WO2021143455A1

WO2021143455A1 - Use of pyroptosis pathway in cell therapy

Info

Publication number: WO2021143455A1
Application number: PCT/CN2020/137498
Authority: WO
Inventors: 黄波; 刘玉英
Original assignee: 中国医学科学院基础医学研究所
Priority date: 2020-01-14
Filing date: 2020-12-18
Publication date: 2021-07-22
Also published as: CN113189340A

Abstract

Disclosed is the use of a pyroptosis pathway in cell therapy, the use comprising: the use of a Gasdermin E-mediated pyroptosis pathway in the prediction and/or treatment of cytokine release syndrome, the use of a reagent for specifically detecting the activity or level of GSDME protein or gene in the preparation of a kit for predicting the risk of cytokine release syndrome occurring in a subject, and the use of a reagent for blocking and/or inhibiting the activity or level of GSDME protein or gene in the preparation of a drug for inhibiting and/or reducing the occurrence of cytokine release syndrome in a subject.

Description

Use of pyrolysis pathway in cell therapy

Technical field

The present invention relates to the technical field of prediction and/or treatment of cytokine release syndrome. Specifically, the present invention relates to the use of Gasdermin E (GSDME) as a marker to predict cytokine release syndrome, and the technical field of blocking or inhibiting Gasdermin E (GSDME) gene expression and/or activating pathways to improve cytokine release syndrome .

Background technique

Genetically engineered T cells (CAR-T) modified with chimeric antigen receptors have achieved significant effects in the treatment of malignant tumors, such as B-cell malignancies (see SSNeelapu et al., Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.N Engl J Med 377,2531-2544 (2017); SL Maude et al., Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoma.N Engl J 17 Med 378,439-448 (2018); and M. Sadelain et al., Therapeutic T cell engineering. Nature 545, 19 423-431 (2017)), but the treatment process is often accompanied by cytokine release syndrome (CRS). This severe systemic inflammatory response hinders the further development of CAR-T cell clinical therapy (see, CL Bonifant et al. Toxicity and management in CAR T-cell therapy. Mol Ther Oncolytics 3,16011 (2016); MLDavila, etc. Human Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 6,224ra225 (2014)). Current studies have shown that CRS is an acute inflammatory response characterized by elevated serum inflammatory cytokines and related clinical symptoms such as fever, hypotension, and respiratory insufficiency (see N. Frey, Cytokine release syndrome: Who is at risk and how to treat.Best Pract 8 Res Clin Haematol 30,336-340 (2017); DTTeachey, et al. Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chicemia 6 Antigen Receptor T-cell Therapy 6According to Therapy 79 (2016); and MLDavila et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 6,224ra225-224ra225 (2014)). CAR-T cells rapidly activate and expand after being infused into patients, and the expanded CAR-T cells cause rapid and massive death of B leukemia cells in a short period of time. The incidence of CRS and the severity of symptoms are positively related to the degree of lysis and destruction of B leukemia cells. However, the molecular mechanism underlying the massive necrosis of acute malignant tumor cells and the occurrence of CRS has not been explored clearly. At the same time, other studies have shown that in the humanized mouse model of CAR-T cell therapy, macrophages are involved in the occurrence of CRS (see T. Giavridis et al., CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade. Nat Med 24,731-738 (2018); and M. Norelli et al. Monocyte-derived IL-1 and IL-6 are differently required for cytokine-release syndrome and neurotoxicity due to CAR T cells. Nat Med 24,739-748 (2018)), but its specific molecular mechanism is still unclear.

Cells can die in many ways. Apoptosis has always been considered the only form of programmed cell death. However, current studies have shown the existence of a new form of programmed death, which is characterized by rapid cell swelling, the appearance of large membrane vesicles on the plasma membrane, and the release of pro-inflammatory factors (see J. Shi, et al., Pyroptosis: Gasdermin- Mediated Programmed Necrotic Cell Death. Trends Biochem Sci 42,245-254 (2017); and D. Wallach et al. Programmed necrosis in inflammation: Toward identification of the efficiency molecules. Science 352, aaf2154 (2016)). So far, at least two new molecular pathways that mediate programmed cell death have been identified. One is necroptosis mediated by mixed-lineage kinase domain-like pseudokinase (MLKL) (see J. Lin et al. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. Nature 540, 124-128 (2016); and J. Yuan, Et al. Neuroptosis and RIPK1-mediated neuroinflammation in CNS diseases. Nat Rev Neurosci 20, 19-33 (2019)), and the other is gasdermin D (GSDMD) or gasdermin E (GSDME) mediated pyrolysis (see Y. Wang et al., Chemotherapy drugsinduce pyroptosis through caspase-3 cleavage of a gasdermin.Nature 547,99-103 (2017); and S.Ruhl et al.ESCRT-dependentmembranerepairnegativelyregulatespyroptosisdownstreamof-GSDMD 362.S.Ruhl et al. (2018)). Among them, MLKL-mediated programmed cell death is mainly through the recruitment of receptor protein kinase 1 (RIP1) and 3 (RIP3) through the TNF-a receptor to form a death complex, and then activate MLKL to produce membrane nanopores, which leads to necrosis. Cell death. Unlike MLKL, GSDMD or GSDME is activated by inflammatory caspase (caspase-1, -4, -5 and -11) or caspase-3, which can form oligomers And it is inserted into the cell membrane to form a hole, thus mediating cell pyrolysis.

In order to overcome the adverse consequences of cytokine release syndrome caused by CAR T in the treatment of tumors in the prior art, new strategies are needed to control the cytokine release syndrome, especially cytokines, while maintaining or improving the efficacy of CAR T cell therapy. Security issues caused by the storm. The inventors provided new ideas for the prediction and treatment of CAR-T-induced CRS and the clinical application of CAR-T cells by explaining the relationship between two new types of programmed cell death and CRS.

Summary of the invention

Our research shows that GSDME is commonly expressed in malignant B-cell tumors that ^{express CD19+ (such as Raji and NALM-6 cells).} In addition, ^{tumor cells expressing HER2 +} (such as SGC-7901 and MCF-7) also express high levels of GSDME. We further analyzed the correlation between GSDME levels in primary B-ALL leukemia cells isolated from human B-cell leukemia patients and the level of CRS that occurred after CD19-CAR T treatment. According to our research results, the following implementation plan is proposed.

In one aspect, the present invention provides the use of a reagent for specifically detecting the activity or level of GSDME protein or gene in the preparation of a kit for predicting the risk of cytokine release syndrome in a subject; preferably, the reagent is used for Detect the expression level of GSDME protein or mRNA.

In the use provided by the present invention, if the subject's GSDME protein or gene activity or level is higher than the reference activity or level, it is predicted that the subject is at risk of developing cytokine release syndrome. Preferably, if the subject The GSDME protein or gene activity or level is higher than the reference activity or level by 2 times, which predicts that the subject is at risk of developing severe cytokine release syndrome. Preferably, according to the present invention, severe cytokine release syndrome refers to a cytokine release syndrome of grade IV (showing life-threatening symptoms and requiring ventilator support) and grade V (death) according to clinical classification.

In a specific embodiment of the present invention, the reference activity or level of GSDME protein or gene refers to the predetermined activity or level of GSDME protein or gene in patients who do not develop cytokine release syndrome after CAR T cell treatment.

In a preferred embodiment, the reference activity or level is a predetermined GSDME protein or gene activity or level in a subject that does not develop cytokine release syndrome.

The use according to the present invention, wherein the subject is a subject suffering from cancer; preferably, the cancer is related to the expression of CD19 and/or HER2; preferably, the subject is suffering from The following cancers: B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia ( CLL), B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large Cell follicular lymphoma, malignant lymphocytosis, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma , Hodgkin’s lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor and Waldenstrom’s macroglobulinemia.

In a further embodiment, the GSDME protein or gene is isolated from cancer cells of the subject, preferably, isolated from cancer cells expressing CD19 and/or HER2 in the subject; further preferably, isolated from cancer cells selected from the following Cells: B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL) , B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell filtration Alveolar lymphoma, malignant lymphocytosis, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, Hodgkin’s Chikin lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor and Waldenstrom macroglobulinemia.

In another embodiment, a method for assessing the risk of cytokine release syndrome in a subject with cancer before receiving CAR T cell therapy is provided, the method comprising detecting cancer cells isolated from the subject GSDME protein or gene activity or level, preferably, the CAR T cells are CD19 and/or HER2 CAR T cells.

On the other hand, the present invention provides the use of an agent for blocking and/or inhibiting the activity or level of GSDME protein or gene in the preparation of a medicament for inhibiting and/or reducing the occurrence of cytokine release syndrome in a subject; preferably, The subject is a cancer patient, more preferably, the subject has a cancer associated with CD19 and/or HER2 expression; preferably, the subject has a cancer selected from the group consisting of: B cell acute Lymphocytic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell premature Granulocyte leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant Lymphocyte proliferation, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, Plasmablast lymphoma, plasmacytoid dendritic cell tumor and Waldenstrom macroglobulinemia.

In a further preferred embodiment, the agent that blocks and/or inhibits the activity or level of GSDME protein or gene is used to inhibit and/or reduce CAR T cell therapy (for example, CD19 and/or HER2 CAR T cell therapy). Factor release syndrome.

According to the present invention, blocking and/or inhibiting GSDME protein or gene activity or level refers to reducing, reducing, inhibiting or eliminating GSDME protein or gene, such as knocking out GSDME gene, reducing mRNA activity or expression level.

It is known that activated caspase-3 can cleave GSDME to produce its active form, which is inserted into the cell membrane to cause pore formation and subsequent pyrolysis (Y. Wang et al., Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547,99-103(2017);).

Therefore, in a preferred embodiment, the agent for blocking and/or inhibiting the activity or level of GSDME protein or gene according to the present invention is selected from agents that inhibit the activity of GSDME protein (e.g., caspase-1, -3 , -4, -5, or 11), GSDME protein antagonists, anti-GSDME antibodies, and agents that delete or reduce the expression of GSDME genes (such as mRNA or DNA) (such as antisense sequences of GSDME mRNA, such as miRNA) , A reagent for knocking out GSDME DNA through CRISPR-Cas9); in a more preferred embodiment, the reagent for blocking and/or inhibiting the activity or level of GSDME protein or gene is a caspase-3 inhibitor More preferably, the agent for blocking and/or inhibiting the activity or level of GSDME protein or gene is belnacasan.

In another aspect, the present invention provides a composition for treating cancer, which comprises CAR T (for example, CD19 CAR T cell, or HER2 CAR T cell) and one or more blocking and/or inhibiting GSDME proteins or genes The activity or level of the agent, preferably, the agent that blocks and/or inhibits the activity or level of the GSDME protein or gene is selected from the group of agents that inhibit the activity of the GSDME protein (for example, caspase-1, -3, -4 , -5, or 11 inhibitors), GSDME protein antagonists, anti-GSDME antibodies, and agents that delete or reduce the expression of GSDME genes (such as mRNA or DNA) (such as GSDME and antisense sequences of mRNA such as miRNA) for Reagent for knocking out GSDME DNA through CRISPR-Cas9). In a more preferred embodiment, the agent for blocking and/or inhibiting the activity or level of GSDME protein or gene is a caspase-3 inhibitor, more preferably, the blocking and/or inhibiting GSDME protein Or the agent of gene activity or level is belnacasan. Preferably, the cancer is a cancer related to the expression of CD19 and/or HER2; preferably, the cancer is selected from: B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL) , Acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma Tumor, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoprolosis, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma Tumors, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumors, and Waldenstrom Macroglobulinemia.

According to the composition provided by the present invention, the CAR T (for example, CD19 CAR T cell, or HER2 CAR T cell) and one or more agents that block and/or inhibit the activity or level of GSDME protein or gene can be simultaneously , Separately or sequentially.

The composition provided according to the present invention can effectively reduce the cytokine syndrome caused by CAR T cell therapy, without changing the killing effect of CAR T cells on tumor cells.

Description of the drawings

Figure 1A to Figure 1G: HER2 CAR T or CD19 CAR T cells and Raji cells or NALM-6 cells were incubated for 6 hours at different target ratios or treated at different time points at a 2:1 ratio, and then the cell membrane was detected. The ratio of protein V single positive and annexin V and PI double positive cells. At the same time, the amount of LDH released in the supernatant was detected.

Figure 2A to Figure 2G: HER2 CAR T or CD19 CAR T cells and wild-type Raji cells or NALM-6 cells or GSDME knockout Raji cells or NALM-6 cells were treated for 6 hours at a 2:1 effective target ratio, and then tested The ratio of annexin V single positive cells and annexin V and PI double positive cells. At the same time, the amount of LDH released in the supernatant was detected.

Figure 3A to Figure 3D: Wild-type Raji cells or NALM-6 cells or GSDME knockout Raji cells or NALM-6 cells were inoculated into immunodeficient mice through the tail vein. After 21 days, the tail vein was injected with 10 times the amount of the tumor cells. T cells were used to detect the serum levels of inflammatory factors SAA, IL-6 and IL-1β before and 48 hours after injection and the survival time of mice.

Figure 4A to Figure 4D: Wild-type Raji cells or NALM-6 cells were inoculated into immunodeficient mice through the tail vein. After 21 days, CAR T cells were injected into the tail vein with 10 times the amount of tumor cells, and at the same time one week before CAR T reinfusion The macrophage scavenger clophosome-A (clophosome-A) or caspase-1 inhibitor benacasan (belnacasan) was injected into the tail vein to detect the serum inflammatory factors SAA, SAA, The content of IL-6 and IL-1β and the survival time of mice.

Figure 5A shows the anti-GSDME Western blot analysis of lysates of primary B leukemia cells (n=11) isolated from human B-ALL patients;

Figure 5B shows the correlation between GSDME expression and CRS score of primary B leukemia cells in human B-ALL patients (n=11);

Figure 5C shows the serum LDH level in human B-ALL patients;

Figure 5D shows the correlation between serum LDH level and CRS level in human B-ALL patients.

Detailed ways

The main purpose of this study is to clarify the underlying mechanism of CRS. For relapsed or refractory B-cell acute lymphoblastic leukemia (R/R ALL), CD19 CAR T and HER2 CAR T cells were produced in vitro, and autologous T cells transduced with CD19 TCR-ζ/4-1BB lentiviral vector Cells express CAR containing CD3ζ domain in vitro to provide T cell activation signals and 4-1BB domain to provide costimulatory signals.

We also analyzed the correlation between GSDME levels in primary B-ALL leukemia cells isolated from human B-cell leukemia patients and the level of CRS that occurred after CD19-CAR T treatment.

In in vitro studies, CAR T or unmodified T cells are used to co-culture with different tumor cells to determine the pyrolysis or apoptosis of tumor cells.

On the other hand, we also used Cas9 technology to knock out different genes to clarify how the macrophage-dependent pathway stimulates CRS caused by pyrosis in a mouse model.

Example 1. Construction of human CD19 or HER2 CAR T cells

The construction of chimeric antigen receptors for HER2 and CD19 was as previously described (RAMorgan, et al., Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.Mol Ther 18, 843-851 (2010 ); and MCMilone, et al., Chimeric receptors containing CD137 signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. Mol Ther 17,1453-1464(2009)). In short, the single-chain Fv fragment of HER2 from mAb 4D5 or the single-chain Fv fragment of CD19 from clone FMC63 is connected to the CD8α chain hinge and transmembrane region with CD3ζ and CD28 intracellular signaling domains, and This cassette is inserted into a lentiviral vector (provided by Obioo Bioscience Company). Finally, according to the manufacturer’s instructions, CD3/CD28 activation beads (Invitrogen) were used to stimulate CD8 ⁺ T cells to initiate transduction. The final recombinant human IL-2 was in Vivo-15 medium (Lonza) containing 5% FBS. The concentration is 100U/ml. The cells were harvested on day 2 for lentiviral transduction and resuspended in the same medium. The supernatant containing the lentivirus was added to the culture medium at the ratio of MOI 1:10, and according to the manufacturer’s instructions, with RetroNectin (CH-296; Takara Bio, Ohtsu, Japan, with 10mg/ml CH-296) Coated) Coated tablet. Then, the cells were centrifuged at 1000 g at 32°C for 2 hours, and incubated at 37°C for 6 hours. After 2 days, the infection rate was quantified by flow cytometry. In this study, the number of CD19-CAR T cells was used according to the ratio of effector cells to target cells (2:1) or in vitro experiments. T cells were transfected with lentivirus-CAR and cultured in vivo for 10 days. On the 3rd and 5th day after lentiviral transduction, and at the end of the culture, the transfection efficiency was evaluated by flow cytometry. The transfection efficiency is about 40%. For in vitro experiments, we culture CAR T cells for 5-7 days. These T cells grow logarithmically during expansion. For clinical trials, we use CD3ζ-4-1BB-CART. Otherwise, use CD3ζ-CD28-CART cells.

Example 2. Construction of mouse CAR T with human CD19 (mCAR hCD19)

The sequence of mCAR-hCD19 includes the antigen receptor of human CD19 or the single-chain variable fragment of HER2, murine CD3ζ, CD28 and/or 4--1-BB, and the N-terminal myc tag, as described above (see, for example, J. Chen Et al., NR4A transcription factors limit CAR T cell function in solid tumours. Nature 567, 530-534 (2019)), which was synthesized by SyngenTech. The chimeric antigen construct was then cloned into MSCV-GFP (Clontech) murine retroviral vector (MSCV-myc-CAR-2A). Then, the Platinum-E retroviral packaging cell line Ecotropic (PlatE) cells (Cell Biolabs, RV-101) were transfected with mCAR-hCD19 plasmid and pCL-Eco retroviral packaging plasmid to obtain reverse transcription containing mCAR-hCD19 Virus. OT-1 CD8 ⁺ T cells were activated by anti-CD3/CD28 magnetic beads (Gibco, 11453D), IL-2 (Perprotech, 212-12) and 55 μM β-mercaptoethanol (Gibco, 21985-023) for 24 hours. Then, in the presence of RetroNectin (Takara Bio), OT-1 CD8 ⁺ T cells were infected with the above virus for 8 hours. After 24 hours, GFP-positive cells were sorted by flow cytometry using BD Biosciences FACSAria II to obtain cells expressing high levels of hCD19 or hHER2.

Example 3. CAR T cells induce tumor cell pyrolysis in vitro

The HER2 CAR T or CD19 CAR T cells prepared in Example 1 above were incubated with Raji cells or NALM-6 cells for 6 hours at different effective target ratios or treated at different time points at a 2:1 effective target ratio. Cytometry was used to detect the ratio of annexin V single positive cells and annexin V and PI double positive cells (see Figure 1A to Figure 1C). An ELISA kit (eBiosence, CA, USA) was used to detect the release of LDH in the supernatant according to the manufacturer's instructions (see Figure 1D to Figure 1G). Through Student's t test or one-way analysis of variance, **p<0.01, ***p<0.001. The data represents the mean ± SD of three independent experiments.

The above results indicate that when CAR T kills tumor cells, it causes pyrolysis rather than apoptosis, which can be seen from the increase in the ratio of annexin V and PI double positive cells and the increase in LDH release.

Example 4. Generating a stable GSDME knockout cell line by CRISPR-Cas9

In order to generate stable GSDME knockout Raji cells or NALM-6 cell lines, the CRISPR-Cas9 method well known in the art was used to knock out the GSDME gene in Raji cells or NALM-6 cell lines. In short, clone the SGRNA corresponding to the GSDME gene into the pL-CRISPR.EFS.GFP vector plasmid, and package the plasmids psPAX2 and pMD2.G to co-transfect HEK293T cells. After 48 hours, the lentivirus was harvested and concentrated, and the Raji and NALM-6 cell lines were co-infected with polyethylene at a final concentration of 8 μg/ml. Two days later, the knock-out cells were confirmed by Western blot hybridization.

Example 5. CAR T cells induce tumor cell pyrolysis in vitro is mediated by GSDME

HER2 CAR T or CD19 CAR T cells and wild-type Raji cells or NALM-6 cells or GSDME knockout Raji cells or NALM-6 cells were treated for 6 hours at a 2:1 target ratio, and then flow cytometry Detect the ratio of annexin V single positive cells and annexin V and PI double positive cells (see Figure 2A to Figure 2E). At the same time, an ELISA kit (eBiosence, CA, USA) was used to detect the release of LDH in the supernatant according to the manufacturer's instructions (see Figure 2F to Figure 2G). Through Student's t test or one-way analysis of variance, **p<0.01, ***p<0.001. The data represents the mean ± SD of three independent experiments.

It can be seen from Figure 2A and 2G that after GSDME is knocked out, the pyrolysis caused by CAR T killing tumor cells is converted into apoptosis, which can be seen from the increase in the ratio of annexin V and PI double positive cells and the increase in LDH release; but it does not change. Killing effect, because the proportion of annexin V single positive cells did not decrease.

Example 6. CAR T cell therapy induces tumor cell pyrolysis and CRS in mice

Wild-type Raji cells or NALM-6 cells or GSDME knock-out Raji cells or NALM-6 cells were inoculated into immunodeficient mice SCID-beige through the tail vein, and the tumor burden was detected 21 days later, and the mice that met the requirements were injected into the tail vein 10 CAR T cells that are twice the amount of tumor cells. ELISA kits (eBiosence, CA, USA) were used to detect the levels of inflammatory factors SAA, IL-6 and IL-1β in mouse serum 4 hours before CAR T cell injection and 48 hours after injection according to the manufacturer's instructions (see figure 3A to 3C), and record the survival period of the mice (see Figure 3D). Through Student's t test or one-way analysis of variance, **p<0.01, ***p<0.001. The data represents the mean ± SD of three independent experiments.

The results show that knockout of GSDME can alleviate the release of inflammatory factors caused by CAR T (see Figure 3A to Figure 3C), reduce death due to CRS, and prolong survival (Figure 3D).

Example 7. Macrophage scavengers or caspase-1 inhibitors reduce CRS induced by CAR T cell therapy in mice

Wild-type Raji cells or NALM-6 cells were inoculated into immunodeficient mice SCID-beige through the tail vein. The tumor burden was measured 21 days later. The mice that met the requirements were injected with CAR T cells in the tail vein of 10 times the amount of tumor cells. One week before the CAR T cell injection, the macrophage scavenger clophosome-A (clophosome-A) (200μl/mouse) or the caspase-1 inhibitor benacasan (100mg) was injected into the tail vein one week before the CAR T cell injection. /kg), once every other day, a total of 3 times. Before CAR T cell detection and 48 hours after injection, use an ELISA kit (eBiosence, CA, USA) to detect the serum levels of inflammatory factors SAA, IL-6 and IL-1β according to the manufacturer's instructions (Figure 4A to Figure 4C) ), and record the survival period of the mice (Figure 4D). Through Student's t test or one-way analysis of variance, **p<0.01, ***p<0.001. The data represents the mean ± SD of three independent experiments.

The results show that the macrophage scavenger cloversone-A or caspase-1 inhibitor Benakasan can alleviate the release of inflammatory factors caused by CAR T (see Figure 4A to Figure 4C) and reduce the risk of CRS Death (see Figure 4D). This is consistent with our findings that tumor cell pyrolysis triggers the release of pro-inflammatory cytokines from macrophages.

Example 8. Correlation between the GSDME expression level of primary B-ALL leukemia cells in human B-cell leukemia patients and the grade of CRS induced by CD19-CAR T.

The patient came from the First Affiliated Hospital of Zhengzhou University. In order to be eligible to participate in the study, patients who were at least 4 years old and not more than 70 years old, and were diagnosed with CD19 ⁺ relapsed or refractory B-cell leukemia, were diagnosed as not. It is in line with autologous or heterologous SCT, and the prognosis is limited according to currently available therapies (several months to <2 years survival); ECOG result is 0, 1 or 2; with stable vital signs. Other eligibility criteria are healthy heart, liver and kidney function. All patients provided written informed consent to participate in the study. Peripheral blood or bone marrow is obtained from these patients. The experiment was approved by the clinical trial ethics committee of the First Affiliated Hospital of Zhengzhou University.

Cytokine Release Syndrome (CRS) grade

The grading system of CRS is based on clinical classification (see, S.S.Neelapu et al., Chimeric antigen receptor T-cell therapy-assessment and management of toxicities. Nat Rev Clin Oncol 15, 47 (2017)). In short, Grade I are non-life-threatening symptoms, such as fever, headache, myalgia, malaise, nausea or fatigue, etc.; Grade II includes those that require intravenous fluids or small doses of vasopressors, or respond to them Symptoms, level II organ toxicity or oxygen fraction is less than 40%; level III includes symptoms that require active intervention (large doses or multiple blood pressure drugs) or respond to it, level III organ toxicity or oxygen fraction equal to Or more than 40%; Grade IV is life-threatening symptoms and requires ventilator support; Grade V is death. Organ toxicity is classified according to CTCAE v4.03 (see Common Terminology Criteria for Adverse Events (CTCAE) Version 5.0 (NIH Publication, 2017). U.S. DEPARTMENT OF HEALTH AND HUMAN 12 SERVICES).

The B-cell leukemia cells were separated from the patient, lysed in M2 lysis buffer, and treated with ultrasound. BCA kit (Applygen Technologies Inc., China) was used to quantify protein. Then, the protein was separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was blocked in 5% BSA, incubated with anti-GSDME (Abcam, UK) antibody overnight, and then incubated with a secondary antibody coupled with horseradish peroxidase, and tested.

We analyzed the GSDME levels in primary B-ALL leukemia cells isolated from 11 patients before CD19-CAR T treatment. Although B leukemia cells from patients generally express GSDME (Figure 5A), in these patients, higher levels of GSDME are associated with more severe CRS cases (Figure 5B).

In addition, we used an ELISA kit (eBiosence, CA, USA) to detect the serum LDH level of patients after CAR T treatment according to the manufacturer's instructions.

It was found that patients with higher CRS grade (n=7) had higher blood LDH levels than low CRS (n=4) (Figure 5C), and the level of LDH was positively correlated with the severity of CRS (Figure 5D) . Together, these indicate that the pyrolysis of B leukemia cells induced by CAR T cell therapy triggers the patient's CRS.

Claims

Use of a reagent for specifically detecting the activity or level of GSDME protein or gene in preparing a kit for predicting the risk of cytokine release syndrome in a subject; preferably, the reagent is used for detecting the expression of GSDME protein or mRNA Level.
The use according to claim 1, wherein if the subject's GSDME protein or gene activity or level is higher than the reference activity or level, it is predicted that the subject is at risk of developing cytokine release syndrome, preferably if the subject is affected The GSDME protein or gene activity or level of the subject is higher than the reference activity or level twice, which predicts that the subject is at risk of developing severe cytokine release syndrome.
The use according to claim 1 or 2, wherein the subject is a subject suffering from cancer; preferably, the cancer is related to the expression of CD19 and/or HER2; preferably, the subject suffers from There are cancers selected from the following: B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell promyelocytic leukemia, blastic plasma cells Dendritic cell tumor, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoprolosis, MALT lymphoma, Mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic Cell tumors and Waldenstrom's macroglobulinemia.
The use according to claim 1 or 2, wherein the risk of cytokine release syndrome in the subject is assessed before receiving CAR T cell therapy; preferably, the CAR T cells are CD19 and/or HER2 CAR T cells.
Use of an agent for blocking and/or inhibiting the activity or level of GSDME protein or gene in the preparation of a medicament for inhibiting and/or reducing the occurrence of cytokine release syndrome in a subject, preferably, the subject has cancer More preferably, the subject has a cancer associated with CD19 and/or HER2 expression; preferably, the subject has a cancer selected from the group consisting of B-cell acute lymphoblastic leukemia, T-cell acute lymphoma Cell leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt lymphoma, diffuse large B cell Lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphocytosis, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia And myelodysplastic syndrome, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor and Waldenstrom macroglobulinemia.
The use according to claim 5, wherein the agent that blocks and/or inhibits the activity or level of GSDME protein or gene is selected from the group consisting of agents that inhibit GSDME protein activity, agents that reduce GSDME protein expression levels, and reduce GSDME mRNA expression levels Preferably, the agent that inhibits the activity of GSDME protein is selected from inhibitors of caspase-1, -3, -4, -5, or 11; the agent that reduces GSDME The agent for protein expression level is preferably selected from GSDME protein antagonist and anti-GSDME antibody; the agent for reducing GSDME mRNA expression level is preferably selected from the antisense sequence of GSDME mRNA; the agent for knocking out GSDME DNA is preferably selected from Reagent for knocking out GSDME DNA through CRISPR-Cas9.
The use according to claim 5 or 6, wherein the agent that blocks and/or inhibits the activity or level of GSDME protein or gene is used to inhibit and/or reduce cytokine release syndrome caused by CAR T cell therapy.
A composition for treating cancer, wherein the composition comprises CAR T and one or more agents that block and/or inhibit the activity or level of GSDME protein or gene, preferably, the block and/or The agent that inhibits the activity or level of GSDME protein or gene is selected from the group consisting of agents that inhibit the activity of GSDME protein, agents that reduce the expression level of GSDME protein, agents that reduce the expression level of GSDME mRNA, and agents that knock out GSDME DNA; preferably, the inhibitor of GSDME protein The active agent is selected from inhibitors of caspase-1, -3, -4, -5, or 11; the agent for reducing the expression level of GSDME protein is preferably selected from GSDME protein antagonists and anti-GSDME antibodies; The reagent for reducing the expression level of GSDME mRNA is preferably selected from antisense sequences of GSDME mRNA; the reagent for knocking out GSDME DNA is preferably selected from reagents for knocking out GSDME DNA through CRISPR-Cas9.
The use of the composition according to claim 8 in the preparation of a medicament for the treatment of cancer in a subject, preferably, the cancer is a cancer related to the expression of CD19 and/or HER2; preferably, the cancer is selected from : B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell promyelocytic leukemia, blastic plasmacytoid dendritic cell tumor , Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphocytosis, MALT lymphoma, mantle cell lymphoma, marginal Regional lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, plasmablast lymphoma, plasmacytoid dendritic cell tumor and Waldens Tron Macroglobulinemia.
The use according to claim 9, wherein the CAR T in the composition and one or more agents that block and/or inhibit the activity or level of GSDME protein or genes are administered simultaneously, separately or sequentially.