RESUMO
Efficient production of large quantities of therapeutic antibodies is becoming a major goal of the pharmaceutical industry. We developed a proprietary expression system using a polyprotein precursor-based approach to antibody expression in mammalian cells. In this approach, the coding regions for heavy and light chains are included within a single open reading frame (sORF) separated by an in-frame intein gene. A single mRNA and subsequent polypeptide are produced upon transient and stable transfection into HEK293 and CHO cells, respectively. Heavy and light chains are separated by the autocatalytic action of the intein and antibody processing proceeds to produce active, secreted antibody. Here, we report advances in sORF technology toward establishment of a viable manufacturing platform for therapeutic antibodies in CHO cells. Increasing expression levels and improving antibody processing by intein and signal peptide selection are discussed.
Assuntos
Expressão Gênica , Vetores Genéticos/genética , Inteínas , Fases de Leitura Aberta , Anticorpos de Cadeia Única , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/biossíntese , Cadeias Leves de Imunoglobulina/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/genéticaRESUMO
We describe a novel polyprotein precursor-based approach to express antibodies from mammalian cells. Rather than expressing heavy and light chain proteins from separate expression units, the antibody heavy and light chains are contained in one single-open reading frame (sORF) separated by an intein gene fused in frame. Inside mammalian cells this ORF is transcribed into a single mRNA, and translated into one polypeptide. The antibody heavy and light chains are separated posttranslationally, assembled into the functional antibody molecule, and secreted into culture medium. It is demonstrated that Pol I intein from P. horikoshii mediates protein splicing and cleavage reactions in mammalian cells, in the context of antibody heavy and light chain amino acid sequences. To allow the separation of antibody heavy chain, light chain, and the intein, we investigated a number of intein mutations designed to inhibit intein-mediated splicing but preserve cleavage reactions. We have also designed constructs in which the signal peptide downstream from intein has altered hydrophobicity. The use of some of these mutant constructs resulted in more efficient antibody secretion, highlighting areas that can be further explored in improving such an expression system. An antibody secreted using one of the sORF constructs was characterized. This antibody has correct N-terminal sequences for both of its heavy and light chains, correct heavy and light chain MW as well as intact MW as measured by mass spectrometry. Its affinity to antigen, as measured by surface plasmon resonance (SPR), is indistinguishable from that of the same antibody produced using conventional method.