Abstract:
Monoclonal antibodies that specifically bind to HCV core antigen. Also provided are hybridoma cell lines which secrete these antibodies, methods for making and using these antibodies, including kits that include these antibodies.

Description:
This application claims the benefit of U.S. Provisional Application Ser. No. 60/337,453, filed Nov. 5, 2001 (now abandoned). 
    
    
     BACKGROUND OF THE INVENTION 
     An estimated 170 million people worldwide have been infected by hepatitis C virus (HCV). In the next few years, the number of U.S. deaths for HCV-caused liver disease and cancer may overtake deaths caused by Acquired Immune Deficiency Syndrome (AIDS). 
     The transmission of HCV seems to require blood-to-blood contact. Carrying a single strand of ribonucleic acid (RNA), HCV contains just one gene, coding for a polyprotein that is subsequently cleaved into at least 10 functional proteins. Clearly, the ability to test the blood supply for HCV is of great importance. A sensitive assay that can detect infection at an early stage. Detection of both HCV antigens and antibodies in a single assay would therefore be advantageous. 
     HCV core detection assays capture HCV antigen from the HCV infected specimens on a solid phase coated with anti-core monoclonal antibodies. The captured core protein is detected by a binding reaction with an anti-core monoclonal antibody labeled with enzyme using standard immunoassay technology. Availability of good monoclonal antibodies to multiple epitopes of HCV core antigen will increase the sensitivity of HCV antigen detection assays. For one thing, multiple antibodies increase the efficiency of the capture of the HCV antigen from specimens onto the solid phase. Second, using multiple antibodies for detection increases the sensitivity of the system by providing a cumulative higher signal. Another advantage is that the use of multiple epitope recognizing antibodies in assays enables the assays to detect specimens infected with different genotypes of the HCV virus. 
     SUMMARY OF THE INVENTION 
     The present invention describes a library of monoclonal antibodies directed against various regions of the HCV core protein. These antibodies recognize ten (10) different epitopes in the HCV core region and are spread throughout the core protein. The epitopes recognized by these monoclonal antibodies are five to eight (5-8) amino acids (“aa”) long. These antibodies in individual pairs or multiple pairs are useful in detecting HCV core protein in patients infected with HCV. Specifically, these antibodies are advantageous in detecting HCV in blood from infected humans. Finally, these antibodies along with properly tailored recombinant proteins can be used for the simultaneous detection of HCV core protein and ant-HCV antibodies. 
     We have developed a number of anti-HCV core monoclonal antibodies. These antibodies have unique properties in that they recognize short sequences, in the region of HCV core that does not have major epitopes recognized by human anti-HCV antibodies. These antibodies can be useful reagents in detecting HCV core antigen in blood specimens from individuals infected with HCV. The recognition of short epitopes by these monoclonal antibodies and the location of these sequences in the core region of HCV make these monoclonal antibodies very useful in the development of a HCV antigen/antibody combination assay. For the combination assay these monoclonal antibodies are used in combination with core antigen that has been modified to eliminate the ability of these antigens to bind to these monoclonal antibodies but maintain their ability to bind human anti-HCV antibodies. Therefore, the HCV core proteins used to capture the antibodies present in infected serum will not react with the HCV core monoclonal antibodies used in the test to simultaneously capture the antigens present in the infected serum. A combination assay can detect HCV infection earlier than the currently employed HCV antibody assay. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The effectiveness and advantages of the invention are further illustrated by the following examples. 
     EXAMPLE 1 
     Immunogens 
     Immunogens used for raising the antibodies were a full length core recombinant protein (“FLC”)(amino acids (aa) 1-191); c22-3, HCV core protein expressed in yeast as an SOD fusion protein (aa 1-120); and large synthetic peptides, such as ODS243(aa 70-110); and keyhole limpet hemacynin (KLH) conjugated 15 mer peptides containing sequences from HCV core antigen. 
     EXAMPLE 2 
     Immunizing Protocol 
     The antibodies were generated using standard procedures. See for example, Ed Harlow and David Lane, Antibodies, A Laboratory Manual, 1988, Cold Spring Harbor, Chapter 6. Mice were immunized with the immunogens described herein. The mice were bled and screened with a number of recombinant proteins and peptides from the HCV core sequence. Pure monoclonal antibodies were prepared by protein A affinity chromatography. The fine specificity of the monoclonal antibodies was determined by binding overlapping octapeptides contained in each of the corresponding 15 mer peptide. Most antibodies were mapped to a 6-8 amino acid region in the HCV core protein sequence. 
     EXAMPLE 3 
     Characterization of the Antibodies 
     The monoclonal antibodies were characterized by ELISA using overlapping synthetic peptides approximately 15 aa long. The antibodies were further characterized for their fine specificity by peptide ELISA using overlapping octapeptides included in the reactive 15 mer peptide. A summary of antibodies generated is included in the following table. 
     TABLE 1 
     The table below identifies 15 novel antibodies. The antibodies were screened at every stage of antibody development with microtitrewell plates coated with immunogen. The immunogen used to immunize the mice that produced each strain of monoclonal antibody is identified as one of the following: peptide ODS 243, a large peptide further defined in Example 1; “FLC” means full length core antigen, defined in Example 1; or KLH conjugated core peptide #8, a short peptide, further defined herein. The specificity of each to a numbered peptide is shown and the amino acid sequences of each numbered peptide is identified herein. Furthermore, the epitope to which the antibody specifically binds is included in the last column, defined by the amino acids that encode for the epitope. 
     The antibodies were deposited on Oct. 24, 2001 with the International Depository Authority: American Type Culture Collection, Manassas, Va. 20110-2209 USA (“ATCC”). The ATCC accession numbers are listed in the first column of the table below. 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 AG-FUSION 
                   
                   
                   
                   
               
               
                 ATCC 
                 #, 
                   
                   
                 SPECIFI- 
               
               
                 # 
                 clone 
                 IMMUNOGEN 
                 ISOTYPE 
                 CITY 
                 AA 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 PTA- 
                 ODS243, 
                 Peptide 
                 IgG2b 
                 HCV core 
                  77- 
               
               
                 3811 
                 7B4F11 
                 ODS 243 
                   
                 peptide #8 
                  91 
               
               
                 PTA- 
                 ODS243, 
                 Peptide 
                 IgG2a 
                 HCV core 
                  86- 
               
               
                 3803 
                 1E3D12 
                 ODS 243 
                   
                 peptide #9 
                 100 
               
               
                 PTA- 
                 ODS243, 
                 Peptide 
                 IgG2b 
                 HCV core 
                  77- 
               
               
                 3802 
                 7C12C4 
                 ODS 243 
                   
                 peptide #8 
                  91 
               
               
                 PTA- 
                 core#3, 
                 FLC 
                 IgG1 
                 HCV core 
                 106- 
               
               
                 3813 
                 2A11C6 
                   
                   
                 peptide #11 
                 120 
               
               
                 PTA- 
                 Core#12, 
                 FLC 
                 IgG1 
                 HCV core 
                  29- 
               
               
                 3809 
                 1B7A1 
                   
                   
                 peptide #3 
                  43 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG1 
                 HCV core 
                  39- 
               
               
                 3805 
                 5A12G12 
                   
                   
                 peptide #4 
                  53 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG1 
                 HCV core 
                 48- 
               
               
                 3812 
                 4H7E7 
                   
                   
                 peptide #5 
                  62 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG1 
                 HCV core 
                  58- 
               
               
                 3806 
                 12F4A11 
                   
                   
                 peptide #6 
                  72 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG1 
                 HCV core 
                  67- 
               
               
                 3804 
                 14D12A12 
                   
                   
                 peptide #7 
                  81 
               
               
                 PTA- 
                 c22-8#4, 
                 KLH 
                 IgG1 
                 HCV core 
                  77- 
               
               
                 3807 
                 6D8E8 
                 Conjugated 
                   
                 peptide #8 
                  91 
               
               
                   
                   
                 core 
               
               
                   
                   
                 peptide 
               
               
                   
                   
                 #8 
               
               
                 PTA- 
                 Core#12, 
                 FLC 
                 IgG2b 
                 HCV core 
                  96- 
               
               
                 3800 
                 4G10G6 
                   
                   
                 peptide #10 
                 110 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG2a 
                 HCV core 
                 106- 
               
               
                 3801 
                 6E7E1 
                   
                   
                 peptide #11 
                 120 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG2b 
                 HCV core 
                 106- 
               
               
                 3810 
                 11D12A6 
                   
                   
                 peptide #11 
                 120 
               
               
                 PTA- 
                 Core#13, 
                 FLC 
                 IgG3 
                 HCV core 
                 106- 
               
               
                 3808 
                 14B7C3 
                   
                   
                 peptide #11 
                 120 
               
               
                 PTA- 
                 Core#12, 
                 FLC 
                 IgG1 
                 HCV core 
                 156- 
               
               
                 3799 
                 4A6H3 
                   
                   
                 peptide #16 
                 170 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 The table below shows HCV core synthetic peptides. 
               
             
          
           
               
                 Peptide 
                 Amino Acid 
                 HCV protein 
                   
               
               
                 ID # 
                 Sequence 
                 AA Location 
               
               
                   
               
             
          
           
               
                 0 
                 MSTNPKPQKKNKRNT 
                  1-15 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:1 
               
               
                 1 
                 KNKRNTNRRPQDVKF 
                 10-24 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:2 
               
               
                 2 
                 QDVKFPGGGQIVGGV 
                 20-34 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:3 
               
               
                 3 
                 QIVGGVYLLPRRGPR 
                 29-43 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:4 
               
               
                 4 
                 RRGPRLGVRATRKTS 
                 39-53 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:5 
               
               
                 5 
                 ATRKTSERSQPRGRR 
                 48-62 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:6 
               
               
                 6 
                 PRGRRQPIPKARRPE 
                 58-72 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:7 
               
               
                 7 
                 KARRPEGRTWAQPGY 
                 67-81 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:8 
               
               
                 8 
                 AQPGYPWPLYGNEGC 
                 77-91 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:9 
               
               
                 9 
                 YGNEGCGWAGWLLSP 
                  86-100 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:10 
               
               
                 10 
                 WLLSPRGSRPSWGPT 
                  96-110 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:11 
               
               
                 11 
                 SWGPTDPRRRSRLNG 
                 106-120 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:12 
               
               
                 12 
                 SRLNGKVIDTLTCGF 
                 116-130 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:13 
               
               
                 13 
                 LTCGFADLMGYIPLV 
                 126-140 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:14 
               
               
                 14 
                 YIPLVGAPLGGAARA 
                 136-150 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:15 
               
               
                 15 
                 GAARALAHGVRVLED 
                 146-160 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:16 
               
               
                 16 
                 RVLEDGVNYATGNLP 
                 156-170 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:17 
               
               
                 17 
                 TGNLPGCSFSIFLLA 
                 166-180 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:18 
               
               
                 18 
                 IFLLALLSCLTVPAS 
                 176-190 
                 SEQ ID 
               
               
                   
                   
                   
                 NO.:19 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 The table below shows the screening of the 
               
               
                 antibody identified as PTA-3811 with ELISA plates coated 
               
               
                 with HCV core peptides. 
               
             
          
           
               
                   
                 Peptide 
                 Optical Density 
               
               
                   
                 ID # 
                 in ELISA 
               
               
                   
                   
               
             
          
           
               
                   
                 0 
                 0 
               
               
                   
                 1 
                 0.04 
               
               
                   
                 2 
                 0 
               
               
                   
                 3 
                 0.01 
               
               
                   
                 4 
                 0.01 
               
               
                   
                 5 
                 0 
               
               
                   
                 6 
                 0 
               
               
                   
                 7 
                 0.01 
               
               
                   
                 8 
                 2.5 
               
               
                   
                 9 
                 0 
               
               
                   
                 10 
                 0.01 
               
               
                   
                 11 
                 0.01 
               
               
                   
                 12 
                 0 
               
               
                   
                 13 
                 0.01 
               
               
                   
                 14 
                 0.01 
               
               
                   
                 15 
                 0 
               
               
                   
                 16 
                 0.01 
               
               
                   
                 17 
                 0.01 
               
               
                   
                 18 
                 0.01 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
           
               
                 TABLE 4 
               
               
                   
               
             
             
               
                 Epitope mapping of monoclonal antibody PTA-3811 
               
               
                 using overlapping octapeptides. 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sequence of 
                 AA 
                   
                   
               
               
                   
                 octapeptides 
                 Location 
                 ALU 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 (7.9) Biotin-Ahx- 
                 75-82 
                 0.57 
                 SEQ ID 
               
               
                   
                 TWAQPGYP 
                   
                   
                 NO.:20 
               
               
                   
                 (7.10) Biotin-Ahx- 
                 76-83 
                 1.05 
                 SEQ ID 
               
               
                   
                 WAQPGYPW 
                   
                   
                 NO.:21 
               
               
                   
                 (8.1) Biotin-Ahx- 
                 77-84 
                 0.61 
                 SEQ ID 
               
               
                   
                 AQPGYPWP 
                   
                   
                 NO.:22 
               
               
                   
                 (8.2) Biotin-Ahx- 
                 78-85 
                 10.84 
                 SEQ ID 
               
               
                   
                 QPGYPWPL 
                   
                   
                 NO.:23 
               
               
                   
                 (8.3) Biotin-Ahx- 
                 79-86 
                 45.89 
                 SEQ ID 
               
               
                   
                 PGYPWPLY 
                   
                   
                 NO.:24 
               
               
                   
                 (8.4) Biotin-Ahx- 
                 80-87 
                 43.03 
                 SEQ ID 
               
               
                   
                 GYPWPLYG 
                   
                   
                 NO.:25 
               
               
                   
                 (8.5) Biotin-Ahx- 
                 81-88 
                 33.81 
                 SEQ ID 
               
               
                   
                 YPWPLYGN 
                   
                   
                 NO.:26 
               
               
                   
                 (8.6) Biotin-Ahx- 
                 82-89 
                 3.11 
                 SEQ ID 
               
               
                   
                 PWPLYGNE 
                   
                   
                 NO.:27 
               
               
                   
                 (8.7) Biotin-Ahx- 
                 83-90 
                 0.27 
                 SEQ ID 
               
               
                   
                 WPLYGNEG 
                   
                   
                 NO.:28 
               
               
                   
                 (8.8) Biotin-Ahx- 
                 84-91 
                 0.3 
                 SEQ ID 
               
               
                   
                 PLYGNEGC 
                   
                   
                 NO.:29 
               
               
                   
                 (8.9) Biotin-Ahx- 
                 85-92 
                 0.42 
                 SEQ ID 
               
               
                   
                 LYGNEGCG 
                   
                   
                 NO.:30 
               
               
                   
                 (9.1) Biotin-Ahx- 
                 86-93 
                 0.49 
                 SEQ ID 
               
               
                   
                 YGNEGCGW 
                   
                   
                 NO.:31 
               
               
                   
                 (9.2) Biotin-Ahx- 
                 87-94 
                 0.6 
                 SEQ ID 
               
               
                   
                 GNEGCGWA 
                   
                   
                 NO.:32 
               
               
                   
                   
               
             
          
         
       
     
     Monoclonal antibody 7B4F11 reacted with Octapeptides 8.2, 8.3, 8.4, 8.5 in a chemiluminescence ELISA measured in arbitrary light units (ALU). Based on the reactivity of the octapeptides the monoclonal epitope is centered around the sequence PWPL (SEQ ID NO.: 33). 
     The above examples are meant to illustrate, but not to limit, the scope and spirit of the invention. 
     It is to be understood that numerous changes and modifications may be made therein without departing from the scope and intent of the invention. 
     For example, it would be understood by one skilled in the art that all of the monoclonal antibodies disclosed herein can be used individually or as combinations as capture regents or as detecting reagents for the detection of HCV core antigen in a HCV core antigen assay or an HCV antibody/antigen combination assay.