CN116617214A - Application of Tim-3 targeted small molecular compound in tumor immunotherapy - Google Patents

Abstract

The invention belongs to the technical field of immunology and antitumor drugs, and particularly relates to application of a small molecular compound of a targeting Tim-3 in tumor immunotherapy. According to the invention, a series of 3, 5-disubstituted-3 a,6 a-dihydrospiro [ furan [3,4-c ] pyrrole-1, 2' -indene ] -1',3',4,6 (3H, 5H) -tetralone small molecular compounds capable of promoting immune cell anti-tumor function through targeting Tim-3 are researched and screened. The compound obviously improves the survival rate and the anti-tumor activity of primary CD8+ cytotoxic T lymphocytes and human chimeric antigen receptor T cells, obviously promotes the killing activity of NK cells and the DC antigen presenting capability, shows the equivalent tumor inhibition effect as Tim-3 blocking antibodies, effectively inhibits tumor progression and synergistically enhances PD-1 blocking-induced anti-tumor response, and has good potential development and application values.

Description

Application of Tim-3 targeted small molecular compound in tumor immunotherapy

Technical Field

The invention belongs to the technical field of immunology and antitumor drugs, and particularly relates to application of a small molecular compound of a targeting Tim-3 in tumor immunotherapy.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Immune checkpoint blockade (Immune checkpoint blockade, ICB) therapy has become a well-established way of tumor treatment following surgical treatment, radiation therapy and chemotherapy, and has completely altered the treatment of a variety of tumors. Notably, therapeutic antibodies targeting PD-1 and CTLA-4 achieve potent and durable anti-tumor responses in a variety of tumor types by restoring the effector function of depleting T cells. However, a significant fraction of cancer patients are poorly responsive to PD-1 or CTLA-4 antibody treatment. Therefore, it is critical to find novel immune checkpoint molecules as therapeutic targets.

Tim-3 is involved in terminal differentiation and depletion of T cells during chronic viral infection and tumor progression, and is one of the very promising immune checkpoint targets at present. Tim-3 is highly expressed on intratumoral depletion T cells, while in a mouse model, antibodies targeting Tim-3 can reverse T cell depletion and promote tumor regression. Furthermore, CD8 co-expressing PD-1 and Tim-3 in melanoma patients and in mouse tumor models ⁺ T cells exhibit a more depleted phenotype, with severely impaired proliferation and cytokine production. In several studies, tim-3 and PD-1 combined blocking treatment significantly enhanced T cell function, inhibited tumor growth, and was more effective than monotherapy. Notably, recent clinical trial data indicate that Tim-3 blockade enhances the anti-tumor effect of PD-1 antibodies.

As a type I membrane protein, tim-3 consists of an N-terminal immunoglobulin variable region (IgV), a glycosylated mucin-like domain, a transmembrane region and a C-terminal cytoplasmic tail. 4 different Tim-3 ligands have been reported, including Galectin-9 (Galectin-9), phosphatidylserine (PtdSer), high mobility group protein B1 (HMGB 1), and carcinoembryonic antigen-related cell adhesion molecule1 (CEA Cell Adhesion Molecule, CEACAM 1). Crystal structure studies showed that there is a common FG-CC 'loop in the IgV domain of mouse and human Tim-3 as a binding site for PtdSer, CEACAM and HMGB1, while Galectin-9 binds to the N-linked glycan on the other side of the FG-CC' loop. Importantly, a common feature of the mouse and human Tim-3 antibodies that are currently demonstrated to be efficacious is to block Tim-3 binding to PtdSer and CEACAM1, but not to block Tim-3 binding to Galectin-9. Thus, the PtdSer binding pocket of Tim-3 is a very critical and promising target for the development of Tim-3 inhibitors.

Although antibodies have been widely used to block immune checkpoint pathways, and some Tim-3 antibodies are currently in preclinical development, tim-3 antibodies have not shown a strong anti-tumor response in early clinical trials to date. Compared with antibodies, small molecule immune checkpoint inhibitors exhibit higher tissue penetration, good biosafety, and better potential for inhibiting tumor growth and migration. Nevertheless, in the field of immune checkpoint blockade, the development of small molecule inhibitors is far behind therapeutic antibodies, and only a few PD-1/PD-L1 small molecule inhibitors are in clinical trials. At present, no small molecule Tim-3 inhibitor with cell activity is reported. Therefore, the identification of the functional small molecule Tim-3 inhibitor for improving tumor immunotherapy has extremely strong clinical transformation significance.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides application of a small molecular compound targeting Tim-3 in tumor immunotherapy. The invention researches and screens a series of 3, 5-disubstituted-3 a,6 a-dihydrospiro [ furan [3,4-c ] pyrrole-1, 2' -indene ] -1',3',4,6 (3H, 5H) -tetraketone small molecular compounds which promote the anti-tumor function of immune cells by targeting Tim-3, and a composition containing the compounds and application thereof to promoting the anti-tumor function of immune cells. Based on the above results, the present invention has been completed.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect of the invention there is provided the use of a compound in the preparation of a Tim-3 inhibitor;

wherein the compound is a 3, 5-disubstituted-3 a,6 a-dihydrospiro [ furo [3,4-c ] pyrrole-1, 2' -indene ] -1',3',4,6 (3H, 5H) -tetraketone compound, which has a structure shown in a general formula I:

wherein, the liquid crystal display device comprises a liquid crystal display device,

R ₁ the method comprises the following steps: c (C) ₃ -C ₆ Cycloalkyl, benzene ring with 0 to 2 substituents, benzyl with 0 to 2 substituents, six-membered heterocycle with 0 to 2 substituents, five-membered heterocycle with 0 to 2 substituents, said substituents each being independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyanoNitro, halomethyl, carboxamide, carboxyl, ester groups;

R ₂ the method comprises the following steps: c (C) ₃ -C ₆ Cycloalkyl, benzene ring with 0 to 2 substituents, six membered heterocycle with 0 to 2 substituents, five membered heterocycle with 0 to 2 substituents, said substituents each being independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

m may be selected from integers of 0, 1 or 2, R ₃ Each occurrence is independently C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

the compound configuration may be racemate or a single chiral configuration.

According to a preferred embodiment of the present invention,

R ₁ the method comprises the following steps: a benzene ring having 0 to 2 substituents, a benzyl having 0 to 2 substituents, each of said substituents being independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

R ₂ the method comprises the following steps: a benzene ring having 0 to 2 substituents each independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

m may be selected from integers of 0, 1 or 2, R ₃ Each occurrence is independently C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

the compound is in a racemate or a single chiral configuration.

It should be noted that, with respect to substituents, the present invention refers to the case where, when more than one substituent may be present, the substituents may be the same or different from each other.

According to a further preferred embodiment of the invention, the 3, 5-disubstituted-3 a,6 a-dihydrospiro, [ furo [3,4-c ] pyrrole-1, 2' -indene ] -1',3',4,6 (3H, 5H) -tetraketone compound is one of the following:

further, the compounds may also include pharmaceutically acceptable salts, isotopic derivatives, solvates thereof, or stereoisomers, geometric isomers, tautomers thereof, or prodrug molecules, metabolites thereof.

As understood by those of ordinary skill in the art, the pharmaceutically acceptable salts include the alkali metal salt forms of the above compounds (particularly sodium or potassium salts), or salts of the compounds with inorganic salts such as hydrochloric, sulfuric, nitric or hydrobromic acid, and salts with organic acids such as methanesulfonic, toluenesulfonic or trifluoroacetic acid. The term "pharmaceutically acceptable" or "pharmaceutically acceptable" used interchangeably therewith, for example when describing a "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to the subject, but may also refer to synthetic materials of pharmaceutical use, for example salts formed as intermediates in the preparation of chiral resolution, which salts may play a role in obtaining the end products of the invention, although such salts cannot be administered directly to the subject.

It should be noted that the above-mentioned compounds of the present invention can be used not only as drugs, but also as experimental reagents, and thus are useful for basic studies related to the Tim-3 signaling pathway.

In a second aspect of the invention, there is provided a medicament for the preparation of a medicament for the prophylaxis and/or treatment of a tumour, of the above-mentioned compounds.

According to the present invention, the concept of "prevention and/or treatment" means any measure suitable for the treatment of a tumor-related disease, or for the prophylactic treatment of such a represented disease or of a represented symptom, or for the avoidance of recurrence of such a disease, e.g. after the end of a treatment period or for the treatment of a symptom of a disease that has already been developed, or for the pre-interventional prevention or inhibition or reduction of the occurrence of such a disease or symptom.

It is noted that tumors are used in the present invention as known to those skilled in the art, including benign tumors and/or malignant tumors. Benign tumors are defined as hyperproliferative cells that are unable to form aggressive, metastatic tumors in vivo. Conversely, a malignancy is defined as a cell with multiple cellular abnormalities and biochemical abnormalities that are capable of developing a systemic disease (e.g., tumor metastasis in a distant organ).

In yet another embodiment of the invention, the medicament of the invention is useful for the treatment of malignant tumors. Examples of malignant tumors that can be treated with the medicament of the invention include solid tumors and hematological tumors. The solid tumors may be tumors of the breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine glands (e.g., thyroid and adrenal cortex), esophagus, endometrium, germ cells, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin (e.g., melanoma), ureter, vagina and vulva. Malignant tumors include hereditary cancers, such as retinoblastoma and Wilms tumor (Wilms tumor). Furthermore, malignant tumors include primary tumors in the organ and corresponding secondary tumors in distant organs (tumor metastasis). Hematological neoplasms can be, for example, aggressive and painless forms of leukemia and lymphoma, i.e., non-hodgkin's disease, chronic and acute myeloid leukemia (CML/AML), acute Lymphoblastic Leukemia (ALL), hodgkin's disease, multiple myeloma, and T-cell lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, oncological syndromes, and cancers of unknown primary sites and AIDS-related malignancies.

In a third aspect of the invention, there is provided a composition comprising at least the compound described above.

In a fourth aspect of the invention, there is provided a pharmaceutical formulation comprising at least the compound described above, and at least one pharmaceutically acceptable adjuvant and/or carrier.

The auxiliary materials refer to components except active ingredients in the composition or the pharmaceutical preparation, and are nontoxic to a subject. Excipients commonly used in the art such as buffers, stabilizers, preservatives or excipients, commonly used excipients such as binders, fillers, wetting agents, disintegrants and the like.

As an example, excipients that may be used in the formulation of the present invention include, but are not limited to: the excipient is selected from calcium phosphate, magnesium stearate, talcum powder, dextrin, starch, gel cellulose, methyl cellulose, sodium carboxymethyl cellulose and polyvinylpyrrolidone.

The pharmaceutical carrier of the present invention may be a pharmaceutically acceptable solvent, suspending agent, vesicle, nanomaterial, etc. for delivering the compound of the first aspect of the present invention into an animal or human. The carrier may be liquid or solid and is selected according to the intended mode of administration. In addition, proteins and liposomes are also drug carriers.

The compounds of the present invention may be formulated into compositions or pharmaceutical preparations by those skilled in the art using well-known techniques. Such as by mixing any of the compounds disclosed in the first aspect of the invention (at least one compound) with a pharmaceutically acceptable adjuvant and then, if desired, shaping the resulting mixture into the desired shape. Except as noted herein, the preparation of pharmaceutical formulations may also be carried out in accordance with modern pharmaceutical formulation related books. And, other than those mentioned in the present invention, suitable pharmaceutical excipients are known in the art, for example, see the pharmaceutical excipients handbook of 2005 edition (original fourth edition), which is not described here in detail.

In a fifth aspect of the invention there is provided the use of a composition or pharmaceutical formulation as hereinbefore described in any one or more of:

(a) Inhibiting a Tim-3 signal pathway or preparing a Tim-3 signal pathway inhibitor;

(b) Promoting immune cell function and preparing a product for promoting immune cell function;

(c) Treating a disease associated with a Tim-3 signaling pathway or preparing a product of a disease associated with a Tim-3 signaling pathway;

wherein in (B), the immune cells include, but are not limited to, T cells, regulatory T cells (Tregs), dendritic Cells (DCs), B cells, macrophages, natural killer cells (NK), and mast cells.

More specifically, the promotion of immune cell function includes:

(b1) Enhancing the survival and effector functions of primary cd8+ cytotoxic T lymphocytes and human chimeric antigen receptor T cells, including but not limited to tumor cell or virus clearance functions;

(b2) Promoting NK cell killing activity and DC antigen presenting ability, including but not limited to promoting tumor cell or virus clearance.

In (c), the diseases related to Tim-3 signaling pathway include but are not limited to tumor, viral infectious diseases and autoimmune diseases.

Wherein the tumor has been explained in detail in the second aspect, and is not described in detail herein.

The viral infectious diseases include, but are not limited to, respiratory tract viral diseases, gastrointestinal viral diseases, liver viral diseases, skin and mucous membrane viral diseases, eye viral diseases, central nervous system viral diseases, lymphocytic viral diseases, insect transmitted viral diseases, and lentiviral infectious diseases.

More specifically, the respiratory viral diseases include, but are not limited to, infections with rhinoviruses, adenoviruses, respiratory syncytial viruses, parainfluenza viruses, and coronaviruses; influenza, mumps, etc.

The gastrointestinal viral diseases include, but are not limited to, viral gastroenteritis, specifically, rotaviral gastroenteritis, norwalk viral gastroenteritis, adenoviral gastroenteritis, astroviral gastroenteritis, coronal viral gastroenteritis, cup-shaped viral gastroenteritis and the like.

The liver viral diseases include, but are not limited to, viral hepatitis A, viral hepatitis B, viral hepatitis C, viral hepatitis D, viral hepatitis E and viral hepatitis EB.

The skin and mucous membrane viral diseases include, but are not limited to, measles, rubella, infant rash, varicella and zoster, smallpox, herpes simplex virus infection, rabies, foot and mouth disease, and the like.

The eye viral diseases include, but are not limited to, epidemic keratoconjunctivitis, follicular conjunctivitis, herpetic keratoconjunctivitis, and the like.

The central nervous system viral diseases comprise epidemic encephalitis B, western equine encephalitis, eastern equine encephalitis, san Louis encephalitis, venezuelan equine encephalitis, murray Valley encephalitis, california encephalitis, forest encephalitis, lymphocytic choriomeningitis, etc.

Such lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection, and acquired immunodeficiency syndrome, among others.

The insect-transmitted viral diseases include, but are not limited to, viral hemorrhagic fever, dengue hemorrhagic fever, and the like.

The lentivirus infection diseases include, but are not limited to, subacute sclerotic encephalitis, kuru disease, progressive multifocal leukoencephalopathy, subacute spongiform encephalopathy, and the like.

The autoimmune diseases include organ-specific autoimmune diseases and systemic autoimmune diseases;

wherein the organ-specific autoimmune disease includes, but is not limited to, chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, lung hemorrhagic nephritis syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral spinal sclerosis, acute idiopathic polyneuritis, and the like.

The systemic autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, pemphigus, dermatomyositis, mixed connective tissue disease, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, and the like.

In particular, the above-described compositions or pharmaceutical formulations of the invention have the following applications:

(c1) Inhibit tumor progression or preparing a product that inhibits tumor progression;

(c2) Synergistically enhancing the anti-tumor response induced by the PD-1 blocking or preparing a product synergistically enhancing the anti-tumor response induced by the PD-1 blocking;

(c3) Tumor immunotherapy or a product for tumor immunotherapy.

Of course, the above-mentioned products may be drugs or experimental reagents which can be used for basic studies, and thus for basic studies related to the Tim-3 signaling pathway.

The beneficial technical effects of the technical scheme are that:

the technical scheme firstly provides the effect of the 3, 5-disubstituted-3 a,6 a-dihydrospiro [ furan [3,4-c ] pyrrole-1, 2' -indene ] -1',3',4,6 (3H, 5H) -tetraketone compound or pharmaceutically acceptable salt, isotopic derivative and solvate thereof, or stereoisomer, geometric isomer and tautomer thereof, or prodrug molecules and metabolite thereof in inhibiting Tim-3 signal path with high efficiency and high selectivity. Specifically, a series of small molecular compounds are screened from a SPECS library through a virtual screening mode, and experiments prove that the compounds remarkably improve the survival rate and the anti-tumor activity of primary CD8+ cytotoxic T lymphocytes and human chimeric antigen receptor T cells, remarkably promote the killing activity of NK cells and the DC antigen presenting capability, show the tumor inhibition effect equivalent to Tim-3 blocking antibodies, effectively inhibit tumor progression and synergistically enhance the anti-tumor response induced by PD-1 blocking. Therefore, the compounds can be used for preparing medicines or medicinal compositions for preventing and/or treating tumors, and have good potential development and application values.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 shows functional screening to identify small molecule compounds ML-T7 targeting Tim-3;

wherein: a is the use of OT-I CD8 ⁺ Functional experimental flow of CTL; b is the effect of 17 candidate small molecule compounds on CTL proliferation; c is CD8 after pretreatment of 17 candidate small molecule compounds for flow detection ⁺ CTL cytokine secretion; d is fluorescence quantitative detection of DMSO and ML-T7 pretreated CD8 ⁺ IFN-gamma, TNF-alpha and IL-2mRNA expression levels in CTL; e is DMSO and ML-T7 pretreated CD8 ⁺ CTL against 2nM OVA _257-264 Killing ability of incubated EL4 tumors; f is DMSO and ML-T7 pretreated CD8 ⁺ CTL against 10nM OVA _257-264 In vivo killing ability of the incubated spleen cells.

FIG. 2 shows that ML-T7 binds to Tim-3 and blocks the interaction of Tim-3 with PtdSer and CEACAM 1;

wherein: a is SPR analysis of the affinity between ML-T7 and purified Tim-3 protein; b is MST assay to detect affinity between ML-T7 and GFP-Tim-3 fusion protein in cell supernatant (n=3); c is the RMSD value of the dynamics simulation analysis ML-T7-hTim3, ML-T7-mTim3 and PtdSer-hTim-3 complex; d calculates binding affinity of ML-T7/PtdSer and Tim-3 for MM/GBSA; e is a combination schematic diagram of dynamic simulation prediction ML-T7 and hTim-3IgV domain FG-CC' ditch; f is a two-dimensional (2D) schematic diagram of the ML-T7-hTim3 complex binding mode, highlighting hydrogen bonding and major hydrophobic interactions; g is purified Tim-3 protein incubated with ML-T7 or anti-Tim-3 antibody, followed by addition of dexamethasone-treated thymocytes, followed by flow cytometry analysis of Tim-3 binding to apoptotic thymocytes (n=3), with increasing concentration of ML-T7, tim-3 binding to apoptotic thymocytes being blocked; h is the purified Tim-3 protein incubated with ML-T7 or anti-Tim-3 antibody, jurkat-CEACAM1 cells were added, and flow cytometry analyzed for Tim-3 binding to Jurkat-CEACAM1 cells (n=3), with binding of Tim-3 to Jurkat-CEACAM1 blocked with increasing concentration of ML-T7.

FIG. 3 shows that ML-T7 enhances TCR/STAT5 signaling and promotes CD8 through Tim-3 ⁺ Anti-tumor activity of T cells;

wherein: a is the flow assay of levels of IFN-gamma, TNF-alpha, IL-2, perforin and Granzyme B produced by DMSO and ML-T7 pre-treated WT and Tim-3KO CTL; b is DMSO and ML-T7 pretreated WT and Tim-3KO CTL killing 2nM OVA _257-264 Ability of incubated EL4 tumors; c is the apoptosis level of WT and Tim-3KO CTL flow-tested in DMSO and ML-T7 pretreatment; d is the proliferation level of WT and Tim-3KO CTL flow-tested in DMSO and ML-T7 pretreatment; e is a flow assay for the levels of PD-1 and CTLA-4 expression by DMSO and ML-T7 pre-treated WT and Tim-3KO CTL; f and G are GESA analysis results of DMSO and ML-T7 pre-treated WT and Tim-3KO CTL; h is the phosphorylation level of corresponding proteins detected by Western blot after the WT pretreated with DMSO and ML-T7 and Tim-3KO CTL are stimulated by 2 mug/mL CD3/CD28 antibody for 15 min; i is a schematic diagram of a mice with B16-MO5 subcutaneous tumor treated by the pretreatment of DMSO and ML-T7 CTL feedback; j is a tumor growth curve; k is the record tumor-bearing mice life cycle; l is a schematic representation of a mice treated with DMSO and ML-T7 pre-treated CTL feedback for B16-MO5-Fluc lung metastasis; m is the luciferase activity of the lung of the detection mouse; n is the survival time of the tumor-bearing mice; o is a schematic representation of DMSO and ML-T7 pretreated Tim-3KO CTL reinfusion treatment of B16-MO5-Fluc lung metastasis mice; p is the luciferase activity of the lung of the detection mouse; q is the record tumor-bearing mice survival.

FIG. 4 shows ML-T7 direct enhancement of CD8 ⁺ Survival and effector function of CTLs;

wherein: a is DMSO (CD 45.1) ⁺ ) Or ML-T7 (CD 45.1) ⁺ CD45.2 ⁺ ) Pretreated OT-I CD8 ⁺ CTL is co-infused back to B16-MO5 tumor-bearing mice at a ratio of 1:1; b is the proportion of CTL pretreated with DMSO or ML-T7 in the tumor monitored by flow on days 7 and 14; c is the proportion of Annexin V+CTL in the flow detection tumor; d is Ki67 in spleen and lymph node by flow detection ⁺ Ratio of CTL; e is the ratio of CD25+ to CD69+ CTL in the flow-detected tumor; f is the flow detection of CD25 in spleen and lymph node ⁺ Ratio of CTL; g is flow type tumor and spleen detectionThe ability of CTLs in the viscera and lymph nodes to secrete IFN-gamma, TNF-alpha and IL-2; h is the expression level of PD-1 and CTLA-4 on CTL in the flow detection tumor.

FIG. 5 shows that ML-T7 enhances effector functions of NK cells and DCs;

wherein: a is the flow assay of IFN-gamma, TNF-alpha, CD107a and granzyme B expression levels of DMSO and ML-T7 pre-treated NK92 cells; b is the in vitro killing ability of NK92 cells treated with DMSO and ML-T7 against K562-Fluc target cells; c is the expression level of CD11C, CD80, CD86 and MHCII in DMSO and ML-T7 pre-treated DC; d is the level of activation of DMSO and ML-T7 pre-treated DC-stimulated CTL; e is the cytokine secretion ability of DMSO and ML-T7 pre-treated DC-stimulated CTL.

FIG. 6 shows anti-tumor activity in HCC models of both wild-type and humanized mice given ML-T7 intraperitoneally;

wherein: a is a schematic of an in situ HCC model induced by intraperitoneal injection of different doses of ML-T7 (DMSO as control, once every 2 days) or Tim-3 antibodies (IgG as control, 3 times a week); b and C are living body imaging detection of liver luciferase activity of mice, and tumor progress is monitored; d is a survival curve of mice; e is CD8 in flow detection tumor ⁺ Percentage and number of T cells; f is flow detection of tumor infiltration CD8 ⁺ Ki67 levels of T cells; g is flow detection of tumor infiltration CD8 ⁺ IFN-gamma, TNF-alpha and IL-2 secretion capacity of T cells; h is flow type detection of tumor infiltration CD8 ⁺ PD-1 expression levels of T cells; i is the IFN-gamma and TNF-alpha expression levels of flow detection tumor infiltrating NK cells; j is the expression level of CD40 and CD86 of the flow-detected intratumoral DCs; k is that an HCC model is built in a Tim-3 humanized mouse and ML-T7 intraperitoneal injection is carried out; l is bioluminescence imaging to monitor tumor growth; m is the survival curve of mice.

FIG. 7 is a graph showing that intraperitoneal administration of ML-T7 promotes an anti-tumor immune response in a mouse model of HCC;

wherein: a is the HCC mice weight change profile after drug administration treatment; b is the liver weight ratio of HCC mice; c is AST and ALT levels in HCC mice; d is the flow detection of CD8 in spleen ⁺ Percentage and number of T cells; e is the flow detection of spleen CD8 ⁺ T cellKi67 level of (a); f is the flow detection of spleen CD8 ⁺ IFN-gamma, TNF-alpha and IL-2 secretion capacity of T cells; g is a flow assay for spleen CD8 ⁺ PD-1 expression levels of T cells; h is the IFN-gamma and TNF-alpha expression level of the spleen NK cells; i is the expression level of CD40 and CD86 of the spleen DC detected in a flow mode; j is the percentage and number of MDSCs in flow-detected tumors; k is the percentage and number of MDSC in the spleen of the flow detection; l is the percentage and number of tregs in the flow detection tumor; m is the percentage and number of tregs in the flow assay spleen.

FIG. 8 shows that ML-T7 peritoneal administration in a mouse model of melanoma shows anti-tumor activity;

wherein: a is 50mg/kg ML-T7 (DMSO is used as control, once every 2 days) for intraperitoneal injection therapy of B16F10 subcutaneous tumor model schematic; b is a mouse tumor growth curve and a tumor photo; c is the flow detection of tumor and spleen CD8 ⁺ T cell ratio; d is CD8 for detecting tumor infiltration by flow ⁺ IFN-gamma, TNF-alpha and IL-2 secretion capacity of T cells; e is flow detection of tumor infiltration CD8 ⁺ T cell PD-1 and TOX expression; f is flow detection of tumor infiltration CD8 ⁺ T cell CD25 and CD69 expression; g is the proportion of NK cells in the flow detection tumor; h is the ability of flow-through detection of IFN-gamma and TNF-alpha secretion by NK cells within tumors.

FIG. 9 is that ML-T7 treatment enhances the anti-tumor activity of human CAR T cells;

wherein: a is flow cytometry to detect the killing capacity of DMSO and ML-T7 pretreated 19BBz CAR T cells to Namalwa cells; b is the apoptosis and proliferation status of 19BBz CAR T cells pretreated by flow detection DMSO and ML-T7; c is the fluorescent quantitative detection of IFN-gamma, TNF-alpha and IL-2 mRNA expression levels in DMSO and ML-T7 pre-treated 19BBz CAR T cells; d is that CAR T cells and Namalwa cells are co-cultured for 18 hours according to E:T ratio of 1:1, 2:1 and 4:1, and the content of IFN-gamma, TNF-alpha and IL-2 in the supernatant is detected by adopting an ELISA method; e is a schematic representation of adoptive transfer of DMSO-or ML-T7-treated 19BBz CAR-T cells to Namalva-Fluc tumor-bearing B-NDG mice; f and G are the conditions for monitoring tumor growth by bioluminescence imaging; h is a survival curve of tumor-bearing mice; i is the CD3 in the peripheral blood of the tumor-bearing B-NDG mice detected in a flow mode ⁺ CAR T cell ratioExamples are; j is CD8 in peripheral blood of tumor-bearing B-NDG mice by flow detection ⁺ CAR T cell ratio; k is CD8 in peripheral blood of tumor-bearing B-NDG mice by flow detection ⁺ Ratio of CAR T cell apoptosis; l is CD8 in flow detection tumor-bearing B-NDG mice ⁺ CAR T cell IFN- γ and TNF- α expression levels.

FIG. 10 shows that ML-T7 treatment has a synergistic effect with PD-1 antibody treatment in HCC model;

Wherein: a is a schematic representation of a model of HCC induced by the treatment of Akt/cMyc alone or in combination with ML-T7 (DMSO as control, once every 2 days) and PD-1 antibodies (IgG as control, 3 times a week); b is the liver weight ratio of HCC mice; c and D are methods for assessing tumor growth using bioluminescence imaging; e is HCC mouse survival curve; f is HCC mouse serum AST and ATL levels; g is HE staining and Ki67 immunohistochemical staining of HCC mouse liver.

FIG. 11 is a graph showing that ML-T7 synergistically promotes tumor immune responses in vivo with PD-1 antibody treatment;

wherein: a is CD45 in flow detection tumor ⁺ Percentage and number of T cells; b is CD8 in flow detection tumor ⁺ Percentage and number of T cells; c is flow detection of tumor infiltration CD8 ⁺ Ki67 levels of T cells; d is the flow detection of spleen CD8 ⁺ Percentage and number of T cells; e is the flow detection of spleen CD8 ⁺ Ki67 levels of T cells; f is the flow detection of spleen CD8 ⁺ IFN-gamma, TNF-alpha and IL-2 secretion capacity of T cells; g is flow detection of tumor infiltration CD8 ⁺ IFN-gamma and TNF-alpha secretion capacity of T cells; h is the IFN-gamma and TNF-alpha expression level of flow detection tumor infiltration NK cells; i is the flow detection of IFN-gamma and TNF-alpha expression levels of spleen NK cells; j is the percentage and number of MDSC cells in the flow detected tumor; k is the percentage and number of MDSC cells in the spleen in the flow assay; l is the percentage and number of tregs in the flow-detected tumor.

FIG. 12 shows that ML-T7 shows good biosafety in mice;

wherein: a is C57BL/6 mice are intraperitoneally injected with 50mg/kg of ML-T7 or DMSO every day, and the weight is changed; b is the weight of heart, spleen and kidney; c is HE slice of heart, liver, spleen, lung, kidney; d is serum AST and ALT levels; e is colon length; f is the whole blood count of hematological analysis Red Blood Cells (RBCs) and White Blood Cells (WBCs); g is the whole blood count of platelets, lymphocytes, monocytes, neutrophils and basophils in the hematological analysis; h is the inhibition of hERG potassium channel by ML-T7; the compound has low inhibition effect on hERG, and the current inhibition rate of ML-T7 at 30 mu M is only 29.04%.

FIG. 13 is a graph of the Derek software prediction of ML-T7 toxicity;

FIG. 14 shows the effect of ML-T7-like compound on enhancing NK effector function;

wherein: a is the ability of NK92 cell line to kill K562 target cells after ML-T7 analog compound treatment; b is the flow detection of NK92 cell CD107 expression; c is the flow detection of NK92 cell TNF-alpha secretion level; d is the flow assay of NK92 cell IFN-gamma secretion levels.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. 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 application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The present application will be further described with reference to examples, but it should be understood that the following description is only for the purpose of illustrating the application and is not to be construed as limiting the application. The compounds used in the examples (including ML-T7 and its series of analogues ML-T7S 1-ML-T7S 17) were all purchased from the Netherlands specs and were all existing compounds in the specs company compound library.

Examples

Experimental method

1. Experimental mice

OT-I transgenic mice (CD 45.1 or CD45.1.2, C57BL/6J background) express H-2Kb/OVA _{257 -264} Specific TCRs. C57BL/6J (CD 45.2) mice and Tim-3 humanized mice (Tim-3-hu) were purchased from Seikang Inc. (Jiangsu Nanjing, china). Tim-3 Knockout (KO) mice (C57 BL/6J background) are constructed by Shanghai Sedan Biotechnology company of China, and are hybridized with OT-I mice to obtain OT-I Tim-3 KO mice. Mice were kept under the conventional 12h light/12 h dark procedure at the university of Shandong laboratory animal center, constant room temperature (20-24 ℃) and no specific pathogen. All mice were used at an age of 8-12 weeks unless otherwise indicated. All animal experiments were performed strictly according to ethical standards and were approved by the institutional animal care and use committee at the university of shandong.

2. Induction of OT-I CTL

Spleens of male OT-I mice of 6-8 weeks old were taken in a sterile environment, and after grinding, resuspended in medium, taking care to supplement the medium while grinding. The cell suspension obtained in the previous step is centrifuged at 1200r/5min, the supernatant is discarded, 5ml of erythrocyte lysate is added, and the mixture is lysed on ice or in a refrigerator at 4 ℃ for 10min. Centrifugation at 1200r/5min, removal of supernatant, cell resuspension of medium, 5ml of medium (RPMI 1640+10% FBS+50. Mu.M. Beta. -mercaptoethanol+double antibody) after centrifugation, counting, 3X10 ⁶ Density board of individual/ml, simultaneously adding OVA _257-264 And (3) stimulating the cells at the concentration of 10nm, centrifuging, changing the liquid after stimulation for 3 days, and continuously culturing for 2 days to obtain CTLs.

3. Flow assay for cytokine secretion

(1) Sufficient EL4 cells were taken to mix with 2nm,200pm,20pm OVA, respectively _257-264 Antigen incubation for 1h, PBS wash once;

(2) Counting CTL cells and EL4 cells to obtain the numbers of the CTL cells and the EL4 cells of 5x10 respectively ⁵ Sum 2.5x10 ⁵ Adding EL4 to CTL and adding a Golgi apparatus inhibitor to the mixture and mixing the mixture uniformly, wherein the sum of the final volumes is 500 mu l;

(3) Mixing CTL cells with EL4, and incubating in an incubator at 37 ℃ for 4-6h;

(4) Collecting cells, centrifuging at 1000r/5min, discarding supernatant, and re-suspending the cells with 50 μl PBS;

(5) Preparing CD8 antibody dilutions (1 sample 0.25. Mu.l antibody), adding 5. Mu.l antibody dilutions to each sample, adding 50. Mu.l cells to the antibody dilutions, and mixing well; mixing the rest cells together as blank control, and staining for 45min at 4 ℃ in a refrigerator;

(6) 500 μl PBS+5mM EDTA resuspended cells, centrifuged at 1000r/5min, the supernatant discarded, 100 μl 2% paraformaldehyde fixative added, and frozen at 4deg.C for 30min;

(7) Resuspension of cells with 500. Mu.l of the membrane-penetrating fluid, centrifugation at 1000r/5min, discarding the supernatant, resuspension of cells with 50. Mu.l of the membrane-penetrating fluid;

(8) Preparing TNF-alpha (PE), IFN-gamma (APC), IL-2 (FITC) antibody dilutions (1 sample 0.25 μl antibody), adding 5 μl antibody dilutions to each sample, adding 50 μl cells to the antibody dilutions, mixing, and staining at 4deg.C for 45min;

(9) Re-suspending the cells with 500 μl of the membrane penetrating solution, centrifuging at 1000r/5min, and discarding the supernatant;

(10) The cells were resuspended in 500. Mu.l PBS, centrifuged at 1000r/5min and the supernatant discarded;

(11) Cells were resuspended in 300 μl PBS and filtered on a flow machine.

4. Surface Plasmon Resonance (SPR) experiments

SPR binding experiments were performed using the Biacore T200 biosensor system (GE Healthcare) at 25 ℃. All buffers were purchased from GE Healthcare. CM5 microsensor chip (GE Healthcare) was incubated with HEPES buffer (10mM HEPES,150mM NaCl,CaCl) ₂ pH 7.4) equilibrated until a stable signal is reached. According to the instrument instructions, his-tagged human Tim-3 protein (cat#tm-H5229, ACROBiosystems Group, beijing, china) was diluted in 10mM sodium acetate buffer (ph=4.0) and immobilized on CM5 chips using an amine coupling kit. The chip is then balanced with the running buffer to stabilize the signal. ML-T7 was diluted in HEPES buffer at 6 spots in a 2-fold gradient starting at a maximum concentration of 25. Mu.M. ML-T7 90s binding and 90s dissociation were serially diluted in HEPES buffer at a flow rate of 30 μl/min. Data analysis was performed using Biacore T200 software and equilibrium constants (Kd) were calculated using a 1:1 kinetic fitting model.

5. Micro thermophoresis experiment (MST)

MST experiments were performed using a Monolith NT.115 instrument (NanoTemper Technologies). Briefly, HEK293T cells were transfected with plasmids expressing GFP-Tim-3 fusion proteins or GFP (control) using Polyethylenimine (PEI) reagents. Cell supernatants were collected 48h after transfection and filtered through 0.45 μm filters. ML-T7 was dissolved in 625. Mu.M PBS containing 5% DMSO concentration, and diluted 2-fold in a gradient. The gradient diluted ML-T7 was incubated with cell supernatant containing GFP-tim-3 fusion protein or GFP (control) containing 5% DMSO at a ratio of 1:1 for 20min, and the samples were loaded into glass capillaries for MST using 20% LED excitation power and 40% MST power. Binding curves were analyzed using mo. Affinity Analysis software (nanotemplate, version 2.3).

6. Assay of Tim-3 binding Using apoptotic thymocytes or Jurkat-CEACAM1

C57BL/6J mouse thymus cells were labeled with 0.1mM Calcein-AM (ThermoFisher), and then apoptosis was induced by treatment with 1mM dexamethasone (Sigma) in DMEM with 10% FBS at 37℃for 6 h. Annexin V (BD Pharmingen) staining confirmed apoptosis. Recombinant human Tim-3 protein (Sanyou organism) was incubated with specified concentrations of ML-T7 or DMSO at room temperature for 30min, then added to apoptotic thymocytes or human T lymphoblastic cell line overexpressing CEACAM1 (Jukrat-Ceacam 1), stained with anti-human Tim-3 APC antibody (BioLegend). Flow cytometry detects Tim-3 binding.

7. Adoptive CTL metastasis therapy in murine melanoma model

Mice were modeled for subcutaneous melanoma and 2X10 mice were treated on day 0 ⁵ The B16-MO5 melanoma cells were inoculated subcutaneously in wild-type C57BL/6J mice. Tumor-bearing mice were injected intravenously with 10. Mu.M ML-T7 or 4X 10 when tumors were accessible on day 10 ⁶ DMSO-treated OT-I CD8 ⁺ CTL. Tumors were measured every 2-3 days with digital calipers, expressed as tumor volume (in width ² X length/2 calculation). Based on ethical consideration, the tumor size is>Mice of 20mm will be treated by CO inhalation ₂ Euthanasia was performed. Survival was recorded daily.

Establishing a lung metastasis model, and intravenous injection of wild C57BL/6J mice on day 0×10 ⁵ The lung metastasis model was established with B16-MO5 melanoma cells (B15-MO 5-Fluc) expressing firefly luciferase. Tumor-bearing mice were intravenously injected on day 10 with ML-T7 or DMSO-treated OT-I CD8+ CTL (4X 10) ⁶ ). Bioluminescence imaging was performed using the IVIS Spectrum system (PerkinElmer) to monitor lung metastases. Luciferase Activity level was expressed as p/sec/cm ² /sr. Survival was recorded daily.

8. Preparation of tumor mouse model

Mouse melanoma model: male C57BL/6 mice aged 5-6 weeks old were subcutaneously injected with 2X10 ⁵ B16-MO5 cells, after tumor growth (about 6 d), were measured every other day for tumor minor (a) and major (B) diameters, and tumor volume v=a was calculated ² b/2; counting the death time of the mice and drawing survival curves of the mice.

Hydrodynamic injection Akt/cMyc plasmid induced hepatocellular carcinoma (HCC) model: the cMyc, akt/Fluc gene plasmids and the plasmid SB100 encoding the Sleeping Beauty transposase were extracted according to the plasmid extraction kit steps, and the cMyc, akt gene plasmids and the SB100 plasmids were combined at a ratio of 12 μg per mouse: 12 μg:1 μg, a volume (10% of body weight, e.g., 20g of body weight using 2ml PBS) of 0.9% sodium chloride solution, vortexing, passing through a 0.22 μm microporous filter membrane, and injecting the prepared plasmid into 8 week old wild type male C57BL6 mice by mouse tail vein hydrodynamic high pressure injection. The liver of the mice can form liver cell liver cancer 3-4 weeks after plasmid injection.

9. Preparation and functional experiments of human CAR T cells

Human 19BBz CAR-T cells targeting human CD19 were prepared. Lentiviral 19BBz CAR expression plasmids were transfected with psPAX2 (Addgene, # 12260) and pMD2G (Addgene, # 12259) into Lenti-X293T cells using Lentifit (Han-animate, china) transfection reagents. The lentiviruses containing the supernatant were collected 48 and 72 hours after transfection, concentrated by ultracentrifugation and filtered through a 0.22 μm filter. Primary human T cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors, activated with anti-CD 3/CD28 beads at a 1:1 ratio (Miltenyi Biotec, bergisch Gladbach, germany) in TexMACS GMP medium (Miltenyi Biotec) supplemented with 10% FBS and 50IU/mL IL-2 for 2 days, and then transduced by centrifugation at 32℃for 2 hours with concentrated supernatants containing human 19BBz CAR lentiviral particles. The transduced cells were passaged daily for 5d with fresh medium containing 50IU/mL IL-2 at a ratio of 1:2. Transfection efficiency was determined by flow cytometry to detect GFP expression of the CAR plasmid.

After expansion, the CAR T cells were treated with 10 μm ML-T7 or DMSO for 48h. ML-T7 or DMSO-treated CAR T cells were co-cultured with Namalwa target cells at a ratio of 1:1, 2:1, 4:1 for 18h. Cell supernatants were collected and assayed for IL-2, IFN-gamma and TNF-alpha levels using an ELISA kit (Dakewe). Flow cytometry was used to examine the cytotoxic capacity of CAR T cells based on the ratio of Dil-labeled target cells to anti-human CD19-APC antibody (cat# 561742, bd) -labeled CAR T cells.

10. CAR T cell therapy

Will be 5X 10 ⁵ Namalwa cells expressing firefly luciferase (Namalwa-Fluc) were tail-injected into B-NDG (NOD-Prkdcsccid IL2rgtm 1) mice in 200. Mu.L PBS and randomly divided into 3 groups on days 5 and 10, receiving PBS, 3X 10, respectively ⁶ 19BBz CAR-T cells and 3X 10 treated with DMSO ⁶ 19BBz CAR-T cells treated with ML-T7. Tumor progression was monitored by bioluminescence imaging using the IVIS Spectrum system (PerkinElmer) on days 10 and 16, respectively, and survival was recorded daily. PBMCs were isolated from recipient mice, stained with the indicated antibodies, and analyzed using flow cytometry.

11. Method for separating and stimulating tumor infiltrating lymphocytes

Removing tumor tissue (subcutaneous tumor or liver cancer) of mice, grinding with a mortar, filtering, collecting cell sap, centrifuging at 600rpm for 1min, pouring supernatant into a clean 50ml centrifuge tube, centrifuging at 1200rpm for 5min, discarding supernatant, adding 8ml percoll working solution, centrifuging at 2000rpm for 20min, discarding supernatant, adding 5ml erythrocyte lysate, centrifuging at ice lysis 10min,1200rpm 5min, discarding supernatant, adding 2ml PBS, and collecting small amount of cells for CD8 ⁺ T cell count, centrifugation of the remaining cells, addition of 1640 complete medium, stimulation with PMA (200 ng/ml) and ionomycin (1 μm) for 4-6h followed by flow antibody staining of the harvested cells, and addition of Golgi apparatus inhibitor 4h before harvesting.

12. Flow cytometry analysis

Surface staining: the cells were labeled with surface markers at 4℃for 30min in the dark. For intracellular staining, cells were fixed using IC fixation buffer (cat#00-8222-49, ebioscience), broken using membrane disruption buffer (cat#00-8333-56, ebioscience), and stained with the indicated antibodies. For transcription factor staining, cells were fixed and broken with fixation/break concentrate and dilution buffer (cat#00-5521, ebioscience). Samples were run using a CytoFLEX S flow cytometer (beckman coulter) and data analysis was performed using the cytexert program (beckman coulter) and FlowJo software.

Experimental results

1. Functional screening to identify small molecule compound ML-T7 for promoting CTL functional cells

In order to find Tim-3 small molecule inhibitors, we used a strategy of virtual screening combined with functional screening. Considering that the amino acid homology of human and murine Tim-3 exceeds 63% and that the structure is highly conserved at PtdSer binding site, we have performed homologous modeling of human Tim-3 (hTim-3) using murine Tim-3 (mTim-3) and PtdSer (PDB: 3 KAA) as templates. Using this model, a virtual screening of 204380 compounds in the SPECS library was performed with PtdSer binding sites as binding pockets, and a total of 192 compounds with predicted affinity for the target greater than-7.4 Kcal/mol were identified (FIG. 1A). These compounds are then further screened by a variety of strategies such as structural similarity clustering, physicochemical property prediction, and binding pattern analysis. Finally, 17 small molecule compounds were selected, designated ML-T1 through ML-T17, for subsequent CD 8-based ⁺ CTL function screening.

We used OT-I T Cell Receptor (TCR) transgenic mice (a type widely used for assessment of CD8 ⁺ Spleen cells and ovalbumin polypeptides (257-264) (SIINFEKL, OVA) of a mouse model of T cell anti-tumor immune function _257-264 ) Incubation was performed in the presence of ML-T1 to ML-T17 (at a concentration of 10. Mu.M) (FIG. 1B). ML-T7 had no significant effect on cell viability (FIG. 1C), significantly enhanced IFN- γ, TNF- α and IL-2 production in OT-I cells at the mRNA and protein levels (FIGS. 1D and E). ML-T7 pretreatmentOT-ⅠCD8 ⁺ CTLs showed greater ability to kill target cells both in vitro and in vivo (fig. 1F and G).

2. ML-T7 binds to Tim-3 and blocks Tim-3 interaction with PtdSer and CEACAM1

To determine whether ML-T7 binds directly to Tim-3 and acts as a Tim-3 inhibitor, we performed Surface Plasmon Resonance (SPR) experiments using the commercially available his-tagged hTim-3 protein. As shown in FIG. 2A, ML-T7 binds to hTim-3 with a KD of 6.98. Mu.M, indicating a moderate interaction between ML-T7 and hTim-3. To verify this interaction we performed a micro thermophoresis (MST) experiment using cell supernatants containing hTim-3 (GFP-hTim-3) fused with green fluorescent protein or control GFP. Only GFP-hTim-3 cell supernatants produced binding curves with ML-T7 (FIG. 2B), further demonstrating that ML-T7 can bind to hTim-3.

Molecular dynamics simulation studies further support the interaction of ML-T7 with hTim-3. As shown in FIG. 2C, the results of the position Root Mean Square Deviation (RMSD) analysis indicate that both mTim-3 and hTim-3 bind stably to ML-T7 with only slight fluctuations compared to the original conformation. Molecular mechanics calculated by MM/GBSA showed that ML-T7 has a higher affinity for both hTim-3 and mTim-3 than PtdSer for Tim-3, meaning that ML-T7 can effectively interrupt PtdSer interactions with Tim-3 (FIG. 2D). Detailed analysis of representative conformations of molecular dynamics results showed that ML-T7 forms a broad interaction with residues of FG-CC' loop of hTim-3: one oxygen in the pyridine from the ligand backbone forms a polar contact with the nitrogen atom of the Glu33 backbone, while the three terminal aromatic rings of ML-T7 have multiple hydrophobic interactions with adjacent residues Val31, phe32, cys34, arg82, gin 84, and Ile88 (fig. 2E and F).

Crystal structure studies indicate that FG-CC' ring is a binding site for PtdSer. To examine the ability of ML-T7 to block binding of Tim-3 to PtdSer, mice were induced to die by thymus cells with dexamethasone to expose PtdSer on their surface, and then incubated with Tim-3 protein in the presence of ML-T7. We observed that ML-T7 inhibited the binding of apoptotic thymocytes to hTim-3 in a dose-dependent manner (figure 2G). In addition, flow cytometry analysis showed that ML-T7 also blocked binding of hTim-3 to CEACAM1 overexpressing Jurkat cells in a concentration-dependent manner (FIG. 2H), consistent with the notion that PtdSer and CEACAM1 bind to Tim-3 in the same pocket.

3. ML-T7 enhances TCR/STAT5 signaling through Tim-3 and promotes CD8 ⁺ T cell antitumor Activity

To confirm that ML-T7 promotes CD8 as a Tim-3 inhibitor ⁺ T cell function, we hybridized Tim-3 knockout (Tim-3 KO) mice with OT-I mice, resulting in Tim-3 knockout (Tim-3 KO) TCR-specific CD8 ⁺ CTL. As expected, ML-T7 significantly promoted production of IFN- γ, TNF- α, IL-2, perforin and granzyme B in wild-type (WT) CTL, but no promotion in Tim-3 KO CTL (FIG. 3A). In addition, tim-3 knockout abrogated the effect of ML-T7 on enhancing CTL killing tumor cells in vitro (FIG. 3B). We have also found that ML-T7 treatment reduced apoptosis and increased proliferation capacity of CTL, whereas ML-T7 had no or little effect on apoptosis and cell proliferation of Tim-3 KO CTL (FIGS. 3C and D). Meanwhile, ML-T7 treatment inhibited the expression of the inhibitory receptors PD-1 and CTLA-4 in WT cells, but had no effect on Tim-3 KO CTL, indicating that ML-T7 treatment could inhibit T cell depletion (FIG. 3E). Further RNA sequencing (RNA-seq) combined with Gene Set Enrichment Analysis (GSEA) showed that the TCR signaling pathway and IL2-STAT5 pathway were significantly enriched in ML-T7 treated CTL (FIGS. 3F and G). Accordingly, ML-T7 treatment increased the phosphorylation levels of PLCγ1, ZAP70, LCK, ERK1/2 and STAT5 following stimulation with anti-CD 3/CD28 antibodies. Notably, ML-T7 did not affect Tim-3 KO OT-I CD8 ⁺ TCR and STAT5 signals in CTLs (fig. 3H). The above results indicate that ML-T7 enhances CD8 through Tim-3 ⁺ TCR and STAT5 signaling pathways of CTLs.

To further verify whether ML-T7 could enhance the anti-tumor function of CTL in vivo, we treated ML-T7 or control OT-I CD8 ⁺ CTL were adoptive returned to B16-MO5 melanoma tumor-bearing mice (fig. 3I). Encouraging that reinfusion of ML-T7-pretreated CTLs significantly inhibited tumor progression, extending survival of recipient mice (fig. 3J and 3K). Similar results were obtained in B16-MO5 (B16-MO 5-Fluc) lung metastasis model mice carrying firefly luciferase (FIG. 3L). Control miceIn contrast, OT-I CD8 treated with ML-T7 ⁺ Recipient mice for CTLs showed lower tumor burden in the lung (fig. 3M) and longer survival (fig. 3N). Importantly, when we treated ML-T7-treated or DMSO-treated Tim-3 KO CD8 ⁺ CTL metastasized into B16-MO5-Fluc lung metastatic mice (fig. 3O), no difference in tumor progression and survival between the two groups was found (fig. 3P and Q). Therefore, the above experiments indicate that ML-T7 modulates CTL anti-tumor function through Tim-3.

4. ML-T7 directly enhances survival and effector function of CD8+ CTL

To verify whether ML-T7 endogenously promoted effector function of CTLs, we reinfused equivalent controls and ML-T7-treated CTLs to B16-MO5 subcutaneously tumor model mice (FIG. 4A). Flow cytometry results showed that ML-T7 treatment significantly promoted CTL aggregation and persistence in tumors (fig. 4B). Consistent with this, ML-T7 treatment reduced apoptosis and increased proliferation of CTLs in tumors (fig. 4C). We also observed an increase in proliferation of ML-T7 treated CTLs in spleen and lymph nodes (fig. 4D). In addition, ML-T7 treated CD8 in tumor, spleen and lymph node of recipient mice ⁺ CTLs not only expressed higher activating molecules CD25 and CD69 (fig. 4E and F), but also produced more IFN- γ, TNF- α and IL-2 (fig. 4G). We also observed that in ML-T7 treated CTLs infiltrating tumors, the expression of PD-1 and CTLA-4 was significantly reduced, suggesting a lower degree of depletion (fig. 4H). Thus, ML-T7 can directly prolong the survival time of CD8+ CTL and enhance its effector function.

5. ML-T7 enhances NK cell and DC functions

Considering that Tim-3 is expressed in large numbers on innate immune cells and plays an important role in the negative regulatory function of Natural Killer (NK) cells and Dendritic Cells (DCs), both of which are critical for tumor immunotherapy. Therefore, we examined the effect of ML-T7 on NK cell function. Flow cytometry analysis showed that 10. Mu.M ML-T7 treatment significantly enhanced the production of effector molecules IFN-. Gamma., TNF-. Alpha., CD107a and granzyme B in NK92 cells of the human NK cell line (FIG. 5A). The killing activity of ML-T7 treated NK92 cells against K562-Fluc cells was significantly increased at different E: T ratios compared to control NK92 cells (fig. 5B).

To examine the regulation of DC function by ML-T7, we used granulocyte-macrophage stimulating colony factor (GM-CSF) and interleukin-4 (IL-4) to culture bone marrow cells for 5 days, and then added 1. Mu.g/mL LPS and 10. Mu.M ML-T7 at day 6 for 24 hours to prepare mature DCs (mDCs). The ML-T7 treatment increased expression of CD11C, CD80, CD86 and mhc ii on the DC surface compared to DMSO (fig. 5C), indicating enhanced DC maturation and enhanced antigen presenting ability. Further functional experiments showed that DC-activated T cells treated by ML-T7 activated more strongly and had a stronger effector function (FIGS. 5D and E). Taken together, our results indicate that ML-T7 treatment can promote DC maturation and anti-tumor function of NK cells.

6. ML-T7 intraperitoneal administration shows anti-tumor activity in both mouse HCC liver cancer model and humanized mice

To assess whether ML-T7 could be used as a direct tumor immunotherapeutic, we administered different doses of ML-T7 intraperitoneally to treat a model of spontaneous in situ hepatocellular carcinoma (HCC) driven by the oncogene Akt/Myc, which expresses firefly luciferase, indicative of tumor burden. To compare the effect of ML-T7 and Tim-3 blocking antibodies, we included a commercial Tim-3 antibody (. Alpha. -Tim-3) as a control (FIG. 6A). There was no significant difference in mouse body weight during different doses of ML-T7 treatment (fig. 7A). Remarkably, in vivo imaging results showed that ML-T7 at therapeutic doses of 20mg/kg and 50mg/kg could significantly inhibit tumor growth (fig. 6B and C). In agreement, the liver weight ratio and AST and ALT levels were also significantly reduced in the 20mg/kg and 50mg/kg treatment groups (FIGS. 7B and C). Notably, the level of ML-T7 at the 20mg/kg dose was comparable to that of the alpha-Tim-3 antibody at tumor inhibition and prolonged survival (FIGS. 6C and D, FIG. 7B).

Further flow cytometry analysis showed that systemic administration of ML-T7 at doses of 20mg/kg and 50mg/kg caused CD8 in both tumor and spleen, similar to the effect of alpha-Tim-3 antibody treatment ⁺ T cell ratio and number (FIGS. 6E and 7D) increased significantly and Ki67 positive CD8 was observed ⁺ T cells (fig. 6F and 7E) increased significantly suggesting stronger proliferation. In addition, ML-T7 enhances tumor infiltration and spleen CD8 ⁺ Production of IFN-gamma, TNF-alpha and IL-2 effector molecules in T cells (FIGS. 6G and 7F) and reduced CD8 ⁺ Expression of PD-1 in T cells suggests inhibition of T cell depletion (fig. 6H and 7G). Furthermore, ML-T7 administration promoted not only the secretion of IFN- γ and TNF- α levels by tumor and spleen NK cells (fig. 6I and 7H), but also infiltration and maturation of DCs in tumors (fig. 6J and 7I). In addition, ML-T7 treatment reduced infiltration of Myeloid Derived Suppressor Cells (MDSCs) and regulatory T cells (tregs) (fig. 7J to M), suggesting that ML-T7 alleviates the tumor immunosuppressive microenvironment. The data indicate that like the alpha-Tim-3 antibody, ML-T7 plays a role in tumor immunotherapy by promoting CTL, NK cell and DC functions, thereby inhibiting the immunosuppressive tumor microenvironment.

To further verify the therapeutic efficacy of ML-T7, an in situ Akt/Myc-HCC model was induced in humanized Tim-3 (Tim-3-HU) knock-in mice whose Tim-3 extracellular domain was replaced by that of human Tim-3, followed by systemic treatment with 20mg/kg ML-T7 (FIG. 6K). As expected, ML-T7 greatly inhibited tumor growth, exhibited weaker luciferase activity and prolonged survival of mice (FIGS. 6L and M). These results indicate that ML-T7 is also a potent candidate for tumor therapy targeting human Tim-3.

7. ML-T7 abdominal cavity administration shows anti-tumor activity in a mouse melanoma model

To assess whether ML-T7 could treat melanoma, we treated B16F10 melanoma model (8A) with 50mg/kg dose of ML-T7 by intraperitoneal injection. 50mg/kg ML-T7 treatment significantly inhibited melanoma growth (FIG. 8B). Flow cytometry analysis showed that 50mg/kg of ML-T7 significantly increased CD8 in both tumor and spleen ⁺ T cell ratio (FIG. 8C) and enhanced tumor infiltration of CD8 ⁺ Production of IFN-gamma, TNF-alpha, IL-2 and like effector molecules in T cells (FIG. 8D). Importantly, ML-T7 significantly reduces CD8 ⁺ Expression of PD-1 and TOX in T cells reverses T cell depletion (TOX) ⁺ TCF1 ^- Reduced ratio) (fig. 8E), and promotes the expression of the activating molecules CD25 and CD69 of T cells (fig. 8F). Furthermore, ML-T7 administration also promotes tumor infiltration of NK cell proportion and secretion of IFN-gamma and TNF-alphaIs shown (FIGS. 8G and H)

8. ML-T7 treatment can enhance anti-tumor activity of human CAR T cells

CAR T cell therapy has achieved tremendous clinical success in hematological malignancies. Since ML-T7 can bind to Tim-3 and enhance the CTL anti-tumor efficacy of mice in a Tim-3 dependent manner, we want to determine whether ML-T7 can also enhance the therapeutic effect of human CAR T cells. To this end, we used lentiviruses to transfect 19BBz CARs into T cells of human Peripheral Blood Mononuclear Cells (PBMCs), construct CD 19-targeted CAR T cells, and treated with DMSO or ML-T7. Consistent with the results of mouse CTLs, ML-T7 in vitro treatment significantly improved the cell killing ability of CAR T cells to express the human B lymphoblast cell line Namalwa of CD19 (fig. 9A). We also found that ML-T7 treatment reduced apoptosis of CAR T cells, increasing proliferation of CAR T cells (fig. 9B). At the same time, ML-T7 treated CAR T cells increased IFN- γ, TNF- α and IL-2 production at both protein and mRNA levels (FIGS. 9C and D). Furthermore, ML-T7 treated 19BBz CAR T cells showed significantly enhanced anti-tumor activity in Namalwa-Fluc tumor bearing B-NDG mice compared to DMSO treated 19BBz CAR T cells. Adoptive transfer of ML-T7 treated 19BBz CAR T cells greatly induced tumor regression and prolonged mouse survival (fig. 9E to H). Furthermore, more CD3 was detected in the peripheral blood of mice receiving ML-T7 pre-treated CAR T cells than mice receiving control CAR T cells ⁺ And CD8 ⁺ CAR T cells (fig. 9I and J). Reinfusion of ML-T7 treated CD8 compared to control CAR T cells ⁺ CAR T cells showed less apoptosis (fig. 9K) and higher IFN- γ and TNF- α secretion levels (fig. 9L), consistent with data for ML-T7 treated mouse CTLs.

Taken together, the above results demonstrate that ML-T7 treatment significantly enhances the therapeutic potential of adoptive transfer of human CAR T cells during CAR T cell preparation, further supporting that ML-T7 is also suitable for human T cells.

9. ML-T7 has synergistic effect with PD-1 antibody treatment in vivo

Previous studies have reported that combined blockade of Tim-3 and PD-1 could synergistically enhance anti-tumor response, we further explored whether ML-T7 could synergistically act with PD-1 antibodies in tumor immunotherapy in AKT/Myc HCC tumor models (fig. 10A). Based on in vivo imaging, survival curves, liver weight measurements, ALT/AST assessment, hematoxylin-eosin (HE) staining and Ki67 staining, the ML-T7 monotherapy at 20mg/kg showed almost similar anti-tumor activity as the PD-1 monoclonal antibody at 100mg/kg (FIGS. 10B to G). Most importantly, the combination ML-T7 and PD-1 antibodies showed stronger anti-tumor activity than either alone (fig. 7B to E, 50% of mice (3/6) receiving the combination survived on day 120, while 0 or 1/6 mice receiving either ML-T7 or PD-1 antibody single drug treatment survived to day 120 (fig. 10E).

Next, we further explored the effect of ML-T7 and PD-1 antibodies alone or in combination on immune cells in HCC tumor models. Similar to PD-1 antibody alone, either ML-T7 alone or in combination with PD-1 antibody significantly promoted CD45 in tumors ⁺ Infiltration of immune cells (FIG. 11A) increased CD8 in tumors and spleen ⁺ The proportion, number and proliferation of T cells (fig. 11B to E) also significantly enhanced CD8 in tumor and spleen ⁺ The ability of T cells to produce cytokines (IFN-. Gamma., TNF-. Alpha.and IL-2) (FIGS. 11F and G). Consistent with the negative modulation of NK cell function by Tim-3 and PD-1, we also observed that both ML-T7 and PD-1 antibody treatment strongly increased production of IFN-gamma and TNF-alpha by NK cells (FIGS. 11H and I), indicating enhanced NK tumor killing activity. Notably, ML-T7 therapy showed tremendous synergy with PD-1 antibody therapy in restoring NK cell viability and inhibiting MDSCs and Treg infiltration (fig. 11J to L).

Taken together, ML-T7 not only shows potent anti-tumor activity in vivo, but also synergistically relieves the inhibitory immune environment in the tumor microenvironment with PD-1 antibody therapy, reactivating CD8 ⁺ Anti-tumor response of T cells and NK cells.

10. ML-T7 shows good biosafety in mice

To determine the biosafety of ML-T7, its clinical transformation prospects are further explored. We intraperitoneally injected C57BL/6 mice with 50mg/kg ML-T for 7 2 weeks per day. ML-T7 treatment did not cause any abnormal changes in body weight or clinical symptoms during the experiment (FIG. 12A). At necropsy, ML-T7 treated mice did not have any significant differences in heart, spleen and kidney weights (fig. 12B). Further histological analysis of heart, liver, spleen, lung and kidney by HE staining showed that ML-T7 treated mice had no significant abnormalities (fig. 12C). In addition, serum biochemical tests showed that ALT and AST levels were normal after ML-T7 treatment, indicating that 50mg/kg ML-T7 had no significant hepatotoxicity to mice (FIG. 12D). At the same time, ML-T7 did not affect colon length (FIG. 12E). Furthermore, in ML-T7 treated mice, hematological evaluation showed no signs of anemia, significant leukopenia, or leukocytosis (fig. 12F). We did not observe significant changes in platelet, lymphocyte, monocyte, neutrophil and basophil counts (figure 12G). We also tested the hERG inhibition of this compound, which is a concurrent cause of severe tachyarrhythmias. The results showed that ML-T7 did not significantly inhibit hERG potassium channels with acceptable IC50>30 μm (fig. 12H). Toxicity assessment of ML-T7 using the well-known toxicity prediction software Derek also supported the safety of ML-T7 in 56 test projects (FIG. 13).

In conclusion, the ML-T7 has good tolerance in mice, does not cause obvious adverse reaction, and indicates that the ML-T7 is a safe and effective antitumor small molecule drug, and deserves further development and application to clinical transformation.

11. ML-T7 series analogue compounds have effect of enhancing NK cell antitumor activity

The present invention further tests the ability of more ML-T7-like compounds to promote NK92 cell killing of tumor cells and secretion of effector IFN-gamma, TNF alpha and CD107 a. The results show that the compounds can remarkably promote NK92 cell killing capacity and secretion effector cell factor capacity (figure 14), and the compounds are suggested to have the similar immunotherapeutic potential of inhibiting Tim-3 as MT-L7.

In conclusion, the invention successfully discovers a small molecule Tim-3 inhibitor which can enhance the adoptive transfer treatment effect of CTL or CAR T cells, and the functions of NK cells and DC, and can directly play anti-tumor activity in a preclinical tumor model to inhibit tumor progression. The efficacy of ML-T7 alone or in combination with PD-1 antibodies supports its further clinical transformation.

It should be noted that the above examples are only for illustrating the technical solution of the present invention and are not limiting thereof. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can make modifications and equivalents to the technical solutions of the present invention as required, without departing from the spirit and scope of the technical solutions of the present invention.

Claims (10)

1. The application of the compound in preparing Tim-3 inhibitor;

wherein the compound is 3, 5-disubstituted-3 a,6 a-dihydrospiro [ furo [3,4-c ] pyrrole-1, 2' -indene ]

1',3',4,6 (3 h,5 h) -tetraones having the structure of formula I:

wherein, the liquid crystal display device comprises a liquid crystal display device,

R ₁ the method comprises the following steps: c (C) ₃ -C ₆ Cycloalkyl, benzene ring with 0 to 2 substituents, benzyl with 0 to 2 substituents, six-membered heterocycle with 0 to 2 substituents, five-membered heterocycle with 0 to 2 substituents, said substituents each being independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

R ₂ the method comprises the following steps: c (C) ₃ -C ₆ Cycloalkyl, benzene ring with 0 to 2 substituents, six membered heterocycle with 0 to 2 substituents, five membered heterocycle with 0 to 2 substituents, said substituents each being independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

m is selected from integers of 0, 1 or 2, R ₃ Each occurrence is independently C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups.

2. The use according to claim 1, wherein,

R ₁ the method comprises the following steps: a benzene ring having 0 to 2 substituents, a benzyl having 0 to 2 substituents, each of said substituents being independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

R ₂ the method comprises the following steps: a benzene ring having 0 to 2 substituents each independently selected from C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

m is selected from integers of 0, 1 or 2, R ₃ Each occurrence is independently C ₁ -C ₂ Alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxamide, carboxyl, ester groups;

the compound is in a racemate or a single chiral configuration.

3. The use according to claim 1, wherein the compound is one of the following:

4. the use according to claims 1-3, wherein the compound further comprises a pharmaceutically acceptable salt, isotopic derivative, solvate thereof, or a stereoisomer, geometric isomer, tautomer thereof, or a prodrug molecule, metabolite thereof.

5. A compound for use according to any one of claims 1 to 4 for the preparation of a medicament for the prophylaxis and/or treatment of tumors.

6. The use of claim 5, wherein the neoplasm comprises benign neoplasm and/or malignant neoplasm; the malignant tumor comprises solid tumor and blood tumor; preferably a solid tumor, including liver cancer.

7. A composition comprising at least a compound for use according to any one of claims 1 to 4.

8. Pharmaceutical formulation, characterized in that it comprises at least a compound for use according to any one of claims 1 to 4.

9. Use of the composition of claim 7 or the pharmaceutical formulation of claim 8 in any one or more of the following:

(a) Inhibiting a Tim-3 signal pathway or preparing a Tim-3 signal pathway inhibitor;

(b) Promoting immune cell function and preparing a product for promoting immune cell function;

(c) Treating a disease associated with a Tim-3 signaling pathway or preparing a product of a disease associated with a Tim-3 signaling pathway;

wherein in said (B), said immune cells comprise T cells, regulatory T cells, dendritic cells, B cells, macrophages, natural killer cells and mast cells;

More specifically, the promotion of immune cell function includes:

(b1) Enhancing the survival and effector functions of primary cd8+ cytotoxic T lymphocytes and human chimeric antigen receptor T cells, including but not limited to tumor cell or virus clearance functions;

(b2) Promoting NK cell killing activity and DC antigen presenting ability, including but not limited to promoting tumor cell or virus clearance;

in said (c), the disease associated with Tim-3 signaling pathway includes tumor, viral infectious disease, and autoimmune disease;

specifically, the (c) application includes:

(c1) Inhibit tumor progression or preparing a product that inhibits tumor progression;

(c2) Synergistically enhancing the anti-tumor response induced by the PD-1 blocking or preparing a product synergistically enhancing the anti-tumor response induced by the PD-1 blocking;

(c3) Tumor immunotherapy or a product for tumor immunotherapy.

10. The use according to claim 9, wherein the product is a pharmaceutical or experimental agent for use in basic research.