Abstract:
The present invention relates to an antibody against C5, and a method for preventing and treating complement-related diseases using the antibody, wherein the antibody against C5 is effectively usable in preventing and treating complement-related diseases by inhibiting complement activation.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to an antibody against Complement component 5 (C5), and a method for preventing and treating complement-related diseases using the antibody. 
       BACKGROUND ART 
       [0002]    A complement system plays a first step in innate immunity to most rapidly recognize and destroy an infection source. In addition, the complement system plays an important role in bridging between innate immunity and adaptive immunity by interaction with immune cells. The complement system is activated by a classical pathway, an alternative pathway and a lectin pathway, and then various kinds of complement proteins are activated. The complement proteins activate secretion of inflammatory substances, control an inflammatory response by interaction with immune cells, and effectively eliminate external infection sources by creating materials attacking the infection source, and the like. It is known that since the complement system inhibits an excessive increase in complementary activity by various kinds of complement regulatory proteins, maintains homeostasis, and plays a critical role through various steps of an inflammatory response and an immune response, when complement protein and complement regulatory protein are not properly controlled, various diseases are caused. 
         [0003]    When the complement system is activated by the classical pathway, the alternative pathway and the lectin pathway, Complement component 5 (C5) convertase cleaves C5 into C5a and C5b. 
         [0004]    C5 is expressed intracellularly as a single pro-C5 peptide of 1676 amino acids consisting of 18 residue signal sequences and an Arg-rich linker sequence (RPRR) between a mature N-terminal β-chain and a C-terminal α-chain. The mature C5 has a molecular weight of about 190 kDa, and consists of two polypeptide chains (α, 115 kDa and β, 75 kDa) which are connected by disulfide bonds. The C5 convertase cleaves C5 between residues 74 and 75 of the alpha chain to release the 74 amino acid C5a peptide and the C5b fragment which are subsequently incorporated into the membrane-attack complex (MAC). 
         [0005]    C5a which is anaphylatoxin, directly activates white blood cells and platelets, and functions as a chemotactic factor of a neutrophil. C5b forms a membrane attack complex together with C6, C7, C8 and C9 in a final step of complement activation to induce hemolysis. 
         [0006]    When the complement system is over-activated, since abnormal immune response, and damage of normal cells occur, abnormal activity of the complement system is related with autoimmune diseases, complement-related diseases, and the like. A hemolytic blood disease is a complement-related disease occurring when blood cells are not protected from attack of complement proteins due to genetic defects. It has been reported that complement activation is also related with vigorous immune response and tissue destruction reaction that occur in rheumatoid arthritis, transplant, and the like, and materials such as VEGF are released by tissue damage as well as the immune reaction by the complement activation to cause angiogenesis, which leads to elderly-related macular degeneration and diabetic retinopathy. 
         [0007]    That is, the complement system plays an important role in maintaining health; however, it potentially causes diseases or contributes to occurrence of diseases. Accordingly, it is preferable to develop a novel antibody, and the like, of a complement system to be used for treating and diagnosing complement-related diseases. 
         [0008]    There are provided a composition comprising a complement inhibitor, a method for treating or preventing complement-related diseases, and a use thereof. 
       SUMMARY OF INVENTION 
     Technical Problem 
       [0009]    The present invention has been made in an effort to provide a complement C5-binding molecule (for example, a C5-binding antibody or antigen-binding fragment thereof), a pharmaceutical composition comprising the molecule, a method for preparing the molecule and the composition, and a method for using the molecule and the composition, and a use of the molecule and the composition. 
         [0010]    In addition, the present invention has been made in an effort to provide an antibody specifically binding to C5 protein, or antigen-binding fragment thereof. 
         [0011]    Further, the present invention has been made in an effort to provide a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a heavy chain variable region having at least 90%, 95%, 97%, 98% or at least 99% sequence identity to any one selected from SEQ ID NOs: 7, 17, 27, 37, 47 or 57. 
         [0012]    In addition, the present invention has been made in an effort to provide a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a light chain variable region having at least 90%, 95%, 97%, 98% or at least 99% sequence identity to any one selected from SEQ ID NO: 8, 18, 28, 38, 48 or 58. 
         [0013]    Further, the present invention has been made in an effort to provide a vector and a host cell comprising the nucleic acid as described above. 
         [0014]    In addition, the present invention has been made in an effort to provide a pharmaceutical composition, comprising: at least one C5-binding molecule (for example, a C5-binding antibody or antigen-binding fragment thereof). 
         [0015]    Further, the present invention has been made in an effort to provide a method for treating or diagnosing complement-related diseases, using a C5-binding molecule. 
         [0016]    In addition, the present invention has been made in an effort to provide a kit for diagnosing complement-related diseases comprising: a C5-binding molecule; and a container. 
         [0017]    Further, the present invention has been made in an effort to provide a use of the C5-binding molecule in preparing a medicament for treating complement-related diseases. 
         [0018]    Further, the present invention has been made in an effort to provide a use of the C5-binding molecule in treating complement-related diseases. 
       Solution to Problem 
       [0019]    An exemplary embodiment of the present invention provides an antibody specifically binding to C5 protein, or antigen-binding fragment thereof. The antibody of the present invention or the antigen-binding fragment thereof may prevent or treat complement-related diseases by inhibiting complement activation by specific binding to C5 protein. 
         [0020]    An “antibody” of the present invention includes whole antibodies and any antigen-binding portion or single chains thereof. A naturally occurring “antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (V H ) and a heavy chain constant region (C H ). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (V L ) and a light chain constant region (C L ). The light chain constant region consists of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDR), interspersed with regions that are more conserved, referred to as framework regions (FR). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. 
         [0021]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention specifically binds to the beta chain (β-chain) of C5, more specifically, to an MG4 domain of the C5 beta-chain, and more specifically, based on amino acid sequences of the beta chain, to 332nd to 398th amino acid residue sequences, preferably, 332nd to 378th amino acid residue sequences, and more preferably 332nd to 364th amino acid residue sequences, more preferably 332nd to 348th amino acid residue sequences and/or 350th to 420th, preferably, 369th to 409th, more preferably, 379th to 398th, and more preferably, 386th to 392nd amino acid residue sequences. For example, as C5 protein capable of being bound, amino acid sequences of human C5 protein are represented by SEQ ID NO. 61, amino acid sequences of the beta chain of human C5 protein are represented by SEQ ID NO. 62, and amino acid sequences of MG4 domain of the beta chain of human C5 protein are represented by SEQ ID NO. 63. Interspecific cross-reactivity with other species such as rabbits, rats, monkeys, and the like is also provided. 
         [0022]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention has an affinity constant (K A ) of at least 1×10 7 M −1 , 1×10 8 M −1 , 1×10 9 M −1 , 1×10 10 M −1 , or 1×10 11 M −1 . 
         [0023]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof according to the present invention is antibodies bound to the same epitope as antibodies shown in Tables 1 to 6 below or antigen-binding fragment thereof, and has at least 90%, 95%, 97%, 98% or at least 99% sequence identity to corresponding sequences. In addition, antibodies having complement inhibiting activity are also included in the scope of the present invention. In addition, in a case of some modifications that are obvious in heavy chain and light chain constant regions, the modifications within a scope in which the same or similar complement inhibitory activity is provided are included in the scope of the present invention. Further, since each of these antibodies is capable of being bound to C5, nucleotide sequences that encode VH, VL, full length heavy chain sequences, and full length light chain sequences (amino acid sequences and nucleotide sequences that encode the amino acid sequences) may be “mixed and matched” to create other C5-binding antibodies of the present invention. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 C5 Antibody (HRA-06-H2-1) 
               
             
          
           
               
                 HRA-06-H2-1 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                 CDRH1 
                 1. GFSFSGRYWIQ 
               
               
                 CDR1 of Heavy Chain 
               
               
                   
               
               
                 CDRH2 
                 2. SVWPGITGDTNYANWAKG 
               
               
                 CDR2 of Heavy Chain 
               
               
                   
               
               
                 CDRH3 
                 3. EPVAWGGGLDL 
               
               
                 CDR3 of Heavy Chain 
               
               
                   
               
               
                 CDRL1 
                 4. QASQSINNQLS 
               
               
                 CDR1 of Light Chain 
               
               
                   
               
               
                 CDRL2 
                 5. YASTLAS 
               
               
                 CDR2 of Light Chain 
               
               
                   
               
               
                 CDRL3 
                 6. QGSYYSGGWDYG 
               
               
                 CDR1 of Light Chain 
               
               
                   
               
               
                 VH 
                 7. EVQLVESGGGLVQPGGSLRLSCAASGFS 
               
               
                 Variable region of 
                 FSGRYWIQWVRQAPGKGLEWVASVWPGITGD 
               
               
                 Heavy Chain 
                 TNYANWAKGRFTISRDDSKNTLYLQMNSLRA 
               
               
                   
                 EDTAVYYCAREPVAWGGGLDLWGQGTLVTVS 
               
               
                   
                 S 
               
               
                   
               
               
                 VL 
                 8. DIQMTQSPSSLSASVGDRVTITCQASQS 
               
               
                 Variable region of 
                 INNQLSWYQQKPGKAPKLLIYYASTLASGYP 
               
               
                 Light Chain 
                 SRFSGSGSGTDFTLTISSLQPEDFATYYCQG 
               
               
                   
                 SYYSGGWDYGFGQGTKVEIK 
               
               
                   
               
               
                 Heavy Chain 
                 9. EVQLVESGGGLVQPGGSLRLSCAASGFS 
               
               
                   
                 FSGRYWIQWVRQAPGKGLEWVASVWPGITGD 
               
               
                   
                 TNYANWAKGRFTISRDDSKNTLYLQMNSLRA 
               
               
                   
                 EDTAVYYCAREPVAWGGGLDLWGQGTLVTVS 
               
               
                   
                 SASTKGPSVFPLAPCSRSTSESTAALGCLVK 
               
               
                   
                 DYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 
               
               
                   
                 LYSLSSVVTVPSSNFGTQTYTCNVDHKPSNT 
               
               
                   
                 KVDKTVERKCCVECPPCPAPPVAGPSVFLFP 
               
               
                   
                 PKPKDTLMISRTPEVTCVVVDVSQEDPEVQF 
               
               
                   
                 NWYVDGVEVHNAKTKPREEQFNSTYRVVSVL 
               
               
                   
                 TVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS 
               
               
                   
                 KAKGQPREPQVYTLPPSQEEMTKNQVSLTCL 
               
               
                   
                 VKGFYPSDIAVEWESNGQPENNYKTTPPVLD 
               
               
                   
                 SDGSFFLYSRLTVDKSRWQEGNVFSCSVMHE 
               
               
                   
                 ALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 Light Chain 
                 10. DIQMTQSPSSLSASVGDRVTITCQASQ 
               
               
                   
                 SINNQLSWYQQKPGKAPKLLIYYASTLASGV 
               
               
                   
                 PSRFSGSGSGTDFTLTISSLQPEDFATYYCQ 
               
               
                   
                 GSYYSGGWDYGFGQGTKVEIKRTVAAPSVFI 
               
               
                   
                 FPPSDEQLKSGTASVVCLLNNFYPREAKVQW 
               
               
                   
                 KVDNALQSGNSQESVTEQDSKDSTYSLSSTL 
               
               
                   
                 TLSKADYEKHKVYACEVTHQGLSSPVTKSFN 
               
               
                   
                 RGEC 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 C5 Antibody (HRA-06-H2-7) 
               
             
          
           
               
                 HRA-06-112-7 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                 CDRH1 
                 11. GFSFSGRYWIQ 
               
               
                   
               
               
                 CDRH2 
                 12. SGWPGATGDTNYANWAKG 
               
               
                   
               
               
                 CDRH3 
                 13. EPVAWGGGLDL 
               
               
                   
               
               
                 CDRL1 
                 14. QASQSINNQLS 
               
               
                   
               
               
                 CDRL2 
                 15. YASTLAS 
               
               
                   
               
               
                 CDRL3 
                 16. QGSYYSGGWDYG 
               
               
                   
               
               
                 VH 
                 17. EVQLVESGGGLVQPGGSLRLSCAASGFSFSGRY 
               
               
                   
                 WIQWVRQAPGKGLEWVASGWPGATGDTNYANWAKGRF 
               
               
                   
                 TISRDDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGG 
               
               
                   
                 GLDLWGQGTLVTVSS 
               
               
                   
               
               
                 VL 
                 18. DIQMTQSPSSLSASVGDRVTITCQASQSINNQL 
               
               
                   
                 SWYQQKPGKAPKLLIYYASTLASGVPSRFSGSGSGTD 
               
               
                   
                 FTLTISSLQPEDFATYYCQGSYYSGGWDYGFGQGTKV 
               
               
                   
                 EIK 
               
               
                   
               
               
                 Heavy Chain 
                 19. EVQLVESGGGLVQPGGSLRLSCAASGFSFSGRY 
               
               
                   
                 WIQWVRQAPGKGLEWVASGWPGATGDTNYANWAKGRF 
               
               
                   
                 TISRDDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGG 
               
               
                   
                 GLDLWGQGTLVTVSSASTKGPSVFPLAPCSRSTSEST 
               
               
                   
                 AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS 
               
               
                   
                 SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDK 
               
               
                   
                 TVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMIS 
               
               
                   
                 RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP 
               
               
                   
                 REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL 
               
               
                   
                 PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL 
               
               
                   
                 TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG 
               
               
                   
                 SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQK 
               
               
                   
                 SLSLSLGK 
               
               
                   
               
               
                 Light Chain 
                 20. DIQMTQSPSSLSASVGDRVTITCQASQSINNQL 
               
               
                   
                 SWYQQKPGKAPKLLIYYASTLASGVPSRFSGSGSGTD 
               
               
                   
                 FTLTISSLQPEDFATYYCQGSYYSGGWDYGFGQGTKV 
               
               
                   
                 EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP 
               
               
                   
                 REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST 
               
               
                   
                 LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                   
               
               
                 C5 Antibody (HRA-06-H2-18) 
               
             
          
           
               
                 HRA-06-H2-18 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                 CDRH1 
                 21. GFSFSGRYWIQ 
               
               
                   
               
               
                 CDRH2 
                 22. SSSLRGTGDTNYANWAKG 
               
               
                   
               
               
                 CDRH3 
                 23. EPVAWGGGLDL 
               
               
                   
               
               
                 CDRL1 
                 24. QASQSLNNQLS 
               
               
                   
               
               
                 CDRL2 
                 25. YASTLAS 
               
               
                   
               
               
                 CDRL3 
                 26.QGSYYSGGWDYG 
               
               
                   
               
               
                 VH 
                 27.EVQLVESGGGLVQPGGSLRLSCAASGFSFSGRYWI 
               
               
                   
                 QWVRQAPGKGLEWVASSSLRGTGDTNYANWAKGRFTIS 
               
               
                   
                 RDDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDL 
               
               
                   
                 WGQGTLVTVSS 
               
               
                   
               
               
                 VL 
                 28.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSW 
               
               
                   
                 YQQKPGKAPKLLIYYASTLASGVPSRFSGSGSGTDFTL 
               
               
                   
                 TISSLQPEDFATYYCQGSYYSGGWDYGFGQGTKVEIK 
               
               
                   
               
               
                 Heavy 
                 29.EVQLVESGGGLVQPGGSLRLSCAASGFSFSGRYWI 
               
               
                 Chain 
                 QWVRQAPGKGLEWVASSSLRGTGDTNYANWAKGRFTIS 
               
               
                   
                 RDDSKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDL 
               
               
                   
                 WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC 
               
               
                   
                 LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL 
               
               
                   
                 SSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC 
               
               
                   
                 VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCV 
               
               
                   
                 VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY 
               
               
                   
                 RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK 
               
               
                   
                 AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD 
               
               
                   
                 IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK 
               
               
                   
                 SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 Light 
                 30.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSW 
               
               
                 Chain 
                 YQQKPGKAPKLLIYYASTLASGVPSRFSGSGSGTDFTL 
               
               
                   
                 TISSLQPEDFATYYCQGSYYSGGWDYGFGQGTKVEIKR 
               
               
                   
                 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV 
               
               
                   
                 QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA 
               
               
                   
                 DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 C5 Antibody (HRA-06-H2-24) 
               
             
          
           
               
                 HRA- 
                   
               
               
                 06- 
                   
               
               
                 H2-24 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                 CDRH1 
                 31.GFSFSGRYWIQ 
               
               
                   
               
               
                 CDRH2 
                 32.SVWPGFTGDTNYANWAKG 
               
               
                   
               
               
                 CDRH3 
                 33.EPVAWGGGLDL 
               
               
                   
               
               
                 CDRL1 
                 34.QASQSINNQLS 
               
               
                   
               
               
                 CDRL2 
                 35.YASTLAS 
               
               
                   
               
               
                 CDRL3 
                 36.QGSYYSGGWDYG 
               
               
                   
               
               
                 VH 
                 37.EVQLVESGGGLVQPGGSLRLSCAASGFSFSGRYWIQWVRQ 
               
               
                   
                 APGKGLEWVASVWPGFTGDTNYANWAKGRFTISRDDSKNTL 
               
               
                   
                 YLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQGTLVTVSS 
               
               
                   
               
               
                 VL 
                 38.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQKPG 
               
               
                   
                 KAPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                 YYCQGSYYSGGWDYGFGQGTKVEIK 
               
               
                   
               
               
                 Heavy 
                 39.EVQLVESGGGLVQPGGSLRLSCAASGFSFSGRYWIQWVRQ 
               
               
                 Chain 
                 APGKGLEWVASVWPGFTGDTNYANWAKGRFTISRDDSKNTL 
               
               
                   
                 YLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQGTLVTVS 
               
               
                   
                 SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN 
               
               
                   
                 SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNV 
               
               
                   
                 DHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD 
               
               
                   
                 TLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK 
               
               
                   
                 PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI 
               
               
                   
                 EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS 
               
               
                   
                 DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW 
               
               
                   
                 QEGNVFSCSVMHEALHNHYTQKSLSLSLGK 
               
               
                   
               
               
                 Light 
                 40.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQKPG 
               
               
                 Chain 
                 KAPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPEDFAT 
               
               
                   
                 YYCQGSYYSGGWDYGFGQGTKVEIKRTVAAPSVFIFPPSDEQ 
               
               
                   
                 LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTE 
               
               
                   
                 QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK 
               
               
                   
                 SFNRGEC 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 C5 Antibody (HRA-06-H1-9-H2-7) 
               
             
          
           
               
                 HRA-06- 
                   
               
               
                 H1-9- 
                   
               
               
                 H2-7 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                 CDRH1 
                 41.GFSLSGRYWIQ 
               
               
                   
               
               
                 CDRH2 
                 42.SGWPGATGDTNYANWAKG 
               
               
                   
               
               
                 CDRH3 
                 43.EPVAWGGGLDL 
               
               
                   
               
               
                 CDRL1 
                 44.QASQSINNQLS 
               
               
                   
               
               
                 CDRL2 
                 45.YASTLAS 
               
               
                   
               
               
                 CDRL3 
                 46.QGSYYSGGWDYG 
               
               
                   
               
               
                 VH 
                 47.EVQLVESGGGLVQPGGSLRLSCAASGFSLSGRYWIQWV 
               
               
                   
                 RQAPGKGLEWVASGWPGATGDTNYANWAKGRFTISRDD 
               
               
                   
                 SKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQ 
               
               
                   
                 GTLVTVSS 
               
               
                   
               
               
                 VL 
                 48.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQK 
               
               
                   
                 PGKAPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPE 
               
               
                   
                 DFATYYCQGSYYSGGWDYGFGQGTKVEIK 
               
               
                   
               
               
                 Heavy 
                 49.EVQLVESGGGLVQPGGSLRLSCAASGFSLSGRYWIQWV 
               
               
                 Chain 
                 RQAPGKGLEWVASGWPGATGDTNYANWAKGRFTISRDD 
               
               
                   
                 SKNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQ 
               
               
                   
                 GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF 
               
               
                   
                 PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS 
               
               
                   
                 NFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPV 
               
               
                   
                 AGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN 
               
               
                   
                 WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL 
               
               
                   
                 NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ 
               
               
                   
                 EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP 
               
               
                   
                 PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN 
               
               
                   
                 HYTQKSLSLSLGK 
               
               
                   
               
               
                 Light 
                 50.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQK 
               
               
                 Chain 
                 PGKAPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPE 
               
               
                   
                 DFATYYCQGSYYSGGWDYGFGQGTKVEIKRTVAAPSVFIF 
               
               
                   
                 PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG 
               
               
                   
                 NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT 
               
               
                   
                 HQGLSSPVTKSFNRGEC 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 C5 Antibody (HRA-06-H1-9-H2-24) 
               
             
          
           
               
                 HRA-06- 
                   
               
               
                 H1-9- 
                   
               
               
                 H2-24 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                 CDRH1 
                 51.GFSLSGRYWIQ 
               
               
                   
               
               
                 CDRH2 
                 52.SVWPGFTGDTNYANWAKG 
               
               
                   
               
               
                 CDRH3 
                 53.EPVAWGGGLDL 
               
               
                   
               
               
                 CDRL1 
                 54.QASQSINNQLS 
               
               
                   
               
               
                 CDRL2 
                 55.YASTLAS 
               
               
                   
               
               
                 CDRL3 
                 56.QGSYYSGGWDYG 
               
               
                   
               
               
                 VH 
                 57.EVQLVESGGGLVQPGGSLRLSCAASGFSLSGRYWIQWV 
               
               
                   
                 RQAPGKGLEWVASVWPGFTGDTNYANWAKGRFTISRDDS 
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQG 
               
               
                   
                 TLVTVSS 
               
               
                   
               
               
                 VL 
                 58.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQK 
               
               
                   
                 PGKAPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPE 
               
               
                   
                 DFATYYCQGSYYSGGWDYGFGQGTKVEIK 
               
               
                   
               
               
                 Heavy 
                 59.EVQLVESGGGLVQPGGSLRLSCAASGFSLSGRYWIQWV 
               
               
                 Chain 
                 RQAPGKGLEWVASVWPGFTGDTNYANWAKGRFTISRDDS 
               
               
                   
                 KNTLYLQMNSLRAEDTAVYYCAREPVAWGGGLDLWGQG 
               
               
                   
                 TLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE 
               
               
                   
                 PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNF 
               
               
                   
                 GTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAG 
               
               
                   
                 PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY 
               
               
                   
                 VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGK 
               
               
                   
                 EYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM 
               
               
                   
                 TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL 
               
               
                   
                 DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT 
               
               
                   
                 QKSLSLSLGK 
               
               
                   
               
               
                 Light 
                 60.DIQMTQSPSSLSASVGDRVTITCQASQSINNQLSWYQQK 
               
               
                 Chain 
                 PGKAPKLLIYYASTLASGVPSRFSGSGSGTDFTLTISSLQPE 
               
               
                   
                 DFATYYCQGSYYSGGWDYGFGQGTKVEIKRTVAAPSVFIF 
               
               
                   
                 PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG 
               
               
                   
                 NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT 
               
               
                   
                 HQGLSSPVTKSFNRGEC 
               
               
                   
               
             
          
         
       
     
         [0024]    The antibody of the present invention is prepared by using all antibodies comprising amino acids that are identical to antibodies shown in Tables 1 to 6; antibodies having heavy chain variable regions comprising CDR1, CDR2 and CDR3 sequences, and light chain variable regions comprising CDR1, CDR2 and CDR3 sequences, wherein at least one of the CDR sequences has the antibody described in the present invention or specific amino acid sequences based on conservative modifications thereof; antibodies having functional properties of the C5-binding antibody of the present invention; antibodies bound to the same epitope as the antibodies shown in Tables 1 to 6; antibodies having at least one VH and/or VL sequences described in the present invention as a starting material to engineer a modified antibody, and includes all of antibodies having properties which are partially modified from the starting antibody, comprising the above-mentioned antibodies. 
         [0025]    In addition, the antibody of the present invention includes those in which modifications have been made to framework residues within VH and/or VL, in order to improve properties of the antibody. 
         [0026]    Further, the antibody of the present invention may be a fully human antibody specifically bound to a C5 protein. When compared to chimeric antibodies, and the like, the antibody of the present invention may have further reduced antigenicity when administered to human subjects. A human antibody includes heavy or light chain variable regions or full length heavy or light chains that are the products of or derived from a particular germline sequence if the variable regions or full length chains of the antibody are obtained from a system that uses human germline immunoglobulin genes. Such systems include immunizing a transgenic mouse carrying human immunoglobulin genes with the antigen of interest or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody that is “the product of” or “derived from” a human germline immunoglobulin sequence may be identified as such by comparing the amino acid sequence of the human antibody with the amino acid sequences of human germline immunoglobulins and selecting the human germline immunoglobulin sequence that is closest in sequence to the sequence of the human antibody. 
         [0027]    In addition, the antibody of the present invention may be a bispecific or a multispecific antibody. The antibody of the present invention or the antigen-binding fragment thereof may be bispecific molecules that are bound to more than two different binding sites or target molecules. 
         [0028]    In some exemplary embodiments, the antibody of the present invention may be a monoclonal antibody specifically bound to the C5 protein. For example, the antibody of the present invention may be a human or humanized monoclonal antibody or a chimeric antibody that specifically binds to the C5 protein, and includes a human heavy chain constant region and a human light chain constant region. In addition, the antibody of the present invention may be a single chain antibody, and may be a Fab fragment, a single-chain variable fragment (scFv), and IgG isotype. Preferable IgG isotpes include IgG2, IgG4 and/or IgG2/4. In some exemplary embodiments, the IgG isotype of the present invention is IgG2/4. IgG2/4 hybrid constant region may have a form in which CH1 and a hinge region of IgG2 are fused with CH2 and CH3 regions of IgG4. 
         [0029]    The monoclonal antibody may be produced by general methods for producing monoclonal antibodies, and may be expressed and purified by inserting a synthesized antibody gene into a vector for expressing an antibody, preferably, pcDNA, pCI, pCMV, pCEP4, and the like. In addition, the monoclonal antibody may be produced by using viral or carcinogenic transformation of B lymphocytes, or on the basis of sequence of a murine monoclonal antibody produced using a murine system. For example, DNA encoding heavy chain and light chain immunoglobulin may be obtained from murine hybridoma and may contain non-murine immunoglobulin sequences together therewith, by standard molecular biology techniques. In addition, the human monoclonal antibody against C5 may be produced by using transgenic or transchromosomic mice having a part of a human immune system rather than a mouse immune system. 
         [0030]    In some exemplary embodiments, the present invention provides an antibody or antigen-binding fragment thereof comprising a framework in which amino acids are substituted into an antibody framework from the respective human VH or VL germline sequences. 
         [0031]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes at least one complementarity determining region (CDR) sequence having at least 95% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 11, 12, 21, 22, 31, 32, 41, 42, 51 or 52. 
         [0032]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes at least one heavy chain complementarity determining region sequence as the same as SEQ ID NO: 1, 2, 3, 11, 12, 21, 22, 31, 32, 41, 42, 51 or 52. 
         [0033]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes at least one light chain complementarity determining region sequence as the same as SEQ ID NO: 4, 5, or 6. 
         [0034]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes any one heavy chain complementarity determining region 1 (CDR1) selected from SEQ ID NO: 1, 11, 21, 31, 41 or 51, any one heavy chain complementarity determining region 2 (CDR2) selected from SEQ ID NO: 2, 12, 22, 32, 42 or 52, and/or any one heavy chain CDR3 selected from SEQ ID NO: 3, 13, 23, 33, 43 or 53. 
         [0035]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes light chain CDR1 of SEQ ID NO: 4, light chain CDR2 of SEQ ID NO: 5, and/or light chain CDR3 of SEQ ID NO: 6. 
         [0036]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes any one heavy chain variable region selected from SEQ ID NO: 7, 17, 27, 37, 47 or 57, or includes a heavy chain variable region having at least 90%, 95%, 97%, or at least 99% sequence identity to any one heavy chain variable region selected from SEQ ID NO: 7, 17, 27, 37, 47 or 57. 
         [0037]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes a light chain variable region of SEQ ID NO: 8 or includes a light chain variable region having at least 90%, 95%, 97% or at least 99% sequence identity to the light chain variable region of SEQ ID NO: 8. 
         [0038]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes any one heavy chain selected from SEQ ID NO: 9, 19, 29, 39, 49 or 59, or includes a heavy chain variable region having at least 90%, 95%, 97%, or at least 99% sequence identity to any one heavy chain selected from SEQ ID NO: 9, 19, 29, 39, 49 or 59. 
         [0039]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes a light chain of SEQ ID NO: 10, or includes a light chain having at least 90%, 95%, 97% or at least 99% sequence identity to the light chain of SEQ ID NO: 10. 
         [0040]    In some exemplary embodiments, the antibody or antigen-binding fragment thereof that specifically binds to the C5 protein according to the present invention includes those bound to an epitope in the beta chain of the C5 protein of SEQ ID No. 62. In detail, the epitope may correspond to 332nd to 398th amino acid residue sequences, preferably, 332nd to 378th, more preferably, 332nd to 364th, and much more preferably, 332nd to 348th, and/or 350th to 420th, preferably, 369th to 409th, more preferably, 379th to 398th, and much more preferably, 386th to 392nd amino acid residue sequences, based on the beta chain amino acid sequence of the C5 protein. 
         [0041]    In addition, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a heavy chain variable region having at least 90%, 95%, 97%, 98% or at least 99% sequence identity to any one selected from SEQ ID NO: 7, 17, 27, 37, 47 or 57. 
         [0042]    In some exemplary embodiments, the nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a heavy chain variable region of the present invention has sequences shown in Table 7 below or has at least 90%, 95%, 97%, 98% or at least 99% sequence identity to any one sequence thereof. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Nucleotide Sequence Encoding Heavy Chain Variable Region 
               
             
          
           
               
                 Variable region of Heavy chan 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                 HRA-06-H2-1 
                 64. GAG GTG CAG CTG GTG GAG TCT GGC GGC 
               
               
                   
                 GGA CTG GTG CAG CCT GGC GGA AGC TTG CGG 
               
               
                   
                 CTG TCC TGC GCC GCC TCC GGA TTC TCC TTC AGT 
               
               
                   
                 GGC AGG TAC TGG ATA CAA TGG GTG CGG CAG 
               
               
                   
                 GCC CCT GGC AAG GGC CTC GAG TGG GTG GCC 
               
               
                   
                 TCT GTG TGG CCT GGT ATT ACT GGT GAC ACT 
               
               
                   
                 AAC TAC GCG AAC TGG GCG AAA GGC CGG TTC 
               
               
                   
                 ACC ATC TCC CGG GAC GAC TCC AAG AAC ACC 
               
               
                   
                 CTG TAC CTG CAG ATG AAC TCC CTG CGG GCC 
               
               
                   
                 GAG GAC ACC GCC GTG TAC TAC TGC GCC AGA 
               
               
                   
                 GAA CCT GTT GCC TGG GGT GGC GGC TTG GAC 
               
               
                   
                 TTG TGG GGC CAG GGC ACA CTA GTG ACC GTG 
               
               
                   
                 TCC TCC 
               
               
                   
               
               
                 HRA-06-H2-7 
                 65. GAG GTG CAG CTG GTG GAG TCT GGC GGC 
               
               
                   
                 GGA CTG GTG CAG CCT GGC GGA AGC TTG CGG 
               
               
                   
                 CTG TCC TGC GCC GCC TCC GGA TTC TCC TTC AGT 
               
               
                   
                 GGC AGG TAC TGG ATA CAA TGG GTG CGG CAG 
               
               
                   
                 GCC CCT GGC AAG GGC CTC GAG TGG GTG GCC 
               
               
                   
                 AGT GGT TGG CCG GGG GCG ACT GGT GAC ACT 
               
               
                   
                 AAC TAC GCG AAC TGG GCG AAA GGC CGG TTC 
               
               
                   
                 ACC ATC TCC CGG GAC GAC TCC AAG AAC ACC 
               
               
                   
                 CTG TAC CTG CAG ATG AAC TCC CTG CGG GCC 
               
               
                   
                 GAG GAC ACC GCC GTG TAC TAC TGC GCC AGA 
               
               
                   
                 GAA CCT GTT GCC TGG GGT GGC GGC TTG GAC 
               
               
                   
                 TTG TGG GGC CAG GGC ACA CTA GTG ACC GTG 
               
               
                   
                 TCC TCC 
               
               
                   
               
               
                 HRA-06-H2-18 
                 66. GAG GTG CAG CTG GTG GAG TCT GGC GGC 
               
               
                   
                 GGA CTG GTG CAG CCT GGC GGA AGC TTG CGG 
               
               
                   
                 CTG TCC TGC GCC GCC TCC GGA TTC TCC TTC AGT 
               
               
                   
                 GGC AGG TAC TGG ATA CAA TGG GTG CGG CAG 
               
               
                   
                 GCC CCT GGC AAG GGC CTC GAG TGG GTG GCC 
               
               
                   
                 AGT TCT AGT TTG CGG GGG ACT GGT GAC ACT 
               
               
                   
                 AAC TAC GCG AAC TGG GCG AAA GGC CGG TTC 
               
               
                   
                 ACC ATC TCC CGG GAC GAC TCC AAG AAC ACC 
               
               
                   
                 CTG TAC CTG CAG ATG AAC TCC CTG CGG GCC 
               
               
                   
                 GAG GAC ACC GCC GTG TAC TAC TGC GCC AGA 
               
               
                   
                 GAA CCT GTT GCC TGG GGT GGC GGC TTG GAC 
               
               
                   
                 TTG TGG GGC CAG GGC ACA CTA GTG ACC GTG 
               
               
                   
                 TCC TCC 
               
               
                   
               
               
                 HRA-06-H2-24 
                 67. GAG GTG CAG CTG GTG GAG TCT GGC GGC 
               
               
                   
                 GGA CTG GTG CAG CCT GGC GGA AGC TTG CGG 
               
               
                   
                 CTG TCC TGC GCC GCC TCC GGA TTC TCC TTC AGT 
               
               
                   
                 GGC AGG TAC TGG ATA CAA TGG GTG CGG CAG 
               
               
                   
                 GCC CCT GGC AAG GGC CTC GAG TGG GTG GCC 
               
               
                   
                 TCG GTG TGG CCG GGG TTT ACT GGT GAC ACT 
               
               
                   
                 AAC TAC GCG AAC TGG GCG AAA GGC CGG TTC 
               
               
                   
                 ACC ATC TCC CGG GAC GAC TCC AAG AAC ACC 
               
               
                   
                 CTG TAC CTG CAG ATG AAC TCC CTG CGG GCC 
               
               
                   
                 GAG GAC ACC GCC GTG TAC TAC TGC GCC AGA 
               
               
                   
                 GAA CCT GTT GCC TGG GGT GGC GGC TTG GAC 
               
               
                   
                 TTG TGG GGC CAG GGC ACA CTA GTG ACC GTG 
               
               
                   
                 TCC TCC 
               
               
                   
               
               
                 HRA-06-H1-9-H2-7 
                 68. GAG GTG CAG CTG GTG GAG TCT GGC GGC 
               
               
                   
                 GGA CTG GTG CAG CCT GGC GGA AGC TTG CGG 
               
               
                   
                 CTG TCC TGC GCC GCC TCC GGA TTC TCC CTC AGT 
               
               
                   
                 GGC AGG TAC TGG ATA CAA TGG GTG CGG CAG 
               
               
                   
                 GCC CCT GGC AAG GGC CTC GAG TGG GTG GCC 
               
               
                   
                 AGT GGT TGG CCG GGG GCG ACT GGT GAC ACT 
               
               
                   
                 AAC TAC GCG AAC TGG GCG AAA GGC CGG TTC 
               
               
                   
                 ACC ATC TCC CGG GAC GAC TCC AAG AAC ACC 
               
               
                   
                 CTG TAC CTG CAG ATG AAC TCC CTG CGG GCC 
               
               
                   
                 GAG GAC ACC GCC GTG TAC TAC TGC GCC AGA 
               
               
                   
                 GAA CCT GTT GCC TGG GGT GGC GGC TTG GAC 
               
               
                   
                 TTG TGG GGC CAG GGC ACA CTA GTG ACC GTG 
               
               
                   
                 TCC TCC 
               
               
                   
               
               
                 HRA-06-H1-9-H2-24 
                 69. GAG GTG CAG CTG GTG GAG TCT GGC GGC 
               
               
                   
                 GGA CTG GTG CAG CCT GGC GGA AGC TTG CGG 
               
               
                   
                 CTG TCC TGC GCC GCC TCC GGA TTC TCC CTC AGT 
               
               
                   
                 GGC AGG TAC TGG ATA CAA TGG GTG CGG CAG 
               
               
                   
                 GCC CCT GGC AAG GGC CTC GAG TGG GTG GCC 
               
               
                   
                 TCG GTG TGG CCG GGG TTT ACT GGT GAC ACT 
               
               
                   
                 AAC TAC GCG AAC TGG GCG AAA GGC CGG TTC 
               
               
                   
                 ACC ATC TCC CGG GAC GAC TCC AAG AAC ACC 
               
               
                   
                 CTG TAC CTG CAG ATG AAC TCC CTG CGG GCC 
               
               
                   
                 GAG GAC ACC GCC GTG TAC TAC TGC GCC AGA 
               
               
                   
                 GAA CCT GTT GCC TGG GGT GGC GGC TTG GAC 
               
               
                   
                 TTG TGG GGC CAG GGC ACA CTA GTG ACC GTG 
               
               
                   
                 TCC TCC 
               
               
                   
               
             
          
         
       
     
         [0043]    In addition, the present invention provides a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a light chain variable region having at least 90%, 95%, 97%, 98% or at least 99% sequence identity to SEQ ID NO: 8. In some exemplary embodiments, the nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising a light chain variable region of the present invention has sequences shown in Table 8 below or has at least 90%, 95%, 97%, 98% or at least 99% sequence identity to any one sequence thereof. 
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Nucleotide Sequence Encoding Light Chain Variable Region 
               
             
          
           
               
                 Variable region of Light chain 
                 SEQ ID NO and Sequence 
               
               
                   
               
               
                   
                 70. GAC ATC CAG ATG ACC CAG TCC CCC TCC TCG 
               
               
                   
                 CTG AGC GCC TCC GTG GGC GAC CGG GTG ACC 
               
               
                   
                 ATC ACC TGC CAG GCC AGT CAG AGC ATT AAC 
               
               
                   
                 AAC CAA CTA TCC TGG TAT CAG CAG AAG CCT 
               
               
                   
                 GGC AAG GCG CCT AAG CTG CTG ATC TAC TAT 
               
               
                   
                 GCA TCC ACT CTG GCA TCT GGC GTG CCT TCC 
               
               
                   
                 CGG TTC TCC GGA TCC GGC TCC GGC ACC GAC 
               
               
                   
                 TTC ACC CTG ACC ATC TCC TCC CTG CAA CCT 
               
               
                   
                 GAG GAC TTC GCC ACC TAC TAC TGC CAA GGC 
               
               
                   
                 AGT TAT TAT AGT GGT GGT TGG GAC TAT GGT 
               
               
                   
                 TTC GGC CAG GGT ACC AAG GTG GAG ATC AAG 
               
               
                   
               
             
          
         
       
     
         [0044]    Further, the present invention provides a vector and a host cell comprising the nucleic acid as described above. In one exemplary embodiment, the present invention provides a host cell comprising (1) a recombinant DNA segment encoding the heavy chain of the antibody of the present invention, and (2) a second recombinant DNA segment encoding the light chain of the antibody of the present invention. In another exemplary embodiment, the present invention provides a host cell comprising a recombinant DNA segment encoding each of the heavy chain and the light chain of the antibody of the present invention. In some exemplary embodiment, the antibody or the antigen-binding fragment thereof is a human monoclonal antibody or antigen-binding fragment thereof. 
         [0045]    In order to express polynucleotide encoding a C5-binding antibody, chain, or binding fragment thereof, various expression vector may be used, and in order to produce antibodies in mammalian host cells, both of virus-based and non-viral expression vector may be used. Vectors such as pcDNA, pCI, pCMV, pCEP4, and the like, and host cells such as HEK293, CHO, CHO-DG44, and the like, may be used. 
         [0046]    The host cell for harboring and expressing the C5-binding antibody may be an eukaryotic cell or a prokaryotic cell.  E. Coli , preferably,  E. coli  ER2738. HB2151, BL21, and the like, may be included as examples, which are eukaryotic host cells useful for cloning and expressing the polynucleotide of the present invention. Other microbial host cells suitable for being used include  bacillus , such as  Bacillus subtilis , and other enteric bacteria, such as  Salmonella, Serratia  and various  Pseudomonas  species. Other microbes, such as yeast, are capable of being employed to express C5-binding polypeptide of the present invention, and insect cells in combination with baculovirus vectors may also be used. 
         [0047]    In some preferred exemplary embodiments, mammalian host cells are used to express and produce the C5-binding polypeptide of the present invention. For example, they may be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector. In addition, for example, as any animal or human cell, a number of suitable host cell lines capable of secreting immunoglobulin comprising CHO cell lines, Cos cell lines, HeLa cells, myeloma cell lines, HEK cell lines, transformed B-cells and hybridomas may be used, preferably, HEK293, CHO, CHO-DG44 may be used. 
         [0048]    Further, the present invention provides a pharmaceutical composition, comprising: at least one C5-binding molecule (for example, a C5-binding antibody or antigen-binding fragment thereof). 
         [0049]    The pharmaceutical composition of the present invention is effective for treating complement-related diseases. The complement-related diseases include all diseases and pathological conditions in which onset of the diseases is related with abnormality of activation of the complement system, for example, complement deficiency. For example, the complement-related diseases include inflammatory diseases and autoimmune diseases, such as rheumatoid arthritis (RA), osteoarthritis, acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis, glomerulonephritis, renal vasculitis, cardiopulmonary by-pass, heart failure-induced coronary endothelial dysfunction, type II membrane-proliferative glomerulonephritis, acute renal failure, antiphospholipid syndrome, macular degeneration, endophthalmitis, new blood vessel disease, allograft transplantation, hyperacute rejection, hemodialysis, chronic obstructive pulmonary disorder (COPD) respiratory distress syndrome, asthma, paroxymal nocturnal hemoglobinuria (PNH) and aspiration pneumonia, but the present invention is not limited thereto. 
         [0050]    The composition may additionally contain one or more other therapeutic agents that are suitable for treating or preventing complement-related diseases. Pharmaceutical carriers enhance or stabilize the composition, or facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. 
         [0051]    The pharmaceutical composition of the present invention may be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. It is preferred that administration may be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. In a specific exemplary embodiment, the antibodies of the present invention are formulated so that they can be administered intravitreally into the eye. Depending on the route of administration, active compounds, that is, antibody, bispecific and multispecific molecules, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. 
         [0052]    The composition needs to be sterile and fluid. Proper fluidity may be maintained, for example, by using the coating materials such as lecithin, or by maintaining required particle size in the case of dispersion liquid and by using surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions may be brought about by comprising an agent which delays absorption, for example, aluminum monostearate or gelatin in the composition. 
         [0053]    The pharmaceutical composition of the present invention may be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., [Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000] and [Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978]. The pharmaceutical composition is preferably prepared under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the C5-binding antibody is employed in the pharmaceutical composition of the present invention. The C5-binding antibodies are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). 
         [0054]    Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, for example, activity of the particular compositions of the present invention employed, or an ester, a salt or an amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and other factors. 
         [0055]    Treatment dosages need to be titrated to optimize safety and efficacy. For systemic administration with an antibody, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 15 mg/kg, of the host body weight. An exemplary treatment regime entails systemic administration once per every two weeks or once a month or once every 3 to 6 months. For intravitreal administration with an antibody, the dosage ranges from about 0.0001 to about 10 mg. An exemplary treatment regime entails systemic administration once per every two weeks or once a month or once every 3 to 6 months. 
         [0056]    In some methods of systemic administration, dosage is adjusted to achieve a plasma antibody concentration of 1-1000 μg/ml and in some methods 25-500 μg/ml. Alternatively, antibody can be administered as a sustained release formulation, in the case in which less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. 
         [0057]    Further, the present invention provides a method for treating or diagnosing complement-related diseases, using a C5-binding molecule. 
         [0058]    The method for treating complement-related diseases using a C5-binding molecule of the present invention includes: administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof or a composition comprising the same. Term “therapeutically effective amount” used in the present invention indicates an amount of the C5-binding molecule of the present invention or an amount of the composition comprising the C5-binding molecule of the present invention which is effective for preventing or treating complement-related diseases. 
         [0059]    When the C5-binding molecule or the composition comprising the same is administered in combination with another agent as a therapeutic agent of the present invention, these two materials may be administered sequentially or simultaneously in any order. Suitable agents for combination treatment with C5-binding antibodies include agents known in the art that are able to modulate activities of complement components. For example, the agents include phosphonate esters, polyanionic substances, sulfonyl fluorides, polynucleotides, pimaric acids, several antiinflammatories, and the like. A combination therapy with at least one therapeutic agent, and the like, may be added, and may bring results of synergism. 
         [0060]    The present invention includes diagnostic assay determining expression of C5 protein and/or nucleic acid and C5 protein function in biological samples (for example, blood, blood serum, cells, tissue) or from a subject suffering from complement-related diseases or a subject having a risk thereof. In the antibody of the present invention, for example, radioimmunoassay (REA), enzyme-linked immunosorbent assay (ELISA), and radial diffusion assay are usable for detecting a complement cleavage product. Further, a diagnostic assay, a prognostic assay, pharmacological genetic and clinical monitoring may be used to prophylactically treat a subject with the purpose of prognosis (prediction). In addition, the present invention provides a prognosis (prediction) assay for determining whether or not the subject is at the risk of onset of diseases related with regulation abnormality of activation of complement pathway. For example, mutation in the C5 gene may be assayed in a biological sample. By using this assay with the purpose of prognosis or prediction, a subject may be prophylactically treated before the onset of diseases characterized by expression or activity of the C5 protein, nucleic acid or diseases related therewith. 
         [0061]    In addition, the present invention provides a kit for diagnosing complement-related diseases comprising: a C5-binding molecule; and a container. The kit for diagnosing of the present invention may include at least any one of the above-mentioned C5-binding molecule. The container may include a solid carrier, and the C5-binding molecule may be bound to the solid carrier, and the solid carrier may be porous or non-porous, flat or non-planar. 
         [0062]    Further, the present invention provides a use of the C5-binding molecule in preparing a medicament for treating complement-related diseases. The C5-binding molecule of the present invention for preparing a medicament or the composition comprising the same may be mixed with acceptable carriers, and the like, and may be prepared as a complex medication together with other agents to have a synergistic effect of the active ingredients. 
         [0063]    In addition, the present invention provides a use of the C5-binding molecule. The C5-binding molecule for treating complement-related diseases of the present invention may be used with the purpose of treatment, and may be used as a use of a prognosis assay for determining expression of the C5 protein and/or nucleic acid or C5 protein function from a subject suffering from complement-related diseases or a subject having a risk thereof. 
         [0064]    The descriptions in the use, the composition, and the treatment method of the present invention are applied as the same as each other unless contradictory. 
         [0065]    The C5-binding molecule of the present invention is effective for diagnosing, preventing, and treating complement-related diseases. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0066]      FIG. 1  shows absorbance of 40 clones randomly selected after bio-panning using immune libraries of rabbit (A) and chicken (B) according to an exemplary embodiment of the present invention. 
           [0067]      FIG. 2  shows absorbance measurement results of five kinds of antibodies selected from the immune libraries of rabbit and the immune libraries of chicken according to an exemplary embodiment of the present invention, and eculizumab which is a comparative antibody, to human C5. 
           [0068]      FIG. 3  shows results of a number of clones having binding affinity to C5, obtained from five mutant sub-libraries. 
           [0069]      FIG. 4  shows comparison results of binding affinity of clones with improved affinity produced by using HRA-06 clone which is humanized clone according to an exemplary embodiment of the present invention as a template. 
           [0070]      FIG. 5  shows that the antibody produced according to an exemplary embodiment of the present invention has high complement dependent cytotoxicity inhibitory ability in complement dependent cytotoxicity assay. 
           [0071]      FIG. 6  shows the antibody produced according to an exemplary embodiment of the present invention has high C5a generation inhibitory ability in C5a generation assay. 
           [0072]      FIG. 7  shows cross-species reactivity of a monoclonal antibody produced according to an exemplary embodiment of the present invention. 
           [0073]      FIG. 8  shows results of antibodies produced and purified according to an exemplary embodiment of the present invention by size-exclusion chromatography. 
           [0074]      FIG. 9  shows that the antibody according to an exemplary embodiment of the present invention is bound to a beta chain of C5. 
           [0075]      FIG. 10  shows that the antibody according to an exemplary embodiment of the present invention is bound to an MG4 domain of the beta chain of C5. 
           [0076]      FIG. 11  shows that the antibody according to an exemplary embodiment of the present invention specifically binds to the MG4 domain in a mutant Fc fusion protein in which one domain is sequentially removed from C terminus of the beta chain. 
           [0077]      FIG. 12  shows that the antibody according to an exemplary embodiment of the present invention is bound to 332nd to 348th amino acid residues at N-terminus of the beta chain in the mutant from which the MG4 domain is sequentially removed, as confirmed by immunoblotting. 
           [0078]      FIG. 13  shows that the antibody according to an exemplary embodiment of the present invention is bound to 379th to 398th amino acid residues at N-terminus of the beta chain, as confirmed by ELISA. 
           [0079]      FIG. 14  shows that the antibody according to an exemplary embodiment of the present invention is bound to 386th to 392nd amino acid residues at N-terminus of the beta chain (55th to 61th amino acid sequences based on the MG4 domain sequence), as confirmed by ELISA. 
       
    
    
     DESCRIPTION OF EMBODIMENTS 
       [0080]    Hereinafter, components and technical features of the present invention are described in more detail through the following examples. However, the following Examples are provided by way of examples, and therefore, the protective scope of the present invention is not limited to only the following Examples. 
         [0081]    Various examples described herein are described with reference to the drawings. In the following description, various specific details, for example, specific forms, compositions, and preparation methods, and the like, are described for complete understanding of the present invention. However, specific Examples may be practiced without at least one specific details or together with other known methods and forms. In another exemplary embodiment, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the present invention. Reference of “one exemplary embodiment” or “Examples” throughout the specification means that specific characteristics, forms, compositions, or properties described associated with Examples are included in one or more Examples of the present invention. Therefore, circumstance of expression “one exemplary embodiment” or “Examples” in various places throughout the specification does not necessarily indicate the same exemplary embodiment of the present invention. In addition, the specific characteristics, forms, compositions, or properties may be combined with each other by any suitable method in at least one exemplary embodiment. 
       Example 1 
     Construction of C5 Immune Antibody Library 
       [0082]    5 μg of human C5 protein (Calbiochem) was mixed with the RIBI MPL+TDM+CWS adjuvant (Sigma, St. Louis, Mo., USA) and injected subcutaneously into NZW rabbits and chickens, and boost immunizations were performed three times in rabbits and four times in chickens with 2-week intervals. Total RNA was isolated from the spleen and bone marrow of the immune-finished rabbit and spleen, bone marrow and bursa of fabricius of immune-finished chicken, by using TRI reagent (Invitrogen, Carlsbad, Calif., USA), and first-strand cDNA was synthesized using oligo-dT primer and SuperScript™ III First-Strand Synthesis System (Invitrogen). Single-chain Fv libraries were constructed by using primers of Table 9 (rabbit) and Table 10 (chicken) below that are specific to heavy chain variable regions and light chain variable regions of immunoglobulin. For rabbit scFv library, 10 primer combinations of V L  (9λ V κ  and 1×V λ )) and 4 combinations of V H  were used to amplify coding sequences. For chicken scFv library, one primer combination of each V λ  and V H  was used to amplify coding sequences. 
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Primers for V κ , V λ  and V H  of 
               
               
                 rabbit single-chain Fv libraries 
               
               
                   
               
             
             
               
                 Vκ 5′ Sense Primers 
               
             
          
           
               
                 RSCVK1 
                 71. GGG CCC AGG CGG CCG AGC TCG TGM TGA CCC 
               
               
                   
                 AGA CTC CA 
               
               
                   
               
               
                 RSCVK2 
                 72. GGG CCC AGG CGG CCG AGC TCG ATM TGA CCC 
               
               
                   
                 AGA CTC CA 
               
               
                   
               
               
                 RSCVK3 
                 73. GGG CCC AGG CGG CCG AGC TCG TGA TGA CCC 
               
               
                   
                 AGA CTG AA 
               
               
                   
               
             
          
           
               
                 V κ 3′Reverse Primers, LongLinker 
               
             
          
           
               
                 RKB9J1o-BL 
                 74. GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC 
               
               
                   
                 GCC CGA GCC ACC GCC ACC AGA GGA TAG GAT CTC 
               
               
                   
                 CAG CTC GGT CCC 
               
               
                   
               
               
                 RKB9Jo-BL 
                 75. GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC 
               
               
                   
                 GCC CGA GCC ACC GCC ACC AGA GGA TAG GAT CTC 
               
               
                   
                 CAG CTC GGT CCC 
               
               
                   
               
               
                 RKB42Jo-BL 
                 76. GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC 
               
               
                   
                 GCC CGA GCC ACC GCC ACC AGA GGA TTT GAC SAC 
               
               
                   
                 CAC CTC GGT CCC 
               
               
                   
               
             
          
           
               
                 V λ 5′Sense Primer 
               
             
          
           
               
                 RSCλJ 
                 77. GGG CCC AGG CGG CCG AGC TCG TGC TGA CTC 
               
               
                   
                 AGT CGC CCT C 
               
               
                   
               
             
          
           
               
                 V λ 3′Reverse Primer, LongLinker 
               
             
          
           
               
                 RJλo-BL 
                 78. GGA AGA TCT AGA GGA ACC ACC CCC ACC ACC 
               
               
                   
                 GCC CGA GCC ACC GCC ACC AGA GGA GCC TGT GAC 
               
               
                   
                 GGT CAG CTG GGT CCC 
               
               
                   
               
             
          
           
               
                 V H 5′Sense Primers 
               
             
          
           
               
                 RSCVH1 
                 79. GGT GGT TCC TCT AGA TCT TCC CAG TCG GTG 
               
               
                   
                 GAG GAG TCC RGG 
               
               
                   
               
               
                 RSCVH2 
                 80. GGT GGT TCC TCT AGA TCT TCC CAG TCG GTG AAG 
               
               
                   
                 GAG TCC GAG 
               
               
                   
               
               
                 RSCVH3 
                 81. GGT GGT TCC TCT AGA TCT TCC CAG TCG YTG GAG 
               
               
                   
                 GAG TCC GGG 
               
               
                   
               
               
                 RSCVH4 
                 82. GGT GGT TCC TCT AGA TCT TCC CAG SAG CAG CTG 
               
               
                   
                 RTG GAG TCC GG 
               
               
                   
               
             
          
           
               
                 V H 3′Reverse Primers 
               
             
          
           
               
                 RSCG-B 
                 83. CCT GGC CGG CCT GGC CAC TAG TGA CTG AYG 
               
               
                   
                 GAG CCT TAG GTT GCC C 
               
               
                   
               
             
          
           
               
                 Overlap Extension Primers 
               
             
          
           
               
                 RSC-F (sense) 
                 84. GAG GAG GAG GAG GAG GAG GCG GGG CCC AGG 
               
               
                   
                 CGG CCG AGC TC 
               
               
                   
               
               
                 RSC-B (reverse) 
                 85. GAG GAG GAG GAG GAG GAG CCT GGC CGG CCT 
               
               
                   
                 GGC CAC TAG TG 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 Primers for V λ  and V H  of chicken single-chain Fv libraries 
               
               
                   
               
             
             
               
                 V λ  Primers 
               
             
          
           
               
                 CSCVK (sense) 
                 86. GTG GCC CAG GCG GCC CTG ACT CAG CCG TCC 
               
               
                   
                 TCG GTG TC 
               
               
                   
               
               
                 CKJo-B (reverse) 
                 87. GGA AGA TCT AGA GGA CTG ACC TAG GAC GGT 
               
               
                   
                 CAG G 
               
               
                   
               
             
          
           
               
                 V H Primers 
               
             
          
           
               
                 CSCVHo-FL (sense) 
                 88. GGT CAG TCC TCT AGA TCT TCC GGC GGT GGT 
               
               
                   
                 GGC AGC TCC GGT GGT GGC GGT TCC GCC GTG ACG 
               
               
                   
                 TTG GAC GAG 
               
               
                   
               
               
                 CSCG-B (reverse) 
                 89. CTG GCC GGC CTG GCC ACT AGT GGA GGA GAC 
               
               
                   
                 GAT GAC TTC GGT CC 
               
               
                   
               
             
          
           
               
                 Overlap Extension Primers 
               
             
          
           
               
                 CSC-F (sense) 
                 90. GAG GAG GAG GAG GAG GAG GTG GCC CAG GCG 
               
               
                   
                 GCC CTG ACT CAG 
               
               
                   
               
               
                 CSC-B (reverse) 
                 91. GAG GAG GAG GAG GAG GAG GAG CTG GCC GGC 
               
               
                   
                 CTG GCC ACT AGT GGA GG 
               
               
                   
               
             
          
         
       
     
         [0083]    In each reaction, 1 μl of cDNA was mixed with 60 pmol of each primer, 10 μl of 10× reaction buffer, 8 μl of 2.5 mM dNTPs, 0.5 μl of Taq DNA polymerase and water to a final volume of 100 μl. The PCR reactions were carried out under the following conditions: 30 cycles of 15 sec at 94° C., 30 sec at 56° C., and 90 sec at 72° C., followed by a final extension for 10 min at 72° C. Amplified fragments with length of approximately 350 base pairs were loaded and run on a 1.5% agarose gel, and purified with QIAEX II Gel Extraction Kit (QIAGEN, Valencia, Calif., USA). In the second round of PCR, the first round V L  products and V H  products were randomly joined by overlap extension PCR. Each PCR reaction was performed in a 100 μl mixture composed of 100 ng of purified V L  product and V H  product, 60 pmol of each primer, 10 μl of 10× reaction buffer, 8 μl of 2.5 mM dNTPs and 0.5 μl of Taq DNA polymerase. The PCR reactions were carried out under the following conditions: 20 cycles of 15 sec at 94° C., 30 sec at 56° C., and 2 min at 72° C., followed by a final extension for 10 min at 72° C. About 700 base pair-sized scFv fragments were purified with QIAEX II Gel Extraction Kit (QIAGEN). The scFv fragments and pComb3XSS vector were digested with SfiI restriction enzyme (Roche Molecular Systems, Pleasanton, Calif., USA) by incubating for 8 hr at 50° C. 700 ng of SfiI-digested scFv was ligated with 1400 ng of pComb3X vector using T4 DNA ligase by incubating the reaction mixture for 12 hr at 16° C., followed by ethanol precipitation. Ligated library was transformed into  E. coli  ER2738 by electoporation. The cells were resuspended with 3 ml of Super Broth (SB) medium and incubated for 1 hr at 37° C. while shaking at 250 rpm. Then 10 ml of SB medium and 3 μl of 100 mg/ml carbenicillin were added to the culture. The library size was determined by plating 0.1, 1 and 10 μl of the culture on Luria Broth (LB) plate containing 50 μg/ml of carbenicillin. After one hour of incubation, 4.5 μl of 100 mg/ml carbenicillin was added to the culture and incubated for an additional hour. The culture was added to 2 ml of VCSM13 helper phage (&gt;10 11  cfu/ml), 183 ml of SB medium and 92.5 μl of 100 mg/ml carbenicillin and incubated for 2 hr at 37° C. while shaking at 250 rpm. Kanamycin (280 μl) was added to the culture, and the culture was shaken overnight at 250 rpm and 37° C. The next day, the culture was centrifuged at 3,000 g for 15 min. The bacterial pellet was saved for phagemid DNA preparation and the supernatant was transferred to clean centrifuge bottle. 8 g of polyethylene glycol-8000 (PEG-8000, Sigma) and 6 g of NaCl (Merck) were added, and the supernatant was stored on ice for 30 min. The supernatant was centrifuged at 15,000 g for 15 min at 4° C. The phage pellet was resuspended in Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA). 
       Example 2 
     Bio-Panning 
       [0084]    3 ug of human C5 antibody was coated with 1×10 7  magnetic beads (Dynabeads M270-Epoxy, Invitrogen) at room temperature for 16 hours. The beads were washed with PBS and blocked with PBS containing 3% BSA at room temperature for 1 hour. The coated beads were washed and incubated together with Phage-displayed scFv for 2 hours at room temperature. The beads were washed with 0.5% TPBS to remove phages which were not bound. The bound phages were eluted with 100 ul of 0.1M glycine-HCl and neutralized with 6 ul of 2M Tris-HCl (pH 9.0). The eluted phages infected  E. coli  ER2738 and were rescued with VCSM13 helper phage for overnight amplification. The input and output phage titer were determined by plating the phage infected bacterial culture at 37° C. on LB plate containing 50 μg/ml of carbenicillin. Next day, phage was precipitated by adding PEG-8000 and NaCl as described in Example 1. 
       Example 3 
     Selection of scFv Clones by Phage ELISA 
       [0085]    ELISA using phages displaying scFvs was performed against human C5 to analyze the selected clones from bio-panning Microtiter 96-well plate was coated with 100 ng of human C5 per well for overnight at 4° C. and blocked with 3% BSA in PBS. Each phage culture was mixed with an equal volume of 6% BSA in PBS, added to human C5-coated 96-well plate, and incubated for 2 hr at 37° C. After the incubation was finished, the plate was washed and incubated with a HRP conjugated anti-M13 antibody (Amersham, USA). After the incubation was finished, the plate was washed, and 1 μg/ml of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, Amresco, Ohio, USA) in 0.05 M Citric acid buffer and 1.0% H 2 O 2  were added to each well, followed by color formation, and the absorbance was measured at 405 nm. 
         [0086]    Results thereof were shown in  FIG. 1 . 
         [0087]      FIG. 1A  shows immune libraries of rabbit and  FIG. 1B  shows immune libraries of chicken. As analysis results of gene sequence of clones exhibiting the absorbance of 0.6 or more to human C5, five scFv clones each having different sequence were obtained from two kinds from rabbit immune libraries and three kinds from chicken immune libraries. 
         [0088]    In addition, the selected five kinds of scFv clones and eculizumab which is a control, were converted to ScFv-Fc fusion protein to compare binding affinity by ELISA against C5. Amounts of the antibodies bound to C5 were determined by using HRP-bound anti-human IgG according to the same method as described above, and results thereof were shown in  FIG. 2 . 
         [0089]    As shown in  FIG. 2 , all of the selected five kinds of scFv clones exhibited higher absorbance than that of eculizumab. 
       Example 4 
     Construction of Affinity-Matured and Humanized Antibodies 
       [0090]    6 CDRs (complementarity determining regions, light chain antigen complementarity determining regions 1-3 [CDRL 1-3], heavy chain antigen complementarity determining regions 1-3 [CDRH1-3]) having binding affinity and activation inhibiting ability to human complement C5 were inserted between 8 framework regions (FRL1-4, FRH1-4) from the human germline Kappa1/IGHV3-23 to synthesize humanized anti-complement C5 scFv gene (HRA-06, Genscript, Piscataway, N.J., USA). 
         [0091]    To generate mutant sub-libraries of HRA-06, oligonucleotides containing degenerate codons NNK or MNN (N=A, T, G or C, K=G or T, M=A or C) were used. ScFv gene of HRA-06 was used for template DNA. Randomized codons were introduced in five CDRs except CDRH3 by PCR. Amplified scFv fragments were purified with QIAEX II Gel Extraction Kit (QIAGEN). The scFv and pComb3XSS vector were digested with SfiI restriction enzyme (Roche Molecular Systems) and ligated, followed by ethanol precipitation. Ligated libraries were transfected into  E. coli  ER2738 by the same method as Example 1 to construct phage libraries. Antigens were selected based on the constructed phage libraries by the same method as Example 2. Lastly, binding affinity to C5 was confirmed by phage ELISA according to the same method as Example 3. 
         [0092]    As shown in  FIG. 3 , a number of clones having binding affinity to C5 were obtained from 5 kinds of mutant sub-libraries. 
       Example 5 
     Construction of Recombinant Anti-C5 Antibodies and Eculizumab as a Control 
       [0093]    1. Sub-Cloning of Anti-C5 Antibody into Full IgG Vector and scFv-Fc Vector 
         [0094]    Gene encoding human IgG2 hinge and IgG2/4 hybrid CH2-CH3 was inserted into pCEP4 vector (Invitrogen) by HindIII (New England Biolabs) and XhoI (New England Biolabs) restriction enzyme. The gene encoding anti-C5 scFv was sub-cloned in the 5′ end of Fc region by two SfiI restriction sites. For the light chain, human immunoglobulin C κ  gene was sub-cloned into a mammalian expression vector. For the heavy chain, the gene from human CH1 and the hinge of human IgG2 to IgG2/4 hybrid CH2-CH3 region was sub-cloned into the mammalian expression vector. Variable light chains and variable heavy chains were sub-cloned into this full IgG vector. Antibody sequence of eculizumab was obtained by synthesizing heavy chain and light chain genes based on antibody sequence stated in examination report of Eculizumab (Product Name: Soliris) posted on ‘Japan Pharmaceuticals and Medical Devices Agency (PMDA)’. 
         [0095]    2. Transfection and Protein Purification 
         [0096]    Transfection was performed to over-express recombinant proteins. 2 μg of mammalian expression vector per ml of culture volume and 4 μg of polyethyleneimine/ml (PEI, Polysciences, Warrington, Pa., USA) were mixed in 150 mM NaCl corresponding to 1/10 of culture volume, and let stand at room temperature for 15 min. The mixture was added to the HEK 293F cells (2×10 6  cells/ml) and incubated for 5 days under the following condition: FreeStyle™ 293 Expression medium containing 100 U/ml penicillin (Invitrogen) and 100 U/ml streptomycin (Invitrogen), 37° C., 7% CO 2 , 135 rpm on an orbital shaker. Cell culture supernatants were harvested and subjected to protein A affinity gel chromatography to purify IgG and Fc fusion protein. 
       Example 6 
     Measurement of Binding Affinity of Monoclonal Antibody 
       [0097]    ELISA (enzyme-linked immunosorbent assay) was performed to measure complement C5 binding affinity of the antibodies produced by Example 5. The antibodies diluted for each concentration were added to 96-well plates coated with C5 to perform reaction. Horseradish peroxidase-labeled anti-human IgG antibody was used as a secondary antibody, followed by color formation with ABTS (2,2′-Azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), and absorbance was measured by the same method as Example 3. 
         [0098]    The monoclonal antibodies used in this experiment were six kinds of affinity-matured and humanized anti-C5 antibodies (HRA-06-H2-1, HRA-06-H2-7, HRA-06-H2-18, HRA-06-H2-24, HRA-06-H1-9-H2-7, HRA-06-H1-9-H2-24) and positive control group antibody Eculizumab, and negative control group antibody Palivizumab, and results thereof were shown in  FIG. 3 . 
         [0099]    As shown in  FIG. 4 , all of the anti-complement C5 antibodies exhibited binding affinity to C5 and six kinds of affinity-matured and humanized anti-C5 antibodies exhibited high absorbance as compared to Eculizumab. 
       Example 7 
     Complement-Dependent Cytotoxicity (CDC) Assay In Vitro 
       [0100]    CD20-expressing human Burkitt&#39;s lymphoma cell line Ran was kept in RPMI 1640 supplemented with 10% FBS (Invitrogen), 100 U/ml penicillin (Invitrogen) and 100 U/ml streptomycin (Invitrogen). Target cells were washed and resuspended at a concentration of 1×10 6  cells/ml. Anti-CD20 human IgG, rituximab (Roche) were diluted with CDC solution at a concentration of 3 μg/ml. Equal volume of target cells and sensitizing antibody were mixed together to make a volume of 100 μl/well in 96-well plate, and let stand at room temperature for 5 min Assay was started by adding human complement serum, resulting in a final volume of 150 μl per well and a final concentration of 4% blood serum. After 2 hours of incubation, 15 μl of Tetrazolium salt (WST-1, Takara Bio, Japan) was added to each well and the plate was incubated for additional 2 hours. The viable cells were analyzed by measuring OD at 450 nm Effect of anti-C5 antibody was evaluated by pre-incubation with serum at 37° C. for 30 min prior to adding to target cells and sensitizing antibody mixture. Same concentration of Palivizumab was used as an IgG control. Percentage of cell viability was calculated with the formula: 
         [0000]      % Viability=(Test antibody −Background)/(Test Without Antibody −Background)×100
 
         [0101]    Results thereof were shown in  FIG. 5 . 
         [0102]    As shown in  FIG. 5 , the affinity-matured and humanized anti-C5 antibodies produced according to the present invention exhibited CDC inhibitory ability, and all of the antibodies exhibited high cell viability as compared to Eculizumab. 
       Example 8 
     Measurement of C5a Production Content In Vitro 
       [0103]    After 2 hours of incubation of target cells, sensitizing antibody and serum, the cells were pelleted by centrifugation and the supernatant was assayed for C5a content by sandwich ELISA using the BD OptiEIA™ Human C5a ELISA Kit II (BD Biosciences, San Jose, Calif., USA) following manufacturer&#39;s instruction. 
         [0104]    Results thereof were shown in  FIG. 6 . 
         [0105]    As shown in  FIG. 6 , all of the affinity-matured and humanized anti-C5 antibodies produced according to the present invention exhibited C5a production inhibitory ability, and all of four kinds of affinity-matured and humanized anti-C5 antibodies exhibited high inhibitory ability as compared to Eculizumab. 
       Example 9 
     Measurement of Cross-Species Reactivity of Monoclonal Antibody 
       [0106]    Immunoblotting was performed to confirm whether or not the monoclonal antibody was bound to the complement C5 of other species rather than human. Blood serums of human C5 protein and human (Sigma), rhesus monkey, BALB/c mouse, Wistar rat, NZW rabbit were diluted and subjected to SDS-PAGE, respectively, and resolved proteins were transferred to nitrocellulose membrane. Immunoblotting was performed by the anti-complement C5 antibody HRA-06-H2-1 produced according to the present invention. 
         [0107]    Results thereof were shown in  FIG. 7 . 
         [0108]    As shown in  FIG. 7 , the anti-complement C5 antibodies exhibited binding affinity to C5 of human (Sigma), rhesus monkey, Wistar rat, and NZW rabbit. 
       Example 10 
     Size-Exclusion Chromatography 
       [0109]    Size-exclusion chromatography (SEC) analysis was performed on purified antibodies by using Waters 2489 system (Waters Corporation, Milford, Mass., USA), and Zenix-C 300 column (Sepax Technologies, Inc., Newark, Del., USA). Mobile phase composition (150 mM sodium phosphate, pH 7.0) and flow rate (1.0 mL/min) were constant in all runs. Concentration of protein was determined by monitoring the absorbance of column eluate at 280 nm Fractional concentration was calculated by dividing individual peak areas by the sum of peak areas. 
         [0110]    Results thereof were shown in  FIG. 8 .  FIGS. 8A to 8D  represent A) HRA-06-H2-1, B) HRA-06-H2-7, C) HRA-06-H2-18, D) HRA-06-H2-24, E) HRA-06-H1-9-H2-7 and F) HRA-06-H1-9-H2-24, respectively. 
         [0111]    As shown in  FIG. 8 , it was confirmed that aggregation was hardly detected in physical chemical properties of the anti-complement C5 antibody. 
       Example 11 
     Epitope Mapping 
       [0112]    1. Confirmation of Binding of Antibody to C5 Beta-Chain 
         [0113]    Complement C5 proteins were subjected to SDS-PAGE under non-reducing condition (lane 1) and reducing condition (lane 2), respectively, followed by immunoblotting using the anti-complement C5 antibody, to confirm whether or not beta-chain binding was formed. Results thereof were shown in  FIG. 9 . 
         [0114]      FIG. 9A  shows the binding when Eculizumab was used as an antibody, and FIG.  9 B shows the binding when HRA-06-H2-1 according to the present invention was used as an antibody. As known in the art, it was confirmed that Eculizumab was bound to C5 (entire complement protein), and bound to alpha-chain in lane 2 under reducing condition. Meanwhile, it was confirmed that HRA-06-H2-1 antibody according to the present invention bound to C5 (entire complement protein), and bound to beta-chain in lane 2 under reducing condition. 
         [0115]    2. Production of C5 Beta Chain Mutant Domains as Fc Fusion Proteins and Identification of Binding Site 
         [0116]    Six domains consisting beta chain of C5 and serial deletion mutant of beta chain were amplified from cDNA. The primers were designed to add SfiI restriction sites at both the 5′ and 3′ ends (Table 11). The serial deletion mutant of beta chain of C5 was amplified by primer combination as described in Table 12. The amplified PCR fragments were digested with SfiI and cloned into modified pCEP4 vector containing the hinge region and CH2-CH3 domain of human IgG1 at the 3′ region of the cloning site. These clones were transfected and Fc fusion proteins were purified as described in Example 5. 
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 Primer sequences for amplification of beta 
               
               
                 chain domains 
               
               
                   
               
             
             
               
                 Forward primers (5′→3′) 
               
             
          
           
               
                 MG1_F 
                 92. GGCCCAGGCGGCCATGGGCCTTTTGGGAATACTTTG 
               
               
                   
               
               
                 MG2_F 
                 93. GGCCCAGGCGGCCAATGGATTTCTCTTCATTCATAC 
               
               
                   
               
               
                 MG3_F 
                 94. GGCCCAGGCGGCCCCACATTTTTCTGTCTCAATC 
               
               
                   
               
               
                 MG4_F 
                 95. GGCCCAGGCGGCCTCTCCCTACAAACTGAATTTG 
               
               
                   
               
               
                 MG5_F 
                 96. GGCCCAGGCGGCCACTGATAACCATAAGGCTTTG 
               
               
                   
               
               
                 Linker_F 
                 97. GGCCCAGGCGGCCTCCTGGGTGGCATTAGC 
               
               
                   
               
             
          
           
               
                 Reverse primers (5′→3′) 
               
             
          
           
               
                 MG1_R 
                 98. GGCCGGCCTGGCCGTCATAGGTTATTGGCATTCT 
               
               
                   
               
               
                 MG2_R 
                 99. GGCCGGCCTGGCCCAAGACATATTCTTTAACTTC 
               
               
                   
               
               
                 MG3_R 
                 100. GGCCGGCCTGGCCGAGGACATATTTGATGCCAG 
               
               
                   
               
               
                 MG4_R 
                 101. GGCCGGCCTGGCCCCAATCAATATAAAGGTAACTTTG 
               
               
                   
               
               
                 MG5_R 
                 102. GGCCGGCCTGGCCATCCATTCCAGTTGCCATATTA 
               
               
                   
               
               
                 Linker_R 
                 103. GGCCGGCCTGGCCGAGAATTTCTTTACAAGGTTC 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Primer combinations for construction of beta chain  
               
               
                 domains and deletion mutant of beta chain 
               
             
          
           
               
                   
                 Domain name 
                 Primer combination 
               
               
                   
                   
               
               
                   
                 MG1 
                 MG1_F/MG1-R 
               
               
                   
                 MG2 
                 MG2_F/MG2-R 
               
               
                   
                 MG3 
                 MG3_F/MG3-R 
               
               
                   
                 MG4 
                 MG4_F/MG4-R 
               
               
                   
                 MG5 
                 MG5_F/MG5-R 
               
               
                   
                 Linker 
                 Linker_F/Linker-R 
               
               
                   
                 MG1-2 
                 MG1_F/MG2-R 
               
               
                   
                 MG1-3 
                 MG1_F/MG3-R 
               
               
                   
                 MG1-4 
                 MG1_F/MG4-R 
               
               
                   
                 MG1-5 
                 MG1_F/MG5-R 
               
               
                   
                   
               
             
          
         
       
     
         [0117]    The proteins comprising each domain were subjected to SDS-PAGE, respectively, and immunoblotting was performed by using anti-complement C5 antibody (HRA-06-H2-1). Results thereof were shown in  FIGS. 10 and 11 . 
         [0118]      FIG. 10A  is schematic diagram showing a structure of the C5 beta chain, and the produced Fc fusion protein, and  FIG. 10B  shows immunoblotting results. As shown in  FIG. 10 , it was confirmed that the HRA-06-H2-1 antibody produced according to the present invention bound to the Fc fusion protein having an MG4 domain. 
         [0119]      FIG. 11A  is schematic diagram showing a structure of the C5 beta chain, and the produced Fc fusion protein, and  FIG. 11B  shows immunoblotting results. As shown in  FIG. 11 , it was confirmed that the HRA-06-H2-1 antibody produced according to the present invention was bound only to the Fc fusion protein comprising an MG4 domain. 
         [0120]    3. Production of MG4 Domains as Fc Fusion Proteins and Identification of Binding Site 
         [0121]    MG4 domain of the beta chain and five mutants from which the MG4 domain is sequentially removed from N-terminus of the MG4 domain were cloned. The primers were designed to add SfiI restriction sites at both the 5′ and 3′ ends (Table 13). The amplified PCR fragments were digested with SfiI and cloned into modified pCEP4 vector containing the hinge region and CH2-CH3 domain of human IgG1 at the 3′ region of the cloning site. These clones were transfected and Fc fusion proteins were purified as described in Example 5. The purified Fc fusion proteins were subjected to SDS-PAGE, and immunoblotting was performed as described above. 
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 Primer sequences for amplification of MG4 domains 
               
               
                   
               
             
             
               
                 Forward primers (5′→3′) 
               
             
          
           
               
                 hMG4_F 
                 104. GGCCCAGGCGGCCTCTCCCTACAAACTGAATTTG 
               
               
                   
               
               
                 d332-348_F 
                 105. GGCCCAGGCGGCCATTCCATATCCCATCAAGG 
               
               
                   
               
               
                 d332-378_F 
                 106. GGCCCAGGCGGCCGTAAACCAAGAGACATCTGAC 
               
               
                   
               
               
                 d332-396_F 
                 107. GGCCCAGGCGGCCGATGGAGTAGCTTCCTTTG 
               
               
                   
               
               
                 d332-424_F 
                 108. GGCCCAGGCGGCCCCAGAAGAAAATCAGGCC 
               
               
                   
               
             
          
           
               
                 Reverse primers (5′→3′) 
               
             
          
           
               
                 hMG4_R 
                 109. 
               
               
                   
                 GGCCGGCCTGGCCCCAATCAATATAAAGGTAACTTTG 
               
               
                   
               
             
          
         
       
     
         [0122]    Results thereof were shown in  FIG. 12 .  FIG. 12A  shows MG4 domain and Fc fusion protein produced by sequentially removing the domain, and  FIG. 12B  shows results of immunoblotting, using HRA-06-H2-1 antibody. As shown in  FIG. 12 , it was confirmed that the binding was not achieved in the mutants from which 332nd-348th amino acid residue sequences were removed from N-terminus of the beta chain, which could be appreciated that 332nd-348th amino acid residue sequences in the MG4 domain of the beta chain were sequences having high antibody-binding possibility. 
         [0123]    4. Confirmation of Antibody-Binding Sites from Human/Mouse Hybrid MG4 Domains 
         [0124]    Human/mouse hybrid MG4 domains of the C5 beta chain were produced in overlap extension PCR. The primers were designed to add SfiI restriction sites at both the 5′ and 3′ ends (Table 14). PCR fragments were digested with SfiI and cloned into Sfi I-digested pComb3X vector. These clones were transfected into  E. coli  ER 2738. Single colony from each human/mouse MG4 hybrid was incubated until absorbance at 600 nm reached about 1.0. Phage infection was performed by adding VCSM13 helper phage (10 11  pfu/ml), followed by incubation at 37° C. for 2 hr. Kanamycin (25 μg/ml) was added and the culture was incubated at 37° C. overnight with constant shaking. Bacteria were removed by centrifugation at 3,000 g for 15 min 
         [0000]    
       
         
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
               
             
               
               
             
           
               
                 TABLE 14 
               
               
                   
               
               
                 Primer sequences for amplification of 
               
               
                 human/mouse hybrid MG4 domains 
               
               
                   
               
             
             
               
                 hMG4-m332-348 Structure 
               
             
          
           
               
                 m332-348_F 
                 110. 
               
               
                   
                 GGCCCAGGCGGCCTCTCCCTACACACTGAATTTGGTC 
               
               
                   
                 GCTACTCCTCTTTTCGTGAAGCCCGGGATTCCATATCC 
               
               
                   
                 CATCAAGGTGC 
               
               
                   
               
               
                 hMG4_R 
                 111. GGCCCAGGCGGCCTCTCCCTACAAACTGAATTTG 
               
               
                   
               
             
          
           
               
                 Amplification of N-terminus of Mouse MG4 
               
               
                 Forward primers (5′→3′) 
               
             
          
           
               
                 mMG4_F 
                 112. TCTCCCTACACACTGAATTTGG 
               
               
                   
               
             
          
           
               
                 Reverse primers (5′→3′) 
               
             
          
           
               
                 m332-359_R 
                 113. GTCAAGCGAATCTTTAACCTGTGCCTTG 
               
               
                   
               
               
                 m332-368_R 
                 114. TGTTACTGGGACCCCTCCTACCGCCTG 
               
               
                   
               
               
                 m332-378_R 
                 115. TTGGTTTACATCGACTGTTTGTGCCATC 
               
               
                   
               
               
                 m332-385_R 
                 116. TGGATCCAAGTCAGATGTCTCTTGATTCAC 
               
               
                   
               
               
                 m332-392_R 
                 117. AACACGTGTGATGCTCCTCTTTGTTTCC 
               
               
                   
               
               
                 m332-398_R 
                 118. GGAAGCTACTCCATCAGTGTCATGAGTG 
               
               
                   
               
               
                 m332-409_R 
                 119. CACCGTCACATTTGATGGGAGGTTCAGC 
               
               
                   
               
             
          
           
               
                 Amplification of C-terminus of Human MG4 
               
               
                 Forward primers (5′→3′) 
               
             
          
           
               
                 m332-359_F 
                 120. GTTAAAGATTCGCTTGACCAGTTGGTAG 
               
               
                   
               
               
                 m332-368_F 
                 121. GGAGGGGTCCCAGTAACACTGAATGCAC 
               
               
                   
               
               
                 m332-378_F 
                 122. ACAGTCGATGTAAACCAAGAGACATCTGAC 
               
               
                   
               
               
                 m332-385_F 
                 123. ACATCTGACTTGGATCCAAGCAAAAGTGT 
               
               
                   
               
               
                 m332-392_F 
                 124. AGGAGCATCACACGTGTTGATGATGGAGTA 
               
               
                   
               
               
                 m332-398_F 
                 125. ACTGATGGAGTAGCTTCCTTTGTGCTTAATC 
               
               
                   
               
               
                 m332-409_F 
                 126. CCATCAAATGTGACGGTGCTGGAGTTTA 
               
               
                   
               
             
          
           
               
                 Reverse primers (5′→3′) 
               
             
          
           
               
                 hMG4_R 
                 127. GGCCCAGGCGGCCTCTCCCTACAAACTGAATTTG 
               
               
                   
               
             
          
           
               
                 mMG4-h332-348           Structure 
               
             
          
           
               
                 h332-348_F 
                 128. GGCCCAGGCGGCCTCTCCCTACAAACTGAATTT 
               
               
                   
                 GGTTGCTACTCCTCTTTTCCTGAAGCCTGGGATTCCA 
               
               
                   
                 TTTTCCATCAAG 
               
               
                   
               
               
                 mMG4_R 
                 129. CCAAGCGATGTAAATGTAAC 
               
               
                   
               
             
          
           
               
                 Amplification of N-terminus of Human MG4 
               
               
                 Forward primers (5′→3′) 
               
             
          
           
               
                 hMG4_F 
                 130. GGCCCAGGCGGCCTCTCCCTACAAACTGAATTTG 
               
               
                   
               
             
          
           
               
                 Reverse primers (5′→3′) 
               
             
          
           
               
                 h332-359_R 
                 131. CTCGAGTGAATCTTTAACCTGCACCTTGA 
               
               
                   
               
               
                 h332-368_R 
                 132. AGTTACTGGGACTCCTCCTACCAACTG 
               
               
                   
               
               
                 h332-378_R 
                 133. TTGATTCACATCAATTGTTTGTGCATTCAG 
               
               
                   
               
               
                 h332-385_R 
                 134. TGTTTCCAAGTCAGATGTCTCTTGGTTTAC 
               
               
                   
               
               
                 h332-392_R 
                 135. GTCATGAGTTACACTTTTGCTTGGATCCA 
               
               
                   
               
               
                 h332-398_R 
                 136. CACAGCTACTCCATCATCAACACGTGTTAC 
               
               
                   
               
               
                 h332-409_R 
                 137. CACCGTCACTCCAGATGGGAGATTAAGCAC 
               
               
                   
               
             
          
           
               
                 Amplification of C-terminus of Mouse MG4 
               
               
                 Forward primers (5′→3′) 
               
             
          
           
               
                 h332-359_F 
                 138. GTTAAAGATTCACTCGAGCAGGCGGT 
               
               
                   
               
               
                 h332-368_F 
                 139. GGAGGAGTCCCAGTAACTCTGATGGCAC 
               
               
                   
               
               
                 h332-378_F 
                 140. ACAATTGATGTGAATCAAGAGACATCTGAC 
               
               
                   
               
               
                 h332-385_F 
                 141. ACATCTGACTTGGAAACAAAGAGGAGCATC 
               
               
                   
               
               
                 h332-392_F 
                 142. CAAAAGTGTAACTCATGACACTGATGGAG 
               
               
                   
               
               
                 h332-398_F 
                 143. GATGATGGAGTAGCTGTGTTTGTGCTGAAC 
               
               
                   
               
               
                 h332-409_F 
                 144. CCATCTGGAGTGACGGTGCTAAAGTTTG 
               
               
                   
               
             
          
           
               
                 Reverse primers (5′→3′) 
               
             
          
           
               
                 mMG4_R 
                 145. CCAAGCGATGTAAATGTAAC 
               
               
                   
               
             
          
         
       
     
         [0125]    Phage ELISA was performed as follows. Anti-C5 IgG2/4, HRA-06-H2-7, was diluted in 0.1 M sodium bicarbonate buffer (pH 8.6) and 100 ng of the antibody was coated on 96 well plate at 4° C. overnight. Each well was blocked by adding 100 μl of 5% skim milk in TBS containing 0.05% Tween 20 and incubated for 1 hr at 37° C. Phage was diluted two fold in 6% BSA/PBS then 50 μl of diluted phage was added to each well, and incubated for 2 hr at 37° C. The plate was washed, and 50 μl of diluted HRP-bound anti-M13 antibody (1:5000) was added, and the plate was incubated for 1 hr at 37° C. The plate was washed, and 50 μl of ABTS substrate solution was added to each well and the absorbance was measured at 405 nm. 
         [0126]    Results thereof were shown in  FIGS. 13 and 14 . 
         [0127]      FIG. 13A  shows human/mouse hybrid MG4 domains, and  FIG. 13B  shows ELISA results. As shown in  FIG. 13 , the HRA-06-H2-7 antibody was bound when 379th to 398th amino acid residue sequences based on the beta chain sequence were human sequences, which shows binding possibility to the corresponding site of the antibody. 
         [0128]      FIG. 14  shows results obtained by more specifically confirming the binding sites of the sequences.  FIG. 14A  shows human/mouse hybrid MG4 domains, and  FIG. 14B  shows ELISA results. As shown in  FIG. 14 , the HRA-06-H2-7 antibody was bound when 386th to 392nd amino acid residue sequences based on the beta chain sequence (55th to 61th amino acid sequences based on the MG4 domain sequence) were human sequences, which shows binding possibility to the corresponding site of the antibody.