Fusion molecule and method for treating immunological diseases

Abstract

The present disclosure relates to a fusion molecule is capable of binding to an antigenic substance of which an elevated level or excessive amount causes or results in an immunological disease and inducing phagocytosis, thereby removing, reducing, or enhancing removal or reduction of the antigenic substance. Therefore, the fusion molecule can be useful in treating an immunological diseases, delaying development or onset of an immunological diseases, or alleviating symptoms of an immunological diseases.

Description

Title of Invention: FUSION MOLECULE AND METHOD FOR TREATING IMMUNOLOGICAL DISEASES Technical Field [1] The present disclosure relates to fusion molecules that are suitable for preventing or treating immunological diseases. The present disclosure also relates to nucleic acid molecules encoding the fusion molecules. The present disclosure further relates to compositions containing the fusion molecules, methods of preventing or treating immunological diseases, uses of the fusion molecules in treating immunological diseases. [2] Background Art [3] Immune diseases are diseases in which components of the mammalian immune system cause, mediate, or otherwise contribute to pathological conditions in mammals, and in particular, inflammatory diseases are diseases that are attracting attention worldwide and are in an urgent need of a therapeutic agent. Inflammation is generally a localized protective response of body tissues against host invasion by foreign substances or harmful stimuli. Causes of inflammation may include infectious causes such as bacteria, viruses and parasites, physical causes such as burns or radioactive irradiation, chemicals such as toxins, drugs or industrial agents, immune responses such as allergies and autoimmune responses, or conditions associated with oxidative stress. [4] In normal cases, the inflammatory response removes external infectious agents and regenerates damaged tissues to restore the functions of the living organism. However, if the inflammatory response occurs excessively or continuously when antigens are not removed or internal substances are the cause, it may cause acute inflammation as a disease that threatens the human life, diseases within joints such as rheumatoid arthritis, skin diseases such as psoriasis, allergic inflammatory diseases such as bronchial asthma, and autoimmune diseases caused by the immune system's attack on autoantigens, and it may be an obstacle to treatment processes such as blood transfusions, drug administration, and organ transplantation. [5] Currently, drugs such as steroids and aspirin have been developed as therapeutic agents for excessive immune responses such as inflammatory diseases and autoimmune diseases, but these are known to cause symptoms such as edema, gastrointestinal diseases, bleeding, and hepatotoxicity as side effects. In addition, there are cases where it fails to act selectively on the cause of inflammation, resulting in severe immunosuppression (Check and Kaliner, Am. Rev. Respir. Dis., 141, p44-51. 1990). In addition, since there is still no therapeutic agent that can completely treat these diseases, an effective therapeutic agent without side effects is necessary. [6] Meanwhile, TAM (Tyro3, Axl, and MerTK) receptors are receptor tyrosine kinases, and it has recently been highlighted that TAM ligands that are capable of activating these receptors are important in controlling tissue homeostasis and inflammation. TAM receptors are known to be particularly involved in anti-inflammatory action and resolution of inflammation, where the anti-inflammatory action refers to reduction and elimination of the activity of pro-inflammatory mediators which may be carried out by inhibition of the synthesis of these mediators, selective antagonistic actions, scavenging, post-translational modification such as cleavage, or decomposition. The resolution of inflammation may be accomplished by such methods as removing the stimulus that causes inflammation, promoting the removal of the causative cells through apoptosis or phagocytosis, enhancing the induction of non-inflammatory macrophages, facilitating macrophage reprogramming, and secreting inflammation-inhibiting substances (e.g. IL-10, etc.). [7] As these characteristics were reported, various attempts have been made to treat inflammatory or autoimmune diseases by administering TAM ligands Gas6 or ProSl, and reports have actually shown that they exhibited effects in reducing the secretion of pro-inflammatory cytokines and alleviating some of the symptoms caused by inflammation. (Peng et al., PLoS One, 14, e0219788, 2019; Waterborg et al., Front. In Immunol., 9, 742, 2018; Jiang et al., J. Cell Mol. Med., 23(4), 2769-2781, 2019). However, the TAM ligands contain only a region that binds to the TAM receptor and a region that binds to PS (phosphatidylserine) as the regions that has activity related to binding to other molecules, thus making it difficult to selectively act on substances that cause inflammation. [8] Therefore, there is a need for improved approaches that are capable of efficiently inducing selective clearance of antigens. [9] Disclosure of Invention Technical Problem

[10] The present disclosure relates to fusion molecules that can induce selective clearance of a target substance that triggers or induces undesired or pathological immune reaction such as autoimmune disease, transplant rejection, or allergic or hyperimmune responses.

[11] Objects to be achieved by the present invention are not limited to the abovementioned object, and other objects not mentioned herein will be clearly understood by those skilled in the art from the following description. Solution to Problem

[13] One aspect of the present disclosure provides a fusion molecule comprising: a first region that is capable of binding to a TAM (Tyro3, Axl and MerTK) receptor; and a second region that specifically binds to a target substance to be cleared or decreased, and the fusion molecule does not induce inflammatory responses and wherein an increased or elevated level of the target inflammation-related substance or an increased or elevated expression of the target inflammation-related substance triggers or induces undesired or pathological immune response such as autoimmune disease, transplant rejection, or allergic or hyperimmune responses. In embodiments, the fusion molecule does not have an effector function and does not induce Fc-mediated inflammatory responses.

[14] In some embodiments, the TAM receptor may be any one selected from the group consisting of Tyro3, Axl, MerTK, or a combination thereof, which are capable of inducing phagocytosis by binding to a laminin G-like domain (or LG domain) of a phagocytic cell including, but not limited to, macrophages or microglial cells. In embodiments, the TAM receptor may be Axl region of TAM receptor.

[15] In embodiments, the first region may comprise Gas6, ProSl, Tubby, Tulpl, Gal3, or an active fragment thereof, which each is capable of specifically binding to a TAM receptor. The first region may be selected from Gas6, ProSl, or an active fragment thereof, which each is capable of specifically binding to a TAM receptor. In embodiments, the first region may comprise or consist essentially of Gas6 or an active fragment thereof that is capable of binding to TAM receptor. In embodiments, the first region comprising or consisting essentially of Gas6 or an active fragment thereof is capable of binding to Axl receptor.

[16] In certain embodiments, the first region may comprise a laminin G-like domain of Gas6 or ProSl, or an active fragment thereof, which contains a laminin G-like domain as a phagocytosis-related bridging molecule which is abundantly expressed in various tissue, and thus is able to induce phagocytosis through a TAM receptor. In embodiments, the laminin G-like domain may comprise an LG1 domain, an LG2 domain, or a combination thereof, and may preferably include both an LG1 domain and an LG2 domain, which are able to induce phagocytosis by binding to the TAM receptor.

[17] Exemplary embodiments are directed to a binding molecule or fusion molecule comprising a first region capable of biding to a TAM receptor and a second region capable of specifically biding to a target inflammation-related substance, said target inflammation-related substance being a substance with an increased or elevated amount or with an increased or elevated expression that triggers, induces, or causes undesired or pathological immune reaction such as autoimmune disease, transplant rejection, or allergic or hyperimmune responses, wherein the first region and the second region are coupled to each other directly or via a linker, wherein the first region comprises

[18] (a) a TAM receptor ligand;

[19] (b) an anti-Axl antibody or an antigen-binding fragment thereof;

[20] (c) an anti-Tyro3 antibody or an antigen-binding fragment thereof; or

[21] (d) an anti-MerTK antibody or an antigen-binding fragment thereof, with proviso that when the first region comprises an anti-MerTK antibody or an antigen-binding fragment thereof, the molecule is not a bispecific antibody; or

[22] (e) combinations thereof.

[23] According to some embodiments, the binding molecule may further comprise a scaffold bound to the first region, to the second, or to both of the first region and the second region at different positions.

[24] In embodiments, the first region is a TAM receptor ligand and the TAM receptor ligand comprises a sequence selected from the group consisting of SEQ ID NOs: 1-113 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto.

[25] In still some embodiments, the first region is capable of binding to an Axl receptor the first region capable of binding to an Axl receptor comprises one or more sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto.

[26] In still some embodiments, the first region is capable of binding to an Axl receptor the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 1 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto, and / or the sequence of SEQ ID NO: 2 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto.

[27] In still another embodiment, the first region capable of binding to an Axl receptor the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 5 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto.

[28] In still some embodiments, the first region is capable of binding to an Axl receptor the first region capable of binding to an Axl receptor comprises one or more sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto.

[29] In still some embodiments, the first region is capable of binding to an Axl receptor the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 3 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto, and / or the sequence of SEQ ID NO: 4 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto.

[30] In still another embodiment, the first region capable of binding to an Axl receptor the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 6 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99% of sequence identity thereto.

[31] In embodiments, the fusion proteins according to present disclosure do not contain the target substance that is to be cleared or reduced by the administration of the fusion proteins.

[32] In embodiments, the first region comprising a laminin G-like domain of Gas6 or ProSl, or an active fragment thereof, does not comprise a Gia domain. Without being bound to a particular theory, it is expected that the lacking of Gia domain in the first region may make the fusion molecule not be able to recognize phosphatidylserine (PS) of TAM receptor, while the second region is able to induce phagocytosis by recognizing a target substance.

[33] In some embodiments, the first region comprising a laminin G-like domain of Gas6 or ProSl, or an active fragment thereof, does not comprise a Gia domain and does not comprise an EGF domain. The lacking of EGF domain in the first region provides an advantage in the manufacturing process of the fusion molecule to increase the yield by suppressing an aggregation of the fusion molecule during the purification step. In some embodiments, the fusion molecule (or binding molecule) may form a homodimer or a heterodimer, or form a linear multimer as a single chain.

[34] According to embodiments, the target substance that is to be cleared or decreased and to which the second region specifically binds, may be a substance of which an increased or elevated amount or with increased or elevated expression triggers, induces or causes undesired or pathological immune reaction such as autoimmune disease, transplant rejection, or allergic or hyperimmune responses.

[35] The target substance may be an inflammation-related substance. The target substance may be one or more selected from autoantigens in Table 1 below, autoantibodies thereof, or a complex of the autoantigens and the autoantibodies thereof; immune cell surface molecules including co-stimulatory molecules and receptors such as CD20, CD 19, CD52, CD80 / 86, CD28, CD40, CD40L, OX40, OX40L, C5 alpha receptor 1, IL-1R, IL-6R, IL-17R, IL-4R, IL-5R, IL-13R, IFN-gamma receptor, IL-12R, IL-21R, IL-22R, TGF-beta receptor, IL-23R, Thymic Stromal LymphoPoietin receptor (TSLPR), IL-31R, IL-33R, IGF-1R, TNFR, FcRn Large Subunit p51, Integrin alpha-D (ITGAD), Toll-like receptors (TLRs) including TLR3, TLR4, TLR5, and TLR7, or the like; complements such as Complement Clq, Complement C3, Complement C5, or the like; chemokines such as CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, or the like; cytokines such as IL-1, TNF-alpha, IL-6, IL-17, IL-4, IL-5, IL-13, IFN-gamma, IL-12, IL-21, IL-22, TGF-beta, IL-23, Thymic Stromal LymphoPoietin (TSLP), IL-31, IL-33, or the like; cell adhesion molecules such as ICAM1, VCAM1, MADCAM1, Integrin alpha 4, Integrin beta 7, LFA-1 (or MAC-1), VLA-4, or the like. In Table 1 below, amino acid sequences of the autoantigens as well as exemplary references disclosing antibodies binding to the target substances are listed. One skilled in the art should understand that ligands or receptors or autoantibodies, which bind to the listed target autoantigen substances, can also be encompassed as the second region in the present disclosure. The entire contents of the references in Table 1 are incorporated by reference herein.

[36]

[37] [Table 1] Target Substance Abbreviati on Disease UniProt (UniProtK B reviewed (Swiss-Pro t)) GenBank or NCBI Accession No. Exemplary References disclosing region binding to target substance, or known binding materials Factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, thrombin, vWF Acquired hemophilia A C9JV37 US201500992 98A1, US202302871 41A1 Calcium-sen sing receptor CaSR Acquired hy-poparathyroidis m, autoimmune P41180 Spermine, Gamma-Gluta myl peptides, hypophysitis, autoimmune oophoritis L- amino acids ACTH ACTH deficiency WO20161008 38A2 21-Hydroxyl ase (CYP21) Addison disease, APS-III P08686 Trichohyalin Alopecia areata Q07283 Oxidized LDL (OxLDL) Atherosclerosis US201101828 16A1 Transcription coactivator p75 Atopic dermatitis 075475 JP02 (Maertens et al., Journal of Cell Sciences, vol 119,Issue 12, 2006) p-80-Coilin Atopic dermatitis, Linear morphea AAB81550.1 Cl inhibitor Autoimmune Cl deficiency P05155 US10080788B 2 AMPA-recep tor Autoimmune encephalitis EP2338492A1 CRMP5 Autoimmune encephalitis Q9BPU6 DPPX / DPP6 Autoimmune encephalitis P42658 GABAA-rec eptor GABAaR Autoimmune encephalitis P34903 and others * GAB AB-rec eptor GABAbR Autoimmune encephalitis Q9UBS5, 075899 Glycine Autoimmune P23415, receptor (GlyR) encephalitis P23416, 075311, P48167 Hu (ANNA-1) Autoimmune encephalitis Mal Autoimmune encephalitis Q8ND90 US7026450B2 US6387639B1 Ma2 Autoimmune encephalitis Q9UL42 US7026450B2 US6387639B1 Ri (ANNA-2) Autoimmune encephalitis Zic4 Autoimmune encephalitis AAI36340.1 Voltage-gate d potassium channel (VGKC)-co mplex Autoimmune encephalitis (Isaac's syndrome / acqui red neuromyotonia, Miller Fisher syndrome, Morvan's syndrome) NMDA-rece ptor Autoimmune encephalitis, systemic lupus erythematosus (SLE) Q05586, 015399, Q14957, Q12879, QI 3224, 060391, Q8TCU5 WO20141878 79 Joi Autoimmune encephalitis, systemic lupus erythematosus (SLE), arthritis, Polymyositis / de rmatomyositis H / K ATPase Autoimmune gastritis, pernicious anemia / atrophic gastritis Thyroid peroxidase Autoimmune Hashimoto thyroiditis, immun-odysregulation, polyendocrinop athy, enteropathy, X-linked P07202 Erythrocyte Ei Autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome Sci Transl Med. (2019) 11(506): eaau8217. F-actin Autoimmune hepatitis US5632991A Asialoglycop rotein receptor Autoimmune hepatitis type 1 AAB58308.1 WO20140237 09 Al Cytochrome P450 2D6 (CYP2D6) Autoimmune hepatitis type 2 P10635 NXP-2 / MOR C3 Autoimmune myopathies Q14149 TIFl-y / TRI M-33 Autoimmune myopathies Q9UPN9 [32 Integrin Autoimmune neutropenia, P05107 Evans syndrome Nuclear au-toantigenic sperm protein (NASP) NASP Autoimmune orchitis, autoimmune polyglandular syndrome 1 / II / III P49321 Lactoferrin Autoimmune pancreatitis P02788 CN103965354 A 17-a-Hydrox ylase (CYP17) Autoimmune polyendocrine syndrome type I & III (APS-I / III) P05093 Cholesterol side-chain cleavage enzyme (CYP11A) Autoimmune polyendocrine syndrome type I (APS-I) P05108 Tryptophan hydroxylase Autoimmune polyendocrine syndrome type I (APS-I) AAA67050.1 Tyrosine hydroxylase Autoimmune polyendocrine syndrome type I (APS-I) AAI43612.1 Aromatic L-amino acid decarboxylas e Autoimmune polyendocrine syndrome type I (APS-I) P20711 Glycoprotein Ilb / IIIa and Ib / IX Autoimmune thrombocytope nia purpura (thrombotic GPIIb (Integrin a2b): P08514 thrombocytope nic purpura (iTTP))) GPIIIa (Integrin [33): P05106 GPlb: P07359 GPIX: P14770 Thyroglobuli n Autoimmune thyroiditis P01266 US10450372B 2 Hemidesmos omal protein 180 BP180 Bullous pemphigoid, herpes ges-tationis, cicatricial pemphigoid Q9UMD9 WO20200729 37A1 CN101333255 A p53 Cancer, systemic lupus erythematosus (SLE) P04637 WO20180749 78 Recoverin Cancer-as sociat ed retinopathy P35243 Actin Chronic active hepatitis, primary biliary cirrhosis IgE receptor Chronic idiopathic urticaria WO20172119 28A1 Myelin-assoc iated glycoprotein (MAG) MAG Chronic inflammatory demyelinating polyneuropathy , Encephalitis lethargica, POEMS P20916 EP2110139A2 syndrome Tubulin Chronic liver disease, visceral leishmaniasis Laminin-332 Cicatricial pemphigoid Laminin subunit a3: Q16787 Tissue transglutaminase TG2 Coeliac disease, Crohn's disease P21980 WO 2006 / 100679 WO20131752 29 Al Desmin Crohn disease, coronary artery disease P17661 aB-crystallin, desmoplakin Bactericidal / permeabilityincreasing protein (BPI) BPI Cystic fibrosis vasculitides P17213 lipopolysacc harides Transglutami nase Dermatitis herpetiformis, Discoid lupus erythematosus AAH03551.1 Melanoma Differentiati on-Associate d gene 5 (MDA5) MDA5 (CADM-14 0) ** Dermatomy ositi s Q9BYX4 SAE-1 Dermatomy ositi s Q9UBE0 SAE-2 Dermatomy ositi s Q9UBT2 DNA-depend ent nucleosome-sti mulated Dermatomy ositi s US6180612B 1 ATPase Chromodom ain-helicase-DNA-bindin g protein 4 (CHD4) CHD4 (Mi-2) Dermatomy ositi s / polymyo sitis Q14839 [3-Adrenorec eptor Dilated cardiomyopathy CAA02051.1 Adenine nucleotide translocator ANT Dilated cardiomyopathy, myocarditis P12235 Collagen type VII Epidermolysis bullosa acquisita AAA58965.1 IgG Essential mixed cryoglobulinem ia AAA02914.1 G-CSF Felty's syndrome P09919 CA3052877A 1 Collagen type IV a3-chain Goodpasture syndrome ABX71213.1 Thyrotropin receptor (TSHR) TSHR Graves disease P16473 EP1565493B1 Sodium iodide symporter (NIS) Graves disease, autoimmune hypothyroidism Q92911 Peripheral myelin protein 22 (PMP22) PMP22 Guillain-Barre syndrome Q01453 GM gangliosides Guillain-Barre syndrome, POEMS syndrome S-antigen HLA-B27-asso ciated acute anterior uveitis, sympathetic ophthalmia P10523 HMGCR Immune-mediat ed necrotizing myopathy P04035 Signal recognition particle 54kDa subunit (SRP54) SRP54 Immune-mediat ed necrotizing myopathy P61011 IgA Immunodeficie ncy, Erythema elevatum diutinum, Kawasaki disease CAA1O818.1 US9688776B2 Synaptotagm in Lambert-Eaton myasthenic syndrome Q8N9I0 Voltage-gate d calcium channels Lambert-Eaton myasthenic syndrome (3IV spectrin Lower motor neuron syndrome Q9H254 SNRNP70 Mixed connective tissues disease P08621 CNPase Multiple sclerosis P09543 Myelin-assoc iated oligodendrocyte basic protein (MOBP) MOBP Multiple sclerosis Q13875 Myelin pro-teolipid protein (PLP) PLP Multiple sclerosis P60201 S100 calcium binding protein B Sloop Multiple sclerosis P04271 WO20161491 16A1 Transaldolas e Multiple sclerosis P37837 Myelin basic protein (MBP) MBP Multiple sclerosis, demyelinating diseases P02686 Myelin-oligo dendrocyte glycoprotein (MOG) MOG Multiple sclerosis, Neuromyelitis optica Q16653 US202200250 44 Al Acetylcholin e receptor Myasthenia gravis CAA26344.1 Low-density lipoprotein receptor-relat ed protein 4 (LRP4) LRP4 *** Myasthenia gravis 075096 Muscle, skeletal receptor tyrosine-prot ein kinase (MuSK) MuSK Myasthenia gravis 015146 WO20212120 53A2 Aminoacyl-t RNA synthetase Myositis, dermatomyositis, Antisynthetase syndrome TRIB2 Narcolepsy, pediatric autoimmune neuropsychiatric disorders associated with streptococci (PANDAS) Q92519 Myeloperoxi dase (MPO) MPO Necrotizing and crescentic glomerulonephr itis (NCGN), systemic vasculitis, microscopic polyantiitis, polyarteritis nodosa P05164 Aquaporin 4 (APQ-4) Neuromyelitis optica P55087 WO20160335 09 Al Amphiphysi n Neuronopathy (autoimmune encephalitis), small lung cell cancer P49418 EXOSC9 Overlap syndrome (scleroderma / po lymyositis) Q06265 EXOSC10 / P MSCL Overlap syndrome (scleroderma / po Q01780 lymyositis) Yo protein Paraneoplastic cerebellar degeneration Hu proteins Paraneoplastic encephalomyeli tis Ri protein Paraneoplastic opsoclonus myoclonus ataxia Desmoplakin Paraneoplastic pemphigus P15924 Gephyrin Paraneoplastic stiff man syndrome Q9NQX3 Desmoglein 1 Pemphigus foliaceus Q02413 Desmoglein 3 Pemphigus vulgaris P32926 Intrinsic factor type 1 Pernicious anemia [32-Glycopro tein I (|32-GPI) Primary antiphospholipid syndrome X Pyruvate dehydrogenase complex-E2 (PDC-E2) Primary biliary cirrhosis Q9HAN0 (unreviewe d) Aggrecan G1 RA P16112 US202101151 17A9 Carbamylate d antigens RA Cartilage glycoprotein- HC-gp39 / C HI3L1 / YK RA P36222 US8673301B2 39 L-40 Fc part of immunoglob ulins RA Glucose-6-p hosphate isomerase RA P06744 Keratin RA CAA7394 3.1 Protein-argin ine deiminase type-4 PAD4 RA Q9UM07 Collagen (multiple types, especially II, IV and IX) RA, systemic lupus erythematosus (SLE), progressive systemic sclerosis, relapsing polychondritis WO20160162 69 Al Fibrinogen, [3a RA, Still's disease CAA5074 0.1 Leukemia inhibitory factor (LIF) LIF RA, systemic sclerosis, normal subjects US10968273B 2 Glutamate receptor (GLUR) Rasmussen encephalitis P42261, P42262, P42263, P48058, P39086, QI 3002, QI3003 US5739291A Myosin Rheumatic fever, Takayasu CAA86293.1 arteritis, temporal arteritis B23 Scleroderma AAA58386.1 Nucleophos min (NPM) NPM scleroderma P06748 Fibrillarm Scleroderma, CREST syndrome P22087 Topoisomera se-I (Scl-70) Scleroderma, Raynaud syndrome, pulmonary fibrosis NCBI Reference Sequence: NP_003277.1 Interferon-y-inducible protein 16 (IFI16) IFI16 Sjogren syndrome, systemic lupus erythematosus (SLE) Q16666 WO20150956 09 Al La phosphoprotein La / SSB Sjogren syndrome, systemic lupus erythematosus (SLE) P05455 Ro60 Sjogren syndrome, systemic lupus erythematosus (SLE) P10155 Ro52 (TRIM21) Sjogren syndrome, systemic lupus erythematosus (SLE), myositis, scleroderma P19474 Golgin (95, 97, 160,180) Sjogren Syndrome, systemic lupus erythematosus (SLE), RA Golgin-95 : AAA35920.1 Golgin-72 variant: BAD92095.1 Golgin-160 : AAL93149.1 Anionic phospholipid / protein complex systemic lupus erythematosus (SLE) Cardiolipin systemic lupus erythematosus (SLE) Components of the Sm splicing ri-bonucleoprot ein (subunits A-G) systemic lupus erythematosus (SLE) dsDNA systemic lupus erythematosus (SLE) Histone H2A-H2B-D NA systemic lupus erythematosus (SLE) Proliferating cell nuclear antigen (PCNA) PCNA systemic lupus erythematosus (SLE) P12004 US11667700B 2 Ribosomal P Rib-P systemic lupus erythematosus (SLE) Sjogren Syndrome A Ro / SSA systemic lupus erythematosus (SLE) Smith Sm systemic lupus erythematosus (SLE) Ul-RNP systemic lupus erythematosus (SLE) P09012 U2 snRNP B systemic lupus erythematosus (SLE) P08579 Vimentin systemic lupus erythematosus (SLE) P08670 Clq systemic lupus erythematosus (SLE), membrane proliferative glomerulonephr itis (MPGN) ClqA: P02745 ClqB: P02746 ClqC: P02747 US11649279B 2 Fibronectin systemic lupus erythematosus (SLE), RA, morphea P02751 EP2236517A1 Ku-DNA-pro tein kinase systemic lupus erythematosus (SLE), scleroderma Carbonic anhydrase II systemic lupus erythematosus (SLE), Sjogren syndrome, systemic sclerosis P00918 Neuronal nicotinic acetylcholine Subacute autonomic neuropathy, AAA59942.1 receptor cancer Centromere-associated proteins Systemic sclerosis, CREST syndrome P49450, P07199, Q02224 RNA polymerase I-III (RNP) Systemic sclerosis, systemic lupus erythematosus (SLE) Thyroid and eye muscle shared protein G2s Thyroid associated ophthalmopathy P31040 Leukocyte function-asso ciated antigen (LFA-1) Treatment-resis tant Lyme arthritis P20701, P05107 US6703018B2 Chromograni nA Type 1 diabetes P10645 IA-2 (ICA512) Type 1 diabetes Q16849 IGRP Type 1 diabetes Q9NQR9 ZnT8 Type 1 diabetes AAP44332.1 WO20200371 74A1 Insulin Type 1 diabetes, insulin hypoglycemic syndrome (Hirata disease) P01308 Glutamate decarboxylas e (GAD65) Type 1 diabetes, stiff man syndrome, autoimmune AAA62367.1 encephalitis, Batten disease / Neuronal ceroid lipofuscinoses Insulin receptor Type B insulin resistance, acanthosis, systemic lupus erythematosus (systemic lupus erythematosus (SLE)) P06213 US202202278 57A1 Heat shock protein (65-kDa heat shock protein) Hsp65 Various immune-related disorders (RA) SOX-10 Vitiligo P56693 US201602162 69 Al Tyrosinase Vitiligo, metastatic melanoma P14679 KUMEL1 / A RMC9 Vogt-Koyanagi -Harada syndrome Q7Z3E5 Proteinase 3 / Myeloblast in PR3 Wegener granulomatosis, Churg-Strauss syndrome, vasculitis P24158

[38] *: Total 35 entries are found at https: / www.uniprot.org / uniprotkb?query=GABAAR+ALPHA&facets=model_organis m%3A9606.

[39] **: MDA5 is coded by the interferon-induced helicase C domain-containing protein 1 (IFIH1) gene.

[40] ***: Lrp4 is a Receptor for Agrin and Forms a Complex with MuSK-Agrin (AGRN) : 000468.

[41]

[42] Amino acid sequences of target substances may be obtained from public database such as UniProtKB / Swiss-Prot or NCBI. For example, exemplary amino acid sequences of the target substances may include, but not are limited to the followings.

[43] CD20: UniProt accession no. Pl 1836 (human) and its variants at www.uniprot.org / uniprotkb / P 11836 / variant-vie wer, and orthologs thereof.

[44] CD19: UniProt accession no. P15391 (human) and its variants at www.uniprot.org / uniprotkb / P15391 / variant-viewer, and orthologs thereof.

[45] CD52: UniProt accession no. P31358 (human) and its variants at www.uniprot.org / uniprotkb / P31358 / variant-vie wer, and orthologs thereof.

[46] CD80: UniProt accession no. P33681 (human) and its variants at www.uniprot.org / uniprotkb / P33681 / variant-viewer, and orthologs thereof.

[47] CD86: UniProt accession no. P42081 (human) and its variants at www.uniprot.org / uniprotkb / P42081 / variant-viewer, and orthologs thereof.

[48] CD28: UniProt accession no. P10747 (human) and its variants at www.uniprot.org / uniprotkb / P10747 / variant-viewer, and orthologs thereof.

[49] CD40: UniProt accession no. P25942 (human) and its variants at www.uniprot.org / uniprotkb / P25942 / variant-viewer. and orthologs thereof.

[50] Complement C3: UniProt accession no. P01024 (human) and its variants at www.uniprot.org / uniprotkb / PO 1024 / variant-viewer, and orthologs thereof.

[51] Complement C5: UniProt accession no. P01031 (human) and its variants at www.uniprot.org / uniprotkb / PO 1031 / variant-viewer, and orthologs thereof.

[52] C5 alpha receptor 1: UniProt accession no. P21730 (human) and its variants at www.uniprot.org / uniprotkb / P21730 / variant-viewer, and orthologs thereof.

[53] IL-1R: UniProt accession no. P14778 (human) and its variants at www.uniprot.org / uniprotkb / P14778 / variant-viewer, and orthologs thereof.

[54] IL-6R: UniProt accession no. P08887 (human) and its variants at www.uniprot.org / uniprotkb / P08887 / variant-viewer, and orthologs thereof.

[55] IL-6ST (Interleukin-6 receptor subunit beta): UniProt accession no. P40189 (human) and its variants at www.uniprot.org / uniprotkb / P40189 / variant-viewer, and orthologs thereof.

[56] IL-17C: UniProt accession no. Q9P0M4 (human) and its variants at www.uniprot.org / uniprotkb / PQ9P0M4 / variant-viewer, and orthologs thereof.

[57] IL- I 7RA: UniProt accession no. Q96F46 (human) and its variants at www.uniprot.org / uniprotkb / Q96F46 / variant-viewer, and orthologs thereof.

[58] IL-17RB: UniProt accession no. Q9NRM6 (human) and its variants at www.uniprot.org / uniprotkb / Q9NRM6 / variant-viewer, and orthologs thereof.

[59] IL-4R: UniProt accession no. P24394 (human) and its variants at www.uniprot.org / uniprotkb / QP24394 / variant-viewer, and orthologs thereof.

[60] IL-5R: UniProt accession no. Q01344 (human) and its variants at www.uniprot.org / uniprotkb / Q01344 / variant-viewer, and orthologs thereof.

[61] IL-5RB: UniProt accession no. P32927 (human) and its variants at www.uniprot.org / uniprotkb / P32927 / variant-viewer, and orthologs thereof.

[62] IL-13R1: UniProt accession no. P78552 (human) and its variants at www.uniprot.org / uniprotkb / P78552 / variant-viewer, and orthologs thereof.

[63] IL-13R2: UniProt accession no. Q14627 (human) and its variants at www.uniprot.org / uniprotkb / Q14627 / variant-viewer, and orthologs thereof; or UniProt accession no. D0EFR8 (human) and its variants at www.uniprot.org / uniprotkb / D0EFR8 / variant-viewer, and orthologs thereof.

[64] IFN-gamma receptor 1: UniProt accession no. Q15260 (human) and its variants at www.uniprot.org / uniprotkb / Q15260 / variant-viewer, and orthologs thereof.

[65] IFN-gamma receptor 2: UniProt accession no. P38484 (human) and its variants at www.uniprot.org / uniprotkb / P38484 / variant-viewer, and orthologs thereof.

[66] Integrin alpha-D (ITGAD): UniProt accession no. Q13349 (human) and its variants at www.uniprot.org / uniprotkb / Q13349 / variant-viewer, and orthologs thereof.

[67] IL-12RB1: UniProt accession no. P42701 (human) and its variants at www.uniprot.org / uniprotkb / P42701 / variant-viewer, and orthologs thereof.

[68] IL-12RB2: UniProt accession no. Q99665 (human) and its variants at www.uniprot.org / uniprotkb / Q99665 / variant-viewer, and orthologs thereof.

[69] IL-21R: UniProt accession no. Q9HBE5 (human) and its variants at www.uniprot.org / uniprotkb / Q9HBE5 / variant-viewer, and orthologs thereof.

[70] IL-22RA1: UniProt accession no. Q8N6P7 (human) and its variants at www.uniprot.org / uniprotkb / Q8N6P7 / variant-viewer, and orthologs thereof.

[71] IL-22RA2: UniProt accession no. Q969J5 (human) and its variants at www.uniprot.org / uniprotkb / Q969J5 / variant-viewer, and orthologs thereof.

[72] TGF-beta receptor type 1: UniProt accession no. P36897 (human) and its variants at www.uniprot.org / uniprotkb / P36897 / variant-viewer, and orthologs thereof; TGF-beta receptor type 2: UniProt accession no. P37173 (human) and its variants at www.uniprot.org / uniprotkb / P37173 / variant-viewer, and orthologs thereof; TGF-beta receptor type 3: UniProt accession no. Q03167 (human) and its variants at www.uniprot.org / uniprotkb / Q03167 / variant-viewer, and orthologs thereof.

[73] IL-23R: UniProt accession no. Q5VWK5 (human) and its variants at www.uniprot.org / uniprotkb / Q5VWK5 / variant-viewer, and orthologs thereof.

[74] Thymic Stromal LymphoPoietin receptor (TSLPR): UniProt accession no. Q9HC73 (human) and its variants at www.uniprot.org / uniprotkb / Q9HC73 / variant-viewer, and orthologs thereof.

[75] IL-31R: UniProt accession no. Q8NI17 (human) and its variants at www.uniprot.org / uniprotkb / Q8NI17 / variant-viewer, and orthologs thereof.

[76] IL-33R: UniProt accession no. Q01638 (human) and its variants at www.uniprot.org / uniprotkb / Q8NB14 / variant-viewer, and orthologs thereof.

[77] IGF-1R: UniProt accession no. P08069 (human) and its variants at www.uniprot.org / uniprotkb / P08069 / variant-viewer, and orthologs thereof.

[78] TNFR1: UniProt accession no. P19438 (human) and its variants at www.uniprot.org / uniprotkb / P19438 / variant-viewer, and orthologs thereof.

[79] TNFR2: UniProt accession no. P20333 (human) and its variants at www.uniprot.org / uniprotkb / P20333 / variant-viewer, and orthologs thereof.

[80] FcRn Large Subunit p51: UniProt accession no. P55899 (human) and its variants at www.uniprot.org / uniprotkb / P55899 / variant-viewer, and orthologs thereof.

[81] CCL14: UniProt accession no. Q16627 (human) and its variants at www.uniprot.org / uniprotkb / Q16627 / variant-viewer, and orthologs thereof.

[82] CCL15: UniProt accession no. Q16663 (human) and its variants at www.uniprot.org / uniprotkb / Q16663 / variant-viewer, and orthologs thereof.

[83] CCL18: UniProt accession no. P55774 (human) and its variants at www.uniprot.org / uniprotkb / P55774 / variant-viewer, and orthologs thereof.

[84] CCL19: UniProt accession no. Q99731 (human) and its variants at www.uniprot.org / uniprotkb / Q99731 / variant-viewer, and orthologs thereof.

[85] CCL20: UniProt accession no. QP78556 (human) and its variants at www.uniprot.org / uniprotkb / P78556 / variant-viewer, and orthologs thereof.

[86] CCL21: UniProt accession no. 000585 (human) and its variants at www.uniprot.org / uniprotkb / 000585 / variant-viewer, and orthologs thereof.

[87] CCL23: UniProt accession no. P55773 (human) and its variants at www.uniprot.org / uniprotkb / P55773 / variant-viewer, and orthologs thereof.

[88] CCL25: UniProt accession no. Q68A93 (human) and its variants at www.uniprot.org / uniprotkb / Q68A93 / variant-viewer, and orthologs thereof.

[89] CCL27: UniProt accession no. Q9Y4X3 (human) and its variants at www.uniprot.org / uniprotkb / Q9Y4X3 / variant-viewer, and orthologs thereof.

[90] CXCL12: UniProt accession no. P48061 (human) and its variants at www.uniprot.org / uniprotkb / P48061 / variant-viewer, and orthologs thereof.

[91] CXCL13: UniProt accession no. 043927 (human) and its variants at www.uniprot.org / uniprotkb / O43927 / variant-viewer, and orthologs thereof.

[92] IL-1A: UniProt accession no. P01583 (human) and its variants at www.uniprot.org / uniprotkb / P01583 / variant-viewer, and orthologs thereof; IL-1B: UniProt accession no. P01584 (human) and its variants at www.uniprot.org / uniprotkb / P01584 / variant-viewer, and orthologs thereof.

[93] TNF-alpha: UniProt accession no. P01375 (human) and its variants at www.uniprot.org / uniprotkb / P01375 / variant-viewer, and orthologs thereof.

[94] CXCL-8: UniProt accession no. P10145 and its variants at www.uniprot.org / uniprotkb / P10145 / variant-viewer, and orthologs thereof.

[95] CCL2: UniProt accession no. P13500 (human) and its variants at www.uniprot.org / uniprotkb / Q13500 / variant-viewer, and orthologs thereof.

[96] CCL3: UniProt accession no. P10147 (human) and its variants at www.uniprot.org / uniprotkb / P10147 / variant-viewer, and orthologs thereof.

[97] CCL4: UniProt accession no. P13236 (human) and its variants at www.uniprot.org / uniprotkb / P13236 / variant-viewer, and orthologs thereof.

[98] CCL5: UniProt accession no. P13501 (human) and its variants at www.uniprot.org / uniprotkb / P13501 / variant-viewer, and orthologs thereof.

[99] CCL11: UniProt accession no. P51671 (human) and its variants at www.uniprot.org / uniprotkb / P51671 / variant-viewer, and orthologs thereof.

[100] CXCL10: UniProt accession no. P02778 (human) and its variants at www.uniprot.org / uniprotkb / P02778 / variant-viewer, and orthologs thereof.

[101] IL-6: UniProt accession no. P05231 (human) and its variants at www.uniprot.org / uniprotkb / P05231 / variant-viewer, and orthologs thereof.

[102] IL-17: UniProt accession no. Q16552 (human) and its variants at www.uniprot.org / uniprotkb / Q16552 / variant-viewer, and orthologs thereof.

[103] IL-4: UniProt accession no. P05112 (human) and its variants at www.uniprot.org / uniprotkb / P05112 / variant-viewer, and orthologs thereof.

[104] IL-5: UniProt accession no. P05113 (human) and its variants at www.uniprot.org / uniprotkb / P05113 / variant-viewer, and orthologs thereof.

[105] IL-13: UniProt accession no. P35225 (human) and its variants at www.uniprot.org / uniprotkb / P35225 / variant-viewer, and orthologs thereof.

[106] IFN-gamma: UniProt accession no. P01579 (human) and its variants at www.uniprot.org / uniprotkb / P01579 / variant-viewer, and orthologs thereof.

[107] IL-12A: UniProt accession no. P29459 (human) and its variants at www.uniprot.org / uniprotkb / P29459 / variant-viewer, and orthologs thereof; IL-12: UniProt accession no. P29460 (human) and its variants at www.uniprot.org / uniprotkb / P29460 / variant-viewer, and orthologs thereof.

[108] IL-21: UniProt accession no. Q9HBE4 (human) and its variants at www.uniprot.org / uniprotkb / Q9HBE4 / variant-viewer, and orthologs thereof.

[109] IL-22: UniProt accession no. Q9GZX6 (human) and its variants at www.uniprot.org / uniprotkb / Q9GZX6 / variant-viewer, and orthologs thereof.

[110] TGF-beta-1: UniProt accession no. P01137 (human) and its variants at www.uniprot.org / uniprotkb / P01137 / variant-viewer, and orthologs thereof; TGF-beta-2: UniProt accession no. P61812 (human) and its variants at www.uniprot.org / uniprotkb / P61812 / variant-viewer, and orthologs thereof; TGF-beta-3: UniProt accession no. P10600 (human) and its variants at www.uniprot.org / uniprotkb / P10600 / variant-viewer, and orthologs thereof. [Ill] IL-23A: UniProt accession no. Q9NPF7 (human) and its variants at www.uniprot.org / uniprotkb / Q9NPF7 / variant-viewer, and orthologs thereof; IL-23B: UniProt accession no. P29460 (human) and its variants at www.uniprot.org / uniprotkb / P29460 / variant-viewer, and orthologs thereof.

[112] Thymic Stromal LymphoPoietin (TSLP): UniProt accession no. Q969D9 (human) and its variants at www.uniprot.org / uniprotkb / Q969D9 / variant-viewer, and orthologs thereof.

[113] IL-31: UniProt accession no. Q6EBC2 (human) and its variants at www.uniprot.org / uniprotkb / Q6EBC2 / variant-viewer, and orthologs thereof.

[114] 0X40 (Tumor necrosis factor receptor superfamily member 4): UniProt accession no. P23510 (human) and its variants at www.uniprot.org / uniprotkb / P23510 / variant-viewer, and orthologs thereof.

[115] OX40L(Tumor necrosis factor receptor superfamily member 4): UniProt accession no. P43489 (human) and its variants at www.uniprot.org / uniprotkb / P43489 / variant-viewer, and orthologs thereof.

[116] IL-33: UniProt accession no. 095760 (human) and its variants at www.uniprot.org / uniprotkb / O95760 / variant-viewer, and orthologs thereof.

[117] CD40L: UniProt accession no. P29965 (human) and its variants at www.uniprot.org / uniprotkb / P29965 / variant-viewer, and orthologs thereof.

[118] ICAM1: UniProt accession no. P05362 (human) and its variants at www.uniprot.org / uniprotkb / P05362 / variant-viewer, and orthologs thereof.

[119] VCAM1: UniProt accession no. P19320 (human) and its variants at www.uniprot.org / uniprotkb / P19320 / variant-viewer, and orthologs thereof.

[120] MADCAM1: UniProt accession no. Q13477 (human) or B9EGE2 and their variants at www.uniprot.org / uniprotkb / O13477 / variant-viewer and www.uniprot.org / uniprotkb / B9EGE2 / variant-viewer, respectively, and orthologs thereof.

[121] Integrin alpha 4: UniProt accession no. P13612 (human) and its variants at www.uniprot.org / uniprotkb / P13612 / variant-viewer, and orthologs thereof.

[122] Integrin beta 7: UniProt accession no. P26010 (human) and its variants at www.uniprot.org / uniprotkb / P26010 / variant-viewer, and orthologs thereof.

[123] LFA-1 or MAC-l(dhner of integrin alpha-M and integrinbeta-2): UniProt accession no. Pl 1215 (human) for integrin alpha-M (ITAM) and its variants at www.uniprot.org / uniprotkb / P11215 / variant-viewer, and orthologs thereof, and UniProt accession no. P05107 (human) for integrinbeta-2 (ITB2) and its variants at www.uniprot.org / uniprotkb / P05107 / variant-viewer, and orthologs thereof.

[124] VLA-4 (dimer of CD49d and CD29): UniProt accession no. P13612 (human) for CD49d and its variants at www.uniprot.org / uniprotkb / P13612 / variant-viewer, and orthologs thereof and UniProt accession no. P05556 (human) for CD29 and its variants at www.uniprot.org / uniprotkb / P05556 / variant-viewer, and orthologs thereof.

[125] TLR3: UniProt accession no. 015455 (human) and its variants at www.uniprot.org / uniprotkb / O15455 / variant-viewer. TLR3 is reported to be associated with inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), colitis, rheumatoid arthritis. Antibodies binding to TLR3 are disclosed in, for example, US Patent No. 8153583B2.

[126] TLR4: UniProt accession no. 000206 (human) and its variants at www.uniprot.org / uniprotkb / 000206 / variant-viewer. TLR4 is reported to be associated with rheumatoid arthritis. Antibodies binding to TLR4 are disclosed in, for example, US Patent No. 7312320B2.

[127] TLR5: UniProt accession no. 060602 (human) and its variants at www.uniprot.org / uniprotkb / 060602 / variant-viewer. TLR5 is reported to be associated with rheumatoid arthritis. Substances binding to TLR5 are disclosed in, for example, US Patent No. 8703146B2 and US Patent Application Publication No. 20200362052A1.

[128] TLR7: UniProt accession no. Q9NYK1 (human) and its variants at www.uniprot.org / uniprotkb / Q9NYKl / variant-viewer. TLR7 is reported to be associated with systemic lupus erythematosus, and cutaneous lupus erythematosus. Antibodies binding to TLR7 are disclosed in, for example, US Patent Application Publication Nos. 20200362052A1 and 20210040225A1.

[129]

[130] In some embodiments, the immunological disease is an autoimmune disease or an inflammatory disease. In another particular embodiment, the autoimmune or inflammatory disease is multiple sclerosis (MS), rheumatoid arthritis, a spondyloarthropathy, systemic lupus erythematosus, an antibody-mediated inflammatory or autoimmune disease, graft versus host disease, sepsis, diabetes type 1, diabetes type 2, psoriasis, atherosclerosis, Sjogren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemic reperfusion, Crohn's Disease, endometriosis, glomerulonephritis, myasthenia gravis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, or inflammatory autoimmune myositis. In specific embodiments, the spondyloarthropathy is selected from ankylosing spondylitis, reactive arthritis, enteropathic arthritis associated with inflammatory bowel disease, psoriatic arthritis, isolated acute anterior uveitis, undifferentiated spondyloarthropathy, Behcet's syndrome, and juvenile idiopathic arthritis. In one embodiment, the immunological disease is caused by or exacerbated by binding of excessive antigenic substance to immunoglobulins or immune cells or by the increased amount of antigenic substance or an elevated expression of antigenic substance. In a certain particular embodiment, the immune cell is a dendritic cell.

[131] In embodiments, the present disclosure is directed to a nucleic acid or polynucleotide encoding the fusion proteins described above.

[132] In embodiments, the present disclosure is directed to a vector containing the nucleic acid or polynucleotide.

[133] Embodiments are directed to a host cell containing the vector.

[134] Another aspect of the present disclosure provides a method of producing a therapeutic fusion molecule for treatment of an immunological diseases in a subject, comprising expressing the fusion molecule by culturing a host cell under a condition for expressing the fusion molecule.

[135] In embodiments, the present disclosure is directed to a method of reducing or enhancing a reduction of a substance that triggers, induces, or causes undesired or pathological immune reaction such as autoimmune disease, transplant rejection, or allergic or hyperimmune responses in a subject, which method comprises administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, wherein the fusion molecule comprises a first region that is capable of binding to a TAM (Tyro3, Axl and MerTK) receptor on surface of a cell in the subject; and a second region that specifically binds to the substance. In nonlimiting embodiments, the substance and the immunological diseases may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have an effector function and does not induce Fc-mediated inflammatory responses. For example, the fusion molecule does not comprise a moiety to bind to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fey receptor). The fusion molecule does not contain the target substance to be cleared or reduced by the administration of the fusion molecule.

[136] In embodiments, the present disclosure is directed to a method of removing or clearing or enhancing clearance of an antigenic substance that triggers, induces, or causes undesired or pathological immune reaction such as autoimmune disease, transplant rejection, or allergic or hyperimmune responses in a subject, which method comprises administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, wherein the fusion molecule comprises a first region that is capable of binding to a TAM (Tyro3, Axl and MerTK) receptor on surface of a cell in the subject; and a second region that specifically binds to the antigenic substance. In non-limiting embodiments, the substance and the undesired or pathological immune reason may be one or more of those listed in Table 1. In nonlimiting embodiments, the fusion molecule does not have an effector function and does not induce inflammatory responses. For example, the fusion molecule does not comprise a moiety to bind to an Fc receptor, and may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fey receptor). The fusion molecule does not contain the target substance to be cleared or reduced by the administration of the fusion molecule.

[137] In embodiments, the present disclosure is directed to a method of treating, preventing, or ameliorating an immunological disease in a subject who has or who is at risk of developing the immunological disease. The method comprises administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, wherein the fusion molecule comprises a first region that is capable of binding to a TAM (Tyro3, Axl and MerTK) receptor on surface of a cell in the subject and a second region that specifically binds to an antigenic substance that triggers, induces, or causes the immunological diseases. In non-limiting embodiments, the antigenic substance and the immunological disease may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have an effector function and does not induce Fc-mediated inflammatory responses. For example, the fusion molecule does not comprise a moiety to bind to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fey receptor). The fusion molecule does not contain the target substance to be cleared or reduced by the administration of the fusion molecule.

[138] In embodiments, the present disclosure is directed to a method of delaying development of a symptom associated with an immunological disease that is caused by or triggered by an antigenic substance, in a subject. The method comprises administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, a vector comprising the polynucleotide, wherein the fusion molecule comprises a first region that is capable of binding to a TAM (Tyro3, Axl and MerTK) receptor on surface of a cell in the subject; and a second region that specifically binds to the antigenic substance. In non-limiting embodiments, the antigenic substance and the immunological disease may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have an effector function and does not induce Fc-mediated inflammatory responses. For example, the fusion molecule does not comprise a moiety to bind to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fey receptor). The fusion molecule does not contain the target substance to be cleared or reduced by the administration of the fusion molecule.

[139] In embodiments, the present disclosure provides a method of reducing an antigenic substance that triggers, induces, or causes undesired or pathological immune reaction such as autoimmune disease, transplant rejection, or allergic or hyperimmune responses, in a subject. The method comprises administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, wherein the fusion molecule comprises a first region that is capable of binding to a TAM (Tyro3, Axl and MerTK) receptor on surface of a cell in the subject, and a second region that specifically binds to the antigenic substance. The antigenic substance may be soluble, oligomeric, or aggregated form. In some embodiments, the undesired or pathological immune responses to the antigenic substance are inhibited and / or reduced. Thus, the method of the disclosure can be used to treat any disease associated with or caused by undesired or pathological immune reaction to the antigenic substance. In non-limiting embodiments, the antigenic substance and the disease may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have an effector function and does not induce Fc-mediated inflammatory responses. For example, the fusion molecule does not comprise a moiety to bind to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fey receptor). The fusion molecule does not contain the target substance to be cleared or reduced by the administration of the fusion molecule.

[140] In embodiments, the present disclosure is directed to a pharmaceutical composition comprising an effective amount of any of the above-disclosed fusion molecule or polynucleotides encoding the fusion molecule, and a pharmaceutical acceptable excipient. In non-limiting embodiments, the fusion molecule does not have an effector function and does not induce Fc-mediated inflammatory responses. For example, the fusion molecule does not comprise a moiety to bind to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fey receptor). The fusion molecule does not contain the target substance to be cleared or reduced by the administration of the fusion molecule.

[141] In embodiments, the present disclosure is directed to a use of any of the abovedisclosed fusion molecule or polynucleotides encoding the fusion molecule, for use in manufacturing a medicament suitable to treat immunological diseases.

[142] In embodiments, the present disclosure is directed to a use of any of the abovedisclosed fusion molecule or polynucleotides encoding the fusion molecule, or pharmaceutical compositions for use in treating or preventing immunological diseases.

[143] In embodiments, the present disclosure is directed to kits comprising an effective amount of any of the above-disclosed fusion molecule or polynucleotides encoding the fusion molecule. The kits are generally in suitable packaging and provided with ap- propriate instructions, are useful for any of the methods described herein.

[144] These and other aspects, objects, features and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of illustrated example embodiments.

[145] Advantageous Effects of Invention

[146] The present disclosure relates to a fusion molecule having phagocytosis-inducing activity, which can solve the problem of tissue damage caused by activation of an inflammatory response, which occurs in the prior art. Accordingly, the fusion molecule is able to effectively clear or reduce the substance that has an increased expression or amount to, for example a normal level or amount, and thus may be used to prevent or treat immunological diseases caused by the increased or elevated substances, for example, those listed in Table 1 or other diseases described herein. The fusion molecule may be administered to a patient in the form of a purified fusion molecule or a gene therapy vector capable of expressing and secreting the fusion molecule when introduced into a cell.

[147] However, it should be understood that effects of the present disclosure are not limited to the above effects, and include all effects that may be inferred from the configuration of the invention described in the detailed description or claims.

[148] Brief Description of Drawings

[149] FIG. 1A to FIG. IC show the effects on the EAE score and body weight change when EAE was induced in mice with astrocyte-specific deletion of the Axl gene.

[150] FIG. 2A to FIG. 2C show the effects on the EAE score and body weight change when EAE was induced in mice with microglia-specific deletion of the Mertk gene.

[151] FIG. 3A shows the schematic diagram of the configurations of the prepared AAV expressing anti-FITC-Gas6 and anti-MOG(8-18C5)-Gas6.

[152] FIG. 3B shows the amino acid sequences of anti-FITC-Gas6 and anti-MOG(8-18C5)-Gas6, constructed in Example 3.

[153] FIG. 3C and FIG. 3D show the sequences of nucleic acid encoding the anti-FITC-Gas6 depicted in FIG. 3B.

[154] FIG. 3E and FIG. 3F show the sequences of nucleic acid encoding the anti-MOG(8-18C5)-Gas6 depicted in FIG. 3B.

[155] FIG. 4A to FIG. 4C show the effects of Anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vitro.

[156] FIG. 5A to FIG. 5C show the effects on the EAE score and body weight change when expressing Anti-MOG(8-18C5)-Gas6 in EAE mice.

[157] FIG. 6A to FIG. 6E show the effects of systemically expressed Anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

[158] FIG. 7A to FIG. 7E show the effects of locally expressed Anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

[159] FIG. 8 shows the schematic diagram of the configurations of the prepared Anti-MOG(01)-Gas6.

[160] FIG. 9A and FIG. 9B show the antigen (human and mouse MOG) binding activity of anti-MOG(01)-Gas6 fusion molecule measured by ELISA.

[161] FIG. 9C shows the results of measuring the degree of binding of anti-MOG(01)-Gas6 fusion molecule to mouse MOG protein on the cell surface by using flow cytometry.

[162] FIG. 10 shows the effect of anti-MOG(01)-Gas6 on the removal of myelin debris in vitro.

[163] FIG. 11 shows the schematic diagram of the configurations of the prepared anti-MBP-Gas6.

[164] FIG. 12A and FIG. 12B show the antigen (human and mouse MBP) binding activity of anti-MBP-Gas6 fusion molecule measured by ELISA.

[165] FIG. 13 shows the effect of anti-MBP-Gas6 on the removal of myelin debris in vitro.

[166] FIG. 14A shows the schematic diagram of the configurations of the prepared Anti-TNFa(Adalimumab)-Gas6 and Anti-TNFa(Infliximab)-Gas6. FIG. 14B and FIG. 14C show the sequence of anti-TNFa(Adalimumab)-Gas6 and anti-TNFa(Infliximab)-Gas6.

[167] FIG. 15A shows the antigen (human TNFa) binding activity of anti-TNFa-Gas6 fusion molecule measured by ELISA and FIG. 15B shows the results of measuring the degree of binding of anti-TNFa-Gas6 fusion molecule and human TNFa protein on the cell surface by using flow cytometry.

[168] FIG. 16 shows the level of inhibition of TNFa signal activation by anti-TNFa-Gas6 fusion molecule in HEK-Blue™ TNFa cells.

[169] FIG. 17 shows the induction of Axl activation on U2OSAxl cell by anti-TNFa-Gas6 fusion molecule.

[170] FIG. 18 shows the induction of Axl-mediated phagocytosis by anti-TNFa-Gas6 using THP-lAxl -derived macrophages as effector cell.

[171] FIG. 19A shows the schematic diagram of the configurations of the prepared anti-CD20(Rituximab)-Gas6. FIG. 19B shows the amino acid sequence of anti-CD20(Rituximab)-Gas6.

[172] FIG. 20A shows the antigen (human CD20) binding activity of anti-CD20-Gas6 fusion molecule measured by ELISA and FIG. 20B shows the degree of binding of anti-CD20-Gas6 fusion molecule and human CD20 protein on the cell surface by using flow cytometry.

[173] FIG. 21 shows the induction of Axl activation on U2OSAxl cell by anti-CD20-Gas6 fusion molecule.

[174] FIG. 22 shows the induction of Axl-mediated phagocytosis by anti-CD20-Gas6 using THP-lAxl-derived macrophages as effector cell.

[175] FIG. 23A to FIG. 23K schematically depicts structures of fusion proteins according to various non-limiting embodiments of the present disclosure.

[176] FIG. 24 to FIG. 34 show the amino acid sequences of exemplary fusion proteins according to various non-limiting embodiments of the present disclosure.

[177] Best Mode for Carrying out the Invention

[178] Methods and compositions are provided for reducing or suppressing formation of or clearing or removing or reducing a target substance of which an increased amount or expression is associated with or characteristic of or causes an immunological disorder or disease via a phagocytosis, preventing or treating an individual having or at risk of developing an immunological disease or disorder, improving symptoms of an immunological disease or disorder.

[179] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context dearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[180]

[181] [DEFINITIONS]

[182] As used herein, the singular forms "a," "an," and "the," refer to both the singular as well as plural, unless the context clearly indicates otherwise. Therefore, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[183] As used herein, the terms "about" and "consisting essentially of" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, "about" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). For example, "about 5 mg" can include any number between 4.5 mg and 5.5 mg (for 10%), between 4.75 mg and 6.25 mg (for 5%), between 4.8 mg and 6.2 mg (for 4%), between 4.85 mg and 6.15 mg (for 3%), between 4.9 mg and 6.1 mg (for 2%), or between 4.95 mg and 6.05 mg (for 1%). Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[184] As used herein, "administration" or "administering" refers to the introduction of a composition into a subject by a chosen route. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. In some examples, the peptides and antibodies disclosed herein are administered to a subject.

[185] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[186] As used herein, "polypeptide," "oligopeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. The terms also encompass an amino acid polymer that has been modified naturally or by in tervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon an antibody, the polypeptides can occur as single chains or associated chains.

[187] As used herein, "polynucleotide," or "nucleic acid," as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These al ternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR2 (“amidate”), P(O)R, P(O)OR', CO or CH2 (“formacetal”), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[188] As used herein, "recipient", "individual", "subject", "host", and "patient", are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In embodiments, the mammal is human.

[189] As used herein, "antibody" refers to single chain, two-chain, and multi-chain proteins and glycoproteins belonging to the classes of polyclonal, monoclonal, chimeric and hetero immunoglobulins (monoclonal antibodies being preferred); it also includes synthetic and genetically engineered variants of these immunoglobulins.

[190] As used herein, "specific binding," "specifically binds," and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides / epitopes). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 105 M or less (e.g., 106 M or less, 107 M or less, 10 8 M or less, 109 M or less, 10 10 M or less, 10 11 M or less, 10 12 M or less, 10 13 M or less, 10 14 M or less, 10 15 M or less, or 10 16 M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. As used herein, the "binding" and "specific binding" of the first region to TAM receptor and the second region to a target substance do not require modulating, changing, affecting, or modifying activity of the bound TAM receptor or the target substance.

[191] As used herein, "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a b-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the b-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[192] "Fv" is the minimum antibody fragment, which contains a complete antigenrecognition and -binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv), one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[193] The term "complementarity determining region" or "CDR," as used herein, refers to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. For example, in general, there are three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, 2023358200   23 Sep 2026 in some embodiments, the CDRs correspond to the amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For instance, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.

[194] The "Fab fragment" also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab‘)2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[195] As used herein, the term "antibody fragment" or "antigen-binding fragment" or "active fragment" is defined as a portion of an intact antibody comprising the antigen binding site or variable region of the intact antibody, wherein the portion is free of the constant heavy chain domains (i.e. CH2, CH3, and CH4, depending on antibody isotype) of the Fc region of the intact antibody. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single chain polypeptide"), including without limitation (1) single-chain Fv (scFv) molecules, (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety, (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety, (4) nanobodies comprising single Ig domains from non-human species or other specific singledomain binding modules; and multispecific or multivalent structures formed from antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and / or can contain any hinge region sequence found in an intact antibody, and / or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s), and (5) an isolated complementarity determining region (CDR).

[196] The terms “phagocytic cells” and “phagocytes” are used inter- changeably herein to refer to a cell that is capable of phagocytosis. There are four main categories of phagocytes: macrophages, mononuclear cells (histiocytes and monocytes), polymorphonuclear leukocytes (neutrophils), and dendritic cells.

[197] As used herein "chimeric" refers to a molecule that includes sequences derived from two different molecules.

[198] The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The numbering of the residues in the Fc region is that of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3.

[199] As used herein, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.

[200] A "native sequence Fc region" or "wile-type Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% sequence identity with a native sequence Fc region and / or with an Fc region of a parent polypeptide, and most preferably at least about 90% sequence identity therewith, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

[201] As used herein, an "effective dosage” or "effective amount" drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as inhibiting, suppressing or reducing the elevation of substance level, reducing, removing, clearing elevated antigenic substance or to reduce to its normal level, sequestering or increasing soluble substance circulating in biological fluids, decreasing one or more symptoms resulting from the disease (biochemical, histological and / or behavioral), including its complications and intermediate pathological phenotypes presenting during development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication, delaying the progression of the disease, and / or prolonging survival of patients. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[202] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: inhibiting, suppressing or reducing the formation of deposit of substance, reducing, removing, or clearing antigenic substance deposits, improving cognition, reversing or slowing cognitive decline, sequestering soluble substance circulating in biological fluids, reducing a substance (including soluble, oligomeric and deposited) in a tissue, inhibiting, slowing and / or reducing an elevation or increased level of antigenic substance in the tissue, inhibiting, slowing and / or reducing toxic effects of a substance peptide in a tissue, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of patients. The tissue may include brain of an individual.

[203] The term "development" of a disease means the onset and / or progression of the disease within an individual. A disease development can be detectable using standard clinical techniques as described herein. However, development also refers to disease progression that may be initially undetectable. For purposes of this invention, progression refers to the biological course of the disease state, in this case, as determined by a standard neurological examination, patient interview, or may be determined by more specialized testing. A variety of these diagnostic tests include, but not limited to, neuroimaging, detecting alterations of levels of specific proteins in the serum or cerebrospinal fluid (e.g., any one of the antigenic substances listed in Table 1 or combinations thereof), computerized tomography (CT), and magnetic resonance imaging (MRI). "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of a disease includes initial onset and / or recurrence.

[204] As used herein, "delaying" development of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a method that delays development of a disease is a method that reduces probability of disease development in a given time frame and / or reduces extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects.

[205] As used herein, "vector" means a construct, which is capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[206] A "host cell" includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

[207] As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[208] As used herein, "pharmaceutically acceptable carrier" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods.

[209]

[210] [TAM RECEPTOR]

[211] TAM receptors (Tyro3, Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors comprise 2 immunoglobulin-like and 2 fibronectin type III repeats in their extracellular domains in tandem. This is connected to a single-pass transmembrane domain and a cytoplasmic protein tyrosine kinase.

[212] TAM receptors enhance phagocytosis of apoptotic cells, also known as efferocytosis.

[213] The Axl protein contains 894 amino acids with a glycine-rich loop (Gly543-Gly548), a catalytic loop (His670-Asn677), and a DFG motif (Asp690-Phe691-Gly692). Although the molecular weight of the full-length Axl is 104 kDa, post-translational modifications of the extracellular domains give rise to two modified forms with molecular weights 120 and 140 kDa. Potential N-linked glycosylation sites include Asn43, Asnl57, Asnl98, Asn339, Asn345, and Asn401. In various embodiments of the present disclosure, the term "Axl" or "Axl receptor" or "Axl protein" includes the full-length Axl of 104 kDa, post-translational modified Axl, and glycosylated Axl. In some embodiments, the human Axl polypeptide corresponds to Genbank accession no. NP_068713, NP_068713.2, SEQ ID NO: 114. In one embodiment, the nucleic acid encoding the human Ax I polypeptide corresponds to Genbank accession no. NM_021913, version no. NM_021913.5. Murine Axl refers to the Axl member of the murine TAM family of receptor tyrosine kinases. In some embodiments, the murine Axl polypeptide corresponds to Genbank accession no. AAH46618, version no. AAH46618.1, SEQ ID NO: 115. In one embodiment, the nucleic acid encoding the murine Axl polypeptide corresponds to Genbank accession no. BC046618, version no. BC046618.1.

[214] MerTK (Mer tyrosine kinase) is a receptor tyrosine kinase that transduces signals from the extracellular matrix into the cytoplasm by binding to several ligands including TULP1 or GAS6. It regulates many physiological processes including cell survival, migration and differentiation. Ligand binding at the cell surface induces dimerization and autophosphorylation of TYRO3 on its intracellular domain that provides docking sites for downstream signaling molecules. Following activation by ligand, MerTK interacts with PIK3R1 and thereby enhances PI3-kinase activity.

[215] Human MerTK contains 999 amino acid residues (Accession nos. Q12866, NP_006334.2). mRNA and genomic DNA sequences are available under accession nos. AAB60430.1 and AAG33129.1, respectively. Various natural variants and posttranslation modifications as well as fragments are reported. ( www.uniprot.org / uniprotkb / 012866 / entry. last visited June 11, 2023).

[216] Human Tyro3 tyrosine kinase receptor contains 890 amino acid residues (Accession Nos. Q06418, NP_001317193.1, NP_006284.2). Polynucleotide sequences encoding human tyro3 are available under accession nos. NM_001330264.1 and NM_006293.3. Various mRNA sequences encoding human tyro3 are reported under accession numbers such as AAA19236.1, BAA04467.1, AAC50070.1, BAA21781.1, AAH49368.1, AAH51756.1, and CAA51396.1. Several natural variants and posttranslation modifications are reported ( www.unipro t.org / uniprotkb / 006418 / entry#sequences. last visited June 11, 2023).

[217] The cells expressing the TAM receptor(s) may be at least one type of professional phagocytes, at least one type of non-professional phagocytes, or a combination thereof. Here, the professional phagocytes refer to cells whose main role is to remove dead cells and accumulated debris through phagocytosis, and examples thereof include macrophages, neutrophils, dendritic cells, and mast cells. Macrophages usually stay in each tissue that can become a path of infection, and in many cases, they are called different names for tissues, including, for example, adipose tissue macrophages, bone marrow or blood monocytes, hepatic Kupffer cells, lymph node sinus histiocytes, alveolar macrophages, connective tissue histiocytes or giant cells, microglia of the central nervous system, placental Hofbauer cells, renal intraglomerular mesangial cells, bone osteoclasts, epithelioid cells of granulomas, red pulp macrophages of the spleen, peritoneal macrophage of the peritoneal cavity, LysoMac (lysozyme-expressing macrophage) of Peyer's patch, and the like. On the other hand, the non-professional phagocytes refer to cells that mainly perform functions specific to the tissue in which the phagocytes reside, but can perform phagocytosis when necessary, and examples thereof epithelial cells, endothelial cells, fibroblasts, mesenchymal cells, some tissuespecific cells, for example, astrocytes or oligodendrocyte of the central nervous system, retinal Muller glia, hepatocytes, muscular satellite cells, testicular Sertoli cells, etc., and some lymphocytes such as natural killer cells, large granular lymphocytes, eosinophils, basophils, B cells, etc. The fusion molecule according to the present disclosure is able to induce phagocytosis in phagocytes specific to a tissue in which a target substance to be cleared increases. For example, when the amount or expression of antigenic proteins are elevated in the brain are to be cleared, the phagocytosis may be induced in astrocytes, microglia, oligodendrocytes, or combinations thereof. It may be induced, for example, by topically administering the fusion molecule according to the present disclosure to this tissue or by manipulating cells in the tissue to express and secrete the fusion molecule.

[218]

[219]

[220] [FIRST REGION COMPRISING A SEQUENCE CAPABLE OF BINDING TO TAM RECEPTOR]

[221] TAM receptors can be activated via their ligands, growth arrest specific 6 protein (Gas6) and Protein S (ProSl), which are members of the family of vitamin K-dependent proteins.

[222] In exemplary embodiments, the first region that is capable of binding to TAM receptors may comprise, consist of, or consist essentially of one or more TAM ligands.

[223] A TAM ligand, protein S contains an amino terminal gamma-carboxyglutamic acid (Gia) domain, followed by a thrombin-sensitive loop region and 4 epidermal growth factor-like domains ending with the carboxy-terminal (C-terminal), consisting of 2 laminin G repeats that together comprise the sex hormone-binding globulin domain (right figure of FIG. 1 A). The C-terminal region is sufficient for TAM receptor binding and phosphorylation. Gas6 is a 75-kDa vitamin K-dependent protein and has high structural homology (approximately 42%) with protein S and the modular composition is the same as shown in FIG. 1A.

[224] In addition to Gas6 (SEQ ID NO: 7) and ProSl (SEQ ID NO: 34), tubby (accession nos. P50607, U54644.1, AAB53494.1, U82467.1, AAB53699.1, CH471064.2, EAW68634.1, BC075031.2, AAH75031.1, BC075032.2, AAH75032.1, NP_003311.2, NP_813977.1, 1S31_A), tubby-like protein 1 (Tulpl) (accession nos. AAB53700.1, AAH32714.1, AAH65261.1, NP_001276324.1, AAB97966.1, EAX03840.1, EAX03839.1, BAJ84064.1, BAJ84063.1, AKU84911.1, NP_813977.1, NP_003311.2), and galectin-3 (Gal3) (accession nos. NP_002297, NP_002297.1) are reported as TAM receptor ligands. Tubby and Gal-3 specifically bind to Mer, whereas Tulpl can activate all 3 of the TAM receptors.

[225] Gas6, one of the ligands for TAM receptors, is reported to show the highest affinity for Axl compared to Tyro3 or Mer. Human Gas6 contains 678 amino acids (SEQ ID NO: 7), with gamma-carboxyglutamic acid (Gia) domains, four epidermal growth factor (EGF)-like domains, and two laminin G-like (LG) domains (Figure 1A, right figure). Various isoforms of GAS6 are reported. For example, S6L, G8R, G8V, R14H, L18Q isoforms have been reported and these isoforms are included in the present disclosure.

[226] In embodiments, the first region that is capable of binding to TAM receptor may be TAM receptor agonists. TAM receptor agonists include agents that significantly increase the biological activity of a TAM receptor in a cell, for instance an agent that specifically binds to and activates a TAM receptor. For example, a TAM receptor agonist may increase the biological TAM receptor activity by at least 25%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, or even at least 500%. Methods of measuring such activity are known in the art. In some examples, an increase in biological activity is indicated by a increase in expression of Tyro3, Axl, or Mer or combinations thereof (at the DMA, RNA, or protein level). In other examples, an increase in biological activity is indicated by a change in a downstream effect, such an increase in TAM autophosphorylation, decrease in TLR-induced cytokine production, decrease in TLR-induced stimulation of MAP kinase activation, decrease in TLR-induced NF-kB activation, or increase in SOCS1 and SOCS 3 expression. Methods of detecting such alternations in expression or activity (which in some examples are quantified) are routine, and can include western blotting, ELISA, flow cytometry, northern blotting, PCR, RT-PCR, and the like. In embodiments, the TAM receptor that is to be activated by the first region according to the present disclosure may be Axl or Mer.

[227] In embodiments, the first region that is capable of binding to TAM receptor may comprise, consist of, or consist essentially of Gas6 protein or an active fragment thereof. The term "active fragment" as used herein denotes a fragment that is capable of binding to TAM receptor, in particular, Axl receptor. For example, an active fragment of Gas6 protein may comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 1, 2, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, or 87. For example, an active fragment of ProSl protein may comprise, consist of, or consist essential of the sequence of SEQ ID NO: 3, 4, 6, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113. The present disclosure encompass the sequences having sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the sequence of any one of SEQ ID NOs. Sequences of SEQ ID NOs: 8-23 show sequence identity of at least 85% to SEQ ID NO: 1 (LG-1 domain of Gas6). Sequences of SEQ ID NOs: 24-33 show sequence identity of at least 85% to SEQ ID NO: 2 (LG-2 domain of Gas6). Sequences of SEQ ID NOs: 35-45 show sequence identity of at least 85% to SEQ ID NO: 3 (LG-1 domain of ProSl). Sequences of SEQ ID NOs: 46-62 show sequence identity of at least 85% to SEQ ID NO: 4 (LG-2 domain of ProSl). Sequences of SEQ ID NOs: 63-87 show sequence identity of at least 85% to SEQ ID NO: 5 (LG domains of Gas6). Sequences of SEQ ID NOs: 88-113 show sequence identity of at least 84% to SEQ ID NO: 6 (LG domains of ProSl).

[228] In other embodiments, the first region may comprise, consist, or consist essentially of variable region or CDRs of an anti-Axl antibody or a full-length anti-Axl antibody of which the effector function, in particular, Fc receptor-binding function is abolished or removed. The antibody or antigen-binding fragments may bind to extracellular domain of Axl, for example expressed on surface of phagocytic cells and induce internalization and phagocytosis without involving inflammatory reaction, in particular Fc-mediated inflammatory reaction. Non-limiting examples of anti-Axl antibody may include those described in, for example, WO2017200493A1, WO2015193430A1, WO2011159980A1, WO2016097370A1, WO2012175691A1, WO2015193428Al, WO2010131733A1, WO2017220695Al, WO2010130751A1, WO2016166302Al, WO2017009258A1, WO2016005593A1, US 20190134193A1, and the like, all of which the contents are incorporated by reference herein in their entireties. According to embodiments of the present disclosure, variable region, CDRs, or scFv, F(ab), or F(ab') of those anti-Axl antibodies may be employed as the first region of the fusion molecule.

[229] In other embodiments, the first region may comprise, consist, or consist essentially of variable region or CDRs of an anti-MerTK (Mer Tyrosine Kinase) antibody or a fulllength anti-MerTK antibody of which the effector function, in particular, Fc receptorbinding function is abolished or removed. The antibody or antigen-binding fragments may bind to extracellular domain of MerTK, for example expressed on surface of phagocytic cells and induce internalization and phagocytosis without involving inflammatory reaction, in particular Fc-mediated inflammatory reaction. Non-limiting examples of MerTK antibody may include those described in, for example, WO2016106221A1, WO2020076799A1, WO2020176497A1, and the like, all of which the contents are incorporated by reference herein in their entireties. According to embodiments of the present disclosure, variable region, CDRs, or scFv, F(ab), or F(ab') of those anti-MerTK antibodies may be employed as the first region of the fusion molecule.

[230] In other embodiments, the first region may comprise, consist, or consist essentially of variable region or CDRs of an anti-Tyro3 antibody or a full-length anti-Tyro3 antibody of which the effector function, in particular, Fc receptor-binding function is abolished or removed. The antibody or antigen-binding fragments may bind to extracellular domain of Tyro3, for example expressed on surface of phagocytic cells and induce internalization and phagocytosis without involving inflammatory reaction, in particular Fc-mediated inflammatory reaction. Non-limiting examples of anti-Tyro3 antibody may include those described in, for example, WO2016166348A1, and the like, all of which the contents are incorporated by reference herein in their entireties. According to embodiments of the present disclosure, variable region, CDRs, or scFv, F(ab), or F(ab') of those anti-Tyro3 antibodies may be employed as the first region of the fusion molecule.

[231] The peptide comprising the sequence of any one of SEQ ID NOs above includes not only the amino acid sequence of the peptide but also an amino acid sequence variant thereof. The term "sequence variant" refers to a protein having a sequence in which one or more amino acid residues differ from the amino acid sequence. As long as the activity of the fusion molecule is maintained, any truncation, deletion, insertion, substitution, or a combination thereof in the final structure of the protein is possible. One example of the sequence variant is a form in which amino acid residues at sites not essential for activity are truncated or deleted, or amino acid residues at sites important for autoinhibition are substituted. In some cases, it may also be modified by phosphorylation, glycosylation, methylation, farnesylation, or the like. These sequence variations and modifications are more preferable when the function and / or stability (thermal stability, pH stability, structural stability, etc.) and / or solubility of the protein are increased by mutation in the amino acid sequence.

[232] The method for mutagenesis of the amino acid sequence is based on a method of producing a nucleic acid molecule comprising a nucleotide sequence corresponding to the amino acid sequence to be mutated by mutating a nucleotide sequence encoding the protein, and a method for obtaining the gene encoding the protein may be performed in vivo or in vitro using any mutagenesis technique well known in the art, for example, site-directed mutagenesis (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 technique (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), or the like.

[233] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

[234] Examples of modified polypeptides include polypeptides with conservative sub- stitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or use of chemical analogs.

[235] Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of "conservative substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 2, or as further described below in reference to amino acid classes, may be introduced and the products screened.

[236]

[237] [Table 2] Amino acid substitutions Original Residue Conservative Substitutions Exemplary Substitutions Ala (A) Vai Vai; Leu; He Arg (R) Lys Lys; Gin; Asn Asn (N) Gin Gin; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gin (Q) Asn Asn; Glu Glu (E) Asp Asp; Gin Gly (G) Ala Ala His (H) Arg Asn; Gin; Lys; Arg He (I) Leu Leu; Vai; Met; Ala; Phe; Norleucine Leu (L) He Norleucine; lie; Vai; Met; Ala; Phe Lys (K) Arg Arg; Gin; Asn Met (M) Leu Leu; Phe; lie Phe(F) Tyr Leu; Vai; lie; Ala; Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr(Y) Phe Trp; Phe; Thr; Ser Vai (V) Leu He; Leu; Met; Phe; Ala;Norleucine

[238]

[239] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

[240] - Non-polar: Norleucine, Met, Ala, Vai, Leu, He;

[241] - Polar without charge: Cys, Ser, Thr, Asn, Gin;

[242] - Acidic (negatively charged): Asp, Glu;

[243] - Basic (positively charged): Lys, Arg;

[244] - Residues that influence chain orientation: Gly, Pro; and

[245] - Aromatic: Trp, Tyr, Phe, His.

[246] Non-conservative substitutions are made by exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[247] Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Changes in the variable region can alter binding affinity and / or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR domain. In still other embodiments, the CDR domain is CDR H3 and / or CDR L3.

[248]

[249] [TARGET INFLAMMATION-RELATED SUBSTANCES AND IMMUNOLOGICAL DISEASES OR IMMUNE-MEDIATED DISEASES]

[250] The target inflammation-related substance may include one or more autoantigens, autoantibodies thereof, or a complex of the autoantigens and the autoantibodies thereof; one or more immune cell surface molecules including co-stimulatory molecules and receptors; one or more complements; one or more chemokines; one or more cytokines; one or more cell adhesion molecules; or a combination thereof.

[251] The non-limiting exemplary antigenic substances that triggers, causes, or induces undesired or unwanted immune response and the related immunological diseases (immune-mediated diseases) are listed in Table 1 above. Non-limiting examples of immune cell surface molecules including co-stimulatory molecules and receptors, complements, chemokines, cytokines, and cell adhesion molecules are disclosed above.

[252] The fusion molecule according to the embodiments of the present disclosure reduces, clears or removes, or enhances the clearance or removal of antigenic substances in the subject and thereby may be useful for treating or preventing an immunological disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder, or a proliferative disease or disorder. In certain embodiments, the fusion molecule according to the present disclosure inhibits, reduces, clears or removes, or enhances the clearance or removal of inflammation-related substances in the subject and thereby may be useful for treating or preventing an immunological disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder, or a proliferative disease or disorder. An immunological disease or disorder may be an autoimmune disease or an inflammatory disease, exemplified in the present disclosure.

[253] In certain embodiments, the immunological disease or disorder is multiple sclerosis, myasthenia gravis, diabetes mellitus type 1, diabetes mellitus type 2, rheumatoid arthritis, neuromyelitis optica, autoimmune encephalitis, fatty liver disease, endometriosis, inflammatory bowel disease, asthma, obesity, ankylosing spondylitis, antiphospholipid antibody syndrome, chronic recurrent multifocal osteomyelitis, gout, henoch-schonlein purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus (sle), juvenile scleroderma, juvenile vasculitis, kawasaki disease, lupus (systemic lupus erythematosus), mixed connective tissue disease, myositis, poststreptococcal inflammatory syndromes, psoriatic arthritis, reactive arthritis, scleroderma, sjogren's syndrome, spondyloarthritis / spondyloarthropathy, systemic juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, celiac disease, thrombotic thrombocytopenic purpura (iTTP), and the like.

[254] The fusion molecules described herein may also be useful for treating a cardiovascular disease or disorder, such as atherosclerosis, endocarditis, hypertension, or peripheral ischemic disease.

[255] The fusion molecules described herein may be useful for treating or preventing, inhibiting, slowing the progression of, or reducing the symptoms associated with, an immunological disease or disorder, a cardiovascular disease or disorder, a metabolic disease or disorder, or a proliferative disease or disorder. An immunological disorder includes an inflammatory disease or disorder and an autoimmune disease or disorder. While inflammation or an inflammatory response is a host's normal and protective response to an injury, inflammation can cause undesired damage. For example, atherosclerosis is, at least in part, a pathological response to arterial injury and the consequent inflammatory cascade. A cardiovascular disease or disorder that may be treated, which may include a disease and disorder that is also considered an immunological disease / disorder, includes for example, atherosclerosis, endocarditis, hypertension, or peripheral ischemic disease. A metabolic disease or disorder includes diabetes, obesity, and diseases and disorders associated with an increased or elevated level of antigenic substances.

[256] Allergens are other antigens for which tolerance of the immune response thereto is also desirable. Even in diseases where the pathogenic autoantigen is unknown, bystander suppression may be induced using antigens present in the anatomical vicinity. For example, autoantibodies to collagen are observed in rheumatoid arthritis and, accordingly, a collagen-encoding gene or collagen may be the target inflammation-related substance that is to be cleared or removed or reduced by the administration of the fusion molecule. In this case, an aptamer specifically binding to the collagen-encoding gene or an antibody binding to the collagen or fragment thereof can be utilized as the second region of the fusion molecule. Furthermore, a fusion protein comprising a second region binding to beta cell autoantigens may be utilized to prevent development or treatment of type 1 diabetes (see e.g. Bach and Chatenoud (2001) Ann Rev Immunol 19: 131-161).

[257] Auto-antibodies directed against myelin oligodendrocyte glycoprotein (MOG) is observed in autoimmune encephalomyelitis and in many other CNS diseases as well as multiple sclerosis. Accordingly, administering a fusion molecule comprising an antiMOG antibody or fragment thereof as a second region allows for treatment of multiple sclerosis as well as related autoimmune disorders of the central nervous system.

[258] In general, an immune response includes (1) a humoral response, in which antibodies specific for antigens are produced by differentiated B lymphocytes known as plasma cells, and (2) a cell mediated response, in which various types of T lymphocytes act to eliminate antigens by a number of mechanisms. For example, helper T cells that are capable of recognizing specific antigens may respond by releasing soluble mediators such as cytokines to recruit additional cells of the immune system to participate in an immune response. Also, cytotoxic T cells that are also capable of specific antigen recognition may respond by binding to and destroying or damaging an antigen-bearing cell or particle.

[259] An immune response in a host or subject may be determined by any number of well-known immunological methods described herein and with which those having ordinary skill in the art will be readily familiar. Such assays include, but need not be limited to, in vivo or in vitro determination of soluble antibodies, soluble mediators such as cytokines (e.g., IFN-y, IL-2, IL-4, IL-10, IL-12, IL-6, IL-23, TNF-a, and TGF-[3), lym-phokines, chemokines, hormones, growth factors, and the like, as well as other soluble small peptide, carbohydrate, nucleotide and / or lipid mediators; cellular activation state changes as determined by altered functional or structural properties of cells of the immune system, for example cell proliferation, altered motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cell maturation, such as maturation of dendritic cells in response to a stimulus; alteration in relationship between a Thl response and a Th2 response; cellular differentiation by cells of the immune system, including altered surface antigen expression profiles or the onset of apoptosis (programmed cell death). Procedures for performing these and similar assays are may be found, for example, in Lefkovits (Immunology Methods Manual. The Comprehensive Sourcebook of Techniques, 1998).

[260] Levels of cytokines may be determined according to methods described herein and practiced in the art, including ELISA, ELISPOT, and flow cytometry (to measure intracellular cytokines). Immune cell proliferation and clonal expansion resulting from an antigen-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as spleen cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and / or cell viability, such as by incorporation of tritiated thymidine or nonradioactive assays, such as MTT assays and the like. The effect of a fusion polypeptide described herein on the balance between a Thl immune response and a Th2 immune response may be examined, for example, by determining levels of Thl cytokines, such as IFN-y, IL-12, IL-2, and TNF-[3, and Type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

[261] In certain embodiments, the antigenic substance and the related immunological diseases do not include the substances of which aberrant accumulation or aggregation in a living tissue is characteristic of or associated with a disease such as a neurological disease or disorder.

[262]

[263] [SECOND REGION OF FUSION MOLECULE]

[264] The second region that specifically binds to the target substance may be selected from among an antibody, an antigen-binding fragment thereof, an antibody-like protein, a peptide, an aptamer, and a soluble receptor, and is not particularly limited as long as it specifically binds to the target substance.

[265] Here, the antibody or an antigen-binding fragment thereof may be selected from among, for example, i) immunoglobulins such as IgGl, IgG2, IgG3 and IgG4; ii) native antibody fragments such as Fv, Fab, Fab', F(ab')2, VHH, VNAR, etc.; and iii) engineered antibodies such as scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, 2023358200   23 Sep 2026 tetrabody, pentabody, etc. The antibody or antigen-binding fragment thereof may be, for example, a Mab, Fab, or single-chain variable fragment (scFv) based on an antibody that specifically binds to a corresponding target substance, or six complementarity-determining regions (CDRs) derived from the antibody. That is, the protein or antigen-binding fragment thereof that specifically binds to the target substance comprises a portion necessary for an activity that specifically binds to the target substance, and the type or range thereof is not particularly limited as long as the protein or antigen-binding fragment thereof is linked to the first region and does not cause an inflammatory response and synaptic damage.

[266] The antibody or antigen-binding fragment thereof may not comprise an Fc region, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fcy receptor). This Fc region variant may serve to improve properties such as purification. Fc variants with a reduced affinity to the human FcyRIIIA and / or FcyRIIA and / or FcyRI compared to a IgG Fc region by way of amino acid substitution are disclosed for example, WO2012130831 and USP 8753628, ofwhich entire content is incorporated by reference herein. Fc regions may be aglycosylated or deglycosylated.

[267] The antibody-like protein refers to a protein scaffold capable of specifically binding to a target substance, like an antibody. Antibody-like proteins may be designed to have a size of about 2 to 20 kDa, which is smaller than antibodies (about 150 kDa on average), and thus target a binding site that antibodies cannot reach. It is known that antibody-like proteins are more stable at high temperatures than antibodies and are much easier to synthesize using nonmammalian cells such as viruses and yeast or synthesize chemically, compared to antibodies.

[268] As used herein, the term "aptamer" refers to a single-stranded DNA (ssDNA) or RNA having high specificity and affinity for a specific substance. Aptamers have a very high affinity for specific substances, are stable, may be synthesized in a relatively simple way, may be modified in various ways to increase the binding affinity thereof, and can target cells, proteins, and even small organic substances. Thus, the aptamers are characterized by having very high specificity and stability compared to antibodies that have already been developed. In addition, the aptamer may be produced through a known SELEX (Systematic Evolution of Ligands by Exponential enrichment) method. As this aptamer, an aptamer that specifically binds to, for example, any one of the listed target substances, may be produced through a known SELEX (Systematic Evolution of Ligands by Exponential enrichment) method and then linked to the first region, thereby producing the fusion molecule according to the present invention.

[269] The aptamer of the present disclosure is not limited as long as it is able to specifically bind to any one of the listed target substances, and bases that are used for the aptamer may be selected from among A, G, C, U, and deoxy forms thereof, unless otherwise specified.

[270] In addition, the aptamer may be modified by linkage of at least one, selected from the group consisting of polyethylene glycol (PEG), inverted deoxy thymidine (idT), locked nucleic acid (LNA), 2'-methoxy nucleoside, 2'-amino nucleoside, 2'F-nucleoside, amine linker, thiol linker, and cholesterol, at the 5'-end region, intermediate region, 3'-end region, or both ends thereof in order to increase the stability thereof. Inverted deoxythymidine (idT) is a molecule that is generally used to prevent nuclease degradation of an aptamer having weak nuclease resistance. In the case of a nucleic acid unit, the 3'-OH of the previous nucleotide is attached to the 5'-OH of the next nucleotide to form a chain, but in the case of idT, the 3'-OH of the previous nucleotide to attached the 3'-OH of the next unit so that 5'-OH, not 3'-OH, is exposed. Thus, idT is a molecule that has the effect of inhibiting degradation by 3' exonuclease, a type of nuclease.

[271] Since the fusion molecule according to the present disclosure induces phagocytosis through interaction with the TAM receptor(s), the phagocytosis may be induced in cells expressing the TAM receptor(s). Phagocytosis generally means ingestion of cells or particles of 0.5 pm or more in size, and includes a process of tethering, engulfing, and degrading the cells or particles. In this case, phagocytosis forms a phagosome that surrounds the internalized cell or particle, and includes degradation within the phagolysosome by fusion of the phagosome and the lysosome. In phagocytosis, the process of cell death by apoptosis or necrosis is also referred to as efferocytosis.

[272] Non-limiting representative examples of the second regions and their target are shown in Table 3. The entire contents of the references listed in Table 3 are incorporated by reference herein. One skilled in the art should understand that not only the antibodies but also ligands to the listed target substance can serve as the second region. For example, a second region this is capable of binding to TGFBR1 (transforming growth factor beta receptor 1) may be TGF beta.

[273]

[274] [Table 3] Abbr eviat ion Diseases associated with or characterized by aberrant accumulation of target substance Target substance (accessio n number, UniProt) Second Region Exemplar y Referenc es Region2 VH (in case when the second region is an antibody) Region2 VL (in case when the second region is an antibody) CD2 0 Multiple Sclerosis, Rheumatoid Arthritis P11836 Rituximab QVQLQQPGAELVK PGASVKMSCKASG YTFTSYNMHWVK QTPGRGLEWIGAIY PGNGDTSYNQKFK GKATLTADKSSST AYMQLSSLTSEDS AVYYCARSTYYGG DWYFNVWGAGTT VTVSA (SEQ ID NO: 145) Rituximab QIVLSQSPAILSAS PGEKVTMTCRAS SSVSYIHWFQQKP GSSPKPWIYATSN LASGVPVRFSGSG SGTSYSLTISRVE AEDAATYYCQQ WTSNPPTFGGGT KLEIK (SEQ ID NO: 146) Critical Reviews in Oncolog y / Hemat ology 64 (2007) 210-225 CD1 9 Immunoglob ulin G4-related Diseases, Systemic Lupus Erythematosus, Systemic Sclerosis P15391 EVQLVESGGGLVK PGGSLKLSCAASG YTFTSYVMHWVR QAPGKGLEWIGYI NPYNDGTKYNEKF QGRVTISSDKSIST AYMELSSLRSEDT AMYYCARGTYYY GSRVFDYWGQGTL VTVSS (SEQ ID NO: 147) DIVMTQSPATLSL SPGERATLSCRSS KSLLNSNGNTYL YWFQQKPGQSPQ LLIYRMSNLASG VPDRFSGSGSGTE FTLTISSLEPEDFA VYYCMQHLEYPF TFGAGTKLEIK (SEQ ID NO: 148) US11618 788B2 C3 Lupus Nephritis, Myasthenia Gravis, Antibody-me diated P01024 EVQLVESGGGLVQ PGGSLRLSCAASGF TFSSYAMSWVRQA PGKGLEWVSAISG SGGSTYYADSVKG RFTISRDNSKNTLY DIVLTQPPSVSGA PGQRVTISCSGSS SNIGSNYVSWYQ QLPGTAPKLLIYD NNQRPSGVPDRF SGSKSGTSASLAI WO2021 159939A 1 Rejection LQMNSLRAEDTAV YYCARNPFYVGVF DVWGQGTLVTVSS A (SEQ ID NO: 149) TGLQSEDEADYY CSAWDGDMLVR VFGGGTKLTVLG (SEQ ID NO: 150) C5 Myasthenia Gravis, Amyotrophic Lateral Sclerosis, ANCA Vasculitis P01031 QVQLVQSGAEVKK PGASVKVSCKASG YTFTDEYMNWVR QAPGQSLEWMGYI NPNNGGADYNOK FOGRVTMTVDOSI STAYMELSRLRSD DTAVYFCARLGYS NPYFDFWGQGTLV TVSS (SEQ ID NO: 151) DIVLTOSPDSLAV SLGERATINCKAS QDVNTAVAWYO QKPDQSPKLLIY WASTRHTGVPAR FTGSGSGTDYTLT ISSLQAEDVAVYF COQHHVSPWTFG GGTKVEIK (SEQ ID NO: 152) US 9932395 B2 C5A RI polyangiitis, ANCA vasculitis, Churg-Straus s Syndrome, Systemic Lupus Erythematosus P21730 QVQLRQPGAELVR PGASVKLSCKASA YTFTSYWMNWFK QRPEQGLEWIGRID PYSDSETRYNQKF EDRALLTVDKSSST AYMQLSSLTSEDS AVYYCARFVIPSG GFAYWGQGTLVT VSA (SEQ ID NO: 153) DVVMTQTPLSLP VSLGDQASISCRS SQSPVHSNGNTY LHWYLQKPGQSP KLLIYKVSNRFSG VPDRFSGSGSGTD FTLKISRVEAEDL GVYFCSQSTHVPP TFGSGTKLEIK (SEQ ID NO: 154) WO2023 129870A 2 CD5 2 Multiple Sclerosis, Graft Versus Host Disease (GVHD) P31358 EVQLVESGGGLVQ PGGSLRLSCAASGF PFSNYWMNWVRQ APGKGLEWVGQIR LKSNNYATHYAES VKGRFTISRDDSKN SLYLQMNSLKTED TAVYYCTPIDYWG QGTTVTVSS (SEQ ID NO: 155) DIVMTQTPLSLSV TPGQPASISCKSS QSLLYSNAKTYL NWVLQKPGQSPQ RLIYLVSKLDSGV PDRFSGSGSGTDF TLKISRVEAEDVG VYYCVQGSHFHT FGQGTKLEIK (SEQ ID NO: 156) US97084 07B2 FCG Myathenia P55899 Rozanolixizumab Rozanolixizumab US 10233 RT Gravis, Rheumatoid Arthritis, Systemic Lupus Erythematosus EVPLVESGGGLVQ PGGSLRLSCAVSGF TFSNYGMVWVRQ APGKGLEWVAYID SDGDNTYYRDSVK GRFTISRDNAKSSL YLQMNSLRAEDTA VYYCTTGIVRPFLY WGQGTLVTVS (SEQ ID NO: 157) DIQMTQSPSSLSA SVGDRVTITCKSS QSLVGASGKTYL YWLFQKPGKAPK RLIYLVSTLDSGIP SRFSGSGSGTEFT LTISSLQPEDFAT YYCLQGTHFPHT FGQGTKLEIK (SEQ ID NO: 158) 243B2 IL1 A Rheumatoid Arthritis, Systemic Sclerosis, Hidradenitis Suppurativa, Ulcerative Colitis P01583 Bermechimab QVQLVESGGGVVQ PGRSLRLSCTASGF TFSMFGVHWVRQ APGKGLEWVAAV SYDGSNKYYAESV KGRFTISRDNSKNI LFLQMDSLRLEDT AVYYCARGRPKV VIPAPLAHWGQGT LVTFSS (SEQ ID NO: 159) Bermechimab IQMTQSPSSVSAS VGDRVTITCRAS QGISSWLAWYQQ KPGKAPKLLIYEA SNLETGVPSRFSG SGSGSDFTLTISSL QPEDFATYYCQQ TSSFLLSFGGGTK VEHKR (SEQ ID NO: 160) KR20230 004638A IL1 B Pyoderma Gangrenosu m, Pulmonary Sarcoidosis, Osteoarthritis Pain P01584 QVQLQESGPGLVK PSQTLSLTCSFSGFS LSTSGMGVGWIRQ PSGKGLEWLAHIW WDGDESYNPSLKS RLTISKDTSKNQVS LKITSVTAADTAV YFCARNRYDPPWF VDWGQGTLVTVSS (SEQ ID NO: 161) DIQMTQSTSSLSA SVGDRVTITCRAS QDISNYLSWYQQ KPGKAVKLLIYY TSKLHSGVPSRFS GSGSGTDYTLTIS SLQQEDFATYFCL QGKMLPWTFGQ GTKLEIK (SEQ ID NO: 162) US75311 66B2 IL1 RI Rheumatoid Arthritis, Intracerebral Hemorrhage, Interstitial P14778 MEFGLSWVFLVAL LRGVQC / QVQLVE SGGGVVQPGRSLR LSCAVSGFTFSNYG MHWVRQAPGKGL MEAPAQLLFLLL LWLPDTTG / EIVL TQSPATLSLSPGE RATLSCRASQSVS SYLAWYQQKPG AU2008 207483B 2 Cystitis EWVAALieWNDGE NKHHAGSVRGRFT LieSRDNSKNTLYL QMNSLRAEDTAVY YCARGRYFDWLLF EYWGQGTLVTVSS (SEQ ID NO: 163) QAPRLLIYDASNR ATGIPARFSGSGS GTDFTLTISSLEPE DFAVYYCQQRSN WPPLTFGGGTKV EIK (SEQ ID NO: 164) IL6 Rheumatoid Arthritis, Giant Lymph Node Hyperplasia, Antibody-Me diated Rejection, Amyotrophic Lateral Sclerosis P05231 EVKLEESGGGLVQ PGGSMKLSCVASG FTFSNYWMNWVR QSPEKGLEWVAEI RLKSNNYATHYAE SVKGRFTISRDDSK SSVYLQMNNLRAE DTGIYYCTREDYY GYPDYWGQGTTLT VSS (SEQ ID NO: 165) DIVLTQSPASLAV SLGQRATISCRAS ESVDNFGISFMN WFQQKPGQPPKL LIYVASNQGSGVP ARFSGSGSGTDFS LNIHPMEEDDTA MYFCQQSKEVPW TFGGGTKLEIK (SEQ ID NO: 166) US85363 08B2 IL6 R Rheumatoid Arthritis, Cytokine Release Syndrome, Giant Lymph Node Hyperplasia P08887 QVQLQESGPGLVK PSETLSLTCAVSGH SISHDHAWSWVRQ PPGEGLEWIGFISY SGITNYNPSLQGRV TISRDNSKNTLYLQ MNSLRAEDTAVYY CARSLARTTAMDY WGEGTLVTVSS (SEQ ID NO: 167) DIQMTQSPSSLSA SVGDSVTITCQAS TDISSHLNWYQQ KPGKAPELLIYYG SHLLSGVPSRFSG SGSGTDFTFTISSL EAEDAATYYCGQ GNRLPYTFGQGT KVEIE (SEQ ID NO: 168) US20220 280532A 1 IL6S T Non-Alcohol ic Steato-hepatitis (NASH); Non Alcoholic Fatty Liver Disease (NAFLD) P40189 QVQLQESGPGLVK PSETLSLTCAVSGH SISHDHAWSWVRQ PPGEGLEWIGFISY SGITNYNPSLQGRV TISRDNSKNTLYLQ MNSLRAEDTAVYY CARSLARTTAMDY WGEGTLVTVSS DIQMTQSPSSLSA SVGDSVTITCQAS TDISSHLNWYQQ KPGKAPELLIYYG SHLLSGVPSRFSG SGSGTDFTFTISSL EAEDAATYYCGQ GNRLPYTFGQGT KVEIE (SEQ ID US20210 017286A 1 (SEQ ID NO: 169) NO: 170) IL17 A Psoriasis, Hidradenitis Suppurativa, Non Alcoholic Fatty Liver Disease (NAFLD), Polymyalgia Rheumatica (PMR) Q16552 EVKLVESGGGLVR PGGTLKLSCAASG FTFSSFDMSWGRQ TPEKRLEWVAFMS SGGSTYYPDSVKG RFTISRDNVRNILY LQMISLRSEDTAM YYCARGERYGSY WGQGTLVTVSA (SEQ ID NO: 171) DIQMTQSSSYLSV SLGGRVTITCKAS DHINNWLAWYQ QKPGNAPRLLISG ATSLETGVPSRFS GSGSGKDYTLSIT SLQTEDVATYYC QQYWSTPFTFGS GTKLEIK (SEQ ID NO: 172) US 1073811 2B2 IL17 C Psoriasis, Atopic Dermatitis Q9P0M4 EVQLLESGGGLVQ PGGSLRLSCAASGF TVSDYAMHWVRQ APGKGLEWVSYIG GVGEGTQYAESVK GRFTISRDNSKNTL YLQMNSLRAEDTA VYYCARGFAIRYY GFDYWGQGTLVT VSS (SEQ ID NO: 173) SYELTQPPSVSVS PGQTASITCSGDK LGDKYAYWYQQ KPGQSPVLVIYQD SKRPSGIPERFSGS NSGNTATLTISGT QAEDEADYYCQV FTFPLVTTVFGGG TKLTVLGQ (SEQ ID NO: 174) US 10259 869B2 IL17 RA Plaque Psoriasis, Psoriasis, Rheumatoid Arthritis Q96F46 SEQ ID NO: 28 of US9073999B2 SEQ ID NO: 2 of US9073999B2 US90739 99B2 IL17 RC Rheumatoid arthritis, psoriasis, inflammatory bowel disease Q8NAC3 CN11381 7058B, CN11389 6793B TNF A Rheumatoid Arthritis, P01375 Adalimumab EVQLVESGGGLVQ Adalimumab DIQMTQSPSSLSA Schroer et al., Crohn's Disease, Hidradenitis Suppurativa, Ulcerative Colitis, Psoriasis PGRSLRLSCAASGF TFDDYAMHWVRQ APGKGLEWVSAIT WNSGHIDYADSVE GRFTISRDNAKNSL YLQMNSLRAEDTA VYYCAKVSYLSTA SSLDYWGQGTLVT VSS (SEQ ID NO: 176) SVGDRVTITCRAS QGIRNYLAWYQQ KPGKAPKLLIYA ASTLQSGVPSRFS GSGSGTDFTLTIS SLQPEDVATYYC QRYNRAPYTFGQ GTKVEIK (SEQ ID NO: 177) (2015)A generic approach to engineer antibody pH-switc hes using combinat orial histidine scanning libraries and yeast display, mAbs, 7:1, 138-151 TNF RSF 1A, TNF RI Multiple sclerosis, psoriasis, NASH, Inflammatory Bowel Disease P19438 QVQLVQSGAEVKK PGSSVKVSCKASG YTFTDFYINWVRQ APGQGLEWIGEIYP YSGHAYYNEKFKA RVTITADKSTSTAY MELSSLRSEDTAV YYCARWDFLDYW GQGTTVTVSS (SEQ ID NO: 178) DIVMTQSPLSLPV TPGEPASISCRSSQ SLLHSNGNTYLH WYLQKPGQSPQL LIYTVSNRFSGVP DRFSGSGSGTDFT LKISRVEAEDVG VYYCSQSTHVPY TFGGGTKVEIK (SEQ ID NO: 179) US88597 39B2 TNF RSF IB, TNF R2 Graft versus Host Disease (GVHD), Multiple sclerosis P20333 Heavy Chain CDRs: DYIMH (SEQ ID NO: 180) WVDPEYGSTDYAE KFKKK (SEQ ID NO: 181) DDGSYSPFDY (SEQ ID NO: 182) Light Chain CDRs: QASQNINKYIA (SEQ ID NO: 183) YTSTLES (SEQ ID NO: 184) LQYVNLLT (SEQ ID NO: 185) US20210 301028A 1 IL4 Atopic P05112 QVQLVESGGGVVQ EIVLTQSPGTLSLS US71868 Dermatitis, Venous Leg Ulcers PGRSLRLSCAASGF AFSSYAIQWVRQA PGKGLEWVAVISY DGSKKYYADSVK GRFTISRDNSKNTL YLQMNSLRAEDTA VYYCAREGRRGSF DYWGQGTLVTVSS (SEQIDNO: 186) PGERATLSCRASQ SVSTSYLAWYQQ KPGQAPRLLIYGA SSRATGIPDRFSG SGSGTDFTLTISR LEPEDFAVYYCQ QYGSSPTFGQGT KVEIK (SEQ ID NO: 187) 09B2 / EP 2292665 Bl IL4 RA Asthma, Atopic Dermatitis, Eosinophilic Esophagitis, Ulcerative Colitis, Systemic Sclerosis, Nasal polyps P24394 EVQLVESGGGLVQ PGGSLRLSCAVSGF TFSSYAMSWVRQA PGKGLEWVSSITG GGGGIYYADSVKG RFTISRDNSKNTVY LQMNSLRAEDTAV YYCAKDRISITIRPR YFGLDFWGQGTTV TVSS (SEQ ID NO: 188) DIVMTQSPLSLPV TPGEPASISCRSSR SVLYGNGYNYLD WYLQKSGQSPQL LIYLGTNVAAGV PDRFSGSGSGTDF TLKISRVEAEDVG FYYCMQSLRTPY TFGQGTKLEIK (SEQIDNO: 189) US02022 0081485 Al IL5 Asthma, Hypereosinophil ic Syndrome, Nasal Polyps, Churg-Straus s Syndrome P05113 QVQLKESGPGLVA PSQSLSITCTVSGFS LTSYSVHWVRQPP GKGLEWLGVIWAS GGTDYNSALMSRL SISKDNSKSQVFLK LNSLQTDDTAMYY CARDPPSSLLRLDY WGQGTTLTVSS (SEQ ID NO: 190) EKVTMSCKSSQS LLNSGNQKNYLA WYQQKPGQPPKL LIYGASTRESGVP DRFTGSGSGTDFT LSISSVQAEDLAV YYCQNVHSFPFTF GSGTELEIK (SEQ ID NO: 191) US98346 00B2 IL5 RA Asthma, Rhinosinusitis, Chronic Obstructive Pulmonary Disease (COPD), Q01344 EVQLVQSGAEVKK PGASVKVSCKASG YTFTSYVIHWVRQ RPGQGLAWMGYI NPYNDGTKYNERF KGKVTITSDRSTST VYMELSSLRSEDT DIQMTQSPSSLSA SVGDRVTITCGTS EDIINYLNWYQQ KPGKAPKLLIYHT SRLQSGVPSRFSG SGSGTDFTLTISSL QPEDFATYYCQQ Benralizu mab Churg-Straus s Syndrome, Nasal Polyps AVYLCGREGIRYY GLLGDYWGQGTL VTVSS (SEQ ID NO: 192) GYTLPYTFGQGT KVEIK (SEQ ID NO: 193) CSF 2RB , IL3 RB, IL5 RB Allergic Asthma, Chronic Obstructive Pulmonary Disease (COPD) P32927 SEQ ID NO: 29 of WO2023027177A1 SEQ ID NO: 30 of WO2023027177A1 WO2023 027177A 1 IL13 Atopic Dermatitis, Eosinophilic Esophagitis, Gastroenterit is P35225 QVOLVQSGAEVKK PGASVKVSCKASG YTFTNYGLSWVRQ APGQGLEWMGWI SANNGDTNYGQEF QGRVTMTTDTSTS TAYMELRSLRSDD TAVYYCARDSSSS WARWFFDLWGRG TLVTVSS (SEQ ID NO: 194) SYVLTOPPSVSVA PGKTARITCGGNI IGSKLVHWYQQK PGQAPVLVIYDD GDRPSGIPERFSG SNSGNTATLTISR VEAGDEADYYCO VWDTGSDPVVFG GGTKLTVL (SEQ ID NO: 195) US98563 17B2 IL13 RAI Atopic Dermatitis, Allergic Asthma P78552 EVQLQQSGGGLVQ PGRSLNLSCAASGF TFNDYYMAWVRQ APKKGLEWVATII YDGTRTYYRDSVK GRFTISRDNAKSTL YLQMDSLRSEDTA TYYCATPWGSWG QGTTVTVSS (SEQ ID NO: 196) DIQMTQSPSSMPA SLGERVTISCRAS QGISNFLNWYQQ KADGTIKPLIYYT SNLQSAVPSRFSG SGSGTDYSLTISS LEPEDFAMYYCQ QYDSSPWTFGGG TKLEITR (SEQ ID NO: 197) US20050 154192A 1 EP14498 51A1 IFN G Systemic Lupus Erythematosus, Arthritis, En-cephalomyeli P01579 EVQLLESGGGLVQ PGGSLRLSCAASGF TFSSYAMSWVRQA PGKGLEWVSAISG SGGSTYYADSVKG NFMLTQPHSVSES PGKTVTISCTRSS GTIASNYVQWYQ QRPGSSPTTVIYE DNQRPSGVPDRF US77000 98B2 / KR 1013805 70B1 tis, Vitiligo RFTISRDNSKNTLY LQMNSLRAEDTAV YYCAKDGSSGWY VPHWFDPWGRGT MVTVSS (SEQ ID NO: 198) SGSIDSSSNSASLT ISGLKTEDEADYY CQSYDNSNHWVF GGGTKVTVLG (SEQ ID NO: 199) IFN GR1 Psoriasis, Rheumatoid Arthritis, Allergic Rhinitis Pl 5260 CN11457 3713A IFN GR2 Atopic Dermatitis, Psoriasis, Rheumatoid Arthritis, Allergic Rhinitis P38484 WO2022 031890A 1 IL12 A Plaque Psoriasis, Psoriatic Arthritis, Crohn's Disease, Ulcerative Colitis P29459 QVQLVESGGGVVQ PGRSLRLSCAASGF TFSSYGMHWVRQ APGKGLEWVAFIR YDGSNKYYADSV KGRFTISRDNSKNT LYLQMNSLRAEDT AVYYCKTHGSHD NWGQGTMVTVSS (SEQ ID NO: 200) QSVLTQPPSVSGA PGQRVTISCSGSR SNIGSNTVKWYQ QLPGTAPKLLIYY NDQRPSGVPDRF SGSKSGTSASLAI TGLQAEDEADYY CQSYDRYTHPAL LFGTGTKVTVLG (SEQ ID NO: 201) US87659 18B2 IL12 RBI Alzheimer's Disease, Rheumatoid Arthritis P42701 SEQ ID NO: 6 of US20220177567A1 SEQ ID NO: 7 of US20220177567A1 US20220 177567A 1 IL12 RB2 Crohn's Disease Q99665 WO2022 031942A 2 IL21 Graft Versus Q9HBE4 QVQLVQSGAEVKK DIQMTQSPSSLSA US93943 Host Disease (GVHD), Celiac Disease PGASVKVSCKASG YTFTDYWMHWVR QAPGQGLEWMGLI DTSDVYTIYNQKF KGRVTMTRDTSTS TVYMELSSLRSED TAVYYCARYGPLA MDYWGQGTLVYV SS (SEQ ID NO: 202) SVGDRVTITCRAS QDISNYLNWYQQ KPGKAPKLLIYYT SRLHSGVPSRFSG SGSGTDFTLTISSL QPEDFATYYCQQ FHTLRTFGGGTK VEIK (SEQ ID NO: 203) 62B2 IL21 R Systemic Lupus Erythematosus Q9HBE5 QVQLVQSGAEVKK PGSSVRVSCKASG GTFNIYSVSWVRQ APGQGLEWMGRII PMRDIANYAQRFQ GRVTLTADKSSGT AYMELRGLRSDDT AVYWCATLAGPL DSWGQGTLVT (SEQ ID NO: 204) SSELTQDPAVSVG LGQTVTITCQGGS LRQYYASWYQQ KPGQAPVVVIYG KNKRPSGIPDRFS GTTSGNTASLTIT GAQAEDEADYYC KSRDSSGNHPLY VFGAGTKLTVLG ES (SEQ ID NO: 205) US 7495085 B2 IL22 Ulcerative Colitis, Psoriasis, Rheumatoid Arthritis Q9GZX6 EVQLVQSGAEVKK PGASVKVSCQASG YTFSDYYIHWVRQ TPGQGFEWMGWV NPDTGGTRYAOKF OGWVTMTRDMSN TTAYMELPRLRDD DTAVYYCARDLTG FDPFDIWGQGTLV TVSS (SEQ ID NO: 206) QSVLTQPPSVSVA PGKTATITCGGN NFRNKRVHWYQ QRPGQAPVLVIY YDSDRPSGIPERF SGSRSGNTATLTI SRVEAGDEADFY COVWDSSTDRPL FGGGTKLTVLG (SEQ ID NO: 207) US78115 67B2 / EP23274 23B1 IL22 RAI Inflammator y Bowel Disease, Non-Alcohol ic Steato-hepatitis Q8N6P7 QVQLVESGGGLVQ PGGSLRLSCAASGF TFSSYDMSWVRQA PGKGLEVWSSIYN DGSNTAYSDSVKG RFTISRDNAKNTLY NFMLTQPSAVSV SLGQTAKITCQG GYYAHWYQQKP GQAPVLVIYGNN NRPSGIPERFSGSS SGNTATLTISGAQ US20230 088269A 1 (NASH), Psoriasis, Multiple Sclerosis, Atopic dermatitis LQMNSLKSEDTAV YYCAKVGGQGTQ VTVSS (SEQ ID NO: 208) AEDEAEYYCQSG SSSANAVFGGGT HLTVL (SEQ ID NO: 209) TGF Bl Venous Leg Ulcers, Osteoarthritis, Liver Fibrosis P01137 SEQ ID NO: 4 of US20030064069A1 SEQ ID NO: 8 of US20030064069A1 US20030 064069A 1 TGF B2 Idiopathic Pulmonary Fibrosis, Systemic Sclerosis, Non-Alcohol ic Steato-hepatitis (NASH) P61812 Fig IB of US20030064069A1 Fig 1A of US20030064069A1 US80124 82B2 TGF B3 Idiopathic Pulmonary Fibrosis, Diabetic Macular Edema, Wet Macular Degeneration, Non-Alcohol ic Steato-hepatitis (NASH) P10600 US52623 19A CD8 0 / 86 Rheumatoid Arthritis, Psoriatic Arthritis, Graft Versus P33681 US20070 065436A 1 Host Disease (GVHD), Kidney Transplantati on Rejection CD2 8 Kidney / Heart / Lung Transplant Rejection, Systemic Lupus Erythematosus, Rheumatoid Arthritis, Multiple Sclerosis P10747 QVQLVQSGAEVKK PGASVKVSCKASG YTFTSYYIHWVRQ APGQGLLWIGCIYP GNVNTNYNEKFKD RATLTVDTSISTAY MELSRLRSDDTAV YFCTRSHYGLDWN FDVWGQGTTVTVS S (SEQ ID NO: 210) DIQMTQSPSSLSA SVGDRVTITVysH ASQNIYVWLNW YQQKPGKAPKLL IYKASNLHTGVPS RFSGSGSGTDFTL TISSLQPEDFATY YCQQGQTYPYTF GGGTKVEIK (SEQ ID NO: 211) WO2006 050949A 2 / EP21710 60B1 IL23 A Crohn's Disease, Psoriatic Arthritis, Ulcerative Colitis, Plaque Psoriasis Q9NPF7 QVQLVQSGAEVKK PGSSVKVSCKASG YKFTRYVMHWVR QAPGQGLEWMGYI NPYNDGTNYNEKF KGRVTITADKSTST AYMELSSLRSEDT AVYYCARNWDTG LWGQGTTVTVSS (SEQ ID NO: 212) DIQMTQSPSSLSA SVGDRVTITCKAS DHILKFLTWYQQ KPGKAPKLLIYG ATSLETGVPSRFS GSGSGTDFTLTIS SLQPEDFATYYC QMYWSTPFTFGG GTKVEIK (SEQ ID NO: 213) US20170 275356A 1 IL23 R Psoriasis, Inflammatory Bowel Disease, Multiple Sclerosis Q5VWK 5 QVQLVESGGGVVQ PGRSLRLSCAASGF DFNSYGMSWVRQ APGKGLEWVADIN SKSYNATYYADSV KDRFTISRDNSKNT LYLQMNSLRAEDT AVYYCARHHSDYF EYWGQGTLVTVSS (SEQ ID NO: 214) DIQMTQSPSSLSA SVGDRVTITCLAS EDIYNNLAWYQQ KPGKAPKLLIYH ASSLQDGVPSRFS GSGSGTDFTLTIS SLQPEDFATYYCL QDSEYPPTFGQGT KVEIKRT (SEQ ID NO: 215) US20110 158992A 1 TSL Asthma, Q969D9 QVQLVQSGAEVKK EIVLTQSPGTLSLS US20120 p Eosinophilic Esophagitis, Rhinosinusiti s, Atopic Dermatitis, Chronic Obstructive Pulmonary Disease (COPD) PGASVKVSCKASG YIFTDYAMHWVR QAPGQGLEWMGT FIPLLDTSDYAQKF QGRVTMTADTSTS TAYMELRSLRSDD TAVYYCARMGVT HSYVMDAWGQGT LVTVSS (SEQ ID NO: 216) PGERATLSCRASQ PISISVHWYQQKP GQAPRLLIYFASQ SISGIPDRFSGSGS GTDFTLTISRLEPE DFAVYYCQQTFS LPYTFGQGTKVEI KRT (SEQ ID NO: 217) 219565A 1 CRL F2, TSL PR Q9HC73 EVQLLESGGGLVQ PGGSLRLSCAASGF TFRSSAMHWVRQ APGKGLKWVSSVS GSGAGTYYADSVK GRFTISRDNPKNTL YLQMNSLRAEDTA VYYCVKEGGSRGF DYWGQGTLVTVSS (SEQ ID NO: 218) DIQMTQSPSSLSA SVGDRVTITCRAS QDISNYLAWFQQ KPGKAPKSLIYTA SSLQSGVPSKFSG SGSGTDFTLTISSL QPEDFATYYCQQ YNLYPPTFGQGT KVEIKR (SEQ ID NO: 219) US 9908941 B2 IL31 Atopic Dermatitis Q6EBC2 QVQLQQSGAELAR PGASVNLSCKASG YTLTRYWMQWVK QRPGQGLEWIGAI YPGLGDTRYSQKF KGKATLTADKSSS TAYMQLNNLASED SAVYYCAFPDGYY AAPYGMDYWGQG TSVTVSS (SEQ ID NO: 220) DIQMTQSPASLSA SVGETVTITCRAS GNTHNYLAWYQ QKQGKSPQLLVY NAKTLADGVPSR FSGSRSETQYSLK INSLQPEDFGSYY CQHFWSTPWTFG GGTKLEIK (SEQ ID NO: 221) US 10273 297B2 IL31 RA Pruritus, Prurigo, Atopic Dermatitis, Systemic Sclerosis, Q8NI17 QVQLVQSGAEVKK PGASVKVSCKASG YTFTGYIMNWVRQ APGQGLEWMGLIN PYNGGTDYNPQFQ DRVTITADKSTSTA DIQMTQSPSSLSA SVGDRVTITCQAS EDIYSFVAWYQQ KPGKAPKLLIYN AQTEAQGVPSRF SGSGSGTDFTLTI Nemoliz umab US20200 385476A 1 End-Stage Kidney Disease YMELSSLRSEDTA VYYCARDGYDDG PYTLETWGQGTLV TVSS (SEQ ID NO: 222) SSLQPEDFATYYC QHHYDSPLTFGG GTKVEIK (SEQ ID NO: 223) TNF RSF 4, 0X4 0 Atopic Dermatitis, Alopecia Areata, Inflammatory Bowel Disease, Systemic Lupus Erythematosus P43489 QVQLVQSGAEVKK PGASVKVSCKVSG YTFTSYWITWVRQ RPGKGLEWMGDIY PGSGSTNQNEKFK SRVTMTVDTSTDT AYMELSSLRSEDT AVYYCATLRPYYF VYWGQGTLVTVSS (SEQ ID NO: 224) DIQMTQSPSSLSA SVGDRVTITCRAS QDISNYLNWYQQ KPGKTPKLLIYYT SRLLSGVPSRFSG SGSGTDYTLTISS LQPEDFATYYCQ QGNTLPLTFGQG TRLEIK (SEQ ID NO: 225) US 10442 866B1 TNF SF4, 0X4 OL Atopic Dermatitis, Hidradenitis Suppurativa, Asthma, Psoriasis, Graft Versus Host Disease P23510 QVQLQQPGAELVR PGASVkLSCKASG YTFTSYWLNWVK QRPGQGLEWIVMI DPSDSETHYNQVF KDKATLTVDKSSS TAYMQLSSLTSED SAVYYCIRGRGNF YGGSHAMEYWGQ GTLLTVSS (SEQ ID NO: 226) DILMTQTPLSLPV SLGDQASISCRSS QSIVHGNGNTYL EWHLQKPGQSPK LLIYRVSNRFSGV PDRFSGSGSGTDF TLKINRVEAEDLG VYYCFQGSHVPY TFGGGTKVEIKR (SEQ ID NO: 227) US20100 272738A 1 IL33 Atopic Dermatitis, Chronic Obstructive Pulmonary Disease (COPD), Asthma 095760 EVQLVESGGGLVQ PGGSLRLSCAASGF TFSSYWMYWVRQ APGKGLEWVAAIT PNAGEDYYPESVK GRFTISRDNAKNSL YLQMNSLRAEDTA VYYCARGHYYYTS YSLGYWGQGTLVT VSS (SEQ ID NO: 228) DIQMTQSPSSLSA SVGDRVTITCKAS QNINKHLDWYQQ KPGKAPKLLIYFT NNLQTGVPSRFS GSGSGTDFTLTIS SLQPEDFATYYCF QYNQGWTFGGG TKVEIK (SEQ ID NO: 229) US20200 308272A 1 IL1 Atopic Q01638 SEQ ID NO: 95 of SEQ ID NO: 29 of WO2013 RL1, ST2 Dermatitis, Chronic Obstructive Pulmonary Disease (COPD), Asthma, Alzheimer's Disease WO2013173761A2 WO2013173761A2 173761A 2 CD4 0 Systemic Lupus Erythematosus, Rheumatoid Arthritis, Ulcerative Colitis, Multiple Sclerosis P25942 QVQLVESGGGVVQ PGRSLRLSCAASGF SFSSTYVCWVRQA PGKGLEWIACIYTG DGTNYSASWAKG RFTISKDSSKNTVY LQMNSLRAEDTAV YFCARPDITYGFAI NFWGPGTLVTVSS (SEQ ID NO: 230) MDMRVPAQLLG LLLLWLRGARCD IQMTQSPSSLSAS VGDRVTIKCQAS QSISSRLAWYQQ KPGKPPKLLIYRA STLASGVPSRFSG SGSGTDFTLTISSL QPEDVATYYCQC TGYGISWPIGGGT KVEIK (SEQ ID NO: 231) US 9994640 B2 CD4 OLG CD1 54 Systemic Lupus Erythematosus, Relapsing Multiple Sclerosis, Rheumatoid Arthritis, Sjogren's Syndrome P29965 QVQLVQSGAEVVK PGASVKLSCKASG YIFTSYYMYWVKQ APGQGLEWIGEINP SNGDTNFNEKFKS KATLTVDKSASTA YMELSSLRSEDTA VYYCTRSDGRND MDSWGQGTLVTV SS (SEQ ID NO: 232) DIVLTQSPATLSV SPGERATISCRAS QRVSSSTYSYMH WYQQKPGQPPKL LIKYASNLESGVP ARFSGSGSGTDFT LTISSVEPEDFAT YYCQHSWEIPPTF GGGTKLEIK (SEQ ID NO: 233) US 1101499 0B2 IGF 1R Bronchopul monary Dysplasia, Graves' Ophthalmopathy, P08069 QVELVESGGGVVQ PGRSQRLSCAASGF TFSSYGMHWVRQ APGKGLEWVAIIW FDGSSTYYADSVR EIVLTQSPATLSLS PGERATLSCRASQ SVSSYLAWYQQK PGQAPRLLIYDAS KRATGIPARFSGS US20120 149879A 1 Acute Ischemic Stroke GRFTISRDNSKNTL YLQMNSLRAEDTA VYFCARELGRRYF DLWGRGTLVSVSS (SEQ ID NO: 234) GSGTDFTLTISsyst emic lupus erythematosus (SLE)PEDFAVYY CQQRSKWPPWTF GQGTKVESK (SEQ ID NO: 235) ICA Ml Keratoconjun ctivitis Sicca, Diabetic Retinopathy P05362 QVQLQESGGGLVQ PGGSLRLSCAASGF TFSSYAMSWVRQA PGKGLEWVSAISG SGGSTYYADSVKG RFTISRDNSKNTLY LQMSSLRAEDTAF YYCANSAYTGGW YDYWGHGTLVTV SS (SEQ ID NO: 236) ASELTQDPAVSV ALGQTVKITCQG DSLRTYYASWYQ QRPGQAPVLVIY GENSRPSGIPDRF SGSSSGNTASLTI TGAQAEDEADYY CNSRDSSGNHLR VFGGGTKLTVL (SEQ ID NO: 237) US20220 002432A 1 VC AM 1 Cholangitis, Liver / Kidney Fibrosis, Systemic Sclerosis P19320 EVQLVESGGGLVK PGGSLKLSCAASGF TFSSYTMSWVRQS PEKRLEWVAEISSG GSYTHYAATVTGR FTISRDNVKNTLYL EMSSLRSEDTAMY YCARGELYWGQG TLVTVSA (SEQ ID NO: 238) DVVLTQIPSTLSV TFGQPASISCKAS QSLLDRGGKTFF NWLLQRPGQSPK RLIYLVSKLDSGV PDRFTGSGSGTDF TLKISRVEAEDLG VYYCWQGTHFP WTFGGGTRLEIK (SEQ ID NO: 239) US20230 212295A 1 MA DC AM 1 Non-Alcohol ic Steato-hepatitis (NASH), Graft Versus Host Disease (GVHD), Ulcerative Colitis, Crohn's QI 3477 MDWTWSILFLVAA ATGAHSQVQLVQS GAEVKKPGASVKV SCKASGYTFTSYGI NWVRQAPGQGLE WMGWISVYSGNT NYAQKVQGRVTM TADTSTSTAYMDL RSLRSDDTAVYYC AREGSSSSGDYYY MRLPAQLLGLLM LWIPGSSADIVMT QTPLSLSVTPGQP ASISCKSSQSLLH TDGTTYLYW YLQKPGQPPQLLI YEVSNRFSGVPD RFSGSGSGTDFTL KISRVEAEDVGIY YCMQNIQLP US93281 69B2 / EP21775 37A2 Disease GMDVWGQGTTVT VSSASTKGPSVFPL APCSRSTSESTAAL GCLVKDYFPEPVT VSWNSGALTSGVH TFPAVLQSSGLYSL SSVVTVPSSNFGTQ TYTCNVDHKPSNT KVDKTVERKCCVE CPPCPAPPVAGPSV FLFPPKPKDTLMIS RTPEVTCVVVDVS HEDPEVQFNWYV DGVEVHNAKTKPR EEQFNSTFRVVSVL TVVHQDWLNGKE YKCKVSNKGLPAP IEKTISKTKGQPRE PQVYTLPPSREEMT KNQVSLTCLVKGF YPSDIAVEWESNG QPENNYKTTPPML DSDGSFFLYSKLTV DKSRWQQGNVFSC SVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 240) WTFGQGTKVEIK RTVAAPSVFIFPPS DEQLKSGTASVV CLLNNFYPREAK VQWKVDNALQ SGNSQESVTEQDS KDSTYSLSSTLTL SKADYEKHKVYA CEVTHQGLSSPVT KSFNRGEC (SEQ ID NO: 241) ITG A4 Ulcerative Colitis, Crohn's Disease, Pouchitis, Graft Versus Host Disease (GVHD), Relapsing Remitting P13612 QVQLVQSGAEVKK PGASVKVSCKGSG YTFTSYWMHWVR QAPGQRLEWIGEID PSESNTNYQKFKG RVTLTVDISASTAY MELSSLRSEDTAV YYCARGGYDGWD YAIDYWGQGTLVT VSS (SEQ ID NO: DVVMTQSPLSLP VTPGEPASISCRSS QSLAKSYGNTYL SWYLQKPGQSPQ LLIYGISNRFSGVP DRFSGSGSGTDFT LKISRVEAEDVG VYYCLQGTHQPY TFGQGTKVEIK (SEQ ID NO: 243) US20170 327584A 1 / EP35815 85A1 Multiple Sclerosis (RRMS) 242) ITG B7 Ulcerative Colitis, Crohn's Disease, Graft Versus Host Disease (GVHD) P26010 EVQLVESGGGLVQ PGGSLRLSCAASGF FITNNYWGWVRQ APGKGLEWVGYIS YSGSTSYNPSLKSR FTISRDTSKNTFYL QMNSLRAEDTAVY YCARTGSSGYFDF WGQGTLVTVSS (SEQ ID NO: 244) DIQMTQSPSSLSA SVGDRVTITCRAS ESVDDLLHWYQQ KPGKAPKLLIKY ASQSISGVPSRFS GSGSGTDFTLTIS SLQPEDFATYYC QQGNSLPNTFGQ GTKVEIKR (SEQ ID NO: 245) US20180 086833A 1 / KR20170 120601A ITG AL, LFA -1 Keratoconjun ctivitis Sicca (Dry Eye), Plaque Psoriasis, Inflammatory Bowel Disease, Allergic asthma P20701 EVQLLESGGGLVQ PGGSLRLSCAASGF TFSRYVMWWVRQ APGKGLEWVSYIW PSGGNTYYADSVK GRFTISRDNSKNTL YLQMNSLRAEDTA VYYCASSYDFWSN AFDIWGQGTMVTV SS (SEQ ID NO: 246) LNWYQQKTGKA PKALIYAASSLQS GVPSRFSGSGSGT DFTLTISSLQLEDF ATYYCQQSYSTP SFGQGTKVEIKRT (SEQ ID NO: 247) US20080 069777A 1 / CA25549 65C ITG AM, MA C-l Lupus Nephritis, Multiple Sclerosis P11215 SEQ ID NO: 13 of WO2016197974A1 SEQ ID NO: 33 of WO2016197974A1 WO201 6197974 Al ITG Bl Keratoconjun ctivitis Sicca (Dry Eye), Liver Fibrosis, Idiopathic Pulmonary Fibrosis P05556 US20220 111045A 1 CCR Multiple B2KIU0 MEWSGVFIFLLSVT MKLPVRLLVLMF US71221 5 Sclerosis, Non-alcoholi c Steato-hepatitis, Inflammatory Bowel Diseases AGVHSEVQLVESG GGLVKPGGSLRLS CAASGYTFSNYWI GWVRQAPGKGLE WIGDIYPGGNYIRN NEKFKDKTTLSAD TSKNTAYLQMNSL KTEDTAVYYCGSS FGSNYVFAWFTY WGQGTLVTVSS (SEQ ID NO: 248) WIPASSSDIVMTQ SPLSLPVTPGEPA SISCRSSQRLLSSY GHTYLHWYLQKP GQSPQLLIYEVSN RFSGVPDRFSGSG SGTDFTLKISRVE AEDVGVYYCSQS THVPLTFGQGTK VEIK (SEQ ID NO: 249) 85B2 CCL 2 Dry (Atrophic) Macular Degeneration, Lupus Nephritis, Heart Transplant Rejection P13500 SEQ ID NO: 71 of US11739142B2 SEQ ID NO: 75 US11739142B2 US 11739 142B2 CCL 3 Acute Febrile Neutrophilic Dermatosis P10147 SEQ ID NO: 51 of US9005617B2 SEQ ID NO: 56 of US9005617B2 US90056 17B2 CCL 4 Multiple Sclerosiss, Rheumatoid Arthritis, Inflammatory Bowel Disease P13236 SEQ ID NO: 21 of US9005617B2 SEQ ID NO: 22 of US9005617B2 US90056 17B2 CCL 5 Dermatitis, Asthma, Chronic Obstructive Pulmonary Disease P13501 SEQ ID NO: 51 of US9005617B2 SEQ ID NO: 56 of US9005617B2 US90056 17B2 (COPD); CCL 11 Allergic Conjunctiviti s, Bullous Pemphigoid, Non-Alcohol ic Steato-hepatitis (NASH), Asthma, Parkinson's Disease P51671 SEQ ID NO: 2 of US20210277103A1 SEQ ID NO: 4 of US20210277103A1 US20210 277103A 1 CCL 14 - QI 6627 SEQ ID NO: 21 of US9005617B2 SEQ ID NO: 22 of US9005617B2 US90056 17B2 CCL 15 - Q16663 SEQ ID NO: 51 of US9005617B2 SEQ ID NO: 56 of US9005617B2 US90056 17B2 CCL 18 - P55774 SEQ ID NO: 21 of US9005617B2 SEQ ID NO: 22 of US9005617B2 US90056 17B2 CCL 19 - Q99731 CN11011 7329A CCL 20 Multiple Sclerosis, Dermatitis (Eczema), Psoriatic Arthritis P78556 SEQ ID NO: 2 of JP6289520B2 SEQ ID NO: 8 of JP6289520B2 JP62895 20B2 CCL 21 - 000585 SEQ ID NO: 14 of WO2022047125A1 SEQ ID NO: 7 of WO2022047125A1 WO2022 047125A 1 CCL 23 - P55773 SEQ ID NO: 51 of US9005617B2 SEQ ID NO: 56 of US9005617B2 US90056 17B2 CCL 25 - 015444 US86583 77B2 CCL 27 - Q9Y4X3 US76018 15B2 cxc L8 Plaque Psoriasis (Psoriasis Vulgaris), Gastric Ulcers P10145 SEQ ID NO: 14 of US 10047156B2 SEQ ID NO: 13 of US 10047156B2 US 10047 156B2 CXC LIO Inflammator y Bowel Disease P02778 US79641 94B2 CXC L12 Diabetic Foot Ulcers, Pancreatic Islet Transplant Rejection P48061 SEQ ID NO: 7 of US10647766B2 SEQ ID NO: 9 of US10647766B2 US 10647 766B2 CXC L13 Rheumatoid Arthritis, Multiple Sclerosis, Sicca Syndrome (Sjogren) 043927 SEQ ID NO: 3 of US 20210087263A1 SEQ ID NO: 8 of US 20210087263A1 US 2021008 7263A1 Integ rin alph a-D (ITG AD) Q13349 EP13250 31A2 IL-1 7RB Atopic Dermatitis (Atopic Eczema), Asthma, Ulcerative Colitis Q9NRM6 SEQ ID NOG of US11505612B2 SEQ ID NO: 4 of US11505612B2 US 11505 612B2 IL-1 - Q14627 SEQ ID NO: 2 of SEQ ID NO: 1 of CN10144 3 RA 2 CN101440130A CN101440130A 0130A IL-2 2RA 2 Graft Versus Host Disease (GVHD), Ulcerative Colitis, Atopic Dermatitis, Multiple Sclerosis, Parkinson's Disease Q969J5 SEQ ID NO: 16 of CN102665759B SEQ ID NO: 17 of CN102665759B CN10266 5759B TGF BRI Degenerative Disc Disease, Crohn's Disease, Pulmonary Arterial Hypertension P36897 TGF-beta TGF BR2 Asthma, Idiopathic Pulmonary Fibrosis, Amyotrophic Lateral Sclerosis P37173 SEQ ID NO: 25 of US 8147834B2 SEQ ID NO: 27 of US 8147834B2 US 8147834 B2 TGF BR3 Post Menopausal Osteoporosis , Obesity Q03167 SEQ ID NO: 95 SEQ ID NO: 96 US 1124688 3B2 Eryt hroc yte Li Autoimmune hemolytic anemia, autoimmune lymphoprolif MGWSCIILFLVAT ATGVHSDIQMTQS PSVLSASVGDRVT LNCKASQNINKYL NWYQQKLGEAPK MGWSCIILFLVA TATGAHSEVKLQ ESGGGLVQPGGS LKLSCVASGFTFR DHWMNWVRQAP Sci Transl Med. (2019) ll(506):e erative syndrome VLIYNTNNLQTGIP SRFSGSGSGTDFTL TISSLQPEDFATYF CFQHYTWPTFGGG TKLEIKRADAAPT VSIFPPSSEQLTSGG ASVVCFLNNFYPK DINVKWKIDGSER QNGVLN SWTDQDSKDSTYS MSSTLTLTKDEYE RHNSYTCEATHKT STSPIVKSFNRNEC (SEQ ID NO: 262) GKTMEWIGDIRP DGSDTNYAPSVR NRFTISRDNARSI LYLQMSNMRSDY TATYYCVRDSPT RAGLMDAWGQG TSVTVSSAKTTAP SVYPLAPGSAAQ TNSMVTLGCLVK GYFPEPVTVTWN SGSLSSGVHTFPA VLQSDLYTLSSSV TVPSSTWPSETVT CNVAHPASSTKV DKKIVPRDCGCK PCICTVPEVSSVFI FPPKPKDVLTITL TPKVTCVVVDISK DDPEVQFSWFVD DVEVHTAQTQPR EEQFNSTFRSVSE LPIMHQDWLNGK EFKCRVNSAAFP APIEKTISKTKGR PKAPQVYTIPPPK EQMAKDKVSLTC MITDFFPEDITVE WQWNGQPAENY KNTQPIMDTDGS YFVYSKLNVQKS NWEAGNTFTCSV LHEGLHNHHTEK SLSHSPGK (SEQ ID NO: 263) aau8217 TLR 3 Inflammator y Bowel Disease, 015455 SEQ ID NO: 6 of US8153583B2 SEQ ID NO: 16 of US8153583B2 US81535 83B2 Chronic Obstructive Pulmonary Disease (COPD), Colitis, Rheumatoid Arthritis TLR 4 Rheumatoid Arthritis 000206 SEQ ID NO: 22 of US7312320B2 SEQ ID NO: 27 of US7312320B2 US73123 20B2 TLR 5 Rheumatoid Arthritis 060602 CBLB502 (Entolimod) Cable Gonzalez. , Com-municati ons biology (2023) 6(31) SEQ ID NO:44, 45, 46, 47, 48, 49, 50, or variants thereof, of US8703146B2 US87031 46B2 TLR 7 Systemic Lupus Erythematosus, Cutaneous Lupus Erythematosus Q9NYK1 SEQ ID NO: 5 of US20210040225A SEQ ID NO: 11 of US20210040225A US20210 040225A 1

[275]

[276] [FUSION MOLECULE OR BINDING MOLECULE]

[277] The induction of phagocytosis by the fusion molecule according to the present disclosure may not involve an inflammatory response. This enables clearance of the target substance without inducing an inflammatory response and tissue damage caused by an inflammatory response to be suppressed so that tissue dysfunction caused by an elevated amount or expression of the target substance can be treated more safely than conventional techniques.

[278] The first region and the second regions, described above, are coupled to each other directly or via a linker to form a fusion molecule.

[279] The fusion molecule may further comprise a tag. When such a label is added to the fusion molecule, it may be used to check the purification, expression, action or mechanism of action of the fusion molecule.

[280] Examples of the tag 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, T7genel0-tag, staphylococcal protein A-tag, streptococcal protein G-tag, glutathione-S-transferase (GST)-tag, dihydrofolate reductase (DHFR)-tag, cellulose binding domains (CBDs)-tag, maltose binding protein (MBP)-tag, galactose-binding protein-tag, calmodulin binding protein (CBP)-tag, hemagglutinin influenza virus (HAI)-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, and the like. The tag may be located at the N-terminus, C-terminus or internally of the target protein.

[281] The fusion molecule may further comprise a signal peptide or leader sequence at the N-terminus. It is known that a signal peptide is a short peptide present at the N-terminus at the initial stage of protein synthesis toward the secretory pathway, and directs the intracellular localization of the corresponding protein, membrane topology (in the case of a membrane protein), and the like. The signal peptide may be cleaved during expression and extracellular secretion of the fusion molecule.

[282] The above-mentioned first region, second region, tag, signal peptide, or regions having minimal functionality (e.g., LG1 and LG2 regions or scFv heavy chain variable region and light chain variable region) included in the fusion molecule may be linked together directly or by a linker comprising a short oligopeptide or polypeptide. In general, the linker may comprise 2 to 500 amino acid residues. The length or type of the linker is not particularly limited as long as the linker can link the above-described regions together so as to have the intended activity, thereby forming the fusion molecule. An example of the linker may be the commonly used oligopeptide linker (GGGGS)n (SEQ ID NO: 116), that is, a linker in which one or more Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 117) units are repeated. Other examples of the linker include, but are not limited to, (GSSGGS)n (SEQ ID NO: 118), KESGSVSSE-QLAQFRSLD (SEQ ID NO: 119), EGKSSGSGSESKST (SEQ ID NO: 120), GSAGSAAGSGEF (SEQ ID NO: 121), (EAAAK)n (SEQ ID NO: 122), CR-RRRRREAEAC (SEQ ID NO: 123), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 124), GGGGGGGG (SEQ ID NO: 125), GGGGGG (SEQ ID NO: 126), AEAAAKEAAAAKA (SEQ ID NO: 127), PAPAP (SEQ ID NO: 128), (Ala-Pro)n, VSQTSKLTRAETVFPDV (SEQ ID NO: 129), PLGLWA (SEQ ID NO: 130), TRHRQPRGWE (SEQ ID NO: 131), AGNRVRRSVG (SEQ ID NO: 132), RRRRRRRR (SEQ ID NO: 133), GFLG (SEQ ID NO: 134), and GSSGGSGSSG-GSGGGDEADGSRGSQKAGVDE (SEQ ID NO: 135. Other suitable linkers comprise the sequences described in WO2012 / 088461A, of which the content is incorporated by reference herein in its entirety.

[283] The fusion molecule according to embodiments of the present disclosure may further comprise a scaffold bound to the first region, to the second, or to both of the first region and the second region at different positions of the scaffold. The scaffold may include, but not limited thereto, a single chain Fc region with reduced or abolished Fc receptor binding affinity, a multimer Fc region with reduced or abolished Fc receptor binding affinity, an antibody without variable region, or an Fc - hinge region with reduced or abolished Fc receptor binding affinity. The first region may be linked or fused to one position of the scaffold and the second region may be linked or fused to another position of the scaffold. The link or fusion between the first / the second region and the scaffold may be a direct bond or via a linker described above.

[284] The fusion molecules according to aspects of the present disclosure may have a structure as schematically shown in non-limiting exemplary illustrations of, for example, FIGS. 23A to FIG. 23K.

[285] Another aspect of the present disclosure provides a nucleic acid molecule encoding the fusion molecule, and an expression vector containing the same.

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

[287] The nucleic acid molecule sequence encoding the fusion molecule may be isolated from nature or artificially produced through synthesis or genetic recombination. The nucleic acid molecule sequence encoding the fusion molecule is operatively linked to an expression vector capable of expressing the same.

[288] The term "expression vector" is a vector capable of expressing a protein or RNA of interest by introducing a nucleic acid sequence encoding a gene of interest into a suitable host cell, and refers to a gene construct containing essential regulatory elements operably linked to express the gene insert. Such expression vectors include all vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors.

[289] A suitable expression vector has expression control elements such as a promoter, a start codon, a stop codon, a polyadenylation signal and an enhancer. The start codon and the stop codon are generally considered to be part of a nucleic acid sequence encoding a protein, and the sequence encoding the protein is designed to be in frame so as to be operable in the vector. The promoter may be constitutive or inducible. In addition, a conventional expression vector contains a selectable marker. Operational linkage with the expression vector can be performed using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[290] The expression vector may preferably be configured to express the fusion molecule in a host cell for isolation and purification of the fusion molecule or such that the vector may be introduced into a cell in vivo and the corresponding cell may express and secrete the fusion molecule. For the purpose of introducing into cells in vivo, the vector may preferably be a non-integrating vector, that is, a vector that is not integrated into the genome of a host cell.

[291] Still another aspect of the present disclosure provides a cell expressing the fusion molecule.

[292] The cells may be transformed to contain the nucleic acid molecule or an expression vector containing the same, and the "transformation" may be performed using suitable standard techniques selected depending on the host cell as known in the art, including any method of introducing the nucleic acid molecule into an organism, cell, tissue, or organ. These 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-, dextran sulfate-, lipofectamine-, and desiccation / inhibition-mediated transformation methods.

[293] Examples of the host cells 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 camosus). Other examples of the host cell include fungal cells such as Aspergillus, yeast cells, including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, and Neurospora crassa, lower eukaryotic cells, or cells derived from higher eukaryotes including insect cells, plant cells, or mammalian cells. Examples of suitable animal cells include, for example, COS, CHO, or HEK293 cells. Examples of plant cells include tobacco, corn, soybean, and rice cells. By using methods known to those having ordinary skill in the art and based on the present disclosure, a nucleic acid vector may be designed for expressing foreign sequences in a particular host system, and then polynucleotide sequences encoding the fusion polypeptide may be inserted. The regulatory elements will vary according to the particular host.

[294] After the fusion molecule is expressed in the cells, it may be isolated and purified using conventional biochemical isolation techniques, such as treatment with a protein precipitating agent (salting out method), centrifugation, sonication, ultrafiltration, dialysis, or various chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, which are generally used in combination in order to isolate proteins with high purity (Sambrook et al., Molecular Cloning: A laboratory 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)).

[295] Thus obtained fusion protein can be assessed to determine whether the fusion protein substantially increases TAM receptor activity. The method can include contacting the cell with the test fusion protein and determining whether contacting the cell with the test fusion protein: alters TAM autophosphorylation, TLR-induced cytokine production, TLR-induced stimulation of MAP kinase activation, and / or TLR-induced NF-kB activation, as compared to a control. In this example, an increase in TAM autophosphorylation, or a decrease in TLR-induced cytokine production, TLR-induced stimulation of MAP kinase activation, or TLR-induced NF-kB activation in the presence of the test fusion protein relative to the control level indicates that the fusion protein stimulates TAM receptor activity.

[296] Autophosphorylation assays are well known in the art. In one example, cells expressing a TAM receptor are cultured and treated with test media, for instance, for 20 minutes at 37° C. Media is aspirated off and cold lysis buffer is added to each sample. The sample is centrifuged to spin down cell nuclei, and the supernatant is mixed with protein A agarose beads and affinity purified anti-TAM receptor antibody, then incubated. Protein A beads are pelleted and washed and separated on Tris-Glycine gels, and transferred (for Western blotting) onto a PVDF membrane (Millipore). The blot is probed with anti-phosphotyrosine as the primary antibody. A substantial decrease in phospho tyro sine labeling relative to control indicates that the test fusion protein is a TAM receptor inhibitor. A control can be a known value indicative of phosphotyrosine labeling in a sample, such as a cell, not treated with a test agent. For example, a substantial increase in phosphotyrosine labeling relative to control indicates that the test fusion protein is a TAM receptor agonist. For example, an increase in TAM phospho tyro sine by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or at least about 200% as compared to such a control indicates that the test fusion protein activates the TAM receptor.

[297] Cytokine assays are also well known in the art. For example, cytokine assays are manufactured by Assay Designs, Inc, Ann Arbor, Mich.; AssayGate, Inc., Ijamsville, Md.; and Panomics, Inc., Fremont, Calif. An increase in TLR-induced cytokine production in the presence of the test agent relative to the control level indicates that the test agent inhibits TAM receptor activity. A control level can be a reference value indicative of the amount of TLR-induced cytokine production in the absence of a test fusion protein or the amount of TLR-induced cytokine production in the absence of a test fusion protein. For example, a substantial decrease in TLR-induced cytokine production relative to control indicates that the test fusion protein is a TAM receptor agonist. For example, a decrease in TLR-induced cytokine production by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to such a control indicates that the test fusion protein activates the TAM receptor, and thus the test fusion protein activates a TAM receptor.

[298] MAP kinase activity can be determined by performing MAP kinase assays. A substantial increase in MAP kinase activation (such as indicated by an increase in phosphorylation of p38) in the presence of the test fusion protein relative to the control level of MAP kinase activity (such as basal levels of MAP kinase activity) indicates that the test fusion protein inhibits TAM receptor activity. For example, a substantial decrease in MAP kinase activity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to such a control indicates that the test fusion protein activates the TAM receptor and thus the test fusion protein activates a TAM receptor.

[299] Fusion proteins can be assessed by determining TLR-induced NF-kB activation. In this example, a substantial decrease in TLR-induced NF-kB activation relative to control indicates that the test agent is a TAM receptor agonist and thus the test fusion protein activates a TAM receptor. For example, a substantial decrease in TLR-induced NF-kB activation by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% as compared to such a control indicates that the test fusion protein activates the TAM receptor.

[300]

[301] [PHARMACEUTICAL COMPOSITION]

[302] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating a disease caused by an increased amount or an increased expression of the target substance in living tissue, the pharmaceutical composition containing the fusion molecule or the expression vector. Here, the composition may be administered topically to a site where the substance that causes the disease, that is, the amount or expression of the target substance has elevated.

[303] A further aspect of the present disclosure provides the use of the fusion molecule for manufacture of a medicament for preventing or treating an immunological disease or disorder.

[304] The fusion molecule, which is an active ingredient in the pharmaceutical composition, is contained in a "pharmaceutically effective amount".

[305] The pharmaceutical composition may be administered orally or parenterally, preferably parenterally. More preferably, it may be administered topically to a tissue in which the target substance to be cleared shows an increased / elevated level or an increased expression.

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

[307] When the pharmaceutical composition is prepared as an injectable formulation, it may be prepared as the injectable formulation a conventional method known in the art. The injectable formulation may be in a form dispersed in a sterile medium so that it may be administered directly to a patient or may be in a form that may be administered after being dispersed in distilled water for injection at an appropriate concentration.

[308] When the pharmaceutical composition is formulated for oral administration, it may contain one or more carriers selected from among diluents, lubricants, binders, disin-tegrants, sweeteners, stabilizers, and preservatives, and may contain one or more additives selected from among flavorings, vitamins, and antioxidants.

[309] Techniques necessary for formulation of the pharmaceutical composition, and pharmaceutically acceptable carriers, additives, etc. are widely known to those skilled in the art (see, for example, 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)).

[310] The appropriate dosage of the pharmaceutical composition may vary depending on factors such as formulation method, administration mode, patient's age, weight, sex, medical condition, diet, administration time, administration route, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition of the present disclosure is 0.0001 to 1,000 qg / kg body weight for an adult.

[311] Mode for the Invention

[312] Hereinafter, the present disclosure will be described in more detail with reference to examples and experimental examples. However, the following examples and experimental examples are illustrative only, and the scope of the invention is not limited thereto.

[313] Astrocytes and microglia are known to play an important role in the progression of multiple sclerosis (MS). These cells have been reported to express TAM receptors and exhibit phagocytosis and anti-inflammatory activity upon activation of TAM receptors. In Examples 1 and 2 below, it was attempted to determine the effect of gene knockout (KO) of the major TAM receptors in the two types of cells above on the progression of MS in EAE (experimental autoimmune encephalomyelitis) mice, which is one of the MS model animals. The EAE model is a disease model that is generated by inducing demyelination of myelin in the central nervous system by inducing the activation of T cells that recognize MOG (Myelin Oligodendrocyte Glycoprotein). Since the model has been reported in many clinical and histopathological applications to be similar to human multiple sclerosis, it is most commonly used as an animal model to study the mechanisms and therapeutic effects of multiple sclerosis.

[314]

[315] Example 1: Role of astrocytes in regulating neuroinflammation through Axl in CNS system

[316] ldhlll-CreERT2;Axl f / f mice were used to remove the astrocyte-specific Axl gene from adult mice. Axl f / f mice were used as controls. Tamoxifen (75 mg / kg) was intraperitoneally administered for five consecutive days to exhibit the activity of CreERT2 in 8-week old female mice. MOG35-55 contained in complete Freund's adjuvant (CFA) and pertussis toxin (PTX) were administered to induce experimental autoimmune encephalomyelitis (EAE) in 9-week old mice. The EAE score and body weight change were observed for 25 days after the EAE induction. Experimental autoimmune encephalomyelitis (EAE) is the most frequently used animal model to study the immunopathogenesis of a chronic inflammatory of the central nervous system (CNS) disease such as multiple sclerosis (MS) and to test the therapeutic efficacy of novel agents.

[317] To remove the Axl gene astrocyte-specifically and induce MS-like disease (EAE) in mice, the mice were manipulated according to the protocol shown in FIG. 1A. In more detail, Aldhlll-CreERT2;Axl f / f female mice including an Aidhill promoter-linked construct and Axl f / f were used to astrocyte-specifically express CreERT2, and Axl f / f female mice were used as controls.

[318] When the mice were 8 weeks old, CreERT2 was activated by intraperitoneal administration of tamoxifen for five consecutive days. Next, to induce EAE in 9-week old mice, MOG35-55 contained in complete Freund's adjuvant (CFA) was injected subcutaneously, and pertussis toxin (PTX) was administered intraperitoneally for two consecutive days. After that, the EAE score and body weight change were confirmed for 25 days.

[319] As a result, it was confirmed that the EAE score and body weight change were more severe in the mice in which the astrocyte-specific Axl gene was deleted than in the controls (FIGS. IB and IC). Through this, it was found that astrocytes play an important role in regulating EAE and that this is accomplished through Axl.

[320]

[321] Example 2: Role of microglia in regulating neuroinflammation through Mertk in CNS system

[322] Cx3crl-CreERT2;Mertk f / f mice were used to remove the microglia-specific Mertk gene from adult mice. Mertk f / f mice were used as controls. Tamoxifen (75 mg / kg) was intraperitoneally administered in five consecutive days to exhibit the activity of CreERT2 in 8-week old female mice. MOG35-55 contained in a complete Freund's adjuvant (CFA) and pertussis toxin (PTX) were administered to induce experimental autoimmune encephalomyelitis (EAE) in 9-week old mice. The EAE score and body weight change were observed for 25 days after the EAE induction. To remove the Mertk gene microglia-specifically and induce MS-like disease in mice, the mice were manipulated according to the protocol shown in FIG. 2A.

[323] In more detail, Cx3crl-CreERT2;Mertk f / f female mice including a Cx3crl promoter-linked construct and Mertk f / f were used to microglia-specifically express CreERT2; and Mertk f / f female mice were used as controls. When the mice were 8 weeks old, CreERT2 was activated by intraperitoneal administration of tamoxifen for five consecutive days. Next, to induce EAE in 9-week old mice, MOG35-55 contained in CFA was injected subcutaneously, and pertussis toxin (PTX) was administered intraperitoneally for two consecutive days. After that, the EAE score and body weight change were confirmed for 25 days. As a result, it was confirmed that the EAE score and body weight change were more severe in the mice in which the Mertk gene was deleted microglia-specifically than in the controls (FIGS. 2B and 2C). Through this, it was found that microglia play an important role in regulating EAE and that this is accomplished through Mertk.

[324]

[325] Example 3:Construction of Fusion protein: Anti-FITC-Gas6 fusion molecule and Anti-MOG(8-18C5)-Gas6 fusion molecule

[326] To effectively remove myelin debris by using the TAM receptor, AAV expressing a fusion molecule based on the human Gas6 protein was prepared.

[327] In more detail, the Gia domain and EGF repeat domain, which are the sites that recognize PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and the antibody single chain Fv fragment for MOG protein, which is highly expressed on myelin, was placed at the sites (anti-MOG(8-18C5)-Gas6). See FIGS. 3A to FIG. 3F.

[328] In addition, as a control, anti-FITC-Gas6 was prepared together by introducing, instead of an anti-MOG scFv, an anti-E2 scFv that selectively recognizes FITC, which is a substance that does not naturally exist in the body.

[329]

[330] Example 4: In vitro Effects of Anti-MOG(8-18C5)-Gas6 fusion molecule in removing myelin debris

[331] To express Anti-FITC-Gas6, Anti-MOG(8-18C5)-Gas6 in HEK293T cell line, transfection of an expression vector was performed. A serum-free media was substituted three days after the transfection, and the supernatant was collected and concentrated one day after later. Western blotting was performed to confirm the protein size and expression. HMC3 (human microglia cell line) was used to confirm the effect of the protein on the removal of myelin debris. To visualize the removal of myelin debris in vitro, myelin-pHrodo was prepared by extracting myelin from mouse brain and conjugating the same with pH-sensitive pHrodo. After treating the HMC3 cells with the concentrated supernatant and the myelin-pHrodo, live cell imaging was performed with IncuCyte.

[332] To confirm the effect of anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vitro, the protein was expressed in the HEK293T cells according to the protocol shown in FIG. 4A. In more detail, to express the protein, transfection of an expression vector was performed in the HEK293T cell line and the supernatant was concentrated. It was confirmed that the protein was expressed in a predicted size of 75-76 kDa (FIG. 4B). To confirm the effect on the removal of myelin debris in an in vitro system, the myelin-pHrodo was used in the HMC3 cell line. As a result, it was confirmed that when anti-MOG(8-18C5)-Gas6 is present in the HMC3 cell line, myelin debris was removed more rapidly, compared to the vehicle or Anti-FITC-Gas6 (FIG. 4C). Through this, it was found that anti-MOG(8-18C5)-Gas6 was expressed properly in a functional form and plays an important role in effectively removing myelin debris.

[333]

[334] Example 5: In vivo Effects of Anti-MOG(8-18C5)-Gas6 fusion molecule in mitigating EAE severity

[335] Experimental Protocol: AAV PHP.eB-CMV-anti-FITC-Gas6-HA, AAV PHP.eB-CMV-Anti-MOG(8-18C5)-Gas6-HA were administered to wild-type C57BL / 6J 6-week old female mice by retro-orbital injection at 1*10" vg. To induce EAE in 9-week old mice, MOG35-55 contained in complete Freund's adjuvant (CFA) and pertussis toxin (PTX) were administered. After the EAE induction, the EAE score and body weight change were observed for 25 days.

[336] To overexpress anti-MOG(8-18C5)-Gas6 and induce MS-like disease (EAE) in mice, the mice were manipulated according to the protocol shown in FIG. 5A. AAV PHP.eB-CMV-anti-FITC-Gas6-HA, AAV PHP.eB-CMV-Anti-MOG(8-18C5)-Gas6-HA were administered to wild-type C57BL / 6J 6-week old female mice by retro-orbital injection at 1*10" vg. Next, to induce EAE in 9-week old mice, MOG35-55 contained in CFA was injected subcutaneously, and pertussis toxin (PTX) was administered intraperitoneally for two con- 2023358200   23 Sep 2026 secutive days. After that, the EAE score and body weight change were confirmed for 25 days. As a result, it was confirmed that the EAE score was lower and the body weight change was less in the mice to which AAV expressing anti-MOG(8-18C5)-Gas6 were administered than in the controls (vehicle) or AAV expressing Anti-FITC-Gas6 (FIG. 5B and 5C). Through this, it was found that Anti-MOG(8-18C5)-Gas6 was expressed properly in a functional form in mice by AAV and plays an important role in mitigating the EAE severity.

[337]

[338] Example 6: Safety of fusion molecule in animals (systemic expression of fusion protein)

[339] Experimental protocol: AAV PHP.eB-CMV-anti-FITC-Gas6-HA, AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were administered to wild-type C57BL / 6J 6-week old female mice by retro-orbital injection at 1*1011 vg. Three weeks later, immunohistochemistry was performed by sampling the brain. See, FIG. 6A.

[340] To confirm the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin, wild-type mice were used and the mice were manipulated according to the protocol shown in FIG. 6A. In more details, AAV PHP.eB-CMV-anti-FITC-Gas6-HA, AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were administered to wild-type C57BL / 6J 6-week old female mice by retro-orbital injection at 1*1011 vg. Three weeks later, immunohistochemistry was performed by sampling the brain to confirm myelin level (MBP), lysosomal contents (Cathepsin D), and glial activation (GFAP, IBA1). As a result, it was confirmed that when anti-MOG(8-18C5)-Gas6 was systemically administered to the wild-type mice, there was no significance changes in myelin level, lysosomal contents, and glial activation compared to anti-FITC-Gas6 (FIG. 6B to 6E). Through this, it was found that there is no side effect in myelination and glial activation when Anti-MOG(8-18C5)-Gas6 was systemically expressed.

[341]

[342] Example 7: Safety of fusion molecule in animals (local expression of fusion protein)

[343] Experimental protocol: AAV PHP.eB-CMV-anti-FITC-Gas6-HA, AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were stereotaxically injected at the corpus callosum of wild-type C57BL / 6J 6-week old female mice at 1*1012 vg / mL (200 nL). Three weeks later, immunohistochemistry was performed by sampling the brain.

[344] To confirm the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin, wild-type mice were used and the mice were manipulated according to the protocol shown in FIG. 7A. In more detail, AAV PHP.eB-CMV-anti-FITC-Gas6-HA, AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were stereotaxically injected at the corpus callosum of wild-type C57BL / 6J 6-week old female mice at 1*1012 vg / mL (200 nL). Three weeks later, immunohistochemistry was performed by sampling the brain to confirm the degree of viral expression (HA), myelin level (MBP), and glial activation (GFAP, IBA1). As a result, it was confirmed that when Anti-MOG(8-18C5)-Gas6 was administered locally to the wild-type mice, the expression occurred at the injection region but not on the contralateral side (FIG. 7B). In addition, it was confirmed that there was no significant change in myelin level and glial activation compared to anti-FITC-Gas6 (FIG. 7C to FIG. 7E). Through this, it was found that there is no side effect in myelination and glial activation when Anti-MOG(8-18C5)-Gas6 was locally expressed.

[345]

[346] Example 8: Binding Activity of Fusion Molecule To Target Substance

[347] To effectively remove myelin fragments by using TAM receptors, a fusion molecule based on the human Gas6 protein was prepared. In more detail, the Gia domain and EGF repeat domain, which are the sites that recognize PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and the antibody single chain Fv fragment for MOG protein, which is highly expressed on myelin, was placed at the sites (Anti-MOG(01)-Gas6). See FIG. 8.

[348] ELISA was performed to evaluate the antigen-binding activity of the anti-MOG(01)-Gas6 prepared above. Human MOG protein (R&D Systems) or mouse MOG protein (R&D Systems) diluted in DPBS at a concentration of 0.5 pg / mL was added to a 96-well plate in an amount of 100 pL per well, incubated overnight at 4°C for coating, and washed with 0.05% Tween-20 / PBS (PBST) for four times. Then, 3% BSA / PBST was added in an amount of 200 pL per well, blocked, and washed with PBST for four times. anti-MOG(01)-Gas6 diluted by concentration were added in an amount of 100 pL per well and incubated for 2 hours at room temperature. The plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. After washing with PBST for four times, 100 pL of Peroxidase AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated for 1 hour at room temperature. After washing, 100 pL of TMB solution was added per well, and color was developed for 10 minutes. After stopping the reaction with a stop solution, the absorbance at 450 nm and 650 nm was analyzed with a spectrophotometer. As shown in FIGS. 9A and 9B, anti-MOG(01)-Gas6 exhibited binding activity to the MOG protein.

[349] To confirm the binding activity of anti-MOG(01)-Gas6 fusion molecule prepared above to the MOG expressed on the cell surface, the HEK293 cell line overexpressing mouse MOG (hereinafter referred to as HEK293-MOG cell line) was treated with the anti-MOG(01)-Gas6 fusion molecule, and then the anti-MOG(01)-Gas6 fusion molecule bound to the MOG on the cell surface was detected by using flow cytometry. 2023358200   23 Sep 2026 Briefly, the HEK293-MOG cell line resuspended in the FACS solution (DPBS + 3% FBS + 10 mM EDTA + 1X Pen / Strep + 20 mM HEPES) was incubated with the anti-MOG-Gas6 fusion molecule for 1 hour at 4°C. After that, to remove the anti-MOG(01)-Gas6 fusion molecule remaining in the supernatant without binding to the cell surface MOG, washing operation was performed twice, in which the FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. To detect the anti-MOG(01)-Gas6 fusion molecule bound to the cell surface MOG, G4S Linker (E7O2V) Rabbit mAb (Cell Signaling Technology) was diluted 1:50 with the FACS solution, and 100 pL was added to each well and incubated for 30 minutes at 4OC. After repeating the washing operation twice, the mean fluorescence intensity (MFI) was analyzed using flow cytometry. The obtained results are shown in FIG. 9C. As shown in FIG. 9C, it was confirmed that anti-MOG(01)-Gas6 was capable of binding to the mouse MOG protein expressed on the cell surface.

[350]

[351] Example 9: Effects of Fusion Protein (anti-MOG(01)-Gas6) Fusion Molecule in Removing Myelin Debris

[352] Anti-MOG(01)-Gas6 was used as purified proteins. To confirm the effect of the proteins on the removal of myelin debris, THP-1Axl (human monocyte cell line in which the Axl gene is overexpressed) was used. To visualize the removal of myelin debris in vitro, myelin-pHrodo was prepared by extracting myelin from mouse brain and conjugating the same with pH-sensitive pHrodo. After treating the THP-1 Axl with the proteins (5 pg mL) and the myelin-pHrodo, live cell imaging was performed with IncuCyte. See FIG. 10.

[353] The effect of anti-MOG(01)-Gas6 on the removal of myelin debris was confirmed in vitro. In more detail, the anti-MOG(01)-Gas6 was purified, and the myelin-pHrodo was used in the THP-1Axl cell line to confirm their effect on the removal of myelin debris in the in vitro system. As a result, it was confirmed that anti-MOG(01)-Gas6 removed myelin effectively in the THP-1Axl cell line compared to the vehicle (FIG. 10).

[354]

[355] Example 10: Binding of Fusion Protein (anti-MBP-Gas6) Fusion Molecule to Target Substance

[356] To effectively remove myelin fragments by using TAM receptors, a fusion molecule based on the human Gas6 protein was prepared. In more detail, the Gla domain and EGF repeat domain, which are the sites that recognize PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and the antibody single chain Fv fragment for MBP protein, which is highly expressed in myelin, was placed at the sites (anti-MBP-Gas6). See FIG. 11.

[357] ELISA was performed to evaluate the antigen-binding activity of the anti-MBP-Gas6 fusion molecule prepared in above. Human MBP protein (Enzo Life Sciences) and mouse MBP protein (Creative BioMart) diluted in DPBS at a concentration of 1 pg / mL was added to a 96-well plate in an amount of 100 pL per well, incubated overnight at 4°C for coating, and washed with 0.05% Tween-20 / PBS (PBST) for four times. Then, 3% BSA / PBST was added in an amount of 200 pL per well, blocked, and washed with PBST for four times. The anti-MBP-Gas6 fusion molecule diluted by concentration were added in an amount of 100 pL per well and incubated for 2 hours at room temperature. The plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. After washing with PBST for four times, 100 pL of Peroxidase AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated for 1 hour at room temperature. After washing, 100 pL of TMB solution was added per well, and color was developed for 10 minutes. After stopping the reaction with a stop solution, the absorbance at 450 nm and 650 nm was analyzed with a spectrophotometer.

[358] The obtained results are shown in FIG. 12A and FIG. 12B. The tested anti-MBP-Gas6 fusion molecule exhibited binding activity to the human and mouse MBP protein.

[359]

[360] Example 11: Effects of Fusion Protein (anti-MBP-Gas6)in Removing Myelin Debris

[361] Anti-MBP-Gas6 was used as purified proteins. To confirm the effect of the proteins on the removal of myelin debris, THP-lAxl (human monocyte cell line in which the Axl gene is overexpressed) was used. To visualize the removal of myelin debris in vitro, myelin-pHrodo was prepared by extracting myelin from mouse brain and conjugating the same with pH-sensitive pHrodo. After treating the THP-lAxl cells with the proteins (5 pg / mL) and the myelin-pHrodo, live cell imaging was performed with IncuCyte, and the MFI values were compared and evaluated after 20 hours.

[362] The effect of anti-MBP-Gas6 on the removal of myelin debris was confirmed in vitro . In more detail, the anti-MBP-Gas6 was purified, and the myelin-pHrodo was used in the THP-lAxl cell line to confirm their effect on the removal of myelin debris in vitro. As a result, it was confirmed that the tested anti-MBP-Gas6 fusion molecule effectively removed the myelin debris in monocyte cell line compared to the vehicle (FIG. 13).

[363]

[364] Example 12: Construction of Anti-TNFa(Adalimumab)-Gas6 Fusion Molecule and Anti-TNFa(Infliximab)-Gas6 Fusion Molecule

[365] To effectively inhibit or remove TNFa by using TAM receptors, a fusion molecule based on the human Gas6 protein was prepared. In more detail, the Gia domain and EGF repeat domain, which are the sites that recognize PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and the antibody single chain Fv fragment for TNFa, Adalimumab or Infliximab, was placed at the sites (Anti-TNFa-Gas6). See FIGS. 14A to 14C.

[366] ELISA was performed to evaluate the antigen-binding activity of the two types of anti-TNFa-Gas6 fusion molecule prepared above. Human TNFa protein (R&D Systems) diluted in DPBS at a concentration of 0.5 p,g / mL was added to a 96-well plate in an amount of 100 p.L per well, incubated overnight at 4°C for coating, and washed with 0.05% Tween-20 / PBS (PBST) for four times. Then, 3% BSA / PBST was added in an amount of 200 p.L per well, blocked, and washed with PBST for four times. The anti-TNFa-Gas6 fusion molecule diluted by concentration were added in an amount of 100 p.L per well and incubated for 2 hours at room temperature. The plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. After washing with PBST for four times, 100 p.L of Peroxidase AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated for 1 hour at room temperature. After washing, 100 p.L of TMB solution was added per well, and color was developed for 10 minutes. After stopping the reaction with a stop solution, the absorbance at 450 nm and 650 nm was analyzed with a spectrophotometer. The obtained results are shown in FIG. 15A. The two tested anti-TNFa-Gas6 fusion molecule exhibited binding activity to the human TNFa protein.

[367]

[368] Example 13: Binding Activity of Anti-TNFa(Adalimumab)-Gas6 Fusion Molecule and Anti-TNFa(Infliximab)-Gas6 Fusion Molecule to Target Substance TNFa

[369] To confirm the binding activity of the two types of anti-TNFa-Gas6 fusion molecules prepared in Example 12 above to the TNFa expressed on the cell surface, the CHO-K1 cell line overexpressing human cell membrane TNFa (hereinafter referred to as CHO-mTNFa cell line; Promega) was treated with the anti-TNFa-Gas6 fusion molecule, and then the anti-TNFa-Gas6 fusion molecule bound to the TNFa on the cell surface was detected by using flow cytometry. Briefly, the CHO-mTNFa cell line resuspended in the FACS solution (DPBS + 3% FBS + 10 mM EDTA + IX Pen / Strep + 20 mM HEPES) was incubated with the anti-TNFa-Gas6 fusion molecule for 1 hour at 4°C. After that, to remove the anti-TNFa-Gas6 fusion molecule remaining in the supernatant without binding to the cell surface TNFa, washing operation was performed twice, in which the FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. To detect the anti-TNFa-Gas6 fusion molecule bound to the cell surface TNF, G4S Linker (E7O2V) Rabbit mAb (Cell Signaling Technology) was diluted 1:50 with the FACS solution, and 100 pL was added to each well and incubated for 30 minutes at 4°C. After repeating the washing operation twice, the mean fluorescence intensity (MFI) was analyzed using flow cytometry.

[370] The obtained results are shown in FIG. 15B. It was confirmed that the two tested anti-TNFa-Gas6 fusion molecules were capable of binding to the human cell membrane TNFa expressed on the cell surface.

[371]

[372] Example 14: Activity of Anti-TNFa(Adalimumab)-Gas6 Fusion Molecule and Anti-TNFa(Infliximab)-Gas6 Fusion Molecule to Activate Axl

[373] To confirm the ability of the anti-TNFa-Gas6 fusion molecule candidate substances prepared in Example 12 above to inhibit signal activation by TNFa, a TNFa activation inhibition test was performed by using HEK-BLUE™ TNFa cells. Briefly, 20 pL of human TNFa protein and 20 pL of anti-TNFa-Gas6 fusion molecule diluted by concentration were added per well on a flat-bottom 96-well plate, and HEK-BLUE™ TNFa cells were dispensed to each well at a density of 5X104 / 160 pL / well. It was dispensed into each well according to the density of the well. After culturing in an 5% CO2 incubator at 37°C for 24 hours, 20 pL of the supernatant was transferred to a new flat-bottom 96-well plate, and 180 pL of QUANTLBLUE™ solution was added per well. After two hours, the absorbance at 655 nm was measured by using a spectrophotometer, and the inhibitory ability (%) was calculated based on the measured value under conditions in which only the human TNFa protein was treated. As shown in FIG. 16, it was confirmed that the two tested anti-TNFa-Gas6 fusion molecules are capable of inhibiting the signal activation by TNFa.

[374] To confirm the ability of the anti-TNFa-Gas6 fusion molecule prepared in Example 12 above to induce Axl activation, TAM receptor dimerization assay was performed by using human osteosarcoma cell line U2OSAxl (Eurofins DiscoverX) in which ProLink-Tagged Axl and Enzyme Acceptor (EA)-tagged SH2 domain were overexpressed. In the assay, the cell line reacts sensitively to Axl receptor activation by Gas6 by producing chemiluminescence, and mTNFa-expressing cells (Promega) are treated together with anti-TNFa-Gas6 fusion molecules to determine whether antigen (TNFa)-specific Axl activation is induced.

[375] As shown in FIG. 17, it was confirmed that Axl activation was induced only when mTNFa-expressing cells were treated together with anti-TNFa-Gas6 fusion molecules.

[376]

[377] Example 15: Activity of Anti-TNFa(Adalimumab)-Gas6 Fusion Molecule and 2023358200   23 Sep 2026 Anti-TNFa(Infliximab)-Gas6 Fusion Molecule to induce Axl-mediated Phagocytosis

[378] To confirm the Axl-mediated phagocytosis of the anti-TNFa-Gas6 fusion molecule candidate prepared in Example 12 above, a phagocytosis assay was performed using macrophages differentiated from THP-1Axl cells. THP-1Axl cells were treated with 25 nM of PMA (phorbol 12-myristate 13-acetate) for 72 hours, cultured in serum- and PMA-free medium for 24 hours, and then incubated with LPS (100 ng / mL) and IFN-y (10 ng / mL) for 24 hours to differentiate into macrophages. [378a]   mTNFa-expressing cells (Promega) stained using CTV (CellTrace Violet) were used as target cells, and effector cells and target cells were mixed in a 1:2 ratio and incubated together for 2 hours. After repeating the washing operation twice using FACS solution, the cell surface was stained with CD11b and the phagocytosis of CD11b+ macrophages toward CTV+ target cells was analyzed using flow cytometry.

[379] As shown in FIG. 18, it was confirmed that the anti-TNFa-Gas6 fusion molecule candidate induces phagocytosis on target cells mediated by the Axl receptor expressed on macrophages.

[380]

[381] Example 16: Construction of Anti-CD20(Rituximab)-Gas6 Fusion Molecule

[382] To effectively remove CD20-expressing immune cells by using TAM receptors, a fusion molecule based on the human Gas6 protein was prepared. See FIG. 19A and FIG. 19B. In more detail, the Gla domain and EGF repeat domain, which are the sites that recognize PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and the antibody single chain Fv fragment for CD20, Rituximab, was placed at the sites (Anti-CD20-Gas6).

[383] ELISA was performed to evaluate the antigen-binding activity of the anti-CD20-Gas6 fusion molecule prepared above. Human CD20 protein (Sino Biological) diluted in DPBS at a concentration of 0.5 pg / mL was added to a 96-well plate in an amount of 100 pL per well, incubated overnight at 4°C for coating, and washed with 0.05% Tween-20 / PBS (PBST) for four times. Then, 3% BSA / PBST was added in an amount of 200 pL per well, blocked, and washed with PBST for four times. The anti-CD20-Gas6 fusion molecule diluted by concentration were added in an amount of 100 pL per well and incubated for 2 hours at room temperature. The plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. After washing with PBST for four times, 100 pL of Peroxidase AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated for 1 hour at room temperature. After washing, 100 pL of TMB solution was added per well, and color was developed for 10 minutes. After stopping the reaction with a stop solution, the absorbance at 450 nm and 650 nm was analyzed with a spectrophotometer. The obtained results are shown in FIG. 20A. The anti-CD20-Gas6 fusion molecule exhibited binding activity to the human CD20 protein.

[384]

[385] Example 17: Binding Activity of Anti-CD20(Rituximab)-Gas6 Fusion Molecule to Target Substance

[386] To confirm the binding activity of anti-CD20-Gas6 fusion molecule prepared in Example 16 above to the human CD20 protein expressed on the cell surface, the Raji cell (ATCC) overexpressing human CD20 was treated with the anti-CD20-Gas6 fusion molecule, and then the anti-CD20-Gas6 fusion molecule bound to the CD20 on the cell surface was detected by using flow cytometry. Briefly, the Raji cell resuspended in the FACS solution (DPBS + 3% FBS + 10 mM EDTA + IX Pen / Strep + 20 mM HEPES) was incubated with the anti-CD20-Gas6 fusion molecule for 1 hour at 4°C. After that, to remove the anti-CD20-Gas6 fusion molecule remaining in the supernatant without binding to the cell surface CD20, washing operation was performed twice, in which the FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. To detect the anti-CD20-Gas6 fusion molecule bound to the cell surface CD20, G4S Linker (E7O2V) Rabbit mAb (Cell Signaling Technology) was diluted 1:50 with the FACS solution, and 100 p.L was added to each well and incubated for 30 minutes at 4°C. After repeating the washing operation twice, the mean fluorescence intensity (MFI) was analyzed using flow cytometry.

[387] The obtained results are shown in FIG. 20B. It was confirmed that the tested anti-CD20-Gas6 fusion molecule was capable of binding to the human CD20 protein expressed on the cell surface.

[388]

[389] Example 18: Activity of Anti-CD20(Rituximab)-Gas6 Fusion Molecule to Activate Axl

[390] To confirm the ability of the anti-CD20-Gas6 fusion molecule prepared in Example 16 above to induce Axl activation, TAM receptor dimerization assay was performed by using human osteosarcoma cell line U2OSAxl (Eurofins DiscoverX) in which ProLink-Tagged Axl and Enzyme Acceptor (EA)-tagged SH2 domain were overexpressed. In the assay, the cell line reacts sensitively to Axl receptor activation by Gas6 by producing chemiluminescence, and CD20-expressing Raji cell line (ATCC) are treated together with anti-CD20-Gas6 fusion molecules to determine whether antigen (CD20)-specific Axl activation is induced. As shown in FIG. 21, it was confirmed that Axl activation was induced only when CD20-expressing Raji cells were treated together with anti-CD20-Gas6 fusion molecules. 2023358200   23 Sep 2026

[391]

[392] Example 19: Activity of Anti-CD20(Rituximab)-Gas6 Fusion Molecule to Induce Axl-mediated Phagocytosis

[393] To confirm the Axl-mediated phagocytosis of the anti-CD20-Gas6 fusion molecule candidate prepared in Example 16 above, a phagocytosis assay was performed using macrophages differentiated from THP-1Axl cells. THP-1Axl cells were treated with 25 nM of PMA (phorbol 12-myristate 13-acetate) for 72 hours, cultured in serum- and PMA-free medium for 24 hours, and then incubated with LPS (100 ng / mL) and IFN-y (10 ng / mL) for 24 hours to differentiate into macrophages. Raji cells stained using CTV (CellTrace Violet) were used as target cells, and effector cells and target cells were mixed in a 1:2 ratio and incubated together for 2 hours. After repeating the washing operation twice using FACS solution, the cell surface was stained with CD11b and the phagocytosis of CD11b+ macrophages toward CTV+ target cells was analyzed using flow cytometry.

[394] As shown in FIG. 22, it was confirmed that the anti-CD20-Gas6 fusion molecule candidate induces phagocytosis on target cells mediated by the Axl receptor expressed on macrophages.

[395]

[396] Example 20: Adalimumab[scFv] - ProSl Fusion Molecule

[397] To prepare a TNFa-specific fusion protein based on ProS1 protein, the Gla domain and the EGF repeat domain are first removed, and a single-chain variable fragment (scFv) of adalimumab, a TNFa-specific antibody, is introduced at that position (aTNFa-ProS1). FIG. 24 shows the amino acid sequence of the chimeric phagocytosis inducer.

[398] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the resulting adalimumab [scFv] -ProS1 fusion molecule are evaluated.

[399]

[400] Example 21: Adalimumab[Fab]-Gas6 (anti-TNFa antibody heavy chain VH-CH1 (Fab)-Gas6-His)

[401] To prepare gas6 protein-based TNFa-specific fusion protein, the Gla domain and the EGF repeat domain are first removed, and an antigen-binding fragment (Fab) or monoclonal antibody (Mab) of the TNFa-specific antibody adalimumab is introduced at that position (aTNFa[Fab]-Gas6, and aTNFa[Mab]-Gas6). The Fc region of the heavy chain of the Mab contains NA mutation to reduce or abolish Fc gamma receptor binding affinity. FIG. 25 shows the amino acid sequence of Adalimumab[Fab]-Gas6 chimeric phagocytosis inducers.

[402] By following the procedure described in one or more of Examples 8-11, Examples 13-15, 2023358200   23 Sep 2026 or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[Fab]-Gas6 (anti-TNFa antibody heavy chain VH-CH1 (Fab)-Gas6-His) fusion molecule are evaluated.

[403]

[404] Example 22: Adalimumab[Mab]-Gas6 (anti-TNFa antibody heavy chain (Mab)-Gas6-His)

[405] To prepare gas6 protein-based TNFa-specific fusion protein, the Gla domain and the EGF repeat domain are first removed, and an antigen-binding fragment (Fab) or monoclonal antibody (Mab) of the TNFa-specific antibody adalimumab is introduced at that position (aTNFa[Fab]-Gas6, and aTNFa[Mab]-Gas6). FIG. 26 shows the amino acid sequence of Adalimumab[Mab]-Gas6 chimeric phagocytosis inducers.

[406] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[Mab]-Gas6 (anti-TNFa antibody heavy chain (Mab)-Gas6-His) fusion molecule are evaluated.

[407]

[408] Example 23: Adalimumab[Mab or Fab]-comprising fusion molecules

[409] By employing the sequence of SEQ ID NO: 253 (FIG. 27) and the sequence of SEQ ID NO: 251 (FIG. 25), the sequence of SEQ ID NO: 252 (FIG. 26), the sequence of SEQ ID NO: 258 (FIG. 32), or the sequence of SEQ ID NO: 260 (FIG. 34) / SEQ ID NO: 261 (FIG. 34) to prepare Adalimumab[Fab]-Gas6 fusion protein, Adalimumab[Mab]-Gas6 fusion protein, Adalimumab[Mab]-anti-Axl (homodimer) fusion protein, or Adalimumab[Mab] - anti-Axl (heterodimer) fusion proteins.

[410] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[Mab] or Adalimumab[Fab]-comprising fusion molecules are evaluated.

[411]

[412] Example 24: Adalimumab[scFv]-MFc-Gas6

[413] As non-limiting exemplary embodiment of the binding molecule containing a scaffold protein between the first region and the second region, wherein the Gas6 and antibody scFv (in this example, adalimumab scFv) are employed as the first region and the second region, respectively, a single chain Fc region with reduced or abolished Fc receptor binding affinity are manufactured. The sequences employed in the construction are shown in FIG. 28. 2023358200   23 Sep 2026

[414] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[scFv]-MFc-Gas6 fusion molecule are evaluated.

[415]

[416] Example 25: Adalimumab[scFv]-Fc(DD)-Gas6 (heterodimer)

[417] As another non-limiting exemplary embodiment of the binding molecule containing a scaffold protein between the first region and the second region, wherein the Gas6 and antibody scFv (in this example, adalimumab scFv) are employed as the first region and the second region, respectively, a heterodimeric binding molecule is manufactured. The first polypeptide of the heterodimeric binding molecule comprises adalimumab scFv, Fc region (DD), and Gas6, and the second polypeptide of the heterodimeric binding molecule comprises adalimumab scFv region and Fc region (KK). The peptide sequences are shown in FIGs. 29 and 30.

[418] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[scFv]-Fc(DD)-Gas6 heterodimer fusion molecule are evaluated.

[419]

[420] Example 26: Adalimumab[scFv]-Fc-Gas6 (homodimer)

[421] Still another non-limiting exemplary embodiment of the binding molecule containing a scaffold protein between the first region and the second region, a homodimer comprising two polypeptides which each comprise adalimumab scFv (as second region), Fc region (scaffold), and Gas6 is manufactured. The peptide sequences are shown in FIG.31.

[422] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[scFv]-Fc-Gas6 homodimer fusion molecule are evaluated.

[423]

[424]

[425]

[426]

[427]

[428] Example 28: Adalimumab[Mab]-anti-Axl (homodimer)

[429] As non-limiting exemplary embodiment of the binding molecule containing a scaffold protein between the first region and the second region, a bispecific antibody, wherein a scFv of 2023358200   23 Sep 2026 anti-Axl antibody and adalimumab are employed as the first region and the second region, respectively, is manufactured. See FIG. 32. A heavy chain of the bispecific antibody has the following sequence of SEQ ID NO: 258 (FIG. 32) and the light chain of adalimumab has the sequence of SEQ ID NO: 253. The Fc region of the heavy chain contains NA mutation to reduce or abolish Fc gamma receptor binding affinity.

[430] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[Mab]-anti-Axl homodimer fusion molecule are evaluated.

[431]

[432] Example 29: Anti-Axl-Fc-adalimumab[scFv] (homodimer)

[433] As non-limiting exemplary embodiment of the binding molecule containing a scaffold protein between the first region and the second region, a homodimeric bispecific antibody, wherein a scFv of anti-Axl antibody and a scFv region of an adalimumab are employed as the first region and the second region, respectively, is manufactured. See FIG. 33. The bispecific antibody comprises a first polypeptide and a second polypeptide, which are identical to each other and each comprises the sequence of SEQ ID NO: 259. The structure of the first / second polypeptide is illustrated in FIG. 33, and the Fc region scaffold contains NA mutation to reduce or abolish Fc gamma receptor binding affinity.

[434] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained anti-Axl-Fc-Adalimumab[scFv] homodimer fusion molecule are evaluated.

[435]

[436] Example 30: Adalimumab[Mab](DD) - anti-Axl (heterodimer)

[437] As another non-limiting exemplary embodiment of the binding molecule containing a scaffold protein between the first region and the second region, a heterodimeric bispecific antibody, wherein a scFv of anti-Axl antibody and adalimumab are employed as the first region and the second region, respectively, is manufactured. The first polypeptide of a heavy chain of the bispecific antibody has the following sequence of SEQ ID NO: 260 of FIG. 34, the second polypeptide of a heavy chain of the bispecific antibody comprises the sequence of SEQ ID NO: 261 of FIG. 34, and adalimumab light chain has the sequence of SEQ ID NO: 253 of FIG. 27. The Fc region contains NA mutation to reduce or abolish Fc gamma receptor binding affinity and the polypeptides of the Fc region form a hetero dimer (DD-KK). 2023358200   23 Sep 2026

[438] By following the procedure described in one or more of Examples 8-11, Examples 13-15, or Examples 17-19, the binding activity to target substance, induction of TAM activation and phagocytosis of the obtained Adalimumab[Mab](DD) - anti-Axl heterodimer fusion molecule are evaluated.

[439]

[440] The scope of the present disclosure is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and equivalents thereto should be construed as being included in the scope of the present invention.

[441] The fusion molecules having phagocytosis-inducing activity according to the embodiment of the present disclosure can solve the problem of tissue damage caused by activation of an inflammatory response, which occurs in the prior art. Accordingly, the fusion molecules could effectively clear or reduce the amount of antigenic substance, and thus may be used to prevent or treat immunological diseases.

[442]

[443] Incorporation by Reference

[444] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated herein by reference in their entireties.

Claims

1. A binding molecule comprising a first region capable of binding to a TAM receptorand a second region capable of specifically binding to a target substance, said target substance being a substance of which an elevated amount or an elevated expression in a living tissue induces or causes an immunological disease,wherein the first region and the second region are coupled to each other directly or via a linker, wherein the first region comprises(a)     a TAM receptor ligand;(b)     an anti-Axl antibody or an antigen-binding fragment thereof;(c)     an anti-Tyro3 antibody or an antigen-binding fragment thereof;(d)     an anti-MerTK antibody or an antigen-binding fragment thereof; or(e)     combinations thereof,wherein the binding molecule does not comprise the target substance or a fragment thereof, and wherein the target substance is not a substance selected from the group consisting of Amyloid precursor protein-derived P-amyloid, a-Synuclein (AaSyn), PrPSc, Microtubule-associated protein tau, Huntingtin exon 1, TAR DNA-binding protein 43 (TDP43), Superoxide dismutase 1 (SOD1), ABri peptide, ADan peptide, Immunoglobulin light-chain fragment, Immunoglobulin heavy-chain fragment, N-terminal fragment of serum amyloid A protein, Transthyretin, P-2 microglobulin, N-terminal fragment of apolipoprotein AI, C-terminally extended apolipoprotein AII, N-terminal fragment of apolipoprotein AIV, apolipoprotein C-II, apolipoprotein C-III, Gelsolin fragment, Lysozyme, Fibrinogen alpha chain fragment, N-terminally truncated cystatin C, Amylin, IAPP, Calcitonin, Atrial natriuretic factor, Prolactin, Lactadherin or medin, ODAM (odontogenic ameloblast-associated protein), SP-C (pulmonary surfactant-associated protein C), LECT-2(Leukocyte cell-derived chemotaxin-2), Galectin-7, Corneodesmosin, C-terminal fragment of TGFBI (or keratoepithelin), SGI (Semenogelin-1), S100 protein (A8 or A9) and Enfuvirtide.

2. The binding molecule according to claim 1, further comprising a scaffold bound to thefirst region, to the second, or to both of the first region and the second region at different positions.2023358200   23 Sep 20263.      The binding molecule according to claim 1, wherein the TAM receptor ligand is oneselected from the group consisting of Gas6, ProS1, Tubby, Tulp1, Gal3, and combinations thereof, or an Axl-binding fragment thereof.

4. The binding molecule according to claim 1, wherein the first region is (a) the TAMreceptor ligand and wherein the TAM receptor ligand comprises a sequence selected from the group consisting of SEQ ID NOs: 1-113 or a sequence having at least 85% of sequence identity thereto.

5. The binding molecule according to claim 1, wherein the first region is (a) the TAMreceptor ligand and wherein the TAM receptor ligand comprises one or more sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ IDNO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ IDNO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 64,SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ IDNO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75,SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86,and SEQ ID NO: 87, or a sequence having at least 85% of sequence identity thereto.

6. The binding molecule according to claim 1, wherein the first region is (a) the TAMreceptor ligand and wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 1 or a sequence having at least 85% of sequence identity thereto and the sequence of SEQ ID NO: 2 or a sequence having at least 85% of sequence identity thereto.

7. The binding molecule according to claim 1, wherein the first region is (a) the TAMreceptor ligand and wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 5 or a sequence having at least 85% of sequence identity thereto.2023358200   23 Sep 20268.      The binding molecule according to claim 1, wherein the first region is (a) the TAMreceptor ligand and wherein the TAM receptor ligand comprises one or more sequences selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40,SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ IDNO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51,SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62,SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ IDNO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113, or a sequence having at least 85% of sequence identity thereto.

9. The binding molecule according to claim 1, wherein the first region is (a) the TAMreceptor ligand and wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 3 or a sequence having at least 85% of sequence identity thereto and the sequence of SEQ ID NO: 4 or a sequence having at least 85% of sequence identity thereto.

10. The binding molecule according to claim 1, wherein the first region is (a) the TAMreceptor ligand and wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 6 or a sequence having at least 85% of sequence identity thereto.

11. The binding molecule according to claim 1, which forms a homodimer, a heterodimer,or a multimer in a single chain.

12. The binding molecule according to claim 1, wherein the target substance is an autoantigen, auto-antibody, a complex of auto-antigen and auto-antibody, a cytokine, a chemokine, a complement, a receptor, an immune cell specific marker, a cell adhesion molecule, or a combination thereof.2023358200   23 Sep 202613.     The binding molecule according to claim 1, wherein the target substance is one or moreselected from the group consisting of Factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, thrombin, vWF, Calcium-sensing receptor, ACTH, 21-Hydroxylase (CYP21),      trichohyalin, oxidized low density lipoprotein (OxLDL),transcription coactivator p75, p-80-Coilin, C1 inhibitor,   AMPA-receptor,   CRMP5,DPPX / DPP6, GABAA-receptor, glycine receptor (GlyR), Hu (ANNA-1), Ma1, Ma2, Ri (ANNA-2), Zic4, voltage-gated potassium channel (VGKC)-complex, NMDA-receptor, Jo1, H / K ATPase, thyroid peroxidase, erythrocyte I / I, F-actin asialoglycoprotein receptor, cytochrome P450 2D6 (CYP2D6), NXP-2 / MORC3, TIF1-y / TRIM-33, p2 integrin, nuclear autoantigenic sperm protein (NASP), lactoferrin 17-a-Hydroxylase (CYP17), cholesterol sidechain cleavage enzyme (CYP11A), tryptophan hydroxylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, glycoprotein IIb / IIIa and Ib / IX, thyroglobulin, hemidesmosomal protein 180, p53, recoverin, actin, IgE receptor, myelin-associated glycoprotein (MAG), tubulin, laminin-332, tissue transglutaminase, desmin, bactericidal / permeability-increasing protein (BPI), transglutaminase, melanoma differentiation-associated gene 5 (MDA5), SUMOactivating enzyme subunit (SAE)-1 (SAE-1), SAE-2, DNA-dependent nucleosome-stimulated ATPase, chromodomain-helicase-DNA-binding protein 4 (CHD4), P-adrenoreceptor, adenine nucleotide translocator, collagen type VII, IgG, G-CSF, collagen type IV a3-chain, thyrotropin receptor (TSHR), sodium iodide symporter (NIS), peripheral myelin protein 22 (PMP22), GM gangliosides, S-antigen, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), signal recognition particle 54kDa subunit (SRP54), IgA, synaptotagmin, voltage-gated calcium channels, pIV spectrin, U1 small nuclear ribonucleoprotein 70 kDa (SNRNP70), CNPase, myelin-associated oligodendrocyte basic protein (MOBP), myelin proteolipid protein (PLP), S100 calcium binding protein B, transaldolase, myelin basic protein (MBP), myelinoligodendrocyte glycoprotein (MOG), acetylcholine receptor, low-density lipoprotein receptor-related protein 4 (LRP4), muscle skeletal receptor tyrosine-protein kinase (MuSK), aminoacyl-tRNA synthetase, Tribbles Pseudokinase 2 (TRIB2), myeloperoxidase (MPO), aquaporin 4 (APQ-4), amphiphysin, Exosome component 9 (EXOSC9), EXOSC10 / PMSCL, Yo protein, Hu protein, Ri protein, desmoplakin, gephyrin, desmoglein 1, desmoglein 3, intrinsic factor type 1, p2-glycoprotein I (p2-GPI), pyruvate dehydrogenase complex-E2 (PDC-E2), aggrecan G1, carbamylated antigens, cartilage glycoprotein-39, Fc part of immunoglobulins, glucose-6-phosphate isomerase, keratin, protein-arginine deiminase type-4, collagen (multiple types, especially II, IV and IX), fibrinogen Pa,lLeukemia inhibitory factor2023358200   23 Sep 2026(LIF), glutamate receptor (GLUR), myosin, B23, nucleophosmin (NPM), fibrillarin, topoisomerase-I (Scl-70), interferon—Y—inducible protein 16 (IFI16), La phosphoprotein, Ro60, Ro52 (TRIM21), golgin (95, 97, 160, 180), anionic phospholipid / protein complex, cardiolipin, components of the Sm splicing ribonucleoprotein (subunits A-G), autologous double-stranded DNA (dsDNA), histone H2A-H2B-DNA, proliferating cell nuclear antigen (PCNA), ribosomal P, Sjogren Syndrome (SSA), Smith, U1-RNP, U2 snRNP B, vimentin, C1q, fibronectin, Ku-DNA-protein kinase, Carbonic anhydrase II, neuronal nicotinic acetylcholine receptor, centromere-associated protein, RNA polymerase I-III (RNP), thyroid and eye muscle shared protein, leukocyte function-associated antigen (LFA-1), chromogranin A, IA-2 (ICA512), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), ZnT8, insulin, glutamate decarboxylase (GAD65), insulin receptor, heat shock protein (65-kDa heat shock protein), SOX-10, tyrosinase, KUMEL1 / ARMC9, proteinase 3 / myeloblastin, CD20, CD19, Complement C3, Complement C5, C5 alpha receptor 1, CD52, FcRn Large Subunit p51, IL-1, IL-1R, IL-6, IL-6R, IL-17, IL-17R, TNF-alpha, TNFR, IL-4, IL-4R, IL-5, IL-5R, IL-13, IL-13R, IFN-gamma, IFN-gamma receptor, IL-12, IL-12R, IL-21, IL-21R, IL-22, IL-22R, TGF-beta, TGF-beta receptor, CD80 / 86, CD28, IL-23, IL-23R, thymic stromal lymphoPoietin (TSLP), TSLPR, IL-31, IL-31R, OX40, OX40L, IL-33, IL-33R, CD40, CD40L, IGF-1R, ICAM1, VCAM1, MADCAM1, Integrin alpha 4, Integrin beta 7, VLA-4, toll-like receptor (TLR)-3, TLR-4, TLR-5, TLR-7, and a combination thereof.

14. The binding molecule according to claim 1, wherein the second region that specificallybinds to the target substance is selected from the group consisting of an antibody or an antigenbinding fragment thereof, an antibody-like protein, a peptide, an aptamer, and a soluble receptor, which each specifically bind to the target substance.

15. The binding molecule according to claim 2, wherein the scaffold is a single chain Fcregion with reduced or abolished Fc receptor binding affinity, a multimer Fc region with reduced or abolished Fc receptor binding affinity, an antibody without variable region, or an Fc - hinge region with reduced or abolished Fc receptor binding affinity.

16. The binding molecule according to claim 1, wherein the immunological disease isinflammatory disease or autoimmune disease is selected from the group consisting ofmultiple sclerosis, myasthenia gravis, diabetes mellitus type 1, diabetes mellitus type 2, rheumatoid2023358200   23 Sep 2026arthritis, neuromyelitis optica, autoimmune encephalitis, fatty liver disease, endometriosis, inflammatory bowel disease, asthma, obesity, ankylosing spondylitis, antiphospholipid antibody syndrome, chronic recurrent multifocal osteomyelitis, gout, henoch-schonlein purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus (sle), juvenile scleroderma, juvenile vasculitis, kawasaki disease, lupus (systemic lupus erythematosus), mixed connective tissue disease, myositis, poststreptococcal inflammatory syndromes, psoriatic arthritis, reactive arthritis, scleroderma, sjogren's syndrome, spondyloarthritis / spondyloarthropathy, systemic juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, celiac disease, thrombotic thrombocytopenic purpura (iTTP), and combinations thereof.

17. A nucleic acid molecule encoding the binding molecule according to claim 1.

18. An expression vector comprising the nucleic acid molecule according to claim 17.

19. A cell expressing the binding molecule according to claim 1.

20. A pharmaceutical composition comprising (i) the binding molecule of claim 1, (ii) apolynucleotide encoding the binding molecule, (iii) an expression vector carrying the polynucleotide, or a combination thereof, as an active ingredient, and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, which is for(i) reducing or enhancing a reduction of an elevated level of the target substance that causes or induces an immunological disease in a subject to a normal level,(ii) removing or clearing or enhancing clearance of a target substance with an elevated expression or increased amount, which elevated expression or increased amount causes or induces an immunological disease in a subject,(iii) suppressing an elevation of expression or amount of a target substance in a subject, (iv) treating or preventing an immunological disease in subject,(v) delaying development of a symptom associated with an immunological disease, and / or(vi) alleviating symptoms of an immunological disease in a subject.2023358200   23 Sep 202622.     Use according to claim 21, wherein the elevated level of the target substance occurs inbrain of the subject.

23. Use of the binding molecule of claim 1, a polynucleotide encoding the bindingmolecule, a vector, or a pharmaceutical composition comprising the binding molecule, the polynucleotide, or the vector, in one or more of the method selected from the group consisting of:reducing or enhancing a reduction of an elevated level of the target substance that causes or induces an immunological disease in a subject to a normal level,removing or clearing or enhancing clearance of a target substance with an elevated expression or increased amount, which elevated expression or increased amount causes or induces an immunological disease in a subject, andsuppressing an elevation of expression or amount of a target substance in a subject.

24. Use according to claim 23, wherein the elevated level of the target substance occurs inbrain of the subject.

25. Use of the binding molecule of claim 1, a polynucleotide encoding the bindingmolecule, a vector, or a pharmaceutical composition comprising the binding molecule, the polynucleotide, or the vector, in one or more of the method selected from the group consisting of:treating or preventing an immunological disease in subject,delaying development of a symptom associated with an immunological disease, and alleviating symptoms of an immunological disease in a subject.

26. Use of the binding molecule of claim 1, a polynucleotide encoding the bindingmolecule, a vector, or a pharmaceutical composition comprising the binding molecule, the polynucleotide, or the vector, in manufacturing a medicament to treat or prevent an immunological disease in subject, to delay development of a symptom associated with an immunological disease, or alleviate symptoms of an immunological disease in a subject.

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