Methods and compositions for improving recombinant protein production

Abstract

Nucleic acid molecules modified to enhance recombinant protein, e.g., antibody, expression and/or reduce or eliminate mis-spliced and/or intron read-through (IRT) by-products are disclosed. The invention also provides methods for producing proteins devoid of mis-spliced and/or intron read-through by-products by the use of such vectors in host cells under cell culture conditions suitable for recombinant protein expression.

1 . A nucleic acid molecule comprising a nucleotide sequence having one or more intron and exon sequences, wherein at least one intron sequence is deleted compared to the naturally-occurring genomic sequence to reduce a mis-spliced or an intron read-through (IRT) by-product, and wherein said nucleotide sequence encodes an antibody heavy chain or a fragment thereof. 
     
     
         2 . A nucleic acid molecule that comprises a nucleotide sequence comprising one or more intron and exon sequences, wherein at least three intron sequences are deleted compared to the naturally-occurring genomic sequence to enhance protein expression, and wherein said nucleotide sequence encodes an antibody heavy chain or a fragment thereof. 
     
     
         3 . The nucleic acid molecule of  claim 1 , wherein the antibody heavy chain or fragment thereof comprises a heavy chain variable region, a hinge region, a first constant region (C H 1), a second constant region (C H 2), and third constant region (C H 3) of a human immunoglobulin G subtype. 
     
     
         4 . The nucleic acid molecule of  claim 2 , wherein the antibody heavy chain or fragment thereof comprises a heavy chain variable region, a hinge region, a first constant region (C H 1), a second constant region (C H 2), and third constant region (C H 3) of a human immunoglobulin G subtype. 
     
     
         5 . The nucleic acid molecule of  claim 3 , wherein the immunoglobulin G subtype is a human IgG1 or human IgG4, or a mutated form thereof. 
     
     
         6 . The nucleic acid molecule of  claim 4 , wherein the immunoglobulin G subtype is a human IgG1 or human IgG4, or a mutated form thereof. 
     
     
         7 . The nucleic acid molecule of  claim 3 , wherein an intron between the C H 2 region and the C H 3 region of the immunoglobulin heavy chain constant region is deleted. 
     
     
         8 . The nucleic acid molecule of  claim 4 , wherein an intron between the C H 1 region and the hinge region, an intron between the hinge region and C H 2 region, and an intron between the C H 2 region and the C H 3 region of the immunoglobulin heavy chain constant region are deleted. 
     
     
         9 . The nucleic acid molecule of  claim 3 , wherein the nucleotide sequence encoding the heavy chain hinge region, and the first, second and third constant regions comprises a sequence at least 95% identical to the nucleotide sequence shown in  FIG. 8  (SEQ ID NO:1), or at least 95% identical to the nucleotide sequence shown in  FIG. 9  (SEQ ID NO:3). 
     
     
         10 . The nucleic acid molecule of  claim 4 , wherein the nucleotide sequence encoding the heavy chain hinge region, and the first, second and third constant regions comprises a sequence at least 95% identical to the nucleotide sequence shown in  FIG. 8  (SEQ ID NO:1), or at least 95% identical to the nucleotide sequence shown in  FIG. 9  (SEQ ID NO:3). 
     
     
         11 . The nucleic acid molecule of  claim 7 , wherein the deletion of the intron between C H 2 and C H 3 corresponds to about nucleotides 1409 to 1505 of human IgG1 as shown in  FIG. 8  (SEQ ID NO:1), or about nucleotides 1401 to 1497 of human IgG4 as shown in  FIG. 9  (SEQ ID NO:3). 
     
     
         12 . The nucleic acid molecule of  claim 8 , wherein the deletion of the intron between C H 2 and C H 3 corresponds to about nucleotides 1409 to 1505 of human IgG1 as shown in  FIG. 8  (SEQ ID NO:1), or about nucleotides 1401 to 1497 of human IgG4 as shown in  FIG. 9  (SEQ ID NO:3). 
     
     
         13 . The nucleic acid molecule of  claim 8 , wherein the deletion of the intron between C H 1 and the hinge region corresponds to about nucleotides 525 to 915 of human IgG1 as shown in  FIG. 8  (SEQ ID NO:1), or about nucleotides 525 to 916 of human IgG4 as shown in  FIG. 9  (SEQ ID NO:3). 
     
     
         14 . The nucleic acid molecule of  claim 8 , wherein the deletion of the intron between the hinge region and C H 2 corresponds to about nucleotides 961 to 1078 of human IgG1 as shown in  FIG. 8  (SEQ ID NO:1), or about nucleotides 953 to 1070 of human IgG4 as shown in  FIG. 9  (SEQ ID NO:3). 
     
     
         15 . A nucleic acid molecule comprising a nucleotide sequence encoding human IgG1, wherein said nucleotide sequence is at least 90% identical to the sequence shown in  FIG. 10  (SEQ ID NO:5), or at least 90% identical to the sequence shown in  FIG. 11  (SEQ ID NO:6). 
     
     
         16 . A genomic nucleotide sequence encoding a human IgG1 or IgG4 heavy chain constant region, or a mutated form thereof, wherein said nucleotide sequence lacks at least one intron present in the naturally-occurring genomic sequence, and wherein said intron facilitates intron-read through. 
     
     
         17 . A nucleic acid molecule comprising a nucleotide sequence represented by the formula:
   V H -Int1-C H 1-Int2-Hinge-Int3-C H 2-C H 3,   wherein V H  is a nucleotide sequence encoding a heavy chain variable region;   C H 1, C H 2, and C H 3 are nucleotide sequences encoding the corresponding heavy chain constant region;   Hinge is a nucleotide sequence encoding a hinge region of a heavy chain constant region; and   Int1, Int2 and Int3 are introns from the heavy chain genomic sequence, wherein the nucleotide sequence encodes a human immunoglobulin G heavy chain.   
     
     
         18 . A nucleic acid molecule comprising a nucleotide sequence represented by the formula:
   V H -Int1-C H 1-Hinge-C H 2-C H 3,   wherein V H  is a nucleotide sequence encoding a heavy chain variable region;   C H 1, C H 2, and C H 3 are nucleotide sequences encoding the corresponding heavy chain constant region;   Hinge is a nucleotide sequence encoding a hinge region of a heavy chain constant region; and   Int1 is an intron from the heavy chain genomic sequence, wherein the nucleotide sequence encodes a human immunoglobulin G heavy chain.   
     
     
         19 . An expression vector comprising the nucleic acid molecule of  claim 3 . 
     
     
         20 . An expression vector comprising the nucleic acid molecule of  claim 4 . 
     
     
         21 . A host cell comprising the nucleic acid molecule of  claim 19 . 
     
     
         22 . A host cell comprising the nucleic acid molecule of  claim 20 . 
     
     
         23 . A method of expressing, in a mammalian host cell, an antibody or fragment thereof substantially free of an intron read-through (IRT) product, comprising:
 identifying an IRT product in a nucleic acid sample from the host cell;   introducing the nucleic acid molecule of  claim 3  into the host cell;   culturing said host cell under conditions that allow expression of the recombinant antibody or fragment thereof, thereby producing a culture of host cells; and   obtaining the recombinant antibody or fragment thereof from the culture of host cells.   
     
     
         24 . A method for enhancing expression of a recombinant antibody or fragment thereof, comprising:
 introducing the nucleic acid molecule of  claim 4 , and a nucleotide sequence encoding a light chain variable region and a constant region into a mammalian host cell;   culturing said host cell under conditions that allow expression of the recombinant antibody, thereby producing a culture of host cells; and   obtaining the recombinant antibody from the culture of host cells.   
     
     
         25 . A method for producing a recombinant antibody or fragment thereof substantially devoid of intron read-through (IRT) heavy chain by-product, comprising:
 culturing a mammalian host cell comprising the nucleic acid molecule of  claim 3  and a nucleic acid encoding an antibody light chain, under conditions such that the heavy and light chains are expressed.   
     
     
         26 . A method for detecting an IRT product, in a sample, comprising:
 obtaining a nucleic acid sample from a recombinant cell;   contacting said nucleic acid sample with nucleic acid probes complementary to an intron and adjacent exon sequence, under conditions that allow hybridization of the nucleic acid sample and the probes;   detecting the resulting complex, wherein detection in said sample of a complex, using the nucleic acid probe complementary to the intron sequence is indicative of the presence of the IRT product.   
     
     
         27 . An antibody, or antigen-binding fragment thereof, made by the method comprising the steps of  claim 23  under suitable conditions to allow expression and assembly of the antibody or fragment. 
     
     
         28 . A pharmaceutical composition comprising the antibody of  claim 25 , and a pharmaceutically acceptable carrier.