Fusion molecules and methods of treating immune diseases

Abstract

The present disclosure relates to a fusion molecule capable of binding to an antigen substance whose level or amount is increased to cause or cause an immune disease and capable of inducing phagocytosis, thereby removing, reducing, or enhancing removal or reduction of an antigen substance. Therefore, the fusion molecule can be used for treating immune diseases, delaying development or onset of immune diseases, or alleviating symptoms of immune diseases.

Description

Technical Field

[0002] The present disclosure relates to fusion molecules suitable for preventing or treating immune diseases. The present disclosure also relates to nucleic acid molecules encoding the fusion molecules. The present disclosure further relates to compositions comprising the fusion molecules, methods for preventing or treating immune diseases, and uses of the fusion molecules in treating immune diseases. Background Art

[0003] Immune diseases are diseases that are caused, mediated, or otherwise contribute to pathological conditions in mammals by components of the mammalian immune system. Inflammatory diseases, in particular, are of worldwide concern and are in urgent need of therapeutic agents. Inflammation is generally a localized protective response of body tissues to host invasion by foreign substances or harmful stimuli. The causes of inflammation may include: infectious causes such as bacteria, viruses, and parasites, physical causes such as burns or radioactive radiation, chemicals such as toxins, drugs, or industrial agents, immune reactions such as allergies and autoimmune reactions, or conditions associated with oxidative stress.

[0004] 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, which can become a life-threatening disease in humans, including joint 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 transfusions, drug administration, and organ transplants.

[0005] 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 disorders, bleeding, and hepatotoxicity 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 treat these diseases, there is a need for an effective therapeutic agent without side effects.

[0006] Meanwhile, TAM (Tyro3, ​​Axl and MerTK) receptors are receptor tyrosine kinases, and what has recently attracted attention is that the TAM ligands that can activate these receptors play an important role in controlling tissue homeostasis and inflammation. Known TAM receptors are particularly involved in anti-inflammatory effects and the disappearance of inflammation, wherein anti-inflammatory effects refer to reducing and eliminating the activity of pro-inflammatory mediators, which can be carried out by inhibiting the synthesis, selective antagonism, removal, post-translational modification such as cutting or decomposition of these mediators. The disappearance of inflammation can be completed 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.).

[0007] 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 shown that they are effective 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 only contain a region that binds to the TAM receptor and a region that binds to PS (phosphatidylserine), which are regions with activity related to binding to other molecules. Therefore, it is difficult to selectively act on substances that cause inflammation.

[0008] Therefore, there is a need for improved methods that can effectively induce selective clearance of antigens. Summary of the Invention

[0009] The present disclosure relates to fusion molecules capable of inducing the selective clearance of target substances that trigger or induce undesirable or pathological immune responses, such as autoimmune diseases, transplant rejection, or allergic or hyperimmune responses.

[0010] 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 that specifically binds to a target substance to be eliminated 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 effector function and does not induce an Fc-mediated inflammatory response.

[0011] In some embodiments, the TAM receptor can 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 can be the Axl region of the TAM receptor.

[0012] In an embodiment, the first region may comprise Gas6, ProS1, Tubby, Tulp1, Gal3 or an active fragment thereof, each of which is capable of specifically binding to a TAM receptor. The first region may be selected from Gas6, ProS1 or an active fragment thereof, each of which is capable of specifically binding to a TAM receptor. In an embodiment, the first region may comprise or consist essentially of Gas6 or an active fragment thereof that is capable of binding to a TAM receptor. In an embodiment, the first region comprising or consisting essentially of Gas6 or an active fragment thereof is capable of binding to an Axl receptor.

[0013] In certain embodiments, the first region may comprise a laminin G-like domain of Gas6 or ProS1 or an active fragment thereof, which comprises a laminin G-like domain as a bridging molecule associated with phagocytosis, which is abundantly expressed in a variety of tissues and is therefore capable of inducing phagocytosis through TAM receptors. In embodiments, the laminin G-like domain may comprise an LG1 domain, an LG2 domain, or a combination thereof, and may preferably comprise both an LG1 domain and an LG2 domain, which is capable of inducing phagocytosis by binding to the TAM receptor.

[0014] Exemplary embodiments relate to a binding molecule or 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 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 via a linker, wherein the first region comprises:

[0015] TAM receptor ligands;

[0016] anti-Axl antibody or antigen-binding fragment thereof;

[0017] an anti-Tyro3 antibody or an antigen-binding fragment thereof; or

[0018] 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

[0019] A combination of them.

[0020] 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.

[0021] In an embodiment, the first region is a TAM receptor ligand, and the TAM receptor ligand comprises a sequence selected from SEQ ID NOs: 1-113, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0022] In some further 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 region selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 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.

[0023] In still 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.

[0024] 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.

[0025] In some further 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 member selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO:113, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0026] In still 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.

[0027] 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.

[0028] In an embodiment, the fusion protein according to the present disclosure does not comprise a target substance that is cleared or reduced by administering the fusion protein.

[0029] In an embodiment, the first region comprising the laminin G-like domain or an active fragment thereof of 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.

[0030] In some embodiments, the first region of the laminin G-like domain or its active fragment comprising Gas6 or ProS1 does not comprise 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 by inhibiting the aggregation of the fusion molecule during the purification step to increase the yield. In some embodiments, the fusion molecule (or binding molecule) can form homodimers or heterodimers, or form linear multimers as single chains.

[0031] According to an 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 an autoimmune disease, transplant rejection, or an allergic or hyperimmune response.

[0032] The target substance can be an inflammation-related substance. The target substance can be one or more selected from the following substances: the autoantigens in Table 1 below, their autoantibodies, or a complex of the autoantigen and its autoantibody; immune cell surface molecules, including costimulatory 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 α-D (ITGAD), Toll-like receptor (TLR), including TLR 3, TLR4, TLR5 and TLR7, etc.; complement, 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 for 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.

[0033] Table 1

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[0045]

[0046]

[0047] *: There are 35 entries at: https: / / www.uniprot.org / uniprotkb?query=GABAAR+ALPHA&facets=model_organism%3A9606.

[0048] **: MDA5 is encoded by the interferon-induced helicase C domain-containing protein 1 (IFIH1) gene.

[0049] ***: Lrp4 is a receptor for Agrin and forms a complex with MuSK Agrin (AGRN): O00468.

[0050] The amino acid sequence of the target substance can be obtained from public databases such as UniProtKB / Swiss-Prot or NCBI. For example, exemplary amino acid sequences of the target substance may include, but are not limited to, the following.

[0051] CD20: UniProt accession number P11836 (human) and its variants at www.uniprot.org / uniprotkb / P11836 / variant-viewer, and their homologous sequences.

[0052] CD19: UniProt accession number P15391 (human) and its variants at www.uniprot.org / uniprotkb / P15391 / variant-viewer, and their homologous sequences.

[0053] CD52: UniProt accession number P31358 (human) and its variants at www.uniprot.org / uniprotkb / P31358 / variant-viewer, and their homologous sequences.

[0054] CD80: UniProt accession number P33681 (human) and its variants at www.uniprot.org / uniprotkb / P33681 / variant-viewer, and their homologous sequences.

[0055] CD86: UniProt accession number P42081 (human) and its variants at www.uniprot.org / uniprotkb / P42081 / variant-viewer, and their homologous sequences.

[0056] CD28: UniProt accession number P10747 (human) and its variants at www.uniprot.org / uniprotkb / P10747 / variant-viewer, and their homologous sequences.

[0057] CD40: UniProt accession number P25942 (human) and its variants at www.uniprot.org / uniprotkb / P25942 / variant-viewer, and their homologous sequences.

[0058] Complement C3: UniProt accession number P01024 (human) and its variants at www.uniprot.org / uniprotkb / P01024 / variant-viewer, and its homologous sequences.

[0059] Complement C5: UniProt accession number P01031 (human) and its variants at www.uniprot.org / uniprotkb / P01031 / variant-viewer, and their homologous sequences.

[0060] C5α receptor 1: UniProt accession number P21730 (human) and its variants at www.uniprot.org / uniprotkb / P21730 / variant-viewer, and their homologous sequences.

[0061] IL-1R: UniProt accession number P14778 (human) and its variants at www.uniprot.org / uniprotkb / P14778 / variant-viewer, and their homologous sequences.

[0062] IL-6R: UniProt accession number P08887 (human) and its variants at www.uniprot.org / uniprotkb / P08887 / variant-viewer, and their homologous sequences.

[0063] 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.

[0064] IL-17C: UniProt accession number Q9P0M4 (human) and its variants at www.uniprot.org / uniprotkb / PQ9P0M4 / variant-viewer, and their homologous sequences.

[0065] IL-17RA: UniProt accession number Q96F46 (human) and its variants at www.uniprot.org / uniprotkb / Q96F46 / variant-viewer, and their homologous sequences.

[0066] IL-17RB: UniProt accession number Q9NRM6 (human) and its variants at www.uniprot.org / uniprotkb / Q9NRM6 / variant-viewer, and their homologous sequences.

[0067] IL-4R: UniProt accession number P24394 (human) and its variants at www.uniprot.org / uniprotkb / QP24394 / variant-viewer, and their homologous sequences.

[0068] IL-5R: UniProt accession number Q01344 (human) and its variants at www.uniprot.org / uniprotkb / Q01344 / variant-viewer, and their homologous sequences.

[0069] IL-5RB: UniProt accession number P32927 (human) and its variants at www.uniprot.org / uniprotkb / P32927 / variant-viewer, and their homologous sequences.

[0070] IL-13R1: UniProt accession number P78552 (human) and its variants at www.uniprot.org / uniprotkb / P78552 / variant-viewer, and their homologous sequences.

[0071] IL-13R2: UniProt Accession No. Q14627 (human) and variants thereof at www.uniprot.org / uniprotkb / Q14627 / variant-viewer, and homologous sequences thereof; or UniProt Accession No. D0EFR8 (human) and variants thereof at www.uniprot.org / uniprotkb / D0EFR8 / variant-viewer, and homologous sequences thereof.

[0072] IFN-γ receptor 1: UniProt accession number Q15260 (human) and its variants at www.uniprot.org / uniprotkb / Q15260 / variant-viewer, and their homologous sequences.

[0073] IFN-γ receptor 2: UniProt accession number P38484 (human) and its variants at www.uniprot.org / uniprotkb / P38484 / variant-viewer, and their homologous sequences.

[0074] Integrin alpha-D (ITGAD): UniProt accession number Q13349 (human) and its variants at www.uniprot.org / uniprotkb / Q13349 / variant-viewer, and their homologous sequences.

[0075] IL-12RB1: UniProt accession number P42701 (human) and its variants at www.uniprot.org / uniprotkb / P42701 / variant-viewer, and their homologous sequences.

[0076] IL-12RB2: UniProt accession number Q99665 (human) and its variants at www.uniprot.org / uniprotkb / Q99665 / variant-viewer, and their homologous sequences.

[0077] IL-21R: UniProt accession number Q9HBE5 (human) and its variants at www.uniprot.org / uniprotkb / Q9HBE5 / variant-viewer, and their homologous sequences.

[0078] IL-22RA1: UniProt accession number Q8N6P7 (human) and its variants at www.uniprot.org / uniprotkb / Q8N6P7 / variant-viewer, and their homologous sequences.

[0079] IL-22RA2: UniProt accession number Q969J5 (human) and its variants at www.uniprot.org / uniprotkb / Q969J5 / variant-viewer, and their homologous sequences.

[0080] 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.

[0081] IL-23R: UniProt accession number Q5VWK5 (human) and its variants at www.uniprot.org / uniprotkb / Q5VWK5 / variant-viewer, and their homologous sequences.

[0082] 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.

[0083] IL-31R: UniProt accession number Q8NI17 (human) and its variants at www.uniprot.org / uniprotkb / Q8NI17 / variant-viewer, and their homologous sequences.

[0084] IL-33R: UniProt accession number Q01638 (human) and its variants at www.uniprot.org / uniprotkb / Q8NB14 / variant-viewer, and their homologous sequences.

[0085] IGF-1R: UniProt accession number P08069 (human) and its variants at www.uniprot.org / uniprotkb / P08069 / variant-viewer, and their homologous sequences.

[0086] TNFR1: UniProt accession number P19438 (human) and its variants at www.uniprot.org / uniprotkb / P19438 / variant-viewer, and their homologous sequences.

[0087] TNFR2: UniProt accession number P20333 (human) and its variants at www.uniprot.org / uniprotkb / P20333 / variant-viewer, and their homologous sequences.

[0088] FcRn large subunit p51: UniProt accession number P55899 (human) and its variants at www.uniprot.org / uniprotkb / P55899 / variant-viewer, and their homologous sequences.

[0089] CCL14: UniProt accession number Q16627 (human) and its variants at www.uniprot.org / uniprotkb / Q16627 / variant-viewer, and their homologous sequences.

[0090] CCL15: UniProt accession number Q16663 (human) and its variants at www.uniprot.org / uniprotkb / Q16663 / variant-viewer, and their homologous sequences.

[0091] CCL18: UniProt accession number P55774 (human) and its variants at www.uniprot.org / uniprotkb / P55774 / variant-viewer, and their homologous sequences.

[0092] CCL19: UniProt accession number Q99731 (human) and its variants at www.uniprot.org / uniprotkb / Q99731 / variant-viewer, and their homologous sequences.

[0093] CCL20: UniProt accession number QP78556 (human) and its variants at www.uniprot.org / uniprotkb / P78556 / variant-viewer, and their homologous sequences.

[0094] CCL21: UniProt accession number O00585 (human) and its variants at www.uniprot.org / uniprotkb / O00585 / variant-viewer, and their homologous sequences.

[0095] CCL23: UniProt accession number P55773 (human) and its variants at www.uniprot.org / uniprotkb / P55773 / variant-viewer, and their homologous sequences.

[0096] CCL25: UniProt accession number Q68A93 (human) and its variants at www.uniprot.org / uniprotkb / Q68A93 / variant-viewer, and their homologous sequences.

[0097] CCL27: UniProt accession number Q9Y4X3 (human) and its variants at www.uniprot.org / uniprotkb / Q9Y4X3 / variant-viewer, and their homologous sequences.

[0098] CXCL12: UniProt accession number P48061 (human) and its variants at www.uniprot.org / uniprotkb / P48061 / variant-viewer, and their homologous sequences.

[0099] CXCL13: UniProt accession number O43927 (human) and its variants at www.uniprot.org / uniprotkb / O43927 / variant-viewer, and their homologous sequences.

[0100] 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.

[0101] TNF-α: UniProt accession number P01375 (human) and its variants at www.uniprot.org / uniprotkb / P01375 / variant-viewer, and their homologous sequences.

[0102] CXCL-8: UniProt accession number P10145 and its variants at www.uniprot.org / uniprotkb / P10145 / variant-viewer, and their homologous sequences.

[0103] CCL2: UniProt accession number P13500 (human) and its variants at www.uniprot.org / uniprotkb / Q13500 / variant-viewer, and their homologous sequences.

[0104] CCL3: UniProt accession number P10147 (human) and its variants at www.uniprot.org / uniprotkb / P10147 / variant-viewer, and their homologous sequences.

[0105] CCL4: UniProt accession number P13236 (human) and its variants at www.uniprot.org / uniprotkb / P13236 / variant-viewer, and their homologous sequences.

[0106] CCL5: UniProt accession number P13501 (human) and its variants at www.uniprot.org / uniprotkb / P13501 / variant-viewer, and their homologous sequences.

[0107] CCL11: UniProt accession number P51671 (human) and its variants at www.uniprot.org / uniprotkb / P51671 / variant-viewer, and their homologous sequences.

[0108] CXCL10: UniProt accession number P02778 (human) and its variants at www.uniprot.org / uniprotkb / P02778 / variant-viewer, and their homologous sequences.

[0109] IL-6: UniProt accession number P05231 (human) and its variants at www.uniprot.org / uniprotkb / P05231 / variant-viewer, and their homologous sequences.

[0110] IL-17: UniProt accession number Q16552 (human) and its variants at www.uniprot.org / uniprotkb / Q16552 / variant-viewer, and their homologous sequences.

[0111] IL-4: UniProt accession number P05112 (human) and its variants at www.uniprot.org / uniprotkb / P05112 / variant-viewer, and their homologous sequences.

[0112] IL-5: UniProt accession number P05113 (human) and its variants at www.uniprot.org / uniprotkb / P05113 / variant-viewer, and their homologous sequences.

[0113] IL-13: UniProt accession number P35225 (human) and its variants at www.uniprot.org / uniprotkb / P35225 / variant-viewer, and their homologous sequences.

[0114] IFN-γ: UniProt accession number P01579 (human) and its variants at www.uniprot.org / uniprotkb / P01579 / variant-viewer, and their homologous sequences.

[0115] 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.

[0116] IL-21: UniProt accession number Q9HBE4 (human) and its variants at www.uniprot.org / uniprotkb / Q9HBE4 / variant-viewer, and their homologous sequences.

[0117] IL-22: UniProt accession number Q9GZX6 (human) and its variants at www.uniprot.org / uniprotkb / Q9GZX6 / variant-viewer, and their homologous sequences.

[0118] TGF-β-1: UniProt accession number P01137 (human) and its variants at www.uniprot.org / uniprotkb / P01137 / variant-viewer, and their homologous sequences; TGF-β-2: UniProt accession number P61812 (human) and its variants at www.uniprot.org / uniprotkb / P61812 / variant-viewer, and their homologous sequences; TGF-β-3: UniProt accession number P10600 (human) and its variants at www.uniprot.org / uniprotkb / P10600 / variant-viewer, and their homologous sequences.

[0119] 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.

[0120] Thymic stromal lymphopoietin (TSLP): UniProt accession number Q969D9 (human) and its variants at www.uniprot.org / uniprotkb / Q969D9 / variant-viewer, and their homologous sequences.

[0121] IL-31: UniProt accession number Q6EBC2 (human) and its variants at www.uniprot.org / uniprotkb / Q6EBC2 / variant-viewer, and their homologous sequences.

[0122] 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.

[0123] 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.

[0124] IL-33: UniProt accession number O95760 (human) and its variants at www.uniprot.org / uniprotkb / O95760 / variant-viewer, and their homologous sequences.

[0125] CD40L: UniProt accession number P29965 (human) and its variants at www.uniprot.org / uniprotkb / P29965 / variant-viewer, and their homologous sequences.

[0126] ICAM1: UniProt accession number P05362 (human) and its variants at www.uniprot.org / uniprotkb / P05362 / variant-viewer, and their homologous sequences.

[0127] VCAM1: UniProt accession number P19320 (homo sapiens) and its variants at www.uniprot.org / uniprotkb / P19320 / variant-viewer, and their homologous sequences.

[0128] MADCAM1: UniProt accession number Q13477 (human) or B9EGE2 and variants thereof at www.uniprot.org / uniprotkb / Q13477 / variant-viewer and www.uniprot.org / uniprotkb / B9EGE2 / variant-viewer, respectively, and homologous sequences thereof.

[0129] Integrin α4: UniProt accession number P13612 (human) and its variants at www.uniprot.org / uniprotkb / P13612 / variant-viewer, and their homologous sequences.

[0130] Integrin β7: UniProt accession number P26010 (human) and its variants at www.uniprot.org / uniprotkb / P26010 / variant-viewer, and their homologous sequences.

[0131] 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.

[0132] 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.

[0133] TLR3: UniProt accession number O15455 (human) and its variants at www.uniprot.org / uniprotkb / O15455 / variant-viewer. TLR3 has been reported to be associated with inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), colitis, and rheumatoid arthritis. Antibodies that bind to TLR3 are disclosed, for example, in U.S. Patent No. 8,153,583 B2.

[0134] TLR4: UniProt accession number 000206 (human) and its variants at www.uniprot.org / uniprotkb / O00206 / variant-viewer. TLR4 has been reported to be associated with rheumatoid arthritis. Antibodies that bind to TLR4 are disclosed in, for example, U.S. Patent No. 7,312,320 B2.

[0135] TLR5: UniProt accession number O60602 (human) and its variants at www.uniprot.org / uniprotkb / O60602 / variant-viewer. TLR5 has been 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.

[0136] TLR7: UniProt accession number Q9NYK1 (human) and its variants at www.uniprot.org / uniprotkb / Q9NYK1 / variant-viewer. TLR7 has been reported to be associated with systemic lupus erythematosus and cutaneous lupus erythematosus. Antibodies that bind to TLR7 are disclosed, for example, in U.S. Patent Application Publication Nos. 20200362052A1 and 20210040225A1.

[0137] 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, spondyloarthropathies, 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, ischemic reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, asthma, acute respiratory distress syndrome (ARDS), vasculitis or inflammatory autoimmune myositis. In a specific embodiment, spondyloarthropathies are selected from: ankylosing spondylitis, reactive arthritis, enteropathic arthritis associated with inflammatory bowel disease, psoriatic arthritis, isolated acute anterior uveitis, undifferentiated spondyloarthropathies, Behçet's syndrome and juvenile idiopathic arthritis. In one embodiment, the immune disease is caused or exacerbated 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.

[0138] In an embodiment, the present disclosure relates to a nucleic acid or polynucleotide encoding the above-mentioned fusion protein.

[0139] In an embodiment, the present disclosure relates to a vector comprising the nucleic acid or polynucleotide.

[0140] Embodiments relate to a host cell comprising the vector.

[0141] 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 under which the fusion molecule is expressed.

[0142] In an embodiment, the present disclosure relates to a method for reducing a substance or enhancing the reduction of a substance, the substance triggering, inducing or causing 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 a 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 the substance. In non-limiting embodiments, the substance and the immune disease may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have 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.

[0143] 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 such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response in a subject, the method comprising administering to a 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. In non-limiting embodiments, the substance and the undesirable or pathological immune cause may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have effector function and does not induce an inflammatory response. For example, the fusion molecule does not include a portion that binds to an Fc receptor and 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 cleared or reduced by administering the fusion molecule.

[0144] In an embodiment, the present disclosure relates to a method for treating, preventing or improving an immune disease in a subject having an immune disease or a risk of having an immune disease. The method includes 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 TAM (Tyro3, ​​Axl and MerTK) receptors on the cell surface of the subject; and a second region specifically bound to an antigenic substance that triggers, induces or causes an immune disease. In non-limiting embodiments, the antigenic substance and the immune disease may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have 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.

[0145] 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 includes administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, a vector comprising the polynucleotide, wherein the fusion molecule comprises: a first region capable of binding to TAM (Tyro3, ​​Axl and MerTK) receptors on the cell surface of the subject; and a second region that specifically binds to an antigenic substance. In non-limiting embodiments, the antigenic substance and the immune disease may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have 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.

[0146] 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 such as an autoimmune disease, transplant rejection, or allergic or hyperimmune response in a subject. The method comprises administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, wherein the fusion molecule comprises: a first region 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, the undesirable or pathological immune response to the antigenic substance is suppressed and / or reduced. Therefore, the method of the present disclosure can be used to treat any disease associated with or caused by an undesirable or pathological immune response to an antigenic substance. In non-limiting embodiments, the antigenic substance and the disease may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule does not have effector function and does not induce an Fc-mediated inflammatory response. For example, the fusion molecule does not contain a portion that binds to an Fc receptor, and preferably may 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.

[0147] In an embodiment, the present disclosure relates to a pharmaceutical composition comprising an effective amount of any 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 effector function and does not induce Fc-mediated inflammatory responses. 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.

[0148] 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 the preparation of a medicament for treating an immune disease.

[0149] 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.

[0150] In an embodiment, the present disclosure relates to a kit comprising an effective amount of any one of the fusion molecules disclosed above or the polynucleotide encoding the fusion molecule. The kit is generally packaged in a suitable manner and accompanied by appropriate instructions, and can be used in any method described in this specification.

[0151] The foregoing and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those having ordinary skill in the art upon consideration of the detailed description of the exemplary embodiments set forth below. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figures 1A to 1C Shown are the effects on EAE scores and body weight changes when EAE is induced in mice with astrocyte-specific deletion of the Axl gene.

[0153] Figures 2A to 2C Shown are the effects on EAE scores and body weight changes when EAE is induced in mice with microglia-specific deletion of the Mer tk gene.

[0154] Figure 3A Schematic diagram showing the configuration of the prepared AAV expressing anti-FITC-Gas6 and anti-MOG (8-18C5)-Gas6.

[0155] Figure 3B The amino acid sequences of anti-FITC-Gas6 and anti-MOG (8-18C5)-Gas6 constructed in Example 3 are shown.

[0156] Figure 3C and Figure 3D shows the encoding as Figure 3B The nucleic acid sequence of anti-FITC-Gas6 is shown.

[0157] Figure 3E and Figure 3F shows the encoding as Figure 3B The nucleic acid sequence of anti-MOG(8-18C5)-Gas6 is shown.

[0158] Figures 4A to 4C Shown are the effects of anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vitro.

[0159] Figures 5A to 5C Shown are the effects on EAE scores and body weight changes when anti-MOG(8-18C5)-Gas6 was expressed in EAE mice.

[0160] Figures 6A to 6E Shown are the effects of systemic expression of anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

[0161] 7A to 7E Shown is the effect of local expression of anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

[0162] Figure 8A schematic diagram showing the configuration of the prepared anti-MOG(01)-Gas6.

[0163] Figure 9A and Figure 9B Shown are the antigen (human and mouse MOG) binding activities of the anti-MOG(01)-Gas6 fusion molecules measured by ELISA.

[0164] Figure 9C The results of measuring the degree of binding of anti-MOG (01)-Gas6 fusion molecules to mouse MOG protein on the cell surface using flow cytometry are shown.

[0165] Figure 10 Shown are the effects of anti-MOG(01)-Gas6 on the removal of myelin debris in vitro.

[0166] Figure 11 A schematic diagram showing the configuration of the prepared anti-MBP-Gas6.

[0167] Figure 12A and Figure 12B Shown are the antigen (human and mouse MBP) binding activities of the anti-MBP-Gas6 fusion molecules measured by ELISA.

[0168] Figure 13 Shown are the effects of anti-MBP-Gas6 on the removal of myelin debris in vitro.

[0169] Figure 14A Schematic diagram showing the configurations of the prepared anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6. Figure 14B and Figure 14C The sequences of anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6 are shown.

[0170] Figure 15A Shown is the antigen (human TNFα) binding activity of the anti-TNFα-GAS6 fusion molecule measured by ELSA, Figure 15B The results of measuring the degree of binding between the anti-TNFα-GAS6 fusion molecule and the human TNFα protein on the cell surface using flow cytometry are shown.

[0171] Figure 16 The figure shows the inhibitory level of anti-TNFα-GAS6 fusion molecule on TNFα signaling in HEK-Blue TNFα cells.

[0172] Figure 17 The results show that the anti-TNFα-GAS6 fusion molecule can be used to inhibit the growth of U2OS cells. AxlInduction of Axl-activated proteins in cells.

[0173] Figure 18 Shown using THP-1 Axl –Derived macrophages act as effector cells for the induction of Axl-mediated phagocytosis by anti-TNFα-Gas6.

[0174] Figure 19A A schematic diagram showing the configuration of the prepared anti-CD20 (rituximab)-Gas6. Figure 19B The amino acid sequence of anti-CD20 (rituximab)-Gas6 is shown.

[0175] Figure 20A Shown is the antigen (human CD20) binding activity of the anti-CD20-Gas6 fusion molecule measured by ELISA, Figure 20B Shown is the extent of binding between anti-CD20-Gas6 fusion molecules and human CD20 protein on the cell surface using flow cytometry.

[0176] Figure 21 The anti-CD20-Gas6 fusion molecule is shown to inhibit the expression of U2OS. Axl Induction of Axl-activated proteins in cells.

[0177] Figure 22 Shown using THP-1 Axl –Derived macrophages act as effector cells for the induction of Axl-mediated phagocytosis by anti-CD20-Gas6.

[0178] Figures 23A to 23K The structures of fusion proteins according to various non-limiting embodiments of the present disclosure are schematically depicted.

[0179] Figures 24 to 34 Shown are the amino acid sequences of exemplary fusion proteins according to various non-limiting embodiments of the present disclosure. DETAILED DESCRIPTION

[0180] Methods and compositions are provided for reducing or inhibiting the formation, clearance, elimination, or reduction of 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 the symptoms of immune diseases or disorders.

[0181] Where a range of values ​​is provided, it will be understood that, unless the context clearly dictates otherwise, each intermediate value of one tenth of the unit of the lower limit between the upper and lower limits of the range is also specifically disclosed. The present invention includes intermediate values ​​within any prescribed value or prescribed range and each smaller range between any other prescribed value or intermediate value within the prescribed range. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope, and each range in which any limit, no limit, or both limits are included within the smaller range is also included within the present invention, subject to any specifically excluded limit in the prescribed range. Where the prescribed range includes one or two limits, the range excluding one or both of the included limits is also included within the present invention.

[0182] [definition]

[0183] 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, 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 forth, as would be known to one of ordinary skill in the art.

[0184] As used herein, the terms "about" and "consisting essentially of" refer to values ​​or components that are within an acceptable error range for the particular value or component as determined by one of ordinary skill in the art, depending 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 more than 1 standard deviation as practiced in the art. Alternatively, "about" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). For example, "about 5 mg" can include any 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%). Furthermore, particularly with respect to biological systems or processes, these terms can 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 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.

[0185] As used herein, "administering" or "administering" refers to introducing a composition into a subject via 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 herein are administered to a subject.

[0186] 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 encoded by the genetic code, as well as those that are later modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bound to a hydrogen, carboxyl, amine, and R group, for example, homoserine, norleucine, methionine sulfoxide, or methionine methylsulfonium. 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 mimetics refer to compounds whose structure is different from the conventional chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids.

[0187] As used in this specification, "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably in this specification to 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 mimetics 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 manipulation or modification, such as binding to a labeling component. Also included in the definition are, for example, polypeptides comprising 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 based on antibodies, the polypeptides can appear in the form of single chains or associated chains.

[0188] As used in this specification, "polynucleotide" or "nucleic acid" as used interchangeably herein refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. 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 can 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, "end-capping," replacing one or more naturally occurring nucleotides with an analog; internucleotide modifications, for example, modifications with uncharged linkages (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) and charged linkages (e.g., phosphorothioate, phosphorodithioate, 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; modifications with modified linkages (e.g., α-anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. In addition, any hydroxyl group typically present in the sugar can be substituted with, for example, a phosphonate group, a phosphate group, protected with standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or can be conjugated to a solid support. The OH at the 5' and 3' terminals can be phosphorylated or partially replaced by an amine or an organic blocking group of 1 to 20 carbon atoms. Other hydroxyls can also be derived into standard protecting groups. Polynucleotides can also comprise similar forms of ribose or deoxyribose well known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleosides. One or more phosphodiester connections can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is substituted with P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("amide"), P(O)R, P(O)OR', CO, or CH2 ("acetal"), wherein R or R' are each independently H or substituted or unsubstituted with 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 be identical. The above description applies to all polynucleotides contemplated herein, including RNA and DNA.

[0189] As used herein, "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, for whom diagnosis, treatment, or therapy is desired. For therapeutic purposes, "mammal" refers to any animal classified as a mammal, including humans, domestic and livestock animals, as well as zoo animals, sports animals, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, and the like. In embodiments, the mammal is a human.

[0190] As used herein, "antibody" refers to single-chain, double-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.

[0191] As used herein, "specific binding," "specifically binds," and the like refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides / epitopes). In some embodiments, the affinity of 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 Below M, 10 -7 Below M, 10 -8 Below M, 10 -9 Below M, 10 -10 M or less, 10 -11 Below M, 10 -12 Below M, 10 -13 M or less, 10 -14 M or less, 10 -15 Below M 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 herein, "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.

[0192] As used herein, "variable" refers to the fact that specific portions 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 domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called frameworks (FRs). The variable domains of native heavy and light chains each contain four FR regions, primarily in a b-sheet configuration connected by three CDRs, which form loops connecting the b-sheet structure and, in some cases, form 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 constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0193] "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 chain and heavy chain 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 to an antigen, although the affinity is lower than that of the entire binding site.

[0194] As used herein, the term "complementarity determining region" or "CDR" 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 (e.g., HCDR1, HCDR2, and HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. 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 a combination 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 combined Kabat and Chothia 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, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.

[0195] A "Fab fragment" also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Throughout this specification, Fab'-SH is the designation for Fab' in which the cysteine ​​residues of the constant domains have a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0196] As used herein, the term "antibody fragment" or "antigen-binding fragment" or "active fragment" is defined as a portion of an intact antibody that contains the antigen binding site or variable region of the intact antibody, wherein the portion does not contain the constant heavy chain domains of the Fc region of the intact antibody (i.e., CH2, CH3 and CH4, depending on the antibody isotype). 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 herein as a “single-chain antibody fragment” or “single-chain polypeptide”), including but not limited to (1) single-chain Fv (scFv) molecules, (2) single-chain polypeptides comprising only one light chain variable domain, or fragments thereof comprising the three CDRs of a light chain variable domain, without the associated heavy chain portion, (3) 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, (4) nanobodies comprising a single Ig domain from a non-human species or other specific single domain binding module; 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 constant domain sequence found in the non-Fc region of an intact antibody (e.g., CH1 in an IgG isotype), and / or may comprise any hinge region sequence found in an intact antibody, and / or may comprise a leucine zipper sequence fused to or located within a hinge region sequence or constant domain sequence of a heavy chain, and (5) isolated complementarity determining regions (CDRs).

[0197] The terms "phagocytic cells," "phagocytes," and "apoptotic cells" are used interchangeably throughout this specification to refer to cells capable of phagocytosis. There are four main types of phagocytes: macrophages, monocytes (histiocytes and mononuclear cells), polymorphonuclear leukocytes (neutrophils), and dendritic cells.

[0198] As used herein, "chimeric" refers to a molecule that includes sequences from two different molecules.

[0199] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of immunoglobulin heavy chains can vary, the human IgG heavy chain Fc region is typically defined as extending from an amino acid residue at position Cys226 or from Pro230 to their carboxyl termini. The residue numbering of the Fc region is the same as that of 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 typically includes two constant domains, CH2 and CH3.

[0200] As used herein, "Fc receptor" and "FcR" describe receptors that bind to the Fc region of an antibody. Preferred FcRs are native sequence human FcRs. Furthermore, preferred FcRs are those 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.

[0201] A "native sequence Fc region" or "Wile-type Fc region" comprises an amino acid sequence identical to an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region due to at least one amino acid modification, but retains at least one effector function of a native sequence Fc region. Preferably, the variant Fc region comprises at least one amino acid substitution in the native sequence Fc region or the Fc region of the parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions, preferably from 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 disclosure preferably have at least about 80% sequence identity to 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%, or at least about 99% sequence identity thereto.

[0202] 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 prophylactic use, beneficial or desired results include results such as eliminating or reducing risk, reducing 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 appear during the course of the disease. For therapeutic use, beneficial or desired results include clinical results, such as inhibiting, suppressing, or reducing elevated levels of a substance, reducing, removing, clearing elevated antigenic substances, or reducing them to their normal levels, sequestering 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 course of the disease, improving the quality of life of patients with the disease, reducing the dose of other drugs required to treat the disease, enhancing the effect of another drug, delaying the progression of the disease, and / or prolonging the patient's survival. An effective dose can be administered once or multiple times. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve a prophylactic or therapeutic treatment. As understood in the clinical setting, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective 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 can or has been achieved in combination with one or more other drugs.

[0203] As used herein, "treatment" or "treatment" is a method for obtaining a beneficial or desired result (including a clinical result). For the purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, one or more of the following: preventing, inhibiting or reducing the formation of deposits of a substance, reducing, removing or clearing deposits of antigenic substances, improving cognition, reversing or slowing cognitive decline, sequestering soluble substances circulating in biological fluids, reducing substances (including soluble, oligomeric and deposited substances) in tissues, inhibiting, slowing and / or reducing increases or elevations in the levels of antigenic substances in tissues, inhibiting, slowing and / or reducing the toxic effects of substance peptides in tissues, reducing symptoms caused by the disease, improving the quality of life of patients with the disease, reducing the dosage of other drugs required to treat the disease, delaying the progression of the disease, and / or prolonging the patient's survival. The tissue may include the brain of an individual.

[0204] 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 the development of a disease. However, development also refers to the 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, in this case, determined by a 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, detecting changes in the 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), computed tomography (CT), and magnetic resonance imaging (MRI). "Development" includes occurrence, recurrence, and onset. As used in this specification, "onset" or "occurrence" of a disease includes initial onset and / or recurrence.

[0205] As used herein, "delaying" the development of a disease means delaying, hindering, slowing, postponing, 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 can actually include prevention, i.e., that the individual will not develop the disease. For example, a method for delaying the development of a disease is a method that reduces the probability of the disease developing within a given timeframe and / or reduces the extent of the disease within a given timeframe, when compared to not using the method. Such comparisons are typically based on clinical studies using statistically significant numbers of subjects.

[0206] As used herein, "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 cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and specific eukaryotic cells such as producer cells.

[0207] "Host cell" includes a single cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny are not necessarily 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.

[0208] As used herein, an "expression control sequence" refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or inducible promoter, or an enhancer. An expression control sequence is operably linked to a nucleic acid sequence to be transcribed.

[0209] As used herein, a "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient, allows the ingredient to maintain biological activity 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, 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 well-known conventional methods.

[0210] [TAM receptors]

[0211] TAM receptors (Tyro3, ​​Axl and Mer) belong to the receptor tyrosine kinase family, which plays an important role in hemostasis and inflammation. In addition, they affect cell proliferation, survival, adhesion and migration. TAM receptors contain two immunoglobulin-like and two fibronectin type III repeats in their extracellular domain. This is connected to a one-way transmembrane domain and a cytoplasmic protein tyrosine kinase.

[0212] TAM receptors enhance phagocytosis of apoptotic cells, also known as efferocytosis.

[0213] 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 Nos. NP_068713, NP_068713.2, and 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, and 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.

[0214] 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 numerous physiological processes, including cell survival, migration, and differentiation. Ligand binding at the cell surface induces dimerization and autophosphorylation of TYRO3 on its intracellular domain, providing docking sites for downstream signaling molecules. Upon ligand activation, MerTK interacts with PIK3R1, thereby enhancing PI3-kinase activity.

[0215] Human MerTK comprises 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 June 11, 2023).

[0216] The human Tyro3 tyrosine kinase receptor comprises 890 amino acid residues (accession numbers Q06418, NP_001317193.1, NP_006284.2). The polynucleotide sequence encoding human tyro3 is available under accession numbers NM_001330264.1 and NM_006293.3. Various mRNA sequences encoding human tyro3 are reported with accession numbers such as 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 on June 11, 2023).

[0217] The cell expressing TAM receptor can be at least one type of full-time phagocyte, at least one type of non-professional phagocyte or their combination.Here, full-time phagocyte refers to that its main function is to remove the cell of dead cells and the debris of accumulation by phagocytosis, and its example includes macrophage, neutrophil, dendritic cell and mast cell.Macrophage usually stays in each tissue that can become infection route, and in many cases, they are referred to as different tissue names, including, for example, adipose tissue macrophage, bone marrow or blood monocyte, liver Kupffer cell (Kupffercells), lymph node sinus tissue cell, alveolar macrophage, connective tissue tissue cell or giant cell, microglia of central nervous system, placenta Hofbauer cell (Hofbauer cells), glomerular mesangial cell, bone osteoclast, the epithelial cell of granuloma, the red pulp macrophage of spleen, peritoneal macrophage of peritoneal cavity, the lysozyme macrophage (macrophage expressing lysozyme) etc. of Peyer's patch (Peyer'spatch). On the other hand, non-professional phagocytes refer to cells that mainly perform the specific functions of the tissue in which the phagocytes are located, but can perform phagocytosis when necessary, and their 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 glia, hepatocytes, muscle satellite cells, testicular supporting cells, etc., and some lymphocytes, such as natural killer cells, large-granular lymphocytes, eosinophils, basophils, B cells, etc. The fusion molecules according to the present disclosure can induce phagocytosis in phagocytes that are specific to the tissue in which the target substance to be cleared is increased. For example, when the amount or expression of the antigen protein in the brain increases and needs to be cleared, phagocytosis can be induced in astrocytes, microglia, oligodendrocytes or a combination thereof. For example, it can be induced by locally 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.

[0218] [First region comprising a sequence capable of binding to a TAM receptor]

[0219] 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.

[0220] In exemplary embodiments, the first region capable of binding to a TAM receptor can comprise, consist of, or consist essentially of one or more TAM ligands.

[0221] The TAM ligand, protein S, consists of an amino-terminal γ-carboxyglutamate (Gla) domain, followed by a thrombin-sensitive loop region and four epidermal growth factor-like domains ending in the carboxyl terminus (C-terminus), consisting of two laminin G repeats, which together include the sex hormone-binding globulin domain ( Figure 1A Gas6 is a 75 kDa vitamin K-dependent protein with high structural homology (approximately 42%) to protein S and a modular organization similar to that of Figure 1A Same as shown.

[0222] 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 No. AAB55 Tubby and Gal-3 specifically bind to Mer, while Tulp1 can activate all three TAM receptors.

[0223] It has been reported that Gas6, one of the ligands of TAM receptors, exhibits the highest affinity for Axl compared to Tyro3 or Mer. Human Gas6 contains 678 amino acids (SEQ ID NO: 7) and has a γ-carboxyglutamate (Gla) domain, four epidermal growth factor (EGF)-like domains, and two laminin G-like (LG) domains ( Figure 1A , right). Various subtypes of GAS6 have been reported. For example, S6L, G8R, G8V, R14H, and L18Q subtypes have been reported, and these subtypes are included in the present disclosure.

[0224] In an embodiment, the first region capable of binding to a TAM receptor can 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 instances, the increase in biological activity is indicated by changes in downstream effects, such as an increase in TAM autophosphorylation, a decrease in the production of TLR-induced cytokines, a decrease in the stimulation initiated by TLR-induced MAP kinases, a decrease in the activation of TLR-induced NF-kB or an increase in SOCS1 and SOCS3 expression. Methods for detecting such changes in expression or activity (in some instances quantitatively) 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.

[0225] In an embodiment, the first region capable of binding to a TAM receptor may comprise, consist of, or consist essentially of a Gas6 protein or an active fragment thereof. The term "active fragment" as used herein 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, consist of, or essentially consist of the sequence of SEQ ID NO: 1, 2, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 or 87. 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 exhibit at least 85% sequence identity to SEQ ID NO: 4 (LG-2 domain of ProS1). SEQ ID NOs: 63-87 exhibit at least 85% sequence identity to SEQ ID NO: 5 (LG domain of Gas6).SEQ ID NOs: 88-113 exhibit at least 85% sequence identity to SEQ ID NO: 6 (LG domain of ProS1).

[0226] In other embodiments, the first region may comprise, consist of, or consist essentially of the variable region or CDR of an anti-Axl antibody or full-length anti-Axl antibody whose effector function, particularly Fc receptor binding function, has been abolished or removed. The antibody or antigen-binding fragment can bind to the extracellular domain of Axl, 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 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.

[0227] In other embodiments, the first region may include, or consist of, or consist essentially of: an anti-MerTK (Mer tyrosine kinase) antibody or a full-length anti-MerTK antibody whose effector function, in particular Fc receptor binding function, is eliminated or removed. The antibody or antigen-binding fragment can 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, in particular 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 regions, CDRs or scFv, F(ab) or F(ab′) of these anti-MerTK antibodies can be used as the first region of the fusion molecule.

[0228] In other embodiments, the first region may include, or consist of, or consist essentially of: an anti-Tyro3 antibody or a variable region or CDR of a full-length anti-Tyro3 antibody whose effector function, in particular, 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, in particular 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 in their entirety. According to embodiments of the present disclosure, the variable regions, CDRs or scFv, F(ab) or F(ab′) of these anti-Tyro3 antibodies can be used as the first region of the fusion molecule.

[0229] A 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 variants. 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 non-essential sites for activity are truncated or deleted, or the amino acid residues at sites important for autoinhibition are substituted. In some cases, sequence variants 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.

[0230] 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 mutagenesis 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 11). 6,pp.61-70) etc.

[0231] The amino acid sequence inserts and comprises that length is from a residue to the amido-and / or carboxyl terminal fusion of the polypeptide that comprises 100 or more residues, and inserts in the sequence of single or multiple amino acid residues.The example that terminal inserts comprises the antibody with N-terminal methionyl residue or is fused to the antibody of epitope tag.Other insertion variants of antibody molecule comprise the fusion of enzyme or polypeptide and antibody N-or C-terminal, and this increases the serum half-life of antibody.

[0232] 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 deleteriously alter functional activity; or polypeptides using chemical analogs.

[0233] Substitution variants have at least one amino acid residue in the antibody molecule that has been removed and a different residue inserted in its place. The sites of greatest interest for substitution mutations include hypervariable regions, but FR changes are also contemplated. Conservative substitutions are shown under the heading "conservative substitutions" in Table 2. If such substitutions result in changes in biological activity, then further changes can be introduced and the products screened, designated as "exemplary substitutions" in Table 2, or as further described below with reference to amino acid classes.

[0234] Table 2: Amino acid substitutions

[0235] 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; norleucine

[0236] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their effect on maintaining: (a) the structure of the polypeptide backbone in the area 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 chain. Based on common side chain properties, naturally occurring residues are classified as:

[0237] Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

[0238] Polar but uncharged: Cys, Ser, Thr, Asn, Gln;

[0239] Acidic (negatively charged): Asp, Glu;

[0240] Basic (positively charged): Lys, Arg;

[0241] Residues that affect chain orientation: Gly, Pro; and

[0242] Aromatic: Trp, Tyr, Phe, His.

[0243] 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, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​linkages may be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

[0244] 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 variable region).The variation of variable region can change binding affinity and / or specificity.In some embodiments, in CDR structural domain, carry out and be no more than one to five conservative amino acid replacements.In other embodiments, in CDR structural domain, carry out and be no more than one to three conservative amino acid replacements.In another other embodiment, CDR structural domain is CDR H3 and / or CDR L3.

[0245] [Targeting inflammation-related substances and immune diseases or immune-mediated diseases]

[0246] 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 complement; one or more chemokines; one or more cytokines; one or more cell adhesion molecules; or a combination thereof.

[0247] 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 and include co-stimulatory molecules and receptors, complement, chemokines, cytokines and cell adhesion molecules.

[0248] Fusion molecules according to embodiments of the present disclosure reduce, remove or remove the antigenic substance in the subject, or enhance the removal or removal of the antigenic substance in the subject, thereby can be used to treat or prevent immune disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder or proliferative disease or disorder. In specific embodiments, fusion molecules according to the present disclosure suppress, reduce, remove or remove inflammation-related substances in the subject, or enhance the removal or removal of inflammation-related substances in the subject, thereby can be used to treat or prevent immune disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder or proliferative disease or disorder. Immune disease or disorder can be the autoimmune disease or inflammatory disease exemplified in the disclosure.

[0249] 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 relapsing multifocal osteomyelitis, gout, Henoch-Schonlein purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus erythematosus (SLE), juvenile scleroderma, juvenile vasculitis, Kawasaki disease, lupus (systemic lupus erythematosus), mixed connective tissue disease, dermatomyositis, poststreptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjögren's syndrome, spondyloarthritis / spondyloarthropathies, systemic juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, celiac disease, thrombotic thrombocytopenic purpura (iTTP), and the like.

[0250] The fusion molecules described herein can also be used to treat cardiovascular diseases or disorders, such as atherosclerosis, endocarditis, hypertension, or peripheral ischemic disease.

[0251] The fusion molecule described in this specification sheets can be used to treat or prevent, suppress, slow down immune disease or disorder, cardiovascular disease or disorder, metabolic disease or disorder or proliferative disease or disorder progress, or alleviate the symptoms associated therewith.Immune disorders include inflammatory diseases or disorders, and autoimmune diseases or disorders.Although inflammation or inflammatory response are normal reactions and protective reactions of the host to damage, inflammation can cause undesirable damage.For example, atherosclerosis is at least in part a pathological reaction to arterial damage and subsequent inflammatory cascade reaction.Cardiovascular disease or disorder that can be treated, it can include and be considered to immune disease / disorder disease and disorder, it includes for example atherosclerosis, endocarditis, hypertension or peripheral ischemic disease.Metabolic disease or disorder include diabetes, obesity and increase or raise relevant disease and disorder with antigenic substance level.

[0252] Allergen is another antigen, and the tolerance to its immune response is also desirable. Even in the case where the pathogenic autoantigen is an unknown disease, the antigen at the anatomical adjacent site can also be used to induce bystander inhibition. For example, autoantibodies to collagen are observed in rheumatoid arthritis. Therefore, the gene encoding collagen or collagen can be the target inflammation-related substance to be cleared or removed or reduced by administering a fusion molecule. In this case, the aptamer specifically bound to the gene encoding collagen or the antibody bound to collagen or its fragment can be used as the second region of the fusion molecule. In addition, the fusion protein comprising the second region combined with the beta cell autoantigen can be used for preventing the development of type 1 diabetes or treatment (see, for example, Bach and Chatenoud (2001) Ann Rev Immunol 19: 131-161).

[0253] 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 moiety can treat multiple sclerosis and related autoimmune diseases of the central nervous system.

[0254] 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 can recognize a specific antigen 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 can also recognize a specific antigen can respond by binding to and destroying or damaging cells or particles carrying the antigen.

[0255] The immune response in a host or subject can be determined by any of the well-known immunological methods described in this specification, which are also familiar to those skilled in the art. Such determinations include, but are not necessarily limited to, in vivo or in vitro determinations 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, and other soluble small peptides, carbohydrates, nucleotides, and / or lipid mediators; changes in cell activation state determined by changes in the functional or structural properties of immune system 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; and cell differentiation of immune system cells, including changes in surface antigen expression profiles 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).

[0256] The level of cytokine can be determined according to that described in this specification sheets and practiced in this area, including ELISA, ELISPOT and flow cytometry (to measure intracellular cytokines). Immune cell proliferation and the selection of colonization amplification caused by the stimulation of antigen-specific induction or immune response can be measured by the following method: separate lymphocytes such as spleen cells or cells from lymph nodes, stimulate cells with antigens, and measure the generation of cytokines, cell proliferation and / or cell viability, such as by incorporating tritiated thymidine pyrimidine or non-radioactive assays, such as MTT assays etc. The effect of the fusion polypeptide described in this specification sheets on the balance between Th1 immune response and Th2 immune response can be by, for example, measuring the level 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.

[0257] In certain embodiments, antigenic substances and associated immune diseases do not include substances whose abnormal accumulation or aggregation in living tissue is characteristic of or associated with diseases such as neurological diseases or disorders.

[0258] [Second region of the fusion molecule]

[0259] 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.

[0260] 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, diabodies, triabodies, tetrabodies, pentabodies, etc. The antibody or its antigen-binding fragment can be, for example, a Mab, Fab or single-chain variable fragment (scFv) based on an antibody that specifically binds to a corresponding target substance, or six complementary determining regions (CDRs) from an antibody. That is, the protein or its antigen-binding fragment that specifically binds to the target substance contains the part necessary for the activity of specific binding to the target substance, and there is no particular limitation on its type or scope, as long as the protein or its antigen-binding fragment is connected to the first region and does not cause inflammatory response and synaptic damage. For example, the target substance can be beta-amyloid protein. In this case, the protein or antigen-binding fragment thereof that specifically binds to the target substance can include aducanumab or a single-chain variable fragment thereof. The second region includes a Mab, Fab, or single-chain variable fragment based on six complementarity determining regions (CDRs) derived from commercially available antibodies such as aducanumab, semorinemab, and cinpanemab.

[0261] 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 FcγRIIIA and / or FcγRIIA and / or FcγRI 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.

[0262] Antibody-like proteins are protein scaffolds that can specifically bind to target substances such as antibodies. Antibody-like proteins can be designed to have a size of approximately 2kDa to 20kDa, which is smaller than antibodies (average of approximately 150kDa), 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 or chemically synthesize using non-mammalian cells such as viruses and yeast than antibodies.

[0263] 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 a very high affinity for a specific substance, 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 to already developed antibodies, 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 connected to the first region to produce a fusion molecule according to the present invention.

[0264] There is no limitation on the aptamer of the present disclosure as long as it can specifically bind to any of the listed target substances, and unless otherwise specified, the base used for the aptamer can be selected from A, G, C, U and their deoxy forms.

[0265] In addition, the aptamer can be modified by connecting 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 in the 5'-terminal region, the middle region, the 3'-terminal region or both ends to increase its stability. Inverted deoxythymidine (idT) is a molecule that is generally used to prevent nuclease degradation of aptamers with weak nuclease resistance. In the case of a nucleic acid unit, the 3'-OH of the previous nucleotide is connected 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 connected 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 effect of inhibiting degradation by 3' exonuclease (a type of nuclease).

[0266] 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 ingestion of cells or particles with a size of 0.5 μm or more, and includes the process of binding, engulfing and degrading cells or particles. In this case, phagocytosis forms phagosomes that surround the internalized cells or particles, and includes degradation within the phagolysosomes by fusion of phagosomes and lysosomes. In phagocytosis, the process of cell death by apoptosis or necrosis is also called efferocytosis.

[0267] Non-limiting representative examples of second regions and their targets 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 TGFβ.

[0268] Table 3

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] [Fusion molecule or binding molecule]

[0283] 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 inflammatory responses, thereby allowing for safer treatment of tissue dysfunction caused by increased amounts or expression of target substances than conventional techniques.

[0284] The first region and the second region are coupled to each other directly or through a linker to form a fusion molecule.

[0285] The fusion molecule may further comprise a tag. When such a tag is incorporated into the fusion molecule, it can be used to examine the purification, expression, action, or mechanism of action of the fusion molecule.

[0286] Examples of tags include, but are not limited to, His tag, T7 tag, S tag, FLAG tag, streptomycin (Strep) tag, thioredoxin (Trx) tag, His-patch thioredoxin tag, lacZ (L-galactosidase) tag, chloramphenicol acetyltransferase tag, trpE tag, avidin / streptavidin / streptomycin tag, T7gene10 tag, Staphylococcal protein A tag, Streptococcal 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, calmodulin 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.

[0287] The fusion molecule may also contain a signal peptide or leader sequence at the N-terminus. Signal peptides are short peptides present at the N-terminus during the initial stages of protein synthesis toward the secretory pathway and are known to guide the corresponding protein's intracellular localization, membrane topology (in the case of membrane proteins), and other functions. The signal peptide may be cleaved during the expression and extracellular secretion of the fusion molecule.

[0288] The above-mentioned first region, second region, tag, signal peptide or region with minimum function (for example, 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 (SEQID 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.

[0289] 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 connector.

[0290] Fusion molecules according to aspects of the present disclosure can have non-limiting exemplary schemes, for example, Figures 23A to 23K The structure is schematically shown in FIG.

[0291] Another aspect of the present disclosure provides a nucleic acid molecule encoding the fusion molecule, and an expression vector comprising the nucleic acid molecule.

[0292] 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 having an activity equivalent thereto.

[0293] The nucleic acid sequence encoding the fusion molecule can be isolated 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.

[0294] 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 genetic construct comprising the necessary regulatory elements operably linked to express the gene. Such expression vectors include all vectors such as plasmid vectors, cosmid vectors, phage vectors, and viral vectors.

[0295] Suitable expression vectors have expression control components, such as promoters, start codons, stop codons, polyadenylation signals and enhancers. Start codons and stop codons are generally considered to be a part of the nucleic acid sequence of the encoded protein, and the sequence of the encoded protein is designed in the frame so that it is operable in the vector. The promoter can be constitutive or inducible. In addition, conventional expression vectors include selective markers. Genetic recombination techniques well known in the art can be used to be operatively connected to the expression vector, and enzymes well known in the art can be used to carry out site-specific DNA cutting and connection.

[0296] The expression vector can preferably be 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 to 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 does not integrate into the genome of the host cell.

[0297] Yet another aspect of the present disclosure provides a cell expressing the fusion molecule.

[0298] 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 organisms, cells, tissues or organs. These methods include, but are not limited to: electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-, dextran sulfate-, liposome- and desiccation / inhibition-mediated transformation methods.

[0299] 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. Using methods known to those skilled in the art and based on the present disclosure, nucleic acid vectors can be designed for expressing exogenous sequences in a particular host system, and then the polynucleotide sequence encoding the fusion polypeptide can be inserted. The regulatory elements will vary depending on the specific host.

[0300] After the fusion molecule is expressed in the cell, it can be isolated and purified using conventional biochemical separation techniques, such as treatment with a protein precipitant (salting out), centrifugation, sonication, ultrafiltration, dialysis, or various chromatographic analyses such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography. These 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)).

[0301] The resulting fusion protein can be evaluated to determine whether the fusion protein significantly increases TAM receptor activity. The method can include contacting a cell with a test fusion protein and determining whether contacting the cell with the test fusion protein alters TAM autophosphorylation, TLR-induced cytokine production, TLR-induced MAP kinase activation, 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 activation, or TLR-induced NF-kB activation in the presence of the test fusion protein relative to the control level indicates that the fusion protein stimulates TAM receptor activity.

[0302] Autophosphorylation assay is well known in the art. In one example, cells expressing TAM receptors are cultured and processed with test culture medium, for example, at 37°C for 20 minutes. The culture medium is aspirated and a cold lysis buffer is added to each sample. The sample is centrifuged to allow the nucleus to spin down, and the supernatant is mixed with protein A agarose beads and affinity-purified anti-TAM receptor antibodies and then cultured. Protein A beads are granulated and washed and separated on a 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 the phosphotyrosine label relative to the control indicates that the test fusion protein is a TAM receptor inhibitor. The control can be a known value of the phosphotyrosine label in the indicator sample, such as cells without test reagent treatment. For example, the significant increase of the phosphotyrosine label relative to the control indicates that the test fusion protein is a TAM receptor agonist. For example, an increase in TAM phosphotyrosine of 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%, compared to such a control, indicates that the test fusion protein activates the TAM receptor.

[0303] 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 the amount of TLR-induced cytokine production in the absence of the test fusion protein. For example, a significant decrease 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 of 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 turns on the TAM receptor.

[0304] MAP kinase activity can be determined by performing a MAP kinase assay. A significant increase in MAP kinase activation (as indicated by increased p38 phosphorylation) in the presence of the test fusion protein relative to a control level of MAP kinase activity (e.g., a basal level of MAP kinase activity) indicates that the test fusion protein inhibits TAM receptor activity. For example, a significant decrease in MAP kinase activity of 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 activating the TAM receptor.

[0305] 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 a control indicates that the test agent is a TAM receptor agonist, and thus, the test fusion protein activates the TAM receptor. For example, a significant reduction in TLR-induced NF-kB activation of 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 such a control indicates that the test fusion protein activates the TAM receptor.

[0306] [Pharmaceutical composition]

[0307] 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 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.

[0308] Another aspect of the present disclosure provides use of a fusion molecule in the preparation of a medicament for preventing or treating an immune disease or disorder.

[0309] The fusion molecule is the active ingredient in the pharmaceutical composition, which is contained in a "pharmaceutically effective amount".

[0310] The pharmaceutical composition can be administered orally or parenterally, preferably parenterally. More preferably, it can be administered locally to a tissue where the target substance to be cleared exhibits increased / elevated levels or increased expression.

[0311] As used in this specification, the term "parenteral administration" includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

[0312] When the pharmaceutical composition is prepared as an injectable preparation, it can be prepared as an injectable preparation using conventional methods known in the art. The injectable preparation can be in the form of being dispersed in a sterile culture medium so that it can be directly administered to a patient, or can be in the form of being administered after being dispersed in distilled water for injection at an appropriate concentration.

[0313] 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.

[0314] 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)).

[0315] The appropriate dosage of the pharmaceutical composition can vary depending on factors such as formulation method, administration mode, patient age, weight, sex, medical condition, diet, administration time, administration route, excretion rate and reaction sensitivity. The dosage of the pharmaceutical composition of the present disclosure is 0.0001 μg / kg to 1,000 μg / kg body weight for adults.

[0316] Beneficial effects

[0317] The present disclosure relates to a fusion molecule with phagocytosis-inducing activity that can address the problem of tissue damage caused by the initiation of inflammatory responses encountered in the prior art. Thus, the fusion molecule can effectively eliminate and reduce substances whose expression or amount is increased to, for example, normal levels or amounts, and can therefore 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 herein. The fusion molecule can be administered to a patient in the form of a purified fusion molecule or a gene therapy vector capable of expressing and secreting the fusion molecule when introduced into cells.

[0318] 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 claims.

[0319] Example

[0320] Hereinafter, the present disclosure will be described in more detail with reference to Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative, and the scope of the present invention is not limited thereto.

[0321] It is known that astrocytes and microglia play an important role in the progress of multiple sclerosis (MS). It is reported that these cells express TAM receptors and show phagocytosis and anti-inflammatory activity when TAM receptors are activated. In Examples 1 and 2 below, attempts were made to determine the impact of the gene knockout (KO) of the main TAM receptors in the cells of the above two types on the MS progression in EAE (experimental autoimmune encephalomyelitis) mice (one of MS model animals). The EAE model is a disease model produced by inducing the demyelination of the myelin sheath in the central nervous system by inducing the activation of T cells that recognize MOG (myelin oligodendrocyte glycoprotein). Due to the fact that the model has been reported to be similar to human multiple sclerosis in many clinical and histopathological applications, it is most often used as an animal model to study the mechanism and therapeutic effect of multiple sclerosis.

[0322] Example 1: Astrocytes regulate inflammation in the CNS system through Axl

[0323] Aldh1l1-CreERT2; Axl f / f mice were used to remove the astrocyte-specific Axl gene from adult mice. Axl f / f mice were used as controls. Tamoxifen (75 mg / kg) was 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 body weight changes were observed 25 days after EAE induction. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model to study the immune pathogenesis of chronic inflammatory diseases of the central nervous system (CNS) such as multiple sclerosis (MS) and to test the efficacy of new drugs.

[0324] To ablate the astrocyte-specific Axl gene and induce MS-like disease (EAE) in mice, we used Figure 1AMice were operated according to the protocol shown. In more detail, Aldh1l1-CreERT2;Axl f / f female mice containing a construct linked to the Aldh1l1 promoter and Axl f / f were used to express CreERT2 specifically in astrocytes, and Axl f / f female mice were used as controls.

[0325] 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 in complete Freund's adjuvant (CFA) was subcutaneously injected, and pertussis toxin (PTX) was administered intraperitoneally for 2 consecutive days. EAE scores and body weight changes were then monitored for 25 consecutive days.

[0326] As a result, it was confirmed that the EAE score and body 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 ). Thus, it was found that astrocytes play an important role in regulating EAE, and this is achieved through Axl.

[0327] Example 2: The role of microglia in regulating inflammation in the CNS system through Mertk

[0328] 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. The EAE scores and body weight changes were observed 25 days after EAE induction. In order to remove the Mertk gene specific to microglia and induce MS-like disease in mice, according to Figure 2A Mice were manipulated according to the protocol shown.

[0329] In more detail, Cx3cr1-CreERT2 comprising a construct connected to the Cx3cr1 promoter and Mertk f / f; Mertk f / f female mice were used to express CreERT2 specifically in microglia; Mertk f / f female mice were used as controls. When the mice were 8 weeks old, CreERT2 was initiated by intraperitoneal administration of tamoxifen for 5 consecutive days. Next, in order to induce EAE in 9-week-old mice, MOG35-55 contained in CFA was subcutaneously injected, and pertussis toxin (PTX) was administered intraperitoneally for two consecutive days. Afterwards, EAE scores and weight changes were confirmed for 25 consecutive days. As a result, it was confirmed that the EAE scores and weight changes of mice in which the Mertk gene was specifically deleted by microglia were more severe than those in the control group ( Figure 2B and Figure 2C ). This suggests that microglia play an important role in regulating EAE, and this is achieved through Mertk.

[0330] Example 3: Construction of fusion proteins: anti-FITC-Gas6 fusion molecules and anti-MOG (8-18C5)-Gas6 fusion molecules

[0331] To efficiently remove myelin debris by using TAM receptors, AAV expressing a fusion molecule based on the human Gas6 protein was prepared.

[0332] More specifically, the Gla domain and EGF repeat domain, which are sites for recognizing PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and a single-chain Fv fragment of an antibody against the MOG protein, which is highly expressed on myelin, was placed on these sites (anti-MOG (8-18C5)-Gas6). Figures 3A to 3F .

[0333] 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.

[0334] Example 4: In vivo effect of anti-MOG (8-18C5)-Gas6 fusion molecule in removing myelin debris

[0335] To express anti-FITC-Gas6 and anti-MOG (8-18C5)-Gas6 in the HEK293T cell line, expression vectors were transfected. Serum-free medium was replaced three days after transfection, and the supernatant was collected and concentrated one day later. Western blotting was performed to confirm protein size and expression. HMC3 (human microglial 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 combining it with pH-sensitive pHrodo. After treating HMC3 cells with concentrated supernatant and myelin-pHrodo, live cell imaging was performed using IncuCyte.

[0336] To confirm the effect of anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vivo, according to Figure 4A The scheme shown expresses the protein in HEK293T cells. In more detail, to express the protein, the expression vector was transfected into the HEK293T cell line and the supernatant was concentrated. The protein was confirmed to be expressed with a predicted size of 75kDa to 76kDa ( Figure 4B To confirm the effect of myelin debris removal in an in vivo system, Myelin-pHrodo was used in the HMC3 cell line. The results showed 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 ( Figure 4C ). Thus, it was found that anti-MOG(8-18C5)-Gas6 was properly expressed in a functional form and played an important role in the efficient removal of myelin debris.

[0337] Example 5: In vivo effect of anti-MOG (8-18C5)-Gas6 fusion molecule in reducing the severity of EAE

[0338] Experimental procedure: AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into the 400 μg / mL PBS at 1×10 11 Vg was administered by retro-orbital injection to wild-type C57BL / 6J 6-week-old female mice. To induce EAE in 9-week-old mice, MOG35-55 and pertussis toxin (PTX) in complete Freund's adjuvant (CFA) were administered. After EAE induction, EAE scores and body weight changes were observed for 25 consecutive days.

[0339] To overexpress anti-MOG(8-18C5)-Gas6 in mice and induce MS-like disease (EAE), we used Figure 5AMice were operated according to the protocol shown. AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG (8-18C5)-Gas6-HA were injected into the mouse body with 1×10 11 Vg was administered to wild-type C57BL / 6J 6-week-old female mice after retro-orbital injection. Next, in order 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. Afterwards, EAE scores and weight changes were confirmed for 25 consecutive days. As a result, it was confirmed that compared with the control (vehicle control group) or the AAV expressing anti-FITC-Gas6, in the mice administered with AAV expressing anti-MOG (8-18C5)-Gas6, EAE scores were lower and weight changes were smaller ( Figure 5B and Figure 5C ). We found that anti-MOG(8-18C5)-Gas6 was properly expressed in a functional form in mice via AAV and played an important role in alleviating the severity of EAE.

[0340] Example 6: Safety of Fusion Molecules in Animals (Systemic Expression of Fusion Proteins)

[0341] Experimental procedure: AV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into the 1×10 11 Vg was administered retro-orbitally to wild-type C57BL / 6J 6-week-old female mice. Three weeks later, the brains were sampled for immunohistochemistry. Figure 6A .

[0342] To confirm the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin, wild-type mice were used and the Figure 8 Mice were operated according to the protocol shown in A. In more detail, AAVPHP.eB-CMV-anti-FITC-Gas6-HA and AAVPHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into the mouse body with 1×10 11 Vg was administered by retro-orbital injection to wild-type C57BL / 6J 6-week-old female mice. Three weeks later, brain samples were taken for immunohistochemistry to confirm myelin levels (MBP), lysosomal content (cathepsin D), and glial activation (GFAP, IBA1). The results confirmed that when anti-MOG (8-18C5)-Gas6 was systemically administered to wild-type mice, there was no significant change 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, it had no adverse effects on myelination and glial cell priming.

[0343] Example 7: Safety of Fusion Molecules in Animals (Local Expression of Fusion Proteins)

[0344] Experimental procedure: AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were added at 1×10 12 vg / mL (200 nL) was stereotaxically injected into the corpus callosum of 6-week-old wild-type C57BL / 6J female mice. Three weeks later, the brains were sampled for immunohistochemical analysis.

[0345] To confirm the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin, wild-type mice were used and the Figure 9A In more detail, AAVPHP.eB-CMV-anti-FITC-Gas6-HA and AAVPHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into the mice at a concentration of 1×10 12 vg / mL (200nL) was stereotactically injected into the corpus callosum of 6-week-old wild-type C57BL / 6J female mice. Three weeks later, the brain was sampled for immunohistochemical analysis to confirm the extent of viral expression (HA), myelin levels (MBP), and glial activation (GFAP, IBA1). The results confirmed that when anti-MOG (8-18C5)-Gas6 was locally administered to wild-type mice, expression occurred in the injected area but not in the contralateral side ( Figure 7B In addition, it was confirmed that myelin levels and glial priming were not significantly changed compared with anti-FITC-Gas6 ( Figures 7C to 7E ). Thus, it was found that when anti-MOG(8-18C5)-Gas6 was locally expressed, it had no adverse effects on myelination and glial cell activation.

[0346] Example 8: Binding activity of fusion molecules to target substances

[0347] In order to effectively remove myelin fragments using TAM receptors, a fusion molecule based on the human Gas6 protein was prepared. More specifically, the Gla domain and EGF repeat domain, which are sites for recognizing PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and a single-chain Fv fragment of an antibody against the MOG protein, which is highly expressed on myelin, was placed on these sites (anti-MOG (01)-Gas6). Figure 8 .

[0348] ELISA was performed to evaluate the antigen binding activity of the anti-MOG(01)-Gas6 prepared above. Human MBP protein (R&D Systems) or mouse MBP protein (R&D Systems) diluted in DPBS at a concentration of 0.5 μg / mL was added to a 96-well plate in an amount of 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 in an amount of 200 μL per well, blocked, and washed four times with PBST. Anti-MOG (01)-Gas 6 diluted according to the concentration was added in an amount of 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 ImmunoResearch) and incubate at room temperature for 1 hour. After washing, 100 μL of TMB solution was added to each well and the color was developed for 10 minutes. After stopping the reaction with stop solution, the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer. Figure 9A and Figure 9B As shown, anti-MOG(01)-Gas6 exhibited binding activity to MOG protein.

[0349] 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 overexpressing mouse MOG (hereinafter referred to as the 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 + 10mM EDTA + 1X Pen / Strep + 20mM HEPES) was incubated with the anti-MOG-Gas6 fusion molecule at 4°C for 1 hour. Afterwards, in order to remove the anti-MOG (01) -Gas6 fusion molecules remaining in the supernatant and not binding to MOG on the cell surface, two washing operations were performed, wherein 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 MOG on the cell surface, 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 by flow cytometry. The results obtained are shown in FIG. Figure 9C As shown. Figure 9C As shown, it was confirmed that anti-MOG(01)-Gas6 was able to bind to the mouse MOG protein expressed on the cell surface.

[0350] Example 9: Effect of fusion protein (anti-MOG (01)-Gas6) fusion molecule in removing myelin debris

[0351] Anti-MOG(01)-Gas6 was used as the purified protein. To confirm the effect of the protein on the removal of myelin debris, THP-1 Axl (a human monocytic cell line in which the Axl gene is overexpressed). To visualize the removal of myelin debris in vivo, myelin-pHrodo was prepared by extracting myelin from mouse brain and combining it with pH-sensitive pHrodo. THP-1 cells were treated with protein (5 μg / mL) and myelin-pHrodo. Axl Afterwards, live cell imaging was performed using the IncuCyte. Figure 10 .

[0352] 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 myelin-pHrodo was used in THP-1 AxlThe results showed that anti-MOG(01)-Gas6 could effectively remove myelin debris in THP-1 cells compared with the vehicle control group. Axl Efficient demyelination in cell lines ( Figures 9A to 9C ).

[0353] Example 10: Binding of fusion protein (anti-MBP-Gas6) fusion molecule to target substance

[0354] In order to effectively remove myelin fragments using TAM receptors, a fusion molecule based on the human Gas6 protein was prepared. More specifically, the Gla domain and EGF repeat domain, which are sites for recognizing PS (phosphatidylserine) in apoptotic cells, were removed from Gas6, and a single-chain Fv fragment of an antibody against the MBP protein, which is highly expressed in myelin, was placed at these sites (anti-MBP-Gas6). Figure 11 .

[0355] ELISA was performed to evaluate the antigen binding activity of the anti-MBP-Gas6 fusion molecules prepared above. Human MBP protein (Enzo Life Sciences) or mouse MBP protein (CreativeBioMart) diluted at a concentration of 1 μg / mL in DPBS was added to a 96-well plate in an amount of 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 to each well in an amount of 200 μL, blocked, and washed four times with PBST. Anti-MBP-Gas6 fusion molecules diluted in concentration were added to each well in an amount of 100 μL, and incubated for 2 hours at room temperature. The culture dish was washed with PBST, processed with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. 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 the color was developed for 10 minutes. After stopping the reaction with stop solution, the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer.

[0356] The results obtained are as follows Figure 12A and Figure 12B The tested anti-MBP-Gas6 fusion molecules exhibited binding activity to human MBP protein and mouse MBP protein.

[0357] Example 11: Effect of fusion protein (anti-MBP-Gas6) in removing myelin debris

[0358] Anti-MBP-Gas6 was used as the purified protein. To confirm the effect of the protein on the removal of myelin debris, THP-1 Axl (a human monocytic cell line in which the Axl gene is overexpressed). To visualize the removal of myelin debris in vivo, myelin-pHrodo was prepared by extracting myelin from mouse brain and combining it with pH-sensitive pHrodo. THP-1 cells were treated with protein (5 μg / mL) and myelin-pHrodo. Axl Cells were then subjected to live cell imaging using the IncuCyte, and MFI values ​​were compared and evaluated after 20 h.

[0359] 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 myelin-pHrodo was used to remove myelin debris from THP-1 cells. Axl The results showed that the anti-MBP-Gas6 fusion molecules tested effectively removed myelin debris in monocytic cell lines compared with the vehicle control group ( Figure 13 ).

[0360] Example 12: Construction of anti-TNFα (adalimumab)-Gas6 fusion molecules and anti-TNFα (infliximab)-Gas6 fusion molecules

[0361] In order to effectively inhibit or remove TNFα by using TAM receptors, a fusion molecule based on the human Gas6 protein was prepared. More specifically, the Gla domain and EGF repeat domain, which are sites for recognizing PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and a single-chain Fv fragment of an antibody of TNFα, adalimumab, or infliximab was placed on these sites (anti-TNFα-Gas6). 14A to 14C .

[0362] 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 in an amount of 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 in an amount of 200 μL per well, blocked, and washed four times with PBST. Anti-TNFα-Gas6 fusion molecules diluted by concentration were added in an amount of 100 μL per well and incubated for 2 hours at room temperature. The culture plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. 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 the color was developed for 10 minutes. After stopping the reaction with stop solution, the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer. The results were as follows: Figure 15A The two tested anti-TNFα-GAS6 fusion molecules exhibited binding activity to human TNFα protein.

[0363] Example 13: Binding activity of anti-TNFα (adalimumab)-Gas6 fusion molecules and anti-TNFα (infliximab)-Gas6 fusion molecules with the target substance TNFα

[0364] To confirm the binding activity of the two types of anti-TNFα-GAS6 fusion molecules prepared in Example 12 above to TNFα expressed on the cell surface, a CHO-K1 cell line overexpressing human cell membrane TNFα (hereinafter referred to as the CHO-mTNFα cell line; Promega) was treated with the anti-TNFα-GAS6 fusion molecules, and then the anti-TNFα-GAS6 fusion molecules binding to TNFα on the cell surface were detected using flow cytometry. Briefly, the CHO-mTNFα cell line resuspended in FACS solution (DPBS + 3% FBS + 10mM EDTA + 1X Pen / Strep + 20mM HEPES) was incubated with the anti-TNFα-Gas6 fusion molecules at 4°C for 1 hour. Thereafter, to remove the anti-TNFα-Gas6 fusion molecules remaining in the supernatant that did not bind to cell surface TNFα, two wash operations were performed, wherein FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. To detect anti-TNFα-Gas6 fusion molecules binding 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. The cells were incubated at 4°C for 30 minutes. After two washes, mean fluorescence intensity (MFI) was analyzed by flow cytometry.

[0365] The results obtained are as follows Figure 15B It was demonstrated that the two tested anti-TNFα-GAS6 fusion molecules were able to bind to human cell membrane TNFα expressed on the cell surface.

[0366] Example 14: Anti-TNFα (adalimumab)-Gas6 fusion molecules and anti-TNFα (infliximab)-Gas6 fusion molecules activate Axl activity

[0367] In order to confirm the ability of the anti-TNFα-GAS6 fusion molecule candidate prepared in Example 12 above to inhibit the initiation of TNFα signaling, HEK-BLUE TM TNFα cells were used for TNFα inhibition assay. Briefly, 20 μL of human TNFα protein and 20 μL of diluted anti-TNFα-GAS6 fusion molecules were added to each well of a flat-bottom 96-well plate and HEK-BLUE cells were plated. TM TNFα cells were collected at 5×10 4 / 160μL / well density was dispensed into each well. It was dispensed into each well according to the density of the well. After culturing at 37°C in a 5% CO2 incubator for 24 hours, 20μL of the supernatant was transferred to a new flat-bottom 96-well plate and 180μL of Quanti-Blue was added to each well.TM After 2 hours, the absorbance at 655 nm was measured using a spectrophotometer, and the inhibitory ability (%) was calculated based on the measured value under the condition of treating only human TNFα protein. Figure 16 As shown in , it was confirmed that the two tested anti-TNFα-Gas6 fusion molecules were able to inhibit the signaling initiation of TNFα.

[0368] In order to confirm the ability of the anti-TNFα-GAS6 fusion molecule prepared in Example 12 above to induce Axl activation, the human osteosarcoma cell line U2OS was used. Axl A TAM receptor dimerization assay was performed using a TAM receptor dimerization assay (Eurofins DiscoverX) in which ProLink-labeled Axl and an enzyme receptor (EA)-labeled SH2 domain were overexpressed. In this assay, the cell line was sensitized to Gas6-activated Axl receptors by producing chemiluminescence, and cells expressing mTNFα (Promega) were treated with an anti-TNFα-Gas6 fusion molecule to determine whether antigen (TNFα)-specific Axl activation was induced.

[0369] like Figure 17 As shown in , Axl activation was induced only when cells expressing mTNFα were treated with an anti-TNFα-GAS6 fusion molecule.

[0370] Example 15: Activity of anti-TNFα (adalimumab)-Gas6 fusion molecules and anti-TNFα (infliximab)-Gas6 fusion molecules in inducing Axl-mediated phagocytosis

[0371] In order to confirm the Axl-mediated phagocytosis of the anti-TNFα-GAS6 fusion molecule candidate prepared in Example 12 above, the cells were isolated from THP-1 cells. Axl Phagocytosis assay of differentiated macrophages was performed. THP-1 cells were treated with 25 nM PMA (phorbol 12-myristate 13-acetate). Axl The cells were cultured for 72 hours in 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. mTNFα-expressing cells (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 operations with FACS solution twice, the cell surface was stained with CD11b and phagocytosis of CD11bd+ macrophages toward CTV+ target cells was analyzed using flow cytometry.

[0372] like Figure 18 As shown in , it was demonstrated that the anti-TNFα-GAS6 fusion molecule candidate induced phagocytosis of target cells mediated by the Axl receptor expressed on macrophages.

[0373] Example 16: Construction of anti-CD20 (rituximab)-Gas6 fusion molecule

[0374] To effectively remove CD20-expressing immune cells by using TAM receptors, a fusion molecule based on human Gas6 protein was prepared. Figure 19A and Figure 19B In more detail, the Gla domain and EGF repeat domain, which are sites for recognizing PS (phosphatidylserine) of apoptotic cells, were removed from Gas6, and rituximab, a single-chain Fv fragment of an antibody to CD20, was placed on these sites (anti-CD20-Gas6).

[0375] ELISA was performed to assess the antigen binding activity of the anti-CD20-Gas6 fusion molecules 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 in an amount of 100 μL per well, incubated overnight at 4°C for coating, and washed four times with 0.05% Tween-20 / PBS (PBST). 3% BSA / PBST was then added to the plate in an amount of 200 μL per well, blocked, and washed four times with PBST. Anti-CD20-Gas6 fusion molecules diluted in concentration were added to the plate in an amount of 100 μL per well, and incubated for 2 hours at room temperature. The culture dish was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. 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 the color was developed for 10 minutes. After stopping the reaction with stop solution, the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer. The results were as follows: Figure 20A As shown, the anti-CD20-Gas6 fusion molecule exhibits binding activity to human CD20 protein.

[0376] Example 17: Binding activity of anti-CD20 (rituximab)-Gas6 fusion molecule to target substance

[0377] 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) overexpressing human CD20 were treated with the anti-CD20-Gas6 fusion molecule, and then the anti-CD20-Gas6 fusion molecule bound to CD20 on the cell surface was detected using flow cytometry. Briefly, Raji cells resuspended in FACS solution (DPBS + 3% FBS + 10mM EDTA + 1X Pen / Strep + 20mM HEPES) were incubated with the anti-CD20-Gas6 fusion molecule at 4°C for 1 hour. Thereafter, in order to remove the anti-CD20-Gas6 fusion molecule remaining in the supernatant and not bound to cell surface CD20, two washing operations were performed, wherein FACS solution was added to each well, and the resulting mixture was centrifuged at 2,000 rpm for 3 minutes to remove the supernatant. To detect anti-CD20-Gas6 fusion molecules bound to cell surface CD20, G4S linker (E7O2V) rabbit mAb (Cell Signaling Technologies) was diluted 1:50 with FACS solution and 100 μL was added to each well and incubated at 4°C for 30 minutes. After repeated washing, the mean fluorescence intensity (MFI) was analyzed by flow cytometry.

[0378] The results obtained are as follows Figure 20B As shown in . It was confirmed that the tested anti-CD20-Gas6 fusion molecules were able to bind to human CD20 protein expressed on the cell surface.

[0379] Example 18: Anti-CD20 (rituximab)-Gas6 fusion molecule activates Axl activity

[0380] In order to confirm the ability of the anti-CD20-Gas6 fusion molecule prepared in Example 16 above to induce Axl activation, the human osteosarcoma cell line U2OS was used. Axl (Eurofins DiscoverX) was used to perform a TAM receptor dimerization assay in which ProLink-labeled Axl and enzyme receptor (EA)-labeled SH2 domain were overexpressed. In the assay, the cell line was sensitive to Axl receptor activation by Gas6 by producing chemiluminescence, and the CD20-expressing Raji cell line (ATCC) was treated with an anti-CD20-Gas6 fusion molecule to determine whether antigen (CD20)-specific Axl activation was induced. Figure 21 As shown in , Axl activation was induced only when CD20-expressing Raji cells were treated with anti-CD20-Gas6 fusion molecules.

[0381] Example 19: Activity of anti-CD20 (rituximab)-Gas6 fusion molecule in inducing Axl-mediated phagocytosis

[0382] To confirm the Axl-mediated phagocytosis of the anti-CD20-Gas6 fusion molecule candidate prepared in Example 16 above, a 1:1 ratio of 1:1 was used to isolate the 1:1 ratio from THP-1 cells. Axl Phagocytosis of differentiated macrophages. THP-1 cells were treated with 25 nM PMA (phorbol 12-myristate 13-acetate). Axl The cells were cultured for 72 hours in serum-free and PMA-free medium for 24 hours, and then cultured for 24 hours with LPS (100 ng / mL) and IFN-γ (10 ng / mL) to differentiate into macrophages. Using cells expressing mTNFα (Promega) stained with CTV (CellTrace Violet) as target cells, effector cells and target cells were mixed in a 1:2 ratio and cultured together for 2 hours. After repeating the washing operation twice with FACS solution, the cell surface was stained with CD11b and the phagocytosis of CD11bd+ macrophages towards CTV+ target cells was analyzed using flow cytometry.

[0383] like Figure 22 As shown in , it was demonstrated that the anti-CD20-Gas6 fusion molecule candidate induced phagocytosis of target cells mediated by the Axl receptor expressed on macrophages.

[0384] Example 20: Adalimumab [scFv]–ProS1 fusion molecule

[0385] To prepare a MOG-specific fusion protein based on ProS1 protein, the Gla domain and EGF repeat domain were first removed, and the single-chain variable fragment (scFv) of adalimumab and a TNFα-specific antibody (αTNFα-ProS1) were introduced in their place. Figure 24 The amino acid and nucleotide sequences of the chimeric phagocytosis inducers are shown.

[0386] 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 target substance binding activity, TAM-activated induction, and phagocytosis.

[0387] Example 21: Adalimumab [Fab]-Gas6 (anti-TNFα antibody heavy chain VH-CH1 (Fab)-Gas6-His)

[0388] To create a TNFα-specific fusion protein based on the gas6 protein, the Gla domain and EGF repeat domain were first removed, and an antigen-binding fragment (Fab) or monoclonal antibody (Mab) of the TNFα-specific antibody adalimumab (αTNFα[Fab]-Gas6 and αTNFα[Mab]-Gas6) was introduced in its place. The Fc region of the Mab heavy chain contained an NA mutation to reduce or eliminate Fcγ receptor binding affinity. Figure 25 The amino acid sequences of two chimeric phagocytosis inducers are shown.

[0389] 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-initiated induction, and phagocytosis.

[0390] Example 22: Adalimumab [Mab]-Gas6 (Anti-TNFα Antibody Heavy Chain (Mab)-Gas6-His)

[0391] To prepare the gas6 protein-like TNFα-specific 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 at this position (αTNFα[Fab]-Gas6 and αTNFα[Mab]-Gas6). Figure 26 The amino acid sequences of two chimeric phagocytosis inducers are shown.

[0392] 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 molecules were evaluated for their target substance binding activity, TAM-initiated induction, and phagocytosis.

[0393] Example 23: Fusion molecules containing adalimumab [Mab or Fab] were generated by using SEQ ID NO: 253 ( Figure 27 ) and SEQ ID NO: 251 ( Figure 25 ) sequence, SEQ ID NO: 252 ( Figure 26 )'s sequence number: 258( Figure 32 ) or SEQ ID NO: 260 ( Figure 34 ) / SEQ ID NO: 261( Figure 34) sequence to prepare adalimumab [Fab]-Gas6 fusion protein, adalimumab [Mab]-Gas6 fusion protein, adalimumab [Mab]-anti-Axl (homodimer) fusion protein or adalimumab [Mab]-anti-Axl (heterodimer) fusion protein.

[0394] 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] were evaluated for binding activity to a target substance, induction of TAM activation, and phagocytosis.

[0395] Example 24: Adalimumab [scFv]–MFc–Gas6

[0396] As a non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between a first region and a second region, wherein Gas6 and an antibody scFv (in this example, 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 sequences used in the structure are as follows Figure 28 As shown in .

[0397] The resulting adalimumab [scFv]-MFc-Gas6 fusion molecule was evaluated for target substance binding activity, TAM-activated induction, and phagocytosis by following the procedures described in one or more of Examples 8 to 11, 13 to 15, or 17-19.

[0398] Example 25: Adalimumab [scFv]–Fc(DD)–Gas6 (heterodimer)

[0399] 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 as shown in FIG. Figure 29 As shown in .

[0400] 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(DD)-Gas6 heterodimeric fusion molecules were evaluated for target substance binding activity, TAM-initiated induction, and phagocytosis.

[0401] Example 26: Adalimumab [scFv]–Fc–Gas6 (homodimer)

[0402] 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 comprising an adalimumab scFv (as the second region), an Fc region (scaffold), and Gas6. The peptide sequences are shown in Figure 30 middle.

[0403] 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-initiated induction, and phagocytosis.

[0404] Example 27: Adalimumab [scFv]–Fc–Gas6 (homodimer)

[0405] 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 comprising an adalimumab scFv (as the second region), an Fc region (scaffold), and Gas6. The peptide sequences are shown in Figure 31 middle.

[0406] 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-initiated induction, and phagocytosis.

[0407] Example 28: Adalimumab [Mab]—anti-Axl (homodimer)

[0408] As a non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between a first region and a second region, a bispecific antibody was prepared in which the scFv of an anti-Axl antibody and adalimumab were used as the first region and the second region, respectively. 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 comprises an NA mutation to reduce or eliminate Fcγ receptor binding affinity. Figure 32 .

[0409] 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 their target substance binding activity, TAM-initiated induction, and phagocytosis.

[0410] Example 29: Anti-Axl-Fc-Adalimumab [scFv] (homodimer)

[0411] As a non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between a first region and a second region, a homodimeric bispecific antibody was prepared in which the scFv region of an anti-Axl antibody and the scFv region of adalimumab were used as the first region and the second region, respectively. 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 as follows Figure 33 As shown in , and the Fc region scaffold contains NA mutations to reduce or eliminate Fcγ receptor binding affinity.

[0412] 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 target substance binding activity, TAM-initiated induction, and phagocytosis.

[0413] Example 30: Adalimumab [Mab] (DD) anti-Axl (heterodimer)

[0414] 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 in which the scFv of an 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 Figure 34 The second polypeptide of the heavy chain of the bispecific antibody comprises Figure 34 SEQ ID NO: 261, and the light chain of the anti-amyloid antibody has Figure 27 SEQ ID NO: 253. The Fc region comprises an NA mutation to reduce or eliminate Fcγ receptor binding affinity, and the polypeptide of the Fc region forms a heterodimer (DD-KK).

[0415] 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.

[0416] The scope of the present disclosure is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be understood to be included within the scope of the present invention.

[0417] The fusion molecules with phagocytosis-inducing activity according to the embodiments of the present disclosure can solve the problem of tissue damage caused by the initiation of inflammatory responses in the prior art. Therefore, the fusion molecules can effectively eliminate or reduce the amount of antigenic substances, and thus can be used to prevent or treat immune diseases.

[0418] Incorporated by Reference

[0419] All publications, patent applications, patents, and other references mentioned in this specification are expressly incorporated herein by reference in their entirety.

Claims

1. A fusion molecule comprising 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 amount or expression in living tissue increases and induces or causes an immune disease, in, The first region and the second region are coupled to each other directly or via a linker, The first area includes: TAM receptor ligands; anti-Axl antibody or antigen-binding fragment thereof; anti-Tyro3 antibodies or antigen-binding fragments thereof; an anti-MerTK antibody or an antigen-binding fragment thereof; or Their combination, Wherein, the fusion molecule does not contain the target substance or a fragment thereof. 2 . The fusion molecule according to claim 1 , further comprising a scaffold bound to the first region, the second region, or both the first region and the second region at different positions.

3. The fusion molecule according to claim 1, wherein The TAM receptor ligand is one selected from Gas 6, ProS1, Tubby, Tulp 1, Gal 3 and a combination thereof, or an Axl binding fragment thereof.

4. The fusion molecule according to 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.

5. The fusion molecule according to claim 1, wherein : SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 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.

6. The fusion molecule according to 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 therewith, and the sequence of SEQ ID NO: 2 or a sequence having at least 85% sequence identity therewith.

7. The fusion molecule according to 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.

8. The fusion molecule according to claim 1, wherein The first region is (a) the TAM receptor ligand, wherein the TAM receptor ligand comprises a member selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64 : 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, and SEQ ID NO: 113, or a sequence having at least 85% sequence identity thereto.

9. The fusion molecule according to 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 therewith, and the sequence of SEQ ID NO: 4 or a sequence having at least 85% sequence identity therewith.

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

12. The fusion molecule according to 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.

13. The fusion molecule according to 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 sensing receptor, ACTH, 21-hydroxylase (CYP21), hair clear protein, oxidized low-density lipoprotein (OxLDL), transcription co-initiator factor p75, p-80-Coilin, C 1 inhibitor, AMPA-receptor, CRMP5, DPPX / DPP6, GABAA receptor, glycine receptor (GlyR), Hu (ANNA-1), Ma 1, Ma2, Ri (ANNA-2), Zi c4, voltage-gated potassium channel (VGKC)-complex, NMDA-receptor, Jo 1, H / K ATPase, thyroid peroxidase, erythrocyte I / I, F-actin asialoglycoprotein receptor, cytochrome P450 2D6 (CYP2D6), NXP-2 / MORC3, TIF 1-γ / TRIM-33, β2 integrin, nuclear autoantigen sperm protein (NASP), lactoferrin 17-α-hydroxylase (CYP 17), cholesterol side-chain cleavage enzyme (CYP 11A), tryptophan hydroxylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, glycoprotein IIb / IIIa and Ib / IX, thyroglobulin, hemidesmosomal protein 180, p53, recoverin, actin, IgE receptor, myelin-associated glycoprotein (MAG), tubulin, laminin-332, tissue transglutaminase, desmin, bactericidal / permeability-increasing protein (BPI), transglutaminase, melanoma differentiation-associated gene 5 (MDA5), SUMO-activated enzyme subunit (SAE)-1 (SAE-1), SAE-2, DNA-dependent nucleosome-2 (DNA-dependent nucleosome-3) receptor. Stimulates ATPase, chromatin domain-helicase-DNA binding protein 4 (CHD4), β-adrenergic 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, synaptotagmin, voltage-gated calcium channel, βIV spectrin, U 1 Small nuclear ribonucleoprotein 70 kDa (SNRNP70), CNPas e, 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, Tribbles pseudokinase 2 (TRIB2), myeloperoxidase (MPO), aquaporin 4 (APQ-4), amphiphilin, exosome component 9 (EXOSC9), EXOSC 10 / PMSCL, Yo protein, Hu protein, Ri protein, desmoplakin, desmocollin, desmoglein 1, desmoglein 3, intrinsic factor type 1, β2-glycoprotein I (β2-GPI), pyruvate dehydrogenase complex-E2 (PDC-E2), aggrecan G1, carbamylated antigen, cartilage glycoprotein-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), glutamate receptor (GLUR), myosin, B23, nucleophosmin (NPM), nucleolar fibrillin, topoisomerase-I (Scl-70), interferon-γ-induced protein 16 (IFI 16), La phosphoprotein, Ro60, Ro52 (TRIM21), high-glucose body proteins (95, 97, 160, 180), anionic phospholipid / protein complex, cardiolipin, component of Sm splicing ribonucleoprotein (subunit AG), self-double-stranded DNA (dsDNA), histone H2A-H2B-DNA, proliferating cell nuclear antigen (PCNA), ribosomal P, Sjögren's syndrome (SSA), Smith, U1-RNP, U2 snRNP B, vimentin, C1q, fibronectin, Ku-DNA-protein kinase, carbonic anhydrase II, neuronal nicotinic acetylcholine receptor, centromere-associated protein, RNA polymerase II II (RNP), thyroid and eye muscle shared protein, leukocyte function-associated antigen (LFA-1), chromogranin A, IA-2 (ICA512), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), ZnT8, insulin, glutamate decarboxylase (GAD65), insulin receptor, heat shock protein (65-kDa heat shock protein), SOX-10, tyrosinase, KUMEL 1 / 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-17, 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 stromal lymphopoietin (TSLP), TSLPR, IL-31, IL-31R, OX40, OX40L, IL-33, IL-33R, CD40, CD40L, IGF-1R, ICAM 1, VCAM 1, MADCAM 1, integrin α4, integrin β7, VLA-4, t TLR-3, TLR-4, TLR-5, TLR-7, and combinations thereof.

14. The fusion molecule according to claim 1, wherein The second region that specifically binds to the target substance is selected from antibodies or antigen-binding fragments thereof, antibody-like proteins, peptides, aptamers and soluble receptors, each of which specifically binds to the target substance.

15. The fusion molecule according to 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.

16. The fusion molecule according to claim 1, wherein The immune disease is an inflammatory disease or autoimmune disease 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 relapsing multifocal osteomyelitis, gout, Henoch-Schonlein purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus (SLE), juvenile scleroderma, juvenile vasculitis, Kawasaki disease, lupus (systemic lupus erythematosus), mixed connective tissue disease, myositis, poststreptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjögren's syndrome, spondyloarthritis / spondyloarthropathies, systemic juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, celiac disease, thrombotic thrombocytopenic purpura (iTTP) and a combination thereof.

17. A nucleic acid molecule encoding the fusion molecule of claim 1. An expression vector comprising the nucleic acid molecule according to claim 17 .

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

20. 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.

21. Use of the pharmaceutical composition according to claim 20, for: (i) reducing the elevated level of a target substance to a normal level, or enhancing the reduction of the elevated level of the target substance to a normal level, (ii) removing or clearing a target substance whose expression is elevated or whose amount is increased, or enhancing the clearance of a target substance whose expression is elevated or whose amount is increased, wherein the elevated expression or increased amount of the target substance causes or induces an immune disease in a subject, (iii) inhibiting the increase in the expression or amount of the target substance in the subject, (iv) treating or preventing an immune disease in a subject, Delay the development of symptoms associated with immune disorders, and / or Alleviating symptoms of an immune disease in a subject.

22. The use according to claim 21, wherein Elevated levels of the target substance occur in the brain of the subject.

23. Use of the fusion molecule, the polynucleotide encoding the fusion molecule, the vector, or the pharmaceutical composition comprising the fusion molecule, the polynucleotide, or the vector according to claim 1 in one or more methods selected from the group consisting of: reducing the elevated level of a target substance to a normal level, or enhancing the reduction of the elevated level of the target substance to a normal level, Removing or clearing a target substance whose expression is elevated or whose amount is increased, or enhancing the clearance of a target substance whose expression is elevated or whose amount is increased, wherein the elevated expression or increased amount of the target substance causes or induces an immune disease in a subject, and Inhibit the increase in the expression or amount of the target substance in the subject.

24. The use according to claim 23, wherein Elevated levels of the target substance occur in the brain of the subject.

25. Use of the fusion molecule, the polynucleotide encoding the fusion molecule, the vector, or the pharmaceutical composition comprising the fusion molecule, the polynucleotide, or the vector according to claim 1 in one or more methods selected from the group consisting of: Treating or preventing an immune disease in a subject, Delaying the development of symptoms associated with immune diseases, and Alleviating symptoms of an immune disease in a subject.

26. Use of the fusion molecule according to claim 1, the polynucleotide encoding the fusion molecule, the vector, or the pharmaceutical composition comprising the fusion molecule, the polynucleotide or the vector in the preparation of a medicament for treating or preventing an immune disease in a subject, for delaying the development of symptoms associated with an immune disease, or for alleviating the symptoms of an immune disease in a subject.

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