Novel modified immunoglobulin fc-fusion protein and use thereof.

Abstract

The present invention relates to a novel immunoglobulin Fc domain variant protein and use thereof, and when expressed in the form of a fusion protein with another biologically active protein, an immunoglobulin Fc domain variant protein 5 according to an embodiment of the present invention can prolong the half-life in vivo of the biologically active protein as well as effectively suppress effector functions such as ADCC or CDC to minimize unexpected side effects. 20 24 20 55 86 07 A ug 2 02 4 A B S T R A C T 2 0 2 4 2 0 5 5 8 6 0 7 A u g 2 0 2 4

Description

[00137] Example 4: Preparation of anti-CD40L hybrid antibody

[00138]     4-1: Preparation of chimeric anti-CD40L antibody 5

[00139] The present inventors prepared an anti-CD40L antibody obtained by introducing an FcyRIIa binding inhibitory variant into heavy-chain CH2 and CH3 domains of an antibody (CIO) in which an Fc region of the mouse-derived anti-CD40L (anti-CD154) antibody described in WO2016 / 182335A1 was substituted with a human IgGl Fc region, and the anti-CD40L antibody was named “C10M”. 10   

[00140]     4-2: Preparation of chimeric anti-CD40L-NTIG hybrid antibody

[00141] The present inventors designed a hybrid heavy chain protein in which a heavy chain moiety in a Fab moiety of the C10M antibody is linked to the N-terminus, does not bind to FcyR, of the NTIG protein prepared in Example 1, and the hybrid heavy chain protein was named “PG-129”. 15

[00142] A polynucleotide encoding a hybrid heavy chain of the hybrid heavy chain protein in which the heavy chain moiety (Vh-CH1, SEQ ID NO: 61) in Fab of the C10M antibody is linked to NTIG (SEQ ID NO: 7) was prepared and inserted into a pAD15 expression vector (WO2015 / 009052A). Moreover, a polynucleotide encoding a light heavy moiety (Vl-Cl, SEQ ID NO: 62) in Fab of the C10M 20 antibody was inserted into the pAD15 expression vector so that the polynucleotide can be regulated under a dual promoter. Thereafter, the vector was co-transfected into a CHO DG44 (from Dr. Chasin, Columbia University, USA) cell using a Neon-transfection system.

[00143] The expression vector completed as above was also transiently 25 expressed using the ExpiCHO kit manufactured by Thermo Fisher Scientific 2024205586   07 Aug 2024 Solutions LLC. Specifically, the vector construct prepared as above and the ExpiFectamine reagent contained in the kit were mixed in the ExpiCHO-S cell, the resultant was cultured for 1 day in an incubator with conditions of 8% CO2 and 37°C, then the temperature was lowered to 32°C, and the culture was performed until the 5 7th day.

[00144] Thereafter, capturing and purification of protein A were performed, it was checked whether a candidate material was purified through PAGE analysis and western blot analysis under non-reducing and reducing conditions, and formulation was performed with a formulation buffer solution according to the pl value of the 10 candidate material. The formulated material was quantified using Nano drop, and the final purity was checked using SEC-HPLC.

[00145] As a result, as shown in FIGS. 20a and 20b, the light chain and the heavy chain were normally expressed, a single band appeared under the nonreducing conditions, and thus it could be found that a normal heterotetramer was 15 formed. Moreover, as a result of SEC-HPLC analysis, the purity was 97.9%, which is very high.

[00146]     4-3: Preparation of humanized anti-CD40L-NTIG hybrid antibody

[00147] The chimeric antibodies have sufficiently improved disadvantages of 20 the conventional human antibodies by introducing the NTIG technique (immunoglobulin Fc domain variant protein) of the present invention, but the present inventors have determined that the antigen-binding site was still a mouse-derived antibody and thus unnecessary immune responses in the human body may occur, and, in order to prepare a safer hybrid antibody, designed a humanized antibody 25 (anti-CD40L Fab-NTIG) in which the framework part except for an antigenic 2024205586   07 Aug 2024 determinant of Fab, which is the antigen-binding fragment of the antibody that is the base of the present invention, was substituted with the sequence of the human antibody (Tables 1 and 2), and the humanized antibody was named “PG-400”.

[00148] Furthermore, the present inventors designed to prepare a bispecific 5 hybrid antibody (anti-CD40L Fab-NTIG-IL-lOVm) in which the IL-10 monomeric variant protein (SEQ ID NO: 56), which is a cytokine having an immunosuppressive activity, was linked to the heavy chain C-terminus of the hybrid antibody (Table 3), and the bispecific hybrid antibody was named “PG-410”. The structure of the light chain of the bispecific hybrid antibody is the same as shown in Table 2. 10

[00149] [Table 1] Configuration of heavy chain of humanized anti-CD40L- NTIG hybrid antibody Constituent element Amino acid sequence SEQ ID NO: Anti-CD40L heavy chain Vh-CH1 QVQLVQ S GAE WKPGAS VKL S CKAS GYIFT S YYMY WVKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATL TVDKSASTAYMELSSLRSEDTAVYYCTRSDGRNDM DSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKV 64 hinge+NTIG EPKSCDKTHT CPPCPSHTQP LGVFLFPPKP KDQLMISRTP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV LHEALHNHYT QKSLSLSLG 68

[00150] [Table 2] Configuration of light chain of humanized anti-CD40L- NTIG hybrid antibody Constituent element Amino acid sequence SEQ ID NO: Light chain of DIVLTQSPATLSVSPGERATISCRASQRVSSSTY 67 2024205586   07 Aug 2024 anti-CD40L antibody SYMHWYQQKPGQPPKLLIKYASNLESGVPARFSG SGSGTDFTLTISSVEPEDFATYYCQHSWEIPPTF GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

[00151] [Table 3] Configuration of heavy chain of bispecific anti-CD40L- NTIG hybrid antibody Constituent element Amino acid sequence SEQ ID NO: Anti-CD40L heavy chain Vh-CH1 QVQLVQ S GAEWKPGASVKL SCKASGYIFTS YYMYW VKQAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTV DKSASTAYMELSSLRSEDTAVYYCTRSDGRNDMDSW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV 64 hinge+NTIG EPKSCDKTHT CPPCPSHTQP LGVFLFPPKP KDQLMISRTP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV LHEALHNHYT QKSLSLSLG 68 Linker peptide AAGSGGGGGS GGGGSGGGGS 26 IL-lOVm SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTF FQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFY LEEVMPQAENQDPDAKAHVNSLGENLKTLRLRLRRC HRFLPCENGGSGGSGGSKSKAVEQVKNAFNKLQEKG IYKAMSEFDIFINYIEAYMTMKIRN 56

[00152] In order to produce humanized anti-CD40L hybrid antibodies (PG400 and PG-410), a polynucleotide encoding each of the heavy chain and the light 5 chain of the antibody was prepared, and then for temporary expression in an animal cell, plasmid DNA was transfected into a HEK293F cell using the N293F vector system (Y-BIOLOGICS). In order to prepare a nucleic acid, 25 pg of plasmid DNA 2024205586   07 Aug 2024 was added to and mixed with 3 ml of the medium, and then 25 pl of 2 mg / ml Polyethylenimine (PEI, PolyPlus, USA) was added thereto, followed by mixing. The reaction solution was allowed to stand at room temperature for 15 minutes, and then put into 40 ml of a culture solution cultured at lx 106 cells / ml, and the resultant was 5 cultured for 24 hours under conditions of 120 rpm, 37°C, and 8% CO2. 24 hours after DNA transfection, a nutritional supplement medium component (Soytone, BD, USA) was added so that the final concentration was 10 g / L. After culturing for 7 days, the cell culture solution was centrifuged at 5000 rpm for 10 minutes to collect a supernatant. 10

[00153] Subsequently, a protein A resin filled a column and was washed with IxDPBS, the collected supernatant was combined with the resin at 4°C at a rate of 0.5 ml / min, and a protein was eluted with 0.1 M glycine. In order to replace a buffer solution, the eluted solution was put into a dialysis tube (GeBAflex tube, Geba, Israel) and dialyzed at 4°C with IxDPBS. The obtained substance was subjected to 15 gel filtration using a Superdex 200 resin, and then formulated with a PBS (pH of 7.4) buffer solution.

[00154] For the purified hybrid antibody, the yield and the purity of the hybrid antibody were assayed through SDS-PAGE analysis, size-exclusion FPLC, and sizeexclusion HPLC analysis. 20

[00155] As a result, as shown in FIGS. 21a, 21b, 22a, and 22b, it could be found that PG-400 and PG-410 to which the humanized anti-CD40L hybrid antibodies of the present invention were applied were produced with high purity.

[00156] 4-4: Design of anti-CD40L scFv-NTIG-IL-10 fusion protein

[00157] The present inventors sorted out a variable region moiety in the Fab of 25 the humanized anti-CD40L antibody, designed scFv using the sorted-out variable 2024205586   07 Aug 2024 region moiety, designed a single chain-based antibody binding-fragment fusion protein by inserting scFv instead of the Vh-CH1 moiety of the heavy chain construct of the anti-CD40L antibody used in Example 4-3, and designed a bispecific fusion protein in which the IL-10 monomeric variant (SEQ ID NO: 56) used in Example 4-3 5 was linked to the C-terminus of the anti-CD40L scFv-NTIG through the linker peptide (SEQ ID NO: 26) (Table 4). In the present Example, the anti-CD40L scFv is composed of the light-chain variable region (Vl), the linker, the heavy-chain variable region (Vh) in this order, but conversely, an anti-CD40L scFv, which is composed of Vh, the linker, and Vl in this order, may be used because the CD40L-binding ability 10 is maintained even in a case of using the latter anti-CD40L scFv, and also regarding the type of linker, various types can be used.

[00158] [Table 4] Configuration of anti-CD40L scFv-NTIG-IL-10 fusion protein Constituent element Amino acid sequence SEQ ID NO: Signal sequence MGWS C11L FLVATAT GVH S 62 Anti-CD40L scFv Vl DIVLTQSPATLSVSPGERATISCRASQRVSSSTYSYMH WYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFT LTISSVEPEDFATYYCQHSWEIPPTFGGGTKLEIKRTV AAPSV 69 Linker 1 GSTSGSGKPGSGEGS 48 Vh QVQLVQ S GAEWKPGASVKL SCKASGYIFTS YYMYWVK QAPGQGLEWIGEINPSNGDTNFNEKFKSKATLTVDKSA STAYMELSSLRSEDTAVYYCTRSDGRNDMDSWGQGTLV TVSS 70 hinge+NTIG EPKSCDKTHT CPPCPSHTQP LGVFLFPPKP KDQLMISRTP EVTCVWDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRWSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 68 2024205586   07 Aug 2024 LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV LHEALHNHYT QKSLSLSLG Linker 2 AAGSGGGGGS GGGGSGGGGS 26 IL-lOVm SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQ MKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEV MPQAENQDPDAKAHVNSLGENLKTLRLRLRRCHRFLPC ENGGSGGSGGSKS KAVE QVKNAFNKLQEKGIYKAMS E F DIFINYIEAYMTMKIRN 56

[00159]     Example 5: Preparation of heterodimer

[00160] The present inventors designed bispecific fusion proteins containing both the GLP-1 analog and the GLP-2 analog as shown in Table 5. In these 5 bispecific fusion proteins, the knobs-into-holes (KIH) structure is introduced so as to form a heterodimer by an intermolecular disulfide bond and a hydrophobic interaction between the first fusion protein and the second fusion protein.

[00161] A difference between Examples 5-1 and 5-2 is whether an N-linked sugar chain attachment site is included so that an N-glycan sugar chain can be linked 10 to the linker linking the GLP-1 analog of the first fusion protein to NTIG. This is because GLP-1 as well as Exendin 4 (Ex4), oxyntomodulin (OXM), GLP-2 all bind to GLP-1 R which is a GLP-1 receptor, whereas only GLP-2 binds to GLP-2R, and GLP-1 R is expressed in various organs such as a brain, a heart, a liver, muscle, and pancreas in addition to gastrointestinal tract (GI tract), and thus it is necessary to 15 lower the affinity with GLP-1 R somewhat in order to allow GLP-1 to function specifically in the gastrointestinal tract. For the reason, in order to lower the binding force of the GLP-1 analog to GLP-1 R somewhat, the present inventors used a protein to which a glycan attachment domain was added so that glycan can be attached to the linker moiety (including the IgD / IgGl hybrid hinge) of the GLP-1 analog (Example 20   5-1). 2024205586   07 Aug 2024

[00162] [Table 5] Design of fusion protein for formation of GLP-l / GLP-2 heterodimer according to one exemplary embodiment of present invention Example Name GLP-1 / 2 analog (SEQ ID NO:) Linker (SEQ ID NO:) Fc region Overall configuration (SEQ ID NO:) 5-1 MG 12-4 1. GLP-1 / Exendin 4 hybrid (49) Glycan linker (21) knob First fusion protein (71) 2. GLP-2-2G (52) Non-glycan linker (14) hole Second fusion protein (72) 5-2 MG12-5 1. GLP-1 / Exendin 4 hybrid (49) Non-glycan linker (14) knob First fusion protein (73) 2. GLP-2-2G (52) Non-glycan linker (14) hole Second fusion protein (72)

[00163] The vector constructs prepared as above were transiently expressed using the ExpiCHO kit manufactured by Thermo Fisher Scientific Solutions LLC. 5 Specifically, the vector constructs prepared as above and the ExpiFectamine reagent contained in the kit were mixed in the ExpiCHO-S cell, the resultant was cultured for 1 day in an incubator with conditions of 8% CO2 and 37°C, then the temperature was lowered to 32°C, and the culture was performed until the 7th day.

[00164] The supernatant obtained through the culturing and the heterodimer 10 proteins (were named ‘MG 12-4’ and ‘MG 12-5’, respectively) of Examples 5-1 and 5-2 purified through a protein A column and a secondary column were appropriately diluted with a 4X LDS sample buffer solution and water for injection to prepare a sample so that the final concentration was 3 to 10 pg / 20 pL. In a case of a sample under the reducing conditions, each substance to be analyzed, the 4X LDS sample 15 buffer solution, a 10X reducing agent, and the water for injection were appropriately diluted to prepare a sample so that the final concentration was 3 to 10 pg / 20 pL, and the sample was heated in a 70°C heating block for 10 minutes. The prepared sample was loaded in an amount of 20 pL in each well of a gel fixed in an electrophoresis 2024205586   07 Aug 2024 apparatus which was installed in advance. For a size marker, the sample was loaded at 3 to 5 p.L / well. After a power supply device was set to 120 V and 90 minutes, electrophoresis was performed. The gel subjected to the electrophoresis was separated, and then stained using a staining solution and a destaining solution, and 5 the results were analyzed. As a result, as shown in FIG. 23, it could be confirmed that the heterodimer according to one exemplary embodiment of the present invention was normally produced.

[00165] Example 6: Analysis of effect of hinge in fusion protein production 10

[00166] The present inventors investigated how the production efficiency of the protein varies depending on the type of hinge when API is linked to the N-terminus of the NTIG protein, while linking various types of API to the N-terminus and / or the C-terminus of the NTIG protein of the present invention.

[00167]     6-1: Preparation of Fc£RIa-NTIG-IL-13a2 15

[00168] The present inventors designed a bispecific fusion protein (FcsRIa- NTIG-IL-13Ra2) in which FcsRIa (SEQ ID NO: 58) was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-13Ra2 (SEQ ID NO: 74) was linked to the C-terminus, and the bispecific fusion protein was named “PG-50”. While producing PG-50, a hinge region linking FcsRIa to NTIG was investigated for protein 20 productivity when using an IgD hinge (SEQ ID NO: 15) or a combination of a (GiSjs linker and an IgGl hinge (SEQ ID NO: 16). In this way, a fusion protein using the IgD hinge was named “PG-50-1”, and a fusion protein using the (GiSjs linker / IgGl hinge was named “PG-50-2”. In a case of the IgD hinge, one intermolecular disulfide bond was formed, and in a case of the (GiSjs linker / IgGl hinge, two 25 intermolecular disulfide bonds were formed. 2024205586   07 Aug 2024

[00169] As a result, as shown in FIGS. 24a and 24b, it was found that the production yield for the target material of the fusion protein (PG-50-2) containing the IgGl hinge was increased by about 15% compared to the PG-50-1 containing the IgD hinge. 5   

[00170]     6-2: Preparation of GLP-2-2G-NTIG-IL-10wt

[00171] The present inventors analyzed the effects of various types of hinges on a protein production yield using different types of API. For the analysis, specifically, a construct was prepared in which GLP-2-2G (SEQ ID NO: 52) was linked to the N-terminus of NTIG (SEQ ID NO: 7) and IL-lOwt (SEQ ID NO: 75) 10 was linked to the C-terminus of NTIG. Here, a bispecific fusion protein (6LP-2-26- NTIG-IL-lOwt) was designed by linking GLP-2-2G to NTIG using a (648)3 linker+IgD / IgGl hybrid hinge (SEQ ID NO: 14), and linking IL-lOwt to the C-terminus of NTIG using a linker peptide consists of the amino acid sequence set forth in SEQ ID NO: 26, and was named “PG-073”. A polynucleotide encoding the fusion 15 protein designed as above was prepared and then inserted into the pBispec expression vector, a fusion protein was expressed using the ExpiCHO system according to the method used in Example 2-1, and then the protein expression level in the culture supernatant was checked by SDS-PAGE. The IgD / IgGl hybrid hinge forms two intermolecular disulfide bonds. 20

[00172] As a result, as shown in FIG. 24c, in a case of PG-073, the size of a main band was 110 kDa under the non-reducing conditions, and it was confirmed that a bispecific fusion protein to which both GLP-2-2G and IL-10 were linked was formed. However, a band in which one or both of the GLP-2 were cleaved was found below the main band. Therefore, the (648)3 linker+IgD / Ig61 hybrid hinge 25 linker used between 6LP-2 and NTI6 was found to be somewhat unstable. 2024205586   07 Aug 2024 Moreover, the amount of the fusion protein produced based on 100 ml of the culture solution was only 0.5 mg (PG-073) and thus protein productivity was decreased, and the purity was also about 66%, which is low.

[00173]     6-3 and 6-4: Preparation of GLP-2-2G(3 point)-NTIG-IL-10Vm 5

[00174] Accordingly, the present inventors designed fusion proteins (GLP-2V (3 point)-NTIG-IL-10Vm) in which in the GLP-2-2G-NTIG-IL-10wt fusion protein, GLP-2-2G was changed to a mutant (SEQ ID NO: 76) containing a 3 point mutation, the hinge portion was replaced with a different type of linker or hinge (each SEQ ID NO: 17 or 18), and IL-lOwt was replaced with IL-lOVm prepared in Example 2-4, 10 and the fusion proteins were named “PG210-5 (Example 6-3)” and “PG210-6 (Example 6-4), respectively.

[00175] After a polynucleotide encoding each of the fusion proteins was inserted into the pBispec expression vector, a fusion protein was expressed using the ExpiCHO system according to the method used in Example 2-1, and then the protein 15 expression level in the culture supernatant was checked by SDS-PAGE.

[00176] As a result, as shown in FIGS. 24d and 24e, in a case of PG-210-5, 3.48 mg of protein was found based on 200 ml of the culture solution and the purity was found to be about 88.3%, and in a case of PG-210-6, 5 mg of protein was found and the purity was found to be 90.7%. 20   

[00177] Experimental Example 1: Analysis of FcRn-binding affinity

[00178] The present inventors compared the binding affinity of the NTIG protein of the present invention with FcRn to IgG4 Fc and IgGl antibodies.

[00179] For the comparison, specifically, the recombinant FcRn (rhFcRn, R&D systems, USA), which is a ligand, was dissolved in an acetate buffer at a 25 concentration of 5 pg / ml, and then fixed to a biosensor chip (Series S CM5 sensor 2024205586   07 Aug 2024 chip, GE Healthcare, USA) using amine coupling.

[00180] Subsequently, in order to compare the FcRn-binding affinity of NTIG of the present invention and the FcRn-binding affinity of IgG4 Fc, the present inventors analyzed, through surface plasmon resonance (SPR), the binding affinity 5 of, with FcRn, PG-11 prepared in Example 2-1 and dulaglutide (trade name Trulicity) which is a fusion protein in which commercial GLP-1 is linked to IgG4 Fc. As the analyzer, Biocore 8K (GE Healthcare, USA) was used, and all reagents used in the analysis were purchased from GE Healthcare. PG-11 and Trulicity were dissolved in a running buffer solution (PBST, pH of 6.0), then dilution was 10 sequentially performed from 1,000 nM by 1 / 2, and multi-cycle kinetic analysis was performed. The experiment was performed at a flow rate of 30 pl / min at 25°C, an association of an analyte was performed for 60 seconds, and then dissociation was performed for 60 seconds. Based on the results of the experiment, the optimal antibody concentration was selected (Trulicity: 8,000 nM and PG-11: 2,000 nM), 15 multi-cycle kinetic analysis was performed, and the equilibrium dissociation constant (Kd) was measured using the Biacore Insight program (GE Healthcare, USA). At this time, the buffer solution was replaced with a PBS buffer solution having a pH of 7.4 during regeneration.

[00181] As shown in Table 6 and FIGS. 25 and 26, as a result of the 20 comparative analysis of steady state affinity Kd, the Kd value of Trulicity was 2,090 nM and the Kd value of PG-11 was 199 nM, and the result indicates that the affinity of PG-11 according to one exemplary embodiment of the present invention with FcRn is about 5.3 times higher than that of Trulicity. The binding affinity with FcRn is known to be related to the pharmacokinetics of the Fc fusion protein, and 25 accordingly, the half-life in vivo of PG-11 is expected to be longer compared to 2024205586   07 Aug 2024 Trulicity (Mackness et al., MAbs. 11(7): 1276-1288, 2019).

[00182] [Table 6] Analysis results of affinity of PG-11 and Trulicity with FcRn Analyte PG-11 Trulicity Steady state affinity (Kd) 397±19.3 nM 2,090±75.5 nM CV% (Steady state affinity, Kd) 4.86 3.61

[00183] In addition, in order to compare the FcRn-binding affinity of NTIG of 5 the present invention and the FcRn-binding affinity of IgG4 Fc, the present inventors analyzed, through surface plasmon resonance (SPR), the binding affinity of, with FcRn, PG-11 and Rituximab which is a commercial recombinant chimeric anti-CD20 antibody of an IgGl family. PG-11 and Rituximab were dissolved in a running PBST buffer solution (pH of 6.0) at various concentrations (2,000 nM to 1.96 nM for 10 PG-11, and 4,000 nM to 3.9 nM for Rituximab), and then similar to the comparative analysis of PG-11 and Trulicity, association, dissociation, and regeneration were performed under the conditions of 60 seconds for the association, 60 seconds for the dissociation, and 30 seconds for the regeneration. During the regeneration, a PBS buffer solution having a pH of 7.4 was used. 15

[00184] As shown in Table 7 and FIG. 27, as a result of the comparative analysis of steady state affinity Kd, the Kd value of Rituximab was 2,390 nM and the Kd value of PG-11 was 256 nM, and the result indicates that the affinity of PG-11 according to one exemplary embodiment of the present invention with FcRn is about 9.3 times higher than that of Rituximab. 20

[00185] [Table 7] Analysis results of affinity of PG-11 and Rituximab with FcRn Analyte PG-11 Rituximab Steady state affinity (Kd) 256 nM 2,390±242 nM 2024205586   07 Aug 2024 CV% Steady state affinity (Kd) - 10.15

[00186] As described above, NTIG according to one exemplary embodiment of the present invention has high affinity with FcRn compared to the Fc region of conventional IgGl or IgG4, and thus when NTIG is administered in vivo, the half-life is expected to be significantly improved. 5

[00187] Subsequently, the present inventors intended to compare the affinity of the modified IgG4 Fc domain protein of SEQ ID NO: 2 with FcRn, on the basis of NTIG (SEQ ID NO: 7) according to one exemplary embodiment of the present invention.

[00188] For the comparison, instead of calculating a simple Kd value, the 10 present inventors calculated a steady state affinity (Kd) by associating the proteins at a pH of 6.0 and performing dissociation at a pH of 7.3 (Piche-Nicholas et al., MAbs. 10(1): 81-94, 2018). Specifically, for the binding affinity of anti-PD-Ll-NTIG-IL-2 and anti-PD-Ll-modified IgG4 Fc-IL-2 prepared in Example 3-1 with rhFcRn, SPR analysis was performed by changing the pH conditions during the association and the 15 dissociation as described above.

[00189] As a result, as shown in Table 8 and FIG. 28, it could be found that the affinity, with the FcRn, of anti-PD-Ll-NTIG-IL-2 to which NTIG was applied tended to be higher than that of anti-PD-Ll-modified IgG4 Fc-IL-2 using the modified IgG4 Fc. However, in the exchange process of the binding buffer solution, 20 a large amount of protein aggregates were generated in the anti-PD-Ll-modified IgG4 Fc-IL-2 protein sample, and thus there was a problem that the reliability of the results was poor. Accordingly, the same experiment was performed again by changing a test material to a ligand and FcRn to a sample.

[00190] These results suggest that the application of NTIG exhibits an effect 2024205586   07 Aug 2024 of enhancing the half-life in vivo through enhancement of the FcRn-b inding force of the produced protein as well as a superior effect on the physical properties the and stability of the protein, compared to the previously known application of the modified IgG4 Fc of SEQ ID NO: 2. 5

[00191] [Table 8] Analysis results of affinity of NTIG and modified IgG4 Fc with FcRn Ligand Analyte Binding ratio ka (1 / ms) kd (1 / s) Kd (M) Steady state affinity Kd (M) Chi2 Rmax %bound FcRn Anti-PD-Ll-NTIG-IL-2 1:1 1.99E +06 5.43 E-01 2.73 E-07 2.95E-07 6.57E-02 7.1 2.04 FcRn Anti-PD- Ll-modified IgG4 Fc-IL-2 1:1 4.37E +06 1.98 E+00 4.5307 8.41-07 1.16E- 01 5 12.12

[00192] As the buffer solution used for the analysis, the running buffer solution (IX PBST, pH of 7.3), the sample dilution buffer solution (IX PBST, pH of 6.0), the dissociation buffer solution (IX PBST, pH of 7.3), and the regeneration 10 buffer solution (IX PBS, pH of 8.0) were used.

[00193] As a result of analysis, as shown in Table 9 and FIG. 29, it was found that both proteins exhibited fast binding and rapid dissociation patterns, both proteins tended to dissociate well from the binding to FcRn under neutral conditions (pH of 7.3), but under the condition of a pH of 6.0, the affinity of the anti-PD-Ll-NTIG-IL- 15   2 protein was 5.06 times higher than that of the anti-PD-Ll-modified IgG4 Fc-IL-2 protein. Consequently, the anti-PD-Ll-NTIG-IL-2 protein showed a tendency to rapidly bind even to a lower concentration of FcRn under the condition of a pH of 6.0, compared to the anti-PD-Ll-modified IgG4 Fc-IL-2 protein. Considering that 2024205586   07 Aug 2024 the Fc-containing protein absorbed into the cell by the endocytosis binds to FcRn under the low pH condition of the endosome, the results strongly suggest that the fusion protein containing NTIG would be more efficiently recycled by FcRn.

[00194] [Table 9] Analysis results of affinity of NTIG and modified IgG4 Fc 5 with FcRn (switching between ligand and analyte) Ligand Analyte Binding ratio Steady state affinity Kd(M) Fixation level (RU) Relative affinity Anti-PD-Ll- NTIG-IL-2 FcRn 1:1 3.91E-07 (397 nM) 242.1 5.06 Anti-PD-Ll-modified IgG4 Fc-IL-2 FcRn 1:1 1.98E-06 (1,980 nM) 236.5 -

[00195] Experimental Example 2: Analysis of binding affinity with various Fey receptors

[00196] The Fc region of IgGl is known to have binding affinity with Fey RI and FcyRIIIa. These Fc receptors are associated with effector functions such as 10 ADCC and CDC, are helpful in anticancer treatment when using antibodies or Fc fusion proteins for the anticancer treatment, but have disadvantages in which side effects are caused when using API for the purpose of treating other diseases other than anti-cancer treatment.

[00197] Accordingly, the present inventors analyzed the degree of the affinity 15 of the NTIG fusion protein according to one exemplary embodiment of the present invention with Fey RI and FcyRIIIa compared to the IgGl antibody through bio-layer interferometry analysis (BLI).

[00198] As a result, as shown in FIG. 30, it was found that Rituximab, which is an IgGl-based antibody, specifically bound to FcyRI and FcyRIIIa, whereas PG20   11, which is the NTIG-containing fusion protein according to one exemplary embodiment of the present invention, did not specifically bind to these antibody 2024205586   07 Aug 2024 receptors.

[00199] Furthermore, the present inventors performed comparative analysis of the binding affinity of Rituximab and PG-11 according to one exemplary embodiment of the present invention with FcyRIIa, FcyRIIb, and FcyRIIIb. 5

[00200] As a result, as shown in FIG. 31, it was found that Rituximab specifically bound to the three Fc receptors. Meanwhile, PG-11 according to one exemplary embodiment of the present invention showed weak binding, but considering the shape of the sensogram, the binding is considered to be non-specific binding. 10   

[00201] Experimental Example 3: Analysis of binding to complement Clq

[00202] The antibody Fc region may induce complement-dependent cytotoxicity (CDC) by binding to the complement Clq, and thus may cause a serious problem in the clinical application of the Fc fusion protein for treating general diseases other than cancer treatment. Therefore, the degree of the binding affinity of 15 the NTIG-containing fusion protein according to one exemplary embodiment of the present invention with the complement Clq was analyzed compared to Rituximab, which is an IgGl-based antibody.

[00203] For the comparison, specifically, PG-11 and Rituximab were each diluted with a coating buffer solution (0.1 M Na2HPO4, pH of 9.6) so that each 20 concentration was 2 pg / ml. Subsequently, 100 pl of each diluted sample was loaded into each well and reacted overnight at 4°C. Next, washing was performed three times by adding 300 pl of a washing buffer solution to the plate each time, 200 pl of a blocking buffer solution (2% BSA / PBS) was added to each well and reacted at room temperature for 2 hours. 300 pl of the washing buffer solution was added to 25 the plate, then washing was repeatedly performed three times, an anti-Clq antibody 2024205586   07 Aug 2024 was diluted with an assay buffer solution tolO, 3, 1, 0.3, and 0.1 pg / ml by 1 / 3, and mixed (duplication) in an amount of 50 pl in each well, and then the resultant was reacted at room temperature for 2 hours. Then, the washing buffer solution was mixed in the plate by 300 pl each time and washing was repeatedly performed eight 5 times. A Clq-HRP conjugate was diluted 400-fold, and 50 pl of the resultant was added to each well and reacted at room temperature for 1 hour. The washing buffer solution was added to the plate by 300 pl each time, washing was repeatedly performed eight times, 50 pl of TMB as a substrate was mixed, and the resultant was reacted at room temperature. When the color development reaction appeared in blue, 10   50 pl of a stop solution was added to each well to stop the reaction, and then optical density was measured using a microplate reader set at 450 nm.

[00204] As a result of the analysis, as shown in FIG. 32, it was found that Rituximab bound to Clq in a concentration-dependent manner, whereas PG-11 according to one exemplary embodiment of the present invention did not bind to 15 Clq.

[00205] Therefore, it was confirmed that NTIG according to one exemplary embodiment of the present invention is a safe protein carrier capable of minimizing side effects due to the Fc region such as ADCC or CDC while improving the half-life of API. 20   

[00206] Experimental Example 4: Analysis of GLP-2 activity

[00207] Whether the GLP-2 activity was properly exhibited with respect to a number of fusion proteins containing GLP-2 among Examples described above was investigated by cAMP Hunter™ eXpress GPCR analysis (Table 10).

[00208] [Table 10] Constructs used for GLP-2 activity analysis Example Code Construct structure 2-2 PG-22 GLP-2G-NTIG 2024205586   07 Aug 2024 5-1 MG 12-4 GLP-1E / GLP-2G-NTIG (GLP-1 w / glycan linker) 5-2 MG12-5 GLP-1E / GLP-2G-NTIG (GLP-1 w / non-glycan linker) 6-3 PG-210-5 GLP-2V(3 point)-NTIG-IL-10Vm (Patent linker) 6-4 PG-210-6 GLP-2 V(3 point)-NTIG-IL-10Vm (GS+IgGl hinge linker)

[00209] As a result of analysis, as shown in FIG. 33 and Table 11, it was found that PG-210-6 among the fusion proteins prepared according to one Example of the present invention exhibited a GLP-2 activity almost similar to those of PG-22 5 as Comparative Example, and PG-210-5 of another Example of the present invention exhibited an activity which was about half an activity of PG-210-6. MG 12-4 and MG 12-5, which are heterodimers, were also found to exhibit a GLP-2 activity which was only about half the GLP-2 activity of PG-22, but since MG 12-4 and MG 12-5 each contain one GLP-2 molecule, in case of MG 12-4 and MG 12-5, a 50% decrease 10 in activity compared to PG-22 in which two GLP-2 molecules bound could be predicted. PG-210-5 was a protein to which the linker peptide used in the prior patent literature (US7,812,121B2) was applied, and it was found that the activity of PG-210-6 using the mixed linker peptide of (G4S)3+IgGl hinge used in the present invention was excellent. 15

[00210] [Table 11] Results of GLP-2 activity analysis Example 2-2 5-1 5-2 6-3 6-4 First ECso (nM) 2.84 5.81 5.49 7.44 2.46 First relative activity (%) 100% 48.8% 51.7% 38.1% 115% Second EC50 (nM) 2.65 6.08 4.85 5.95 2.93 Second relative activity (%) 100% 43.5% 54.6% 44.5% 90% Average activity (%) 100% 46.15% 53.15% 41% 102.5%

[00211] Experimental Example 5: Analysis of immunosuppressive activity of IL-10Vm fusion protein

[00212] 5-1: Analysis of affinity with IL-10 receptor 2024205586   07 Aug 2024

[00213] The present inventors analyzed the affinity of the fusion proteins of Examples 2-3 and 2-4 with the IL-10 receptor 1 (IL-10R1) through the bio-layer interferometry analysis (BLI).

[00214] For the analysis, the present inventors attached an IL-10R His-tag 5 protein to a 96-well plate using a Dip and Read™ Amine Reactive 2nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, 200 pl of D. W. was dispensed into a 96-well plate, and an amine biosensor included in the kit was inserted to perform hydration for 10 minutes. Subsequently, after 200 pl of D. W. was additionally dispensed into a new 96-well plate, EDC and NHS were mixed at 10   1:1 in an amount of 1 / 20 of the required sample amount and then diluted with D. W., and 200 pl of the resultant was dispensed into the 96-well plates. Next, the IL-10R His-tag protein was diluted to 10 pg / ml in a 10 mM acetate solution having a pH of 5.0, and then 200 pl of the resultant was added to the 96-well plates. After 200 pl of 1 M ethanolamine was added to the 96-well plates, the biosensor plate and the 15 sample plate were placed in an Octet®-K2 instrument and measured. After the measurement was completed, 200 pl of a 1 x kinetics buffer solution was added to the sample plate, and then a baseline was determined. Thereafter, the prepare NTIG-IL-10 fusion protein was diluted with the lx kinetics buffer solution to various concentrations (0, 62.5, 125, 250, 500, and 1,000 nM), 200 pl of the resultant was 20 dispensed to the sample plate, and then bio-layer interferometry (BLI) was measured using the Octet®-K2 instrument.

[00215] [Table 12] Parameter Example 2-3 Example 2-4 Kd (nM) 11.H0.9 29.2±8.85 Kon (1 / MS) 71100±6670 20550±2312 Kiis (1 / s) 0.0008 0.0006 R2 0.97 0.97 2024205586   07 Aug 2024

[00216] As a result, as shown in FIG. 34 and Table 12, the Kd value of the fusion protein (NTIG-IL-lOVm) according to Example 2-4 of the present invention was 29.2 nM, which was about three times the Kd value (11.1 nM) of the dimeric IL-10 fusion protein of Example 2-3. The result may be interpreted that the monomeric 5   IL-10 variant fusion protein has a lower affinity with the receptor than the dimeric IL-10 protein, but it could be predicted that the affinity could be reduced due to the monomer formation.

[00217]     5-2: Analysis of immunosuppressive activity of IL-lOVm fusion protein 10

[00218] The present inventors analyzed immunosuppressive activities in vitro of various fusion proteins (PG-088m, PG-110, PG-410, and PG-210-6) containing the monomeric IL-10 variant protein (IL-lOVm) according to one exemplary embodiment of the present invention.

[00219] For the analysis, specifically, after a supernatant was removed from a 15   T75 flask including a macrophage Raw264.7 in culture, washing was performed once with PBS, then 3 ml of TrypLE™ Select Enzyme was added, and the resultant was reacted at 37°C for 3 to 5 minutes. Thereafter, 10 ml of a fresh medium was added to the T75 flask, and a cell suspension from the bottom of the flask was transferred to a 50 ml conical tube and then centrifuged at 1,500 rpm for 5 minutes. Next, the 20 supernatant was removed and the tube was gently tapped to release cells. Then, 5 ml of a fresh medium were mixed and the cells were counted. The cell concentration was adjusted to be lx 105 cells / ml, and 100 pl of the resultant was dispensed into a 96-well flat-bottom plate.

[00220] Thereafter, PG-088m prepared in Example 2-4, PG-110 prepared in 25 Example 3-3, PG-410 prepared in Example 4-3, and PG-210-6 prepared in Example 2024205586   07 Aug 2024 6-4 were sequentially diluted to various concentrations (0 to 1,000 nM for PG-210-6 and PG-410, and 0 to 1,507 nM for PG-110 and PG-088m), and then 50 pl of each protein was dispensed into the 96-well plate into which the macrophage Raw264.7 was dispensed. After 20 minutes, a lipopolysaccharide (LPS) was treated in an 5 increment of 50 pl so that the final concentration was 400 ng / ml, and the resultant was cultured at 36°C for 16 hours. The next day, the supernatant was collected, and then TNF-a ELISA (Biolegend, USA) analysis was performed according to the protocol of the manufacturer.

[00221] As a result, as shown in FIGS. 35a to 35c, all fusion proteins 10 containing IL-lOVm according to one exemplary embodiment of the present invention were found to inhibit TNF-a secretion in macrophages in a concentrationdependent manner. The significant difference according to the type of fusion protein was not found. The results indicate that the monomeric IL-10 variant protein (IL-lOVm) according to one exemplary embodiment of the present invention properly 15 works even when the monomeric IL-10 variant protein is linked to the C-terminus of the NTIG protein.

[00222]      5-3: Analysis of IgE-binding ability

[00223] The present inventors analyzed the affinity of the fusion protein (FcaRIa-NTIG-IL-lOVm) prepared in Example 3-3 with mouse IgE and human IgE 20 through the bio-layer interferometry analysis (BLI).

[00224] For the analysis, the present inventors first attached the FcaRIa-NTIG protein having no IL-lOVm as a control group and the fusion protein prepared in Example 3-3 to a 96-well plate using a Dip and Read™ Amine Reactive 2nd Generation (AR2G) Reagent Kit (forteBio, Cat No. 18-5092). Specifically, 200 pl of 25 D. W. was dispensed into a 96-well plate, and then an amine biosensor included in 2024205586   07 Aug 2024 the kit was inserted to perform hydration for 10 minutes. Subsequently, after 200 pl of D. W. was additionally dispensed, EDC and NHS were mixed at 1:1 in an amount of 1 / 20 of the required sample amount and then diluted with D. W., and 200 pl of the resultant was dispensed into the 96-well plate. Next, the FcaRIa-modified IgG4 Fc 5 protein and the FcaRIa-NTIG-IL-lOVm fusion protein were diluted to 10 pg / ml in a 10 mM acetate solution having a pH of 5.0, and then 200 pl of the resultant was added to the 96-well plate. After 200 pl of 1 M ethanolamine was added to the 96-well plate, the biosensor plate and the sample plate were placed in an Octet®-K2 instrument and measured. After the measurement was completed, 200 pl of a lx 10 kinetics buffer solution was added to the sample plate, and then a baseline was determined. Thereafter, anti-DNP mouse IgE (Sigma) was diluted with the lx kinetics buffer solution to various concentrations (50 pM to 3.125 nM), 200 pl of the resultant was dispensed to the sample plate, and then bio-layer interferometry (BLI) was measured using the Octet®-K2 instrument. 15

[00225] [Table 13] Parameter FcaRIa-modified IgG4 Fc PG-110 (FcaRIa-NTIG-IL-lOVm) First Second Third First Second Third Average Kd (nM) 0.364 0.478 0.940 0.224 0.282 0.346 Average: 0.594 Average: 0.284 Average Kon (1 / MS) 1.23xl05 1.49xl05 1.66xl05 1.52xl05 1.44xl05 1.39xl05 Average Kiis (1 / s) 4.48X10'5 7.10X10'5 1.56X10'4 3.40x10'5 4.06X10'5 4.82xl0'5 Average R2 0.996 0.999 0.997 0.999 0.999 0.999

[00226] As a result, as shown in FIG. 36a and Table 13, the Kd value of the fusion protein (FcaRIa-NTIG-IL-lOVm) according to one exemplary embodiment of the present invention was 0.284 nM, which was slightly lower than that of FcaRIa-modified IgG4 F as a control group. However, the difference is not a significant 2024205586   07 Aug 2024 difference, and thus it was found that the binding ability to IgE was not decreased by the addition of the monomeric IL-10 protein.

[00227] In addition to the above results, in order to measure the binding degree of human FcaRIa and human IgE used in the present invention, the present 5 inventors analyzed the affinity of the fusion protein (PG-110) of Example 3-3 with human IgE using the abovementioned BLI method.

[00228] [Table 14] PG-110 (FcaRIa-NTIG-IL-lOVm) Average Kd (nM) 0.346 Average Kon (1 / Ms) 2.19^105 Average Kais (1 / s) 7.59X10'5 Average R2 0.9955

[00229] As a result, as shown in FIG. 36b and Table 14, the affinity of the fusion protein according to one exemplary embodiment of the present invention with 10 human IgE was also similar to the affinity with mouse IgE.

[00230]      5-4: Analysis of inhibitory activity against IgE

[00231] Subsequently, the present inventors investigated whether the fusion protein (FcaRIa-NTIG-IL-lOVm) prepared in Example 3-3 exhibited inhibitory activity against IgE by an in vitro experiment. 15

[00232] For the investigation, specifically, FcaRIa-modified IgG4 Fc as a control group and the fusion protein (PG-110) of Example 3-3 as an experimental material were sequentially diluted to various concentrations to prepare samples, and the samples were mixed with 1 pl / ml of anti-DNP mouse IgE and reacted at room temperature for 30 minutes. Then, marrow-derived mast cells obtained from a 20 mouse were washed with an HBSS buffer solution, adjusted so that the concentration was 5><105 cells / 60 pl, and then dispensed into 96-well plate. 20 pl of the sample reacted with IgE and the test drug was added to previously prepared mast cells, and 2024205586   07 Aug 2024 the resultant was cultured for 25 minutes in an incubator with conditions of 37°C and 5% CO2. Next, 20 pl of DNP BSA(500 ng / ml) was added, the resultant was cultured again for 30 minutes in the incubator with conditions of 37°C and 5% CO2 and centrifuged at 4°C at a rate of 1,500 rpm, and 30 pl of a supernatant was separated. 5   30 pl of the separated supernatant and 30 pl of a substrate (4-nitrophenyl N-acetyl-P- glucosaminide, 5.84 mM) were mixed and cultured for 25 minutes in the incubator with conditions of 37°C and 5% CO2. 400 pl of a 0.1 M sodium carbonate buffer solution (pH of 10) was mixed and the reaction was terminated. Thereafter, the relative amounts of P-hexominidase secreted from the mast cells were compared by 10 measuring the absorbance at 405 nm, and an effect of suppressing mast cells according to the concentration of each drug was checked. The analysis was independently conducted three times and an average thereof was obtained.

[00233] As a result, as shown in FIG. 37 and Table 15, the fusion protein PG110 according to one exemplary embodiment of the present invention inhibited the 15 activity of IgE in a concentration-dependent manner, the degree thereof was similar to that of FcaRIa-modified IgG4 Fc as the control group, but IC50 of the fusion protein PG-110 according to one exemplary embodiment of the present invention was slightly lower than that of the FcaRIa-modified IgG4 Fc.

[00234] [Table 15] Test material IC50 (nM) First test Second test Third test Fourth test FcaRIa-modified IgG4 Fc 19.58 17.88 22.31 19.92 PG-110 21.64 14.78 14.42 16.947 20   

[00235] Experimental Example 6: Analysis of binding to CD40L

[00236] The present inventors investigated the binding affinity of PG-400 and PG-410 respectively prepared in Examples 4-2 and 4-3 of the present invention with 2024205586   07 Aug 2024 human CD40L through the BLI analysis.

[00237] For the investigation, specifically, 200 pl of distilled water was dispensed onto a 96-well black flat-bottom plate, a biosensor tray was placed on the hydrated black plate, and then an amine biosensor was inserted to perform hydration 5 for 10 minutes. Then, 200 pl of distilled water was dispensed onto a new 96-well black sample plate, EDC and NHS were mixed at 1:1 in an amount of 1 / 20 of the required amount and then diluted with distilled water, and the 200 pl of the resultant was dispensed into the 96-well black sample plate. Next, human CD40L (Peptrotech, Korea) was diluted to 5 pg / ml in a 10 mM acetate solution (pH of 6.0), 10 and then 200 pl of the resultant was added to the 96-well black sample plate. 200 pl of 1 M ethanolamine was added to the plate, the biosensor plate and the sample plate were placed in an Octet®-K2 instrument, and the degree of binding of CD40L was measured on the amine biosensor. After the measurement was completed, the 96-well black sample plate was taken out from the measuring instrument, 200 pl of a 1 x 15 kinetics buffer solution was mixed, and a baseline was set. PG-410 and PG-400, which are analytes, were sequentially diluted with the 1x kinetics buffer solution so that the concentrations were 200 to 12.5 nM, and then 200 pl of the resultant was dispensed into the 96-well black sample plate. The sample plate was placed again in the Octet®-K2 instrument, and then the degree of binding to CD40L was measured 20 by the BLI analysis.

[00238] [Table 16] Analyte PG-400 PG-410 Kd (nM) 0.36 nM <1E-12 Kon(l / Ms) 8.47E+05 1.96E+05 Kdis(l / s) 3.11E-04 <1E-O7

[00239] As a result, as shown in FIG. 38 and Table 16, the fusion protein (PG- 2024205586   07 Aug 2024 410) according to one exemplary embodiment of the present invention had a KOn value for CD40L which is 4.3 times lower than that of the PG-400, but had a Kd value of fM or less (<1E-12), which indicates a very high binding affinity, and consequently, it was confirmed that the PG-400 and PG-410 proteins can bind to 5 human CD40L well. Here, it was found that PG-410, which is the fusion protein (anti-CD40L-NTIG-IL-10Vm) according to one exemplary embodiment of the present invention, bound to CD40L with a very high binding affinity.

[00240] Experimental Example 7: Pharmacokinetic analysis of PG-110

[00241] The present inventors checked the pharmacokinetic profile (PK) by 10 comparing the half-life, the area under the curve (AUC), the highest concentration in the blood (Cmax), and the like of the bispecific fusion protein (PG-110), which was prepared above, according to Example 3-3.

[00242] Specifically, the fusion protein (PG-110) to be tested was administered to three male Sprague Dawley (SD) rats per group by subcutaneous 15   (SC), intravenous (IV), intraperitoneal (IP), and intramuscular (IM) routes at a dose of 1 mg / kg. Blood was obtained prior to injection and after 0.5, 1, 5, 10, 24, 48, 72, 120, 168, 240, and 336 hours after injection, and then stored at room temperature for 30 minutes to cause aggregation. Aggregated blood was centrifuged at 3,000 rpm for 10 minutes, and then a serum of each sample was obtained and stored in a cryogenic 20 freezer. The concentrations of the fusion proteins according to one exemplary embodiment of the present invention in sera were analyzed using an IgG-Fc Quantitation set (Bethyl, Cat# E80-104, Lot# E80-104-30), which can specifically detect Fc among the administered proteins.

[00243] As a result, as shown in FIG. 39, it was found that the physiologically 25 active protein to which NTIG was fused can be maintained in the body for a long 2024205586   07 Aug 2024 time regardless of the administration route.

[00244] The present invention has been described with reference to the abovementioned Examples and Experimental Examples, but the description is merely exemplary, and those skilled in the art could understand that various modifications 5 and other equivalent embodiments can be made therefrom. Therefore, the real technical protection scope of the present invention shall be determined by the technical spirit of the appended claims.

[00245] Industrial availability

[00246] The NTIG protein according to one exemplary embodiment of the 10 present invention is a fusion partner of various biologically active proteins, and increases the therapeutic effect of the biologically active protein by effectively increasing the half-life of the biologically active protein as well as enables safer drug delivery by minimizing effector functions such as ADCC and CDC, which are disadvantages of the Fc fusion protein. Therefore, the NTIG protein according to 15 one exemplary embodiment of the present invention can be effectively used in the preparation of protein medicines.

Claims

1. A fusion protein comprising an antibody mimetic and an immunoglobulin Fc domain variant protein;wherein the immunoglobulin Fc domain variant protein has a T18Q and M196L mutation in the amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4,wherein the antibody mimetic is an Affiibody; Affilin; Affimer; Affitin; Alphabody; Anticalin; Avimer; DARPin; Fynomer; Kunitz domain peptides; a monobody; nanoCLAMP; a variable lymphocyte receptor (VLR); a repebody; or an antigen-binding fragment of an antibody selected from the group consisting of Fab, F(ab’)2, diabody, triabody, sdAb, and VhH.

2. The fusion protein according to claim 1, wherein the immunoglobulin Fc domain variant protein comprises a Knobs-into-Holes structure.

3. The fusion protein according to claim 2, wherein the Knobs-into-Holes structure comprises a Knob structure in which the 10th amino acid of the CH3 domain of the modified IgG4 Fc region, serine (S), is substituted with cysteine (C), and the 22nd amino acid of the CH3 domain, threonine (T), is substituted with tryptophan (W); or the 22nd amino acid of said CH3 domain, threonine (T), is substituted with tryptophan (W), or tyrosine (Y).

4. The fusion protein according to claim 2, wherein the Knobs-into-Holes structure comprises a Hole structure in which the 5th amino acid of the CH3 domain, tyrosine (Y), is substituted with cysteine (C), the 22nd amino acid, threonine (T), is substituted with serine (S), the 24th amino acid, leucine (L), is substituted with alanine (A), and the 63rd amino acid, tyrosine (Y), is substituted with valine (V); or the 22nd amino acid of the CH3 domain, threonine (T), is substituted with serine (S), the 24th amino acid, leucine (L), is substituted with alanine (A), and the 63rd amino acid, tyrosine (Y), is substituted for valine (V); or the2024205586   07 Aug 202463rd amino acid of the CH3 domain, tyrosine (Y), is substituted for threonine (T).

5. The fusion protein according to claim 1, wherein the antibody mimetic is linked to the N-terminus and / or C-terminus of the immunoglobulin Fc domain variant protein.

6. The fusion protein of claim 5, wherein the antibody mimetic is linked to the N-terminus or the C-terminus of the immunoglobulin Fc domain variant protein through a linker peptide or a hinge region of an antibody.

7. A polynucleotide which encodes the fusion protein of claim 1.

8. A recombinant vector comprising the polynucleotide of claim 7.

9. A homodimer comprising the fusion protein of claim 1.

10. A heterodimer comprising the fusion protein of claim 1.

11. A composition comprising, as an active ingredient, at least one selected from the group consisting of the fusion protein of claim 1, the homodimer of claim 9, and the heterodimer of claim 10.

12. A method for treating a disease in a subject, the method comprising a step of administering to the subject, in a therapeutically effective amount, the fusion protein of claim 1, or a homodimer or a heterodimer which contains the fusion protein.

13. Use of the fusion protein of claim 1, or a homodimer or a heterodimer which contains the fusion protein, in the manufacture of a medicament for treating a disease in a subject.

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