TW202417520A - Fusion molecule and method for treating immunological diseases - Google Patents

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

Fusion molecules and methods for treating immune diseases

The present disclosure relates to a fusion molecule suitable for preventing or treating an immune disease. The present disclosure also relates to a nucleic acid molecule encoding the fusion molecule. The present disclosure further relates to a composition comprising the fusion molecule, a method for preventing or treating an immune disease, and the use of the fusion molecule in treating an immune disease.

Immune diseases are diseases that are caused, mediated or otherwise contribute to pathological conditions in mammals by components of the mammalian immune system. In particular, inflammatory diseases are of worldwide concern and for which treatments are urgently needed. Inflammation is usually a local protective response of body tissues to 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 radiation, chemical substances such as toxins, drugs or industrial agents, immune responses such as allergies and autoimmune reactions, or conditions associated with oxidative stress.

Under normal circumstances, the inflammatory response clears external infectious agents and regenerates damaged tissues to restore the functions of living organisms. However, if the inflammatory response is excessive or persistent when antigens are not cleared or internal substances are the cause, it may cause acute inflammation, becoming a disease threatening human life, intra-articular diseases such as rheumatoid arthritis, skin diseases such as psoriasis, allergic inflammatory diseases such as bronchial asthma, and autoimmune diseases caused by the immune system attacking self-antigens, and it may become an obstacle to treatment procedures such as blood transfusion, drug administration and organ transplantation.

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 drugs are known to cause symptoms such as edema, gastrointestinal diseases, bleeding and liver toxicity as side effects. In addition, in some cases, they cannot selectively act on the cause of inflammation, resulting in severe immunosuppression (Check and Kaliner, Am. Rev. Respir. Dis., 141, pp. 44-51, 1990). In addition, since there is currently no therapeutic agent that can completely cure these diseases, an effective therapeutic agent without side effects is needed.

Meanwhile, TAM (Tyro3, Axl and MerTK) receptors are receptor tyrosine kinases, and it has recently been noted that TAM ligands that can activate these receptors play an important role in controlling tissue homeostasis and inflammation. TAM receptors are known to be particularly involved in anti-inflammatory effects and resolution of inflammation, wherein anti-inflammatory effects refer to the reduction and elimination of the activity of pro-inflammatory mediators, which can be achieved by inhibiting the synthesis of these mediators, selective antagonism, clearance, post-translational modifications such as cleavage, or decomposition. Resolution of inflammation can be achieved by methods such as removing the stimulus that causes inflammation, promoting the removal of pathogenic cells by apoptosis or phagocytosis, enhancing the induction of non-inflammatory macrophages, promoting macrophage reprogramming, and secreting inflammation-inhibiting substances (e.g., IL-10, etc.).

With the reports of these characteristics, various attempts have been made to treat inflammatory or autoimmune diseases by administering TAM ligands Gas6 or ProS1, and reports have actually shown their effectiveness in reducing the secretion of proinflammatory cytokines and alleviating some 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, TAM ligands consist only of a region that binds to TAM receptors and a region that binds to PS (phosphatidylserine), which are regions that have activity related to binding to other molecules. Therefore, it is difficult to selectively act on substances that cause inflammation.

Therefore, there is a need for improved methods that can effectively induce selective depletion of antigens.

The present disclosure relates to fusion molecules that are capable of inducing the selective elimination of target substances that trigger or induce undesirable or pathological immune responses, such as autoimmune diseases, transplant rejection, or allergic or hyperimmune responses.

One aspect of the present disclosure provides a fusion molecule comprising: a first region capable of binding to a TAM (Tyro3, Axl, and MerTK) receptor; and a second region specifically binding to a target substance to be removed or reduced, and the fusion molecule does not induce an inflammatory response, wherein the level of the targeted inflammation-related substance increases or rises, or the expression of the targeted inflammation-related substance increases or rises, triggering or inducing an undesirable or pathological immune response, such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response. In one embodiment, the fusion molecule does not have an effector function and does not induce an Fc-mediated inflammatory response.

In some embodiments, the TAM receptor may be any one selected from Tyro3, Axl, MerTK or a combination thereof, which can induce phagocytosis by binding to the laminin G-like domain (or LG domain) of phagocytic cells, including but not limited to macrophages or microglia. In embodiments, the TAM receptor may be the Axl region of the TAM receptor.

In embodiments, the first region may comprise Gas6, ProS1, Tubby, Tulp1, Gal3 or active fragments thereof, each of which is capable of specifically binding to a TAM receptor. The first region may be selected from Gas6, ProS1 or active fragments thereof, each of which is capable of specifically binding to a TAM receptor. In embodiments, the first region may comprise or consist essentially of Gas6 or its active fragment capable of binding to a TAM receptor. In embodiments, the first region comprising or consisting essentially of Gas6 or its active fragment capable of binding to an Axl receptor.

In a specific embodiment, the first region may include a laminin G-like domain of Gas6 or ProS1 or an active fragment thereof, which includes a laminin G-like domain as a bridge molecule associated with phagocytosis, which is expressed in large quantities in various tissues and is therefore capable of inducing phagocytosis through TAM receptors. In an embodiment, the laminin G-like domain may include a LG1 domain, a LG2 domain, or a combination thereof, and may preferably include both a LG1 domain and a LG2 domain, which is capable of inducing phagocytosis by binding to the TAM receptor.

Exemplary embodiments relate to a binding molecule or a fusion molecule comprising a first region capable of binding to a TAM receptor and a second region capable of specifically binding to a targeted inflammation-related substance, wherein the targeted inflammation-related substance is a substance with an increased or elevated amount or with an increased or elevated expression, which triggers, induces or causes an undesirable or pathological immune response, such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response, wherein the first region and the second region are coupled to each other directly or by 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; or (d) an anti-MerTK antibody or an antigen-binding fragment thereof, provided that when the first region comprises an anti-MerTK antibody or an antigen-binding fragment thereof, the molecule is not a bispecific antibody; or (e) a combination thereof.

According to some embodiments, the binding molecule may further comprise a scaffold that binds to the first region, the second region, or both the first region and the second region at different positions.

In an embodiment, the first region is a TAM receptor ligand, and the TAM receptor ligand comprises a sequence selected from SEQ ID NO: 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 97%, at least 98% or at least 99% sequence identity thereto.

In some further embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to an Axl receptor comprises a sequence selected from 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 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 97%, at least 98%, or at least 99% sequence identity thereto.

In some other embodiments, the first region is capable of binding to the Axl receptor, and the first region capable of binding to the Axl receptor comprises: a 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 97%, at least 98% or at least 99% sequence identity thereto; and/or a 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 97%, at least 98% or at least 99% sequence identity thereto.

In another embodiment, the first region is capable of binding to the Axl receptor, and the first region capable of binding to the 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 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

In some further embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to an Axl receptor comprises a region selected from 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: : 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, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123. 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 97%, at least 98%, or at least 99% sequence identity thereto.

In some other embodiments, the first region is capable of binding to the Axl receptor, and the first region capable of binding to the Axl receptor comprises: a 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 97%, at least 98% or at least 99% sequence identity thereto; and/or a 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 97%, at least 98% or at least 99% sequence identity thereto.

In another embodiment, the first region is capable of binding to the Axl receptor, and the first region capable of binding to the 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 97%, at least 98% or at least 99% sequence identity thereto.

In an embodiment, a fusion protein according to the present disclosure does not comprise a target substance that is eliminated or reduced by administering the fusion protein.

In an embodiment, the first region of the laminin G-like domain or its active fragment comprising Gas6 or ProS1 does not comprise a Gla domain. Without being bound by a particular theory, it is expected that the lack of a Gla domain in the first region will render the fusion molecule unable to recognize phosphatidylserine (PS) of the TAM receptor, while the second region is able to induce phagocytosis by recognizing the target substance.

In some embodiments, the first region of the laminin G-like domain or its active fragment containing Gas6 or ProS1 does not contain a Gla domain or an EGF domain. The lack of an EGF domain in the first region provides an advantage in the preparation of the fusion molecule, increasing the yield by inhibiting the aggregation of the fusion molecule during the purification step. In some embodiments, the fusion molecule (or binding molecule) can form a homodimer or a heterodimer, or form a linear multimer as a single chain.

According to the embodiment, the target substance to be eliminated or reduced and specifically bound to the second region can be a substance whose increased or elevated amount or increased or elevated expression triggers, induces or causes an undesirable or pathological immune response, such as autoimmune disease, transplant rejection, or allergic or hyperimmune response.

The target substance may be an inflammation-related substance. The target substance may be one or more selected from the following substances: autoantigens, their autoantibodies, or complexes of autoantigens and their autoantibodies in Table 1 below; immune cell surface molecules, including co-stimulatory molecules and receptors such as CD20, CD19, CD52, CD80/86, CD28, CD40, CD40L, OX40, OX40L, C5α receptor 1, IL-1R, IL-6R, IL-17R, IL-4R, IL-5R, IL-13R, IFN-γ receptor, IL-12R, IL-21R, IL-22R, TGF-β 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 receptor (TLR), including TLR 3, TLR4, TLR5 and TLR7, etc.; complements, such as complement C1q, complement C3, complement C5, etc.; chemokines, such as CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10, etc.; cytokines, such as IL-1, TNF-α, I L-6, IL-17, IL-4, IL-5, IL-13, IFN-γ, IL-12, IL-21, IL-22, TGF-β, IL-23, thymic stromal lymphopoietin (TSLP), IL-31, IL-33, etc.; cell adhesion molecules, such as ICAM1, VCAM1, MADCAM1, integrin α4, integrin β7, LFA-1 (or MAC-1), VLA-4, etc. In Table 1 below, the amino acid sequences of autoantigens are listed, as well as exemplary references that disclose antibodies that bind to target substances. It should be understood by those with ordinary knowledge in the art that ligands or receptors or autoantibodies that bind to the listed target autoantigen substances can also be included in the second region of the present disclosure. The entire contents of the references in Table 1 are incorporated into this specification by reference.

Table 1 Target substance Abbreviation disease UniProt (reviewed by UniProtKB ( Swiss-Prot )) GenBank or NCBI accession number Exemplary references disclosing regions that bind to a target substance or known binding substances Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Thrombin, vWF Acquired hemophilia A C9JV37 US20150099298A1, US20230287141A1 Calcium sensitive receptor CaSR Acquired hypoparathyroidism, autoimmune hypophysitis, autoimmune oophoritis P41180 Spermine, γ-Glutathione, L-Amino Acid ACTH ACTH deficiency WO2016100838A2 21-Hydroxycycline (CYP21) Addison's disease, APS-III P08686 Hair Hyalin Spotted baldness Q07283 Oxidized LDL (OxLDL) Atherosclerosis US20110182816A1 Transcriptional co-activator p75 Atopic dermatitis O75475 JP02 (Maertens et al., Journal of Cell Sciences, vol 119, Issue 12, 2006) p-80-Coilin Atopic dermatitis, scleroderma zoster AAB81550.1 C1 inhibitors Autoimmune C1 deficiency P05155 US10080788B2 AMPA receptor Autoimmune encephalitis EP2338492A1 CRMP5 Autoimmune encephalitis Q9BPU6 DPPX/DPP6 Autoimmune encephalitis P42658 GABAA-receptor GABA _A R Autoimmune encephalitis P34903 and others* GABAB receptor GABA _B R Autoimmune encephalitis Q9UBS5、O75899 Glycine receptor (GlyR) Autoimmune encephalitis P23415, P23416, O75311, P48167 Hu (ANNA-1) Autoimmune encephalitis Ma1 Autoimmune encephalitis Q8ND90 US7026450B2, US6387639B1 Ma2 Autoimmune encephalitis Q9UL42 US7026450B2, US6387639B1 Ri (ANNA-2) Autoimmune encephalitis Zic4 Autoimmune encephalitis AAI36340.1 Voltage-gated potassium channel (VGKC)-complex Autoimmune encephalitis (Isaac syndrome/acquired neuromyotonia, Miller Fisher syndrome, Morvan syndrome) NMDA-receptors Autoimmune encephalitis, systemic lupus erythematosus (SLE) Q05586, O15399, Q14957, Q12879, Q13224, O60391, Q8TCU5 WO2014187879 Jo1 Autoimmune encephalitis, systemic lupus erythematosus (SLE), arthritis, polymyositis/dermatomyositis H/K ATPase Autoimmune gastritis, pernicious anemia/atrophic gastritis Thyroid peroxidase Autoimmune Hashimoto's thyroiditis, immune disorders, polyendocrinopathy, intestinal diseases, X-linked P07202 Red blood cell I/i Autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome Sci Transl Med.(2019) 11(506):eaau8217. F-actin Autoimmune hepatitis US5632991A Asialoglycoprotein receptor Autoimmune hepatitis type 1 AAB58308.1 WO2014023709A1 Cytochrome P450 2D6 (CYP2D6) Autoimmune hepatitis type 2 P10635 NXP-2/MORC3 Autoimmune myopathy Q14149 TIF1-γ/TRIM-33 Autoimmune myopathy Q9UPN9 β2 integrin Autoimmune neutropenia, Evans syndrome P05107 Nuclear autoantigen sperm protein (NASP) NASP Autoimmune orchitis, autoimmune polyglandular syndrome I/II/III P49321 Lactoferrin Autoimmune pancreatitis P02788 CN103965354A 17-α-Hydroxytransferase (CYP17) Autoimmune polyendocrine syndrome type I and 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 decarboxylase Autoimmune polyendocrine syndrome type I (APS-I) P20711 Glycoprotein IIb/IIIa and Ib/IX Autoimmune thrombocytopenic purpura (iTTP) GPIIb (integrin α2b): P08514 GPIIIa (integrin β3): P05106 GP1b: P07359 GPIX: P14770 Thyroglobulin Autoimmune thyroiditis P01266 US10450372B2 Hemibasin 180 BP180 Large blisters of conspicuous acne, herpes of pregnancy, cicatricial conspicuous acne Q9UMD9 WO2020072937A1 CN101333255A p53 Cancer, systemic lupus erythematosus (SLE) P04637 WO2018074978 Recoverin Cancer-related retinopathy P35243 Actin Chronic active hepatitis, primary biliary cirrhosis IgE receptor Chronic idiopathic urticaria WO2017211928A1 Myelin-associated glycoprotein (MAG) MAG Chronic inflammatory demyelinating polyneuropathy, encephalitis lethargica, POEMS syndrome P20916 EP2110139A2 Tubulin Chronic liver disease, visceral leishmaniasis Laminin-332 Cicatricial acne Laminin subunit α3: Q16787 Tissue transglutaminase TG2 Chylous diarrhea, Crohn's disease P21980 WO 2006/100679 WO2013175229A1 Desmin Crohn's disease, coronary artery disease P17661 αB crystallin, desmoplakin Bactericidal/permeability-increasing protein (BPI) BPI Cystic fibrosing vasculitis P17213 Lipopolysaccharide Transglutaminase Herpetiform dermatitis, discoid lupus erythematosus AAH03551.1 Melanoma differentiation-associated gene 5 (MDA5) MDA5 (CADM-140)** Dermatomyositis Q9BYX SAE-1 Dermatomyositis Q9UBE0 SAE-2 Dermatomyositis Q9UBT2 DNA-dependent nucleosome-stimulated ATPase Dermatomyositis US6180612B1 Chromodomain-helicase-DNA-binding protein 4 (CHD4) CHD4 (Mi-2) Dermatomyositis/polymyositis Q14839 β-adrenaline receptor Dilated cardiomyopathy CAA02051.1 Adenine nucleotide transporter ANT Dilated cardiomyopathy, myocarditis P12235 Type VII collagen Acquired epidermolysis bullosa AAA58965.1 IgG Essential mixed cryoglobulinemia AAA02914.1 G-CSF Felty syndrome P09919 CA3052877A1 Type IV collagen α3 chain Goodpasture's syndrome ABX71213.1 Thyrotropin receptor (TSHR) TSHR Graves' disease P16473 EP1565493B1 Sodium-iodine transporter (NIS) Graves' disease, autoimmune hypothyroidism Q92911 Peripheral myelin protein 22 (PMP22) PMP22 Guillain-Barré syndrome Q01453 GM ganglioside Guillain-Barré syndrome, POEMS syndrome S-antigen HLA-B27-related acute anterior uveitis, sympathetic ophthalmia P10523 HMGCR Immune-mediated necrotic myopathy P04035 Signal recognition particle 54kDa subunit (SRP54) SRP54 Immune-mediated necrotic myopathy P61011 IgA Immunodeficiency, erythema protuberans, Kawasaki disease CAA10818.1 US9688776B2 Synaptic binding protein Lambert-Eaton myasthenia syndrome Q8N9I0 Voltage-gated calcium channel Lambert-Eaton myasthenia syndrome βIV spectrin Lower motor neuron syndrome Q9H254 SNRNP70 Mixed connective tissue disease P08621 CNPase Multiple sclerosis P09543 Myelin-associated oligodendrocyte basic protein (MOBP) MOBP Multiple sclerosis Q13875 Myelin proteolipid protein (PLP) PLP Multiple sclerosis P60201 S100 calcium binding protein B S100β Multiple sclerosis P04271 WO2016149116A1 Transaldolase Multiple sclerosis P37837 Myelin basic protein (MBP) MBP Multiple sclerosis, demyelinating disease P02686 Myelin oligodendrocyte glycoprotein (MOG) MOG Multiple sclerosis, neuromyelitis optica Q16653 US20220025044A1 Acetylcholine receptor Myasthenia gravis CAA26344.1 Low-density lipoprotein receptor-related protein 4 (LRP4) LRP4*** Myasthenia gravis O75096 Muscle and bone receptor tyrosine protein kinase (MuSK) MuSK Myasthenia gravis O15146 WO2021212053A2 Aminoacyl-tRNA synthetase Myositis, dermatomyositis, antisynthetase syndrome TRIB2 Narcolepsy, Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS) Q92519 Myeloperoxidase (MPO) MPO Necrotic and crescentic nephritis (NCGN), systemic vasculitis, microscopic polyarteritis, nodular polyarteritis P05164 Aquaporin 4 (APQ-4) Neuromyelitis optica P55087 WO2016033509A1 Amphiphysin Neurological disease (autoimmune encephalitis), small cell lung cancer P49418 EXOSC9 Overlap syndrome (scleroderma/polymyositis) Q06265 EXOSC10/PMSCL Overlap syndrome (scleroderma/polymyositis) Q01780 Yo Protein Paraneoplastic cerebellar degeneration Hu protein Paraneoplastic encephalomyelitis Ri protein Paraneoplastic strabismus, ocular plexus, muscle plexus, ataxia Desmoplakin Paraneoplastic ulcer P15924 Gephyrin Paraneoplastic Stiff-Person Syndrome Q9NQX3 Desmoglin 1 Deciduous ulcer Q02413 Desmoglin 3 Common acne vulgaris P32926 Intrinsic Factor Type 1 Pernicious anemia β2-glycoprotein I (β2-GPI) Primary antiphospholipid syndrome X Pyruvate dehydrogenase complex-E2 (PDC-E2) Primary biliary cirrhosis Q9HAN0 (unverified) Proteoglycan (Aggrecan G1) RA P16112 US20210115117A9 Aminoformyl antigen RA Human cartilage glycoprotein-39 HC-gp39/CHI3L1/YKL-40 RA P36222 US8673301B2 Immunoglobulin Fc part RA Glucose-6-phosphate isomerase RA P06744 Keratin RA CAA73943.1 Protein-arginine deiminase type 4 PAD4 RA Q9UM07 Collagen (various types, especially types II, IV, and IX) RA, systemic lupus erythematosus (SLE), progressive systemic sclerosis, recurrent polychondritis WO2016016269A1 Fibrinogen, βα RA, Still's disease CAA50740.1 Leukemia inhibitory factor (LLIF) LIF RA, systemic sclerosis, normal subjects US10968273B2 Glutamine receptor (GLUR) Lasamson encephalitis P42261, P42262, P42263, P48058, P39086, Q13002, Q13003 US5739291A myosin Rheumatoid artery disease, aortic artery inflammation, temporal artery inflammation CAA86293.1 B23 scleroderma AAA58386.1 Nucleolar phosphoprotein (NPM) NPM scleroderma P06748 Fibrin Scleroderma, CREST syndrome P22087 Topoisomerase-I (Scl-70) Scleroderma, Raynaud's syndrome, pulmonary fibrosis NCBI reference sequence: NP_003277.1 Interferon-γ-inducing protein 16 (IFI16) IFI16 Sjogren's syndrome, systemic lupus erythematosus (SLE) Q16666 WO2015095609A1 La phosphoprotein La/SSB Sjogren's syndrome, systemic lupus erythematosus (SLE) P05455 Ro60 Sjogren's syndrome, systemic lupus erythematosus (SLE) P10155 Ro52 (TRIM21) Sjogren's syndrome, systemic lupus erythematosus (SLE), myositis, scleroderma P19474 Homoglia protein (95, 97, 160, 180) Sjogren's syndrome, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) Homoglia protein-95: AAA35920.1 Homoglia protein-72 variant: BAD92095.1 Homoglia protein-160: AAL93149.1 Anionic phospholipid/protein complex Systemic lupus erythematosus (SLE) Cardiolipin Systemic lupus erythematosus (SLE) Component of Sm splicing ribonucleoprotein (subunit AG) Systemic lupus erythematosus (SLE) dsDNA Systemic lupus erythematosus (SLE) Histone H2A-H2B-DNA Systemic lupus erythematosus (SLE) Proliferating cell nuclear antigen (PCNA) PCNA Systemic lupus erythematosus (SLE) P12004 US11667700B2 Ribosome P Rib-P Systemic lupus erythematosus (SLE) Sjogren's syndrome A Ro/SSA Systemic lupus erythematosus (SLE) Smith (anti-Sm antibody) Sm Systemic lupus erythematosus (SLE) U1-RNP Systemic lupus erythematosus (SLE) P09012 U2 snRNP B Systemic lupus erythematosus (SLE) P08579 Vimentin Systemic lupus erythematosus (SLE) P08670 Q1 Systemic lupus erythematosus (SLE), membranoproliferative glomerulonephritis (MPGN) C1qA:P02745 C1qB:P02746 C1qC:P02747 US11649279B2 Fibronectin Systemic lupus erythematosus (SLE), RA, morphea P02751 EP2236517A1 Ku-DNA-protein kinase Systemic lupus erythematosus (SLE), scleroderma Carbonic anhydrase II Systemic lupus erythematosus (SLE), Sjögren's syndrome, systemic sclerosis P00918 Neuronal Nicotine Acetylcholine Receptor Subacute autonomic neuropathy, cancer AAA59942.1 Centromere-associated protein Systemic sclerosis, CREST syndrome P49450, P07199, Q02224 RNA polymerase I-III (RNP) Systemic sclerosis, systemic lupus erythematosus (SLE) Thyroid and eye muscle common protein G2s Thyroid-related eye disease P31040 Leukocyte function-associated antigen (LFA-1) Treatment-resistant Lyme disease arthritis P20701, P05107 US6703018B2 Chromogranin A Type 1 diabetes P10645 IA-2 (ICA512) Type 1 diabetes Q16849 IGRP Type 1 diabetes Q9NQR9 ZnT8 Type 1 diabetes AAP44332.1 WO2020037174A1 Insulin Type 1 diabetes, hypoglycemia syndrome (Hirata disease) P01308 Glutamine decarboxylase (GAD65) Type 1 diabetes, stiff person syndrome, autoimmune encephalitis, Batten disease/neurovascular lipofuscinosis AAA62367.1 Insulin receptor Type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE) P06213 US20220227857A1 Heat shock protein (65-kDa heat shock protein) Hsp65 Various immune-related diseases (RA) SOX-10 Vitiligo P56693 US20160216269A1 Tyrosinase Vitiligo, metastatic melanoma P14679 KUMEL1/ARMC9 Vogt-Koyanagi-Harada syndrome Q7Z3E5 Proteinase 3/Myeloblastin PR3 Wegener's granuloma, Chargar-Strauss syndrome, vasculitis P24158 *: There are 35 entries at: https://www.uniprot.org/uniprotkb?query=GABAAR+ALPHA&facets=model_organism%3A9606. **: MDA5 is encoded by the interferon-induced helicase C domain-containing protein 1 (IFIH1) gene. ***: • Lrp4 is a receptor for Agrin and forms a complex with MuSK • Agrin (AGRN): O00468.

The amino acid sequence of the target substance can be obtained from a public database such as UniProtKB/Swiss-Prot or NCBI. For example, an exemplary amino acid sequence of the target substance may include but is not limited to the following.

CD20: UniProt accession number P11836 (human) and its variants at www.uniprot.org/uniprotkb/P11836/variant-viewer, and their homologous sequences.

CD19: UniProt accession number P15391 (human) and its variants at www.uniprot.org/uniprotkb/P15391/variant-viewer, and their homologous sequences.

CD52: UniProt accession number P31358 (human) and its variants at www.uniprot.org/uniprotkb/P31358/variant-viewer, and their homologous sequences.

CD80: UniProt accession number P33681 (human) and its variants at www.uniprot.org/uniprotkb/P33681/variant-viewer, and their homologous sequences.

CD86: UniProt accession number P42081 (human) and its variants at www.uniprot.org/uniprotkb/P42081/variant-viewer, and their homologous sequences.

CD28: UniProt accession number P10747 (human) and its variants at www.uniprot.org/uniprotkb/P10747/variant-viewer, and their homologous sequences.

CD40: UniProt accession number P25942 (human) and its variants at www.uniprot.org/uniprotkb/P25942/variant-viewer, and their homologous sequences.

Complement C3: UniProt accession number P01024 (human) and its variants at www.uniprot.org/uniprotkb/P01024/variant-viewer, and its homologous sequences.

Complement C5: UniProt accession number P01031 (homologous) and its variants at www.uniprot.org/uniprotkb/P01031/variant-viewer, and their homologous sequences.

C5 alpha receptor 1: UniProt accession number P21730 (human) and its variants at www.uniprot.org/uniprotkb/P21730/variant-viewer, and their homologous sequences.

IL-1R: UniProt accession number P14778 (human) and its variants at www.uniprot.org/uniprotkb/P14778/variant-viewer, and their homologous sequences.

IL-6R: UniProt accession number P08887 (human) and its variants at www.uniprot.org/uniprotkb/P08887/variant-viewer, and their homologous sequences.

IL-6ST (interleukin-6 receptor subunit beta): UniProt accession number P40189 (human) and its variants at www.uniprot.org/uniprotkb/P40189/variant-viewer, and their homologous sequences.

IL-17C: UniProt accession number Q9P0M4 (human) and its variants at www.uniprot.org/uniprotkb/PQ9P0M4/variant-viewer, and their homologous sequences.

IL-17RA: UniProt accession number Q96F46 (human) and its variants at www.uniprot.org/uniprotkb/Q96F46/variant-viewer, and their homologous sequences.

IL-17RB: UniProt accession number Q9NRM6 (human) and its variants at www.uniprot.org/uniprotkb/Q9NRM6/variant-viewer, and their homologous sequences.

IL-4R: UniProt accession number P24394 (human) and its variants at www.uniprot.org/uniprotkb/QP24394/variant-viewer, and their homologous sequences.

IL-5R: UniProt accession number Q01344 (human) and its variants at www.uniprot.org/uniprotkb/Q01344/variant-viewer, and their homologous sequences.

IL-5RB: UniProt accession number P32927 (human) and its variants at www.uniprot.org/uniprotkb/P32927/variant-viewer, and their homologous sequences.

IL-13R1: UniProt accession number P78552 (human) and its variants at www.uniprot.org/uniprotkb/P78552/variant-viewer, and their homologous sequences.

IL-13R2: UniProt Accession No. Q14627 (human) and its variants at www.uniprot.org/uniprotkb/Q14627/variant-viewer, and their homologous sequences; or UniProt Accession No. D0EFR8 (human) and its variants at www.uniprot.org/uniprotkb/D0EFR8/variant-viewer, and their homologous sequences.

IFN-γ receptor 1: UniProt accession number Q15260 (human) and its variants at www.uniprot.org/uniprotkb/Q15260/variant-viewer, and their homologous sequences.

IFN-γ receptor 2: UniProt accession number P38484 (human) and its variants at www.uniprot.org/uniprotkb/P38484/variant-viewer, and their homologous sequences.

Integrin alpha-D (ITGAD): UniProt accession number Q13349 (human) and its variants at www.uniprot.org/uniprotkb/Q13349/variant-viewer, and their homologous sequences.

IL-12RB1: UniProt accession number P42701 (human) and its variants at www.uniprot.org/uniprotkb/P42701/variant-viewer, and their homologous sequences.

IL-12RB2: UniProt accession number Q99665 (human) and its variants at www.uniprot.org/uniprotkb/Q99665/variant-viewer, and their homologous sequences.

IL-21R: UniProt accession number Q9HBE5 (human) and its variants at www.uniprot.org/uniprotkb/Q9HBE5/variant-viewer, and their homologous sequences.

IL-22RA1: UniProt accession number Q8N6P7 (human) and its variants at www.uniprot.org/uniprotkb/Q8N6P7/variant-viewer, and their homologous sequences.

IL-22RA2: UniProt accession number Q969J5 (human) and its variants at www.uniprot.org/uniprotkb/Q969J5/variant-viewer, and their homologous sequences.

TGF-β receptor type 1: UniProt accession number P36897 (human) and its variants at www.uniprot.org/uniprotkb/P36897/variant-viewer, and their homologous sequences; TGF-β receptor type 2: UniProt accession number P37173 (human) and its variants at www.uniprot.org/uniprotkb/P37173/variant-viewer, and their homologous sequences; TGF-β receptor type 3: UniProt accession number Q03167 (human) and its variants at www.uniprot.org/uniprotkb/Q03167/variant-viewer, and their homologous sequences.

IL-23R: UniProt accession number Q5VWK5 (human) and its variants at www.uniprot.org/uniprotkb/Q5VWK5/variant-viewer, and their homologous sequences.

Thymic stromal lymphopoietin receptor (TSLPR): UniProt accession number Q9HC73 (human) and its variants at www.uniprot.org/uniprotkb/Q9HC73/variant-viewer, and their homologous sequences.

IL-31R: UniProt accession number Q8NI17 (human) and its variants at www.uniprot.org/uniprotkb/Q8NI17/variant-viewer, and their homologous sequences.

IL-33R: UniProt accession number Q01638 (human) and its variants at www.uniprot.org/uniprotkb/Q8NB14/variant-viewer, and their homologous sequences.

IGF-1R: UniProt accession number P08069 (human) and its variants at www.uniprot.org/uniprotkb/P08069/variant-viewer, and their homologous sequences.

TNFR1: UniProt accession number P19438 (human) and its variants at www.uniprot.org/uniprotkb/P19438/variant-viewer, and their homologous sequences.

TNFR2: UniProt accession number P20333 (human) and its variants at www.uniprot.org/uniprotkb/P20333/variant-viewer, and their homologous sequences.

FcRn large subunit p51: UniProt accession number P55899 (human) and its variants at www.uniprot.org/uniprotkb/P55899/variant-viewer, and their homologous sequences.

CCL14: UniProt accession number Q16627 (human) and its variants at www.uniprot.org/uniprotkb/Q16627/variant-viewer, and their homologous sequences.

CCL15: UniProt accession number Q16663 (human) and its variants at www.uniprot.org/uniprotkb/Q16663/variant-viewer, and their homologous sequences.

CCL18: UniProt accession number P55774 (human) and its variants at www.uniprot.org/uniprotkb/P55774/variant-viewer, and their homologous sequences.

CCL19: UniProt accession number Q99731 (human) and its variants at www.uniprot.org/uniprotkb/Q99731/variant-viewer, and their homologous sequences.

CCL20: UniProt accession number QP78556 (human) and its variants at www.uniprot.org/uniprotkb/P78556/variant-viewer, and their homologous sequences.

CCL21: UniProt accession number O00585 (human) and its variants at www.uniprot.org/uniprotkb/O00585/variant-viewer, and their homologous sequences.

CCL23: UniProt accession number P55773 (human) and its variants at www.uniprot.org/uniprotkb/P55773/variant-viewer, and their homologous sequences.

CCL25: UniProt accession number Q68A93 (human) and its variants at www.uniprot.org/uniprotkb/Q68A93/variant-viewer, and their homologous sequences.

CCL27: UniProt accession number Q9Y4X3 (human) and its variants at www.uniprot.org/uniprotkb/Q9Y4X3/variant-viewer, and their homologous sequences.

CXCL12: UniProt accession number P48061 (human) and its variants at www.uniprot.org/uniprotkb/P48061/variant-viewer, and their homologous sequences.

CXCL13: UniProt accession number O43927 (human) and its variants at www.uniprot.org/uniprotkb/O43927/variant-viewer, and their homologous sequences.

IL-1A: UniProt accession number P01583 (human) and its variants at www.uniprot.org/uniprotkb/P01583/variant-viewer, and their homologous sequences; IL-1B: UniProt accession number P01584 (human) and its variants at www.uniprot.org/uniprotkb/P01584/variant-viewer, and their homologous sequences.

TNF-α: UniProt accession number P01375 (human) and its variants at www.uniprot.org/uniprotkb/P01375/variant-viewer, and their homologous sequences.

CXCL-8: UniProt accession number P10145 and its variants at www.uniprot.org/uniprotkb/P10145/variant-viewer, and their homologous sequences.

CCL2: UniProt accession number P13500 (human) and its variants at www.uniprot.org/uniprotkb/Q13500/variant-viewer, and their homologous sequences.

CCL3: UniProt accession number P10147 (human) and its variants at www.uniprot.org/uniprotkb/P10147/variant-viewer, and their homologous sequences.

CCL4: UniProt accession number P13236 (human) and its variants at www.uniprot.org/uniprotkb/P13236/variant-viewer, and their homologous sequences.

CCL5: UniProt accession number P13501 (human) and its variants at www.uniprot.org/uniprotkb/P13501/variant-viewer, and their homologous sequences.

CCL11: UniProt accession number P51671 (human) and its variants at www.uniprot.org/uniprotkb/P51671/variant-viewer, and their homologous sequences.

CXCL10: UniProt accession number P02778 (homo sapiens) and its variants at www.uniprot.org/uniprotkb/P02778/variant-viewer, and their homologous sequences.

IL-6: UniProt accession number P05231 (human) and its variants at www.uniprot.org/uniprotkb/P05231/variant-viewer, and their homologous sequences.

IL-17: UniProt accession number Q16552 (human) and its variants at www.uniprot.org/uniprotkb/Q16552/variant-viewer, and their homologous sequences.

IL-4: UniProt accession number P05112 (human) and its variants at www.uniprot.org/uniprotkb/P05112/variant-viewer, and their homologous sequences.

IL-5: UniProt accession number P05113 (human) and its variants at www.uniprot.org/uniprotkb/P05113/variant-viewer, and their homologous sequences.

IL-13: UniProt accession number P35225 (human) and its variants at www.uniprot.org/uniprotkb/P35225/variant-viewer, and their homologous sequences.

IFN-γ: UniProt accession number P01579 (human) and its variants at www.uniprot.org/uniprotkb/P01579/variant-viewer, and their homologous sequences.

IL-12A: UniProt accession number P29459 (human) and its variants at www.uniprot.org/uniprotkb/P29459/variant-viewer, and their homologous sequences; IL-12: UniProt accession number P29460 (human) and its variants at www.uniprot.org/uniprotkb/P29460/variant-viewer, and their homologous sequences.

IL-21: UniProt accession number Q9HBE4 (human) and its variants at www.uniprot.org/uniprotkb/Q9HBE4/variant-viewer, and their homologous sequences.

IL-22: UniProt accession number Q9GZX6 (human) and its variants at www.uniprot.org/uniprotkb/Q9GZX6/variant-viewer, and their homologous sequences.

TGF-β-1: UniProt Accession No. P01137 (human) and its variants at www.uniprot.org/uniprotkb/P01137/variant-viewer, and their homologous sequences; TGF-β-2: UniProt Accession No. P61812 (human) and its variants at www.uniprot.org/uniprotkb/P61812/variant-viewer, and their homologous sequences; TGF-β-3: UniProt Accession No. P10600 (human) and its variants at www.uniprot.org/uniprotkb/P10600/variant-viewer, and their homologous sequences.

IL-23A: UniProt accession number Q9NPF7 (human) and its variants at www.uniprot.org/uniprotkb/Q9NPF7/variant-viewer, and their homologous sequences; IL-23B: UniProt accession number P29460 (human) and its variants at www.uniprot.org/uniprotkb/P29460/variant-viewer, and their homologous sequences.

Thymic stromal lymphopoietin (TSLP): UniProt accession number Q969D9 (human) and its variants at www.uniprot.org/uniprotkb/Q969D9/variant-viewer, and their homologous sequences.

IL-31: UniProt accession number Q6EBC2 (human) and its variants at www.uniprot.org/uniprotkb/Q6EBC2/variant-viewer, and their homologous sequences.

OX40 (tumor necrosis factor receptor superfamily member 4): UniProt accession number P23510 (human) and its variants at www.uniprot.org/uniprotkb/P23510/variant-viewer, and their homologous sequences.

OX40L (tumor necrosis factor receptor superfamily member 4): UniProt accession number P43489 (human) and its variants at www.uniprot.org/uniprotkb/P43489/variant-viewer, and their homologous sequences.

IL-33: UniProt accession number O95760 (human) and its variants at www.uniprot.org/uniprotkb/O95760/variant-viewer, and their homologous sequences.

CD40L: UniProt accession number P29965 (human) and its variants at www.uniprot.org/uniprotkb/P29965/variant-viewer, and their homologous sequences.

ICAM1: UniProt accession number P05362 (human) and its variants at www.uniprot.org/uniprotkb/P05362/variant-viewer, and their homologous sequences.

VCAM1: UniProt accession number P19320 (homo sapiens) and its variants at www.uniprot.org/uniprotkb/P19320/variant-viewer, and their homologous sequences.

MADCAM1: UniProt accession number Q13477 (homologous) or B9EGE2 and their variants at www.uniprot.org/uniprotkb/Q13477/variant-viewer and www.uniprot.org/uniprotkb/B9EGE2/variant-viewer, respectively, and their homologous sequences.

Integrin α4: UniProt accession number P13612 (human) and its variants at www.uniprot.org/uniprotkb/P13612/variant-viewer, and their homologous sequences.

Integrin β7: UniProt accession number P26010 (human) and its variants at www.uniprot.org/uniprotkb/P26010/variant-viewer, and their homologous sequences.

LFA-1 or MAC-1 (dimer of integrin α-M and integrin β-2): UniProt accession number P11215 (human) of integrin α-M (ITAM) and its variants at www.uniprot.org/uniprotkb/P11215/variant-viewer, and their homologous sequences, and UniProt accession number P05107 (human) of integrin β-2 and its variants at www.uniprot.org/uniprotkb/P05107/variant-viewer, and their homologous sequences.

VLA-4 (dimer of CD49d and CD29): UniProt accession number P13612 (human) of CD49d and its variants at www.uniprot.org/uniprotkb/P13612/variant-viewer, and their homologous sequences, and UniProt accession number P05556 (human) of CD29 and its variants at www.uniprot.org/uniprotkb/P05556/variant-viewer, and their homologous sequences.

TLR3: UniProt accession number O15455 (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, and rheumatoid arthritis. Antibodies that bind to TLR3 are disclosed in, for example, U.S. Patent No. 8153583B2.

TLR4: UniProt accession number O00206 (human) and its variants at www.uniprot.org/uniprotkb/O00206/variant-viewer. TLR4 is reported to be associated with rheumatoid arthritis. Antibodies that bind to TLR4 are disclosed in, for example, U.S. Patent No. 7312320B2.

TLR5: UniProt accession number O60602 (human) and its variants at www.uniprot.org/uniprotkb/O60602/variant-viewer. TLR5 is reported to be associated with rheumatoid arthritis. Substances that bind to TLR5 are disclosed in, for example, U.S. Patent No. 8703146B2 and U.S. Patent Application Publication No. 20200362052A1.

TLR7: UniProt accession number Q9NYK1 (human) and its variants at www.uniprot.org/uniprotkb/Q9NYK1/variant-viewer. TLR7 is reported to be associated with systemic lupus erythematosus and cutaneous lupus erythematosus. Antibodies that bind to TLR7 are disclosed in, for example, U.S. Patent Application Publication Nos. 20200362052A1 and 20210040225A1.

In some embodiments, the immune disease is an autoimmune disease or an inflammatory disease. In another specific embodiment, the autoimmune or inflammatory disease is multiple sclerosis (MS), rheumatoid arthritis, spondylosis, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune diseases, graft-versus-host disease, sepsis, type 1 diabetes, type 2 diabetes, psoriasis, atherosclerosis, Sjögren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia-reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, or inflammatory autoimmune myositis. In a specific embodiment, the spondyloarthropathies are selected from ankylosing spondylitis, reactive arthritis, enteropathic arthritis associated with inflammatory bowel disease, psoriatic arthritis, isolated acute anterior uveitis, undifferentiated spondyloarthropathies, Behcet's syndrome, and juvenile idiopathic arthritis. In one embodiment, the immune disease is caused or aggravated by excessive antigenic substances binding to immunoglobulins or immune cells or by an increase in the amount of antigenic substances or an increase in the expression of antigenic substances. In a specific embodiment, the immune cells are dendritic cells.

In an embodiment, the present disclosure relates to a nucleic acid or polynucleotide encoding the above-mentioned fusion protein.

In an embodiment, the disclosure relates to a vector comprising the nucleic acid or polynucleotide.

Embodiments relate to a host cell comprising the vector.

Another aspect of the present disclosure provides a method of producing a therapeutic fusion molecule for treating an immune disease in a subject, the method comprising expressing the fusion molecule by culturing a host cell under conditions where the fusion molecule is expressed.

In an embodiment, the present disclosure relates to a method for reducing a substance or enhancing the reduction of a substance that triggers, induces or causes an undesirable or pathological immune response such as an autoimmune disease, transplant rejection, or allergic or hyperimmune response in a subject, the method comprising 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 capable of binding to a TAM (Tyro3, Axl, and MerTK) receptor on the cell surface of the subject; and a second region that specifically binds to the substance. In a non-limiting embodiment, the substance and the immune disease can be one or more of those listed in Table 1. In a non-limiting embodiment, the fusion molecule does not have effector function and does not induce Fc-mediated inflammatory response. For example, the fusion molecule does not include a portion that binds to an Fc receptor, and preferably may include an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not include a target substance that is eliminated or reduced by administering the fusion molecule.

In an embodiment, the present disclosure relates to a method for removing or clearing an antigenic substance, or enhancing the clearance of an antigenic substance, wherein the antigenic substance triggers, induces or causes an undesirable or pathological immune response in a subject, such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response, the method comprising 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 capable of binding to a TAM (Tyro3, Axl, and MerTK) receptor on the cell surface of the subject; and a second region that specifically binds to the antigenic substance. In a non-limiting embodiment, the substance and the undesirable or pathological immune cause may be one or more of those listed in Table 1. In a non-limiting embodiment, the fusion molecule does not have effector function and does not induce an inflammatory response. For example, the fusion molecule does not comprise a portion that binds to an Fc receptor, and may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not comprise a target substance that is eliminated or reduced by administering the fusion molecule.

In an embodiment, the present disclosure relates to a method for treating, preventing or improving an immune disease in a subject suffering from an immune disease or at risk of suffering from an immune 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 capable of binding to a TAM (Tyro3, Axl and MerTK) receptor on the cell surface of the subject; and a second region specifically binding to an antigenic substance that triggers, induces or causes an immune disease. In a non-limiting embodiment, the antigenic substance and the immune disease may be one or more of those listed in Table 1. In a non-limiting embodiment, the fusion molecule does not have an effector function and does not induce an Fc-mediated inflammatory response. For example, the fusion molecule does not include a portion that binds to an Fc receptor, and may preferably include an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not comprise the target substance that is eliminated or reduced by administration of the fusion molecule.

In an embodiment, the present disclosure relates to a method for delaying the development of symptoms associated with an immune disease caused 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, or a vector comprising the polynucleotide, wherein the fusion molecule comprises: a first region capable of binding to a TAM (Tyro3, Axl, and MerTK) receptor on the cell surface of the subject; and a second region specifically binding to an antigenic substance. In a non-limiting embodiment, the antigenic substance and the immune disease may be one or more of those listed in Table 1. In a non-limiting embodiment, the fusion molecule does not have an effector function and does not induce an Fc-mediated inflammatory response. For example, the fusion molecule does not comprise a portion that binds to an Fc receptor, and may preferably comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not comprise the target substance that is eliminated or reduced by administration of the fusion molecule.

In an embodiment, the present disclosure provides a method for reducing an antigenic substance that triggers, induces or causes an undesirable or pathological immune response in a subject, such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response. The method comprises administering an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule to the subject, wherein the fusion molecule comprises: a first region capable of binding to a TAM (Tyro3, Axl, and MerTK) receptor on the cell surface of the subject; and a second region that specifically binds to the antigenic substance. The antigenic substance may be in a soluble, oligomeric, or aggregated form. In some embodiments, an undesirable or pathological immune response to the antigenic substance is suppressed and/or reduced. Therefore, the method disclosed herein can be used to treat any disease associated with or caused by an undesirable or pathological immune response to an antigenic substance. In a non-limiting embodiment, the antigenic substance and the disease may be one or more of those listed in Table 1. In a non-limiting embodiment, the fusion molecule does not have effector function and does not induce Fc-mediated inflammatory response. For example, the fusion molecule does not contain a portion that binds to an Fc receptor, and may preferably contain an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not contain a target substance that is eliminated or reduced by administering the fusion molecule.

In an embodiment, the present disclosure relates to a pharmaceutical composition comprising an effective amount of any one of the above disclosed fusion molecules or polynucleotides encoding the fusion molecules, and a pharmaceutically acceptable excipient. In a non-limiting embodiment, the fusion molecule does not have an effector function and does not induce an Fc-mediated inflammatory response. For example, the fusion molecule does not include a portion that binds to an Fc receptor, and preferably may include an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not include a target substance that is removed or reduced by administering the fusion molecule.

In an embodiment, the present disclosure relates to the use of any of the above-disclosed fusion molecules or polynucleotides encoding the fusion molecules for preparing a drug suitable for treating immune diseases.

In an embodiment, the present disclosure relates to the use of any of the above-disclosed fusion molecules or polynucleotides encoding the fusion molecules or pharmaceutical compositions for treating or preventing immune diseases.

In embodiments, the present disclosure relates to a kit comprising an effective amount of any of the above disclosed fusion molecules or polynucleotides encoding the fusion molecules. The kit is usually packaged in a suitable manner and provided with appropriate instructions, and can be used for any method described in the specification.

The above and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those having ordinary knowledge in the art after considering the detailed description of the exemplary embodiments shown below.

Methods and compositions are provided for reducing or inhibiting the formation, or clearing, or eliminating, or reducing target substances associated with, characteristic of, or causing immune disorders or diseases through phagocytosis, preventing or treating individuals who have or may develop immune diseases or disorders, and improving symptoms of immune diseases or disorders.

Where a range of values is provided, it is to be understood that, unless the context clearly dictates otherwise, each intermediate value between the upper and lower limits of the range that is one-tenth of the unit of the lower limit is also specifically disclosed. The present invention includes each smaller range between any specified value or intermediate value within the specified range and any other specified value or intermediate value within the specified range. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which any limit, no limit, or both limits are included in the smaller range is also included in the present invention, subject to any specifically excluded limit in the specified range. Where the specified range includes one or two limits, ranges excluding one or both of the included limits are also included in the present invention.

[Definition]

As used in this specification, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. Thus, as known to those of ordinary skill in the art, for example, reference to "a cell" includes a plurality of such cells, reference to "the peptide" includes reference to one or more peptides and their equivalents, such as polypeptides, and so on.

As used in this specification, the terms "about" and "consisting essentially of" refer to values or components that are within an acceptable error range for a particular value or component as determined by one of ordinary skill in the art, which depends in part on how the value or component is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within 1 or greater than 1 standard deviation per 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" may include any value between 4.5 mg and 5.5 mg (10%), 4.75 mg and 6.25 mg (5%), 4.8 mg and 6.2 mg (4%), 4.85 mg and 6.15 mg (3%), 4.9 mg and 6.1 mg (2%), or 4.95 mg and 6.05 mg (1%). In addition, particularly with respect to biological systems or processes, these terms may mean up to an order of magnitude or up to 5 times a value. When specific values or components are provided in the application and patent claims, unless otherwise stated, the meaning of "about" or "consisting essentially of..." should be considered to be within an acceptable error range for the specific value or component.

As used in this specification, "administering" or "using" refers to introducing a composition into a subject's body by a selected route. For example, if the selected route is intravenous injection, the composition is administered by introducing the composition into the subject's vein. In some examples, the peptides and antibodies disclosed in this specification are administered to a subject.

As used in this specification, "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 naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, γ-carboxyglutamine, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bound to hydrogen, a carboxyl group, an amine group, and an R group, for example, homoserine, norleucine, methionine sulfoxide, and methionine methylsulfoxide. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid analogs are chemical compounds that have structures that are different from the conventional chemical structures of amino acids, but function in a manner similar to naturally occurring amino acids.

As used in this specification, "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably in this specification and refer to polymers of amino acid residues. These terms also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. These terms also include amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other operation or modification, such as binding to a marker component. The definition also includes, for example, polypeptides containing analogs of one or more amino acids (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that because the polypeptides of the present invention are antibody-based, the polypeptides may appear in the form of a single chain or in a linked chain.

As used in this specification, "polynucleotide" or "nucleic acid" as used interchangeably in this specification refers to a polymer of nucleotides of any length, and includes 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. The polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. If present, the nucleotide structure can be modified before or after assembly of the polymer. The sequence of nucleotides will be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "capping", replacing one or more naturally occurring nucleotides with an analog; internucleotide modifications, for example, modifications with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); modifications comprising side chain moieties, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, polylysine, etc.); modifications with intercalators (e.g., acridine, psoralen, etc.); modifications comprising chelators (e.g., metals, radioactive metals, boron, oxidized metals, etc.); modifications comprising alkylating agents; modified linkages (e.g., alpha isomeric nucleic acids, etc.); and unmodified forms of polynucleotides. In addition, any hydroxyl group commonly present in sugar can be by, for example, phosphonate group, phosphate group replacement, protected by standard protecting groups, or activated to prepare additional connection with additional nucleotide, or can be covalently connected with solid support. OH at 5' and 3' ends can be phosphorylated or partially replaced by amine or organic end-capping groups of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived into standard protecting groups. Polynucleotide can also comprise similar forms of ribose or deoxyribose known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro or 2'-azido-ribose, carbocyclic sugar analogs, α-isomer sugars, epimers such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and debasing nucleoside analogs such as methyl nucleosides. One or more phosphodiester linkages may be replaced by alternative linker groups. These alternative linker groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O) _NR2 ("amide"), P(O)R, P(O)OR', CO or _CH2 ("formal"), wherein R or R' is independently H or substituted or unsubstituted by an alkyl (1-20C) group optionally containing an ether (-O-) linker, an aryl, an alkenyl, a cycloalkyl, a cycloalkenyl or an aralkyl group. Not all linkers in a polynucleotide need to be identical. The above description applies to all polynucleotides referred to in this specification, including RNA and DNA.

As used in this specification, "recipient", "individual", "subject", "host" and "patient" are used interchangeably in this specification and refer to any mammalian subject, especially human, that requires diagnosis, treatment or therapy. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic animals and livestock, as well as zoo animals, sports animals or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In an embodiment, the mammal is a human.

As used in this specification, "antibody" refers to single-chain, dual-chain and multi-chain proteins and glycoproteins belonging to the classes of polyclonal, monoclonal, chimeric and heterologous immunoglobulins (monoclonal antibodies are preferred); it also includes synthetic and genetically engineered variants of these immunoglobulins.

As used herein, "specifically binds", "specifically binds", etc. refers 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 a molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10 ^{− 5} M or less (e.g., 10 ^{− 6} M or less, 10 ^{− 7} M or less, 10 ^{− 8} M or less, 10 ^{− 9} 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, and increased binding affinity is associated with a lower KD. As used in this specification, "binding" of the first region to the TAM receptor and "specific binding" of the second region to the target substance do not require regulation, alteration, influence or modification of the activity of the bound TAM receptor or target substance.

As used in this specification, "variable" refers to the fact that specific parts of the variable domain differ greatly in sequence between antibodies, and are used for the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed in the variable domain of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved parts in the variable domain are called frameworks (FRs). The variable domains of natural heavy and light chains each contain four FR regions, mainly adopting a b-sheet configuration connected by three CDRs, which form a loop connecting the b-sheet structure and in some cases form a part of the b-sheet structure. The CDRs in each chain are tightly bound together by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The homeostatic domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, such as the participation of antibodies in antibody-dependent cellular cytotoxicity.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. In two-chain Fv species, this region consists of a dimer of one heavy chain and one light chain variable domain in tight non-covalent association. In single-chain Fv species (scFv), one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimer" structure similar to that in two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define the 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 containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

The term "complementary determining region" or "CDR" as used herein refers to the amino acid sequence within the variable region of an antibody that confers antigen specificity and binding affinity. For example, typically, 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 in 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 combinations thereof. According to 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); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia numbering scheme, in some embodiments, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the Kabat and Chothia combination numbering schemes, in some embodiments, a CDR corresponds to an amino acid residue that is part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, a CDR corresponds 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.

The "Fab fragment" also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that some residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. In this specification, Fab'-SH is the name of Fab' in which the cysteine residues of the constant domains have free thiol groups. F(ab')2 antibody fragments were originally produced as a pair of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

As used in this specification, the term "antibody fragment" or "antigen-binding fragment" or "active fragment" is defined as a portion of an intact antibody that includes the antigen-binding site or variable region of the intact antibody, wherein the portion does not contain the constant heavy chain domains (i.e., CH2, CH3 and CH4, depending on the 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; dimers; any antibody fragment that is a polypeptide having a primary structure consisting of an uninterrupted sequence of consecutive amino acid residues (referred to as a "single-chain antibody fragment" or "single-chain polypeptide" in this specification), including but not limited to (1) single-chain Fv (scFv) molecules, (2) single-chain Fv molecules that contain only one light chain variable domain, and (3) single-chain Fv molecules that contain only one light chain variable domain. (1) polypeptides, or fragments thereof comprising the three CDRs of a light chain variable domain without the associated heavy chain portion, (2) single chain polypeptides comprising only one heavy chain variable region, or fragments thereof comprising the three CDRs of a heavy chain variable region without the associated light chain portion, (3) nanobodies comprising a single Ig domain from a non-human species or other specific single domain binding modules; and multispecific or multivalent structures formed by antibody fragments. In an antibody fragment comprising one or more heavy chains, the heavy chain may comprise any of the invariant domain sequences found in the non-Fc region of an intact antibody (e.g., CH1 in an IgG isotype), and/or may comprise any of the hinge region sequences found in an intact antibody, and/or may comprise a leucine zipper sequence fused to or located within a hinge region sequence or invariant domain sequence of the heavy chain, and (5) isolated complementarity determining regions (CDRs).

The terms "phagocytic cells," "phagocytes," and "apoptotic cells" are used interchangeably in this manual to refer to cells that are capable of phagocytosis. There are four main types of phagocytes: macrophages, mononuclear cells (tissue cells and monocytes), polymorphonuclear leukocytes (neutrophils), and dendritic cells.

As used herein, "chimeric" refers to a molecule that includes sequences from two different molecules.

The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "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 may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to their carboxyl termini. The residue numbering of the Fc region is the same as the EU index 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 includes two constant structural domains, CH2 and CH3.

As used in this specification, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. In addition, preferred FcRs are receptors that bind to IgG antibodies (gamma receptors) and include FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains.

A "native sequence Fc region" or "Wile-type Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution in the native sequence Fc region or the Fc region of the parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, compared to the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc regions of the present specification preferably have at least about 80% sequence identity with a native sequence Fc region and/or the Fc region of a parent polypeptide, and most preferably have at least about 90% sequence identity, 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 thereto.

As used herein, an "effective dose" or "effective amount" of a drug, compound or pharmaceutical composition is an amount sufficient to produce a beneficial or desired result. For preventive use, beneficial or desired results include results such as eliminating or reducing the risk, reducing the severity or delaying the onset of a disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes that occur during the course of disease development. For therapeutic use, beneficial or desired results include clinical results, such as inhibiting, suppressing or reducing the increase in the level of a substance, reducing, removing, clearing elevated antigenic substances or reducing them to their normal levels, isolating or increasing soluble substances circulating in biological fluids, reducing one or more symptoms (biochemical, histological and/or behavioral) caused by the disease, including its complications and intermediate pathological phenotypes that appear during the development of the disease, improving the quality of life of patients with the disease, reducing the dosage of other drugs required to treat the disease, enhancing the effect of another drug, delaying the progression of the disease, and/or prolonging the survival of the patient. The effective dose can be administered once or multiple times. For the purpose of the present invention, the effective dose of a drug, compound or drug composition is an amount sufficient to directly or indirectly complete preventive treatment or therapeutic treatment. As understood in a clinical setting, an effective dose of a drug, compound, or drug composition may or may not be achieved in conjunction with another drug, compound, or drug composition. Thus, an "effective dose" may be considered in the context of administering one or more therapeutic agents, and a single drug may be considered to be administered in an effective amount if the desired result may be or has been achieved in conjunction with one or more other drugs.

As used in this specification, "treatment" or "treatment" is an approach intended to obtain beneficial or desired results, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: preventing, inhibiting or reducing the formation of deposits of substances, reducing, removing or clearing deposits of antigenic substances, improving cognition, reversing or slowing cognitive decline, isolating soluble substances circulating in biological fluids, reducing substances in tissues (including soluble, oligomeric and deposited), inhibiting, slowing and/or reducing the increase or elevation of antigenic substance levels in tissues, inhibiting, slowing and/or reducing the toxic effects of substance peptides in tissues, reducing symptoms caused by diseases, improving the quality of life of patients with diseases, reducing the dosage of other drugs required to treat diseases, delaying the progression of diseases, and/or prolonging the survival of patients. The tissues may include the brain of an individual.

The term "development" of a disease refers to the onset and/or progression of a disease in an individual. As described in this specification, standard clinical techniques can be used to detect disease progression. However, development also refers to progression of a disease that may not be detected initially. For the purposes of the present invention, progression refers to the biological course of the disease state, which in this case is determined by standard neurological examination, patient interview, or can be determined by more specialized tests. Various of these diagnostic tests include, but are not limited to, neuroimaging, detection of changes in levels of specific proteins in serum or cerebrospinal fluid (e.g., any one of the antigenic substances listed in Table 1 or a combination thereof), computer tomography (CT), and magnetic resonance imaging (MRI). "Development" includes occurrence, relapse, and attack. As used in this specification, "onset" or "occurrence" of a disease includes initial attack and/or relapse.

As used in this specification, "delaying" the development of a disease means delaying, hindering, slowing, delaying, stabilizing and/or postponing the development of a disease. This delay can be of varying lengths of time, depending on the history of the disease and/or the individual being treated. It will be apparent to one of ordinary skill in the art that a sufficient or significant delay may actually include prevention, i.e., that the individual will not develop the disease. For example, a method of delaying the development of a disease is a method that reduces the probability of disease development within a given time frame and/or reduces the extent of the disease within 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.

As used in this specification, "vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with a cation condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and specific eukaryotic cells such as production cells.

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

As used in this specification, "expression control sequence" refers to a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

As used in this specification, "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient, keeps the ingredient biologically active and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or regular saline (0.9%). Compositions containing such carriers are formulated by known conventional methods.

[TAM receptors]

TAM receptors (Tyro3, Axl and Mer) belong to the family of receptor tyrosine kinases, which play an important role in hemostasis and inflammation. In addition, they affect cell proliferation, survival, adhesion and migration. TAM receptors contain 2 immunoglobulin-like and 2 fibronectin type III repeat sequences in tandem in their extracellular domain. This is linked to a single-pass transmembrane domain and a cytoplasmic protein tyrosine kinase.

TAM receptors enhance the phagocytosis of apoptotic cells, also known as efferocytosis.

The Axl protein comprises 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 full-length Axl is 104 kDa, post-translational modifications of the extracellular domain produce two modified forms with molecular weights of 120 kDa and 140 kDa. Potential N-linked glycosylation sites include Asn43, Asn157, Asn198, Asn339, Asn345, and Asn401. In various embodiments of the present disclosure, the term "Axl" or "Axl receptor" or "Axl protein" includes full-length Axl of 104 kDa, post-translationally 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 Axl 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.

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

Human MerTK contains 999 amino acid residues (accession numbers Q12866, NP_006334.2). The mRNA and genomic DNA sequences are available under accession numbers AAB60430.1 and AAG33129.1, respectively. Various natural variants and post-translational modifications as well as fragments have been reported. (www.uniprot.org/uniprotkb/Q12866/entry, last accessed on 11 June 2023).

The 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 Nos. AAA19236.1, BAA04467.1, AAC50070.1, BAA21781.1, AA49368.1, AAH51756.1, and CAA51396.1. Several natural variants and post-translational modifications have been reported (www.uniprot.org/uniprotkb/Q06418/entry#sequences, last accessed 11 June 2023).

The cells expressing TAM receptors can be at least one type of professional phagocyte, at least one type of non-professional phagocyte, or a combination thereof. Here, professional phagocytes refer to cells whose main function is to clear dead cells and accumulated debris by phagocytosis, examples of which include macrophages, neutrophils, dendritic cells, and mast cells. Macrophages are usually found in various tissues that can serve as a pathway for infection, and in many cases they are referred to by different tissue names, including, for example, adipose tissue macrophages, bone marrow or blood monocytes, liver Kupffer cells, lymph node sinus tissue cells, alveolar macrophages, connective tissue tissue cells or giant cells, microglia of the central nervous system, placental Hofbauer cells, mesangial cells in the kidney glomeruli, osteoclasts of bones, epithelioid cells of granulomas, red pulp macrophages of the spleen, peritoneal macrophages of the peritoneal cavity, Peyer's patches, and mitochondrial macrophages. patch) of lysozyme macrophages (macrophages expressing lysozyme), etc. On the other hand, non-professional phagocytes refer to cells that mainly perform specific functions of the tissue in which the phagocyte is located, but can perform phagocytosis when necessary. Examples are epithelial cells, endothelial cells, fibroblasts, mesenchymal cells, some tissue-specific cells such as astrocytes or oligodendrocytes of the central nervous system, retinal Muller's jelly, hepatocytes, myosatellites, testicular supporting cells, etc., and some lymphocytes such as natural killer cells, large granulocytes, eosinophils, basophils, B cells, etc. Fusion molecules according to the present disclosure are capable of inducing phagocytosis in phagocytes specific for tissues in which the target substance to be removed is increased. For example, when the amount or expression of antigenic proteins in the brain is elevated and needs to be removed, phagocytosis can be induced in astrocytes, microglia, oligodendrocytes, or a combination thereof. For example, phagocytosis can be induced by topically administering the fusion molecules according to the present disclosure to the tissue or by manipulating cells in the tissue to express and secrete the fusion molecules.

[The first region comprising a sequence capable of binding to a TAM receptor]

TAM receptors can be activated by their ligands, growth arrest specificity 6 protein (Gas6) and protein S (ProS1), which are members of the vitamin K-dependent protein family.

In exemplary embodiments, the first region capable of binding to a TAM receptor can include, consist of, or consist essentially of one or more TAM ligands.

The TAM ligand, protein S, contains an amino-terminal γ-carboxyglutamic acid (Gla) domain, followed by a thrombin-sensitive loop region and four epidermal growth factor-like domains ending in a carboxyl terminus (C-terminus), composed of two laminin G repeat sequences, which together include the sex hormone-binding globulin domain (right panel of Figure 1A). The C-terminal region is sufficient for TAM receptor binding and phosphorylation. Gas6 is a 75 kDa vitamin K-dependent protein with high structural homology (approximately 42%) to protein S and a modular organization identical to that shown in Figure 1A.

In addition to Gas6 (SEQ ID NO: 7) and ProS1 (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 (Tulp1) (accession nos. AAB53494.1, AAB53499.1, CH471064.2, EAW68634.1, BC075031.2, AAH75031.1, BC075032.2, AAH75032.1, NP_003311.2, NP_813977.1, 1S31_A), and tubby-like protein 1 (Tulp1) (accession nos. AAB53494.1, AAB53499.1, CH471064.2, EAW68634.1, BC075031.2, AAH75031.1, BC075032.2, AAH75032.1, NP_003311.2, NP_813977.1, 1S31_A). Tubby and Gal-3 specifically bind to Mer, while Tulp1 can activate all three TAM receptors.

Gas6, one of the ligands of TAM receptors, has been reported to show the highest affinity for Axl compared to Tyro3 or Mer. Human Gas6 contains 678 amino acids (SEQ ID NO: 7), has a γ-carboxyglutamic acid (Gla) domain, four epidermal growth factor (EGF)-like domains, and two laminin G-like (LG) domains ( FIG. 1A , right). Various subtypes of GAS6 have been reported. For example, S6L, G8R, G8V, R14H, L18Q subtypes have been reported, and these subtypes are included in the present disclosure.

In embodiments, the first region capable of binding to a TAM receptor may be a TAM receptor agonist. TAM receptor agonists include reagents that significantly increase the biological activity of TAM receptors in cells, such as reagents that specifically bind to and activate TAM receptors. For example, TAM receptor agonists can increase the biological activity of TAM receptors 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 for measuring this activity are known in the art. In some instances, the increase in biological activity is indicated by an increase in the expression of Tyro3, Axl or Mer or a combination thereof (at DMA, RNA or protein levels). In other examples, the increase in biological activity is indicated by changes in downstream effects, such as an increase in TAM autophosphorylation, a decrease in TLR-induced cytokine production, a decrease in TLR-induced MAP kinase-activated stimulation, a decrease in TLR-induced NF-kB activation, or an increase in SOCS1 and SOCS3 expression. Methods for detecting such changes in expression or activity (quantitative in some examples) are conventional and may include western blotting, ELISA, flow cytometry, northern blotting, PCR, RT-PCR, etc. In embodiments, the TAM receptor activated by the first region according to the present disclosure may be Axl or Mer.

In an embodiment, the first region capable of binding to a TAM receptor may comprise Gas6 protein or an active fragment thereof, or consist of Gas6 protein or an active fragment thereof, or consist essentially of Gas6 protein or an active fragment thereof. The term "active fragment" used in this specification refers to a fragment capable of binding to a TAM receptor, particularly an Axl receptor. For example, the active fragment of Gas6 protein can include 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, or consist of them, or consist essentially of them. For example, the active fragment of the ProS1 protein may include, consist of, or essentially consist 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 includes sequences having 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% sequence identity to any of the sequences in SEQ ID NOs. SEQ ID NOs: 8-23 exhibit at least 85% sequence identity to SEQ ID NO: 1 (LG-1 domain of Gas6). SEQ ID NOs: 24-33 exhibit at least 85% sequence identity to SEQ ID NO: 2 (LG-2 domain of Gas6). SEQ ID NOs: 35-45 exhibit at least 85% sequence identity to SEQ ID NO: 3 (LG-1 domain of ProS1). SEQ ID NOs: 46-62 show at least 85% sequence identity to SEQ ID NO: 4 (LG-2 domain of ProS1). SEQ ID NOs: 63-87 show at least 85% sequence identity to SEQ ID NO: 5 (LG domain of Gas6). SEQ ID NOs: 88-113 show at least 85% sequence identity to SEQ ID NO: 6 (LG domain of ProS1).

In other embodiments, the first region may include, consist of, or consist essentially of: a variable region or CDR of an anti-Axl antibody or full-length anti-Axl antibody whose effector function, particularly Fc receptor binding function, is abolished or removed. The antibody or antigen-binding fragment may bind to the extracellular domain of Axl, for example expressed on the surface of phagocytic cells and induce internalization and phagocytosis without involving an inflammatory response, particularly an Fc-mediated inflammatory response. Non-limiting examples of anti-Axl antibodies may include, for example, those described in WO2017200493A1, WO2015193430A1, WO2011159980A1, WO2016097370A1, WO2012175691A1, WO2015193428A1, WO2010131733A1, WO2017220695A1, WO2010130751A1, WO2016166302A1, WO2017009258A1, WO2016005593A1, US20190134193A1, etc., all of which are incorporated herein by reference in their entirety. According to embodiments of the present disclosure, the variable region, CDR or scFv, F(ab) or F(ab') of these anti-Axl antibodies can be used as the first region of the fusion molecule.

In other embodiments, the first region may include, consist of, or consist essentially of: a variable region or CDR of an anti-MerTK (Mer tyrosine kinase) antibody or full-length anti-MerTK antibody whose effector function, particularly Fc receptor binding function, is eliminated or removed. The antibody or antigen-binding fragment may bind to the extracellular domain of MerTK, for example, expressed on the surface of phagocytes and induce internalization and phagocytosis without involving an inflammatory response, particularly an Fc-mediated inflammatory response. Non-limiting examples of MerTK antibodies may include, for example, those described in WO2016106221A1, WO2020076799A1, WO2020176497A1, etc., the contents of all of which are incorporated herein by reference in their entirety. According to embodiments of the present disclosure, the variable region, CDR or scFv, F(ab) or F(ab') of these anti-MerTK antibodies can be used as the first region of the fusion molecule.

In other embodiments, the first region may include, consist of, or consist essentially of: a variable region or CDR of an anti-Tyro3 antibody or full-length anti-Tyro3 antibody whose effector function, especially Fc receptor binding function, is eliminated or removed. The antibody or antigen-binding fragment can bind to the extracellular domain of Tyro3, for example, expressed on the surface of phagocytes and induce internalization and phagocytosis without involving an inflammatory response, especially an Fc-mediated inflammatory response. Non-limiting examples of anti-Tyro3 antibodies may include, for example, those described in WO2016166348A1, etc., all of which are incorporated herein by reference. According to the embodiments disclosed herein, the variable regions, CDRs or scFvs, F(ab) or F(ab′) of these anti-Tyro3 antibodies can be used as the first region of the fusion molecule.

The peptide comprising any of the above-mentioned SEQ ID NOs includes not only the amino acid sequence of the peptide, but also its amino acid sequence variant. The term "sequence variant" refers to a protein having a sequence in which one or more amino acid residues are different from the amino acid sequence. As long as the activity of the fusion molecule remains unchanged, any truncation, deletion, insertion, substitution or combination thereof in the final structure of the protein is possible. An example of a sequence variant is a form in which the amino acid residues at the non-essential sites of activity are truncated or deleted, or the amino acid residues at the sites important for autoinhibition are substituted. In some cases, the sequence variant can also be modified by phosphorylation, glycosylation, methylation, farnesylation, etc. When the function and/or stability (thermal stability, pH stability, structural stability, etc.) and/or solubility of the protein are improved by mutations in the amino acid sequence, these sequence variations and modifications are more preferred.

The method for mutating an amino acid sequence is based on the following method: a method for generating a nucleic acid molecule comprising a nucleotide sequence corresponding to the amino acid sequence to be mutated by mutating a nucleotide sequence encoding a protein, and the method for obtaining a gene encoding a protein can be performed in vivo or in vitro using any mutation technique 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. USA ., 83:710, 1986), TAB linker (Pharmacia), PCR technology (Higuchi, 1989, "Using PCR to Engineer DNA" in PCR Technology: Principles and Applications for DNA Amplification , H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70) etc.

The amino acid sequence insertion includes amino- and/or carboxyl terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues or antibodies fused to epitope markers. Other insertion variants of antibody molecules include fusions of enzymes or polypeptides to the N- or C-terminals of antibodies, which increase the serum half-life of antibodies.

Examples of modified polypeptides include: polypeptides having conservative substitutions of amino acid residues; polypeptides having one or more amino acid deletions or additions that do not significantly and adversely alter functional activity; or polypeptides using chemical analogs.

Substitution variants have at least one amino acid residue in the removed antibody molecule, and a different residue is inserted in its place. The most interesting positions for substitution mutations include hypervariable regions, but FR changes are also considered. Conservative substitutions are shown under the heading of "conservative substitutions" in Table 2. If such substitutions result in changes in biological activity, more changes can be introduced and the products screened, named "exemplary substitutions" in Table 2, or further described below with reference to amino acid classes.

Table 2: Amino Acid Substitutions Original residue Conservative substitution Exemplary substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn(N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe; norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; 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 Val (V) Leu Ile; Leu; Met; Phe; Ala; nor-leucine

Substantial modification of the biological properties of an antibody is achieved by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the region of the substitution, e.g., a folded or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the bulk of the side chains. Based on common side-chain properties, naturally occurring residues are classified as: - nonpolar: norleucine, Met, Ala, Val, Leu, Ile; - polar but uncharged: Cys, Ser, Thr, Asn, Gln; - acidic (negatively charged): Asp, Glu; - basic (positively charged): Lys, Arg; - residues affecting chain orientation: Gly, Pro; and - aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are achieved by exchanging a member of one of these classes for another. Any cysteine residue that is not involved in maintaining the proper conformation of the antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

The scope of amino acid modification can be from changing or modifying one or more amino acids to completing the redesign of a region (such as a variable region). The change of the variable region can change the binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are carried out in the CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are carried out in the CDR domain. In yet another other embodiment, the CDR domain is CDR H3 and/or CDR L3.

[Targeting inflammation-related substances and immune diseases or immune-mediated diseases]

Targeted inflammation-related substances may include: one or more autoantigens, their autoantibodies, or complexes of autoantigens and their autoantibodies; one or more immune cell surface molecules, including co-stimulatory molecules and receptors; one or more complements; one or more trending factors; one or more cytokines; one or more cell adhesion molecules; or combinations thereof.

Non-limiting exemplary antigenic substances that trigger, cause or induce undesirable or unwanted immune responses and related immune diseases (immune-mediated diseases) are listed above in Table 1. Non-limiting examples of immune cell surface molecules are disclosed above, including co-stimulatory molecules and receptors, complements, chemokines, cytokines and cell adhesion molecules.

Fusion molecules according to embodiments of the present disclosure reduce, eliminate or remove antigenic substances in a subject, or enhance the elimination or removal of antigenic substances in a subject, thereby being useful for treating or preventing immune diseases or disorders, cardiovascular diseases or disorders, metabolic diseases or disorders, or proliferative diseases or disorders. In specific embodiments, fusion molecules according to the present disclosure inhibit, reduce, eliminate or remove inflammation-related substances in a subject, or enhance the elimination or removal of inflammation-related substances in a subject, thereby being useful for treating or preventing immune diseases or disorders, cardiovascular diseases or disorders, metabolic diseases or disorders, or proliferative diseases or disorders. Immune diseases or disorders may be autoimmune diseases or inflammatory diseases exemplified in the present disclosure.

In certain embodiments, the immune disease or disorder is multiple sclerosis, myasthenia gravis, type 1 diabetes, type 2 diabetes, rheumatoid arthritis, neuromyelitis optica, autoimmune encephalitis, fatty liver, endometriosis, inflammatory bowel disease, asthma, obesity, ankylosing spondylitis, antiphospholipid antibody syndrome, chronic recurrent multifocal osteomyelitis, gout, allergic purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus erythematosus SLE, juvenile scleroderma, juvenile vasculitis, Kawasaki disease, lupus erythematosus (systemic lupus erythematosus), mixed connective tissue disease, dermatomyositis, post-streptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren's syndrome, spondylarthritis/spondylosis, systemic juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, chylous diarrhea, thrombotic thrombocytopenic purpura (iTTP), etc.

The fusion molecules described herein can also be used to treat cardiovascular diseases or disorders, such as atherosclerosis, endocarditis, hypertension or peripheral ischemic disease.

The fusion molecules described in this specification can be used to treat or prevent, inhibit, slow the progression of, or alleviate symptoms associated with, immune diseases or disorders, cardiovascular diseases or disorders, metabolic diseases or disorders, or proliferative diseases or disorders. Immune disorders include inflammatory diseases or disorders, and autoimmune diseases or disorders. Although inflammation or inflammatory responses are normal and protective responses of the host to injury, inflammation can cause undesirable damage. For example, atherosclerosis is at least in part a pathological response to arterial injury and the subsequent inflammatory cascade. Cardiovascular diseases or disorders that can be treated can include diseases and disorders that are also considered immune diseases/disorders, including, for example, atherosclerosis, endocarditis, hypertension, or peripheral ischemic disease. Metabolic diseases or disorders include diabetes, obesity, and diseases and disorders associated with increased or elevated levels of antigenic substances.

Allergen is another antigen, and tolerance to its immune response is also desirable. Even in the case of an unknown disease in which the pathogenic autoantigen is unknown, antigens in anatomically adjacent sites can also be used to induce bystander inhibition. For example, autoantibodies to collagen are observed in rheumatoid arthritis, and therefore, the gene encoding collagen or collagen can be a target inflammation-related substance to be removed or removed or reduced by administering a fusion molecule. In this case, an adaptor that specifically binds to the gene encoding collagen or an antibody that binds to collagen or its fragment can be used as the second region of the fusion molecule. In addition, a fusion protein comprising a second region that is bound to a β cell autoantigen can be used to prevent the development or treatment of type 1 diabetes (see, for example, Bach and Chatenoud (2001) Ann Rev Immunol 19: 131-161).

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are observed in autoimmune encephalomyelitis and many other CNS diseases as well as multiple sclerosis. Therefore, administration of a fusion molecule comprising an anti-MOG antibody or fragment thereof as a second region can treat multiple sclerosis and related autoimmune diseases of the central nervous system.

In general, immune responses include (1) humoral responses, in which antigen-specific antibodies are produced by differentiated B lymphocytes known as plasma cells, and (2) cell-mediated responses, in which various types of T lymphocytes eliminate the antigen through a variety of mechanisms. For example, helper T cells that are capable of recognizing specific antigens can respond by releasing soluble mediators such as cytokines to recruit additional cells of the immune system to participate in the immune response. In addition, cytotoxic T cells that are also capable of recognizing specific antigens can respond by binding to and destroying or damaging cells or particles carrying the antigen.

The immune response in the host or subject can be determined by any of the well-known immunological methods described in this specification, and those skilled in the art are also very familiar with these methods. Such assays include, but are not necessarily limited to, in vivo or in vitro assays of soluble antibodies, soluble mediators such as cytokines (e.g., IFN-γ, IL-2, IL-4, IL-10, IL-12, IL-6, IL-23, TNF-α and TGF-β), lymphokines, chemokines, hormones, growth factors, etc., as well as other soluble small peptides, carbohydrates, nucleotides and/or lipid mediators; Changes in the activation state of cells determined by changes in the functional or structural properties of cells, such as cell proliferation, changes in motility, induction of specialized activities such as specific gene expression or cytolytic behavior; cell maturation, such as maturation of dendritic cells in response to stimulation; changes in the relationship between Th1 and Th2 responses; cell differentiation of cells of the immune system, including changes in the surface antigen expression spectrum or the occurrence of apoptosis (programmed cell death). Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual. The Comprehensive Sourcebook of Techniques, 1998).

The level of cytokines can be determined according to the methods described in this specification and practiced in the art, including ELISA, ELISPOT and flow cytometry (to measure intracellular cytokines). Proliferation and selective expansion of immune cells caused by antigen-specific induction or stimulation of immune response can be measured by the following methods: isolating lymphocytes such as spleen cells or cells from lymph nodes, stimulating cells with antigen, and measuring cytokine production, cell proliferation, and/or cell viability, such as by incorporation of tritiated thymidine or non-radioactive assays, such as MTT assays, etc. The effect of the fusion polypeptide described in the present specification on the balance between Th1 immune response and Th2 immune response can be determined by, for example, measuring the levels of Th1 cytokines such as IFN-γ, IL-12, IL-2 and TNF-β, and type 2 cytokines such as IL-4, IL-5, IL-9, IL-10 and IL-13.

In certain embodiments, antigenic substances and associated immune diseases do not include substances whose abnormal accumulation or aggregation in living tissues is characteristic of or associated with diseases such as neurological diseases or disorders.

[Second region of fusion molecule]

The second region that specifically binds to the target substance can be selected from antibodies, antigen-binding fragments thereof, antibody-like proteins, peptides, aptamers and soluble receptors, and is not particularly limited as long as the second region specifically binds to the target substance.

Here, the antibody or its antigen-binding fragment can be selected from, for example: i) immunoglobulins, such as IgG1, IgG2, IgG3 and IgG4; ii) natural 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, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, pentaspecific antibodies, etc. The antibody or its antigen-binding fragment can be, for example, a Mab, Fab or single-chain variable fragment (scFv) of an antibody that specifically binds to a corresponding target substance, or six complementary determining regions (CDRs) from an antibody. That is, the protein or antigen-binding fragment thereof that specifically binds to the target substance contains a part necessary for the activity of specifically binding to the target substance, and there is no particular limitation on its type or range, as long as the protein or antigen-binding fragment thereof is linked to the first region and does not cause inflammatory response and synaptic damage. For example, the target substance may be β-amyloid protein, in which case the protein or antigen-binding fragment thereof that specifically binds to the target substance may contain aducanumab or a single-chain variable fragment thereof. The second region contains Mab, Fab or a single-chain variable fragment based on six complementary determining regions (CDRs) from commercially available antibodies such as aducanumab, semorinemab and cinpanemab.

The antibody or its antigen-binding fragment may not contain an Fc region, and preferably may contain an Fc region receptor that does not bind to an Fc receptor (particularly an Fcγ receptor). The Fc region variant can be used to improve performance such as purification. For example, WO2012130831 and USP8753628 disclose Fc variants with reduced affinity for human FcyRIIIA and/or FcyRIIA and/or FcyRI by amino acid substitution compared to the Fc region of IgG, the entire contents of which are incorporated into this specification by reference. The Fc region may be glycosylated or deglycosylated.

Antibody-like proteins refer to protein scaffolds that are able to specifically bind to a target substance such as an antibody. Antibody-like proteins can be designed to have a size of about 2 kDa to 20 kDa, which is smaller than antibodies (average about 150 kDa), thereby targeting binding sites that antibodies cannot reach. Antibody-like proteins are known to be more stable than antibodies at high temperatures and are easier to synthesize using non-mammalian cells such as viruses and yeast or chemically synthesized than antibodies.

As used in this specification, the term "aptamer" refers to a single-stranded DNA (ssDNA) or RNA with high specificity and affinity for a specific substance. Aptamers have very high affinity for specific substances, are stable, can be synthesized in a relatively simple manner, can be modified in a variety of ways to increase their binding affinity, and can target cells, proteins, and even small organic substances. Therefore, compared with antibodies that have been developed, aptamers are characterized by having very high specificity and stability. In addition, aptamers can be produced by the known SELEX (systematic evolution of ligands by exponential enrichment) method. As such an aptamer, for example, an aptamer that specifically binds to any of the listed target substances can be produced by the known SELEX (systematic evolution of ligands by exponential enrichment) method, and then linked to the first region, thereby producing a fusion molecule according to the present invention.

The aptamers disclosed herein are not limited as long as they can specifically bind to any of the listed target substances, and unless otherwise specified, the base group used for the aptamer can be selected from A, G, C, U and their deoxy forms.

In addition, the aptamer may be modified by the connection of at least one selected from polyethylene glycol (PEG), inverted deoxythymidine (idT), locked nucleic acid (LNA), 2′-methoxy nucleoside, 2′-amino nucleoside, 2′F-nucleoside, amine linker, thiol linker and cholesterol at the 5′-terminal region, the middle region, the 3′-terminal region or both ends thereof to increase its stability. Inverted deoxythymidine (idT) is a molecule generally used to prevent nuclease degradation of aptamers having weak nuclease resistance. In the case of a nucleic acid unit, the 3′-OH of the previous nucleotide is linked 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 is linked to the 3′-OH of the next unit, so that the 5′-OH is exposed instead of the 3′-OH. Therefore, idT is a molecule that has the ability to inhibit degradation by 3′ exonuclease (a type of nuclease).

Since the fusion molecules according to the present disclosure induce phagocytosis by interacting with TAM receptors, phagocytosis can be induced in cells expressing TAM receptors. Phagocytosis generally refers to the uptake of cells or particles with a size of 0.5 μm or more, and includes the process of confining, engulfing and degrading cells or particles. In this case, phagocytosis forms a phagosome that surrounds the internalized cell or particle, and includes degradation within the phagolysosome through fusion of the phagosome and the lysosome. In phagocytosis, the process of cell death by apoptosis or necrosis is also called efferocytosis.

Non-limiting representative examples of second regions and their targeting are shown in Table 3. The entire contents of the references listed in Table 3 are incorporated into this specification by reference. It should be understood by those skilled in the art that not only antibodies but also ligands of the listed target substances can serve as second regions. For example, the second region capable of binding to TGFBR1 (transforming growth factor beta receptor 1) can be TGF beta.

table 3 Abbreviation Diseases associated with or characterized by abnormal accumulation of target substances Target substance (accession number, UniProt) Second area Exemplary References Region 2 VH (if the second region is an antibody) Region 2 VL (if the second region is an antibody) CD20 Multiple sclerosis, rheumatoid arthritis P11836 Rituximab QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA (SEQ ID NO: 145) Rituximab QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK (SEQ ID NO: 146) Critical Reviews in Oncology/Hematology 64 (2007) 210–225 CD19 Immunoglobulin G4-related diseases, systemic lupus erythematosus, systemic sclerosis P15391 EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGSRVFDYWGQGTLVTVSS (SEQ ID NO: 147) DIVMTQSPATLSLSPGERATLSCRSSKSLLNSNGNTYLYWFQQKPGQSPQLLIYRMSNLASGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPFTFGAGTKLEIK (SEQ ID NO: 148) US11618788B2 C3 Lupus nephritis, myasthenia gravis, antibody-mediated rejection P01024 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARNPFYVGVFDVWGQGTLVTVSSA (SEQ ID NO: 149) DIVLTQPPSVSGAPGQRVTISCSGSSSNIGSNYVSWYQQLPGTAPKLLIYDNNQRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCSAWDGDMLVRVFGGGTKLTVLG (SEQ ID NO: 150) WO2021159939A1 C5 Myasthenia gravis, amyotrophic lateral sclerosis, ANCA vasculitis P01031 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDEYMNWVRQAPGQSLEWMGYINPNNGGADYNOKFOGRVTMTVDOSISTAYMELSRLRSDDTAVYFCARLGYSNPYFDFWGQGTLVTVSS (SEQ ID NO: 151) DIVLTOSPDSLAVSLGERATINCKASQDVNTAVAWYOQKPDQSPKLLIYWASTRHTGVPARFTGSGSGTDYTLTISSLQAEDVAVYFCOQHHVSPWTFGGGTKVEIK (SEQ ID NO: 152) US9932395 B2 C5AR1 Polyangiitis, ANCA vasculitis, Chargar-Strauss syndrome, systemic lupus erythematosus P21730 QVQLRQPGAELVRPGASVKLSCKASAYTFTSYWMNWFKQRPEQGLEWIGRIDPYSDSETRYNQKFEDRALLTVDKSSSTAYMQLSSLTSEDSAVYYCARFVIPSGGFAYWGQGTLVTVSA (SEQ ID NO: 153) DVVMTQTPLSLPVSLGDQASISCRSSQSPVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPTFGSGTKLEIK (SEQ ID NO: 154) WO2023129870A2 CD52 Multiple sclerosis, graft-versus-host disease P31358 EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSS (SEQ ID NO: 155) DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNAKTYLNWVLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCVQGSHFHTFGQGTKLEIKk (SEQ ID NO: 156) US9708407B2 FCGRT Myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus P55899 Rozanolixizumab EVPLVESGGGLVQPGGSLRLSCAVSGFTFSNYGMVWVRQAPGKGLEWVAYIDSDGDNTYYRDSVKGRFTISRDNAKSSLYLQMNSLRAEDTAVYYCTTGIVRPFLYWGQGTLVTVS (SEQ ID NO: 157) Rozanolixizumab DIQMTQSPSSLSASVGDRVTITCKSSQSLVGASGKTYLYWLFQKPGKAPKRLIYLVSTLDSGIPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQGTHFPHTFGQGTKLEIK (SEQ ID NO: 158) US10233243B2 IL1A Rheumatoid arthritis, systemic sclerosis, hidradenitis suppurativa, ulcerative colitis P01583 Bermekimab QVQLVESGGGVVQPGRSLRLSCTASGFTFSMFGVHWVRQAPGKGLEWVAAVSYDGSNKYYAESVKGRFTISRDNSKNILFLQMDSLRLEDTAVYYCARGRPKVVIPAPLAHWGQGTLVTFSS (SEQ ID NO: 159) Bermekimab IQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYEASNLETGVPSRFSGSGSGSDFTLTISSLQPEDFATYYCQQTSSFLLSFGGGTKVEHKR (SEQ ID NO: 160) KR20230004638A IL1B Gangrenous pyoderma, pulmonary sarcoidosis, osteoarthritis pain P01584 QVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCARNRYDPPWFVDWGQGTLVTVSS (SEQ ID NO: 161) DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPWTFGQGTKLEIK (SEQ ID NO: 162) US7531166B2 IL1R1 Rheumatoid arthritis, cerebral hemorrhage, interstitial cystitis P14778 MEFGLSWVFLVALLRGVQC/QVQLVESGGGVVQPGRSLRLSCAVSGFTFSNYGMHWVRQAPGKGLEWVAALieWNDGENKHHAGSVRGRFTLieSRDNSKNTLYLQMNSLRAEDTAVYYCARGRYFDWLLFEYWGQGTLVTVSS (SEQ ID NO: 163) MEAPAQLLFLLLLWLPDTTG/EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFGGGTKVEIK (SEQ ID NO: 164) AU2008207483B2 IL6 Rheumatoid arthritis, giant lymphadenopathy, antibody-mediated rejection, amyotrophic collateral sclerosis P05231 EVKLEESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTREDYYGYPDYWGQGTTLTVSS (SEQ ID NO: 165) DIVLTQSPASLAVSLGQRATISCRASESVDNFGISFMNWFQQKPGQPPKLLIYVASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIK (SEQ ID NO: 166) US8536308B2 IL6R Rheumatoid arthritis, cytokine release syndrome, giant lymphadenopathy P08887 QVQLQESGPGLVKPSETLSLTCAVSGHSISHDHAWSWVRQPPGEGLEWIGFISYSGITNYNPSLQGRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGEGTLVTVSS (SEQ ID NO: 167) DIQMTQSPSSLSASVGDSVTITCQASTDISSHLNWYQQKPGKAPELLIYYGSHLLSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCGQGNRLPYTFGQGTKVEIE (SEQ ID NO: 168) US20220280532A1 IL6ST Nonalcoholic steatohepatitis (NASH); Nonalcoholic fatty liver disease (NAFLD) P40189 QVQLQESGPGLVKPSETLSLTCAVSGHSISHDHAWSWVRQPPGEGLEWIGFISYSGITNYNPSLQGRVTISRDNSKNTLYLQMNSLRAEDTAVYYCARSLARTTAMDYWGEGTLVTVSS (Serial number: 169) DIQMTQSPSSLSASVGDSVTITCQASTDISSHLNWYQQKPGKAPELLIYYGSHLLSGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCGQGNRLPYTFGQGTKVEIE (Serial number: 170) US20210017286A1 IL17A Psoriasis, hidradenitis purulentis, non-alcoholic fatty liver disease (NAFLD), polymyalgia rheumatica (PMR) Q16552 EVKLVESGGGLVRPGGTLKLSCAASGFTFSSFDMSWGRQTPEKRLEWVAFMSSGGSTYYPDSVKGRFTISRDNVRNILYLQMISLRSEDTAMYYCARGERYGSYWGQGTLVTVSA (SEQ ID NO: 171) DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPFTFGSGTKLEIK (SEQ ID NO: 172) US10738112 B2 IL17C Psoriasis, atopic dermatitis Q9P0M4 EVQLLESGGGLVQPGGSLRLSCAASGFTVSDYAMHWVRQAPGKGLEWVSYIGGVGEGTQYAESVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFAIRYYGFDYWGQGTLVTVSS (SEQ ID NO: 173) SYELTQPPSVSVSPGQTASITCSGDKLGDKYAYWYQQKPGQSPVLVIYQDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQVFTFPLVTTVFGGGTKLTVLGQ (SEQ ID NO: 174) US10259869B2 IL17RA Plaque psoriasis, Psoriasis, Rheumatoid arthritis Q96F46 SEQ ID NO: 28 in US9073999B2 SEQ ID NO: 2 in US9073999B2 US9073999B2 IL17RC Rheumatoid arthritis, psoriasis, inflammatory bowel disease Q8NAC3 CN113817058B, CN113896793B TNFA Rheumatoid arthritis, Crohn's disease, hidradenitis suppurativa, ulcerative colitis, psoriasis P01375 Adalimumab EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS (SEQ ID NO: 176) Adalimumab DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK (SEQ ID NO: 177) Schröter et al., (2015) A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display, mAbs, 7:1, 138-151 TNFRSF1A, TNFR1 Multiple sclerosis, psoriasis, NASH, inflammatory bowel disease P19438 QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDFYINWVRQAPGQGLEWIGEIYPYSGHAYYNEKFKARVTITADKSTSTAYMELSSLRSEDTAVYYCARWDFLDYWGQGTTVTVSS (SEQ ID NO: 178) DIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGNTYLHWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPYTFGGGTKVEIK (SEQ ID NO: 179) US8859739B2 TNFRSF1B, TNFR2 Graft-versus-host disease (GVHD), multiple sclerosis P20333 Rechain CDRs: DYIMH (SEQ ID NO: 180) WVDPEYGSTDYAEKFKKK (SEQ ID NO: 181) DDGSYSPFDY (SEQ ID NO: 182) Light chain CDR: QASQNINKYIA (SEQ ID NO: 183) YTSTLES (SEQ ID NO: 184) LQYVNLLT (SEQ ID NO: 185) US20210301028A1 IL4 Atopic dermatitis, venous ulcer of lower limbs P05112 QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYAIQWVRQAPGKGLEWVAVISYDGSKKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGRRGSFDYWGQGTLVTVSS (SEQ ID NO: 186) EIVLTQSPGTLSLSPGERATLSCRASQSVSTSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPTFGQGTKVEIK (SEQ ID NO: 187) US7186809B2/ EP 2292665 B1 IL4RA Asthma, atopic dermatitis, eosinophilic esophagitis, ulcerative colitis, systemic sclerosis, nasal polyps P24394 EVQLVESGGGLVQPGGSLRLSCAVSGFTFSSYAMSWVRQAPGKGLEWVSSITGGGGGIYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAKDRISITIRPRYFGLDFWGQGTTVTVSS (SEQ ID NO: 188) DIVMTQSPLSLPVTPGEPASISCRSSRSVLYGNGYNYLDWYLQKSGQSPQLLIYLGTNVAAGVPDRFSGSGSGTDFTLKISRVEAEDVGFYYCMQSLRTPYTFGQGTKLEIK (SEQ ID NO: 189) US020220081485A1 IL5 Asthma, hypereosinophilic syndrome, nasal polyps, Chug-Strauss syndrome P05113 QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYSVHWVRQPPGKGLEWLGVIWASGGTDYNSALMSRLSISKDNSKSQVFLKLNSLQTDDTAMYYCARDPPSSLLRLDYWGQGTTLTVSS (SEQ ID NO: 190) EKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLSISSVQAEDLAVYYCQNVHSFPFTFGSGTELEIK (SEQ ID NO: 191) US9834600B2 IL5RA Asthma, sinusitis, chronic obstructive pulmonary disease (COPD), Chargar-Strauss syndrome, nasal polyps Q01344 EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVIHWVRQRPGQGLAWMGYINPYNDGTKYNERFKGKVTITSDRSTSTVYMELSSLRSEDTAVYLCGREGIRYYGLLGDYWGQGTLVTVSS (SEQ ID NO: 192) DIQMTQSPSSLSASVGDRVTITCGTSEDIINYLNWYQQKPGKAPKLLIYHTSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGYTLPYTFGQGTKVEIK (SEQ ID NO: 193) Benralizumab CSF2RB, IL3RB, IL5RB Allergic asthma, chronic obstructive pulmonary disease (COPD) P32927 SEQ ID NO: 29 of WO 2023027177A1 SEQ ID NO: 30 of WO2023027177A WO2023027177A1 IL13 Atopic dermatitis, eosinophilic esophagitis, gastroenteritis P35225 QVOLVQSGAEVKKPGASVKVSCKASGYTFTNYGLSWVRQAPGQGLEWMGWISANNGDTNYGQEFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSSSSWARWFFDLWGRGTLVTVSS (SEQ ID NO: 194) SYVLTOPPSVSVAPGKTARITCGGNIIGSKLVHWYQQKPGQAPVLVIYDDGDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCOVWDTGSDPVVFGGGTKLTVL (SEQ ID NO: 195) US9856317B2 IL13RA1 Atopic dermatitis, allergic asthma P78552 EVQLQQSGGGLVQPGRSLNLSCAASGFTFNDYYMAWVRQAPKKGLEWVATIIYDGTRTYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCATPWGSWGQGTTVTVSS (SEQ ID NO: 196) DIQMTQSPSSMPASLGERVTISCRASQGISNFLNWYQQKADGTIKPLIYYTSNLQSAVPSRFSGSGSGTDYSLTISSLEPEDFAMYYCQQYDSSPWTFGGGTKLEITR (SEQ ID NO: 197) US20050154192A1 EP1449851A1 IFNG Systemic lupus erythematosus, arthritis, encephalomyelitis, vitiligo P01579 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDGSSGWYVPHWFDPWGRGTMVTVSS (SEQ ID NO: 198) NFMLTQPHSVSESPGKTVTISCTRSSGTIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDNSNHWVFGGGTKVTVLG (SEQ ID NO: 199) US7700098B2/ KR101380570B1 IFNGR1 Psoriasis, rheumatoid arthritis, allergic rhinitis P15260 CN114573713A IFNGR2 Atopic dermatitis, psoriasis, rheumatoid arthritis, allergic rhinitis P38484 WO2022031890A1 IL12A Plaque psoriasis, psoriatic arthritis, Crohn's disease, ulcerative colitis P29459 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSS (SEQ ID NO: 200) QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLG (SEQ ID NO: 201) US8765918B2 IL12RB1 Alzheimer's disease, rheumatoid arthritis P42701 SEQ ID NO: 6 of UA20220177567A1 SEQ ID NO: 7 of US20220177567A1 US20220177567A1 IL12RB2 Crohn's disease Q99665 WO2022031942A2 IL21 Graft-versus-host disease (GVHD), chylous diarrhea Q9HBE4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGLIDTSDVYTIYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYGPLAMDYWGQGTLVYVSS (SEQ ID NO: 202) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFHTLRTFGGGTKVEIK (SEQ ID NO: 203) US9394362B2 IL21R Systemic lupus erythematosus Q9HBE5 QVQLVQSGAEVKKPGSSVRVSCKASGGTFNIYSVSWVRQAPGQGLEWMGRIIPMRDIANYAQRFQGRVTLTADKSSGTAYMELRGLRSDDTAVYWCATLAGPLDSWGQGTLVT (SEQ ID NO: 204) SSELTQDPAVSVGLGQTVTITCQGGSLRQYYASWYQQKPGQAPVVVIYGKNKRPSGIPDRFSGTTSGNTASLTITGAQAEDEADYYCKSRDSSGNHPLYVFGAGTKLTVLGES (SEQ ID NO: 205) US7495085 B2 IL22 Ulcerative colitis, psoriasis, rheumatoid arthritis Q9GZX6 EVQLVQSGAEVKKPGASVKVSCQASGYTFSDYYIHWVRQTPGQGFEWMGWVNPDTGGTRYAOKFOGWVTMTRDMSNTTAYMELPRLRDDDTAVYYCARDLTGFDPFDIWGQGTLVTVSS (SEQ ID NO: 206) QSVLTQPPSVSVAPGKTATITCGGNNFRNKRVHWYQQRPGQAPVLVIYYDSDRPSGIPERFSGSRSGNTATLTISRVEAGDEADFYCOVWDSSTDRPLFGGGTKLTVLG (SEQ ID NO: 207) US7811567B2/ EP2327423B1 IL22RA1 Inflammatory bowel disease, nonalcoholic steatohepatitis (NASH), psoriasis, multiple sclerosis, atopic dermatitis Q8N6P7 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEVWSSIYNDGSNTAYSDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKVGGQGTQVTVSS (SEQ ID NO: 208) NFMLTQPSAVSVSLGQTAKITCQGGYYAHWYQQKPGQAPVLVIYGNNNRPSGIPERFSGSSSGNTATLTISGAQAEDEAEYYCQSGSSSANAVFGGGTHLTVL (SEQ ID NO: 209) US20230088269A1 TGFB1 Lower limb venous ulcer, osteoarthritis, liver fibrosis P01137 SEQ ID NO: 4 in US20030064069A1 SEQ ID NO: 8 of US20030064069A1 US20030064069A1 TGFB2 Idiopathic pulmonary fibrosis, systemic sclerosis, nonalcoholic steatohepatitis (NASH) P61812 FIG. 1B of US20030064069A1 FIG. 1A of US20030064069A1 US8012482B2 TGFB3 Idiopathic pulmonary fibrosis, diabetic macular edema, wet macular degeneration, nonalcoholic steatohepatitis (NASH) P10600 US5262319A CD80/86 Rheumatoid arthritis, psoriatic arthritis, graft-versus-host disease (GVHD), renal transplant rejection P33681 US20070065436A1 CD28 Kidney/heart/lung transplant rejection, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis P10747 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLLWIGCIYPGNVNTNYNEKFKDRATLTVDTSISTAYMELSRLRSDDTAVYFCTRSHYGLDWNFDVWGQGTTVTVSS (SEQ ID NO: 210) DIQMTQSPSSLSASVGDRVTITVysHASQNIYVWLNWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGQTYPYTFGGGTKVEIK (SEQ ID NO: 211) WO2006050949A2/ EP2171060B1 IL23A Crohn's disease, psoriatic arthritis, ulcerative colitis, plaque psoriasis Q9NPF7 QVQLVQSGAEVKKPGSSVKVSCKASGYKFTRYVMHWVRQAPGQGLEWMGYINPYNDGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNWDTGLWGQGTTVTVSS (SEQ ID NO: 212) DIQMTQSPSSLSASVGDRVTITCKASDHILKFLTWYQQKPGKAPKLLIYGATSLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQMYWSTPFTFGGGTKVEIK (SEQ ID NO: 213) US20170275356A1 IL23R Psoriasis, inflammatory bowel disease, multiple sclerosis Q5VWK QVQLVESGGGVVQPGRSLRLSCAASGFDFNSYGMSWVRQAPGKGLEWVADINSKSYNATYYADSVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHHSDYFEYWGQGTLVTVSS (SEQ ID NO: 214) DIQMTQSPSSLSASVGDRVTITCLASEDIYNNLAWYQQKPGKAPKLLIYHASSLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDSEYPPTFGQGTKVEIKRT (SEQ ID NO: 215) US20110158992A1 TSLP Asthma, eosinophilic esophagitis, sinusitis, atopic dermatitis, chronic obstructive pulmonary disease (COPD) Q969D9 QVQLVQSGAEVKKPGASVKVSCKASGYIFTDYAMHWVRQAPGQGLEWMGTFIPLLDTSDYAQKFQGRVTMTADTSTSTAYMELRSLRSDDTAVYYCARMGVTHSYVMDAWGQGTLVTVS (SEQ ID NO: 216) EIVLTQSPGTLSLSPGERATLSCRASQPISISVHWYQQKPGQAPRLLIYFASQSISGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTFSLPYTFGQGTKVEIKRT (SEQ ID NO: 217) US20120219565A1 CRLF2,TSLPR - Q9HC73 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSSAMHWVRQAPGKGLKWVSSVSGSGAGTYYADSVKGRFTISRDNPKNTLYLQMNSLRAEDTAVYYCVKEGGSRGFDYWGQGTLVTVSS (SEQ ID NO: 218) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQKPGKAPKSLIYTASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQYNLYPPTFGQGTKVEIKR (SEQ ID NO: 219) US9908941B2 IL31 Atopic dermatitis Q6EBC2 QVQLQQSGAELARPGASVNLSCKASGYTLTRYWMQWVKQRPGQGLEWIGAIYPGLGDTRYSQKFKGKATLTADKSSSTAYMQLNNLASEDSAVYYCAFPDGYYAAPYGMDYWGQGTSVTVSS (SEQ ID NO: 220) DIQMTQSPASLSASVGETVTITCRASGNTHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSRSETQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK (SEQ ID NO: 221) US10273297B2 IL31RA Itching, rashes, atopic dermatitis, systemic sclerosis, end-stage kidney disease Q8NI17 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYIMNWVRQAPGQGLEWMGLINPYNGGTDYNPQFQDRVTITADKSTSTAYMELSSLRSEDTAVYYCARDGYDDGPYTLETWGQGTLVTVSS (SEQ ID NO: 222) DIQMTQSPSSLSASVGDRVTITCQASEDIYSFVAWYQQKPGKAPKLLIYNAQTEAQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYDSPLTFGGGTKVEIK (SEQ ID NO: 223) Nemolizumab US20200385476A1 TNFRSF4, OX40 Atopic dermatitis, alopecia areata, inflammatory bowel disease, systemic lupus erythematosus P43489 QVQLVQSGAEVKKPGASVKVSCKVSGYTFTSYWITWVRQRPGKGLEWMGDIYPGSGSTNQNEKFKSRVTMTVDTSTDTAYMELSSLRSEDTAVYYCATLRPYYFVYWGQGTLVTVSS (SEQ ID NO: 224) DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKTPKLLIYYTSRLLSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPLTFGQGTRLEIK (SEQ ID NO: 225) US10442866B1 TNFSF4, OX40L Atopic dermatitis, hidradenitis suppurativa, asthma, psoriasis, graft-versus-host disease P23510 QVQLQQPGAELVRPGASVkLSCKASGYTFTSYWLNWVKQRPGQGLEWIVMIDPSDSETHYNQVFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCIRGRGNFYGGSHAMEYWGQGTLLTVSS (SEQ ID NO: 226) DILMTQTPLSLPVSLGDQASISCRSSQSIVHGNGNTYLEWHLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYYCFQGSHVPYTFGGGTKVEIKR (SEQ ID NO: 227) US20100272738A1 IL33 Atopic dermatitis, chronic obstructive pulmonary disease (COPD), asthma O95760 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMYWVRQAPGKGLEWVAAITPNAGEDYYPESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGHYYYTSYSLGYWGQGTLVTVSS (SEQ ID NO: 228) DIQMTQSPSSLSASVGDRVTITCKASQNINKHLDWYQQKPGKAPKLLIYFTNNLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQYNQGWTFGGGTKVEIK (SEQ ID NO: 229) US20200308272A1 IL1RL1,ST2 Atopic dermatitis, chronic obstructive pulmonary disease (COPD), asthma, Alzheimer's disease Q01638 SEQ ID NO: 95 of WO2013173761A2 SEQ ID NO: 29 of WO2013173761A2 WO2013173761A2 CD40 Systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, multiple sclerosis P25942 QVQLVESGGGVVQPGRSLRLSCAASGFSFSSTYVCWVRQAPGKGLEWIACIYTGDGTNYSASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYFCARPDITYGFAINFWGPGTLVTVSS (SEQ ID NO: 230) MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLSASVGDRVTIKCQASQSISSRLAWYQQKPGKPPKLLIYRASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQCTGYGISWPIGGGTKVEIK (SEQ ID NO: 231) US9994640B2 CD40LG, CD154 Systemic lupus erythematosus, recurrent multiple sclerosis, rheumatoid arthritis, Sjogren's syndrome P29965 QVQLVQSGAEVVKPGASVKLSCKASGYIFTSYYMYWVKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSASTAYMELSSLRSEDTAVYYCTRSDGRNDMDSWGQGTLVTVSS (SEQ ID NO: 232) DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTISSVEPEDFATYYCQHSWEIPPTFGGGTKLEIK (SEQ ID NO: 233) US11014990B2 IGF1R Bronchopulmonary dysplasia, Graves' ophthalmopathy, acute ischemic stroke P08069 QVELVESGGGVVQPGRSQRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAIIWFDGSSTYYADSVRGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCARELGRRYFDLWGRGTLVSVSS (SEQ ID NO: 234) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASKRATGIPARFSGSGSGTDFTLTIS Systemic lupus erythematosus (SLE) PEDFAVYYCQQRSKWPPWTFGQGTKVESK (SEQ ID NO: 235) US20120149879A1 ICAM1 Keratoconjunctivitis sicca, diabetic retinopathy P05362 QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAFYYCANSAYTGGWYDYWGHGTLVTVSS (SEQ ID NO: 236) ASELTQDPAVSVALGQTVKITCQGDSLRTYYASWYQQRPGQAPVLVIYGENSRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHLRVFGGGTKLTVL (SEQ ID NO: 237) US20220002432A1 VCAM1 Cholangitis, hepato/renal fibrosis, systemic sclerosis P19320 EVQLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQSPEKRLEWVAEISSGGSYTHYAATVTGRFTISRDNVKNTLYLEMSSLRSEDTAMYYCARGELYWGQGTLVTVSA (SEQ ID NO: 238) DVVLTQIPSTLSVTFGQPASISCKASQSLLDRGGKTFFNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTRLEIK (SEQ ID NO: 239) US20230212295A1 MADCAM1 Nonalcoholic steatohepatitis (NASH), graft-versus-host disease (GVHD), ulcerative colitis, Crohn's disease Q13477 MDWTWSILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGINWVRQAPGQGLEWMGWISVYSGNTNYAQKVQGRVTMTADTSTSTAYMDLRSLRSDDTAVYYCAREGSSSSGDYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKV DKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKk (SEQ ID NO: 240) MRLPAQLLGLLMLWIPGSSADIVMTQTPLSLSVTPGQPASISCKSSQSLLHTDGTTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQNIQLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 241) US9328169B2/ EP2177537A2 ITGA4 Ulcerative colitis, Crohn's disease, ulcerative colitis, graft-versus-host disease (GVHD), relapsing remitting multiple sclerosis (RRMS) P13612 QVQLVQSGAEVKKPGASVKVSCKGSGYTFTSYWMHWVRQAPGQRLEWIGEIDPSESNTNYQKFKGRVTLTVDISASTAYMELSSLRSEDTAVYYCARGGYDGWDYAIDYWGQGTLVTVSS (SEQ ID NO: 242) DVVMTQSPLSLPVTPGEPASISCRSSQSLAKSYGNTYLSWYLQKPGQSPQLLIYGISNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCLQGTHQPYTFGQGTKVEIK (SEQ ID NO: 243) US20170327584A1/ EP3581585A1 ITGB7 Ulcerative colitis, Crohn's disease, graft-versus-host disease (GVHD) P26010 EVQLVESGGGLVQPGGSLRLSCAASGFFITNNYWGWVRQAPGKGLEWVGYISYSGSTSYNPSLKSRFTISRDTSKNTFYLQMNSLRAEDTAVYYCARTGSSGYFDFWGQGTLVTVSS (SEQ ID NO: 244) DIQMTQSPSSLSASVGDRVTITCRASESVDDLLHWYQQKPGKAPKLLIKYASQSISGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNSLPNTFGQGTKVEIKR (SEQ ID NO: 245) US20180086833A1/ KR20170120601A ITGAL, LFA-1 Keratoconjunctivitis sicca (dry eyes), plaque psoriasis, inflammatory bowel disease, allergic asthma P20701 EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSYDFWSNAFDIWGQGTMVTVSS (SEQ ID NO: 246) LNWYQQKTGKAPKALIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQLEDFATYYCQQSYSTPSFGQGTKVEIKRT (SEQ ID NO: 247) US20080069777A1/ CA2554965C ITGAM, MAC-1 Lupus nephritis, multiple sclerosis P11215 SEQ ID NO: 13 in WO2016197974A1 SEQ ID NO: SEQ ID NO: 33 in WO2016197974A1 WO2016197974A1 ITGB1 Keratoconjunctivitis sicca (dry eyes), hepatic fibrosis, idiopathic pulmonary fibrosis P05556 US20220111045A1 CCR5 Multiple sclerosis, nonalcoholic steatohepatitis, inflammatory bowel disease B2KIU MEWSGVFIFLLSVTAGVHSEVQLVESGGGLVKPGGSLRLSCAASGYTFSNYWIGWVRQAPGKGLEWIGDIYPGGNYIRNNEKFKDKTTLSADTSKNTAYLQMNSLKTEDTAVYYCGSSFGSNYVFAWFTYWGQGTLVTVSS (SEQ ID NO: 248) MKLPVRLLVLMFWIPASSSDIVMTQSPLSLPVTPGEPASISCRSSQRLLSSYGHTYLHWYLQKPGQSPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHVPLTFGQGTKVEIK (SEQ ID NO: 249) US7122185B2 CCL2 Dry (atrophic) macular degeneration, lupus nephritis, heart transplant rejection P13500 SEQ ID NO: 71 in US11739142B2 SEQ ID NO: 75 in US11739142B2 US11739142B2 CCL3 Acute febrile neutrophilic dermatosis P10147 SEQ ID NO: 51 in US9005617B2 SEQ ID NO: 56 in US9005617B2 US9005617B2 CCL4 Multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease P13236 SEQ ID NO: 21 in US9005617B2 SEQ ID NO: 22 in US9005617B2 US9005617B2 CCL5 Dermatitis, asthma, chronic obstructive pulmonary disease (COPD); P13501 SEQ ID NO: 51 in US9005617B2 SEQ ID NO: 56 in US9005617B2 US9005617B2 CCL11 Allergic conjunctivitis, blisters, nonalcoholic steatohepatitis (NASH), asthma, Parkinson's disease P51671 SEQ ID NO: 2 in US20210277103A1 SEQ ID NO: 4 in US20210277103A1 US20210277103A1 CCL14 - Q16627 SEQ ID NO: 21 in US9005617B2 SEQ ID NO: 22 in US9005617B2 US9005617B2 CCL15 - Q16663 SEQ ID NO: 51 in US9005617B2 SEQ ID NO: 56 in US9005617B2 US9005617B2 CCL18 - P55774 SEQ ID NO: 21 in US9005617B2 SEQ ID NO: 22 in US9005617B2 US9005617B2 CCL19 - Q99731 CN110117329A CCL20 Multiple sclerosis, dermatitis (eczema), psoriatic arthritis P78556 SEQ ID NO: 2 in JP6289520B2 SEQ ID NO: 8 in JP6289520B2 JP6289520B2 CCL21 - O00585 SEQ ID NO: 14 in WO2022047125A1 SEQ ID NO: 7 in WO2022047125A1 WO2022047125A1 CCL23 - P55773 SEQ ID NO: 51 in US9005617B2 SEQ ID NO: 56 in US9005617B2 US9005617B2 CCL25 - O15444 US8658377B2 CCL27 - Q9Y4X3 US7601815B2 CXCL8 Plaque psoriasis (psoriasis vulgaris), gastric ulcer P10145 SEQ ID NO: 14 in US10047156B2 SEQ ID NO: 13 of US10047156B2 US10047156B2 CXCL10 Inflammatory bowel disease P02778 US7964194B2 CXCL12 Diabetic foot ulcer, islet transplant rejection P48061 SEQ ID NO: 7 in US10647766B2 SEQ ID NO: 9 in US10647766B2 US10647766B2 CXCL13 Rheumatoid arthritis, multiple sclerosis, Sjögren's syndrome (Sjögren's syndrome) O43927 SEQ ID NO: 3 of US20210087263A1 SEQ ID NO: 8 of US20210087263A1 US20210087263A1 Integrin alpha-D (ITGAD) - Q13349 EP1325031A2 IL-17RB Atopic dermatitis (atopic eczema), asthma, ulcerative colitis Q9NRM6 SEQ ID NO: 3 of US11505612B2 SEQ ID NO: 4 in US11505612B2 US11505612B2 IL-13RA2 - Q14627 SEQ ID NO: 2 in CN101440130A SEQ ID NO: 1 in CN101440130A CN101440130A IL-22RA2 Graft-versus-host disease (GVHD), ulcerative colitis, atopic dermatitis, multiple sclerosis, Parkinson's disease Q969J5 SEQ ID NO: 16 in CN102665759B SEQ ID NO: 17 in CN102665759B CN102665759B TGFBR1 Degenerative disc disease, Crohn's disease, pulmonary hypertension P36897 TGF-β TGFBR2 Asthma, idiopathic pulmonary fibrosis, amyotrophic lateral sclerosis P37173 SEQ ID NO: 25 in US8147834B2 SEQ ID NO: 27 in US8147834B2 US8147834B2 TGFBR3 Postmenopausal osteoporosis, obesity Q03167 SEQ ID NO: 95 SEQ ID NO: 96 US11246883B2 Red blood cell I/i Autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome MGWSCIILFLVATATGVHSDIQMTQSPSVLSASVGDRVTLNCKASQNINKYLNWYQQKLGEAPKVLIYNTNNLQTGIPSRFSGSGSGTDFTLTISSLQPEDFATYFCFQHYTWPTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 262) MGWSCIILFLVATATGAHSEVKLQESGGGLVQPGGSLKLSCVASGFTFRDHWMNWVRQAPGKTMEWIGDIRPDGSDTNYAPSVRNRFTISRDNARSILYLQMSNMRSDYTATYYCVRDSPTRAGLMDAWGQGTSVTVSSAKTTAPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVD KKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 263) Sci Transl Med. (2019) 11(506):eaau8217 TLR3 Inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), colitis, rheumatoid arthritis O15455 SEQ ID NO: 6 in US8153583B2 SEQ ID NO: 16 in US8153583B2 US8153583B2 TLR4 Rheumatoid arthritis O00206 SEQ ID NO: 22 in US7312320B2 SEQ ID NO: 27 in US7312320B2 US7312320B2 TLR5 Rheumatoid arthritis O60602 CBLB502 (Entolimod) Cable Gonzalez , Communications biology (2023) 6(31) SEQ ID NO: 44, 45, 46, 47, 48, 49, 50 in US8703146B2 or variants thereof US8703146B2 TLR7 Systemic lupus erythematosus, cutaneous lupus erythematosus Q9NYK1 SEQ ID NO: 5 in US20210040225A SEQ ID NO: 11 in US20210040225A US20210040225A1

[Fusion molecule or binding molecule]

The induction of phagocytosis by the fusion molecules disclosed herein does not involve an inflammatory response. This enables the removal of target substances without inducing an inflammatory response and inhibits tissue damage caused by an inflammatory response, thereby making it possible to treat tissue dysfunction caused by an increase in the amount or expression of a target substance more safely than conventional techniques.

The first region and the second region are coupled to each other directly or through a linker to form a fusion molecule.

The fusion molecule may further comprise a tag. When such a tag is added to the fusion molecule, it can be used to examine the purification, expression, action or mechanism of action of the fusion molecule.

Examples of tags include, but are not limited to: His tag, T7 tag, S tag, FLAG tag, Strep tag, Trx tag, His-pbutanethioredoxin tag, lacZ (L-galactosidase) tag, chloramphenicol acetyltransferase tag, trpE tag, avidin/streptoavidin/streptomycin tag, T7gene10 tag, Staphylococcus protein A tag, Streptococcus protein G tag, glutathione- S -transferase (GST) tag, dihydrofolate reductase (DHFR) tag, cellulose binding domain (CBD) tag, maltose binding protein (MBP) tag, galactose binding protein tag, calcitonin binding protein (CBP) tag, hemagglutinin influenza virus (HAI) tag, HSV tag, B- (VP7 protein region of blue tongue virus) tag, polycysteine tag, polyphenylalanine tag, (Ala-Trp-Trp-Pro) _n tag, polyaspartic acid tag, c-myc tag, lac repressor tag, etc. The tag can be located at the N-terminus, C-terminus or internally of the target protein.

The fusion molecule may also contain a signal peptide or leader sequence at the N-terminus. Signal peptides are known to be short peptides present at the N-terminus at the initial stage of protein synthesis toward the secretory pathway and direct the intracellular localization element, membrane topology (in the case of membrane proteins), etc. of the corresponding protein. The signal peptide may be cleaved during the expression and extracellular secretion of the fusion molecule.

The above-mentioned first region, second region, marker, signal peptide or region with minimum function (e.g., LG1 and LG2 regions or scFv heavy chain variable region and light chain variable region) contained in the fusion molecule can be directly or through a linker comprising a short oligopeptide or polypeptide and connected to each other. Generally, the linker can contain 2 to 500 amino acid residues. There is no particular limitation on the length or type of the linker, as long as the linker can connect the above-mentioned regions together so as to have the expected activity, thereby forming a fusion molecule. An example of a linker can be a 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) monomers are repeated. Other examples of linkers include, but are not limited to: (GSSGGS)n (SEQ ID NO: 118), KESGSVSSEQLAQFRSLD (SEQ ID NO: 119), EGKSSGSGSESKST (SEQ ID NO: 120), GSAGSAAGSGEF (SEQ ID NO: 121), (EAAAK)n (SEQ ID NO: 122), CRRRRRREAEAC (SEQ ID NO: 123), A(EAAAK) _4ALEA (EAAAK) _4A (SEQ ID NO: 124), GGGGGGGG (SEQ ID NO: 125), GGGGGG (SEQ ID NO: 126), AEAAAAKEAAAAKA (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). NO: 131), AGNRVRRSVG (SEQ ID NO: 132), RRRRRRRRR (SEQ ID NO: 133), GFLG (SEQ ID NO: 134) and GSSGGSGSSGGSGGGGDEADGSRGSQKAGVDE (SEQ ID NO: 135). Other suitable linkers include the sequences described in WO2012/088461A, the contents of which are incorporated herein by reference in their entirety.

The fusion molecule according to the embodiment of the present disclosure may further include a scaffold that is bound to the first region, the second region, or both the first region and the second region at different positions of the scaffold. The scaffold may include, but is not limited to, a single-chain Fc region with reduced or eliminated Fc receptor binding affinity, a multimeric Fc region with reduced or eliminated Fc receptor binding affinity, an antibody without a variable region, or an Fc hinge region with reduced or eliminated Fc receptor binding affinity. The first region may be connected or fused to one position of the scaffold, and the second region may be connected or fused to another position of the scaffold. The connection or fusion between the first region/second region and the scaffold may be direct binding or through the above-mentioned linker.

Fusion molecules according to various aspects of the present disclosure can have structures schematically shown in non-limiting exemplary drawings, for example, Figures 23A to 23K.

Another aspect of the present disclosure provides a nucleic acid molecule encoding the fusion molecule, and an expression vector comprising the nucleic acid molecule.

As described above, the nucleic acid sequence encoding the fusion molecule can be mutated by substitution, deletion, insertion or a combination thereof of one or more nucleotide residues, as long as it encodes a protein with equivalent activity.

The nucleic acid sequence encoding the fusion molecule can be separated from nature, or can be artificially produced by synthesis or gene recombination. The nucleic acid sequence encoding the fusion molecule is operably linked to an expression vector capable of expressing the fusion molecule.

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 comprising necessary regulatory elements operably linked to express the gene embedded. Such expression vectors include all vectors such as plasmid vectors, cosmid vectors, phage vectors and viral vectors.

Suitable expression vectors have expression control elements, such as promoters, start codons, stop codons, polyadenylation signals and enhancers. Start codons and stop codons are generally considered to be part of the nucleic acid sequence encoding the protein, and the sequence encoding the protein is designed in a frame so as to be operable in the vector. The promoter can be constitutive or inducible. In addition, conventional expression vectors contain selectable markers. Operative connection with the expression vector can be performed using genetic recombination techniques known in the art, and site-specific DNA cutting and connection can be performed using enzymes known in the art.

The expression vector can be preferably configured to express the fusion molecule in the host cell to isolate and purify the fusion molecule, or to allow the vector to be introduced into cells in vivo and the corresponding cells can express and secrete the fusion molecule. For the purpose of introduction into cells in vivo, the vector can preferably be a non-integrating vector, that is, a vector that is not integrated into the genome of the host cell.

Another aspect of the present disclosure provides a cell expressing the fusion molecule.

Cells can be transformed with expression vectors containing nucleic acid molecules or nucleic acid molecules, and can be "transformed" using appropriate standard techniques based on host cell selection known in the art, including any method for introducing nucleic acid molecules into an organism, cell, tissue or organ. These methods include, but are not limited to: electroporation, protoplast fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-, dextran sulfate-, liposome- and desiccation/inhibition-mediated transformation methods.

Examples of 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., Staphylococcus carnosus ). Other examples of host cells include fungal cells, such as Aspergillus , yeast cells, including Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces , and Neurospora crassa ; lower eukaryotic cells; or cells from higher eukaryotic organisms, 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 of ordinary skill in the art and based on the present disclosure, a nucleic acid vector for expressing an exogenous sequence in a specific host system can be designed, and then a polynucleotide sequence encoding a fusion polypeptide can be inserted. The regulatory elements will vary depending on the specific host.

After the fusion molecule is expressed in cells, it can be isolated and purified using conventional biochemical separation techniques, such as treatment with a protein precipitant (salting out), centrifugation, ultrasonic treatment, ultrafiltration, dialysis or various chromatographic analyses such as molecular sieving chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography, which are usually used in combination to isolate high-purity proteins (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)).

The resulting fusion protein can be evaluated to determine whether the fusion protein significantly increases TAM receptor activity. The method can include contacting cells with a test fusion protein and determining whether contacting cells with the test fusion protein: alters TAM autophosphorylation, TLR-induced cytokine production, TLR-induced MAP kinase-activated stimulation, and/or TLR-induced NF-kB activation compared to a control. In this example, an increase in TAM autophosphorylation, or a decrease in TLR-induced cytokine production, TLR-induced MAP kinase-activated stimulation, or TLR-induced NF-kB activation in the presence of the test fusion protein relative to a control level indicates that the fusion protein stimulates TAM receptor activity.

Autophosphorylation assays are well known in the art. In one example, cells expressing TAM receptors are cultured and treated with assay medium, for example, at 37°C for 20 minutes. The medium is aspirated and cold lysis buffer is added to each sample. The samples are centrifuged to spin down the nuclei, and the supernatant is mixed with protein A agarose beads and affinity purified anti-TAM receptor antibodies and then cultured. The protein A beads are pelleted and washed and separated on Tris-glycine gel and transferred (for Western blotting) to a PVDF membrane (Millipore). Anti-phosphotyrosine is used as the first antibody to detect the blot. The significant reduction of phosphotyrosine labeling relative to control indicates that the test fusion protein is a TAM receptor inhibitor. The control can be a known value of the phosphotyrosine labeling in the indicator sample, such as cells not treated with the test reagent. For example, the significant increase of phosphotyrosine labeling relative to control indicates that the test fusion protein is a TAM receptor agonist. For example, compared with this control, TAM phosphotyrosine increases 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%, indicating that the test fusion protein activates the TAM receptor.

Cytokine assays are also well known in the art. For example, cytokine assays are manufactured by the following companies: Assay Designs, Inc, Ann Arbor, Mich; AssayGate, Inc., Ijamsville, Md.; and Panomics, Inc., Fremont, Calif. Relative to the control level, in the presence of the test agent, an increase in TLR-induced cytokine production indicates that the test agent inhibits TAM receptor activity. The control level can be a reference value indicating the amount of TLR-induced cytokine production in the absence of the test fusion protein, or indicating the amount of TLR-induced cytokine production in the absence of the test fusion protein. For example, a significant reduction in TLR-induced cytokine production relative to the control indicates that the test fusion protein is a TAM receptor agonist. For example, a reduction 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% compared to such a control indicates that the test fusion protein activates the TAM receptor, thereby, the test fusion protein activates the TAM receptor.

MAP kinase activity can be determined by performing a MAP kinase assay. A significant increase in MAP kinase activation in the presence of a test fusion protein relative to a control level of MAP kinase activity (such as a basal level of MAP kinase activity) (as indicated by an increase in p38 phosphorylation) indicates that the test fusion protein inhibits TAM receptor activity. For example, a significant 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% compared to such a control indicates that the test fusion protein activates the TAM receptor, thereby, the test fusion protein activates the TAM receptor.

The fusion protein can be evaluated by determining TLR-induced NF-kB activation. In this example, a significant reduction in TLR-induced NF-kB activation relative to the control indicates that the test agent is a TAM receptor agonist, whereby the test fusion protein activates the TAM receptor. For example, a significant reduction 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% relative to this control indicates that the test fusion protein activates the TAM receptor.

[Drug Combination]

Another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating a disease caused by an increase in the amount or expression of a target substance in a living tissue, the pharmaceutical composition comprising the fusion molecule or the expression vector. Here, the composition can be locally administered to a site where the amount or expression of the disease-causing substance, i.e., the target substance, is increased.

Another aspect of the present disclosure provides the use of a fusion molecule in the preparation of a medicament for preventing or treating an immune disease or disorder.

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

The pharmaceutical composition can be administered orally or parenterally, preferably parenterally. More preferably, it can be administered locally to a tissue in which the target substance to be eliminated shows an increased level/elevation or increased expression.

As used in this specification, the term "parenteral administration" includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

When the pharmaceutical composition is prepared as an injectable preparation, it can be prepared as an injectable preparation by conventional methods known in the art. The injectable preparation may be in a form dispersed in a sterile medium so that it can be directly administered to a patient, or may be in a form of administration after being dispersed in distilled water of an appropriate concentration for injection.

When the pharmaceutical composition is formulated for oral administration, it may contain one or more carriers selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives, and may contain one or more additives selected from flavorings, vitamins and antioxidants.

The techniques required for the preparation of the pharmaceutical composition and pharmaceutically acceptable carriers, additives, etc. are well known to those skilled in the art (see, for example, 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)).

The appropriate dosage of the pharmaceutical composition may vary depending on factors such as the preparation method, administration route, patient age, weight, sex, medical condition, diet, administration time, administration route, excretion rate and reaction sensitivity. The dosage of the pharmaceutical composition disclosed herein is 0.0001 μg/kg to 1,000 μg/kg body weight for adults.

Beneficial Effects

The present disclosure relates to a fusion molecule with phagocytosis-inducing activity, which can solve the problem of tissue damage caused by the activation of inflammatory response in the prior art. Therefore, the fusion molecule can effectively remove and reduce the substance whose expression or amount is increased to, for example, a normal level or amount, and therefore, can be used to prevent or treat immune diseases caused by increased or elevated substances, such as those listed in Table 1 or other diseases described in this specification. The fusion molecule can be administered to a patient in the form of a purified fusion molecule or a gene therapy vector that can express and secrete the fusion molecule when introduced into a cell.

However, it should be understood that the effects of the present disclosure are not limited to the above-mentioned effects and include all effects that can be inferred from the configuration of the present invention described in the detailed description or the scope of application.

Embodiment

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

Astrocytes and microglia are known to play an important role in the progression of multiple sclerosis (MS). It is reported that these cells express TAM receptors and exhibit phagocytosis and anti-inflammatory activity upon activation of TAM receptors. In the following Examples 1 and 2, attempts were made to determine the effect of gene knockout (KO) of the major TAM receptors in the above two types of cells on the progression of MS in EAE (experimental autoimmune encephalomyelitis) mice (one of the MS model animals). The EAE model is a disease model generated by inducing demyelination of myelin in the central nervous system by inducing the activation of T cells that recognize MOG (myelin oligodendrocyte glycoprotein). This model is most commonly used as an animal model to study the mechanisms and therapeutic effects of multiple sclerosis, as its similarities to human MS have been reported in many clinical and histopathological applications.

Embodiment 1 ：Astrocytes through Axl exist CNS Role of the system in regulating inflammation

Aldh1l1-CreERT2;Axl f/f mice were used to eliminate 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 demonstrate the activity of CreERT2 in 8-week-old female mice. MOG35-55 and pertussis toxin (PTX) contained in complete Freund's adjuvant (CFA) were administered to induce experimental autoimmune encephalomyelitis (EAE) in 9-week-old mice. EAE scores and body weight changes were observed 25 days after EAE induction. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model to study the immunopathogenesis of chronic inflammatory diseases of the central nervous system (CNS) such as multiple sclerosis (MS) and to test the efficacy of novel therapeutic agents.

To remove the Axl gene specific to astrocytes and induce MS-like disease (EAE) in mice, mice were operated according to the scheme shown in Figure 1A. In more detail, a construct containing the Aldh1l1 promoter ligation and Axl f/f Aldh1l1-CreERT2;Axl f/f female mice were used to express CreERT2 specifically in astrocytes, and Axl f/f female mice were used as controls.

When mice were 8 weeks old, CreERT2 was activated by intraperitoneal administration of tamoxifen for 5 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 2 consecutive days. Thereafter, EAE scores and body weight changes were confirmed for 25 consecutive days.

As a result, it was confirmed that the EAE score and weight changes of mice in which the astrocyte-specific Axl gene was deleted were more severe than those of the control group (Figure 1B and Figure 1C). It was found that astrocytes play an important role in regulating EAE, and this is achieved through Axl.

Embodiment 2 ：Microglia through Mertk exist CNS Role of the system in regulating inflammation

Cx3cr1-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 administered intraperitoneally for five consecutive days to demonstrate the activity of CreERT2 in 8-week-old female mice. MOG35-55 and pertussis toxin (PTX) contained in complete Freund's adjuvant (CFA) were administered to induce experimental autoimmune encephalomyelitis (EAE) in 9-week-old mice. EAE scores and weight changes were observed 25 days after EAE induction. In order to remove the microglia-specific Mertk gene and induce MS-like disease in mice, mice were operated according to the scheme shown in Figure 2A.

In more detail, a construct containing a Cx3cr1 promoter-linked construct and Mertk f/f Cx3cr1-CreERT2; Mertk f/f female mice were used to express CreERT2 specifically in microglia; Mertk f/f female mice were used as controls. When mice were 8 weeks old, CreERT2 was activated by intraperitoneal administration of tamoxifen for 5 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. Thereafter, EAE scores and body weight changes were confirmed for 25 consecutive days. As a result, it was confirmed that the EAE score and body weight changes of mice in which the Mertk gene was specifically deleted by microglia were more severe than those of the control group (Figure 2B and Figure 2C). It was found that microglia play an important role in regulating EAE, and this is achieved through Mertk.

Embodiment 3 ： Construction of fusion protein： Anti -FITC-Gas6 Fusion molecules and antibodies -MOG(8-18C5)-Gas6 Fusion molecules

To efficiently remove myelin debris by using TAM receptors, AAV expressing fusion molecules based on human Gas6 protein were prepared.

In more detail, the Gla domain and EGF repeat domain, which are sites for PS (phosphatidylserine) recognition of apoptotic cells, were removed from Gas6, and a single-chain Fv fragment of an antibody to the MOG protein highly expressed on myelin was placed on these sites (anti-MOG (8-18C5)-Gas6). See Figures 3A to 3D.

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

Embodiment 4 :anti -MOG(8-18C5)-Gas6 In vivo role of fusion molecules in removing myelin debris

To express anti-FITC-Gas6, anti-MOG(8-18C5)-Gas6 in the HEK293T cell line, transfection of the expression vector was performed. The serum-free medium was replaced three days after transfection, and the supernatant was collected and concentrated one day thereafter. Western blotting was performed to confirm 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 vivo, Myelin-pHrodo was prepared by extracting myelin from mouse brain and conjugating it to pH-sensitive pHrodo. After treating HMC3 cells with concentrated supernatant and Myelin-pHrodo, live cell imaging was performed using IncuCyte.

To confirm the effect of anti-MOG (8-18C5) -Gas6 on the removal of myelin debris in vivo, the protein was expressed in HEK293T cells according to the scheme shown in FIG. 4A . In more detail, to express the protein, transfection of the expression vector was performed in the HEK293T cell line and the supernatant was concentrated. It was confirmed that the protein was expressed at a predicted size of 75 kDa to 76 kDa ( FIG. 4B ). To confirm the effect on the removal of myelin debris in the in vivo system, Myelin-pHrodo was used in the HMC3 cell line. As a result, it was confirmed that when anti-MOG (8-18C5) -Gas6 was present in the HMC3 cell line, myelin debris was removed faster than in the vehicle control group (vehicle) or anti-FITC-Gas6 ( FIG. 4C ). We found that anti-MOG(8-18C5)-Gas6 was appropriately expressed in a functional form and played an important role in the efficient removal of myelin debris.

Embodiment 5 :anti -MOG(8-18C5)-Gas6 Fusion molecules are reducing EAE Effects in the body according to severity

Experimental procedures: AAV PHP.eB-CMV-anti-FITC-Gas6-HA and 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 ¹¹ vg. To induce EAE in 9-week-old mice, MOG35-55 and pertussis toxin (PTX) were administered in complete Freund's adjuvant (CFA). After EAE induction, EAE scores and body weight changes were observed for 25 consecutive days.

To overexpress anti-MOG(8-18C5)-Gas6 in mice and induce MS-like disease (EAE), mice were operated according to the protocol shown in FIG5A . 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 ¹¹ 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 consecutive days. Thereafter, EAE scores and weight changes were confirmed for 25 consecutive days. The results showed that mice administered with AAV expressing anti-MOG(8-18C5)-Gas6 had lower EAE scores and smaller changes in body weight compared with the control (vehicle control group) or AAV expressing anti-FITC-Gas6 (Figure 5B and Figure 5C). Thus, it was found that anti-MOG(8-18C5)-Gas6 was properly expressed in a functional form in mice by AAV and played an important role in reducing the severity of EAE.

Embodiment 6 ：Safety of fusion molecules in animals (systemic expression of fusion proteins)

Experimental procedure: AV PHP.eB-CMV-anti-FITC-Gas6-HA and 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 ¹¹ vg. Three weeks later, brain samples were taken for immunohistochemistry. See Figure 6A.

To confirm the effect of systemically expressed anti-MOG (8-18C5) -Gas6 on normal myelin, wild-type mice were used and operated according to the protocol shown in FIG8A . In more detail, 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 ¹¹ vg. Three weeks later, immunohistochemistry was performed by sampling the brain to confirm myelin levels (MBP), lysosomal content (cathepsin D), and neuronal activation (GFAP, IBA1). The results confirmed that when anti-MOG(8-18C5)-Gas6 was systemically administered to wild-type mice, there were no significant changes in myelin levels, lysosomal content, and glial activation compared to anti-FITC-Gas6 (Figures 6B to 6E). Thus, it was found that when anti-MOG(8-18C5)-Gas6 was expressed systemically, there were no side effects on myelination and glial cell activation.

Embodiment 7 ：Safety of fusion molecules in animals (local expression of fusion proteins)

Experimental procedure: AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were stereotactically injected into the corpus callosum of wild-type C57BL/6J 6-week-old female mice at 1×10 ¹² vg/mL (200 nL). Three weeks later, the brain was sampled for immunohistochemical analysis.

To confirm the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin, wild-type mice were used and operated according to the protocol shown in Figure 9A. In more detail, AAV PHP.eB-CMV-anti-FITC-Gas6-HA, AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were stereotaxically injected into the corpus callosum of wild-type C57BL/6J 6-week-old female mice at 1×10 ¹² vg/mL (200 nL). Three weeks later, immunohistochemical analysis was performed by sampling the brain to confirm the extent of viral expression (HA), myelin levels (MBP), and neuronal activation (GFAP, IBA1). The results confirmed that when anti-MOG(8-18C5)-Gas6 was topically administered to wild-type mice, expression occurred in the injected area rather than on the contralateral side (Fig. 7B). In addition, it was confirmed that myelin levels and glial activation did not change significantly compared with anti-FITC-Gas6 (Fig. 7C to Fig. 7E). Thus, it was found that when anti-MOG(8-18C5)-Gas6 was topically expressed, there was no adverse effect on myelination and glial cell activation.

Embodiment 8 ：Binding activity of fusion molecule and target substance

In order to effectively remove myelin fragments by using TAM receptors, a fusion molecule based on human Gas6 protein was prepared. More specifically, the Gla domain and EGF repeat domain, which are sites for PS (phosphatidylserine) recognition of apoptotic cells, were removed from Gas6, and a single-chain Fv fragment of an antibody to MOG protein highly expressed on myelin was placed on these sites (anti-MOG (01)-Gas6). See Figure 8.

ELISA was performed to evaluate the antigen binding activity of anti-MOG(01)-Gas6 prepared above. Human MBP protein (R&D Systems) or mouse MBP protein (R&D Systems) diluted at 0.5 μg/mL in DPBS was added to a 96-well plate at 100 μL per well, incubated overnight at 4°C for coating, and washed four times with 0.05% Tween-20/PBS (PBST). Then, 3% BSA/PBST was added at 200 μL per well, blocked, and washed four times with PBST. Anti-MOG(01)-Gas6 diluted at the concentration was added at 100 μL per well and incubated at room temperature for 2 hours. The plate was washed with PBST and incubated with anti-human Gas6 antibody (R&D Systems). Systems) and incubated at room temperature for 1 hour. After washing four times with PBST, 100 μL of peroxidase-affinity pure bovine anti-goat IgG (H+L) antibody (Jackson Immuno Research) was added to each well and incubated at room temperature for 1 hour. After washing, 100 μL of TMB solution was added to each well and color was developed for 10 minutes. After stopping the reaction with the stop solution, the absorbance at 450 nm and 650 nm was analyzed by a spectrophotometer. As shown in Figures 9A and 9B, anti-MOG(01)-Gas6 showed binding activity to MOG protein.

In order to confirm the binding activity of the anti-MOG(01)-Gas6 fusion molecule prepared above to MOG expressed on the cell surface, the HEK293 cell line expressing 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 MOG on the cell surface was detected using flow cytometry. Briefly, the HEK293-MOG cell line resuspended in FACS solution (DPBS+3%FBS+10 mM EDTA+1X Pen/Strep+20 mM HEPES) was incubated with the anti-MOG-Gas6 fusion molecule at 4°C for 1 hour. Thereafter, in order to remove the anti-MOG(01)-Gas6 fusion molecules remaining in the supernatant and not bound to cell surface MOG, two washing operations were performed, in which FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. In order to detect the anti-MOG(01)-Gas6 fusion molecules bound to cell surface MOG, G4S linker (E7O2V) rabbit mAb (Cell Signaling Technology) was diluted 1:50 with FACS solution, and 100 μL was added to each well and incubated at 4°C for 30 minutes. After repeating the washing operation twice, the mean fluorescence intensity (MFI) was analyzed using flow cytometry. The results obtained are shown in Figure 9C. As shown in FIG. 9C , it was confirmed that anti-MOG(01)-Gas6 was able to bind to the mouse MOG protein expressed on the cell surface.

Embodiment 9 : Fusion protein (anti -MOG(01)-Gas6 ) The role of fusion molecules in the removal of myelin debris

Anti-MOG(01)-Gas6 was used as purified protein. To confirm the effect of protein on the removal of myelin debris, THP-1 ^Axl (a human monocytic cell line in which the Axl gene is overexpressed) was used. To visualize the removal of myelin debris in vivo, Myelin-pHrodo was prepared by extracting myelin from mouse brain and conjugating it to pH-sensitive pHrodo. After THP-1 ^Axl was treated with protein (5 µg/mL) and Myelin-pHrodo, live cell imaging was performed using the IncuCyte. See Figure 10.

The effect of anti-MOG(01)-Gas6 on the removal of myelin debris was confirmed in vitro. In more detail, anti-MOG(01)-Gas6 was purified and used in the THP-1 ^Axl cell line to confirm their effect on the removal of myelin debris in an in vitro system. The results confirmed that anti-MOG(01)-Gas6 effectively removed myelin in the THP-1 ^Axl cell line compared with the vehicle control group (Figure 9A to Figure 9C).

Embodiment 10 : Fusion protein (anti -MBP-Gas6 ) Binding of fusion molecules to target substances

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

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

The results obtained are shown in Figure 12A and Figure 12B. The tested anti-MBP-Gas6 fusion molecules showed binding activity to human MBP protein and mouse MBP protein.

Embodiment 11 : Fusion protein (anti -MBP-Gas6 ) in the removal of myelin debris

Anti-MBP-Gas6 was used as purified protein. To confirm the effect of protein on the removal of myelin debris, THP-1 ^Axl (a human monocytic cell line in which the Axl gene is overexpressed) was used. To visualize the removal of myelin debris in vivo, Myelin-pHrodo was prepared by extracting myelin from mouse brain and conjugating it to pH-sensitive pHrodo. After THP-1 ^Axl cells were treated with protein (5 µg/mL) and Myelin-pHrodo, live cell imaging was performed using IncuCyte, and MFI values were compared and evaluated after 20 hours.

The effect of anti-MBP-Gas6 on the removal of myelin debris was confirmed in vitro. In more detail, anti-MBP-Gas6 was purified and used in the THP-1 ^Axl cell line to confirm their effect on the removal of myelin debris in vitro. The results confirmed that the tested anti-MBP-Gas6 fusion molecules effectively removed myelin debris in the monocytic cell line compared to the vehicle control group (Figure 13).

Embodiment 12 :anti -TNFα( Adalimumab )-Gas6 Fusion molecules and antibodies -TNFα( Infliximab )-Gas6 Construction of fusion molecules

In order to effectively inhibit or remove TNFα by using TAM receptors, fusion molecules based on human Gas6 protein were prepared. In more detail, the Gla domain and EGF repeat domain, which are sites for PS (phosphatidylserine) that recognize apoptotic cells, were removed from Gas6, and antibody single-chain Fv fragments of TNFα, adalimumab or infliximab were placed at these sites (anti-TNFα-Gas6). See Figures 14A to 14C.

ELISA was performed to evaluate the antigen binding activity of the two types of anti-TNFα-GAS6 fusion molecules prepared above. Human TNFα protein (R&D Systems) diluted at a concentration of 0.5 μg/mL in DPBS was added to a 96-well plate at 100 μL per well, incubated overnight at 4°C for coating, and washed four times with 0.05% Tween-20/PBS (PBST). Then, 3% BSA/PBST was added at 200 μL per well, blocked, and washed four times with PBST. Anti-TNFα-Gas6 fusion molecules diluted at the concentration were added at 100 μL per well and incubated at room temperature for 2 hours. The culture plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated at room temperature for 1 hour. After washing four times with PBST, 100 μL of peroxidase-affinity pure bovine anti-goat IgG (H+L) antibody (Jackson Immuno Research) was added to each well and incubated at room temperature for 1 hour. After washing, 100 μL of TMB solution was added to each well and color was developed for 10 minutes. After stopping the reaction with the stop solution, the absorbance at 450 nm and 650 nm was analyzed by a spectrophotometer. The results are shown in Figure 15A. The anti-TNF α-GAS6 fusion molecules of the two tests showed binding activity to the human TNFα protein.

Embodiment 13 :anti -TNFα( Adalimumab )-Gas6 Fusion molecules and antibodies -TNFα( Infliximab )-Gas6 Fusion molecules and target substances TNFα Binding activity

In order to confirm the binding activity of the two types of anti-TNFα-GAS6 fusion molecules prepared in Example 12 above with TNFα expressed on the cell surface, the CHO-K1 cell line expressing human cell membrane TNFα (hereinafter referred to as CHO-MTNFα cell line; Promega) was treated with anti-TNFα-GAS6 fusion molecules, and then the anti-TNFα-GAS6 fusion molecules bound to TNFα on the cell surface were detected using flow cytometry. Briefly, the CHO-mTNFα cell line resuspended in FACS solution (DPBS+3%FBS+10 mM EDTA+1X Pen/Strep+20 mM HEPES) was cultured with the anti-TNFα-Gas6 fusion molecules at 4°C for 1 hour. Afterwards, in order to remove the anti-TNFα-Gas6 fusion molecules remaining in the supernatant and not bound to cell surface TNFα, two washing operations were performed, in which FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. In order to detect the anti-TNFα-Gas6 fusion molecules bound to cell surface TNF, G4S linker (E7O2V) rabbit mAb (Cell Signaling Technology) was diluted 1:50 with FACS solution, and 100 μL was added to each well and incubated at 4°C for 30 minutes. After repeating the washing operation twice, the mean fluorescence intensity (MFI) was analyzed using flow cytometry.

The results obtained are shown in Figure 15B, which demonstrated that the anti-TNFα-GAS6 fusion molecules in both tests were able to bind to human cell membrane TNFα expressed on the cell surface.

Embodiment 14 :anti -TNFα( Adalimumab )-Gas6 Fusion molecules and antibodies -TNFα( Infliximab )-Gas6 Fusion molecules activate Axl Activity

In order to confirm the ability of the anti-TNFα-GAS6 fusion molecule candidate prepared in Example 12 above to inhibit TNFα signal activation, a TNFα activation inhibition test was performed using HEK-BLUE™ TNFα cells. Briefly, 20 μL of human TNFα protein and 20 μL of anti-TNFα-GAS6 fusion molecules diluted by concentration were added to each well of a flat-bottom 96-well plate, and HEK-BLUE™ TNFα cells were distributed to each well at a density of 5×10 ⁴ /160 μL/well. They were distributed to each well according to the density of the well. After culturing at 37°C in a 5% CO ₂ incubator for 24 hours, 20 μL of the supernatant was transferred to a new flat-bottom 96-well plate, and 180 μL of Quanti-Blue™ solution was added to each well. After 2 hours, the absorbance at 655 nm was measured using a spectrophotometer, and the inhibitory capacity (%) was calculated based on the measured value under the condition of treating only human TNFα protein. As shown in FIG16 , it was confirmed that the anti-TNFα-Gas6 fusion molecules of both tests were able to inhibit the signal initiation of TNFα.

To confirm the ability of the anti-TNFα-GAS6 fusion molecule prepared in Example 12 above to induce Axl activation, a TAM receptor dimerization assay was performed using the human osteosarcoma cell line U2OS ^Axl (Eurofins DiscoverX), in which ProLink-tagged Axl and enzyme receptor (EA)-tagged SH2 domain were overexpressed. In the assay, the cell line was sensitive to Gas6-activated Axl receptor response by generating chemiluminescence, and cells expressing mTNFα (Promega) were treated with the anti-TNFα-Gas6 fusion molecule to determine whether antigen (TNFα)-specific Axl activation was induced.

As shown in FIG. 17 , it was confirmed that Axl activation was induced only when cells expressing mTNFα were treated with anti-TNFα-GAS6 fusion molecules.

Embodiment 15 :anti -TNFα( Adalimumab )-Gas6 Fusion molecules and antibodies -TNFα( Infliximab )-Gas6 Fusion molecules induce Axl Mediated phagocytosis activity

To confirm the Axl-mediated phagocytosis of the anti-TNFα-GAS6 fusion molecule candidate prepared in Example 12 above, a phagocytosis assay was performed using macrophages differentiated from THP-1 ^Axl cells. THP-1 ^Axl cells were treated with 25 nM PMA (phorbol 12-myristate 13-acetate) for 72 hours, cultured in a serum-free and PMA-free medium for 24 hours, and then cultured with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 hours to differentiate into macrophages. Cells expressing mTNFα (Promega) stained with CTV (CellTrace Violet) were used as target cells, and effector cells and target cells were mixed at a ratio of 1:2 and cultured together for 2 hours. After repeated washing with FACS solution twice, the cell surface was stained with CD11b and flow cytometry was used to analyze the phagocytosis of CD11bd+ macrophages toward CTV+ target cells.

As shown in FIG. 18 , it was demonstrated that the anti-TNFα-GAS6 fusion molecule candidate induced phagocytosis of target cells mediated by the Axl receptor expressed on macrophages.

Embodiment 16 :anti -CD20( Rituximab )-Gas6 Construction of fusion molecules

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

ELISA was performed to evaluate the antigen binding activity of the anti-CD20-Gas6 fusion molecule prepared above. Human CD20 protein (Sino Biological) diluted at a concentration of 0.5 μg/mL in DPBS was added to a 96-well plate at 100 μL per well, incubated overnight at 4°C for coating, and washed four times with 0.05% Tween-20/PBS (PBST). Then, 3% BSA/PBST was added at 200 μL per well, blocked, and washed four times with PBST. Anti-CD20-Gas6 fusion molecules diluted at the concentration were added at 100 μL per well and incubated at room temperature for 2 hours. The culture plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated at room temperature for 1 hour. After washing four times with PBST, 100 μL of peroxidase-affinity pure bovine anti-goat IgG (H+L) antibody (Jackson Immuno Research) was added to each well and incubated at room temperature for 1 hour. After washing, 100 μL of TMB solution was added to each well and color was developed for 10 minutes. After stopping the reaction with the stop solution, the absorbance at 450 nm and 650 nm was analyzed by a spectrophotometer. The results are shown in Figure 20A. The anti-CD20-Gas6 fusion molecule exhibits binding activity to the human CD20 protein.

Embodiment 17 :anti -CD20( Rituximab )-Gas6 Binding activity of fusion molecules to target substances

In order to confirm the binding activity of the anti-CD20-Gas6 fusion molecule prepared in Example 16 above to the human CD20 protein expressed on the cell surface, Raji cells (ATCC) expressing human CD20 were treated with anti-CD20-Gas6 fusion molecules, and then anti-CD20-Gas6 fusion molecules bound to CD20 on the cell surface were detected using flow cytometry. Briefly, Raji cells resuspended in FACS solution (DPBS + 3% FBS + 10 mM EDTA + 1X Pen/Strep + 20 mM HEPES) were cultured with anti-CD20-Gas6 fusion molecules at 4°C for 1 hour. Afterwards, in order to remove the anti-CD20-Gas6 fusion molecules remaining in the supernatant without binding to the cell surface CD20, two washing operations were performed, in which FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. In order to detect the anti-CD20-Gas6 fusion molecules bound to the cell surface CD20, G4S linker (E7O2V) rabbit mAb (Cellular Information Transduction Technology) was diluted 1:50 with FACS solution, and 100 μL was added to each well and incubated at 4°C for 30 minutes. After repeating the washing operation twice, the mean fluorescent intensity (MFI) was analyzed using flow cytometry.

The results obtained are shown in Figure 20B, which demonstrates that the tested anti-CD20-Gas6 fusion molecule is able to bind to the human CD20 protein expressed on the cell surface.

Embodiment 18 :anti -CD20( Rituximab )-Gas6 Fusion molecules activate Axl Activity

To confirm the ability of the anti-CD20-Gas6 fusion molecule prepared in Example 16 above to induce Axl activation, a TAM receptor dimerization assay was performed using the human osteosarcoma cell line U2OS ^Axl (Eurofins DiscoverX), in which ProLink-tagged Axl and enzyme receptor (EA)-tagged SH2 domain were overexpressed. In the assay, the cell line was sensitive to the Axl receptor activated by Gas6 by producing chemiluminescence, and the CD20-expressing Raji cell line (ATCC) was treated with the anti-CD20-Gas6 fusion molecule to determine whether antigen (CD20)-specific Axl activation was induced. As shown in FIG. 21 , it was confirmed that Axl activation was induced only when Raji cells expressing CD20 were treated with anti-CD20-Gas6 fusion molecules.

Embodiment 19 :anti -CD20( Rituximab )-Gas6 Fusion molecules induce Axl Mediated phagocytosis activity

To confirm the Axl-mediated phagocytosis of the anti-CD20-Gas6 fusion molecule candidate prepared in Example 16 above, phagocytosis was performed using macrophages differentiated from THP-1 ^Axl cells. THP-1 ^Axl cells were treated with 25 nM PMA (phorbol 12-myristate 13-acetate) for 72 hours, cultured in a serum-free and PMA-free medium for 24 hours, and then cultured with LPS (100 ng/mL) and IFN-γ (10 ng/mL) for 24 hours to differentiate into macrophages. Using mTNFα-expressing cells (Promega) stained with CTV (Cell Trace Violet) as target cells, effector cells and target cells were mixed at a ratio of 1:2 and cultured together for 2 hours. After repeated washing with FACS solution twice, the cell surface was stained with CD11b and the phagocytosis of CD11bd+ macrophages toward CTV+ target cells was analyzed by flow cytometry.

As shown in FIG. 22 , it was demonstrated that the anti-CD20-Gas6 fusion molecule candidate induced phagocytosis of target cells mediated by the Axl receptor expressed on macrophages.

Embodiment 20 ：Adalimumab [scFv]–ProS1 Fusion molecules

To prepare a MOG-specific fusion protein based on the ProS1 protein, the Gla domain and the EGF repeat domain were first removed, and a single-chain variable fragment (scFv) of adalimumab and a TNFα-specific antibody (αTNFα-ProS1) were introduced at this position. Figure 24 shows the amino acid sequence and nucleotide sequence of the chimeric phagocytic inducer.

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [scFv]-ProS1 fusion molecules were evaluated for binding activity to target substances, TAM-activated induction, and phagocytosis.

Embodiment twenty one ：Adalimumab [Fab]-Gas6 (anti -TNFα Antibody heavy chain VH-CH1(Fab)-Gas6-His ）

To prepare the gas6 protein-specific TNFα fusion protein, the Gla domain and EGF repeat domain were first removed, and the antigen-binding fragment (Fab) or monoclonal antibody (Mab) of the TNFα-specific antibody adalimumab was introduced in this position (αTNFα[Fab]-Gas6 and αTNFα[Mab]-Gas6). The Fc region of the heavy chain of the Mab contained NA mutations to reduce or eliminate Fcγ receptor binding affinity. Figure 25 shows the amino acid sequences of the two chimeric phagocytic inducers.

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [Fab]-Gas6 (anti-TNFα antibody heavy chain VH-CH1 (Fab) -Gas6-His) fusion molecules were evaluated for their target substance binding activity, TAM-activated induction, and phagocytosis.

Embodiment twenty two ：Adalimumab [Mab]-Gas6 (anti -TNFα Antibody heavy chain (Mab)-Gas6-His ）

To prepare the gas6 protein-TNFα-specific fusion protein, the Gla domain and the EGF repeat domain were first removed, and the antigen-binding fragment (Fab) or monoclonal antibody (Mab) of the TNFα-specific antibody adalimumab was introduced at this position (αTNFα[Fab]-Gas6 and αTNFα[Mab]-Gas6). Figure 26 shows the amino acid sequences of the two chimeric phagocytic inducers.

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [Mab]-Gas6 (anti-TNFα antibody heavy chain (Mab)-Gas6-His) fusion molecule was evaluated for its target substance binding activity, TAM-activated induction, and phagocytosis.

Embodiment twenty three : Contains adalimumab [Mab or Fab] Fusion molecules

Adalimumab [Fab]‒Gas6 fusion protein, adalimumab [Mab]‒Gas6 fusion protein, adalimumab [Mab]-anti-Axl (homodimer) fusion protein or adalimumab [Mab]-anti-Axl (heterodimer) fusion protein is prepared by using the sequence of SEQ ID NO: 253 (Figure 27) and the sequence of SEQ ID NO: 251 (Figure 25), the sequence of SEQ ID NO: 252 (Figure 26), the sequence of SEQ ID NO: 258 (Figure 32), or the sequence of SEQ ID NO: 260 (Figure 34)/SEQ ID NO: 261 (Figure 34).

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [Mab] or fusion molecules comprising adalimumab [Fab] are evaluated for binding activity to a target substance, TAM-activated induction, and phagocytosis.

Embodiment twenty four ：Adalimumab [scFv]–MFc–Gas6

As a non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first region and the second region, wherein Gas6 and an antibody scFv (in this embodiment, adalimumab scFv) are used as the first region and the second region, respectively, a single-chain Fc region with reduced or eliminated Fc receptor binding affinity is prepared. The sequence used in the structure is shown in Figure 28.

By following the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17-19, the resulting adalimumab [scFv]-MFc-Gas6 fusion molecule was evaluated for its binding activity to the target substance, TAM-activated induction, and phagocytosis.

Embodiment 25 ：Adalimumab [scFv]–Fc(DD)–Gas6 (Heterodimer)

As another non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first region and the second region, wherein Gas6 and an antibody scFv (in this embodiment, adalimumab scFv) are used as the first region and the second region, respectively, a heterodimeric binding molecule is prepared. The first polypeptide of the heterodimeric binding molecule comprises adalimumab scFv, an Fc region (DD) and Gas6, and the second polypeptide of the heterodimeric binding molecule comprises an adalimumab scFv region and an Fc region (KK). The peptide sequence is shown in Figure 29.

By following the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19, the resulting adalimumab [scFv]-Fc (DD) -Gas6 heterodimeric fusion molecules were evaluated for target substance binding activity, TAM-activated induction, and phagocytosis.

Embodiment 26 ：Adalimumab [scFv]–Fc–Gas6 (homodimer)

In another non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first region and the second region, a homodimer comprising two polypeptides was prepared, each of which comprises an adalimumab scFv (as the second region), an Fc region (scaffold) and Gas6. The peptide sequences are shown in FIG30 .

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [scFv]-Fc-Gas6 homodimer fusion molecules were evaluated for target substance binding activity, TAM-activated induction, and phagocytosis.

Embodiment 27 ：Adalimumab [scFv]–Fc–Gas6 (homodimer)

In another non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first region and the second region, a homodimer comprising two polypeptides was prepared, each of which comprises an adalimumab scFv (as the second region), an Fc region (scaffold) and Gas6. The peptide sequence is shown in Figure 31.

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [scFv]-Fc-Gas6 homodimer fusion molecules were evaluated for target substance binding activity, TAM-activated induction, and phagocytosis.

Embodiment 28 ：Adalimumab [Mab]– anti- -Axl (homodimer)

As a non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first region and the second region, a bispecific antibody was prepared in which an anti-Axl antibody and the scFv of adalimumab were used as the first region and the second region, respectively. See Figure 32. The heavy chain of the bispecific antibody has the following sequence SEQ ID NO: 258 (Figure 32) and the light chain of the adalimumab light chain has SEQ ID NO: 253. The Fc region of the heavy chain contains NA mutations to reduce or eliminate Fcγ receptor binding affinity. See Figure 32.

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [Mab]-anti-Axl homodimer fusion molecules were evaluated for binding activity to target substances, TAM-initiated induction, and phagocytosis.

Embodiment 29 :anti -Axl–Fc– Adalimumab [scFv] (homodimer)

As a non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first region and the second region, a homodimeric bispecific antibody was prepared in which the scFv of the anti-Axl antibody and the scFv region of adalimumab were used as the first region and the second region, respectively. See Figure 33. The bispecific antibody comprises a first polypeptide and a second polypeptide that are identical to each other and each comprises SEQ ID NO: 259. The structure of the first/second polypeptide is shown in Figure 33, and the Fc region scaffold comprises NA mutations to reduce or eliminate Fcγ receptor binding affinity.

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting anti-Axl-Fc-adalimumab [scFv] homodimer fusion molecules were evaluated for binding activity to target substances, TAM-activated induction, and phagocytosis.

Embodiment 30 ：Adalimumab [Mab](DD) anti- -Axl (Heterodimer)

As another non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first region and the second region, a heterodimeric bispecific antibody was prepared, wherein the scFv of anti-Axl antibody and adalimumab were used as the first region and the second region, respectively. The first polypeptide of the heavy chain of the bispecific antibody has SEQ ID NO: 260 of the following sequence of Figure 34, the second polypeptide of the heavy chain of the bispecific antibody comprises SEQ ID NO: 261 of Figure 34, and the light chain of the anti-amyloid antibody has SEQ ID NO: 253 of Figure 27. The Fc region comprises NA mutations to reduce or eliminate Fcγ receptor binding affinity, and the polypeptides of the Fc region form heterodimers (DD-KK).

By following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17 to 19, the resulting adalimumab [Mab] (DD)-anti-Axl heterodimer fusion molecules were evaluated for target substance binding activity, TAM-initiated induction, and phagocytosis.

The scope of the present disclosure is defined by the appended patent application scope, and all changes or modifications derived from the meaning and scope of the patent application scope and its equivalents should be understood to be included in the scope of the present invention.

The fusion molecule with phagocytosis inducing activity according to the embodiments of the present disclosure can solve the problem of tissue damage caused by the activation of inflammatory response in the prior art. Therefore, the fusion molecule can effectively remove or reduce the amount of antigenic substances, thereby being used to prevent or treat immune diseases.

Incorporate by reference

All publications, patent applications, patents and other references mentioned in this specification are expressly incorporated into this specification by reference in their entirety.

without

FIG. 1A to FIG. 1C show the effects on EAE scores and body weight changes when EAE is induced in mice with astrocyte-specific deletion of the Axl gene.

FIG. 2A to FIG. 2C show the effects on EAE scores and body weight changes when EAE is induced in mice with microglia-specific deletion of the Mertk gene.

FIG. 3A shows a schematic diagram of the configuration of the prepared AAV expressing anti-FITC-Gas6 and anti-MOG (8-18C5)-Gas6.

Figure 3B shows the amino acid sequences of anti-FITC-Gas6 and anti-MOG (8-18C5) -Gas6 constructed in Example 3, and Figure 3C and Figure 3D show the nucleic acid sequences encoding anti-FITC-Gas6 and anti-MOG (8-18C5) -Gas6 as shown in Figure 3B, respectively.

Figures 4A to 4C show the effects of anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vitro.

5A to 5C show the effects on EAE scores and body weight changes when anti-MOG(8-18C5)-Gas6 is expressed in EAE mice.

Figures 6A to 6E show the effects of systemic expression of anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

Figures 7A to 7E show the effects of local expression of anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

FIG8 shows a schematic diagram of the configuration of the prepared anti-MOG(01)-Gas6.

Figures 9A and 9B show the antigen (human and mouse MOG) binding activity of anti-MOG(01)-Gas6 fusion molecules measured by ELISA.

FIG9C shows the results of measuring the binding degree of anti-MOG (01) -Gas6 fusion molecule to mouse MOG protein on the cell surface using flow cytometer.

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

FIG11 shows a schematic diagram of the configuration of the prepared anti-MBP-Gas6.

Figures 12A and 12B show the antigen (human and mouse MBP) binding activity of anti-MBP-Gas6 fusion molecules measured by ELISA.

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

Figure 14A shows a schematic diagram of the configuration of the prepared anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6. Figures 14B and 14C show the sequences of anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6.

Figure 15A shows the antigen (human TNFα) binding activity of the anti-TNFα-GAS6 fusion molecule measured by ELSA, and Figure 15B shows the results of the binding degree of the anti-TNFα-GAS6 fusion molecule and human TNFα protein on the cell surface measured using a flow cytometer.

FIG. 16 shows the level of inhibition of TNFα signaling activation by anti-TNFα-GAS6 fusion molecules in HEK-Blue™ TNFα cells.

FIG. 17 shows the induction of Axl activation in U2OS ^Axl cells by anti-TNFα-GAS6 fusion molecules.

FIG. 18 shows the induction of Axl-mediated phagocytosis by anti-TNFα-Gas6 using THP-1 ^Axl -derived macrophages as effector cells.

Figure 19A shows a schematic diagram of the configuration of the prepared anti-CD20 (rituximab)-Gas6. Figure 19B shows the amino acid sequence of anti-CD20 (rituximab)-Gas6.

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

FIG. 21 shows the induction of Axl activation in U2OS ^Axl cells by anti-CD20-Gas6 fusion molecules.

FIG. 22 shows the induction of Axl-mediated phagocytosis by anti-CD20-Gas6 using THP-1 ^Axl -derived macrophages as effector cells.

Figures 23A to 23K schematically depict the structures of fusion proteins according to various non-limiting embodiments of the present disclosure.

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

TW202417520A_112139239_SEQL.xml

Claims (26)

A fusion molecule comprises a first region capable of binding to a TAM receptor and a second region capable of specifically binding to a target substance, wherein the target substance is a substance whose increased amount or increased expression in living tissues induces or causes immune diseases, wherein the first region and the second region are coupled to each other directly or through 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) a combination thereof, wherein the fusion molecule does not comprise the target substance or a fragment thereof. The fusion molecule as described in claim 1 further comprises a support that is bound to the first region, the second region, or both the first region and the second region at different positions. The fusion molecule as described in claim 1, wherein the TAM receptor ligand is one selected from Gas6, ProS1, Tubby, Tulp1, Gal3 and a combination thereof, or an Axl-binding fragment thereof. A fusion molecule as described in claim 1, wherein the first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises a sequence selected from SEQ ID NOs: 1-113 or a sequence having at least 85% sequence identity therewith. A fusion molecule as described in claim 1, wherein the first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises a sequence selected from 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 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 sequences having at least 85% sequence identity thereto. A fusion molecule as described in claim 1, wherein the first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 1 or a sequence having at least 85% sequence identity thereto, and the sequence of SEQ ID NO: 2 or a sequence having at least 85% sequence identity thereto. The fusion molecule of claim 1, wherein the first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 5 or a sequence having at least 85% sequence identity thereto. A fusion molecule as described in claim 1, wherein the first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises a ligand selected from 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: 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% sequence identity thereto. A fusion molecule as described in claim 1, wherein the first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 3 or a sequence having at least 85% sequence identity thereto, and the sequence of SEQ ID NO: 4 or a sequence having at least 85% sequence identity thereto. The fusion molecule of claim 1, wherein the first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises the sequence of SEQ ID NO: 6 or a sequence having at least 85% sequence identity thereto. The fusion molecule as described in claim 1 forms a homodimer, a heterodimer or a multimer in a single chain. The fusion molecule as described in claim 1, wherein the target substance is an autoantigen, an autoantibody, a complex of an autoantigen and an autoantibody, a cytokine, a chemokine, a complement, a receptor, an immune cell-specific marker, a cell adhesion molecule, or a combination thereof. A fusion molecule as described in claim 1, wherein the target substance is one or more selected from the following: factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, thrombin, vWF, calcium-sensitive receptor, ACTH, 21-hydroxylase (CYP21), hair transparent protein, oxidized low-density lipoprotein (OxLDL), transcription co-activator 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-γ/TRIM-33, β2 integrin, nuclear autoantigen sperm protein (NASP), lactoferrin 17-α-hydroxylase (CYP17), cholesterol side chain cleavage enzyme (CYP11A), tryptophan hydroxylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, glycoprotein IIb/IIIa and Ib/IX, thyroglobulin, hemiglobin 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), S UMO-activating enzyme subunit (SAE)-1 (SAE-1), SAE-2, DNA-dependent nucleosome-stimulated ATPase, chromatin domain-helicase-DNA binding protein 4 (CHD4), β-adrenaline receptor, adenine nucleotide transporter, type VII collagen, IgG, G-CSF, type IV collagen α3 chain, thyrotropin receptor (TSHR), sodium iodide symporter (NIS), peripheral myelin protein 22 (PMP22), GM ganglioside, S-antigen, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), signal recognition particle 54kDa subunit (SRP54), IgA, synaptic binding protein, voltage-gated calcium channel, βIV 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), myelin oligodendrocyte glycoprotein (MOG), acetylcholine receptor, low-density lipoprotein receptor-related protein 4 (LRP4), musculoskeletal receptor tyrosine-protein kinase (MuSK), aminoacyl-tRNA synthetase, Tribble TRIB2, myeloperoxidase (MPO), aquaporin 4 (APQ-4), amphiphilin, exosome component 9 (EXOSC9), EXOSC10/PMSCL, Yo protein, Hu protein, Ri protein, desmoplakin, desmoplakin, desmoplakin 1, desmoplakin 3, intrinsic factor type 1, β2-glycoprotein I (β2-GPI), pyruvate dehydrogenase complex-E2 (PDC-E2), aggrecan G1 , aminoformylated antigen, chondroitin-39, Fc portion of immunoglobulin, glucose-6-phosphate isomerase, keratin, protein-arginine deiminase type 4, collagen (various types, especially types II, IV, and IX), fibrinogen beta-alpha, leukemia inhibitory factor (LIF), glutamine receptor (GLUR), myosin, B23, nucleophosmin (NPM), nucleolar fibrosis, topoisomerase-I (Scl-70), interferon-gamma-inducing protein 16 (IFI16), La phosphoprotein, Ro60, Ro52 (TRIM21), Homozygous proteins (95, 97, 160, 180), anionic phospholipid/protein complex, cardiolipin, components of Sm splicing ribonucleoprotein (subunits A-G), self-double-stranded DNA (dsDNA), histone H2A-H2B-DNA, proliferating cell nuclear antigen (PCNA), ribosomal P, Sjogren's syndrome (SSA), Smith, U1-RNP, U2 snRNP B, vimentin, C1q, fibronectin, Ku-DNA-protein kinase, carbonic anhydrase II, neuronal nicotinic acid acetylcholine receptor, centromere-associated protein, RNA polymerase I-III (RNP), thyroid and eye muscle common protein, leukocyte function-associated antigen (LFA-1), chromaffin protein A, IA-2 (ICA512), islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), Zn T8, insulin, glutamine 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, C5α receptor 1, CD52, FcRn large subunit p51, IL-1, IL-1R, IL-6, IL-6R, IL-1 7. IL-17R, TNF-α, TNFR, IL-4, IL-4R, IL-5, IL-5R, IL-13, IL-13R, IFN-γ, IFN-γ receptor, IL-12, IL-12R, IL-21, IL-21R, IL-22, IL-22R, TGF-β, TGF-β receptor, CD80/86, CD28, IL-23, IL-23R, thymic stroma TSLP, TSLPR, IL-31, IL-31R, OX40, OX40L, IL-33, IL-33R, CD40, CD40L, IGF-1R, ICAM1, VCAM1, MADCAM1, integrin α4, integrin β7, VLA-4, toll-like receptor (TLR)-3, TLR-4, TLR-5, TLR-7, and their combinations. A fusion molecule as described in claim 1, wherein the second region that specifically binds to the target substance is selected from antibodies or their antigen-binding fragments, antibody-like proteins, peptides, aptamers and soluble receptors, each of which specifically binds to the target substance. A fusion molecule as described in claim 2, wherein the scaffold is a single-chain Fc region with reduced or eliminated Fc receptor binding affinity, a multimeric Fc region with reduced or eliminated Fc receptor binding affinity, an antibody without a variable region, or an Fc-hinge region with reduced or eliminated Fc receptor binding affinity. The fusion molecule of claim 1, wherein the immune disease is selected from multiple sclerosis, myasthenia gravis, type 1 diabetes, type 2 diabetes, 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, allergic 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 syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren's syndrome, spondyloarthritis/spondyloarthropathies, systemic juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, chylous diarrhea, thrombotic thrombocytopenic purpura (iTTP), and combinations thereof, inflammatory or autoimmune diseases. A nucleic acid molecule encoding the fusion molecule as described in claim 1. An expression vector comprising the nucleic acid molecule as described in claim 17. A cell expressing the fusion molecule as described in claim 1. A pharmaceutical composition comprising: (i) the fusion molecule of claim 1, (ii) a polynucleotide encoding the fusion molecule, (iii) an expression vector carrying the polynucleotide, or a combination thereof as an active ingredient, and a pharmaceutically acceptable carrier. A use of the pharmaceutical composition as described in claim 20, which is used to: (i) reduce the elevated level of a target substance to a normal level, or increase the reduction of the elevated level of the target substance to a normal level, wherein the target substance causes or induces an immune disease in a subject, (ii) remove or clear a target substance whose expression is elevated or increased, or enhance the clearance of a target substance whose expression is elevated or increased, wherein the elevated expression or increased amount of the target substance causes or induces an immune disease in a subject, (iii) inhibit the increase in the expression or amount of the target substance in a subject, (iv) treat or prevent an immune disease in a subject, (v) delay the development of symptoms associated with an immune disease, and/or (vi) alleviate the symptoms of an immune disease in a subject. The use as described in claim 21, wherein the elevated level of the target substance occurs in the brain of the subject. A use of a fusion molecule as described in claim 1, a polynucleotide encoding the fusion molecule, a vector, or a pharmaceutical composition comprising the fusion molecule, the polynucleotide or the vector in one or more methods selected from the following: reducing the elevated level of a target substance to a normal level, or increasing the reduction of the elevated level of the target substance to a normal level, wherein the target substance causes or induces an immune disease in a subject, removing or clearing a target substance with elevated expression or increased amount, or enhancing the clearance of a target substance with elevated expression or increased amount, wherein the elevated expression or increased amount of the target substance causes or induces an immune disease in a subject, and inhibiting the elevated expression or amount of the target substance in a subject. The use of claim 23, wherein the elevated level of the target substance occurs in the brain of the subject. A use of a fusion molecule as described in claim 1, a polynucleotide encoding the fusion molecule, a vector, or a pharmaceutical composition comprising the fusion molecule, the polynucleotide or the vector in one or more methods selected from the following: treating or preventing an immune disease in a subject, delaying the development of symptoms associated with an immune disease, and alleviating symptoms of an immune disease in a subject. A use of a fusion molecule as described in claim 1, a polynucleotide encoding the fusion molecule, a vector, or a pharmaceutical composition comprising the fusion molecule, the polynucleotide or the vector in the preparation of a drug for treating or preventing an immune disease in a subject, for delaying the development of symptoms associated with an immune disease, or for alleviating symptoms of an immune disease in a subject.