Method for producing protein capable of binding to TAM receptor

Abstract

The present invention relates to: a medium composition for producing a protein capable of binding to a TAM receptor, comprising an antioxidant, a copper compound, calcium chloride (CaCl2), and / or EDTA; a composition for inhibiting cleavage of a protein capable of binding to a TAM receptor; and a method for producing a protein capable of binding to a TAM receptor. A phenolic antioxidant, a copper compound, calcium chloride, or EDTA is suitable for high-efficiency production of the protein by inhibiting cleavage of the protein capable of binding to a TAM receptor, and thus can be usefully employed for protein production.

Description

Method for producing a protein capable of binding to TAM receptors

[0001] The present invention relates to a medium composition for producing a protein capable of binding to a TAM receptor comprising an antioxidant or a copper compound, a composition for inhibiting the cleavage of a protein capable of binding to a TAM receptor, and a method for producing a protein capable of binding to a TAM receptor.

[0002]

[0003] TAM receptors such as Axl, Mertk, and Tyro3 are known to play important roles in the immune, nervous, reproductive, and vascular systems (Nature Reviews Immunology volume 8, pages 327-336, 2008). Proteins closely related to the above TAM receptors function as TAM ligands, and TAM ligands known include GAS6 (Growth arrest specific factor 6), ProS1 (Protein S), SHBG (Sex hormone-binding globulin), Tubby, Tulp1 (Tubby-related protein 1), or Gal3 (Galectin-3). In particular, Axl, one of the TAM receptors, is expressed in various types of cells and is known to be involved in cell survival and proliferation by regulating various signaling pathways such as phosphatidylinositol 3-kinase-Akt, extracellular signal-regulated kinase (ERK) 1 / 2, P38 MAP kinases, JNK, and nuclear factor (NF)-kB when activated by binding to its ligand, GAS6.

[0004] Accordingly, the inventors have previously developed a fusion molecule through prior research that recognizes a target substance through a region such as an antibody or antigen-binding fragment and eliminates the target substance by inducing phagocytosis through Axl activation via a TAM ligand region without accompanying an inflammatory response. However, it was confirmed that there are problems with the high-efficiency production or manufacturing of such TAM ligands, fragments thereof, or variants thereof; or fusion proteins containing them, as the protein is cleaved during their preparation.

[0005] Accordingly, the inventors have completed the present invention by discovering a culture medium composition for high-efficiency production or manufacturing of a TAM ligand, a fragment thereof, or a variant thereof, which is a protein capable of binding to a TAM receptor; or a fusion protein comprising these.

[0006]

[0007] The object of the present invention is to provide a medium composition for producing a protein capable of binding to a TAM receptor, comprising one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA.

[0008] Another objective of the present invention is to provide a composition for inhibiting the cleavage of a protein capable of binding to a TAM receptor, comprising one or more selected from the group consisting of antioxidants, copper compounds, calcium chloride (CaCl2), and EDTA.

[0009] Another objective of the present invention is to provide a method for producing a protein capable of binding to a TAM receptor, comprising the steps of: (a) transfecting a cell line with a recombinant expression vector comprising a polynucleotide encoding a protein capable of binding to a TAM receptor; (b) adding one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA to the cell line transfected in step (a) and culturing it; and (c) isolating the protein capable of binding to a TAM receptor from the cell culture medium of step (b).

[0010]

[0011] To achieve the above objective, the present invention provides a medium composition for producing a protein capable of binding to a TAM receptor, comprising one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA.

[0012] In addition, the present invention provides a composition for inhibiting the cleavage of a protein that can bind to a TAM receptor, comprising one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA.

[0013] In addition, the present invention provides a method for producing a protein capable of binding to a TAM receptor, comprising: (a) transfecting a cell line with a recombinant expression vector comprising a polynucleotide encoding a protein capable of binding to a TAM receptor; (b) adding one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA to the cell line transfected in step (a) and culturing it; and (c) isolating the protein capable of binding to a TAM receptor from the cell culture medium of step (b).

[0014]

[0015] The composition according to the present invention is suitable for high-efficiency production of proteins by inhibiting the cleavage of proteins that can bind to TAM receptors, and can be usefully used for protein production.

[0016]

[0017] Figure 1 shows the structure of a protein that can be bound to a TAM receptor according to the present invention.

[0018] Figure 2 shows the results of confirming the expression pattern of the fusion protein during the production of the GAS6 fusion protein using CHO cells (Lane 1: Purified scFv-GAS6; Lane 2: Standard Marker; Lane 3: scFv-Fc(mono)-GAS6 expressed by CHO cells; and Lane 4: scFv-GAS6 expressed by CHO cells).

[0019] Figure 3 shows the results of analyzing the degree of cleavage of the fusion protein after adding a protease inhibitor cocktail to the culture medium during the production of the GAS6 fusion protein (Lane 1: Marker; Lane 2: scFv-Fc(mono)-GAS6 expressed by CHO cell; Lane 3: scFv-Fc(mono)-GAS6 (Protease inhibitor cocktail: 1 / 200 fold); and Lane 4: scFv-Fc(mono)-GAS6 (Protease inhibitor cocktail: 1 / 1000 fold)).

[0020] Figure 4 shows the results of analyzing the degree of cleavage of the fusion protein after adding a protease inhibitor cocktail and each component included therein to the culture medium during the production of the GAS6 fusion protein.

[0021] Figures 5a to 5c show the results of analyzing the degree of cleavage of the fusion protein after adding apigenin (a), baicalein (b), and kaempferol (c) to the culture medium at concentrations of 25 μM, 50 μM, 75 μM, and 100 μM, respectively, during the production of the GAS6 fusion protein (Lanes 1–3: control group (DMSO only); Lanes 4–6: each antioxidant 25 μM; Lanes 7–9: each antioxidant 50 μM; Lanes 10–12: each antioxidant 50 μM; and Lanes 13–15: each antioxidant 50 μM), and Figure 5d shows the results of analyzing the protein cleaved ratio according to concentration based on the results of Figures 5a to 5c (n=3).

[0022] Figures 6a and 6b show the results of analyzing the degree of cleavage of the fusion protein after adding divalent ions to the culture medium during the production of the GAS6 fusion protein (Lane 1: Control (Day 5); Lane 2: Standard Marker; Lane 3: CuSO4; Lane 4: CuCl2; Lane 5: CuBr2; Lane 6: NiSO4; Lane 7: MnSO4; Lane 8: MgSO4; and Lane 9: MgCl2).

[0023] Figure 7a shows the results of analyzing the degree of cleavage of the fusion protein after adding copper sulfate (CuSO4) at 1, 5, and 10 μM during the production of the GAS6 fusion protein (Lanes 1–3: control; Lanes 4–6: 1 μM; Lanes 7–9: 5 μM; and Lanes 10–12: 10 μM), Figure 7b shows the results of analyzing the degree of cleavage of the fusion protein after adding calcium chloride (CaCl2) at 1, 4, 10, 25, and 50 mM (Lanes 1–3: control; Lanes 4–6: 1 mM; Lanes 7–9: 4 mM; Lanes 10–12: 10 mM; Lanes 13–15: 25 mM; and Lanes 16–18: 50 mM), and Figure 7c shows the results of analyzing the fusion protein after adding EDTA to the culture medium at 5, 15, 30, and 50 μM. Figure 7d shows the results of analyzing the degree of protein cleavage (Lane 1–3: control group; Lane 4–6: 5 μM; Lane 7–9: 15 μM; Lane 10–12: 30 μM; and Lane 13–15: 50 μM), and Figure 7d shows the results of analyzing the protein cleaved ratio according to concentration based on the results of Figures 7a to 7c (n=3).

[0024] Figure 8a shows the results of analyzing the degree of cleavage of the fusion protein after adding copper sulfate (CuSO4) alone or a mixture of copper sulfate (CuSO4) with calcium chloride (CaCl2) or EDTA to the culture medium during the production of the GAS6 fusion protein (Lane 1: Marker; Lane 2: Control (3 Days); Lane 3: Control (5 Days); Lane 4: CuSO4 25 μM; Lane 5: CuSO4 50 μM; Lane 6: CuSO4 25 μM + CuCl2 2 mM; Lane 7: CuSO4 25 μM + CuCl2 4 mM; and Lane 8: CuSO4 25 μM + EDTA 10 μM), and Figure 8b shows the results of analyzing the protein cleavage rate according to the results of Figure 8a.

[0025] Figure 9a shows the results of analyzing the degree of cleavage of the fusion protein after adding copper sulfate (CuSO4) to the culture medium at concentrations of 0.01, 0.1, 0.25, 0.5, 1, 5, 10, 50, 100, 200, 500, and 1000 μM during the production of the GAS6 fusion protein (Lane 1: control group; Lanes 2–13: each concentration), Figure 9b shows the results of analyzing the protein cleavage ratio according to the results of Figure 9a, and Figure 9c shows the results of comparing the total expression amount (relative value) of the intact form and the cleaved form of the GAS6 fusion protein from the results of Figure 9a.

[0026] Figure 10 shows the results of analyzing the degree of cleavage of TAM ligands or variants after adding copper sulfate (CuSO4) to the culture medium during the temporary expression of TAM ligands or TAM ligand variants without scFv fusion (Lanes 1–3: TAM ligand (LG12(Wild type GAS6)) test group treated with 50 μM copper sulfate; Lanes 4–6: TAM ligand (LG12(Wild type GAS6)) test group not treated with copper ions; Lane 7: Test group loaded with 0.5 μg of purified wild-type TAM ligand as a control; Lane 8: Test group loaded with 0.5 μg of TAM ligand variant (V2; variant); Lanes 9–11: TAM ligand variant (LG12(V2)) test group not treated with copper ions; Lanes 12–14: copper sulfate 50 μM treated TAM ligand variant (LG12(V2)) test group; and n=3).

[0027]

[0028] The present invention will be described in detail below.

[0029] The terms used in this invention have been selected based on currently widely used general terms whenever possible, taking into account the functions of the invention; however, these terms may vary depending on the intent of those skilled in the art or the emergence of new technologies. Additionally, in specific cases, terms may be selected arbitrarily, and in such cases, their meanings will be described in detail in the description section of the relevant embodiments. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0030] In the present invention, when a component or step is described as "comprising," this means that, unless specifically stated otherwise, it does not exclude other components or steps but may include additional components or steps.

[0031]

[0032] The present invention provides a medium composition for producing a protein capable of binding to a TAM receptor, comprising one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA.

[0033] In the present invention, the term "medium" refers to a cell culture medium for the production of proteins.

[0034] In the present invention, the antioxidant may be a phenolic antioxidant, preferably selected from the group consisting of baicalein, quercetin, kaempferol, luteolin, apigenin, chrysin, wogonin, and galangin, but is not limited thereto.

[0035] The above antioxidant may be present at a concentration of 1 μM to 1,000 μM, preferably at a concentration of 10 μM to 500 μM, 10 μM to 400 μM, 10 μM to 300 μM, 10 μM to 200 μM, or 10 μM to 100 μM, and may be present at a concentration of at least 1 μM, 2 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, or 50 μM, or at least 60 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, or 500 μM or less, but is not limited thereto.

[0036] For example, baicalein may be included at a concentration of 10 μM to 100 μM, preferably at a concentration of 25 μM to 100 μM, and more preferably at a concentration of 50 μM to 100 μM, but is not limited thereto.

[0037] Kaempferol may be included at a concentration of 10 μM to 100 μM, preferably at a concentration of 25 μM to 100 μM, but is not limited thereto.

[0038] Apigenin may be included at a concentration of 1 μM to 50 μM, preferably at a concentration of 1 μM to 25 μM, but is not limited thereto.

[0039] Quercetin, luteolin, chrysin, wogonin, and galangin may be included at a concentration of 1 μM to 100 μM, but are not limited thereto.

[0040] In the present invention, the copper compound may be selected from the group consisting of copper sulfate (CuSO4), copper phosphate (Cu3(PO4)2), copper oxide (Cu2O), copper II oxide (CuO), copper acetate (Cu(OAc)2), copper carbonate (CuO3), copper hydroxide (Cu(OH)2), copper nitrate (Cu(NO3)2), copper chloride (CuCl2), copper sulfide (CuS), and copper bromide (CuBr2), but is not limited thereto.

[0041] In the present invention, the copper compound may have a concentration of 0.01 μM to 1,000 μM, preferably 0.01 μM to 500 μM, and may be included at a concentration of 0.01 μM, 0.02 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM or more, and may be included at a concentration of 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 75 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM or It may be included at a concentration of 500 μM or less, preferably at a concentration of 1 μM to 200 μM, and more preferably at a concentration of 5 μM to 200 μM, but is not limited thereto.

[0042] In the present invention, the calcium chloride may be 0.1 mM to 100 mM, may be included at a concentration of 0.1 mM, 0.2 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, or 10 mM or higher, may be included at a concentration of 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM or lower, preferably at a concentration of 1 mM to 50 mM, and more preferably at a concentration of 1 mM to 20 mM, but is not limited thereto.

[0043] In the present invention, the EDTA may be at a concentration of 1 μM to 200 μM, may be included at a concentration of 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, or 30 μM or higher, and may be included at a concentration of 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, or 200 μM or lower, and preferably may be included at a concentration of 1 μM to 100 μM, a concentration of 5 μM to 50 μM, or a concentration of 10 μM to 50 μM, but is not limited thereto.

[0044] In the present invention, the copper compound may be treated by mixing it with calcium chloride or EDTA. In the present invention, it was confirmed that treating with a copper compound and calcium chloride, or treating with a combination of a copper compound and EDTA, has the effect of inhibiting the cleavage of a protein capable of binding to a TAM receptor (see Examples).

[0045] In the present invention, the TAM receptor may be selected from the group consisting of MerTK, Axl, and Tyro3.

[0046] In the present invention, the protein capable of binding to the TAM receptor may be (a) a TAM ligand, a fragment thereof, or a variant thereof; or (b) a fusion protein comprising the TAM ligand, a fragment thereof, or a variant thereof.

[0047] The term "TAM binder" in the present invention refers to a TAM ligand, a fragment thereof, or a variant thereof. That is, a protein capable of binding to the TAM receptor of the present invention may be a TAM binder or a fusion protein comprising a TAM binder as a partial region.

[0048] In the present invention, the TAM ligand may be selected from the group consisting of GAS6 (Growth arrest specific factor 6), ProS1 (Protein S), SHBG (Sex hormone-binding globulin), Tubby, Tulp1 (Tubby-related protein 1), and Gal3 (Galectin-3), but is not limited thereto.

[0049] The above "fragment" refers to a partial fragment of a TAM ligand having the function of a TAM ligand that binds to and activates a TAM receptor. Preferably, the fragment of the TAM ligand may include a laminin G-like domain or a part thereof, and may be a sequence having at least 85% homology with a wild-type TAM ligand, for which reference may be made to U.S. Patent Publication No. 2024 / 0018204.

[0050] The above "variant" refers to a protein or (poly)peptide having a sequence in which one or more amino acid residues differ from the original amino acid sequence of the protein or (poly)peptide, for example, the sequence of the wild-type protein or (poly)peptide, and any cleavage, deletion, insertion, substitution, etc., and combinations thereof are possible in the final structure as long as the activity of the protein or (poly)peptide is maintained. An example of a variant is a form in which amino acid residues in a site not essential for activity are cleaved or deleted, or an amino acid residue in a site important for function or physical properties is substituted. In addition, in some cases, modifications such as phosphorylation, glycosylation, methylation, or farnesylation may be made. It is desirable if, through such sequence variation and modification, the productivity, function and / or stability (thermal stability, pH stability, structural stability, etc.) and / or solubility of the protein are increased by the variation in the amino acid sequence.

[0051] In the present invention, the TAM ligand, a fragment thereof, or a variant thereof comprises the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology therewith, and the amino acid sequence of SEQ ID NO. 2 or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% It may include an amino acid sequence having the above homology. In addition, the TAM ligand, a fragment thereof, or a variant thereof comprises the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology therewith, and the amino acid sequence of SEQ ID NO. 4 or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology therewith. It may include an amino acid sequence having homology. Additionally, the TAM ligand, a fragment thereof, or a variant thereof may include the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6, or may include an amino acid sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology with SEQ ID NO. 5 or SEQ ID NO. 6.

[0052] In the present invention, the fragment of GAS6 capable of binding to a TAM receptor may be SEQ ID NOs 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. In addition, fragments of ProS1 capable of binding to TAM receptors are SEQ ID NOs. 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, It may be 111, 112, or 113. The sequences of sequence no. 8 to 23 show at least 85% homology with sequence no. 1 (LG1 domain of GAS6). The sequences of sequence no. 24 to 33 show at least 85% homology with sequence no. 2 (LG2 domain of GAS6). The sequences of sequence no. 35 to 45 show at least 85% homology with sequence no. 3 (LG1 domain of ProS1). The sequences of sequence no. 46 to 62 show at least 85% homology with sequence no. 4 (LG2 domain of ProS1). The sequences of sequence no. 63 to 87 show at least 85% homology with sequence no. 5 (LG domain of GAS6). The sequences of sequence no. 88 to 113 show at least 85% homology with sequence no. 6 (LG domain of ProS1).

[0053] Preferably, the TAM ligand, a fragment or variant thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 113 and 115 to 120, but is not limited thereto.

[0054] In addition, the above TAM ligand, its fragment, or variant may include the amino acid sequence of SEQ ID NO. 114, which is an SHBG sequence capable of binding to a TAM receptor.

[0055] In the present invention, the fusion protein may further comprise an antibody or antigen-binding fragment that binds to a specific antigen.

[0056] The antibody or antigen-binding fragment may be selected from the group consisting of immunoglobulins such as IgG, IgM, IgD, IgE, IgA, IgY, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2; basic antibody fragments such as Fv, Fab, Fab', F(ab')2, VHH, VNAR, etc.; and engineered antibodies such as scFv, dsFv, ds-scFv, (scFv)2, diabody, triabody, tetrabody, and pentabody, etc. The antibody of the present invention or its antigen-binding fragment may be a Mab, Fab, or single-chain variable fragment (scFv) based on an antibody that specifically binds to a corresponding target substance, or a six complementarity determining region (CDR) derived from an antibody.

[0057] The above antigen may be a target substance that accumulates in biological tissues and induces or causes disease. The above target substance may be selected from beta-amyloid (β-amyloid), tau, alpha-synuclein (α-synuclein), huntingtin, prion, and the abnormal accumulation substances listed in Table 1 below, but is not limited thereto. Refer to Korean Registered Patent No. 10-2549520 for this.

[0058]

[0059] Abnormal accumulation substances Weaknesses Diseases Beta-Amyloid A (derived from Amyloid precursor protein): Alzheimer's disease, heterologous cerebral hemorrhage with amyloidosis, etc. α-Synuclein A (αSyn): Parkinson's disease, Parkinsonian dementia, Dementia with Lewy bodies, Multiple System Atrophy, etc. PrPScAPrP: Transmissible Spongiform Encephalopathy (Fatal familial insomnia, Gerstmann-Strussler-Scheinker disease, Creutzfeldt-Jacob disease, New variant Creutzfeldt-Jacob disease, etc.) Microtubule-associated protein Tau (ATau): Various tauopathies (Pick's disease, Progressive supranuclear palsy, Corticobasal degeneration, frontotemporal dementia with parkinsonism linked to chromosome 17, argyrophilic grain disease, etc.), Alzheimer's disease, Parkinson's disease, etc. Huntington's disease, etc. TAR DNA-binding protein 43 (TDP43) (None) Frontotemporal dementia, amyotrophic lateral sclerosis (ALS), etc. Superoxide dismutase 1 (SOD1) (None) amyotrophic lateral sclerosis,ALS) etc. ABri peptide ABri Familial British dementia ADan peptide ADan Familial Danish dementia Immunoglobulin light chain fragment AL Light chain amyloidosis Immunoglobulin heavy chain fragment AH Heavy chain amyloidosis Total N-terminal fragment of plasma amyloid A (Serum amyloid A) protein AAAA amyloidosis (AA amyloidosis) Transthyretin ATTR Senile systemic amyloidosis, Familial amyloid polyneuropathy, Familial amyloid cardiomyopathy, Leptomeningeal amyloidosis Beta-2 microglobulin Aβ2M Dialysis-related amyloidosis,Hereditary visceral amyloidosis N-terminal fragment of Apolipoprotein AI (AApoAI) AIApoAI amyloidosis C-terminal elongated Apolipoprotein AII AApoAIIApoAIII amyloidosis N-terminal fragment of Apolipoprotein AIV (AApoAIVApoAIV amyloidosis) Apolipoprotein C-II AApoCIIApoCII amyloidosis Apolipoprotein C-III AApoCIII ApoCIII amyloidosis Gelsoliin fragment (AGel) Finnish type familial amyloidosis Lysozyme (ALys) Hereditary non-neuropathic systemic amyloidosis Fibrinogen alpha chain fragment (AFib) Fibrinogen amyloidosis amyloidosis) N-terminally truncated Cystatin C (ACys) Icelandic type amyloidosis associated with hereditary cerebral hemorrhage Amylin (IAPP) AIAPP Diabetes mellitus type 2, Insulinoma Calcitonin ACal Medullary carcinoma of the thyroid Atrial natriuretic factor AANF Cardiac arrhythmias,Isolated atrial amyloidosis Prolactin APro Pituitary prolactinoma Insulin AIns Injection-localized amyloidosis Lactadherin (or Medin) AMed Aortic medial amyloidosis Lactotransferrin (or Lactoferrin) ALac Gelatinous drop-like corneal dystrophy ODAM (Odontogenic ameloblast-associated protein) AOAAP Calcifying epithelial odontogenic tumors SP-C (Pulmonary surfactant-associated protein C) ASPC Pulmonary alveolar proteinosis LECT-2 (Leukocyte cell-derived chemotaxin-2) ALECT2 Renal LECT2 amyloidosis Galectin-7 Agal7 Lichen amyloidosis, Macular amyloidosis Corneodesmosin AC or Hypotrichosis simplex of the scalp C-terminal fragment of TGFBI (or Keratoepithelin) AKer Lattice corneal dystrophy (type I,3A or Avellino present) SGI (Semenogelin-1) ASem1 Seminal vesicle amyloidosis S100 Protein (A8 or A9) (Absent) Prostate cancer Enfuvirtide AEnf Injection-localized amyloidosis,

[0060]

[0061] In addition, the above target substance may be a substance in which increased amounts or expression in biological tissues induce or cause immune diseases. The target substances are Factor II, factor V, factor VII, factor VIII, factor IX, factor X, factor CRMP5, DPPX / DPP6, GABAA-receptor, glycine receptor (GlyR), Hu (ANNA-1), Ma1, Ma2, Ri (ANNA-2), Zic4, voltage-gated potassium channel (VGKC)-complex, NMDA-receptor, Jo1, H / K ATPase, thyroid peroxidase, erythrocyte I / I, F-actin asialoglycoprotein receptor, cytochrome P450 2D6 (CYP2D6), NXP-2 / MORC3, TIF1-γ / TRIM-33, β2 integrin, nuclear 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-associated 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-adrenoreceptor, adenine nucleotide translocator, collagen type VII, IgG, G-CSF, collagen type IV α3-chain, thyrotropin receptor (TSHR), sodium iodide symporter (NIS), peripheral myelin protein 22 (PMP22), GM gangliosides, S-antigen, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), signal recognition particle 54kDa subunit (SRP54), IgA, synaptotagmin, voltage-gated calcium channels, 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 skeletal receptor tyrosine-protein kinase (MuSK), aminoacyl-tRNA synthetase, Tribbles Pseudokinase 2 (TRIB2), myeloperoxidase (MPO), aquaporin 4 (APQ-4), amphiphysin, Exosome component 9 (EXOSC9), EXOSC10 / PMSCL, Yo protein, Hu protein, Ri protein, desmoplakin, gephyrin, desmoglein 1, desmoglein 3, intrinsic factor type 1, β2-glycoprotein I (β2-GPI), pyruvate dehydrogenase complex-E2 (PDC-E2), aggrecan G1, carbamylated antigens, cartilage glycoprotein-39, Fc part of immunoglobulins, glucose-6-phosphate isomerase, keratin, protein-arginine deiminase type-4, collagen (multiple types, especially II, IV and IX), fibrinogen βα,lLeukemia 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 the Sm splicing ribonucleoprotein (subunits A-G), autologous double-stranded DNA (dsDNA),histone H2A-H2B-DNA, proliferating cell nuclear antigen (PCNA), ribosomal P, Sjogren Syndrome (SSA),Smith,U1-RNP, U2 snRNP B, vimentin, C1q, fibronectin, Ku-DNA-protein kinase, Carbonic anhydrase II, neuronal nicotinic acetylcholine receptor, centromere-associated protein, RNA polymerase I-III (RNP), thyroid and eye muscle shared protein, leukocyte function-associated antigen (LFA-1), chromogranin A, IA-2 (ICA512), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), ZnT8, insulin, glutamate decarboxylase (GAD65), insulin receptor, heat shock protein (65-kDa heat shock protein), SOX-10, tyrosinase, KUMEL1 / ARMC9, proteinase 3 / myeloblastin, CD20, CD19, Complement C3, Complement C5, C5 alpha receptor 1, CD52, FcRn Large Subunit p51, IL-1, IL-1R, IL-6, IL-6R, IL-17,IL-17R, TNF-alpha, TNFR, IL-4, IL-4R, IL-5, IL-5R, IL-13, IL-13R, IFN-gamma, IFN-gamma receptor, IL-12, IL-12R, IL-21, IL-21R, IL-22, IL-22R, TGF-beta, TGF-beta receptor, CD80 / 86, CD28, IL-23, IL-23R, thymic stromal lymphopoietin (TSLP), TSLPR, IL-31, IL-31R, OX40, OX40L, IL-33, IL-33R, CD40, CD40L, IGF-1R, ICAM1, VCAM1, MADCAM1, Integrin alpha 4, Integrin beta 7, VLA-4, toll-like receptor (TLR)-3, TLR-4, It may be selected from TLR-5, TLR-7, Phosphatidylserine (PS), and combinations thereof, but is not limited thereto. Reference may be made to International Patent Publication WO2024080854A1.

[0062] In the present invention, the antibody or antigen-binding fragment may be connected to the N-terminus or C-terminus of a TAM ligand, a fragment thereof, or a variant thereof, and preferably, the antibody or antigen-binding fragment may be connected to the N-terminus of a TAM ligand, a fragment thereof, or a variant thereof, but is not particularly limited thereto.

[0063] In the present invention, the antibody or antigen-binding fragment that binds to the specific antigen may be directly or through a linker connected to a TAM ligand, a fragment thereof, or a variant thereof.

[0064] The above "linker" is (GGGGS)n (n is an integer from 1 to 10, SEQ NO. 134), (GSSGGS)n (n is an integer from 1 to 10, SEQ NO. 135), (EAAAK)n (n is an integer from 1 to 10, SEQ NO. 136), KESGSVSSEQLAQFRSLD (SEQ NO. 137), EGKSSGSGSESKST (SEQ NO. 138), GSAGSAAGSGEF (SEQ NO. 139), CRRRRRREAEAC (SEQ NO. 140), GGSGGSGGS (SEQ NO. 141), GGGGGGGG (SEQ NO. 142), GGGGGG (SEQ NO. 143), GGGSGGS (SEQ NO. 144), GGGSGG (SEQ NO. 145), GGSGG (SEQ NO. 146), AEAAAKEAAAAKA (SEQ NO. 147), It may consist of the amino acid sequences of PAPAP (SEQ No. 148), (Ala-Pro)n (n is an integer from 1 to 10), VSQTSKLTRAETVFPDV (SEQ No. 149), PLGLWA (SEQ No. 150), TRHRQPRGWE (SEQ No. 151), AGNRVRRSVG (SEQ No. 152), RRRRRRRR (SEQ No. 153), GFLG (SEQ No. 154), or GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE (SEQ No. 155), but is not limited thereto.

[0065] In the present invention, the fusion protein may be a monomer, a dimer, or a multimer.

[0066] Preferably, the fusion protein of the present invention may be a scFv-TAM binder structure comprising an antigen-binding fragment scFv and a TAM binder, a Fab-TAM binder structure comprising Fab and a TAM binder, or a scFv-Fc(mono)-TAM binder structure comprising scFv, Fc(mono), and a TAM binder. Additionally, the fusion protein of the present invention may be a scFv-Fc-TAM binder(monovalent) structure comprising scFv, Fc, and a TAM binder(monovalent), an IgG-TAM binder(monovalent) structure comprising IgG and a TAM binder(monovalent), a scFv-Fc-TAM binder(bivalent) structure comprising scFv, Fc, and a TAM binder(bivalent), or an IgG-TAM binder(bivalent) structure comprising IgG and a TAM binder(bivalent).

[0067] In the above, the antibody IgG or the antigen-binding fragment scFv, Fab used were anti-amyloid beta antibodies such as aducanumab, gantenerumab, lecanemab, donanemab, anti-TNFa antibody adalimumab, or anti-MOG antibody 8-18C5, but are not limited thereto.

[0068] In the scFv-Fc(mono)-TAM binder structure above, Fc may be an IgG4-based CH2 and CH3, and CH2 may have YTE (M252Y / S254T / T256E) and L351F, S354E, T366R, P395K, F405R, Y407E, L432C, H433S, N434W, Y436L, T437C, and dQ438 variants introduced, but is not limited thereto.

[0069] In the scFv-Fc-TAM binder (monovalent) structure, IgG-TAM binder (monovalent) structure, scFv-Fc-TAM binder (bivalent) structure and IgG-TAM binder (bivalent) structure, a YTE (M252Y / S254T / T256E) variant may be introduced into CH2 of Fc to increase the binding affinity to FcRn and thereby increase the antibody half-life, and a LALA (L234A / L235A), LALAPG (L234A / L235A / P329G), or NA (N297A) variant may be introduced to significantly reduce the affinity for the Fc gamma receptor and thereby reduce effector functions such as ADCC and CDC effects.

[0070] In addition, in the scFv-Fc-TAM binder (monovalent) structure and the IgG-TAM binder (monovalent) structure, to induce two Ig heavy chains to form a heterodimer, DD (K392D / K409D) and KK (E356K,D399K), and Knob-into-Hole (T366W,T366S / L368A / Y407V) mutations may be introduced into the CH3 of Fc.

[0071] In addition, the present invention provides a composition for inhibiting the cleavage of a protein that can bind to a TAM receptor, comprising one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA.

[0072] The above cleavage occurs in a portion of the TAM ligand, and cleavage may also occur in its fragments or variants, and cleavage may also occur in fusion proteins containing the TAM ligand, its fragments, or its variants. The composition of the present invention has the effect of inhibiting the cleavage of the TAM ligand, its fragments, or its variants, and also has the effect of inhibiting the cleavage of fusion proteins containing the same.

[0073] In addition, the present invention provides a method for producing a protein capable of binding to a TAM receptor, comprising: (a) transfecting a cell line with a recombinant expression vector comprising a polynucleotide encoding a protein capable of binding to a TAM receptor; (b) adding one or more selected from the group consisting of an antioxidant, a copper compound, calcium chloride (CaCl2), and EDTA to the cell line transfected in step (a) and culturing it; and (c) isolating the protein capable of binding to a TAM receptor from the cell culture medium of step (b).

[0074] In the present invention, the cell line of step (a) may be selected from the group consisting of CHO (Chinese hamster ovary) cells, BHK (Baby hamster kidney) cells, NSO (Mouse myeloma) cells, SP2 / 0 (Mouse myeloma) cells and HEK293 (Human embryonic kidney) cells, but is not limited thereto.

[0075] In the present invention, the culture in step (b) may be fed batch culture, batch culture, or perfusion culture, but is not limited thereto.

[0076]

[0077] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0078]

[0079] Example 1. Preparation of a protein capable of binding to a TAM ligand

[0080] In the present invention, a protein capable of binding to a TAM receptor was produced. The protein capable of binding to the TAM receptor is a TAM ligand that is a TAM binder, a fragment thereof, or a variant thereof, or a fusion protein comprising the TAM ligand that is a TAM binder, a fragment thereof, or a variant thereof. The TAM ligand may be GAS6 (Growth arrest specific factor 6), ProS1 (Protein S), SHBG (Sex hormone-binding globulin), Tubby, Tulp1 (Tubby-related protein 1), or Gal3 (Galectin-3).

[0081] The above TAM ligand, a fragment thereof, or a variant thereof may comprise an amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 85% or more homology thereto, or an amino acid sequence of SEQ ID NO. 2 or an amino acid sequence having 85% or more homology thereto, or an amino acid sequence of SEQ ID NO. 3 or an amino acid sequence having 85% or more homology thereto, or an amino acid sequence of SEQ ID NO. 4 or an amino acid sequence having 85% or more homology thereto. Additionally, the above TAM ligand, a fragment thereof, or a variant thereof may comprise an amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 6, or an amino acid sequence having 85% or more homology with SEQ ID NO. 5 or SEQ ID NO. 6.

[0082] More specifically, the TAM ligand, a fragment thereof, or a variant thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 113 and 115 to 120, and it can be confirmed that this is a sequence having at least 85% homology with a wild-type TAM ligand. Reference may be made to U.S. Patent Publication No. 2024 / 0018204. Additionally, the TAM ligand may be the SHBG sequence of SEQ ID NO. 114.

[0083] In addition, a fusion protein comprising a TAM ligand, a fragment thereof, or a variant thereof further comprises an antibody or antigen-binding fragment capable of binding to a specific antigen. The antibody or antigen-binding fragment may bind to a target substance that accumulates in biological tissues and induces or causes disease, as may be referenced in Korean Registered Patent No. 10-2549520. Furthermore, the antibody or antigen-binding fragment may be a substance in which increased amounts or expression in biological tissues induce or cause immune diseases, as may be referenced in International Published Patent WO2024080854A1.

[0084] In the present invention, as examples of proteins capable of binding to a TAM receptor, a TAM binder structure, a scFv-TAM binder structure, a Fab-TAM binder structure, a scFv-Fc(mono)-TAM binder structure, a scFv-Fc-TAM binder (monovalent) structure, an IgG-TAM binder (monovalent) structure, a scFv-Fc-TAM binder (bivalent) structure, and an IgG-TAM binder (bivalent) structure were constructed (Fig. 1). The specific sequences of protein structures capable of binding to a TAM receptor are as follows.

[0085]

[0086] 1.1.TAM binder structure

[0087] The TAM binder structures are as shown in Tables 2 to 4 below. As shown in Table 2 below, a laminin G-like domain among GAS6, which is a TAM ligand, may be used, and a signal sequence (or signal peptide) may be connected to its N-terminus, and six histidines may be connected to its C-terminus. In addition, the TAM ligand protein of the present invention may be replaced with an amino acid sequence selected from the group consisting of SEQ ID NO. 6 to 113, or the amino acid sequence of SEQ ID NO. 5, instead of the laminin G-like domain containing the amino acid sequence of SEQ ID NO. 5 in Table 2. In addition, as shown in Table 3, it may be replaced with the SHBG sequence of SEQ ID NO. 114.

[0088]

[0089] Construct 1: TAM binder (서열번호 121)ORF1: Signal Sequence-GAS6-Taq(Hexa-histidine)-Stop(Singal Sequence)METDTLLLWVLLLWVPGSTG(GAS6_Laminin G-like domain)DILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA(서열번호 5)(Taq_Hexa-histidine)HHHHHH

[0090]

[0091] SHBG SequenceDPPAVHLSNGPGQEPIAVMTFDLTKITKTSSSFEVRTWDPEGVIFYGDTNPKDDWFMLGLRDGRPEIQLHNHWAQLTVGAGPRLDDGRWHQVEVKMEGDSVLLEVDGEEVLRLRQVSGPLTSKRHPIMRIALGGLLLFPASNLRLPLVPALDGCLRRDSWLDKQAEISASAPTSLRSCDVESN PGIFLPPGTQAEFNLRDIPQPHAEPWAFSLDLGLKQAAGSGHLLALGTPENPSWLSLHLQDQKVVLSSGSGPGLDLPLVLGLPLQLKLSMSRVVLSQGSKMKALALPPLGLAPLLNLWAKPQGRLFLGALPGEDSSTSFCLNGLWAQGQRLDVDQALNRSHEIWTHSCPQSPGNGTDASH (SEQ ID NO: 114)

[0092]

[0093] In addition, as shown in Table 4, the TAM ligand may be replaced with an amino acid sequence selected from the group consisting of SEQ ID NOs 115 to 120, which are variants of the TAM ligand, instead of the laminin G-like domain of Table 2.

[0094]

[0095] TAM ligand variant 1 (Seq. No. 115)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRQQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELDRNLVIKVNRDAVMKIAVAGDLFQPERGQYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWAPDLRAVLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPTSAPVTAYYRGCMTLEVNRRRLLDLDEAEYKHSDITAHSCPPVEVTAM ligand variant 2 (Seq. No. 116)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRQQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELDRNLVIKVNRDAVMKIAVAGDLFQPERGQYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWNPDLRAVPLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPTSAPVTAYYRGCMTLEVNRRLLLDLDEAAYKHSDITAHSCPPVEVTAM ligand variant 3 (serial number117)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRQQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGQYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCYSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWAPDLRAVLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPTSAPVTAYYRGCMTLEVNRRLLLDLDEAAYKHSDITAHSCPPVEVTAM ligand variant 4 (serial number 118)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRQQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELDRNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCYSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWNPDLRTVPLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPTSAPVTAYYRGCMTLEVNRRLLLDLDEAEYKHSDITSHSCPPVEPTAM ligand variant 5 (serial number119)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRQQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELDRNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWNPDLRTVPLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPDSAPVTAYYRGCMTLEVNRRLLLDLDEAEYKHSDITSHSCPPVEPTAM ligand variant 6 (serial number 120)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWAPDLRAVPLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPTSAPVTAYYRGCMTLEVNRRLLLDLDEAAYKHSDITAHSCPPVEP

[0096]

[0097] 1.2. The scFv-TAM binds the structure

[0098] The scFv-TAM binder structure is as shown in Table 5 below. As shown in Table 5 below, the laminin G-like domain of GAS6, a TAM ligand, may be used, and the scFv of the anti-amyloid beta antibody aducanumab may be linked to its N-terminus, a signal sequence (or signal peptide) may be linked to the N-terminus of the scFv, and six histidines may be linked to the C-terminus of the laminin G-like domain. In addition, the scFv and the TAM ligand may be linked via a linker sequence, and the VL (heavy chain variable region) and VH (light chain variable region) of the scFv may also be linked via the linker sequence (G4S)2.

[0099] As described in Section 1.1. TAM binder above, other TAM ligands, fragments thereof, or variants may be used in place of the laminin G-like domain in the TAM binder of Table 3. Additionally, the VL and VH of scFv may be changed to the order of VH and VL, and scFv stabilization techniques may be applied. The linker sequence may be replaced with (GGGGS)n, (GSSGGS)n, (EAAAK)n, etc.

[0100] In addition, the scFv of aducanumab in Table 5 can be replaced with scFvs of other anti-amyloid beta antibodies such as gantenerumab, lecanemab, and donanemab. It can also be replaced with the anti-TNFa antibody adalimumab and the scFv of the anti-MOG antibody 8-18C5.

[0101]

[0102] Construct 2: scFv-TAM binds (서열번호 122)ORF1: Signal Sequence-Aducanumab scFv(hVL-(G4S)3-hVH)-(G4S)2)-GAS6-Taq(Hexa-histidine)-Stop(Signal Sequence)METDTLLLWVLLWVPGSTGD(Aducanumab_hVL)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK(Linker_(G4S)3)GGGGSGGGGSGGGGS(Aducanumab_hVH)EVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKLEWVAVIWFDGTTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGKTTVTVSS(Linker_(G4S)2)GGGGSGGGGS(GAS6_Laminin G-like domain)DILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDTTIQETVKVNTRMQCFSVTERG SFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA(서열번다 5)(Taq_Hexa-histidine)HHHHHH

[0103]

[0104] 1.3. Fab-TAM binder structure

[0105] The Fab-TAM binder structure is as shown in Table 6 below. As shown in Table 6 below, the laminin G-like domain of GAS6, which is the TAM ligand, may be used, and the Fab of the anti-MOG antibody 8-18C5 may be linked to its N-terminus, a signal sequence (or signal peptide) may be linked to the N-terminus of the Fab, and six histidines may be linked to the C-terminus of the laminin G-like domain. In addition, the Fab and TAM ligand may be linked via the linker sequence (G4S)2 or (G4S)3.

[0106] As described in 1.1. TAM binder above, other TAM ligands, fragments thereof, or variants may be used in place of the laminin G-like domain in the TAM binder of Table 6. Additionally, the TAM ligand may be attached to ORF1 instead of ORF2. The linker sequence may be replaced with (GGGGS)n, (GSSGGS)n, (EAAAK)n, etc.

[0107] In addition, Fab of 8-18C5 in Table 6 can be replaced with Fabs such as aducanumab, gantenerumab, lecanemab, and donanemab, which are anti-amyloid beta antibodies. It can also be replaced with adalimumab, which is an anti-TNFa antibody.

[0108]

[0109] Construct 3: Fab-TAM binderORF1: Signal Sequence-8-18C5 Fab HC(mVH-hCH1_hIgG1)-Partial hinge-Stop(서열번호 123)(Signal Sequence)METDTLLLWVLLLWVPGSTGD(ch8-18C5_mVH)EVKLHESGAGLVKPGASVEISCKATGYTFSSFWIEWVKQRPGHGLEWIGEILPGRGRTNYNEKFKGKATFTAETSSNTAYMQLSSLTSEDSAVYYCATGNTMVNMPYWGQGTTVTVSS(hCH1)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(Partial hinge)EPKSCORF2:Signal Sequence-8-18C5 Fab LC(mVL-hCL_hkappa)-(G4S)3-GAS6-Taq(Hexa-histidine)-Stop (서열번호 124)(Signal Sequence)METDTLLLWVLLLWVPGSTGD(ch8-18C5_mVL)DIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGLPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHSYPLTFGAGTKLEIK(ch8-18C5_hCL)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Linker_(G4S)3)GGGGSGGGGSGGGGS(GAS6_Laminin G-likedomain)DILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVI NHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDDTTIQETVKVNTRMQCFSVTERG SFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVS LRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA (SEQ ID NO: 5)(Taq_Hexa-histidine)HHHHHH

[0110]

[0111] 1.4. scFv-Fc(mono)-TAM binder structure

[0112] The scFv-Fc(mono)-TAM binder structure is as shown in Table 7 below. As shown in Table 7 below, the laminin G-like domain of GAS6, which is a TAM ligand, may be used, and the scFv and Fc(monovalent) of the anti-amyloid beta antibody aducanumab may be linked to its N-terminus, a signal sequence (or signal peptide) may be linked to the N-terminus of scFv, and six histidines may be linked to the C-terminus of the laminin G-like domain. Additionally, the VL and VH of scFv, scFv and Fc(mono), Fc(mono) and TAM ligands may be linked via the linker sequence (G4S)2 or (G4S)3.

[0113] As described in Section 1.1. TAM binder above, other TAM ligands, fragments thereof, or variants may be used in place of the laminin G-like domain in the TAM binder of Table 7. Additionally, the VL and VH of scFv may be changed to the order of VH and VL, and scFv stabilization techniques may be applied. The linker sequence may be replaced with (GGGGS)n, (GSSGGS)n, (EAAAK)n, etc.

[0114] In addition, the scFv of aducanumab in Table 7 can be replaced with scFvs of other anti-amyloid beta antibodies such as gantenerumab, lecanemab, and donanemab. It can also be replaced with the anti-TNFa antibody adalimumab and the scFv of the anti-MOG antibody 8-18C5.

[0115] Fc can be IgG4-based CH2 and CH3, and CH2 may have introduced YTE (M252Y / S254T / T256E) and L351F, S354E, T366R, P395K, F405R, Y407E, L432C, H433S, N434W, Y436L, T437C, and dQ438 variants.

[0116]

[0117] Construct 4: scFv-Fc(mono)-TAM binds (서열벨벨 125)ORF1: Signal Sequence-Aducanumab scFv(hVL-(G4S)3-hVH)-(G4S)2-Fc mono(hCH2(YTE)-hCH3_hIgG4)-(G4S)3-GAS6-Taq(Hexa-histidine)-Stop(Singal). Sequence)METDTLLLWVLLLWVPGSTGD(Aducanumab_hVL)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK(Linker_(G4S)3)GGGGSGGGGSGGGGS(Aducanumab_hVH)EVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMD VWGKGTTVTVSS(Linker_(G4S)2)GGGGSGGGGS(hCH2_YTE)APEFLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK(hCH3)GQPREPQVYTFPPEQEEMTKNQVSLRCLVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFRLESRLTVDKSRWQEGNVFSCSVMHEACSWHLCKSLSLSLGK(Linker_(G4S)3)GGGGSGGGGSGGGGS(GAS6_Laminin G-likedomain)DILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVI NHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDDTTIQETVKVNTRMQCFSVTERG SFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVS LRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA (SEQ ID NO: 5)(Taq_Hexa-histidine)HHHHHH

[0118]

[0119] 1.5. scFv-Fc-TAM binder(monovalent) structure

[0120] The scFv-Fc-TAM binder (monovalent) structure is as shown in Table 8 below. As shown in Table 8 below, the laminin G-like domain of GAS6, which is a TAM ligand, may be used, and the scFv and Fc of the anti-amyloid beta antibody Gantenerumab may be linked to its N-terminus, a signal sequence (or signal peptide) may be linked to the N-terminus of scFv, and six histidines may be linked to the C-terminus of the laminin G-like domain. Additionally, the VL and VH of scFv, scFv and Fc, and Fc and TAM ligands may each be linked via the linker sequence (G4S)2 or (G4S)3.

[0121] As described in Section 1.1. TAM binder above, other TAM ligands, fragments thereof, or variants may be used in place of the laminin G-like domain in the TAM binder of Table 8. Additionally, the VL and VH of scFv may be changed to the order of VH and VL, and scFv stabilization techniques may be applied. The linker sequence may be replaced with (GGGGS)n, (GSSGGS)n, (EAAAK)n, etc.

[0122] In addition, the scFv of gantenerumab in Table 8 can be replaced with scFvs of other anti-amyloid beta antibodies such as aducanumab, lecanemab, and donanemab. It can also be replaced with the anti-TNFa antibody adalimumab and the scFv of the anti-MOG antibody 8-18C5.

[0123] In the CH2 of Fc, the YTE (M252Y / S254T / T256E) variant may be introduced to increase the binding affinity to FcRn and thereby increase the antibody's half-life, and LALA (L234A / L235A), LALAPG (L234A / L235A / P329G), or NA (N297A) variants may be introduced to significantly decrease the affinity to the Fc gamma receptor, thereby reducing effector functions such as ADCC and CDC effects. Additionally, in the CH3 of Fc, DD (K392D / K409D), KK (E356K / D399K), and Knob-into-Hole (T366W, T366S / L368A / Y407V) variants may be introduced to induce two Ig heavy chains to form a heterodimer.

[0124] The TAM binder can be attached to ORF2 instead of ORF1 in Table 8. In this case, the TAM binder can be attached to either DD or KK, or to either Knob or Hole, in CH3 of Fc.

[0125]

[0126] Construct 5: scFv-Fc-TAM binds (monovalent)ORF1: Signal Sequence-Gantenerumab scFv(hVL-(G4S)3-hVH)-(G4S)2-Fc(Partial hinge-CH2(YTE, NA)-CH3(K392D, K409D))-(G4S)3-eGAS6(Variant 6)-Taq(Hexa-histidine)-Stop (서열번벨) 126)(Signal Sequence)METDTLLLWVLLWVPGSTGD(Gantenerumab_hVL)DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYCLQIYNMPITFGQGTKVEIK(Linker_(G4S)3)GGGGSGGGGSGGGGS(Gantenerumab_hVH)QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS(Linker_(G4S)2)GGGGSGGGGS(Partial hinge)DKTHTCPPCP(CH2_YTE, N297A)APELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(CH3_K392D,K409D)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYDTPPVLDSDGSFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Linker_(G4S)3)GGGGSGGGGSGGGGS(eGAS6(Variant 6)_Laminin G-like domain)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWAPDLRAVLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPTSAPVTAYYRGCMTLEVNRRLLLDLDEAAYKHSDITAHSCPPVEP(서열번호 5)(Taq_Hexa-histidine)HHHHHHORF2: Signal Sequence-Gantenerumab scFv(hVL-(G4S)3-hVH)-(G4S)2-Fc(Partial hinge-CH2(YTE,NA)-CH3(E356K / D399K))-Stop(서열번호 127)(Signal Sequence)METDTLLLWVLLLWVPGSTGD(Gantenerumab_hVL)DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIK(Linker_(G4S)3)GGGGSGGGGSGGGGS(Gantenerumab_hVH)QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS(Linker_(G4S)2)GGGGSGGGGS(Partial hinge)DKTHTCPPCP(CH2_YTE, NA)APELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(CH3_E356K, D399K)GQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,

[0127]

[0128] 1.6. IgG-TAM binder(monovalent) 구조체

[0129] The IgG-TAM binder (monovalent) construct is as shown in Table 9 below. As shown in Table 9 below, a variant of the TAM ligand may be used for the TAM binder, and the IgG of the anti-amyloid beta antibody Gantenerumab may be linked to its N-terminus, a signal sequence (or signal peptide) may be linked to the N-terminus of the IgG, and six histidines may be linked to the C-terminus of the variant of the TAM ligand. Additionally, the IgG and TAM ligands may be linked via the linker sequence (G4S)3.

[0130] As described in 1.1. TAM binder above, other TAM ligands, fragments thereof, or variants may be used in place of variants of the TAM ligand in the TAM binder of Table 9. Additionally, the linker sequence may be replaced with (GGGGS)n, (GSSGGS)n, (EAAAK)n, etc.

[0131] In addition, the IgG of gantenerumab in Table 9 may be replaced with IgG of other anti-amyloid beta antibodies such as aducanumab, lecanemab, and donanemab. It may also be replaced with IgG of adalimumab, an anti-TNFa antibody, or IgG of 8-18C5, an anti-MOG antibody.

[0132] In the CH2 of Fc, the YTE (M252Y / S254T / T256E) variant may be introduced to increase the binding affinity to FcRn and thereby increase the antibody's half-life, and LALA (L234A / L235A), LALAPG (L234A / L235A / P329G), or NA (N297A) variants may be introduced to significantly decrease the affinity to the Fc gamma receptor, thereby reducing effector functions such as ADCC and CDC effects. Additionally, in the CH3 of Fc, DD (K392D / K409D), KK (E356K / D399K), and Knob-into-Hole (T366W, T366S / L368A / Y407V) variants may be introduced to induce two Ig heavy chains to form a heterodimer.

[0133] The TAM binder can be attached to ORF2 instead of ORF1 in Table 9. In this case, the TAM binder can be attached to either DD or KK, or to either Knob or Hole, in CH3 of Fc.

[0134]

[0135] Construct 6: IgG-TAM binder(monovalent)ORF1: Signal Sequence-Gantenerumab IgG(hVH-CH1-Hinge-CH2(LALAPG)-CH3(K392D, K409D)_hIgG1)-(G4S)3-eGAS6(Variant 1)-Taq(Hexa-histidine)-Stop(서열번호 128)(Signal Sequence)METDTLLLWVLLLWVPGSTG(Gantenerumab_hVH)QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS(CH1)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(Hinge)EPKSCDKTHTCPPCP(CH2_L234A / L235A / P329G)APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK(CH3_K392D,K409D)GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Linker_(G4S)3)GGGGSGGGGSGGGGS(eGAS6(Variant 1)_Laminin G-like domain)DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRQQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELDRNLVIKVNRDAVMKIAVAGDLFQPERGQYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWAPDLRAVPLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPDTSAPVTAYYRGCMTLEVNRRLLDLDEAEYKHSDITAHSCPPVEP (서열번호 115)(Tag: Hexa-histidine)HHHHHHORF2: Signal Sequence-Gantenerumab IgG(hVH-CH1-Hinge-CH2(NA)-CH3(E356K,D399K)_hIgG1)-Stop (서열번호 129)(Signal Sequence)METDTLLLWVLLLWVPGSTG(Gantenerumab_hVH)QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS(CH1)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(Hinge)EPKSCDKTHTCPPCP(CH2_L234A / L235A / P329G))APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK(CH3_E356K, D399K)GQPREPQVYTLPPSRKELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKORF3: Signal Sequence-Gantenerumab IgG(hVL-hCL_hKappa)-Stop (서열번호 130)(Signal Sequence)METDTLLLWVLLLWVPGSTG(Gantenerumab_hVL)DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIK(hCL)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC,

[0136]

[0137] 1.7. scFv-Fc-TAM binder(bivalent) structure

[0138] The scFv-Fc-TAM binder (bivalent) construct is as shown in Table 10 below. As shown in Table 10 below, the laminin G-like domain of GAS6, which is a TAM ligand, may be used, and the scFv and Fc of the anti-amyloid beta antibody aducanumab may be linked to its N-terminus, a signal sequence (or signal peptide) may be linked to the N-terminus of scFv, and six histidines may be linked to the C-terminus of the laminin G-like domain. In addition, the VL and VH of scFv, Fc, and TAM ligand may be linked via the linker sequence (G4S)3.

[0139] As described in Section 1.1. TAM binder above, other TAM ligands, fragments thereof, or variants may be used in place of the laminin G-like domain in the TAM binder of Table 10. Additionally, the VL and VH of scFv may be changed to the order of VH and VL, and scFv stabilization techniques may be applied. The linker sequence may be replaced with (GGGGS)n, (GSSGGS)n, (EAAAK)n, etc.

[0140] In addition, the scFv of aducanumab in Table 10 can be replaced with scFvs of other anti-amyloid beta antibodies such as gantenerumab, lecanemab, and donanemab. It can also be replaced with the anti-TNFa antibody adalimumab and the scFv of the anti-MOG antibody 8-18C5.

[0141] In the CH2 of Fc, a YTE (M252Y / S254T / T256E) variant may be introduced to increase the binding affinity to FcRn and thus increase the antibody's half-life, and LALA (L234A / L235A), LALAPG (L234A / L235A / P329G), or NA (N297A) variants may be introduced to significantly decrease the affinity to the Fc gamma receptor and thereby reduce effector functions such as ADCC and CDC effects.

[0142]

[0143] Construct 7: scFv-Fc-TAM binder(bivalent) (Section 131)ORF1: Signal Sequence-Aducanumab scFv(hVL-(G4S)3-hVH)-Hinge-CH2(NA)-CH3-(G4S)3-GAS6-Taq(Hexa-histidine)-Stop(Signal Sequence)METDTLLLWVLLWVPGSTGD(Aducanumab_hVL)DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK(Linker_(G4S)3)GGGGSGGGGSGGGGS(Aducanumab_hVH)QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYM DVWGKGTTVTVSS(Hinge)EPKSCDKTHTCPPCP(CH2_N297A)APELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA K(CH3)GQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Linker_(G4S)3)GGGGSGGGGSGGGGS(GAS6_Laminin G-likedomain)DILPCVPFSVAKSVKSLYLGRMFSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNGVGRVTSSGPVI NHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNGEDDTTIQETVKVNTRMQCFSVTERG SFYPGSGFAFYSLDYMRTPLDVGTESTWEVEVVAHIRPAADTGVLFALWAPDLRAVPLSVALVDYHSTKKLKKQLVVLAVEHTALALMEIKVCDGQEHVVTVS LRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPVTSAPVTAFYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEPAAA (SEQ ID NO: 5)(Tag_Hexa-histidine)HHHHHH

[0144]

[0145] 1.8. IgG-TAM binder (bivalent) construct

[0146] The IgG-TAM binder (bivalent) construct is as shown in Table 11 below. As shown in Table 11 below, a variant of the TAM ligand may be used, and the IgG of the anti-amyloid beta antibody Gantenerumab may be linked to the N-terminus of the TAM ligand, a signal sequence (or signal peptide) may be linked to the N-terminus of the IgG, and six histidines may be linked to the C-terminus of the variant of the TAM ligand. In addition, the IgG and TAM ligands may be linked via the linker sequence (G4S)3.

[0147] As described in 1.1. TAM binder above, other TAM ligands, fragments thereof, or variants may be used in place of the TAM ligand variants in the TAM binder of Table 11. Additionally, the linker sequence may be replaced with (GGGGS)n, (GSSGGS)n, (EAAAK)n, etc.

[0148] In addition, the IgG of gantenerumab in Table 11 may be replaced with IgG of other anti-amyloid beta antibodies such as aducanumab, lecanemab, and donanemab. It may also be replaced with IgG of adalimumab, an anti-TNFa antibody, or IgG of 8-18C5, an anti-MOG antibody.

[0149] In the CH2 of Fc, a YTE (M252Y / S254T / T256E) variant may be introduced to increase the binding affinity to FcRn and thus increase the antibody's half-life, and LALA (L234A / L235A), LALAPG (L234A / L235A / P329G), or NA (N297A) variants may be introduced to significantly decrease the affinity to the Fc gamma receptor and thereby reduce effector functions such as ADCC and CDC effects.

[0150]

[0151] Construct 8: IgG-TAM binder(bivalent)ORF1: Signal Sequence-Gantenerumab IgG(hVH-CH1-Hinge-CH2(NA)-CH3_hIgG1)-(G4S)3-eGAS6(Variant 6)-Taq(Hexa-histidine)-Stop (서열번호 132)(Signal Sequence)METDTLLLWVLLLWVPGSTGD(Gantenerumab_hVH)QVELVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGTLVTVSS(CH1)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV(Hinge)EPKSCDKTHTCPPCP(CH2_N297A)APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(CH3)GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Linker_(G4S)3)GGGGSGGGGSGGGGS(eGAS6(Variant 6)_Laminin G-likedomain) DPCVPFSVAKSVKSLYLGRLYSGTPVIRLRFKRLQPTRLVAEFDFRTFDPEGILLFAGGHQDSTWIVLALRAGRLELQLRYNKRGRVYSSGPVINHGMWQTISVEELARNLVIKVNRDAVMKIAVAGDLFQPERGLYHLNLTVGGIPFHEKDLVQPINPRLDGCMRSWNWLNDEETNTTIQETVKVNTRMQCFSVTERGSFYPGSGFAFYSLDYMRGSTWEVEVVAHIRPAQDTGVLFALWAPDLRAVPLSVALKDRLVVLAVEHTPLALMEIKVCDGQEHVVTVSLRDGEATLEVDGTRGQSEVSAAQLQERLAVLERHLRSPVLTFAGGLPDVPDTSAPVTAYYRGCMTLEVNRRLLDLDEAAYKHSDITAHSCPPVEP(SEQ ID NO: 120)(Tag_Hexa-histidine) HHHHHHORF2: Signal Sequence - Gantenerumab IgG(hVL-hCL-_hKappa)-Stop(SEQ ID NO: 133)(Signal Sequence) METDTLLLWVLLLWVPGSTGD(Gantenerumab_hVL) DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIK(Gantenerumab_hCL) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0152]

[0153] Example 2. Confirmation of the Expression Pattern of the Fusion Protein

[0154] In order to produce a fusion protein (hereinafter referred to as "GAS6 fusion protein") having a structure in which scFv, an antigen-binding fragment of the fusion protein of the present invention, is combined with GAS6, one of the TAM ligands, the fusion protein was temporarily expressed by transfecting CHO (Chinese hamster ovary) cells with a recombinant vector containing a polynucleotide encoding the fusion protein using the Lipofectamine method.

[0155] Specifically, a GAS6 fusion protein containing GAS6 linked to the C-terminus of an antibody fragment was temporarily expressed using the ExpiCHO expression system (Thermofisher, A29133). ExpiCHO-S cells were expressed in ExpiCHO expression medium at a volume of 0.2 × 10⁶ 6 ~ 0.3×10 6 Subculture cells at a concentration of cell / mL in an incubator at 37°C, humidity above 80%, and 8% CO2, 4×10⁶ cells at 3-day intervals 6 ~ 6×10 6 Cells were maintained at a concentration of 3.0 × 10⁶ cells / mL. For the temporary expression of the fusion protein containing GAS6, ExpiCHO cells were prepared at a concentration of 3.0 × 10⁶ cells one day prior to transfection. 6 Passings were performed in ExpiCHO expression medium at a cell / mL cell concentration, and 6.0×10⁶ on the day of transfection 6The cells were prepared to achieve a cell / mL ratio. 25 mL of cells were placed in a 125 mL Erlenmeyer flask, and suspension culture was performed in a 37°C incubator. For transfection, 20 μg of GAS6-containing fusion protein DNA was mixed into 1 mL of OptiPRO SFM medium (included in the ExpiCHO expression system kit) in one test tube, and 80 μL of Expifectamine CHO reagent was mixed into 1 mL of OptiPRO SFM medium in another test tube. The contents of each test tube were combined and added to the 125 mL Erlenmeyer flask containing 25 mL of cells. The Erlenmeyer flask was then suspended in a 37°C incubator. 6 mL of ExpiCHO feed was added one day after transfection.

[0156] Afterward, the culture medium from which cells had been removed by centrifugation was recovered after 3 days of culture or on the 5th day of culture, SDS-PAGE was performed, and the presence or absence of cleavage of the GAS6 fusion protein was confirmed through Western blot analysis using a Human anti-GAS6 antibody (R&D system, AF885).

[0157] As a result, it was confirmed that when GAS6 fusion proteins were transiently expressed using CHO cells, a small amount of the protein with the same molecular weight as expected (intact form) was detected (gray arrows in Lanes 3 and 4), while a large amount of GAS6 with a smaller molecular weight of about 20 kDa was detected (black arrows in Lanes 3 and 4) (Fig. 2). This indicates that GAS6 was cleaved during the transient expression of GAS6 fusion proteins in CHO cells.

[0158]

[0159] Example 3. Evaluation of the inhibitory effect of a protease inhibitor on the cleavage of a fusion protein

[0160] To determine whether cleavage of the fusion protein by protease occurs during the production of the GAS6 fusion protein, an experiment was conducted to check the degree of cleavage of the fusion protein after adding a protease inhibitor cocktail (P8340, Sigma-Aldrich) to the culture medium. For the temporary expression of the GAS6 fusion protein using CHO cells, the protease inhibitor cocktail was added on day 1 of culture after transfection, diluted to 200 times (Lane 3) or 1000 times (Lane 4) the culture volume. Cells were cultured at 37°C, and on day 4 of culture, the culture medium was collected and Western blot analysis was performed.

[0161] As a result, it was confirmed that the test groups (Lane 3 and Lane 4) with added protease inhibitor cocktail had a concentration-dependent effect of inhibiting the cleavage of the fusion protein compared to the control group (Lane 2) without addition (Fig. 3).

[0162] Next, the inhibitory effect of each component in the protease inhibitor cocktail on the cleavage of the GAS6 fusion protein was evaluated. The components used in the protease inhibitor cocktail were aprotinin (serine protease inhibitor: A6103, Sigma-Aldrich), AEBSF (serine protease inhibitor: A8456, Sigma-Aldrich), bestatin (aminopeptideidase inhibitor: B:8385, Sigma-Aldrich), leupeptin (serine and cysteine ​​protease inhibitor: L8511, Sigma-Aldrich), and pepstein A (acid protease inhibitor: BP5318, Sigma-Aldrich). For the temporary expression of the GAS6 fusion protein using CHO cells, each component was added on day 1 of culture after transfection, and Western blot analysis was performed on day 3 of culture to evaluate the inhibitory effect on the cleavage of the GAS6 fusion protein.

[0163] As a result, it was confirmed that each protease inhibitor component included in the protease inhibitor cocktail differs in the efficiency of inhibiting the cleavage of the GAS6 fusion protein (Fig. 4). Thus, it was confirmed that the GAS6 fusion protein of the present invention is cleaved by various proteases.

[0164]

[0165] Example 4. Evaluation of the inhibitory effect of antioxidant addition on the cleavage of fusion proteins

[0166] Reactive oxygen species (ROS) are generated during animal cell culture due to cellular respiration or external stress. Since these ROS can lead to protein oxidation or cleavage, as well as reduced stability and activity, regulating oxidative stress by reducing increased ROS in animal cell culture can influence cellular metabolism, protein synthesis, and productivity. Accordingly, the inhibitory effect of various antioxidants on the cleavage of GAS6 fusion proteins was evaluated.

[0167] As antioxidants, the phenolic antioxidants apigenin (Apigenin, 10798, Sigma-Aldrich), baicalein (Baicalein, 465119, Sigma-Aldrich), and kaempferol (Kaempferol, 60010, Sigma-Aldrich) were used at concentrations of 25 μM, 50 μM, 75 μM, and 100 μM, respectively. DMSO was used as the solvent for each antioxidant in the experiment. The experiment was performed 3 times for each concentration condition (n = 3).

[0168] For the temporary expression of GAS6 fusion proteins using CHO cells, the culture conditions were set at 200 rpm at 37°C and 8% CO2 conditions with an initial culture volume of 15 mL in a 50 mL TPP culture tube. On day 1 after transfection, three types of antioxidants were added at different concentrations and culture was performed, and on day 5, the culture medium was collected and Western blot analysis was conducted.

[0169] As a result, compared to the control group (Lane 1 to 3), apigenin and kaempferol showed a significant inhibitory effect on the cleavage of GAS6 fusion proteins at concentrations of 25 μM or higher, and baicalein showed a significant inhibitory effect on the cleavage of GAS6 fusion proteins at concentrations of 50 μM or higher (Figs. 5a to 5d).

[0170] From the above results, the present invention confirmed that the addition of phenolic antioxidants such as apigenin, baicalein, and kaempferol during the expression of GAS6 fusion protein has the effect of improving the production of GAS6 fusion protein by inhibiting the cleavage of the fusion protein.

[0171]

[0172] Example 5. Evaluation of the inhibitory effect of divalent ions on the cleavage of a fusion protein

[0173] The inhibitory effect of divalent ions on the cleavage of GAS6 fusion proteins was evaluated. Copper(II) sulfate (CuSO4), copper(II) chloride (CuCl2), copper(II) bromide (CuBr2), nickel(II) sulfate (NiSO4), manganese(II) sulfate (MnSO4), or magnesium sulfate (MgSO4) were used as divalent ions. For the temporary expression of GAS6 fusion proteins in CHO cells, each divalent ion was added at a concentration of 50 μM on day 1 of culture after transfection, and the cells were cultured at 37°C. On day 5 of culture, the culture medium was collected and Western blot analysis was performed.

[0174] As a result, among the divalent ions, copper ions (Cu 2+ It was confirmed that in the test groups (Lanes 3 to 5) containing ), the cleavage of the GAS6 fusion protein was significantly inhibited compared to the control group (Lane 1) (Figs. 6a and 6b). On the other hand, nickel ions (Ni 2+ ), manganese ions (Mn 2+ ) or magnesium ions (Mg 2+It was confirmed that the cleavage of the GAS6 fusion protein was not inhibited in the test groups (Lanes 6 to 9) containing ) (Figs. 6a and 6b).

[0175] From the above results, the present invention relates to copper ions (Cu) during the expression of the GAS6 fusion protein. 2+ It was confirmed that the addition of ) has the effect of improving the production of GAS6 fusion proteins by inhibiting the cleavage of GAS6 fusion proteins.

[0176]

[0177] Example 6. Evaluation of the inhibitory effect of copper ions, etc. on the cleavage of fusion proteins

[0178] Copper ions (Cu 2+ The inhibitory efficacy of cleavage on GAS6 fusion proteins was evaluated according to the addition concentrations of various copper sulfate (CuSO4), calcium chloride (CaCl2), and EDTA (ethylenediaminetetraacetic acid), including EDTA. For the temporary expression of GAS6 fusion proteins using CHO cells, post-transfection culture was set at 200 rpm at 37°C and 8% CO2 conditions with an initial culture volume of 15 mL in a 50 mL TPP culture tube. Copper sulfate (CuSO4) was treated at concentrations of 1 μM to 10 μM (1, 5, and 10 μM), calcium chloride (CaCl2) at concentrations of 1 mM to 50 mM (1, 4, 10, 25, and 50 mM), and EDTA at concentrations of 5 μM to 50 μM (5, 15, 30, and 50 μM). Each condition was repeated 3 times (n = 3). After culturing for 5 days, Western blot analysis was performed using the culture medium.

[0179] As a result, copper sulfate (CuSO4) showed an inhibitory effect on the cleavage of the fusion protein at concentrations of 1 μM or higher compared to the control group (Lanes 1–3), and in particular, it was confirmed that the inhibitory effect increased in a concentration-dependent manner (Figs. 7a and 7d). In addition, calcium chloride (CaCl2) showed an inhibitory effect on the cleavage of the fusion protein at concentrations of 1 mM or higher compared to the control group (Lanes 1–3), and in particular, it showed similar levels of inhibitory effect at 1 mM and 4 mM, and was confirmed to show a significantly inhibitory effect at high concentrations of 10 mM or higher (Figs. 7b and 7d). EDTA was confirmed to show a concentration-dependent inhibitory effect on the cleavage of the fusion protein at concentrations of 30 μM or higher (Figs. 7c and 7d).

[0180] In addition, for temporary expression of GAS6 fusion protein using CHO cells, on day 1 of culture after transfection, 25 μM or 50 μM copper sulfate (CuSO4) alone, a mixture of 25 μM copper sulfate (CuSO4) and 2 mM or 4 mM calcium chloride (CaCl2), or a mixture of 25 μM copper sulfate (CuSO4) and 10 μM EDTA was added, and the cells were cultured at 37°C, and on day 5 of culture, the culture medium was collected and Western blot analysis was performed.

[0181] As a result, as shown in Figures 8a and 8b, it was confirmed that the cleavage of the GAS6 fusion protein was significantly inhibited in a concentration-dependent manner in the test groups treated with copper sulfate (CuSO4) alone (Lanes 4 and 5) compared to the control groups (Lanes 2 and 3) on day 3 or day 5. In addition, in the test groups treated with a mixture of copper sulfate (CuSO4) and calcium chloride (CaCl2) (Lanes 6 and 7), the cleavage of the GAS6 fusion protein was inhibited even more significantly compared to the test group treated with copper sulfate (CuSO4) alone at the same concentration (Lane 4), and it was confirmed that the cleavage of the GAS6 fusion protein was inhibited in a concentration-dependent manner by calcium chloride (CaCl2). In particular, it was confirmed that the cleavage of the GAS6 fusion protein was most significantly inhibited in the test group treated with a mixture of copper sulfate (CuSO4) and EDTA (Lane 8) compared to the other test groups.

[0182] From the above results, the present invention confirmed that the addition of copper sulfate (CuSO4), calcium chloride (CaCl2), and EDTA, either individually or in combination, during the production of GAS6 fusion proteins using CHO cells has an inhibitory effect on the cleavage of GAS6 fusion proteins, thereby improving the production of fusion proteins.

[0183]

[0184] Example 7. Determination of the minimum effective concentration of copper ions having a cleavage inhibitory effect

[0185] Copper ions (Cu) showing inhibitory effect on the cleavage of GAS6 fusion proteins 2+To determine the minimum effective concentration of ), various treatment concentrations were set to evaluate the cleavage inhibitory effect. Temporary expression using CHO cells was performed for 5 days as in Example 6 above. Copper sulfate (CuSO4) was treated at concentrations ranging from 0.01 to 1,000 μM (0.01, 0.1, 0.25, 0.5, 1, 5, 10, 50, 100, 200, 500, and 1,000 μM). Each condition was repeated 3 times (n = 3).

[0186] As a result, copper sulfate (CuSO4) showed an inhibitory effect on the cleavage of GAS6 fusion proteins at concentrations ranging from 0.01 μM to 500 μM compared to the control group (Lane 1), and it was confirmed that the inhibitory effect increased in a concentration-dependent manner (Figs. 9a and 9b). In particular, it was confirmed that the total expression amount of all GAS6 fusion proteins increased as the concentration of copper sulfate (CuSO4) increased from 0.01 μM to 500 μM (Fig. 9c).

[0187] From the above results, it was confirmed that the addition of 0.01 μM or more of copper ions in the present invention has an inhibitory effect on the cleavage of GAS6 fusion proteins and, from this, an effect of improving the production of fusion proteins.

[0188]

[0189] Example 8. Confirmation of TAM ligand expression pattern and evaluation of the inhibitory effect of copper ions on TAM ligand cleavage.

[0190] Temporary expression of the TAM ligand structure without scFv fusion and copper ions (Cu 2+The cleavage inhibition effect following the addition of ) was evaluated. LG1 / LG2 domains (GAS6 wild type) and variants of LG1 / LG2 domains (V2) were used as TAM ligands. The provisional expression and verification procedures were performed in the same manner as in Example 6 above, except that on day 1 of culture after transfection, copper sulfate (CuSO4) was not treated or copper sulfate (CuSO4) was treated at 50 μM. The number of replicates for each condition was 3 (n = 3).

[0191] As a result, when compared to the LG1 / LG2 domain (GAS6 wild type) protein (Lane 7) cultured and purified after treatment with 50 μM copper sulfate (CuSO4), copper ions (Cu 2+ In the untreated groups (Lanes 4–6), only cleaved proteins were identified and no proteins of the target size were observed, whereas in the groups treated with 50 μM copper sulfate (CuSO4) (Lanes 1–3), proteins of the target size were identified and a significant reduction in cleaved proteins was confirmed (Fig. 10). Similarly, when compared to the LG1 / LG2 domain variant (V2) protein (Lane 8) cultured and purified after treatment with 50 μM copper sulfate (CuSO4), copper ions (Cu) in the groups treated with 50 μM copper sulfate (Lanes 12–14) were observed during the production of the LG1 / LG2 domain variant (V2). 2+ It was confirmed that the expression level of total protein was significantly increased compared to the untreated group (Lane 9~11) (Fig. 10).

[0192] From the above results, it was confirmed that the addition of copper ions in the present invention has the effect of enhancing the production of GAS6 fusion proteins through the inhibitory effect of cleavage of GAS6 fusion proteins.

[0193]

[0194] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0195] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. A medium composition for producing a protein capable of binding to a TAM receptor, comprising one or more selected from the group consisting of antioxidants, copper compounds, calcium chloride (CaCl2), and EDTA (ethylenediaminetetraacetic acid).

2. In Paragraph 1, A composition in which the above antioxidant is a phenolic antioxidant.

3. In Paragraph 2, A composition in which the above-mentioned phenolic antioxidant is selected from the group consisting of baicalein, quercetin, kaempferol, luteolin, apigenin, chrysin, wogonin, and galangin.

4. In Paragraph 3, A composition in which the above-mentioned phenolic antioxidant is included at a concentration of 1 μM to 1,000 μM.

5. In Paragraph 1, A composition in which the copper compound is selected from the group consisting of copper sulfate (CuSO4), copper phosphate (Cu3(PO4)2), copper oxide (Cu2O), copper II oxide (CuO), copper acetate (Cu(OAc)2), copper carbonate (CuO3), copper hydroxide (Cu(OH)2), copper nitrate (Cu(NO3)2), copper chloride (CuCl2), copper sulfide (CuS), and copper bromide (CuBr2).

6. In Paragraph 5, A composition in which the copper compound is included at a concentration of 0.01 μM to 1,000 μM.

7. In Paragraph 1, A composition in which the calcium chloride is included at a concentration of 0.1 mM to 100 mM.

8. In Paragraph 1, A composition in which the above EDTA is included at a concentration of 1 μM to 100 μM.

9. In Paragraph 1, A composition in which the above TAM receptor is selected from the group consisting of MerTK, Axl, and Tyro3.

10. In Paragraph 1, The protein capable of binding to the above TAM receptor is (a) a TAM ligand, a fragment thereof, or a variant thereof; or (b) A composition comprising a fusion protein including the above TAM ligand, a fragment thereof, or a variant thereof.

11. In Paragraph 10, A composition in which the above TAM ligand is selected from the group consisting of GAS6 (Growth arrest specific factor 6), ProS1 (Protein S), SHBG (Sex hormone-binding globulin), Tubby, Tulp1 (Tubby-related protein 1), and Gal3 (Galectin-3).

12. In Paragraph 10, A composition wherein the above TAM ligand, a fragment thereof, or a variant thereof comprises an amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having 85% or more homology thereto, and comprises an amino acid sequence of SEQ ID NO. 2 or an amino acid sequence having 85% or more homology thereto.

13. In Paragraph 10, A composition wherein the above TAM ligand, a fragment thereof, or a variant thereof comprises the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence having 85% or more homology thereto, and comprises the amino acid sequence of SEQ ID NO. 4 or an amino acid sequence having 85% or more homology thereto.

14. In Paragraph 10, A composition wherein the above TAM ligand, a fragment thereof, or a variant thereof comprises the amino acid sequence of SEQ ID NO. 5 or an amino acid sequence having 85% or more homology thereto.

15. In Paragraph 10, A composition wherein the above TAM ligand, a fragment thereof, or a variant thereof comprises the amino acid sequence of SEQ ID NO. 6 or an amino acid sequence having 85% or more homology thereto.

16. In Paragraph 10, A composition wherein the above TAM ligand, a fragment thereof, or a variant thereof comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 113, 115 to 120.

17. In Paragraph 10, A composition wherein the above TAM ligand, a fragment thereof, or a variant thereof comprises the amino acid sequence of SEQ ID NO.

114.

18. In Paragraph 10, A composition wherein the fusion protein further comprises an antibody or antigen-binding fragment that binds to a specific antigen.

19. In Paragraph 18, A composition in which the above antibody or antigen-binding fragment is connected to the N-terminus or C-terminus of a TAM ligand, a fragment thereof, or a variant thereof.

20. In Paragraph 18, A composition in which an antibody or antigen-binding fragment binding to the specific antigen is linked directly or through a linker to a TAM ligand, a fragment thereof, or a variant thereof.

21. In Paragraph 18, A composition in which the fusion protein is a monomer, dimer, or polymer.

22. A composition for inhibiting the cleavage of a protein capable of binding to a TAM receptor, comprising one or more selected from the group consisting of antioxidants, copper compounds, calcium chloride (CaCl2), and EDTA. 23.(a) Transfecting a cell line with a recombinant expression vector comprising a polynucleotide encoding a protein capable of binding to a TAM receptor; (b) a step of adding one or more selected from the group consisting of antioxidants, copper compounds, calcium chloride (CaCl2), and EDTA to the cell line transfected in step (a) and culturing it; and (c) A method for producing a protein capable of binding to a TAM receptor, comprising the step of isolating a protein capable of binding to a TAM receptor from the cell culture medium of step (b) above.

24. In Paragraph 23, A method in which the cell line of step (a) above is selected from the group consisting of CHO (Chinese hamster ovary) cells, BHK (Baby hamster kidney) cells, NSO (Mouse myeloma) cells, SP2 / 0 (Mouse myeloma) cells and HEK293 (Human embryonic kidney) cells.

25. In Paragraph 23, A method in which the culture of step (b) above is performed as fed batch culture, batch culture, or perfusion culture.

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