Patent
This disclosure provides methods and compositions related to producing full-length antibodies in E. coli having an oxidative cytoplasm, which is helpful for maintaining the three-dimensional structure and stability of proteins having disulfide bonds. The E. coli is used to produce full length antibody in high yield.
1 . An E. coli cell with an oxidative cytoplasm, wherein the E. coli cell comprises
(a) a coding sequence encoding a full-length antibody heavy chain (HC); and
(b) a coding sequence encoding a full-length antibody light chain (LC);
wherein the cell comprises an expression vector comprising a first ribosome-binding site for translation of the HC and a second ribosome-binding site for translation of the LC, and wherein the HC and LC are not exported outside of the cytoplasm of the cell, and wherein the cell produces more LC than HC.
2 . The E. coli cell of claim 1 , wherein the molar ratio of HC and LC produced ranges from 1:1 to 1:3.
3 . The E. coli cell of claim 1 , wherein the first ribosomal binding site is SEQ ID NO: 28 and the second ribosomal binding site is SEQ ID NO: 29.
4 . The E. coli cell of claim 1 , wherein the coding sequences for the HC and LC are operably linked to regulatory elements that direct expression in the cytoplasm of the E. coli cell.
5 . The E. coli cell of claim 1 , wherein the cell is capable of assembling the expressed HC and LC under non-reducing conditions to form a full-length antibody.
6 . The E. coli cell of claim 1 , wherein the E. coli cell, when cultured under conditions permitting expression, produces a full-length antibody comprising said HC and LC in an amount of at least about 200 mg per liter of culture medium.
7 . The E. coli cell of claim 1 , wherein expression of the HC and/or LC is controlled by a promoter selected from the group consisting of a T7 promoter, a promoter having substantially similar promoter strength as a T7 promoter, a T5 promoter, or a promoter having substantially similar promoter strength as a T5 promoter.
8 . The E. coli cell of claim 1 , wherein the cell comprises:
(a) a plasmid comprising a bicistronic operon including the coding sequence for the HC and the coding sequence for the LC, or
(b) a plasmid comprising a first monocistronic operon for the HC and a second monocistronic operon for the LC.
9 . The E. coli cell of claim 8 , wherein:
(a) the bicistronic operon comprises a promoter that drives expression of both the HC and LC,
wherein the promoter is a T7 promoter or a promoter having substantially similar promoter strength as a T7 promoter, or
(b) the first or second monocistronic operon comprises a T7 promoter.
10 . The E. coli cell of claim 9 , wherein:
(a) the bicistronic operon comprises a T7 terminator, or
(b) the first or second monocistronic operon comprises a T7 terminator.
11 . An E. coli cell with an oxidative cytoplasm, wherein the E. coli cell comprises an expression vector comprising
(a) a coding sequence encoding a full-length antibody heavy chain (HC); and
(b) a coding sequence encoding a full-length antibody light chain (LC)
(c) a first ribosome-binding site for translation of the HC, wherein
wherein:
the first ribosomal binding site is transcribed from a DNA sequence selected from the group consisting of SEQ ID NO: 17-19, and
(d) a second ribosome-binding site for translation of the LC, wherein the second ribosomal binding site is transcribed from a DNA sequence selected from the group consisting of SEQ ID NO: 20-23,
and wherein the cell produces more LC than HC.
12 . The E. coli cell of claim 11 , wherein the first ribosomal binding site has a sequence of SEQ ID NO: 17 or SEQ ID NO: 18.
13 . The E. coli cell of claim 1 , wherein the HC and/or LC of the full-length antibody comprises at least one non-natural amino acid.
14 . The E. coli cell of claim 13 , wherein the coding sequence for the HC and/or the coding sequence for the LC has been modified to comprise at least one non-natural amino acid codon, said codon not resulting in the incorporation of any naturally occurring amino acid.
15 . The E. coli cell of claim 13 , further comprising at least one tRNA charged with a non-natural amino acid, said tRNA containing an anticodon complementary to at least one non-natural amino acid codon present in the coding sequence for the HC and/or LC.
16 . The E. coli cell of claim 15 , wherein said non-natural amino acid codon is the amber codon TAG.
17 . The E. coli cell of claim 15 , wherein said non-natural amino acid is selected from the group consisting of para-methylazido-L-phenylalanine (pAMF), AEK, and pAcF.
18 . The E. coli cell of claim 14 , wherein the codon immediately 3′ to at least one non-natural amino acid codon is codon optimized.
19 . The E. coli cell of claim 1 , wherein the full-length antibody produced is selected from the group consisting of a B10 antibody, an H01 antibody, a 7219 antibody, an anti-PD1 antibody, an anti-Tim3 antibody, an anti-LAG3 antibody, and an anti-Her2 antibody.
20 . The E. coli cell of claim 19 , wherein the coding sequence for the HC of the B10 antibody contains a mutation relative to SEQ ID NO: 1, said mutation resulting in a codon for a natural amino acid selected from F412, Y188, and F249, being substituted with a non-natural amino acid codon, and wherein a non-natural amino acid is introduced to the HC by charging a tRNA complementary to the non-natural amino acid codon.
21 . The E. coli cell of claim 19 , wherein the coding sequence for the LC of the B10 antibody or the coding sequence for the LC of trastuzumab contains at least one mutation relative to SEQ ID NO: 2, said mutation resulting in a codon for a natural amino acid selected from K43 or E162 being substituted with a non-natural amino acid codon.
22 . A method to produce a full-length antibody comprising a heavy chain (HC) and a light chain (LC), wherein the method comprises:
culturing E. coli cells of claim 1 , thereby producing the full-length antibody.
23 . The E. coli cell of claim 1 , wherein the HC and/or LC of the full-length antibody comprises at least one non-natural amino acid.
24 . The E. coli cell of claim 1 , wherein said non-natural amino acid is selected from the group consisting of para-methylazido-L-phenylalanine (pAMF), AEK, and pAcF.
25 . The E. coli cell of claim 1 , wherein the signal peptides of HC and LC are modified such that they are not exported outside the cytoplasm.