CN116617214B - 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 a small molecule compound targeting Tim-3 in tumor immunotherapy

Technical Field

The present invention belongs to the technical field of immunology and anti-tumor drugs, and specifically relates to the application of a small molecule compound targeting Tim-3 in tumor immunotherapy.

Background Art

The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

Immune checkpoint blockade (ICB) therapy has become a recognized effective tumor treatment method after surgery, radiotherapy and chemotherapy, and has completely changed the treatment of many tumors. It is worth noting that therapeutic antibodies targeting PD-1 and CTLA-4 achieve strong and lasting anti-tumor responses in various tumor types by restoring the effector function of exhausted T cells. However, a large proportion of cancer patients have poor response to PD-1 or CTLA-4 antibody treatment. Therefore, it is critical to find new immune checkpoint molecules as therapeutic targets.

Tim-3 is involved in the terminal differentiation and exhaustion of T cells during chronic viral infection and tumor progression, and is currently one of the most promising immune checkpoint targets. Tim-3 is highly expressed on exhausted T cells in tumors, and in mouse models, antibodies targeting Tim-3 can reverse T cell exhaustion and promote tumor regression. In addition, in melanoma patients and mouse tumor models, CD8 ⁺ T cells co-expressing PD-1 and Tim-3 exhibit a more exhausted phenotype, and their proliferation and cytokine production capabilities are severely impaired. In multiple studies, combined blockade of Tim-3 and PD-1 can significantly enhance T cell function, inhibit tumor growth, and is more effective than single therapy. It is worth noting that recent clinical trial data show 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. Four different Tim-3 ligands have been reported, including galectin-9, phosphatidylserine (PtdSer), high mobility group protein B1 (HMGB1), and carcinoembryonic antigen-related cell adhesion molecule 1 (CEA Cell Adhesion Molecule 1, CEACAM1). Crystal structure studies have shown that there is a common FG-CC' loop in the IgV domain of mouse and human Tim-3 as a binding site for PtdSer, CEACAM1, and HMGB1, while Galectin-9 binds to the N-linked glycan on the other side of the FG-CC' loop. Importantly, the common feature of the mouse and human Tim-3 antibodies that have been proven to be effective is that they block the binding of Tim-3 to PtdSer and CEACAM1, but do not block the binding of Tim-3 to Galectin-9. Therefore, 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 strong anti-tumor responses in early clinical trials to date. Compared with antibodies, small molecule immune checkpoint inhibitors show higher tissue penetration, good biosafety, and better potential to inhibit tumor growth and migration. Despite this, in the field of immune checkpoint blockade, the development of small molecule inhibitors lags far behind therapeutic antibodies, with only a few PD-1/PD-L1 small molecule inhibitors undergoing clinical trials. There are currently no reports of small molecule Tim-3 inhibitors with cell activity. Therefore, identifying functional small molecule Tim-3 inhibitors to improve tumor immunotherapy has extremely strong clinical translational significance.

Summary of the invention

In view of the deficiencies in the above-mentioned prior art, the present invention provides an application of a small molecule compound targeting Tim-3 in tumor immunotherapy. The present invention studies and screens a series of 3,5-disubstituted-3a,6a-dihydrospiro[furan[3,4-c]pyrrole-1,2'-indene]-1',3',4,6(3H,5H)-tetraketone small molecule compounds that promote the anti-tumor function of immune cells by targeting Tim-3, as well as a composition containing the above-mentioned compounds and its application in promoting the anti-tumor function of immune cells. Based on the above research results, the present invention is completed.

To achieve the above object, the present invention adopts the following technical solutions:

The first aspect of the present invention provides the use of a compound in the preparation of a Tim-3 inhibitor;

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

in,

_R1 can be: _C3 - _C6 cycloalkyl, a benzene ring having 0 to 2 substitutions, a benzyl having 0 to 2 substitutions, a six-membered heterocyclic ring having 0 to 2 substitutions, a five-membered heterocyclic ring having 0 to 2 substitutions, and the substituents are each independently selected from _C1 - _C2 alkyl, halogen, hydroxyl, methoxy, amino, methylamino, cyano, nitro, halomethyl, formamide, carboxyl, and ester groups;

_R2 can be: _C3 - _C6 cycloalkyl, a benzene ring having 0 to 2 substitutions, a six-membered heterocyclic ring having 0 to 2 substitutions, a five-membered heterocyclic ring having 0 to 2 substitutions, and the substituents are each independently selected from _C1 - _C2 alkyl, halogen, hydroxyl, methoxy, amino, methylamino, cyano, nitro, halomethyl, formamide, carboxyl, and ester groups;

m can be selected from an integer of 0, 1 or 2, R ₃ is independently C ₁ -C ₂ alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxyl, ester at each occurrence;

The compound may be in the form of a racemate or a single chiral configuration.

According to the preferred embodiment of the present invention,

_R1 can be: a benzene ring with 0 to 2 substitutions, a benzyl group with 0 to 2 substitutions, wherein the substituents are each independently selected from _C1 - _C2 alkyl, halogen, hydroxyl, methoxy, amino, methylamino, cyano, nitro, halomethyl, formamide, carboxyl, and ester groups;

R ₂ can be: a benzene ring with 0 to 2 substituents, wherein the substituents are each independently selected from C ₁ -C ₂ alkyl, halogen, hydroxyl, methoxy, amino, methylamino, cyano, nitro, halomethyl, formamide, carboxyl, and ester groups;

m can be selected from an integer of 0, 1 or 2, R ₃ is independently C ₁ -C ₂ alkyl, halogen, hydroxy, methoxy, amino, methylamino, cyano, nitro, halomethyl, carboxyl, ester at each occurrence;

The compound is in racemic or single chiral configuration.

It should be noted that, regarding substituents, the term "independently" in the present invention means that when there may be more than one substituent, the substituents may be the same as or different from each other.

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

Furthermore, the compound may also include its pharmaceutically acceptable salts, isotope derivatives, solvates, or stereoisomers, geometric isomers, tautomers, or prodrug molecules, metabolites.

According to the understanding of those skilled in the art, the pharmaceutically acceptable salts include alkali metal salt forms of the above compounds (specific examples include sodium salts or potassium salts), or salts formed by the compounds and inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid or hydrobromic acid, and salts formed by organic acids such as methanesulfonic acid, toluenesulfonic acid or trifluoroacetic acid. The term "pharmaceutically acceptable" or "pharmaceutically acceptable" used interchangeably therewith, for example, when describing "pharmaceutically acceptable salts", means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance with pharmaceutical use value, such as a salt formed as an intermediate when performing chiral resolution, although the salt of this intermediate cannot be directly administered to the subject, the salt can play a role in obtaining the final product of the present invention.

It should be noted that the above compounds of the present invention can not only be used as drugs, but also can obviously be used as experimental reagents, so as to be used in basic research related to the Tim-3 signaling pathway.

The second aspect of the present invention provides the above compounds for use in the preparation of drugs for preventing and/or treating tumors.

According to the present invention, the concept of "prevention and/or treatment" refers to any measure applicable to the treatment of tumor-related diseases, or preventive treatment of such diseases or symptoms, or avoiding the recurrence of such diseases, such as recurrence after the end of the treatment period or treating the symptoms of the disease that has already occurred, or pre-intervention to prevent, inhibit or reduce the occurrence of such diseases or symptoms.

It should be noted that tumor is used in the present invention as known to those skilled in the art, and includes benign tumors and/or malignant tumors. Benign tumors are defined as excessive proliferation of cells that cannot form aggressive, metastatic tumors in the body. Conversely, malignant tumors are defined as cells with multiple cellular abnormalities and biochemical abnormalities that can form systemic diseases (e.g., tumor metastasis in distant organs).

In another specific embodiment of the present invention, medicine of the present invention can be used for treating malignant tumor.The example of malignant tumor of available medicine therapy of the present invention comprises solid tumor and hematological tumor.Solid tumor can be the tumor of breast, bladder, bone, brain, central and peripheral nervous system, colon, endocrine gland (such as thyroid and adrenal cortex), esophagus, endometrium, germ cell, head and neck, liver, lung, larynx and hypopharynx, mesothelioma, ovary, pancreas, prostate, rectum, kidney, small intestine, soft tissue, testis, stomach, skin (such as melanoma), ureter, vagina and vulva.Malignant tumor comprises hereditary cancer, for example retinoblastoma and nephroblastoma (Wilms tumor).In addition, malignant tumor comprises primary tumor in described organ and corresponding secondary tumor (tumor metastasis) in distal organ. Hematological tumors can be, for example, aggressive and indolent forms of leukemia and lymphoma, i.e., non-Hodgkin's disease, chronic and acute myeloid leukemia (CML/AML), acute lymphocytic leukemia (ALL), Hodgkin's disease, multiple myeloma, and T-cell lymphoma. Also included are myelodysplastic syndromes, plasmacytomas, tumoroid syndromes, and cancers of unknown primary site and AIDS-related malignancies.

The third aspect of the present invention provides a composition, which at least comprises the above compound.

The fourth aspect of the present invention provides a pharmaceutical preparation comprising at least the above compound and at least one pharmaceutically acceptable excipient and/or carrier.

The excipients of the present invention refer to ingredients other than the active ingredients in the composition or pharmaceutical preparation, which are non-toxic to the subject. Commonly used excipients in the art include buffers, stabilizers, preservatives or excipients, and commonly used excipients include binders, fillers, wetting agents, disintegrants, etc.

As an example, the excipients that can be used in the preparation of the present invention include but are not limited to: the excipient is selected from calcium phosphate, magnesium stearate, talc, dextrin, starch, gel cellulose, methyl cellulose, carboxymethyl cellulose sodium salt and polyvinyl pyrrolidone.

The drug carrier of the present invention can be a pharmaceutically acceptable solvent, suspending agent, vesicle, nanomaterial, etc., which is used to deliver the compound described in the first aspect of the present invention to an animal or human body. The carrier can be liquid or solid and is selected according to the planned administration method. In addition, proteins and liposomes are also drug carriers.

Those skilled in the art can prepare the compounds of the present invention into compositions or pharmaceutical preparations using known techniques. For example, any compound (at least one compound) disclosed in the above-mentioned first aspect of the present invention is mixed with a pharmaceutical excipient, and then, if necessary, the resulting mixture is formed into a desired shape. Except for those mentioned in the present invention, pharmaceutical preparations can also be prepared according to books related to modern pharmaceutical preparations. And, except for those mentioned in the present invention, suitable pharmaceutical excipients are known in the art, for example, see the 2005 edition of the Handbook of Pharmaceutical Excipients (original fourth edition), which will not be repeated here.

The fifth aspect of the present invention provides the use of the above composition or pharmaceutical preparation in any one or more of the following:

(a) inhibiting the Tim-3 signaling pathway or preparing an inhibitor of the Tim-3 signaling pathway;

(b) promoting immune cell function and preparing products that promote immune cell function;

(c) treating diseases related to the Tim-3 signaling pathway or preparing products for treating diseases related to the Tim-3 signaling pathway;

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

More specifically, the promotion of immune cell function includes:

(b1) improving the survival rate and effector function of primary CD8+ cytotoxic T lymphocytes and human chimeric antigen receptor T cells, including but not limited to the function of clearing tumor cells or viruses;

(b2) Promoting the killing activity of NK cells and the antigen presenting ability of DC, including but not limited to promoting the clearance of tumor cells or viruses.

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

Among them, the tumor has been explained in detail in the second aspect and will not be repeated here.

The viral infectious diseases include, but are not limited to, respiratory 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-borne viral diseases and slow virus infectious diseases.

More specifically, the respiratory viral diseases include, but are not limited to, infections by rhinovirus, adenovirus, respiratory syncytial virus, parainfluenza virus and coronavirus; as well as influenza and mumps, etc.

The gastrointestinal viral diseases include, but are not limited to, viral gastroenteritis, specifically, rotavirus gastroenteritis, Norwalk virus gastroenteritis, adenovirus gastroenteritis, astrovirus gastroenteritis, coronavirus gastroenteritis and calicivirus gastroenteritis, etc.

The viral liver diseases include, but are not limited to, hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E and Epstein-Barr virus hepatitis.

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

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

The central nervous system viral diseases include epidemic encephalitis B, western equine encephalitis, eastern equine encephalitis, St. Louis encephalitis, Venezuelan equine encephalitis, Murray Valley encephalitis, California encephalitis, forest encephalitis and lymphocytic choriomeningitis, etc.

The lymphocytic viral diseases include infectious mononucleosis, cytomegalovirus infection and acquired immunodeficiency syndrome, etc.

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

The lentivirus-infected diseases include, but are not limited to, subacute sclerosing panencephalitis, kuru, progressive multifocal leukoencephalopathy, and subacute spongiform encephalopathy.

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

Among them, the organ-specific autoimmune diseases include but are not limited to chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, etc.

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-mentioned composition or pharmaceutical preparation of the present invention has the following applications:

(c1) inhibiting tumor progression or preparing products that inhibit tumor progression;

(c2) synergistically enhancing the anti-tumor response induced by PD-1 blockade or preparing a product that synergistically enhances the anti-tumor response induced by PD-1 blockade;

(c3) Tumor immunotherapy or products for use in tumor immunotherapy.

Of course, the above-mentioned products can be drugs or experimental reagents, and the experimental reagents can be used for basic research, thereby being used for basic research related to the Tim-3 signaling pathway.

Beneficial technical effects of the above technical solution:

The above technical scheme first proposed the role of 3,5-disubstituted-3a,6a-dihydrospiro[furan[3,4-c]pyrrole-1,2'-indene]-1',3',4,6(3H,5H)-tetraketone compounds or their pharmaceutically acceptable salts, isotope derivatives, solvates, or their stereoisomers, geometric isomers, tautomers, or their prodrug molecules and metabolites in the efficient and highly selective inhibition of the Tim-3 signaling pathway. Specifically, the present invention screened out a series of small molecule compounds from the SPECS library by virtual screening. It was verified by experiments that such compounds significantly improved the survival rate and anti-tumor activity of primary CD8+ cytotoxic T lymphocytes and human chimeric antigen receptor T cells, significantly promoted the killing activity of NK cells and DC antigen presentation ability, and showed an inhibitory effect on tumors comparable to Tim-3 blocking antibodies, effectively inhibiting tumor progression and synergistically enhancing the anti-tumor response induced by PD-1 blockade. Therefore, this type of compound can be used to prepare drugs or pharmaceutical compositions for preventing and/or treating tumors, and has good potential development and application value.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

Figure 1 shows the functional screening and identification of the small molecule compound ML-T7 targeting Tim-3;

Among them: A is the functional experimental process using OT-ⅠCD8 ⁺ CTL; B is the effect of 17 candidate small molecule compounds on the proliferation of CTL; C is the flow cytometry detection of CD8 ⁺ CTL cytokine secretion after pretreatment with 17 candidate small molecule compounds; D is the fluorescence quantitative detection of IFN-γ, TNF-α and IL-2 mRNA expression levels in CD8 ⁺ CTL pretreated with DMSO and ML-T7; E is the killing ability of CD8 ⁺ CTL pretreated with DMSO and ML-T7 against EL4 tumors incubated with 2nM OVA _257-264 ; F is the in vivo killing ability of CD8 ⁺ CTL pretreated with DMSO and ML-T7 against spleen cells incubated with 10nM OVA _257-264 .

FIG2 shows that ML-T7 binds to Tim-3 and blocks the interaction between Tim-3 and PtdSer and CEACAM1;

Among them: A is the SPR analysis of the affinity between ML-T7 and purified Tim-3 protein; B is the MST method to detect the affinity between ML-T7 and GFP-Tim-3 fusion protein in the cell supernatant (n=3); C is the RMSD value of ML-T7-hTim3, ML-T7-mTim3 and PtdSer-hTim-3 complexes analyzed by dynamic simulation; D is the MM/GBSA calculation of the binding affinity of ML-T7/PtdSer and Tim-3; E is a schematic diagram of the dynamic simulation prediction of the binding of ML-T7 to the FG-CC' groove of the hTim-3IgV domain; F is a two-dimensional (2D) schematic diagram of the binding mode of the ML-T7-hTim3 complex, highlighting the hydrogen bonds and The main hydrophobic interaction; G is the purified Tim-3 protein incubated with ML-T7 or anti-Tim-3 antibody, and then dexamethasone-treated thymocytes were added, and then flow cytometry was used to analyze the binding of Tim-3 to apoptotic thymocytes (n=3). As the concentration of ML-T7 increased, the binding of Tim-3 to apoptotic thymocytes was blocked; H is the purified Tim-3 protein incubated with ML-T7 or anti-Tim-3 antibody, and then Jurkat-CEACAM1 cells were added, and flow cytometry was used to analyze the cell binding of Tim-3 to Jurkat-CEACAM1 (n=3). As the concentration of ML-T7 increased, the binding of Tim-3 to Jurkat-CEACAM1 was blocked.

FIG3 shows that ML-T7 enhances TCR/STAT5 signaling and promotes the anti-tumor activity of CD8 ⁺ T cells through Tim-3;

Among them: A is the flow cytometry detection of the levels of IFN-γ, TNF-α, IL-2, Perforin and Granzyme B produced by WT and Tim-3 KO CTLs pretreated with DMSO and ML-T7; B is the ability of WT and Tim-3 KO CTLs pretreated with DMSO and ML-T7 to kill EL4 tumors incubated with 2nMOVA _257-264 ; C is the flow cytometry detection of the apoptosis level of WT and Tim-3 KO CTLs pretreated with DMSO and ML-T7; D is the flow cytometry detection of the proliferation level of WT and Tim-3 KO CTLs pretreated with DMSO and ML-T7; E is the flow cytometry detection of the expression of PD-1 and CTLA-4 levels of WT and Tim-3 KO CTLs pretreated with DMSO and ML-T7; F and G are the GESA analysis results of WT and Tim-3 KO CTLs pretreated with DMSO and ML-T7; H is the results of 2μg/mL After 15 minutes of CD3/CD28 antibody stimulation, the phosphorylation levels of the corresponding proteins were detected by Western blot; I is a schematic diagram of the treatment of B16-MO5 subcutaneous tumor mice by CTL infusion pretreated with DMSO and ML-T7; J is the detection of tumor growth curve; K is the recording of the survival of tumor-bearing mice; L is a schematic diagram of the treatment of B16-MO5-Fluc lung metastasis mice by CTL infusion pretreated with DMSO and ML-T7; M is the detection of luciferase activity in the mouse lungs; N is the recording of the survival of tumor-bearing mice; O is a schematic diagram of the treatment of B16-MO5-Fluc lung metastasis mice by Tim-3KO CTL infusion pretreated with DMSO and ML-T7; P is the detection of luciferase activity in the mouse lungs; Q is the recording of the survival of tumor-bearing mice.

FIG4 shows that ML-T7 directly enhances the survival and effector function of CD8 ⁺ CTLs;

Among them: A is a schematic diagram of the process of infusing OT-ⅠCD8 ⁺ CTLs pretreated with DMSO (CD45.1 ⁺ ) or ML-T7 (CD45.1 ⁺ CD45.2 ⁺ ) into B16-MO5 tumor-bearing mice at a ratio of 1:1; B is the proportion of CTLs pretreated with DMSO or ML-T7 in tumors monitored by flow cytometry on days 7 and 14; C is the proportion of Annexin V+CTLs in tumors detected by flow cytometry; D is the proportion of Ki67 ⁺ CTLs in spleen and lymph nodes detected by flow cytometry; E is the proportion of CD25+ and CD69+CTLs in tumors detected by flow cytometry; F is the proportion of CD25 ⁺ CTLs in spleen and lymph nodes detected by flow cytometry; G is the ability of CTLs in tumors, spleen and lymph nodes to secrete IFN-γ, TNF-α and IL-2 by flow cytometry; H is the expression levels of PD-1 and CTLA-4 on CTLs in tumors detected by flow cytometry.

Figure 5 shows that ML-T7 can enhance the effector function of NK cells and DCs;

Among them: A is the expression level of IFN-γ, TNF-α, CD107a and granzyme B in NK92 cells pretreated with DMSO and ML-T7 by flow cytometry; 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 DCs pretreated with DMSO and ML-T7; D is the activation level of CTL stimulated by DCs pretreated with DMSO and ML-T7; E is the cytokine secretion ability of CTL stimulated by DCs pretreated with DMSO and ML-T7.

FIG6 shows that ML-T7 exhibits anti-tumor activity in HCC models of wild-type and humanized mice after intraperitoneal administration;

Among them: A is a schematic diagram of the in situ HCC model induced by Akt/cMyc treated with intraperitoneal injection of different doses of ML-T7 (DMSO as control, once every 2 days) or Tim-3 antibody (IgG as control, 3 times a week); B and C are in vivo imaging to detect the luciferase activity in the mouse liver to monitor tumor progression; D is the mouse survival curve; E is the flow cytometry detection of the percentage and number of CD8 ⁺ T cells in the tumor; F is the flow cytometry detection of the Ki67 level of tumor-infiltrating CD8 ⁺ T cells; G is the flow cytometry detection of the IFN-γ, TNF-α and IL-2 secretion capacity of tumor-infiltrating CD8 ^{+ T cells; H is the flow cytometry detection of the PD-1 expression level of tumor-infiltrating CD8+ T} cells; I is the flow cytometry detection of the IFN-γ and TNF-α expression levels of tumor-infiltrating NK cells; J is the flow cytometry detection of the CD40 and CD86 expression levels of DCs in the tumor; K is the establishment of the HCC model in Tim-3 humanized mice and the intraperitoneal injection of ML-T7; L is the bioluminescence imaging to monitor tumor growth; M is the mouse survival curve.

FIG7 shows that intraperitoneal administration of ML-T7 promotes anti-tumor immune response in HCC mouse model;

Among them: A is the body weight change curve of HCC mice after drug treatment; B is the liver weight to body weight ratio of HCC mice; C is the AST and ALT levels of HCC mice; D is the percentage and number of CD8 ⁺ T cells in the spleen detected by flow cytometry; E is the Ki67 level of spleen CD8 ⁺ T cells detected by flow cytometry; F is the IFN-γ, TNF-α and IL-2 secretion capacity of spleen CD8 ⁺ T cells detected by flow cytometry; G is the PD-1 expression level of spleen CD8 ⁺ T cells detected by flow cytometry; H is the IFN-γ and TNF-α expression levels of spleen NK cells detected by flow cytometry; I is the CD40 and CD86 expression levels of spleen DC detected by flow cytometry; J is the percentage and number of MDSC in tumor detected by flow cytometry; K is the percentage and number of MDSC in spleen detected by flow cytometry; L is the percentage and number of Treg in tumor detected by flow cytometry; M is the percentage and number of Treg in spleen detected by flow cytometry.

FIG8 shows that intraperitoneal administration of ML-T7 exhibits anti-tumor activity in a mouse melanoma model;

Among them: A is a schematic diagram of the B16F10 subcutaneous tumor model treated with intraperitoneal injection of 50 mg/kg ML-T7 (DMSO as control, once every 2 days); B is the mouse tumor growth curve and tumor photos; C is the ratio of tumor and spleen CD8 ⁺ T cells detected by flow cytometry; D is the IFN-γ, TNF-α and IL-2 secretion capacity of tumor-infiltrating CD8 ⁺ T cells detected by flow cytometry; E is the PD-1 and TOX expression of tumor-infiltrating CD8 ⁺ T cells detected by flow cytometry; F is the CD25 and CD69 expression of tumor-infiltrating CD8 ⁺ T cells detected by flow cytometry; G is the ratio of NK cells in the tumor detected by flow cytometry; H is the ability of NK cells in the tumor to secrete IFN-γ and TNF-α.

FIG9 shows that ML-T7 treatment enhances the anti-tumor activity of human CAR T cells;

Among them: A is the flow cytometry detection of the killing ability of 19BBz CAR T cells pretreated with DMSO and ML-T7 on Namalwa cells; B is the flow cytometry detection of the apoptosis and proliferation of 19BBz CAR T cells pretreated with DMSO and ML-T7; C is the fluorescence quantitative detection of the mRNA expression levels of IFN-γ, TNF-α and IL-2 in 19BBz CAR T cells pretreated with DMSO and ML-T7; D is the co-culture of CAR T cells and Namalwa cells at an E:T ratio of 1:1, 2:1 and 4:1 for 18 hours, and the ELISA method is used to detect the content of IFN-γ, TNF-α and IL-2 in the supernatant; E is a schematic diagram of the adoptive transfer of 19BBz CAR-T cells treated with DMSO or ML-T7 into Namalva-Fluc tumor-bearing B-NDG mice; F and G are the bioluminescence imaging to monitor tumor growth; H is the survival curve of tumor-bearing mice; I is the flow cytometry detection of CD3 ⁺ CAR in the peripheral blood of tumor-bearing B-NDG mice T cell ratio; J is the ratio of CD8 ⁺ CAR T cells in the peripheral blood of tumor-bearing B-NDG mice detected by flow cytometry; K is the ratio of CD8 ⁺ CAR T cells apoptosis in the peripheral blood of tumor-bearing B-NDG mice detected by flow cytometry; L is the expression level of IFN-γ and TNF-α of CD8 ⁺ CAR T cells in tumor-bearing B-NDG mice detected by flow cytometry.

FIG10 shows that ML-T7 treatment and PD-1 antibody treatment have synergistic effects in HCC models;

Among them: A is a schematic diagram of the Akt/cMyc-induced HCC model treated with ML-T7 (DMSO as control, once every 2 days) and PD-1 antibody (IgG as control, 3 times a week) alone or in combination; B is the liver weight to body weight ratio of HCC mice; C and D are the evaluation of tumor growth using bioluminescence imaging; E is the survival curve of HCC mice; F is the serum AST and ATL levels of HCC mice; G is HE staining and Ki67 immunohistochemical staining of the liver of HCC mice.

FIG11 shows that ML-T7 synergizes with PD-1 antibody therapy to promote tumor immune response in vivo;

Among them: A is the percentage and number of CD45 ⁺ T cells in the tumor detected by flow cytometry; B is the percentage and number of CD8 ⁺ T cells in the tumor detected by flow cytometry; C is the Ki67 level of tumor-infiltrating CD8 ⁺ T cells detected by flow cytometry; D is the percentage and number of spleen CD8 ⁺ T cells detected by flow cytometry; E is the Ki67 level of spleen CD8 ⁺ T cells detected by flow cytometry; F is the IFN-γ, TNF-α and IL-2 secretion capacity of spleen CD8 ⁺ T cells detected by flow cytometry; G is the IFN-γ and TNF-α secretion capacity of tumor-infiltrating CD8 ⁺ T cells detected by flow cytometry; H is the IFN-γ and TNF-α expression levels of tumor-infiltrating NK cells detected by flow cytometry; I is the IFN-γ and TNF-α expression levels of spleen NK cells detected by flow cytometry; J is the percentage and number of MDSC cells in the tumor detected by flow cytometry; K is the percentage and number of MDSC cells in the spleen detected by flow cytometry; L is the percentage and number of Treg in the tumor detected by flow cytometry.

FIG12 shows that ML-T7 exhibits good biosafety in mice;

Among them: A is the weight change of C57BL/6 mice injected intraperitoneally with 50 mg/kg ML-T7 or DMSO every day; B is the weight of the heart, spleen and kidney; C is the HE section of the heart, liver, spleen, lung and kidney; D is the serum AST and ALT levels; E is the colon length; F is the complete blood count of red blood cells (RBC) and white blood cells (WBC) in hematological analysis; G is the complete blood count of platelets, lymphocytes, monocytes, neutrophils and basophils in hematological analysis; H is the inhibitory effect of ML-T7 on hERG potassium channel; the compound has a low inhibitory effect on hERG, and the current inhibition rate of ML-T7 at 30 μM is only 29.04%.

Figure 13 shows the prediction of ML-T7 toxicity by Derek software;

FIG14 shows that ML-T7 analog compounds have the effect of enhancing NK effector function;

Among them: A is the ability of NK92 cell line to kill K562 target cells after treatment with ML-T7 similar compounds; B is the flow cytometry detection of CD107 expression in NK92 cells; C is the flow cytometry detection of TNF-α secretion level in NK92 cells; D is the flow cytometry detection of IFN-γ secretion level in NK92 cells.

DETAILED DESCRIPTION

It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and/or combinations thereof.

The present invention is further described in conjunction with the examples. It should be noted that the following description is only for the purpose of explaining the present invention and does not limit its content. The compounds used in the examples (including ML-T7 and its series of similar compounds ML-T7S1 to ML-T7S17) were purchased from specs company in the Netherlands and are all existing compounds in the compound library of specs company.

Example

Experimental methods

1. Experimental mice

OT-Ⅰ transgenic mice (CD45.1 or CD45.1.2, C57BL/6J background) express H-2Kb/OVA _{257 -264} -specific TCR. C57BL/6J (CD45.2) mice and Tim-3 humanized mice (Tim-3-hu) were purchased from Jicui Pharmaceutical Co., Ltd. (Nanjing, Jiangsu, China). Tim-3 knockout (KO) mice (C57BL/6J background) were constructed by Shanghai Sidansai Biotechnology Co., Ltd., China, and hybridized with OT-Ⅰ mice to obtain OT-ⅠTim-3 KO mice. Mice were housed in the Experimental Animal Center of Shandong University under a constant room temperature (20-24°C) and specific pathogen-free conditions with a routine 12 h light/12 h dark schedule. All mice were used at 8-12 weeks of age unless otherwise stated. All animal experiments were performed in strict accordance with ethical standards and approved by the Animal Care and Use Committee of Shandong University.

2. Induction of OT-ICTL

Take the spleen of a 6-8 week old male OT-Ⅰ mouse in a sterile environment, grind it and resuspend it in culture medium, and pay attention to adding culture medium while grinding. Centrifuge the cell suspension obtained in the previous step at 1200r/5min, discard the supernatant, add 5ml red blood cell lysis buffer, and lyse it on ice or in a refrigerator at 4℃ for 10min. Centrifuge at 1200r/5min, discard the supernatant, resuspend the cells in culture medium, resuspend in 5ml culture medium (RPMI 1640+10% FBS+50μMβ-mercaptoethanol+double antibody) after centrifugation, count, and plate at a density of 3x10 ⁶ /ml. At the same time, add OVA _257-264 to stimulate the cells at a concentration of 10nm. After 3 days of stimulation, centrifuge and change the medium. Continue to culture for 2 days to obtain CTLs.

3. Flow cytometry detection of cytokine secretion

(1) Take enough EL4 cells, incubate with 2 nm, 200 pm, and 20 pm OVA _257-264 antigen for 1 h, and wash once with PBS;

(2) Count the CTL cells and EL4 cells to make the number of CTL cells and EL4 cells 5x10 ⁵ and 2.5x10 ⁵ , respectively. The final volume is 500 μl. Add EL4 to CTL and Golgi inhibitor and mix well.

(3) Mix CTL cells and EL4 and incubate at 37°C for 4-6 hours;

(4) Collect cells, centrifuge at 1000 r/5 min, discard the supernatant, and resuspend the cells in 50 μl PBS;

(5) Prepare CD8 antibody diluent (0.25 μl antibody per sample), add 5 μl antibody diluent to each sample, add 50 μl cells to the antibody diluent, and mix well; mix the remaining cells together as a blank control and stain in a 4°C refrigerator for 45 min;

(6) Resuspend the cells in 500 μl PBS + 5 mM EDTA, centrifuge at 1000 r/5 min, discard the supernatant, add 100 μl 2% paraformaldehyde fixative, and fix in a 4°C refrigerator for 30 min;

(7) Resuspend the cells in 500 μl of transmembrane buffer, centrifuge at 1000 r/5 min, discard the supernatant, and resuspend the cells in 50 μl of transmembrane buffer;

(8) Prepare TNF-α (PE), IFN-γ (APC), IL-2 (FITC) antibody diluents (0.25 μl antibody per sample), add 5 μl antibody diluent to each sample, add 50 μl cells to the antibody diluent, mix well, and stain in a 4°C refrigerator for 45 min;

(9) Resuspend the cells in 500 μl of transmembrane buffer, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

(10) Resuspend the cells in 500 μl PBS, centrifuge at 1000 rpm for 5 min, and discard the supernatant;

(11) Resuspend the cells in 300 μl PBS, filter, and then analyze on a flow cytometer.

4. Surface plasmon resonance (SPR) experiment

SPR binding experiments were performed using a Biacore T200 biosensor system (GE Healthcare) at 25°C. All buffers were purchased from GE Healthcare. A CM5 microsensor chip (GE Healthcare) was equilibrated with HEPES buffer (10 mM HEPES, 150 mM NaCl, CaCl ₂ , pH 7.4) until a stable signal was achieved. According to the instrument manual, his-tagged human Tim-3 protein (Cat# TM-H5229, ACROBiosystems Group, Beijing, China) was diluted in 10 mM sodium acetate buffer (pH = 4.0) and immobilized on the CM5 chip using an amine coupling kit. The chip was then equilibrated with running buffer to stabilize the signal. ML-T7 was diluted 2-fold in HEPES buffer starting from a maximum concentration of 25 μM for 6 points. ML-T7 was serially diluted in HEPES buffer at a flow rate of 30 μL/min for 90 s binding and 90 s dissociation. Data were analyzed using Biacore T200 software, and the equilibrium constant (Kd) was calculated using a 1:1 kinetic fitting model.

5. Microscale thermophoresis (MST)

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

6. Determination of Tim-3 binding using apoptotic thymocytes or Jurkat-CEACAM1

C57BL/6J mouse thymocytes were labeled with 0.1 mM Calcein-AM (ThermoFisher), and then treated with 1 mM dexamethasone (Sigma) in DMEM containing 10% FBS at 37°C for 6 h to induce cell apoptosis. Annexin V (BD Pharmingen) staining confirmed cell apoptosis. Recombinant human Tim-3 protein (Sanyou Bio) was incubated with the specified concentration of ML-T7 or DMSO at room temperature for 30 min, then added to apoptotic thymocytes or human T lymphoblastoid cell line overexpressing CEACAM1 (Jukrat-Ceacam1) and stained with anti-human Tim-3 APC antibody (BioLegend). Tim-3 binding was detected by flow cytometry.

7. Adoptive CTL transfer therapy in mouse melanoma model

A subcutaneous melanoma model was established in mice. Wild-type C57BL/6J mice were subcutaneously inoculated with 2×10 ⁵ B16-MO5 melanoma cells on day 0. Tumor-bearing mice were intravenously injected with 10 μM ML-T7 or 4×10 ⁶ DMSO-treated OT-ⅠCD8 ⁺ CTLs when tumors were palpable on day 10. Tumors were measured with a digital caliper every 2 to 3 days and expressed as tumor volume (calculated as width ² ×length/2). Based on ethical considerations, mice with tumors >20 mm in size were euthanized by CO ₂ inhalation. Survival rates were recorded daily.

The lung metastasis model was established by intravenously injecting 2×10 ⁵ B16-MO5 melanoma cells expressing firefly luciferase (B15-MO5-Fluc) into wild-type C57BL/6J mice on day 0 to establish the lung metastasis model. Tumor-bearing mice were intravenously injected with OT-ⅠCD8+CTL (4×10 ⁶ ) treated with ML-T7 or DMSO on day 10. Bioluminescence imaging was performed using the IVIS Spectrum system (PerkinElmer) to monitor lung metastases. The luciferase activity level was expressed as p/sec/cm ² /sr. The survival rate was recorded every day.

8. Preparation of tumor mouse model

Mouse melanoma model: 5-6 week old male C57BL/6 mice were subcutaneously injected with 2x10 ⁵ B16-MO5 cells. After tumors grew (approximately 6 days), the short diameter (a) and long diameter (b) of the tumors were measured with a vernier caliper every other day, and the tumor volume V=a ² b/2 was calculated; the time of mouse death was counted, and the mouse survival curve was drawn.

Hydrodynamic injection of Akt/cMyc plasmid to induce hepatocellular carcinoma (HCC) model: cMyc, Akt/Fluc gene plasmids and SB100 plasmid encoding Sleeping Beauty transposase were extracted according to the steps of the plasmid extraction kit. The cMyc, Akt gene plasmids and SB100 plasmids were added to a certain volume (10% of body weight, for example, 20g body weight uses 2ml PBS) of 0.9% sodium chloride solution at a ratio of 12μg:12μg:1μg per mouse, vortexed and mixed, and filtered through a 0.22μm microporous membrane. The prepared plasmids were injected into 8-week-old wild-type male C57BL6 mice through the mouse tail vein hydrodynamic high-pressure injection method. Hepatocellular carcinoma can be seen in the mouse liver 3 to 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. Lenti-X 293T cells were transfected with lentiviral 19BBz CAR expression plasmids together with psPAX2 (Addgene, #12260) and pMD2G (Addgene, #12259) using Lentifit (Han Bio, China) transfection reagent. The lentivirus-containing supernatant was collected at 48 and 72 h after transfection, concentrated by ultracentrifugation, and filtered with a 0.22 μm filter. Primary human T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors, activated with anti-CD3/CD28 beads for 2 days in TexMACS GMP medium (Miltenyi Biotec) supplemented with 10% FBS and 50 IU/mL IL-2 at a ratio of 1:1 (Miltenyi Biotec, Bergisch Gladbach, Germany), and then centrifuged at 32°C to transduce the concentrated supernatant containing human 19BBz CAR lentiviral particles for 2 hours. The transduced cells were passaged daily for 5 days with fresh culture medium containing 50 IU/mL IL-2 at a ratio of 1:2. The transfection efficiency was determined by detecting the GFP expression of the CAR plasmid by flow cytometry.

After expansion, CAR T cells were treated with 10 μM ML-T7 or DMSO for 48 h. ML-T7 or DMSO-treated CAR T cells were co-cultured with Namalwa target cells at a ratio of 1:1, 2:1, and 4:1 for 18 h. Cell supernatants were collected and ELISA kits (Dakewe) were used to detect IL-2, IFN-γ, and TNF-α levels. Flow cytometry was used to detect the cytotoxicity of CAR T cells based on the ratio of Dil-labeled target cells to CAR T cells labeled with anti-human CD19-APC antibody (cat#561742, BD).

10. CAR T-cell therapy

5 × 10 ⁵ Namalwa cells expressing firefly luciferase (Namalwa-Fluc) in 200 μL PBS were injected into B-NDG (NOD-Prkdcscid IL2rgtm1) mice via the tail vein and randomly divided into three groups on days 5 and 10 to receive PBS, 3 × 10 ⁶ DMSO-treated 19BBz CAR-T cells, and 3 × 10 ⁶ ML-T7-treated 19BBz CAR-T cells, respectively. Tumor progression was monitored by bioluminescence imaging using the IVIS Spectrum system (PerkinElmer) on days 10 and 16, and survival was recorded daily. PBMCs were isolated from recipient mice, stained with the indicated antibodies, and analyzed using flow cytometry.

11. Isolation and stimulation of tumor infiltrating lymphocytes

The mouse tumor tissue (subcutaneous tumor or liver cancer) was peeled off, crushed in a mortar, filtered, and the cell fluid was collected. Centrifuged at 600rpm for 1min, the supernatant was poured into a clean 50ml centrifuge tube, centrifuged at 1200rpm for 5min, and after discarding the supernatant, 8ml of Percoll working solution was added, and centrifuged at 2000rpm for 20min. After discarding the upper layer, 5ml of red blood cell lysis solution was added, lysed on ice for 10min, centrifuged at 1200rpm for 5min, the supernatant was discarded, and 2ml of PBS was added. A small amount of cells were taken for CD8 ⁺ T cell counting. After centrifugation of the remaining cells, 1640 complete medium was added, and PMA (200ng/ml) and ionomycin (1μm) were stimulated for 4-6h, and the cells were harvested for flow cytometry antibody staining. Golgi inhibitors were added 4h before harvesting the cells.

12. Flow cytometry analysis

Surface staining: Cells were labeled with surface markers for 30 min at 4°C in the dark. For intracellular staining, cells were fixed with IC Fixation Buffer (cat#00-8222-49, eBioscience), permeabilized with Permeabilization Buffer (cat#00-8333-56, eBioscience), and stained with the indicated antibodies. For transcription factor staining, cells were fixed and permeabilized with Fixation/Permeabilization Concentrate and Dilution Buffer (cat#00-5521, eBioscience). Samples were run using a CytoFLEX S flow cytometer (Beckman Coulter), and data were analyzed using the CytExpert program (Beckman Coulter) and FlowJo software.

Experimental Results

1. Functional screening identified the small molecule compound ML-T7 that promotes CTL functional cells

In order to find small molecule inhibitors of Tim-3, we adopted a strategy of virtual screening combined with functional screening. Considering that the amino acid homology of Tim-3 between humans and mice exceeds 63%, and the structure of the PtdSer binding site is highly conserved, we used the complex formed by mouse Tim-3 (mTim-3) and PtdSer (PDB: 3KAA) as a template to build a homology model of human Tim-3 (hTim-3). Using this model, 204,380 compounds in the SPECS library were virtually screened with the PtdSer binding site as the binding pocket, and a total of 192 compounds with a predicted affinity greater than -7.4 Kcal/mol to the target were identified (Figure 1A). Then, these compounds were further screened by multiple strategies such as structural similarity clustering, physicochemical property prediction, and binding mode analysis. Finally, 17 small molecule compounds were selected, named ML-T1 to ML-T17, for subsequent CD8 ⁺ CTL functional screening.

We incubated splenocytes from OT-Ⅰ T cell receptor (TCR) transgenic mice (a mouse model widely used to evaluate CD8 ⁺ T cell antitumor immunity) with ovalbumin peptide (257-264) (SIINFEKL, OVA _257-264 ) in the presence of ML-T1 to ML-T17 (10 μM concentration) (Figure 1B). ML-T7 had no significant effect on cell viability (Figure 1C) and significantly enhanced the production of IFN-γ, TNF-α, and IL-2 in OT-Ⅰ cells at the mRNA and protein levels (Figures 1D and E). OT-Ⅰ CD8 ⁺ CTLs pretreated with ML-T7 showed enhanced ability to kill target cells both in vitro and in vivo (Figures 1F and G).

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

To determine whether ML-T7 directly binds to Tim-3 and functions as a Tim-3 inhibitor, we performed surface plasmon resonance (SPR) experiments using commercially available his-tagged hTim-3 protein. As shown in Figure 2A, ML-T7 bound to hTim-3 with a KD value of 6.98 μM, indicating that there is a moderate degree of interaction between ML-T7 and hTim-3. To verify this interaction, we performed microscale thermophoresis (MST) experiments using cell supernatants containing hTim-3 fused to green fluorescent protein (GFP-hTim-3) or control GFP. Only the GFP-hTim-3 cell supernatant produced a binding curve with ML-T7 (Figure 2B), further demonstrating that ML-T7 can bind to hTim-3.

Molecular dynamics simulation studies further supported the interaction between ML-T7 and hTim-3. As shown in Figure 2C, the results of the position root mean square deviation (RMSD) analysis showed that both mTim-3 and hTim-3 were stably bound to ML-T7, with only slight fluctuations compared to the original conformation. Molecular mechanics calculated by MM/GBSA showed that the affinity of ML-T7 for both hTim-3 and mTim-3 was higher than the affinity of PtdSer for Tim-3, which means that ML-T7 can effectively interrupt the interaction between PtdSer and Tim-3 (Figure 2D). Detailed analysis of representative conformations from the molecular dynamics results showed that ML-T7 forms extensive interactions with residues of the 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 neighboring residues Val31, Phe32, Cys34, Arg82, Gln84, and Ile88 (Fig. 2E and F).

Crystal structure studies have shown that the FG-CC' loop is the binding site for PtdSer. To test the ability of ML-T7 to block the binding of Tim-3 to PtdSer, mouse thymocytes were induced to apoptosis 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 cytometric analysis showed that ML-T7 also blocked the binding of hTim-3 to CEACAM1-overexpressing Jurkat cells in a concentration-dependent manner (Figure 2H), which is consistent with the view that PtdSer and CEACAM1 bind to the same pocket with Tim-3.

3. ML-T7 enhances TCR/STAT5 signaling and promotes CD8 ⁺ T cell anti-tumor activity through Tim-3

To confirm that ML-T7 promotes CD8 ⁺ T cell function as a Tim-3 inhibitor, we crossed Tim-3 knockout (Tim-3 KO) mice with OT-Ⅰ mice to generate Tim-3 knockout (Tim-3 KO) TCR-specific CD8 ⁺ CTLs. As expected, ML-T7 significantly promoted the production of IFN-γ, TNF-α, IL-2, perforin, and granzyme B in wild-type (WT) CTLs, but not in Tim-3 KO CTLs (Figure 3A). In addition, Tim-3 knockout abolished the effect of ML-T7 on enhancing the ability of CTLs to kill tumor cells in vitro (Figure 3B). We also found that ML-T7 treatment reduced CTL apoptosis and increased proliferation capacity, while ML-T7 had no or minimal effect on apoptosis and cell proliferation of Tim-3 KO CTLs (Figures 3C and D). Meanwhile, ML-T7 treatment suppressed the expression of inhibitory receptors PD-1 and CTLA-4 in WT cells, but had no effect in Tim-3 KO CTLs, indicating that ML-T7 treatment could inhibit T cell exhaustion (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 CTLs (Fig. 3F and G). Correspondingly, ML-T7 treatment increased the phosphorylation levels of PLCγ1, ZAP70, LCK, ERK1/2, and STAT5 after anti-CD3/CD28 antibody stimulation. Notably, ML-T7 did not affect TCR and STAT5 signals in Tim-3 KO OT-ⅠCD8 ⁺ CTLs (Fig. 3H). The above results indicate that ML-T7 enhances the TCR and STAT5 signaling pathways of CD8 ⁺ CTLs through Tim-3.

To further verify whether ML-T7 can enhance the antitumor function of CTL in vivo, we adoptively transferred ML-T7-treated or control OT-ⅠCD8 ⁺ CTL into B16-MO5 melanoma-bearing mice (Figure 3I). Encouragingly, the infusion of ML-T7-pretreated CTL significantly inhibited tumor progression and prolonged the survival of recipient mice (Figures 3J and 3K). Similar results were obtained in the B16-MO5 (B16-MO5-Fluc) lung metastasis tumor model mice carrying firefly luciferase (Figure 3L). Compared with control mice, recipient mice receiving ML-T7-treated OT-ⅠCD8 ⁺ CTL showed lower tumor burden in the lung (Figure 3M) and longer survival time (Figure 3N). Importantly, when we transferred ML-T7-treated or DMSO-treated Tim-3 KO CD8 ⁺ CTL into B16-MO5-Fluc lung metastasis mice (Figure 3O), no difference in tumor progression and survival time was found between the two groups (Figures 3P and Q). Therefore, the above experiments indicate that ML-T7 regulates CTL anti-tumor function through Tim-3.

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

To verify whether ML-T7 endogenously promotes the effector function of CTL, we infused equal amounts of control and ML-T7-treated CTLs back into B16-MO5 subcutaneous tumor model mice (Figure 4A). Flow cytometry results showed that ML-T7 treatment significantly promoted the aggregation and persistence of CTLs in tumors (Figure 4B). Consistent with this, ML-T7 treatment reduced CTL apoptosis in tumors and increased proliferation (Figure 4C). We also observed an increase in the proliferation of ML-T7-treated CTLs in the spleen and lymph nodes (Figure 4D). In addition, in the tumors, spleens, and lymph nodes of recipient mice, ML-T7-treated CD8 ⁺ CTLs not only expressed higher activation molecules CD25 and CD69 (Figures 4E and F), but also produced more IFN-γ, TNF-α, and IL-2 (Figure 4G). We also observed that the expression of PD-1 and CTLA-4 was significantly reduced in ML-T7-treated CTLs that infiltrated tumors, suggesting that they were less exhausted (Figure 4H). Therefore, ML-T7 can directly prolong the survival time of CD8+CTL and enhance its effector function.

5. ML-T7 enhances the function of NK cells and DCs

Considering that Tim-3 is abundantly expressed 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 crucial for tumor immunotherapy. Therefore, we examined the effect of ML-T7 on NK cell function. Flow cytometry analysis showed that 10 μM ML-T7 treatment significantly enhanced the production of effector molecules IFN-γ, TNF-α, CD107a, and granzyme B in NK92 cells, a human NK cell line (Figure 5A). Compared with control NK92 cells, the cytotoxic activity of ML-T7-treated NK92 cells against K562-Fluc cells was significantly increased at different E:T ratios (Figure 5B).

To detect the regulatory effect of ML-T7 on DC function, we cultured bone marrow cells with granulocyte-macrophage stimulating colony factor (GM-CSF) and interleukin-4 (IL-4) for 5 days, and then added 1μg/mL LPS and 10μM ML-T7 on the 6th day for 24h to prepare mature DC (mDC). Compared with DMSO, ML-T7 treatment increased the expression of CD11c, CD80, CD86 and MHCII on the surface of DC (Figure 5C), indicating that DC maturation was enhanced and antigen presentation ability was enhanced. Further functional experiments showed that T cells activated by ML-T7-treated DC were more strongly activated and had stronger effector function (Figure 5D and E). In summary, our results show that ML-T7 treatment can promote DC maturation and the anti-tumor function of NK cells.

6. Intraperitoneal administration of ML-T7 showed anti-tumor activity in both mouse HCC liver cancer models and humanized mice

To evaluate whether ML-T7 can be used as a direct tumor immunotherapy, we treated the spontaneous orthotopic hepatocellular carcinoma (HCC) mouse model driven by the oncogene Akt/Myc, which expresses firefly luciferase to indicate tumor burden, with different doses of ML-T7 administered intraperitoneally. To compare the effects of ML-T7 and Tim-3 blocking antibodies, we included a commercial Tim-3 antibody (α-Tim-3) as a control (Figure 6A). There was no significant difference in the body weight of mice during treatment with different doses of ML-T7 (Figure 7A). Strikingly, the results of in vivo imaging showed that ML-T7 at treatment doses of 20 mg/kg and 50 mg/kg could significantly inhibit tumor growth (Figure 6B and C). Consistently, the liver weight to body weight ratio and AST and ALT levels were also significantly reduced in the 20 mg/kg and 50 mg/kg treatment groups (Figure 7B and C). Notably, the 20 mg/kg dose of ML-T7 and α-Tim-3 antibodies achieved comparable levels of tumor inhibition and survival extension (Fig. 6C and D, Fig. 7B).

Further flow cytometry analysis showed that, similar to the effect of α-Tim-3 antibody treatment, systemic administration of ML-T7 at doses of 20 mg/kg and 50 mg/kg caused a significant increase in the proportion and number of CD8 ⁺ T cells in both tumors and spleens (Figures 6E and 7D), and a significant increase in Ki67-positive CD8 ⁺ T cells (Figures 6F and 7E) was observed, suggesting stronger proliferation. In addition, ML-T7 enhanced the production of effector molecules such as IFN-γ, TNF-α, and IL-2 in tumor-infiltrating and splenic CD8 ⁺ T cells (Figures 6G and 7F), and reduced the expression of PD-1 in CD8 ⁺ T cells, suggesting the inhibition of T cell exhaustion (Figures 6H and 7G). In addition, ML-T7 administration not only promoted the levels of IFN-γ and TNF-α secretion by tumor and splenic NK cells (Figures 6I and 7H), but also promoted the infiltration and maturation of DCs in tumors (Figures 6J and 7I). In addition, ML-T7 treatment reduced the infiltration of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) (Figure 7J to M), suggesting that ML-T7 alleviated the tumor immunosuppressive microenvironment. The above data show that, similar to the α-Tim-3 antibody, ML-T7 promotes CTL, NK cell and DC functions, thereby inhibiting the immunosuppressive tumor microenvironment and exerting tumor immunotherapy.

To further verify the therapeutic efficiency of ML-T7, an orthotopic Akt/Myc-HCC model was induced in humanized Tim-3 (Tim-3-HU) knock-in mice (the extracellular domain of Tim-3 in this mouse was replaced by the extracellular domain of human Tim-3), followed by systemic treatment with 20 mg/kg of ML-T7 (Figure 6K). As expected, ML-T7 greatly inhibited tumor growth, as shown by weaker luciferase activity and prolonged survival of mice (Figure 6L and M). These results indicate that ML-T7 is also an effective candidate drug for tumor therapy targeting human Tim-3.

7. Intraperitoneal administration of ML-T7 showed anti-tumor activity in a mouse melanoma model

To evaluate whether ML-T7 can treat melanoma, we treated the B16F10 melanoma model with 50 mg/kg of ML-T7 by intraperitoneal injection (8A). Treatment with 50 mg/kg ML-T7 significantly inhibited the growth of melanoma (Figure 8B). Flow cytometric analysis showed that 50 mg/kg of ML-T7 significantly increased the proportion of CD8 ⁺ T cells in both tumors and spleens (Figure 8C) and enhanced the production of effector molecules such as IFN-γ, TNF-α, and IL-2 in tumor-infiltrating CD8 ⁺ T cells (Figure 8D). Importantly, ML-T7 significantly reduced the expression of PD-1 and TOX in CD8 ⁺ T cells, reversed T cell exhaustion (reduced TOX ⁺ TCF1 ^- ratio) (Figure 8E), and promoted the expression of T cell activation molecules CD25 and CD69 (Figure 8F). In addition, ML-T7 administration also promoted the proportion of tumor-infiltrating NK cells and the levels of secretion of IFN-γ and TNF-α (Figures 8G and H)

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

CAR T cell therapy has achieved great clinical success in hematological malignancies. Since ML-T7 can bind to Tim-3 and enhance the antitumor efficacy of mouse CTL in a Tim-3-dependent manner, we wanted to determine whether ML-T7 can also enhance the therapeutic effect of human CAR T cells. To this end, we used lentivirus to transfect 19BBz CAR into T cells of human peripheral blood mononuclear cells (PBMCs), constructed CD19-targeted CAR T cells, and treated them with DMSO or ML-T7. Consistent with the results of mouse CTL, ML-T7 treatment in vitro significantly enhanced the cell killing ability of CAR T cells against Namalwa, a human B lymphoblastoid cell line expressing CD19 (Figure 9A). We also found that ML-T7 treatment reduced the apoptosis of CAR T cells and increased the proliferation of CAR T cells (Figure 9B). At the same time, ML-T7-treated CAR T cells increased the production of IFN-γ, TNF-α, and IL-2 at the protein and mRNA levels (Figures 9C and D). In addition, ML-T7-treated 19BBzCAR T cells showed significantly enhanced antitumor activity in B-NDG mice bearing Namalwa-Fluc tumors compared with DMSO-treated 19BBz CAR T cells. Adoptive transfer of ML-T7-treated 19BBz CAR T cells greatly induced tumor regression and prolonged mouse survival (Figures 9E to H). In addition, more CD3+ and CD8 ⁺ CAR T cells were detected in the peripheral blood of mice receiving ML-T7-pretreated CAR T cells compared with mice receiving control CAR T cells ^{(Figures 9I and J). Compared with control CAR T cells, the infused ML-T7-treated CD8+ CAR} T cells showed less apoptosis (Figure 9K) and higher IFN-γ and TNF-α secretion levels (Figure 9L), which is consistent with the data of ML-T7-treated mouse CTLs.

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

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

Previous studies have reported that combined blockade of Tim-3 and PD-1 can synergistically improve antitumor responses, and we further explored whether ML-T7 can synergize with PD-1 antibody in tumor immunotherapy in the AKT/Myc HCC tumor model (Figure 10A). According to in vivo imaging, survival curves, liver weight measurement, ALT/AST evaluation, hematoxylin-eosin (HE) staining, and Ki67 staining, 20 mg/kg of ML-T7 monotherapy showed almost similar antitumor activity to 100 mg/kg of PD-1 monoclonal antibody (Figure 10B to G). Most importantly, the combination of ML-T7 and PD-1 antibody showed stronger antitumor activity than either treatment alone (Figure 7B to E, 50% of mice (3/6) receiving combined treatment survived on day 120, while 0 or 1/6 mice receiving ML-T7 or PD-1 antibody monotherapy survived to day 120 (Figure 10E). These results indicate that ML-T7 combination therapy can significantly enhance the antitumor efficacy of PD-1 antibody.

Next, we further explored the effects of ML-T7 and PD-1 antibody treatment alone or in combination on immune cells in HCC tumor models. Similar to PD-1 antibody treatment alone, ML-T7 treatment alone or in combination with PD-1 antibody treatment significantly promoted the infiltration of CD45 ⁺ immune cells in tumors (Figure 11A), increased the proportion, number, and proliferation of CD8 ⁺ T cells in tumors and spleens (Figures 11B to E), and significantly enhanced the ability of CD8 ⁺ T cells in tumors and spleens to produce cytokines (IFN-γ, TNF-α, and IL-2) (Figures 11F and G). Consistent with the negative regulatory effects of Tim-3 and PD-1 on NK cell function, we also observed that both ML-T7 and PD-1 antibody treatment strongly increased the production of IFN-γ and TNF-α by NK cells (Figures 11H and I), indicating enhanced NK tumor killing activity. Notably, ML-T7 therapy showed great synergy with PD-1 antibody therapy in restoring NK cell viability and inhibiting MDSCs and Treg infiltration (Figure 11J to L).

In summary, ML-T7 not only showed potent antitumor activity in vivo, but also synergized with PD-1 antibody treatment to alleviate the suppressive immune environment in the tumor microenvironment and reactivated the antitumor response of CD8 ⁺ T cells and NK cells.

10. ML-T7 shows good biosafety in mice

In order to determine the biosafety of ML-T7 and further explore its prospects for clinical transformation. We intraperitoneally injected C57BL/6 mice with 50 mg/kg ML-T7 every day for 2 weeks. ML-T7 treatment did not cause any abnormal changes in body weight or clinical symptoms during the experiment (Figure 12A). At autopsy, ML-T7-treated mice did not show any significant differences in the weight of the heart, spleen, and kidneys (Figure 12B). Further histological analysis of the heart, liver, spleen, lungs, and kidneys by HE staining showed that there were no obvious abnormalities in ML-T7-treated mice (Figure 12C). In addition, serum biochemical tests showed that ALT and AST levels were normal after ML-T7 treatment, indicating that 50 mg/kg ML-T7 had no obvious hepatotoxicity to mice (Figure 12D). At the same time, ML-T7 did not affect colon length (Figure 12E). In addition, hematological evaluation did not show signs of anemia, significant leukopenia, or leukocytosis in ML-T7-treated mice (Figure 12F). We did not observe significant changes in platelet, lymphocyte, monocyte, neutrophil and basophil counts (Figure 12G). We also tested the hERG inhibition of the compound, which is a complication of severe tachyarrhythmias. The results showed that ML-T7 did not significantly inhibit the hERG potassium channel with an acceptable IC50>30μM (Figure 12H). The toxicity assessment of ML-T7 using the well-known toxicity prediction software Derek also supported the safety of ML-T7 in 56 test items (Figure 13).

In summary, ML-T7 was well tolerated in mice and did not cause obvious adverse reactions, suggesting that ML-T7 is a safe and effective anti-tumor small molecule drug that deserves further development for clinical transformation.

11. ML-T7 series of similar compounds have the effect of enhancing the anti-tumor activity of NK cells

The present invention further tested the ability of more ML-T7-like compounds to promote NK92 cells to kill tumor cells and secrete effector factors IFN-γ, TNFα and CD107a. The results showed that this type of compound can significantly promote the killing ability of NK92 cells and the ability to secrete effector cytokines (Figure 14), suggesting that this type of compound has the immunotherapy potential of inhibiting Tim-3 similar to MT-L7.

In summary, the present invention successfully discovered a small molecule Tim-3 inhibitor, which can enhance the adoptive transfer therapeutic effect of CTL or CAR T cells, as well as the function of NK cells and DCs, and directly exert anti-tumor activity and inhibit tumor progression in preclinical tumor models. 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 used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims (4)

1. Use of the compound in the preparation of drugs for preventing and/or treating tumors; Wherein, the structural formula of the compound is as follows: The tumors include liver cancer and melanoma. 2. The use according to claim 1, characterized in that the compound also includes a pharmaceutically acceptable salt thereof. 3. Use of a composition in the preparation of a drug for synergistically enhancing the anti-tumor response induced by PD-1 blockade, the composition comprising at least the compound of any one of claims 1 to 2; The tumor is liver cancer. 4. Use of a pharmaceutical preparation in the preparation of a drug for synergistically enhancing the anti-tumor response induced by PD-1 blockade, comprising at least the compound of any one of claims 1 to 2; The tumor is liver cancer.