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:
BACKGROUND OF THE INVENTION  
         [0001]    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).  
           [0002]    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.  
           [0003]    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  
         [0004]    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.  
           [0005]    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  
         [0006]    The effectiveness and advantages of the invention are further illustrated by the following examples. 
       
    
    
     EXAMPLE 1  
       [0007]    Immunogens  
         [0008]    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 15mer peptides containing sequences from HCV core antigen.  
       EXAMPLE 2  
       [0009]    Immunizing Protocol  
         [0010]    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 15mer peptide. Most antibodies were mapped to a 6-8 amino acid region in the HCV core protein sequence.  
       EXAMPLE 3  
       [0011]    Characterization of the Antibodies  
         [0012]    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 15mer peptide. A summary of antibodies generated is included in the following table.  
         [0013]    Table 1  
         [0014]    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.  
                                                   ATCC   AG-FUSION #,                       #   clone   IMMUNOGEN   ISOTYPE   SPECIFICITY   AA                   PTA-   ODS243, 7B4F11   Peptide   IgG 2b   HCV core   77-91       3811       ODS 243       peptide #8       PTA-   ODS243, 1E3D12   Peptide   IgG2a   HCV core    86-100       3803       ODS 243       peptide #9       PTA-   ODS243, 7C12C4   Peptide   IgG2b   HCV core   77-91       3802       ODS 243       peptide #8       PTA-   core #3,   FLC   IgG1   HCV core   106-120       3813   2A11C6           peptide #11       PTA-   Core #12,   FLC   IgG1   HCV core   29-43       3809   1B7A1           peptide #3       PTA-   Core #13,   FLC   IgG1   HCV core   39-53       3805   5A12G12           peptide #4       PTA-   Core #13,   FLC   IgG1   HCV core   48-62       3812   4H7E7           peptide #5       PTA-   Core #13, 12F4A   FLC   IgG1   HCV core   58-72       3806   11           peptide #6       PTA-   Core #13,   FLC   IgG1   HCV core   67-81       3804   14D12A12           peptide #7       PTA-   c22-8#4,   KLH   IgG1   HCV core   77-91       3807   6D8E8   Conjugate       peptide #8               d core               peptide               #8       PTA-   Core #12,   FLC   IgG2b   HCV core    96-110       3800   4G10G6           peptide #10       PTA-   Core #13,   FLC   IgG2a   HCV core   106-120       3801   6E7E1           peptide #11       PTA-   Core #13,   FLC   IgG2b   HCV core   106-120       3810   11D12A6           peptide #11       PTA-   Core #13,   FLC   IgG3   HCV core   106-120       3808   14B7C3           peptide #11       PTA-   Core #12,   FLC   IgG1   HCV core   156-170       3799   4A6H3           peptide #16                  
 
         [0015]    [0015]                                                           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                    
         [0016]    [0016]                                           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                        
         [0017]    [0017]                                                               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               TWAQPGYP                       (7.10) Biotin-Ahx-   76-83   1.05           WAQPGYPW                       (8.1) Biotin-Ahx-   77-84   0.61           AQPGYPWP                       (8.2) Biotin-Ahx-   78-85   10.84           QPGYPWPL                       (8.3) Biotin-Ahx-   79-86   45.89           PGYPWPLY                       (8.4) Biotin-Ahx-   80-87   43.03           GYPWPLYG                       (8.5) Biotin-Ahx-   81-88   33.81           YPWPLYGN                       (8.6) Biotin-Ahx-   82-89   3.11           PWPLYGNE                       (8.7) Biotin-Ahx-   83-90   0.27           WPLYGNEG                       (8.8) Biotin-Ahx-   84-91   0.3           PLYGNEGC                       (8.9) Biotin-Ahx-   85-92   0.42           LYGNEGCG                       (9.1) Biotin-Ahx-   86-93   0.49           YGNEGCGW                       (9.2) Biotin-Ahx-   87-94   0.6           GNEGCGWA                        
         [0018]    Monoclonal antibody 7B4F11 reacted with Octapeptides 8.2, 8.3, 8.4, 8.5 in a chemiluminiscence ELISA measured in arbitrary light units (ALU). Based on the reactivity of the octapeptides the monoclonal epitope is centered around the sequence PWPL.  
         [0019]    The above examples are meant to illustrate, but not to limit, the scope and spirit of the invention.  
         [0020]    It is to be understood that numerous changes and modifications may be made therein without departing from the scope and intent of the invention.  
         [0021]    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.  
     
       
       
         1 
         
           
             19  
           
           
             1  
             15  
             PRT  
             Hepatitis C virus  
           
            1 

Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr 
1               5                   10                  15 

 
           
             2  
             15  
             PRT  
             Hepatitis C virus  
           
            2 

Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe 
1               5                   10                  15 

 
           
             3  
             15  
             PRT  
             Hepatitis C virus  
           
            3 

Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val 
1               5                   10                  15 

 
           
             4  
             15  
             PRT  
             Hepatitis C virus  
           
            4 

Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg 
1               5                   10                  15 

 
           
             5  
             15  
             PRT  
             Hepatitis C virus  
           
            5 

Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser 
1               5                   10                  15 

 
           
             6  
             15  
             PRT  
             Hepatitis C virus  
           
            6 

Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg 
1               5                   10                  15 

 
           
             7  
             15  
             PRT  
             Hepatitis C virus  
           
            7 

Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu 
1               5                   10                  15 

 
           
             8  
             15  
             PRT  
             Hepatitis C virus  
           
            8 

Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr 
1               5                   10                  15 

 
           
             9  
             15  
             PRT  
             Hepatitis C virus  
           
            9 

Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys 
1               5                   10                  15 

 
           
             10  
             15  
             PRT  
             Hepatitis C virus  
           
            10 

Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro 
1               5                   10                  15 

 
           
             11  
             15  
             PRT  
             Hepatitis C virus  
           
            11 

Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr 
1               5                   10                  15 

 
           
             12  
             15  
             PRT  
             Hepatitis C virus  
           
            12 

Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Leu Asn Gly 
1               5                   10                  15 

 
           
             13  
             15  
             PRT  
             Hepatitis C virus  
           
            13 

Ser Arg Leu Asn Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe 
1               5                   10                  15 

 
           
             14  
             15  
             PRT  
             Hepatitis C virus  
           
            14 

Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val 
1               5                   10                  15 

 
           
             15  
             15  
             PRT  
             Hepatitis C virus  
           
            15 

Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Arg Ala 
1               5                   10                  15 

 
           
             16  
             15  
             PRT  
             Hepatitis C virus  
           
            16 

Gly Ala Ala Arg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp 
1               5                   10                  15 

 
           
             17  
             15  
             PRT  
             Hepatitis C virus  
           
            17 

Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro 
1               5                   10                  15 

 
           
             18  
             15  
             PRT  
             Hepatitis C virus  
           
            18 

Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala 
1               5                   10                  15 

 
           
             19  
             15  
             PRT  
             Hepatitis C virus  
           
            19 

Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser 
1               5                   10                  15