WO2020020089A1

WO2020020089A1 - Uses of specific antibody, implantable medical instrument, and preparation method therefor

Info

Publication number: WO2020020089A1
Application number: PCT/CN2019/096999
Authority: WO
Inventors: 王玄; 陈陆; 李俊菲
Original assignee: 上海微创医疗器械（集团）有限公司
Priority date: 2018-07-27
Filing date: 2019-07-22
Publication date: 2020-01-30
Also published as: CN110755609A

Abstract

Provided are the uses of a specific antibody, an implantable medical instrument, and a preparation method therefor. The uses are those of a specific antibody of an ATP-binding cassette transporter in preparing a drug for promoting endothelial repair. The presenthe surface of the implantable medical instrument is loaded with the specific antibody of the ATP-binding cassette transporter. The preparation method comprises loading the specific antibody of the ATP-binding cassette transporter on at least a part of the surface of the implantable medical instrument; and/or binding the specific loaded antibody of the ATP-binding cassette transporter into the hole or the groove of the implantable medical instrument.

Description

Use of specific antibody, implanted medical device and preparation method thereof

Technical field

The invention belongs to the technical field of biomedicine, and particularly relates to the use of a specific antibody, an implanted medical device and a preparation method thereof.

Background technique

Atherosclerotic diseases can cause damage to the vascular endothelium, which can cause diseases such as thrombosis and inflammation, and cause myocardial infarction. If the damaged tissue cannot be repaired during treatment, it will lead to intimal hyperplasia and restenosis of the blood vessels, thus endangering the patient's life. Hematopoietic stem cells are adult stem cells in the blood system with long-term self-renewal capabilities and the potential to differentiate into various types of mature blood cells. The surface of medical devices implanted in the blood system is loaded with substances that can capture hematopoietic stem cells, and induce hematopoietic stem cells to differentiate into the required types of cells in situ as required, which can improve the device's treatment and tissue repair effects.

The left atrial appendage is an auricular sac protruding from the left atrium and belongs to the left atrium. It is the main component of the left atrium. Left atrial appendage occlusion was developed by Boston Scientific and approved for marketing in Europe in 2006. On December 31, 2013, it was officially approved by the China Food and Drug Administration for listing and officially launched in China in March 2014. Through this interventional surgery, the left atrial appendage, the root site of thrombosis in patients with AF, can be used to reduce the risk of stroke in patients with AF. The surface of the occluder is covered with an expandable polytetrafluoroethylene film, which is similar to general implanted medical devices. It also needs to be loaded with drugs that repair damaged tissues to prevent the adverse consequences of surgery.

In 1996, Margaret A. Goodell used the fluorescent dye Hoechst 33342 to infect bone marrow cells and performed flow cytometry to obtain a group of double-negative cell groups isolated from the main group. The experiments proved that this group of cells has the characteristics of stem cells. Hoechst 33342 is a fluorescent dye that can stain the DNA of living cells. Its excitation light is 350 nm and the emitted light is divided into two bands. When analyzing the emitted light of Hoechst33342 in two different bands in whole bone marrow cells at the same time, there is a small group of cells that are significantly different from the main group in the double negative region. After analysis, the surface molecular markers of this group of cells are consistent with hematopoietic stem cells. This phenomenon is due to the presence of proteins on the membrane of hematopoietic stem cells that pump Hoechst 33342 dye out.

At present, medical devices that promote endothelial repair are generally achieved by loading small molecular compounds or compositions on the surface of medical devices, which are all based on the mechanism of promoting cell growth. However, this method is generally poor in specificity. While promoting endothelial growth, it also promotes the growth of other cells, including smooth muscle and fibroblasts, resulting in adverse consequences such as restenosis of blood vessels and thrombosis. In addition, the side effects of the drugs in this way are large, which can bring other adverse effects.

Summary of the Invention

The invention provides the use of a specific antibody, an implanted medical device and a preparation method thereof, which are used to provide a new way to promote endothelial repair.

The invention provides a use of a specific antibody, the specific antibody is a specific antibody of an ATP-binding box protein, and the use is in the preparation of a medicament for promoting endothelial repair. There are proteins on the membrane of hematopoietic stem cells that pump Hoechst 33342 dye, including the ATP-binding box protein superprotein family. Therefore, these proteins can be considered as markers of hematopoietic stem cells.

Optionally, the ATP-binding cassette protein is selected from MDR-1 or Bcrp-1. Multidrug resistance protein-1 (MDR-1 or ABCB1 or P-glycoprotein 1 or CD243) and breast cancer resistance protein-1 (Bcrp-1 or Bcrp or ABCG2, or CD338), is very effective in promoting endothelial repair.

Optionally, the MDR-1 specific antibody is Clone REA495. Clone REA495 was purchased from Miltenyi Biotech Co., Ltd., Germany.

Optionally, the Bcrp-1 specific antibody is Clone 5D3. Clone 5D3 was purchased from Miltenyi Biotech Co., Ltd., Germany.

The invention also provides an implanted medical device, wherein the surface of the implanted medical device is loaded with a specific antibody of an ATP-binding box protein.

Optionally, the ATP-binding cassette protein is selected from MDR-1 or Bcrp-1.

Optionally, the MDR-1 specific antibody is Clone REA495.

Optionally, the Bcrp-1 specific antibody is Clone 5D3.

Optionally, a specific antibody of the ATP-binding cassette protein is supported on at least one side of the implanted medical device.

Optionally, the implanted medical device is a vascular prosthesis, a prosthetic valve membrane, a prosthetic valve stent, a left atrial appendage occluder membrane, a left atrial appendage occluder stent, a heart occluder membrane, a heart occluder stent, or an artificial Patch.

Optionally, the implanted medical device is a left atrial appendage occluder membrane or a cardiac occluder membrane.

Optionally, the concentration range of the specific antibody of the ATP-binding cassette protein on the surface of the implanted medical device is: 10-1000 g / mm ² . When the concentration is too low, the antibody is quickly dissolved in the blood; when the concentration reaches a certain level, the antibody binding site is saturated, and the above effect is no longer increased.

The present invention also provides a method for preparing the above-mentioned implanted medical device, which comprises supporting a specific antibody of the ATP-binding box protein on at least a part of the surface of the implanted medical device and / or specificity of the ATP-binding box protein. Loaded in a hole or groove of the implanted medical device.

Optionally, the specific antibody of the ATP-binding cassette protein is loaded on the surface of the implanted medical device by at least one of a direct coating method, a chemical grafting method, and an electrostatic adsorption method.

Among them, the direct coating method is to directly apply the antibody solution to the surface of the device, and the coating method may be spray coating, dip coating or spin coating.

Among them, the chemical grafting method may be to chemically modify the antibody molecules so that they can be chemically connected to the surface of the device or the surface of the device coating, or it may use an antibody linker or a cross-linking agent to bind the antibody molecules to the surface of the device. .

Among them, the electrostatic adsorption method is to coat the surface of the device with an oppositely charged coating, so that the antibody molecules are coated on the surface of the device by electrostatic adsorption.

The technical scheme of the present invention creatively uses the specific antibody of the ATP-binding box protein to achieve the effect of promoting endothelial repair of the medical device. Compared with existing endothelial repair methods, the technical scheme of the present invention captures the human hematopoietic stem cells and induces them to produce cells to achieve targeted endothelial repair. Therefore, the side effect of this method is small, and it will not bring other adverse effects, and the comprehensive effect is more ideal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a fluorescence diagram of a sample loaded with a specific antibody of an ATP-binding box protein in a antibody loading experiment of Example 5 after staining with a secondary antibody fluorescent dye, and the three diagrams from left to right correspond to Examples 1, 2, and 4 sample;

FIG. 2 is a fluorescence diagram of a sample loaded with a specific antibody against an ATP-binding box protein in a cell capture experiment of Example 5 to capture cells in the blood, and the cells were fluorescently stained with specific antibody fluorescence, and the three maps from left to right correspond sequentially Samples of Examples 1, 2 and 4;

FIG. 3 is a SEM characterization diagram of a stent and a drug stent loaded with a specific antibody to an ATP-binding cassette protein in an animal experiment of Example 14 after implantation into the iliac artery of a rabbit 14 days, and FIG. 3a to FIG. 3c are Examples 1, 2 and 4 stent sample, Figure 3d is a stent sample of the control group;

4 is a fluorescence diagram of the membrane of Example 3 after being stained with a secondary antibody fluorescent dye in the antibody loading experiment of Example 6;

5 is a fluorescence diagram of a cell capture experiment in Example 6 using a membrane of Example 3 to capture cells in the blood and perform specific antibody fluorescent staining on the cells.

FIG. 6 is a SEM characterization image of the sample film sewn in the left atrial appendage occluder stent in the animal experiment of Example 6 14 days after implantation in the body. An untreated sample film was used as a control.

detailed description

In order to facilitate understanding, the use of the specific antibody of the ATP-binding cassette protein is explained below with reference to the examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Unless otherwise specified, the raw materials and instruments used in the present invention are commercially available products.

The "medical device" referred to in the present invention may be a device implanted in a body. The device can be used temporarily for short periods or permanently implanted for long periods. In certain embodiments, suitable devices are commonly used for arrhythmia, heart failure, valvular disease, vascular disease, diabetes, neurological diseases and disorders, orthopedics, neurosurgery, oncology, ophthalmology, and ENT Surgery provides medical and / or diagnostic equipment. The medical devices involved in the present invention include, but are not limited to, the following devices: stents, stent grafts, anastomotic connectors, synthetic patches, leads, electrodes, needles, wires, catheters, sensors, surgical instruments, angioplasty balls, and wound drainage tubes , Shunt, tube, infusion sleeve, urethral intubation, pellet, implant, blood oxygen generator, pump, vascular graft, vascular access box port), heart valves, annuloplasty rings, sutures, surgical clips, surgical staples, pacemakers, implantable defibrillators, neurostimulators, orthopedic surgical instruments, cerebrospinal fluid shunt tubes, implantable Medication pumps, cages, artificial discs, alternative devices to the nucleus pulposus, ear canals, intraocular lenses, and any tubes used in interventional procedures. Preferably, the medical devices according to the present invention are in particular: stents, balloons, occluders, valves or artificial blood vessels.

Example 1

100g Bcrp1 monoclonal antibody Clone 5D3 (purchased from Miltenyi Biotech Co., Ltd., Germany) was dissolved in 10 mL of antibody dilution solution (Wuhan Ph.D.) to prepare an antibody solution with a concentration of 10 g / mL. Polyelectrolyte sodium hyaluronate (HA) 1mg / mL dissolved in 0.1% NaCl solution, polyelectrolyte chitosan (CS) 1mg / mL dissolved in 0.1% NaCl solution, polyelectrolyte polyimide for base coating Amine (PEI) 5 mg / mL was dissolved in a 0.1% NaCl solution.

Wash and dry the bare stent. First immersed in polyacetimide (PEI) solution to obtain the PEI layer; immersed the PEI-treated stent in sodium hyaluronate (HA) solution to obtain the outer layer of HA; then dipped the stent in chitosan (CS ) Solution, the CS outer layer was obtained. The above-mentioned HA / CS electrostatic assembly process was repeated 7 times to obtain a sodium hyaluronate / chitosan self-assembled multilayer film, which is denoted as PEI (HA / CS) ₇ . The stent treated as above was immersed in the antibody solution for 30 min, and N _{2 was} taken out and blow-dried. The preparation of the Clone 5D3 loaded scaffold was completed. After weighing calculation, the concentration of Clone 5D3 on the surface of the stent was finally measured to be 21 g / mm ² .

Example 2

100g of MDR1 monoclonal antibody Clone REA495 (purchased from Miltenyi Biotech Co., Ltd., Germany) was dissolved in 1 mL of antibody dilution solution (Wuhan Ph.D.) to prepare an antibody solution with a concentration of 100 g / mL. The laser-cut L605 bare stent was immersed in a solution of H ₂ SO ₄ : H ₂ O ₂ = 3: 1 for 30 minutes, rinsed with purified water 3 times, and blown dry with nitrogen. The stent after the above treatment was immersed in the antibody solution, left for 30 minutes, and then taken out. After being naturally dried, it was stored at 4 ° C. The preparation of the Clone REA495 loaded scaffold was completed. After weighing calculation, the concentration of Clone REA495 on the surface of the stent was finally measured to be 89 g / mm ² .

Example 3

1mg of MDR1 monoclonal antibody Clone REA495 (purchased from Miltenyi Biotech Co., Ltd., Germany) was dissolved in 1 mL of antibody dilution solution (Wuhan Ph.D.) to prepare an antibody solution with a concentration of 1 mg / mL. The antibody solution was sprayed onto the surface of the left atrial appendage occluder membrane using an ultrasonic sprayer, and was naturally dried and stored at 4 ° C. Preparation of Clone REA495 loaded membrane was completed. After weighing calculation, the concentration of Clone REA495 on the film surface was finally measured to be 780 g / mm ² .

Example 4

1mg Bcrp1 monoclonal antibody Clone 5D3 (purchased from Miltenyi Biotechnology Co., Ltd., Germany) was dissolved in 1 mL of antibody dilution solution (Wuhan Ph.D.) to prepare an antibody solution with a concentration of 1 mg / mL. Polyelectrolyte sodium hyaluronate (HA) 1mg / mL dissolved in 0.1% NaCl solution, polyelectrolyte chitosan (CS) 1mg / mL dissolved in 0.1% NaCl solution, polyelectrolyte polyimide for base coating Amine (PEI) 5 mg / mL was dissolved in a 0.1% NaCl solution. The antibody linker NHS (100 mM) / EDC (400 mM) was dissolved in a 0.1% NaCl solution.

Wash and dry the bare stent. First immersed in polyacetimide (PEI) solution to obtain the PEI layer; immerse the PEI-treated stent in sodium hyaluronate (HA) solution to obtain the outer layer of HA; then immerse the stent in chitosan (CS) solution In, the CS outer layer is obtained. The above-mentioned HA / CS electrostatic assembly process was repeated 7 times to obtain a sodium hyaluronate / chitosan self-assembled multilayer film, which is denoted as PEI (HA / CS) ₇ . The stent was immersed in an antibody linker NHS (100 mM) / EDC (400 mM) solution and reacted at 4 ° C for 2 hours, then taken out into the antibody solution and reacted at 37 ° C for 2 hours, and N _{2 was} taken out and blow dried. The preparation of the Clone 5D3 loaded scaffold was completed. The concentration of Clone 5D3 on the surface of the obtained stent was 813 g / mm ² .

Example 5

This example is the in vivo and in vitro function evaluation of the scaffolds loaded with Clone 5D3 or Clone REA495 antibody in three ways in Examples 1, 2, and 4.

5.1 Evaluation of antibody loading results

The antibody-loaded scaffold was fluorescently stained with a TRITC-labeled anti-mouse IgG secondary antibody to identify whether the antibody was successfully loaded. FIG. 1 shows the fluorescence pictures of the samples of Example 1, Example 2, and Example 4 stained with a secondary antibody fluorescent dye in order from left to right. It can be seen from the figure that the scaffold can be stained with fluorescent colors after being loaded with antibodies. This indicates that the antibody can be successfully loaded on the surface of the scaffold.

5.2 Cell Capture Experiment

Collect 5 mL of rabbit venous blood and mix it with PBS. The volume ratio of rabbit venous blood to PBS is 1: 1. The diluted blood is slowly added to the surface of Ficoll lymphocyte separation fluid, and the volume ratio of diluted blood to separation fluid is 1. Centrifuge for 30 minutes at 2500 rpm; aspirate the lymphocyte layer rich in platelet-rich plasma into a clean centrifuge tube, add 5 mL PBS, pipette and mix well, centrifuge at 2000 rpm for 10 minutes; remove the clear solution from the upper layer, and then add 5 mL PBS Mix with a pipette, centrifuge at 2000 rpm for 10 minutes, remove the supernatant to obtain the bottom cells, cover the centrifuge tube cap, and transfer to a sterile operating table. Open the centrifuge tube cover, add 6 mL of EGM-2 medium to the bottom of the tube, and mix by pipetting to obtain a cell suspension. The 6-well plate covered with fibronectin was used, and the antibody-supported scaffolds in Examples 1, 2, and 4 were placed in different 6-well plate wells. Take the mixed cell suspension and add 2mL of cell suspension to each well. The six-well plate was placed in a 37 ° C incubator with a CO ₂ concentration of 5% with shaking culture. After 2 hours, the 6-well plate was taken out, the cells were fixed, and the cells were stained with antibody dyes (CD338-PE, CD243-PE) grafted with phycoerythrin fluorescent groups, respectively, and the cell performance was identified.

Because Bcrp1 cells captured by Bcrp1 antibody must express Bcrp-1, MDR1 cells captured by MDR1 antibody must express MDR-1. Therefore, the antibody fluorescent dye CD338-PE corresponding to Bcrp-1 and the CD243-PE fluorescent dye corresponding to MDR-1 are used. Dyes can fluorescently capture captured cells. The samples in Examples 1, 2, and 4 captured cells in the blood, and the cells captured on the surface of the samples could be stained with red fluorescence by the phycoerythrin fluorescent dye. Figure 2 shows the fluorescence images of the cells captured by the samples of Examples 1, 2, and 4 after specific antibody fluorescence staining in sequence from left to right, which shows that samples loaded with Bcrp1 antibody or MDR1 antibody can successfully capture hematopoietic stem cells.

5.3 Animal experiments

(A) sample preparation

Bcrp-1 and MDR-1 antibody-loaded scaffolds were prepared according to the methods in Examples 1, 2, and 4. The tools and reagents used in the preparation process need to be sterilized beforehand, and the entire preparation process is completed in a sterile environment. The obtained stent is crimped, blow molded, and packed in a sterile environment. Finally, it was stored at 4 ° C until use.

(Two) sample implantation

Nine New Zealand white rabbits weighing 2.2-2.6 kg were prepared, and the animal weight matched the age. The experimental group was three different cell capture scaffolds, and the control group was a rapamycin drug scaffold with 3 animals in each group. Each rabbit implanted a stent in the left and right iliac arteries as a parallel sample. The device was implanted according to the ratio of target stent: arterial size = 1.10: 1.0 to 1.20: 1.0. Prior to stent implantation, target vessels will be evaluated by angiography. After the stent is implanted, all target vessels will be evaluated again by angiography.

(Three) experimental results

Fourteen days after the stent was implanted, the blood vessels in the stent segment were taken out, cut along the axial direction of the blood vessel, rinsed with normal saline containing heparin for 1 minute, and then transferred to 2.5% glutaraldehyde to fix. The endothelium coverage was observed with a scanning electron microscope. The stent samples were characterized by SEM 14 days after implantation into the iliac artery of the rabbit. FIG. 3a to FIG. 3c are SEM images of the stent samples of Examples 1, 2 and 4, in order, and FIG. 3d is a SEM image of the stent samples of the control group. It can be found from FIG. 3 that the vascular endothelium had complete endothelial tissue coverage and the endothelial cells had a good morphology 14 days after the stent samples of Examples 1, 2 and 4 were implanted. In the control group, a large amount of fibrin and lymphocytes covered the inner wall of the blood vessel 14 days after the implantation of the drug stent, and the endothelial growth was poor. The results show that the prepared stem cell capture scaffold can achieve better effect of promoting endothelial repair.

Example 6

This example is an evaluation of the function of the left atrial appendage occluder membrane loaded with Clone REA495 in vivo and in vivo in Example 3.

6.1 Evaluation of antibody loading results

The antibody-loaded membrane was fluorescently stained with a TRITC-labeled anti-mouse IgG secondary antibody to identify whether the antibody was successfully loaded. As shown in Figure 4, the left atrial appendage occluder membrane can be stained with fluorescent color after being loaded with antibodies. This indicates that antibodies can be successfully loaded on the membrane surface.

6.2 Cell Capture Experiment

Cell capture experiments were performed on the membrane surface according to the cell capture method in Example 5. As shown in Figure 5, the cells captured on the sample surface could be stained with a phycoerythrin fluorescent dye to a fluorescent color, indicating that the antibody-loaded sample could be successfully captured. Hematopoietic stem cells.

6.3 Left atrial appendage occluder implantation

(1) Sample implantation

The antibody-loaded membrane was sewn on the surface of the left atrial appendage occluder stent, and was sterilized for use. Six beagle dogs with a weight of about 30 kg were prepared, and three in each of the experimental group and the control group. The left atrial appendage was loaded with antibodies in the experimental group, and the left atrial appendage was untreated in the control group. After the animals were anesthetized, atrial septal puncture was performed under X-ray fluoroscopy. After the puncture was successful, the puncture sheath was pushed into the left atrium, the puncture needle was withdrawn, the superhard guide wire was inserted along the dilator, the left atrial appendage occluder sheath was exchanged, and a 6F pigtail catheter was sent along the sheath to the left atrial appendage for imaging. By observing the shape of the left atrial appendage and measuring the width and depth of the opening of the left atrial appendage, an occluder of appropriate specifications was selected for implantation in the left atrial appendage, and the occlusive effect was observed by angiography and the occlusion stability was tested by traction. If the occlusion effect is good and the occluder is stable, release the occluder. Withdraw the sheath tube and complete the operation to complete the left atrial appendage occluder implantation operation.

(2) Experimental results

Fourteen days after the occluder was implanted, the animals were euthanized. The left atrial appendage occluder was dissected out and the left atrial appendage occluder membrane was removed from the stent. After fixation and dehydration, the endothelialization of the membrane surface was observed by SEM. As shown in FIG. 6, the left picture is the sample film loaded with Clone REA 495 in Example 3, and the right picture is the untreated sample film in the control group. The image on the left shows that the membrane surface is completely covered by endothelial cells after the antibody is loaded, and the image on the right shows a large amount of fibrin and blood cells deposited on the untreated membrane surface. This shows that the left atrial appendage occluder using an antibody-loaded membrane can rapidly achieve endothelialization.

The technical scheme provided by the present invention creatively uses the specific antibody of the ATP-binding box protein to achieve the effect of promoting endothelial repair of the medical device. Compared with existing endothelial repair methods, the technical scheme provided by the present invention captures the human hematopoietic stem cells and induces them to produce cells to achieve targeted endothelial repair. Therefore, the side effect of this method is small, and it will not bring other adverse effects, and the comprehensive effect is more ideal.

It should be noted that the above embodiments are only used to explain the technical solution of the present invention, rather than limiting the protection scope of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, a person of ordinary skill in the art can still modify the technical solutions described in the foregoing embodiments, or equivalently replace some or all of the technical features, and these modifications or Instead, the essence of the corresponding technical solution does not depart from the protection scope of the technical solutions of the embodiments of the present invention.

Claims

A use of a specific antibody, characterized in that the specific antibody is a specific antibody of an ATP-binding box protein MDR-1 or Bcrp-1, and the use is in the preparation of a medicament for promoting endothelial repair.
The use according to claim 1, wherein the specific antibody of the ATP-binding cassette protein MDR-1 or Bcrp-1 is Clone REA495 or Clone 5D3.
An implanted medical device, characterized in that the surface of the implanted medical device is loaded with a specific antibody against ATP-binding box protein MDR-1 or Bcrp-1.
The implantable medical device according to claim 3, wherein the specific antibody for the ATP-binding cassette protein MDR-1 or Bcrp-1 is Clone REA495 or Clone 5D3.
The implanted medical device according to claim 4, wherein a specific antibody of the ATP-binding cassette protein is supported on at least one side of the implanted medical device.
The implanted medical device according to claim 4, wherein the implanted medical device is a prosthetic blood vessel, a prosthetic valve membrane, a prosthetic valve stent, a left atrial appendage occluder membrane, a left atrial appendage occluder stent, and a heart Occluder membrane, cardiac occluder stent, or artificial patch.
The implantable medical device according to claim 5, wherein the implantable medical device is a left atrial appendage occluder membrane or a cardiac occluder membrane.
The implanted medical device according to any one of claims 3 to 7, characterized in that, on the surface of the implanted medical device, the concentration range of the specific antibody of the ATP-binding box protein is: 10 to 1000g / mm 2 .
A method for preparing an implanted medical device according to any one of claims 3 to 8, comprising:

Loading at least a portion of the surface of the implanted medical device with a specific antibody to the ATP-binding cassette protein; and / or

A specific antibody of the ATP-binding cassette protein is loaded in a well or a groove of the implanted medical device.
The preparation method according to claim 9, wherein the specific antibody of the ATP-binding box protein is loaded onto the implant by at least one of a direct coating method, a chemical grafting method, and an electrostatic adsorption method. The surface of a medical device.