Methods of producing full-length antibodies using E. coli
Inventors
Groff, Daniel • Hanson, Jeffrey
Assignees
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Publication Number
US-12351850-B2
Publication Date
2025-07-08
Expiration Date
Abstract
This disclosure provides a method of producing full-length antibodies in E. coli with a yield of at least about 200 mg/L. The E. coli cells typically have an oxidative cytoplasm, which is helpful for maintaining the three-dimensional structure and stability of proteins having disulfide bonds. In some cases, the molar ratio of the produced HC and LC amount from culturing the E. coli ranges from about 1:1 to about 1:3 (e.g., from about 1:1 to about 1:2.5, from about 1:1 to about 1:2).
Core Innovation
The invention provides a method to produce a full-length antibody comprising a heavy chain and a light chain in the cytoplasm of E. coli cells. An expression vector comprises an HC coding sequence and an LC coding sequence, including a first ribosome-binding site for translation of the HC and a second ribosomal binding site for translation of the LC, and the HC and LC are not exported outside of the cytoplasm of the cell.
The expression vector is configured to produce more LC than HC and to produce HC and LC at a molar ratio ranging between 1:1 and 1:3 from translating the HC coding sequence and the LC coding sequence. The full-length antibody is produced by culturing the E. coli cells under conditions permissible for producing the HC and LC in a culture medium, and the E. coli cells contain an oxidative cytoplasm.
In specific embodiments, the method uses defined ribosome-binding site DNA sequences for the HC and LC, and is described for producing a full-length B10 antibody in the cytoplasm. The HC coding sequence contains a mutation relative to SEQ ID NO: 1 that results in a codon for a natural amino acid being substituted with a non-natural amino acid codon, and the non-natural amino acid is introduced to the HC by charging a tRNA complementary to the non-natural amino acid codon.
Claims Coverage
The partial document contains three independent claims. Across these independent claims, the coverage focuses on cytoplasmic production of full-length antibodies in oxidative E. coli, controlled LC-over-HC stoichiometry, specified translation ribosome-binding sites, cytoplasmic retention, performance thresholds, and, in the B10 method, HC mutations that implement non-natural amino acid codons via tRNA charging.
Cytoplasmic oxidative production of full-length HC+LC antibody in E. coli
Producing a full-length antibody comprising an HC and an LC in the cytoplasm of E. coli cells using an expression vector and culturing under conditions permissible for producing the HC and LC, wherein the E. coli cells contain an oxidative cytoplasm and the HC and LC are not exported outside of the cytoplasm.
LC-over-HC stoichiometry via ribosome-binding sites producing HC:LC 1:1 to 1:3
Configuring the expression vector to produce more LC than HC and to produce HC and LC at a molar ratio ranging between 1:1 and 1:3 from translating the HC coding sequence and the LC coding sequence, using a first ribosome-binding site for translation of the HC and a second ribosomal binding site for translation of the LC.
Yield or cell-pellet weight percentage thresholds for full-length antibody
Producing the full-length antibody in an amount of about 200 mg per liter or more of the culture medium, or wherein the weight percentage of the full-length antibody produced relative to the weight of cell pellet produced from the E. coli cells is in a range from 0.05% to 20%.
Defined ribosome-binding site DNA sequences for HC and LC translation in oxidative cytoplasm without export
Culturing E. coli cells expressing coding sequences for an HC and an LC, where at least some cells contain an oxidative cytoplasm and each of the at least some cells contains a first ribosome-binding site for translation of the HC and a second ribosomal binding site for translation of the LC, with the first ribosome-binding site transcribed from DNA sequences selected from SEQ ID NO: 17-19 and the second ribosomal binding sequence transcribed from DNA sequences selected from SEQ ID NO: 20-23, while maintaining HC and LC not exported outside the cytoplasm and producing HC:LC at a molar ratio between 1:1 and 1:3 with greater LC than HC.
B10 HC mutation implementing non-natural amino acid codon with complementary charged tRNA
In a method to produce a full-length B10 antibody in the cytoplasm, providing an expression vector comprising a B10 antibody HC coding sequence and LC coding sequence with ribosome-binding sites SEQ ID NO: 28 and SEQ ID NO: 29, configuring LC-over-HC at a molar ratio between 1:1 and 1:3, and wherein the HC coding sequence contains a mutation relative to SEQ ID NO: 1 resulting in substitution of a codon for a natural amino acid with a non-natural amino acid codon, with the natural amino acid selected from F412, Y188, and F249, and introducing the non-natural amino acid by charging a tRNA complementary to the non-natural amino acid codon.
Overall, the independent claims define cytoplasmic, oxidative E. coli production of full-length antibodies with controlled LC-over-HC stoichiometry, cytoplasmic retention, specified ribosome-binding site elements, and yield/performance thresholds; a further independent claim for B10 adds an HC mutation that substitutes natural-amino-acid codons with non-natural amino acid codons implemented by complementary charged tRNA.
Stated Advantages
High-yield production of full-length antibody in E. coli, characterized by about 200 mg per liter or more, or 0.05% to 20% weight percentage relative to cell pellet weight.
Improved control of assembly stoichiometry by producing more LC than HC at an HC:LC molar ratio ranging between 1:1 and 1:3 while maintaining oxidative cytoplasm and preventing export outside the cytoplasm.
Site-specific incorporation of non-natural amino acids into the B10 HC by substituting selected natural amino acids with non-natural amino acid codons introduced via complementary charged tRNA.
Documented Applications
Production of a full-length B10 antibody in the cytoplasm of E. coli using an expression vector with defined ribosome-binding sites and LC-over-HC stoichiometry.
Antibody-drug conjugate/bioconjugation approaches using click chemistry and oxime ligation, with LCMS verification and reported antibody examples including B10, FolRa-B10 IgG, trastuzumab, anti-PD1, anti-Tim3, anti-LAG3, and others.
Production and verification of engineered antibody examples including B10, FolRa-B10 IgG, trastuzumab, and antibodies listed in the partial content via example titers and figures.
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