Recombinant glial cell comprising chimeric antigen receptor and use thereof

Abstract

The present invention relates to a recombinant glial cell and, more particularly, to a recombinant glial cell comprising a chimeric antigen receptor consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 10, wherein the recombinant glial cell is capable of specifically recognizing an alpha-synuclein aggregate and binding thereto, thereby inducing clearance of the alpha-synuclein aggregate.

Description

Recombinant glial cells containing chimeric antigen receptors and their uses

[0001] The present invention relates to recombinant glial cells comprising chimeric antigen receptors and the use thereof.

[0002] Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by motor dysfunction resulting from the specific death of dopaminergic neurons in the substantia nigra (SNc) of the midbrain. Consequently, while symptoms can be improved by administering dopamine precursors such as L-DOPA, dopamine agonists, and MAO inhibitors to restore motor function, their effectiveness is limited due to the disease's progression and drug resistance associated with long-term use. Furthermore, it is known that these treatments cannot slow disease progression because they fail to address the underlying cause. A key pathological feature of Parkinson's disease is the extracellular accumulation of alpha-synuclein (α-Syn) protein, which is known to promote neuronal death. While many therapies targeting alpha-synuclein have been developed, they face limitations such as difficulty penetrating the blood-brain barrier and inducing immune responses, necessitating new therapeutic strategies.

[0003] Astrocytes and microglia are major glial cells responsible for maintaining homeostasis and performing neuroprotective functions in the central nervous system. These glial cells possess the ability to remove harmful substances through phagocytosis and have the potential to be utilized for the removal of alpha-synuclein aggregates; however, under natural conditions, their specific recognition and removal capabilities for alpha-synuclein aggregates are limited.

[0004] Chimeric antigen receptors (CARs) are a technology composed of a single-chain antibody that recognizes a specific antigen and an intracellular signaling domain, exhibiting high efficiency in specifically recognizing and eliminating target antigens. When expressed in glial cells, they can confer the ability to specifically recognize and eliminate alpha-synuclein aggregates. This can serve as an innovative therapeutic strategy that overcomes the limitations of existing treatments and enables the direct elimination of alpha-synuclein within the central nervous system.

[0005] The inventors of the present invention developed a chimeric antigen receptor comprising an extracellular domain that specifically binds to alpha-synuclein aggregates and a phagocytic signaling domain, and expressed it in astrocytes to complete the present invention.

[0006] The present invention aims to provide recombinant glial cells comprising a chimeric antigen receptor.

[0007] The present invention aims to provide a pharmaceutical composition for the treatment or prevention of neurodegenerative diseases comprising recombinant glial cells.

[0008] 1. A recombinant glial cell comprising a chimeric antigen receptor composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

[0009] 2. In the above 1, the glial cell is a recombinant glial cell that is an astrocyte or a microglia.

[0010] 3. The recombinant glial cell of 1 above, comprising a chimeric antigen receptor composed of any one of the amino acid sequences of SEQ ID NOs 2 to 4.

[0011] 4. In the above 1, the chimeric antigen receptor is introduced into a recombinant glial cell at the neural progenitor cell stage.

[0012] 5. The recombinant glial cell of 1 above, wherein the chimeric antigen receptor comprises a CD8 cell membrane-penetrating domain.

[0013] 6. The recombinant glial cell of 1 above, wherein the chimeric antigen receptor comprises an intracellular domain of MEGF10.

[0014] 7. In 1 above, the chimeric antigen receptor is a recombinant glial cell that is an alpha-synuclein aggregate-specific binding receptor.

[0015] 8. A pharmaceutical composition for the treatment or prevention of neurodegenerative diseases comprising any one of the recombinant glial cells of 1 to 7 above.

[0016] 9. In the above 8, the neurodegenerative disease is Parkinson's disease, and the pharmaceutical composition for the treatment or prevention of a neurodegenerative disease.

[0017] Recombinant glial cells expressing the chimeric antigen receptor of the present invention can specifically recognize alpha-synuclein aggregates and bind to them.

[0018] Recombinant glial cells expressing the chimeric antigen receptor of the present invention can recognize alpha-synuclein aggregates as antigens, induce their removal, and exhibit phagocytosis.

[0019] The pharmaceutical composition of the present invention can prevent or treat neurodegenerative diseases by removing accumulated alpha-synuclein aggregates or inhibiting the accumulation of alpha-synuclein aggregates.

[0020] Figure 1 is a schematic diagram showing the gene structure of an alpha-synuclein aggregate-specific chimeric antigen receptor.

[0021] Figure 2 shows the results of analyzing the alpha-synuclein aggregate removal ability of chimeric antigen receptors expressed in astrocyte cell lines using a pH-sensitive fluorescent probe.

[0022] Figure 3 shows the results of analyzing the alpha-synuclein aggregate removal ability of chimeric antigen receptors expressed in microglia cell lines using a pH-sensitive fluorescent probe.

[0023] Figure 4 shows the results of the analysis of alpha-synuclein aggregate resolution in a chimeric antigen receptor-expressing cell line.

[0024] Figure 5 shows the results of comparing expression efficiency according to the astrocyte differentiation stage and the timing of chimeric antigen receptor introduction.

[0025] Figure 6 shows the results of confirming intracellular changes in alpha-synuclein aggregates in astrocytes differentiated from human pluripotent stem cells.

[0026] Figure 7 confirms the inhibitory effect of chimeric antigen receptor-expressing astrocytes on neuronal damage.

[0027] Figure 8 shows the results confirming the effect of reducing alpha-synuclein aggregates in chimeric antigen receptor-expressing astrocytes.

[0028] The present invention provides a recombinant glial cell comprising a chimeric antigen receptor.

[0029] The present invention relates to a recombinant glial cell comprising a chimeric antigen receptor, and more specifically, to a recombinant glial cell comprising a chimeric antigen receptor composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10, which can specifically recognize alpha-synuclein aggregates and bind to them to induce the removal of alpha-synuclein aggregates.

[0030] The present invention provides a recombinant glial cell comprising a chimeric antigen receptor composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

[0031] The "Chimeric antigen receptor (CAR)" of the present invention includes a single-chain antibody that recognizes a specific antigen and an intracellular signaling domain, and has high efficiency in specifically recognizing and removing target antigens.

[0032] The chimeric antigen receptor of the present invention may comprise an extracellular domain (ECD), a transmembrane domain, and an intracellular signaling domain. The specific function or activity of the chimeric antigen receptor may vary depending on the selection or combination of the extracellular domain, the transmembrane domain, and the intracellular signaling domain.

[0033] In addition, the chimeric antigen receptor may include an antibody, a single domain antibody, or a single chain variable fragment (scFv) that specifically binds to alpha-synuclein aggregates.

[0034] In addition, the chimeric antigen receptor may further include one or more of a signal peptide, a GS linker, a cell membrane-penetrating domain, and an intracellular domain, and is not limited thereto if it is a generally known component of the chimeric antigen receptor.

[0035] The extracellular domain may include any one of the amino acid sequences represented by SEQ ID NOs 11 to 20, and preferably may include an amino acid sequence having sequence homology of 90% or more, more preferably 95% or more, and most preferably 98% or more with the amino acid sequence.

[0036] Sequence homology for amino acid sequences is confirmed by comparing the optimally arranged sequence with a comparison region, and a portion of the amino acid sequence in the comparison region may include additions or deletions of amino acids (i.e., gaps) when compared with a reference sequence for the optimal arrangement (which does not include additions or deletions).

[0037] The aforementioned short-chain variable fragment may refer to a protein that links the variable regions of the light and heavy chains of an antibody. The two variable regions are connected by a linker peptide composed of approximately 15 amino acids and may have a structure of light-chain variable region-linker-heavy-chain variable region or heavy-chain variable region-linker-light-chain variable region. This short-chain variable fragment retains the antigen recognition properties of the original antibody. The linker primarily uses a flexible hydrophilic peptide chain (GS linker) composed of glycine and serine, and generally has a 15-amino acid sequence such as "(Gly-Gly-Gly-Gly-Ser)3" or a similar structure.

[0038] The extracellular domain of the present invention has excellent binding affinity with alpha-synuclein aggregates.

[0039] The above extracellular domain may include a short-chain variable fragment with high binding affinity to alpha-synuclein aggregates, and the above extracellular domain may include a sequence encoding a receptor with high binding affinity to alpha-synuclein aggregates.

[0040] The above signal peptide may include, for example, a CD8α signal peptide sequence.

[0041] The above transmembrane domain may include, for example, a CD8 hinge or a CD8 transmembrane domain.

[0042] The above intracellular signaling domain may include, for example, an intracellular domain of MEGF10.

[0043] The chimeric antigen receptor containing the above-mentioned MEGF10 intracellular domain can induce potent phagocytosis.

[0044] The recombinant glial cells of the present invention express a chimeric antigen receptor containing a MEGF10 signal sequence that induces the most superior phagocytosis, so they can effectively phagocytize alpha-synuclein aggregates.

[0045] The chimeric antigen receptor of the present invention may be composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

[0046] A chimeric antigen receptor comprising any one of the amino acid sequences of SEQ ID NOs 1 to 10 can specifically recognize alpha-synuclein aggregates and bind to alpha-synuclein aggregates by including a predetermined extracellular domain (ECD). The chimeric antigen receptor may be an alpha-synuclein aggregate-specific binding receptor.

[0047] In one embodiment, the chimeric antigen receptor of the present invention effectively recognized alpha-synuclein aggregates and transported them to lysosomes.

[0048] Therefore, recombinant glial cells containing a chimeric antigen receptor composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10 can perform enhanced alpha-synuclein aggregate-specific phagocytosis by including a signal transduction domain that induces phagocytic activation of the glial cells.

[0049] The chimeric antigen receptor of the present invention can be expressed on the surface of a phagocyte to recognize and bind to a target antigen, thereby inducing activation of the phagocyte. The receptor may be intended to selectively recognize a specific antigen of a target cell to be eliminated, and the target cell may be a cell present in a specific tissue or a cell in a lesion site that causes disease.

[0050] In addition, the chimeric antigen receptor of the present invention may be composed of any one of the amino acid sequences of SEQ ID NOs 2 to 4. The receptor can effectively recognize alpha-synuclein aggregates and transport them to lysosomes.

[0051] In addition, the chimeric antigen receptor composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10 of the present invention includes a functionally equivalent variant. The functionally equivalent variant is a protein produced by the insertion, modification, or removal of amino acids, which exhibits at least 70% sequence similarity to the chimeric antigen receptor protein composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10, and may have sequence similarities of 80%, 90%, or 95% or more. The variant performs essentially the same biological function as the chimeric antigen receptor protein composed of any one of the amino acid sequences of SEQ ID NOs 1 to 10, and specifically, can maintain the ability to selectively recognize and bind to alpha-synuclein aggregates.

[0052] In addition, the present invention includes fragments, variants, and similar molecules of a chimeric antigen receptor.

[0053] “Fragment,” “modifier,” and “similar molecule” may refer to polypeptides that maintain the essential biological function and activity of the chimeric antigen receptor of the present invention. The fragment, modifier, and similar molecule may exist in the following forms: (1) a polypeptide in which one or more amino acids are substituted with other amino acids, wherein the substitution is preferably to an amino acid having similar characteristics, and the said amino acid may or may not be naturally occurring; (2) a polypeptide in which one or more amino acid residues are chemically modified; (3) a polypeptide to which a substance that increases the stability of the polypeptide (e.g., a substance that extends the half-life, such as polyethylene glycol) is conjugated; (4) a polypeptide to which an additional amino acid sequence for a specific purpose is conjugated, wherein such additional sequence may include a signal peptide, a secretion sequence, a label sequence for purification, a precursor protein sequence, or a sequence for a fusion protein.

[0054] The term "glial cells" (neuroglia) in this specification refers to cells that play a role in protecting, assisting, or supporting nerve cells (neurons) in the nervous system, and may be used interchangeably with the term "glial cells."

[0055] The glial cells mentioned above may be astrocytes, microglia, ependymocytes, or oligodendrocytes, and preferably may be astrocytes or microglia.

[0056] The recombinant glial cells of the present invention may be those that have been differentiated into glial cells by introducing the chimeric antigen receptor into neural progenitor cells.

[0057] The above chimeric antigen receptor may be introduced at the neural progenitor cell stage.

[0058] In one embodiment, the cell may be obtained from or manufactured from human pluripotent stem cells.

[0059] In one embodiment, the chimeric antigen receptor was most effectively expressed when the chimeric antigen receptor gene of the present invention was introduced into neural progenitor cells differentiated from human pluripotent stem cells and then differentiated into astrocytes.

[0060] The term "recombinant glial cell" of the present invention may refer to a recombinant glial cell in which a chimeric antigen receptor consisting of any one of the amino acid sequences of SEQ ID NOs 1 to 10 is introduced and expressed. However, it is not limited thereto.

[0061] The above introduction may mean transfection or transduction, for example, transforming glial cells with a recombinant vector.

[0062] The recombinant vector may be a combination of expression regulatory sequences, such as promoters, terminators, and enhancers, and signal sequences for membrane targeting or secretion of proteins, appropriately selected according to the characteristics of the host cell for the expression of the chimeric antigen receptor. The vector includes, but is not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. The vector may include regulatory elements for gene expression regulation, such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and may also be manufactured in various ways depending on the purpose to include signal sequences or leader sequences for membrane targeting or secretion of proteins.

[0063] The recombinant vector may be a viral vector containing a gene encoding a chimeric antigen receptor, and the viral vector may be, for example, a lentivirus, retrovirus, adenovirus, adeno-associated virus, sendaivirus, episomal vector, or BAC vector.

[0064] Glial cells transfected with the chimeric antigen receptor of the present invention can recognize alpha-synuclein aggregates as antigens and bind strongly to them. Through the above introduction, the glial cells can specifically respond to alpha-synuclein aggregates, rather than the receptors that the glial cells originally phagocytated prior to introduction.

[0065] Glial cells transduced with the chimeric antigen receptor of the present invention can exhibit phagocytosis by inducing the removal of alpha-synuclein aggregates.

[0066] The chimeric antigen receptor of the present invention can be introduced into cells exhibiting phagocytosis, for example, it can be expressed in the glial cells.

[0067] The chimeric antigen receptor of the present invention can be expressed in the glial cells to promote the phagocytosis of alpha-synuclein aggregates.

[0068] In addition, the present invention comprises a pharmaceutical composition for the treatment or prevention of neurodegenerative diseases comprising recombinant glial cells.

[0069] The aforementioned neurodegenerative disease refers to a disease in which central nervous system cells cease to function or die due to structural changes in specific parts of the brain, and includes, for example, Alzheimer's disease, Parkinson's disease, Huntington's disease, or atrophic lateral sclerosis.

[0070] “Treatment” means any act in which the symptoms of an alpha-synuclein aggregate-related disease are improved or beneficially altered by the administration of a pharmaceutical composition containing the recombinant glial cells of the present invention.

[0071] “Prevention” refers to any act of suppressing the occurrence of or delaying the onset of alpha-synuclein aggregate-related diseases by administering a pharmaceutical composition containing recombinant glial cells of the present invention.

[0072] In this specification, the term "pharmaceutical composition" may be in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition may be intended for use in humans. The pharmaceutical composition is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injectable solutions according to conventional methods.

[0073] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a flavoring agent, etc.; for injectables, it may include a mixture of a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, a stabilizer, etc.; and for topical administration, a base, an excipient, a lubricant, a preservative, etc.

[0074] The formulations of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be prepared in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release formulations, etc.

[0075] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0076] The routes of administration of the pharmaceutical composition according to the present invention are not limited to but may include oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal, and oral or parenteral administration is preferred. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0077] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, elimination rate, drug combination, and the severity of the disease associated with alpha-synuclein aggregates to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 500 mg / kg or 0.001 to 500 mg / kg per day. Administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0078] The present invention will be explained in more detail below with reference to examples.

[0079]

[0080] Examples

[0081] Example 1. Preparation of a chimeric antigen receptor expression vector that specifically binds to alpha-synuclein aggregates

[0082] To prepare the chimeric antigen receptor of the present invention, 10 types of chimeric antigen receptor extracellular domain protein-coding sequences were synthesized, each having a CD8α signaling peptide and 7 different short-chain variable fragments or 3 types of receptor extracellular domains. By inserting the coding sequence of the CD8α transmembrane domain and the coding sequence of the intracellular signal transduction domain of MEGF10 into the synthesized extracellular domain protein-coding sequences, an alpha-synuclein aggregate-specific chimeric antigen receptor expression vector was prepared.

[0083] The gene and protein sequence combinations of each prepared vector are shown in Table 1, and a schematic diagram of the gene arrangement is shown in Figure 1. The corresponding amino acid sequences are shown in Table 2.

[0084] Sequence Number Gene Sequence Number 1306C7B3-hMEGF10 Sequence Number 2Cinpanemab_scFv-hMEGF10 Sequence Number 3Prasinezumab_scFv-hMEGF10 Sequence Number 4LAG3-hMEGF10 Sequence Number 5NRX1b-hMEGF10 Sequence Number 610H-hMEGF10 Sequence Number 7D5E-hMEGF10 Sequence Number 8D10-hMEGF10 Sequence Number 9DG10-hMEGF10 Sequence Number 10APLP1-hMEGF10

[0085] 서열번호아미노산 서열서열번호 1MALPVTALLLPLALLLHAARPEVQLVESGGGLVKPGGSRKLSCAASGFTFSDHGMHWVRQAPEKGLEWVAYISSGSSTIYYADTVKGRFTISRDNAKNTLFLQMTSLRSEDTAMYYCARSYIPYWGQGTLVTVSAGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호2MALPVTALLLPLALLLHAARPEVQLVESGGGLVEPGGSLRLSCAVSGFDFEKAWMSWVRQAPGQGLQWVARIKSTADGGTTSYAAPVEGRFIISRDDSRNMLYLQMNSLKTEDTAVYYCTSAHWGQGTLVTVSGGGGSGGGGSGGGGSSYELTQPPSVSVSPGQTARITCSGEALPMQFAHWYQQRPGKAPVIVVYKDSERPSGVPERFSGSSSGTTATLTITGVQAEDEADYYCQSPDSTNTYEVFGGGTKLTVLSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호3MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVASISSGGGSTYYPDNVKGRFTISRDDAKNSLYLQMNSLRAEDTAVYYCARGGAGIDYWGQGTLVTVSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKSIQTLLYSSNQKNYLAWFQQKPGKAPKLLIYWASIRKSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQYYSYPLTFGGGTKLEITTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호4MALPVTALLLPLALLLHAARPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호5MALPVTALLLPLALLLHAARPASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNATLQVDSWPVIERYPAGNNDNERLAIARQRIPYRLGRVVDEWLLDKGRQLTIFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLNMAAENDANIAIVGNVRLVGEVPSSMTTESTATAMQSEMSTSIMETTTTLATSTARRGKPPTKEPISQTTDDILVASAECPSDDEDIDPCEPSSGGLANPTRAGGREPYPGSAEVIRESSSTTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호6MALPVTALLLPLALLLHAARPAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSNISSAGKGLEWVSSIDDSGASTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSASFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYTASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSAASPSTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호7MALPVTALLLPLALLLHAARPAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGQKGGGTEYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHFENFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASHLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRRPPSTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호8MALPVTALLLPLALLLHAARPAEVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQVNSLRAEDTAVYYCARINAKWGQGTLVTVSSGGGGSGGGGSGGSALDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPGDFATYYCQQSYSTPTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호9MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYAMHWVRQAPGQRLEWMGWINAGNGKRKYSQKFQDRVTINRDTSASTIYMELSSLGSEDTAVYYCAREEDHAGSGSYLSMDVWGQGSTVTVSSAGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQNVLYSSNNKNYLAWYQQKPGHPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTITSLQTEDVAVYYCQQYYSSPLTFGGGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE서열번호10MALPVTALLLPLALLLHAARPSLAGGSPGAAEAPGSAQVAGLCGRLTLHRDLRTGRWEPDPQRSRRCLRDPQRVLEYCRQMYPELQIARVEQATQAIPMERWCGGSRSGSCAHPHHQVVPFRCLPGEFVSEALLVPEGCRFLHQERMDQCESSTRRHQEAQEACSSQGLILHGSGMLLPCGSDRFRGVEYVCCPPPGTPDPSGTAVGDPSTRSWPPGSRVEGAEDEEEEESFPQPVDDYFVEPPQAEEEEETVPPPSSHTLAVVGKVTPTPRPTDGVDIYFGMPGEISEHEGFLRAKMDLEERRMRQINEVMREWAMADNQSKNLPKADRQALNEHFQSILQTLEEQVSGERQRLVETHATRVIALINDQRRAALEGFLAALQADPPQAERVLLALRRYLRAEQKEQRHTLRHYQHVAAVDPEKAQQMRFQVHTHLQVIEERVNQSLGLLDQNPHLAQELRPQIQELLHSEHLGPSELEAPAPGGSSEDKGGLQPPDSKDDTPMTLPKGSTEQDAASPEKEKMNPLEQYERKVNASVPRGFPFHSSEIQRDELAPAGTGVSRETTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCTSYRHKQKGKESSMPAVTYTPAMRVVNADYTISGTLPHSNGGNANSHYFTNPSYHTLTQCATSPHVNNRDRMTVTKSKNNQLFVNLKNVNPGKRGPVGDCTGTLPADWKHGGYLNELGAFGLDRSYMGKSLKDLGKNSEYNSSNCSLSSSENPYATIKDPPVLIPKSSECGYVEMKSPARRDSPYAEINNSTSANRNVYEVEPTVSVVQGVFSNNGRLSQDPYDLPKNSHIPCHYDLLPVRDSSSSPKQEDSGGSSSNSSSSSE

[0086]

[0087] Example 2. Preparation of a recombinant lentivirus for expressing a chimeric antigen receptor that specifically binds to alpha-synuclein aggregates

[0088] To prepare recombinant lentiviruses for expressing chimeric antigen receptors that specifically bind to alpha-synuclein aggregates, the HEK293FT cell line (Invitrogen) was prepared as follows. For cell culture, complete medium was prepared by adding 10% fetal bovine serum (FBS) and 1X Penicillin-Streptomycin (Gibco) to DMEM medium (Gibco), mixing uniformly by induction, and preheating to 37°C. For the HEK293FT cell line, frozen cell stock was rapidly thawed in a 37°C water bath for 2–3 minutes, inoculated into 5 mL of complete medium, and cultured at 37°C under 5% CO2 conditions. Once cell saturation reached 80% or higher, it was maintained through subculture. For lentivirus transfection, 1–2 x 10⁶ HEK293FT cell lines were placed in a 10 cm diameter culture dish. 6 Transfection was performed after inoculating 10 mL of complete medium at the concentration of cells and culturing for 16 hours.

[0089] Lentivirus transfection was performed using Lipofectamine 3000 (Invitrogen). 4 μg of the expression vector prepared in the same manner as in Example 1, and 1.3 μg each of the pRSV-Rev, pMDLg / pRRE, and pMD2.G packaging vectors were added to 500 μL of Opti-mem (Invitrogen) along with 30 μL of p3000. Separately, 30 μL of lipofectamine 3000 was added to 500 μL of Opti-mem, and the two solutions were mixed and reacted at room temperature for 20 minutes. The mixture thus prepared was dropwise added to the prepared HEK293FT cells and mixed uniformly. After incubation for 16 hours, the solution was replaced with 9 mL of fresh complete medium and incubated for an additional 32 hours.

[0090] After incubation, the supernatant was collected and stored at 4°C, and 9 mL of fresh complete medium was added to the cells. After 24 hours, all collected supernatants were combined and filtered through a 0.45 μm filter to remove cell residues, and Lenti-X concentrator (Takara) was added in an amount equal to 1 / 3 of the total supernatant volume and treated for at least 16 hours. The treated supernatant was centrifuged at 1500g for 45 minutes to concentrate the lentivirus, and the concentrated lentivirus was dissolved in phosphate buffered saline (PBS) and stored at -80°C until use.

[0091]

[0092] Example 3. Preparation of immortalized mouse astrocytes and microglia HMC3 cells expressing a chimeric antigen receptor that specifically binds to alpha-synuclein aggregates

[0093] To prepare immortalized mouse astrocyte cell lines and HMC3 cells expressing a chimeric antigen receptor that specifically binds to alpha-synuclein aggregates, recombinant lentiviruses prepared in the same manner as in Example 2 were transfected into each cell line. More specifically, recombinant lentiviruses at a concentration of 0.1 to 10 MOI were inoculated into complete medium supplemented with 8 μg / mL of polybrene (Merk) and inverted to ensure uniform mixing. After adding 1 mL of the mixture to each cell line prepared in a 6-well plate, the mixture was centrifuged at 1,000 g for 60 minutes and incubated for 24 hours in a 37 ℃, 5% CO2 incubator. After 24 hours, the culture medium was replaced with fresh medium.

[0094] To confirm the expression of a chimeric antigen receptor that specifically binds to alpha-synuclein aggregates in transduced cell lines, transduced immortalized mouse astrocyte cell lines and HMC3 cells were collected, washed with PBS, and the expression of the P2A sequence-linked fluorescent protein mRuby3 was confirmed using a flow cytometry analyzer (FACS).

[0095] Flow cytometry confirmed that immortalized mouse astrocyte cell lines and HMC3 cells expressing a chimeric antigen receptor that specifically binds to alpha-synuclein aggregates were produced.

[0096]

[0097] Example 4. Screening of Alpha-Synuclein Aggregate Removal Ability of Chimeric Antigen Receptors Using a pH-Sensitive Fluorescent Probe

[0098] To track the intracellular migration pathway of alpha-synuclein aggregates, the pH-dependent fluorescent dye Protonex Green was conjugated to the aggregates. First, recombinant human alpha-synuclein protein was dissolved in DPBS at a concentration of 1 mg / mL, and alpha-synuclein aggregates were prepared by stirring in a thermomixer (Eppendorf) at 1,000 rpm and 37°C for 7 days. The prepared alpha-synuclein aggregates were reacted with the pH-dependent fluorescent dye Protonex Green (AAT Bioquest) at room temperature for 3 hours according to the manufacturer's protocol. After the reaction, the aggregates were washed using a 3 kDa cut-off centrifuge filter to remove excess fluorescent dye. The fluorescently labeled alpha-synuclein aggregates were homogenized by treating them three times for 10 seconds each using a probe sonicator.

[0099] The 10 types of chimeric antigen receptor-expressing astrocyte cell lines prepared in Example 3 were each 1 x 10 5 Cells were seeded into 24-well plates at a cell / well concentration, and after 24 hours, alpha-synuclein aggregates labeled with Protonex Green at a concentration of 1 μg / mL were treated. Cells were collected at 4, 8, 12, and 24 hours after treatment, washed twice with PBS, and the intracellular fluorescence intensity of Protonex Green was measured using a flow cytometer. Since Protonex Green exhibits increased fluorescence intensity in lysosomes at low pH, an increase in fluorescence intensity indicates the migration of alpha-synuclein aggregates to the lysosomes.

[0100] Analysis results showed that among 10 chimeric antigen receptor-expressing astrocyte cell lines, fluorescence intensity increased significantly in a time-dependent manner in four chimeric antigen receptor-expressing cell lines, including the short-chain variable fragment of cinpanemab, the short-chain variable fragment of 306C7B3, the extracellular domain of LAG3, and the extracellular domain of NRX1B. In particular, the highest fluorescence intensity was observed at 24 hours after treatment, indicating that the corresponding chimeric antigen receptors can effectively recognize alpha-synuclein aggregates and transport them into lysosomes (Fig. 2).

[0101] The four types of chimeric antigen receptor-expressing microglia cell lines prepared in Example 3 were analyzed after being treated for 24 hours in the same manner as the astrocyte cell lines. As a result, among the four types of chimeric antigen receptor-expressing microglia cell lines, the ability to invaginate alpha-synuclein aggregates and move lysosomes was confirmed in three types of chimeric antigen receptor-expressing cell lines containing the short-chain variable fragment of cinpanemab, the short-chain variable fragment of 306C7B3, and the extracellular domain of LAG3 (Fig. 3).

[0102]

[0103] Example 5. Analysis of Alpha-Synuclein Aggregate Resolution in Chimeric Antigen Receptor-Expressing Cell Lines

[0104] Western blot analysis was performed to evaluate the alpha-synuclein aggregate degradation ability of cell lines expressing the chimeric antigen receptor selected in Example 4. Each cell line was treated with 1 μg / mL of alpha-synuclein aggregate and cultured for 24 hours with or without treatment with the lysosomal degradation inhibitor chloroquine (25 μM).

[0105] After culture, cells were collected and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, protease inhibitor cocktail), and the soluble fraction was separated by centrifugation at 13,000 rpm for 20 minutes. The insoluble pellet was redissolved in 2% SDS buffer (50 mM Tris-HCl pH 7.4, 2% SDS).

[0106] Protein samples were subjected to electrophoresis using a 4-12% Bis-Tris polyacrylamide gel. The Bis-Tris gel was prepared with a thickness of 1.5 mm, and for the separation gel, based on a final volume of 18 mL, 6 mL of 3x bis-Tris gel buffer (1 M bis-Tris, pH 6.5-6.8), 3.6 mL of 40% acrylamide stock solution (29:1), and 8.4 mL of distilled water were mixed, and 150 μL of 10% APS and 60 μL of TEMED were added for polymerization.

[0107] Proteins that had undergone electrophoresis were transferred to a PVDF membrane and detected using an anti-alpha-synuclein antibody. In the soluble fraction, no difference in the amount of alpha-synuclein protein was observed depending on whether or not chloroquine was treated; however, in the insoluble fraction, alpha-synuclein aggregates in the form of dimers and oligomers, including 15 kDa monomers, accumulated in cells treated with chloroquine (Fig. 4). This indicates that cells expressing chimeric antigen receptors are transporting alpha-synuclein aggregates to lysosomes to degrade them.

[0108]

[0109] Example 6. Analysis of Chimeric Antigen Receptor Expression and Alpha-Synuclein Aggregate Removal Ability in Human Pluripotent Stem Cell-Derived Astrocytes

[0110] As presented in previous literature (J Cell Physiol. 236:7625 (2021) and Korean Patent 10-2534488), astrocytes were differentiated from human pluripotent stem cells as follows. Human pluripotent stem cell-derived neural progenitor cells (hPSC-NPCs) were placed in a Matrigel-coated 6-well plate at a passage count of 5 or less at a rate of 3 × 10⁶ 4 Cell count / cm² 2 The cells were inoculated at a density of [value]. Subsequently, rtTA-expressing lentivirus, NFIB-expressing lentivirus, and the chimeric antigen receptor-expressing lentivirus selected in Example 4 were treated with 1 μg / ml polybrene (Millipore Sigma) (multiplicity of infection [MOI]: 1.0 each). To increase transfection efficiency, centrifugation was performed at 1,000g for 1 hour at room temperature. After 18 hours of incubation, the medium containing the viruses was removed and replaced with fresh neural progenitor cell medium containing 2.5 μg / ml doxycycline (DOX) to induce NFIB expression. The day of DOX treatment was designated as Day 0.

[0111] On Day 1, the medium was replaced with neural progenitor cell medium containing 10 ng / ml ciliary neurotrophic factor and 10 ng / ml BMP4 (Peprotech), and on Day 3, it was replaced with commercial astrocyte medium (ScienCell) to proceed with differentiation until Day 14. To select virus-infected cells, 1.25 μg / ml puromycin was administered from Day 1 to Day 5.

[0112] 1x10 on day 14 of the eruption 5Cells were inoculated into a 24-well plate at a concentration of cell-to-well, and after 24 hours, Protonex Green-labeled alpha-synuclein aggregates prepared in the same manner as in Example 4 were treated at a concentration of 1 μg / mL. After 24 hours of treatment, the cells were collected, washed twice with PBS, and the fluorescence intensity of Protonex Green within the cells was measured using a flow cytometer. As a result of the analysis, a significant increase in fluorescence intensity was observed in human pluripotent stem cell-derived astrocytes expressing chimeric antigen receptors compared to the control group, indicating that the prepared cells can effectively transport alpha-synuclein aggregates into lysosomes.

[0113] To effectively express chimeric antigen receptors in astrocytes, the timing of introducing the gene for the chimeric antigen receptor during the differentiation process of astrocytes was tested. During the differentiation process from human pluripotent stem cells to astrocytes, a chimeric antigen receptor-expressing lentivirus was introduced at the human embryonic stem cell stage (ES-CAR), neural progenitor cell stage (NPC-CAR), and astrocyte stage (AST-CAR), respectively.

[0114] Flow cytometry analysis showed that ES-CAR cells exhibited a chimeric antigen receptor expression rate of approximately 20%, while NPC-CAR cells showed the highest expression rate of approximately 75%. AST-CAR cells showed an expression rate of approximately 25%. This suggests that introducing the chimeric antigen receptor gene at the neural progenitor cell stage is the most efficient (Fig. 5).

[0115] Furthermore, NPC-CAR cells exhibited the highest expression levels in mRNA expression analysis via qRT-PCR. As a result of qRT-PCR analysis using scFv-specific primers, NPC-CAR cells displayed significantly higher mRNA expression levels compared to ES-CAR or AST-CAR cells. Specifically, NPC-CAR cells showed expression levels approximately 3 times higher than ES-CAR cells and 5 times higher than AST-CAR cells. A similar trend was observed in qRT-PCR analysis using EGFP-specific primers, with the highest mRNA expression levels observed in NPC-CAR cells (Fig. 5). These results indicate that introducing the gene at the neural progenitor cell stage is optimal for the efficient expression of chimeric antigen receptors, and suggest that neural progenitor cells are in a cellular state more suitable for the introduction and expression of foreign genes.

[0116] Finally, the function of chimeric antigen receptors was verified in astrocytes differentiated from human pluripotent stem cells. Alpha-synuclein aggregates labeled with Protonex Green were treated with a control group (mRuby3) and astrocytes expressing 306C7B3 and Cinpanemab scFv chimeric antigen receptors, and intracellular fluorescence changes over time were measured by flow cytometry (Fig. 6).

[0117] When comparing the time immediately after treatment (0h) and after 24 hours (24h), the fluorescence intensity in astrocytes expressing 306C7B3 and Cinpanemab scFv chimeric antigen receptors increased significantly. This means that even in astrocytes differentiated from stem cells, chimeric antigen receptors can effectively capture alpha-synuclein aggregates and deliver them to lysosomes.

[0118]

[0119] Example 7. Confirmation of the inhibitory effect of chimeric antigen receptor-expressing astrocytes on neuronal damage

[0120] To determine whether chimeric antigen receptor-expressing astrocytes (CAR-Ast) can inhibit neuronal damage, a co-culture experiment was performed using a dopamine neuron damage model. Dopamine neurons differentiated from human pluripotent stem cells were inoculated into a 24-well plate, and neuronal damage was induced by treating them with 6-OHDA (6-hydroxydopamine), a dopamine neuron-specific toxin. Subsequently, fibroblasts or chimeric antigen receptor-expressing astrocytes (CAR-Ast) prepared in Example 6 were co-cultured as controls, respectively.

[0121] To evaluate the degree of neuronal damage, immunofluorescence staining for tyrosine hydroxylase (TH), a marker of dopamine neurons, was performed, and TH fluorescence intensity was quantitatively analyzed (Fig. 7). As a result of the analysis, the highest TH fluorescence intensity was observed in the group of dopamine neurons cultured alone without 6-OHDA treatment (Only neuron). Neuronal damage was confirmed in the group co-cultured with fibroblasts after 6-OHDA treatment (+6-OHDA, +Fibroblast), where TH fluorescence intensity decreased significantly. On the other hand, significantly higher TH fluorescence intensity was observed in the group co-cultured with chimeric antigen receptor-expressing astrocytes after 6-OHDA treatment (+6-OHDA, +CAR-Ast) compared to the fibroblast co-culture group (p<0.05).

[0122] These results indicate that astrocytes expressing chimeric antigen receptors can effectively inhibit dopaminergic neuronal damage induced by 6-OHDA, and that treatment for dopaminergic neuronal degenerative diseases such as Parkinson's disease is possible.

[0123]

[0124] Example 8. Confirmation of the effect of reducing alpha-synuclein aggregates in chimeric antigen receptor-expressing astrocytes

[0125] To investigate the therapeutic effect of chimeric antigen receptor-expressing astrocytes on the intracellular accumulation of pathogenic alpha-synuclein, a major pathological feature of Parkinson's disease, experiments were conducted using an alpha-synuclein preformed fibrils (PFF) model. Human pluripotent stem cell-derived dopaminergic neurons were co-cultured with control astrocytes (mCherry-Ast) or chimeric antigen receptor-expressing astrocytes (CAR-Ast) or cultured alone, and treated with PFF to induce the accumulation of pathogenic alpha-synuclein.

[0126] The protein level of alpha-synuclein phosphorylated at serine residue 129 (pS129 αSyn), a representative indicator of pathogenic alpha-synuclein, was measured via Western blot analysis and quantified by normalizing to TH expression levels (Fig. 8). As a result of the analysis, accumulation of pS129 alpha-synuclein was confirmed in the group treated only with PFF, while the group co-cultured with control astrocytes (mCherry-Ast) showed a tendency for pS129 alpha-synuclein levels to increase. On the other hand, in the group co-cultured with chimeric antigen receptor-expressing astrocytes (CAR-Ast), the level of pS129 alpha-synuclein was significantly reduced compared to the group treated only with PFF, and the level decreased to about one-fifth of that of the control astrocyte co-culture group.

[0127] It was confirmed that astrocytes expressing chimeric antigen receptors can effectively remove alpha-synuclein aggregates, thereby effectively inhibiting the production of pathogenic pS129 alpha-synuclein within neurons. This demonstrates that the lysosomal transport function of alpha-synuclein aggregates identified in Example 6 can be applied in the same way in actual pathological environments to produce therapeutic effects.

Claims

1. A recombinant glial cell comprising a chimeric antigen receptor consisting of any one of the amino acid sequences of SEQ ID NOs 1 to 10.

2. The recombinant glial cell of Claim 1, wherein the glial cell is an astrocyte or a microglia.

3. The recombinant glial cell of claim 1 comprising a chimeric antigen receptor composed of any one of the amino acid sequences of SEQ ID NOs 2 to 4.

4. The recombinant glial cell of claim 1, wherein the chimeric antigen receptor is introduced into a cell at the neural progenitor cell stage.

5. A recombinant glial cell according to claim 1, wherein the chimeric antigen receptor comprises a CD8 cell membrane-penetrating domain.

6. A recombinant glial cell according to claim 1, wherein the chimeric antigen receptor comprises an intracellular domain of MEGF10.

7. The recombinant glial cell of claim 1, wherein the chimeric antigen receptor is an alpha-synuclein aggregate-specific binding receptor.

8. A pharmaceutical composition for the treatment or prevention of neurodegenerative diseases comprising recombinant glial cells according to any one of claims 1 to 7.

9. A pharmaceutical composition for the treatment or prevention of a neurodegenerative disease, wherein the neurodegenerative disease is Parkinson's disease, in accordance with claim 8.

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