Expression of immunoglobulin-cytokine fusion proteins in malignant B cells

Abstract

According to the invention, there is provided a vector for the expression of immunoglobulin-cytokin fusion proteins in malignant B cells at least containing operably linked to each other (a) a region of at least 1.5 kb which is homologous to a region of the μ intron or the κ intron and which lacks a functional C μ or C κ enhancer or contains a non-functional C μ or C κ enhancer; (b) at least one DNA sequence encoding a domain of an immunoglobulin or a part thereof; (c) a DNA sequence encoding a cytokin; and (d) a marker gene selectable in eukaryotic B cells and lacking a functional enhancer region wherein the expression of said marker following integration is controlled by the cellular C μ or C κ enhancer.

1 . Vector for the expression of immunoglobulin-cytokin fusion proteins in malignant B cells, at least containing operably linked to each other 
 (a) a region of at least 1.5 kb which is homologous to a region of the μ intron or the κ intron;    (b) at least one DNA sequence encoding a domain of an immunoglobulin or a part thereof;    (c) a DNA sequence encoding a cytokin; and    (d) a marker gene which is selectable in eukaryotic B cells and contains a functional enhancer region.    
     
     
         2 . Vector according to  claim 1 , wherein said region of at least 1.5 kb contains a functional C μ  or C κ  enhancer.  
     
     
         3 . Vector according to  claim 1 , wherein said region of at least 1.5 kb contains a non-functional C μ  or C κ  enhancer.  
     
     
         4 . Vector according to  claim 1 , wherein the marker gene selectable in eukaryotic B cells contains a non-functional enhancer region.  
     
     
         5 . Vector according to  claim 1 , wherein the marker gene selectable in eukaryotic B cells lacks an enhancer region.  
     
     
         6 . Vector according to  claim 1 , wherein the DNA sequence of (b) encodes a constant region or a part thereof.  
     
     
         7 . Vector according to  claim 1 , wherein the region homologous to a region comprising the C μ  or the C κ  enhancer of the μ or the κ intron comprises at least 1.9 kb.  
     
     
         8 . Vector according to  claim 1 , wherein the region homologous to a region comprising the C μ  or the C κ  enhancer of the μ or the κ intron comprises at least 2.0 kb.  
     
     
         9 . Vector according to  claim 1 , said vector containing a regulatory unit which is compatible with bacteria.  
     
     
         10 . Vector according to  claim 1 , wherein the immunoglobulin is a chimeric immunoglobulin.  
     
     
         11 . Vector according to  claim 1 , wherein the DNA sequence of (b) encodes the domain of a human immunoglobulin chain.  
     
     
         12 . Vector according to  claim 1 , wherein the DNA sequence of (b) encodes domains derived from mouse, rat, goat, horse or sheep.  
     
     
         13 . Vector according to  claim 1 , wherein the DNA sequence of (b) encodes all the C domains of a secretory antibody.  
     
     
         14 . Vector according to  claim 1 , wherein the DNA sequence according to (b) encodes all the C domains of a membrane-bound antibody.  
     
     
         15 . Vector according to  claim 1 , characterized in that said DNA sequence of (c) encodes interleukins, interferons, colony-stimulating factors, lymphokins or growth factors.  
     
     
         16 . Vector according to  claim 15 , characterized in that said DNA sequence of (c) encodes IL-2, IL-4, IL-7, IL-12, IL-13, GM-CSF or interferon γ.  
     
     
         17 . Vector according to  claim 1 , wherein the selectable marker gene is gpt, neo or a marker gene encoding hygromycin resistance.  
     
     
         18 . Method for the expression of an immunoglobulin-cytokin fusion protein in malignant B cells in vitro including the following steps of: 
 (a) introduction of a vector according to  claim 1  into a malignant B cell;    (b) selection and identification of cells stably expressing the fusion protein;    (c) treatment of the cells to render them replication-incompetent.    
     
     
         19 . Method according to  claim 18 , wherein step (b) is ommitted.  
     
     
         20 . Method according to claims  18  or  19 , wherein step (a) is performed by means of transfection.  
     
     
         21 . Method according to  claim 20 , wherein said transfection is performed by electroporation, calciumphosphate co-precipitation, lipofection, the DEAE dextran technique or by retroviral gene transfer.  
     
     
         22 . Method according to  claim 18 , wherein the selection is carried out in a medium containing mycophenolic acid, G418, or hygromycin as a selective agent.  
     
     
         23 . Method according to  claim 18 , wherein following introduction of a vector according to  claim 1  by homologous recombination a site-specific integration of said vector 3′ of the heavy chain V gene of the malignant B cell is performed.  
     
     
         24 . Method according to  claim 18 , wherein the expression is controlled by the endogenous V H  promoter.  
     
     
         25 . Use of a vector according to  claim 1  in the expression of immunoglobulin-cytokin fusion proteins in malignant B cells.  
     
     
         26 . Use according to  claim 25 , wherein the malignant B cell is a B cell leukemia cell, a B cell lymphoma cell or a plasmacytoma cell.  
     
     
         27 . Use according to  claim 25 , wherein by expression of the immunoglobulin-cytokin fusion proteins the activation of T cells is achieved.  
     
     
         28 . Use of a vector according to  claim 1  in malignant B cells for the vaccination of patients having malignant B cell diseases.  
     
     
         29 . Malignant B cell containing a vector according to  claim 1  in integrated form, wherein an immunoglobulin-cytokin fusion protein is expressed by said cell.  
     
     
         30 . Use of a malignant B cell which has been rendered replication-incompetent and contains a vector according to  claim 1  in integrated form and is capable of expression of an immunoglobulin-cytokin fusion protein in the treatment of patients having malignant B cell diseases.