Monoclonal antibody to the clonotypic structure of a T-cell receptor, a pharmaceutical composition and a diagnostic reagent comprising the same

Abstract

The present invention relates to an efficient method of producing monoclonal antibodies against surface antigens of cells and viruses. The method accommodates for antigens which are present in only relatively small amounts, or antigens of which only very small amounts are available or antigens which easily lose their in vivo conformation. Thus the method according to the invention comprises a series of steps, comprising a step in which B-cells from a mammal injected with surface antigen-comprising material are enriched with respect to the relative number of specific B-cells and a step which comprises a small-scale fusion technique.

1 . Method of preparing a monoclonal antibody against a cell surface antigen, comprising the steps of 
 1) injecting a mammal with cell surface antigen-comprising material, said material being chosen from the group consisting of i) whole cells and ii) a membrane fraction obtained by treating whole cells;    2) isolating a B-cells-containing cell fraction from the spleen of said mammal;    3) enriching said cell fraction obtained in step 2 in B-cells specific for said cell surface antigen by contacting the cell fraction with carrier-bound material of cells related to said whole cells, said carrier-bound related material of cells lacking said cell surface antigen, and separating B-cells bound to carrier-bound material of related cells from the enriched unbound B-cells-containing cell fraction to be used in the next step;    4) subjecting the enriched B-cells-containing cell fraction obtained in the previous step to limiting dilution followed by clonal expansion;    5) selecting a B-cell clone and immortalising said selected B-cell clone using a small-scale fusion technique; and    6) selecting and cloning a hybridoma capable of producing antibodies which specifically bind said cell surface antigen, followed by isolating a monoclonal antibody-comprising fraction from supernatant of said hybridoma.    
     
     
         2 . Method according to  claim 1 , characterized in that the mammal injected with surface antigen is of a different species than the mammalian species from which the surface antigen originates.  
     
     
         3 . Method according to  claim 1  or  2 , characterized in that the surface antigen has a constant section and a variable section, wherein at least a part of said variable section defines a specificity determining part of said surface antigen.  
     
     
         4 . Method according to  claim 3 , characterized in that receptor molecule-comprising material is used as the surface antigen-comprising material.  
     
     
         5 . Method according to  claim 4 , characterized in that a T-cell clone is used to prepare the receptor molecule-comprising material.  
     
     
         6 . Method according to any of the preceding claims, characterized in that the membrane fraction in step 1 is obtained by mechanical treatment of the whole cells.  
     
     
         7 . Method according to any of the preceding claims, characterized the cell surface antigen-comprising material is injected in the mammal in the absence of an adjuvant.  
     
     
         8 . Method according to any of the preceding claims, characterized in that the enriched B-cells-containing cell fraction of step 3 which is to be used in step 4 is further enriched by contacting the cell fraction with carrier-bound cell surface antigen comprising material chosen from the group of j) whole cells jj) a membrane fraction obtained from said whole cells and jjj) substantially purified cell surface antigen, and subsequently separating B-cells not bound to said carrier-bound material from B-cells bound to said carrier-bound material, said B-cells bound to said carrier-bound material comprising the further enriched cell fraction to be used in step 4.  
     
     
         9 . Method according to any of the preceding claims, characterized in that paramagnetic beads are used as the carrier.  
     
     
         10 . Method according to any of the preceding claims, characterized in that the selection in at least one of the steps 5 and 6 is conducted using an agglutination assay wherein supernatant of the B-cell clone is contacted with a carrier coated with antibodies capable of binding antibodies of the species of the injected mammal used in step 1 and whole cells bearing the cell surface antigen, and agglutination is detected.  
     
     
         11 . Method according to  claim 10 , characterized in that whole cells related to said whole cells but lacking said cell surface antigen are used as a control.  
     
     
         12 . Method according to any of the preceding claims, characterized in that the selected B-cell clone in step 5 is mixed with myeloma cells and subjected to mini-electrofusion.  
     
     
         13 . Monoclonal antibody reactive with the clonotypic structure of a T-cell receptor.  
     
     
         14 . Monoclonal antibody according to  claim 13 , characterized in that the T-cell receptor is a T-cell receptor associated with an auto-immune disease.  
     
     
         15 . Monoclonal antibody according to  claim 14 , characterized in that the auto-immune disease is rheumatoid arthritis.  
     
     
         16 . Monoclonal antibody according to  claim 15 , characterized in that said monoclonal antibody is reactive with the T-cell receptor of a HC gp-39 reactive T-cell clone.  
     
     
         17 . Monoclonal antibody according to  claim 16 , characterized in that the T-cell clone is H.243 (ECACC accession No. 96103122).  
     
     
         18 . Monoclonal antibody according to  claim 17 , characterized in that it is produced by a hybridoma chosen from the group consisting of TCR 69 (ECACC accession No. 96103118), TCR 70 (ECACC accession No. 96103119), TCR 72 (ECACC accession No. 96103120) and TCR 83 (ECACC accession No. 96103121).  
     
     
         19 . Pharmaceutical composition comprising a monoclonal antibody prepared according to any of the conclusions  1  to  12 , admixed with a suitable excipient.  
     
     
         20 . Pharmaceutical composition comprising a monoclonal antibody according to any of the  claims 13  to  18  admixed with a suitable excipient, suitable for the treatment of rheumatoid arthritis.  
     
     
         21 . Diagnostic reagent comprising a monoclonal antibody chosen from the group consisting of a monoclonal antibody prepared according to any of the  claims 1  to  12  and a monoclonal antibody according to any of the  claims 13  to  18 .