WO2020009482A1 - Anticorps anti-alpha-synucléine et son utilisation - Google Patents

Anticorps anti-alpha-synucléine et son utilisation Download PDF

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WO2020009482A1
WO2020009482A1 PCT/KR2019/008170 KR2019008170W WO2020009482A1 WO 2020009482 A1 WO2020009482 A1 WO 2020009482A1 KR 2019008170 W KR2019008170 W KR 2019008170W WO 2020009482 A1 WO2020009482 A1 WO 2020009482A1
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synuclein
alpha
antibody
seq
nos
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Korean (ko)
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김동인
김연주
안성원
안진형
손용규
이승재
김태경
최민선
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ABL Bio Inc
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ABL Bio Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to anti-alpha-synuclein antibodies or antigen binding fragments thereof, alpha-synuclein detection and diagnosis of related diseases using the same.
  • Alpha-synuclein ( ⁇ -Synuclein, ⁇ -syn) is expressed mainly at the presynaptic ends of neurons and is present as a monomer in a naturally unfolded state in normal state. Alpha-synuclein helps regulate the release of dopamine, an important neurotransmitter that controls the start and stop of spontaneous and involuntary movements. In particular, the function of alpha-synuclein is important with increasing synaptic activity and with age, and is an important factor of neurodegeneration.
  • alpha-synuclein binds to and interacts with droplets, phospholipid bilayers, or lipid membranes, resulting in structural changes resulting in the formation of a secondary structure in the folded or folded ⁇ -helical form. It forms aggregates comprising molecules in the form of dimers, oligomers and / or fibrous forms.
  • alpha-synuclein aggregates are known to cause toxicity to cells, such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), multiple system atrophy (MSA), and Louis Dementia with Lewy bodies (DLB), a major component of Lewy bodies, abnormal protein aggregates found in neurons of various diseases.
  • Post-translational modifications such as phosphorylation of alpha-synuclein, or ubiquitination are also known to be involved in the aggregation and neurotoxicity of alpha-synuclein.
  • Alpha-synuclein is known to kill dopamine neurons and cause inflammatory responses in animal and cell experiments, and to cause motor symptoms similar to Parkinson's disease in experimental animals.
  • alpha-synuclein aggregation is associated with the pathogenesis of a group of neurodegenerative diseases called synucleinopathy, including Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, multiple systemic atrophy and many other neuroaxonic diseases. It is known that this is.
  • alpha-synuclein has become a target for the treatment of synuclein disease, and furthermore, there is a need for developing a technology for further improving the therapeutic effect by targeting and early diagnosis.
  • Another object of the present invention is to provide a polynucleotide encoding an anti alpha-synuclein antibody or antigen binding fragment thereof, a recombinant vector comprising the same, and a recombinant cell comprising the same.
  • Still another object of the present invention is to provide a composition for detecting alpha-synuclein, a kit for detecting alpha-synuclein and a method for detecting alpha-synuclein.
  • Still another object of the present invention is to provide a kit for diagnosing a-synocleinopathy and a method for providing information for diagnosing synuclein disease.
  • One embodiment of the present invention is an anti-alpha-synuclein ( ⁇ -syn) antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises (i) complementarity determining regions of CDRH1, CDRH2 and CDRH3.
  • a light chain variable region comprising the complementarity determining regions of CDRL1, CDRL2 and CDRL3, wherein the CDRH1 is selected from SEQ ID NOs: 1 to 5, the CDRH2 is selected from SEQ ID NOs: 6 to 10, and the CDRH3 is Selected from SEQ ID NOs: 11 to 15;
  • the CDRL1 is selected from SEQ ID NOs: 16 to 20, the CDRL2 is selected from SEQ ID NOs: 21 to 25, and the CDRL3 is selected from SEQ ID NOs: 26 to 30, to an anti alpha-synuclein antibody or antigen binding fragment thereof It is about.
  • CDRH1, CDRH2 and CDRH3 of the heavy chain variable region; And the sequences of CDRL1, CDRL2 and CDRL3 of the light chain variable region may be any one of the following, an anti-alpha-synuclein antibody or an antigen binding fragment thereof.
  • the CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 1, 6 and 11, respectively, and the CDRL1, CDRL2 and CDRL3 are SEQ ID NOs: 16, 21 and 26, respectively;
  • the CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 2, 7 and 12, respectively, and the CDRL1, CDRL2 and CDRL3 are SEQ ID NOs: 17, 22 and 27, respectively;
  • the CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 3, 8 and 13, respectively, and the CDRL1, CDRL2 and CDRL3 are SEQ ID NOs: 18, 23 and 28, respectively;
  • the CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 4, 9 and 14, respectively, and the CDRL1, CDRL2 and CDRL3 are SEQ ID NOs: 19, 24 and 29, respectively; or
  • the CDRH1, CDRH2 and CDRH3 are SEQ ID NOs: 5, 10 and 15, respectively, and the CDRL1, CDRL2 and CDRL3 are SEQ ID NOs: 20, 25 and 30, respectively.
  • the heavy chain variable region may be an anti-alpha-synuclein antibody or antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOs: 31 to 35.
  • the light chain variable region may be an anti-alpha-synuclein antibody or antigen binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOs: 36 to 40.
  • the heavy chain variable region and light chain variable region are SEQ ID NOs: 31 and 36, respectively; SEQ ID NOs: 32 and 37; SEQ ID NOs: 33 and 38; SEQ ID NOs: 34 and 39; Or it may be to include a sequence represented by SEQ ID NO: 35 and 40, but is not limited thereto.
  • the CDR1 to CDR3 sequences of the anti-alpha-synuclein antibody heavy chain variable region of the present invention may be selected from the amino acid sequences shown in Table 1 below, or may include amino acid sequences having substantial sequence identity with the selected amino acid sequences.
  • the CDR1 to CDR3 sequence of the anti-alpha-synuclein antibody light chain variable region of the present invention may be selected from the amino acid sequences shown in Table 2 below, or may include an amino acid sequence having substantial sequence identity with the selected amino acid sequence.
  • sequence of the antibody heavy chain variable region of the present invention may be selected from the amino acid sequences shown in Table 3 below, or may include an amino acid sequence having substantial sequence identity with the selected amino acid sequence.
  • sequence of the antibody light chain variable region of the present invention may be selected from the amino acid sequences shown in Table 4 below, or may include an amino acid sequence having substantial sequence identity with the selected amino acid sequence.
  • sequence homology more preferably, 80% homology or 90% homology.
  • Alignment methods for sequence comparison are known in the art. In one embodiment it may have about 90%, 95%, or 99% identity with the disclosed heavy chain variable region. In addition, it may have about 90%, 95%, or 99% identity with the light chain variable region disclosed in other embodiments. For example, in the case of a variant that exhibits 90%, 95%, or 99% identity with the sequence of an antibody or antigen binding fragment disclosed herein, any variation may occur in the backbone of the variable region rather than the CDR.
  • antibody refers to a substance produced by stimulation of an antigen in the immune system, and may be generated in vivo, recombinantly generated, or artificially synthesized. It is not particularly limited. Antibodies in the present invention include all animal antibodies, chimeric antibodies, humanized antibodies, and human antibodies. In addition, the antibody in the present invention may include all antigen-binding fragments of the antibody having the antigen-binding ability.
  • the antibody may be selected from all subtypes of immunoglobulins (eg, IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).
  • the antibody in IgG form may be in the form of an IgG1, IgG2, IgG3, or IgG4 subtype, such as an IgG1 or IgG2 subtype.
  • an antigen binding fragment means a portion or polypeptide comprising an antibody having a specific binding ability to an antigen.
  • an antigen binding fragment may be a portion or polypeptide of an antibody comprising amino acid residues that interact with an antigen (eg, an epitope) to confer specificity and / or affinity for the antigen.
  • the antigen binding fragment may be one selected from the group consisting of antibody fragments including one or more of the complementarity determining regions, such as scFv, (scFv) 2, scFv-Fc, Fab, Fab 'and F (ab') 2 . This is not restrictive.
  • Such biologically active fragments may be produced by recombinant DNA techniques or may be produced, for example, by enzymatic or chemical cleavage of an intact antibody. Immunologically functional immunoglobulin fragments are not limited thereto.
  • CDRs complementarity-determining regions
  • alpha-synuclein is human alpha-synuclein, monkey alpha-synuclein (eg, Rhesus Alpha-synuclein), mouse alpha-synuclein, rat alpha-synuclein, and the like, and may be selected from mammal alpha-synuclein, eg, human alpha-synuclein is alpha-synuclein (NCBI ID: NP_000336). It may be, but is not limited thereto.
  • the alpha-synuclein may refer to human alpha-synuclein, and the antibody or antigen-binding fragment thereof provided in the present invention is not only human alpha-synuclein, but also monkey (eg Rhesus), rat, and / or mouse. It may also have a specific binding capacity to alpha-synuclein. Furthermore, it may be recombinant rather than naturally produced (non-natyurally ocurring; for example, chemically synthesized or recombinantly produced), and the recombination may use techniques well known in the art.
  • the antibody or antigen-binding fragment thereof binds to the C-terminal portion of the alpha-synuclein, and specifically, in the amino acid sequence of SEQ ID NO: 41 of the human alpha-synuclein protein, residues C, for example, residues 110 to Or a C-terminal region comprising a peptide consisting of at least 11 or 12 contiguous amino acids comprising residues 120 or 111-122. It was confirmed that the antibody or antigen-binding fragment thereof according to the present invention recognizes the antigen recognition site and binds with high affinity for alpha-synuclein aggregate.
  • affinity or affinity is the strength of the interaction between an antibody or antigen-binding fragment thereof and an antigen, the CDR sequence of the antibody or antigen-binding fragment, and / or the physicochemical of the antibody or antigen-binding fragment. Properties (hydrophilic / hydrophobic, electrostatic properties, etc.), the size, shape of the antigen, and / or characteristics of the antigen such as charge. Methods for determining such affinity are known in the art and may be represented by a dissociation constant (KD), but are not limited thereto.
  • KD dissociation constant
  • light chain includes full length light chains and fragments thereof having variable region sequences sufficient to provide binding specificity for the antigen or epitope.
  • the full length light chain includes the variable region domain VL, and the constant region domain CL.
  • the variable region domain of the light chain is at the amino terminus of the light chain polypeptide.
  • Types of light chains include kappa and lambda chains.
  • “heavy chain” includes full length heavy chains and fragments thereof having variable region sequences sufficient to provide binding specificity for the antigen or epitope.
  • the full length heavy chain comprises the variable region domain VH and three constant region domains CH1, CH2 and CH3.
  • the VH domain is at the amino terminus of the heavy chain polypeptide
  • the CH domain is at the carboxy terminus
  • CH3 is located closest to the carboxy-terminus.
  • Heavy chains include isotypes of IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
  • alpha-synuclein or alpha-synuclein aggregate means that the affinity for alpha-synuclein monomer or alpha-synuclein aggregate is relatively high compared to other antigens.
  • the affinity for alpha-synuclein aggregates can exhibit a dissociation constant (K D ) of 4.0 x 10 -8 M or less in all ELISA, BIAcore and Octet assays, more specifically 1.0 x 10 -13 M to 2 x 10 ⁇ 8 M, but is not limited thereto.
  • the anti-alpha-synuclein antibody or antigen-binding fragment thereof of the present invention is present in the nervous system of the subject, the nerve cell outside of the target nervous system, extracellular space, etc. It was confirmed that it can bind with alpha-synuclein or aggregates thereof with high affinity, in particular, exhibited a high affinity for the aggregates and confirmed that it can bind more specifically (FIGS. 1 to 4).
  • Another embodiment of the present invention relates to a polynucleotide encoding the anti alpha-synuclein antibody or antigen-binding fragment.
  • the polynucleotides can be prepared by known chemical synthesis, and include both DNA, RNA or variants thereof as single or double stranded nucleotide polymers encoding the anti alpha-synuclein antibody or antigen-binding fragment.
  • Another example of the present invention relates to a recombinant vector comprising the polynucleotide.
  • the "recombinant vector” is an expression vector capable of expressing a protein of interest in a suitable host cell, and refers to a gene construct comprising essential regulatory elements operably linked to express the gene insert. Operable linkage with recombinant vectors can be made using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can employ enzymes commonly known in the art.
  • the vector may be an expression vector, and may include a selective marker for selecting a host cell, and in the case of a replicable expression vector, may include a replication origin.
  • Another example of the invention relates to a cell transformed with the recombinant vector.
  • the "transformed cell” means a cell into which a polynucleotide or a recombinant vector of interest is introduced into a host cell. Transformation may be a foreign DNA introduced into the cell by the 'introduction' method, and can be carried out by selecting a suitable standard technology according to the host cell as known in the art. In addition, the type of the transformed cells is not limited.
  • Another embodiment of the present invention relates to a composition for detecting alpha-synuclein, comprising the anti-alpha-synuclein antibody or antigen-binding fragment.
  • the alpha-synuclein may be in the form of aggregates.
  • the antibody included in the detection composition may typically be labeled with a detectable labeling substance.
  • suitable labeling materials include radioisotopes or radionuclides (eg, 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent materials (eg, FITC, rhodamine, lanthanide phosphors), enzymes (e.g., horseradish peroxidase, ⁇ -galactosidase, luciferase, alkaline phosphatase), chemiluminators, biotinyl groups, or secondary reporters Recognized polypeptide epitopes (eg, leucine zipper pair sequences, binding sites for antibodies, metal binding domains, epitope tags) are included, but are not limited to these.
  • the labeling substance can be coupled to the antibody through spacer arms of various lengths to reduce potential steric hindrance.
  • Various methods for labeling proteins are known
  • the antibodies of the present invention can be used for identification of tissues comprising alpha-synuclein aggregates.
  • the antibody may be labeled with a label, and binding of the labeled antibody to alpha-synuclein aggregates may be detected.
  • kits comprising the alpha-synuclein detection composition.
  • the kit may be used together with various labels, buffers, and the like, as well as agents capable of detecting alpha-synuclein, synuclein aggregates, etc. of the present invention, and may include all kits known in the art. However, the present invention is not limited thereto.
  • Another embodiment of the present invention comprises the steps of: a) reacting the anti alpha-synuclein antibody or antigen-binding fragment with a sample; And b) detecting the reactant in step a).
  • the sample may include tissue, cells, whole blood, plasma, serum, blood, saliva, sputum, lymph, cerebrospinal fluid, intercellular fluid, tear or urine, as well as samples of cell culture components in vitro, for example, cellular components, Cell culture media, recombinant cells and the like.
  • the detection method may be performed in vitro or in vivo.
  • In vivo imaging can be performed using, for example, positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging or magnetic resonance imaging (MRI).
  • PET positron emission tomography
  • SPECT single photon emission tomography
  • NIR near infrared optical imaging
  • MRI magnetic resonance imaging
  • In Vitro methods can also be performed using Western blot, immunoprecipitation, Enzyme Linkied Immuno Sorbent Assay (ELISA), Radioimmunoassay (RIA) or immunohistochemical methods well known to those skilled in the art.
  • ELISA Enzyme Linkied Immuno Sorbent Assay
  • RIA Radioimmunoassay
  • the anti-alpha-synuclein antibody or antigen-binding fragment thereof of the present invention can detect a small amount of alpha-synuclein aggregate by binding to alpha-synuclein or alpha-synuclein aggregate with high affinity. It was confirmed (FIGS. 2-4). From this, by using the anti-alpha-synuclein antibody or antigen-binding fragment thereof of the present invention, the presence of alpha-synuclein aggregates in the brain, central nervous system, etc. can be detected early before the concentration of aggregates increases, thereby premature synuclein disease is detected. I can diagnose it.
  • kits for diagnosing synocleinopathy comprising the composition for detecting alpha-synuclein.
  • the kit may be used in addition to the agent capable of detecting alpha-synuclein, synuclein aggregates, etc. of the present invention or the agent capable of diagnosing Synuclein disease, various labels, buffers, and the like, and are known in the art. It may include the configuration of all kits, but is not limited thereto.
  • “synocleinopathy” includes all neurodegenerative disorders characterized by pathological synuclein aggregates. Parkinson's disease, Parkinson's disease dementia (PDD), dementia with Lewy bodies (DLB), Lewy body disease, dementia with Lewy bodies, Parkinson's syndrome with dementia, multiple system atrophy Several neurodegenerative disorders, including system atrophy (MSA), multiple nervous system atrophy, and neurodegenerative type I (NBIA Type I) with brain iron deposition, are grouped collectively as synuclein disease. Alpha-synuclein aggregation is also secondary to Alzheimer's disease (Kim et al. Alzheimer's Research & Therapy 2014, 6:73).
  • Synuclein disease is a diverse group of neurodegenerative disorders that share common pathological characteristics: In neuropathological experiments, distinctive lesions are alpha-synuclein in selected populations of neurons and oligodendrocytes. It includes abnormal aggregation of proteins and can be detected.
  • Alpha-synuclein is a 140 amino acid protein that is widely expressed in the neocortex, hippocampus, dentate gyrus, posterior neurosphere, striatum, thalamus and cerebellum.
  • Alpha-synuclein is also highly expressed in hematopoietic cells, including protein and platelets, including B-cells, T-cells and NK cells. The exact role of alpha-synuclein in these cells is unknown, but is believed to be involved in the differentiation of megakaryocytes (platelet precursors).
  • a disease associated with alpha-synuclein aggregate is a group of neurodegenerative diseases called synuclein disease, and is characterized by the discovery of alpha-synuclein aggregates in lesions including neuronal and glia populations.
  • diseases include, but are not limited to, Parkinson's disease, Parkinson's disease dementia, Lewy body dementia, Alzheimer's Lewy body disease, complex Alzheimer's and Parkinson's disease, multisystem atrophy, and many other neuroaxons.
  • the antibody can be effectively used for the treatment of Parkinson's disease.
  • Another embodiment of the present invention comprises the steps of: a) detecting the alpha-synuclein by reacting the anti-alpha-synuclein antibody or antigen-binding fragment with a sample sample; And b) comparing the detected alpha-synuclein concentration or intracellular location with the results of the control sample.
  • Specific synuclein disease is as described above.
  • the alpha-synuclein may be in the form of aggregates.
  • the alpha-synuclein aggregates are due to changes in the alpha-synuclein structure, and include oligomers, protofibrils, fibrils, and / or structures or aggregates comprising one or more of the above structures, wherein the alpha-synuclein aggregates All forms or structures may be included without limitation.
  • sample sample' refers to any substance that has, or is estimated to be, a synuclein disease.
  • the sample may be natural or synthetic and may be obtained by any means known to those skilled in the art.
  • Samples may include tissues, cells, whole blood, plasma, serum, blood, saliva, sputum, lymph, cerebrospinal fluid, intercellular fluid, tears or urine, as well as samples of cell culture components in vitro, e.g., cell components, cell culture media, Recombinant cells and the like.
  • control sample refers to a biological sample obtained from an individual who does not develop Synuclein disease.
  • the concentration of alpha-synuclein aggregates is increased by comparing the sample with the control sample, it can be diagnosed as synuclein disease.
  • the diagnosis may include age, PET (DATscan) image data for dopamine transporters, cognitive-related questions such as Montreal cognitive assessment (moca), Unified Parkinson's Disease Rating Scale (UPDRS), and Hoehn & Yahr stage. Questionnaire data related to the same motor / non-motor symptoms may be matched.
  • age PET
  • DPTscan PET
  • cognitive-related questions such as Montreal cognitive assessment (moca), Unified Parkinson's Disease Rating Scale (UPDRS), and Hoehn & Yahr stage.
  • Questionnaire data related to the same motor / non-motor symptoms may be matched.
  • antibodies can typically be labeled with a detectable labeling substance, the forms of which can be labeled as described above.
  • the anti-alpha-synuclein antibody or antigen-binding fragment thereof of the present invention can detect alpha-synuclein aggregates that cause synuclein disease with high sensitivity (FIGS. 1 to FIG. 1). 4) It can be usefully used for the diagnosis of synuclein disease.
  • Another embodiment of the present invention comprises the steps of: a) treating a test sample with a candidate drug for synuclein disease; b) reacting the sample sample before and after the candidate with the anti-alpha-synuclein antibody or antigen-binding fragment; And c) selecting a synuclein disease therapeutic agent if the alpha-synuclein aggregate is reduced after treatment with the candidate substance.
  • a) treating a test sample with a candidate drug for synuclein disease b) reacting the sample sample before and after the candidate with the anti-alpha-synuclein antibody or antigen-binding fragment
  • selecting a synuclein disease therapeutic agent if the alpha-synuclein aggregate is reduced after treatment with the candidate substance.
  • the synuclein disease treatment agent may be in the form of a small molecule, a compound, an antibody, an anticancer agent, a virus, or the like, and is limited to an agent capable of treating or improving the synuclein disease through the effect of reducing synuclein aggregates. All can be applied without.
  • the present invention by measuring the change in the degree of binding of the anti-alpha-synuclein antibody or antigen-binding fragment of the present invention and the synuclein, in particular the aggregate form of the synuclein to the sample treated with the candidate drug for synuclein disease,
  • the ability to suppress synuclein disease and the effects of treating synuclein disease can be determined.
  • the candidate may be selected as a therapeutic agent for synuclein.
  • whether the reduction of the alpha-synuclein aggregate can be determined by including the step of detecting the amount of free antibody.
  • Increasing the concentration of free antibodies, ie, antibodies that are not bound to alpha-synuclein aggregates suggests that candidates can compete with the antibody for binding to alpha-synuclein aggregates, even if the free antibodies increase after candidate treatment. It can be determined that the candidate substance can be applied as a therapeutic agent for synuclein disease.
  • Anti-alpha-synuclein antibodies or antigen-binding fragments thereof of the present invention can detect alpha-synuclein with high accuracy and sensitivity by binding with high affinity to alpha-synuclein, especially alpha-synuclein aggregates.
  • the antibody of the present invention can be detected even if the concentration of alpha-synuclein aggregates is low through high affinity, so that it is possible to diagnose synuclein disease early and thus have high utility in diagnosing synuclein disease.
  • Figure 1 shows a dot blot result of determining whether the anti alpha-synuclein antibody prepared in one example of the present invention specifically recognizes the native alpha-synuclein in aggregate form.
  • Figure 2 shows the result of measuring the affinity of the anti-alpha-synuclein antibody prepared in one example of the present invention by ELISA.
  • Figure 3 shows the results of analyzing the binding specificity and affinity for the alpha-synuclein aggregate of the anti-alpha-synuclein antibody prepared in one embodiment of the present invention by BIAcore (7B7).
  • Figure 4 shows the results of analysis by Octet the preferred binding specificity for the alpha-synuclein aggregate of the anti alpha-synuclein antibody prepared in one example of the present invention (A: 5A7, B: D3C6).
  • Figure 5 shows the results confirming that the anti-alpha-synuclein antibodies 5A7 and 7B7 prepared in one example of the present invention recognize and bind to human cell expression alpha-synuclein protein.
  • an alpha-synuclein monomer obtained by cleaving full-length (140 residues) or 21 C-terminal residues was added to 37 ° C. thermomixer C, shaken at 1050 rpm for 14 days, and used for sonication.
  • 140 and 119 residues of alpha-synuclein fibrils prepared at a concentration of 1 mg / ml was admixed with adjuvant and 1: 1 (vol: vol).
  • the spleen of the immunized mouse was removed to obtain cells therefrom. Spleen cells were then suspended in Hybridoma-SFM medium (Thermo Fisher Scientific, USA) with 10% FBS. To prepare hybridomas, murine myeloma cells, SP2 / 0-Ag14, and spleen cells were mixed in a hybridoma-SFM medium containing no serum and centrifuged to remove the medium. Subsequently, PEG was added to the cell pellet, followed by incubation at 37 ° C for 1 minute to induce cell fusion.
  • a 5A7 (IgG1 kappa) clone was obtained using the full-length (140 residues) alpha-synuclein aggregate as an antigen, and 4G3 and 7B7 (IgG1, respectively) using the alpha-synuclein aggregate from which the C-terminal 21 residues were cleaved as antigen.
  • clone of kappa, IgG2b kappa) was obtained.
  • each hybridoma was cultured in RPMI1640 medium containing 10% FBS. After the culture medium was replaced with serum-free SFM medium for antibody production, the culture medium was incubated for about 4 days. Cell culture supernatants were separated and centrifuged, filtered through a 0.22 ⁇ m filter, and IgG1 type was purified by protein G column, and the remaining antibodies were purified by protein A column.
  • Variable region and CDR sequences are described in Ahn et al, Mol. Cells 2004, 18 (2): 237-241. After culturing the hybridomas, only cells were separated by centrifugation. RNA was isolated by adding trizol to the isolated hybridoma, and cDNA was synthesized using the template, and the variable region and CDR sequences were confirmed by sequencing.
  • gblock m.biotech
  • pcDNA3.4 animal cell culture vector
  • the prepared vector was maxi-prep (Qiagen) to secure a large amount of plasmid DNA, and then introduced into cells as follows.
  • ExpiCHO TM Gibco, Cat: A29127
  • ExpiCHO TM expression medium Gibco, Cat: A29100-01
  • the EExpiFectamine TM CHO and plasmid DNA complexes were prepared using an ExpiFectamine TM CHO transfection kit (Gibco, Cat: A29129) for transfection into the prepared parent cells. Cool OptiPRO TM SFM ®: by dividing the (Gibco, Cat 12309019) medium, respectively, and then each inoculated with the DNA and ExpiFectamine TM CHO reagent prepared in an appropriate concentration, and mixed for 5 minutes at room temperature, and was inoculated on a cell culture after transfection injection Started.
  • the enhancers and feeds included in the ExpiFectamine TM CHO transfection kit were inoculated into the transfected cells, and after 5 days, the feed was further inoculated, and then cultured at 8% CO 2 , 37 ° C. and 120 rpm for 10 days. Completed.
  • the culture solution was transferred to a centrifuge bottle, centrifuged at 4 ° C and 6500 rpm for 30 minutes, filtered through a filter having a size of 0.2 ⁇ m, to secure a culture medium except for suspended solids, and then purified. Proceeded.
  • Cultures were purified using HiTrap MabSelectSure (GE Healthcare, 11-0034-94). After equilibration with equilibration buffer (50 mM Tris-HCl pH7.2, 100 mM NaCl), the recovered culture was loaded onto a column. When the loading was completed, the medium was washed with 50 mM sodium citrate pH 5.0 and eluted with 50 mM sodium citrate pH 3.4. The eluate was neutralized to pH 6.0 by adding 1M Tris-HCl pH 9.0. The eluate was then buffer exchanged and concentrated in PBS (phosphate buffered saline, pH 7.4) and stored at 4 ° C. until use.
  • PBS phosphate buffered saline, pH 7.4
  • the second purification was performed based on the size of the eluted sample by passing the first purification portion with 1X PBS buffer in a HiLoad 26/600 superdex 200 column.
  • the amino acid sequence of the purified antibody was analyzed by mass spectrometry and confirmed to match the variable region of the mouse-derived monoclonal antibody.
  • the chimeric forms of human IgG1 antibodies were prepared by substituting the variable regions of the 4G3, 5A7, and 7B7 antibodies identified by the above method in the backbone variable region portion of the human IgG1 isotype.
  • a mini library was prepared in which a human or human-derived sequence was introduced at each CDR residue while binding the human framework to the CDR1, CDR2 and CDR3 residues of the chimeric antibody.
  • Competent cells of the library were subjected to 17 ⁇ g 2X YT Tryptone (CONDA, 1612.00) containing 34 ⁇ g / ml chloramphenicol (Sigma, C0857), 2% glucose (Sigma, G5400) and 5 mM MgCl 2 (Sigma, M2393). 10 g of extract (CONDA, 1702.00), 5 g of NaCl (Sigma, S7653)], and then incubated at 37 ° C.
  • alpha-synuclein aggregates were added to PBS in an immunotube (immunotube, maxisorp 444202), and the protein was adsorbed on the test tube surface overnight at 4 ° C., followed by bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • a 3% solution was added to the test tube to protect the surface where alpha-synuclein aggregates were not adsorbed.
  • an antibody phage library of 10 12 CFU dispersed in 3% BSA solution was placed in an immunoassay tube containing alpha-synuclein monomer protein and reacted at room temperature for 1 hour (negative selection).
  • Phages unbound to the alpha-synuclein monomer were recovered and bound to the immunoassay tube to which the alpha-synuclein aggregate was attached for 2 hours at room temperature. Subsequently, non-specifically bound phages were washed 5 to 30 times with PBS-T (0.05% Tween 20) solution, and the remaining antigen-specific phage antibodies were recovered using 100 mM triethylamine solution. The recovered phages were neutralized with 1M Tris buffer (pH 7.4), then infected with ER2537 Escherichia coli at 37 ° C for 1 hour, and the infected E.
  • PBS-T 0.05% Tween 20
  • coli was plated in 2X YT agar medium containing carbenicillin and incubated overnight at 37 ° C. Escherichia coli cultured the next day was suspended in 4 ml of 2X YT carbenicillin culture and 15% glycerol was added to store some at -80 ° C and the rest to produce phage for the next round of panning. This process was repeated three rounds in total to amplify antigen specific antibodies. As the panning round progressed, the number of washings using PBS-T was increased to amplify and concentrate the antigen-specific phage.
  • the following experiment was performed to select monoclonal antibodies that specifically bind to alpha-synuclein aggregates from the phage pool obtained through the panning.
  • the phage pool was plated in LB-tetracycline / carbenicillin agar medium and cultured to obtain a single colony. Monoclones were then inoculated in 96 well plates containing 400 ⁇ l of 2X YT-tetracycline / cabenicillin medium per well and grown overnight, and then 10 ⁇ l of the culture solution contained fresh 390 ⁇ l of 2X YT-tetracycline / carbenicillin medium. Into a 96 well plate was incubated for 4 hours at 37 °C. 1mM IPTG was added to the culture and incubated overnight at 30 ° C. The culture broth cultured overnight was centrifuged to obtain the supernatant.
  • Clones expressing monoclonal soluble scFv that bind to alpha-synuclein aggregates were then selected using the ELISA method. Specifically, the 7B7 antibody selected in Example 1-2 was put into a 96 well plate and coated overnight at 4 ° C. 200% of 3% BSA was added to each well and blocked for 2 hours at 37 ° C. Subsequently, alpha-synuclein aggregates and monomers were respectively loaded at a concentration of 100 ng / well and reacted at 37 ° C. for 2 hours. Then washed 5 times with PBS-T 300 ⁇ L.
  • the prepared monoclonal supernatant was mixed with 3% BSA 1: 1 (vol: vol), and the mixed solution was loaded onto the plate combined with the aggregate and the monomer by 100 ⁇ L and reacted at 37 ° C. for 2 hours. After washing 5 times with PBS-T 300 ⁇ L, the anti-HA HRP binding antibody was added and reacted for 1 hour at 37 °C, then washed 5 times with PBS-T. After 100 ⁇ L of TMB (Tetramethylbenzidine, Sigma, T0440) was added and developed, 50 ⁇ L of 1N H 2 SO 4 was added to stop the reaction, and the absorbance was measured at 450 nm. Clones with absorbance greater than 0.5 were considered positive by binding, and clones that did not bind BSA nonspecifically were excluded.
  • TMB Tetramethylbenzidine
  • the CDR residues of the clones found in the library were analyzed in in silico, causing serious problems in binding to the framework, or clones containing no T-cell epitope, B cell epitope, or MHCII epitope in the framework except the CDRs. Screened.
  • a human antibody of IgG1 backbone was prepared by substituting the variable region of the humanized antibody for the backbone variable region of the human IgG1 isotype. In this manner, D3C6 and A3F10 were obtained.
  • Dot blot experiments were performed to analyze whether the antibodies according to the invention bind to monomers or aggregates in their native state.
  • 50 ng or 100 ng of alpha-synuclein monomer or fibril protein (manufactured by Prof. Seung-Jae Lee, Seoul National University; Bae et al., J. Neurosci 32: 13454, 2012) was used as a dot blot apparatus (BioRad).
  • BioRad dot blot apparatus
  • Two-fold dilutions of monomer or fibril protein were loaded sequentially from right to left of the membrane (12.5, 25, 50, 100 ng or 25, 50, 100 ng).
  • the alpha-synuclein antibody prepared in Examples 1, 2 and 3 was 1 mg / ml.
  • the membrane and the antibody were incubated at room temperature for 1 hour in TBST containing% bovine serum albumin.
  • signals were analyzed using a chemiluminscence substrate (NEN) as a secondary antibody and substrate bound to HRP (horse radish peroxidase).
  • NNN chemiluminscence substrate
  • HRP horse radish peroxidase
  • the alpha-synuclein antibody according to the present invention was shown to bind preferentially to aggregates as compared to the alpha-synuclein monomer.
  • 5A7 was shown to specifically bind to aggregates
  • D3C6, 4G3 and A3F10 were found to bind preferentially to aggregates.
  • ELISA was performed to quantitatively analyze the binding capacity of the antibody according to the present application to the antigen.
  • the mouse anti-alpha-synuclein antibody obtained in Example 1 was coated on a 96 well plate at a concentration of 1 ug / ml, followed by alpha-synuclein blood at a concentration of 10, 100, 1000, 10,000 ng / ml.
  • Humanized antibodies obtained from phage libraries were also subjected to ELISA in the same manner and the measurement results are shown in FIG. 2.
  • the antibody according to the present invention was found to have a high binding force to the aggregates and to bind the aggregates preferentially.
  • 4G3 and A3F10 showed an affinity of 1 x 10 -9 M to 3 x 10 -9 M.
  • the instrument used T200 (GE Healthcare, S / N: 1565888). Chip used Protein A (GE Healthcare, Cat. 29-1275-56), Regeneration buffer 10 mM Glycine-HCl pH1.5 (GE Healthcare, Cat. BR-1003-54), Running buffer and analyte dilution, HBS-EP was used as sample dilution buffer.
  • the anti alpha-synuclein antibodies (5A7 and 7B7) prepared in Example 1 were diluted with 1 ⁇ HBS-EP (GE Healthcare, Cat.
  • alpha-synuclein monomer (1 mg / ml) or Fibrillated protein (3 mg / ml) (analyte) was serially diluted 2 fold and analyzed at a total of 6 concentrations (0, 0.39, 1.56, 6.25, 25, 100 nM) including 0 nM.
  • monomers set the target RU to 800 (theoretical)
  • fibrils set the target RU to 100 (theoretical)
  • capture phase to 60 seconds contact time
  • flow rate to 30 ⁇ l / min
  • stabilization period to 180 seconds. Proceeded.
  • the association time was 120 seconds
  • the flow rate was 30 ⁇ l / min.
  • the dissociation time was 360 seconds and the flow rate was 30 ⁇ l / min.
  • the regeneration phase the regeneration time was performed twice at 240 sec (first), 60 sec (second) at a flow rate of 30 ⁇ l / min.
  • the bivalent model was used for the fitting and the evaluation software was BIACore T200 Evaluation software (GE healthcare). The results are described in FIG.
  • Figure 3 is a BIAcore analysis showing that the monoclonal antibody prepared in one example of the present invention has a very high affinity for the aggregate of alpha-synuclein.
  • the dissociation constant (K D ) of the monoclonal antibody prepared in one example of the present invention for the alpha-synuclein aggregate measured as shown in Table 6 was 1 x 10 -12 M or less.
  • the monoclonal antibody prepared in one example of the present invention has a high binding affinity for alpha-synuclein aggregates, and thus effectively detects a causative agent of neurodegenerative diseases with alpha-synuclein etiology such as Parkinson's disease. To show that the antibody is applicable.
  • mouse alpha-synuclein antibodies 7B7 and 5A7 prepared in Example 1 can recognize alpha-synuclein expressed in human cells was analyzed by immunoprecipitation method.
  • Human neuroblastoma cell line SH-SY5Y was transfected with a vector that overexpresses alpha-synuclein to express alpha-synuclein, and then cultured cells were treated with PBS, 1% Triton X-100, 1%. Cell membranes were disrupted by administration of 1X PBS solution containing (v / v) protease inhibitor mixture (Sigma). The lysate was centrifuged at 16,000 g at 4 ° C. for 10 minutes to remove cell shells and nuclei and use supernatant.
  • the heavy and light chains of antibodies ie IgG, 7B7, 5A7 that bind to protein A / G in the immunoprecipitation sample and bind to the target were thick at 50 kDa and 25 kDa, respectively (Fig. 5, asterisk).
  • the bands present in E of the mouse IgG treatment group were considered to be non-specific. Since the alpha-synuclein monomer size was 15 kDa, the bands near the 15 kDa and the bands not shown in the mouse IgG negative control (black arrowheads) were all judged to be alpha-synuclein monomers and aggregates recognized by 7B7 or 5A7. In the case of the mouse IgG treatment group, there is no band corresponding to 15 kDa in the eluant, so it can be determined that alpha-synuclein in the input does not exist in the lane. Both 5A7 and 7B7 showed distinct alpha-synuclein monomer bands of 15 kDa.
  • the 7B7 treatment group showed an oligomer-sized band not present in the mouse IgG lanes.
  • the results indicate that the 7B7 clone is particularly sensitive to the recombinant alpha-synuclein protein commonly used as well as the alpha-synuclein that is actually produced by mammalian cells, that is found in the actual physiological conditions of mammalian cells. (FIG. 5).
  • One of ordinary skill in the art would expect to be able to bind oligomers or aggregates to other clones depending on the specific experimental conditions of the immunoprecipitation assay (heating, etc.).
  • the 7B7 clone is the most sensitive to the highest affinity to the alpha-synuclein found in the actual physiological conditions can be detected sensitively.

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Abstract

La présente invention concerne un anticorps anti-α-Syn ou un fragment de liaison à l'antigène de celui-ci, ainsi que la détection de α-Syn et le diagnostic de maladies associées à l'aide de celui-ci.
PCT/KR2019/008170 2018-07-03 2019-07-03 Anticorps anti-alpha-synucléine et son utilisation Ceased WO2020009482A1 (fr)

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CN116057382A (zh) * 2020-05-18 2023-05-02 安培里翁公司 α-突触核蛋白底物及其制备和使用方法
CN116102646A (zh) * 2023-02-13 2023-05-12 贵安新区康顺生物科技有限公司 抗人-磷酸化突触核蛋白抗体及其应用
CN118506873A (zh) * 2024-05-29 2024-08-16 北京百度网讯科技有限公司 抗体生成方法、装置及电子设备
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US11142570B2 (en) 2017-02-17 2021-10-12 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
US11827695B2 (en) 2017-02-17 2023-11-28 Bristol-Myers Squibb Company Antibodies to alpha-synuclein and uses thereof
CN111544584A (zh) * 2020-04-16 2020-08-18 首都医科大学 一种单克隆抗体对帕金森病的治疗作用
CN116057382A (zh) * 2020-05-18 2023-05-02 安培里翁公司 α-突触核蛋白底物及其制备和使用方法
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EP4340881A4 (fr) * 2021-05-12 2025-05-14 ABL Bio Inc. Anticorps pour le traitement d'alpha-synucléinopathies
CN116102646A (zh) * 2023-02-13 2023-05-12 贵安新区康顺生物科技有限公司 抗人-磷酸化突触核蛋白抗体及其应用
CN118506873A (zh) * 2024-05-29 2024-08-16 北京百度网讯科技有限公司 抗体生成方法、装置及电子设备

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