Abstract:
A framework fragment derived from a chicken antibody, in which an amino acid at a particular position is replaced by cysteine, a heavy chain variable region or a light chain variable region including the framework fragment, or an antibody including the heavy chain variable region or the light chain variable region does not induce or prevents formation of disulfide bonds between antibody molecules while maintaining activity and reactivity of the antibody. The antibody introduced with cysteine or an antigen-binding fragment thereof easily binds with a conjugation compound such as a chemotherapeutic drug, an enzyme, an aptamer, a toxin, an affinity ligand, or a detection label, thereby being applied to various fields for diagnosis or treatment of diseases.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to engineered chicken antibodies which are modified with cysteine residues, and more particularly, to antibodies with application for treatment or diagnosis of diseases. The cysteine-modified antibodies may be conjugated with chemotherapeutic drugs, toxins, peptides, affinity ligands such as biotin, and detection labels such as radioisotopes and fluorophores, and used in the diagnosis or treatment of diseases. 
       BACKGROUND ART 
       [0002]    Antibodies are widely used for diagnosis and treatment. Antibodies may have improved functions or may be provided with new functions by chemical binding with drugs, enzymes, dyes, peptides, aptamers, linkers, toxins, isotopes, etc. In the chemical binding with these substances, functional groups such as OH group of a tyrosine residue, NH 3  group of a lysine residue, or a carboxyl group of aspartic acid or glutamic acid of antibodies are involved (literature [Goldenberg D. M. et al., J. Med. (1978) 298:1384-1386; Rainsbury R. M. et al., Lancet (1983) 2:934-938; and Yamada H. et al., Biochemistry (1981) 20:4836-4842]). 
         [0003]    However, these functional groups are also present in the antigen-binding sites. Therefore, if the residues in the antigen-binding sites are involved in the chemical binding, affinity of antibody to antigen may be affected. Further, in the binding reaction using cysteine, reduction of disulfide bonds within a chain (polypeptide) of an antibody molecule or between the chains is a prerequisite, which may affect activity of the protein (literature [Stimmel J. B. et al., J. Biol. Chem. (2000) 273: 30445-30450]). Further, proteins may be rendered inactive or non-specific by misfolding or loss of a tertiary structure (literature [Zhang W. et al., Anal. Biochem. (2002) 311:1-9]). 
         [0004]    Site-specific conjugation is preferred over random amino modification as it enables chemical modification of a site away from the binding site, promoting complete retention of biological activity and allowing control over the possible number of prosthetic groups added. Cysteine-engineered antibodies have been conjugated through linkers at the newly introduced cysteine thiols with thiol-reactive linker reagents and drug-linker reagents to prepare cysteine-engineered antibody drug conjugates with anti-cancer properties, for example, anti-MUC16 (US 2008/0311134), anti-CD22 (US 2008/0050310), anti-ROBO4 (US 2008/0247951), anti-TENB2 (US 2009/0117100), anti-CD79B (US 2009/0028856; US 2009/0068202) thio ADC. However, one or more antibody heavy-chain and light-chain variable region genes exist in human or other animals, and as a result, each different antibody has a different framework sequence. Accordingly, there is a problem that the cysteine introduction site should be optimized with respect to respective antibodies. 
       DISCLOSURE 
     Technical Problem 
       [0005]    An aspect of the present invention provides a framework fragment of a chicken antibody, in which a cysteine residue is introduced into a framework region present in a heavy chain variable region or a light chain variable region, a heavy chain variable region or a light chain variable region including the framework fragment, or an antibody including the heavy chain variable region or the light chain variable region. 
         [0006]    Another aspect of the present invention provides a conjugate including the antibody or an antigen-binding fragment thereof and a conjugation compound, the conjugate being prepared by conjugating the conjugation compound, such as a chemotherapeutic drug, an enzyme, an aptamer, a toxin, an affinity ligand, or a detection label, to the introduced cysteine residue which is present in the framework fragment of the chicken antibody, the heavy chain variable region or light chain variable region including the framework fragment, or the chicken antibody including the heavy chain variable region or light chain variable region. 
         [0007]    Still another aspect provides use of the conjugate including the antibody or the antigen-binding fragment thereof and the conjugation compound in the diagnosis or treatment of a disease. 
       Technical Solution 
       [0008]    The present inventors have selected a residue which is able to maintain antibody activity and affinity although replaced by cysteine, from various amino acid residues present in framework regions in heavy chain variable region and light chain variable region of a chicken antibody, and also provided a framework fragment in a heavy chain variable region and light chain variable region of a chicken antibody, in which a particular amino acid of the framework fragment is modified with cysteine, a heavy chain variable region or light chain variable region including the framework, and an antibody including the heavy chain variable region or light chain variable region, or an antigen-binding fragment thereof. 
         [0009]    Preferably, the cysteine-introduced site does not induce formation of disulfide bonds between antibody molecules so that no dimers are formed or a small amount of dimers is formed. Therefore, reactivity of cysteine is maintained. The antibody introduced with the cysteine or the antigen-binding fragment thereof easily binds with a conjugation compound such as a chemotherapeutic drug, an enzyme, an aptamer, a toxin, an affinity ligand, or a detection label, thereby being applied to various fields for diagnosis or treatment of diseases. 
         [0010]    Further, the cysteine-modified framework according to the present invention, the heavy chain variable region or light chain variable region including the framework, and the antibody including the heavy chain variable region or light chain variable region or the antigen-binding fragment thereof are derived from a chicken antibody, and the chicken antibody possess one heavy chain variable region gene and one light chain variable region, unlike antibodies of humans or other animals. For this reason, all antibodies have the identical frameworks. Therefore, in the cysteine-modified framework according to the present invention or the antibody including the same, cysteines are located at the same positions, and thus equally applied to all antibodies produced in chickens. 
         [0011]    Hereinafter, the present invention will be described in more detail. 
         [0012]    An antibody according to an embodiment of the present invention is an antibody including a framework, in which one or more amino acid residues present in a framework region in a heavy chain variable region and a light chain variable region derived from a chicken antibody are replaced by cysteines. 
         [0013]    An antibody according to another embodiment of the present invention is an antibody including the framework, in which one or more amino acid residues present in a framework region in a heavy chain variable region and a light chain variable region derived from a chicken antibody are replaced by cysteines, and one or more of the two amino acids flanking both sides of the replaced cysteine are additionally replaced by one or more charged amino acids selected from the group consisting of aspartic acid, glutamic acid, arginine, and lysine. 
         [0014]    In the present invention, preparation of the modified antibody including the framework having the replaced cysteine is performed by using a chicken antibody possessing one heavy chain variable region gene and one light chain variable region gene, and thus the cysteine-modified antibody may be rapidly prepared without respective optimization of the introduction site of cysteine residue, unlike antibodies of humans or other animals having a plurality of heavy chain and light chain variable region genes. 
         [0015]    In the present invention, the antibody (Cys), in which one or more amino acid residues present in the framework region in the heavy chain variable region and the light chain variable region derived from the chicken antibody are replaced by cysteine, is advantageous in that two or more amino acids as well as one amino acid are replaced by cysteines, and therefore, two conjugation compounds are conjugated thereto, or two or more different conjugation compounds are conjugated thereto to prepare a conjugate having various functions. 
         [0016]    Further, the modified antibody according to the present invention is also advantageous in that in addition to the cysteine replacement, at least one amino acid of the two amino acids flanking both sides of the replaced cysteine is replaced by one or more charged amino acids selected from the group consisting of aspartic acid, glutamic acid, arginine, and lysine, thereby effectively inducing a covalent bond with a substance having an opposite charge, and inhibiting formation of disulfide bonds between the cysteine-modified antibodies having the same local charges to prevent antibody aggregation, and replacement of the amino acid with negatively charged aspartic acid or glutamic acid inhibits the breakage of maleimide bonds(literature [Shen B. Q. et al., Nat. Biotechnol. (2012) 30: 184-189]). 
         [0017]    In the present invention, the amino acid at position replaceable with cysteine is an amino acid other than cysteine. Any amino acid located within the framework may be replaced, as long as it is able to maintain antibody activity and affinity even though replaced by cysteine. 
         [0018]    The amino acids to be replaced, satisfying the above conditions, are located within the framework which is present in the light chain variable region of a chicken antibody, including framework 1 of SEQ ID NO: 1, framework 2 of SEQ ID NO: 2, framework 3 of SEQ ID NO: 3, and framework 4 of SEQ ID NO: 4, and structures of these frameworks are shown in detail in  FIG. 1   a.    
         [0019]    In an embodiment of the present invention, the modified antibody may be an antibody including only a modified framework of the light chain variable region, only a modified framework of the heavy chain variable region, or both of the modified framework of the light chain variable region and the modified framework of the heavy chain variable region. 
         [0020]    In the framework present in the light chain variable region of the chicken antibody, the amino acid to be replaced by cysteine is located at one or more positions selected from the group consisting of positions 3, 4, 5, 12, and 16 in the amino acid sequence of SEQ ID NO: 1 by sequential numbering (corresponding to positions 5, 6, 7, 14, and 18 by Kabat numbering), 
         [0021]    positions 31 and 40 in the amino acid sequence of SEQ ID NO: 2 by sequential numbering (corresponding to positions 37 and 45 by Kabat numbering), 
         [0022]    positions 52, 53, 56, 58, 59, 60, 61, 64, 65, 71, 78, and 82 in the amino acid sequence of SEQ ID NO: 3 by sequential numbering (corresponding to positions 57, 58, 61, 63, 64, 65, 66, 69, 70, 76, 83, and 87 by Kabat numbering), and 
         [0023]    positions 98 and 102 in the amino acid sequence of SEQ ID NO: 4 by sequential numbering (corresponding to positions 102 and 106 by Kabat numbering). 
         [0024]    In the framework present in the light chain variable region, the position of the amino acid replaceable by cysteine is shown in Table 1a. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1a 
               
             
             
               
                   
               
               
                 Light chain framework 
               
             
          
           
               
                   
                   
                 Sequence 
                 Kabat 
                 Wild type 
               
               
                 Light chain 
                 Cys mutant 
                 numbering 
                 numbering 
                 amino acid 
               
               
                   
               
               
                 Framework 1 
                 LF1-2 
                 L3 
                 L5 
                 T 
               
               
                   
                 LF1-3 
                 L4 
                 L6 
                 Q 
               
               
                   
                 LF1-4 
                 L5 
                 L7 
                 P 
               
               
                   
                 LF1-11 
                 L12 
                 L14 
                 N 
               
               
                   
                 LF1-15 
                 L16 
                 L18 
                 T 
               
               
                 Framework 2 
                 LF2-3 
                 L31 
                 L37 
                 Q 
               
               
                   
                 LF2-12 
                 L40 
                 L45 
                 V 
               
               
                 Framework 3 
                 LF3-1 
                 L52 
                 L57 
                 N 
               
               
                   
                 LF3-2 
                 L53 
                 L58 
                 I 
               
               
                   
                 LF3-5 
                 L56 
                 L61 
                 R 
               
               
                   
                 LF3-7 
                 L58 
                 L63 
                 S 
               
               
                   
                 LF3-8 
                 L59 
                 L64 
                 G 
               
               
                   
                 LF3-9 
                 L60 
                 L65 
                 S 
               
               
                   
                 LF3-10 
                 L61 
                 L66 
                 T 
               
               
                   
                 LF3-13 
                 L64 
                 L69 
                 S 
               
               
                   
                 LF3-14 
                 L65 
                 L70 
                 T 
               
               
                   
                 LF3-20 
                 L71 
                 L76 
                 T 
               
               
                   
                 LF3-27 
                 L78 
                 L83 
                 A 
               
               
                   
                 LF3-31 
                 L82 
                 L87 
                 Y 
               
               
                 Framework 4 
                 LF4-5 
                 L98 
                 L102 
                 T 
               
               
                   
                 LF4-9 
                 L102 
                 L106 
                 V 
               
               
                   
               
             
          
         
       
     
         [0025]    The amino acids to be replaced, satisfying the above conditions, are located within the framework which is present in the heavy chain variable region of a chicken antibody, including framework 1 of SEQ ID NO: 5, framework 2 of SEQ ID NO: 6, framework 3 of SEQ ID NO: 7, and framework 4 of SEQ ID NO: 8, and structures of these frameworks are shown in detail in  FIG. 1   a.    
         [0026]    In the framework present in the heavy chain variable region of the chicken antibody, the amino acid to be replaced by cysteine is located at one or more positions selected from the group consisting of positions 1, 3, 5, 6, 7, 11, 12, 13, 16, 18, 19, 24, and 25 in the amino acid sequence of SEQ ID NO: 5 by sequential numbering (corresponding to positions 1, 3, 5, 6, 7, 11, 12, 13, 16, 18, 19, 24, and 25 by Kabat numbering), 
         [0027]    positions 42 and 48 in the amino acid sequence of SEQ ID NO: 6 by sequential numbering (corresponding to positions 42 and 48 by Kabat numbering), 
         [0028]    positions 70, 73, 75, 76, 78, 87, 88, 89, 91, and 93 in the amino acid sequence of SEQ ID NO: 7 by sequential numbering (corresponding to positions 69, 72, 74, 75, 77, 83, 84, 85, 87, and 89 by Kabat numbering), and 
         [0029]    positions 123 and 126 in the amino acid sequence of SEQ ID NO: 8 by sequential numbering (corresponding to positions 110 and 113 by Kabat numbering). 
         [0030]    In the framework present in the heavy chain variable region, the position of the amino acid replaceable by cysteine is shown in Table 1b. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1b 
               
             
             
               
                   
               
               
                 Heavy chain framework 
               
             
          
           
               
                   
                   
                 Sequence 
                 Kabat 
                 Wild type 
               
               
                 Light chain 
                 Cys mutant 
                 numbering 
                 numbering 
                 amino acid 
               
               
                   
               
               
                 Framework 1 
                 HF1-1 
                 H1 
                 H1 
                 A 
               
               
                   
                 HF1-3 
                 H3 
                 H3 
                 T 
               
               
                   
                 HF1-5 
                 H5 
                 H5 
                 D 
               
               
                   
                 HF1-6 
                 H6 
                 H6 
                 E 
               
               
                   
                 HF1-7 
                 H7 
                 H7 
                 S 
               
               
                   
                 HF1-11 
                 H11 
                 H11 
                 L 
               
               
                   
                 HF1-12 
                 H12 
                 H12 
                 Q 
               
               
                   
                 HF1-13 
                 H13 
                 H13 
                 T 
               
               
                   
                 HF1-16 
                 H16 
                 H16 
                 G 
               
               
                   
                 HF1-18 
                 H18 
                 H18 
                 L 
               
               
                   
                 HF1-19 
                 H19 
                 H19 
                 S 
               
               
                   
                 HF1-24 
                 H24 
                 H24 
                 A 
               
               
                   
                 HF1-25 
                 H25 
                 H25 
                 S 
               
               
                 Framework 2 
                 HF2-7 
                 H42 
                 H42 
                 G 
               
               
                   
                 HF2-13 
                 H48 
                 H48 
                 V 
               
               
                 Framework 3 
                 HF3-4 
                 H70 
                 H69 
                 I 
               
               
                   
                 HF3-7 
                 H73 
                 H72 
                 D 
               
               
                   
                 HF3-9 
                 H75 
                 H74 
                 G 
               
               
                   
                 HF3-10 
                 H76 
                 H75 
                 Q 
               
               
                   
                 HF3-12 
                 H78 
                 H77 
                 T 
               
               
                   
                 HF3-21 
                 H87 
                 H83 
                 R 
               
               
                   
                 HF3-22 
                 H88 
                 H84 
                 A 
               
               
                   
                 HF3-23 
                 H89 
                 H85 
                 E 
               
               
                   
                 HF3-25 
                 H91 
                 H87 
                 T 
               
               
                   
                 HF3-27 
                 H93 
                 H89 
                 T 
               
               
                 Framework 4 
                 HF4-8 
                 H123 
                 H110 
                 I 
               
               
                   
                 HF4-11 
                 H126 
                 H113 
                 S 
               
               
                   
               
             
          
         
       
     
         [0031]    Further, in the light chain variable region, the amino acid to be replaced by cysteine may be preferably located at one or more positions selected from the group consisting of positions 3, 4 and 5 in the amino acid sequence of SEQ ID NO: 1 by sequential numbering (corresponding to positions 5, 6, and 7 by Kabat numbering). In the heavy chain variable region, the amino acid to be replaced by cysteine may be preferably located at one or more positions selected from the group consisting of positions 13 and 16 in the amino acid sequence of SEQ ID NO: 5 by sequential numbering (corresponding to positions 13 and 16 by Kabat numbering). 
         [0032]    In an embodiment of the present invention, at least one of the two amino acids flanking both sides of the replaced cysteine may be replaced by one or more charged amino acids selected from the group consisting of aspartic acid, glutamic acid, arginine, and lysine, in addition to replacement of cysteine at the preferred amino acid position. With respect to the modified antibody, for example, when the amino acid to be replaced by cysteine in the light chain variable region is preferably located at one or more positions selected from the group consisting of positions 3, 4 and 5 in the amino acid sequence of SEQ ID NO: 1 by sequential numbering, or the amino acid to be replaced by cysteine in the heavy chain variable region is preferably located at one or more positions selected from the group consisting of positions 13 and 16 in the amino acid sequence of SEQ ID NO: 5 by sequential numbering, the framework in which the amino acids flanking both sides of the replaced cysteine are replaced by charged amino acids may include modified sequences of Tables 3a and 3b. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3a 
               
               
                   
               
               
                   
                 Sequence 
                 SEQ 
                   
                 Sequence 
                 SEQ 
                   
                 Sequence 
                 SEQ 
               
               
                 Mutant 
                 Kabat numbering 
                 ID 
                 Mutant 
                 Kabat numbering 
                 ID 
                 Mutant 
                 Kabat numbering 
                 ID 
               
               
                 name 
                 (L3-L4-L5-L6-L7) 
                 NO 
                 name 
                 (L4-L5-L6-L7-L8) 
                 NO 
                 name 
                 (L5-L6-L7-L8-L9) 
                 NO 
               
               
                   
               
             
             
               
                 L2CR 
                 ALCRP 
                  9 
                 T3CR 
                 LTCRS 
                 25 
                 Q4CR 
                 TQCRS 
                 41 
               
               
                   
               
               
                 L2CK 
                 ALCKP 
                 10 
                 T3CK 
                 LTCKS 
                 26 
                 Q4CK 
                 TQCKS 
                 42 
               
               
                   
               
               
                 R2CQ 
                 ARCQP 
                 11 
                 R3CP 
                 LRCPS 
                 27 
                 R4CS 
                 TRCSS 
                 43 
               
               
                   
               
               
                 R2CR 
                 ARCRP 
                 12 
                 R3CR 
                 LRCRS 
                 28 
                 R4CR 
                 TRCRS 
                 44 
               
               
                   
               
               
                 R2CK 
                 ARCKP 
                 13 
                 R3CK 
                 LRCKS 
                 29 
                 R4CK 
                 TRCKS 
                 45 
               
               
                   
               
               
                 K2CQ 
                 AKCQP 
                 14 
                 K3CP 
                 LKCPS 
                 30 
                 K4CS 
                 TKCSS 
                 46 
               
               
                   
               
               
                 K2CR 
                 AKCRP 
                 15 
                 K3CR 
                 LKCRS 
                 31 
                 K4CR 
                 TKCRS 
                 47 
               
               
                   
               
               
                 K2CK 
                 AKCKP 
                 16 
                 K3CK 
                 LKCKS 
                 32 
                 K4CK 
                 TKCKS 
                 48 
               
               
                   
               
               
                 L2CD 
                 ALCDP 
                 17 
                 T3CD 
                 LTCDS 
                 33 
                 Q4CD 
                 TQCDS 
                 49 
               
               
                   
               
               
                 L2CE 
                 ALCEP 
                 18 
                 T3CE 
                 LTCES 
                 34 
                 Q4CE 
                 TQCES 
                 50 
               
               
                   
               
               
                 D2CQ 
                 ADCQP 
                 19 
                 D3CP 
                 LDCPS 
                 35 
                 D4CS 
                 TDCSS 
                 51 
               
               
                   
               
               
                 D2CD 
                 ADCDP 
                 20 
                 D3CD 
                 LDCDS 
                 36 
                 D4CD 
                 TDCDS 
                 52 
               
               
                   
               
               
                 D2CE 
                 ADCEP 
                 21 
                 D3CE 
                 LDCES 
                 37 
                 D4CE 
                 TDCES 
                 53 
               
               
                   
               
               
                 E2CQ 
                 AECQP 
                 22 
                 E3CP 
                 LECPS 
                 38 
                 E4CS 
                 TRCSS 
                 54 
               
               
                   
               
               
                 E2CD 
                 AECDP 
                 23 
                 E3CD 
                 LECDS 
                 39 
                 E4CD 
                 TECDS 
                 55 
               
               
                   
               
               
                 E2CE 
                 AECEP 
                 24 
                 E3CE 
                 LECES 
                 40 
                 E4CE 
                 TECES 
                 56 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3b 
               
             
             
               
                   
               
               
                   
                 Sequence 
                 SEQ 
                   
                 Sequence 
                 SEQ 
               
             
          
           
               
                 Mutant 
                 Kabat numbering 
                 ID 
                 Mutant 
                 Kabat numbering 
                 ID 
               
               
                 name 
                 (H11-H12-H13-H14-H15) 
                 NO 
                 name 
                 (H14-H15-H16-H17-H18) 
                 NO 
               
               
                   
               
               
                 Q13CR 
                 LQCRG 
                 57 
                 G16CR 
                 PGCRL 
                 73 
               
               
                   
               
               
                 Q13CK 
                 LQCKG 
                 58 
                 G16CK 
                 PGCKL 
                 74 
               
               
                   
               
               
                 R13CP 
                 LRCPG 
                 59 
                 R16CA 
                 PRCAL 
                 75 
               
               
                   
               
               
                 R13CR 
                 LRCRG 
                 60 
                 R16CR 
                 PRCRL 
                 76 
               
               
                   
               
               
                 R13CK 
                 LRCKG 
                 61 
                 R16CK 
                 PRCKL 
                 77 
               
               
                   
               
               
                 K13CP 
                 LKCPG 
                 62 
                 K16CA 
                 PKCAL 
                 78 
               
               
                   
               
               
                 K13CR 
                 LKCRG 
                 63 
                 K16CR 
                 PKCRL 
                 79 
               
               
                   
               
               
                 K13CK 
                 LKCKG 
                 64 
                 K16CK 
                 PKCKL 
                 80 
               
               
                   
               
               
                 Q13CD 
                 LQCDG 
                 65 
                 G16CD 
                 PGCDL 
                 81 
               
               
                   
               
               
                 Q13CE 
                 LQCEG 
                 66 
                 G16CE 
                 PGCEL 
                 82 
               
               
                   
               
               
                 D13CP 
                 LDCPG 
                 67 
                 D16CA 
                 PDCAL 
                 83 
               
               
                   
               
               
                 D13CD 
                 LDCDG 
                 68 
                 D16CD 
                 PDCDL 
                 84 
               
               
                   
               
               
                 D13CE 
                 LDCEG 
                 69 
                 D16CE 
                 PDCEL 
                 85 
               
               
                   
               
               
                 E13CP 
                 LECPG 
                 70 
                 E16CA 
                 PECAL 
                 86 
               
               
                   
               
               
                 E13CD 
                 LECDG 
                 71 
                 E16CD 
                 PECDL 
                 87 
               
               
                   
               
               
                 E13CE 
                 LECEG 
                 72 
                 E16CE 
                 PECEL 
                 88 
               
               
                   
               
             
          
         
       
     
         [0033]    Therefore, the modified antibody including replacement of adjacent amino acids as well as replacement of cysteine may be an antibody including a modified light chain variable region having one or more selected from the group consisting of amino acid sequences of SEQ ID NO: 57 to SEQ ID NO: 88, a modified heavy chain variable region having one or more selected from the group consisting of amino acid sequences of SEQ ID NO: 57 to SEQ ID NO: 88, or both of the modified light chain variable region and the modified heavy chain variable region. 
         [0034]    Unless stated otherwise, the terms and phrases as used herein are intended to have the following meanings. As used herein, the term “antibody” is used in the broadest sense, and specifically, covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting the desired biological activity. Antibodies may be murine, human, humanized, chimeric antibodies, or derived from other species. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule containing an antigen binding site that immunospecifically binds a target antigen (such targets including, but not limited to, cancer cells or cells producing autoimmune antibodies associated with an autoimmune disease) or part thereof. The immunoglobulin disclosed herein may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins may be derived from any species. 
         [0035]    As used herein, the term “antibody fragments” includes a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′) 2 , and Fv fragments; diabodies; linear antibodies; minibodies (literature [Olafsen T. et al. Protein Eng. Design &amp; Sel. (2004) 17:315-323]), fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. 
         [0036]    In the present invention, the cysteine-substituted antibody may be prepared by a method including 1) inducing a mutation in a nucleic acid sequence encoding the cysteine-substituted antibody; 2) expressing the cysteine-substituted antibody; and 3) isolating and purifying the cysteine-substituted antibody. Further, the replacement of amino acids flanking both sides of the cysteine may be also performed in the same manner as in the replacement of cysteine. 
         [0037]    In another specific embodiment of the present invention, provided is a conjugate including the modified antibody according to the present invention or an antigen-binding fragment thereof and a conjugation compound, the conjugate being prepared by conjugating the conjugation compound, such as a chemotherapeutic drug, an enzyme, a peptide, an aptamer, a toxin, an affinity ligand, or a detection label, to the modified antibody. According to the type of the conjugation compound, the conjugate may be variously applied to diagnosis or treatment of diseases. 
         [0038]    In still another embodiment of the present invention, the antibody chemically binding to a peptide may further have a function of contributing to peptide affinity, such as effective penetration across the blood brain barrier via its receptor (literature [Chacko A. M et al., Expert Opin. Drug Deliv. (2013) 10: 907-26; patent: EP2 233 156 B 1]). 
         [0039]    The conjugation compound may functions to (i) provide a detectable signal, (ii) interact with a second label to modify the detectable signal provided by the first or second label, e.g. FRET (fluorescence resonance energy transfer), (iii) stabilize interactions or increase affinity of binding, with antigen or ligand, (iv) affect mobility, e.g. electrophoretic mobility, or cell-permeability, by charge, hydrophobicity, shape, or other physical parameters, or (v) provide a capture moiety, in order to modulate ligand affinity, antibody/antigen binding, or ionic complexation. 
         [0040]    Labeled cysteine-substituted antibodies may be useful in diagnostic assays, e.g., for detecting expression of an antigen of interest in specific cells, tissues, or serum. For diagnostic applications, the antibody will typically be labeled with a detectable moiety. Numerous labels are available which may be generally grouped into the following categories: 
         [0041]    (a) Radioisotopes (radionuclides), such as  3 H,  11 C  14   C,    18 F,  32 P,  35  S,  64 Cu,  68 Ga,  86 Y,  89 Zr,  99 Tc,  111 In,  123 I,  124 I,  125 I,  131 I,  133 Xe,  177 Lu,  211 At, or  213 Bi. Radioisotope-labeled antibodies are useful in receptor targeted imaging experiments. The antibody may be labeled with ligand reagents that bind, chelate or otherwise complex a radioisotope metal where the reagent is reactive with the engineered cysteine thiol of the antibody. 
         [0042]    (b) Fluorescent labels such as rare earth chelates (europium chelates), fluorescein types including FITC, 5-carboxyfluorescein, 6-carboxy fluorescein, etc.; rhodamine types including TAMRA, etc.; dansyl; Lissamine; cyanines; phycoerythrins; Texas Red; and analogs thereof. Fluorescent dyes and fluorescent label reagents include those which are commercially available from Invitrogen/Molecular Probes (Eugene, Oreg., USA) and Pierce Biotechnology, Inc. (Rockford, Ill., USA). 
         [0043]    (c) Various enzyme-substrate labels are available or disclosed in literatures (U.S. pat. No. 4,275,149). The enzyme generally catalyzes a chemical alteration of a chromogenic substrate that may be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which may be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. Examples of enzymatic labels include luciferase, urease, peroxidase such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucoamylase, lysozyme, saccharide oxidase, etc. Techniques for conjugating enzymes to antibodies may be performed by a general method. 
         [0044]    In still another embodiment, the cysteine-substituted antibody may be labeled through the cysteine thiol with radionuclides, fluorescent dyes, bioluminescence-triggering substrate moieties, chemiluminescence-triggering substrate moieties, enzymes, and other detection labels for imaging experiments with diagnostic, pharmacodynamic, and therapeutic applications. Generally, the labeled cysteine engineered antibody, i.e. “biomarker” or “probe”, is administered by injection, perfusion, or oral ingestion to a living organism, e.g. human, rodent, or other small animal, a perfused organ, or tissue sample. The distribution of the probe is detected over a time course and represented by an image. 
       Effect of the Invention 
       [0045]    A framework fragment of a chicken antibody according to the present invention, a heavy chain variable region or a light chain variable region including the framework fragment, or an antibody including the heavy chain variable region or the light chain variable region does not induce or prevents formation of disulfide bonds between antibody molecules while maintaining activity and reactivity of the antibody. The antibody introduced with cysteine or an antigen-binding fragment thereof easily binds with a conjugation compound such as a chemotherapeutic drug, an enzyme, an aptamer, a toxin, an affinity ligand, or a detection label, thereby being applied to various fields for diagnosis or treatment of mammalian cells or related diseases. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0046]      FIG. 1 a    shows a sequential numbering scheme (top row) in comparison with the Kabat numbering scheme (bottom row) for base sequences of wild-type and germ-line of a chicken antibody used in the present experiment; 
           [0047]      FIG. 1 b    shows a cartoon depiction of Cys-modified antibody phage binding to immobilized PSA with binding of anti-phage HRP antibody for absorbance detection; 
           [0048]      FIGS. 1 c  and 1 d    show binding measurements with detection of absorbance at 405 nm of Cys-modified antibody phages (upper: modified light chain, lower: modified heavy chain); 
           [0049]      FIG. 1 e    shows a three-dimensional representation of a chicken antibody fragment derived by X-ray crystal coordinates, in which the structure positions of 5 types of the engineered Cys residues of the heavy and light chains are shown; 
           [0050]      FIG. 2  shows binding measurements with detection of absorbance at 405 nm of antibody phages including two cysteine residues having thiol reactivity; 
           [0051]      FIGS. 3 a  and 3 b    show binding measurements with detection of absorbance at 405 nm of Cys-modified antibody phages with replaced with cationic amino acids (upper: modified light chain, lower: modified heavy chain); 
           [0052]      FIGS. 3 c  and 3 d    show binding measurements with detection of absorbance at 405 nm of Cys-modified antibody phages with replacement of anionic amino acid (upper: modified light chain, lower: modified heavy chain); 
           [0053]      FIG. 4 a    shows coomassie blue staining of Cys-modified single chain Fv (scFv)-type antibodies expressed from cell culture for conjugation; 
           [0054]      FIG. 4 b    shows coomassie blue staining of cationic or anionic Cys-modified scFv-type antibodies expressed from cell culture for conjugation; 
           [0055]      FIG. 5 a    shows coomassie blue staining of Cys-modified full IgG-type antibodies expressed from cell culture for conjugation; 
           [0056]      FIG. 5 b    shows coomassie blue staining of cationic or anionic Cys-modified full IgG-type antibodies expressed from cell culture for conjugation; 
           [0057]      FIG. 6  shows a cartoon depiction of conjugation of maleimide activated PEG to Cys-modified antibody; 
           [0058]      FIG. 7 a    shows coomassie blue staining of maleimide PEG-conjugated scFv; 
           [0059]      FIG. 7 b    shows iodine staining of maleimide PEG-conjugated scFv; 
           [0060]      FIG. 7 c    shows immunoblotting of maleimide PEG-conjugated scFv; 
           [0061]      FIG. 8 a    shows coomassie blue staining of maleimide PEG-conjugated full IgG; 
           [0062]      FIG. 8 b    shows iodine staining of maleimide PEG-conjugated full IgG; 
           [0063]      FIG. 8 c    shows immunoblotting of maleimide PEG-conjugated full IgG; 
           [0064]      FIG. 9 a    shows coomassie blue staining of maleimide PEG-conjugated scFv with replacement of cationic or anionic residue; 
           [0065]      FIG. 9 b    shows iodine staining of maleimide PEG-conjugated scFv with replacement of cationic or anionic residue; 
           [0066]      FIG. 9 c    shows immunoblotting of maleimide PEG-conjugated scFv with replacement of cationic or anionic residue; 
           [0067]      FIG. 10 a    shows coomassie blue staining of maleimide PEG-conjugated full IgG with replacement of cationic or anionic residue; 
           [0068]      FIG. 10 b    shows iodine staining of maleimide PEG-conjugated full IgG with replacement of cationic or anionic residue; 
           [0069]      FIG. 10 c    shows immunoblotting of maleimide PEG-conjugated full IgG with replacement of cationic or anionic residue; 
           [0070]      FIG. 11 a    shows a cartoon depiction of conjugation of GMBS-cotinine to Cys-modified antibody; 
           [0071]      FIG. 11 b    shows a cartoon depiction of GMBS-cotinine-conjugated, Cys-modified antibody binding to immobilized PSA with binding of anti-cotinine HRP antibody for absorbance detection; 
           [0072]      FIG. 12  shows detection of absorbance at 450 nm of binding of GMBS-cotinine to C kappa -fused, Cys-modified scFv (upper: detection with anti-cotinine IgG-HRP, lower: detection with anti-C kappa  IgG-HRP); 
           [0073]      FIG. 13 a    shows injection time of cotinine-Cys into mice and blood collection time; and 
           [0074]      FIG. 13 b    shows detection of absorbance at 450 nm to examine the presence or absence of cotinine-Cys-modified antibody in the serum before and after injection of cotinine-Cys-modified antibody into mice. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0075]    The present invention will be described in more detail with reference to specific Examples of the present invention. However, the present invention is not limited to the following Examples, and it will be apparent to those skilled in the art that various modifications and changes may be made thereto without departing from the scope and spirit of the present invention. Therefore, the appended claims should be interpreted broadly, as may be consistent with the spirit and scope of the present invention. 
       EXAMPLE 1 
     Preparation of Antibody Having One Cys Modification 
       [0076]    1-1. Preparation of Modified Antibody Phage 
         [0077]    An antibody used in Example was a chicken antibody against PSA (prostate specific antigen), and  FIG. 1 a    shows base sequences of frameworks of anti-PSA antibody (wild-type) and chicken germ-line ([literature [Andris-Widhopf J. et al., J Immunol. Methods (2000) 242: 159-181]). Kabat numbering system is available at http://www.bioinf.org.uk/abs/. 
         [0078]    For preparation of Cys-modified antibody, PCR was first performed to insert NNK (N:A/C/G/T, K:G/T) into 78 light chain domain framework (V L  domain framework) residues and 84 heavy chain domain framework (V H  domain framework) residues in order to randomize the respective residues, in accordance with the literature [Chung J. et al., FASEB J. (2004) 18: 361-383]. Thereafter, in accordance with a method described in Barbas, C. F. (2001) Phage display: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, the obtained scFv (single chain Fv) DNA was inserted into a phagemid vector pcomb3X, which was transformed into  E. coli  ER2738. Then, 92 colonies were selected according to each residue to obtain a culture supernatant containing phage-displayed scFv (single chain Fv). 
         [0079]    1-2. Selection of Modified Antibodies and Measurement of Their Affinity to Antigen 
         [0080]    The culture supernatant containing the phage antibodies was used to perform enzyme-linked immunosorbent assay (ELISA) as in a method illustrated in  FIG. 1   b.  A microtiter plate was coated with an antigen PSA, and the culture supernatant containing phages was added thereto, followed by incubation for 1 hour. After washing three times, HRP (horse radish peroxidase)-conjugated anti-M13 antibody was added thereto. After incubation for 1 hour and washing three times, H 2 O 2 -added ABTS was added to examine color development. Then, absorbance at 405 nm was measured. As a control group, a wild-type phage was used. Even after 21 light chain residues and 27 heavy chain residues were replaced by cysteine, they showed affinity similar to that of the wild-type antibody ( FIGS. 1 c    and 1d). In Tables 1a and 1b, sequential numbering and Kabat numbering of the Cys-modified antibodies, and a base sequence of the wild-type of each modified antibody are given. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1a 
               
             
             
               
                   
               
               
                 Light chain framework 
               
             
          
           
               
                   
                   
                 Sequence 
                 Kabat 
                 Wild type 
               
               
                 Light chain 
                 Cys mutant 
                 numbering 
                 numbering 
                 amino acid 
               
               
                   
               
               
                 Framework 1 
                 LF1-2 
                 L3 
                 L5 
                 T 
               
               
                   
                 LF1-3 
                 L4 
                 L6 
                 Q 
               
               
                   
                 LF1-4 
                 L5 
                 L7 
                 P 
               
               
                   
                 LF1-11 
                 L12 
                 L14 
                 N 
               
               
                   
                 LF1-15 
                 L16 
                 L18 
                 T 
               
               
                 Framework 2 
                 LF2-3 
                 L31 
                 L37 
                 Q 
               
               
                   
                 LF2-12 
                 L40 
                 L45 
                 V 
               
               
                 Framework 3 
                 LF3-1 
                 L52 
                 L57 
                 N 
               
               
                   
                 LF3-2 
                 L53 
                 L58 
                 I 
               
               
                   
                 LF3-5 
                 L56 
                 L61 
                 R 
               
               
                   
                 LF3-7 
                 L58 
                 L63 
                 S 
               
               
                   
                 LF3-8 
                 L59 
                 L64 
                 G 
               
               
                   
                 LF3-9 
                 L60 
                 L65 
                 S 
               
               
                   
                 LF3-10 
                 L61 
                 L66 
                 T 
               
               
                   
                 LF3-13 
                 L64 
                 L69 
                 S 
               
               
                   
                 LF3-14 
                 L65 
                 L70 
                 T 
               
               
                   
                 LF3-20 
                 L71 
                 L76 
                 T 
               
               
                   
                 LF3-27 
                 L78 
                 L83 
                 A 
               
               
                   
                 LF3-31 
                 L82 
                 L87 
                 Y 
               
               
                 Framework 4 
                 LF4-5 
                 L98 
                 L102 
                 T 
               
               
                   
                 LF4-9 
                 L102 
                 L106 
                 V 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1b 
               
             
             
               
                   
               
               
                 Heavy chain framework 
               
             
          
           
               
                   
                   
                 Sequence 
                 Kabat 
                 Wild type 
               
               
                 Light chain 
                 Cys mutant 
                 numbering 
                 numbering 
                 amino acid 
               
               
                   
               
               
                 Framework 1 
                 HF1-1 
                 H1 
                 H1 
                 A 
               
               
                   
                 HF1-3 
                 H3 
                 H3 
                 T 
               
               
                   
                 HF1-5 
                 H5 
                 H5 
                 D 
               
               
                   
                 HF1-6 
                 H6 
                 H6 
                 E 
               
               
                   
                 HF1-7 
                 H7 
                 H7 
                 S 
               
               
                   
                 HF1-11 
                 H11 
                 H11 
                 L 
               
               
                   
                 HF1-12 
                 H12 
                 H12 
                 Q 
               
               
                   
                 HF1-13 
                 H13 
                 H13 
                 T 
               
               
                   
                 HF1-16 
                 H16 
                 H16 
                 G 
               
               
                   
                 HF1-18 
                 H18 
                 H18 
                 L 
               
               
                   
                 HF1-19 
                 H19 
                 H19 
                 S 
               
               
                   
                 HF1-24 
                 H24 
                 H24 
                 A 
               
               
                   
                 HF1-25 
                 H25 
                 H25 
                 S 
               
               
                 Framework 2 
                 HF2-7 
                 H42 
                 H42 
                 G 
               
               
                   
                 HF2-13 
                 H48 
                 H48 
                 V 
               
               
                 Framework 3 
                 HF3-4 
                 H70 
                 H69 
                 I 
               
               
                   
                 HF3-7 
                 H73 
                 H72 
                 D 
               
               
                   
                 HF3-9 
                 H75 
                 H74 
                 G 
               
               
                   
                 HF3-10 
                 H76 
                 H75 
                 Q 
               
               
                   
                 HF3-12 
                 H78 
                 H77 
                 T 
               
               
                   
                 HF3-21 
                 H87 
                 H83 
                 R 
               
               
                   
                 HF3-22 
                 H88 
                 H84 
                 A 
               
               
                   
                 HF3-23 
                 H89 
                 H85 
                 E 
               
               
                   
                 HF3-25 
                 H91 
                 H87 
                 T 
               
               
                   
                 HF3-27 
                 H93 
                 H89 
                 T 
               
               
                 Framework 4 
                 HF4-8 
                 H123 
                 H110 
                 I 
               
               
                   
                 HF4-11 
                 H126 
                 H113 
                 S 
               
               
                   
               
             
          
         
       
     
         [0081]    In order to examine solvent accessibility of the selected Cys-modified antibodies, solvent accessible surface area was examined by a three-dimensional structure of a chicken antibody fragment derived by X-ray crystal coordinates (literature [Shih H. H. et al., J Biol. Chem. (2012) 287:44425-44434; PDB ID: 4GLR]), as shown in Tables 2a and 2b. Of them, structural positions of 5 types of base (LF1-2, LF1-3, LF1-4, HF1-13, HF1-16) residues are shown in  FIG. 1   e.    
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2a 
               
             
             
               
                   
               
               
                 Solvent accessibility of light chain 
               
             
          
           
               
                   
                 Light chain 
                 Cys mutant 
                 Solvent accessibility area (A 2 ) 
               
               
                   
                   
               
             
          
           
               
                   
                 Framework 1 
                 LF1-2 
                 95.52 
               
               
                   
                   
                 LF1-3 
                 17 
               
               
                   
                   
                 LF1-4 
                 55.89 
               
               
                   
                   
                 LF1-11 
                 116.46 
               
               
                   
                   
                 LF1-15 
                 7.5 
               
               
                   
                 Framework 2 
                 LF2-3 
                 17.89 
               
               
                   
                   
                 LF2-12 
                 69.64 
               
               
                   
                 Framework 3 
                 LF3-1 
                 139.13 
               
               
                   
                   
                 LF3-2 
                 26.94 
               
               
                   
                   
                 LF3-5 
                 42.19 
               
               
                   
                   
                 LF3-7 
                 46.25 
               
               
                   
                   
                 LF3-8 
                 12.95 
               
               
                   
                   
                 LF3-9 
                 62.79 
               
               
                   
                   
                 LF3-10 
                 45.55 
               
               
                   
                   
                 LF3-13 
                 48.57 
               
               
                   
                   
                 LF3-14 
                 49.06 
               
               
                   
                   
                 LF3-20 
                 72.89 
               
               
                   
                   
                 LF3-27 
                 59.26 
               
               
                   
                   
                 LF3-31 
                 10.64 
               
               
                   
                 Framework 4 
                 LF4-5 
                 0 
               
               
                   
                   
                 LF4-9 
                 6.5 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2b 
               
             
             
               
                   
               
               
                 Solvent accessibility of heavy chain 
               
             
          
           
               
                   
                 Heavy chain 
                 Cys mutant 
                 Solvent accessibility area (A 2 ) 
               
               
                   
                   
               
             
          
           
               
                   
                 Framework 1 
                 HF1-1 
                 127.31 
               
               
                   
                   
                 HF1-3 
                 73.16 
               
               
                   
                   
                 HF1-5 
                 71.13 
               
               
                   
                   
                 HF1-6 
                 6.42 
               
               
                   
                   
                 HF1-7 
                 62.7 
               
               
                   
                   
                 HF1-11 
                 147.06 
               
               
                   
                   
                 HF1-12 
                 29.81 
               
               
                   
                   
                 HF1-13 
                 91.96 
               
               
                   
                   
                 HF1-16 
                 21.38 
               
               
                   
                   
                 HF1-18 
                 12.61 
               
               
                   
                   
                 HF1-19 
                 42.57 
               
               
                   
                   
                 HF1-24 
                 0.88 
               
               
                   
                   
                 HF1-25 
                 45.66 
               
               
                   
                 Framework 2 
                 HF2-7 
                 80.67 
               
               
                   
                   
                 HF2-13 
                 1.53 
               
               
                   
                 Framework 3 
                 HF3-4 
                 4.97 
               
               
                   
                   
                 HF3-7 
                 67.97 
               
               
                   
                   
                 HF3-9 
                 72.49 
               
               
                   
                   
                 HF3-10 
                 121.23 
               
               
                   
                   
                 HF3-12 
                 28.12 
               
               
                   
                   
                 HF3-21 
                 110.03 
               
               
                   
                   
                 HF3-22 
                 70.68 
               
               
                   
                   
                 HF3-23 
                 118.19 
               
               
                   
                   
                 HF3-25 
                 28.23 
               
               
                   
                   
                 HF3-27 
                 5.82 
               
               
                   
                 Framework 4 
                 HF4-8 
                 89.14 
               
               
                   
                   
                 HF4-11 
                 101.82 
               
               
                   
                   
               
             
          
         
       
     
       EXAMPLE 2 
     Preparation of Antibody Having Two Cys Modifications 
       [0082]    From the residues of light chain (L3, L4, L5) and heavy chain (H13, H16) according to sequential numbering, each one residue of the light chain and the heavy chain was replaced by cysteine in combination, and total 6 types (L3H13, L3H16, L4H13, L4H16, L5H13, L5H16) of Cys-modified antibody genes with two reactive cysteines were prepared by PCR. Next, they were expressed in the form of phage-displayed scFv as in Example 1, and then their affinity to PSA antigen was measured by enzyme-linked immunosorbent assay ( FIG. 2 ). 
       EXAMPLE 3 
     Modified Antibody Having Replacement of Amino Acid Residues Adjacent to Cys 
       [0083]    To replace amino acids flanking both sides of 5 types of Cys-modified antibodies (LF1-2, LF1-3, LF1-4, HF1-13 and HF1-16) prepared in Example 1-1 for efficient conjugation by cationic amino acids (arginine, lysine) or anionic amino acids (aspartic acid, glutamic acid), DNA base sequences (arginine; CGT, lysine; AAG, aspartic acid; GAT, glutamic acid; GAA) corresponding to the respective residues were inserted by PCR. scFv DNAs thus obtained were inserted into a phagemid vector pcomb3X, respectively and then transformed into  E. coli  ER2738. Next, the antibodies were incubated in the form of phage-displayed scFv in SB culture medium. Base sequences of the Cys-modified antibodies with replacement of cationic or anionic residues are given in Tables 3a and 3b. 
         [0084]    For measurement of antigen affinity, a microtiter plate was coated with an antigen PSA, and then the culture supernatant containing phages was added. After incubation for 1 hour and washing three times, HRP (horse radish peroxidase)-conjugated anti-M13 antibody was added thereto. After incubation for 1 hour and washing three times, H 2 O 2 -added ABTS was added to examine color development. Then, absorbance at 405 nm was measured. The result of cationic Cys-modified antibodies is shown in  FIGS. 3 a  and 3 b    and the result of anionic Cys-modified antibodies are shown in  FIGS. 3 c  and 3 d   . 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Sequence 
                 SEQ 
                   
                 Sequence 
                 SEQ 
                   
                 Sequence 
                 SEQ 
               
               
                 Mutant 
                 Kabat numbering 
                 ID 
                 Mutant 
                 Kabat numbering 
                 ID 
                 Mutant 
                 Kabat numbering 
                 ID 
               
               
                 name 
                 (L3-L4-L5-L6-L7) 
                 NO 
                 name 
                 (L4-L5-L6-L7-L8) 
                 NO 
                 name 
                 (L5-L6-L7-L8-L9) 
                 NO 
               
               
                   
               
             
             
               
                 L2CR 
                 ALCRP 
                  9 
                 T3CR 
                 LTCRS 
                 25 
                 Q4CR 
                 TQCRS 
                 41 
               
               
                   
               
               
                 L2CK 
                 ALCKP 
                 10 
                 T3CK 
                 LTCKS 
                 26 
                 Q4CK 
                 TQCKS 
                 42 
               
               
                   
               
               
                 R2CQ 
                 ARCQP 
                 11 
                 R3CP 
                 LRCPS 
                 27 
                 R4CS 
                 TRCSS 
                 43 
               
               
                   
               
               
                 R2CR 
                 ARCRP 
                 12 
                 R3CR 
                 LRCRS 
                 28 
                 R4CR 
                 TRCRS 
                 44 
               
               
                   
               
               
                 R2CK 
                 ARCKP 
                 13 
                 R3CK 
                 LRCKS 
                 29 
                 R4CK 
                 TRCKS 
                 45 
               
               
                   
               
               
                 K2CQ 
                 AKCQP 
                 14 
                 K3CP 
                 LKCPS 
                 30 
                 K4CS 
                 TKCSS 
                 46 
               
               
                   
               
               
                 K2CR 
                 AKCRP 
                 15 
                 K3CR 
                 LKCRS 
                 31 
                 K4CR 
                 TKCRS 
                 47 
               
               
                   
               
               
                 K2CK 
                 AKCKP 
                 16 
                 K3CK 
                 LKCKS 
                 32 
                 K4CK 
                 TKCKS 
                 48 
               
               
                   
               
               
                 L2CD 
                 ALCDP 
                 17 
                 T3CD 
                 LTCDS 
                 33 
                 Q4CD 
                 TQCDS 
                 49 
               
               
                   
               
               
                 L2CE 
                 ALCEP 
                 18 
                 T3CE 
                 LTCES 
                 34 
                 Q4CE 
                 TQCES 
                 50 
               
               
                   
               
               
                 D2CQ 
                 ADCQP 
                 19 
                 D3CP 
                 LDCPS 
                 35 
                 D4CS 
                 TDCSS 
                 51 
               
               
                   
               
               
                 D2CD 
                 ADCDP 
                 20 
                 D3CD 
                 LDCDS 
                 36 
                 D4CD 
                 TDCDS 
                 52 
               
               
                   
               
               
                 D2CE 
                 ADCEP 
                 21 
                 D3CE 
                 LDCES 
                 37 
                 D4CE 
                 TDCES 
                 53 
               
               
                   
               
               
                 E2CQ 
                 AECQP 
                 22 
                 E3CP 
                 LECPS 
                 38 
                 E4CS 
                 TRCSS 
                 54 
               
               
                   
               
               
                 E2CD 
                 AECDP 
                 23 
                 E3CD 
                 LECDS 
                 39 
                 E4CD 
                 TECDS 
                 55 
               
               
                   
               
               
                 E2CE 
                 AECEP 
                 24 
                 E3CE 
                 LECES 
                 40 
                 E4CE 
                 TECES 
                 56 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Sequence 
                 SEQ  
                   
                 Sequence 
                 SEQ 
               
             
          
           
               
                 Mutant 
                 Kabat numbering 
                 ID 
                 Mutant 
                 Kabat numbering 
                 ID 
               
               
                 name 
                 (H11-H12-H13-H14-H15) 
                 NO 
                 name 
                 (H14-H15-H16-H17-H18) 
                 NO 
               
               
                   
               
               
                 Q13CR 
                 LQCRG 
                 57 
                 G16CR 
                 PGCRL 
                 73 
               
               
                   
               
               
                 Q13CK 
                 LQCKG 
                 58 
                 G16CK 
                 PGCKL 
                 74 
               
               
                   
               
               
                 R13CP 
                 LRCPG 
                 59 
                 R16CA 
                 PRCAL 
                 75 
               
               
                   
               
               
                 R13CR 
                 LRCRG 
                 60 
                 R16CR 
                 PRCRL 
                 76 
               
               
                   
               
               
                 R13CK 
                 LRCKG 
                 61 
                 R16CK 
                 PRCKL 
                 77 
               
               
                   
               
               
                 K13CP 
                 LKCPG 
                 62 
                 K16CA 
                 PKCAL 
                 78 
               
               
                   
               
               
                 K13CR 
                 LKCRG 
                 63 
                 K16CR 
                 PKCRL 
                 79 
               
               
                   
               
               
                 K13CK 
                 LKCKG 
                 64 
                 K16CK 
                 PKCKL 
                 80 
               
               
                   
               
               
                 Q13CD 
                 LQCDG 
                 65 
                 G16CD 
                 PGCDL 
                 81 
               
               
                   
               
               
                 Q13CE 
                 LQCEG 
                 66 
                 G16CE 
                 PGCEL 
                 82 
               
               
                   
               
               
                 D13CP 
                 LDCPG 
                 67 
                 D16CA 
                 PDCAL 
                 83 
               
               
                   
               
               
                 D13CD 
                 LDCDG 
                 68 
                 D16CD 
                 PDCDL 
                 84 
               
               
                   
               
               
                 D13CE 
                 LDCEG 
                 69 
                 D16CE 
                 PDCEL 
                 85 
               
               
                   
               
               
                 E13CP 
                 LECPG 
                 70 
                 E16CA 
                 PECAL 
                 86 
               
               
                   
               
               
                 E13CD 
                 LECDG 
                 71 
                 E16CD 
                 PECDL 
                 87 
               
               
                   
               
               
                 E13CE 
                 LECEG 
                 72 
                 E16CE 
                 PECEL 
                 88 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 4 
     Cys-Modified scFv Antibody 
       [0085]    4-1. Expression and Purification of Cys-Modified scFv Antibody 
         [0086]    To prepare a fusion protein of Cys-modified scFv and human C kappa , Cys-modified scFv was inserted into a mammalian expression vector pCEP4 containing human C kappa  by cloning, and its expression was induced in HEK293F cells to obtain a final culture broth. Purification of the scFv-C kappa  fusion protein from the culture broth was performed using kappa select beads. After purification, the protein was loaded on a polyacrylamide gel (SDS-PAGE gel), followed by electrophoresis. Expression and purification thereof were confirmed by Coomassie blue staining  FIG. 4 a    shows the result of examining expression and purification of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv proteins. 
         [0087]    4-2. Expression and Purification of Cys-Modified scFv Antibody Having Replacement of Amino Acid Residues Adjacent to Cys 
         [0088]    From the Cys-modified antibodies having replacement of amino acid residues adjacent to Cys with cationic or anionic amino acids, clones maintaining antigen affinity were selected and their fusion proteins with human C kappa  were expressed in HEK293F cells as in Example 4-1, and purified using kappa select beads. Thereafter, electrophoresis was performed on a polyacrylamide gel, and their expression and purification were confirmed by coomassie blue staining  FIG. 4 b    shows the result of examining expression and purification of wild-type, HF1-13, R13CK, D13CE, E13CD, and E13CE scFv-C kappa  fusion proteins. 
       EXAMPLE 5 
     Cys-Modified Full IgG Antibody 
       [0089]    5-1. Expression and Purification of Cys-Modified Full Antibody 
         [0090]    To prepare chicken-human chimeric full IgG protein, light chain and heavy chain DNAs of the variable region of Cys-modified antibody prepared in Example 1 were obtained by PCR and inserted into an IgG expression vector, followed by expression and culture in HEK293F cells. Thereafter, full IgG antibodies were purified using protein A beads. The purified proteins were electrophoresed, and their expression and purification were confirmed by coomassie blue staining.  FIG. 5 a    shows the result of examining expression and purification of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG proteins. 
         [0091]    5-2. Expression and Purification of Cys-Modified Full IgG Antibody Having Replacement of Amino Acid Residues Adjacent to Cys 
         [0092]    To express Cys-modified antibody having replacement of amino acid residues adjacent to Cys prepared in Example 3 in the form of chicken-human chimeric full IgG antibody as in Example 5-1, the antibody gene was inserted into an IgG expression vector, and expressed and cultured in HEK293F cells. Next, full IgG antibodies were purified from the culture broth using protein A beads. The purified proteins were loaded on a polyacrylamide gel, followed by electrophoresis. Expression and purification thereof were confirmed by coomassie blue staining.  FIG. 5 b    shows the result of examining expression and purification of R13CK, E13CD, and E13CE full IgG proteins. 
       EXAMPLE 6 
     PEG-Conjugated, Modified Antibody 
       [0093]    6-1. Preparation of PEG-Cys-Modified Antibody by PEG Conjugation and Assay Thereof 
         [0094]    In accordance with a method described in the literature [Junutula et al., (2008) nature biotechnology 925-32], Cys-modified antibody prepared in Example 1 was reduced by addition of 10-fold TCEP, and then TCEP was removed. For partial oxidation, DHA was added in an amount corresponding to 2-fold of TCEP. Thereafter, maleimide PEG was added in an amount corresponding to 10-fold of the antibody, and incubated at 4° C. for 12 hours to induce conjugation of antibody with PEG.  FIG. 6  shows a cartoon depiction of conjugation of maleimide PEG to sulfhydryl antibody. 
         [0095]    Antibody-PEG was loaded on a polyacrylamide gel, and then electrophoresed. Next, the gel was subjected to coomassie or iodine staining and immunoblotting to examine conjugation of PEG to antibody. For coomassie staining, the gel was stained with coomassie brilliant blue R250, and destained with a destaining solution containing methanol and acetic acid to identify the corresponding protein.  FIG. 7 a    shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv, and  FIG. 8 a    shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG. 
         [0096]    For iodine staining, the polyacrylamide gel was washed with distilled water for 15 minutes, and then left in 5% B1Cl 2  for 15 minutes. Next, the gel was stained with 0.1 M iodine for 10 minutes, and destained with distilled water for 10 minutes to detect only PEG.  FIG. 7 b    shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv, and  FIG. 8 b    shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG. 
         [0097]    For immunoblotting, the polyacrylamide gel was separated according to the size of the protein, and then full IgG was transferred onto an NC membrane, and scFv-C kappa  fusion protein and full IgG light chain region were detected using HRP-conjugated anti-C kappa  antibody and full IgG heavy chain region was detected using HRP-conjugated anti-human Fc antibody.  FIG. 7 c    shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 scFv, and  FIG. 8 c    shows the result of wild-type, LF1-2, LF1-3, LF1-4, HF1-13, and HF1-16 full IgG. 
         [0098]    6-2. Preparation of PEG-Cys-Modified Antibody Having Replacement of Amino Acid Residues Adjacent to Cys by PEG Conjugation and Assay Thereof 
         [0099]    The antibody having replacement of cationic or anionic residue was reduced using TCEP as in Example 6-1, and partially oxidized by addition of DHA. Next, PEG was added to the antibody to induce conjugation of PEG to the antibody. To examine antibody-PEG conjugation, coomassie blue staining, iodine staining and immunoblotting were performed. 
         [0100]      FIG. 9 a    shows the result of coomassie blue staining to examine conjugation of PEG to wild-type, HF1-13, R13CK, D13CE, E13CD, or E13CE scFv, and  FIG. 10 a    shows the result of coomassie blue staining to examine conjugation of PEG to wild-type, HF1-13, R13CK, or E13CE full IgG. 
         [0101]      FIG. 9 b    shows the result of iodine staining to examine conjugation of PEG to wild-type, HF1-13, R13CK, D13CE, E13CD, or E13CE scFv, and  FIG. 10 b    shows the result of iodine staining of conjugation of PEG to HF1-13, R13CK, or E13CE full IgG. 
         [0102]      FIG. 9 c    shows the result of immunoblotting to examine conjugation of PEG to wild-type, HF1-13, R13CK, D13CE, E13CD, or E13CE scFv, and  FIG. 10 c    shows the result of immunoblotting of conjugation of PEG to HF1-13, R13CK, or E13CE full IgG. 
       EXAMPLE 7 
     Preparation of Antibody-Cotinine by Cotinine Conjugation and Assay Thereof 
       [0103]    Cys-modified antibody prepared in Example 1 was reduced by addition of 10-fold TCEP, and then TCEP was removed. For partial oxidation, DHA was added in an amount corresponding to 2-fold of TCEP. Thereafter, GMBS-cotinine was added in an amount corresponding to 10-fold of the antibody, and incubated at 4° C. for 12 hours to induce conjugation of antibody with cotinine.  FIG. 11 a    shows a cartoon depiction of conjugation of GMBS-cotinine to sulfhydryl antibody. 
         [0104]    Antibody-cotinine was assayed by enzyme-linked immunosorbent assay (ELISA) ( FIG. 11 b   ). A plate was coated with PSA antigen, and then antibody-cotinine was added thereto, followed by incubation for 1 hour. After washing three times, HRP-conjugated anti-cotinine IgG or anti-C kappa  IgG antibody was added. After incubation for 1 hour and washing three times, TMB (3,3′,5,5′-Tetramethylbenzidine) was added to examine color development, and then 2M H 2 SO 4  was used to terminate the reaction. Absorbance at 450 nm was measured. In the result of  FIG. 12 , cotinine conjugation was confirmed by anti-cotinine IgG, and anti-C kappa  IgG was used to detect only antibody, irrespective of cotinine conjugation. 
       EXAMPLE 8 
     Injection of Antibody-Cotinine into Mouse and Assay of Cotinine-Antibody 
       [0105]    6-week-old Balb/c mice were randomly divided into three groups (HF1-13, E13CD, E13CE), and then retroorbital blood collection was performed. After 24 hours, each 100 μg of 3 types of cotinine-scFv antibody C kappa  fusion proteins prepared in Example 7 was intravenously injected into mice. 1 hour after cotinine-antibody injection, retroorbital blood collection was performed, and sera were separated using a centrifuge. The separated sera were diluted 50-fold in order to examine the presence or absence of cotinine-antibody in the sera, and enzyme-linked immunosorbent assay was performed as in Example 9. All procedures using mice were performed after inhalation anesthesia in order to minimize pain.