Enhanced production of immunoglobulins

Abstract

The present invention provides methods and compositions for generating transgenic animals, including transgenic mammals, as well as plasma cells that allow for cell surface capture of secreted immunoglobulin molecules produced endogenously in the plasma cells.

We claim: 
     
         1 . A method for generating an antibody-secreting cell capable of expressing a membrane-bound immunoglobulin-capturing molecule that can bind, retain, and display endogenously produced immunoglobulin molecules at its cell surface, comprising the step of introducing into the antibody-secreting cell, or the progenitor of an antibody-secreting cell, a nucleic acid vector comprising a promoter, nucleic acid sequence coding for an immunoglobulin-binding peptide, and nucleic acid sequence coding for a cell surface tether peptide. 
     
     
         2 . The method of  claim 1 , wherein the immunoglobulin-binding peptide is derived from:
 a. one or more bacterial proteins that naturally have affinity for an immunoglobulin; or   b. one or more variable domains of an immunoglobulin that has affinity for any part of another immunoglobulin.   
     
     
         3 . The method of  claim 2 , wherein the bacterial protein is Protein A or Protein G. 
     
     
         4 . The method of  claim 1 , wherein the antibody-secreting cell is a hybridoma cell or a cell of B lymphocyte lineage. 
     
     
         5 . The method of  claim 1 , wherein the promoter is a constitutive promoter or an inducible promoter. 
     
     
         6 . The method of  claim 5 , wherein the promoter expresses the immunoglobulin-capturing molecule preferentially in antibody-secreting cells with minimal expression during B cell development prior to antigen encounter. 
     
     
         7 . The method of  claim 6 , wherein the promoter is selected from B Lymphocyte-Induced Maturation Protein 1, Syndecan 1, Tumor Necrosis Factor Receptor Superfamily Member 17, or Fucosyltransferase 1 genes. 
     
     
         8 . The method of  claim 5 , where in the inducible promoter is a tetracycline-responsive promoter or a tamoxifen-responsive promoter. 
     
     
         9 . The method of  claim 1 , wherein the cell surface tether peptide is:
 a. a transmembrane peptide; or   b. a peptide sequence that can be post-translationally modified to tether the immunoglobulin-binding peptide to the cell surface of the antibody-secreting cell.   
     
     
         10 . The method of  claim 9 , wherein the transmembrane peptide is derived from human Lymphocyte Activation Gene 3, human CD58, rat CD2, or human CD7. 
     
     
         11 . The method of  claim 10 , wherein the C-terminal peptide sequence mediates glycosylphosphatidylinositol linkage to the plasma membrane. 
     
     
         12 . The method of  claim 1 , wherein the nucleic acid vector further comprises:
 a. a nucleic acid sequence coding for a stalk structure;   b. a nucleic acid sequence coding for a reporter peptide;   c. an IRES sequence or a picornavirus 2A ribosomal skip sequence;   d. a nucleic acid sequence coding for a signal peptide; or   e. a combination thereof.   
     
     
         13 . The method of  claim 12 , wherein the reporter peptide is a fluorescent peptide. 
     
     
         14 . The method of  claim 12 , wherein the nucleic acid vector further comprises nucleic acid sequences coding for a stalk structure and a reporter peptide linked to an IRES sequence or picornavirus 2A ribosomal skip sequence. 
     
     
         15 . An antibody-secreting cell produced by the method of  claim 1 . 
     
     
         16 . Part of or whole immunoglobulin molecules derived from the antibody-secreting cell of claim  24 . 
     
     
         17 . The method of  claim 1 , wherein the antibody-secreting cell is used for large-scale production of antibodies. 
     
     
         18 . A genetically modified animal comprising antibody-secreting cells comprising a gene encoding an immunoglobulin-capturing molecule comprising a cell surface tether portion and an immunoglobulin-binding portion, wherein the immunoglobulin-capturing molecule can bind, retain, and display endogenously produced immunoglobulin molecules at a cell surface of the antibody-secreting cells. 
     
     
         19 . A vector for expressing a membrane-bound immunoglobulin-capturing molecule that can bind, retain, and display immunoglobulin molecules at a cell surface of an antibody-secreting cell, comprising a promoter, nucleic acid sequence coding for an immunoglobulin-binding peptide, and nucleic acid sequence coding for a cell surface tether peptide. 
     
     
         20 . The vector of  claim 19 , further comprising nucleic acid sequences coding for a signal peptide, a stalk structure, and a reporter peptide linked to an IRES sequence or picornavirus 2A ribosomal skip sequence.