Use of an antibody in the preparation of a medicament for the treatment of HUS caused by Shiga toxin type II

Abstract

The application discloses application of an antibody in preparation of a drug for treating HUS caused by Shiga toxin type II. Application of an antibody for mononuclear cells in preparation of a drug for treating hemolytic uremic syndrome. The antibody for mononuclear cells is a CD14 antibody. Through single cell sequencing technology, the application finds positive cells combined with Stx2 in peripheral blood, further bioinformatics analysis obtains that most of the cells combined with Stx2 are mononuclear cells, and then the CD14 antibody for mononuclear cells is used to treat HUS caused by Stx2, and the result shows that the CD14 antibody has good treatment effect. Therefore, the antibody for mononuclear cells, especially the CD14 antibody, can be applied in preparation of a drug for treating HUS caused by Stx2.

Description

TECHNICAL FIELD

[0001] The application relates to an antibody in the preparation of treating HUS caused by Shiga toxin type 2, belonging to the field of medicine. BACKGROUND

[0002] Hemolytic uremic syndrome (HUS) is a syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. Due to the susceptibility of renal vascular endothelial cell damage, the kidney becomes the main organ involved during the onset of HUS. HUS is mainly caused by Shiga toxin 2 (Stx2) producing E. coli, and Stx2 is transmitted to target organs to cause serious complications. At present, the number of patients caused by Shiga toxin reaches 2.8 million per year, among which children are the main patient group, especially children under the age of 5, and the pathological manifestation is severe acute kidney injury (AKI), which is also the main cause of morbidity and mortality in elderly patients.

[0003] Current research shows that Shiga toxin-producing E. coli is not tissue invasive, that is, in the absence of Shiga toxin, their pathological effects are the same as those of enteropathogenic E. coli, that is, they invade the colon, destroy tight junctions and eliminate microvilli, leading to watery diarrhea. Stx2 can enter the blood circulation from the intestine, but the transport carrier of Stx2 from the intestine to the kidney or other target organs is not very clear, and free Stx2 has never been detected in the blood of patients, but Shiga toxin can be detected in the kidney biopsy of HUS patients. Further exploration of toxin transport media is crucial for seeking more effective treatment methods.

[0004] Current research shows that Stx2 has certain binding capacity on monocytes. After binding to its receptor, Stx2 can be internalized and targeted to lysosomes for final degradation. Monocytes isolated from human peripheral blood can inhibit protein synthesis in Vero cells after co-incubation with Stx2 in vitro, and Shiga toxin has been detected on monocytes or platelet-monocyte aggregates in patient samples. In addition to possibly serving as a delivery vehicle for toxins, Stx2 can also induce monocytes to release proinflammatory cytokines, including IL-6, IL-8, TNF-alpha, IL-1 beta, CCL5, and thrombogenic tissue factor expression, thereby activating the extrinsic coagulation pathway to cause thrombin generation and blood clotting.

[0005] Currently, there is no specific treatment for HUS caused by Stx2-producing E. coli, and there is no effective treatment method, and the treatment is still mainly for complications, mainly including the following four complications: gastrointestinal tract (severe abdominal pain, colitis), blood (symptomatic anemia and active bleeding), blood vessels (vascular volume depletion, capillary leakage or edema), and kidney (electrolyte, volume and acid-base disorders) and the like. Therefore, preventive measures and effective treatment methods are still urgent problems to be solved, which are summarized into three categories: 1) infection prevention (reducing contact, using bacterial antigens and Stx2 for active immunization); 2) intervention of E. coli using probiotics and the like; 3) therapy targeting downstream molecules (inhibitors of SAPK, apoptosis, thrombosis cascade). Although health measures and education can help reduce the incidence of Stx2-producing E. coli infection and HUS, it seems impractical to immunize humans against Stx2-producing E. coli-related diseases on a large scale.

[0006] CD14, a lipopolysaccharide receptor, is a phosphatidyl inositol anchor membrane protein, and CD14 is a leukocyte differentiation antigen present on the surface of monocytes, macrophages and the like. There is no report on the use of CD14 neutralizing antibodies for the treatment of HUS. SUMMARY

[0007] The purpose of the present application is to overcome the above-mentioned deficiencies in the prior art, and to provide the use of antibodies against monocytes in the preparation of drugs for treating hemolytic uremic syndrome.

[0008] Another purpose of the present application is to provide the use of monocytes as drug screening targets.

[0009] The purpose of the present application can be achieved by the following technical solutions:

[0010] The use of antibodies against monocytes in the preparation of drugs for treating hemolytic uremic syndrome.

[0011] As a preferred embodiment of the present application, the hemolytic uremic syndrome is hemolytic uremic syndrome caused by Shiga toxin type II.

[0012] As a preferred embodiment of the present application, the antibodies against monocytes are CD14 antibodies.

[0013] The CD14 antibody is a synthetic murine CD14 antibody.

[0014] The use of monocytes as targets in the preparation of drugs for treating hemolytic uremic syndrome caused by Shiga toxin type II.

[0015] A pharmaceutical composition for treating hemolytic uremic syndrome caused by Shiga toxin type II, comprising a CD14 antibody and a pharmaceutically acceptable excipient.

[0016] The CD14 antibody is a synthetic murine CD14 neutralizing antibody (low endotoxin, no sodium azide).

[0017] The administration mode of the Stx2-induced HUS treatment product includes, but is not limited to, intravenous injection, intraperitoneal injection, and intramuscular injection.

[0018] Further, the use cycle of the pharmaceutical composition is 50ug per injection every 24 hours.

[0019] Beneficial effects:

[0020] The present application analyzes the mononuclear cell infiltration, toxin infiltration and pathological severity of the kidney, fully evaluates the therapeutic effect of the CD14 neutralizing antibody on the Stx2-induced HUS, and proves that the CD14 neutralizing antibody has good therapeutic effect.

[0021] The present application finds the positive cells combined with Stx2 in the peripheral blood through single cell sequencing technology, further obtains the cells combined with Stx2 through bioinformatics analysis, and then treats the Stx2-induced HUS by using the CD14 neutralizing antibody for the mononuclear cells, and the result shows that the CD14 neutralizing antibody has good therapeutic effect. Therefore, the antibody for the mononuclear cells, especially the CD14 neutralizing antibody, can be applied to the preparation of the treatment of the Stx2-induced HUS. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0023] Figure 1 The positive cells combined with Stx2b-FITC in the fresh peripheral blood of human are analyzed by single cell analysis.

[0024] A) The flow chart for identifying the positive cells combined with Stx2 (10 ng / ml) in the human peripheral blood cells is obtained.

[0025] B) The Stx2b positive cells are sorted by using a flow cytometer.

[0026] C) The types of the Stx2b positive cells are identified by using the method of bioinformatics.

[0027] D) The proportion of various cells in the Stx2b positive cells.

[0028] Figure 2: LPS related Stx2 induced HUS model.

[0029] A) LPS related Stx2 induced HUS model.

[0030] B) LPS related Stx2 induced HUS survival curve.

[0031] C) HE staining of LPS related Stx2 induced HUS model.

[0032] Figure 3 : CD14 neutralizing antibody treatment of LPS related Stx2 induced HUS results.

[0033] A) CD14 neutralizing antibody treatment of LPS related Stx2 induced HUS model.

[0034] B) CD14 neutralizing antibody treatment of LPS related Stx2 induced HUS survival curve.

[0035] C) HE staining of CD14 neutralizing antibody treatment of LPS related Stx2 induced HUS model.

[0036] D) Monocyte / macrophage infiltration of CD14 neutralizing antibody treatment of LPS related Stx2 induced HUS model. DETAILED DESCRIPTION

[0037] The present application screens cells that bind to Stx2 in blood by using the currently more advanced detection method of single cell sequencing, and performs relevant antibody neutralization treatment on the screened cells, and tests the treatment effect of the neutralizing antibody on HUS. The method uses CD14 neutralizing antibody against monocytes to effectively prevent and treat HUS by preventing monocyte / macrophage infiltration in the kidney and inhibiting further deterioration of HUS caused by Stx2, so the present application has certain clinical guiding value for clinically dealing with acute HUS caused by Shiga toxin.

[0038] The present application is further described below by examples.

[0039] Example 1: Screening and identifying cells that bind to Shiga toxin Stx2 based on single cell sequencing technology using human peripheral blood cells.

[0040] Fresh peripheral blood of healthy people was taken with an anticoagulant tube, incubated with Stx2b-FITC (10 ng / ml) at room temperature for 30 minutes, and then sorted with FITC-labeled cells by flow cytometry (BD FACSAria SORP). The cells were collected with a sterile collection tube and stored in PBS containing serum to maintain cell activity. Cell viability was determined by trypan blue staining, and when the viability reached more than 95%, 2 x 10 5 cells were collected for cell pretreatment. Single cells were labeled by microfluidic technology, and a PCR instrument was used for library construction and resequencing. After data quality control and statistics, and cell filtration, the relevant data Figure 1 A, B) were generated. The obtained data were further analyzed by bioinformatics, and three major cell populations were obtained in Stx2-positive cells. Through marker gene screening, it was found that the three types of cells were monocytes, neutrophils, and DC cells, and their proportions were 62.8%, 30.38%, and 1.57% ( Figure 1 C, D).

[0041] The specific experimental steps involved are as follows:

[0042] 1. Stx2b-FITC preparation process

[0043] 1) Add recombinant protein Stx2b to an ultrafiltration tube, add 200-150 μL PBS, centrifuge at 12000 g at 4°C for 10 min,

[0044] 2) Discard the filtrate, and add PBS again, centrifuge at 12000 g at 4°C for 10 min.

[0045] 3) After centrifugation, remove the inner core of the ultrafiltration tube and invert it in a clean outer tube, centrifuge at 4000 g at 4°C for 2 min, and collect the sample after buffer replacement.

[0046] 4) Add 5 μL AbFluorTM 488 labeling solution to the sample solution to be labeled, and mix gently with a pipette.

[0047] 5) Take 2.5 μL of Activated AbFluorTM 488 solution and add it to the reaction solution, add deionized water to 50 μL (Note: This volume is the labeling system), mix gently, and incubate at 37°C in the dark for 1 h.

[0048] 6) Add an appropriate amount of PBS to the reaction solution (to about 500 μL), mix gently, and transfer the solution to a purification column, centrifuge at 12000 g at 4°C for 10 min.

[0049] 7)Discard the filtrate, add an appropriate amount of PBS (constant volume to about 500 μL) to the purification column, 12000g, 4°C centrifugation for 10min.

[0050] 8)Take out the purification column and invert it in a clean centrifuge tube, centrifuge at 4°C, 4000g for 2min, and the solution collected in the centrifuge tube is the coupling product Stx2b-FITC.

[0051] 2. Human peripheral blood cell sorting and single cell sequencing

[0052] 1) Human whole blood cells were incubated with Stx2b-FITC (10 ng / ml) at room temperature for 30 minutes;

[0053] 2) 400g centrifugation for 5min, discard the supernatant, add 5mL red blood cell lysis solution, blow the cells evenly, lyse on ice for 10min;

[0054] 3) 4°C, 400g centrifugation for 5min;

[0055] 4) Discard the supernatant, resuspend the cells with 3mL PBS containing 5% serum, centrifuge at 4°C, 400g for 5min;

[0056] 5) Discard the supernatant, resuspend the cells with 1mL 5% serum PBS;

[0057] 6) Use a sorting flow cytometer (BD FACSAria SORP) to sort out Stx2b-FITC cells and perform single cell sequencing.

[0058] Example 2 Example of Stx2-induced mouse HUS model after LPS stimulation and intervention experiment using CD14 neutralizing antibody against monocytes.

[0059] First, a mouse HUS model was constructed, and LPS (300 μg / kg, Sigma-Aldrich) was injected intraperitoneally 24 hours in advance, and then Stx2 protein (purchased from TOXIN TECHOLOGY) was injected every 24 hours for 3 consecutive days, and the survival of the mice was observed Figure 2 A, B), and then the kidney tissue of the mice was taken out for HE pathological analysis, which showed that the immune cell infiltration of the kidney increased, the renal tubular epithelial cells fell off, and the tubules expanded and were damaged, indicating that the LPS-Stx2-induced mouse HUS model was successfully constructed Figure 2 C).

[0060] On the basis of this model, the present application takes the monocyte with the largest Stx2 binding ratio as the target cell, takes CD14 specifically expressed by the monocyte as the target point, and uses CD14 neutralizing antibody (BioLegend) for treatment intervention. According to the Figure 3A shows the flow of relevant experimental verification, the treatment group in the first injection of type II Shiga toxin protein at the same time, intraperitoneal injection of 50 μg / CD14 neutralizing antibody, 24 h after giving again, a total of two times, the model group (LPS+Stx2 group) each time to give 100 μl of sterile PBS. Results show that after CD14 neutralizing antibody treatment, the mortality of mice appears a certain decline Figure 3 B), the model group of renal immune cell infiltration increased, renal tubular epithelial cell exfoliation, tubular expansion, damage; while the CD14 treatment group renal tissue damage was significantly reduced Figure 3 C). The mouse kidney was embedded in OCT (embedding agent, Sakura) for frozen section observation of Stx2 and immune cell infiltration fluorescence results, the results showed that the model group of kidney Stx2 positive fluorescence signal (green) more, F4 / 80 positive fluorescence signal (red) more, while the treatment group signal both signal was significantly weakened Figure 3 D), which shows that CD14 neutralizing antibody blocks monocyte activation and migration to the kidney, and reduces the progression of mouse HUS.

[0061] In summary, the CD14 neutralizing antibody for monocytes can effectively prevent and treat HUS caused by Shiga toxin, which provides guidance for clinical treatment.

[0062] The specific experimental steps involved are as follows:

[0063] 1. The mice used in the present application are purchased from Ziyuan Biological male 6 weeks, C57, placed in the animal room of Nanjing University for feeding, and the animal experiments involved comply with the regulations of Nanjing University Ethics Committee.

[0064] 2. Mouse kidney tissue HE staining

[0065] After fixing the mouse kidney tissue with paraformaldehyde, paraffin embedding section, soaking in xylene, 10 min→absolute ethanol, 3 min→95% ethanol, 3 min→90% ethanol, 3 min→80% ethanol, 3 min→70% ethanol, 3 min→60% ethanol, 3 min→50% ethanol, 3 min→ddH2O, 3 min→hematoxylin, 3 min→water rinse until colorless→30% ethanol, 3 min→50% ethanol, 3 min→60% ethanol, 3 min→70% ethanol, 3 min→80% ethanol, 3 min→90% ethanol, 3 min→eosin staining 5 min→95% ethanol, 3 min→absolute ethanol, 3 min→xylene soaking 10 min→neutral balsam mounting→overnight drying. Use brightfield microscope to take pictures to observe the kidney structure for histopathological evaluation.

[0066] 3. Kidney tissue immunofluorescence

[0067] 1) OCT embed kidney tissue, cut into 6um with cryostat, mount on slide.

[0068] 2) Wash with PBS three times, fix with 4% paraformaldehyde for 30min, wash with PBS three times.

[0069] 3) Block with 5% BSA for 1h.

[0070] 4) Add antibody (Stx A, Thermo; F4 / 80, CST) at 1:100, incubate overnight at 4C.

[0071] 5) Wash with PBS three times, add secondary fluorescent antibody at 1:1000, incubate for 1h at room temperature in the dark.

[0072] 6) Wash with PBS three times, add DAPI at 1:1000, incubate for 15min at room temperature in the dark.

[0073] 7) Wash with ddH2O three times, air dry, mount with anti-fluorescent quenching agent.

[0074] 8) Take pictures with laser confocal microscope (Leica).

Claims

1. Use of a CD14 neutralizing antibody for the preparation of a medicament for the treatment of hemolytic uremic syndrome caused by Shiga toxin type II.

Images