Patent Publication Number: US-2023133381-A1

Title: Anti-dr5 antibody and use thereof

Description:
TECHNICAL FIELD 
     The present disclosure pertains to an antibody binding specifically to death receptor 5 (DR5) and having the function of killing cancer cells and, more particularly, to an anti-DR5 antibody or an antigen-binding fragment thereof, and use of the antibody or the antigen-binding fragment in preventing or treating cancer. 
     BACKGROUND ART 
     The cell apoptosis pathway through TNF-related apoptosis inducing ligand (TRAIL or Apo2L) and death receptor 5 (DR5), which is one of the receptors thereof, induces cancer cell-selective apoptosis without affecting normal cells and is regarded as an important target for the development of cancer therapeutic agents (Ashkenazi et al., Nat Rev Cancer 2: 420,2002). 
     Currently, recombinant TRAIL and death receptor-specific antibodies, which both target DR5, are developed as therapeutic agents against cancer cells. 
     As for TRAIL, the problem therewith is the low selectivity for DR5 because the ligand binds not only to DR4 (Death receptor 4, TRAIL-Receptor 1) and DR5 (Death receptor 5, TRAIL-Receptor 2), which transduce apoptotic signals, but also to DcR1 (Decoy Receptor 1, TRAIL-Receptor 3) and DcR2 (Decoy Receptor 1, TRAIL-Receptor 4), which cannot transduce apoptotic signals. In addition, recombinant TRAIL is poor in stability and has the adverse effect of inducing apoptosis in normal cells including astrocytes, hepatocytes, keratinocytes etc. (Jo et al., Nature Medicine 6, 564-567, 2000). Hence, active research has been made into the development of anti-DR5 and anti-DR4 antibodies that induces the selective apoptosis of cancer cells, with little adverse effects 
     In relation to death receptor-specific antibodies, clinical trials have been conducted to evaluate cytotoxic activity of the anti-DR5 antibody developed by Genentech Incorporated (U.S.A.) and Amgen (U.S.A.). Human Genome Sciences (U.S.A.) conducted phase II clinical trials for the anti-DR5 antibody HGS-ETR2 and the anti-DR4 antibody HGS-ETR1, but has since stopped development. In addition, development was terminated for anti-DR5 antibodies such as Apomab, Conatumumab, tigatuzumab, etc. 
     Cell apoptosis occurs largely through two major mechanisms, the extrinsic apoptosis pathway and the intrinsic apoptosis pathway. Most chemotherapeutic agents and radiotherapy induce cancer cell death via p53-mediated intrinsic apoptosis, while DR5-mediated apoptosis is induced through p53-non-dependent extrinsic apoptosis pathway and intrinsic apoptosis pathway (Takeda et al., Oncogene, 26, 3745-3757, 2007). In the extrinsic apoptosis pathway, TRAIL or antibodies bind to DR5 to form a Death-Inducing Signaling Complex (DISC), which through the adaptor protein (FADD) activates the apoptosis-initiating proteases caspase-8 and caspase-10. Then, caspase-8 activates the downstream proteases caspase-3 and caspase-7 while inducing the intrinsic apoptosis through the mitochondria (Ohtsuka et al., Oncogene, 22, 2034-2044, 2003). 
     With the growing importance of TRAIL as a target for use in cancer therapy, development of more effective and potent TRAIL-targeted agents is required. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Technical Problem 
     The present disclosure aims to provide an anti-DR5 antibody that binds specifically to death receptor 5 (DR5) to effectively kill various cancer cells, and a use thereof. 
     One aspect provides an anti-DR5 antibody or an antigen-binding fragment thereof. 
     Another aspect provides a pharmaceutical composition for prevention or treatment of cancer, the composition comprising the anti-DR5 antibody and/or the antigen-binding fragment. 
     Another aspect provides a method for preventing or treating cancer, the method comprising a step of administering the anti-DR5 antibody and/or the antigen-binding fragment to a subject in need thereof. 
     Another aspect provides a use of the anti-DR5 antibody and/or the antigen-binding fragment in preventing or treating cancer or in preparing an anticancer agent. 
     Another aspect provides a polynucleotide molecule encoding the anti-DR5 antibody or the antigen-binding fragment, a recombinant vector carrying the polynucleotide, and a recombinant cell harboring the recombinant vector. 
     Another aspect provides a method for preparing an anti-DR5 antibody or an antigen-binding fragment thereof, the method comprising a step of expressing the polynucleotide molecule. 
     Technical Solution 
     Provided are an anti-DR5 antibody that binds specifically to death receptor 5 (DR5) to effectively induce the death of various cancer cells, and a pharmaceutical composition comprising the same for prevention or treatment of cancer. 
     The antibody provided herein is a TRAIL-uncompetitive antibody that retains a constant level of antigen binding activity irrespective of TRAIL (TNF-related apoptosis inducing ligand) concentrations. Hence, the antibody can be effectively developed into innovative novel antibody drugs or diagnostic agents exhibiting superior efficacy compared to previously developed antibody drugs. 
     Death receptor 5 (DR5), also known as TRAIL receptor 2 (TRAILR2) or tumor necrosis factor receptor superfamily member 10B (TNFRSF10B), is a cell surface receptor of the TNF-receptor superfamily that binds TRAIL and mediates apoptosis by transducing an apoptosic signal. DR5 is shown to interact with Caspase 8, Caspase 10, FADD (Fas-Associated protein with Death Domain), and TRAIL. The DR5 may be derived from mammals and may be, for example, a human DR5 (e.g., NCBI accession no. UniProtKB/Swiss-Prot: Q6FH58). 
     An aspect provides a polypeptide that specifically recognizes and/or binds to DR5. The polypeptide may be one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 1 to 16. More specifically, the polypeptide may have the amino acid sequence of one selected from the group consisting of SEQ ID NOS: 1, 2, 4, and 7 to 16. 
     As used herein, the expression “a protein, polypeptide, or polynucleotide having an amino acid sequence or a nucleotide sequence” is intended to encompass all the cases in which a protein, polypeptide, or polynucleotide includes, consists essentially of, or consists of the sequence. 
     Those polypeptides may be used as complementarity determining regions (CDRs) of the anti-DR5 antibody. 
     The polypeptides and their applicable complementarity determining regions are summarized in Table 1, below: 
     
       
         
           
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                 ID 
               
               
                   
                   
                 Sequence 
                 NO 
               
               
                   
               
             
            
               
                   
                 V H -CDR1 
                 GFTFSSFNML 
                  1 
               
               
                   
               
               
                   
                 V H -CDR2 
                 GIGKSDRYTGYGSAVKG 
                  2 
               
               
                   
               
               
                   
                 V H -CDR3 
                 DAGS X1 CG X2 GGWTGACIDT 
                  3 
               
               
                   
                   
                 (X1 = G or P;  
                   
               
               
                   
                   
                 X2 = S or K) 
                   
               
               
                   
                   
                 DAGSGCGSGGWTGACIDT 
                  7 
               
               
                   
                   
                 DAGSPCGSGGWTGACIDT 
                  8 
               
               
                   
                   
                 DAGSPCGKGGWTGACIDT 
                  9 
               
               
                   
               
               
                   
                 V L -CDR1 
                 SGGDSYAGSYYYG 
                  4 
               
               
                   
               
               
                   
                 V L -CDR2 
                 NNNN X3X4X5   
                  5 
               
               
                   
                   
                 (X3 = R, L, or K; 
                   
               
               
                   
                   
                 X4 = P, M, or A;  
                   
               
               
                   
                   
                 X5 = S, P, or K) 
                   
               
               
                   
                   
                 NNNNRPS 
                 10 
               
               
                   
                   
                 NNNNLMP 
                 11 
               
               
                   
                   
                 NNNNKAK 
                 12 
               
               
                   
               
               
                   
                 V L -CDR3 
                 GSRDS X6X7X8GX9     
                  6 
               
               
                   
                   
                 (X6= S, A, or D; 
                   
               
               
                   
                   
                 X7= Y or G; 
                   
               
               
                   
                   
                 X8 = V, M, G, or A; 
                   
               
               
                   
                   
                 X9 = I, A, R, or G) 
                   
               
               
                   
                   
                 GSRDSSYVGI 
                 13 
               
               
                   
                   
                 GSRDSAGMGA 
                 14 
               
               
                   
                   
                 GSRDSDGGGR 
                 15 
               
               
                   
                   
                 GSRDSSGAGG 
                 16 
               
               
                   
               
            
           
         
       
     
     (In Table 1, V H -CDR1, V H -CDR2, and V H -CDR3 represent heavy-chain complementarity determining regions and V L -CDR1, V L -CDR2, and V L -CDR3 represent light-chain complementarity determining regions) 
     Another aspect provides a DR5-targeting polypeptide molecule comprising at least one selected from the group consisting of the polypeptides described above. The DR5-targeting polypeptide molecule has the characteristic of acting to trigger cancer cell death (e.g., apoptosis of cancer cells) without competing with the DR5 ligand TRAIL. 
     The DR5-targeting polypeptide molecule may comprise the aforementioned heavy-chain complementarity determining regions or light-chain complementarity determining regions of the anti-DR5 antibody, or a combination thereof; or a heavy-chain variable region including the heavy-chain complementarity determining region, a light-chain variable region including the light-chain complementarity determining region, or a combination thereof. 
     The DR5-targeting polypeptide molecule may function as, but is not limited to, an anti-DR5 antibody, an antigen-binding fragment of the antibody, or an anti-DR5 antibody analog (a structure having a similar scaffold and function to an antibody; e.g., peptibody, nanobody, and the like), or as a precursor or component (e.g., CDR) of a multi-specific antibody. 
     The term “peptibody”, as used herein, refers to a fusion protein (peptide+antibody) mimicking an antibody in terms of framework and function in which a peptide is fused to a partial or entire constant region, e.g., Fc, of an antibody. In this context, one or more peptides as described above may serve as an antigen-binging fragment (heavy chain and/or light chain CDR or variable region). 
     The term “nanobody,” also called a single-domain antibody, as used herein, refers to an antibody fragment which possesses a monomeric single variable domain of an antibody and shows selectivity for certain antigens, similar to a full length antibody. Its molecular weights generally ranges from about 12 kDa to about 15 kDa, which is much smaller than that (about 150 kDa to about 160 kDa) of a full length antibody (inclusive of two heavy chains and two light chains) and, in some cases, even than that of an Fab or scFv fragment. 
     As used herein, the term “multi-specific antibody” (inclusive of bispecific antibody) refers to an antibody recognizing and/or binding to two or more different antigens, or recognizing and/or binding to different sites of the same antigen, and one of the antigen binding sites of the multi-specific antibody may include the polypeptide described above. 
     One aspect provides an anti-DR5 antibody comprising as a complementarity determining region at least one polypeptide selected from the group of the polypeptides described above, or an antigen-binding fragment thereof. The anti-DR5 antibody provided herein, which has the efficacy of a DR5 agonist, acts to cluster DR5 molecules that exist separately on the cell surface, to generate and transduce an apoptotic signal within the cell, thereby inducing cell death. 
     The anti-DR5 antibody or the antigen-binding fragment thereof may comprise as a heavy-chain complementarity determining region at least one selected from the group consisting of: 
     a polypeptide having the amino acid sequence of SEQ ID NO: 1 (V H -CDR1), 
     a polypeptide having the amino acid sequence of SEQ ID NO: 2 (V H -CDR2), and 
     a polypeptide having the amino acid sequence of SEQ ID NO: 3 (V H -CDR3). 
     The amino acid sequence of SEQ ID NO: 3 is the same as that of the following General Formula 1: 
     
       
         
           
               
               
            
               
                   
                 [General Formula 1] 
               
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 D-A-G-S- X1 -C-G- X2 -G-G-W-T-G-A-C-I-D-T 
               
            
           
         
       
     
     wherein, 
     X1 is G or P, and 
     X2 is S or K. 
     In one embodiment, the polypeptide of SEQ ID NO: 3 usable as the heavy-chain CDR3 of the anti-DR5 antibody or the antigen-binding fragment thereof may have at least one selected from the group consisting of the amino acid sequences of, for example, SEQ ID NOS: 7, 8, and 9 (in the following amino acid sequences, the bold and underlined letters represent amino acid residues modified from the amino acid sequence of SEQ ID NO: 7): 
     
       
         
           
               
               
            
               
                   
                 SEQ ID NO: 7:  
               
               
                   
                 DAGSGCGSGGWTGACIDT 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 8:  
               
               
                   
                 DAGS   P   CGSGGWTGACIDT 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 9:  
               
               
                   
                 DAGS   P   CG   K   GGWTGACIDT 
               
            
           
         
       
     
     In another embodiment, the anti-DR5 antibody or the antigen-binding fragment thereof may comprise as a light-chain complementarity determining region at least one selected from the group consisting of: 
     a polypeptide having the amino acid sequence of SEQ ID NO: 4 (V L -CDR1), 
     a polypeptide having the amino acid sequence of SEQ ID NO: 5 (V L -CDR2), and 
     a polypeptide having the amino acid sequence of SEQ ID NO: 6 polypeptide(V L -CDR3). 
     The amino acid sequences of SEQ ID NOS: 5 and 6 are the same as those of the following General Formulas 2 and 3, respectively. 
     
       
         
           
               
               
            
               
                   
                 [General Formula 2]  
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 N-N-N-N- X3-X4-X5   
               
            
           
         
       
     
     wherein, 
     X3 is R, L, or K, 
     X4 is P, M, or A, and 
     X5 is S, P, or K; 
     
       
         
           
               
               
            
               
                   
                 [General Formula 3] 
               
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 G-S-R-D-S- X6-X7-X8 -G- X9   
               
            
           
         
       
     
     wherein, 
     X6 is S, A, or D, 
     X7 is Y or G, 
     X8 is V, M, G, or A, and 
     X9 is I, A, R, or G. 
     In one embodiment, the polypeptide of SEQ ID NO: 5 available as the light-chain CDR2 of the anti-DR5 antibody or the antigen-binding fragment thereof may have at least one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 10, 11, and 12 (in the following amino acid sequences, the bold and underlined letters represent amino acid residues modified from the amino acid sequence of SEQ ID NO: 10): 
     
       
         
           
               
               
            
               
                   
                 SEQ ID NO: 10:  
               
               
                   
                 NNNNRPS 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 11:  
               
               
                   
                 NNNN   LMP     
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 12:  
               
               
                   
                 NNNN   KAK     
               
            
           
         
       
     
     In one embodiment, the polypeptide of SEQ ID NO: 6 available as the light-chain CDR3 of the anti-DR5 antibody or the antigen-binding fragment thereof may have at least one selected from the group consisting of the amino acid sequences of SEQ ID NOS: 13, 14, 15, and 16 (in the following amino acid sequences, the bold and underlined letters represent amino acid residues modified from the amino acid sequence of SEQ ID NO: 13): 
     
       
         
           
               
               
            
               
                   
                 SEQ ID NO: 13:  
               
               
                   
                 GSRDSSYVGI 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 14:  
               
               
                   
                 GSRDS   AGM   G   A     
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 15:  
               
               
                   
                 GSRDS   DGG   G   R     
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 16:  
               
               
                   
                 GSRDSS   GA   G   G     
               
            
           
         
       
     
     In one embodiment, the anti-DR5 antibody or the antigen-binding fragment thereof may comprise: 
     at least one heavy-chain complementarity determining region selected from the group consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 1 (V H -CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 2 (V H -CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO: 3 (V H -CDR3), or a heavy-chain variable region including the at least one heavy-chain complementarity determining region described above; 
     at least one light-chain complementarity determining region selected from the group consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 4 (V L -CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 5(V L -CDR2), and a polypeptide having the amino acid sequence of SEQ ID NO: 6(V L -CDR3), or a light-chain variable region including the at least one light-chain complementarity determining region described above; 
     a combination of the heavy-chain complementarity determining region and the light-chain complementarity determining region described above; or 
     a combination of the heavy-chain variable region and the light-chain variable region described above. 
     More specifically, the anti-DR5 antibody or the antigen-binding fragment thereof may comprise: 
     at least one heavy-chain complementarity determining region selected from the group consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 1(V H -CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 2 (V H -CDR2), and a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 7 to SEQ ID NO: 9 (V H -CDR3), or a heavy-chain variable region including the at least one heavy-chain complementarity determining region described above; 
     at least one light-chain complementarity determining region selected from the group consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 4(V L -CDR1), a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 12 (V L -CDR2), and a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 16 (V L -CDR3), or a light-chain variable region including the at least one light-chain complementarity determining region described above; 
     a combination of the heavy-chain complementarity determining region and the light-chain complementarity determining region described above; or 
     a combination of the heavy-chain variable region and the light-chain variable region described above. 
     In one embodiment, the anti-DR5 antibody or the antigen-binding fragment may comprise: 
     a heavy-chain variable region comprising a polypeptide having the amino acid sequence of SEQ ID NO: 1(V H -CDR1), a polypeptide having the amino acid sequence of SEQ ID NO: 2(V H -CDR2), and a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 7 to SEQ ID NO: 9 (V H -CDR3); and 
     a light-chain variable region comprising a polypeptide having the amino acid sequence of SEQ ID NO: 4 (V L -CDR1), a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 10 to SEQ ID NO: 12 (V L -CDR2), and a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 16 (V L -CDR3). 
     In one embodiment, the anti-DR5 antibody or the antigen-binding fragment thereof may comprise a combination of the heavy-chain variable regions and light-chain variable regions given in Tables 2 and 3 below. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Sequences of Heavy Chain Complementarity- 
               
               
                 Determining Region (CDR) 
               
            
           
           
               
               
               
               
               
               
            
               
                 SEQ 
                   
                 SEQ 
                   
                 SEQ 
                   
               
               
                 ID 
                   
                 ID 
                   
                 ID 
                   
               
               
                 NO 
                 V H -CDR1 
                 NO 
                 V H -CDR2 
                 NO 
                 V H -CDR3 
               
               
                   
               
               
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGWT 
               
               
                   
                 NML 
                   
                 GSAVKG 
                   
                 GACIDT 
               
               
                   
               
               
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 8 
                 DAGSPCGSGGWT 
               
               
                   
                 NML 
                   
                 GSAVKG 
                   
                 GACIDT 
               
               
                   
               
               
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 9 
                 DAGSPCGKGGWT 
               
               
                   
                 NML 
                   
                 GSAVKG 
                   
                 GACIDT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Sequences of Light Chain CDR 
               
            
           
           
               
               
               
               
               
               
            
               
                 SEQ ID 
                   
                 SEQ ID 
                   
                 SEQ ID 
                   
               
               
                 NO 
                 V L -CDR1 
                 NO 
                 V L -CDR2 
                 NO 
                 V L -CDR3 
               
               
                   
               
               
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 4 
                 SGGDSYAGSY 
                 11 
                 NNNNL 
                 13 
                 GSRDSSY 
               
               
                   
                 YYG 
                   
                 MP 
                   
                 VGI 
               
               
                   
               
               
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 14 
                 GSRDSAG 
               
               
                   
                 YYG 
                   
                 PS 
                   
                 MGA 
               
               
                   
               
               
                 4 
                 SGGDSYAGSY 
                 12 
                 NNNNK 
                 13 
                 GSRDSSY 
               
               
                   
                 YYG 
                   
                 AK 
                   
                 VGI 
               
               
                   
               
               
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 15 
                 GSRDSDG 
               
               
                   
                 YYG 
                   
                 PS 
                   
                 GGR 
               
               
                   
               
               
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 16 
                 GSRDSSG 
               
               
                   
                 YYG 
                   
                 PS 
                   
                 AGG 
               
               
                   
               
            
           
         
       
     
     Provided in another aspect is a heavy-chain variable region comprising the heavy-chain complementarity determining region, a light-chain variable region comprising the light-chain complementarity determining region, or a combination thereof. For example, the heavy-chain variable region may comprise the amino acid sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 37 and the light-chain variable region may comprise the amino acid sequence selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 49. 
     Therefore, the anti-DR5 antibody or the antigen-binding fragment thereof may comprise: 
     a heavy-chain variable region having the amino acid sequence selected from the group consisting of SEQ ID NO: 17 to SEQ ID NO: 37; 
     a light-chain variable region having the amino acid sequence selected from the group consisting of SEQ ID NO: 38 to SEQ ID NO: 49; or 
     a combination thereof. 
     In an embodiment, the anti-DR5 antibody may be an animal-derived antibody (e.g., mouse-derived antibody), a chimeric antibody (e.g., mouse-human chimeric antibody), or a humanized antibody. The antibody or antigen-binding fragment may be isolated from a living body or non-naturally occurring. The antibody or antigen-binding fragment may be recombinant or synthetic. 
     In another embodiment, the antibody may be derived (isolated) from any animal, such as mammals including humans, birds, etc. For example, the antibody may be a human antibody, a mouse antibody, a donkey antibody, a sheep antibody, a rabbit antibody, a goat antibody, a guinea pig antibody, a camel antibody, a horse antibody, or a chicken antibody. Herein, a human antibody is an antibody having an amino acid sequence of human immunoglobulin and includes an antibody isolated from a library of human immunoglobulins or from an animal that has been transgenic for at least one human immunoglobulin and does not include endogenous immunoglobulins. 
     The anti-DR5 antibody may be monoclonal or polyclonal and may be, for example, a monoclonal antibody. A monoclonal antibody can be prepared using a method widely known in the art, for example, using a phage display technique. Alternatively, the anti-DR5 antibody may be constructed in the form of a mouse-derived monoclonal antibody. 
     Except for the heavy-chain CDR and light-chain CDR portions or the heavy-chain variable and light-chain variable regions as defined above, the anti-DR5 antibody or the antigen-binding fragment thereof may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like), for example, from the framework portion, and/or light-chain constant region and/or heavy-chain constant region. 
     A full length antibody (e.g., IgG type) has a structure with two full-length light chains and two full-length heavy chains, in which each light chain is linked to a corresponding heavy chain via a disulfide bond. The constant region of an antibody is divided into a heavy-chain constant region and a light-chain constant region, and the heavy-chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) type and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2) as its subclass. The light chain constant region is of either a kappa (κ) or lambda (λ) type. 
     As used herein, the term “heavy chain” is intended to encompass a full-length heavy chains and fragments thereof, the full-length heavy chain comprising a variable region V H  inclusive of amino acid sequences sufficient to provide specificity to antigens, three constant regions, C H1 , C H2 , and C H3 , and a hinge. The term “light chain” is intended to encompass full-length light chains and fragments thereof, the full-length light chain comprising a variable region V L  inclusive of amino acid sequences sufficient to provide specificity to antigens, and a constant region C L . 
     The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. As used herein, the terms “specifically binding” and “specifically recognizing” have the same general meaning as known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological reaction. 
     The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including a portion of a polypeptide accounting for an antigen-binding site. The antigen-binding fragment may be scFv, (scFv) 2 , scFvFc, Fab, Fab′, or F(ab′) 2 , but is not limited thereto. 
     Among the antigen-binding fragments, Fab, which includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region C H1 , has one antigen-binding site. 
     Fab′ is different from Fab in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of C H1 . 
     An F(ab′) 2  antibody is formed through disulfide bridging of the cysteine residues in the hinge region of Fab′. Fv is a minimal antibody fragment composed of only a heavy chain variable region and a light chain variable region. Recombination techniques of generating an Fv fragment are widely known in the art. 
     Two-chain Fv includes a heavy chain variable region and a light chain region which are linked to each other by a non-covalent bond. Single-chain Fv generally includes a heavy-chain variable region and a light-chain variable region which are linked to each other by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. 
     The antigen-binding fragments may be obtained using protease (for example, Fab may be obtained by restrictively cleaving a whole antibody with papain, and an F(ab′) 2  fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique. 
     The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody, which functions to provide flexibility for the antigen-binding site. 
     Another aspect provides a pharmaceutical composition comprising the anti-DR5 antibody or the antigen-binding fragment thereof as an effective ingredient for prevention and/or treatment of cancer. 
     Another aspect provides a method for prevention and/or treatment of cancer, the method comprising a step of administering a pharmaceutically effective amount of the anti-DR5 antibody or the antigen-binding fragment thereof to a subject in need thereof. The method for prevention and/or treatment of cancer may further comprise a step of identifying a patient in need of prevention and/or treatment of cancer (for example, diagnosing or selecting a subject to be treated) prior to the administering step. 
     The anti-DR5 antibody can be usefully applied to the prevention or treatment of cancer. Because, as described above, the anti-DR5 antibody provided herein functions as a DR5 agonist, it may be advantageous that the cancer cells express DR5 in order for the anti-DR5 antibody to exert sufficient efficacy (e.g., anticancer efficacy such as cancer cell death) when applied thereto. In addition, the cancer may be a TRAIL-sensitive cancer or a TRAIL-resistant cancer. In one embodiment, concrete examples of the cancer include blood cancer, lung cancer, stomach cancer, liver cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin melanoma, uterine cancer, ovarian cancer, rectal cancer, colorectal cancer, colon cancer, breast cancer, uterine sarcoma, fallopian tube carcinoma, endometrial carcinoma, uterine cervical carcinoma, vaginal carcinoma, vulva carcinoma, esophageal cancer, laryngeal cancer, small-intestine cancer, thyroid cancer, parathyroid cancer, soft-tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, solid tumors in juvenile stage, differentiated lymphoma, bladder cancer, renal cancer, renal cell carcinoma, renal pelvic carcinoma, primary central nervous system lymphoma, spinal axis tumors, brain stem glioma, and pituitary adenoma, but are not limited thereto. 
     In one embodiment, the use of the anti-DR5 antibody or the antigen-binding fragment thereof in combination of TRAIL brings about a synergistic effect, compared to the use of the anti-DR5 antibody or the antigen-binding fragment thereof alone, and elicits an anticancer effect on TRAIL-resistant cancer as well as TRAIL-sensitive cancer (see Example 13). 
     Therefore, the pharmaceutical composition may comprise TRAIL (TNF-related apoptosis inducing ligand) in addition to the anti-DR5 antibody or the antigen-binding fragment thereof. 
     That is, the pharmaceutical composition may comprise, as effective ingredients, 
     (1) the anti-DR5 antibody or the antigen-binding fragment thereof; and 
     (2) TRAIL. 
     The pharmaceutical composition may comprise (1) the anti-DR5 antibody or the antigen-binding fragment thereof and (2) TRAIL in one consolidated formulation or in respective formulations. 
     In addition, the method for prevention and/or treatment of cancer may comprise a step of administering a pharmaceutically effective amount of TRAIL in addition to the step of administering the anti-DR5 antibody or the antigen-binding fragment thereof. 
     That is, the method for prevention and/or treatment of cancer may comprise a step of administering (1) a pharmaceutically effective amount of the anti-DR5 antibody or the antigen-binding fragment thereof and (2) a pharmaceutically effective amount of TRAIL, in combination, to a patient in need of prevention and/or treatment of cancer. 
     The combined-administration step may be conducted by administering (1) a pharmaceutically effective amount of the anti-DR5 antibody or the antigen-binding fragment thereof and (2) a pharmaceutically effective amount of TRAIL, as formulated into one dosage form, simultaneously, or (1) a pharmaceutically effective amount of the anti-DR5 antibody or the antigen-binding fragment thereof and (2) a pharmaceutically effective amount of TRAIL, as formulated into respective dosage forms, simultaneously or sequentially without regard for the order thereof. 
     TRAIL (TNF-related apoptosis-inducing ligand), also designated CD253 (cluster of differentiation 253) or TNFSF10 (tumor necrosis factor (ligand) superfamily, member 10), is a protein functioning as a ligand that induces the process of cell death (apoptosis). TRAIL is a cytokine that is widely produced and secreted by normal tissue cells. In one embodiment, the TRAIL may be human-derived and may be represented by, for example, NCBI Accession number NP_001177871.1, NP_003801.1, etc., but is not limited thereto. 
     The anti-DR5 antibody or the antigen-binding fragment thereof (or in combination with TRAIL), which takes advantage of the apoptosis pathway, may be used in combination with at least one chemotherapy agent, such as carboplatin or paclitaxel (Recka et al., Lung Cancer, 82, 441-448, 2013), gemcitabine (Torres et al., Cancer Medicine, 2(6), 925-932, 2013), etc., thereby enhancing an anticancer/anti-tumor effect (see Example 15). 
     In order to exert prophylactic and/or therapeutic effects on cancer, the anti-DR5 antibody or the antigen-binding fragment thereof, or a pharmaceutical composition comprising the same may be administered alone or in combination with surgery, hormone therapy, pharmacotherapy, and/or a biological response modifier. 
     In one embodiment, the pharmaceutical composition may comprise at least one well-known effective ingredient having an anticancer effect (chemotherapy medication) in addition to the anti-DR5 antibody or the antigen-binding fragment thereof (or in combination with TRAIL). Furthermore, the method for prevention and/or treatment of cancer may comprise a step of administering at least one well-known effective ingredient having an anticancer effect (chemotherapy medication) in addition to the anti-DR5 antibody or the antigen-binding fragment thereof (or in combination with TRAIL). 
     The chemotherapy medication that may be used with the anti-DR5 antibody or the antigen-binding fragment thereof may be at least one selected from the group consisting of: alkylating anticancer agents, such as carboplatin, paclitaxel (Recka et al., Lung Cancer, 82, 441-448, 2013), etc.; metabolism antagonist-based anticancer agents, such as gemcitabine (Torres et al., Cancer Medicine, 2(6), 925-932, 2013), etc.; anthracycline-based anticancer agents, such as doxorubicin (H M Amm et al., Mol Cancer Res April, 9; 403, 2011), etc.; and proteasome inhibitor-based anticancer agents, such as bortezomib (Shanker A et al., J Natl Cancer Inst, May 7; 100(9): 649-62, 2008), etc., but is not limited thereto. 
     For proper administration of the effective ingredients such as the anti-DR5 antibody, the antigen-binding agent, and the like, at least one pharmaceutically acceptable carrier may be included in the pharmaceutical composition or may be administered along with the effective ingredients. Examples of pharmaceutically acceptable carriers may include saline, sterilized water, Ringer&#39;s solution, buffered saline, a dextrose solution, a maltodextrin solution, glycerol, ethanol and a mixture of one or more components thereof. Other conventional additive such as antioxidants, buffers, or bacteriostats may be further added, as necessary. In addition, the pharmaceutical composition may be formulated into injectable dosage forms, such as an aqueous solution, suspension or emulsion, or into a pill, a capsule, a granule, or a tablet by adding a diluent, a dispersant, a surfactant, a binder, or a lubricant. Furthermore, the pharmaceutical composition may be properly formulated according to diseases or components, using a method pertinent to the art or disclosed in Remington&#39;s Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, Pa. 
     The effective ingredient such as the anti-DR5 antibody, the antigen-binding fragment, etc. or the pharmaceutical composition may be administered orally or parenterally. For parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and intrarectal administration may be conducted. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration may be coated or formulated to prevent digestion in the stomach. In addition, the composition may be administered using an optional device that enables an active ingredient to be delivered to target cells. 
     The pharmaceutically effective amount of the anti-DR5 antibody or the antigen-binding fragment thereof may be prescribed in various amounts, depending on factors such as preparation (formulation) methods, method of administration, the patient&#39;s age, body weight, gender, pathologic conditions and diet, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, a daily dosage of the anti-DR5 antibody or the antigen-binding fragment thereof may be within the range of 0.001 to 1000 mg/kg, particularly 0.01 to 100 mg/kg, more particularly 0.1 to 50 mg/kg, and even more particularly 0.1 to 20 mg/kg, but is not limited thereto. The daily dosage may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container. The pharmaceutical composition may be administered in combination with other medications, and proper prescriptions may be made on the dose, the administration method, and kinds of the other medications, depending on patients&#39; states. 
     The pharmaceutical composition may be formulated into a form of a solution in oil or an aqueous medium, a suspension, syrup, an emulsion, an extract, powder, granules, a tablet, or a capsule, and may further include a dispersing or a stabilizing agent for formulation. 
     Particularly, the pharmaceutical composition comprising the anti-DR5 antibody or the antigen-binding fragment thereof may be formulated into an immunoliposome since it contains an antibody or an antigen-binding fragment. An antibody-containing liposome may be prepared using any of the methods widely known in the art. The immunoliposome may be a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction. 
     Meanwhile, as the anti-DR5 antibody or the antigen-binding fragment thereof specifically binds to DR5, the antibody or fragment can be used to detect DR5 (i.e., the presence and/or level (concentration)) and/or to diagnose DR5-related diseases (i.e., the presence or changes in DR5 levels (for diseases that show increased or diseased levels relative to normal state)). 
     Accordingly, one embodiment envisages a DR5 detection composition comprising the anti-DR5 antibody or the antigen-binding fragment thereof. Another aspect provides a DR5 detection method comprising the steps of: treating a biological sample with the anti-DR5 antibody or the antigen-binding fragment thereof; and identifying the presence of an antigen-antibody reaction. If an antigen-antibody reaction is identified, the biological sample can be determined to contain DR5, and the level (concentration) of DR5 may be measured by measuring the extent of the antigen-antibody reaction in the biological sample. 
     The biological sample may be selected from the group consisting of cells, tissues, and body fluids obtained from patients (e.g., mammals such as humans), and cultures thereof. A normal sample may be selected from the group consisting of cells, tissues, and body fluids obtained from normal subjects (e g , mammals such as humans) not suffering from a DR5-related disease, and cultures thereof. 
     The step of identifying the presence of an antigen-antibody reaction or the step of measuring an antigen-antibody reaction can be carried out using various methods known in the art. By way of example, an antigen-antibody reaction may be detected through an ordinary enzyme reaction, fluorescence, luminescence, and/or radioactivity detection, and particularly may be measured by a method selected from the group consisting of immunochromatography, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), western blotting, etc., but is not limited thereto. 
     Another aspect provides: 
     a polynucleotide coding for the heavy-chain complementarity determining region, a polynucleotide coding for the light-chain complementarity determining region, or a combination thereof; a polynucleotide coding for the heavy-chain variable region, a polynucleotide coding for the light-chain variable region, or a combination thereof; or a polynucleotide coding for the heavy chain, a polynucleotide coding for the light chain, or a combination thereof, wherein the complementarity determining regions, the heavy- and light-chain variable regions, and the heavy and light chains are as described for the anti-DR5 antibody above; 
     a recombinant vector carrying the polynucleotides or a combination thereof; and 
     a recombinant cell harboring the recombinant vector. 
     In one embodiment, the recombinant vector described above may contain polynucleotides coding respectively for a heavy-chain complementarity determining region and a light-chain complementarity determining region; for a heavy-chain variable region and a light-chain variable region; or for a heavy chain and a light chain in the anti-DR5 antibody, in a single vector or in separate vectors carrying each of the polynucleotides. 
     The term “vector” refers to a means for expressing a target gene in a host cell, as exemplified by a plasmid vector, a cosmid vector, and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The recombinant vector may be constructed from plasmids frequently used in the art (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, μME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), phages (for example, λgt4λB, λ-Charon, λΔz1, and M13) or by manipulating viruses (for example, SV40, etc.). 
     In the recombinant vector, the polynucleotide may be operatively linked to a promoter. The term “operatively linked” is intended to pertain to a functional linkage between a nucleotide sequence of interest and an expression regulatory sequence (for example, a promoter sequence). When being “operatively linked”, the regulatory element can control the transcription and/or translation of the nucleotide of interest. 
     The recombinant vector may be constructed typically as a cloning vector or an expression vector. For recombinant expression vectors, a vector generally available in the art for expressing a foreign protein in plant, animal, or microbial cells may be employed. Various methods well known in the art may be used for the construction of recombinant vectors. 
     For use in hosts, such as prokaryotic or eukaryotic cells, the recombinant vector may be constructed accordingly. For example, when a vector is constructed as an expression vector for use in a prokaryotic host, the vector typically includes a strong promoter for transcription (e.g., a pLκλ promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, a T7 promoter, etc.), a ribosomal binding site for initiating translation, and transcriptional/translational termination sequences. On the other hand, an expression vector for use in a eukaryotic host includes an origin of replication operable in a eukaryotic cell, such as an f1 origin of replication, an SV40 origin of replication, a μMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, and a BBV origin of replication, but is not limited thereto. In addition, the expression vector typically includes a promoter derived from genomes of mammalian cells (for example, metallothionein promoter) or from mammalian viruses (for example, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV), and a polyadenylation sequence as a transcription termination sequence. 
     The recombinant cell may be prepared by introducing the recombinant vector into a suitable host cell. As long as it allows the sequential cloning and expression of the recombinant vector in a stable manner, any host cell known in the art may be employed in the present disclosure. Examples of the prokaryotic host cell available for the present disclosure include  E. coli, Bacillus  spp. such as  Bacillus subtilis  and  Bacillus thuringiensis,  and enterobacteriaceae strains such as  Salmonella typhimurium, Serratia marcescens  and various  Pseudomonas  species. Eukaryotic host cells that may be used for transformation may include, but are not limited to,  Saccharomyce cerevisiae,  insect cells, and animal cells, such as Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK. 
     The nucleic acid molecule or a recombinant vector carrying the same may be introduced (transfected) into a host cell using a method well known in the art. This transfection may be carried out using a CaCl2 or electroporation method when the host cell is prokaryotic. For eukaryotic host cells, the genetic introduction may be achieved using, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or particle bombardment. 
     To select a transformed host cell, advantage may be taken of a phenotype associated with a selection marker according to methods well known in the art. For example, when the selection marker is a gene conferring resistance to a certain antibiotic, the host cells may be grown in the presence of the antibiotic in a medium to select a transformant of interest. 
     Another aspect provides a method for production of an anti-DR5 antibody or an antigen-binding fragment thereof, the method comprising a step of expressing the polynucleotide or the recombinant vector in a pertinent host cell. In one embodiment, the production method may comprise culturing a recombinant cell harboring the polynucleotide or the recombinant vector thereat, and optionally isolating and/or purifying the antibody from the culture medium. 
     Advantageous Effects 
     The present disclosure provides an anti-DR5 antibody for the targeted therapy of DR5-expressing diseases. The antibody of the present disclosure is a TRAIL-uncompetitive antibody that retains a constant level of antigen binding affinity irrespective of TRAIL concentrations. Hence, the antibody can be effectively developed into innovative novel antibody drugs or diagnostic agents exhibiting superior efficacy compared to previously developed antibody drugs. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1    is an association and dissociation sensorgram depicting association and dissociation rates of the antibody D0. 
         FIGS.  2   a  to  2   f    are graphs comparing antibodies D0 and DA1 to DA35 with the control antibody AP with regard to binding activity for DR5. 
         FIGS.  3   a  and  3   b    exhibit SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results of representative antibodies according to the present disclosure. 
         FIGS.  4   a  to  4   h    are size exclusion chromatograms (SEC) of representative antibodies according to the present disclosure. 
         FIG.  5    is shows the pharmacokinetic profile of the antibodies according to the present disclosure. 
         FIGS.  6   a  to  6   r    demonstrate that the antibodies according to the present disclosure have cytotoxic activity in tumor cells. 
         FIGS.  7   a  and  7   b    show the results of tumor growth inhibition of the antibodies according to the present disclosure in xenograft animal models. 
         FIGS.  8   a  and  8   b    demonstrate that the antibodies according to the present disclosure do not compete with TRAIL for binding to DR5. 
         FIGS.  9   a  and  9   b    are graphs delineating synergistic effects of the antibodies according to the present disclosure and TRAIL in assays for inducing cell death. 
         FIGS.  10   a  to  10   c    are graphs demonstrating that the antibodies according to the present disclosure induce cell death via apoptosis. 
         FIGS.  11   a  and  11   b    show the cytotoxic activity of the antibodies according to the present disclosure when used in combination with certain concentrations of gemcitabine. 
     
    
    
     MODE FOR CARRYING OUT THE INVENTION 
     Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples. 
     EXAMPLE 1 
     DR5 Immunization and cDNA Library Construction 
     In order to select antibodies binding specifically to DR5, animal-immunized antibody libraries were constructed. The libraries were constructed by obtaining mRNA from the immune cells of animals immunized with an antigen, amplifying an antibody gene through PCR using a combination of primers for the antibody gene, and cloning the antibody gene into phage display vectors. 
     Briefly, human DR5 (R&amp;D systems, U.S.A.) in mixture with complete Freund&#39;s adjuvant and incomplete Freund&#39;s adjuvant (Sigma, U.S.A.) was subcutaneously injected to three White Leghorn chickens five times at regular intervals of three weeks. Sera from the immunized animals were diluted at a concentration of 1:100, 1:500, 1:2500, and 1:12500 in PBSB (3% bovine serum albumin in phosphate buffered saline) before being stored. Enzyme-linked immunosorbent assay was conducted to examine whether the sera bound human DR5. ELISA plates were coated overnight with 1 μg/ml human DR5 (R&amp;D systems, U.S.A.) at 4° C., followed by reaction with the diluted sera for 2 hours. The plates were washed three times with PB ST (0.1% Tween-20 in PBS) and then incubated with anti-chicken immunoglobulin-HRP (horse radish peroxidase) (1:3000) for one hour. After three rounds of washing with PBST, color development was made for 20 min with ABTS (Thermo, U.S.A.). Absorbance at 405 nm was read on a microplate reader. Sera before immunization did not bind to DR5. Animals that produced sera with strong binding to human DR5 were selected. 
     Five days after the last injection, the bone marrow, spleens, and bursa of Fabricius were collected from the selected chicken. The tissues were mixed with 10 ml of TRI reagent (Molecular research center, U.S.A.) and homogenized with a homogenizer. Addition of 20 ml of TRI reagent was followed by centrifugation. The supernatant thus obtained was mixed with 3 ml of 1-bromo-3-chloropropane (BCP) and centrifuged to obtain supernatant. Total RNA was precipitated by addition of 15 ml of isopropanol. Reverse transcription was conducted using the SuperScript Transcription System (Invitrogen, U.S.A.) with random hexamers as primers (5 min at 65° C.; 5 min at 4° C.; 50 min at 50° C.; 5 min aft 85° C.; and 4° C.). 5 ul (microliters) of the reverse transcription reaction mixture containing the resulting cDNA was loaded onto 1% agarose gel and electrophoresed to detect bands of cDNA of various lengths. 
     EXAMPLE 2 
     Construction of Antibody Library 
     (2-1) Amplification of Immune Antibody Gene 
     In order to amplify the heavy- and light-chain variable regions V H  and V L  of the chicken antibody, PCR was performed as follows. For PCR, the cDNA prepared in Example 1 served as a template and combinations of primers designed for heavy-chain variable regions, light-chain variable regions, and scFv (single chain Fv) linking the heavy- and light-chain variable regions were used as shown in Table 4 below. 0.5 μl of each of the V H  and V L  cDNA libraries, 30 pmoles of the forward primer, 30 pmoles of the reverse primer, 10× PCR buffer, 200 μM dNTPs, and 0.5 μl Taq DNA polymerase was mixed and adjusted to a final volume of 50 pi and subjected to PCR starting with denaturation at 94° C. for 5 min, followed by 30 cycles of 94° C. for 15 sec, 56° C. for 30 sec, and 72° C. for 90 sec. PCR-amplified antibody DNA was separated according to size by 1% agarose gel electrophoresis and purified using a gel extraction kit (Qiagen, U.S.A.). 
     For obtaining scFv DNA, 50 ng of each of the purified V H  and V L  DNAs was used as a template and mixed with 30 pmoles of the forward primer, 30 pmoles of the reverse primers, 10× PCR buffer, 200 μM dNTPs, and 0.5 μl Taq DNA polymerase to a final volume of 50 μl. PCR was conducted by denaturation at 94° C. for 5 min, followed by 20 cycles of 94° C. for 30 sec, 56° C. for 30, and 72° C. for 2 min The PCR-amplified DNA was separated according to size on a 1% agarose gel electrophoresis and purified using a gel extraction kit (Qiagen, U.S.A.). 
     The primers used in the PCR are summarized in Table 4 below. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Primers used in PCR 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                 ID 
               
               
                   
                 Primer 
                 Sequence 
                 NO 
               
               
                   
               
               
                 V H   
                 Forward 
                 GGT CAG TCC TCT AGA TCT TCC GGC GGT 
                 50 
               
               
                   
                   
                 GGTcGGC AGC TCC GGT GGT GGC GGT TCC 
                   
               
               
                   
                   
                 GCC GTGcACG TTG GAC GAG 
                   
               
               
                   
                 Reverse 
                 CTG GCC GGC CTG GCC ACT AGT GGA GGA 
                 51 
               
               
                   
                   
                 GACcGAT GAC TTC GGT CC 
                   
               
               
                   
               
               
                 V L   
                 Forward 
                 GTG GCC CAG GCG GCC CTG ACT CAG CCG 
                 52 
               
               
                   
                   
                 TCCcTCG GTG TC 
                   
               
               
                   
                 Reverse 
                 GGA AGA TCT AGA GGA CTG ACC TAG GAC 
                 53 
               
               
                   
                   
                 GGTcCAGG 
                   
               
               
                   
               
               
                 scFV 
                 Forward 
                 GAG GAG GAG GAG GAG GAG GTG GCC CAG 
                 54 
               
               
                   
                   
                 GCG GCC CTG ACT CAG 
                   
               
               
                   
                 Reverse 
                 GAG GAG GAG GAG GAG GAG GAG CTG GCC 
                 55 
               
               
                   
                   
                 GGC CTG GCC ACT AGT GGA GG 
               
               
                   
               
            
           
         
       
     
     (2-2) Restriction Enzyme Digestion of Antibody DNA 
     The scFV prepared above and the phagemid vector pComb3× (the Scripps Research Institute, CA, U.S.A.) were digested with the restriction enzyme SfiI (Roche, U.S.A.). A mixture of 10 μg of the scFv-encoding PCR fragment, 360 units SfiI (Roche, U.S.A.), and 20 μl of 10× buffer was volumetrically adjusted to have a final volume of 200 μl and allowed to react overnight at 50° C. In addition, a mixture of 20 μg of the pComb3× vector, 120 units SfiI, and 20 μl of 10× buffer was volumetrically adjusted to 200 μl and reacted overnight at 50° C. Each of the resulting digests was electrophoresed on agarose gel and purified using a gel extraction kit (Qiagen, U.S.A.). 
     (2-3) Ligation of Antibody DNA and Library Construction 
     In order to insert scFv fragments into pComb3×, a mixture of 700 ng of the scFV-encoding PCR fragments digested with restriction enzyme SfiI in (2-2) and 1.4 μg of pComb3× was reacted overnight at 16° C. in the presence of T4 DNA ligase (Invitrogen, U.S.A.). The ligation mixture thus obtained was purified by ethanol precipitation and transformed into  E. coli ER 2738(New England Biolab, U.S.A.) by electroporation. The  E. coli were cultured in the presence of  46 μg/ml carbenicillin and 70 ug/ml kanamycin to construct a library having a complexity of 5×10 9 . 
     EXAMPLE 3 
     Selection of Phage Clone Carrying Anti-DR5 scFv 
     From the library obtained in Example 2, having randomized heavy and light chains in the form of scFV, antibodies binding to human DR5 were selected using solid phase immobilized DR5. 
     (3-1) Selection of Antibody Binding to DR5 
     First, 10 μg of human DR5 (R&amp;D systems, U.S.A.) was conjugated to magnetic beads. An antibody DNA library was constructed by fusing the scFv-type antibodies obtained in Example 2 to phage coat protein PIII to enable expression of the antibodies on the phage surface. After being transformed with the antibody library DNA by electroporation,  E. coli ER 2738(New England Biolab) was cultured at 37° C. and then incubated overnight with VCSM13 helper phage (Stratagene, U.S.A.) in the presence of 46 μg/ml carbenicillin and 70 μg/ml kanamycin in SB medium (30 g/L Tryptone, 20 g/L yeast extract, and 10 g/L MOPS, pH 7.0). The resulting culture broth containing  E. coli  and phages was centrifuged to precipitate and remove  E. coli.  The supernatant was recovered and centrifuged after addition of 40 mg/ml polyethylene glycol 8000 and 30 mg/ml NaCl. The PEG precipitated phages were collected and resuspended in PBS. The phages were reacted with human DR5 conjugated to magnetic beads at room temperature for 2 hours to capture phages having affinity for DR5. Thereafter, the beads were washed with 0.5% Tween 20 in PBS and the bound phages were eluted with 0.1M glycine (pH 2.2) and neutralized with 2M Tris. The eluted phages were allowed to infect  E. coli  ER2738 and cultured overnight for the next round of panning This panning procedure was repeated four times. The repeated rounds of panning resulted in the accumulation of phages with high binding affinity. Individual clones selected from the plates of the fourth panning were incubated overnight with VCSM13 helper phage (1:1000) at 37° C. in the presence of 100 μg/ml carbenicillin and 70 μg/ml kanamycin in 96-deep well plates to induce the amplification of phages which express the antibody. After centrifugation of the resulting culture broth, the phages in the supernatant were pre-bound with TRAIL and then plated into DR5-coated ELISA plates. Incubation at 37° C. for 2 hours was followed by ELISA using an HRP-conjugated anti-M13 antibody to identify DR5-binding antibodies. 
     (3-2) Sequencing of Selected Antibody 
       E. coli  ER2738 that were shown to harbor DR5-reactive clones under the selection conditions of Example (3-1) were cultured overnight in SB medium and harvested by centrifugation. Plasmid DNA was prepared using a DNA min-prep kit (GeneAll, Korea) and sequenced. For sequencing, the sequencing primers given in Table 5 below were used. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Primers used in PCR 
               
            
           
           
               
               
               
            
               
                 Primer 
                 Sequence 
                 SEQ ID NO 
               
               
                   
               
               
                 Forward 
                 ACA CTT TAT GCT TCC GGC TC 
                 56 
               
               
                   
               
               
                 Reverse 
                 CAA AAT CAC CGG AAC CAG AG 
                 57 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 4 
     Antibody Optimization—Humanization and Affinity Improvement 
     Among the antibody clones obtained from the animal immunized antibody libraries, a parental clone D0 (chimeric antibody) with high affinity and activity was selected. The framework of the antibody was switched to a human antibody framework (Nishibori et al.,  Molecular Immunology,  43 (2006)). For affinity improvement, a new phage library was constructed with random mutations in the CDR sequences of the heavy and light chain regions. The phage library was obtained in the same manner as in Example 2, reacted for 2 hours at room temperature with 10 μg of human DR5 immobilized onto magnetic beads, and washed five times with 0.5% Tween 20 in PBS. Thereafter, the bound phages were eluted with 0.1 M glycine (pH 2.2) and then neutralized with 2M Tris solution. To increase the selection pressure, 1 μg of the human DR5 was immobilized to magnetic beads and washed 10 times with Tween 20 in PBS in the second round of panning, while 0.1 μg of human DR5 was immobilized and washed 20 times with Tween 20 in PBS in the third round of panning In addition to the above humanization and affinity improvement methods, deimmunized variants were also constructed by substituting amino acids in the variable region framework sequences of the parental clone s that were predicted to have a high immunogenicity in immune cells. The humanized or deimmunized variants of the parental clone obtained as described above were designated “DA1 to DA35”. 
     Sequences of the complementarity determining regions and variable regions obtained from D0 and DA1 to DA35 clones are summarized in Tables 6 to 9 below: 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Sequences of Heavy Chain CDRs 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 SEQ 
                   
                 SEQ 
                   
                 SEQ 
                   
               
               
                 Clone 
                 ID 
                   
                 ID 
                   
                 ID 
                   
               
               
                 no. 
                 NO 
                 V H -CDR1 
                 NO 
                 V H -CDR2 
                 NO 
                 V H -CDR3 
               
               
                   
               
               
                 D0 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA1 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA2 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA3 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA4 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA5 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA6 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA7 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA8 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA9 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA10 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA11 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA12 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA13 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA14 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA15 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA16 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA17 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA18 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA19 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA20 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA21 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA22 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 8 
                 DAGSPCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA23 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 9 
                 DAGSPCGKGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA24 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA25 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA26 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA27 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA28 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA29 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA30 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA31 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA32 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA33 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA34 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
               
                 DA35 
                 1 
                 GFTFSSF 
                 2 
                 GIGKSDRYTGY 
                 7 
                 DAGSGCGSGGW 
               
               
                   
                   
                 NML 
                   
                 GSAVKG 
                   
                 TGACIDT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Sequences of Light Chain CDRs 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 SEQ 
                   
                 SEQ 
                   
                 SEQ 
                   
               
               
                 Clone 
                 ID 
                   
                 ID 
                   
                 ID 
                   
               
               
                 no. 
                 NO 
                 V L -CDR1 
                 NO 
                 V L -CDR2 
                 NO 
                 V L -CDR3 
               
               
                   
               
               
                 D0 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA1 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA2 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA3 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA4 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA5 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA6 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA7 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA8 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA9 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA10 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA11 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA12 
                 4 
                 SGGDSYAGSY 
                 11 
                 NNNNL 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 MP 
                   
                 VGI 
               
               
                   
               
               
                 DA13 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 14 
                 GSRDSAG 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 MGA 
               
               
                   
               
               
                 DA14 
                 4 
                 SGGDSYAGSY 
                 12 
                 NNNNK 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 AK 
                   
                 VGI 
               
               
                   
               
               
                 DA15 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 15 
                 GSRDSDG 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 GGR 
               
               
                   
               
               
                 DA16 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 16 
                 GSRDSSG 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 AGG 
               
               
                   
               
               
                 DA17 
                 4 
                 SGGDSYAGSY 
                 12 
                 NNNNK 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 AK 
                   
                 VGI 
               
               
                   
               
               
                 DA18 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 15 
                 GSRDSDG 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 GGR 
               
               
                   
               
               
                 DA19 
                 4 
                 SGGDSYAGSY 
                 11 
                 NNNNL 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 MP 
                   
                 VGI 
               
               
                   
               
               
                 DA20 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 14 
                 GSRDSAG 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 MGA 
               
               
                   
               
               
                 DA21 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 16 
                 GSRDSSG 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 AGG 
               
               
                   
               
               
                 DA22 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA23 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA24 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA25 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA26 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA27 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA28 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA29 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA30 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA31 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA32 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA33 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA34 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
               
                 DA35 
                 4 
                 SGGDSYAGSY 
                 10 
                 NNNNR 
                 13 
                 GSRDSSY 
               
               
                   
                   
                 YYG 
                   
                 PS 
                   
                 VGI 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Sequences of Heavy Chain Variable Regions 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                 SEQ 
               
               
                   
                   
                 VH- 
                 VH- 
                 VH- 
                   
                 VH- 
                 VH- 
                 ID 
               
               
                 Clone 
                 VH-FW1 
                 CDR1 
                 FW2 
                 CDR2 
                 VH-FW3 
                 CDR3 
                 FW4 
                 NO 
               
               
                   
               
               
                 D0 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 17 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTVRL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QLNNLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA1 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA2 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA3 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 19 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA4 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 20 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA5 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 21 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA6 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 22 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CAR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA7 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 23 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CSR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA8 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA9 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 24 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTLYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA10 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 25 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CSR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA11 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 26 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CAR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA12 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 27 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CSR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA13 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 27 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CSR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA14 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 27 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CSR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA15 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 27 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CSR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA16 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDT 
                 DAGS 
                 WGQ 
                 27 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKNTAYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CSR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA17 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA18 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA19 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA20 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA21 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGS 
                 WGQ 
                 18 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 GCGS 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA22 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGSP 
                 WGQ 
                 28 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 CGSG 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GWTG 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 ACIDT 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                   
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA23 
                 EVQLV 
                 GFT 
                 WV 
                 GIGK 
                 RFTISRDD 
                 DAGSP 
                 WGQ 
                 29 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 SKSTVYL 
                 CGKG 
                 GTLV 
                   
               
               
                   
                 LVQPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GWTG 
                 TVSS 
                   
               
               
                   
                 GSLRLS 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 ACIDT 
                   
                   
               
               
                   
                 CAAS 
                   
                 WV 
                 VKG 
                 CVR 
                   
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA24 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 17 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTVRL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QLNNLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA2 
                 5AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 30 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTVYL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA26 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 30 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTVYL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA27 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 17 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTVRL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QLNNLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA28 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 31 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTARL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QLNNLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA29 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 31 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTARL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QLNNLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA30 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 32 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTAYL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA31 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 33 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTARL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTGTYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA32 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 34 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTVRL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QLNNLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA33 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 35 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTARL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QLNNLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA34 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 36 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTAYL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 DA35 
                 AVTLD 
                 GFT 
                 WV 
                 GIGK 
                 RATISRDD 
                 DAGS 
                 WGH 
                 37 
               
               
                   
                 ESGGG 
                 FSS 
                 RQA 
                 SDRY 
                 GQSTARL 
                 GCGS 
                 GTEV 
                   
               
               
                   
                 LQTPG 
                 FN 
                 PGK 
                 TGY 
                 QMNSLRA 
                 GGWT 
                 IVSS 
                   
               
               
                   
                 GGLSL 
                 ML 
                 GLE 
                 GSA 
                 EDTAVYY 
                 GACID 
                   
                   
               
               
                   
                 VCKGS 
                   
                 WV 
                 VKG 
                 CVK 
                 T 
                   
                   
               
               
                   
                   
                   
                 A 
               
               
                   
               
            
           
         
       
     
     (In Table 8 above, VH-FW1, VH-FW2, and VH-FW3 represent frameworks of the heavy-chain variable region) 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Sequences of Light Chain Variable Regions 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 VL- 
                   
                   
                   
                 SEQ 
               
               
                   
                   
                 VL- 
                 VL- 
                 CDR 
                   
                 VL- 
                 VL- 
                 ID 
               
               
                 Clone 
                 VL-FW1 
                 CDR1 
                 FW2 
                 2 
                 VL-FW3 
                 CDR3 
                 FW4 
                 NO 
               
               
                   
               
               
                 D0 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 38 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEAVYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA1 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 39 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA2 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 40 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA3 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 39 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA4 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 39 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA5 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 39 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA6 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 39 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA7 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 39 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA8 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 41 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SGSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA9 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 40 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA10 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 40 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA11 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 40 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA12 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 42 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNL 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 MP 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA13 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 43 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSA 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 GM 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GA 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA14 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 44 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NN 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 KA 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                 K 
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA15 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 45 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSD 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 GG 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GR 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA16 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 46 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 GA 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GG 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA17 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 44 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NN 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 KA 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                 K 
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA18 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 45 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSD 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 GG 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GR 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA19 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 42 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNL 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 MP 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA20 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 43 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSA 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 GM 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GA 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA21 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 46 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 SRSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 GA 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GG 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA22 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 47 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA23 
                 DIQMT 
                 SGG 
                 WYQ 
                 NN 
                 GVPSRFSG 
                 GSR 
                 FGQ 
                 47 
               
               
                   
                 QSPSSL 
                 DSY 
                 QKP 
                 NNR 
                 STSGTDFT 
                 DSS 
                 GTK 
                   
               
               
                   
                 SASVG 
                 AGS 
                 GKA 
                 PS 
                 LTISSLQP 
                 YV 
                 VEI 
                   
               
               
                   
                 DRVTIT 
                 YY 
                 PKTL 
                   
                 EDFATYY 
                 GI 
                 K 
                   
               
               
                   
                 C 
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA24 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 48 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYY 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA25 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 38 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEAVYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA26 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 48 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYY 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA27 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 49 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA28 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 38 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEAVYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA29 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 49 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA30 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 48 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYY 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA31 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 49 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA32 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 38 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEAVYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA33 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 49 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA34 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 48 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYY 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
                   
                   
                   
               
               
                   
               
               
                 DA35 
                 LTQPSS 
                 SGG 
                 WYQ 
                 NN 
                 NIPSRFSG 
                 GSR 
                 FGA 
                 49 
               
               
                   
                 VSANL 
                 DSY 
                 QKA 
                 NNR 
                 STSGSTAT 
                 DSS 
                 GTT 
                   
               
               
                   
                 GGTVEI 
                 AGS 
                 PGSA 
                 PS 
                 LTITGVQA 
                 YV 
                 LTV 
                   
               
               
                   
                 TC 
                 YY 
                 PVTV 
                   
                 EDEATYF 
                 GI 
                 L 
                   
               
               
                   
                   
                 YG 
                 IY 
                   
                 C 
               
               
                   
               
            
           
         
       
     
     (In Table 9, VL-FW1, VL-FW2, and VL-FW3 represent frameworks of the light-chain variable region) 
     EXAMPLE 5 
     Production of Antibody 
     Immunoglobulin (IgG) proteins were produced for use in assaying the affinity and activity of the antibodies obtained above. From pComb3× carrying the scFv nucleotide, variable and constant region fragments of the heavy and light chains were obtained by PCR using the primer combinations shown in Table 10 and the condition used in Example 2. 
     Genes of variable regions (V H  to V L ) and constant regions (C H  to C k ) in the heavy and light chains were amplified by PCR using the combinations of HC and LC primers in Table 10 and inserted into mammalian cell expression plasmids using the pcDNATM3.3-TOPO® TA cloning kit and pOptiTMVEC-TOPO® TA cloning kit (Invitrogen, U.S.A.). 1 μl of each of the vectors (pcDNATM3.3-TOPO® vector and pOptiTM VEC-TOPO® vector) and fragments were added to a buffer containing 200 mM NaCl and 10 mM MgCl 2  to a final volume of 6 μl and reacted at room temperature for 5 min. The vectors were transformed into DH5α  E. coli  competent cells by heat shock. The resultant colonies were mass cultured to obtain plasmids. The plasmids prepared above were transfected into HEK293F cells (Invitrogen, U.S.A.), which were than cultured for 7 days to express the antibodies. The antibodies were purified using a protein A column (GE, U.S.A.). Cell culture supernatants were loaded onto the column to bind the antibodies (IgG) to protein A, followed by elution with 20 mM sodium citrate buffer (pH 3.0). The antibodies were confirmed by SDS-PAGE to have high purity and light chain and heavy chain molecular weights in accordance with the theoretical size. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                   
                 SEQ 
               
               
                   
                   
                   
                 ID 
               
               
                   
                 Primer 
                 Sequence 
                 NO 
               
               
                   
               
             
            
               
                 V H   
                 Forward 
                 GCT AGC CGC CAC CAT GGG C 
                 58 
               
               
                   
                 Reverse 
                 AGG GGC CCT TGG TGG AGG CCT GGC 
                 59 
               
               
                   
                   
                 CGG CCT GGC CAC T 
                   
               
               
                   
               
               
                 C H   
                 Forward 
                 GCC TCC ACC AAG GGC CCC TC 
                 60 
               
               
                   
                 Reverse 
                 CGG GAT CCC TTG CCG GCC GT 
                 61 
               
               
                   
               
               
                 HC 
                 Forward 
                 GCT AGC CGC CAC CAT GGG C 
                 62 
               
               
                 (Heavy 
                 Reverse 
                 CGG GAT CCC TTG CCG GCC GT 
                 63 
               
               
                 Chain) 
                   
                   
                   
               
               
                   
               
               
                 V L   
                 Forward 
                 AAG CTT GCC GCC ACC ATG 
                 64 
               
               
                   
                 Reverse 
                 AGG GGG CGG CCA CGG TCC GGG AAG 
                 65 
               
               
                   
                   
                 ATC TAG AGG ACT G 
                   
               
               
                   
               
               
                 c k   
                 Forward 
                 CGG ACC GTG GCC GCC CCC TC 
                 66 
               
               
                   
                 Reverse 
                 GCT CTA GAC TAG CAC TCG C 
                 67 
               
               
                   
               
               
                 LC 
                 Forward 
                 AAG CTT GCC GCC ACC ATG 
                 68 
               
               
                 (Light 
                 Reverse 
                 GCT CTA GAC TAG CAC TCG C 
                 69 
               
               
                 Chain) 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 6 
     Assay of Binding Affinity of Antibody to Human DR5 
     In order to measure affinity, human DR5 was coupled to carboxylated dextran biosensor chips (CM5, GE) according to the manufacturer&#39;s instruction. Association and dissociation rates were assessed by injecting IgG proteins that were 2-fold serially diluted to 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.313 nM, and 0.156 nM. 
     Association and dissociation rates were depicted in association and dissociation sensorgrams and calculated using a simple 1:1 Langmuir binding model (BIAcore X100 Evaluation Software, ver. 2.0). The equilibrium dissociation constants (KD), which is the ratio of dissociation constant (Kd) to association constant (Ka), were confirmed to be in the sub-nanomolar range. Measurements of the binding of antibody D0 and DA1 to DA35 of the present disclosure to DR5 are given in Table 11 below (antibody names are given as the names of the clones from which the antibodies were derived). A representative sensorgram for D0 is depicted in  FIG.  1   . 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                   
                 Binding 
                   
                   
                   
               
               
                 Antibody 
                 Molecule 
                 Ka(1/Ms) 
                 Kd(1/s) 
                 KD(M) 
               
               
                   
               
             
            
               
                 D0 
                 Human DR5 
                 1.49 × 10{circumflex over ( )}6  
                 2.26 × 10{circumflex over ( )}−5 
                 1.52 × 10{circumflex over ( )}−11 
               
               
                 DA1 
                 Human DR5 
                 4.90 × 10{circumflex over ( )}6  
                 1.80 × 10{circumflex over ( )}−9 
                 3.80 × 10{circumflex over ( )}−16 
               
               
                 DA2 
                 Human DR5 
                 1.90 × 10{circumflex over ( )}6  
                  4.90 × 10{circumflex over ( )}−10 
                 2.10 × 10{circumflex over ( )}−16 
               
               
                 DA3 
                 Human DR5 
                 4.90 × 10{circumflex over ( )}6  
                 1.80 × 10{circumflex over ( )}−9 
                 3.80 × 10{circumflex over ( )}−16 
               
               
                 DA4 
                 Human DR5 
                 1.20 × 10{circumflex over ( )}6  
                 1.20 × 10{circumflex over ( )}−7 
                 1.00 × 10{circumflex over ( )}−13 
               
               
                 DA5 
                 Human DR5 
                 1.20 × 10{circumflex over ( )}6  
                 2.60 × 10{circumflex over ( )}−9 
                 2.10 × 10{circumflex over ( )}−15 
               
               
                 DA6 
                 Human DR5 
                 7.80 × 10{circumflex over ( )}6  
                 4.20 × 10{circumflex over ( )}−6 
                 5.30 × 10{circumflex over ( )}−13 
               
               
                 DA7 
                 Human DR5 
                 1.40 × 10{circumflex over ( )}6  
                 2.90 × 10{circumflex over ( )}−6 
                 1.90 × 10{circumflex over ( )}−12 
               
               
                 DA8 
                 Human DR5 
                 1.60 × 10{circumflex over ( )}6  
                 2.20 × 10{circumflex over ( )}−8 
                 1.30 × 10{circumflex over ( )}−14 
               
               
                 DA9 
                 Human DR5 
                 5.60 × 10{circumflex over ( )}6  
                 4.00 × 10{circumflex over ( )}−7 
                 7.10 × 10{circumflex over ( )}−14 
               
               
                 DA10 
                 Human DR5 
                 2.30 × 10{circumflex over ( )}6  
                 1.60 × 10{circumflex over ( )}−5 
                 7.00 × 10{circumflex over ( )}−12 
               
               
                 DA12 
                 Human DR5 
                 2.37 × 10{circumflex over ( )}06 
                 2.99 × 10{circumflex over ( )}−5 
                 1.27 × 10{circumflex over ( )}−11 
               
               
                 DA13 
                 Human DR5 
                 2.62 × 10{circumflex over ( )}06 
                 2.94 × 10{circumflex over ( )}−5 
                 1.12 × 10{circumflex over ( )}−11 
               
               
                 DA14 
                 Human DR5 
                 4.27 × 10{circumflex over ( )}06 
                 1.00 × 10{circumflex over ( )}−4 
                 2.35 × 10{circumflex over ( )}−11 
               
               
                 DA15 
                 Human DR5 
                 2.56 × 10{circumflex over ( )}06 
                 2.78 × 10{circumflex over ( )}−6 
                 1.09 × 10{circumflex over ( )}−12 
               
               
                 DA16 
                 Human DR5 
                 2.11 × 10{circumflex over ( )}06 
                 5.53 × 10{circumflex over ( )}−5 
                 2.61 × 10{circumflex over ( )}−11 
               
               
                 DA17 
                 Human DR5 
                 2.54 × 10{circumflex over ( )}06 
                 5.97 × 10{circumflex over ( )}−5 
                 2.35 × 10{circumflex over ( )}−11 
               
               
                 DA18 
                 Human DR5 
                 1.33 × 10{circumflex over ( )}06 
                 3.67 × 10{circumflex over ( )}−5 
                 2.77 × 10{circumflex over ( )}−11 
               
               
                 DA20 
                 Human DR5 
                 1.00 × 10{circumflex over ( )}06 
                 1.21 × 10{circumflex over ( )}−5 
                 1.20 × 10{circumflex over ( )}−11 
               
               
                 DA21 
                 Human DR5 
                 1.11 × 10{circumflex over ( )}06 
                 7.17 × 10{circumflex over ( )}−6 
                 6.43 × 10{circumflex over ( )}−12 
               
               
                 DA22 
                 Human DR5 
                 9.32 × 10{circumflex over ( )}05 
                 3.19 × 10{circumflex over ( )}−5 
                 3.42 × 10{circumflex over ( )}−11 
               
               
                 DA23 
                 Human DR5 
                 6.96 × 10{circumflex over ( )}05 
                 2.80 × 10{circumflex over ( )}−5 
                 4.03 × 10{circumflex over ( )}−11 
               
               
                 DA24 
                 Human DR5 
                 1.20 × 10{circumflex over ( )}06 
                 1.23 × 10{circumflex over ( )}−6 
                 1.02 × 10{circumflex over ( )}−12 
               
               
                 DA25 
                 Human DR5 
                 1.05 × 10{circumflex over ( )}06 
                 1.71 × 10{circumflex over ( )}−6 
                 1.62 × 10{circumflex over ( )}−12 
               
               
                 DA26 
                 Human DR5 
                 1.09 × 10{circumflex over ( )}06 
                 1.10 × 10{circumflex over ( )}−6 
                 1.01 × 10{circumflex over ( )}−12 
               
               
                 DA27 
                 Human DR5 
                 1.17 × 10{circumflex over ( )}06 
                 2.24 × 10{circumflex over ( )}−6 
                 1.92 × 10{circumflex over ( )}−12 
               
               
                 DA28 
                 Human DR5 
                 1.16 × 10{circumflex over ( )}06 
                 4.52 × 10{circumflex over ( )}−6 
                 3.89 × 10{circumflex over ( )}−12 
               
               
                 DA29 
                 Human DR5 
                 1.21 × 10{circumflex over ( )}06 
                 8.47 × 10{circumflex over ( )}−5 
                 6.98 × 10{circumflex over ( )}−11 
               
               
                 DA30 
                 Human DR5 
                 1.03 × 10{circumflex over ( )}06 
                 7.97 × 10{circumflex over ( )}−5 
                 7.74 × 10{circumflex over ( )}−11 
               
               
                 DA31 
                 Human DR5 
                 1.24 × 10{circumflex over ( )}06 
                 8.79 × 10{circumflex over ( )}−5 
                 7.06 × 10{circumflex over ( )}−11 
               
               
                 DA32 
                 Human DR5 
                 1.14 × 10{circumflex over ( )}06 
                 8.59 × 10{circumflex over ( )}−5 
                 7.51 × 10{circumflex over ( )}−11 
               
               
                 DA33 
                 Human DR5 
                 1.24 × 10{circumflex over ( )}06 
                 9.77 × 10{circumflex over ( )}−5 
                 7.88 × 10{circumflex over ( )}−11 
               
               
                 DA34 
                 Human DR5 
                 1.00 × 10{circumflex over ( )}06 
                 9.50 × 10{circumflex over ( )}−5 
                 9.42 × 10{circumflex over ( )}−11 
               
               
                 DA35 
                 Human DR5 
                 1.13 × 10{circumflex over ( )}06 
                  9.12 × 10{circumflex over ( )}−05 
                 8.01 × 10{circumflex over ( )}−11 
               
               
                   
               
            
           
         
       
     
     EXAMPLE 7 
     Assay of Binding Activity of Antibodies for Human DR5 
     The binding activities of the antibodies of the present disclosure were for human DR5 were confirmed by enzyme-linked immunosorbent assay. Human DR5 protein at a concentration of 10 ng/ml was plated at a volume of 150 μl per well onto 96-well immune plates (Nunc, U.S.A.) and adsorbed at room temperature for one hour. After the plates were washed three times with a buffer solution, serial dilutions (0.1-2000 ng/ml) of the antibodies were added to the wells at 150 μl per well and incubated at room temperature for 1-2 hours. The plates were washed again with a buffer solution. Then, HRP (horseradish peroxidase)-conjugated antibody (Serotec, U.S.A.) against anti-human immunoglobulin Fc was 1:20,000 diluted and plated at a volume of 150 μl per well, followed by incubation at room temperature for 1 hour. After washing, a 3,3′,5,5′-tetramethylbenzidine (TMB; Sigma, U.S.A.) solution was added at an amount of 100 μl per well and incubated for 3 to 10 min for color development. When the color development reached a certain level, the reaction was terminated with 1 N sulfuric acid (H 2 SO 4 ). Absorbance at 450 nm was read using a spectrophotometer (Molecular Device, U.S.A.) and the results are depicted in  FIGS.  2   a  to  2   f   . In this assay, the antibody AP was synthesized according to the anti-DR5 antibody sequence disclosed in PCT/US2006/03577 (WO 2006/083971) and used as a control antibody. As can be seen in  FIGS.  2   a  to  2   f   , antibodies D0 and DA1 to DA35 were observed to have higher antigen binding activity compared to the control antibody AP. 
     EXAMPLE 8 
     Analysis of Physicochemical Properties 
     [8-1] Confirmation of Antibody Size 
     The sizes of the antibodies according to the present disclosure were analyzed by SDS-PAGE using NuPAGE 4-12% Bis-Tris gel (Invitrogen, U.S.A.). The prepared anti-DR5 antibodies (D0, DA1, DA4, DA16, DA18, DA20, DA23, DA26, and DA29) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at reduced conditions after treatment with DTT (Invitrogen, U.S.A.) to remove disulfide bonds, and at non-reduced conditions without DTT treatment, to identify the presence of light and heavy chains of the complete antibody. The results are depicted in  FIGS.  3   a    (non-reduced condition) and  3   b  (reduced condition). As shown in  FIG.  3   a   , for samples analyzed at non-reduced conditions, a band was detected at a position between a 98 kDa size marker and a 188 kDa size marker, which corresponds to the size of the complete antibody. As shown in  FIG.  3   b   , for samples analyzed at reduced conditions, two bands were detected at vicinities of a 49 kDa size marker and a 28 kDa size marker, which correspond to the heavy and light chain of the antibodies, respectively. Thus, bands corresponding to the entire antibody, the heavy chain, and the light chain were each detected by SDS-polyacrylamide gel electrophoresis. 
     [8-2] Confirmation of Antibody Purity 
     Antibody purity and (soluble) aggregates were analyzed by size exclusion chromatography (SEC) using a TSKgel G3000SWx1 column (Tosoh, Japan). The results are depicted in  FIGS.  4   a    (control antibody AP),  4   b  (D0),  4   c  (DA1), 4d (DA4),  4   e  (DA15),  4   f  (DA23),  4   g  (DA26), and  4   h  (DA29). SEC was operated with isocratic elution using 100 mM phosphate buffer (pH 6.6) as the mobile phase and measurement of absorbance at 280 nm. In general, a high purity of over 94% was observed, with monomers accounting for 94% or more of the total peak area and aggregate peaks accounting for less than 6%. 
     EXAMPLE 9 
     Pharmacokinetics of Antibody 
     In order to examine in vivo kinetics of the antibodies constructed above, blood half-life was evaluated in mice. Antibodies were intravenously injected at doses of 1-10 mg/kg into nude mice. Blood was sampled from the orbital vein from 5 min to 14 days after injection. The blood collection was conducted using heparinized capillary tubes. Plasma obtained by centrifugation of the blood samples was diluted at certain ratios and used as samples for ELISA evaluation. Human DR5 diluted to a concentration of 10 ng/ml was plated at 100 μl per well onto 96-well immune plates (Nunc, U.S.A.) and adsorbed to the wells for 12-14 hours. The plates were washed three times with buffer solution containing 0.1% Tween 20 and blocked with buffer solution containing bovine serum albumin (Sigma, U.S.A.) at room temperature for 1 hour. After three rounds of washing with buffer solution, the plates were incubated with each of the mouse plasma dilutions and a serial dilution of standard substance at room temperature for 2 to 3 hours. After three washes with buffer solution, a 1:20000 dilution of an HRP-conjugated anti-human immunoglobulin Fc (Fc-HRP) antibody (Bethyl, U.S.A.) was added at a volume of 100 μl per well and incubated at room temperature for 1 hour. After washing three times, 100 μl of a TMB solution (Sigma, U.S.A.) was added to each well and allowed to react. When the color development reached a certain level, the reaction was terminated with 0.2 N sulfuric acid solution. Absorbance at 450 nm was measured using a spectrophotometer (Molecular Device, U.S.A.). The antibody concentration in each plasma sample was calculated from the optical density (OD) measurements, and pharmacokinetic parameters were estimated using WinNonLin ver. 6.2.0.495. The results are shown in  FIG.  5    and Table 12 below. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                   
                 D0 
                 DA22 
                 DA23 
               
               
                   
                   
                 10 
                 10 
                 10 
               
               
                   
                 Unit 
                 mg/kg 
                 mg/kg 
                 mg/kg 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Cmax 
                 ug/ml 
                 286.9 
                 227.9 
                 195.8 
               
               
                 AUCinf 
                 hr*ug/ml 
                 914.1 
                 1025.3 
                 685.3 
               
            
           
           
               
               
               
               
               
               
            
               
                 t½ 
                 initial (−1 d) 
                 day 
                 0.57 
                 0.70 
                 0.58 
               
               
                   
                 Terminal (2 d~14 d) 
                 day 
                 6.68 
                 8.37 
                 7.97 
               
               
                 Rsq 
                 initial (−1 d) 
                   
                 1.000 
                 1.000 
                 1.000 
               
               
                   
                 Terminal (2 d~14 d) 
                   
                 1.000 
                 0.988 
                 0.990 
               
               
                   
               
            
           
         
       
     
     As shown in  FIG.  5    and Table 12, the antibodies (D0, DA22, and DA23) showed blood half-lifes of 6.68 to 8.37 days in the mice employed. 
     EXAMPLE 10 
     Confirmation of Cytotoxicity of Antibody in Tumor Cells 
     The biological activity of the prepared antibodies was determined using in vitro cell death assays. For this assays, the human colorectal cancer cell line Colo205 (ATCC, U.S.A.), the human pancreatic cancer cell line Miapaca-2(ATCC, U.S.A.), and the human lung cancer cell line H2122 (ATCC, U.S.A.) were employed. 
     Each tumor cell line was suspended at a density of 1×10 5  cells/ml, plated at 50 μl per well into 96-well cell culture plates, and cultured for 16-24 hours in a constant temperature and humidity chamber. The antibodies having certain concentrations (0.06-66,000 μM) were applied alone (Ab alone) or in combination with a crosslinking antibody (anti-human Fc) (Serotec, U.S.A.) (Ab linked) and then incubated for 48 hours. 10 μl of resazurin solution (Invitrogen, U.S.A.) was added to each well and incubated for 1 to 2 hours in a constant temperature and humidity chamber. Thereafter, fluorescence was read at 590 nm using a spectrophotometer (Molecular Device). Cell viability was calculated as the percentage of relative fluorescence units (RFU) of test substance-treated group (treated RFU) to relative fluorescence units of a medium-treated group (untreated RFU) (Cell viability (%)=treated RFU/Untreated RFU×100). The results are depicted in  FIGS.  6   a  to  6   r   . For comparison, the same assay as described above was performed on the control antibody AP. 
     As can be seen in  FIGS.  6   a  to  6   r   , the antibodies constructed above showed excellent cell death activity in all the three human cancer cell lines. 
     EXAMPLE 11 
     Evaluation of Anti-Tumor Activity in Xenograft Models 
     Anti-tumor activity was evaluated by injecting the antibodies i nude mice to which human cancer cell lines had been implanted. For human DR5-expressing cancer cell lines, the human lung cancer cell line H2122(ATCC, U.S.A.) and the human pancreatic cancer cell line Miapaca-2(ATCC, U.S.A.) were employed. Two to five million tumor cells were implanted to lateral subcutaneous sites of female BALB/c nude mice 6-8 weeks old (OrientBio), and then tumor volumes were measured using calipers. When the tumors grew to an average volume of 100 to 300 mm 3 , the mice were separated into groups, and the above constructed antibodies (control antibody AP and antibody D0) were injected to the assigned groups. The antibodies were given as three weekly intraperitoneal injections (on the day of group separation, on day 7 and on day 14) at doses of 0.05-2 mg/kg. Tumor volumes were measured twice a week over a period of 28 days to 50 days following initial injection. Buffer solution (PBS) was injected as the negative control. 
     The results are depicted in  FIGS.  7   a    (H 2122  implanted mice) and  7   b  (Miapaca-2-implanted mice). As shown in  FIGS.  7   a  and  7   b   , the antibody D0 showed anti-tumoral activity superior to the control antibody AP. 
     EXAMPLE 12 
     TRAIL-Uncompetitive Binding 
     In order to examine antigen-binding characteristics of the antibodies selected in Example 3, a TRAIL competitive enzyme-linked immunosorbent assay was conducted. ELISA plates were coated with 0.1 μg/ml human DR5 (R&amp;D systems, U.S.A.) at 4° C. Thereafter, the plates were incubated with 0.01 nM-1440 nM of antibodies (D0, DA20, DA21, DA21, DA22, and DA23) together with 0.01-400 nM of TRAIL protein for 2 hours. After the plates were washed three times with PBST (0.1% bovine serum albumin and Tween-20 in PBS), HRP (horse radish peroxidase)-conjugated anti-human immunoglobulin Fc was added to each well and incubated for 1 hour. After three washes with PBST, color development was performed for 20 min with ABTS (Thermo, U.S.A.). When the color development reached to a certain level, the reaction was terminated with 1 N sulfuric acid. Absorbance at 405 nm was measured using a spectrophotometer (Molecular Device, U.S.A.) and is depicted in  FIGS.  8   a  and  8   b   . The antibody AP disclosed in Example (7-1) was used as a control. 
     In  FIGS.  8   a  and  8   b   , antigen binding of the control antibody AP decreased in a TRAIL dose-dependent manner whereas the antibodies D0, DA20, DA21, DA21, DA22, and DA23 maintained a certain level of antigen binding irrespective of TRAIL concentrations, implying that the antibodies of the present invention do not compete with TRAIL for binding to DR5. 
     EXAMPLE 13 
     Effect of Antibody in Combination with TRAIL 
     Antibody properties according to epitopes were analyzed in terms of cell death activity upon co-treatment with the constructed antibodies and TRAIL. In this test, the human colorectal cancer cell line Colo205 (ATCC, U.S.A.) and the human pancreatic cell line Miapaca-2 (ATCC, U.S.A.) were employed. The tumor cells were suspended at a concentration of 1×10 5  cells/ml and the suspensions were plated at 50 μl per well into 96-well culture plates, followed by incubation for 16-24 hours in a constant temperature and humidity chamber. Serial dilutions of the antibodies (0.1-10000 ng/ml) and TRAIL protein at determined concentrations (0.5, 1, 10 ng/ml) were applied in combination to the 96-well plates. After incubation for 48 to 72 hours at a constant temperature in a humidified condition, a resazurin solution (Invitrogen, U.S.A.) was added at an amount of 10 μl to per well. When the reaction reached a certain level after 1 to 2 hours, fluorescence was read at 590 nm using a spectrophotometer (Molecular Device). Cell viability was calculated as the percentage of relative fluorescence units (RFU) of a test substance-treated group (treated RFU) to the relative fluorescence units of a culture medium-treated group (untreated RFU) (Cell viability (%)=treated RFU/Untreated RFU×100). 
     Representative results are depicted in  FIGS.  9   a    (Colo205) and  9   b  (Miapaca-2). For comparison, the above described antibody AP was used as a control, based on the observation in literature (Cell Death and Differentiation, 15, 751-761, 2008) that this antibody AP has a similar binding site to that of TRAIL. 
     As can be seen in  FIGS.  9   a  and  9   b   , higher cell death activity was detected when the antibody D0 or DA23 at concentrations as low as 10 ng/ml were used in combination with TRAIL at certain concentrations, compared to antibody alone, whereas no such increase in activity was observed for the control antibody AP. 
     EXAMPLE 14 
     Assay for Intracellular Caspase Activity 
     In order to investigate the cell death mechanism thereof, the constructed antibodies were assayed for caspase activity. The activity of caspase-3/7 was measured using Apo-ONE® Homogeneous Caspase-3/7 Assay kit (Promega, U.S.A.). In this assay, the human colorectal cancer cell line Colo205(ATCC, U.S.A.), the human pancreatic cancer cell line Miapaca-2 (ATCC, U.S.A.), and the lung cancer cell line H2122 (ATCC, U.S.A.) were employed. A suspension of each tumor cell line having a concentration of 1×10 5  cells/ml was plated at a volume of 50 μl per well into 96-well cell culture plates and cultured for 16-24 hours in a constant temperature and humidity chamber. The cells were treated with predetermined concentrations (0.01-10000 ng/ml) of the antibody alone (Ab alone) or in combination with a crosslinking antibody (anti-human Fc) (Serotec, U.S.A.) (Ab linked) and then cultured for 4 hours. Subsequently, the cells were incubated for 3 to 16 hours with 100 μl of Apo-ONE® Caspase-3/7 Reagent (Promega, U.S.A.) per well in a constant temperature and humidity chamber. When the reaction reached a predetermined level, fluorescence was read at 520 nm using a spectrophotometer (Molecular Device). Caspase activity was calculated as a fold increase of relative fluorescence units (RFU) of a test substance-treated group (treated RFU) to over the relative fluorescence units of a culture medium-treated group (untreated RFU) (Caspase activity=treated RFU/Untreated RFU). 
     Representative results are depicted in  FIGS.  10   a    (Colo205),  10   b  (H2122), and  10   c  (Miapaca-2). For comparison, the antibody AP was treated in the same manner 
     As can be seen in  FIGS.  10   a  and  10   c   , the application of the antibody D0 or DA29 to the cancer cell lines elicited an increase in the activity of casapse-3/7, implying that the cell death mechanism of the antibody of the present disclosure involves the apoptosic pathway. 
     EXAMPLE 15 
     Combination Effect of Antibody and Drug 
     Effects obtained from co-treatment with the above constructed antibody and gemcitabine were evaluated using in vitro tumor cell death assay. This assay was based on the observation in the literature (J Gastrointest Surg. 2006 November; 10(9):1291-300) wherein the co-treatment of pancreatic cancer cells with gemcitabine and an anti-DR5 antibody resulted in greater tumor cell death and caspase activity. 
     The human pancreatic cancer cell lines Miapaca-2 (ATCC, U.S.A.) and Panc-1 (ATCC, U.S.A.) were each suspended at a density of 1×10 5  cells/ml. The suspensions were added in an amount of 50 μl to each well of 96-well cell culture plates and incubated for 16-24 hours in a constant temperature and humidity chamber. The cancer cell lines were treated with the anti-DR5 antibodies of the present disclosure including D0 in combination with certain concentrations (3 nM-300 nM) of gemcitabine and cultured for 48 to 72 hours. The cells were further incubated for 1 to 3 hours after addition of 10 μl of a resazurin solution (Invitrogen, U.S.A.) per well. When the reaction proceeded to a predetermined level, fluorescence was read at 590 nm using a spectrophotometer (Molecular Device). Cell viability was calculated as the percentage of relative fluorescence units (RFU) of a test substance-treated group (treated RFU) to relative fluorescence units of a culture medium-treated group (untreated RFU) (Cell viability (%)=treated RFU/Untreated RFU×100). The results are depicted in  FIGS.  6   a  to  6   r   . Representative results are depicted in  FIGS.  11   a    (Miapaca-2) and  11   b  (Panc-1). For comparison, the control antibody AP was treated in the same manner 
     As can be seen in  FIGS.  11   a    and  11   b,  increased apoptotic activity was detected upon co-treatment with the antibody D0 or DA29 and gemcitabine relative to treatment with each individual substance. 
     Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.