CN112716957A - Application of CDK7 targeted inhibitor in preparation of medicine for treating cytokine release syndrome caused by CAR-T treatment - Google Patents

Abstract

The invention claims an application of a CDK7 targeted inhibitor in preparation of a medicine for treating CAR-T-induced cytokine release syndrome, and the invention verifies through biochemical and animal model experiments that: (1) under a proper concentration, the CDK7 targeted inhibitor THZ1 can specifically regulate and control an inflammation signal pathway of immune cells such as macrophages, T cells, endothelial cells and the like by inhibiting the expression of super enhancers and transcription factors related to inflammatory factors such as stat1, IL-1, IL-6 and the like; (2) can obviously reduce the death of mice caused by acute inflammatory storm caused by CAR-T treatment, and reduce the functional failure caused by inflammation of related organs, and no obvious toxic or side effect is found in the period. Is of great significance in the treatment of CAR-T therapy causing cytokine release syndrome.

Description

Application of CDK7 targeted inhibitor in preparation of medicine for treating cytokine release syndrome caused by CAR-T treatment

Technical Field

The invention relates to the technical field of medicines, in particular to application of a CDK7 targeted inhibitor in preparation of a medicine for treating cytokine release syndrome caused by CAR-T treatment.

Background

Immunotherapy (immunotherapy) refers to a therapeutic method for artificially enhancing or suppressing the immune function of the body to treat diseases in response to a low or high immune state of the body. Immunotherapy of tumors aims to activate the human immune system, relying on autoimmune functions to kill cancer cells and tumor tissues. Among them, the immune checkpoint therapies represented by PD-L1, PD-1, CTLA4 and the like, and the cellular Immunotherapy represented by CAR-T therapy (Chimeric Antigen receptor T-Cell Immunotherapy) bring new hopes to tumor patients. But immunotherapy also has related side effects, especially the occurrence of Cytokine storm or Cytokine Release Syndrome (CRS), which seriously brings life danger to patients.

Cytokine Release Syndrome (CRS), also known as "Cytokine storm", is a phenomenon in which a large amount of cytokines are rapidly produced in the body after the immune system of the body is strongly stimulated (such as by virus, bacteria, immune intervention therapy, and the like), and is a severe inflammatory response syndrome. Among them, immunotherapy, such as CAR-T therapy, is accompanied by a certain percentage of patients with cytokine release syndrome, especially in critically ill patients.

The CAR-T technology is characterized in that genetic materials with specific antigen recognition domains and T cell activation signals are transferred into T cells through a gene modification technology, the T cells are directly combined with specific antigens on the surfaces of tumor cells to be activated, the tumor cells are directly killed by releasing perforin, granzyme B and the like, endogenous immune cells of a human body are recruited to kill the tumor cells by releasing cytokines, the purpose of treating tumors is achieved, immune memory T cells can be formed, and a specific anti-tumor long-acting mechanism is obtained. In the above process, macrophage, neutrophil and other immune cells release cytokines to recruit phase immune cells (macrophages, granulocytes, DC cells, T cells, B cells) to the site of infection, and the cytokines further activate these cells, stimulating them to produce more cytokines. Typically, this positive feedback loop is controllable by the body itself. However, in cytokine storms, a large number of immune cells are activated and secrete more cytokines, which recruit to a larger number of immune cells, creating an uncontrolled cascade of amplification effects. Therefore, CRS is a disease that the over-activation of immune system causes multiple cytokines to be over-expressed in tissues and organs, and finally causes single-organ or multiple-organ damage and death due to exhaustion of function. FIG. 1A shows a graph of the pattern of inflammatory storms, CRS, which can cause a variety of diseases (including infection, acute respiratory distress syndrome ARDS, sepsis, acute pancreatitis, rheumatic diseases, etc.). Severe CRS, which causes systemic inflammatory reaction of the host, may cause damage to various tissues and organs, causing multiple organ failure and even death.

During the formation of the cytokine storm, macrophages play a key role, being the engine of the cytokine storm, and also being the amplifier of the cytokine storm (see fig. 1B). Two documents were published in 5 months 2020, Nature Medicine, and the mechanism of the onset of cytokine storm induced by CAR-T therapy was revealed. We have previously thought that the production of CRS was due to massive CAR-T cell reinfusion. However, these two recent studies have taught us that what causes the cytokine storm is actually macrophages in humans, and in the same case of CAR-T cells, the probability of CRS occurring is greatly increased when macrophages are present near the tumor; when we inhibited macrophage activity, the probability of CRS occurrence was greatly reduced, indicating that macrophages are an important source of inflammatory factor storm in CAR-T therapy.

At present, the treatment means for CRS caused by CAR-T therapy mainly comprises the following aspects: (1) IL-6 receptor antagonists: IL-6 is mainly secreted by macrophages, and has pleiotropic activity, and becomes a biomarker for judging the severity of a cytokine storm and a prognostic index, so that the function of blocking IL-6 becomes a key strategy for inhibiting CRS. Clinically, the Touzumab is used as an inhibitor to block key cell factors of inflammatory storm induced by new coronavirus infection, so that the damage of inflammatory reaction to lung tissues and multiple organs of a patient is effectively reduced. It should be noted that IL-6 is only one of many inflammatory cytokines in cytokine storms, and IL-1, a chemokine, is expressed higher in some kinds of cytokine storms. Therefore, this method has certain limitations; (2) glucocorticoids: in clinic, nonspecific combination treatment measures such as anti-infective drugs, glucocorticoids, nutritional support and the like are mostly adopted for the cytokine storm. Among them, glucocorticoids are commonly used clinically because of their potent anti-inflammatory effects, but glucocorticoids have large side effects. The clinical CRS treatments described above have different limitations or side effects. Therefore, the key problem to be solved urgently is to deeply research the relevant molecular action mechanism, develop safe and efficient targeted drugs, more effectively treat CRS and simultaneously reduce side effects.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention is realized by the following technical scheme:

use of a targeted inhibitor of CDK7 in the preparation of a medicament for the treatment of cytokine release syndrome caused by CAR-T therapy;

further, the targeted inhibitor of CDK7 is THZ1, BS-181 or SY-1365; further preferably, the targeted inhibitor of CDK7 is THZ 1.

Drawings

FIG. 1 is a graph of the cytokine storm formation pattern triggered by CAR-T therapy

FIG. 2 is a schematic representation of the CDK7 regulatory pattern and the structural formula of compound THZ1

FIG. 3 shows the anti-inflammatory molecular mechanism experiment results of THZ1, SY-1365 and BS-181

FIG. 4 is a schematic diagram of THZ1 targeting CDK 7-RNAPOSymerase II complex regulation gene transcription and a molecular signaling pathway western diagram

FIG. 5 is a schematic diagram of the construction of CAR-T complex and related models in CAR-T inflammatory storm model

FIG. 6 is an experiment of THZ1 targeting CDK7-RNA Polymerase II complex to inhibit CAR-T-Raji-Macrophage acute inflammation model

FIG. 7 is an experiment that THZ1 targeting CDK 7-RNAPOSymease II complex does not inhibit the proliferation of CAR-T cells and the clearance of CAR-T cells from lymphoma Raji cells

FIG. 8 is a diagram of whole genome expression profiling of THZ1 targeting CDK 7-RNAPOSymerase II complex

FIG. 9 shows the results of analysis of THZ1 targeting CDK 7-RNAPOSymerase II complex genome-wide CHIP-Seq (RNA Poly II, H3K27AC)

FIG. 10 level experiment of animal experiments with THZ1 targeting CDK7-RNA Polymerase II complex to inhibit CAR-T-Raji-Macrophage-induced inflammatory storm

FIG. 11 immunofluorescence assay of tumor and mouse survival experiments

Advantageous effects

The invention is proved by experiments that:

targeted inhibitors of CDK7 specifically inhibited the acute inflammatory response elicited in CAR-T therapy, and in particular THZ1 was best. The biochemical and animal model experiments prove that: (1) under a proper concentration, the CDK7 targeted inhibitor THZ1 can specifically regulate and control inflammatory signal pathways of immune cells such as macrophages and monocytes, and the like, and can inhibit the expression of inflammatory factors such as IL-1, IL-6, stat1, and the like and related super enhancers and transcription factors; (2) THZ1 can remarkably kill mice caused by acute inflammatory storm caused by CAR-T treatment, reduce function failure caused by inflammation of related organs, and has no obvious toxic or side effect.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

The targeted inhibitor THZ1 of CDK7 is a covalent inhibitor of targeted CDK7 developed by Syros, wherein THZ1 is irreversibly covalently bound with cysteine residues (Cys312) outside a kinase domain of CDK7 protein kinase (see FIG. 2, A shows a pattern diagram that CDK7 regulates DNA transcription into mRNA and CDK7 regulates cell cycle progression; B shows a structural formula of a compound THZ1 of a formula I; and C shows a schematic diagram that THZ1 is irreversibly covalently bound with cysteine residues (Cys312) outside a kinase domain of CDK7 protein kinase), so that phosphorylation of 2 nd, 5 th and 7 th serine residues of RNA polymerase II is prevented, activity of RNA polymerase II is inhibited, and transcription of mRNA is inhibited.

The CAS No. of THZ1 is 1604810-83-4, and the chemical name is: (E) -N- (3- ((5-chloro-4- (1H-indole-3-yl) pyrimidin-2-yl) amino) phenyl) -4- (4- (dimethylamino) but-2-enamido) benzamide with the molecular formula of C₃₁H₂₈ClN₇O₂The structural formula is shown as formula I:

BS-181(CAS No.:1397219-81-6) is a highly selective CDK7 inhibitor, IC₅₀21nmol/L, more than 40 times more selective than CDK1, 2, 4, 6 and 9. In human gastric cancer cell BGC-823, BS-181 significantly reduces the activity of CDK7, and simultaneously down-regulates the level of anti-apoptotic proteins (cyclinD1, XIAP and Bcl-2), thereby further inhibiting the proliferation, invasion and migration of gastric cancer cells.

SY-1365(CAS No.:1816989-16-8) is a highly selective CDK7 inhibitor that inhibits cell growth in vitro for a number of different cancer types. SY-1365 treatment reduced MCL1 protein levels, cancer cells with low expression of BCL2L1(BCL-XL) were more sensitive to SY-1365, SY-1365 showed significant anti-tumor effects as a single drug in multiple AML xenograft models; SY-1365-induced growth inhibition was enhanced in combination with the BCL2 inhibitor venetoclax. Antitumor activity was also observed in the ovarian cancer xenograft model, indicating that SY-1365 has potential application prospects in hematology and clinical studies of solid tumors. Our findings support CDK7 as a novel approach to the treatment of transcription dependent cancers.

Example 1: anti-inflammatory molecular mechanism experiment of THZ1, SY-1365 and BS-181

Monocytes (THP-1, human monocyte cell line) in exponential growth phase are treated with 100ng/ml PMA for 24 hours to form macrophages (mTHP-1, human monocyte induced macrophages), then 30nM THZ1, 30nM SY-1365 and 10 μ M BS-181 are respectively used for treating a 500ng/ml LPS induced inflammation model, cells are collected and relevant protein western detection is carried out, and the results are shown in figure 3, wherein THZ1, SY-1365 and BS-181 have the effect of inhibiting LPS induced inflammation (shown in figure 3A), and THZ1-CDK7, SY-1365-CDK7 and BS-181-CDK7 complexes have certain inhibition effect on the serine phosphorylation at RNAPoly II 2 and 7 sites (shown in figure 3B); in conclusion, THZ1 is the most effective anti-inflammatory agent.

Example 2: THZ1 Targeted CDK7-RNApolymerase II complex regulated gene transcription and molecular signaling pathway western experiment

Monocytes in exponential growth phase were treated with 100ng/ml PMA for 24 hours to form macrophages, then treated with different concentrations of THZ1(0, 20, 40, 100, 200nM) for 30 minutes, then sampled at different time points (8, 24 hours), the cells collected and subjected to western detection of the relevant proteins. FIG. 4 is a schematic diagram of THZ1 targeting CDK 7-RNAPOSymerase II complex regulating gene transcription. Wherein, figure 4A shows a schematic representation of THZ1 targeted regulation of the mRNA transcription complex RNAPoly of CDK 7; FIG. 4B shows the inhibition of serine phosphorylation at RNAPoly II 2,7 site by the THZ1-CDK7 complex in western experiments at different concentrations of THZ1 and different action times: over time, the inhibitory effect diminishes; the inhibitory effect increases with increasing concentration.

Example 3 construction of CAR-T Complex and CAR-T inflammation storm model experiment

FIG. 5A shows a CD19 scFv-4-1BB-CD3 zeta model map of CAR lentivirus; FIG. 5B shows Raji growth pictures of tumor cell line lymphoma cells in CAR-T inflammatory storm model; FIG. 5C shows the culture construction scheme of CAR-T cells: peripheral blood of healthy volunteers was isolated by Ficoll method and cultured by gradient time adherence, and then activated by 2U/ml of CD3, CD28 antibody for proliferation of T cells for 2 days, then transfected with CAR disease and stimulated by IL-17, IL-15 for growth to day 13 for subsequent related experiments.

Example 4: experiment of THZ1 targeting CDK7-RNA Polymerase II complex to inhibit CAR-T-lymphoma cell (Raji) -Macrophage (Macrophage) acute inflammation model

FIG. 6 reflects experiments in which THZ1 targets the CDK7-RNA Polymerase II complex to inhibit the CAR-T-Raji acute inflammation model. Wherein, A shows a flow schematic diagram of the whole experiment, and the experimental process is as follows: taking supernatant obtained after the CAR-T cells and the Raji cells are co-cultured for 24 hours and co-culturing macrophages (mTHP-1 or primary monoclonal-derived macrophages (MDMs)) induced by peripheral blood mononuclear cells of healthy volunteers for 24 hours, and then detecting the content of inflammatory factors in a macrophage culture medium through a multi-factor flow quantitative detection experiment; or culturing CAR-T, lymphoma cells Raji and macrophages in a simulated in vivo environment together, and detecting the content of inflammatory factors in macrophage culture medium by a multi-factor flow quantitative detection experiment, wherein the NCT is normal control T cell and has no specific CD19 molecule targeting lymphoma, and the CAR-T is chimeric antigen receptor T cell; b1 and B2 showed that CAR-T co-cultured with Raji, and that CAR-T significantly released large amounts of cytokines during Raji clearance (IL-1. beta., IL-6, etc.); B3-B5 show that CAR-T, lymphoma cells (Raji) and macrophages (macrophage) are co-cultured, and a large amount of cytokines (IL-1 beta, IL-6 and the like) are remarkably released; C1-C3 show that when CAR-T and Raji are cultured together, a large amount of cytokines (IL-1 beta, IL-6 and the like) are released into a culture medium in the process of clearing Raji, the cytokines in the culture medium can more remarkably activate macrophages (mTHP-1) at the center of an inflammatory storm and continuously release huge amounts of inflammatory factors (IL-6, TNF-alpha, IFN-gamma) to the outside, and THZ1(30nM) can remarkably inhibit the inflammatory factors released by the macrophages: the results of using MDMs in panels D1-D3 are similar to those of panels C1-C3, indicating that THZ1(30nM) can also significantly inhibit the release of peripheral blood monocyte-derived macrophage inflammatory factor in healthy volunteers.

Example 5 experiments that THZ1 targeting CDK 7-RNAPOSymease II Complex did not inhibit the proliferation of CAR-T cells and the clearance of lymphoma Raji cells by CAR-T cells

FIG. 7 shows that THZ1 targets CDK7-RNA Polymerase II complex in inhibiting cytokine storm of CAR-T-Raji-macrophages without affecting CAR-T proliferation and functioning to target tumor cell clearance. Among them, FIG. 7A shows that 30nM THZ1 did not affect the clearance of CAR-T cells from lymphoma Raji cells, and there was no significant difference, compared to the control group; FIG. 7B shows that by measuring the proliferation of cells treated with 30nM THZ1 for 24,48 and 72 hours, respectively, through CFDA SE assay, no significant difference in proliferation rate was observed between the THZ1 group and the control group at each time point, indicating that the THZ1 treatment at 30nM did not affect the proliferation of CAR-T cells.

Example 6: whole genome expression profiling of THZ 1-targeted CDK 7-RNApolymease II complex

Peripheral blood from healthy volunteers was isolated by Ficoll method and gradient time adherence, cultured monocytes were isolated and then treated with 100ng/ml PMA for 24 hours to form macrophages (MDMs), then treated with 30nM THZ1 for 30 minutes and then treated with 500ng/ml LPS for 8 hours, sampled, RNA extracted and RNA transcriptome sequenced. FIG. 8 is the results of genome-wide expression profiling of THZ1 targeting CDK 7-RNAPOSymerase II complex, A: results of mRNA sequencing analysis showed that the gene expression of the relevant inflammatory factors was significantly activated by LPS (in the inflammation model, the expression of the relevant inflammatory genes was up-regulated, and when THZ1 was added to the group, the gene expression of the relevant inflammatory factors was suppressed), and the results showed that the expression of the relevant genes was significantly activated by LPS (as shown in FIG. 8A, the expression of the inflammatory genes of the different groups was significantly suppressed in LPS/Control, but after the addition of THZ1, the up-regulated genes were significantly suppressed, and the expression of the THZ1/LPS group was reduced compared to the corresponding group; B: the differentially expressed genes were mainly enriched in the RNAPOLY II signaling pathway and the inflammatory stress Control signaling pathway by GO enrichment analysis; C: the inhibition of the acute inflammatory response by THZ1 was found by whole genome differential expression analysis comparison, by CCL8,15,5,2,7,4,1, CXCL9,10,11,23,8), cytokines (IL12B, 27,7,23A,1B,1A,6, TNF, CD70, etc.), receptors (CCL15,2, etc.), etc., thereby achieving inhibition of cytokine storm.

Example 7: analysis of THZ1 targeting CDK 7-RNApolymease II Complex Whole genome CHIP-Seq (RNApoly II, H3K27AC)

Monocytes were treated with 100ng/ml PMA for 24 hours to form macrophages, then treated with 0 or 30nM THZ1 for 30 minutes, then co-treated with 0 or 500ng/ml LPS for 8 hours, crosslinked with 1% formaldehyde at room temperature for 10 minutes, quenched with 0.125M glycine for 5 minutes, and then harvested by scraping. The collected samples were subjected to DNA fragmentation and enrichment of related antibody-bound DNA fragments as required by the Kit (SimpleChIP Enzymatic protein IP Kit, CST). then, the Library was constructed with the Kit (ChIP-seq Library materials with construction of NEBNext Ultra II DNA Library Prep Kit for Illumina, NEB), and finally, sequencing and analysis were performed. FIG. 9 shows the results of analysis of THZ1 targeting CDK 7-RNAPOSymerase II complex genome-wide CHIP-Seq (RNAPOLY II, H3K27AC), and FIG. 9A1 shows that THZ1 targets inhibition of CDK7 binding to RNAPLY II, and the comparison of abundance of enriched DNA fragments is described as a whole, and the comparison shows that the abundance of DNA bound by LPS-treated group and RNAPLY II antibody is increased, indicating that more genes are being transcribed; after THZ1 treatment, the abundance of enriched DNA was reduced, indicating that the gene being transcribed was reduced; FIG. 9A2 shows that DNA is overall at the statistical level of transcription initiation and transcription extension; FIG. 9B1 shows that THZ1 inhibits the binding of RNApoly II to inflammatory transcriptional regulators STAT1, IRF1, inhibiting the expression of the genes involved; FIG. 9B2 shows that THZ1 has significant inhibitory activity on the super enhancers STAT1, STAT4 upstream of the inflammatory factors IL- α, IL-1 β, as analyzed by CHIP-Seq data on the super enhancer marker molecule H3K27 ac; FIG. 9B3 reflects that the super enhancer sequence that regulates the expression of IL-1a, IL-1B genes, a cell inflammatory factor, is also inhibited by THZ1 small molecule compounds. This suggests that THZ1 targets CDK 7-RNAPILE II complex, inhibits the binding ability of inflammatory related genes and super enhancers upstream of inflammatory related transcription factor genes, and directly inhibits the binding of RNAPILE II with inflammatory related factor gene sequences and transcription factor gene sequences, inhibits the expression of genes, and the above-mentioned composite action, thereby changing the expression of transcription regulatory factors, and finally influencing the expression of inflammatory cytokines, thereby specifically regulating the production of cytokines and inhibiting the occurrence of inflammatory storms.

Example 8: animal level experiments with THZ1 targeting CDK 7-RNAPOSymerase II complex

FIG. 10 is an animal level experiment of THZ1 targeting CDK7-RNA Polymerase II complex to inhibit CAR-T-Raji-Macrophage induced inflammatory storm, which reflects THZ1 targeting complex, specifically inhibits the release of inflammatory cytokines and reduces organ damage in the process of removing tumor Raji cells by CAR-T cells. Among them, fig. 10A: mouse CAR-T-Raji inflammatory storm model and THZ1 dosing schematic: the experimental process comprises the following steps: mice were given an intraperitoneal injection of THZ110mg/kg, 0.5h followed by LPS40 mg/kg, and then the mice were sacrificed 20 hours for the following analyses: (1) serum was taken for cytokine analysis, (2) peritoneal lavage: cell analysis, (3) organ toxicity assessment; b: in the CAR-T treatment process, the detection of the level inflammation indexes of mice of different groups (the left graph is the body temperature change, and the right graph is the body weight change) finds that the THZ1 treatment group can obviously inhibit the weight loss and the body temperature reduction caused by CAR-T-Raji inflammation storm; c: the multi-factor flow quantitative detection experiment of a mouse blood sample shows that the CAR-T-Raji treatment group mice can obviously initiate cytokine storm, the expression of related cytokines such as IL-1 beta and IL-6 is high, and the expression of the related cytokines is obviously reduced after THZ1 treatment; d: tissue dissection and staining experiments: LPS treatment of mice can remarkably cause inflammatory reaction (tissue congestion) of related organs (lung, liver, kidney and spleen), and THZ1 treatment can remarkably reduce the tissue congestion and acute inflammatory reaction; e: peripheral blood flow cytosubtype analysis shows that: the mice treated by LPS can obviously activate the immune stress of the mice, induce the increase of the number of macrophages, and the ratio of the macrophages can be obviously reduced by the treatment of THZ 1.

Example 9: immunofluorescence detection of mouse tumor and survival experiment result

FIG. 11 reflects tumor immunofluorescence detection and mouse survival experiments, which show that THZ1 can significantly improve the survival rate of mice lethal to cytokine storm during CAR-T treatment and improve tumor suppression efficiency; FIG. 11A reflects: mouse Raji tumor burden and mouse survival monitoring (tumor size of different groups of mice, and mouse survival). It was found that, in the group to which the drug was administered (THZ1+ CAR-T + Raji), mice survived much and tumors were small compared to the other groups (Raji, NCT + Raji, CAR-T + Raji); FIG. 11B reflects: the inflammatory cytokines in the administration group (THZ1+ CAR-T + Raji) were significantly reduced in comparison with those in the other groups (Raji group, NCT + Raji group, CAR-T + Raji group) by the mouse peripheral blood cytokine assay in the above group; c is a statistical graph of survival scores of mice in the experimental group, the death rate of the administration group (THZ1+ CAR-T + Raji) is obviously reduced, and the THZ1 can obviously reduce the death rate of the mice caused by CAR-T inflammatory storm and has significance.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (2)

1. Use of a CDK7 targeted inhibitor in the preparation of a medicament for treating cytokine release syndrome caused by CAR-T therapy (i.e., chimeric antigen receptor T cell immunotherapy).

2. The use according to claim 1 wherein the CDK7 targeted inhibitor is THZ1, BS-181 or SY-1365; further preferably, the targeted inhibitor of CDK7 is THZ 1.

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