treated for 96h, and analyzing the expression condition of the PD-L1 on the cell surface by using a flow experiment.

The flow detection method comprises the following steps: the cells were removed from the medium, washed 2 times with PBS, 0.5ml of pancreatin was added to each well, digested for 2-3min, neutralized with medium, collected by centrifugation at 1000g for 5min, incubated with PBS containing 0.5% BSA at room temperature for 10min, incubated with PE-conjugated PD-L1 antibody and matched isotype control for 30min at 4℃in dark. After three washes with PBS, the cells were analyzed by flow cytometry (Beckman-Colter-Cytofelx) and the data was analyzed using CytExpert2.3 and Flowjo software.

As shown in fig. 2, the IFN-gamma stimulation can significantly increase the expression of the cell membrane PD-L1, the treatment with the PD-L1 monoclonal antibody for 24 hours can significantly reduce the amount of the cell membrane PD-L1, the treatment with the PD-L1 monoclonal antibody for 96 hours can inhibit the reversion of the cell membrane PD-L1 expression caused by the long-time treatment of the PD-L1 monoclonal antibody, and the treatment with the AUY-922 can inhibit the reversion of the cell membrane PD-L1 expression caused by the long-time treatment of the PD-L1 monoclonal antibody, so that the treatment with the AUY-922 can further reduce the cell membrane PD-L1 expression, and the treatment with the AUY-922 can relieve the drug resistance of the PD-L1 monoclonal antibody.

Example 3 AUY-922 treatment in different cell lines promotes PD-L1 degradation and enhances Hsc70 expression levels

Breast cancer cells MCF-7, pancreatic cancer cells PANC1 and lymphoma cells U937 and melanoma cells A375 were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin double antibody, and passaged with 4X 10 ⁵ The cells were inoculated in 6-well plates, after the cells grew to a logarithmic growth phase and the administration was started at an AUY-922 concentration of 1. Mu.M, and after 24 hours of administration, the cells were washed twice with PBS, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10min, and the supernatant was centrifuged and subjected to Western blotting.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skim milk at room temperature for 1h, monoclonal antibodies Tubulin (# M1305-2,1:5000, HUABIO), hsc70 (# 10654-1-AP,1:2000, proteintech), PD-L1 (# 66248-1-lg,1:1000, proteintech) were diluted with PBST+5% skim milk, the corresponding membranes were placed in dilutions, incubated overnight at 4 ℃, washed with PBST 3 times for 10min each, incubated with secondary antibodies anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) for 1h each, washed with PBST 3 times for 10min each, and developed with ECL developing solution.

AUY-922 treatment as shown in FIG. 3 was able to significantly increase the expression of Hsc70 in MCF-7, PANC1, U937 and A375 cells, while reducing the amount of PD-L1.

Example 4 AUY-922 treatment did not affect the transcript levels of PD-L1

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Inoculating the cells with the density of cells in a 6-well plate, adding a complete culture medium to a volume of 2ml, carrying out drug administration treatment when the cells are grown on the wall until the fusion degree is 70% -80%, treating the cells with 1 mu M AUY-922 for 12h and 24h, slowly adding 1ml PBS (phosphate buffered saline) into each well for washing the cells twice, adding 0.5ml pancreatin into each well, digesting for 2-3min, adding the complete culture medium for neutralization, centrifuging for 5min by 1000g to collect the cells, adding 500 mu L Buffer RL with beta-mercaptoethanol for cell lysis, and blowing and sucking until no obvious cell mass exists. Extracting corresponding RNA with RNA extraction kit, removing genome DNA, measuring DNA concentration with spectrophotometer, synthesizing cDNA with 1ug total RNA, and placing the synthesized cDNA in-80 deg.C refrigerator for use. The transcript level of PD-L1 was detected by qPCR with LC 480.

As shown in FIG. 4, AUY-922 treatment did not affect the transcript levels of PD-L1, indicating that AUY-922 reduced the levels of PD-L1 independent of changes in transcript levels.

Example 5 degradation of PD-L1 by AUY-922 depends on lysosomes

MCF-7 and PANC1 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Is inoculated in a 6-well plate, and after the cells grow to the initial administration of the logarithmic growth phase, the cells are treated with 1 mu M AUY-922 for 12 hours, and simultaneously with the AUY-922 treatment, the cells are treated with the respective proteasome inhibitor MG132 (10 mu M), the lysosome inhibitor Leupeptin (100 nM) +NH ₄ Cl (20 mM), E-64D (10. Mu.M) and autophagy inhibitor 3-MA (10 mM) were treated for 12 hours, cells were washed with PBS after the end of administration, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10 minutes, centrifuged, and supernatants were collected for Western blotting.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skim milk at room temperature for 1h, monoclonal antibodies HK2 (# 22029-1-AP,1:1000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), PD-L1 (# 66248-1-lg,1:1000, proteintech) were diluted with PBST+5% skim milk, the corresponding membranes were placed in the dilutions, incubated overnight at 4 ℃, washed with PBST 3 times for 10min each, incubated with secondary antibodies anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-light IgG (# 31460,1:20000,Thermo Fisher Scientific) for 1h at room temperature, washed with PBST 3 times for 10min each, followed by development with ECL development liquid.

AUY-922 treatment as shown in FIG. 5 significantly reduced the amount of PD-L1, with the proteasome inhibitor MG132 treatment having no effect on the amount of PD-L1, but with the lysosomal inhibitor (Leup+NH) ₄ Cl) and E-64D treatments were able to significantly inhibit the degradation of PD-L1, while autophagy inhibitor 3-MA treatment had no effect on the degradation of PD-L1, indicating that AUY-922-promoted degradation of PD-L1 was lysosomal dependent.

Example 6 degradation of PD-L1 by AUY-922 is dependent on Hsc70

Example 3 shows that AUY-922 is able to increase expression of Hsc70 while degrading PD-L1, and therefore in order to determine whether AUY-922 is dependent on Hsc70 for PD-L1 degradation, the following experiment was performed:

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Inoculating the cells to 2 6-hole plates, adding a complete culture medium to a volume of 2ml, carrying out Hsc70 siRNA transfection by using Lipofectamine 2000 when the cells are grown to be adhered to the wall and have a fusion degree of 70% -80%, mixing the corresponding siRNA with the Lipofectamine 2000, standing for 15min, dropwise adding the mixed solution into the cultured cells by using a pipette, and gently shaking a culture dish to uniformly mix the transfection solution with the culture medium. Placed at 37 ℃ and 5% CO ₂ Is cultured in a cell culture incubator for 48 hours, and then treated with 1. Mu.M AUY-922 for 24 hours. After one of the 6-well plates was dosed, the cells were washed with PBS and pooled with 250. Mu.L of a 2 Xloading buffer Heating at 100deg.C for 10min, centrifuging, and collecting supernatant for western blotting detection. After cells were collected in another 6-well plate, the expression of cell surface PD-L1 was measured by flow cytometry.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skim milk at room temperature for 1h, monoclonal antibodies HK2 (# 22029-1-AP,1:1000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), PD-L1 (# 66248-1-lg,1:1000, proteintech), hsc70 (# 10654-1-AP,1:2000, proteintech) were diluted with PBST+5% skim milk, the corresponding membranes were placed in the dilutions, incubated overnight at 4 ℃, washed 3 times with PBST, 10min each time with secondary antibody anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-rabit IgG (# 31460,1:20000,Thermo Fisher Scientific) for 3 times with PBST at room temperature, 10min each time, after which development was performed with ECL developing solution.

Flow cytometry: cells were washed 2 times with PBS after treatment, 0.5ml of pancreatin was added to each well, digested for 2-3min, neutralized with medium, collected by centrifugation at 1000g for 5min, incubated with PBS containing 0.5% BSA at room temperature for 10min, incubated with PE-conjugated PD-L1 antibody (# 329706,1:200, bioleged) and matched isotype control IgG (# 400312,1:200, bioleged) under dark conditions for 30min at 4 ℃. After three washes with PBS, the cells were analyzed by flow cytometry (Beckman-Colter-Cytofelx) and the data was analyzed using CytExpert2.3 and Flowjo software.

As shown in fig. 6A, AUY-922 treatment significantly reduced the amount of PD-L1, and knockdown of Hsc70 prevented degradation of PD-L1 by AUY-922; as shown in FIG. 6B, AUY-922 treatment reduced cell membrane PD-L1 expression, knockdown of Hsc70 prevented AUY-922 from decreasing cell membrane PD-L1 expression, indicating that AUY-922 degradation of PD-L1 was dependent on Hsc70 expression.

EXAMPLE 7 degradation of PD-L1 by AUY-922 by endosomal microautoautophagy

Hsc70 is a cytoplasmic chaperone protein that plays a key role in Chaperone Mediated Autophagy (CMA) and endosomal microalbumin (eMI). Approximately 40% of mammalian proteins contain KFERQ-like motifs, which can serve as substrates for Hsc 70. Although CMA and eMI have similar motifs, their substrates do not overlap completely, and proteins with KFERQ-like motifs are in a semi-aggregated state, forming high molecular weight complexes, or cannot be unfolded and degraded by CMA, but still can be degraded by eMI. Chaperone mediated autophagy CMA recognizes a substrate protein by Hsc70 and carries the substrate protein to the lysosome membrane, which is refolded and degraded into the lysosome with the aid of lysosome membrane protein Lamp2a, lamp2a being considered as the rate limiting step of CMA. Late endosomes are key entry points to eMI, whose membranes contain a specialized transport machinery (ESCRT protein complex) that coordinates the inward budding of endosomal membranes to form endoluminal vesicles. The interaction between Hsc70 and Phosphatidylserine (PS) is critical for the transfer of cytoplasmic proteins to late endosomes via eMI. In this example, degradation of PD-L1 by AUY-922 through endosomal microalbumination was confirmed by several experiments as follows.

(1) The degradation of PD-L1 by AUY-922 is independent of CMA

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin double antibody, passaged and inoculated in 2 6-well plates at a cell density of 4X 105, the whole medium was added to a volume of 2ml, si-Lamp2a transfection was performed with Lipofectamine 2000 when the cells had grown to a confluence of 70% -80% by adherence, the corresponding si-Lamp2a or si-Control was mixed with Lipofectamine 2000 and allowed to stand for 15min, the mixed solution was added dropwise to the cultured cells with a pipette, and the culture dish was gently shaken to mix the transfection solution with the medium. Then placed at 37 ℃ and 5% CO ₂ Is cultured in a cell culture incubator for 48 hours, and then is treated with 1 mu M AUY-922 for 24 hours. One of the 6-well plates was washed with PBS, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10min, centrifuged, and the supernatant was taken for Western blotting. After cells were collected in another 6-well plate, the expression of cell surface PD-L1 was measured by flow cytometry.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skim milk at room temperature for 1h, monoclonal antibodies HK2 (# 22029-1-AP,1:1000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), PD-L1 (# 66248-1-lg,1:1000, proteintech), lamp2a (# ab18528,1:1000, abcam) were diluted with PBST+5% skim milk, the corresponding membranes were placed in the dilutions, incubated overnight at 4℃with PBST for 3 times, 10min each, with secondary antibodies anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-rabit IgG (# 31460,1:20000,Thermo Fisher Scientific) for 1h at room temperature, PBST for 3 times, 10min each, after which development was performed with ECL developing solution.

As shown in FIG. 7A, AUY-922 treatment can significantly reduce the level of PD-L1 protein, and knocking down CMA key protein Lamp2a prevents degradation of CMA substrate protein HK2, but does not affect degradation of PD-L1 protein by AUY-922. As shown in FIG. 7B, AUY-922 treatment can significantly reduce the expression of cell membrane PD-L1, and knocking down expression Lamp2a prevents AUY-922 from reducing the expression of cell membrane PD-L1, which indicates that AUY-922 does not depend on CMA for PD-L1 expression and degradation.

(2) Inhibition of endosomal microalbuminiscence prevents AUY-922-promoted PD-L1 degradation

Cholesterol transport inhibitor U8666A is known to block MVB kinetics and thereby inhibit the occurrence of endosomal microalbumina, so in this experiment inhibition of endosomal microalbumina by U18666A detects whether degradation of PD-L1 by AUY-922 is dependent on endosomal microalbumina by the following method:

Addition of MCF-7 cellsDMEM medium containing 10% FBS and 1% penicillin/streptomycin double antibody was used for culturing, and 4X 10 after passage ⁵ Is inoculated in 2 6-hole plates, added with complete culture medium to 2ml, placed at 37 ℃ and 5% CO ₂ Is cultured in a cell culture incubator of (1) and is treated with U18666A (endosomal microalgal inhibitor) for 24 hours when cells are grown on the wall to a confluence of 70% -80%, and then treated with 1. Mu.M AUY-922 for 12 hours. One of the 6-well plates was washed with PBS, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10min, centrifuged, and the supernatant was taken for Western blotting. After cells were collected in another 6-well plate, the expression of cell surface PD-L1 was measured by flow cytometry.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after blocking the membranes with 5% skim milk for 1h at room temperature, mab PD-L1 (# 66248-1-lg,1:1000, proteintech), tubulin (# M1305-2,1:5000, HUABIO) was diluted with PBST+5% skim milk, the corresponding membranes were placed in dilutions, incubated overnight at 4 ℃, PBST washed 3 times for 10min each, incubated with secondary anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-rabit IgG (# 31460,1:20000,Thermo Fisher Scientific) for 1h at room temperature, PBST washed 3 times for 10min each, followed by development with ECL development solution.

As shown in FIG. 8A, AUY-922 treatment can promote PD-L1 degradation, and U18666A treatment prevents AUY-922 from degrading PD-L1; as shown in FIG. 8B, AUY-922 is capable of reducing the expression of cell membrane PD-L1, and U18666A treatment restores the expression of cell membrane PD-L1.

(3) Degradation of PD-L1 by AUY-922 is dependent on an ESCRT machine

ESCRT complex TSG101 plays an important role in MVB formation and in promoting cytoplasmic protein transport to endosomes. VPS4 is another key component of endosomal microalbumina and is a regulated ATPase for ESCRT-III, which together mediate a variety of membrane remodeling events. To determine whether these key mediators of eMI are involved in the degradation of PD-L1 by AUY-922, in this experiment, whether the degradation of PD-L1 by AUY-922 is dependent on the ESCRT machine was determined by knocking down TSG101 and VPS4 in MCF-7 cells as follows:

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Inoculating the cells to 2 6-well plates, adding a complete culture medium to a volume of 2ml, transfecting the cells by using Lipofectamine 2000 when the cells are grown to be adhered to the wall and have a fusion degree of 70% -80%, mixing the corresponding si-TSG101, si-VPS4 or si-Control with the Lipofectamine 2000, standing for 15min, dropwise adding the mixed solution into the cultured cells by using a pipette, and gently shaking the culture dish to uniformly mix the transfected solution with the culture medium. Then placed at 37℃in 5% CO ₂ Is cultured in a cell culture incubator for 48 hours, and then is treated with 1 mu M AUY-922 for 24 hours. One of the 6-well plates was washed with PBS, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10min, centrifuged, and the supernatant was taken for Western blotting. After cells were collected in another 6-well plate, the expression of cell surface PD-L1 was measured by flow cytometry.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skim milk at room temperature for 1h, mab PD-L1 (# 66248-1-lg,1:1000, proteintech), TSG101 (# ab83,1:1000, abcam), VPS4 (# ab229806,1:1000, abcam), tubulin (# M1305-2,1:5000, HUABIO) were diluted with PBST+5% skim milk, the corresponding membranes were placed in the dilutions, incubated overnight at 4 ℃, PBST was washed 3 times for 10min each, with secondary antibody anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) was incubated 1h at room temperature, PBST was washed 3 times for 10min each, and then developed with ECL developing solution.

As shown in FIGS. 9A and C, AUY-922 treatment can promote PD-L1 degradation, and knocking down expression of TSG101 or VPS4 can prevent AUY-922 from degrading PD-L1; as shown in FIGS. 9B, D, AUY-922 treatment reduced the expression of cell membrane PD-L1, and knock-down of TSG101 or VPS4 restored the expression of cell membrane PD-L1. These results indicate that AUY-922 degradation of PD-L1 and inhibition of membrane PD-L1 expression is dependent on TSG101 and VPS4.

(4) AUY-922 enhances the interaction of PD-L1 with Hsc70

HEK-293T cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody and placed at 37℃with 5% CO ₂ Is cultured in a cell culture chamber of (2X 10) after passage ⁶ Is inoculated in a 10cm dish, added with complete culture medium to 8ml, and is transfected with Flag or pCDNA-PD-L1-Flag by lip2000 when the cells are grown on the wall to 70% -80% fusion, and added with 1 mu M AUY-922 for 12h after 24h of transfection. The medium in the dish was discarded, the cells were washed 2 times by slowly adding 2ml of pre-chilled PBS along the edge of the dish wall, 1ml of pre-chilled TAP lysate (20 mM Tris-HCl (pH 7.5), 150mM NaCl,0.5% NP-40,1mM NaF,1mM Na3VO4,1Mm EDTA,1 XCocktail) was added to each dish after blotting the PBS, and after shaking at 4℃for 10min, the cells were transferred to a 1.5ml centrifuge tube for 30min for shaking. At 4℃after completion of cleavage, 120Centrifuging at 00g for 10min, and collecting supernatant.

The Input samples were prepared as follows: 100. Mu.L of the supernatant was added to the same volume of 2×loading buffer, and the mixture was heated at 100℃for 10 minutes.

The IP samples were prepared as follows; 20. Mu.L of Flag Beads are sucked into a clean 1.5mL EP tube, 600. Mu.L of TAP lysate is added, the mixture is fully mixed and kept stand for 5min, centrifugation is carried out for 2min at 2500rpm/min at 4 ℃, the supernatant is discarded, the washing is repeated for 3 times for 5min each time, and the mixture is placed on ice for standby. After the Input sample is taken, adding the rest cell lysis supernatant into an EP tube containing Flag Beads, rotating at 4 ℃ for incubation for 4 hours after marking, centrifuging at 2500rpm/min for 2 minutes at 4 ℃ after incubation, adding 600 mu L TAP lysate, mixing uniformly, repeatedly cleaning for three times, 5 minutes each time, and discarding the supernatant. 50. Mu.L of 2 XSDS-PAGE Loading Buffer was added to the Beads, and the mixture was heated in a metal bath at 100℃for 10 minutes to perform Western blotting detection.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skim milk at room temperature for 1h, monoclonal antibodies Flag (# M1403-2,1:2000, HUABIO), hsc70 (# 10654-1-AP,1:2000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), after dilution with PBST+5% skim milk, the corresponding membranes were placed in dilutions, incubated overnight at 4 ℃, PBST washed 3 times for 10min each, anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific) and anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) at room temperature for 1h, PBST washed 3 times for 10min each, followed by development with ECL development liquid.

The results are shown in fig. 10, and the IP results show that AUY-922 treatment can significantly enhance the interaction of Hsc70 with PD-L1. The degradation of PD-L1 by AUY-922 is shown to be dependent on the interaction of Hsc70 with PD-L1.

(5) AUY-922 enhances co-localization of PD-L1 with endosomes (Rab 7) and lysosomes (Lamp 1), and reduces co-localization of PD-L1 with circulating endosomes (Rab 11).

30 mu L of 1 XPBS solution is dripped in the center of a 12-orifice plate hole, and a cell climbing sheet is clamped by forceps and placed in the 12-orifice plate, so that the climbing sheet is tightly attached to the 12-orifice plate. MCF-7 cells were washed, digested, neutralized, centrifuged, resuspended in complete medium, and concentrated at 1X 10 ⁵ cells/well were plated into 12-well plates, and complete medium was added to a volume of 1mL. When the cell wall is grown to 60% -70%, 1 mu M AUY-922 is added for 12h. The medium was then discarded and each well was rinsed twice with 1mL of PBS solution for 3min. After the rinsing is finished, 1mL of 4% paraformaldehyde fixing solution is added into each hole, and the solution is rinsed twice for 3min by using PBS solution after 20 min; 1mL of closed punch liquid (5% fetal bovine serum+0.1% Triton X-100) is added to each well, and the mixture is incubated for 1h at room temperature; cutting a sealing film with proper length and width, spreading the sealing film in an immunofluorescence incubation box, and placing wet absorbent cotton into a clamping groove. Diluting the primary antibody with a sealing perforating liquid according to an antibody instruction, sucking 40 mu L of liquid, dripping the liquid on a sealing film, placing the cell surface of the climbing slice on the liquid, enabling the cell to be fully contacted with the antibody, taking no bubbles, covering a cover, and incubating at 4 ℃ overnight; the climbing sheet is clamped back into a new 12-hole plate again, so that the cell surface faces upwards, and 1mL of PBS solution is added into each hole for rinsing twice for 3min each time; under the condition of avoiding light, the corresponding fluorescent secondary antibody is diluted by using a sealing perforating liquid according to an antibody instruction book, 40 mu L of liquid is sucked and dripped on a sealing film of a wetting incubation box, and the cell surface of a climbing slice is placed on the liquid, so that the cell is fully contacted with the antibody, and no bubbles are required to be generated. Covering a cover and incubating for 1h at room temperature; under the condition of avoiding light, the climbing sheet is clamped back into a new 12-hole plate again, so that the cell surface faces upwards, and 1mL of PBS solution is added into each hole for rinsing twice for 3min each time; after the slide glass is marked, 30 mu L of sealing tablet is dripped into the center of the slide glass, the cell surface of the climbing tablet is placed on the sealing tablet, the slide glass is pressed into tablets, no bubbles are generated, and the climbing tablet is fixed by coating nail polish on the edge after the pressing. The image analysis was subsequently performed using an LSM880Zeiss laser confocal microscope. The tablet can be stored at 4deg.C in dark place.

The results are shown in FIG. 11, showing that AUY-922 enhances co-localization of PD-L1 with late endosome RAB 7A; as shown in fig. 12, immunofluorescence results showed that AUY-922 enhanced co-localization of PD-L1 with lysosomal LAMP 1; as shown in FIG. 13, immunofluorescence results showed that AUY-922 attenuated co-localization of PD-L1 with circulating endosome RAB 11.

Example 8 knockdown of expressed HSP90 promotes PD-L1 degradation and enhances expression of Hsc70

AUY-922 is known to promote PD-L1 degradation and enhance expression of Hsc70 as an inhibitor of HSP90, and whether or not it degrades PD-L1 depends on the level of HSP 90. This example was verified by knockdown of HSP90 expression in MCF-7 cells. The specific experimental method is as follows:

the specific experimental method is as follows:

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Inoculating the cells to 2 6-hole plates, adding a complete culture medium to a volume of 2ml, carrying out si-HSP90 alpha/beta transfection by using Lipofectamine 2000 when the cells are grown to be adhered to the wall until the fusion degree is 70% -80%, mixing the corresponding si-Control or si-HSP90 alpha/beta with the Lipofectamine 2000, standing for 15min, dropwise adding the mixed solution into the cultured cells by using a pipette, and gently shaking the culture dish to uniformly mix the transfection solution with the culture medium. Then placed at 37 ℃ and 5% CO ₂ Is cultured in a cell culture incubator for 48 hours, and then is treated with 1 mu M AUY-922 for 24 hours. One of the 6-well plates was washed with PBS, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10min, centrifuged, and the supernatant was taken for Western blotting. After cells were collected in another 6-well plate, the expression of cell surface PD-L1 was measured by flow cytometry.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skimmed milk for 1h at room temperature, mab PD-L1 (# 66248-1-lg,1:1000, proteintech), HSP 90. Alpha. (# 8165,1:1000,Cell Signaling Technology), HSP 90. Beta. (# 7411,1:1000,Cell Signaling Technology), hsc70 (# 10654-1-AP,1:2000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), after dilution with PBST+5% skimmed milk, the corresponding membranes were placed in dilutions, incubated overnight at 4℃and washed 3 times with PBST, each time for 10min, incubated 1h with secondary antibody anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) at room temperature, washed 3 times with PBST, each time for 10min, after which development was performed with ECL developing solution.

As shown in fig. 14, knockdown of HSP90 α/β expression enhanced Hsc70 levels and simultaneously promoted degradation of PD-L1, reducing cell membrane PD-L1 levels.

Experimental example 9 knockdown of expressed HSP90 promotion of PD-L1 degradation was dependent on Hsc70

Knockdown of expressed HSP90 is known to promote PD-L1 degradation and enhance Hsc70 expression, and whether or not the degradation of PD-L1 by knockdown expressed HSP90 is dependent on the level of Hsc 70. This example was validated by simultaneous knockdown of HSP90 and Hsc70 expression in MCF-7 cells. The specific experimental method is as follows:

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Inoculating the cells to 2 6-hole plates, adding a complete culture medium to a volume of 2ml, transfecting the cells with siHSP 90 alpha/beta and siHsc 70 by using Lipofectamine 2000 when the cells are grown to be adhered to the wall and have a fusion degree of 70% -80%, mixing the corresponding siControl or siHSP 90 alpha/beta, siHsc 70 with the Lipofectamine 2000, standing for 15min, dropwise adding the mixed solution into the cultured cells by using a pipetting gun, and gently shaking the culture dish to uniformly mix the transfection solution with the culture medium. Then placed at 37℃in 5% CO ₂ Is cultured in a cell culture incubator for 48 hours, and then is treated with 1 mu M AUY-922 for 24 hours. One of the 6-well plates was washed with PBS, sampled with 250. Mu.L of a 2 Xloading buffer, heated at 100deg.C for 10min, and detachedAnd taking the supernatant after the heart for western blotting detection. After cells were collected in another 6-well plate, the expression of cell surface PD-L1 was measured by flow cytometry.

As shown in fig. 15, knockdown of HSP90 α/β expression can enhance Hsc70 levels and simultaneously promote PD-L1 degradation, reducing cell membrane PD-L1 levels; while knocking down the expression HSP90 alpha/beta and knocking down the expression Hsc70 at the same time prevent the degradation of PD-L1 and the reduction of cell membrane PD-L1. The results indicate that the degradation of PD-L1 by knockdown expression of HSP90 is dependent on high expression of Hsc 70.

Experimental example 10 inhibition of HSP90 (AUY-922 treatment or knockdown of expressed HSP 90) can reduce CMTM6 levels

Given that CMTM6 is a negative regulator of PD-L1, knockdown of CMTM6 expression promotes PD-L1 degradation, and to verify whether inhibition of HSP90 can affect CMTM6 levels, this example was validated by knockdown of CMTM6 expression in MCF-7 cells and AUY-922 treatment. The specific experimental method is as follows:

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Inoculating the cells to 3 6-hole plates, adding a complete culture medium to a volume of 2ml, when the cells are grown on the wall to a fusion degree of 70% -80%, carrying out si-HSP90 alpha/beta transfection on one 6-hole plate by using Lipofectamine 2000, mixing the corresponding si-Control or si-HSP90 alpha/beta with the Lipofectamine 2000, standing for 15min, dropwise adding the mixed solution into the cultured cells by using a pipette, and gently shaking a culture dish to uniformly mix the transfection solution with the culture medium. Then placed at 37 ℃ and 5% CO ₂ Culturing for 60 hours in a cell culture box; one 6-well plate was treated with 1. Mu.M AUY-922 for 0,2,4,8, 12 and 24 hours; one 6-well plate was treated with AUY-922 at various concentrations for 24 hours; cells were washed with PBS from 6-well plates, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100deg.C for 10min, centrifuged and the supernatant was taken for Western blotting.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skimmed milk for 1h at room temperature, the corresponding membranes were placed in dilutions after dilution with PBST+5% skimmed milk, incubation overnight at 4℃with PBST washed 3 times for 10min each with secondary antibody anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) incubated for 1h at room temperature, PBST washed 3 times for 10min each with ECL chromogenic solution after HSP90 alpha (# 8165,1:1000,Cell Signaling Technology), HSP90 beta (# 7411,1:1000,Cell Signaling Technology), hsc70 (# 10654-1-AP,1:2000, proteontech), tubulin (# M1305-2,1:5000, HUABIO) and CMTM6 (# HPA026980,1:1000, sigma) were incubated at room temperature for 1h each with secondary antibody anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) and room temperature for 3 h each with PBST).

As shown in fig. 16, knockdown of HSP90 a/β expression reduced CMTM6 levels. As shown in FIG. 17, both the time gradient and the concentration gradient of AUY-922 treatment were able to reduce CMTM6 levels.

Experimental example 11 knock-down of CMTM6 expressed degradation of PD-L1 dependent on Hsc70

Knockdown of CMTM6 is known to promote PD-L1 degradation, and in order to verify whether knockdown of CMTM6 is dependent on Hsc70 levels, this experiment was verified by knockdown of CMTM6 and Hsc70 simultaneously in MCF-7 cells. The specific experimental method is as follows:

MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Inoculating the cells to 2 6-hole plates, adding a complete culture medium to a volume of 2ml, transfecting the cells by using Lipofectamine 2000 when the cells are grown to be adhered to the wall and have a fusion degree of 70% -80%, mixing the corresponding si-Control or si-CMTM6, si-Hsc70 and Lipofectamine 2000, standing for 15min, dropwise adding the mixed solution into the cultured cells by using a pipette, and gently shaking the culture dish to uniformly mix the transfection solution with the culture medium. Then placed at 37 ℃ and 5% CO ₂ Is cultured in a cell culture incubator for 60 hours. One of the 6-well plates was washed with PBS, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10min, centrifuged, and the supernatant was taken for Western blotting. After cells were collected in another 6-well plate, the expression of cell surface PD-L1 was measured by flow cytometry.

HEK-293T cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody and placed at 37℃with 5% CO ₂ Is cultured in a cell culture chamber of (2X 10) after passage ⁶ Is inoculated in a 10cm dish, a complete culture medium is added to a volume of 8ml, and after the cell wall-attached growth is carried out until the fusion degree is 70% -80%, the lip2000 is used for transfection of si-Control or si-CMTM for 6 h, and then lip2000 is used for transfection of Flag or pCDNA-PD-L1-Flag for 12h. The medium in the dish was discarded, the cells were washed 2 times with 2ml of pre-chilled PBS slowly added along the edge of the dish wall, 1ml of pre-chilled TAP lysate (20 mM Tris-HCl pH 7.5), 150mM NaCl,0.5% NP-40,1mM NaF,1mM Na3VO4,1Mm E was added to each dish after the PBS was blotted dryDTA,1 XCocktail), shaking at 4℃for 10min, and transferring to a 1.5ml centrifuge tube for 30min. After completion of the lysis, 12000g was centrifuged for 10min at 4℃and the supernatant was taken for use. The preparation method of the IP and Input samples is shown in experimental example 7

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skimmed milk for 1h at room temperature, mab PD-L1 (# 66248-1-lg,1:1000, proteintech), CMTM6 (# HPA026980,1:1000, sigma), hsc70 (# 10654-1-AP,1:2000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), after dilution with PBST+5% skimmed milk, the corresponding membranes were placed in dilutions, incubated overnight at 4℃with PBST for 3 times, 10min each, incubated with secondary anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) for 1h at room temperature, PBST for 3 times, 10min each, followed by development with ECL development solution.

As shown in figure 18, knockdown of CMTM6 expression promotes cell total PD-L1 degradation. As shown in fig. 19, simultaneous knockdown of expression Hsc70 prevented the total PD-L1 degradation and decrease in cell membrane PD-L1 induced by knockdown of CMTM 6. It is shown that knocking down CMTM6 promotes PD-L1 degradation dependent on Hsc70.

Experimental example 12 knockdown of CMTM6 expression promoted the interaction of PD-L1 with Hsc70.

It is known that knockdown of CMTM6 promotes PD-L1 degradation, and that degradation is Hsc70 dependent, and whether knockdown of CMTM6 affects PD-L1 interactions with Hsc70. The experimental example was verified by means of an immunoprecipitation experiment in 293T cells. The specific experimental method is as follows:

HEK-293T cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody and placed at 37℃with 5% CO ₂ Is cultured in a cell culture chamber of (2X 10) after passage ⁶ Is inoculated in a 10cm dish, added with complete culture medium to 8ml, and transfected with Flag or pCDNA-PD-L1-Flag or si-Control or si-CMTM6 by lip2000 when the cells are grown on the wall to a confluence of 70% -80%. The medium in the dish was discarded, the cells were washed 2 times with 2ml of pre-chilled PBS slowly added along the edge of the dish wall, 1ml of pre-chilled TAP lysate (20 mM Tris-HCl (pH 7.5), 150mM NaCl,0.5% NP-40,1mM NaF,1mM Na) was added to each dish after the PBS was blotted dry ₃ VO4,1mM EDTA,1 XCocktail), shaking at 4℃for 10min and transferring to a 1.5ml centrifuge tube for 30min. After completion of the lysis, 12000g was centrifuged for 10min at 4℃and the supernatant was taken for use.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membranes were blocked with 5% skimmed milk at room temperature for 1h, mab Flag (#M 1403-2,1:2000, HUABIO), hsc70 (#10654-1-AP, 1:2000, proteintech), CMTM6 (#HPA 026980,1:1000, sigma), tubulin (#M 1305-2,1:5000, HUABIO), after dilution with PBST+5% skimmed milk, the corresponding membranes were placed in dilutions, incubated overnight at 4 ℃, washed 3 times with PBST for 10min each, incubated 1h with secondary antibody anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-mouse IgG (# 31460,1:20000,Thermo Fisher Scientific) at room temperature, washed 3 times with PBST for 10min each, followed by development with ECL development liquid.

The results are shown in figure 20, where the IP results show that knockdown of CMTM6 (si-CMTM 6) can significantly enhance the interaction of Hsc70 with PD-L1. It was shown that the degradation of PD-L1 by knockdown expression of CMTM6 is also dependent on the interaction of Hsc70 with PD-L1.

Experimental example 13 treatment with other HSP90 inhibitors such as 17-AAG, 17-DMAG also promotes PD-L1 degradation, enhancing Hsc70 levels

To test whether other inhibitors of HSP90 are also present to degrade PD-L1 and to enhance the efficacy of Hsc70 levels, we used other inhibitors of HSP90 such as 17-AAG and 17-DMAG. This example was verified by treatment with 17-AAG and 17-DMAG in MCF-7 cells.

The specific experimental method is as follows:

MCF-7 was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin diabody, and passaged at 4X 10 ⁵ Cells were inoculated in 6-well plates, after cells were grown to a concentration of 1. Mu.M at the beginning of the logarithmic growth phase, washed twice with PBS 24h after the administration, collected with 250. Mu.L of a 2 Xloading buffer, heated at 100℃for 10min, and the supernatants were centrifuged and subjected to Western blotting.

Both 17-AAG and 17-DMAG treatments were able to increase the expression of Hsc70 in MCF-7 cells while reducing the amount of PD-L1 as shown in FIGS. 21-22.

Example 14 overexpression of Hsc70 promotes the degradation of PD-L1 by lysosomes

Examples 3 and 8 show that AUY-922 treatment or knockdown of expressed HSP90 can enhance the amount of Hsc70, and whether AUY-922 and knockdown of expressed HSP90 are both related to high levels of Hsc70 in PD-L1 degradation, and this example is verified by over-expression of Hsc70 in MCF-7, PANC1 and other cells. The specific experimental method is as follows:

MCF-7, PANC1 cells were incubated with DMEM medium (from hyclone) +penicillin/streptomycin diabodies (from Gibco, # 15140-122), 37℃and 5% CO ₂ Culturing under conditions to logarithmic phase, digestion with pancreatin (Gibco) and counting, cell density according to 1×10 ⁶ Each well is inoculated into a 6-well plate, and the volume of each well is 2ml, so that the uniform distribution of cells is ensured. After 24h, 2 ug/well of Hsc70-Flag or empty Flag was transfected with lip3000, and after 24h the corresponding proteasome inhibitor (MG 132, 10. Mu.M), lysosome inhibitor (NH) ₄ Cl+Leup; E64D) and a large autophagy inhibitor (3-MA) for 12h, washing the cells twice with PBS, adding 2×loading buffer to digest the cells, transferring into a 1.5ml centrifuge tube, heating at 100deg.C for 10min, centrifuging 12000g for 10min, collecting supernatant, and performing corresponding western blotting detection.

Western blotting technique: preparing 10% polyacrylamide gel, carrying out electrophoresis on 10 mu L of each gel Kong Zhongshang sample for 2h by running 90V gel, carrying out film transfer treatment after bromophenol blue runs to a gel bottom layer, and carrying out film transfer for 1h by using PVDF film under the film transfer condition of 300 mA; after the membrane was blocked with 5% skimmed milk at room temperature for 1h, mab PD-L1 (# 66248-1-lg,1:1000, proteintech), flag (# M1403-2,1:2000, HUABIO), HK2 (# 22029-1-AP,1:1000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), after dilution with PBST+5% skimmed milk, the corresponding membrane was placed in the dilution, incubated overnight at 4℃with PBST for 3 times, 10min each, with secondary anti-mouse IgG (# 31430,1:20000,Thermo Fisher Scientific), anti-rabit IgG (# 31460,1:20000,Thermo Fisher Scientific) for 1h at room temperature, PBST for 3 times, 10min each, after which development with ECL development solution was performed.

As shown in fig. 23, the overexpression of Hsc70 significantly promoted the degradation of PD-L1, while inhibiting the proteasome pathway (MG 132) did not affect the level of PD-L1, while inhibiting the lysosomal pathway (NH ₄ Cl+Leup; E64D) is capable of significantly preventing degradation of PD-L1. Experimental results indicate that enhancing the Hsc70 level can promote the degradation of PD-L1 through lysosomes.

Example 15 Hsc70 promotes PD-L1 degradation, inhibits tumor growth

Binding of the carboxyl-terminal domain of Hsc70 to phosphatidylserine on late endosomal membranes is critical for the development of endosomal microalbumination. Hsc70-3KA is a mutant of Hsc70, and the mutation site of Hsc70 is located at the carboxyl end lysine of Hsc70, so that the combination with phosphatidylserine on late endosomal membrane can be destroyed, and the occurrence of endosomal microalbumination can be inhibited. On the basis, stable transgenic cell lines which over express Hsc70-WT and Hsc70-3KA are constructed on 4T1 cells in the embodiment, and in vivo experiments are carried out. The specific experimental method is as follows:

(1) Construction of 4T1 stable transgenic cell line over-expressing Flag and Hsc70-Flag

HEK293T cells were transfected with packing plasmids of PLVTH-Flag, PLVTH-Hsc70-WT-Flag, PLVTH-Hsc70-3KA-Flag, and medium containing secreted viruses was collected at 48h, 72h and 96h, respectively, filtered with 0.45 μm filter, and virus was precipitated by PEG 8000. The obtained virus is used for infecting 4T1 cells, the culture medium is changed after 24 hours of infection, 4 mug/mL puromycin is used for screening and flow sorting the infected 4T1 cells, and a stable expression single cell stable transgenic strain is obtained, and as shown in figure 24, western immunoblotting detection shows that the construction of the stable transgenic strain with the overexpression of Hsc70 is successful.

(2) Construction of mouse 4T1 breast cancer tumor model

Female BALB/c mice and nude mice of 6-8 weeks are selected, and SPF-class animal feeding conditions (feeding temperature is 20.5-24.5 ℃, humidity is 40-75 percent, and lighting period is 12 hours, namely 12 hours, dark) are provided. Tumor cell inoculation is performed after mice are raised to 8-10 weeks. Selecting 4T1 stable transfer cells over-expressing Flag, hsc70-WT-Flag, hsc70-3KA-Flag, growing to logarithmic phase, digesting and collecting cells with pancreatin, diluting cells with PBS to concentration of 5×10 ⁶ Ml and with matrigel 1:1, mixing and placing on ice for standby.

Mouse breast cancer tumor model: mice were anesthetized and shavers shaved the hair near the second pair of nipples of the mice, with 75% alcohol sterilized. After mixing the 4T1 cell suspension, 100. Mu.l was aspirated, and the mixture was inoculated onto a mammary gland pad using a 1ml syringe, each group was inoculated with 12 mice, and tumor sizes were measured and recorded daily, and a corresponding tumor model construction schematic is shown in FIG. 25.

(3) In vivo anti-tumor effect detection

BALB/c mice and nude mice were each divided into three groups, each of which was inoculated with different 4T1 cells, control group (4T 1 breast cancer cells inoculated with overexpressed Flag), hsc70-WT group (4T 1 breast cancer cells inoculated with overexpressed Hsc 70-Flag), hsc70-3KA group (4T 1 breast cancer cells inoculated with overexpressed Hsc70-3 KA), the mice were observed daily after the cells were inoculated, tumor volumes of mice were recorded daily starting on day 3, tumor Volumes (TV) =1/2×a×b ² Wherein a and b respectively represent the long diameter and the short diameter of the tumor. After the experiment was completed, the tumor tissue of the mice was peeled off, the tumor weight was weighed, and a tumor picture was left.

As shown in fig. 26, in BALB/c of immunized normal mice, overexpression of Hsc70 was able to significantly inhibit tumor growth, whereas in nude mice of immunodeficiency, overexpression of Hsc70 had no inhibitory effect on tumor growth. As shown in fig. 27, 28, the tumor weight and size of the overexpressed Hsc70-WT was significantly reduced after the end of administration in dissected BALB/c mice, while there was no significant difference in the weight and size of the overexpressed Hsc70-WT in nude mice of immunodeficient mice. These results indicate that Hsc 70-promoted tumor suppression is dependent on the function of the immune system.

(4) Tumor tissue immune microenvironment flow detection analysis

Weighing 50mg of tumor tissue, placing in a 6-well plate, adding 10ml of PBS, washing twice, shearing the tumor tissue by using sterile scissors, adding a DMEM culture medium containing type I collagenase (# 2350118, gibco) and DNase I (# 143582, roche), and digesting the tumor tissue in an incubator at 37 ℃ for 1h; the digested cells and tissue fragments were collected, placed in a 45 μm pore-size filter, milled with PBS (0.5% BSA) to prepare a single cell suspension, the isolated cells were stained with a specific surface marker antibody Zombie Violet (# 423114; biolegend) for 30min to isolate viable cells, the cells were washed with PBS, and then incubated with anti-CD 45-PerCP-Cy5.5 (# 103132; biolegend), anti-CD 3-PE-Cy7 (# 100320; biolegend) and anti-CD 8-FITC (# 100706; biolegend) antibodies were diluted with PBS and incubated at 4℃for 30 min. Cells were washed with PBS, fixed and permeabilized with Fix/Perm kit (# 421403; biolegend), and finally stained with anti-APC GzmB (# 372204; biolegent).

Flow antibody labeling was as follows:

Marker CD45 CD3 CD8 GZMB Alive/dead

Colour PE-Cy5 PE-Cy7 FITC APC Zombie Violet

as shown in FIG. 29, flow analysis of immune cells in BALB/c mouse tumor tissue showed that overexpression of Hsc70-WT significantly increased CD8 ⁺ And GzmB ⁺ The proportion of cells shows that Hsc70 enhances the proportion of immune cells in tumor tissues and enhances the anti-tumor immunity capability.

(5) Tumor tissue immune microenvironment fluorescent staining analysis

The end point of the experiment was to strip tumor tissue, 4% paraformaldehyde was fixed for 24h,30% sucrose solution was dehydrated overnight, OCT was used to embed tumor tissue, frozen microtomes were used to section tumor tissue, permeabilized with PBS solution containing 0.1% Triton X-100 and blocked with serum, and the corresponding primary antibody was incubated overnight at 4 ℃. After PBS washing, the tumor tissues are incubated for 1h with a secondary antibody with fluorescent markers, a DAPI-containing sealing liquid sealing sheet is used, and different types of immune cells in the tumor tissues are recorded by a laser confocal microscope and analyzed for proportion and distribution.

As shown in FIG. 30, immunocytes in BALB/c mouse tumor tissues were immunizedThe results of the histochemical analysis also showed that overexpression of Hsc70-WT increased CD8 ⁺ And GzmB ⁺ The number of cells enhances the anti-tumor immunity.

(6) Western immunoblot analysis

Tumor tissues were washed twice with PBS, incubated with RIPA lysate on ice for 30min, centrifuged for 10min 12000g, the supernatant was taken and added with an equal volume of 2×loading buffer, heated at 100deg.C for 10min, centrifuged and transferred with 10% -12% SDS-PAGE gel onto PVDF membrane, membrane was blocked with 5% skim milk at room temperature for 1h, monoclonal antibodies PD-L1 (# 66248-1-lg,1:1000, proteintech), hsc70 (# 10654-1-AP,1:2000, proteintech), tubulin (# M1305-2,1:5000, HUABIO), diluted with PBST+5% skim milk, the corresponding membrane was placed in dilution, incubated overnight at 4deg.C, PBST washed 3 times for 10min each time, secondary antibodies IgG 31430,1:20000,Thermo Fisher Scientific), and antibodies Abb 31460,1:20000,Thermo Fisher Scientific) incubated at room temperature for 3 times, and developed with ECL 1:2000, and developed with image color by means of each time of 80J for 1.J color, and then quantified by using image color development.

As shown in fig. 31, the results of western blotting detection on tumor tissues showed that the expression level of PD-L1 in the tumor tissues overexpressing Hsc70-WT was significantly reduced.

Example 16 AUY-922 has better tumor growth inhibition and anti-tumor immunity promotion than Ganetespib in vivo

(1) Mouse 4T1 breast cancer tumor model construction and grouping

Female BALB/c mice and nude mice of 6-8 weeks are selected, and SPF-class animal feeding conditions (feeding temperature is 20.5-24.5 ℃, humidity is 40-75 percent, and lighting period is 12 hours, namely 12 hours, dark) are provided. Tumor cell inoculation is performed after mice are raised to 8-10 weeks. Selecting 4T1 cells, culturing to logarithmic phase, collecting cells by pancreatin digestion, diluting cells with PBS to concentration of 5×10 ⁶ Ml and with matrigel 1:1, mixing and placing on ice for standby. Mice were anesthetized and shavers shaved the hair near the second pair of nipples of the mice, with 75% alcohol sterilized. After mixing the 4T1 cell suspension, 100. Mu.l was aspirated, and 1m was usedl syringe was inoculated on mammary gland pad, 12 mice were inoculated in each group, tumor size was measured and recorded daily until tumor growth to a volume of 75mm ³ Group dosing was started at this time, BALB/c mice and nude mice were each divided into two groups, a Control group (Control, vehicle), a Gantespib treated group (Gantespib, 25 mg/kg) and an AUY-922 treated group (AUY-922, 25 mg/kg), intraperitoneal injections of 5% DMSO+30% PEG400+65% ddH2O, once daily injections for 14 days, tumor volumes were measured daily, euthanasia was performed on the mice after the end of dosing, tumors were peeled off and tumor weights were weighed. A certain weight of tumor tissue was digested into single cells with collagenase and subjected to a corresponding flow assay, the analytical method of which was described in example 15.

The corresponding tumor model construction scheme is shown in fig. 32.

As shown in FIG. 33, in BALB/c of immunized normal mice, gantespib and AUY-922 can inhibit tumor growth, and AUY-922 has more remarkable effect of inhibiting tumor growth. In nude mice with immunodeficiency, gantespib and AUY-922 have no inhibition on tumor growth. As shown in FIG. 34, the tumor weights of both Gantespib and AUY-922 treated groups in BALB/c mice were significantly reduced after end of dosing dissection, with AUY-922 tumor weights reduced to a greater extent than Gantespib; there was no significant difference in tumor weight in the ganespib and AUY-922 treated groups in nude mice with immunodeficiency. These results indicate that AUY-922 treatment promotes tumor suppression better than ganespib and is dependent on immune system function.

As shown in FIG. 35, flow analysis of immunocytes in tumor tissue of BALB/c mice showed significant increases in CD8 by both Gantespib and AUY-922 treatment ⁺ And GzmB ⁺ Ratio of cells, and AUY-922 treatment on CD8 ⁺ And GzmB ⁺ The proportion of increased cells is superior to Gantespib, indicating that AUY-922 treatment enhances the proportion of immune cells in tumor tissue, enhancing the anti-tumor immunity.

EXAMPLE 17 AUY-922 enhanced anti-tumor Effect of PD-L1 mab

In order to detect whether AUY-922 can enhance the anti-tumor effect of PD-L1 monoclonal antibody, a combination experiment is performed by using a mouse breast cancer tumor model, and meanwhile, the combination effect of Gantespib and PD-L1 monoclonal antibody is compared.

(1) Mouse 4T1 breast cancer tumor model construction and grouping

Female BALB/c mice and nude mice of 6-8 weeks are selected, and SPF-class animal feeding conditions (feeding temperature is 20.5-24.5 ℃, humidity is 40-75 percent, and lighting period is 12 hours, namely 12 hours, dark) are provided. Tumor cell inoculation is performed after mice are raised to 8-10 weeks. 4T1 cells were selected, and after cells were grown to logarithmic growth phase, cells were collected by digestion with pancreatin, diluted to a concentration of 5X 106/ml with PBS and mixed with matrigel 1:1, mixing and placing on ice for standby. Mice were anesthetized and shavers shaved the hair near the second pair of nipples of the mice, with 75% alcohol sterilized. After mixing 4T1 cell suspension, 100 μl was aspirated, inoculated onto mammary gland pad with 1ml syringe, each group inoculated with 12 mice, tumor size was measured and recorded daily until tumor growth to a volume of 75mm ³ Grouping dosing was started at this time, and the groups were divided into four groups, control (vehicle) and PD-L1 mab-treated groups (anti-PD-L1, 100. Mu.g/dose), gantespib and PD-L1 mab-combined group (Gantespib+anti-PD-L1, 12.5mg/kg Gantespib+100. Mu.g/dose anti-PD-L1) and AUY-922 and PD-L1 mab-combined group (AUY-922+anti-PD-L1, 12.5mg/kg AUY-922+100. Mu.g/dose anti-PD-L1), gantespib and AUY-922 were intraperitoneally injected, 5% DMSO+30% PEG400+65% ddO were injected once daily, the PD-L1 mab dose was 100 ug/dose was weighed once every seven days, the tumor volume was measured daily, and the mice were euthanized and tumors were dissected and weight killed. Taking a certain weight of tumor tissue, digesting the tumor tissue into single cells by collagenase, and carrying out corresponding flow detection, wherein the flow detection analysis method is as described in example 15;

The corresponding tumor model construction scheme is shown in fig. 36.

As shown in fig. 37, in the immunized normal mice BALB/c, both PD-L1 mab treatment and combination treatment can inhibit tumor growth, and AUY-922 combined PD-L1 mab can further increase tumor inhibition compared to Ganetespib combined PD-L1 mab and PD-L1 mab. As shown in fig. 38, tumor weight and size were reduced in both PD-L1 mab group and combination group after administration of BALB/c mice was completed, wherein AUY-922 combined PD-L1 mab was able to further reduce tumor weight compared to Ganetespib combined PD-L1 mab and PD-L1 mab alone. These results indicate that the anti-tumor effect of AUY-922 combined with PD-L1 mab is superior to that of PD-L1 mab and Ganetespib combined with PD-L1 mab.

As shown in FIG. 39, flow analysis of immunocytes in tumor tissue of BALB/c mice showed that both PD-L1 mab-treated and combined groups increased CD8 ⁺ And GzmB ⁺ Proportion of cells, wherein AUY-922 increases CD8 in combination with PD-L1 mab ⁺ And GzmB ⁺ The proportion of cells is superior to that of Ganetespib combined PD-L1 monoclonal antibody and PD-L1 monoclonal antibody, which shows that AUY-922 enhances the anti-tumor immunity of PD-L1 monoclonal antibody, and is hopeful to solve the drug resistance of PD-L1 monoclonal antibody.

As shown in fig. 40, flow analysis of tumor cell membrane surface PD-L1 in BALB/c mouse tumor tissue showed that cell membrane PD-L1 levels recovered to normal levels after seven days of treatment with PD-L1 mab, AUY-922 combined PD-L1 mab was able to reduce cell membrane PD-L1 levels and was superior to Ganetespib combined PD-L1 mab group. AUY-922 treatment was shown to reduce the cell membrane lift of PD-L1 after prolonged use of PD-L1 mab.

EXAMPLE 18 AUY-922 enhancement of anti-tumor Effect of CTLA4 monoclonal antibody

In order to detect whether AUY-922 can enhance the anti-tumor effect of an immunosuppressant, a mouse breast cancer tumor model is used for carrying out a combination experiment of AUY-922 and anti-CTLA4 monoclonal antibody, and meanwhile, the combination effect of Gantespib and CTLA4 monoclonal antibody is compared.

(1) Mouse 4T1 breast cancer tumor model construction and grouping

Female BALB/c mice and nude mice of 6-8 weeks are selected, and SPF-class animal feeding conditions (feeding temperature is 20.5-24.5 ℃, humidity is 40-75 percent, and lighting period is 12 hours, namely 12 hours, dark) are provided. Tumor cell inoculation is performed after mice are raised to 8-10 weeks. 4T1 cells were selected, and after cells were grown to logarithmic growth phase, cells were collected by digestion with pancreatin, diluted to a concentration of 5X 106/ml with PBS and mixed with matrigel 1:1, mixing and placing on ice for standby. Anesthetizing the mice, shaving the second pair of papilla-adjacent hairs by a shaverAnd (3) sterilizing with 75% alcohol. After mixing 4T1 cell suspension, 100 μl was aspirated, inoculated onto mammary gland pad with 1ml syringe, each group inoculated with 12 mice, tumor size was measured and recorded daily until tumor growth to a volume of 75mm ³ The group administration was started, and the group was divided into four groups, namely, a Control group (Control, vehicle), a CTLA-4 mab-treated group (anti-PD-L1, 100 ug/vehicle), a Ganetespib-anti-CTLA 4-combined group (Ganetespib+anti-CTLA 4, 12.5mg/kg AUY-922+100 ug/anti-CTLA 4-alone) and an AUY-922-combined group (AUY-922+anti-CTLA 4, 12.5mg/kg AUY-922+100 ug/anti-CTLA 4-alone), ganetespib-and AUY-922 were intraperitoneally injected, 5% DMSO+30% PEG400+65% ddH2O were injected once daily, the tumor volume was measured once daily, and the mice were euthanized after the end of the administration, and the tumor was weighed by peeling. Taking a certain weight of tumor tissue, digesting the tumor tissue into single cells by collagenase, and carrying out corresponding flow detection, wherein the flow detection analysis method is as described in example 15;

As shown in fig. 41, both CTLA4 mab-treated and combined groups were able to inhibit tumor growth in immunized normal mice BALB/c, wherein AUY-922 combined CTLA4 mab was able to further increase tumor inhibition compared to Ganetespib combined CTLA4 mab and CTLA4 mab alone. As shown in fig. 42, both CTLA4 mab group and the combination group showed a decrease in tumor weight and size after administration of the end-dissected BALB/c mice, wherein the effect of AUY-922 combined CTLA4 mab on tumor inhibition was superior to that of CTLA4 mab alone and Ganetespib combined CTLA4 mab. These results indicate that AUY-922 combined CTLA4 monoclonal antibody has more effective anti-tumor effect, and can solve the drug resistance of the CTLA4 monoclonal antibody.

As shown in FIG. 43, flow analysis of immune cells in tumor tissue of BALB/c mice showed that both CTLA4 mab-treated and combined groups increased CD8 ⁺ And GzmB ⁺ Proportion of cells in which AUY-922 increases CD8 in combination with CTLA4 mab ⁺ And GzmB ⁺ The proportion of cells is superior to that of the single CTLA4 antibody and the combined Ganetespib and CTLA4 antibody, which shows that AUY-922 and CTLA4 antibody have more effective capability of enhancing anti-tumor immunity.

As shown in fig. 44, flow analysis of tumor cell membrane surface PD-L1 in BALB/c mouse tumor tissue showed that CTLA4 mab treated cell membrane PD-L1 levels were not significantly different from control group, and AUY-922 combined with CTLA4 mab was able to significantly reduce cell membrane PD-L1 levels.

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims

1. Use of a pharmaceutical combination for:

(iv) Enhancing the interaction of PD-L1 and Hsc 70;

(v) Increasing CD8 in immune cells ⁺ And/or GzmB ⁺ Proportion of cells;

(vi) Treating breast cancer; and/or

(vii) Preparing a medicament for treating breast cancer;

2. the use according to claim 1, wherein the amount of the first medicament in the pharmaceutical combination is 10-1000mg;

And the amount of the second drug is 10-5000mg.

3. The use according to claim 1, wherein the immune checkpoint inhibitor comprises at least one of a PD-1/PD-L1 inhibitor and a CTLA-4 inhibitor.

4. The use according to claim 3, wherein the PD-1/PD-L1 inhibitor is a PD-1/PD-L1 antibody; and/or

The CTLA-4 inhibitor CTLA-4 antibodies or antigen-binding fragments thereof.

5. The use according to claim 1, wherein the use in (i) - (v) is an in vitro non-therapeutic use.

6. A method of treating breast cancer, the method comprising the steps of:

administering to a patient a therapeutically effective amount of the pharmaceutical combination of claim 1.

7. A method of inducing in vitro tumor cell endosomal microalbumina comprising the steps of:

culturing the tumor cells in a culture system comprising the pharmaceutical combination of claim 1.

8. The use according to claim 1, wherein the induction of endosomal microalbumina in tumor cells is by up-regulating the level of Hsc70 expression in the tumor cells.

9. The use according to claim 1, wherein tumor cell endosomal microalbumination is induced by enhancing the interaction of PD-L1 and Hsc 70.

10. A method of up-regulating the expression level of Hsc70 in a tumor cell in vitro, comprising the steps of: