CN106955356B

CN106955356B - Combined medicine composition for treating tumor

Info

Publication number: CN106955356B
Application number: CN201610015530.2A
Authority: CN
Inventors: 许琛琦; 李伯良; 杨魏; 白轶冰; 熊缨
Original assignee: Center for Excellence in Molecular Cell Science of CAS
Current assignee: Center for Excellence in Molecular Cell Science of CAS
Priority date: 2016-01-11
Filing date: 2016-01-11
Publication date: 2021-06-22
Anticipated expiration: 2036-01-11
Also published as: CN106955356A

Abstract

The invention provides a combined medicine composition for treating tumors, which comprises effective dose of acyl coenzyme A: a cholesterol acyltransferase ACAT 1inhibitor and an effective amount of dacarbazine. The invention discloses acyl coenzyme A: the cholesterol acyltransferase ACAT 1inhibitor can enhance the killing activity of CD8T cells on tumors, and has synergistic effect when being combined with dacarbazine, thereby further enhancing the inhibition on tumors.

Description

Combined medicine composition for treating tumor

Technical Field

The invention belongs to the field of biotechnology, and particularly relates to a combined drug for treating tumors.

Background

Malignant tumor is one of the most lethal diseases at present, and the conventional treatment means such as surgical excision, radiotherapy, chemotherapy and the like are mostly applied to tumor treatment, but the conventional treatment means has limitations in tumor treatment and is difficult to completely cure tumor, especially some metastatic malignant tumors.

Chemotherapy is one of the effective means for treating tumors at present. Dacarbazine is known by the chemical name: 5- (3, 3-dimethyl-1-triazenyl) -4-amidoimidazole citrate. It can release methyl positive ion (CH3) + to play the role of alkylation when decomposed in vivo; and at the same time, it becomes a substance similar to an intermediate product of purine biosynthesis, possibly interfering with purine biosynthesis. The traditional Chinese medicine composition is clinically used for treating melanoma at present, and also has some treatment effects on squamous cell carcinoma of lung, undifferentiated carcinoma, leiomyosarcoma, fibrosarcoma and the like. As a tumor chemotherapy medicament, the side effect is large, and the administration is often accompanied by leukopenia, thrombocytopenia, inappetence, nausea, vomit, general malaise, muscle ache, high fever and the like. There may also be numbness of the face and alopecia, and some patients may have dysfunction of liver and kidney.

Tumor immunotherapy has been the focus of research on tumor therapy. At present, the approaches of tumor Immunotherapy mainly include T cell Adoptive therapy (ACT), checkpoint immune therapy (anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibody therapy), tumor vaccine and Chimeric Antigen Receptor T cell therapy (Chimeric Antigen Receptor T-cell Immunotherapy), etc., which have advanced in clinical research, but the tumor Immunotherapy still faces a lot of important obstacles due to the heterogeneity of tumor cells and the complexity of tumor microenvironment.

The CD8T cell is the core of tumor immunotherapy, is a tumor specific killer cell, the killing capability of the tumor specific killer cell is strong and weak, and the infiltration condition in tumor tissues is directly related to the prognosis of tumors, and is directly related to the success or failure of tumor immunotherapy. Since the tumor microenvironment is an immunosuppressive environment, the function of CD8T cells is inhibited by the tumor microenvironment, and the current immunodetection point blocking antibodies anti-PD-1, anti-PD-L1 and anti-CTLA-4 have good overall effect in clinical trials for treating melanoma, but the Objective Response rates of the antibodies (Objective Response Rate) alone and the antibodies (PD-1 and CTLA-4) in combination are unsatisfactory. This means that there is an urgent need to develop new targets to further enhance the function of CD8T cells in tumors.

The intracellular cholesterol metabolic pathway comprises the pathways of cholesterol synthesis, transportation, storage and the like, wherein the cholesterol synthesis pathway is mainly regulated and controlled by a SCAP/SREBP compound and a downstream cholesterol synthesis rate-limiting enzyme HMGCR; the regulation of the cholesterol transport pathway is mainly regulated by proteins and compounds such as NPC1/2 in low-density lipoprotein receptor (LDL receptor), endosome (endosome) and lysosome (lysosome); the storage of cholesterol is regulated by esterification modification and de-esterification modification of cholesterol, and key regulatory proteins comprise acyl coenzyme A: cholesterol acyltransferase ACAT1/2 and Cholesterol Ester Hydrolase (CEH). The ACAT1 has been shown to be a target for treating atherosclerosis and Alzheimer's disease, and some small molecule inhibitors have been developed based on the target, such as Avasimibe, which have good clinical safety in clinical experiments. The prior art does not disclose the association of ACAT1 with tumor therapy.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a combination useful for the treatment of tumors.

In a first aspect, the present invention provides a combination for use in the treatment of tumours, comprising an effective amount of acyl-coa: a cholesterol acyltransferase ACAT 1inhibitor and an effective amount of dacarbazine.

The acyl-coenzyme A: inhibitors of cholesterol acyltransferase ACAT1 refer to compounds which are specific for acyl-coa: a compound having an inhibitory effect on cholesterol acyltransferase ACAT 1.

For acyl-coa: the cholesterol acyltransferase ACAT1 has inhibitory effect including but not limited to: inhibition of acyl-coa: cholesterol acyltransferase ACAT1 activity, or inhibiting acyl-coa: the gene transcription or expression of cholesterol acyltransferase ACAT 1.

The acyl coenzyme A: the cholesterol acyltransferase ACAT 1inhibitor can be siRNA, shRNA, antibody, small molecule compound.

As exemplified in the examples of the present invention, the acyl-coa: the cholesterol acyltransferase ACAT 1inhibitor is selected from: avasimibe, K604, CP113,818, the acyl-coa: the cholesterol acyltransferase ACAT 1inhibitor may also be selected from: purpuracts, manassantin A, diphenylpyridazine derivitives, glisoprenin A, pacitmibe, CI 976, TMP-153, YM 750, GERI-BP002-A, Sandoz 58-035, VULM 1457, CL-283,546, CI-999, E5324, YM17E, FR182980, PD132301-2, F-1394, HL-004, F-12511(eflucimibe), cinnamic acid derivitives, Dup 128, RP-73163, pyripyropene C, FO-1289, AS-183, SPC-49, BeauI, BeauIII, FO-6979, Angelica, ginseng, Decurnsin, terdopentol C, auciveverin, spcyline, betacidides, shinatidine, shiticine-283,546, shiividerivitives, Wriederiv-23, and the like.

The combination therapy drug combination may be in any one of the following forms:

one) reacting acyl-CoA: the cholesterol acyltransferase ACAT 1inhibitor and dacarbazine are prepared into independent preparations respectively, the preparation forms can be the same or different, and the administration routes can be the same or different.

Two) reacting acyl coenzyme A: the cholesterol acyltransferase ACAT 1inhibitor and dacarbazine are prepared into a compound preparation. After converting acyl-CoA: when the cholesterol acyltransferase ACAT 1inhibitor and dacarbazine are administered by the same route of administration and are administered simultaneously, they may be formulated as a combined preparation.

In a second aspect, the present invention provides a method for combined treatment of tumor immunotherapy by administering to a subject an effective amount of an acyl-coa: a cholesterol acyltransferase ACAT 1inhibitor, and an effective amount of dacarbazine.

The subject is a mammal or a CD8T cell of the mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, or the like. Preferably, the primate is a monkey, ape or homo sapiens.

The subject may be a patient suffering from a tumor or an individual expected to improve anti-tumor immunity, or an ex vivo CD8T cell of a patient suffering from a tumor or an individual expected to improve anti-tumor immunity.

An effective amount of acyl-coa: a cholesterol acyltransferase ACAT 1inhibitor and an effective amount of dacarbazine.

Based on acyl-coa: the cholesterol acyltransferase ACAT1 is a newly discovered tumor immunotherapy target, and the research of the invention finds that the cholesterol acyltransferase ACAT1 and dacarbazine can play a synergistic effect in the combined administration of the two drugs, thereby further enhancing the inhibition on tumors.

Tumors targeted by the tumor therapy include solid tumors and non-solid tumors, including but not limited to: malignant tumors of nasal cavity and nasal sinuses, nasopharyngeal carcinoma, oral cancer, laryngeal carcinoma, salivary gland tumor, intracranial tumor, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, cardiac cancer, breast cancer, mediastinal tumor, stomach cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampulla cancer, biliary tract cancer, small intestine tumor, kidney cancer, prostate cancer, bladder cancer, testicular malignancy, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrocytoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, ewing's sarcoma, lymphoma, multiple myeloma, leukemia and the like.

In some embodiments, the tumor therapy is for melanoma, soft tissue sarcoma, lymphoma.

The acyl-coenzyme A: the cholesterol acyltransferase ACAT 1inhibitor and the immunodetection point blocking therapy medicament can be administered to a subject before, during or after receiving tumor immunotherapy.

The invention has the advantages that:

(1) through a series of in vitro experiments, ACAT1 is found to play a key role in regulating and controlling CD8T cell immune response reaction, and inhibition of ACAT1 activity can enhance antigen-specific immune response reaction of CD8T cells and enhance killing capability of the cells to target cells without affecting response of the CD8T cells to self antigens;

(2) in vitro experiments show that the ACAT1 activity is inhibited to enhance the anti-tumor capacity of CD8T cells;

(3) on an animal level, the immune response capability of CD8T cells can be enhanced by knocking out ACAT1 through a T cell specific gene or inhibiting the ACAT1 activity through an inhibitor, and the anti-tumor activity of CD8T cells is enhanced in a mouse melanoma model and a mouse lung cancer model; in the experiment of mouse melanoma treated by combining dacarbazine, the Avasimibe serving as an ACAT 1inhibitor can be used for promoting the antitumor activity of CD8T cells in cooperation with the dacarbazine.

(4) While the prior art demonstrates acyl-coa: the safety of administration of cholesterol acyltransferase ACAT1 inhibitors, however, acyl-coa: the clinical efficacy of the cholesterol acyltransferase ACAT 1inhibitor is not ideal for the primary indication. The present invention provides an acyl-coa: the cholesterol acyltransferase ACAT 1inhibitor provides a new clinical application value, opens up a wide prospect for further market application, and provides a more effective treatment scheme for tumor patients.

Drawings

FIG. 1ACAT1 knock-out enhances the immune response of OT-I CD8T cells to specific antigens, and does not elicit an immune response to self-antigens.

C57BL/6 mouse-derived spleen cells after presenting foreign antigens (N4, A2, T4 and G4) or autoantigens (R4 and Catnb) as Antigen-presenting cells (APC) stimulation

Wild type (WT OT-I) or Acat1^CKOOT-I CD8T cells (CKO OT-I). Intracellular staining and flow assay for the expression of the cytokine IFN γ (n-4).

C57BL/6 mouse-derived spleen cells presenting OVA antigen (OVA)_257-264) Post-as antigen presenting cell stimulation

Wild type (WT OT-I) or Acat1^CKOOT-I CD8T cells (CKO OT-I), produced mature killer T Cells (CTLs) after 5 days. To examine the cell killing ability of CTLs, the killing efficiency was determined by detecting the release of LDH (lactate Dehydrogenase) after mixed culture with CTLs for 4 hours using EL-4 presenting OVA antigen as a target cell.

c. Serum of a mouse with the age of 3 months is taken, and the level of a cytokine IFN gamma and the level of an autoantibody anti-double-stranded DNA antibody in the serum are detected by an ELISA method.

FIG. 2 ACAT1 knockout enhances anti-tumor activity of CD8T cells.

a-b. wild type mouse and Acat1^CKOGrowth rate of melanoma in mice and survival curve analysis of mice. Tumor growth curves were analyzed using the two-way ANOVA statistical method, and survival curves of mice were analyzed using the log-rank (Mantel-Cox) statistical method;

c-d. wild type and Acat1^CKOMice were injected with B16F10 melanoma cells, tumors were removed on day 16, CD8T cells were analyzed by flow analysis for expression of CD44, granzyme B, IFN γ and TNF α, while tumor-infiltrating CD8T cells were counted, CD8/CD4T cells were detected by flow detectionThe ratio and expression of the CD8T cell proliferation marker Ki-67;

e. detecting the expression of tumor infiltration CD8T cell surface immunodetection point receptors PD-1 and CTLA-4 by flow;

f. intracellular staining and flow detection of tumor-infiltrating Treg cells (CD 4)⁺FoxP3⁺) The ratio of (A) to (B);

flow-detecting the expression of CD44 and IFN gamma in CD8T cells in draining lymph nodes, simultaneously counting CD8T cells, and detecting the ratio of CD8/CD4T cells by flow detection;

i-k. melanoma lung metastasis model, detecting the number of tumors in the lung on day 20 after melanoma cell injection, and meanwhile, continuously recording the survival rate and survival time of the remaining mice, wherein the survival curve of the mice is analyzed by using a log-rank (Mantel-Cox) statistical method;

in a melanoma lung metastasis model, hematoxylin-eosin staining is used for observing the infiltration condition of tumor cells in the lung;

flow assay of CD8T cell CD44, granzyme B, IFN γ and TNF α expression in lung in melanoma lung metastasis model;

n-p. wild type and Acat1 that will induce differentiation maturation in vitro^CKOOT-I CTL tail vein injection was performed to C57BL/6 wild type mice bearing tumor B16F10-OVA (Ovalbumin is expressed in B16F10 melanoma cells), and tumor growth and survival rate of the mice were examined.

FIG. 3.ACAT inhibitor Avasimibe treatment enhances the anti-tumor capacity of CD8T cells.

a. Wild-type CD8T cells were treated with Avasimibe (37 ℃,6 hours, i.e., after dosing in RPMI1640 complete medium, CO at 37 ℃)₂Incubate for 6 hours in an incubator, after which they are washed 3 times by centrifugation in culture medium or PBS), followed by 24 hours of stimulation by plating antibody α -CD3(5 μ g/ml) + α -CD28(5 μ g/ml), followed by intracellular staining and flow-testing for the expression of granzyme B, cytokines IFN γ and TNF α;

b, performing Avasimibe treatment on OT-I CTL (37 ℃,6 hours) in the same way as a, and analyzing the killing efficiency of the OT-I CTL on the target cells EL-4 by detecting the release of LDH;

the MTS method detects the influence of Avasimibe treatment on the cell viability of melanoma cells B16F 10;

avasimibe treats B16F10 melanoma-bearing C57BL/6 wild-type mice, and detects a tumor growth curve and mouse survival rate;

tumors were removed on day 18 after tumor injection, tumor-infiltrating CD8T cells were analyzed by flow analysis for expression of CD44, granzyme B, IFN γ, and TNF α, while tumor-infiltrating CD8T cells were counted, and the CD8/CD4T cell ratio and expression of CD8T cell proliferation marker Ki-67 were detected by flow;

i. detecting the expression of tumor infiltration CD8T cell surface immunodetection point receptors PD-1 and CTLA-4 by flow;

j. intracellular staining and flow detection of tumor-infiltrating Treg cells (CD 4)⁺FoxP3⁺) The ratio of (A) to (B);

k. flow detection of MDSC cells in tumor tissue (Gr 1)⁺CD11b⁺CD45⁺) The ratio of (a) to (b).

Figure 4Avasimibe treatment up-regulates cholesterol levels on the plasma membrane of CD8T cells, promoting TCR micro-cluster and immune synapse formation, enhancing the activation of TCR signaling pathways.

Avasimibe treated on CD8T cells (37 ℃,6 hours), and Filipin III staining detects the distribution of free cholesterol in CD8T cells; the relative quantitative result of the plasma membrane cholesterol of the cell is obtained by the processing of Leica LAS AF software;

c, analyzing the distribution of TCR on the plasma membrane surface of the CD8T cell by STORM after the CD8T cell is treated by Avasimibe;

performing Ripley' K function analysis on the imaging information obtained from panel c, wherein r represents the size of the TCR micro-cluster and L (r) -r represents the degree of TCR micro-cluster formation;

after Avasimibe treated on CD8T cells, the formation of immune synapses of CD8T cells was detected by TIRFM;

after OT-I CTL cells are treated by Avasimibe, CTL is activated by 2 mu g/ml alpha-

CD

3 and 2 mu g/ml alpha-CD 28, and then activation of a TCR near-end signal channel and a downstream signal channel is detected by Western-blotting;

storm images tumor-infiltrating CD8T cells and Ripley' K function analysis.

FIG. 5 ACAT1 gene knock-out and Avasimibe inhibit the progression of LLC lung cancer in mice.

LLC lung cancer progresses in wild type mice and ACAT1 gene knockout mice, and the survival curve of the mice is analyzed by a Log-rank (Mantel-Cox) statistical method;

avasimibe treated LLC (Lewis Lung Carcinoma) Lung cancer tumor-bearing mice, Avasimibe is evaluated for efficacy in treating Lung cancer in mice, administration route, administration days and daily dose are shown in figure d, and mouse survival curves are analyzed by Log-rank (Mantel-Cox) statistical method;

FIG. 6 ACAT inhibitor potentiates CD8 in human PBMCs⁺Effector function of T cells

*P<0.05；**P<0.01；***P<0.001。

FIG. 7 shows that Avasimibe combined with the chemotherapeutic Dacarbazine inhibits the progression of melanoma in mice.

a-C, performing Avasimibe and chemotherapy Dacarbazine combined treatment on wild type C57BL/6 mice with melanoma of B16F10, detecting tumor growth curve and mouse survival rate, comparing the difference of the combined treatment and monotherapy effect, and showing the administration route, administration days and daily dose in figure a, wherein the mouse survival curve is analyzed by using Log-rank (Mantel-Cox) statistical method.

i.g. -gavage, i.p. -intraperitoneal injection

Detailed Description

The research of the invention finds that the inhibitor can inhibit the acyl coenzyme A: the cholesterol acyltransferase ACAT1 can enhance the killing activity of CD8T cells on tumors. Acyl-coa is therefore believed to be: the cholesterol acyltransferase ACAT 1inhibitor can be used as tumor immunotherapy agent to be combined with other tumor immunotherapy agents to improve the curative effect of tumor immunotherapy. Further studies have shown that the anti-PD-L1 antibody and the anti-PD-L1 antibody can act synergistically to further enhance tumor inhibition.

Acyl-coenzyme A: cholesterol acyltransferase ACAT

Acyl-coenzyme A: the cholesterol acyltransferase ACAT is an enzyme for regulating and controlling the balance of cholesterol metabolism in cells, and catalyzes cholesterol and long-chain fatty acid to form cholesterol ester in organisms. Two isoisomerases, ACAT1 and ACAT2, are found in mammalian cells. ACAT1 is selectively expressed in tissues and cells, and is involved in intracellular cholesterol metabolic balance; ACAT2 is widely found in liver, gastrointestinal tract and other cells, and is mainly involved in absorption of cholesterol and assembly of apolipoprotein in the diet.

Acyl-coenzyme A: cholesterol acyltransferase ACAT 1inhibitor

Means that for acyl-coa: a compound having an inhibitory effect on cholesterol acyltransferase ACAT 1. For acyl-coa: the cholesterol acyltransferase ACAT1 has inhibitory effect including but not limited to: inhibition of acyl-coa: cholesterol acyltransferase ACAT1 activity, or inhibiting acyl-coa: the gene transcription or expression of cholesterol acyltransferase ACAT 1.

The acyl coenzyme A: cholesterol acyltransferase ACAT1 inhibitors include but are not limited to siRNA, shRNA, antibodies, small molecule compounds.

The acyl-coenzyme A: the cholesterol acyltransferase ACAT 1inhibitor may be an ACAT inhibitor which is not selective for ACAT1 and ACAT2, known as, for example, purpurins, manassantin A, diphenylpyridazine derivitives, glisporpenin A, CP113,818, Avasimibe, Pactibe, etc., or an inhibitor which is selective for ACAT1, such as, for example, K604, etc.

Inhibition of acyl-coa: cholesterol acyltransferase ACAT1 activity is the conversion of acyl-coa: the activity of cholesterol acyltransferase ACAT1 enzyme is reduced. Preferably, the acyl-coa: the cholesterol acyltransferase ACAT1 enzyme activity is reduced by at least 10%, preferably by at least 30%, more preferably by at least 50%, even more preferably by at least 70%, most preferably by at least 90%.

Inhibition of acyl-coa: the gene transcription or expression of cholesterol acyltransferase ACAT1 refers to: reacting an acyl-coenzyme A: the gene for cholesterol acyltransferase ACAT1 does not transcribe, or reduces acyl-coa: the transcriptional activity of the gene of cholesterol acyltransferase ACAT1, or the transcription of acyl-coa: the gene of cholesterol acyltransferase ACAT1 does not express, or reduces the expression of acyl-coa: expression activity of the gene of cholesterol acyltransferase ACAT 1.

The transcription or expression of the ACAT1 gene can be modulated by one skilled in the art using conventional methods, such as gene knock-out, homologous recombination, interfering RNA, etc.

Acyl-coenzyme A: the inhibition of the gene transcription or expression of cholesterol acyltransferase ACAT1 can be verified by PCR and Western Blot detection of expression level.

Preferably, the ACAT1 gene transcription or expression is reduced by at least 10%, preferably by at least 30%, still more preferably by at least 50%, even more preferably by at least 70%, still more preferably by at least 90%, most preferably the ACAT1 gene is not expressed at all, compared to wild type.

Small molecule compounds

The invention refers to a compound which is composed of several or dozens of atoms and has the molecular mass of less than 1000.

Avasimibe(CI-1011)

Chemical name: 2,6-diisopropylphenyl 2- (2,4,6-triisopropylphenyl) acetylsulfonate

The molecular formula is as follows: c₂₉H₄₃NO₄S

IC50:ACAT,3.3μM；CYP2C9,2.9μM；CYP1A2,13.9μM；CYP2C19,26.5μM

Cas No.166518-60-1

The structural formula is as follows

K604

Chemical name:

2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide

IC50:ACAT1,0.45μM；ACAT2,102.85μM

structural formula (xvi):

in the documents Ikenoya, M.et al.A. selective ACAT-1inhibitor, K-604, rendering failure flow losses in a fat-fed hamsters with out extraction of plasmid levels 191, 290. and297,

doi:10.1016/j. atherosclerosis.2006.05.048 (2007).

CP113,818

Chemical name: (-) -N- (2,4-bis (methylpyridin-3-yl) -2- (hexylthio) decanoic amide

IC50:17-75nM

Structural formula (xvi):

in the document Hutter-Paier, B.et al.the ACAT inhibitor CP-113,818 market reduction pathology in a mouse model of Alzheimer's disease. neuron 44,227-,

doi:10.1016/j. neuron.2004.08.043(2004)

U18666A

Cas No.3039-71-2

CI 976

Chemical name: 2,2-Dimethyl-N- (2,4, 6-trimethophenyl) dodecanamide

The molecular formula is as follows: c₂₃H₃₉NO₄

Cas No.114289-47-3；

IC50:SOAT1:IC₅₀＝73nM(human)；Acyl coenzyme A:cholesterol acyltransferase 1:IC₅₀＝73nM(rat)；Sterol O-acyltransferase,Soat:IC₅₀＝110nM(rat)；Foam cell formation:IC₅₀＝3.8μM(rat peritoneal macrophages)；Golgi-associated LPAT activity:IC₅₀＝15μM

Structural formula (I)

TMP-153

The molecular formula is as follows: c₂₄H₁₈CIF₂N₃O

Cas No.128831-46-9

IC50:cholesterol acyltransferase(ACAT):IC₅₀＝5-10nM；Acyl coenzyme A:cholesterol acyltransferase 1:IC₅₀＝5.8nM(Rattus norvegicus)

YM 750

The molecular formula is as follows: c₃₁H₃₆N₂O

Cas No.138046-43-2

IC50:0.18μM

GERI-BP002-A

Chemical name: 2,2' -Methylenbis (6-tert-butyl-4-methyl-phenol);

molecular formula C₂₃H₃₂O₂

Cas No.119-47-1

Sandoz 58-035

Chemical name:

3-[Decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenethyl]propanamiide

SA 58-035

the molecular formula is as follows: c₃₀H₄₇NOSi

Cas No.78934-83-5

IC50:ACAT1,0.3μM(Rat)

VULM 1457

Chemical name:

N-[2,6-bis(1-Methylethyl)phenyl]-N”-[4-[(4-nitrophenyl)thio]phenyl]urea；

the molecular formula is as follows: c₂₅H₂₇N₃O₃S

Cas No.228544-65-8

CTL cell

The CTL cell, i.e., Cytotoxic T Lymphocyte (CTL), is an terminally differentiated CD8T cell that recognizes a corresponding antigen through its TCR and kills tumor cells expressing the corresponding antigen or infected cells.

CD8T cell

CD8T cells are CD8 positive T cells. CD8 (cluster differentiation 8) is a transmembrane glycoprotein of the T Cell Receptor (TCR) which is a co-receptor.

Combination therapeutic drug combinations and methods of administration

one) reacting acyl-CoA: the cholesterol acyltransferase ACAT 1inhibitor and dacarbazine are prepared into independent preparations respectively, the preparation forms can be the same or different, and the administration routes can be the same or different. When in use, the two medicines can be used simultaneously or sequentially. When administered sequentially, the other drugs should be administered to the body during the period that the first drug is still effective in the body.

The commonly used administration of dacarbazine is intravenous injection, intravenous drip or arterial infusion. The usage and the dosage can refer to the prior art.

The small molecule compounds are usually administered by either gastrointestinal or parenteral administration. The siRNA, shRNA and antibody are generally administered parenterally. Can be administered locally or systemically. Avasimibe is a known drug for the treatment of atherosclerosis and alzheimer's disease, and its usage amounts can be referred to the prior art, other acyl-coenzymes a: reference is also made to Avasimibe for the use of the cholesterol acyltransferase ACAT1 inhibitor.

An effective amount of acyl-coa: a cholesterol acyltransferase ACAT 1inhibitor and an effective amount of dacarbazine. In some embodiments, the acyl-coa: the cholesterol acyltransferase ACAT 1inhibitor is administered parenterally, and dacarbazine is administered parenterally. Alternatively, acyl-coa: both the cholesterol acyltransferase ACAT 1inhibitor and dacarbazine are administered parenterally. Alternatively, acyl-coa: the cholesterol acyltransferase ACAT 1inhibitor is used for treating isolated CTL cells, and is returned to the organism after in vitro expansion culture of the CTL cells, while the dacarbazine is directly administered to the organism parenterally. When in use, the two medicines can be used simultaneously or sequentially. When administered sequentially, the other drug should be administered to the organism during the period that the first drug is still effective for the organism.

With acyl-coa: the cholesterol acyltransferase ACAT 1inhibitor is used as a main active ingredient or one of the main active ingredients for preparing a medicament. Generally, the medicament may comprise one or more pharmaceutically acceptable carriers or excipients in addition to the active ingredient, according to the requirements of different dosage forms.

By "pharmaceutically acceptable" is meant that the molecular entities and compositions do not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with acyl-coa: the cholesterol acyltransferase ACAT 1inhibitor is compatible, i.e. can be blended therewith, without the effect of the pharmaceutical composition being substantially reduced as is normally the case. Specific examples of some substances that can serve as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethylcellulose and methylcellulose; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyhydric alcohols such as glycerol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer, and the like. These materials are used as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration.

In the present invention, unless otherwise specified, the pharmaceutical dosage form is not particularly limited, and may be prepared into injection, oral liquid, tablet, capsule, dripping pill, spray, etc., and may be prepared by a conventional method. The choice of the pharmaceutical dosage form should be matched to the mode of administration.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

1. Materials and reagents

Cell culture medium (DMEM), Fetal Bovine Serum (FBS) was purchased from Life Technologies; filipin III, M β CD-cholestrol purchased from Sigma; α -CD3 and α -CD28 were purchased from Biolegend; flow assay antibodies α -mCD4(RM4-5), α -mCD8(53-6.7), α -mCD3 ε (145-2C11), α -IFN- γ (XMG1.2), α -TNF- α (MP6-XT22), α -Granzyme B (NGZB), α -CD44(IM7), α -CD69(H1.2F3), α -PD-1(J43), α -CTLA-4(UC10-4B9), α -Ki67(16A8), α -Foxp3(FJK-16s), α -Gr1(RB6-8C5), α -CD11B (M1/70) and α -CD45(30-F11) were purchased from eBioscience; western-blotting antibodies α -pCD3 ζ was purchased from Abcam, α -CD3 ζ was purchased from Santa Cruze, α -pZAP70, α -ZAP70, α -pLAT, α -LAT, α -pErk1/2 and α -Erk1/2 were purchased from Cell Signaling; MTS assay kit was purchased from Promega; avasimibe from selelck; k604 was synthesized by Shanghai pharmaceutical research institute of Chinese academy of sciences; CP113,818 is supplied by Pierre Fabre; the B16F10 cell line and the LLC (Lewis Lung Carcinoma) cell line were originally derived from ATCC and were supplied from the cell bank of the Chinese academy of sciences

2. Laboratory animal

C57BL/6 mice were purchased from SLAC; OT-I TCR transgenic mice are from Jackson laboratories; CD4^creThe transgenic mice were provided by the national academy of sciences biochemistry and cell biology institute, Liu Xiaolong research group (Jax mie also provided the mice); acat1^flox/floxFrom InGeneius Labs, this testThe experimental animal has a LoxP site in two introns adjacent to 14 exon of Acat1 gene, which encodes His460 site essential for ACAT1 enzyme activity, and Acat1^flox/floxCan be constructed using conventional techniques. All animals were housed in an SPF facility during the experiment.

T cell isolation, culture and flow assay

T cells were isolated from mouse spleen and lymph nodes using CD8 or CD4 negative selection magnetic beads (Stem cells). For tumor-infiltrating T cell isolation, mice were treated 14-18 days after tumor injection, tumors were removed, cut into 1-2mm fragments, digested with collagenase IV (Sigma) for 1 hour, and centrifuged through a 40-70% Percoll (GE) density gradient to obtain a tumor-infiltrating leukocyte population. To further obtain tumor-infiltrated CD8T cells, further isolation was performed using a CD8 positive magnetic bead sorting kit (Stem Cell).

The isolated cells were cultured in RPMI1640 complete medium containing 50. mu.M beta-mercaptoethanol at 37 ℃ in CO₂Culturing in an incubator. To further analyze the functional phenotype of T cells, cells were directly stained with PERCP- α -CD8, FITC- α -CD44, PE- α -CD69 after centrifugation for cell surface markers such as CD8, CD44, CD69, and then flow-assayed; and aiming at the detection of the intracellular granzyme and the cell factor, the separated cells are continuously stimulated to be cultured for 4 hours by 1 mu M ionomycin and 50ng/ml PMA, and 5 mu g/ml BFA is added in the culture process to block the secretion of the granzyme and the cell factor to the outside of the cells. After stimulation was complete, 4% Paraformaldehyde (PFA) was fixed for 10 min at room temperature, and intracellular staining was used to detect intracellular granzyme and cytokine levels.

4. Cholesterol quantification

The relative quantification of plasma membrane cholesterol by Filipin staining and confocal laser imaging

Filipin III dissolved in methanol to 5 mg/ml. T cells were fixed with 4% PFA, then stained with 50. mu.g/ml Filipin III for 30 minutes at 4 ℃ and then washed 5 times with PBS to remove free Filipin. The imaging pictures are obtained by a Leica SP8 laser confocal microscopic imaging system, and the relative quantitative results are obtained by analyzing Leica LAS AF software.

b. Cholesterol quantification method based on cholesterol oxidation

After the cells were fixed for 15 minutes with 0.1% glutaraldehyde, the cells were washed 3 times with PBS, and then treated with 2U/ml cholesterol oxidase at room temperature for 15 minutes to oxidize free cholesterol on the plasma membrane of the cells; after the oxidation reaction was completed, PBS was washed 3 times to remove residual Cholesterol oxidase, and intracellular non-oxidized free Cholesterol was extracted with methanol/chloroform (V: V ═ 1:2), and then quantified using Amplex Red Cholesterol assay kit (Life technology). Plasma membrane cholesterol levels were determined by subtracting the cholesterol levels of the oxidase-treated samples from the cholesterol levels of the non-oxidase-treated samples.

OVA antigen-activated OT-I CD8T cells

T cells in spleen cells of C57BL/6 mice were removed using a T cell positive magnetic bead sorting kit (Miltenyibitech), and then presented with OVA antigen OVA, respectively_257-264(SIINFEKL, abbreviated as N4), mutant SAINFEKL (abbreviated as A2), SIITFEKL (abbreviated as T4), SIIGFEKL (abbreviated as G4), autoantigen RTYTYEKL (abbreviated as Catnb) and positive selection antigen SIIRFEKL (abbreviated as R4) as Antigen Presenting Cells (APC). Specifically, OVA antigen OVA is respectively added_257-264，A2, T4, G4, R4 and autoantigen Catnb were incubated with the previously treated mouse spleen cells at 37 ℃ to give antigen-presenting cells; OT-I CD8T cells sorted from negative magnetic beads were co-cultured (1: 5) with antigen presenting cells for 24 hours, 5. mu.g/ml BFA (being brefeldin A) was added in the last 4 hours of culture to prevent the secretion of cytokines to the outside of the cells, and finally the expression of cytokines was detected by intracellular staining and flow-type detection.

6. The cell killing ability of CD8T Cells (CTL) was examined.

Isolation of the spleen of OT-I transgenic mice, erythrocyte lysis and erythrocyte depletion with 5nM OVA_257-264(N4) and adding 10ng/ml IL-2 into the culture medium, after 3 days, the liquid change is supplemented with IL-2, and the culture is continued for 2 days to obtain differentiated and mature killer CD8T Cells (CTL). The EL is formed by-4 mouse lymphoma cells were incubated with 2nM of the antigenic peptide OVA257-264 and its mutant A2, T4, G4, R4 and autoantigen Catnb, respectively, at 37 ℃ for 30 minutes to present the relevant antigen, and after 3 washes with PBS, the antigen-presenting EL-4 cells and CTL were resuspended in phenol red-free RPMI1640 medium (containing 2% FBS) and then cultured in 1: 1,1: 2,1: 5 ratio mixing (EL-4 cell count 1X 10)⁵) Add to 96 well plate and centrifuge at 200g for 2 min. Culturing in a CO2 incubator at 37 ℃ for 4 hours, centrifuging at 200g for 5 minutes, taking culture supernatant, and detecting the release of lactate dehydrogenase LDH in the culture medium by a CytoTox 96Non-Radioactive cytoxicity kit (Promega) to calculate the killing efficiency of CTL to target cells.

7. Mouse B16 melanoma model

B16F10 melanoma cells were trypsinized, washed 3 times with PBS and then filtered through a 40 μm filter, counted and diluted to 2X 10 with PBS⁶Per ml, then injected subcutaneously (s.c.) to the left of the back (100 μ l volume) of 8-10 weeks male mice, starting on day 10, tumor size was measured with a vernier caliper every two days, length x width. When tumors exceeded 20mm in their longest axis, mice were euthanized and time points of death were recorded.

8. Adoptive therapy of mouse melanoma T cells

B16F10-OVA melanoma cells (Ref) expressing chicken Ovalbumin (Ref) (cf. Yang J et al., Kuper-type immunological synthesis chromatography characteristics do not predict anti-i-blue tumor cell function In vivo. Proc Natl Acad Sci U A.2010.197(10): 4716-21; obtained Zhou P et al., In vivo discovery targets In the tumor micro-surgery. Nature.2014,506 (PBS 7486): 52-7) were trypsinized, washed 3 times with 40 μm and counted In PBS to 2X 10. mu.m⁶Perml, then injected subcutaneously (s.c.) to the left dorsal side (100. mu.l) of 8-10 weeks male mice. On day 10 after injection of tumor cells, the tumorigenic and tumor-size-consistent mice were randomly divided into 3 groups, and 200ul PBS, wild-type OT-I CTL and ACAT1 gene-knocked-out OT-I CTL (1.5X 10) were injected into the tail vein (i.v.)⁶) Tumor size measurements were started 3 days later, once every two days. When the longest axis of the tumor exceeds 20mm, the tumor will be smallMice were euthanized and the time point of death was recorded.

Obtaining of OT-I mice with ACAT1 Gene knockout: CD4^CreMouse and Acat1^flox/floxMating mice to obtain CD4^Cre-Acat1^flox/floxThen mating with OT I TCR transgenic mice to finally obtain OT-I-CD4^Cre-Acat1^flox/floxMice and corresponding control mice OT-I-^e-Acat1^flox/floxA mouse.

The CTL inducing method comprises the following steps: spleen of wild-type or ACAT1 gene knock-out OT I mice is taken, 10nM OVA257-264(N4) peptide fragment is respectively used for stimulation, 10ng/ml IL-2 is provided, and after 2 days of stimulation, culture medium without N4 peptide fragment (containing 10ng/ml IL-2) is replaced on the 3 rd day for continuous culture for 2 days to obtain mature CTL.

9. Mouse LLC lung cancer model

LLC (Lewis Lung Carcinoma) Lung cancer cells are digested with pancreatin, washed 3 times with PBS, filtered through a 40 μm sieve, counted and diluted to 10 with PBS⁷/ml, then injected tail vein (i.v.) into 8-10 week male mice (200 μ l volume). On day 11 after tumor cell injection, tumor-bearing mice were randomly divided into 2 groups, and the mice were injected intraperitoneally (i.p.) with Avasimibe at a dose of 15mg/kg once every 3 days for 9 consecutive administrations. At 7 weeks after tumor cell injection, a portion of the mice lungs were taken and tumor spots were counted to evaluate the tumor treatment effect, while the remaining mice were recorded for time points of death.

10. Ultrahigh resolution random Optical Reconstruction Microscopy (Super-resolution random Optical Reconstruction Microscopy) detects the distribution of TCR (antigen receptor) on the plasma membrane of cells.

Ultrahigh resolution stochastic optical reconstruction microscopy STORM imaging was performed on a Nikon N-STORM imaging system equipped with an ECLIPSE Ti-E electrokinetic phase contrast microscope, Apochromat 100 times TIRF oil lens, and EMCCD. The fluorescent dye is Alexa647, and is excited by a647 nm continuous visible light laser (200mW) and a 450nm diode laser. The TIRF angle parameter is 3900-4000 during imaging so as to ensure that the imaging depth of the sample is about 1 mu m.

CD8T cells and activated CD8T cells (10. mu.g/ml. alpha. -CD3 antibody stimulated at 37 ℃ for 10 minutes) were attached to Ibidi 35 mm. mu. -Dish by polylysine and then fixed with 4% PFA; after 3 washes with PBS, it was labeled with 2. mu.g/ml Alexa 647-. alpha. -CD34 ℃ for 2 hours. Before imaging, PBS was replaced with imaging buffer (TBS, containing 100mm MEA). The imaging speed was maintained at 90-95 frames/sec, the reconstructed images were acquired using NIS Elements AR, and the image information consisted of 20,000-25,000 frames of pictures.

In order to analyze the clustering condition of TCR on a plasma membrane, the position information of TCR micro-clusters in the picture is analyzed by Ripley's K function and Matlab, the value of L (r) -r represents the probability of molecular aggregation in a certain radius (r), and the r corresponding to the maximum value of L (r) -r is the radius of the TCR micro-clusters in the analysis selected area.

11. TIRFM (Total Internal Reflection Fluorescence Microscopy) method is used for dynamically detecting the T cell immune synapse formation process.

DOPC (1, 2-dioleoyl-sn-glycerol-3-phosphocholine) and biotin-labeled DOPE (1, 2-dioleoyl-sn-glycerol-3-phosphoethanomine-cap-biotin) were mixed at a molar ratio of 25:1, and sonicated to form liposomes. Using a cell culture dish with a glass bottom of No.1.5H thickness, the glass bottom surface was treated with piranha acid solution and washed clean with double distilled water. The slides were incubated with biotin-labeled liposomes at a concentration of 0.1mM for 20 minutes. After extensive washing with PBS, the cells were incubated with streptavidin (streptavidin) for 30 minutes. After extensive washing with PBS, the cells were incubated for 30 min with biotin-labeled mouse CD3 epsilon antibody (biotinylated. alpha. -mCD3 epsilon, 145-2C 11). After extensive washing with PBS, blocking was performed with 1% casein for 20 minutes. PBS was washed thoroughly and the planar lipid bilayer was prepared.

The α -mTCR β antibody (Biolegend) was labeled with a fluorescent dye (Alexa Fluor 568NHS Ester, Life Technologies), desalted on a desalting Column, digested with Papain protease (Pierce Fab Micro Preparation Kit, Thermo Scientific), and finally separated on a Protein A Column (Protein A Plus Spin Column, Thermo Scientific) to yield Alexa568- α -mTCR β -Fab.

Freshly isolated mouse spleen lymphocytes were stained with Alexa568- α -mTCR β -Fab and FITC- α -mCD8a (eBioscience) on ice and washed 2 times with PBS. Resuspended cells were added to a flat lipid bilayer-plated petri dish as described above, placed on a 37 ℃ heating stage, photographed at 3 seconds per frame, and imaged by a live cell total internal reflection fluorescence microscope. Or adding the resuspended cells into the culture dish paved with the planar lipid bilayer, placing the culture dish in an incubator at 37 ℃, stimulating for a certain time, fixing the cells by 4% PFA, and imaging by a total internal reflection fluorescence microscope. FITC positive CD8T cells were subjected to data analysis using Image Pro Plus software (Media Cybernetics), ImageJ (NIH), and MATLAB (MathWorks) software.

The MTS method detects the survival rate of tumor cells.

B16F10 cells (5X 10)³) Resuspended in 100. mu.l of medium containing 1. mu.M Avasimibe or DMSO control. Incubation at 37 ℃ for 24, 48, or 72 hours. Mu.l of MTS reagent (Promega) was added and after 1-3 hours of incubation, the absorbance at 490nm was measured. The absorbance at 490nm is proportional to the number of viable cells in culture. The effect of Avasimibe on cell activity was obtained by normalization treatment using DMSO-treated cells as a control group, and the cell activity of the DMSO-treated group was set to 1.

13. Statistical analysis of data

All data statistics herein were statistically analyzed using GraphPad Prism (GraphPad Software, Inc.) using two-labeled unpaired Student's t-test; p <0.05, P <0.01, P <0.001, ns: there was no significant difference (no significant difference).

Construction of T cell-specific knockout ACAT1 mice and Acat1^CKO CD8T、Acat1^CKOAcquisition of CD4T cells

Mixing ACAT1^flox/floxMouse and CD4^CreThe mice are mated, and the obtained mice are verified to be T cell specific ACAT1 knockout mice (CD 4) through PCR and Western Blot detection of expression level^Cre-Acat1^flox/floxAcat1 for short^CKO)。

Acat1^CKOAcquisition of CD8T cells: sorting with CD8 magnetic beads (sorting kit from Stemcell)

Acat1^CKO CD4T cell acquisition: sorting with CD4 magnetic beads (sorting kit from Stemcell)

Method for activating CD8T cells

Fresh T cells sorted by magnetic beads, resuspended in RPMI1640 complete medium, plated for stimulation with α -CD3(5 μ g/ml) and α -CD28(5 μ g/ml), CO at 37 ℃₂After 24 hours of incubation in an incubator, to further detect cytokine expression by intracellular staining-flow cytometry, 5mg/ml Brefeldin A was added over the last 4 hours to block cytokine secretion to the outside of the cells.

Example 1ACAT1 Gene knockout did not elicit CD8T cell responses to autoantigens and autoimmunity

The purpose of this example was to investigate whether enhanced effector function of CD8T cells after ACAT1 depletion would lead to CD8T cells responding to autoantigens and thus induce autoimmune diseases. To investigate this, Acat1 was used^CKOThe mice are further mated with OT-I TCR transgenic mice (Jax mice) to obtain the OT-I background ACAT1 specific gene knockout mice (OT-I Acat 1)^CKO) And corresponding control mice (OT-I). The vast majority of T cells of OT-I transgenic mice are OT-I CD8T cells, the TCR of which specifically recognizes H2K^bPresented Ovalbumin (OVA)_257-264) Antigen (abbreviated as N4) and generates a corresponding CD8T cellular immune response; meanwhile, the OT-I TCR can also recognize a series of OVA mutants (A2, T4, G4 and R4) besides the wild-type antigen (N4) and generate corresponding strong and weak immune response responses. It was found that, in addition to promoting the immune response of OT-I CD8T cells to strong and weak antigens, ACAT1 deficiency was manifested by increased secretion of the cytokine IFN γ and killing effect on target cells (fig. 1a, b). However, the ACAT1 deficiency does not cause OT-I CD8T cells to react against the autoantigen Catnb (. beta. -Catenin fragment, which may be replaced by H2K^bPresentation) and the autoantigen-like R4 to generate an immune response. This result indicates that ACAT1 gene knock-out does not affect T cell affinity (affinity) recognition ability. In order to further verify the autoimmune condition of the mice after ACAT1 gene knockout, an ELISA method is used for detecting a cytokine IFN gamma and an autoantibody anti-double-chain DNA antibody in the serum of the mice. Result display, andcompared with mice, the two indexes are not changed (fig. 1c), which indicates that the ACAT1 gene knockout does not cause autoimmune reaction, and further indicates the safety of the ACAT1 as a tumor immunotherapy target.

Example 2 study of ACAT1 Gene knockout to promote antitumor Activity of CD8T cells Using three melanoma models

Immunotherapy is the hot spot for the current treatment of tumors, and the killing ability of CD8T cells on tumor cells directly relates to the effect of tumor immunotherapy. To verify the effect of ACAT1 knock-out on the anti-tumor ability of CD8T cells, this example first established a subcutaneous melanoma model at Acat1^CKOB16F10 melanoma cells are injected subcutaneously to induce melanoma in mice and wild-type mice, and experiments show that Acat1^CKOProgression of melanoma in mice was significantly delayed, as indicated by slower tumor growth and longer survival and survival of mice (fig. 2.a, b). T cell phenotype of tumor-bearing mice was analyzed and found to be Acat1^CKOTumor-infiltrating CD8T cells were more active in mice, as evidenced by Acat1^CKOIn mouse tumor infiltration CD8T cells, more CD44 high-expression effector cells are provided, and the ratio of the expression granzyme B, cytokine IFN gamma and TNF alpha is higher; and Acat1^CKOThe number of mouse tumor infiltrating CD8T cells and the proportion of CD8/CD4 cells were also higher than the expression level of the wild-type, cell proliferation marker Ki-67 (FIG. 2.c, d). Immunodetection point receptors such as PD-1 and CTLA-4 are currently hot targets for tumor immunotherapy, and experiments also detected the expression level of PD-1 and CTLA-4 on the surface of tumor-infiltrating CD8T cells, which was found to be in Acat1^CKONo significant difference from wild type mice (fig. 2. e); at the same time, Treg cells in CD4T cells in tumors (CD 4) are detected in the experiment⁺FoxP3⁺) Was found that ACAT1 knock-out did not alter the proportion of Treg cells in CD4T cells (fig. 2. f). The above results indicate that ACAT1 knock-out enhances the anti-tumor effector function of CD8T cells.

To further verify that the response of CD8T cells in this tumor model was regulated by ACAT1, experiment was performed with ACAT1 on day 7 after subcutaneous injection of B16F10 melanoma cells^CKOAnd wild type miceThe draining lymph node (draining lymph node) was removed and the phenotype of the T cells was analyzed. The experiment finds that Acat1^CKOCD8T cells in the draining lymph nodes of mice had elevated CD44 levels and cytokine IFN γ expression (fig. 2.g), and the number of CD8T cells and the ratio of CD8/CD4 were also significantly elevated (fig. 2.h), indicating that ACAT1 deficiency promotes the initial immune response of CD8T cells.

Compared with subcutaneous solid tumors, metastatic tumors progress more complicated and are more difficult to treat. In order to further detect whether ACAT1 deficiency affects immune response of CD8T cells to metastatic tumor, a melanoma lung metastasis mouse model is established in an experiment, and the experiment is carried out to Acat1^CKOAnd wild type mice were injected intravenously with B16F10 melanoma cells at the tail, and then the progression of melanoma in the lungs was examined. The experiment found that Acat1^CKOIn mice, lung tumor numbers were significantly reduced and mice were more survivable and longer in life compared to wild-type control mice (fig. 2. i-k). By hematoxylin-eosin (H)&E) Lung tissue was stained and melanoma infiltration in the lungs was significantly reduced following ACAT1 gene knockout (fig. 2. l). Analysis of the phenotype of lung T cells also found Acat1^CKOThe mouse CD8T cells showed more effector cells with high expression of CD44, and also showed a higher ratio of granzyme B and cytokines IFN γ and TNF α (fig. 2. m). These results indicate that ACAT1 deficiency enhances the anti-tumor immune response effect of CD8T cells in non-metastatic and metastatic tumors.

Due to Acat1^CKOAll T cells in mice lack ACAT1, and in order to verify that ACAT1 gene knockout affects the functions of tumor-infiltrating CD8T cells but not CD4T cells to obtain a stronger anti-tumor effect, B16F10-OVA melanoma cells are injected subcutaneously in wild-type C57 mice, and express Ovalbumin, which can be presented on the cell surface by H2Kb of the melanoma cells and then recognized by OT-I CD8T cells. From Acat1 by tail vein injection 10 days after tumor injection after successful induction of melanoma in wild type mice^CKOOT-I CTL from mouse and wild type mice were injected into tumor-bearing wild type mice, tumor growth was monitored and survival of mice was recorded (FIG. 2.n), nodulesAs a result, Acat1 was injected^CKOThe experimental group of OT-I CTLs showed slower tumor growth and longer survival of mice than the control group injected with wild-type OT-I CTLs (FIG. 2.o, p), further indicating that ACAT 1-specific knockout of CD8T cells had better antitumor effects.

Example 3 the ACAT inhibitor Avasimibe enhances the anti-tumor function of CD8T cells.

The purpose of this example is to further validate the application of ACAT 1inhibitor in tumor immunotherapy. ACAT1 is used as a target for treating atherosclerosis, and a series of small molecule drugs are currently used in animal and clinical experiments. Avasimibe is one of them, and the safety of Avasimibe is verified by a plurality of clinical experiments at present. Therefore, Avasimibe is selected to verify the possibility of using ACAT1 as a tumor immunotherapy target. In vitro experiments showed that Avasimibe, like other ACAT inhibitors such as CP113,818 and K604, can promote the release of CD8T cell granzyme B and the expression of cytokines IFN γ and TNF α (fig. 3. a). In vitro killing experiments also showed that Avasimibe enhanced the target cell killing ability of CTLs with a dose effect (fig. 3. b). Experiments further demonstrated the effect of Avasimibe in a mouse melanoma model, by treatment of melanoma tumor-bearing wild-type mice with intraperitoneal administration (15mg/kg), tumor growth was slowed and survival time was significantly prolonged in the Avasimibe-administered mice compared to control mice (fig. 3. d-f). To verify that Avasimibe does not directly inhibit tumor cell growth, B16F10 melanoma cells were treated with Avasimibe in vitro experiments, and it was found that Avasimibe treatment did not affect the activity of B16F10 cells (fig. 3. c). Analysis of mouse tumor-infiltrated immune cells revealed that in mice dosed with Avasimibe: the activity of the tumor-infiltrated CD8T cells is higher, which is shown in that the expression ratio of a CD8T cell surface activation marker CD44 is higher, and the cell ratio of cells expressing granzyme B, cytokines IFN gamma and TNF alpha is also higher. Tumor-infiltrating T cells were counted in mice and the Avasimibe-administered group was found to have a higher number of tumor-infiltrating CD8T cells and a higher CD8/CD4 ratio. In addition, the expression level of the cell proliferation marker Ki-67 in tumor-infiltrating CD8T cells was also higher in the Avasimibe administration group (FIG. 3. g-h). Experiments also examined the expression levels of immunosuppressive receptors PD-1 and CTLA-4 on CD8T cells, taking into account the immunosuppressive effects of the tumor microenvironment, and it was found that the expression of these immunodetection point receptors was not altered by the administration of Avasimibe (fig. 3. i).

Some suppressive immune cells such as treg (regulatory T cell) and MDSC (myoid-derived killer cell) exist in the tumor microenvironment, and play a key role in the development process of tumor by inhibiting the activity of tumor killer cells such as CD8T cells. Thus, experiments further examined the proportion of Treg and MDSC in tumor tissues, Treg (CD 4) following administration of Avasimibe⁺FoxP3⁺) The proportion of cells in CD4T cells did not change significantly, whereas MDSC (Gr 1)⁺CD11b⁺CD45⁺) In CD45⁺There was a slight decrease in the proportion of immune cells (FIG. 3.j, k), and the MDSC was at CD45, considering that the number of CD8T cells in the tumor tissue of the Avasimibe-administered group was significantly increased⁺The reduced proportion of cells was probably due to CD8T cells in CD45⁺The proportion in the cells increased (FIG. 3. h). These evidence suggest that the ACAT inhibitor Avasimibe may play a role in tumor therapy by specifically up-regulating the CD8T cell killing effect.

Example 4Avasimibe promotes anti-tumor Activity of CD8T cells by inhibiting ACAT1

The purpose of this example was to verify whether Avasimibe exerts its tumor immune competence by inhibiting ACAT 1. To verify this, CD8T cells were treated in vitro with Avasimibe and examined for changes in plasma membrane cholesterol levels. Cells were stained by Filipin III and found to have significantly elevated plasma membrane free cholesterol levels in CD8T cells following Avasimibe treatment (fig. 4.a, b). Analysis of the localization of TCR on the CD8T cell surface by STORM revealed that Avasimibe treatment promoted the formation of TCR micro-clusters (fig. 4. c-e); analysis of immune synapse formation during T cell activation by TIRFM also showed that Avasimibe treatment promoted immune synapse formation in CD8T cells (fig. 4.f, g). The activation of the TCR signaling pathway after Avasimibe treatment was further detected by the western-blotting method, and the results also indicate that Avasimibe treatment enhances the activation of the TCR signaling pathway (fig. 4. h). These knotsThe results were consistent with the performance of ACAT1 knockout CD8T cells. Meanwhile, to further verify the mechanism in animals, experiments were conducted to isolate tumor-infiltrating CD8T cells from tumors of tumor-bearing mice administered with Avasimibe, and image analysis of TCR distribution on tumor-infiltrating CD8T cells by STORM revealed Avasimibe treatment (after addition of drugs in RPMI1640 complete medium, CO at 37 ℃.)₂Incubation in incubator for 6 hours, 3 washes with media or PBS centrifugation after treatment) tumor-infiltrated CD8T cell surface TCR micro-clusters were larger (fig. 4. i-k). These evidence suggests that Avasimibe promotes the activation of CD8T cells by inhibiting ACAT1, thereby enhancing its anti-tumor activity.

Example 5 efficacy of Avasimibe in mouse Lung cancer model

The purpose of this example is to further validate ACAT1 as a target for tumor immunotherapy. A mouse LLC (Lewis Lung Carcinoma) Lung cancer model is established through experiments, the development process of Lung cancer of LLC in wild mice and ACAT1 gene knockout mice is detected, the result shows that the ACAT1 gene knockout in T cells obviously inhibits the growth of tumors, and the life cycle of the tumor-bearing mice is also obviously longer than that of the wild mice of a control (fig. 5. a-c). In order to verify the curative effect of the ACAT 1inhibitor Avasimibe on LLC lung cancer, Avasimibe administration is further carried out on mice successfully modeled, and the result shows that Avasimibe has a certain curative effect on the treatment of the lung cancer of the mice, and the Avasimibe has the advantages that the number of tumor cells is smaller than that of a control group, and the survival time of the mice is prolonged (figure 5. d-g).

Example 6 ACAT inhibitors enhance CD8 in human PBMCs⁺Effector function of T cells

The aim of the experiment is to verify the potential value of the ACAT1 as a drug target for clinical use, ACAT inhibitors Avasimibe and CP113,818 are respectively used for inhibiting the ACAT activity in human CD8T cells, then the CD8T cells are activated in an antibody cross-linking mode, and the expression of cytokines IFN gamma and TNF alpha of CD8T cells is detected by flow cytometry.

The test method comprises the following steps: PBMC were isolated from peripheral blood obtained from healthy volunteers by Ficoll and then stimulated with 5. mu.g/ml PHA to obtain CD8⁺Killer T cells. 3 days later, the culture medium was changed to a culture medium without PHAIncubation was continued for 24 hours to reduce background signal. The cells were then incubated for 12 h with the corresponding concentrations of Avasimibe or CP113,818, the cells were changed and stimulated by plating with 5. mu.g/ml α -CD3+ 5. mu.g/ml α -CD28 antibody for 24 h, 5. mu.g/ml Brefeldin A was added for the last 4 h to block cytokine secretion, and finally CD8 was detected by intracellular staining and flow cytometry⁺Expression of the cytokines IFN γ and TNF α in T cells. Two-tailed unpaired t-test was used for differential significance analysis.

And (3) test results: both the ACAT inhibitors CP113,818 and Avasimibe promoted the expression of CD8T cytokines with dose-effect, indicating that ACAT1 as a target for enhancing CD8T cell effector function is equally applicable to human CD8T cells (fig. 6).

Example 7 efficacy of Avasimibe in combination with Dacabazine (Dacarbazine) for the treatment of melanoma in mice

The purpose of this example is to demonstrate the effectiveness of ACAT1 as an immunotherapeutic target in combination with chemotherapeutic agents. Chemotherapy is one of the effective current approaches to treating tumors, and Dacarbazine (Dacarbazine) is the current FDA approved chemotherapeutic agent for the treatment of metastatic melanoma. In this example, in a mouse melanoma model, a combination of Dacarbazine and Avasimibe was administered to mice, and the results showed that the growth of melanoma in mice was significantly inhibited, while the antitumor effect of Avasimibe was significantly enhanced by the combination (fig. 7).

The test animals were divided into 4 groups of 9-13 animals each, see in particular fig. 7b, c, the n-value, i.e. the number of mice, and statistically analyzed, the significance of the differences between the groups, i.e. the p-value, is shown in fig. 7b, c.

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.

Claims

1. Use of a combination comprising an effective amount of Avasimibe and an effective amount of dacarbazine for the preparation of a medicament for the treatment of a tumour selected from melanoma or lung cancer.

2. The use of claim 1, wherein said combination is administered to a mammal or to a CD8T cell from said mammal.

3. The use of claim 2, wherein the mammal is a human.