Method for generating diversity

Abstract

The invention relates to a method for preparing an antibody-producing cell line capable of directed constitutive hypermutation of a specific nucleic acid region, comprising the steps of: a) screening a clonal cell population for V gene diversity; b) isolating one or more cells which display V gene diversity and comparing the rate of accumulation of mutations in the V genes and other genes of the selected cells; and c) selecting a cell in which the rate of V gene mutation exceeds that of other gene mutation.

1 . A method for preparing a gene product having a desired activity, said method comprising the steps of:
 (a) in vitro expression of a nucleic acid encoding said gene product operably linked to control sequences which direct hypermutation in a clonal population of cells, wherein said clonal population of cells are capable of directed constitutive hypermutation and exhibit a rate of target nucleic acid mutation that exceeds the rate of non-target nucleic acid mutation by a factor of 100 or more, wherein the rate of mutation is modulated by genetic manipulation of one or more DNA repair genes, and wherein said genetic manipulation is selected from the group consisting of gene deletion, conversion and insertion;   (b) identifying a cell or cells within said clonal population of cells which expresses a mutated gene product having the desired activity; and   (c) establishing one or more clonal populations of cells from the cell or cells identified in step (b), and selecting from the one or more clonal populations a cell or cell population which expresses a gene product having an improved desired activity.   
     
     
         2 . The method of  claim 1 , wherein said clonal populations of cells of step (a) are selected from a population of cells capable of directed constitutive hypermutation by screening said cell population for on-going target sequence diversification. 
     
     
         3 . The method of  claim 1 , wherein said clonal population of cells is derived from a lymphoid cell line. 
     
     
         4 . The method of  claim 1 , wherein said clonal population of cells is derived from an immunoglobulin-expressing cell line. 
     
     
         5 . The method according to  claim 1 , wherein said clonal populations of cells directs constitutive hypermutation to an endogenous V gene region coding sequence. 
     
     
         6 . The method according to  claim 1 , wherein said control sequences which direct hypermutation are selected from sequences occurring downstream of a J gene cluster. 
     
     
         7 . The method according to  claim 1 , wherein said control sequences comprise elements Ei/MAR, C-plus flanking regions and E3′ as defined according to Klix et al., (1998) Eur. J. Immunol. 28:317-326. 
     
     
         8 . The method according to  claim 1 , wherein said nucleic acid encoding said gene product operably linked to control sequences which direct hypermutation is an exogenous heterologous coding sequence inserted into said cell or cell population. 
     
     
         9 . The method of  claim 8 , wherein an endogenous V gene region coding sequence is replaced by said exogenous heterologous coding sequence. 
     
     
         10 . The method of  claim 1 , wherein said gene product is an immunoglobulin. 
     
     
         11 . The method of  claim 1 , wherein said gene product is a DNA binding protein. 
     
     
         12 . The method of  claim 1 , wherein said desired activity is a binding activity. 
     
     
         13 . The method of  claim 1 , wherein said gene product is an enzyme. 
     
     
         14 . The method of  claim 1 , wherein steps (b) and (c) of  claim 1  are iteratively repeated. 
     
     
         15 . The method of  claim 1 , wherein said identifying of step (b) is based on FACS. 
     
     
         16 . The method of  claim 1 , wherein said identifying of step (b) is based on binding to magnetic beads. 
     
     
         17 . The method of  claim 1 , wherein said identifying of step (b) is based on affinity selection. 
     
     
         18 . The method of  claim 1 , wherein said identifying of step (b) is based on enzymatic activity. 
     
     
         19 . The method of  claim 1 , wherein said selecting of step (c) is based on FACS. 
     
     
         20 . The method of  claim 1 , wherein said selecting of step (c) is based on binding to magnetic beads. 
     
     
         21 . The method of  claim 1 , wherein said selecting of step (c) is based on affinity selection. 
     
     
         22 . The method of  claim 1 , wherein said selecting of step (c) is based on enzymatic activity. 
     
     
         23 . The method according to  claim 2 , wherein said clonal population of cells are selected from a group of cell lines comprising Burkitt lymphoma, follicular lymphoma or diffuse large cell lymphoma cell lines. 
     
     
         24 . The method according to  claim 1 , wherein said one or more DNA repair genes are Rad51 analogues and/or paralogues. 
     
     
         25 . The method according to  claim 24 , wherein said one or more DNA repair genes are selected from the group consisting of Rad51b, Rad51c and analogues and/or paralogues thereof. 
     
     
         26 . The method according to  claim 2 , wherein said cell is an avian cell. 
     
     
         27 . The method according to  claim 26 , wherein said cell is a genetically manipulated chicken DT40 cell. 
     
     
         28 . (The method according to  claim 27 , wherein said cell is a selected from the group consisting of Δ xrcc2 DT40 and Δ xrcc3 DT40.