Fusion proteins containing IDS and their uses

Abstract

The present invention relates to an asymmetric fusion protein in which an antibody fragment binding to an insulin receptor, an IDS enzyme and an Fc region are fused, and uses thereof. The fusion protein can cross the blood-brain barrier (BBB) ​​to deliver the IDS enzyme to the brain. Therefore, a pharmaceutical composition comprising the fusion protein as an active ingredient can be used as a therapeutic agent for central nervous system diseases, and in particular, it is expected to prevent and treat various diseases caused by ribosome accumulation.

Description

Technical Field

[0001] The present invention relates to a fusion protein comprising iduronate-2-sulfatase (IDS) and uses thereof, and more particularly, to a fusion protein in which an IDS enzyme and an antibody fragment binding to an insulin receptor are fused and uses thereof. Background Art

[0002] Hunter syndrome (Mucopolysaccharidosis II) is one of the diseases occurring in the central nervous system (CNS) and is caused by mutations in iduronate-2-sulfatase (IDS). IDS is an enzyme essential for the decomposition of glycosaminoglycans (GAGs). When the activity of IDS is reduced or lost, GAGs accumulate in tissues and cells, which causes disease. Currently, Hunterase and Elaprase are used as therapeutic agents for Hunter syndrome. However, the problem with these therapeutic agents for central nervous system diseases is that the delivery of therapeutic agents to the brain cannot be achieved correctly because they cannot pass through the blood-brain-barrier (BBB) ​​after being injected into the patient's vein.

[0003] At the same time, cerebrovascular cells limit the transfer of substances between the blood and the brain through the BBB, thereby protecting the brain. Therefore, it is difficult to use substances (such as antibodies or enzymes) that circulate in the body through blood vessels as therapeutic agents for diseases occurring in the central nervous system (CNS), because such substances usually do not pass through the BBB, and therefore their transfer to the brain is limited.

[0004] It was subsequently discovered that transferrin or insulin can be delivered to the brain using a receptor-mediated transcytosis (RMT) mechanism via proteins expressed on the surface of cerebrovascular cells, such as transferrin receptor (TfR) or insulin receptor (IR). This mechanism has been used to develop methods for delivering therapeutic agents to the brain. Recently, a technology has been developed that can deliver a variety of substances (such as IDS therapeutic agents) to the brain using anti-TfR antibodies that can pass through the BBB (Korean Patent Publication Nos. 10-2016-0011198 and 10-2015-0039798).

[0005] Therefore, there is a need to develop IDS therapeutic agents that have high affinity for BBB receptors and excellent BBB crossing efficiency. Summary of the invention

[0006] Technical issues

[0007] Therefore, the present inventors have developed a therapeutic agent for IDS enzyme that can pass through the BBB. Specifically, an object of the present invention is to provide an asymmetric fusion protein in which an antibody fragment binding to an insulin receptor, an IDS enzyme and an Fc region are fused, and uses thereof.

[0008] Solution to the problem

[0009] In order to achieve the above objectives, the present invention provides: a fusion protein comprising: a first domain comprising an antibody fragment that binds to an insulin receptor, a second domain comprising a protein having iduronate-2-sulfatase (IDS) activity, and an Fc region; and a method for preparing the fusion protein.

[0010] In addition, the present invention provides a pharmaceutical composition for preventing or treating central nervous system diseases, which comprises the fusion protein as an active ingredient.

[0011] In addition, the present invention provides a method for preparing a fusion protein, which comprises the following steps: combining an antibody fragment that binds to an insulin receptor with an Fc region to prepare a first monomer; combining iduronate-2-sulfatase (IDS) with a heterodimeric Fc region to prepare a second monomer; and mixing the first monomer with the second monomer.

[0012] In addition, the present invention provides use of the fusion protein for preventing or treating central nervous system diseases.

[0013] In addition, the present invention provides use of the fusion protein in manufacturing a drug for preventing or treating central nervous system diseases.

[0014] In addition, the present invention provides a method for preventing or treating a central nervous system disease, comprising the step of administering the fusion protein or a pharmaceutical composition comprising the fusion protein to an individual.

[0015] Advantageous Effects of the Invention

[0016] The fusion protein according to the present invention can effectively pass through the BBB so that the IDS enzyme therapeutic agent is delivered to the brain. Therefore, the pharmaceutical composition containing the fusion protein as an active ingredient can be used as a therapeutic agent for central nervous system diseases. In particular, it is expected that the pharmaceutical composition can prevent or treat a variety of diseases caused by the accumulation of lysosomes. In addition, from the viewpoint that the fusion protein shows excellent cross-reactivity with animal BBB receptors and human BBB receptors, the advantage of the fusion protein is that the fusion protein that has been applied to a variety of disease animal models can be directly applied to clinical research. <110> MOGAM INSTITUTE FOR BIOMEDICAL RESEARCH <120> Fusion protein containing IDS and its use <130> PCB909076MOG <150> KR 2018 / 0129201 <151> 2018-10-26 <160> twenty two <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> H-CDR1 of fusion protein <400> 1 Ser Gly Phe Thr Phe Ser Ser Tyr Tyr Met Ser 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <223> L-CDR1 of fusion protein <400> 2 Gly Ala Ser Arg Asp Val Ser Ser Tyr Leu Ala 1 5 10 <210> 3 <211> 15 <212> PRT <213> Artificial sequence <220> <223> H-CDR2 of fusion protein <400> 3 Ala Ile Ser Tyr Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 <210> 4 <211> 7 <212> PRT <213> Artificial sequence <220> <223> L-CDR2 of fusion protein <400> 4 Asp Ala Asn Ile Leu Gln Ser 1 5 <210> 5 <211> 14 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 of fusion protein <400> 5 His Pro Ser Tyr Gly Thr Val Asn His Ala Tyr Phe Asp Val 1 5 10 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 of fusion protein <400> 6 Leu Glu Tyr Asn Asn Leu Pro Leu Thr 1 5 <210> 7 <211> 14 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 of fusion protein <400> 7 His Pro Arg Tyr Gly Thr Val Asn His Ala Tyr Phe Asp Val 1 5 10 <210> 8 <211> 9 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 of fusion protein <400> 8 Leu Glu Tyr Pro Asn Leu Pro Leu Thr 1 5 <210> 9 <211> 14 <212> PRT <213> Artificial sequence <220> <223> H-CDR3 of fusion protein <400> 9 His Pro Thr Tyr Gly Thr Val Asn His Ala Tyr Phe Asp Val 1 5 10 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 of fusion protein <400> 10 Leu Glu Tyr Asn Val Leu Pro Leu Thr 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial sequence <220> <223> L-CDR3 of fusion protein <400> 11 Leu Glu Tyr Asn Ser Leu Pro Leu Thr 1 5 <210> 12 <211> 452 <212> PRT <213> Artificial sequence <220> <223> Heavy chain of fusion protein <400> 12 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Tyr Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Pro Ser Tyr Gly Thr Val Asn His Ala Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 13 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of the fusion protein <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Arg Asp Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Asn Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Glu Tyr Asn Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 14 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of the fusion protein <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Tyr Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Pro Arg Tyr Gly Thr Val Asn His Ala Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Arg Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Val Ser Asp Gly Ser Phe Thr Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 15 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of the fusion protein <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Arg Asp Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Asn Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Glu Tyr Pro Asn Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 16 <211> 452 <212> PRT <213> Artificial sequence <220> <223> Heavy chain of the fusion protein <400> 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Tyr Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Pro Ser Tyr Gly Thr Val Asn His Ala Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Arg Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Val Ser Asp Gly Ser Phe Thr Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 17 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Heavy Chain of the Fusion Protein <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Tyr Gly Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Pro Thr Tyr Gly Thr Val Asn His Ala Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Arg Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Val Ser Asp Gly Ser Phe Thr Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly 450 <210> 18 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Light chain of the fusion protein <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Arg Asp Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Asn Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Glu Tyr Asn Val Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 19 <211> 214 <212> PRT <213> Artificial sequence <220> <223> Light chain of the fusion protein <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Arg Asp Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Asn Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Glu Tyr Asn Ser Leu Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 20 <211> 776 <212> PRT <213> Artificial Sequence <220> <223> IDS-Fc Region of the Fusion Protein <400> 20 Ser Glu Thr Gln Ala Asn Ser Thr Thr Asp Ala Leu Asn Val Leu Leu 1 5 10 15 Ile Ile Val Asp Asp Leu Arg Pro Ser Leu Gly Cys Tyr Gly Asp Lys 20 25 30 Leu Val Arg Ser Pro Asn Ile Asp Gln Leu Ala Ser His Ser Leu Leu 35 40 45 Phe Gln Asn Ala Phe Ala Gln Gln Ala Val Cys Ala Pro Ser Arg Val 50 55 60 Ser Phe Leu Thr Gly Arg Arg Pro Asp Thr Thr Arg Leu Tyr Asp Phe 65 70 75 80 Asn Ser Tyr Trp Arg Val His Ala Gly Asn Phe Ser Thr Ile Pro Gln 85 90 95 Tyr Phe Lys Glu Asn Gly Tyr Val Thr Met Ser Val Gly Lys Val Phe 100 105 110 His Pro Gly Ile Ser Ser Asn His Thr Asp Asp Ser Pro Tyr Ser Trp 115 120 125 Ser Phe Pro Pro Tyr His Pro Ser Ser Glu Lys Tyr Glu Asn Thr Lys 130 135 140 Thr Cys Arg Gly Pro Asp Gly Glu Leu His Ala Asn Leu Leu Cys Pro 145 150 155 160 Val Asp Val Leu Asp Val Pro Glu Gly Thr Leu Pro Asp Lys Gln Ser 165 170 175 Thr Glu Gln Ala Ile Gln Leu Leu Glu Lys Met Lys Thr Ser Ala Ser 180 185 190 Pro Phe Phe Leu Ala Val Gly Tyr His Lys Pro His Ile Pro Phe Arg 195 200 205 Tyr Pro Lys Glu Phe Gln Lys Leu Tyr Pro Leu Glu Asn Ile Thr Leu 210 215 220 Ala Pro Asp Pro Glu Val Pro Asp Gly Leu Pro Pro Val Ala Tyr Asn 225 230 235 240 Pro Trp Met Asp Ile Arg Gln Arg Glu Asp Val Gln Ala Leu Asn Ile 245 250 255 Ser Val Pro Tyr Gly Pro Ile Pro Val Asp Phe Gln Arg Lys Ile Arg 260 265 270 Gln Ser Tyr Phe Ala Ser Val Ser Tyr Leu Asp Thr Gln Val Gly Arg 275 280 285 Leu Leu Ser Ala Leu Asp Asp Leu Gln Leu Ala Asn Ser Thr Ile Ile 290 295 300 Ala Phe Thr Ser Asp His Gly Trp Ala Leu Gly Glu His Gly Glu Trp 305 310 315 320 Ala Lys Tyr Ser Asn Phe Asp Val Ala Thr His Val Pro Leu Ile Phe 325 330 335 Tyr Val Pro Gly Arg Thr Ala Ser Leu Pro Glu Ala Gly Glu Lys Leu 340 345 350 Phe Pro Tyr Leu Asp Pro Phe Asp Ser Ala Ser Gln Leu Met Glu Pro 355 360 365 Gly Arg Gln Ser Met Asp Leu Val Glu Leu Val Ser Leu Phe Pro Thr 370 375 380 Leu Ala Gly Leu Ala Gly Leu Gln Val Pro Pro Arg Cys Pro Val Pro 385 390 395 400 Ser Phe His Val Glu Leu Cys Arg Glu Gly Lys Asn Leu Leu Lys His 405 410 415 Phe Arg Phe Arg Asp Leu Glu Glu Asp Pro Tyr Leu Pro Gly Asn Pro 420 425 430 Arg Glu Leu Ile Ala Tyr Ser Gln Tyr Pro Arg Pro Ser Asp Ile Pro 435 440 445 Gln Trp Asn Ser Asp Lys Pro Ser Leu Lys Asp Ile Lys Ile Met Gly 450 455 460 Tyr Ser Ile Arg Thr Ile Asp Tyr Arg Tyr Thr Val Trp Val Gly Phe 465 470 475 480 Asn Pro Asp Glu Phe Leu Ala Asn Phe Ser Asp Ile His Ala Gly Glu 485 490 495 Leu Tyr Phe Val Asp Ser Asp Pro Leu Gln Asp His Asn Met Tyr Asn 500 505 510 Asp Ser Gln Gly Gly Asp Leu Phe Gln Leu Leu Met Pro Gly Gly Gly 515 520 525 Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly 530 535 540 Gly Gly Gly Ser Gly Gly Gly Gly Ser His Thr Cys Pro Pro Cys Pro 545 550 555 560 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 565 570 575 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 580 585 590 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 595 600 605 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 610 615 620 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 625 630 635 640 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 645 650 655 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 660 665 670 Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu 675 680 685 Thr Glu Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 690 695 700 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 705 710 715 720 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 725 730 735 Tyr Ser Trp Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 740 745 750 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 755 760 765 Lys Ser Leu Ser Leu Ser Pro Gly 770 775 <210> 21 <211> 525 <212> PRT <213> Artificial Sequence <220> <223> IDS <400> 21 Ser Glu Thr Gln Ala Asn Ser Thr Thr Asp Ala Leu Asn Val Leu Leu 1 5 10 15 Ile Ile Val Asp Asp Leu Arg Pro Ser Leu Gly Cys Tyr Gly Asp Lys 20 25 30 Leu Val Arg Ser Pro Asn Ile Asp Gln Leu Ala Ser His Ser Leu Leu 35 40 45 Phe Gln Asn Ala Phe Ala Gln Gln Ala Val Cys Ala Pro Ser Arg Val 50 55 60 Ser Phe Leu Thr Gly Arg Arg Pro Asp Thr Thr Arg Leu Tyr Asp Phe 65 70 75 80 Asn Ser Tyr Trp Arg Val His Ala Gly Asn Phe Ser Thr Ile Pro Gln 85 90 95 Tyr Phe Lys Glu Asn Gly Tyr Val Thr Met Ser Val Gly Lys Val Phe 100 105 110 His Pro Gly Ile Ser Ser Asn His Thr Asp Asp Ser Pro Tyr Ser Trp 115 120 125 Ser Phe Pro Pro Tyr His Pro Ser Ser Glu Lys Tyr Glu Asn Thr Lys 130 135 140 Thr Cys Arg Gly Pro Asp Gly Glu Leu His Ala Asn Leu Leu Cys Pro 145 150 155 160 Val Asp Val Leu Asp Val Pro Glu Gly Thr Leu Pro Asp Lys Gln Ser 165 170 175 Thr Glu Gln Ala Ile Gln Leu Leu Glu Lys Met Lys Thr Ser Ala Ser 180 185 190 Pro Phe Phe Leu Ala Val Gly Tyr His Lys Pro His Ile Pro Phe Arg 195 200 205 Tyr Pro Lys Glu Phe Gln Lys Leu Tyr Pro Leu Glu Asn Ile Thr Leu 210 215 220 Ala Pro Asp Pro Glu Val Pro Asp Gly Leu Pro Pro Val Ala Tyr Asn 225 230 235 240 Pro Trp Met Asp Ile Arg Gln Arg Glu Asp Val Gln Ala Leu Asn Ile 245 250 255 Ser Val Pro Tyr Gly Pro Ile Pro Val Asp Phe Gln Arg Lys Ile Arg 260 265 270 Gln Ser Tyr Phe Ala Ser Val Ser Tyr Leu Asp Thr Gln Val Gly Arg 275 280 285 Leu Leu Ser Ala Leu Asp Asp Leu Gln Leu Ala Asn Ser Thr Ile Ile 290 295 300 Ala Phe Thr Ser Asp His Gly Trp Ala Leu Gly Glu His Gly Glu Trp 305 310 315 320 Ala Lys Tyr Ser Asn Phe Asp Val Ala Thr His Val Pro Leu Ile Phe 325 330 335 Tyr Val Pro Gly Arg Thr Ala Ser Leu Pro Glu Ala Gly Glu Lys Leu 340 345 350 Phe Pro Tyr Leu Asp Pro Phe Asp Ser Ala Ser Gln Leu Met Glu Pro 355 360 365 Gly Arg Gln Ser Met Asp Leu Val Glu Leu Val Ser Leu Phe Pro Thr 370 375 380 Leu Ala Gly Leu Ala Gly Leu Gln Val Pro Pro Arg Cys Pro Val Pro 385 390 395 400 Ser Phe His Val Glu Leu Cys Arg Glu Gly Lys Asn Leu Leu Lys His 405 410 415 Phe Arg Phe Arg Asp Leu Glu Glu Asp Pro Tyr Leu Pro Gly Asn Pro 420 425 430 Arg Glu Leu Ile Ala Tyr Ser Gln Tyr Pro Arg Pro Ser Asp Ile Pro 435 440 445 Gln Trp Asn Ser Asp Lys Pro Ser Leu Lys Asp Ile Lys Ile Met Gly 450 455 460 Tyr Ser Ile Arg Thr Ile Asp Tyr Arg Tyr Thr Val Trp Val Gly Phe 465 470 475 480 Asn Pro Asp Glu Phe Leu Ala Asn Phe Ser Asp Ile His Ala Gly Glu 485 490 495 Leu Tyr Phe Val Asp Ser Asp Pro Leu Gln Asp His Asn Met Tyr Asn 500 505 510 Asp Ser Gln Gly Gly Asp Leu Phe Gln Leu Leu Met Pro 515 520 525 <210> twenty two <211> 28 <212> PRT <213> Artificial sequence <220> <223> Connectors <400> twenty two Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Gly 1 5 10 15 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram showing the structure and mechanism of action of the fusion protein.

[0019] Figure 2 The sites where mutations were introduced into the antibody (IR739) that binds to the insulin receptor are shown.

[0020] Figure 3 The CDR3 sequences of antibody variants (H031, HL009, L016, L113 and H118) that bind to the insulin receptor are shown.

[0021] Figure 4 Shown are the results obtained by performing FACS analysis to evaluate the cross-reactivity of IR739 in humans, monkeys and mice.

[0022] Figure 5Shown are the results obtained by comparing the binding affinities of IR739 and variants of IR739 (H031, HL009, L016, L113 and H118) to the human insulin receptor and the mouse insulin receptor.

[0023] Figure 6 Shown are the results obtained by comparing the binding affinities of IR739 and fusion proteins (IDS-IR739, IDS-HL009, and IDS-H031) to the human insulin receptor and the mouse insulin receptor.

[0024] Figure 7 Shown are the results obtained by comparing the interactions of insulin and IR739 with the insulin receptor according to changes in IR739 concentration.

[0025] Figure 8 Shown are the results obtained by comparing the interactions of insulin and IR739 with the insulin receptor according to changes in insulin concentration.

[0026] Fig. 9 The half-life of IR739 in mouse blood is shown.

[0027] Fig.10 Shown are the half-lives of IDS and IDS-HL009 in mouse blood.

[0028] Fig.11 The brain uptake efficiency of IR739 is shown.

[0029] Fig.12 Shown are the results obtained by comparing the brain uptake efficiency of variants of IR739 (H031, HL009, L113, and H118).

[0030] Fig.13 Shown are the results obtained by comparing the brain uptake efficiency of the fusion proteins.

[0031] Best Mode for Carrying Out the Invention

[0032] In one aspect of the present invention, a fusion protein is provided, which comprises: a first domain comprising an antibody fragment that binds to an insulin receptor; and a second domain comprising a protein having iduronate-2-sulfatase (IDS) activity.

[0033] The term "insulin receptor" used herein refers to a transmembrane receptor protein expressed on cells and capable of transporting insulin. Here, the insulin receptor can serve as one of the blood-brain barrier (BBB) ​​receptors. Blood-brain barrier receptors include insulin receptor, insulin-like growth factor receptor (IGF-R), transferrin receptor, low-density lipoprotein (LDL) receptor, glucose transporter 1 (Glut1), heparin-binding epidermal growth factor-like growth factor (HB-EGF), etc.; and these are extracellular transmembrane receptor proteins that can cross the BBB transport molecule or can be used to transport the exogenous molecules administered.

[0034] In one embodiment of the present invention, the fusion protein can bind to the extracellular domain (ECD) of the human insulin receptor and pass through the BBB, so that the fused IDS can be delivered to the brain.

[0035] The term "antibody" used herein refers to an immunoglobulin (Ig) molecule that immunoreacts with a certain antigen, which is a protein molecule that functions as a receptor that specifically recognizes an antigen. Antibody is intended to cover both intact antibodies and fragments thereof (antibody fragments).

[0036] The term "iduronate-2-sulfatase (IDS)" as used herein refers to an enzyme essential for the decomposition of lysosomal glycosaminoglycans (GAGs). When a mutation occurs in IDS or when the activity of IDS is reduced or becomes inactive, GAGs accumulate in most tissues and cells, which can cause diseases. Here, IDS may have the amino acid sequence of SEQ ID NO: 21.

[0037] In one embodiment of the present invention, the fusion protein may further comprise an Fc region, and the Fc region may be derived from the heavy chain constant region (CH) of IgG1, IgG2, IgG3 or IgG4. In addition, the Fc region may be a heterodimer; and one of the heterodimer Fcs may have a knob structure, and the other may have a hole structure. The knob or hole structure may be located at CH3 in the Fc region.

[0038] The term "Fc region" as used herein refers to the C-terminal region of an immunoglobulin heavy chain, which includes a portion of the constant region. The Fc region generally includes CH2 and CH3 of the antibody heavy chain constant region. In addition, the Fc region includes a wild-type Fc region and a variant Fc region.

[0039] In one embodiment of the present invention, the first domain may be a Fab or scFv region of an antibody that binds to the insulin receptor. The term "Fab" as used herein refers to a Fab comprising a variable region and a constant region of a light chain and a variable region and a CH1 region of a heavy chain. The term "scFv" as used herein refers to an scFv comprising a heavy chain variable region and a light chain variable region of an antibody, and may be composed of a "light chain variable region (VL)-linker-heavy chain variable region (VH)". A linker refers to an amino acid sequence of a certain length, the function of which is to artificially connect a heavy chain variable region to a light chain variable region.

[0040] The first domain contains CDRs, and these CDRs confer binding specificity to a certain antigen, wherein a group of CDRs (CDR1, CDR2, CDR3) can provide the antigen binding site.

[0041] In one embodiment of the present invention, the first domain may comprise: a heavy chain variable region (VH), comprising an H-CDR1 having an amino acid sequence of SEQ ID NO: 1; an H-CDR2 having an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 having any one of the amino acid sequences selected from SEQ ID NOs: 5, 7 and 9; and a light chain variable region (VL), comprising an L-CDR1 having an amino acid sequence of SEQ ID NO: 2; an L-CDR2 having an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 having any one of the amino acid sequences selected from SEQ ID NOs: 6, 8, 10 and 11. Preferably, the first domain may comprise: a heavy chain variable region (VH), which comprises an H-CDR1 having an amino acid sequence of SEQ ID NO: 1; an H-CDR2 having an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 having an amino acid sequence of SEQ ID NO: 7; and a light chain variable region (VL), which comprises an L-CDR1 having an amino acid sequence of SEQ ID NO: 2; an L-CDR2 having an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 having an amino acid sequence of SEQ ID NO: 8.

[0042] In one embodiment of the present invention, the first monomer obtained by combining the C-terminus of the first domain with the N-terminus of the Fc region may have: a heavy chain having any one of the amino acid sequences selected from SEQ ID NOs: 12, 14, 16 and 17; and a light chain having any one of the amino acid sequences selected from SEQ ID NOs: 13, 15, 18 and 19.

[0043] In one embodiment of the present invention, the second domain may have an amino acid sequence of SEQ ID NO: 21. In one embodiment of the present invention, the fusion protein may have a structure in which the C-terminus of the second domain is bound to the N-terminus of the Fc region; and the second monomer obtained by the above binding may have an amino acid sequence of SEQ ID NO: 20.

[0044] More specifically, the first domain and the Fc region can be bound to each other through a joint. In addition, the second domain and the Fc region can be bound to each other through a joint. As a joint, a single-chain peptide joint consisting of 5 to 20 amino acids connected together by a peptide bond can be used. Amino acid can be one or more selected from the following: glycine, serine, alanine, proline, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, threonine, cysteine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid and glutamic acid. Specifically, the joint can be composed of glycine and serine, and can be a joint of (G4S)n (wherein n is 1 to 5). In one embodiment of the invention, the joint can have the amino acid sequence of SEQ ID NO: 22.

[0045] In one embodiment of the present invention, the fusion protein may have an asymmetric structure, wherein the C-terminus of the first domain binds to the N-terminus of any one of the dimer Fc regions, and the C-terminus of the second domain binds to the other N-terminus of the dimer Fc region. Since the fusion protein has an asymmetric structure, the problem that the bivalent antibody may be biased toward the degradation pathway can be prevented.

[0046] In one embodiment of the present invention, the fusion protein can be a human antibody, a humanized antibody or a chimeric antibody. The term "human antibody" refers to a complete antibody with a variable region, the structure of which and the CDR region are derived from an immunoglobulin sequence; the term "humanized antibody" refers to a chimeric antibody containing a minimal sequence derived from a non-human antibody (e.g., a mouse antibody) immunoglobulin; and the term "chimeric antibody" refers to an antibody derived from a combination of different mammals (e.g., mice, rats, rabbits, goats, or humans).

[0047] In one embodiment of the present invention, the fusion protein can be prepared by methods commonly practiced in the art, for example, the knob-in-hole method (Ridgway et al., Protein Engineering, 617-621 (1996)), the phage antibody library method (Clackson et al., Nature, 352: 624-628 (1991)), the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)) and the recombinant DNA method (U.S. Pat. No. 4,816,567).

[0048] In one embodiment of the present invention, phage antibody library method and well-in-hole method were used to prepare IDS-fused fusion proteins (IDS-IR739, IDS-HL009 and IDS-H031). The fusion proteins can bind to the insulin receptor and pass through the BBB through the receptor-mediated transcytosis (RMT) mechanism, so that the IDS-fused fusion partner site can act on the target in the brain ( Figure 1 ).

[0049] For antibodies that pass through the BBB via the RMT mechanism, their affinity for BBB receptors is very important. Specifically, when the affinity is lower than an appropriate level, there is a problem of reduced efficiency in binding to the BBB. On the other hand, when the affinity is higher than an appropriate level, the rate at which the therapeutic agent is released after passing through the BBB is reduced, resulting in reduced BBB passing efficiency.

[0050] In one embodiment of the present invention, the fusion protein may not inhibit the binding of insulin to the insulin receptor.

[0051] In addition, in one aspect of the present invention, a pharmaceutical composition for preventing or treating a central nervous system (CNS) disease is provided, which comprises the fusion protein as an active ingredient.

[0052] As used herein, the term "central nervous system (CNS)" refers to the complex of nervous tissue that controls body functions and includes the brain and spinal cord. Central nervous system diseases refer to diseases or conditions that affect the CNS and / or have a CNS etiology. Some specific examples of diseases include lysosomal storage diseases (LSDs), Huntington's disease Epilepsy, Parkinson's disease, Alzheimer's disease, stroke, corticobasal degeneration (CBD), corticobasal ganglionic degeneration (CBGD), frontotemporal dementia (FTD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and brain cancer. Some specific examples of lysosomal storage diseases include Hunter syndrome, Tay-Sachs disease Niemann-Pick disease Pompe disease Krabbe disease Gaucher disease Fabry disease Wolman disease Morquio syndrome Menkes syndrome Galactosialidosis, glycogen storage disease, Fanconi-Bickel syndrome Lesch-Nyhan syndrome Zellweger syndrome wait.

[0053] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For oral administration, adhesives, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents, etc. may be used. For injection, buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used. For topical administration, substrates, excipients, lubricants, preservatives, etc. may be used.

[0054] The pharmaceutical composition can be prepared into various preparations by mixing with the above-mentioned pharmaceutically acceptable carriers. For injection, the pharmaceutical composition can be prepared into unit dose ampoules or various dosage forms.

[0055] In addition, the pharmaceutical composition may include a surfactant that can enhance its membrane permeability. The surfactant includes, but is not limited to, those derived from steroids, cationic lipids such as N-[1-(2,3-dioleyl)propyl-N,N,N-trimethylammonium chloride (DOTMA) or various compounds such as cholesterol hemisuccinate and phosphatidylglycerol.

[0056] Regarding the administration route and dosage of the pharmaceutical composition, it can be administered in various ways and amounts according to the individual's condition and the presence or absence of adverse effects, and the optimal administration method and dosage can be selected within an appropriate range by those skilled in the art.

[0057] Specifically, for parenteral administration, the pharmaceutical composition can be administered by any one of the following routes selected from, but not limited to, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intraarteriolar, intraventricular, intralesional, intrathecal and topical administration, and combinations thereof. In addition, the daily dose of the composition is about 0.0001 mg / kg to 100 mg / kg, and preferably 0.001 mg / kg to 10 mg / kg; and the dosage is preferably administered once to multiple times per day. However, the dosage can vary according to individual weight, age, sex, health status, diet, administration time, administration method, excretion rate and disease severity.

[0058] When administered parenterally, the pharmaceutical composition can be administered in a variety of formulations; and in the case of being prepared as a formulation, commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants and surfactants can be used to prepare the pharmaceutical composition. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. In addition, for non-aqueous solutions or suspensions, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, injectable esters such as ethyl oleate, etc. can be used. For the base of the suppository, Witepsol, Macrogol, Tween 61, cocoa butter, lauric acid fat can be used. Glycerin gelatin, etc.

[0059] The term "subject" as used herein refers to a mammal, preferably a human, suffering from or at risk of developing a condition or disease that can be alleviated, inhibited or treated by administering a pharmaceutical composition.

[0060] As used herein, the term "prevention" refers to any action that blocks a disease or inhibits or delays the symptoms of a disease using a pharmaceutical composition.

[0061] As used herein, the term "treatment" refers to any act of using a pharmaceutical composition to ameliorate or beneficially alter a disease.

[0062] In addition, in one aspect of the present invention, a method for preparing a fusion protein is provided, which comprises the following steps: binding an antibody that binds to an insulin receptor to an Fc region to prepare a first monomer; binding iduronate-2-sulfatase (IDS) to a heterodimeric Fc region to prepare a second monomer; and mixing the first monomer with the second monomer.

[0063] In one embodiment of the present invention, the Fc region may be derived from the heavy chain constant region (CH) of IgG1, IgG2, IgG3 or IgG4.

[0064] In one embodiment of the present invention, the antibody may comprise: a heavy chain variable region (VH) comprising an H-CDR1 having an amino acid sequence of SEQ ID NO: 1; an H-CDR2 having an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 having any one of the amino acid sequences selected from SEQ ID NOs: 5, 7 and 9; and a light chain variable region (VL) comprising an L-CDR1 having an amino acid sequence of SEQ ID NO: 2; an L-CDR2 having an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 having any one of the amino acid sequences selected from SEQ ID NOs: 6, 8, 10 and 11. Specifically, the antibody fragment may comprise: a heavy chain variable region (VH), which comprises an H-CDR1 having an amino acid sequence of SEQ ID NO: 1; an H-CDR2 having an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 having an amino acid sequence of SEQ ID NO: 7; and a light chain variable region (VL), which comprises an L-CDR1 having an amino acid sequence of SEQ ID NO: 2; an L-CDR2 having an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 having an amino acid sequence of SEQ ID NO: 8.

[0065] In one embodiment of the present invention, when the CH3 in the Fc region of the first monomer has a node structure, the CH3 in the second monomer may have a hole structure; or when the CH3 in the Fc region of the first monomer has a hole structure, the CH3 in the second monomer may have a node structure.

[0066] In one embodiment of the present invention, the IDS may have the amino acid sequence of SEQ ID NO:21.

[0067] In one embodiment of the present invention, the first monomer may comprise a heavy chain having any one of the amino acid sequences selected from SEQ ID NOs: 12, 14, 16 and 17 and a light chain having any one of the amino acid sequences selected from SEQ ID NOs: 13, 15, 18 and 19; and the second monomer may have the amino acid sequence of SEQ ID NO: 20.

[0068] More specifically, the fusion protein IDS-IR739 according to the present invention may include: a first monomer comprising a heavy chain having an amino acid sequence of SEQ ID NO: 12 or 16 and a light chain having an amino acid sequence of SEQ ID NO: 13; and a second monomer having an amino acid sequence of SEQ ID NO: 20.

[0069] The fusion protein IDS-HL009 according to the present invention may comprise: a first monomer comprising a heavy chain having an amino acid sequence of SEQ ID NO: 14 and a light chain having an amino acid sequence of SEQ ID NO: 15; and a second monomer having an amino acid sequence of SEQ ID NO: 20.

[0070] The fusion protein IDS-H031 according to the present invention may include: a first monomer comprising a heavy chain having an amino acid sequence of SEQ ID NO: 14 and a light chain having an amino acid sequence of SEQ ID NO: 13; and a second monomer having an amino acid sequence of SEQ ID NO: 20.

[0071] The fusion protein IDS-H118 according to the present invention may include: a first monomer comprising a heavy chain having an amino acid sequence of SEQ ID NO: 17 and a light chain having an amino acid sequence of SEQ ID NO: 13; and a second monomer having an amino acid sequence of SEQ ID NO: 20.

[0072] The fusion protein IDS-L016 according to the present invention may comprise: a first monomer comprising a heavy chain having an amino acid sequence of SEQ ID NO: 12 or 16 and a light chain having an amino acid sequence of SEQ ID NO: 18; and a second monomer having an amino acid sequence of SEQ ID NO: 20.

[0073] The fusion protein IDS-L113 according to the present invention may comprise: a first monomer comprising a heavy chain having an amino acid sequence of SEQ ID NO: 12 or 16 and a light chain having an amino acid sequence of SEQ ID NO: 19; and a second monomer having an amino acid sequence of SEQ ID NO: 20.

[0074] The present invention provides use of the fusion protein for preventing or treating central nervous system diseases.

[0075] In addition, the present invention provides use of the fusion protein in manufacturing a drug for preventing or treating central nervous system diseases.

[0076] In addition, the present invention provides a method for preventing or treating a central nervous system disease and / or a method for enhancing a treatment effect, the method comprising the step of administering the fusion protein or a pharmaceutical composition comprising the fusion protein to an individual.

[0077] The subject may be a subject suffering from a central nervous system disease. In addition, the subject may be a mammal, and preferably a human.

[0078] For the route of administration, dosage and frequency of administration of the fusion protein or pharmaceutical composition, administration to the subject can be carried out in a variety of ways and amounts according to the subject's condition and the presence or absence of adverse effects, and the optimal method of administration, dosage and frequency of administration can be selected by those skilled in the art within a suitable range. In addition, the fusion protein or pharmaceutical composition can be administered in combination with other drugs or physiologically active substances whose therapeutic effects are known for the disease to be treated, or can be formulated into a combined preparation with other drugs.

[0079] Invention Mode

[0080] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

[0081] Preparation Example 1. Preparation of IR739 Antibody

[0082] Anti-IR antibodies were prepared using a human synthetic scFv library (MIDAS, MOGAM). The phage library of MIDAS was placed in a tube coated with human IR at a concentration of 5 μg / ml and treated at 37°C for 2 hours. Washed 4 times with PBS buffer to which 0.05% Tween 20 was added. Then, 1% BSA / 0.1M glycine (pH 2.0) buffer was used at room temperature for 10 minutes to elute the phage bound to IR. The eluted phage was neutralized with 70 μl 2M Tris-HCl (pH 9.0) and used to infect 9 ml of pre-cultured Escherichia coli (E.coli) (XL1-Blue) for 30 minutes. Super broth (SB) culture medium, tetracycline and carbenicillin were added to the XL1-Blue infected with phage, and cultured for 1 hour at 37°C. Then, helper phage was added thereto. Cultured for another hour. The culture medium was added thereto to make up to 100 ml, and culture was performed at 37°C overnight.

[0083] The culture medium was harvested by centrifugation, and the phages were precipitated with a 20% PEG solution. After centrifugation, the phages were collected with a PBS solution to which 1% BSA had been added. The panning process was repeated alternately with a mouse IR coated tube and a human IR coated tube until the fourth time using the solution in which the phages were collected. Each XL1-Blue colony infected with a tertiary phage and a quaternary phage was cultured to obtain a culture fluid containing phages respectively. Using culture fluid and IR coated plates, the XL1-Blue clones expressing phages that were combined with human IR and mouse IR were selected by ELISA. The plasmids of the selected clones were sequenced to obtain VH and VL sequences. Based on these sequences, antibodies with conventional IgG forms were prepared.

[0084] IR739, an antibody binding to human IR, monkey IR and mouse IR, was selected among the antibodies obtained by screening the MIDAS human scFv library through cross-reactivity evaluation in the following Experimental Example 1. The protein sequence of IR739 prepared as described above is shown in Table 1 below.

[0085] [Table 1]

[0086]

[0087] Preparation Example 2. Preparation of IR739 antibody variants

[0088] Using random mutagenesis, mutations were introduced into the Asn-Asn sequence in LCDR3 and the Ser-Tyr-Gly-Thr-Val-Asn-His sequence in HCDR3 ( Figure 2 ). In order to introduce mutations, PCR was performed on the gene of IR739 using primers obtained by replacing the codons of the corresponding sequence with NNK degenerate codons (N = A, T, G or C, and K = G or T) to prepare mutant scFv DNA fragments. 1 μg of pCIw vector (Promega) connected with each mutant scFv DNA fragment was transformed into 100 μl XL1-Blue by electroporation. Then, the resultant was placed in 10 ml of super broth (SB) medium and cultured at 37°C for 1 hour.

[0089] 10ml of helper phage was added thereto, and cultured at 37°C for 1 hour. Then, 80ml culture medium supplemented with tetracycline and carbenicillin was added thereto, and cultured overnight. The culture solution of the XL1-Blue with mutation introduced was harvested by centrifugation, and the phage was precipitated with 20% PEG solution. After centrifugation, 1% BSA / 0.1M glycine (pH 2.0) buffer was used to culture for 10 minutes at room temperature to elute the phage bound to IR. The eluted phage was neutralized with 70 μl 2M Tris-HCl (pH 9.0) and used to infect 9ml pre-cultured Escherichia coli (XL1-Blue) for 30 minutes. Super broth (SB) culture medium, tetracycline and carbenicillin were added to the XL1-Blue infected by phage, and cultured at 37°C for 1 hour. Then, helper phage was added thereto. Cultured for another hour. The culture medium was added thereto to make up to 100 ml, and culture was performed at 37°C overnight.

[0090] The culture medium was harvested by centrifugation and the phages were precipitated with a 20% PEG solution. After centrifugation, the phages were collected with a PBS solution to which 1% BSA had been added. The panning process was repeated alternately with a mouse IR-coated tube and a human IR-coated tube using the solution in which the phages were collected until the fourth time. Each XL1-Blue colony infected with a tertiary phage and a quaternary phage was cultured to obtain a culture fluid containing phages. Using the culture fluid and IR-coated plates, clones with a higher or lower absorbance than IR739 were selected by ELISA. The selected clones were sequenced to select unique sequences ( Figure 3 ), and cloned to produce H031, H118, HL009, L016, and L113, which are variants with conventional IgG formats.

[0091] Preparation Example 3. Preparation of asymmetric fusion antibodies

[0092] To prepare each IDS fusion antibody in which one Fab was replaced with IDS (IDS-IR739, IDS-H031, IDS-HL009, and IDS-8D3), a knob in the hole was introduced in the heavy chain of the immunoglobulin. In the heavy chain in which the knob was introduced, Lys409 was replaced with Trp409; and in the heavy chain in which the hole was introduced, Asp399 and Phe405 were replaced with Val399 and Thr405, respectively. By cloning, the VH in IR739, HL009, and 8D3 was replaced with the VH of the heavy chain in which the hole was introduced, and the C-terminus of IDS was fused to the N-terminus of the CH2 of the heavy chain in which the knob was introduced. The antibody 8D3 against the transferrin receptor was used as a control.

[0093] Plasmids expressing the light chain and the heavy chain into which the hole was introduced, and plasmids expressing CH2-CH3 into which the knot was introduced and fused with IDS were co-transfected into CHO cells. Cultivation was performed at 37°C for 10 days. The culture fluid was separated by centrifugation. Then, IDS-IR739, IDS-HL009, IDS-H031, and IDS-8D3 were purified using a protein A column. The IDS fusion antibody was eluted from the protein A column using a 1M L-arginine solution (pH 4). The structure of the asymmetric fusion antibody prepared as described above is shown in FIG. Figure 1 (left) is shown.

[0094] Experimental Example 1. Cross-reactivity assessment

[0095] Each Expi-293 cell in which human IR, monkey IR, and mouse IR were overexpressed was harvested and plated at 5×10 6Cells / ml were placed in each tube. Then, the tubes were treated with 1 μg / ml of each antibody obtained by screening the MIDAS human scFv library. The tubes were cultured at 4°C for 30 minutes and then washed 3 times with cold PBS using a centrifuge. Fluorescent dye-labeled anti-human antibodies (FITC-conjugated anti-human IgG) in 3% BSA / PBS buffer were added thereto and cultured in the dark at 4°C for 30 minutes. Using a centrifuge, the cells were washed 3 times with cold buffer of 3% BSA / 1% sodium azide / PBS. Analyzed using a FACS instrument. As a result, it was identified that antibody IR739 bound to all of human IR, monkey IR, and mouse IR ( Figure 4 ).

[0096] Experimental Example 2. Evaluation of Binding Affinity to IR

[0097] Experimental Example 2.1. Evaluation of Binding Affinity of IR739

[0098] The binding affinity of IR739 to IR was analyzed by surface plasmon resonance (SPR). 1 μg / ml of human IR or mouse IR was fixed on a CM5 sensor chip with acetate (pH 4.0). IR739 was flowed through a CM5 sensor chip fixed with human IR or mouse IR at a concentration of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM or 1.563nM, and its association and dissociation were measured simultaneously. For measurement conditions, the flow rate was set to 30 μl / min, and the association time and dissociation time were set to 120 seconds and 600 seconds, respectively. In order to regenerate, 10mM glycine-HCl (pH 1.5) was flowed for 30 seconds. The results of the binding affinity of IR739 to human IR and mouse IR are shown in Table 2 below.

[0099] [Table 2]

[0100] pH 7.0 Ka( / Ms) Kd( / s) KD(M) People 1R <![CDATA[9.595x10 4 ]]> <![CDATA[14.35x10 -4 ]]> <![CDATA[14.96x10 -9 ]]> Mouse 1R <![CDATA[9.532x10 4 ]]> <![CDATA[6.100x10 -4 ]]> <![CDATA[6.4x10 -9 ]]>

[0101] Experimental Example 2.2. Comparison of Binding Affinity of IR739 and IR739 Variants

[0102] ELISA was performed to compare the binding affinity of IR739 and its variants to IR. The ELISA plate coated with IR at a concentration of 5 μg / ml was blocked with 3% skim milk for 1 hour, and each well was then treated with IR739 and its variants at various concentrations. The treatment was performed at 37°C for 1 hour, and then washed 3 times with 0.05% Tween 20 / PBS solution. In order to detect IR739 and its variants, anti-human Fc IgG conjugated with peroxidase was treated and incubated at 37°C for 1 hour. Then, 0.05% Tween 20 / PBS solution was used to wash 3 times. Developed with tetramethylbenzidine (TMB) ELISA solution, and the absorbance at 450nm was analyzed. As a result, the binding affinity of H031 and HL009 to human IR and mouse IR was higher than that of IR739, and the binding affinity of L016 and H118 to human IR and mouse IR was lower than that of IR739. L113 exhibited similar binding affinity to IR739 ( Figure 5 ).

[0103] Experimental Example 2.3. Binding affinity evaluation of asymmetric fusion antibodies

[0104] ELISA was performed to identify the binding affinity of IDS-HL009 to IR. The ELISA plate coated with IR at a concentration of 5 μg / ml was blocked with 3% skim milk for 1 hour, and then each well was treated with various concentrations of IR739, IDS-IR739, IDS-HL009 or IDS-H031. The treatment was performed at 37°C for 1 hour, and then washed 3 times with 0.05% Tween 20 / PBS solution. In order to detect IR-739, IDS-IR739, IDS-HL009 and IDS-H031, peroxidase-conjugated anti-human Fc IgG was treated and incubated at 37°C for 1 hour. Then, it was washed 3 times with 0.05% Tween20 / PBS solution. The color was developed with TMBELISA solution, and the absorbance at 450nm was analyzed. As a result, IDS-IR739, IDS-H031, and IDS-HL009 had lower binding affinities to human IR and mouse IR than IR739 because in such antibodies, the avidity effect was eliminated by replacing one Fab with IDS ( Figure 6 ).

[0105] Experimental Example 3. Identification of competition between insulin and IR739 for IR

[0106] In order to identify the interaction of IR739 and insulin on IR, a competitive ELISA was performed. The ELISA plate coated with IR at a concentration of 5 μg / ml was blocked with 3% skim milk for 1 hour. Then, a mixture of IR739 and biotinylated insulin at various concentrations was added to each well. Incubated at 37°C for 1 hour and then washed 3 times with 0.05% Tween 20 / PBS solution. Anti-human Fc IgG conjugated with peroxidase was processed to detect IR739, and streptavidin conjugated with peroxidase was processed to detect biotinylated insulin.

[0107] The cells were incubated at 37°C for 1 hour. The cells were then washed three times with 0.05% Tween 20 / PBS solution and developed with TMB ELISA solution. The absorbance at 450 nm was analyzed. As a result, the binding pattern of IR739 to IR was not affected by changes in insulin concentration. Specifically, no changes were observed in the binding curves for each concentration of IR739 when the insulin concentration was changed from 0.6 nM to 150 nM ( Figure 7 ). In addition, the binding pattern of insulin to IR was also unaffected by changes in IR739 concentration; and specifically, no changes were observed in the binding curves for each insulin concentration when IR739 concentration was changed from 0.03 nM to 7 nM ( Figure 8 ).

[0108] Experimental Example 4. Evaluation of pharmacokinetics in mouse blood

[0109] Experimental Example 4.1. Evaluation of the pharmacokinetics of IR739 in mouse blood

[0110] In order to analyze the stability of IR739 in mouse blood, 5 mg / kg of IR739 or 8D3 antibody (anti-TfR antibody) as a control was applied to the tail vein of each mouse. After administration, 50 μl of blood was collected by eye bleeding at 30 minutes, 1 hour, 2 hours, 6 hours and 24 hours, respectively. The blood was centrifuged and the antibody concentration in the supernatant was analyzed by ELISA. For ELISA, the ELISA plate was coated with 1 μg / ml of anti-human Fab IgG, blocked with 3% skim milk, and then treated with 50 μl of 1 / 10 diluted supernatant. Incubate at 37°C for 1 hour and then wash 3 times with 0.05% Tween20 / PBS solution. Treat with peroxidase-conjugated anti-human Fc IgG. Treat at 37°C for 1 hour. Then, wash 3 times with 0.05% Tween 20 / PBS solution and detect with TMB ELISA solution. The concentration was calculated using the results obtained by performing ELISA in the same manner as above with IR739 at 10,000 ng / ml to 4.5 ng / ml (10,000 ng / ml, 3,333 ng / ml, 1,111 ng / ml, 370 ng / ml, 123 ng / ml, 41.2 ng / ml, 13.7 ng / ml, 4.5 ng / ml) as a standard. The half-life of IR739 was determined to be about 24 hours ( Fig. 9 ).

[0111] Experimental Example 4.2. Pharmacokinetic evaluation of IDS-HL009 in mouse blood

[0112] To analyze the stability of IDS-HL009 in mouse blood, 5 mg / kg of IDS, IR739, IDS-HL009 or IDS-8D3 was administered to the tail vein of each mouse. After administration, 50 μl of blood was collected by eye bleeding at 30 minutes, 1 hour, 2 hours, 6 hours and 24 hours, respectively. The blood was centrifuged and the antibody concentration in the supernatant was analyzed by ELISA. For ELISA, ELISA plates were coated with 1 μg / ml of anti-human Fab IgG, blocked with 3% skim milk, and then treated with 50 μl of 1 / 10 diluted supernatant.

[0113] Incubate at 37°C for 1 hour and then wash 3 times with 0.05% Tween 20 / PBS solution. Treat with peroxidase-conjugated anti-human Fc IgG. Treat at 37°C for 1 hour. Then, wash 3 times with 0.05% Tween 20 / PBS solution and detect with TMB ELISA solution. Use the results obtained by performing ELISA in the same manner as above with IR739, IDS-HL009 and IDS-8D3 from 10,000 ng / ml to 4.5 ng / ml (10,000 ng / ml, 3,333 ng / ml, 1,111 ng / ml, 370 ng / ml, 123 ng / ml, 41.2 ng / ml, 13.7 ng / ml, 4.5 ng / ml) as standard to calculate concentration. To measure IDS concentration, ELISA was performed using biotin-conjugated anti-IDS and HRP-conjugated streptavidin on ELISA plates coated with anti-IDS IgG. The half-life of IDS-HL009 was determined to be approximately 14 hours ( Fig.10 ).

[0114] Experimental Example 5. Evaluation of Brain Uptake

[0115] Experimental Example 5.1. Evaluation of brain uptake efficiency of IR739

[0116] To analyze the brain uptake efficiency of IR739, 5 mg / kg of negative control IgG (anti-mesothelin (MSLN) IgG), 8D3 or IR739 was injected into the tail vein of each mouse. Here, anti-mesothelin IgG was used as a negative control, and 8D3 was used as a positive control. After 1 hour and 24 hours, for each substance, each of 3 mice was perfused, and the brain was then removed therefrom. 1 ml of lysis buffer was added to every 100 mg of brain, and crushed. Then, centrifuged at 13,000 rpm. The ELISA plate was coated with 1 μg / ml of anti-human Fab IgG, blocked with 3% skim milk, and then treated with 50 μl of supernatant. Incubated at 37°C for 1 hour, and then washed 3 times with 0.05% Tween 20 / PBS solution. Treated with peroxidase-conjugated anti-human Fc IgG. Incubated at 37°C for 1 hour. Then, washing was performed 3 times with 0.05% Tween 20 / PBS solution, and detection was performed with TMB ELISA solution. The results obtained by performing ELISA in the same manner as above with anti-MSLN IgG, 8D3 and IR739 at 1,000 ng / ml to 0.45 ng / ml (1,000 ng / ml, 333 ng / ml, 111 ng / ml, 37.0 ng / ml, 12.3 ng / ml, 4.12 ng / ml, 1.37 ng / ml, 0.45 ng / ml) were used as the standard to calculate the concentration. IR739 showed a lower brain uptake efficiency than 8D3, while showing a higher brain uptake efficiency than anti-MSLN IgG ( Fig.11 ).

[0117] Experimental Example 5.2. Evaluation of brain uptake efficiency of IR739 variants

[0118] In order to analyze the brain uptake efficiency of IR739 variants, 5mg / kg of anti-MSLN IgG, 8D3, IR739, H031, HL009, L113 or H118 was injected into the tail vein of each mouse. After 1 hour and 24 hours, for each substance, each of 3 mice was perfused and the brain was subsequently removed therefrom. 1ml lysis buffer was added to every 100mg brain and crushed. Then, centrifuged at 13,000rpm. The ELISA plate was coated with 1μg / ml of anti-human Fab IgG, blocked with 3% skim milk, and then treated with 50μl supernatant. Incubated at 37°C for 1 hour and then washed 3 times with 0.05% Tween 20 / PBS solution. Treated with peroxidase-conjugated anti-human Fc IgG. Incubated at 37°C for 1 hour. Then, washing was performed 3 times with 0.05% Tween 20 / PBS solution, and detection was performed with TMB ELISA solution. The results obtained by performing ELISA in the same manner as above with anti-MSLN IgG, 8D3, IR739, H031, HL009, L113 and H118 at 1,000 ng / ml to 0.45 ng / ml (1,000 ng / ml, 333 ng / ml, 111 ng / ml, 37.0 ng / ml, 12.3 ng / ml, 4.12 ng / ml, 1.37 ng / ml, 0.45 ng / ml) were used as standards to calculate the concentration. As a result of the analysis, among the variants, HL009 exhibited a higher brain uptake efficiency than IR739, while H031, L113 and H118 exhibited a lower brain uptake efficiency than IR739 ( Fig.12 ).

[0119] Experimental Example 5.3. Evaluation of brain uptake efficiency of IDS-HL009

[0120] In order to analyze the brain uptake efficiency of IDS-HL009, 5 mg / kg of IDS-HL009 was injected into the tail vein of each mouse. After 1 hour and 24 hours, for each substance, each of 3 mice was perfused and the brain was subsequently removed therefrom. 1 ml of lysis buffer was added to every 100 mg of brain and crushed. Then, centrifuged at 13,000 rpm. The ELISA plate was coated with 1 μg / ml of anti-human Fab IgG, blocked with 3% skim milk, and then treated with 50 μl of supernatant. Incubated at 37°C for 1 hour and then washed 3 times with 0.05% Tween 20 / PBS solution. Treated with peroxidase-conjugated anti-human Fc IgG. Incubated at 37°C for 1 hour. Then, washed 3 times with 0.05% Tween 20 / PBS solution and detected with TMB ELISA solution. The concentration was calculated using the results obtained by performing ELISA in the same manner as above with IDS-HL009 at 1,000 ng / ml to 0.45 ng / ml (1,000 ng / ml, 333 ng / ml, 111 ng / ml, 37.0 ng / ml, 12.3 ng / ml, 4.12 ng / ml, 1.37 ng / ml, 0.45 ng / ml) as a standard. IDS-HL009 showed a higher brain uptake efficiency than IDS, IDS-IR739, and IDS-H031 ( Fig.13 ).

[0121] Experimental Example 6. Characteristic Evaluation of IDS-HL009

[0122] To analyze the properties of IDS and IDS-HL009, 10 ng / ml IDS or 10 μl of IDS-HL009 and 20 μl of 100 mM 4-methylumbelliferyl α-L-pyraniduronic acid-2-sulfate / ester (4MU-α-IdopyraA-2) were placed in a 96-well plate (black) and incubated at 37°C for 4 hours. The reaction was terminated by adding 20 μl of Pi / Ci solution (pH 4.5), and 10 μl of 25 μg / ml recombinant human α-L-iduronidase was added thereto. Incubation was performed at 37°C for 24 hours. Then, 200 μl of 0.25 M sodium carbonate / sodium bicarbonate (pH 10.0) was added thereto and fluorescence was measured (absorption: 355 nm / emission: 460 nm). The activity was analyzed using 4-methylumbelliferone as a standard and the results are shown in Table 3 below.

[0123] [Table 3]

[0124] IDS IDS-HL009 nmol / min / μg 51.4 13.0 nmol / min / p mole 3,084 2,105 % of IDS 68.3

[0125] As shown in Table 3, the IDS activity of IDS-HL009 indicated IDS:IDS-HL009=51.4:13.0 nmol / min based on μg of protein, and indicated IDS:IDS-HL009=3,084:2,105 nmol / min based on moles of protein. From the above results, it can be determined that IDS-HL009 has about 70% of the IDS activity relative to IDS based on moles of protein.

Claims

1. A fusion protein comprising: A first domain comprising an antibody fragment that binds to an insulin receptor; and a second domain comprising a protein having iduronate-2-sulfatase (IDS) activity, wherein the amino acid sequence of the second domain is SEQ ID NO: 21, and The antibody fragment is composed of: A heavy chain variable region (VH), comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 5; a light chain variable region (VL), comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 6; A heavy chain variable region (VH), comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 7; a light chain variable region (VL), comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 8; A heavy chain variable region (VH) comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 7; a light chain variable region (VL) comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 6; A heavy chain variable region (VH) comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 9; a light chain variable region (VL) comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 6; A heavy chain variable region (VH) comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 5; a light chain variable region (VL) comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 10; or A heavy chain variable region (VH), which comprises an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 5; a light chain variable region (VL), which comprises an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO:

11.

2. The fusion protein of claim 1, further comprising: Fc region.

3. The fusion protein of claim 2, wherein the Fc region is a heterodimer. The fusion protein of claim 1 , wherein the first domain is a Fab or scFv region of an antibody that binds to the insulin receptor. The fusion protein of claim 2 , wherein the Fc region is derived from the heavy chain constant region (CH) of IgG1, IgG2, IgG3 or IgG4. The fusion protein of claim 3 , wherein one of the heterodimeric Fcs has a knob structure and the other has a hole structure.

7. The fusion protein of claim 2, wherein the C-terminus of the first domain is bound to the N-terminus of the Fc region.

8. The fusion protein of claim 2, wherein the C-terminus of the second domain is bound to the N-terminus of the Fc region.

9. The fusion protein of claim 1, wherein the fusion protein does not inhibit the binding between insulin receptor and insulin.

10. A pharmaceutical composition for preventing or treating Hunter syndrome, comprising the following as an active ingredient: The fusion protein according to any one of claims 1 to 9.

11. A method for preparing a fusion protein, comprising the following steps: allowing an antibody fragment that binds to an insulin receptor to bind to an Fc region to prepare a first monomer; allowing iduronate-2-sulfatase (IDS) to bind to the heterodimeric Fc region to prepare a second monomer; wherein the amino acid sequence of the IDS is SEQ ID NO: 21, and mixing the first monomer and the second monomer, The antibody fragment is composed of: A heavy chain variable region (VH), comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 5; a light chain variable region (VL), comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 6; A heavy chain variable region (VH), comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 7; a light chain variable region (VL), comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 8; A heavy chain variable region (VH) comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 7; a light chain variable region (VL) comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 6; A heavy chain variable region (VH) comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 9; a light chain variable region (VL) comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 6; A heavy chain variable region (VH) comprising an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 5; a light chain variable region (VL) comprising an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO: 10; or A heavy chain variable region (VH), which comprises an H-CDR1 with an amino acid sequence of SEQ ID NO: 1; an H-CDR2 with an amino acid sequence of SEQ ID NO: 3; and an H-CDR3 with an amino acid sequence of SEQ ID NO: 5; a light chain variable region (VL), which comprises an L-CDR1 with an amino acid sequence of SEQ ID NO: 2; an L-CDR2 with an amino acid sequence of SEQ ID NO: 4; and an L-CDR3 with an amino acid sequence of SEQ ID NO:

11.

12. The method of claim 11, wherein in the case where the CH3 in the Fc region of the first monomer has a knob structure, the CH3 in the second monomer has a hole structure; or In the case where CH3 in the Fc region of the first monomer has a hole structure, CH3 in the second monomer has a knob structure.

13. The method of claim 11, wherein the first monomer comprises: A heavy chain having an amino acid sequence of SEQ ID NO: 12 and a light chain having an amino acid sequence of SEQ ID NO: 13, A heavy chain having an amino acid sequence of SEQ ID NO: 16 and a light chain having an amino acid sequence of SEQ ID NO: 13, A heavy chain having an amino acid sequence of SEQ ID NO: 14 and a light chain having an amino acid sequence of SEQ ID NO: 15, A heavy chain having an amino acid sequence of SEQ ID NO: 14 and a light chain having an amino acid sequence of SEQ ID NO: 13, A heavy chain having an amino acid sequence of SEQ ID NO: 17 and a light chain having an amino acid sequence of SEQ ID NO: 13, A heavy chain having an amino acid sequence of SEQ ID NO: 12 and a light chain having an amino acid sequence of SEQ ID NO: 18, A heavy chain having an amino acid sequence of SEQ ID NO: 16 and a light chain having an amino acid sequence of SEQ ID NO: 18, A heavy chain having an amino acid sequence of SEQ ID NO: 12 and a light chain having an amino acid sequence of SEQ ID NO: 19, or A heavy chain having an amino acid sequence of SEQ ID NO: 16 and a light chain having an amino acid sequence of SEQ ID NO: 19, and The amino acid sequence of the second monomer is SEQ ID NO:

20.

14. Use of the fusion protein according to any one of claims 1 to 9 for the manufacture of a medicament for preventing or treating Hunter syndrome.

15. Use of the pharmaceutical composition according to claim 10 for manufacturing a medicament for preventing or treating Hunter syndrome.

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