Patent Publication Number: US-7914790-B2

Title: Human IgM antibody lysing activated lymphocytes under mediation by homologous complement

Description:
TECHNICAL FIELD 
     The invention relates to a human IgM monoclonal antibody reactive to HIV-infected cells that lyses activated lymphocytes by reacting with a differentiated antigen of the activated lymphocytes under medication by homologous human complements, and a remedy for autoimmune diseases containing the monoclonal antibody. 
     BACKGROUND ART 
     Various immunosuppressants such as cyclosporin and FK506 have been developed for controlling biological immunoreactions in collagen diseases, autoimmune diseases and rejection for organ transplantation. However, since such immunosuppressants are reactive to cells other than immunocompetent cells, side effects of these agent should be taken into consideration. 
     Various methods have been investigated for using antibodies that specifically react with target cells. For example, the target cell with which the antibody has reacted is expected to be lysed by reacting with a complement. While there are species-specific complement control membrane factors (such as DAF, decay accelerating factor; MCP, membrane cofactor protein; and HRF20, 20 kDa homologous restriction factor) on human cell membranes, they can induce no cytolysis reaction via complement reactions for preventing reactions among homologous human complements. 
     On the other hand, it was found that IgM antibodies in human serum that react with the HIV-infected cells are able to yield the cytolysis reaction of the HIV-infected cells via the human complement by overcoming the complement control membrane factors. It was revealed that the IgM antibody can exhibit such action as described above against gangliosides such as GM2 and Gg4 whose expression is enhanced by HIV-infection (Japanese Patent Application Laid-Open No. 9-227409). 
     L55 has been reported as the human IgM monoclonal antibody against GM2 of the gangliosides, wherein L55 is produced by immortalizing human B lymphoblast strain with EB virus. The HIV-infected cells after treating with this human IgM monoclonal antibody have been found to yield cytolysis via a reaction with the human complement. 
     DISCLOSURE OF INVENTION 
     An object of the invention is to provides a remedy for controlling immunological response containing a human IgM monoclonal antibody that specifically reacts with activated lymphocytes to induce cytolysis under mediation by homologous complements. 
     As a result of intensive studies for solving the problems above, the invention provides a human IgM monoclonal antibody reactive to HIV-infected cells that lyse activated human lymphocytes under mediation by homologous human complements. 
     The invention for solving the problems above provides an HIV remedy that cures transplantation rejection response and autoimmune diseases caused by excessive response of T-lymphocytes as well as HIV infection diseases by eliminating activated lymphocytes by cytolyses using a human IgM monoclonal antibody that reacts with HIV-infected cells and activated lymphocytes. 
     More specifically, the invention for solving the problems above provides the human IgM monoclonal antibody according to any one of first to second aspects, wherein the human IgM monoclonal antibody that reacts with the activated lymphocytes and the HIV-infected cells is 9F11 antibody having a base sequence of the H-chain variable region represented by sequence number 1. 
     More specifically, the invention for solving the problems above provides the human IgM monoclonal antibody according to any one of first to third aspects, wherein the human IgM monoclonal antibody that reacts with the activated lymphocytes and the HIV-infected cells is 9F11 antibody having a base sequence of the L-chain variable region represented by sequence number 2. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows specificity of 9F11 antibody. 
       The result of flow cytometory analysis shows that HIV-infected cells are stained with 9F11 antibodies while non-infected cells are not. 
         FIG. 2  shows specificity of 9F11 antibody to peripheral blood lymphocytes. 
       The drawing shows that 9F11 antibody reacts with the activated lymphocytes stimulated with PHA while it does not react with normal peripheral blood lymphocytes (PBMC: peripheral blood lymphocyte). 
         FIG. 3  shows a cell impairing reaction by 9F11 antibody under mediation by the complement. 
       (A) Almost all cells are destroyed 4 hours after addition of 2 μg/ml of 9F11 antibody and fresh human serum (including a complement component) to HIV-1 infected cells MOLT-4/IIIB. No effects have been observed at all when the serum is not added or on non-infected cells MOLT-4. 
       (B) 9F11 antigen is induced by activating the peripheral blood lymphocytes using PHA, and the cells are impaired by 9F11 antibody and the complement as in the HIV-1 infected cells (FHS: fresh human serum (used as a complement source), PHA: human lymphocyte activating agent, %  51 Cr release=mortality of cells, PBMC: peripheral blood lymphocyte). 
         FIG. 4  schematically illustrates 9F11 pt-chain expression plasmid construct. 
     
    
    
     BEST MODE FOR CARRYING OUT THE INVENTION 
     While the invention is described in detail with reference to examples, the technical scope of the invention is by no means restricted to these examples. 
     For solving the problems above, the inventors of the invention immunized HIV-infected cells of a mouse (TC mouse: trans-chromosome mouse; prepared by Kirin Brewery Co., Ltd.) into which human immunoglobulin gene-containing chromosomes had been introduced, and obtained a mouse that produces human antibodies that specifically react with HIV-infected cells. Hybridoma was prepared by a conventional method by fusing spleen cells of the immunized mouse with a mouse myeloma cell strain. Clones producing the monoclonal antibody that react with the HIV-infected cells and lead the infected cells to cytolysis in the presence of a human complement were selected from the hybridoma. The selected hybridomas were named as 9F11 cell strain. A 9F11 antibody that is a monoclonal antibody produced by 9F11 cell strain is a human IgM monoclonal antibody comprising human μ-chain and human κ-chain. While 9F11 antibody was able to yield cytolysis by reacting with the HIV-infected cells under mediation by the human complement, the antibody also caused cytolysis for activated lymphocytes of non-infected lymphocytes. Accordingly, the response to the HIV-infected cells may be comprehended that the lymphocyte is brought to a certain kind of activated state by HIV infection, and the HIV-infected cells are lysed under mediation by the human complement by expressing an antigen (9F11 antigen) against 9F11 as a differentiated antigen. In other words, 9F11 antigen is a differentiated antigen that is expressed by activation of the lymphocyte, and 9F11 antibody that induces cytolysis under mediation by the complement by reacting with the antigen specifically leads the activated lymphocyte to cytolysis under mediation by the complement. It was therefore made clear that a remedy containing 9F11 antibody can be utilized in the treatment for suppressing the activated lymphocyte. The invention has been completed based on the discoveries described above. The cell strain 9F11 that produces 9F11 antibodies of the invention was deposited with National Institute of Advanced Industrial Science and Technology, International Patent Organism Depository (Chuo-6, Higashi 1-1-1, Tsukuba City, Ibaraki Pref.), on May 8, 2003, with an accession number of FERM BP-8379. 
     Table 1 shows the results of base sequence analysis of the genes in the variable regions in κ-chain and μ-chain, respectively, encoding 9F11 antibody. The base sequence of the constant region is approximately the same as the base sequence of reported genes. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
             
            
               
                 Base Sequence of ti-Chain Variable Region 
               
            
           
           
               
               
            
               
                 GCTGAATTCTGGCTGACCAGGGCAGTCACCAGAGC 
                 SEQ ID NO: 1 
               
               
                   
               
               
                 TCCAGACAATGTCTGTCTCCTTCCTCATCTTCCTG 
                   
               
               
                   
               
               
                 CCCGTGCTGGGCCTCCCATGGGGTGTCCTGTCACA 
                   
               
               
                   
               
               
                 GGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGA 
                   
               
               
                   
               
               
                 AGCCCGCGCAGACCCTCTCACTCACCTGTGCCATC 
                   
               
               
                   
               
               
                 TCCGGGGACAGTGTCTCTAGCAACAGTGCTACTTG 
                   
               
               
                   
               
               
                 GAACTGGATCAGGCAGTCCCCATTGAGAGGCCTTG 
                   
               
               
                   
               
               
                 AGTGGCTGGGAAGGACATACTACAGGTCCAAGTGG 
                   
               
               
                   
               
               
                 TATAATGATTATGCAGTATCTGTGAAAAGTCGAAT 
                   
               
               
                   
               
               
                 AACCATCAACCCAGACACATCCAAGAACCAGTTCT 
                   
               
               
                   
               
               
                 CCCTGCAGCTGAACTCTGTGACTCCCCGAGGACAC 
                   
               
               
                   
               
               
                 GGCTGTGTATTACTGTGCAAGAGAGAATTACTATG 
                   
               
               
                   
               
               
                 GTTCGGGGAGGTACAACTGGTTCGACCCCTGGGGC 
                   
               
               
                   
               
               
                 CAGGGAACCCTGGTCACCGTCTCCTCA 
                   
               
               
                   
               
            
           
           
               
            
               
                 Base Sequence of is-Chain Variable Region 
               
            
           
           
               
               
            
               
                 TGTCAGGACACAGCATGGACATGAGGGTCCCCGCT 
                 SEQ ID NO: 2 
               
               
                   
               
               
                 CAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGG 
                   
               
               
                   
               
               
                 TTCCAGATGCGACATCCAGATGACCCAGTCTCCAT 
                   
               
               
                   
               
               
                 CTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACC 
                   
               
               
                   
               
               
                 ATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTG 
                   
               
               
                   
               
               
                 GTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCC 
                   
               
               
                   
               
               
                 CTAAGCTCCTGATCTATGATGCATCCAGTTTGCAA 
                   
               
               
                   
               
               
                 AGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATC 
                   
               
               
                   
               
               
                 TGGGACAGATTTCACTCTCACCATCAGCAGCCTGC 
                   
               
               
                   
               
               
                 AGCCTGAAGATTTTGCAACTTACTATTGTCAACAG 
                   
               
               
                   
               
               
                 GCTAACAGTTTCCCTCTCACTTTCGGCGGAGGGAC 
                   
               
               
                   
               
               
                 CAAGGTGGAGATCAAA 
               
               
                   
               
            
           
         
       
     
     The composition of the invention, which is used for an immunological reaction control agent containing the human IgM monoclonal antibody that specifically reacts with the activated lymphocytes and induces cytolysis under mediation by the homologous complement, may be obtained by combining with physiologically acceptable carriers. The physiologically acceptable carrier is known in the art, and includes physiological buffered saline or other aqueous solutions having a buffer action, or solvents such as glycols, glycerol, oils (for example olive oil) or injectable organic esters. The physiologically acceptable carrier may include compounds that stabilize human IgM antibody or enhance absorption thereof. Examples of the physiologically acceptable compounds include sugars such as glucose, sucrose and dextran; antioxidants such as ascorbic acid and glutathione; chelating agents; proteins such as albumin; and other stabilizers and excipients. Various immunosuppressants such as cyclosporin and FK506 as well as other immunosuppressants may be added to the composition. Any combinations of the physiologically acceptable carriers may be selected depending on administration path and disease of object. 
     Example 1 
     Specificity of 9F11 Antibody 
     Test cells were suspended in a culture medium in a concentration of 1×10 6  cells/ml, and an equal volume of a 9F11 antibody solution in a concentration of 10 μg/ml was added to the suspension followed by reacting for 30 minutes. The test cells were washed, linked 9F11 was stained with fluorescence-labeled antihuman IgM antibody, and the cells were analyzed by flow cytometory. The results showed that 9F11 antigen is expressed by HIV infection since, although MOLT-4 cells and CEM cells as human cell strains were not stained, the cells infected with HIV-1 IIIB strain and MN strain were strongly stained ( FIG. 1 ). Since HIV infection induces activation of the lymphocytes, staining of the activated lymphocytes was investigated after cultivating for 3 days by adding phytohemagglutinin (PHA) to the lymphocyte in the peripheral blood cells of a normal person. It was made clear that 9F11 antigen is a differentiated antigen expressed in the activated T-lymphocytes ( FIG. 2 ), since the activated T-lymphocytes stimulated with PHA was strongly stained, although no staining was observed in the peripheral blood cells and peripheral blood lymphocytes not stimulated. 
     Example 2 
     Cell Impairing Reaction by 9F11 Antibody Under Mediation by Complement 
     Test cells were labeled with  51 Cr as a radioactive isotope in advance, and 40 μl of 9F11 solutions in various concentrations and 20 μl of human fresh serum (complement serum) were added to the labeled test cells (suspended in a culture medium at a concentration of 5×10 5 /ml) to allow the mixture to react for 4 hours on a microtiter plate. After the reaction, the cells were precipitated by centrifugation of the plate, and the radioactivity of  51 Cr released in the supernatant by cytolysis was measured as an index of the cytolysis reaction. The cells expressing 9F11 antigen such as MOLT-4/IIIB (MOLT-4 cells infected with HIV-1) and peripheral blood derived lymphoblasts activated with PHA were lysed under mediation by the complement in the presence of 2 μg/ml of 9F11. On the contrary, no cytolysis was observed by using an inactivated serum after heating human serum at 56° C. or C9 deficient human fresh serum. Since cytolysis occurs by adding purified C9 to the C9 deficient human fresh serum, it was concluded that the human complement reaction is essential for the cytolysis reaction ( FIG. 3 ). 
     Example 3 
     Reconstruction of Antibody by Gene Engineering 
     An example of the method for reconstruction of 9F11 antibody based on the base sequence of the variable region of 9F11 antibody shown in TABLE 1 will be described below, wherein 9F11 antibody-producing cell strains were established using gene engineering such as a shot-gun ligation method (Grundstrom, T. et al., Nucleic Acid Res. 13, 3305-3316, 1985). 
     Amino acid sequences of the variable region of 9F11 antibody were obtained by translating the base sequence in the table. There are many base sequences encoding the amino acid sequences in the variable region of 9F11 antibody as shown in Table 2, such as the base sequence of the variable region of original 9F11 antibody as well as those obtained by changing used codons. Base sequences having certain kinds of restriction enzyme recognition fragment were selected from the sequences for every length capable of chemically synthesizing as oligonucleotides (Table 2). 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Examples of cDNA (i.e., SEQ ID NOs: 3 and 4 and conservatively modified variants 
               
               
                  thereof), encoding the amino acid sequence of 9F11 antibody (SEQ ID NO: 7) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   1 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 M 
                 S 
                 V 
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                 F 
                 L 
                 I 
                 F 
                 L 
                 P 
                 V 
                 L 
                 G 
                 L 
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                 V 
                 L 
                 S 
                 SEQ ID NO: 7 
               
               
                 ATA 
                 TCT 
                 GTT 
                 TCT 
                 TTT 
                 TTA 
                 ATT 
                 TTT 
                 TTA 
                 CCT 
                 GTT 
                 TTA 
                 GGT 
                 TTA 
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                 TGA 
                 GGT 
                 GTT 
                 TTA 
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                 SEQ ID NO: 3 
               
               
                 ATG 
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                 TTG 
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                 GTC 
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                 SEQ ID NO: 4 
               
               
                   
                 TCA 
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                  21 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
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                 V 
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                 CAA 
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                  41 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 T 
                 C 
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                 GCT 
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                 TGA 
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                 ATT 
                 CGT 
                   
               
               
                 ACC 
                 TGC 
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                 TGG 
                 AAC 
                 TGG 
                 ATC 
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                 ACA 
                   
                 GCA 
                   
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                 TCA 
                 TCA 
                   
                 TCA 
                 GCA 
                 ACA 
                   
                   
                   
                   
                 CGA 
                   
               
               
                 ACG 
                   
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                 TCG 
                 GGG 
                   
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                 TCG 
                   
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                 ACG 
                   
                   
                   
                   
                 CGG 
                   
               
               
                   
                   
                   
                   
                 AGT 
                   
                   
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                 AGC 
                   
                   
                 AGC 
                   
                 AGC 
                 AGC 
                   
                 AGC 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                  61 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Q 
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                 CAA 
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                 CAG 
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                 TCA 
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                 TCG 
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                 ACG 
                   
                   
                 CGG 
                 TCG 
                   
                   
                   
                   
               
               
                   
                 AGT 
                   
                 CTA 
                   
                   
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                 CTA 
                   
                   
                   
                   
                   
                   
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                 AGC 
                   
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                  81 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
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                 ACG 
                   
                   
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                 AGT 
                   
                   
                 AGT 
                   
                   
                   
                   
                   
                   
                   
                   
                 AGT 
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                 AGC 
                   
                   
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                 AGC 
                   
                   
                   
               
               
                   
               
               
                 101 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Q 
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                 CAG 
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                 GAG 
                 GAC 
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                 TCA 
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                 GCG 
                   
               
               
                   
                   
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                 CTA 
                   
                 CTA 
                   
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                 AGC 
                 CTG 
                   
                 CTG 
                   
                 AGC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
               
                 121 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 R 
                 E 
                 N 
                 Y 
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                 Y 
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                 Q 
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                 CGT 
                 GAA 
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                 TAT 
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                 TGA 
                 TTT 
                 GAT 
                 CCT 
                 TGA 
                 GGT 
                 CAA 
                 GGT 
                 ACT 
                   
               
               
                 CGC 
                 GAG 
                 AAC 
                 TAC 
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                 GGC 
                 TCC 
                 GGC 
                 CGC 
                 TAC 
                 AAC 
                 TGG 
                 TTC 
                 GAC 
                 CCC 
                 TGG 
                 GGC 
                 CAG 
                 GGC 
                 ACC 
                   
               
               
                 CGA 
                   
                   
                   
                   
                 GGA 
                 TCA 
                 GGA 
                 CGA 
                   
                   
                   
                   
                   
                 CCA 
                   
                 GGA 
                   
                 GGA 
                 ACA 
                   
               
               
                 CGG 
                   
                   
                   
                   
                 GGG 
                 TCG 
                 GGG 
                 CGG 
                   
                   
                   
                   
                   
                 CCG 
                   
                 GGG 
                   
                 GGG 
                 ACG 
                   
               
               
                   
                   
                   
                   
                   
                   
                 AGT 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                 AGC 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 141 
                   
                   
                   
                   
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 L 
                 V 
                 T 
                 V 
                 S 
                 S 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 TTA 
                 GTT 
                 ACT 
                 GTT 
                 TCT 
                 TCT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 TTG 
                 GTC 
                 ACC 
                 GTC 
                 TCC 
                 TCC 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 CTT 
                 GTA 
                 ACA 
                 GTA 
                 TCA 
                 TCA 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 CTC 
                 GTG 
                 ACG 
                 GTG 
                 TCG 
                 TCG 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 CTA 
                   
                   
                   
                 AGT 
                 AGT 
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 CTG 
                   
                   
                   
                 AGC 
                 AGC 
               
               
                   
               
            
           
         
       
     
     Oligonucleotides were chemically synthesized based on the base sequence divided for each restriction enzyme-recognition fragment. After sequentially digesting the synthesized oligonucleotide with a corresponding restriction enzyme, a full length of a base sequence encoding the amino acid sequence of the 9F11 antibody variable region by ligation was obtained. cDNA fragments of the 9F11 antibody variable regions of the H-chain and L-chain obtained by the same method with each other (named as rVμ9F11 and rVκ9F11, respectively) were integrated into vectors having constant region gene sequences of H-chain and L-chain of the human IgM antibody (Cμ and Cκ, respectively) by the same method as forming chimera antibodies to obtain recombinant 9F11μ-chain and κ-chain expression plasmids (rVμ9F11-Cμ and rVκ9F11-Cκ, respectively;  FIG. 4 ). 
     Example 4 
     Expression of Recombinant Antibody 
     Activities of the antibodies obtained using the plasmids expressing the reconstructed 9F11 antibody genes were investigated with a temporary expression system in COST cells (ATCC CRL 1651). Genes were introduced using a mixture of two plasmids (rVμ9F11-Cμ and rVκ9F11-Cκ) and an expression plasmid (Cj) for J-chain of human IgM antibody using a lipofectamine reagent according to the protocol by GIBCO Co. Cultivation was continued for two days thereafter under a usual culture condition, and the supernatant of the gene-introduced cell culture was retrieved. Recombinant 9F11 antibodies secreted in the culture supernatant were confirmed by subjecting the culture supernatant to an assay system by the sandwich ELISA method using antihuman μ-antibody and antihuman κ-antibody. The antibodies were confirmed to have specificity as described above by FACS analysis of the culture supernatant using cells such as U937/IIIB and MOLT-4/IIIB prepared by infection of U937 cells and MOLT-4 with IIIB strain of HIV-1. Further, the activity of recombinant 9F11 antibody was confirmed by a competitive inhibition test by allowing fluorescence-labeled original 9F11 antigen and the culture supernatant to simultaneously react with U937/IIIB or MOLT-4/IIIB. 
     Accordingly, it was confirmed that the base sequences of the μ-chain and κ-chain of 9F11 antibody listed in Table 1 are quite important regions responsible for antibody activity against differentiated antigens of the activated lymphocytes expressed in the HIV-infected cells. 
     It was also confirmed from these results that genes encoding the base sequences of the μ-chain variable region and κ-chain variable region are quite crucial genes for preparing the recombinant anti-HIV antibody and anti-activated lymphocytes. 
     INDUSTRIAL APPLICABILITY 
     Since the human IgM monoclonal antibody of the invention against the differentiated antigens expressed in the activated lymphocytes displays a cytolysis function for the activated lymphocytes under mediation by the complement, the antibody can be used as a remedy for controlling abnormally activated lymphocytes in the body. The invention also provides genes encoding the base sequences of the v chain variable region and x-chain variable regions that are quite crucial for preparing the recombinant anti-activated lymphocytes antibody.