Fusion molecule having non-inflammatory phagocytosis inducing activity

Abstract

To provide a fusion molecule having phagocytosis inducing activity, and to provide applications for prevention or treatment of a disease caused by abnormal accumulation of a target substance.SOLUTION: The present invention relates to a fusion molecule having phagocytosis inducing activity, which can address the issue of tissue damage caused by activation of an inflammatory reaction of the prior art, and thus accumulated abnormal proteins, such as β-amyloid, tau, α-synuclein, huntingtin, or prion, or the like, are effectively removed, so that the present invention can be applied for prevention or treatment of proteinosis caused by the abnormal accumulation of such substances.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a fusion molecule having non-inflammatory phagocyte action-inducing activity, and presents the possibility of being utilized for the prevention or treatment of diseases caused by abnormal accumulation of substances such as proteinosis.

Background Art

[0002] Many degenerative diseases are characterized by abnormal folding, polymerization, and accumulation of specific proteins. Such proteopathies include various types of amyloidosis.

[0003] Amyloidosis is a group of diseases in which abnormal proteins known as amyloid fibrils accumulate in tissues. Amyloid is a protein mass with a diameter of 7 to 13 nm that appears fibrous under a microscope and has a β-sheet structure, and is characterized by being stained with thioflavin T (thio-T) and congo red. Amyloid is not found in the normal body, and it has been reported to date that 36 types of proteins can form amyloid (Non-Patent Document 1: Picken, Acta Haematol. (2020), 143: 322-334). Representative amyloidosis includes neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington disease, and prion disease. In addition, there are multiple amyloidosis with various aspects depending on the causative protein and the affected organ.

[0004] Alzheimer's disease is the leading cause of dementia and a fatal disease accompanied by learning and memory impairments. It is predicted that 130 million people in the world's population will suffer from Alzheimer's disease by 2050, and already one in nine people aged 65 and over has been diagnosed with Alzheimer's disease.

[0005] Alzheimer's disease is characterized by the deposition and accumulation of β-amyloid (Aβ) protein, which is produced by the abnormal degradation of amyloid precursor protein (APP), outside the brain cell membrane, and at the same time shows abnormal binding due to the hyperphosphorylation of tau protein that binds to microtubules.

[0006] Among them, oligomers and fibrils generated by the aggregation of β-amyloid cause a decline in synapse function and cytotoxicity through various pathways, and it has recently been reported that a vicious cycle occurs in which the functional changes of astrocytes and microglia (microglia), which are responsible for immunity in the brain, have an adverse effect on neurons again.

[0007] To date, FDA-approved Alzheimer's disease treatments are drugs that inhibit the breakdown of acetylcholine or the activity of NMDA receptors, and these aim only to temporarily relieve symptoms rather than provide a fundamental cure for the disease. Therefore, there has been no way to fundamentally treat Alzheimer's disease, and it is known as a disease that incurs the highest costs for the treatment and care of patients in the era of population aging.

[0008] For the fundamental treatment of Alzheimer's disease, drug development has been focused on suppressing and removing β-amyloid for decades. However, unfortunately, most Alzheimer's disease therapeutics developed for suppressing and removing β-amyloid have failed to be effective at the clinical stage. For example, in the case of BACE inhibitors for reducing β-amyloid, in Alzheimer's patients with cognitive decline, β-amyloid plaques have already accumulated and neuronal death has occurred, so strategies to further interfere with production are not very effective.

[0009] In recent years, research results have been reported that monoclonal antibodies specifically binding to β-amyloid oligomers and fibrils induced β-amyloid removal and restored cognitive function in Alzheimer's disease patients, and the treatment strategy for Alzheimer's disease using β-amyloid antibodies has suddenly emerged as a new hope.

[0010] The proposed mechanisms of action of β-amyloid monoclonal antibodies to date include that β-amyloid antibodies bind to β-amyloid oligomers and fibrils to prevent their aggregation, or that microglia cause phagocytosis of β-amyloid through Fc receptors that recognize monoclonal antibodies.

[0011] However, despite the progress in the development of such Alzheimer's disease therapeutics, current immunotherapy using β-amyloid monoclonal antibodies shows amyloid-related imaging abnormalities (ARIA: Amyloid-Related-Imaging-Abnormalities) with severe edema in 55% of antibody-treated patients, and for this reason, actually about 35% of ARIA patients were excluded from the clinical trial midway. The ARIA phenomenon is known to be caused by synapse and cytotoxicity due to an inflammatory reaction that is inevitably activated when β-amyloid monoclonal antibodies stimulate Fc receptors.

[0012] Since synapses and neurons present in the brain are sensitive to inflammatory cytokines, treatment using β-amyloid monoclonal antibodies ultimately has an inevitable and essential problem in that even if β-amyloid is removed to some extent, it will simultaneously damage neurons and synapses. In addition, together with monoclonal antibodies, companies such as Alector and Denali have attracted attention by presenting a strategy to improve the β-amyloid removal ability of microglia by activating targets such as TREM2 that regulate the immunological mechanism of microglia. However, if microglia are overly activated in this way, synaptic damage due to an increase in the overall phagocytic ability is expected.

[0013] Therefore, an important future issue in the treatment of Alzheimer's disease is to develop a method that can selectively remove only β-amyloid oligomers and fibrils without causing an inflammatory reaction and synaptic damage, and such a drug is expected to greatly contribute to the treatment of Alzheimer's disease.

[0014] Furthermore, as described above, a method that can selectively remove only the target abnormal accumulation substances, such as abnormal accumulation proteins that cause proteinopathy, without causing an inflammatory reaction and accompanying additional tissue damage can be widely applied to develop a method that can selectively remove abnormal accumulation proteins such as tau, α-synuclein, and huntingtin. Such a drug is expected to greatly contribute to the treatment of not only neurodegenerative diseases such as Huntington's disease but also all diseases related to abnormal accumulation of specific substances.

Prior Art Documents

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0016] The present invention relates to a fusion molecule having phagocyte action-inducing activity, and aims to present the possibility of utilization for the prevention or treatment of diseases caused by abnormal accumulation of a target substance. The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Means for Solving the Problems

[0017] One aspect of the present invention provides a fusion molecule having phagocyte action-inducing activity, which comprises a first region having TAM receptor-binding ability and a second region specifically binding to a target substance.

[0018] Here, the TAM receptor may specifically be one or more selected from the group consisting of Tyro3, Axl, and MerTK, and these can bind to a laminin G-like domain (laminin G-like domain or LG domain) to induce phagocytosis.

[0019] The first region may include Gas6, ProS1, Tubby, Tulp1, Gal3, or active fragments thereof, and there is no particular limitation on the form and range thereof as long as it is a protein in a form in which the ability to induce phagocytosis by interaction with its specific TAM receptor is preserved. The first region may preferably be selected from Gas6, ProS1, or active fragments thereof.

[0020] More specifically, the first region may contain the laminin G-like domain of Gas6 or ProS1, or an active fragment thereof, which contain the laminin G-like domain as a bridging molecule related to phagocytic cell function that is strongly expressed in various tissues, and thereby may induce phagocytic cell function via the TAM receptor.

[0021] Specifically, the laminin G-like domain may contain an LG1 domain, an LG2 domain, or a combination thereof, and preferably may contain both an LG1 domain and an LG2 domain, which may bind to the TAM receptor and induce phagocytosis.

[0022] The first region may be a peptide containing one or more sequences of SEQ ID NO: 1 and SEQ ID NO: 2, or a peptide containing one or more sequences of SEQ ID NO: 3 and SEQ ID NO: 4. Preferably, the first region may be a peptide containing either one of the sequences containing both the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 or the sequences containing both the sequences of SEQ ID NO: 3 and SEQ ID NO: 4. More preferably, it may be a peptide containing the sequence of SEQ ID NO: 5 or SEQ ID NO: 6. The peptide containing the sequence of the SEQ ID NO includes not only the amino acid sequence but also amino acid sequence variants. The sequence variant means a protein having a sequence in which one or more amino acid residues are different from the amino acid sequence. As long as the activity of the fusion molecule is maintained, any cleavage, deletion, insertion, substitution, etc. in the final structure of the protein, and combinations thereof are also possible. An example of a sequence variant is a form in which an amino acid residue at a site not essential for activity is cleaved or deleted, or a form in which an amino acid residue at a site important for autoinhibition is substituted. Also, in some cases, it can be modified by phosphorylation, glycosylation, methylation, farnesylation, etc. It is more preferable that such sequence variations and modifications increase the function and / or stability (thermal stability, pH stability, structural stability, etc.) and / or solubility of the protein due to the variation in the amino acid sequence.

[0023] The method for inducing mutations in the amino acid sequence utilizes a method of producing a nucleic acid molecule containing a nucleotide sequence corresponding to the changing amino acid sequence by mutating the nucleotide sequence encoding the protein, and the method for obtaining the gene encoding this can introduce mutations in vivo or in vitro using all mutagenesis techniques well-known in the art. For example, site-directed mutagenesis (Non-Patent Documents 2-4: Hutchinson et al., J. Biol. Chem., 253: 6551, 1978; Zoller and Smith, DNA, 3: 479-488, 1984; Oliphant et al., Gene, 44: 177, 1986; Hutchinson et al., Proc. Natl. Acad. Sci. U.S.A., 83: 710, 1986), TAB linker (Pharmacia), PCR technology (Non-Patent Document 5: Higuchi, 1989, "Using PCR to Engineer DNA" in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70), etc. can be used.

[0024] Also, when the first region contains the laminin G-like domain of Gas6 or ProS1 or an active fragment thereof, the first region may not contain the Gla domain, which may be for the purpose of preventing the first region from recognizing phosphatidylserine (PS) and allowing the second region to recognize the target substance and induce phagocytic cell action.

[0025] Also, when the first region contains the laminin G-like domain of Gas6 or ProS1, or an active fragment thereof, the first region may not contain both the Gla domain and the EGF domain, which may be for the purpose of suppressing the aggregation phenomenon during the purification process of the fusion molecule and increasing the yield, together with the technical effect obtained by not containing the aforementioned Gla domain.

[0026] The target substance may be a substance that accumulates in living tissue and causes disease. For example, it may be a substance that accumulates in affected, i.e., diseased, tissue of a patient. The substance that accumulates in the disease may be a protein, i.e., the disease may be a proteinopathy, but is not limited thereto. For example, the target substance may be amyloid, i.e., the proteinopathy may be amyloidosis. The target substance may be selected from the abnormal accumulation substances in Table 1 below, and at this time, the disease may be a disease in which each abnormal accumulation substance is detected. For example, the proteinopathy may be selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, and prion disease, and at this time, the target substance may be the abnormal accumulation protein that causes the disease, i.e., β-Amyloid, Tau, α-Synuclein, Huntingtin, and prion protein, respectively.

[0027]

Table 1

[0028] The second region that specifically binds to the target substance may be selected from an antibody that specifically binds to the target substance, an active fragment thereof, an antibody-like protein, a peptide, an aptamer, and a soluble receptor, but there is no particular limitation as long as it can specifically bind to the target substance in a form capable of specifically binding to the target substance.

[0029] Here, the antibody or its active fragment may be, for example, i) an immunoglobulin selected from IgG1, IgG2, IgG3, and IgG4; ii) native antibody fragments such as Fv, Fab, Fab’, F(ab’)2, VHH, VNAR; iii) engineered antibodies such as scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, tetrabody, pentabody. The antibody or its active fragment may be, for example, a Mab, Fab, or its single-chain Fv fragment (scFv) based on six complementarity-determining regions (CDRs) derived from an antibody that specifically binds to the target substance. That is, the protein or its active fragment that specifically binds to the target substance contains a part essential for activity that specifically binds to the target substance, is linked to the first region, and shows an effect of causing no synaptic damage without an inflammatory reaction. There is no particular limitation in its form and scope. For example, the target substance may be β-amyloid. In this case, the protein or its active fragment that specifically binds to the target substance may contain aducanumab or its single-chain Fv fragment. The second region may contain a Mab, Fab, or single-chain Fv fragment based on six complementarity-determining regions (CDRs) derived from any one selected from the group consisting of aducanumab, semorinemab, and cinpanemab.

[0030] The antibody or its active fragment may not contain an Fc region, and preferably may contain an Fc region variant that does not bind to an Fc receptor (especially, an Fcγ receptor). Such an Fc region variant may be included for improving physical properties such as tablets.

[0031] The antibody-like protein refers to a protein scaffold that can specifically bind to a target substance like an antibody. Since the antibody-like protein is smaller than an antibody with an average size of about 150 kDa, being 2 - 20 kDa, it can be designed to target binding sites inaccessible to antibodies. It is known to be more stable at higher temperatures than antibodies and much easier to synthesize using non-mammalian cells such as viruses and yeasts or by chemical synthesis.

[0032] In the present invention, an aptamer refers to single-stranded DNA (ssDNA) or RNA having high specificity and affinity for a specific substance. Since aptamers have high affinity and stability for a specific substance, they can be synthesized by a relatively simple method and can be modified in various ways to enhance the binding force. Since cells, proteins, and even small organic substances can be target substances, they are characterized by extremely high specificity and stability compared to antibodies whose specificity and stability have already been developed. Also, aptamers can be produced by a known method of systematic evolution of ligands by exponential enrichment (SELEX). For example, after producing an aptamer that specifically binds to β-amyloid, tau, or α-synuclein by a known SELEX (Systematic Evolution of Ligands by Exponential enrichment) method, it can be linked to the first region, thereby generating a fusion molecule according to the present invention.

[0033] The aptamer of the present invention is not particularly limited as long as it can specifically bind to β-amyloid, tau, or α-synuclein. The bases used for the aptamer may be selected from the group consisting of A, G, C, U, and their deoxy forms, unless otherwise specified.

[0034] In addition, in order to enhance stability, the aptamer may be modified by binding one or more selected from the group consisting of polyethylene glycol (PEG), inverted deoxythymidine (idT), locked nucleic acid (LNA), 2'-methoxynucleoside, 2'-aminonucleoside, 2'F-nucleoside, amine linker, thiol linker, and cholesterol at the 5'-terminal site, middle site, 3'-terminal site, or both terminal sites. IdT (inverted deoxythymidine) is generally one of the molecules used to prevent degradation of aptamers that are generally vulnerable to nucleases by nucleases. In nucleic acid units (monomers), the 3'-OH of the previous unit binds to the 5'-OH of the next unit to form a chain, but idT artificially changes by binding the 3'-OH of the previous unit to the 3'-OH of the next unit so that the 5'-OH other than the 3'-OH is exposed, and has the effect of suppressing degradation by 3'exonuclease, a type of nuclease.

[0035] The soluble receptor of the present invention includes a region having an activity capable of binding to a target substance, that is, an endogenous ligand. The region may be derived from an endogenous membrane receptor or an intracellular receptor or a derivative thereof. At this time, as the soluble receptor included in the second region of the fusion molecule of the present invention, preferably, a region having an activity other than binding to the target substance in the endogenous receptor is removed and used.

[0036] In the present invention, the peptide that can be the second region means the remainder excluding the antibody or its active fragment, antibody-like protein, and soluble receptor among polypeptides having amino acids capable of specifically binding to a target substance as monomers.

[0037] Since the fusion molecule according to the present invention induces the phagocytic action through the interaction with the TAM receptor, the phagocytic action can be induced in cells expressing the TAM receptor. Phagocytosis generally refers to engulfing cells or particles of 0.5 μm or larger, and includes the processes of tethering, engulfing, and degrading the cells or particles. At this time, phagocytosis may include forming a phagosome that surrounds the internalized cells or particles and degrading them in a phagolysosome by fusion of the phagosome and lysosome. At this time, cells that die or are dying by apoptosis or necrosis mechanisms during phagocytosis are also called efferocytosis.

[0038] The cells expressing the TAM receptor can be one or more professional phagocytes, one or more non-professional phagocytes, or a combination thereof. Here, professional phagocytes refer to cells whose main function is to remove cells killed by phagocytosis and accumulated debris, etc., and include macrophages, neutrophils, dendritic cells, and mast cells. Macrophages usually reside in each tissue that can be an infection route and are often called by different names depending on the tissue. Examples include adipose tissue macrophages in adipose tissue, monocytes in bone marrow and blood, Kupffer cells in the liver, sinus histiocytes in lymph nodes, alveolar macrophages in alveoli, histiocytes in connective tissue or giant cells which are their aggregates, microglia in the central nervous system, Hofbauer cells in the placenta, intraglomerular mesangial cells in the kidney, osteoclasts in bone, epithelioid cells in granulomas, red pulp macrophages in the red pulp of the spleen, peritoneal macrophages in the peritoneal cavity, LysoMac in Peyer's patch, etc.On the one hand, non-professional phagocytes refer to cells that mainly perform functions specific to the tissue in which the phagocytes are present, but can perform phagocytosis as needed. Epithelial cells, endothelial cells, fibroblasts, mesenchymal cells, etc. are applicable, and include some tissue-specific cells, such as astrocytes and oligodendrocytes in the central nervous system, Muller glia in the retina, hepatocytes in the liver, satellite cells in muscle, Sertoli cells in the testis, etc., and also include some lymphocytes such as natural killer cells, large granular lymphocytes, eosinophils, basophils, and B cells. The fusion molecule according to the present invention can induce phagocytic action in phagocytes specific to the tissue in which the target substance to be removed accumulates. For example, when attempting to remove abnormal proteins accumulated in the brain, the phagocytic action can be induced in astrocytes, microglia, oligodendrocytes, or a combination thereof. This can be induced, for example, by locally administering the fusion molecule according to the present invention to such a tissue, or by manipulating the cells in the corresponding tissue to express and secrete the fusion molecule.

[0039] The induction of the phagocytic action may not be accompanied by an inflammatory reaction. This means that it is possible to more safely treat the decline in tissue function caused by the accumulation of the target substance compared to existing technologies in that it can suppress tissue damage caused by the inflammatory reaction without inducing an inflammatory reaction while removing the target substance.

[0040] The fusion molecule may contain a label (tag). When such a label is added to the fusion molecule, it can be used to confirm the purification, expression, presence or absence of action, or action process, etc. of the fusion molecule.

[0041] The said labels include, but are not limited to, His-tag, T7-tag, S-tag, FLAG-tag, Strep-tag, thioredoxin (Trx)-tag, His-patch thioredoxin-tag, lacZ (L-Galactosidase)-tag, chloramphenicol acetyltransferase-tag, trpE-tag, avidin / streptavidin / Strep-tag, T7 gene 10-tag, staphylococprotein A-tag, streptococcal protein G-tag, glutathione-S-transferase (GST) tag, dihydrofolate reductase (DHFR), cellulose binding domains (CBD’s) tag, maltose binding protein (MBP) tag, galactose-binding protein-tag, calmodulin binding protein (CBP) tag, HSV-tag, B- (VP7 protein region of bluetongue virus)-tag, polycysteine-tag, polyphenyalanine-tag, (Ala-Trp-Trp-Pro) n -tag, polyaspartic acid-tag, c-myc-tag, lac repressor-tag, etc. The said labels may be located at the N-terminus, C-terminus or inside of the target protein.

[0042] The fusion molecule may further contain a signal peptide (signal peptide or leader sequence) at the N-terminus. A signal peptide is a short peptide present at the N-terminus at the initial stage of protein synthesis directed towards the secretory pathway, and is known to specify the intracellular location of the protein, (in the case of a membrane protein) the membrane topology, etc. The signal peptide may be cleaved during the process in which the fusion molecule is expressed and secreted extracellularly.

[0043] The aforementioned first region, second region, label, signal peptide, or region having minimal functionality (e.g., the LG1 and LG2 regions, or the heavy chain variable region and light chain variable region of the scFv) contained in the fusion molecule may be directly linked to each other, or may be linked by a linker containing a short oligopeptide or polypeptide. Generally, the linker may contain 2 to 500 amino acid residues. The linker is not particularly limited in terms of its length or type as long as it can link the aforementioned regions so that each region has the intended activity to constitute the fusion molecule. Examples of commonly used oligopeptide linkers include (GGGGS)n, that is, a linker in which one or more Gly-Gly-Gly-Gly-Ser units are repeated. In addition, (GSSGGS)n, KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSAGSAAGSGEF, (EAAAK)n, CRRRRRREAEAC, A(EAAAK)4ALEA(EAAAK)4A, GGGGGGGG, GGGGGG, AEAAAKEAAAAKA, PAPAP, (Ala-Pro)n, VSQTSKLTRAETVFPDV, PLGLWA, TRHRQPRGWE, AGNRVRRSVG, RRRRRRRR, GFLG, or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE, etc. can also be used as linkers, but are not limited thereto.

[0044] In one aspect of the present invention, there is provided a nucleic acid molecule encoding the fusion molecule and an expression vector containing the same.

[0045] As described above, the nucleic acid molecule sequence encoding the fusion molecule can be mutated by substitution, deletion, insertion, or a combination thereof of one or more nucleobases, as long as it encodes a protein having equivalent activity thereto.

[0046] The nucleic acid molecule sequence encoding the fusion molecule can be isolated from nature, artificially synthesized, or prepared by genetic recombination methods. The nucleic acid molecule sequence encoding the fusion molecule is provided by being operably linked to an expression vector capable of expressing it.

[0047] The "expression vector" refers to a vector capable of introducing a nucleic acid sequence encoding a target gene into an appropriate host cell to express a target protein or target RNA, and is a gene construct containing essential regulatory elements operably linked so that the gene insert is expressed. Such expression vectors include all vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors.

[0048] An appropriate expression vector has expression regulatory elements such as a promoter, start codon, stop codon, polyadenylation signal, and enhancer. The start codon and stop codon are generally regarded as part of the nucleic acid sequence encoding the protein, and the protein coding sequence is created to be in frame so as to be operable in the vector. The promoter can be constitutive or inducible. Furthermore, a normal expression vector contains a selectable marker. The operable linkage with the expression vector can be achieved using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes known in the art.

[0049] The expression vector may preferably be constructed such that it is introduced into cells during in vivo injection in order to purify and isolate the fusion molecule after expressing the fusion molecule in host cells or so that the cells can express and secrete the fusion molecule. When it is necessary to introduce it into cells in vivo, the vector is preferably a non-integrating vector, that is, a vector that is not integrated into the genome of the host cell.

[0050] As one aspect of the present invention, there is provided a cell that expresses the fusion molecule.

[0051] The cell may be transformed to contain the nucleic acid molecule or an expression vector containing the same, and the term "transformation" includes any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and as is known in the art, this may be carried out by selecting an appropriate standard technique depending on the host cell. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG (polyethylenglycol), dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0052] Examples of the host cell include, but are not limited to, prokaryotic host cells such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, or Staphylococcus (e.g., Staphylocus carnosus). In addition, cells derived from fungi such as Aspergillus, yeasts including Pichia pastoris, Saccharomyces visiae, Schizosaccharomyces, Neurospora crassa, Schacchomycasaromyces, other lower eukaryotic cells or insect cells, plant cells, and higher eukaryotes such as mammals can be used as the host cell.

[0053] After expressing the fusion molecule in the cells, for separation and purification, ordinary biochemical separation techniques can be used, such as treatment with a protein precipitant (salting-out method), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, various chromatographies such as affinity chromatography (affinity chromatography), etc. Usually, these are used in combination to separate proteins with high purity (Non-Patent Documents 6-7: Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)).

[0054] As one aspect of the present invention, there is provided a pharmaceutical composition (medicinal composition) for preventing or treating a disease caused by the accumulation of the target substance in a living tissue, which contains the fusion molecule or the expression vector. Here, the composition is also one that is locally administered to the location where the causative substance of the disease, that is, the target substance accumulates.

[0055] Also, as one aspect of the present invention, there is provided a method for preventing or treating proteinosis, which includes the step of administering a pharmaceutically effective amount of the fusion molecule to an individual.

[0056] Furthermore, as one aspect of the present invention, there is provided the use of the fusion molecule for manufacturing a medicament for preventing or treating proteinosis.

[0057] The fusion molecule as an active ingredient in the pharmaceutical composition is included in a "pharmaceutically effective amount". The term "pharmaceutically effective amount" means an amount sufficient to achieve the effectiveness or activity of the above fusion molecule.

[0058] The pharmaceutical composition can be administered orally or parenterally, preferably parenterally, more preferably locally to the tissue in which the target substance to be removed has accumulated.

[0059] As used herein, the term "parenteral administration" includes subcutaneous injection, intravenous, intramuscular, intracardiac injection or infusion techniques.

[0060] When formulating the pharmaceutical composition as an injection, it can be produced by the usual methods for producing injections known in the art. The injection may be in a form dispersed in a sterile medium so that it can be used as it is when administered to a patient, or in a form that is dispersed at an appropriate concentration after adding distilled water for injection at the time of administration and then administered.

[0061] When formulating the pharmaceutical composition as an oral dosage form, one or more selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives can be used as carriers, and one or more selected from flavors, vitamins and antioxidants can be used as additives.

[0062] Regarding the techniques necessary for formulating the pharmaceutical composition and pharmaceutically appropriate carriers, additives, etc., they are widely known to those with ordinary knowledge in the field of pharmaceutical formulation, and in this regard, references such as non-patent documents 8 - 10: the Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds., American Pharmaceuticals Association (2003); Remington: the Science and Practice of Pharmacy, 20th edition, Gennaro, Ed., Lippincott Williams & Wilkins (2000); Remington’s Pharmaceutical Sciences (19th ed., 1995), etc. may be referred to.

[0063] The appropriate dosage of the pharmaceutical composition can be formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, gender, medical condition, diet, administration time, administration route, excretion rate, and responsiveness. The dosage of the pharmaceutical composition of the present invention is 0.0001 to 1000 μg / kg (body weight) based on adult standards.

Advantages of the Invention

[0064] The present invention relates to a fusion molecule having phagocyte action-inducing activity, and can solve the problem of tissue damage caused by the activation of the inflammatory response of the prior art, thereby effectively removing abnormal accumulated substances such as β-amyloid, tau, α-synuclein, huntingtin or prion, etc., and can be used for the prevention or treatment of diseases caused by such accumulation, such as Alzheimer's disease, Parkinson's disease, Huntington's disease or prion disease. Such a fusion molecule can be administered to a patient in the form of a purified fusion molecule or in the form of a gene therapy agent vector capable of expressing and secreting the fusion molecule upon intracellular introduction.

[0065] However, the effects of the present invention are not limited to the above effects, and include all effects inferable from the configuration of the invention described in the detailed description or claims of the present invention.

Brief Description of the Drawings

[0066]

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Best Mode for Carrying Out the Invention

[0068] Hereinafter, for more specific explanation, embodiments and experimental examples will be given for detailed description. However, the following embodiments and experimental examples are exemplary and the scope of the invention is not limited thereto.

[0069] Production Example 1. Production of a Fusion Molecule Based on Gas6 Having β-Amyloid Removal Activity (I): β-Amyloid Binding Region in scFv Form To create a β-amyloid (Aβ)-specific chimeric phagocytosis inducer based on the Gas6 protein, first, the Gla domain, which is the site that recognizes phosphatidylserine (PS) of apoptotic cells, was removed, and a single-chain variable fragment (scFv) of aducanumab, a β-amyloid-specific antibody, was introduced at that position [αAβ-Gas6(E)].

[0070] Also, for the efficiency of protein production, the EGF repeat domain present in the internal residue of the Gas6 protein was also removed together, and αAβ-Gas6, which introduced the scFv of aducanumab, was also created (Figure 1).

[0071] Furthermore, as a control group for confirming the β-amyloid-specific binding of the scFv of aducanumab, αFITC-Gas6(E) and αFITC-Gas6, which introduced the E2 scFv that selectively recognizes FITC instead of the scFv of aducanumab, were created together.

[0072] Table 2 below is the amino acid sequence related to the production of the fusion molecule, and Table 3 below is the nucleotide sequence related to the production of the fusion molecule (the underlined sequence is the Flag tag).

[0073]

Table 2

[0074]

Table 3

[0075] Production Example 2. Fusion Molecule Based on Gas6 Targeting Tau To prepare a tau-specific chimeric phagocytosis inducer based on the Gas6 protein, first, the Gla domain and the EGF repeat domain were removed, and a single-chain variable fragment (scFv) of semorinemab, a tau-specific antibody, was introduced at that position (αTau-Gas6). The amino acid sequence and nucleotide sequence of the chimeric phagocytosis inducer are as shown in Table 4.

[0076] [Table 4] TIFF2025106371000020.tif248166TIFF2025106371000021.tif70166

[0077] Production Example 3. Fusion Molecule Based on Gas6 Targeting α-Synuclein To prepare an α-synuclein-specific chimeric phagocytosis inducer based on the Gas6 protein, first, the Gla domain and the EGF repeat domain were removed, and a single-chain variable fragment (scFv) of cinpanemab, an α-synuclein-specific antibody, was introduced at that position (ααSyn-Gas6). The amino acid sequence and nucleotide sequence of the chimeric phagocytosis inducer are as shown in Table 5.

[0078] [Table 5] TIFF2025106371000023.tif248166TIFF2025106371000024.tif51166

[0079] Production Example 4. Fusion Molecule Based on ProS1 Targeting β-Amyloid To prepare a β-amyloid (Aβ)-specific chimeric phagocytosis inducer based on the ProS1 protein, first, the Gla domain and the EGF repeat domain were removed, and a single-chain variable fragment (scFv) of aducanumab, a β-amyloid-specific antibody, was introduced at that position (αAβ-ProS1). The amino acid sequence and nucleotide sequence of the chimeric phagocytosis inducer are as shown in Table 6.

[0080] [Table 6] TIFF2025106371000026.tif248166

[0081] Production Example 5. Fusion Molecule Based on Gas6 Targeting β-Amyloid (II): β-Amyloid Binding Region in Fab and Mab Forms To prepare a β-amyloid (Aβ)-specific chimeric phagocytosis inducer based on the Gas6 protein, first, the Gla domain, which is the site that recognizes phosphatidylserine (PS) of apoptotic cells, was removed, and an antigen-binding fragment (Fab) or monoclonal antibody (Mab) of aducanumab, a β-amyloid-specific antibody, was introduced at that position (αAβ[Fab]-Gas6, αAβ[Mab]-Gas6). The amino acid sequences and nucleotide sequences of the two chimeric phagocytosis inducers are as shown in Tables 7 and 8.

[0082] [Table 7] TIFF2025106371000028.tif166166

[0083]

Table 8

[0084] Experimental Example 1. Fusion Molecule Based on Gas6 Targeting β-Amyloid (I): β-Amyloid Binding Region in scFv Form 1-1. Confirmation of the expression of the fusion molecule in the transformed cells After transfection of the plasmid into HEK293 cells, the expression of the fusion molecule containing the Flag tag according to Production Example 1 was confirmed by Western blot using the Flag tag, and the results are shown in Figure 2.

[0085] 1-2. Confirmation of the β-amyloid specific binding ability of the produced fusion molecule To confirm whether αAβ-Gas6(E), αAβ-Gas6, αFITC-Gas6(E), and αFITC-Gas6 can selectively recognize β-amyloid and FITC, respectively, the cell culture supernatants secreted from HEK293 cells transfected with each plasmid were collected and experimented using beads attached with β-amyloid oligomers and FITC. As a result, as shown in Figure 4, αAβ-Gas6(E) and αAβ-Gas6 recognized only the β-amyloid oligomer beads, and αFITC-Gas6(E) and αFITC-Gas6 recognized only the FITC beads, and it was confirmed that they induced phagocytosis.

[0086] It was confirmed that αAβ-Gas6(E) and αAβ-Gas6 showed similar activities, but it was discovered that αAβ-Gas6, from which the EGF domain of Gas6 was further removed, could be obtained in a high yield without aggregation during the protein purification process, and it was determined to use αAβ-Gas6 in future experiments.

[0087] 1-3. Confirmation of the mechanism of action of the produced fusion molecule (1) Confirmation using cell lines An in vitro Aβ engulfment assay was developed such that β-amyloid oligomers conjugated with a pH indicator emit red fluorescence in lysosomes within cells when taken up (uptake) by phagocytosis.

[0088] As a result of performing the in vitro Aβ engulfment assay using HMC3 cells, a human microglial cell line that expresses the TAM receptor, it was confirmed that β-amyloid oligomers were selectively removed by αAβ-Gas6 (Figs. 5 and 6).

[0089] In particular, experiments in which an antibody that inhibits the function of the TAM receptor was co-treated confirmed that αAβ-Gas6 removes β-amyloid oligomers mainly via Axl among Tyro3, Mertk, and Axl (Figs. 7 - 9). In fact, when Axl was removed from HMC3 cells, the activity of αAβ-Gas6 was significantly reduced. Also, THP-1, a human monocytic cell line that does not express the TAM receptor, did not show an increase in the ability to remove β-amyloid by αAβ-Gas6, but THP-Axl cells overexpressing Axl were confirmed to have a greatly increased ability to take up β-amyloid fibrils in an αAβ-Gas6-dependent manner.

[0090] Next, since THP-Axl cells overexpressing Axl possess both Axl receptor and Fc receptor, the degree of inflammatory response induced when taking up β-amyloid via αAβ-Gas6 and aducanumab was confirmed. For this purpose, first, an NF-kB reporter was expressed in THP-Axl cells. When the control group, αAβ-Gas6, and aducanumab were each added together with β-amyloid oligomers, it was confirmed that the NF-kB reporter increased significantly when aducanumab was added, but was expressed at the same level as or lower than the control group in the case of αAβ-Gas6 (Figure 10). Also, when measuring the secreted protein amounts of the three most representative inflammatory cytokines, IL-1b, IL-6, and TNF, when treated with aducanumab, in THP-Axl cells, the protein amounts of these inflammatory cytokines increased significantly compared to the control group (Figure 11). However, importantly, it was confirmed that with αAβ-Gas6, the amounts of these inflammatory cytokines did not increase compared to the control group. This is a core result that, as suggested by our hypothesis, when the αAβ-Gas6 fusion phagocytosis-inducing derivative protein acts on phagocytes via the TAM receptor on the target substance, it does not induce an inflammatory reaction in the same way as the recognition of natural dead cells and efferocytosis.

[0091] Also, it was confirmed that αAβ-Gas6, unlike aducanumab, increases the expression of the Twist1 / 2 gene, which is known as a mechanism for suppressing the inflammatory response (Figure 12).

[0092] (2) Confirmation using astrocytes and microglia To investigate whether astrocytes and microglia, which are cells expressing the TAM receptor in the brain, can remove β-amyloid via αAβ-Gas6, primary astrocytes and microglia collected from the brains of mice were each purified and then cultured. After that, the purified αAβ-Gas6 and aducanumab were put together with β-amyloid fibrils, and the degree of removal of β-amyloid fibrils was observed in real time.

[0093] As a result, similar to the results obtained with the Axl-expressing cell line HMC3, it was confirmed that αAβ-Gas6 increased the β-amyloid removal ability of microglia in a concentration-dependent manner (Figure 13). Importantly, when aducanumab was added, the β-amyloid removal ability of astrocytes did not change at all, but in the case of αAβ-Gas6, it was confirmed that the β-amyloid removal ability of astrocytes increased significantly in a concentration-dependent manner (Figure 14). This means that αAβ-Gas6 significantly improves the previously weak β-amyloid removal ability due to the fact that astrocytes do not express Fc receptors but express TAM receptors.

[0094] After αAβ-Gas6 and aducanumab were each added together with β-amyloid fibrils to astrocytes and the microglial cell line BV2 cells to increase β-amyloid uptake, the mRNA levels of TNF, IL-1α, and IL-1β were measured to examine the degree of inflammatory response in each cell (Figures 15 and 16). As a result, similar to the results obtained with the cell lines, when treated with aducanumab, the amounts of transcripts and proteins of the above inflammatory cytokines were significantly increased in astrocytes and BV2 cells compared to the control group, but in the case of αAβ-Gas6, it was confirmed that the amounts of these inflammatory cytokines did not increase compared to the control group.

[0095] As described above, it was discovered that using the αAβ-Gas6 fusion phagocytosis inducer can be an epoch-making method to effectively remove β-amyloid plaques accumulated in the patient's brain via astrocytes and microglia without the inflammatory response, which is a major side effect of existing monoclonal antibody therapeutic agents. This is a very promising result and is considered to have the potential to greatly improve the current treatment strategy.

[0096] 1-4. In Vivo Efficacy Evaluation (1) Efficacy (Effectiveness) by Introduction of Fusion Molecule or Its Expression Vector 5XFAD was used as an Alzheimer's disease model mouse. Since 5XFAD simultaneously expresses five mutant genes, the production of β-amyloid plaques occurs early, and it is possible to study the pathological symptoms caused by β-amyloid plaques from 3 to 4 months after birth regardless of aging.

[0097] To verify the effect of αAβ-Gas6 in vivo through the 5XFAD model, αAβ-Gas6 was delivered to the brain in two different ways. It is already known from previous studies that in Alzheimer's disease model mice, aducanumab does not reach the brain well by intravenous injection or intraperitoneal injection. Therefore, in order to accurately compare and analyze the effect of αAβ-Gas6 with the effect of aducanumab, 1) a cannulation surgery was performed to directly insert a cannula into the mouse brain, and purified αAβ-Gas6 and aducanumab were injected into the ventricle of the brain once a day for 3 weeks each, 2) αAβ-Gas6 and aducanumab were each prepared in the form of a lentivirus and expressed using stereotaxic injection in the hippocampus of the mouse. Importantly, it was found that the number of β-amyloid plaques decreased significantly when either adding the purified αAβ-Gas6 protein or expressing the gene in the form of a lentivirus (Figures 17 and 18).

[0098] In addition, as a result of quantifying the amount of β-amyloid in the lysosomes of microglia and astrocytes when αAβ-Gas6 was delivered to the brain in the form of protein and virus respectively, it was revealed that the β-amyloid removal ability was significantly increased in both cells (Figures 19 to 22).

[0099] This means that, similar to the in vitro research results, since the TAM receptor is expressed in both microglia and astrocytes, when αAβ-Gas6 is introduced, microglia and astrocytes can recognize and remove β-amyloid.

[0100] (2) Comparison of the effects of the antibody therapeutic agent and the fusion molecule of the present invention In Alzheimer's disease, it is known that synapses are indiscriminately removed by microglia, resulting in a decrease in the number of synapses. Surprisingly, this phenomenon was exacerbated when aducanumab was delivered to Alzheimer's disease model mice, but when αAβ-Gas6 was expressed in viral form, abnormal synapse removal by microglia was restored to normal levels (Figs. 23 and 24).

[0101] Moreover, in Alzheimer's disease model mice, when cognitive and memory tests for remembering the shape and position of new objects were respectively performed according to the protocol of Fig. 25, the expression of αAβ-Gas6 showed a significantly better cognitive and memory recovery effect compared to aducanumab (Fig. 26).

[0102] Furthermore, in order to confirm whether the chimeric phagocytosis-inducing derivative protein of the present invention is also effective in removing various target substances, in addition to β-amyloid, phagocytosis-inducing proteins specific for tau (Tau) and α-synuclein (αSyn) were produced in the same manner as in Production Examples 2 and 3, and the removal effects of each target substance were confirmed as shown in Experimental Examples 2 and 3.

[0103] Experimental Example 2. Fusion Molecule Based on Gas6 Targeting Tau An in vitro tau engulfment assay was developed by conjugating tau oligomers with a pH indicator so that they can emit red fluorescence in lysosomes in cells when taken up by phagocytosis. HMC3 cells, a human microglial cell line expressing the TAM receptor, were treated with a culture solution expressing the phagocytosis-inducing derivative protein [αTau-Gas6] according to Production Example 2 to perform the in vitro tau engulfment assay. As a result, as shown in Fig. 27, it was confirmed that tau oligomers were selectively removed by αTau-Gas6.

[0104] Experimental Example 3. Fusion Molecule Based on Gas6 Targeting α-Synuclein An in vitro αSyn engulfment assay was developed by conjugating α-synuclein oligomers (αSyn oligomer) with a pH indicator so that it can emit red fluorescence in lysosomes in cells when taken up (uptake) by phagocytosis. HMC3 cells, a human microglial cell line expressing the TAM receptor, were treated with a culture solution expressing the phagocytosis-inducing derivative protein [ααSyn-Gas6] according to Production Example 3 to perform the in vitro αSyn engulfment assay. As a result, as shown in Fig. 28, it was confirmed that α-synuclein oligomers were selectively removed by ααSyn-Gas6.

[0105] Experimental Example 4. Fusion Molecule Based on ProS1 Targeting β-Amyloid Next, in the preparation of the chimeric phagocytosis-inducing protein, to confirm whether the use of other ligands of the TAM receptor instead of Gas6 is effective, αAβ-ProS1 was prepared in the same manner as in Production Example 4 using the ProS1 ligand, and its effectiveness was evaluated. For this purpose, in primary cultured mouse astrocytes expressing the TAM receptor, a culture solution expressing αAβ-ProS1 was treated to perform the in vitro Aβ engulfment assay used in Experimental Examples 1-3. As a result, as shown in Fig. 29, it was confirmed that β-amyloid oligomers were selectively removed by αAβ-ProS1.

[0106] Experimental Example 5. Fusion Molecule Based on Gas6 Targeting β-Amyloid (II): β-Amyloid Binding Region in Fab and Mab Forms Next, in the production of the chimeric phagocytosis-inducing protein, in order to confirm whether various target binding sites other than the scFv can be used as the binding domain of the target protein, instead of scFv, an antigen-binding fragment (Fab) or a full-form monoclonal antibody (Mab) was used to produce a phagocytosis-inducing protein according to Production Example 5 and experiments were conducted (αAβ[Fab]-Gas6 and αAβ[Mab]-Gas6). For this purpose, in vitro Aβ engulfment assay used in Experimental Examples 1-3 was performed by treating the culture solution expressing αAβ[Fab]-Gas6 and αAβ[Mab]-Gas6 on HMC3 cells, a human microglial cell line expressing the TAM receptor. As a result, as shown in FIGS. 30 and 31, it was confirmed that β-amyloid oligomers were selectively removed by each of αAβ[Fab]-Gas6 and αAβ[Mab]-Gas6.

[0107] The scope of the present invention is defined by the claims described below, and all changes or modifications derived from the meaning, scope, and equivalent concepts of the claims are included in the scope of the present invention.

[0108] The embodiments for carrying out the invention are described together with the best mode for carrying out the above invention.

Industrial Applicability

[0109] The fusion molecule having phagocytic cell action-inducing activity according to the embodiment of the present invention can solve the problem of tissue damage caused by the activation of the inflammatory reaction of the prior art, and thereby effectively remove abnormally accumulated substances such as β-amyloid, tau, α-synuclein, huntingtin or prion, etc. It can be used for the prevention and treatment of diseases caused by such accumulations, such as Alzheimer's disease, Parkinson's disease, Huntington's disease or prion disease, and can be used in the therapeutic agent industry for the above diseases.

Claims

1. A fusion molecule having phagocyte action-inducing activity, comprising a first region having TAM receptor-binding ability and a second region specifically binding to a target substance.

2. The fusion molecule according to claim 1, wherein the TAM receptor is one or more selected from the group consisting of Tyro3, Axl, and MerTK.

3. The fusion molecule according to claim 1, wherein the first region comprises Gas6, ProS1, Tubby, Tulp1, Gal3, or an active fragment thereof.

4. The fusion molecule according to claim 1, wherein the first region comprises a laminin G-like domain of Gas6 or ProS1, or a fragment binding to the TAM receptor.

5. The fusion molecule according to claim 1, wherein the first region is a laminin G-like domain comprising the sequences of SEQ ID NOs: 1 and 2, or a laminin G-like domain comprising the sequences of SEQ ID NOs: 3 and 4.

6. The fusion molecule according to claim 1, wherein the target substance is a substance that accumulates in living tissue and causes disease.

7. The fusion molecule according to claim 6, wherein the target substance is amyloid.

8. The fusion molecule according to claim 6, wherein the disease is amyloidosis.

9. The fusion molecule according to claim 1, wherein the target substance is selected from β-amyloid, Tau, α-synuclein, huntingtin, prion, and the abnormal accumulation substances in Table 1.

10. The fusion molecule according to claim 1, wherein the second region specifically binding to the target substance is selected from an antibody specifically binding to the target substance, an antigen-binding fragment, an antibody-like protein, a peptide, an aptamer, and a soluble receptor.

11. The fusion molecule according to claim 1, wherein the phagocyte action is induced in cells expressing the TAM receptor.

12. The fusion molecule according to claim 11, wherein the cells expressing the TAM receptor are one or more professional phagocytes, one or more non-professional phagocytes, or a combination thereof.

13. The fusion molecule according to claim 1, wherein the induction of the phagocyte action does not induce an inflammatory reaction.

14. A nucleic acid molecule encoding the fusion molecule according to claim 1.

15. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ An expression vector comprising the nucleic acid molecule according to claim 1. Characterized in that.

16. Expressing the fusion molecule according to claim 1 A cell characterized by that.

17. A pharmaceutical composition for the prevention or treatment of a disease caused by the accumulation of the target substance in a living tissue, comprising the fusion molecule according to claim 1 or the expression vector according to claim 15.

18. A pharmaceutical composition for the removal of a target substance, comprising the fusion molecule according to claim 1 or the expression vector according to claim 15, wherein the target substance is amyloid A pharmaceutical composition for the removal of a target substance, characterized by that.

19. A pharmaceutical composition for the reduction of a target substance, comprising the fusion molecule according to claim 1 or the expression vector according to claim 15, wherein the target substance is amyloid A pharmaceutical composition for the reduction of a target substance, characterized by that.

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