CN117599177A - Application of medicine for removing IgG/IgM antibody or B cell - Google Patents
Application of medicine for removing IgG/IgM antibody or B cell Download PDFInfo
- Publication number
- CN117599177A CN117599177A CN202311520235.9A CN202311520235A CN117599177A CN 117599177 A CN117599177 A CN 117599177A CN 202311520235 A CN202311520235 A CN 202311520235A CN 117599177 A CN117599177 A CN 117599177A
- Authority
- CN
- China
- Prior art keywords
- dmd
- mice
- muscle
- igg
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000003719 b-lymphocyte Anatomy 0.000 title claims abstract description 54
- 239000003814 drug Substances 0.000 title claims abstract description 32
- 206010013801 Duchenne Muscular Dystrophy Diseases 0.000 claims abstract description 75
- 210000003205 muscle Anatomy 0.000 claims abstract description 44
- 229940079593 drug Drugs 0.000 claims abstract description 14
- 210000001087 myotubule Anatomy 0.000 claims abstract description 10
- 238000011282 treatment Methods 0.000 claims abstract description 7
- 230000009693 chronic damage Effects 0.000 claims abstract description 4
- 230000035876 healing Effects 0.000 claims abstract description 4
- 230000007704 transition Effects 0.000 claims abstract description 3
- 230000001737 promoting effect Effects 0.000 claims abstract 2
- 210000002540 macrophage Anatomy 0.000 claims description 14
- 230000035772 mutation Effects 0.000 claims description 12
- 102000001039 Dystrophin Human genes 0.000 claims description 8
- 108010069091 Dystrophin Proteins 0.000 claims description 8
- 208000026350 Inborn Genetic disease Diseases 0.000 claims description 6
- 208000016361 genetic disease Diseases 0.000 claims description 6
- 230000008595 infiltration Effects 0.000 claims description 6
- 238000001764 infiltration Methods 0.000 claims description 6
- 229940124291 BTK inhibitor Drugs 0.000 claims description 2
- 238000009825 accumulation Methods 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 239000007972 injectable composition Substances 0.000 claims 1
- 201000006938 muscular dystrophy Diseases 0.000 abstract description 23
- 230000002776 aggregation Effects 0.000 abstract description 12
- 238000004220 aggregation Methods 0.000 abstract description 12
- 238000000034 method Methods 0.000 abstract description 10
- 230000000694 effects Effects 0.000 abstract description 9
- 230000008506 pathogenesis Effects 0.000 abstract description 4
- 238000010171 animal model Methods 0.000 abstract description 3
- 230000004761 fibrosis Effects 0.000 abstract description 3
- 206010048654 Muscle fibrosis Diseases 0.000 abstract description 2
- 208000029549 Muscle injury Diseases 0.000 abstract description 2
- 230000002000 scavenging effect Effects 0.000 abstract description 2
- 201000002481 Myositis Diseases 0.000 abstract 1
- 230000004054 inflammatory process Effects 0.000 abstract 1
- 230000008685 targeting Effects 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 155
- 101150015424 dmd gene Proteins 0.000 description 129
- 241000699666 Mus <mouse, genus> Species 0.000 description 35
- 102000004420 Creatine Kinase Human genes 0.000 description 18
- 108010042126 Creatine kinase Proteins 0.000 description 18
- 210000000663 muscle cell Anatomy 0.000 description 17
- 238000010186 staining Methods 0.000 description 15
- 210000002966 serum Anatomy 0.000 description 13
- 108060003951 Immunoglobulin Proteins 0.000 description 12
- 102000018358 immunoglobulin Human genes 0.000 description 12
- 238000010172 mouse model Methods 0.000 description 12
- 230000004220 muscle function Effects 0.000 description 12
- 201000006935 Becker muscular dystrophy Diseases 0.000 description 10
- 230000006872 improvement Effects 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 210000002027 skeletal muscle Anatomy 0.000 description 9
- 101150114401 Fcer1g gene Proteins 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 206010049816 Muscle tightness Diseases 0.000 description 7
- 210000000988 bone and bone Anatomy 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 210000003314 quadriceps muscle Anatomy 0.000 description 7
- 230000002861 ventricular Effects 0.000 description 7
- 230000000747 cardiac effect Effects 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 210000002865 immune cell Anatomy 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 241000195634 Dunaliella Species 0.000 description 5
- 238000005452 bending Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000003125 immunofluorescent labeling Methods 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 230000002107 myocardial effect Effects 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- 102000018682 Interleukin Receptor Common gamma Subunit Human genes 0.000 description 4
- 108010066719 Interleukin Receptor Common gamma Subunit Proteins 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 229940072221 immunoglobulins Drugs 0.000 description 4
- 238000010603 microCT Methods 0.000 description 4
- 210000000107 myocyte Anatomy 0.000 description 4
- 210000005259 peripheral blood Anatomy 0.000 description 4
- 239000011886 peripheral blood Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 208000024891 symptom Diseases 0.000 description 4
- 230000001225 therapeutic effect Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 3
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 3
- 208000007271 Substance Withdrawal Syndrome Diseases 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000004904 shortening Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000011269 treatment regimen Methods 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 2
- 201000008162 B cell deficiency Diseases 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 241001059810 Cantharellula umbonata Species 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 108020004518 RNA Probes Proteins 0.000 description 2
- 239000003391 RNA probe Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000003444 anaesthetic effect Effects 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 230000003176 fibrotic effect Effects 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 210000003141 lower extremity Anatomy 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 238000009256 replacement therapy Methods 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 230000027530 B cell deletion Effects 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 241001456553 Chanodichthys dabryi Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 206010056370 Congestive cardiomyopathy Diseases 0.000 description 1
- 201000010046 Dilated cardiomyopathy Diseases 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 1
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000253999 Phasmatodea Species 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 206010070833 Respiratory muscle weakness Diseases 0.000 description 1
- 208000000875 Spinal Curvatures Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000011281 clinical therapy Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 210000003194 forelimb Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000008802 morphological function Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000002363 skeletal muscle cell Anatomy 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 208000002320 spinal muscular atrophy Diseases 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- YFDSDPIBEUFTMI-UHFFFAOYSA-N tribromoethanol Chemical compound OCC(Br)(Br)Br YFDSDPIBEUFTMI-UHFFFAOYSA-N 0.000 description 1
- 229950004616 tribromoethanol Drugs 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000012285 ultrasound imaging Methods 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
Abstract
The invention provides an application of a medicine for removing IgG/IgM antibodies or B cells. The present invention has found that ectopic aggregation of antibodies on muscle fibers plays a critical role in the pathogenesis of duchenne muscular dystrophy. By drug clearance of IgG antibodies or B cells, aggregation of antibodies is reduced and muscle fibers can gradually transition from a chronic injury state to a healing promoting state. By constructing an animal model, the method for eliminating the IgG antibody or the B cell medicine can effectively reduce the muscle injury, inflammation and fibrosis level of the Du's muscular dystrophy mouse, improve the muscle strength and have no obvious side effect. Therefore, targeting B cells and antibody aggregation by scavenging antibodies or B cell drugs can be used as a novel approach to the treatment of untreated DMD.
Description
Technical Field
The invention relates to the field of treatment of genetic diseases caused by muscular dystrophy protein mutation, and provides an antibody or B cell medicament for effectively treating or relieving Du's muscular dystrophy without obvious side effects for the first time.
Background
The genetic disease caused by muscular dystrophy protein mutation is a recessive muscular dystrophy associated with X, including duchenne muscular dystrophy (Duchenne Muscular Dystrophy, DMD) and becker muscular dystrophy (Becker Muscular Dystrophy, BMD). Thus, the patient is generally male, DMD has a incidence rate of about 1/3500-1/5000 in boys, women are mostly carriers, the incidence rate is rare (1/45000-1/100000), and symptoms are light. Du's muscular dystrophy is due to a mutation in the DMD gene resulting in a deletion of the encoded protein Dystrophin. Patients begin to develop disease about 2-3 years of age, with muscle injury necrosis gradually aggravating as age increases, and the symptoms are mainly represented by continuous degradation of muscle fibers and significant increase of Creatine Kinase (CK) level, and weakness of proximal lower limbs. Generally, patients can not walk independently about 10 years old, need to rely on walking sticks and wheelchairs, and most patients can suffer from spinal curvature, respiratory muscle weakness, dilated cardiomyopathy and the like, and finally die from heart-lung failure about 30-40 years old.
Currently, exon skipping strategies and gene replacement therapies are considered to be the most promising treatment strategies for duchenne muscular dystrophy, but currently there are still major limitations such as: corticosteroid therapy is the only treatment currently available for all types of duchenne muscular dystrophy patients, but has limited efficacy and significant side effects; stop codon readthrough therapies currently lack effective clinical evidence; the efficiency of clinically inducing exon skipping in duchenne muscular dystrophy patients by antisense nucleotides remains relatively low and only for patients with mutations in the hot spot region; strategies to induce exon skipping in duchenne muscular dystrophy patients by gene editing currently have no clinically successful cases (one clinical treatment case, but the patient has died). Gene replacement therapy can successfully induce the generation of artificially designed truncated Dystrophin protein clinically at present, but whether the generated truncated Dystrophin protein has functions or not is still to be further studied, and in addition, the generated truncated Dystrophin protein can possibly cause self body fluid or cell immune response and the like. Thus, existing treatment regimens are optimized, further understanding of the pathogenesis of duchenne muscular dystrophy, and developing new effective and safe therapies for duchenne muscular dystrophy, which are critical to the treatment of duchenne muscular dystrophy.
B cells, also known as B lymphocytes, are differentiated from hematopoietic stem cells in the bone marrow. B cells play an important role in the immune system, and morphological function abnormalities of B cells can cause a variety of diseases, such as B cell tumors, autoimmune diseases, multiple sclerosis, and the like. B cells differentiate into plasma cells upon antigen stimulation, which further produce and secrete antibodies. Antibodies perform humoral immunity primarily by neutralizing toxins and preventing pathogen invasion, activating complement to produce an tapping complex that lyses and destroys cells, opsonophagocytosis, and antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies play an important role in a number of clinical diagnostic, prophylactic and therapeutic procedures. B cell removal therapies are effective in transiently removing B cells and reducing antibody production by B cells, thereby ameliorating the disease processes caused by B cell morphological dysfunction, and are now widely used in clinical therapies. However, the therapeutic effect of antibody or B cell-clearing drugs in duchenne muscular dystrophy has not been reported yet.
Disclosure of Invention
The present invention aims to develop new effective and safe duchenne muscular dystrophy treatment strategies by further understanding the pathogenesis of duchenne muscular dystrophy. The present invention has found that ectopic aggregation of antibodies on muscle fibers plays a critical role in the pathogenesis of duchenne muscular dystrophy. By reducing aggregation of antibodies by scavenging antibodies or B cell drugs, muscle fibers can gradually transition from a chronic injury state to a state that promotes healing. And the phenomenon and the therapeutic effect of the drug are effectively verified in a DMD mouse model.
The technical scheme adopted by the invention is as follows:
the application of a medicine for eliminating IgG/IgM antibodies or B cells is used for preparing medicines for treating or delaying genetic diseases caused by muscular dystrophy protein mutation. The medicine for removing IgG/IgM antibody or B cell specifically refers to a medicine for removing IgG antibody, a medicine for removing IgM antibody, a medicine for removing B cell, or a medicine for simultaneously removing two or more of IgG antibody, igM antibody and B cell.
The IgG/IgM antibody or B cell removal drug reduces accumulation of the IgG/IgM antibody in the muscle of a patient through the antibody or B cell removal, reduces infiltration of M1 type macrophages, and improves the rate of M2 type macrophages so as to gradually convert myofibers from a chronic injury state to a healing promotion state.
The genetic diseases caused by the muscular dystrophy protein mutation comprise Du muscular dystrophy and Becky muscular dystrophy with various mutation types.
The IgG/IgM antibody or B cell eliminating medicine is injection.
The IgG/IgM antibody or B cell eliminating medicine is anti-CD20, anti-CD19 CART, anti-FcRn, fcgamma RI inhibitor or BTK inhibitor, etc.
The invention has the following effective effects: the medicine of the invention reduces the aggregation level of the antibody on the muscle fiber of the DMD animal model by using the antibody clearing or B cell medicine, reduces the infiltration of M1 type macrophages in the muscle of the DMD animal model, improves the ratio of M2 type macrophages, realizes the effect of treating or delaying Dunaliella muscular dystrophy, and has obvious effect of treating or delaying the Dunaliella muscular dystrophy. The invention provides a better strategy for clinically treating Du's muscular dystrophy with different mutation types, and has better clinical prospect.
Drawings
FIG. 1 is a graph showing the result of aggregation of immunoglobulin on the myomembrane of a mouse model of Du's muscular dystrophy. Wherein A is Dmd of 2 weeks of age E4* And immunofluorescence staining of IgG and IgM on tibialis anterior myomembrane of Mdx mice, where Laminin is a marker of myomembrane (scale: 20 μm); b is Dmd of 8 weeks of age E4* And immunofluorescence staining patterns of IgG and IgM on tibialis anterior myomembrane of Mdx mice (scale: 100 μm); dmd with B cell deletion C E4* Mouse (Dmd) E4* Δb) staining pattern of IgG on tibialis anterior myomembrane after intraperitoneal injection of IgG from normal mice (15 mg/kg, twice weekly for 6 weeks) (scale: 50 μm).
Fig. 2 is a graph showing the results of immunoglobulin aggregation on the myomembranes of Duchenne Muscular Dystrophy (DMD) patients and Becker Muscular Dystrophy (BMD) patients. The muscle samples of 7 patients with DMD (including the muscle samples of 1 patient with 3 years old DMD, the muscle samples of 3 patients with 4 years old DMD, the muscle samples of 2 patients with 6 years old DMD, the muscle samples of 1 patient with 11 years old DMD), the muscle samples of 3 patients with BMD (the ages of 10 years old, 21 years old, 23 years old, and the 21 years old BMD patient with severe symptoms) were included, the control group was 4 relatively normal, and the other muscle patients with lighter symptoms (1 patient with spinal muscular atrophy, about 20 years old; 1 patient with ataxia, 23 years old; 1 patient with limb muscular dystrophy type 4, 34 years old; 1 patient with tonic muscular dystrophy, 30 years old).
FIG. 3 is a graph of B cell deficiency at week 8 years Dmd E4* Mouse (Dmd) E4* Δb) compared to 8 week old Dmd E4* Results of improvement of muscle function in mice. Wherein A is Dmd detected by Western blotting E4* Results of immunoglobulin expression in skeletal muscle of Δb mice; b is Dmd with the age of 8 weeks E4* Δb mice and Dmd E4* Comparison graph of the residual muscle tension of mice; c is Dmd with the age of 8 weeks E4* Δb mice and Dmd E4* A comparative bar graph of creatine kinase content in mouse serum, wherein the creatine kinase content is derived from myocardial and skeletal muscle and reflects damage to the myocardial and skeletal muscle; d is Dmd which is 8 weeks old as observed by micro-CT E4* Δb mice and Dmd E4* A photograph of the back bone bending condition of the mice; e is Dmd which is counted by micro-CT and is 8 weeks old E4* Δb mice and Dmd E4* Comparative bar graph of the degree of dorsal bone bending (humpback coefficient) of mice; f is Dmd which is observed by hematoxylin-eosin staining and counted for 8 weeks of age E4* Δb mice and Dmd E4* Ratio bar graph of central nucleated muscle cells in the tibial anterior and rectus femoris of mice.
FIG. 4 is a 12 month-old Dmd B cell deficiency E4* Mouse (Dmd) E4* Δb) compared to 12 months of age Dmd E4* Results of improvement in myocardial and skeletal muscle function in mice. Wherein A is Dmd E4* Δb mice and Dmd E4* Comparing the survival condition of mice; b is Dmd E4* Δb mice and Dmd E4* Comparative bar graph of creatine kinase content in mouse serum; c is Dmd E4* Δb mice and Dmd E4* Comparison graph of the residual muscle tension of mice; d is Dmd by Marsonian dyeing E4* Δb mice and Dmd E4* Mouse cardiac muscleA histogram of tibial anterior, rectus, diaphragmatic fibrotic ratio; e is Dmd counted by micro-CT E4* Δb mice and Dmd E4* Comparative histogram of the degree of dorsal bone bending in mice; f is counted Dmd by an ultrasonic imaging system E4* Δb mice and Dmd E4* Comparison histogram of left heart ejection fraction and left ventricular short axis shrinkage in mice.
FIG. 5 is a graph showing the results of improvement in muscle function in B cell-deficient 12 month-old Mdx mice (MdxDeltaB) compared to 12 month-old Mdx mice. A is a comparison graph of the residual muscle tension of the Mdx delta B mouse and the Mdx mouse; b is a comparative bar graph of creatine kinase content in serum of Mdx delta B mice and Mdx mice; c is a histogram of the ratio of central nucleated muscle cells in tibialis anterior and rectus femoris of 12 month old MdxDeltaB mice and Mdx mice observed by hematoxylin-eosin staining and counted; d is a histogram of mdxΔb mice and Mdx myocardium, tibialis anterior, rectus femoris, diaphragmatic muscle fibrosis ratio counted by masson staining for 12 months of age.
Fig. 6 is a graph of the results of ectopic aggregation of antibodies on muscle fibers resulting from expression of fcyri in a DMD mouse model. Wherein A is a method comprising the steps of combining Protein A/G beads with Dmd of 8 weeks of age E4* ，Dmd E4* Incubating the lysate of the mouse skeletal muscle with delta B and WT delta B, enriching the protein possibly combined with immunoglobulin in the mouse muscle, and analyzing the result by means of mass spectrum; b is Dmd of 8 weeks of age E4* And immunofluorescence staining of fcyri on tibialis anterior myomembrane of Mdx mice, where Laminin is a marker of myomembrane (scale: 100 μm); c is Dmd for detecting 8 weeks old by RNA probe acrobatics E4* The result of the expression of FcγRI in tibialis anterior muscle cells of mice is shown in which Laminin is a marker of muscle cell membrane, DAPI marks the nucleus, and signals in cytoplasm pointed by arrows in the figure are FcγRI mRNA marked by RNA probes (scale: 20 μm); d is Dmd of 8 weeks of age E4* Dmd and Fc common gamma chain knockout E4* (Fcer1g -/- Dmd E4* ) Immunofluorescence staining results for fcyri on tibialis anterior myomembrane of mice (scale: 100 μm).
FIG. 7 is a 6-7 week old Fcer1g -/- Dmd E4* Mice are compared to 6-7 weeks of age Dmd E4* Results of improvement of muscle function in mice. Wherein A is Fcer1g which is observed by hematoxylin-eosin staining and is counted at 6-7 weeks of age -/- Dmd E4* Mice and 6-7 week old Dmd E4* Ratio bar graph of central nucleated muscle cells in the mouse tibialis anterior and rectus femoris; b is Fcer1g of 6-7 weeks old -/- Dmd E4* Mice and 6-7 week old Dmd E4* Comparative bar graph of creatine kinase content in mouse serum; c is Fcer1g of 6-7 weeks old -/- Dmd E4* Mice and 6-7 week old Dmd E4* Comparison bar graph of the residual muscle tension of mice.
FIG. 8 is Dmd of Anti-CD20 administered initially at 1 week of age E4* Mice compared with control Dmd E4* Results of improvement of skeletal muscle function in mice. Wherein A is Dmd E4* Time of administration of mice versus time of detection of myocardial and skeletal muscle function; b is Dmd E4* A plot of the change in the proportion of B cells in blood following administration to mice; c is Dmd E4* A graph of the detection of IgG and IgM content in muscle after 4 weeks of mice administration; d is Dmd E4* Administration group mice and Dmd E4* A comparison graph of creatine kinase content in serum of mice in the control group; e is Dmd E4* Administration group mice and Dmd E4* Comparison of the remaining muscle tension of mice in the control group; f is Dmd counted by micro-CT E4* Administration group mice and Dmd E4* Comparison bar graph of the degree of dorsal bone bending of control mice; g is Dmd E4* Administration group mice and Dmd E4* Comparison of survival of mice in control group.
FIG. 9 is Dmd of Anti-CD20 administered initially at 1 week of age E4* Mice compared with control Dmd E4* Results of myocardial and skeletal muscle pathology in mice. Wherein A is Dmd E4* Administration group mice and Dmd E4* A histogram of the rate of rectus femoris and diaphragmatic myofibrosis in the control mice; b is Dmd counted by an ultrasonic imaging system E4* Administration group mice and Dmd E4* A comparative histogram of left heart ejection fraction and left ventricular short axis shrinkage for control mice; c is flow staining by isolation of immune cells in mouse muscle, statistics Dmd E4* Administration group mice and Dmd E4* Representative graph of the change in M1/M2 type macrophage ratio in the control group mouse muscle; d is Dmd E4* Administration group mice and Dmd E4* Statistical plots of M1/M2 type macrophage ratio in the muscle and number of infiltrating immune cells in the muscle of control mice.
FIG. 10 is a graph showing the results of improvement in muscle function in Mdx mice initially dosed with Anti-CD20 at 1 week of age compared to control Mdx mice. Wherein, A is a dosing time chart of a 1-week-old Mdx mouse; b is a comparison graph of creatine kinase content in serum of mice of the 1-week-old Mdx administration group and mice of an Mdx control group; c is a graph of the comparison of the residual muscle tension of mice in the 1-week-old Mdx administration group and mice in the Mdx control group.
FIG. 11 is a Dmd of Anti-CD20 administered initially at 8 weeks of age E4* Mice compared with control Dmd E4* Results of improvement of muscle function in mice. Wherein A is Dmd at 8 weeks of age E4* Time chart of mice dosing; b is Dmd with the age of 8 weeks E4* A plot of the change in the proportion of B cells in blood following administration to mice; c is Dmd with the age of 8 weeks E4* Administration group mice and Dmd E4* A comparison graph of creatine kinase content in serum of mice in the control group; d is Dmd with the age of 8 weeks E4* Administration group mice and Dmd E4* Comparison of remaining muscle tension in control mice.
Detailed Description
The invention provides application of an antibody or B cell removal drug in preparing a drug for treating or relieving Dunaliella Muscular Dystrophy (DMD), wherein in the application, the antibody or B cell removal drug can effectively reduce the aggregation level of the antibody on muscle fibers of a DMD mouse model, reduce infiltration of M1 type macrophages in the muscle of the DMD mouse model, improve the ratio of M2 type macrophages in the muscle, and realize the effect of treating or delaying the Dunaliella muscular dystrophy.
The invention is further described below with reference to the drawings and examples; of these, in the examples below, the C57BL/10-Mdx mouse is a model of a Dystrophin deletion (due to the presence of a point mutation on exon 23 of the Dmd gene) purchased from Jackson laboratories (Jackson Laboratory); WT ΔB mice, i.e., μMT mice, are a B cell deficient mouse model (Ighmtm1Cgn targeted mutant homozygote mice), purchased from Jackson laboratories (Jackson Laboratory); c57BL/10-Dmd E4* The mice are a Dystrophin-deleted mouse model (caused by the deletion of 4 bases on exon 4 of Dmd gene) and are derived from the national model mouse resource center (Nanjing, china); fcer1g -/- Mice are one type of Fc common gamma chain (encoded by Fcer1 g) knockout mice, given by the university of Shanghai transportation, available in Jackson laboratories (Jackson Laboratory); wild type C57BL/6 mice were purchased from Fukang Biotechnology Co., ltd. C57BL/10-Mdx mice and C57BL/10-Dmd E4* Mice were backcrossed to the C57BL/6 background for at least ten passages. In addition, mdxΔB and Dmd E4* ΔB is obtained from Mdx mice and Dmd, respectively E4* Two passages of mice were hybridized with μMT mice to obtain B cell deficient DMD mice. Fcer1g -/- Dmd E4* By Dmd E4* Mice and Fcer1g -/- Mice were crossed for two generations to obtain mice with double deletions of the Fc common gamma chain and Dystrophin.
In addition, unless otherwise specified, percentages in this application are mass percentages.
Example 1: in vivo experiments in mice demonstrated that significant improvement in disease progression in B cell and antibody knockout DMD mice for Du's muscular dystrophy occurred
DMD mice Dmd were obtained at 2 and 8 weeks of age E4* And tibialis anterior myomembrane of Mdx mice, respectively, were stained with IgG and IgM immunofluorescence, and found to have significant aggregation of immunoglobulins on the surface of the myomembrane of DMD mice at 2 and 8 weeks of age compared to WT mice (as in fig. 1a,1 b). Likewise, obtaining muscle membranes from Du's Muscular Dystrophy (DMD) and Becky's Muscular Dystrophy (BMD) patients for immunofluorescent staining of IgG and IgM, respectively, the same phenomenon was observed in muscle sample sections of DMD and BMD clinical patients, namely: compared to control patients (fig. 2A), DMD and BMD clinical patients had significant aggregation of immunoglobulins on the surface of the muscle cell membrane. Furthermore, by giving Dmd of 1 week of age E4* Delta B mice (B cell deficient DMD mice) were continuously intraperitoneally injected with WT mice IgG (15 mg/kg, administered for 6 weeks, twice weekly) and found to bind to WT mice IgGIs combined with Dmd E4* The surface of Δb mouse muscle cell membrane (see fig. 1C). These results indicate that non-anti-autoantibodies can bind to the surface of the muscle cell membrane of duchenne muscular dystrophy mice.
Dmd by Western blot detection E4* Whether there is immunoglobulin residue in the Δb muscle. The abdominal cavity was filled with 2.5% Avertin 300-500. Mu.l, and 10ml 1 XPBS was used by syringe for heart perfusion. A proper amount of skeletal muscle tissue was cut by surgical scissors, RIPA lysate (150 mM NaCl,1% NP-40,2mM EDTA,50mM Tris pH 7.4,0.1% SDS) was added, and the tissue was homogenized. Standing at 4deg.C for 30min, and shaking once for 5min. Centrifuge 4 ℃,14000rpm, centrifuge 30min. Taking the supernatant, adding a certain volume of 6 XSDS loading dye, uniformly mixing, and denaturing at 95 ℃ for 10min. Subsequently, the mixture was allowed to stand at 4℃to conduct SDS-PAGE gel electrophoresis. Transferring film, sealing skimmed milk, and developing ECL chemiluminescent solution. Western blotting showed Dmd compared to WT mice E4* There was a significant enrichment of immunoglobulins (including IgG and IgM) in the muscle, dmd E4* Immunoglobulin could not be detected in the Δb muscle (fig. 3A).
The tension of the mice forelimb muscles was measured by a mouse muscle tensile tester and the maximum force reading was recorded, 10 times at 10 second intervals. The residual tension experimental result shows that Dmd with the age of 8 weeks E4* Δb,12 month age Dmd E4* Δb and mdxΔb, compared to control group Dmd, which was 8 weeks old E4* Dmd of 12 months of age E4* And Mdx mice showed significant improvement in muscle function (fig. 3b,4c,5 a).
The creatine kinase content in the mouse serum was quantified by the CREATINE KINASE-SL ASSAY (326-10, sekisui) kit. Firstly, carrying out gradient dilution on a standard substance for drawing standard curves, simultaneously adding the standard substance and a sample to be tested into a prepared creatine kinase detection reaction liquid (CK-SL Buffer Reagent (R1) and CK-SL Substrate Reagent (R2) are uniformly mixed according to a ratio of 4:1), reacting for 5-10 minutes at room temperature, detecting the absorbance of the standard substance and the sample to be tested under the absorbance of 340nm by using an enzyme-labeling instrument, drawing a standard curve, and measuring the creatine kinase content of the sample to be tested. The creatine kinase content measurement experiment result shows that Dmd with the age of 8 weeks E4* Δb,12 month age Dmd E4* Δb and mdxΔb, compared to control group Dmd, which was 8 weeks old E4* Dmd of 12 months of age E4* And the creatine kinase content in Mdx serum was significantly reduced (fig. 3c,4b,5 b).
Mice of 8 weeks or 12 months of age were intraperitoneally injected with 2.5% Avertin300 μl-500 μl, fixed by tape after the mice were anesthetized, and placed in a Micro CT machine for photography. Subsequently, 3D reconstruction was performed on the multiple photographs taken using analytical software from bruke SkyScan 1276Micro CT to obtain complete bone images of the mice. Wherein, the calculation of the humpback coefficient means that a line (A1) is drawn between the trailing edge (C7) of the last cervical vertebra and the trailing edge of the sixth lumbar vertebra (L6). Another line (A2) is drawn perpendicular to the dorsal margin of the vertebrae at the point of maximum curvature. The humpback coefficient is calculated as A1/A2. The results of Micro CT show that: dmd of 8 weeks of age E4* Δb,12 month age Dmd E4* Δb, compared to control group Dmd of 8 weeks of age E4* Dmd of 12 months of age E4* The degree of curvature of the dorsal bone is significantly improved (see fig. 3d,3e,4 e).
The muscle tissue of the mice with the age of 8 weeks or 12 months is obtained, then is added into 10 percent neutral formalin for fixation, dehydrated and sliced after paraffin embedding. Subsequently, paraffin sections were subjected to hematoxylin-eosin staining and masson staining, and the degree of newly generated myocytes (central nuclear myocytes) (see fig. 3f,5 c) and fibrosis (see fig. 4d,5 d) in the mouse muscle tissue were observed. Hematoxylin-eosin staining statistics indicated that the ratio of tibialis anterior and rectus femoris central nuclear muscle cells was significantly reduced in DMD mice with B cell knockdown at 8 weeks of age, more closely to WT groups (fig. 3f,5 c) than in DMD mice. The masson staining statistics showed that the rate of fibrotic changes in the anterior tibial, rectus femoris and diaphragmatic muscles was significantly reduced in the 12 month old B cell knockout DMD mice compared to DMD mice (fig. 4d,5 d).
After the 12 month old mice were dehaired with the dehairing paste, the mice were placed in an anesthesia induction box for anesthesia. The anesthetized mice were fixed in a supine position on a cardiac superoperator's station while the mouth and nose of the mice were plugged into an anesthetic mask at a anesthetic flow rate of 0.8L/min at a concentration of about 1% -1.5% to maintain the anesthetized state of the mice. Ultrasound imaging platform acquisition using Vevo 3100 small animalsThe electrocardiogram of the mice was collected and B-model, M-model data was saved. Using the LV Trace tool, in M-model mode, 3 cardiac cycles are continuously traced along the left ventricular anterior wall endocardial tracing. The same procedure was followed to trace the left ventricular posterior wall, followed by 3 cardiac cycles. Thereafter, the LV Trace automatically generates a left heart ejection fraction, left ventricular short axis shortening rate (see FIG. 4F). Results of cardiac ultrasound showed Dmd with 12 month old B cell knockdown E4* Mice are compared with Dmd E4* The mice, left heart ejection fraction and left ventricular short axis shortening rate were significantly improved, closer to WT group.
In conclusion, the invention achieves the purpose of delaying the disease process of different DMD mice models by knocking out the B cells and the antibodies of the DMD mice.
Example 2: the knockout of Fcgamma can effectively reduce the accumulated IgG on the surface of the muscle cell membrane of the Du's muscular dystrophy mice
By combining Protein A/G beads with Dmd E4* Incubation of mouse muscle lysate (negative control Dmd) E4* Δb and WT Δb mice), 4 degrees incubation for 3 hours, enrichment Dmd E4* IgG and IgG complexes in the mouse muscle lysate were blotted off the supernatant and washed 5 times with wash buffer for 5min each time. Then adding loading buffer, boiling at 95 deg.C for 10min, and preserving sample at-80 deg.C. By subjecting the above to mass spectrometry, the results of the mass spectrometry showed that, compared with the negative control group, at Dmd E4* In the group, protein A/G bead binds to immunoglobulins and complement proteins in a large number of muscles. Furthermore, fcgamm and Trim21 at Dmd E4* Are significantly enriched in the group (as in figure 6A). Further immunofluorescence experiments showed that Mdx and Dmd compared to WT mice E4* The expression of fcyri was detected on both mouse muscle cell membrane surfaces (see fig. 6B). RNAscope experiments showed that FcgRI mRNA probe signals could be detected in the muscle cells of the DMD mice, indicating the presence of FcgRI transcripts in the muscle cells of the DMD mice (FIG. 6C). Furthermore, dmd by constructing Fc common gamma chain knockout E4* (Fcer1g -/- Dmd E4* ) Mice, muscle tissue was taken for immunofluorescence experiments, and as a result, found: fcer1g -/- Dmd E4* Skeletal muscle cell surface of mice is freeThe method detects fcyri expression and significantly reduces IgG binding (see fig. 6D). The results show that the expression of the FcgammaRI exists in the muscle cells of the DMD mice, and the knockout of the FcgammaRI can effectively reduce the accumulated IgG on the surface of the muscle cell membrane of the Dunaliella muscular dystrophy mice.
Further study results showed that hematoxylin-eosin staining of 6-7 week old mouse muscle tissue was performed to observe newly generated myocytes (central nuclear myocytes) in mouse muscle tissue (as shown in FIG. 7A), fcer1g -/- Dmd E4* Mice are compared with Dmd E4* The rate of central nucleated muscle cells was significantly reduced in mice. Furthermore, fcer1g -/- Dmd E4* Mice are compared with Dmd E4* The creatine kinase content in the serum of mice was significantly reduced (fig. 7B), and the muscle function was significantly improved (fig. 7C).
In conclusion, the invention can effectively reduce the combination of IgG on the muscle cell surface of the DMD mouse by knocking out the FcgammaRI of the DMD mouse, thereby achieving the purpose of delaying the disease process of the DMD mouse model.
Example 3: the disease process of DMD can be effectively relieved for a long time by eliminating and knocking out B cells through Anti-CD20 antibody in young mice
Anti-CD20 antibody (2 mg/kg) was intraperitoneally injected into DMD mice of 1 week old, wherein DMD mice of 1 week old were intraperitoneally injected with IgG (2 mg/kg) as a control, 1 time per week, and were continuously administered for 8 weeks (see FIGS. 8A, 10A). After 4 weeks of administration, mice were bled by submaxillary vein, 50 μl of peripheral blood was collected in EP tubes containing EDTA solution for detecting B cells in peripheral blood, and the remainder was used for collecting serum. Centrifugation at 1500rpm for 5 minutes, discarding the supernatant, adding 200. Mu.l of erythrocyte lysate, blowing and mixing well, and standing for 5 minutes. Then 1ml PBS stopped cracking red, 1500rpm centrifugal 5min, collect cell precipitation. 50 μl live/dead fix violet kit (thermo, L34964) was added and dissolved in PBS at a volume ratio of 1:1000 and incubated at 4deg.C for 30 minutes in the absence of light; add 50. Mu.l of anti-CD16/CD32 (2.4G2,BD Pharmingen) in a 1:400 volume ratio in a starting buffer (PBS+1% FBS+1mM EDTA) and block for 10min at 4deg.C; mu.l of a stabilizing buffer (1:200 volume ratio) containing the corresponding surface antibodies anti-mouse CD45-APC/Cyanine7 (30-F11, biolegend,103115 and anti-mouse CD19-FITC (1D 3/CD19, biolegend, 152403) was added, protected from light at 4 ℃Incubate for 30min. The starting buffer was washed twice and 200. Mu.l of the stained starting buffer was resuspended and then run on-machine. It was found by CD45 and CD19 staining that during the administration of DMD mice intraperitoneally injected with Anti-CD20 antibody, the ratio of B cells to immune cells was almost 0, and gradually recovered after withdrawal, and the B cell ratio was recovered to normal level 2-4 weeks after withdrawal (see FIG. 8B). Experimental results of simultaneous Western blot detection show that Dmd is 1 week old E4* Mice were intraperitoneally injected with Anti-CD20 antibody for 4 weeks, compared to Dmd E4* The immunoglobulin content in the mouse muscle was significantly reduced (see fig. 8C).
In addition, the experimental results of both DMD mouse models show that creatine kinase content in serum is continuously reduced in Anti-CD20 group compared to Ctrl IgG group (fig. 8d,10 b), and the levels are kept lower for a longer period of time after drug withdrawal. And a significant improvement in muscle function occurs (as in fig. 8e,10 c). The results of Micro CT show that: the extent of dorsal bone bending was still significantly improved in Anti-CD20 mice compared to Ctrl IgG mice 8 months after withdrawal (fig. 8F). Compared with Ctrl IgG group Dmd E4* Mice, anti-CD20 group Dmd E4* The survival time of mice was also significantly prolonged (see FIG. 8G). Further Marsonian staining showed that compared to Ctrl IgG group Dmd E4* Mice, anti-CD20 group Dmd E4* The rate of fibrosis was significantly reduced in the rat rectus and diaphragmatic muscles (figure 9A). Results of cardiac ultrasound showed that, 8 months after drug withdrawal, the Anti-CD20 group mice had significantly improved left heart ejection fraction and left ventricular short axis shortening rate compared to Ctrl IgG group mice, more closely to WT group (fig. 9B).
To further reveal the reason for the sustained therapeutic effect of Anti-CD20 antibodies, the present invention utilized Percoll (GE Healthcare) density gradient centrifugation to isolate Anti-CD20 and Ctrl IgG Dmd groups 10 weeks after drug withdrawal E4* Immune cells in mouse muscle tissue were compared as follows: euthanized mice, the muscle tissue of the upper and lower limbs of the mice were removed and placed in pre-chilled PBS buffer, the muscle was minced by surgical scissors, and the tissue digest (containing 1640 medium, 1% FBS,10mM HEPES,1mM MgCl) 2 ，1mM CaCl 2 0.1mg/ml Dnase I, collagenase II:1 mg/ml) for 30min at 37 ℃. Grinding with 100 μm pore size cell screen, centrifuging at 1500rpm at 4deg.C for 10min, re-suspending with 5mL 40vol%Percoll solution, transferring onto 70vol% Percoll solution, and performing density gradient centrifugation at 600×g at 22deg.C for 30min at a speed of 2 and deceleration of 0. And (3) centrifuging, taking the middle white cloud layer cells, washing twice by using precooled PBS, counting cells, staining by using a flow antibody, and detecting on the machine. The results of flow staining indicate that Anti-CD20 antibodies can continuously reduce the number of immune cells infiltrating the muscle of DMD mice model, mainly reducing M1 type macrophages infiltration, while increasing the rate of M2 type macrophages in the muscle, resulting in a sustained therapeutic effect (fig. 9c,9 d).
In conclusion, the Anti-CD20 antibody is injected into the abdominal cavity of the DMD mouse at the age of 1 week to reduce the level of immunoglobulin in the muscle of the DMD mouse, reduce the infiltration of M1 type macrophages and improve the ratio of M2 type macrophages in the muscle, so that a sustainable treatment effect is generated, the disease characteristics of the DMD mouse are improved, and the effect of delaying the disease process of a DMD mouse model is achieved.
Example 4: adult mice can effectively relieve disease process of DMD by eliminating and knocking out B cells through Anti-CD20 antibody
Dmd of 8 weeks of age E4* Mice were intraperitoneally injected with Anti-CD20 antibody (2 mg/kg), dmd at 8 weeks of age E4* Mice were given IgG (2 mg/kg) intraperitoneally as a control, 1 time per week, for 8 weeks (see fig. 11A). The peripheral blood of the mouse was collected by the submaxillary vein, and the flow type result showed that the ratio of B cells in the peripheral blood to immune cells was almost 0 as in the case of the intraperitoneal injection of Anti-CD20 antibody to the young mouse, and gradually recovered after stopping the drug, and the ratio of B cells recovered to the normal level 2-4 weeks after stopping the drug (see FIG. 11B). Further, further experimental results showed that Dmd, which was 8 weeks old E4* The creatine kinase content in serum was continuously decreased (fig. 11C) and maintained at a lower level for a longer period of time after discontinuation of the drug administration in mice when the Anti-CD20 antibody was intraperitoneally administered compared to Ctrl IgG group for about 6-8 weeks. The muscle function was significantly improved in the Anti-CD20 group compared to Ctrl IgG group (fig. 11D). The results show that the removal of the knocked-out B cells by the Anti-CD20 antibody can effectively relieve adult smallDMD disease progression in mice.
Claims (5)
1. The use of a medicament for the removal of IgG/IgM antibodies or B cells for the preparation of a medicament for the treatment or delay of a genetic disorder caused by a mutation in a dystrophin protein.
2. The use according to claim 1, wherein the strategy of eliminating IgG/IgM antibodies or B-cell drugs reduces accumulation of IgG/IgM antibodies in the patient's muscle, reduces infiltration of M1 type macrophages, and increases the rate of M2 type macrophages by eliminating antibodies or B-cells, achieves a gradual transition of myofibers from chronic injury status to healing promoting status.
3. The use according to claim 1, wherein said genetic diseases caused by mutations in dystrophin include duchenne muscular dystrophy and becker muscular dystrophy of various mutation types.
4. The use according to claim 1, wherein the IgG/IgM antibody-scavenging or B cell drug is an injectable formulation.
5. The use according to claim 1, wherein the IgG/IgM antibody-clearing or B-cell medicament is an anti-CD20, anti-CD19 CART, anti-FcRn, fcyri inhibitor or BTK inhibitor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311520235.9A CN117599177A (en) | 2023-11-15 | 2023-11-15 | Application of medicine for removing IgG/IgM antibody or B cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311520235.9A CN117599177A (en) | 2023-11-15 | 2023-11-15 | Application of medicine for removing IgG/IgM antibody or B cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117599177A true CN117599177A (en) | 2024-02-27 |
Family
ID=89943522
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311520235.9A Pending CN117599177A (en) | 2023-11-15 | 2023-11-15 | Application of medicine for removing IgG/IgM antibody or B cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117599177A (en) |
-
2023
- 2023-11-15 CN CN202311520235.9A patent/CN117599177A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Carpenter et al. | Feline muscular dystrophy with dystrophin deficiency | |
| Liang et al. | Mesenchymal stem cell transplantation for diffuse alveolar hemorrhage in SLE | |
| JP6353073B2 (en) | Composition for cell recovery and method for producing and using the same | |
| EA001379B1 (en) | Method of treating a neurological disorder in a mammal, method of stimulating and promoting growth of damaged peripheral nerves, method, for promoting neuronal regeneration and growth and method for preventing neurodegeneration in a mammal | |
| US10561623B2 (en) | Compositions and methods for treating muscular dystrophy and other disorders | |
| EP4190336A1 (en) | Application of mesenchymal stem cells in preparation of drug for repairing lung damage caused by covid-19 | |
| Shirai et al. | Effects of natural thymocytotoxic autoantibody of NZB mice and of specifically prepared antilymphocyte serum on the tissue distribution of 51Cr-labeled lymphocytes | |
| Antonius et al. | Steroid-responsive Rosai-Dorfman disease | |
| Gu et al. | A biomimetic adipocyte mesenchymal stem cell membrane-encapsulated drug delivery system for the treatment of rheumatoid arthritis | |
| CN103405751A (en) | Composition with cell repairing function and preparation method and application thereof | |
| CN117599177A (en) | Application of medicine for removing IgG/IgM antibody or B cell | |
| US20050249708A1 (en) | Cerebral Intraventricular Transplantation as Method of Treating Amyotrophic Lateral Sclerosis (ALS) | |
| Festen et al. | Multifocal recurrent periostitis responsive to colchicine | |
| WO2023236345A1 (en) | Genetic engineering drug for treating inflammatory arthritis and preparation method therefor | |
| CN115671137A (en) | Application of human deciduous tooth pulp stem cell exosome in preparation of tendon injury medicine | |
| US20220403415A1 (en) | Effective dosages of an adenoviral-based biological delivery and expression system for use in the treatment of osteoarthritis in humans, and compositions comprising the same | |
| Zhao et al. | Stimulating factors and origins of precursor cells in traumatic heterotopic ossification around the temporomandibular joint in mice | |
| KR20180115297A (en) | The use of BYL719 (alpericib) for the treatment of PIK3CA-related overgrowth spectra (PROS-CLOVES syndrome) | |
| JP2023552443A (en) | Danon disease treatment | |
| CN108392624B (en) | Activity promoting peptide and application of mesenchymal stem cells in treating rheumatoid arthritis | |
| CN105326844A (en) | Application of tofacitinib citrate and pharmaceutical composition thereof in preparation of medicine for treating sjogren syndrome | |
| WO2020175954A1 (en) | METHOD FOR TREATING TNFα-RELATED DISEASES | |
| Rewald et al. | Behcet's syndrome treated with high-dose intravenous IgG and low-dose aspirin | |
| Malik et al. | Clinicopathological features and management of immunoproliferative small intestinal disease and primary small intestinal lymphoma in Pakistan | |
| Walker et al. | Immunomodulators |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |