Fusion molecules and methods for treating immune diseases

Abstract

The present disclosure relates to fusion molecules that can bind to and induce phagocytosis of antigenic substances whose elevated levels or excessive amounts cause or contribute to immune disorders, thereby removing, reducing, or enhancing the removal or reduction of the antigenic substances. Thus, the fusion molecules may be useful for treating immune disorders, delaying the development or onset of immune disorders, or alleviating the symptoms of immune disorders.

Description

[Technical Field]

[0001] 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.

[0002] [Background technology]

[0003] Immunologic disorders are conditions in which components of a mammal's immune system cause, mediate, or otherwise contribute to pathology in mammals. Inflammatory diseases, in particular, are a topic of global attention and for which therapeutic drugs are urgently needed. Inflammation is generally a localized defense response of body tissues to host invasion by foreign or harmful stimuli. Causes of inflammation include infectious causes such as bacteria, viruses, and parasites; physical causes such as burns and radiation; chemicals such as toxins, drugs, and industrial agents; immune responses such as allergies and autoimmune responses; and conditions associated with oxidative stress.

[0004] Normally, the inflammatory response removes external infectious agents, regenerates damaged tissues, and restores the body's functions. However, if antigens are not removed or if the inflammatory response is caused by internal substances, excessive or persistent inflammatory responses can lead to life-threatening diseases such as acute inflammation, intra-articular diseases such as rheumatoid arthritis, skin diseases such as psoriasis, allergic inflammatory diseases such as bronchial asthma, and autoimmune diseases caused by the immune system attacking self-antigens, which can interfere with treatment processes such as blood transfusions, medication, and organ transplants.

[0005] Currently, drugs such as steroids and aspirin have been developed as treatments for excessive immune responses in inflammatory and autoimmune diseases. However, these drugs are known to cause side effects such as edema, gastrointestinal disorders, bleeding, and liver toxicity. Furthermore, they are unable to selectively act on the cause of inflammation, and in some cases can cause severe immunosuppression (Check and Kaliner, Am. Rev. Respir. Dis., 141, pp. 44-51, 1990). Furthermore, because no therapeutic drugs are yet available that can completely treat these diseases, effective therapeutic drugs without side effects are needed.

[0006] TAM (Tyro3, ​​Axl, MerTK) receptors are receptor tyrosine kinases, and TAM ligands that can activate these receptors have recently been shown to be important in regulating tissue homeostasis and inflammation. TAM receptors are particularly known to be involved in anti-inflammatory effects and resolution of inflammation. Here, anti-inflammatory effects refer to the reduction and elimination of inflammatory mediators through inhibition of their synthesis, selective antagonism, post-translational modifications such as scavenging and cleavage, and degradation. Resolution of inflammation can be achieved by removing inflammatory stimuli, promoting the elimination of causative cells through apoptosis or phagocytosis, enhancing the induction of non-inflammatory macrophages, promoting macrophage reprogramming, and secreting anti-inflammatory substances (e.g., IL-10).

[0007] Based on these reported properties, various attempts have been made to treat inflammatory and autoimmune diseases by administering the TAM ligands Gas6 or ProS1, and they have been shown to reduce the secretion of inflammatory cytokines and alleviate some of the symptoms caused by inflammation (Peng et al., PLoS One, 14, e0219788, 2019; Waterborg et al., Front. In Immunol., 9, 742, 2018; Jiang et al., J. Cell Mol. Med., 23(4), 2769-2781, 2019). However, because TAM ligands only contain the domains that bind to TAM receptors and the domain that binds to phosphatidylserine (PS), it has been difficult to selectively target inflammatory substances.

[0008] Therefore, there is a need for improved approaches that can efficiently induce selective clearance of antigens.

[0009] Summary of the Invention [Problem to be solved by the invention]

[0010] The present disclosure relates to fusion molecules that can induce the selective clearance of targeted substances that induce or induce unwanted or pathological immune responses, such as autoimmune diseases, transplant rejection, or allergic or hyperimmune responses.

[0011] The problems to be solved by the present invention are not limited to those mentioned above, and other problems not mentioned in this specification will be clearly understood by those skilled in the art from the following description.

[0012] [Means for solving the problem]

[0013] One aspect of the present disclosure provides a fusion molecule comprising: a first region capable of binding to a TAM (Tyro3, ​​Axl, and MerTK) receptor; and a second region specifically binding to a target substance to be cleared or reduced; wherein the fusion molecule does not induce an inflammatory response, and an increase or elevation of the level or expression of the target inflammation-related substance induces or induces an undesirable or pathological immune response, such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response. In an embodiment, the fusion molecule does not have an effector function and does not induce an Fc-mediated inflammatory response.

[0014] In some embodiments, the TAM receptor may be any one selected from the group consisting of Tyro3, ​​Axl, MerTK, or a combination thereof, which induce phagocytosis by binding to the laminin G-like domain (or LG domain) of phagocytes, including, but not limited to, macrophages or microglial cells. In some embodiments, the TAM receptor may be the Axl domain of a TAM receptor.

[0015] In embodiments, 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 embodiments, the first region may comprise or essentially consist of Gas6 or an active fragment thereof capable of binding to a TAM receptor. In embodiments, the first region may comprise or essentially consist of Gas6 or an active fragment thereof capable of binding to an Axl receptor.

[0016] In certain embodiments, the first region may comprise the laminin G-like domain of Gas6 or ProS1, or an active fragment thereof, which contains the laminin G-like domain as a phagocytosis-associated bridging molecule abundantly expressed in various tissues, thereby inducing phagocytosis via a TAM receptor. In certain embodiments, the laminin G-like domain may comprise an LG1 domain, an LG2 domain, or a combination thereof, preferably both the LG1 domain and the LG2 domain, which may bind to the TAM receptor and induce phagocytosis.

[0017] Exemplary embodiments are directed to binding molecules or fusion molecules comprising a first region capable of binding to a TAM receptor and a second region capable of specifically binding to a target inflammation-associated substance, the target inflammation-associated substance being a substance with increased or elevated amount or increased or elevated expression that induces, 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 and second regions are linked to each other directly or via a linker, and the first region comprises:

[0018] (a) TAM receptor ligand;

[0019] (b) an anti-Axl antibody or an antigen-binding fragment thereof;

[0020] (c) an anti-Tyro3 antibody or antigen-binding fragment thereof; or

[0021] (d) an anti-MerTK antibody or antigen-binding fragment thereof, provided that if the first region comprises an anti-MerTK antibody or antigen-binding fragment thereof, the molecule is not a bispecific antibody; or

[0022] (e) combinations thereof;

[0023] According to some embodiments, the binding molecule may further comprise a scaffold attached at different positions to the first region, the second region, or both the first region and the second region.

[0024] In an embodiment, the first region is a TAM receptor ligand, and the TAM receptor ligand comprises a sequence selected from the group consisting of SEQ ID NOs: 1-113, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0025] In some further embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to an Axl receptor is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0026] In some further embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 1 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and / or the sequence of SEQ ID NO: 2 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0027] In yet other embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 5 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0028] In some further embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to the Axl receptor is selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO: 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.

[0029] In some further embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 3 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, and / or the sequence of SEQ ID NO: 4 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0030] In yet other embodiments, the first region is capable of binding to an Axl receptor, and the first region capable of binding to an Axl receptor comprises the sequence of SEQ ID NO: 6 or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0031] In embodiments, a fusion protein according to the present disclosure does not comprise the target substance to be cleared or reduced by administration of the fusion protein.

[0032] In some embodiments, the first region containing the laminin G-like domain of Gas6 or ProS1, or an active fragment thereof, does not contain a Gla domain. Without being bound by theory, it is believed that the lack of a Gla domain in the first region may prevent the fusion molecule from recognizing phosphatidylserine (PS) of a TAM receptor, while the second region can induce phagocytosis by recognizing a target substance.

[0033] In some embodiments, the first region containing the laminin G-like domain of Gas6 or ProS1, or an active fragment thereof, does not contain the Gla domain and does not contain the EGF domain. The deletion of the EGF domain in the first region provides the advantage of increasing yield in the production process of the fusion molecule by suppressing aggregation of the fusion molecule during the purification step. In some embodiments, the fusion molecule (or binding molecule) may form a homodimer or heterodimer, or may form a linear multimer as a single chain (single chain).

[0034] According to an embodiment, the target substance to be cleared or reduced and to which the second region specifically binds may be a substance whose increased or elevated amount, or increased or elevated expression, induces, induces, or causes an undesirable or pathological immune response, such as an autoimmune disease, transplant rejection, or an allergic or hyperimmune response.

[0035] The target substance may be an inflammation-related substance. The target substance may be one or more selected from the autoantigens, their autoantibodies, and complexes of the autoantigens and their autoantibodies listed in Table 1 below: 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), and Toll-like receptors. Immune cell surface molecules, including costimulatory molecules and receptors such as TLRs (including TLR3, TLR4, TLR5, and TLR7); complement factors such as C1q, C3, and C5; chemokines such as CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10; IL-1, TNF-α, IL-6, IL-17, IL-4, IL-5, IL-13, IFN-γ, IL-12, IL-21, IL-22, TGF-β, IL-23, and thymic stromal lymphopoietin (THF)-dependent cytokines (TGF-β). cytokines such as IL-31, IL-33, etc.; and cell adhesion molecules such as ICAM1, VCAM1, MADCAM1, integrin α4, integrin β7, LFA-1 (or MAC-1), and VLA-4. Table 1 below lists exemplary references that disclose the amino acid sequences of the autoantigens and antibodies that bind to the target substances. Those skilled in the art will understand that ligands, receptors, or autoantibodies that bind to the listed target autoantigen substances may also be included as the second region in the present disclosure. The entire contents of the references in Table 1 are incorporated herein by reference.

[0036]

[0037] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0038] *:A total of 35 entries can be found at https: / www.uniprot.org / uniprotkb?query=GABAAR+ALPHA&facets=model_organism%3A9606

[0039] **: MDA5 is encoded by the interferon-induced helicase C domain-containing protein 1 (IFIH1) gene.

[0040] ***: Lrp4 is a receptor for agrin and forms a complex with MuSK-agrin (AGRN): O00468.

[0041]

[0042] 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 include, but are not limited to, the following:

[0043] CD20: UniProt accession number P11836 (human) and its variants (www.uniprot.org / uniprotkb / P11836 / variant-viewer ), and their orthologs.

[0044] CD19: UniProt accession number P15391 (human) and its variants (www.uniprot.org / unip rotkb / P15391 / variant-viewer), and its orthologs.

[0045] CD52: UniProt accession number P31358 (human) and its variants (www.uniprot.org / uniprotkb / P31358 / variant-viewer) , and its orthologs.

[0046] CD80: UniProt accession number P33681 (human) and its variants (www.uniprot.org / unip rotkb / P33681 / variant-viewer), and its orthologs.

[0047] CD86: UniProt accession number P42081 (human) and its variants (www.uniprot.org / uniprotkb / P42081 / variant-viewer), and its orthologs.

[0048] CD28: UniProt accession number P10747 (human) and its variants (www.uniprot.org / uniprotkb / P10747 / variant-viewer), and its orthologs.

[0049] CD40: UniProt accession number P25942 (human) and its variants ( www.uniprot.org / uniprotkb / P25942 / variant-viewer), andits ortholog.

[0050] Complement C3: UniProt accession number P01024 (human) and its variants ( www.uniprot.org / uniprotkb / P01024 / variant-viewer), and its orthologs .

[0051] Complement C5: UniProt accession number P01031 (human) and its variants (www.uniprot.org / uniprotkb / P01031 / variant-viewer) , and its orthologs.

[0052] C5α receptor 1: UniProt accession number P21730 (human) and its variants ( www.uniprot.org / uniprotkb / P21730 / variant-viewer), and its orthologs .

[0053] IL-1R: UniProt accession number P14778 (human) and its variants (www.uniprot.org / uniprotkb / P14778 / variant-viewer), and its orthologs.

[0054] IL-6R: UniProt accession number P08887 (human) and its variants (www.uniprot.org / uniprotkb / P08887 / variant-viewer), and its orthologs.

[0055] IL-6ST (Interleukin-6 receptor subunit β): UniProt accession number P40189 (human) and its variants (www.uniprot.org / uniprotkb / P40189 / variant-viewer), and its orthologs.

[0056] IL-17C: UniProt accession number Q9P0M4 (human) and its variants (www.uniprot.org / uniprotkb / PQ9P0M4 / variant-viewer), and its orthologs.

[0057] IL-17RA: UniProt accession number Q96F46 (human) and its variants (www.uniprot.org / uniprotkb / Q96F46 / variant-viewer), and its orthologs.

[0058] IL-17RB: UniProt accession number Q9NRM6 (human) and its variants (www.uniprot.org / uniprotkb / Q9NRM6 / variant-viewer), and its orthologs.

[0059] IL-4R: UniProt accession number P24394 (human) and its variants (www.uniprot.org / uniprotkb / QP24394 / variant-viewer), and its orthologs.

[0060] IL-5R: UniProt accession number Q01344 (human) and its variants (www.uniprot.org / uniprotkb / Q01344 / variant-viewer), and its orthologs.

[0061] IL-5RB: UniProt accession number P32927 (human) and its variants (www.uniprot.org / uniprotkb / P32927 / variant-viewer), and its orthologs.

[0062] IL-13R1: UniProt accession number P78552 (human) and its variants (www.uniprot.org / uniprotkb / P78552 / variant-viewer), and its orthologs.

[0063] IL-13R2: UniProt accession number Q14627 (human) and its variants (www.uniprot.org / uniprotkb / Q14627 / variant-viewer), and its orthologs; or UniProt accession number D0EFR8 (human) and its variants (www.uniprot.org / uniprotkb / D0EFR8 / variant-viewer), and its orthologs.

[0064] IFN-γ receptor 1: UniProt accession number Q15260 (human) and its variants (www.uniprot.org / uniprotkb / Q15260 / variant-viewer), and its orthologs.

[0065] IFN-γ receptor 2: UniProt accession number P38484 (human) and its variants (www.uniprot.org / uniprotkb / P38484 / variant-viewer), and its orthologs.

[0066] Integrin α-D (ITGAD): UniProt accession number Q13349 (human) and its variants (www.uniprot.org / uniprotkb / Q13349 / variant-viewer), and its orthologs.

[0067] IL-12RB1: UniProt accession number P42701 (human) and its variants (www.uniprot.org / uniprotkb / P42701 / variant-viewer), and its orthologs.

[0068] IL-12RB2: UniProt accession number Q99665 (human) and its variants (www.uniprot.org / uniprotkb / Q99665 / variant-viewer), and its orthologs.

[0069] IL-21R: UniProt accession number Q9HBE5 (human) and its variants (www.uniprot.org / uniprotkb / Q9HBE5 / variant-viewer), and its orthologs.

[0070] IL-22RA1: UniProt accession number Q8N6P7 (human) and its variants (www.uniprot.org / uniprotkb / Q8N6P7 / variant-viewer), and its orthologs.

[0071] IL-22RA2: UniProt accession number Q969J5 (human) and its variants (www.uniprot.org / uniprotkb / Q969J5 / variant-viewer), and its orthologs.

[0072] TGF-β receptor type 1: UniProt accession number P36897 (human) and its variants (www.uniprot.org / uniprotkb / P36897 / variant-viewer) and their orthologs; TGF-β receptor type 2: UniProt accession number P37173 (human) and its variants (www.uniprot.org / uniprotkb / P37173 / variant-viewer) and its orthologs; TGF-β receptor type 3: UniProt accession number Q03167 (human) and its variants (www.uniprot.org / uniprotkb / Q03167 / variant-viewer) and its orthologs.

[0073] IL-23R: UniProt accession number Q5VWK5 (human) and its variants (www.uniprot.org / uniprotkb / Q5VWK5 / variant-viewer), and its orthologs.

[0074] Thymic stromal lymphopoietin receptor (TSLPR): UniProt accession number Q9HC73 (human) and its variants (www.uniprot.org / uniprotkb / Q9HC73 / variant-viewer), and its orthologs.

[0075] IL-31R: UniProt accession number Q8NI17 (human) and its variants (www.uniprot.org / uniprotkb / Q8NI17 / variant-viewer), and its orthologs.

[0076] IL-33R: UniProt accession number Q01638 (human) and its variants (www.uniprot.org / uniprotkb / Q8NB14 / variant-viewer), and its orthologs.

[0077] IGF-1R: UniProt accession number P08069 (human) and its variants (www.uniprot.org / uniprotkb / P08069 / variant-viewer), and its orthologs.

[0078] TNFR1: UniProt accession number P19438 (human) and its variants (www.uniprot.org / uniprotkb / P19438 / variant-viewer), and its orthologs.

[0079] TNFR2: UniProt accession number P20333 (human) and its variants (www.uniprot.org / uniprotkb / P20333 / variant-viewer), and its orthologs.

[0080] FcRn large subunit p51: UniProt accession number P55899 (human) and its variants (www.uniprot.org / uniprotkb / P55899 / variant-viewer), and its orthologs.

[0081] CCL14: UniProt accession number Q16627 (human) and its variants (www.uniprot.org / uniprotkb / Q16627 / variant-viewer), and its orthologs.

[0082] CCL15: UniProt accession number Q16663 (human) and its variants (www.uniprot.org / uniprotkb / Q16663 / variant-viewer), and its orthologs.

[0083] CCL18: UniProt accession number P55774 (human) and its variants (www.uniprot.org / uniprotkb / P55774 / variant-viewer), and its orthologs.

[0084] CCL19: UniProt accession number Q99731 (human) and its variants (www.uniprot.org / uniprotkb / Q99731 / variant-viewer), and its orthologs.

[0085] CCL20: UniProt accession number QP78556 (human) and its variants (www.uniprot.org / uniprotkb / P78556 / variant-viewer), and its orthologs.

[0086] CCL21: UniProt accession number O00585 (human) and its variants (www.uniprot.org / uniprotkb / O00585 / variant-viewer), and its orthologs.

[0087] CCL23: UniProt accession number P55773 (human) and its variants (www.uniprot.org / uniprotkb / P55773 / variant-viewer), and its orthologs.

[0088] CCL25: UniProt accession number Q68A93 (human) and its variants (www.uniprot.org / uniprotkb / Q68A93 / variant-viewer), and its orthologs.

[0089] CCL27: UniProt accession number Q9Y4X3 (human) and its variants (www.uniprot.org / uniprotkb / Q9Y4X3 / variant-viewer), and its orthologs.

[0090] CXCL12: UniProt accession number P48061 (human) and its variants (www.uniprot.org / uniprotkb / P48061 / variant-viewer), and its orthologs.

[0091] CXCL13: UniProt accession number O43927 (human) and its variants (www.uniprot.org / uniprotkb / O43927 / variant-viewer), and its orthologs.

[0092] IL-1A: UniProt accession number P01583 (human) and its variants (www.uniprot.org / uniprotkb / P01583 / variant-viewer), and its orthologs; IL-1B: UniProt accession number P01584 (human) and its variants (www.uniprot.org / uniprotkb / P01584 / variant-viewer), and its orthologs.

[0093] TNF-α: UniProt accession number P01375 (human) and its variants (www.uniprot.org / uniprotkb / P01375 / variant-viewer), and its orthologs.

[0094] CXCL-8: UniProt accession number P10145 and its variants (www.uniprot.org / uniprotkb / P10145 / variant-viewer), and its orthologs.

[0095] CCL2: UniProt accession number P13500 (human) and its variants (www.uniprot.org / uniprotkb / Q13500 / variant-viewer), and its orthologs.

[0096] CCL3: UniProt accession number P10147 (human) and its variants (www.uniprot.org / uniprotkb / P10147 / variant-viewer), and its orthologs.

[0097] CCL4: UniProt accession number P13236 (human) and its variants (www.uniprot.org / uniprotkb / P13236 / variant-viewer), and its orthologs.

[0098] CCL5: UniProt accession number P13501 (human) and its variants (www.uniprot.org / uniprotkb / P13501 / variant-viewer), and its orthologs.

[0099] CCL11: UniProt accession number P51671 (human) and its variants (www.uniprot.org / uniprotkb / P51671 / variant-viewer), and its orthologs.

[0100] CXCL10: UniProt accession number P02778 (human) and its variants (www.uniprot.org / uniprotkb / P02778 / variant-viewer), and its orthologs.

[0101] IL-6: UniProt accession number P05231 (human) and its variants (www.uniprot.org / uniprotkb / P05231 / variant-viewer), and its orthologs.

[0102] IL-17: UniProt accession number Q16552 (human) and its variants (www.uniprot.org / uniprotkb / Q16552 / variant-viewer), and its orthologs.

[0103] IL-4: UniProt accession number P05112 (human) and its variants (www.uniprot.org / uniprotkb / P05112 / variant-viewer), and its orthologs.

[0104] IL-5: UniProt accession number P05113 (human) and its variants (www.uniprot.org / uniprotkb / P05113 / variant-viewer), and its orthologs.

[0105] IL-13: UniProt accession number P35225 (human) and its variants (www.uniprot.org / uniprotkb / P35225 / variant-viewer), and its orthologs.

[0106] IFN-γ: UniProt accession number P01579 (human) and its variants (www.uniprot.org / uniprotkb / P01579 / variant-viewer), and its orthologs.

[0107] IL-12A: UniProt accession number P29459 (human) and its variants (www.uniprot.org / uniprotkb / P29459 / variant-viewer), and its orthologs; IL-12: UniProt accession number P29460 (human) and its variants (www.uniprot.org / uniprotkb / P29460 / variant-viewer), and its orthologs.

[0108] IL-21: UniProt accession number Q9HBE4 (human) and its variants (www.uniprot.org / uniprotkb / Q9HBE4 / variant-viewer), and its orthologs.

[0109] IL-22: UniProt accession number Q9GZX6 (human) and its variants (www.uniprot.org / uniprotkb / Q9GZX6 / variant-viewer), and its orthologs.

[0110] TGF-β-1: UniProt accession number P01137 (human) and its variants (www.uniprot.org / uniprotkb / P01137 / variant-viewer), and its orthologs; TGF-β-2: UniProt accession number P61812 (human) and its variants (www.uniprot.org / uniprotkb / P61812 / variant-viewer), and its orthologs; TGF-β-3: UniProt accession number P10600 (human) and its variants (www.uniprot.org / uniprotkb / P10600 / variant-viewer), and its orthologs.

[0111] IL-23A: UniProt accession number Q9NPF7 (human) and its variants (www.uniprot.org / uniprotkb / Q9NPF7 / variant-viewer), and its orthologs; IL-23B: UniProt accession number P29460 (human) and its variants (www.uniprot.org / uniprotkb / P29460 / variant-viewer), and its orthologs.

[0112] Thymic stromal lymphopoietin (TSLP): UniProt accession number Q969D9 (human) and its variants (www.uniprot.org / uniprotkb / Q969D9 / variant-viewer), and its orthologs.

[0113] IL-31: UniProt accession number Q6EBC2 (human) and its variants (www.uniprot.org / uniprotkb / Q6EBC2 / variant-viewer), and its orthologs.

[0114] OX40 (tumor necrosis factor receptor superfamily member 4): UniProt accession number P23510 (human) and its variants (www.uniprot.org / uniprotkb / P23510 / variant-viewer), and its orthologs.

[0115] OX40L (tumor necrosis factor receptor superfamily member 4): UniProt accession number P43489 (human) and its variants (www.uniprot.org / uniprotkb / P43489 / variant-viewer), and its orthologs.

[0116] IL-33: UniProt accession number O95760 (human) and its variants (www.uniprot.org / uniprotkb / O95760 / variant-viewer), and its orthologs.

[0117] CD40L: UniProt accession number P29965 (human) and its variants (www.uniprot.org / uniprotkb / P29965 / variant-viewer), and its orthologs.

[0118] ICAM1: UniProt accession number P05362 (human) and its variants (www.uniprot.org / uniprotkb / P05362 / variant-viewer), and its orthologs.

[0119] VCAM1: UniProt accession number P19320 (human) and its variants (www.uniprot.org / uniprotkb / P19320 / variant-viewer), and its orthologs.

[0120] MADCAM1: UniProt accession number Q13477 (human) or B9EGE2 and its variants (respectively) www.uniprot.org / uniprotkb / Q13477 / variant-viewer and www.uniprot.org / uniprotkb / B9EGE2 / variant-viewer ), and their orthologs.

[0121] Integrin α4: UniProt accession number P13612 (human) and its variants (www.uniprot.org / uniprotkb / P13612 / variant-viewer), and its orthologs.

[0122] Integrin β7: UniProt accession number P26010 (human) and its variants (www.uniprot.org / uniprotkb / P26010 / variant-viewer), and its orthologs.

[0123] LFA-1 or MAC-1 (a dimer of integrin α-M and integrin β-2): UniProt accession number P11215 (human) and its variants (www.uniprot.org / uniprotkb / P11215 / variant-viewer) for integrin α-M (ITAM) and its orthologs, and UniProt accession number P05107 (human) and its variants (www.uniprot.org / uniprotkb / P05107 / variant-viewer) for integrin β-2 (ITB2) and its orthologs.

[0124] VLA-4 (a dimer of CD49d and CD29): UniProt accession number P13612 (human) for CD49d and its variants (www.uniprot.org / uniprotkb / P13612 / variant-viewer) and its orthologs, and UniProt accession number P05556 (human) for CD29 and its variants (www.uniprot.org / uniprotkb / P05556 / variant-viewer) and its orthologs.

[0125] TLR3: UniProt accession number O15455 (human) and its variants (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.

[0126] TLR4: UniProt accession number O00206 (human) and its variants (www.uniprot.org / uniprotkb / O00206 / variant-viewer). TLR4 has been reported to be associated with rheumatoid arthritis. Antibodies that bind to TLR4 are disclosed, for example, in U.S. Patent No. 7,312,320 B2.

[0127] TLR5: UniProt Accession No. O60602 (human) and its variants (www.uniprot.org / uniprotkb / O60602 / variant-viewer). TLR5 has been reported to be associated with rheumatoid arthritis. Substances that bind to TLR5 are disclosed, for example, in U.S. Patent No. 8,703,146 B2 and U.S. Patent Application Publication No. 20200362052 A1.

[0128] TLR7: UniProt Accession No. Q9NYK1 (human) and its variants (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.

[0129]

[0130] In some embodiments, the immune disease is an autoimmune disease or an inflammatory disease. In other specific embodiments, the autoimmune disease or inflammatory disease is multiple sclerosis (MS), rheumatoid arthritis, spondyloarthropathy, systemic lupus erythematosus, antibody-mediated inflammatory or autoimmune disease, graft-versus-host disease, sepsis, type 1 diabetes, type 2 diabetes, psoriasis, atherosclerosis, Sjögren's syndrome, progressive systemic sclerosis, scleroderma, acute coronary syndrome, ischemia-reperfusion, Crohn's disease, endometriosis, glomerulonephritis, myasthenia gravis, asthma, acute respiratory distress syndrome (ARDS), vasculitis, or inflammatory autoimmune myositis. In specific embodiments, the spondyloarthropathy is selected from ankylosing spondylitis, reactive arthritis, enteropathic arthritis associated with inflammatory bowel disease, psoriatic arthritis, isolated acute anterior uveitis, undifferentiated spondyloarthropathy, Behcet's syndrome, and juvenile idiopathic arthritis. In one embodiment, the immune disorder is caused or exacerbated by excess antigenic material binding to immunoglobulins or immune cells, or by increased amounts or expression of antigenic material, hi certain embodiments, the immune cells are dendritic cells.

[0131] In embodiments, the present disclosure is directed to nucleic acids or polynucleotides encoding the above-described fusion proteins.

[0132] In embodiments, the present disclosure is directed to a vector comprising the nucleic acid or polynucleotide.

[0133] Embodiments are directed to host cells comprising the vectors.

[0134] Another aspect of the present disclosure provides a method of producing a therapeutic fusion molecule for the treatment of an immune disorder in a subject, the method comprising expressing the fusion molecule by culturing a host cell under conditions conducive to expression of the fusion molecule.

[0135] In embodiments, the present disclosure is directed to a method for reducing or enhancing the reduction of a substance that induces, 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 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 surface of a cell in the subject and a second region that specifically binds 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 has no effector function and does not induce an Fc-mediated inflammatory response. For example, the fusion molecule may not comprise a portion that binds to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not comprise the target substance to be cleared or reduced by administration of the fusion molecule.

[0136] In embodiments, the present disclosure is directed to a method for removing, clearing, or enhancing clearance of an antigenic substance that induces, 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 the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, the fusion molecule comprising a first region capable of binding to a TAM (Tyro3, ​​Axl, and MerTK) receptor on the surface of a cell in the subject, and a second region that specifically binds to the antigenic substance. In non-limiting embodiments, the substance and the undesirable or pathological immune response may be one or more of those listed in Table 1. In non-limiting embodiments, the fusion molecule has no effector function and does not induce an inflammatory response. For example, the fusion molecule may 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 contain the target substance to be cleared or reduced by administration of the fusion molecule.

[0137] In embodiments, the present disclosure is directed to a method for treating, preventing, or ameliorating an immune disease in a subject having or at risk of developing the immune disease. The method comprises administering to the subject an effective amount of a fusion molecule or a polynucleotide encoding the fusion molecule, wherein the fusion molecule comprises a first region capable of binding to a TAM (Tyro3, ​​Axl, and MerTK) receptor on the cell surface of the subject and a second region capable of specifically binding to an antigenic substance that induces, induces, or causes the 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 has no effector function and does not induce an Fc-mediated inflammatory response. For example, the fusion molecule may not comprise a portion that binds to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not comprise the target substance to be cleared or reduced by administration of the fusion molecule.

[0138] In an embodiment, the present disclosure is directed to a method for delaying the development of symptoms associated with an immune disease caused or induced 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, or a vector containing the polynucleotide, wherein the fusion molecule comprises a first region capable of binding to a TAM (Tyro3, ​​Axl, and MerTK) receptor on the surface of a cell in the subject, and a second region that specifically binds to the antigenic substance. In a non-limiting embodiment, the antigenic substance and the immune disease may be one or more of those listed in Table 1. In a non-limiting embodiment, the fusion molecule has no effector function and does not induce an Fc-mediated inflammatory response. For example, the fusion molecule may not comprise a portion that binds to an Fc receptor, and preferably may comprise an Fc region variant that does not bind to an Fc receptor (particularly an Fcγ receptor). The fusion molecule does not comprise the target substance to be cleared or reduced by administration of the fusion molecule.

[0139] In embodiments, the present disclosure provides a method for reducing an antigenic substance that induces, 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 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 a TAM (Tyro3, ​​Axl, and MerTK) receptor on the surface of a cell in 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. Thus, 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 the antigenic substance. In a non-limiting embodiment, the antigenic substance and the disease may be one or more of those listed in Table 1. In a non-limiting embodiment, the fusion molecule has no 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 the target substance to be cleared or reduced by administration of the fusion molecule.

[0140] In some embodiments, the present disclosure is directed to a pharmaceutical composition comprising an effective amount of any of the aforementioned fusion molecules or polynucleotides encoding the fusion molecules, and a pharmaceutically acceptable excipient. In a non-limiting embodiment, the fusion molecule has no 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 the target substance to be cleared or reduced by administration of the fusion molecule.

[0141] In embodiments, the present disclosure is directed to the use of any of the aforementioned fusion molecules or polynucleotides encoding said fusion molecules for use in the manufacture of a medicament suitable for the treatment of an immune disorder.

[0142] In embodiments, the present disclosure is directed to the use of any of the aforementioned fusion molecules or polynucleotides encoding said fusion molecules, or pharmaceutical compositions, for use in treating or preventing immune diseases.

[0143] In embodiments, the present disclosure is directed to a kit comprising an effective amount of any of the aforementioned fusion molecules or polynucleotides encoding the fusion molecules, generally in suitable packaging and provided with suitable instructions, useful for any of the methods described herein.

[0144] These and other aspects, objects, features and advantages of the exemplary embodiments will become apparent to those skilled in the art upon consideration of the following detailed description of the illustrated exemplary embodiments.

[0145] [Effects of the Invention]

[0146] The present disclosure relates to fusion molecules with phagocytosis-inducing activity, which can solve the problem of tissue damage caused by the activation of inflammatory responses encountered in the prior art. Therefore, the fusion molecules can effectively clear or reduce substances whose expression or amount is elevated, for example, to normal levels or amounts, and can be used to prevent or treat immune diseases caused by elevated or increased substances, such as those listed in Table 1, or other diseases described herein. The fusion molecules can be administered to patients in the form of purified fusion molecules or in the form of gene therapy vectors that can express and secrete the fusion molecules when introduced into cells.

[0147] However, it should be understood that the effects of the present disclosure are not limited to the above effects, but include all effects that can be inferred from the configuration of the present invention described in the detailed description or the claims.

[0148] [Brief explanation of the drawings]

[0149] [Figure 1A] FIG. 1 shows the effect on experimental autoimmune encephalomyelitis (EAE) scores and changes in body weight when EAE was induced in mice with astrocyte-specific deletion of the Axl gene. [Figure 1B] FIG. 1 shows the effect on experimental autoimmune encephalomyelitis (EAE) scores and changes in body weight when EAE was induced in mice with astrocyte-specific deletion of the Axl gene. [Figure 1C] FIG. 1 shows the effect on experimental autoimmune encephalomyelitis (EAE) scores and changes in body weight when EAE was induced in mice with astrocyte-specific deletion of the Axl gene.

[0150] [Figure 2A] FIG. 1 shows the effect on EAE score and body weight change when EAE is induced in mice with microglia-specific Mertk gene deletion. [Figure 2B] FIG. 1 shows the effect on EAE score and body weight change when EAE is induced in mice with microglia-specific Mertk gene deletion. [Figure 2C] FIG. 1 shows the effect on EAE score and body weight change when EAE is induced in mice with microglia-specific Mertk gene deletion.

[0151] [Figure 3A] FIG. 1 is a schematic diagram showing the construction of the prepared AAVs expressing anti-FITC-Gas6 and anti-MOG(8-18C5)-Gas6.

[0152] [Figure 3B] FIG. 1 shows the amino acid sequences of anti-FITC-Gas6 and anti-MOG(8-18C5)-Gas6 constructed in Example 3.

[0153] [Figure 3C] FIG. 3C shows the sequence of the nucleic acid encoding anti-FITC-Gas6 shown in FIG. 3B. [Figure 3D] FIG. 3C shows the sequence of the nucleic acid encoding anti-FITC-Gas6 shown in FIG. 3B.

[0154] [Figure 3E] FIG. 3C shows the sequence of the nucleic acid encoding anti-MOG(8-18C5)-Gas6 shown in FIG. 3B. [Figure 3F] FIG. 3C shows the sequence of the nucleic acid encoding anti-MOG(8-18C5)-Gas6 shown in FIG. 3B.

[0155] [Figure 4A] FIG. 1 shows the effect of anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vitro. [Figure 4B] FIG. 1 shows the effect of anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vitro. [Figure 4C] FIG. 1 shows the effect of anti-MOG(8-18C5)-Gas6 on the removal of myelin debris in vitro.

[0156] [Figure 5A] FIG. 1 shows the effect of expressing anti-MOG(8-18C5)-Gas6 in EAE mice on EAE scores and body weight changes. [Figure 5B] FIG. 1 shows the effect of expressing anti-MOG(8-18C5)-Gas6 in EAE mice on EAE scores and body weight changes. [Figure 5C] FIG. 1 shows the effect of expressing anti-MOG(8-18C5)-Gas6 in EAE mice on EAE scores and body weight changes.

[0157] [Figure 6A] FIG. 1 shows the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 6B] FIG. 1 shows the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 6C] FIG. 1 shows the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 6D] FIG. 1 shows the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 6E] FIG. 1 shows the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

[0158] [Figure 7A] FIG. 1 shows the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 7B] FIG. 1 shows the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 7C] FIG. 1 shows the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 7D] FIG. 1 shows the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice. [Figure 7E] FIG. 1 shows the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin in wild-type mice.

[0159] [Figure 8] FIG. 1 is a schematic diagram showing the structure of the prepared anti-MOG(01)-Gas6.

[0160] [Figure 9A] FIG. 1 shows the antigen (human and mouse MOG) binding activity of anti-MOG(01)-Gas6 fusion molecules measured by enzyme-linked immunosorbent assay (ELISA). [Figure 9B] FIG. 1 shows the antigen (human and mouse MOG) binding activity of anti-MOG(01)-Gas6 fusion molecules measured by enzyme-linked immunosorbent assay (ELISA).

[0161] [Figure 9C] FIG. 1 shows the results of measuring the binding of anti-MOG(01)-Gas6 fusion molecules to mouse MOG protein on the cell surface using flow cytometry.

[0162] [Figure 10] FIG. 1 shows the effect of anti-MOG(01)-Gas6 on the removal of myelin debris in vitro.

[0163] [Figure 11] FIG. 1 is a schematic diagram showing the construction of the prepared anti-MBP-Gas6 antibody.

[0164] [Figure 12A] FIG. 1 shows the antigen (human and mouse MBP) binding activity of anti-MBP-Gas6 fusion molecules measured by ELISA. [Figure 12B] FIG. 1 shows the antigen (human and mouse MBP) binding activity of anti-MBP-Gas6 fusion molecules measured by ELISA.

[0165] [Figure 13] FIG. 1 shows the effect of anti-MBP-Gas6 on the removal of myelin debris in vitro.

[0166] [Figure 14A] FIG. 1 is a schematic diagram showing the configuration of the prepared anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6. [Figure 14B]FIG. 1 shows the sequences of anti-TNFα(adalimumab)-Gas6 and anti-TNFα(infliximab)-Gas6. [Figure 14C] FIG. 1 shows the sequences of anti-TNFα(adalimumab)-Gas6 and anti-TNFα(infliximab)-Gas6.

[0167] [Figure 15A] FIG. 1 shows the antigen (human TNFα) binding activity of the anti-TNFα-Gas6 fusion molecule measured by ELISA. [Figure 15B] FIG. 1 shows the results of measuring the binding level between the anti-TNFα-Gas6 fusion molecule and human TNFα protein on the cell surface using flow cytometry.

[0168] [Figure 16] FIG. 1 shows the level of inhibition of TNFα signal activation by anti-TNFα-Gas6 fusion molecules in HEK-Blue™ TNFα cells.

[0169] [Figure 17] FIG. 1 shows induction of Axl activation on U2OSAxl cells by anti-TNFα-Gas6 fusion molecules.

[0170] [Figure 18] FIG. 1 shows the induction of Axl-mediated phagocytosis by anti-TNFα-Gas6 using THP-1Axl-derived macrophages as effector cells.

[0171] [Figure 19A] FIG. 1 is a schematic diagram showing the configuration of the prepared anti-CD20 (rituximab)-Gas6. [Figure 19B] FIG. 1 shows the amino acid sequence of anti-CD20 (rituximab)-Gas6.

[0172] [Figure 20A]FIG. 1 shows the antigen (human CD20) binding activity of anti-CD20-Gas6 fusion molecules measured by ELISA. [Figure 20B] FIG. 1 shows the degree of binding between the anti-CD20-Gas6 fusion molecule and human CD20 protein on the cell surface, as determined by flow cytometry.

[0173] [Figure 21] FIG. 1 shows induction of Axl activation on U2OSAxl cells by anti-CD20-Gas6 fusion molecules.

[0174] [Figure 22] FIG. 1 shows induction of Axl-mediated phagocytosis by anti-CD20-Gas6 using THP-1Axl-derived macrophages as effector cells.

[0175] [Figure 23A] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23B] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23C] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23D] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23E] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23F] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23G] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23H] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23I] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23J] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure. [Figure 23K] FIG. 1 is a schematic diagram illustrating the structure of a fusion protein according to a non-limiting embodiment of the present disclosure.

[0176] [Figure 24] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 25] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 26] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 27] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 28] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 29] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 30] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 31] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 32] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 33] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure. [Figure 34] FIG. 1 shows the amino acid sequences of exemplary fusion proteins according to non-limiting embodiments of the present disclosure.

[0177] DETAILED DESCRIPTION OF THE INVENTION

[0178] Methods and compositions are provided for reducing or inhibiting the formation of, or clearing, removing or reducing, target substances whose increased amount or increased expression is associated with, characteristic of, or causes an immune disorder or immune disease via phagocytosis, which methods and compositions prevent or treat individuals having or at risk of developing an immune disease or immune disorder, and ameliorate the symptoms of the immune disease or immune disorder.

[0179] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, is also specifically disclosed. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range where either or both limits are included in the smaller range, or where neither limit is included in the smaller range, is also encompassed within the invention, provided that any specifically excluded limit is within the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0180]

[0181] [Definition]

[0182] As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural unless the context clearly indicates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells, and a reference to a "peptide" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, such as polypeptides.

[0183] As used herein, the terms "about" and "consisting essentially of" refer to a value or composition that falls within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "consisting essentially of" can mean within one standard deviation or more than one standard deviation, as is customary in the art. Alternatively, "about" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). For example, "about 5 mg" may include any number between 4.5 mg and 5.5 mg (10%), between 4.75 mg and 6.25 mg (5%), between 4.8 mg and 6.2 mg (4%), between 4.85 mg and 6.15 mg (3%), between 4.9 mg and 6.1 mg (2%), or between 4.95 mg and 6.05 mg (1%). Moreover, particularly with respect to biological systems or processes, these terms can mean values ​​up to an order of magnitude or up to 5-fold. When specific values ​​or compositions are provided in the specification and claims, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value or composition.

[0184] As used herein, the term "administration" or "administering" refers to introducing a composition into a subject by a selected route. For example, if the selected route is intravenous, 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 the subject.

[0185] As used herein, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs (amino acid analogs) and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those that are subsequently modified (e.g., hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine). Amino acid analogs (amino acid analogs) refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium)). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics are chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0186] As used herein, "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably to refer to a polymer 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 naturally occurring amino acid, as well as to natural and unnatural amino acid polymers. These terms also encompass amino acid polymers that are naturally or artificially modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or other manipulation or modification (such as conjugation with a labeling moiety). Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides of the present invention are based on antibodies, it is understood that the polypeptides can occur as single chains or associated chains.

[0187] As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling moiety. Other types of modifications include, for example, "caps," substitution of one or more analogs of naturally occurring nucleotides; internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), those containing alkylating agents, those with modified linkages (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of polynucleotides. Additionally, any of the hydroxyl groups normally present in the sugar may be replaced by, for example, phosphonate or phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly 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 sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs (such as methyl riboside). One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C) that may contain an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0188] As used herein, the terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired. A "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. In an embodiment, the mammal is a human.

[0189] As used herein, the term "antibody" refers to single-, double-, and multi-chain proteins and glycoproteins belonging to the classes of polyclonal, monoclonal, chimeric, and heteroimmunoglobulins (monoclonal antibodies are preferred); it also includes synthetic and genetically engineered variants of these immunoglobulins.

[0190] As used herein, the terms "specific binding," "specifically binds," and the like refer to the preferential non-covalent or covalent binding of a molecule relative to other molecules or sites 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 greater than or equal to 10. -5 M or less (e.g., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, 10 -15 M or less, or 10 -16The binding affinity is characterized by a KD (dissociation constant) of 0.01 M or less. "Affinity" refers to the strength of binding, with increased binding affinity correlated with a decreased KD. As used herein, the "binding" and "specific binding" of the first region to the TAM receptor and the "binding" and "specific binding" of the second region to the target substance need not regulate, alter, affect, or modify the activity of the bound TAM receptor or target substance.

[0191] As used herein, the term "variable" refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used in the binding and specificity of each particular antibody for a particular antigen. However, variability is not evenly distributed throughout the entire length of the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Naturally occurring heavy-chain and light-chain variable domains each contain four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the β-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequence of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0192] An "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. In two-chain Fv species, this region consists of a non-covalently bound dimer of one heavy-chain variable domain and one light-chain variable domain. In single-chain Fv species (scFv), one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that of two-chain Fv species. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) is capable of recognizing and binding antigen, albeit with lower affinity than the entire binding site.

[0193] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid sequence in antibody variable region that confers antigen specificity and binding affinity.For example, there are generally three CDRs (for example, HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.The precise amino acid sequence boundary of a given CDR can be determined using any of several well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residues of the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In the combined Kabat and Chothia numbering scheme, in some embodiments, a CDR refers to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26 to 35 (HCDR1), 50 to 65 (HCDR2), and 95 to 102 (HCDR3) of a VH (e.g., a mammalian VH, e.g., a human VH), and to amino acid residues 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3) of a VL (e.g., a mammalian VL, e.g., a human VL).

[0194] The "Fab fragment" also contains the constant domain of the light chain and the first constant domain, CH1, of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0195] 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, but 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; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of a single uninterrupted sequence of contiguous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including, but not limited to, (1) a single-chain Fv (scFv) molecule, (2) a single-chain polypeptide containing only one light-chain variable domain, or a fragment thereof containing the three CDRs of a light-chain variable domain without the associated heavy-chain portion, (3) a single-chain polypeptide containing only one heavy-chain variable region, or a fragment thereof containing the three CDRs of a heavy-chain variable region without the associated light-chain portion, and (4) a nanobody comprising a single Ig domain or other specific single-domain binding module from a non-human species; and multispecific or multivalent structures formed from antibody fragments. In antibody fragments comprising one or more heavy chains, the heavy chain can comprise any constant domain sequence found in the non-Fc region of an intact antibody (e.g., CHI of an IgG isotype), and / or can comprise any hinge region sequence found in an intact antibody, and / or can comprise a leucine zipper sequence fused or located to the hinge region or constant domain sequence of the heavy chain, and (5) an isolated complementarity-determining region (CDR).

[0196] As used herein, the terms "phagocytic cells," "phagocytes," and "apoptotic cells" are used interchangeably to refer to cells capable of phagocytosis. There are four main categories of phagocytes: macrophages, mononuclear cells (histiocytes and monocytes), polymorphonuclear leukocytes (neutrophils), and dendritic cells.

[0197] As used herein, the term "chimeric" refers to a molecule that contains sequences derived from two different molecules.

[0198] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The term "Fc region" may refer to a native-sequence Fc region or a mutant Fc region (variant Fc region). Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The numbering of residues within the Fc region is that of the EU index, as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The Fc region of an immunoglobulin usually comprises two constant domains, CH2 and CH3.

[0199] As used herein, the terms "Fc receptor" and "FcR" refer to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Additionally, a preferred FcR is one that binds an IgG antibody (gamma receptor), which includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses (including allelic variants and alternatively spliced ​​forms of these receptors). FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains.

[0200] A "native sequence Fc region" or "wild-type Fc region" comprises an amino acid sequence identical to that of a naturally occurring Fc region. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, yet retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, preferably about one to about five amino acid substitutions, in the native sequence Fc region or the Fc region of a parent polypeptide. The variant Fc regions herein 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, such as 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.

[0201] As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to produce a beneficial or desired result. For prophylactic uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or slowing the progression of a disease (including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifesting during the development of the disease). For therapeutic uses, beneficial or desired results include clinical results such as inhibiting, suppressing, or reducing elevated levels of a substance; reducing, removing, clearing, or reducing elevated antigenic substances to normal levels; sequestering or increasing soluble substances circulating in biological fluids; reducing one or more symptoms resulting from a disease (biochemical, histological, and / or behavioral), including disease complications and intermediate pathological phenotypes manifesting during the progression of the disease; improving the quality of life of those afflicted with the disease; reducing the dosage of other drugs required to treat the disease; enhancing the effectiveness of another drug; slowing the progression of the disease; and / or prolonging patient survival. An effective dose can be administered in one or more administrations. For purposes of this invention, an effective dose of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in clinical contexts, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination 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 agent may be considered to be administered in an effective amount if the desired result can or does occur in combination with one or more other agents.

[0202] As used herein, the term "treatment" or "treating" refers to an approach to achieving beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: inhibiting, suppressing, or reducing the formation of substance deposits; reducing, removing, or clearing antigenic substance deposits; improving cognition; reversing or slowing cognitive decline; sequestrating soluble substances circulating in biological fluids; reducing substances (including soluble, oligomeric, and deposits) in tissues; inhibiting, slowing, and / or reducing elevated or increased levels of antigenic substances in tissues; inhibiting, slowing, and / or reducing the toxic effects of substance peptides in tissues; alleviating symptoms resulting from a disease; improving the quality of life of those suffering from a disease; reducing the dosage of other medications required to treat the disease; slowing the progression of the disease; and / or extending the patient's survival. The tissue may include the individual's brain.

[0203] The term "development" of a disease refers to the onset and / or progression of the disease in an individual. Disease development can be detected using standard clinical techniques described herein. However, development also refers to disease progression that may not be initially detectable. For purposes of the present invention, progression refers to the biological course of the disease state, as determined by standard neurological examination, patient interview, or more professional testing. Such diagnostic tests include, but are not limited to, neuroimaging, detection of changes in the levels of specific proteins in serum or cerebrospinal fluid (e.g., any one or a combination of the antigenic substances listed in Table 1), computed tomography (CT), and magnetic resonance imaging (MRI). "Development" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a disease includes initial onset and / or recurrence.

[0204] As used herein, "delaying the development (progression)" of a disease means postponing, preventing, slowing, retarding, stabilizing, and / or postponing the development (progression) of the disease. The duration of this delay varies depending on the history of the disease and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can effectively encompass prevention, in that the individual does not develop the disease. For example, a method for delaying the development (progression) of a disease is one that reduces the probability of the disease developing within a given time frame and / or reduces the extent of the disease within a given time frame compared to the absence of the method. Such comparisons are usually based on clinical studies using a statistically significant number of subjects.

[0205] As used herein, the term " vector " refers to the construct that can deliver and preferably express one or more gene or sequence of interest into host cell.Examples of vector include but are not limited to virus vector, naked DNA or RNA expression vector, plasmid, cosmid or phage vector, DNA or RNA expression vector associated with cationic condensing agent, DNA or RNA expression vector encapsulated in liposome, and certain eukaryotic cells such as producer cells.

[0206] A "host cell" includes an individual cell or cell culture that can be or has been the recipient of a vector for incorporating a polynucleotide insert. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide of the invention.

[0207] As used herein, the term "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. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0208] As used herein, the term "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient, maintains the biological activity of the ingredient and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers, such as 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 normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods.

[0209]

[0210] [TAM receptor]

[0211] TAM receptors (Tyro3, ​​Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. They also affect cell proliferation, survival, adhesion, and migration. TAM receptors contain two immunoglobulin-like repeats and two fibronectin type III repeats in tandem in their extracellular domains. TAM receptors are linked to a single transmembrane domain and a cytoplasmic protein tyrosine kinase.

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

[0213] The Axl protein contains 894 amino acids, including a glycine-rich loop (Gly543-Gly548), a catalytic loop (His670-Asn677), and a DFG motif (Asp690-Phe691-Gly692). The full-length Axl has a molecular weight of 104 kDa, but post-translational modification of the extracellular domain produces 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 terms "Axl" or "Axl receptor" or "Axl protein" include the 104 kDa full-length Axl, post-translationally modified Axl, and glycosylated Axl. In some embodiments, the human Axl polypeptide corresponds to Genbank accession number NP_068713, NP_068713.2, SEQ ID NO: 114. In one embodiment, a nucleic acid encoding a human Axl polypeptide corresponds to Genbank accession number NM_021913, version number NM_021913.5. Murine (mouse) Axl refers to the Axl member of the Murine (mouse) TAM family of receptor tyrosine kinases. In some embodiments, the Murine Axl polypeptide corresponds to Genbank accession number AAH46618, version number AAH46618.1, SEQ ID NO: 115. In one embodiment, a nucleic acid encoding a Murine Axl polypeptide corresponds to Genbank accession number BC046618, version number 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. MerTK regulates many physiological processes, including cell survival, migration, and differentiation. Ligand binding at the cell surface induces dimerization and autophosphorylation of TYRO3 on the intracellular domain, which provides docking sites for downstream signaling molecules. After ligand activation, MerTK interacts with PIK3R1, thereby enhancing PI3 kinase activity.

[0215] Human MerTK contains 999 amino acid residues (Accession nos. Q12866, NP_006334.2). The mRNA and genomic DNA sequences are available under accession numbers AAB60430.1 and AAG33129.1, respectively. Various naturally occurring 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 contains 890 amino acid residues (accession numbers Q06418, NP_001317193.1, NP_006284.2). Polynucleotide sequences encoding human Tyro3 are available under accession numbers NM_001330264.1 and NM_006293.3. Various mRNA sequences encoding human Tyro3 have been reported under accession numbers such as AAA19236.1, BAA04467.1, AAC50070.1, BAA21781.1, AAH49368.1, AAH51756.1, and CAA51396.1. Several natural variants and post-translational modifications have been reported. (www.uniprot.org / uniprotkb / Q0641 8 / entry#sequences, last accessed June 11, 2023).

[0217] Cells expressing TAM receptors may be at least one type of professional phagocyte, at least one type of non-professional phagocyte, or a combination thereof. Here, professional phagocytes refer to cells whose primary role is to remove dead cells and accumulated debris through phagocytosis, and examples include macrophages, neutrophils, dendritic cells, and mast (mast) cells. Macrophages are typically present in various tissues that may be a route of infection, and are often referred to by different names depending on the tissue, such as adipose tissue macrophages, bone marrow or blood monocytes, Kupffer cells in the liver, lymph node sinus histiocytes, alveolar macrophages, connective tissue histiocytes or giant cells, central nervous system microglia, placental Hofbauer cells, kidney glomerular mesangial cells, bone osteoclasts, epithelioid cells in granulomas, splenic red pulp macrophages, peritoneal macrophages in the peritoneal cavity, and LysoMac (lysozyme-expressing macrophages) in Peyer's patches. Non-professional phagocytes, on the other hand, primarily perform functions specific to the tissue in which they reside but are capable of phagocytosis as needed. Examples include epithelial cells, endothelial cells, fibroblasts, mesenchymal cells, some tissue-specific cells (e.g., astrocytes or oligodendrocytes in the central nervous system, Müller glia in the retina, hepatocytes, muscle satellite cells, Sertoli cells in the testes, etc.), and some lymphocytes (e.g., natural killer cells, large granular lymphocytes, eosinophils, basophils, B cells, etc.). Fusion molecules according to the present disclosure can induce phagocytosis in specific phagocytes in tissues with increased amounts of target substances to be cleared. For example, if the amount or expression of antigenic proteins is elevated in the brain, phagocytosis can be induced in astrocytes, microglia, oligodendrocytes, or a combination thereof. This can be induced, for example, by locally administering a fusion molecule according to the present disclosure to the tissue or by engineering cells in the tissue to express and secrete the fusion molecule.

[0218]

[0219]

[0220] [First region containing a sequence capable of binding to a TAM receptor]

[0221] TAM receptors are activated through their ligands, growth arrest-specific 6 protein (Gas6) and protein S (ProS1), which are members of a family of vitamin K-dependent proteins.

[0222] In exemplary embodiments, the first region capable of binding to a TAM receptor may comprise, consist of, or consist essentially of one or more TAM ligands.

[0223] The TAM ligand, protein S, contains an amino-terminal gamma-carboxyglutamic acid (Gla) domain followed by a thrombin-sensitive loop region and four epidermal growth factor-like domains at the carboxy-terminus (C-terminus), which together consist of two laminin G repeats containing a sex hormone-binding globulin domain (Figure 1A, right panel). The C-terminal region is sufficient for TAM receptor binding and phosphorylation. Gas6 is a 75 kDa vitamin K-dependent protein that shares high structural identity (approximately 42%) with protein S, with a modular organization identical to that shown in Figure 1A.

[0224] In addition to Gas6 (SEQ ID NO: 7) and ProS1 (SEQ ID NO: 34), tubby (accession numbers 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 numbers NP_003311.2, NP_813977.1, ... The three TAM receptor ligands reported are Tubby and Gal3 (accession numbers AAB53700.1, AAH32714.1, AAH65261.1, NP_001276324.1, AAB97966.1, EAX03840.1, EAX03839.1, BAJ84064.1, BAJ84063.1, AKU84911.1, NP_813977.1, and NP_003311.2). Galectin-3 (Gal3) (accession numbers NP_002297 and NP_002297.1) specifically bind to Mer, whereas Tulp1 can activate all three TAM receptors.

[0225] Gas6, one of the ligands for TAM receptors, has been reported to exhibit the highest affinity for Axl compared to Tyro3 or Mer. Human Gas6 contains 678 amino acids (SEQ ID NO: 7) with a gamma-carboxyglutamic acid (Gla) domain, four epidermal growth factor (EGF)-like domains, and two laminin G-like (LG) domains (Figure 1A, right panel). Various isoforms of Gas6 have been reported. For example, the S6L, G8R, G8V, R14H, and L18Q isoforms have been reported, and these isoforms are included in the present disclosure.

[0226] In some embodiments, the first region capable of binding to a TAM receptor can be a TAM receptor agonist. TAM receptor agonists include agents that significantly increase the biological activity of a TAM receptor in a cell, such as agents that specifically bind to and activate a TAM receptor. For example, a TAM receptor agonist can increase biological TAM receptor activity by at least 25%, at least 50%, at least 70%, at least 80%, at least 90%, at least 95%, at least 100%, at least 200%, or even at least 500%. Methods for measuring such activity are known in the art. In some embodiments, increased biological activity is indicated by increased expression of Tyro3, ​​Axl, Mer, or a combination thereof (at the DNA, RNA, or protein levels). In other examples, increased biological activity is indicated by changes in downstream effects, such as increased TAM autophosphorylation, decreased TLR-induced cytokine production, decreased TLR-induced stimulation of MAP kinase activation, decreased TLR-induced NF-kB activation, or increased SOCS1 and SOCS3 expression. Methods for detecting such changes in expression or activity (which in some examples are quantified) are routine and include Western blotting, enzyme-linked immunosorbent assay (ELISA), flow cytometry, Northern blotting, PCR, RT-PCR, and the like. In embodiments, the TAM receptor to be activated by the first domain according to the present disclosure can be Axl or Mer.

[0227] In embodiments, 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. As used herein, the term "active fragment" refers to a fragment capable of binding to a TAM receptor, particularly an Axl receptor. For example, an active fragment of a Gas6 protein can comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 1, 2, 5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, or 87. For example, an active fragment of ProS1 protein can comprise, consist of, or consist essentially of the sequence of SEQ ID NO: 3, 4, 6, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113. The present disclosure encompasses 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 one of the sequences set forth in SEQ ID NOs: 8-23 share at least 85% sequence identity with SEQ ID NO: 1 (the LG-1 domain of Gas6). SEQ ID NOs: 24-33 share at least 85% sequence identity with SEQ ID NO: 2 (the LG-2 domain of Gas6). SEQ ID NOs: 35-45 share at least 85% sequence identity with SEQ ID NO: 3 (the LG-1 domain of ProS1).The sequences of SEQ ID NOs: 46 to 62 show at least 85% sequence identity to SEQ ID NO: 4 (LG-2 domain of ProS1). The sequences of SEQ ID NOs: 63 to 87 show at least 85% sequence identity to SEQ ID NO: 5 (LG domain of Gas6). The sequences of SEQ ID NOs: 88 to 113 show at least 84% sequence identity to SEQ ID NO: 6 (LG domain of ProS1).

[0228] In other embodiments, the first region can comprise, consist of, or consist essentially of the variable region or CDRs of an anti-Axl antibody or a full-length anti-Axl antibody with eliminated or ablated effector function, particularly Fc receptor binding function. The antibody or antigen-binding fragment can bind, for example, to the extracellular domain of Axl expressed on the surface of phagocytes and induce internalization and phagocytosis without an inflammatory response, particularly an Fc-mediated inflammatory response. Non-limiting examples of anti-Axl antibodies include those described in, for example, WO2017200493A1, WO2015193430A1, WO2011159980A1, WO2016097370A1, WO2012175691A1, WO2015193428A1, WO2010131733A1, WO2017220695A1, WO2010130751A1, WO2016166302A1, WO2017009258A1, WO2016005593A1, US20190134193A1, etc., the contents of all of which are incorporated herein by reference. According to embodiments of the present disclosure, the variable regions, CDRs, or scFv, F(ab), or F(ab') of these anti-Axl antibodies can be employed as the first region of the fusion molecule.

[0229] In other embodiments, the first region may comprise, consist of, or essentially consist of the variable region or CDRs of an anti-MerTK (Mer tyrosine kinase) antibody or full-length anti-MerTK antibody with effector function, particularly Fc receptor binding function, abolished or removed. The antibody or antigen-binding fragment may, for example, bind to the extracellular domain of MerTK expressed on the surface of phagocytes and induce internalization and phagocytosis without an inflammatory response, particularly an Fc-mediated inflammatory response. Non-limiting examples of MerTK antibodies include those described in WO2016106221A1, WO2020076799A1, WO2020176497A1, etc., the contents of which are all incorporated herein by reference. According to embodiments of the present disclosure, the variable regions, CDRs, or scFv, F(ab), or F(ab') of these anti-MerTK antibodies may be employed as the first region of a fusion molecule.

[0230] In other embodiments, the first region may comprise, consist of, or essentially consist of the variable region or CDRs of an anti-Tyro3 antibody or a full-length anti-Tyro3 antibody with effector functions, particularly Fc receptor binding functions, abolished or removed. The antibody or antigen-binding fragment may, for example, bind to the extracellular domain of Tyro3 expressed on the surface of phagocytes and induce internalization and phagocytosis without an inflammatory response, particularly an Fc-mediated inflammatory response. Non-limiting examples of anti-Tyro3 antibodies include those described in, for example, WO2016166348A1, the entire contents of which are incorporated herein by reference. According to embodiments of the present disclosure, the variable regions, CDRs, or scFv, F(ab), or F(ab') of these anti-Tyro3 antibodies may be employed as the first region of a fusion molecule.

[0231] A peptide containing any one of the above 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 differ from the amino acid sequence. Any truncation, deletion, insertion, substitution, or combination thereof in the final protein structure is possible as long as the activity of the fusion molecule is maintained. An example of a sequence variant is a form in which an amino acid residue not essential for activity is truncated or deleted, or an amino acid residue important for autoinhibition is substituted. In some cases, the protein may be modified by phosphorylation, glycosylation, methylation, farnesylation, etc. It is more preferable if such sequence mutations and modifications result in increased protein function and / or stability (thermal stability, pH stability, structural stability, etc.) and / or solubility.

[0232] Mutagenesis of amino acid sequences is based on the method of producing a nucleic acid molecule containing a nucleotide sequence corresponding to the amino acid sequence to be mutated by mutating a nucleotide sequence encoding a protein. The method of obtaining a gene encoding a protein can be performed in vivo or in vitro using any mutagenesis technique well known in the art, such as 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 linkers (Pharmacia), PCR technology (Higuchi, 1989, "Using PCR to Engineer DNA" in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70) can be used.

[0233] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0234] Examples of modified polypeptides include polypeptides in which conservative substitutions of amino acid residues have been made, deletions or additions of one or more amino acids that do not significantly result in adverse changes in functional activity, or the use of chemical analogs.

[0235] Substitutional variants involve removing at least one amino acid residue in an antibody molecule and inserting another residue in its place. The most attractive sites for substitutional mutagenesis are hypervariable regions, although FR changes are also considered. Conservative substitutions are shown under the heading "conservative substitutions" in Table 2. If such substitutions result in a change in biological activity, more substantial changes, designated "exemplary substitutions" in Table 2 or further described below in terms of amino acid classes, can be introduced and the products screened.

[0236]

[0237] [Table 2]

[0238]

[0239] Substantial modification of antibody biological properties can be achieved by selecting substitutions that differ significantly in their effectiveness in (a) maintaining the structure of the polypeptide backbone in the area of ​​the substitution, e.g., in a sheet or helical conformation, (b) maintaining the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are classified based on common side chain properties as follows:

[0240] -Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile;

[0241] - uncharged polar: Cys, Ser, Thr, Asn, Gln;

[0242] - Acidic (negatively charged): Asp, Glu;

[0243] -Basic (positively charged): Lys, Arg;

[0244] -residues that influence chain orientation: Gly, Pro; and

[0245] -Aromatic: Trp, Tyr, Phe, His.

[0246] Non-conservative substitutions are made by exchanging a member of one of these classes for another. Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) can be added to an antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0247] Amino acid modifications range from changing or modifying one or more amino acids to completely redesigning a region, such as a variable region. Alterations in the variable region can alter binding affinity and / or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made within the CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within the CDR domain. In yet other embodiments, the CDR domain is CDR H3 and / or CDR L3.

[0248]

[0249] [Target inflammation-related substances and immune or immune-mediated diseases]

[0250] The target inflammation-related substances may include one or more autoantigens, their autoantibodies, or complexes of an autoantigen and its autoantibody; one or more immune cell surface molecules including costimulatory molecules and receptors; one or more complements; one or more chemokines; one or more cytokines; one or more cell adhesion molecules; or combinations thereof.

[0251] Non-limiting exemplary antigenic substances that induce, cause, or induce unwanted or undesired immune responses and associated immune diseases (immune-mediated diseases) are listed above in Table 1. Non-limiting examples of immune cell surface molecules, including costimulatory molecules and receptors, complement, chemokines, cytokines, and cell adhesion molecules, are disclosed above.

[0252] Fusion molecules according to embodiments of the present disclosure may be useful for reducing, removing, or enhancing the clearance or removal of antigenic substances in a subject, thereby treating or preventing immune diseases or disorders, cardiovascular diseases or disorders, metabolic diseases or disorders, or proliferative diseases or disorders. In certain embodiments, fusion molecules according to the present disclosure may be useful for suppressing, reducing, clearing, or removing inflammation-related substances in a subject, or enhancing the clearance or removal of inflammation-related substances, thereby treating or preventing immune diseases or disorders, cardiovascular diseases or disorders, metabolic diseases or disorders, or proliferative diseases or disorders. The immune disease or disorder is an autoimmune disease or inflammatory disease exemplified in the present disclosure.

[0253] 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 disease, endometriosis, inflammatory bowel disease, asthma, obesity, ankylosing spondylitis, antiphospholipid syndrome, chronic relapsing multifocal osteomyelitis, gout, Henoch-Schonlein purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus These include systemic lupus erythematosus (SLE), juvenile scleroderma, juvenile vasculitis, Kawasaki disease, lupus (systemic lupus erythematosus), mixed connective tissue disease, myositis, post-streptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren's syndrome, spondyloarthritis / spondyloarthropathy, systemic juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, celiac disease, and thrombotic thrombocytopenic purpura (iTTP).

[0254] The fusion molecules described herein may also be useful in the treatment of cardiovascular diseases or disorders such as atherosclerosis, endocarditis, hypertension, and peripheral ischemic disease.

[0255] The fusion molecules described herein may be useful for treating, preventing, inhibiting, slowing the progression of, or alleviating symptoms associated with immune, cardiovascular, metabolic, or proliferative diseases or disorders. Immune disorders include inflammatory and autoimmune diseases or disorders. Inflammation or the inflammatory response is a normal and protective host response to injury, but inflammation can also cause unwanted damage. For example, atherosclerosis is, at least in part, a pathological response to arterial injury and the associated inflammatory cascade. Treatable cardiovascular diseases or disorders may also include diseases and disorders considered immune diseases / disorders, but include, for example, atherosclerosis, endocarditis, hypertension, or peripheral ischemic disease. Metabolic diseases or disorders include diabetes, obesity, and diseases and disorders associated with increased or elevated levels of antigenic substances.

[0256] Allergens are other antigens for which tolerance to immune responses is also desirable. Even in diseases where the causative autoantigen is unknown, bystander suppression may be induced by using antigens present in anatomically nearby sites. For example, autoantibodies against collagen are observed in rheumatoid arthritis. Therefore, collagen-encoding genes or collagen may be target inflammation-related substances that should be cleared, removed, or alleviated by administering fusion molecules. In this case, an aptamer that specifically binds to a collagen-encoding gene or an antibody that binds to collagen or its fragments can be used as the second domain of the fusion molecule. Furthermore, fusion proteins consisting of a second domain that binds to a beta cell autoantigen can also be used to prevent or treat the onset of type 1 diabetes (see, for example, Bach and Chatenoud (2001) Ann Rev Immunol 19:131-161).

[0257] Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are observed not only in multiple sclerosis but also in autoimmune encephalomyelitis and many other central nervous system diseases. Therefore, administration of fusion molecules containing an anti-MOG antibody or its fragment as the second domain may enable the treatment of multiple sclerosis as well as related autoimmune diseases of the central nervous system.

[0258] In general, the immune response includes (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 work to eliminate antigens by a number of mechanisms. For example, helper T cells, which are capable of recognizing specific antigens, may respond by releasing soluble mediators such as cytokines to recruit additional cells of the immune system to participate in the immune response. Cytotoxic T cells, which are also capable of specific antigen recognition, may respond by binding to and destroying or damaging antigen-bearing cells or particles.

[0259] The immune response in a host or subject can be determined by several well-known immunological methods described herein and readily apparent to those skilled in the art. Such assays include, but are not limited to, in vivo or in vitro measurements 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 peptide, carbohydrate, nucleotide, and / or lipid mediators; changes in the activation state of cells of the immune system, as determined by changes in their functional or structural properties, 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 a stimulus; changes in the relationship between Th1 and Th2 responses; and cell differentiation by cells of the immune system, including changes in the expression profile of surface antigens or the initiation 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).

[0260] Cytokine levels can be measured according to methods described herein and practiced in the art, including ELISA, ELISPOT, and flow cytometry (measuring intracellular cytokines). Immune cell proliferation and clonal expansion resulting from the induction or stimulation of an antigen-specific immune response can be determined by isolating lymphocytes, such as spleen cells or cells from lymph nodes; stimulating the cells with antigen; and measuring cytokine production, cell proliferation, and / or cell viability, such as by tritiated thymidine incorporation or non-radioactive assays such as the MTT assay. The effect of the fusion polypeptides described herein on the balance between Th1 and Th2 immune responses can be determined by measuring the levels of Th1 cytokines, such as IFN-γ, IL-12, IL-2, and TNF-β, and type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

[0261] In certain embodiments, antigenic substances and associated immune disorders do not include substances whose abnormal accumulation or aggregation in biological tissue is characteristic of or associated with diseases such as neurological diseases or disorders.

[0262]

[0263] [Second domain of fusion molecules]

[0264] The second region that specifically binds to the target substance may be selected from among an antibody, an antigen-binding fragment thereof, an antibody-like protein, a peptide, an aptamer, and a soluble receptor, and is not particularly limited as long as it can specifically bind to the target substance.

[0265] Here, the antibody or antigen-binding fragment thereof may 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, and VNAR; and iii) engineered antibodies such as scFv, dsFv, ds-scFv, (scFv)2, diabodies, triabodies, tetrabodies, and pentabodies. The antibody or antigen-binding fragment thereof may be, for example, a Mab, Fab, or single-chain variable fragment (scFv) based on an antibody that specifically binds to the target substance, or six complementarity-determining regions (CDRs) derived from an antibody. That is, the type and scope of the protein or antigen-binding fragment that specifically binds to the target substance are not particularly limited, as long as it contains the portion necessary for the activity of specifically binding to the target substance, is linked to a first region, and does not cause inflammatory responses and synaptic damage. For example, the target substance may be β-amyloid, and in this case, the protein or antigen-binding fragment thereof that specifically binds to the target substance may comprise aducanumab or a single-chain variable fragment thereof. The second region comprises a Mab, Fab, or single-chain variable fragment based on six complementarity-determining regions (CDRs) derived from commercially available antibodies such as aducanumab, semolinemab, and simpanemab.

[0266] The antibody or antigen-binding fragment thereof may not comprise an Fc region, or preferably may comprise an Fc region variant that does not bind to Fc receptors (particularly Fcγ receptors). This Fc region variant may serve to improve properties such as purification. Fc variants with reduced affinity for human FcyRIIIA and / or FcyRIIA and / or FcyRI compared to IgG Fc regions due to amino acid substitutions are disclosed, for example, in WO2012130831 and USP8753628, the entire contents of which are incorporated herein by reference. The Fc region may be aglycosylated or deglycosylated.

[0267] Antibody-like proteins are protein scaffolds that can specifically bind to target substances like antibodies. They can be designed to be smaller in size, approximately 2-20 kDa, than antibodies (average size approximately 150 kDa), allowing them to target binding sites that antibodies cannot reach. Antibody-like proteins are known to be more stable at higher temperatures than antibodies, and are much easier to synthesize using non-mammalian cells such as viruses or yeast, or to synthesize chemically.

[0268] As used herein, the term "aptamer" refers to single-stranded DNA (ssDNA) or RNA that has high specificity and affinity for a particular substance. Aptamers have very high affinity for a particular substance, are stable, can be synthesized relatively easily, can be modified in various ways to increase binding affinity, and can target cells, proteins, and even small organic substances. Thus, aptamers are characterized by their significantly higher specificity and stability than previously developed antibodies. Aptamers can also be produced by the known SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method. For example, an aptamer that specifically binds to any one of the listed target substances can be produced by the known SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method, and then linked to a first region to produce a fusion molecule of the present invention.

[0269] The aptamers of the present disclosure are not particularly limited as long as they can specifically bind to any of the listed target substances, and the bases used in the aptamers may be selected from the group consisting of A, G, C, U, and their deoxy forms, unless otherwise specified.

[0270] To enhance stability, aptamers may be modified by attaching one or more of the following to the 5'-terminal, middle, 3'-terminal, or both terminals: polyethylene glycol (PEG), inverted deoxythymidine (idT), locked nucleic acid (LNA), 2'-methoxynucleoside, 2'-aminonucleoside, 2'F-nucleoside, amine linker, thiol linker, and cholesterol. Inverted deoxythymidine (idT) is a molecule typically 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 binds to the 5'-OH of the next nucleotide to form a chain. However, in the case of idT, the 3'-OH of the previous nucleotide binds to the 3'-OH of the next unit, exposing the 5'-OH instead of the 3'-OH. Therefore, idT is a molecule that inhibits degradation by 3' exonucleases, a type of nuclease.

[0271] The fusion molecules of the present disclosure induce phagocytosis through interaction with TAM receptors, and thus phagocytosis can be induced in cells expressing TAM receptors. Phagocytosis generally refers to the uptake (engulfment) of cells or particles of 0.5 μm or larger, and includes the processes of tethering, engulfing, and degrading the cells or particles. In this case, phagocytosis involves the formation of phagosomes surrounding the internalized cells or particles, followed by degradation within the phagolysosomes through fusion of the phagosomes with lysosomes. In phagocytosis, the process of cell death by apoptosis or necrosis is also called efferocytosis.

[0272] Non-limiting representative examples of second domains and their targets are shown in Table 3. The entire contents of the references listed in Table 3 are incorporated herein by reference. Those skilled in the art will understand that not only antibodies but also ligands for the listed targets can function as second domains. For example, a second domain capable of binding to TGFBR1 (transforming growth factor beta receptor 1) includes TGFβ.

[0273]

[0274] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16] [Table 3-17] [Table 3-18] [Table 3-19] [Table 3-20]

[0275]

[0276] [Fused or combined molecules]

[0277] The induction of phagocytosis by the fusion molecules of the present disclosure may not be accompanied by an inflammatory response, which allows target substances to be cleared without inducing an inflammatory response and suppresses tissue damage caused by an inflammatory response, thereby enabling safer treatment of tissue dysfunction caused by increased levels or expression of target substances than conventional techniques.

[0278] The first and second domains are bound to each other directly or via a linker to form a fusion molecule.

[0279] The fusion molecule may further comprise a tag, which, when added to the fusion molecule, can be used to verify the purification, expression, action, or mechanism of action of the fusion molecule.

[0280] Examples of tags include His-tag, T7-tag, S-tag, FLAG-tag, Strep-tag, thioredoxin (Trx)-tag, His-patch thioredoxin-tag, L-galactosidase (lacZ)-tag, chloramphenicol acetyltransferase-tag, trpE-tag, avidin / streptavidin / strep-tag, T7 gene 10-tag, staphylococcal protein A-tag, streptococcal protein G-tag, glutathione-S-transferase (GST)-tag, dihydrofolate reductase (DHFR)-tag, and the like. reductase-tag, cellulose binding domains (CBD's)-tag, maltose binding protein (MBP)-tag, galactose-binding protein-tag, calmodulin binding protein (CBP)-tag, hemagglutinin influenza virus (HAI)-tag, HSV-tag, B-(VP7 protein region of bluetongue virus)-tag, polycysteine-tag, polyphenylalanine-tag, (Ala-Trp-Trp-Pro) n Examples of suitable tags include, but are not limited to, a nucleotide-tag, a polyaspartic acid-tag, a c-myc-tag, a lac repressor-tag, etc. The tag may be located at the N-terminus, C-terminus, or internally of the target protein.

[0281] The fusion molecule may further contain a signal peptide or leader sequence at its N-terminus. A signal peptide is a short peptide present at the N-terminus of a protein during the initial synthesis stage of the protein destined for the secretory pathway, and is known to specify the intracellular localization, membrane topology (in the case of a membrane protein), and the like of the protein. The signal peptide may be cleaved during the process of the fusion molecule being expressed and secreted outside the cell.

[0282] The first region, second region, tag, signal peptide, or minimally functional region (e.g., the LG1 and LG2 regions, or the heavy and light chain variable regions of an scFv) contained in the fusion molecule may be directly linked to each other or may be linked via a linker comprising a short oligopeptide or polypeptide. Generally, the linker may contain 2 to 500 amino acid residues. The length and type of the linker are not particularly limited, as long as the linker is capable of linking the aforementioned regions in a manner that provides the intended activity and thereby forming a fusion molecule. An example of a linker is the commonly used oligopeptide linker (GGGGS)n (SEQ ID NO: 116), i.e., a linker consisting of one or more repeated Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 117) units. Other examples of linkers include (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), AEAAAKE Examples of suitable linkers include, but are not limited to, AAAAKA (SEQ ID NO: 127), PAPAP (SEQ ID NO: 128), (Ala-Pro)n, VSQTSKLTRAETVFPDV (SEQ ID NO: 129), PLGLWA (SEQ ID NO: 130), TRHRQPRGWE (SEQ ID NO: 131), AGNRVRRSVG (SEQ ID NO: 132), RRRRRRRR (SEQ ID NO: 133), GFLG (SEQ ID NO: 134), and GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (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.

[0283] Fusion molecules according to embodiments of the present disclosure may further comprise a scaffold bound to the first region, the second region, or both the first and second regions at different positions on 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 linked or fused to one position on the scaffold, and the second region may be linked or fused to another position on the scaffold. The link or fusion between the first region / second region and the scaffold may be a direct bond or may be via a linker as described above.

[0284] Fusion molecules according to embodiments of the present disclosure can have, for example, the structures shown generally in the non-limiting illustrative diagrams of Figures 23A through 23K.

[0285] Another aspect of the present disclosure provides nucleic acid molecules encoding the fusion molecules and expression vectors containing same.

[0286] As mentioned above, the nucleic acid molecule sequence encoding the fusion molecule can be mutated by substitution, deletion, insertion, or a combination thereof of one or more nucleotide residues, as long as it encodes a protein having equivalent activity.

[0287] The nucleic acid molecule sequence encoding the fusion molecule can be isolated from nature, artificially synthesized, or produced by genetic recombination methods. The nucleic acid molecule sequence encoding the fusion molecule is operably linked to an expression vector capable of expressing it.

[0288] The term "expression vector" refers to a vector that can express a protein or RNA of interest by introducing a nucleic acid sequence encoding a gene of interest into a suitable host cell, and that contains the necessary regulatory elements operably linked to express the gene insert. Such expression vectors include all vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors and virus vectors.

[0289] Suitable expression vectors have expression control elements such as a promoter, initiation codon, stop codon, polyadenylation signal, enhancer, etc. The initiation codon and stop codon are generally considered part of the nucleic acid sequence encoding the protein, and the protein-encoding sequence is designed to be in frame so that it is operably within the vector. The promoter can be constitutive or inducible. Furthermore, conventional expression vectors contain a selectable marker. Operational linkage with an expression vector can be performed using recombinant genetic techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes well known in the art.

[0290] The expression vector may be configured to be introduced into cells during in vivo injection, preferably for the purpose of expressing the fusion molecule in the host cell followed by isolation and purification, or for the cell to express and secrete the fusion molecule. If the purpose is to introduce the fusion molecule into a cell in vivo, the vector is preferably a non-integrating vector, i.e., a vector that does not integrate into the genome of the host cell.

[0291] In yet another aspect of the present disclosure, a cell expressing the fusion molecule is provided.

[0292] Cells may be transformed to contain a nucleic acid molecule or an expression vector containing the same, where "transformation" includes any method of introducing a nucleic acid molecule into an organism, cell, tissue, or organ, and may be carried out using standard techniques appropriate to the host cell, as known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO) precipitation, calcium chloride (CaCl) precipitation, silicon carbide fiber agitation, Agrobacterium-mediated transformation, polyethylene glycol (PEG), dextran sulfate, lipofectamine, and desiccation / suppression-mediated transformation.

[0293] 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, Neurospora crassa, etc.; lower eukaryotic cells; or cells derived from higher eukaryotes, including insect cells, plant cells, or mammalian cells. Examples of suitable animal cells include, for example, COS, CHO, or HEK293 cells. Examples of plant cells include tobacco, corn, soybean, and rice cells. Using methods known to those skilled in the art, a nucleic acid vector can be designed based on the present disclosure to express a foreign sequence in a specific host system, and then a polynucleotide sequence encoding a fusion polypeptide can be inserted. Regulatory elements will vary depending on the specific host.

[0294] After expressing the fusion molecule in cells, isolation and purification can be performed using conventional biochemical separation techniques, such as treatment with protein precipitants (salting out), centrifugation, sonication, ultrafiltration, dialysis, or various types of chromatography, such as gel filtration, adsorption chromatography, ion exchange chromatography, and affinity chromatography, which are usually used in combination to isolate highly pure 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)).

[0295] The resulting fusion protein can then be evaluated to determine whether it substantially increases TAM receptor activity. This method may include contacting a cell with the test fusion protein and determining whether contacting the cell with the test fusion protein alters TAM autophosphorylation, TLR-induced cytokine production, TLR-induced stimulation of MAP kinase activation, and / or TLR-induced NF-kB activation, compared to a control group. In this example, an increase in TAM autophosphorylation or a decrease in TLR-induced cytokine production, TLR-induced stimulation of MAP kinase activation, or a decrease in TLR-induced NF-kB activation in the presence of the test fusion protein, compared to control group levels, indicates that the fusion protein stimulates TAM receptor activity.

[0296] Autophosphorylation assays are well known in the art. In one example, cells expressing a TAM receptor are cultured and treated with test medium, e.g., at 37°C for 20 minutes. The medium is aspirated, and cold lysis buffer is added to each sample. The samples are centrifuged to spin down the cell nuclei, and the supernatant is mixed with protein A agarose beads and affinity-purified anti-TAM receptor antibody and incubated. The protein A beads are pelleted, washed, separated on a Tris-glycine gel, and transferred to a PVDF membrane (Millipore) for Western blotting. The blot is probed with anti-phosphotyrosine as the primary antibody. A significant decrease in phosphotyrosine labeling compared to the control indicates that the test fusion protein is an inhibitor of the TAM receptor. The control can be a known value representing phosphotyrosine labeling in a sample, such as cells not treated with the test agent. For example, a significant increase in phosphotyrosine labeling relative to a control group indicates that the test fusion protein is an agonist of the TAM receptor. 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% relative to such a control group indicates that the test fusion protein activates the TAM receptor.

[0297] Cytokine assays are also well known in the art. For example, cytokine assays are manufactured by Assay Designs (Ann Arbor, Michigan, USA), AssayGate (Jamesville, Maryland, USA), and Panomics (Fremont, California, USA). An increase in TLR-induced cytokine production in the presence of a test agent compared to the control level indicates that the test agent inhibits the activity of TAM receptors. The control level can be the amount of TLR-induced cytokine production in the absence of a test fusion protein, or a reference value representing the amount of TLR-induced cytokine production in the absence of a test fusion protein. For example, a significant decrease in TLR-induced cytokine production compared to the control group indicates that the test fusion protein is a TAM receptor agonist. For example, a reduction in TLR-induced cytokine production by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to such a control group indicates that the test fusion protein activates the TAM receptor, and thus, that the test fusion protein activates the TAM receptor.

[0298] MAP kinase activity can be measured by performing a MAP kinase assay. A significant increase in MAP kinase activation (as indicated by increased phosphorylation of p38) in the presence of the test fusion protein compared 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 by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to such a control indicates that the test fusion protein activates the TAM receptor. .

[0299] The fusion protein can be evaluated by measuring TLR-induced NF-kB activation. In this example, a significant reduction in TLR-induced NF-kB activation compared to a control indicates that the test agent is a TAM receptor agonist and, therefore, that the test fusion protein activates the TAM receptor. For example, a significant reduction in TLR-induced NF-kB activation by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to such a control indicates that the test fusion protein activates the TAM receptor.

[0300]

[0301] [Pharmaceutical composition]

[0302] Yet another aspect of the present disclosure provides a pharmaceutical composition for preventing or treating a disease caused by an increased amount or expression of a target substance in biological tissue, the pharmaceutical composition comprising a fusion molecule or an expression vector, wherein the composition can be administered locally to the site where the amount or expression of the disease-causing substance, i.e., the target substance, is elevated.

[0303] A further aspect of the present disclosure provides the use of the fusion molecule for the manufacture of a medicament for the prevention or treatment of an immune disease or disorder.

[0304] The fusion molecule, which is the active ingredient in the pharmaceutical composition, is contained in a "pharmaceutically effective amount."

[0305] The pharmaceutical composition can be administered orally or parenterally, preferably parenterally, and more preferably locally to a tissue showing increased levels / elevated or increased expression of the target substance to be cleared.

[0306] The term "parenteral administration" as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.

[0307] When the pharmaceutical composition is prepared as an injectable formulation, it can be prepared as an injectable formulation by a conventional method known in the art. The injectable formulation may be in the form of a dispersion in a sterile medium so that it can be administered directly to a patient, or it may be in a form that can be administered after being dispersed in distilled water for injection at an appropriate concentration.

[0308] When formulated for oral administration, the pharmaceutical composition may contain one or more carriers selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers, and preservatives, and may also contain one or more additives selected from flavorings, vitamins, and antioxidants.

[0309] The techniques required for formulating pharmaceutical compositions, as well as pharmaceutically acceptable carriers, excipients, etc., are well known to those skilled in the art (see, for example, the Handbook of Pharmaceutical Excipients, 4 th edition,Rowe et al.,Eds.,American Pharmaceuticals Association(2003);Remington:the Science and Practice of Pharmacy,20 th edition,Gennaro,Ed.,Lippincott Williams & Wilkins(2000);Remington's Pharmaceutical Sciences(19 th ed., 1995).

[0310] The appropriate dosage of the pharmaceutical composition may vary depending on factors such as the formulation method, mode of administration, the patient's age, body weight, sex, medical condition, diet, administration time, administration route, excretion rate, and reaction sensitivity. The dosage of the pharmaceutical composition of the present disclosure for adults is 0.0001 to 1,000 μg / kg body weight.

[0311] [Example]

[0312] The present disclosure will be described in more detail below with reference to examples and experimental examples. However, the following examples and experimental examples are merely illustrative and do not limit the scope of the present invention.

[0313] Astrocytes and microglia are known to play important roles in the progression of multiple sclerosis (MS). These cells express TAM receptors, and activation of these receptors has been reported to exhibit phagocytic and anti-inflammatory activities. In Examples 1 and 2 below, we aimed to clarify the effect of genetic knockout (KO) of key TAM receptors in these two cell types on the progression of MS in EAE (experimental autoimmune encephalomyelitis) mice, an MS model animal. The EAE model induces demyelination of myelin in the central nervous system by inducing T cell activation that recognizes myelin oligodendrocyte glycoprotein (MOG). This model has been reported to resemble human multiple sclerosis in many clinical and histopathological applications, making it the most commonly used animal model for studying the mechanisms and therapeutic effects of multiple sclerosis.

[0314]

[0315] Example 1: Role of astrocytes in regulating Axl-mediated neuroinflammation in the central nervous system (CNS)

[0316] Astrocyte-specific Axl gene deletion was performed in adult mice using ldh1l1-CreERT2;Axl f / f mice. Axl f / f mice served as a control group. Tamoxifen (75 mg / kg) was administered intraperitoneally for 5 consecutive days to demonstrate CreERT2 activity in 8-week-old female mice. Experimental autoimmune encephalomyelitis (EAE) was induced in 9-week-old mice by administering MOG35-55 in complete Freund's adjuvant (CFA) and pertussis toxin (PTX). EAE scores and body weight changes were monitored for 25 days after EAE induction. Experimental autoimmune encephalomyelitis (EAE) is the most frequently used animal model to study the immunopathogenesis of chronic inflammation in central nervous system (CNS) diseases such as multiple sclerosis (MS) and to test the therapeutic efficacy of novel agents.

[0317] To ablate the Axl gene specifically in astrocytes and induce MS-like disease (EAE) in mice, mice were manipulated according to the protocol shown in Figure 1A. More specifically, Aldh1l1-CreERT2, which contains the Aldh1l1 promoter-linked construct and Axl f / f;Axl f / f female mice were used to express CreERT2 specifically in astrocytes, and Axl f / f female mice were used as a control group.

[0318] At 8 weeks of age, mice were intraperitoneally injected with tamoxifen for 5 consecutive days to activate CreERT2. Next, to induce EAE in 9-week-old mice, MOG35-55 in complete Freund's adjuvant (CFA) was injected subcutaneously, followed by intraperitoneal injection of pertussis toxin (PTX) for 2 consecutive days. EAE scores and body weight changes were then monitored for 25 days.

[0319] The results confirmed that mice lacking the astrocyte-specific Axl gene had more severe EAE scores and weight loss than controls (Figures 1B and 1C), indicating that astrocytes play an important role in regulating EAE, and that this is achieved via Axl.

[0320]

[0321] Example 2: Role of microglia in regulating Mertk-mediated neuroinflammation in the central nervous system

[0322] Cx3cr1-CreERT2;Mertk f / f mice were used to ablate the Mertk gene specifically in microglia from adult mice. Mertk f / f mice served as a control group. Tamoxifen (75 mg / kg) was administered intraperitoneally for five consecutive days to demonstrate CreERT2 activity in 8-week-old female mice. Experimental autoimmune encephalomyelitis (EAE) was induced in 9-week-old mice by administering MOG35-55 in complete Freund's adjuvant (CFA) and pertussis toxin (PTX). EAE scores and body weight changes were monitored for 25 days after EAE induction. To ablate the Mertk gene specifically in microglia and induce MS-like disease in mice, mice were manipulated according to the protocol shown in Figure 2A.

[0323] More specifically, we used Cx3cr1-CreERT2;Mertk f / f female mice containing a Cx3cr1 promoter-linked construct and Mertk f / f to express CreERT2 specifically in microglia, while Mertk f / f female mice served as controls. When the 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 CFA was injected subcutaneously, followed by intraperitoneal administration of pertussis toxin (PTX) for 2 consecutive days. EAE scores and weight changes were then monitored for 25 days. Results confirmed that mice with microglia-specific Mertk deletion had more severe EAE scores and weight changes than the control group (Figure 2B and Figure 2C). This indicates that microglia play an important role in regulating EAE, and that this is mediated by Mertk.

[0324]

[0325] Example 3: Construction of fusion proteins: anti-FITC-Gas6 fusion molecule and anti-MOG(8-18C5)-Gas6 fusion molecule

[0326] To effectively remove myelin debris using TAM receptors, we prepared adeno-associated viruses (AAVs) expressing a fusion molecule based on the human Gas6 protein.

[0327] Specifically, the Gla domain and EGF repeat domain, which recognize phosphatidylserine (PS) in 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 in its place (anti-MOG(8-18C5)-Gas6). See Figures 3A to 3F.

[0328] Furthermore, as a control, anti-FITC-Gas6 was also prepared by incorporating anti-E2 scFv, which selectively recognizes FITC, a substance not naturally occurring in the body, instead of anti-MOG scFv.

[0329]

[0330] Example 4: In vitro effect of anti-MOG(8-18C5)-Gas6 fusion molecule on myelin debris removal

[0331] The HEK293T cell line was transfected with expression vectors to express anti-FITC-Gas6 and anti-MOG(8-18C5)-Gas6. Three days after transfection, serum-free medium was introduced, and one day later, the supernatant was collected and concentrated. Western blotting was performed to confirm protein size and expression. The effect of the proteins on myelin debris clearance was confirmed using HMC3 (a human microglial cell line). To visualize myelin debris clearance in vitro, myelin was extracted from mouse brain and conjugated with pH-sensitive pHrodo to prepare myelin-pHrodo. After treating HMC3 cells with the concentrated supernatant and myelin-pHrodo, live cell imaging was performed using an IncuCyte.

[0332] To confirm the effect of anti-MOG(8-18C5)-Gas6 on myelin debris removal in vitro, we expressed the protein in HEK293T cells according to the protocol shown in Figure 4A. Specifically, to express the protein, HEK293T cells were transfected with the expression vector, and the supernatant was concentrated. The protein was confirmed to be expressed at the predicted size of 75 kDa to 76 kDa (Figure 4B). To confirm the effect of myelin debris removal in vitro, we used Myelin-pHrodo in the HMC3 cell line. The results confirmed that myelin debris was removed more rapidly in the presence of anti-MOG(8-18C5)-Gas6 compared to vehicle or anti-FITC-Gas6 in the HMC3 cell line (Figure 4C). This demonstrates that anti-MOG(8-18C5)-Gas6 is properly expressed in a functional form and plays an important role in effectively removing myelin debris.

[0333]

[0334] Example 5: In vivo efficacy of anti-MOG(8-18C5)-Gas6 fusion molecule in reducing EAE severity

[0335] Experimental protocol: AAV PHP.eB-CMV-Anti-FITC-Gas6-HA and AAV PHP.eB-CMV-Anti-MOG(8-18C5)-Gas6-HA were injected into wild-type C57BL / 6J 6-week-old female mice at 1 x 10 11 The mice were administered MOG35-55 in complete Freund's adjuvant (CFA) and pertussis toxin (PTX) via retro-orbital injection. After EAE induction, the mice were monitored for EAE scores and body weight changes for 25 days.

[0336] To induce MS-like disease (EAE) in mice by overexpressing anti-MOG(8-18C5)-Gas6, mice were manipulated according to the protocol shown in Figure 5A. AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into wild-type C57BL / 6J 6-week-old female mice at 1 x 10 11 The mice were administered anti-MOG(8-18C5)-Gas6 AAVs via retroorbital injection. To induce EAE in 9-week-old mice, MOG35-55 in CFA was injected subcutaneously and pertussis toxin (PTX) was administered intraperitoneally for two consecutive days. EAE scores and body weight changes were then monitored for 25 days. Mice administered anti-MOG(8-18C5)-Gas6 AAVs had lower EAE scores and less body weight change than mice administered the control group (vehicle) or anti-FITC-Gas6 AAVs (Figure 5B and Figure 5C). This demonstrates that anti-MOG(8-18C5)-Gas6 is properly and functionally expressed in mice by AAVs and plays an important role in reducing the severity of EAE.

[0337]

[0338] Example 6: Safety of fusion molecules in animals (systemic expression of fusion proteins)

[0339] Experimental protocol: AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into wild-type C57BL / 6J 6-week-old female mice at 1 x 10 11 vg by retro-orbital injection. After 3 weeks, brains were sampled for immunohistochemistry. See Figure 6A.

[0340] To confirm the effect of systemically expressed anti-MOG(8-18C5)-Gas6 on normal myelin, wild-type mice were used and operated according to the protocol shown in Figure 8A. More specifically, AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into wild-type C57BL / 6J 6-week-old female mice at 1 x 10 11 The mice were administered anti-MOG(8-18C5)-Gas6 via retro-orbital injection. Three weeks later, brain samples were collected and immunohistochemistry was performed to examine myelin levels (MBP), lysosomal content (cathepsin D), and glial activation (GFAP, IBA1). Systemic administration of anti-MOG(8-18C5)-Gas6 to wild-type mice showed no significant changes in myelin levels, lysosomal content, or glial activation compared to anti-FITC-Gas6 (Figures 6B to 6E). This indicates that systemic expression of anti-MOG(8-18C5)-Gas6 has no adverse effects on myelination and glial activation.

[0341]

[0342] Example 7: Safety of fusion molecules in animals (local expression of fusion proteins)

[0343] Experimental protocol: AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into the corpus callosum of wild-type C57BL / 6J 6-week-old female mice at 1 x 10 12 Three weeks later, brains were sampled for immunohistochemistry.

[0344] To confirm the effect of locally expressed anti-MOG(8-18C5)-Gas6 on normal myelin, wild-type mice were used and operated according to the protocol shown in Figure 9A. More specifically, AAV PHP.eB-CMV-anti-FITC-Gas6-HA and AAV PHP.eB-CMV-anti-MOG(8-18C5)-Gas6-HA were injected into the corpus callosum of 6-week-old wild-type C57BL / 6J female mice at 1 x 10 ng / mL.12 The mice were stereotactically injected at a concentration of 200 nL (vg / mL). Three weeks later, brain samples were taken and immunohistochemistry was performed to assess the degree of viral expression (HA), myelin levels (MBP), and glial activation (GFAP, IBA1). Local administration of anti-MOG(8-18C5)-Gas6 in wild-type mice demonstrated expression at the injection site but not in the contralateral side (Figure 7B). Furthermore, compared with anti-FITC-Gas6, no significant changes in myelin levels or glial activation were observed (Figure 7C-7E). This indicates that local administration of anti-MOG(8-18C5)-Gas6 has no adverse effects on myelination or glial activation.

[0345]

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

[0347] To effectively remove myelin fragments using TAM receptors, we prepared a fusion molecule based on the human Gas6 protein. Specifically, the Gla domain and EGF repeat domain, which recognize PS (phosphatidylserine) in 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 in its place (anti-MOG(01)-Gas6). See Figure 8.

[0348] ELISA was performed to evaluate the antigen binding activity of the anti-MOG(01)-Gas6 antibody prepared above. Human MOG protein (R&D Systems) or mouse MOG protein (R&D Systems) diluted in DPBS at 0.5 μg / mL was added to a 96-well plate at 100 μL / well. The plate was incubated overnight at 4°C for coating and then washed four times with 0.05% Tween-20 / PBS (PBST). Next, 3% BSA / PBST was added at 200 μL / well for blocking and washed four times with PBST. The diluted anti-MOG(01)-Gas6 antibody was added at 100 μL / well and incubated for 2 hours at room temperature. The plate was washed with PBST and 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 AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated at room temperature for 1 hour. After washing, 100 μL of TMB solution was added per well and allowed to develop for 10 minutes. The reaction was stopped with stop solution, and the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer. As shown in Figure 9A and Figure 9B, anti-MOG(01)-Gas6 exhibited binding activity to MOG protein.

[0349] To confirm the binding activity of the anti-MOG(01)-Gas6 fusion molecule to MOG expressed on the cell surface, HEK293 cells overexpressing mouse MOG (hereafter referred to as HEK293-MOG cell line) were treated with the anti-MOG(01)-Gas6 fusion molecule and then detected by flow cytometry. Briefly, HEK293-MOG cell line resuspended in FACS solution (DPBS + 3% FBS + 10 mM EDTA + 1X Pen / Strep + 20 mM HEPES) was incubated with the anti-MOG-Gas6 fusion molecule for 1 hour at 4°C. To remove any remaining anti-MOG(01)-Gas6 fusion molecules in the supernatant that had not bound to cell surface MOG, each well was washed twice with FACS solution. The resulting mixture was centrifuged at 2,000 rpm for 3 minutes and the supernatant was removed. To detect cell surface MOG-bound anti-MOG(01)-Gas6 fusion molecules, a G4S linker (E7O2V) rabbit mAb (Cell Signaling Technology) was diluted 1:50 in FACS solution, added in 100 μL portions to each well, and incubated at 4°C for 30 minutes. After two washes, the mean fluorescence intensity (MFI) was analyzed by flow cytometry. The results are shown in Figure 9C. As shown in Figure 9C, anti-MOG(01)-Gas6 was confirmed to bind to mouse MOG protein expressed on the cell surface.

[0350]

[0351] Example 9: Effect of fusion protein (anti-MOG(01)-Gas6) fusion molecule on myelin debris removal

[0352] Anti-MOG(01)-Gas6 was used as the purified protein. To confirm the effect of the protein on the clearance of myelin debris, THP-1 AxlTo visualize myelin debris clearance in vitro, we used a human monocytic cell line (Axl-overexpressing cell line) and extracted myelin from mouse brain and combined it with pH-sensitive pHrodo to prepare myelin-pHrodo. THP-1 with protein (5 μg / mL) and myelin-pHrodo Axl After processing, live cell imaging was performed using IncuCyte (see Figure 10).

[0353] The effect of anti-MOG(01)-Gas6 on myelin debris removal was confirmed in vitro. More specifically, anti-MOG(01)-Gas6 was purified and myelin-pHrodo was administered to THP-1 cells. Axl The effect of anti-MOG(01)-Gas6 on myelin debris removal in vitro was confirmed using a cell line. The results showed that anti-MOG(01)-Gas6 significantly reduced myelin debris removal in THP-1 cells compared to vehicle. Axl It was confirmed that the compound effectively removed myelin in the cell line (FIGS. 9A to 9C).

[0354]

[0355] Example 10: Binding of fusion protein (anti-MBP-Gas6) fusion molecule to target substance

[0356] To effectively remove myelin fragments using TAM receptors, we prepared a fusion molecule based on the human Gas6 protein. Specifically, the Gla domain and EGF repeat domain, which recognize PS (phosphatidylserine) in apoptotic cells, were removed from Gas6, and a single-chain Fv fragment of an antibody against MBP protein, which is highly expressed in myelin, was placed in its place (anti-MBP-Gas6). See Figure 11.

[0357] ELISA was performed to evaluate the antigen binding activity of the anti-MBP-Gas6 fusion molecules prepared above. Human MBP protein (Enzo Life Sciences) and mouse MBP protein (Creative BioMart) diluted to a concentration of 1 μg / mL in DPBS were added to a 96-well plate at 100 μL / well and incubated overnight at 4°C. The plate was then washed four times with 0.05% Tween-20 / PBS (PBST). 3% BSA / PBST was then added at 200 μL / well for blocking, followed by four washes with PBST. The diluted anti-MBP-Gas6 fusion molecules were added at 100 μL / well and incubated for 2 hours at room temperature. The plate was then washed with PBST and 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 AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated at room temperature for 1 hour. After washing, 100 μL of TMB solution was added per well and allowed to develop for 10 minutes. The reaction was stopped with stop solution, and the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer.

[0358] The results obtained are shown in Figures 12A and 12B. The tested anti-MBP-Gas6 fusion molecules showed binding activity to human and mouse MBP proteins.

[0359]

[0360] Example 11: Effect of fusion protein (anti-MBP-Gas6) on myelin debris removal

[0361] Anti-MBP-Gas6 was used as the purified protein. To confirm the effect of the protein on the clearance of myelin debris, THP-1 AxlWe used a human monocytic cell line (Axl overexpression cell line) to visualize myelin debris clearance in vitro. Myelin was extracted from mouse brain and combined with pH-sensitive pHrodo to prepare myelin-pHrodo. The protein (5 μg / mL) and myelin-pHrodo were used to visualize myelin debris clearance in vitro. Axl After treatment, live cell imaging was performed using IncuCyte, and MFI values ​​were compared and evaluated 20 hours later.

[0362] The effect of anti-MBP-Gas6 on myelin debris removal was confirmed in vitro. More specifically, anti-MBP-Gas6 was purified and myelin-pHrodo was administered to THP-1 cells. Axl The effects of the anti-MBP-Gas6 fusion molecule on myelin debris removal in vitro were confirmed in monocytic cell lines, and the results showed that the tested anti-MBP-Gas6 fusion molecule effectively removed myelin debris in monocytic cell lines compared with vehicle (Figure 13).

[0363]

[0364] Example 12: Construction of anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6 fusion molecules

[0365] To effectively suppress or eliminate TNFα using TAM receptors, we prepared fusion molecules based on the human Gas6 protein. Specifically, the Gla domain and EGF repeat domain, which recognize PS (phosphatidylserine) in apoptotic cells, were removed from Gas6, and the GlcNAc domain was replaced with the single-chain Fv fragments of antibodies against TNFα, adalimumab or infliximab (anti-TNFα-Gas6). See Figures 14A to 14C.

[0366] ELISA was performed to evaluate the antigen-binding activity of the two anti-TNFα-Gas6 fusion molecules prepared above. Human TNFα protein (R&D Systems) diluted in DPBS at a concentration of 0.5 μg / mL was added to a 96-well plate at 100 μL / well. The plate was incubated overnight at 4°C for coating and then washed four times with 0.05% Tween-20 / PBS (PBST). 3% BSA / PBST was then added at 200 μL / well for blocking and washed four times with PBST. The diluted anti-TNFα-Gas6 fusion molecule was added at 100 μL / well and incubated for 2 hours at room temperature. The plate was washed with PBST, treated with anti-human Gas6 antibody (R&D Systems), and incubated for 1 hour at room temperature. After washing four times with PBST, 100 μL of Peroxidase AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated at room temperature for 1 hour. After washing, 100 μL of TMB solution was added per well and allowed to develop for 10 minutes. The reaction was stopped with stop solution, and the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer. The results are shown in Figure 15A. The two anti-TNFα-Gas6 fusion molecules tested exhibited binding activity to human TNFα protein.

[0367]

[0368] Example 13: Binding activity of anti-TNFα (adalimumab)-Gas6 fusion molecules and anti-TNFα (infliximab)-Gas6 fusion molecules to the target substance TNFα

[0369] To confirm the binding activity of the two anti-TNFα-Gas6 fusion molecules prepared in Example 12 above to TNFα expressed on the cell surface, a CHO-K1 cell line overexpressing human plasma membrane TNFα (hereafter referred to as the CHO-mTNFα cell line; Promega) was treated with the anti-TNFα-Gas6 fusion molecule, and then the anti-TNFα-Gas6 fusion molecule bound to the cell surface TNFα was detected using flow cytometry. Briefly, the CHO-mTNFα cell line resuspended in FACS solution (DPBS + 3% FBS + 10 mM EDTA + 1X Pen / Strep + 20 mM HEPES) was incubated with the anti-TNFα-Gas6 fusion molecule at 4°C for 1 hour. To remove any remaining anti-TNFα-Gas6 fusion molecules in the supernatant that had not bound to cell surface TNFα, each well was washed twice with FACS solution. The resulting mixture was centrifuged at 2,000 rpm for 3 minutes and the supernatant was removed. To detect the anti-TNFα-Gas6 fusion molecules bound to cell surface TNFα, a G4S linker (E7O2V) rabbit mAb (Cell Signaling Technology) was diluted 1:50 in FACS solution, added in 100 μL portions to each well, and incubated at 4°C for 30 minutes. After two washes, the mean fluorescence intensity (MFI) was analyzed by flow cytometry.

[0370] The results are shown in Figure 15B. It was confirmed that the two anti-TNFα-Gas6 fusion molecules tested were able to bind to human membrane TNFα expressed on the cell surface.

[0371]

[0372] Example 14: Activity of anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6 fusion molecules to activate Axl

[0373] To confirm the ability of the anti-TNFα-Gas6 fusion molecule candidate substances prepared in Example 12 to inhibit TNFα signal activation, a TNFα activation inhibition test was performed using HEK-BLUE™ TNFα cells. Briefly, 20 μL of human TNFα protein and 20 μL of diluted anti-TNFα-Gas6 fusion molecule were added per well to a flat-bottom 96-well plate, and 5×10 HEK-BLUE™ TNFα cells were cultured. 4 The cells were dispensed into each well at a density of 160 μL / well. Cells were dispensed into each well according to the well density. After 24 hours of culture at 37°C in a 5% CO2 incubator, 20 μL of the supernatant was transferred to a new flat-bottom 96-well plate, and 180 μL of QUANTI-BLUE™ solution was added per well. After 2 hours, absorbance at 655 nm was measured using a spectrophotometer, and the inhibitory activity (%) was calculated based on the measurement value obtained with human TNFα protein alone. As shown in Figure 16, the two tested anti-TNFα-Gas6 fusion molecules were confirmed to be able to inhibit TNFα-mediated signal activation.

[0374] To confirm the ability of the anti-TNFα-Gas6 fusion molecule prepared in Example 12 to induce Axl activation, we used the human osteosarcoma cell line U2OS in which ProLink-tagged Axl and enzyme receptor (EA)-tagged SH2 domain were overexpressed. Axl We performed a TAM receptor dimerization assay using a cell line sensitive to Axl receptor activation by Gas6 by generating chemiluminescence (Eurofins DiscoverX), and treated mTNFα-expressing cells (Promega) with an anti-TNFα-Gas6 fusion molecule to determine whether antigen (TNFα)-specific Axl activation was induced.

[0375] As shown in FIG. 17, it was confirmed that Axl activation was induced only when mTNFα-expressing cells were treated with the anti-TNFα-Gas6 fusion molecule.

[0376]

[0377] Example 15: Activity of anti-TNFα (adalimumab)-Gas6 and anti-TNFα (infliximab)-Gas6 fusion molecules to induce Axl-mediated phagocytosis

[0378] To confirm the Axl-mediated phagocytic activity of the anti-TNFα-Gas6 fusion molecule candidate prepared in Example 12, THP-1 Axl Phagocytosis assay was performed using macrophages differentiated from THP-1 cells. Axl Cells were treated with 25 nM PMA (phorbol 12-myristate 13-acetate) for 72 hours, cultured in serum-free and PMA-free medium for 24 hours, and then incubated 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. Effector and target cells were mixed at a 1:2 ratio and incubated together for 2 hours. After two washes with FACS solution, the cell surface was stained with CD11b. Phagocytosis of CD11b+ macrophages toward CTV+ target cells was analyzed by flow cytometry.

[0379] As shown in Figure 18, it was confirmed that the anti-TNFα-Gas6 fusion molecule candidate induced phagocytosis against target cells mediated by the Axl receptor expressed on macrophages.

[0380]

[0381] Example 16: Construction of anti-CD20 (rituximab)-Gas6 fusion molecule

[0382] To effectively eliminate CD20-expressing immune cells using TAM receptors, we prepared a fusion molecule based on the human Gas6 protein. (See Figures 19A and 19B.) More specifically, the Gla domain and EGF repeat domain, which recognize PS (phosphatidylserine) on apoptotic cells, were removed from Gas6, and rituximab, a single-chain Fv fragment of an antibody against CD20, was inserted in its place (anti-CD20-Gas6).

[0383] ELISA was performed to evaluate the antigen-binding activity of the anti-CD20-Gas6 fusion molecule prepared above. Human CD20 protein (Sino Biological) diluted in DPBS at a concentration of 0.5 μg / mL was added to a 96-well plate at 100 μL / well and incubated overnight at 4°C for coating. The plate was then washed four times with 0.05% Tween-20 / PBS (PBST). 3% BSA / PBST was then added at 200 μL / well for blocking, followed by four washes with PBST. The diluted anti-CD20-Gas6 fusion molecule was added at 100 μL / well and incubated for 2 hours at room temperature. The plate was washed with PBST and 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 AffiniPure Bovine Anti-Goat IgG (H+L) antibody (Jackson ImmunoResearch) was added per well and incubated at room temperature for 1 hour. After washing, 100 μL of TMB solution was added per well and allowed to develop for 10 minutes. The reaction was stopped with stop solution, and the absorbance at 450 nm and 650 nm was analyzed using a spectrophotometer. The results are shown in Figure 20A. The anti-CD20-Gas6 fusion molecule exhibited binding activity to human CD20 protein.

[0384]

[0385] Example 17: Binding activity of anti-CD20 (rituximab)-Gas6 fusion molecule to target substance

[0386] To confirm the binding activity of the anti-CD20-Gas6 fusion molecule prepared in Example 16 above to 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 cell surface CD20 was detected using flow cytometry. Briefly, Raji cells resuspended in FACS solution (DPBS + 3% FBS + 10 mM EDTA + 1X Pen / Strep + 20 mM HEPES) were incubated with the anti-CD20-Gas6 fusion molecule at 4°C for 1 hour. To remove anti-CD20-Gas6 fusion molecules remaining in the supernatant without binding to cell surface CD20, each well was washed twice with FACS solution. The resulting mixture was centrifuged at 2,000 rpm for 3 minutes, and the supernatant was removed. To detect anti-CD20-Gas6 fusion molecules bound to CD20 on the cell surface, G4S linker (E7O2V) rabbit mAb (Cell Signaling Technology) was diluted 1:50 in FACS solution and added in 100 μL portions to each well. o The cells were incubated at C for 30 minutes. After washing twice, the mean fluorescence intensity (MFI) was analyzed by flow cytometry.

[0387] The results are shown in Figure 20B. It was confirmed that the tested anti-CD20-Gas6 fusion molecule was able to bind to human CD20 protein expressed on the cell surface.

[0388]

[0389] Example 18: Activity of anti-CD20 (rituximab)-Gas6 fusion molecules to activate Axl

[0390] To confirm the ability of the anti-CD20-Gas6 fusion molecule prepared in Example 16 to induce Axl activation, we used the human osteosarcoma cell line U2OS, in which ProLink-tagged Axl and Enzyme Acceptor (EA)-tagged SH2 domain were overexpressed. AxlA TAM receptor dimerization assay was performed using a Eurofins DiscoverX™ TAM receptor assay. In this assay, cell lines sensitively respond to Axl receptor activation by Gas6 by generating chemiluminescence. The CD20-expressing Raji cell line (ATCC) was treated with anti-CD20-Gas6 fusion molecules to determine whether antigen (CD20)-specific Axl activation was induced. As shown in Figure 21, Axl activation was confirmed only when CD20-expressing Raji cells were treated with anti-CD20-Gas6 fusion molecules.

[0391]

[0392] Example 19: Activity of anti-CD20 (rituximab)-Gas6 fusion molecules to induce Axl-mediated phagocytosis

[0393] To confirm the Axl-mediated phagocytosis of the anti-CD20-Gas6 fusion molecule candidate prepared in Example 16, THP-1 Axl Phagocytosis assay was performed using macrophages differentiated from THP-1 cells. Axl Cells were treated with 25 nM PMA (phorbol 12-myristate 13-acetate) for 72 hours, cultured in serum-free and PMA-free medium for 24 hours, and then incubated 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. Effector and target cells were mixed at a 1:2 ratio and incubated together for 2 hours. After two washes with FACS solution, the cell surface was stained with CD11b. Phagocytosis of CD11b+ macrophages toward CTV+ target cells was analyzed by flow cytometry.

[0394] As shown in Figure 22, the anti-CD20-Gas6 fusion molecule candidate was confirmed to induce phagocytosis of target cells mediated by the Axl receptor expressed on macrophages.

[0395]

[0396] Example 20: Adalimumab [scFv]-ProS1 fusion molecule

[0397] To prepare a MOG-specific fusion protein based on the ProS1 protein, the Gla domain and EGF repeat domain were first removed, and a single-chain variable fragment (scFv) of the TNFα-specific antibody adalimumab was introduced in its place (αTNFα-ProS1). Figure 24 shows the amino acid and nucleotide sequences of the chimeric phagocytic derivative.

[0398] The binding activity of the resulting adalimumab [scFv]-ProS1 fusion molecule to a target substance, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0399]

[0400] Example 21: Adalimumab [Fab]-Gas6 (anti-TNFα antibody heavy chain VH-CH1(Fab)-Gas6-His)

[0401] To prepare Gas6-based TNFα-specific fusion proteins, the Gla domain and EGF repeat domain were first removed, and then the antigen-binding fragment (Fab) or monoclonal antibody (Mab) of the TNFα-specific antibody adalimumab was introduced in its place (αTNFα[Fab]-Gas6, αTNFα[Mab]-Gas6). The Fc region of the MAb heavy chain contains an NA mutation that reduces or eliminates Fcγ receptor binding affinity. Figure 25 shows the amino acid sequences of two chimeric phagocytic derivatives.

[0402] The binding activity of the resulting adalimumab [Fab]-Gas6 (anti-TNFα antibody heavy chain VH-CH1(Fab)-Gas6-His) fusion molecule to a target substance, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, and Examples 17 to 19.

[0403]

[0404] Example 22: Adalimumab [Mab]-Gas6 (anti-TNFα antibody heavy chain (Mab)-Gas6-

[0405] To prepare Gas6-based TNFα-specific fusion proteins, the Gla domain and EGF repeat domain were first removed, and then an antigen-binding fragment (Fab) or monoclonal antibody (Mab) of the TNFα-specific antibody adalimumab was introduced in their place (αTNFα[Fab]-Gas6 and αTNFα[Mab]-Gas6). Figure 26 shows the amino acid sequences of the two chimeric phagocytic derivatives.

[0406] The binding activity of the resulting adalimumab [Mab]-Gas6 (anti-TNFα antibody heavy chain (Mab)-Gas6-His) fusion molecule to a target substance, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0407]

[0408] Example 23: Fusion molecules containing adalimumab [Mab or Fab]

[0409] The sequence of SEQ ID NO: 253 (Figure 27), the sequence of SEQ ID NO: 251 (Figure 25), the sequence of SEQ ID NO: 252 (Figure 26), the sequence of SEQ ID NO: 258 (Figure 32), or the sequence of SEQ ID NO: 260 (Figure 34) / SEQ ID NO: 261 (Figure 34) is employed to prepare an adalimumab [Fab]-Gas6 fusion protein, an adalimumab [Mab]-Gas6 fusion protein, an adalimumab [Mab]-anti-Axl (homodimer) fusion protein, or an adalimumab [Mab]-anti-Axl (heterodimer) fusion protein.

[0410] The binding activity of the resulting fusion molecules containing adalimumab [Mab] or adalimumab [Fab] to a target substance, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0411]

[0412] Example 24: Adalimumab [scFv]-MFc-Gas6

[0413] As a non-limiting example of a binding molecule comprising a scaffold protein between the first and second domains, Gas6 and an antibody scFv (in this example, adalimumab scFv) are employed as the first and second domains, respectively, to produce a single-chain Fc domain with reduced or eliminated Fc receptor binding affinity. The sequences employed are shown in Figure 28.

[0414] The binding activity of the resulting adalimumab[scFv]-MFc-Gas6 fusion molecule to a target substance, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0415]

[0416] Example 25: Adalimumab [scFv]-Fc(DD)-Gas6 (heterodimer)

[0417] In another non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first and second regions, Gas6 and an antibody scFv (in this example, adalimumab scFv) are employed as the first and second regions, respectively, to produce a heterodimeric binding molecule. The first polypeptide of the heterodimeric binding molecule comprises adalimumab scFv, an Fc region (DD), and Gas6, and the second polypeptide of the heterodimeric binding molecule comprises an adalimumab scFv region and an Fc region (KK). The peptide sequence is shown in Figure 29.

[0418] The binding activity of the resulting adalimumab[scFv]-Fc(DD)-Gas6 heterodimeric fusion molecules to target substances, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0419]

[0420] Example 26: Adalimumab [scFv]-Fc-Gas6 (homodimer)

[0421] As yet another non-limiting illustrative embodiment of a binding molecule comprising a scaffold protein between the first and second domains, a homodimer is produced comprising two polypeptides, each having an adalimumab scFv (second domain), an Fc domain (scaffold), and Gas6. The peptide sequences are shown in Figure 30.

[0422] The binding activity of the resulting adalimumab[scFv]-Fc-Gas6 homodimer fusion molecules to target substances, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0423]

[0424] Example 27: Adalimumab [scFv]-Fc-Gas6 (homodimer)

[0425] As yet another non-limiting illustrative embodiment of a binding molecule comprising a scaffold protein between the first and second domains, a homodimer is produced comprising two polypeptides, each having an adalimumab scFv (second domain), an Fc domain (scaffold), and Gas6. The peptide sequences are shown in Figure 31.

[0426] The binding activity of the resulting adalimumab[scFv]-Fc-Gas6 homodimer fusion molecules to target substances, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0427]

[0428] Example 28: Adalimumab [Mab]-anti-Axl (homodimer)

[0429] As a non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first and second regions, a bispecific antibody is produced in which an scFv of an anti-Axl antibody and adalimumab are employed as the first and second regions, respectively. See Figure 32. The heavy chain of the bispecific antibody has the sequence of SEQ ID NO: 258 (Figure 32), and the light chain of adalimumab has the sequence of SEQ ID NO: 253. The Fc region of the heavy chain contains an NA mutation that reduces or eliminates Fcγ receptor binding affinity. See Figure 32.

[0430] The binding activity of the resulting adalimumab [Mab]-anti-Axl homodimer fusion molecules to target substances, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0431]

[0432] Example 29: Anti-Axl-Fc-adalimumab [scFv] (homodimer)

[0433] As a non-limiting example of a binding molecule comprising a scaffold protein between the first and second regions, a homodimeric bispecific antibody is produced in which the scFv region of an anti-Axl antibody and the scFv region of adalimumab are employed as the first and second regions, respectively. See Figure 33. The bispecific antibody comprises a first polypeptide and a second polypeptide, which are identical to each other and each comprise the sequence of SEQ ID NO: 259. The structure of the first / second polypeptide is shown in Figure 33, and the Fc region scaffold comprises an NA mutation that reduces or eliminates Fcγ receptor binding affinity.

[0434] The binding activity of the resulting anti-Axl-Fc-adalimumab [scFv] homodimeric fusion molecules to target substances, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0435]

[0436] Example 30: Adalimumab [Mab] (DD)-anti-Axl (heterodimer)

[0437] As another non-limiting exemplary embodiment of a binding molecule comprising a scaffold protein between the first and second regions, a heterodimeric bispecific antibody is produced in which an scFv of an anti-Axl antibody and adalimumab are employed as the first and second regions, respectively. The first polypeptide of the heavy chain of the bispecific antibody has the sequence of SEQ ID NO: 260 in Figure 34, the second polypeptide of the heavy chain of the bispecific antibody has the sequence of SEQ ID NO: 261 in Figure 34, and the light chain of the anti-amyloid antibody has the sequence of SEQ ID NO: 253 in Figure 27. The Fc region contains an NA mutation that reduces or eliminates Fcγ receptor binding affinity, and the polypeptides of the Fc region form a heterodimer (DD-KK).

[0438] The binding activity of the resulting adalimumab [Mab](DD)-anti-Axl heterodimer fusion molecules to target substances, induction of TAM activation, and phagocytosis are evaluated according to the procedures described in one or more of Examples 8 to 11, Examples 13 to 15, or Examples 17 to 19.

[0439]

[0440] The scope of the present disclosure is defined by the appended claims, and all changes and modifications that come within the meaning and range of the claims and their equivalents should be construed as being within the scope of the invention.

[0441] 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 activation of inflammatory responses, which occurs in conventional technologies. Therefore, the fusion molecules can effectively clear or reduce the amount of antigenic substances, and thus can be used to prevent or treat immune diseases.

[0442]

[0443] Incorporation by Reference

[0444] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety.

Claims

1. a first region capable of binding to a TAM receptor; a second region capable of specifically binding to a target substance; Including, the target substance is a substance that induces or causes an immune disease by increasing its amount or expression in biological tissue, the first region and the second region are bound to each other directly or via a linker; The first region is (a) a TAM receptor ligand; (b) an anti-Axl antibody or an antigen-binding fragment thereof; (c) an anti-Tyro3 antibody or an antigen-binding fragment thereof; (d) an anti-MerTK antibody or an antigen-binding fragment thereof; or (e) including combinations thereof, A binding molecule that does not contain the target substance or a fragment thereof.

2. The binding molecule of claim 1 , further comprising a scaffold attached at a different position to the first region, the second region, or both the first region and the second region.

3. 2. The binding molecule of claim 1, wherein the TAM receptor ligand is one selected from the group consisting of Gas6, ProS1, Tubby, Tulp1, Gal3, and combinations thereof, or an Axl-binding fragment thereof.

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

5. the first region is (a) the TAM receptor ligand, and the TAM receptor ligand is 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, 2. The binding molecule of claim 1, comprising one or more sequences selected from the group consisting of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, and SEQ ID NO: 87, or a sequence having at least 85% sequence identity thereto.

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

7. The binding molecule of claim 1, wherein the first region is (a) the TAM receptor ligand, and the TAM receptor ligand comprises the sequence of SEQ ID NO: 5 or a sequence having at least 85% sequence identity thereto.

8. the first region is (a) the TAM receptor ligand, and the TAM receptor ligand is 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:8 8, 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% sequence identity thereto.

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

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

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

12. The binding molecule of claim 1 , wherein the target substance is an autoantigen, an autoantibody, a complex of an autoantigen and an autoantibody, a cytokine, a chemokine, complement, a receptor, an immune cell-specific marker, a cell adhesion molecule, or a combination thereof.

13. The target substance is selected from the group consisting of factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, thrombin, vWF, calcium-sensing receptor, ACTH, 21-hydroxylase (CYP21), trichohyalin, oxidized low-density lipoprotein (OxLDL), transcriptional coactivator p75, p-80-coilin, C1 inhibitor, AMPA receptor, CRMP5, DPPX / DPP6, GABAA receptor, glycine receptor (GlyR), Hu (ANNA-1), Ma1, Ma2, Ri (ANNA-2), Zic4, voltage-gated potassium channel (VGKC) complex, NMDA receptor, Jo1, H / K ATPase, thyroid peroxidase, erythrocyte I / I, F-actin asialoglycoprotein receptor, and cytochrome P450. 2D6 (CYP2D6), NXP-2 / MORC3, TIF1-γ / TRIM-33, β2 integrin, nuclear autoantigenic sperm protein (NASP), lactoferrin 17-α-hydroxylase (CYP17), cholesterol side-chain cleavage enzyme (CYP11A), tryptophan hydroxylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, glycoprotein IIb / IIIa and Ib / IX, thyroglobulin, 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 related gene 5 (MDA5), SUMO-activating enzyme subunit (SAE)-1 (SAE-1), SAE-2, DNA-dependent nucleosome-stimulated ATPase, chromodomain helicase DNA-binding protein 4 (CHD4), β-adrenergic receptor, adenine nucleotide translocator (adenine nucleotide transporter), collagen type VII, IgG, G-CSF, collagen type IV α3 chain, thyroid-stimulating hormone 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 54 kDa subunit (SRP54), IgA, synaptotagmin,Voltage-gated calcium channel, βIV spectrin, U1 small nuclear ribonucleoprotein 70 kDa (SNRNP70), CNPase, myelin-associated oligodendrocyte basic protein (MOBP), myelin proteolipid protein (PLP), S100 calcium-binding protein B, transaldolase, myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), acetylcholine receptor, low-density lipoprotein receptor-related protein 4 (LRP4), muscle-specific receptor tyrosine kinase (MuSK), aminoacyl-tRNA synthetase, tribbles pseudokinase 2 (TRIB2), myeloperoxidase (MPO), aquaporin 4 (APQ-4), amphiphysin, exosome component 9 (EXOSC9), EXOSC10 / PMSCL, Yo protein, Hu protein, Ri protein, desmoplakin, gephyrin, desmoglein 1, desmoglein 3, intrinsic factor type 1, β2-glycoprotein I (β2-GPI), Pill Pyrate 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 (multiple types, especially II, IV, and IX), fibrinogen βα, leukemia inhibitory factor (LIF), glutamate receptor (GLUR), myosin, B23, nucleophosmin (NPM), fibrillarin, topoisomerase I (Scl-70) , interferon-γ-inducible protein 16 (IFI16), La phosphoprotein, Ro60, Ro52 (TRIM21), golgin (95, 97, 160, 180), anionic phospholipid / protein complex, cardiolipin, components of Sm splicing ribonucleoprotein (subunits A-G), self-double-stranded DNA (dsDNA), histone H2A-H2B-DNA, proliferating cell nuclear antigen (PCNA), ribosomal P, 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 I-III (RNP), thyroid and eye muscle shared protein,Leukocyte function-associated antigen (LFA-1), chromogranin A, IA-2 (ICA512), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), ZnT8, insulin, glutamic acid decarboxylase (GAD65), insulin receptor, heat shock protein (65-kDa heat shock protein), SOX-10, tyrosinase, KUMEL1 / ARMC9, proteinase 3 / myeloblastin, CD20, CD19, complement C3, complement C5, C5α receptor 1, CD52, FcRn large subunit p51, IL-1, IL-1R, IL-6, IL-6R, IL-17, IL-17R, TNF-α, TNFR, IL-4, IL-4R, IL-5, IL-5R, I 2. The binding molecule of claim 1, wherein the binding molecule is one or more selected from the group consisting of 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, ICAM1, VCAM1, MADCAM1, integrin α4, integrin β7, VLA-4, Toll-like receptor (TLR)-3, TLR-4, TLR-5, TLR-7, and combinations thereof. ,

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

15. 3. The binding molecule of 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 with no variable region, or an Fc-hinge region with reduced or eliminated Fc receptor binding affinity.

16. The immune disease is selected from the group consisting of 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 syndrome, chronic relapsing multifocal osteomyelitis, gout, Henoch-Schonlein purpura, juvenile dermatomyositis, juvenile idiopathic arthritis, juvenile lupus (SLE), juvenile scleroderma, juvenile vasculitis, Kawasaki disease, and lupus (systemic erythematosus).

10. The binding molecule of claim 1, wherein the inflammatory or autoimmune disease is selected from the group consisting of psoriatic arthritis, mixed connective tissue disease, myositis, post-streptococcal inflammatory syndrome, psoriatic arthritis, reactive arthritis, scleroderma, Sjogren's syndrome, spondyloarthritis / spondyloarthropathy, systemic onset juvenile idiopathic arthritis, undifferentiated connective tissue disease, uveitis, vasculitis, celiac disease, thrombotic thrombocytopenic purpura (iTTP), and combinations thereof.

17. A nucleic acid molecule encoding the binding molecule of claim 1.

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

19. A cell expressing the binding molecule of claim 1.

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

21. (i) reducing to normal levels or enhancing the reduction of elevated levels of said target substance that causes or induces an immune disease in a subject; (ii) removing, clearing, or enhancing clearance of a target substance that causes or induces an immune disease in a subject due to increased expression or amount; (iii) suppressing an increase in the expression or amount of a target substance in a subject (iv) treating or preventing an immune disorder in a subject; (v) delaying the onset of symptoms associated with immune disorders, and / or (vi) alleviating symptoms of an immune disease in a subject, the pharmaceutical composition of claim 20.

22. 22. The use of claim 21, wherein the elevated level of the target substance is in the brain of the subject.

23. Use of the binding molecule of claim 1; a polynucleotide encoding said binding molecule; a vector; or a pharmaceutical composition comprising said binding molecule, said polynucleotide, or said vector, in one or more methods selected from the group consisting of: reducing or enhancing the reduction of elevated levels of said target substance that causes or induces an immune disease in a subject to normal levels; Removing or clearing or enhancing clearance of a target substance that causes or induces an immune disease in a subject due to increased expression or amount; and Suppressing the increase in the expression or amount of a target substance in a subject.

24. 24. The use of claim 23, wherein the elevated level of the substance is in the brain of the subject.

25. Use of the binding molecule of claim 1; a polynucleotide encoding said binding molecule; a vector; or a pharmaceutical composition comprising said binding molecule, said polynucleotide, or said vector, in one or more methods selected from the group consisting of: Treating or preventing an immune disease in a subject; delaying the onset of symptoms associated with an immune disease; and Alleviating symptoms of an immune disorder in a subject.

26. 10. Use of a binding molecule of claim 1; a polynucleotide encoding said binding molecule; a vector; or a pharmaceutical composition comprising said binding molecule, said polynucleotide, or said vector; in the manufacture of a medicament for treating or preventing an immune disease in a subject, delaying the development of symptoms associated with an immune disease, or alleviating symptoms of an immune disease in a subject.

Images