CN118318039A

CN118318039A - DNA constructs and host cells for expression of recombinant proteins

Info

Publication number: CN118318039A
Application number: CN202280076114.7A
Authority: CN
Inventors: K·米尔扎德; D·维克斯特罗姆; N·伊斯梅尔; P·萨缪尔森; M·巴拉什基维奇; T·萨利赫; K·塞克尔; D·崔; S·古迪斯; P·埃德布林克; M·卡多; K·史蒂芬森; K·斯特兰伯格
Original assignee: Xbrane Biopharmaceutical Co
Current assignee: Xbrane Biopharmaceutical Co
Priority date: 2021-09-24
Filing date: 2022-09-23
Publication date: 2024-07-09

Abstract

A DNA construct for expressing a recombinant protein, wherein the DNA construct comprises: -at least one of the nucleotide sequences of SEQ ID nos 20 and 21, wherein the nucleotide sequences of SEQ ID nos 20 and 21 are TIR sequences; -a nucleotide sequence encoding a signal peptide; and wherein the nucleotide sequences of SEQ ID Nos 20 and 21 contain at least the first 9 nucleotides of the signal peptide coding sequence.

Description

DNA constructs and host cells for expression of recombinant proteins

Technical Field

The present invention relates to DNA constructs suitable for expression of recombinant proteins in bacterial host cells. The invention also relates to vectors and bacterial host cells containing the DNA construct and to methods for producing the recombinant protein by exposing the bacterial host cell to rhamnose and thereby inducing the expression of the recombinant protein.

Background

Metabolism of rhamnose involves uptake of L-rhamnose into cells by the permease RhaT, followed by isomerization to L-rhamnose by L-rhamnose isomerase (RhaA), followed by further phosphorylation of L-rhamnose by rhamnose kinase (RhaB), and finally hydrolysis by rhamnose-1-phosphate aldolase (RhaD) to produce dihydroxyacetone phosphate and L-lactaldehyde [1]. Genes rhaA, rhaB and rhaD form an operon called rhaBAD and are transcribed with the aid of the rhaBAD promoter [1]. Compared to other systems, the rhamnose metabolic pathway differs in that two transcriptional activators called RhaS and RhaR are required for regulation, as specified below [1].

The rhaBAD operon is a positive regulated catabolic operon which transcribes the rhaB, rhaA and rhaD genes described above, respectively, from the rhaSR operon, with about 240bp DNA between the transcription initiation sites of each [1]. The rhaSR operon encodes RhaS and RhaR, wherein the respective monomers of the dimeric RhaS and RhaR proteins contain two helix-turn-helix motifs and are contacted with two major grooves of DNA. rhaR regulates transcription of rhaSR by binding to promoter DNA spanning bases-32 to-82 relative to the rhaSR transcription initiation site [1]. After rhaSR expression, rhaS binds DNA upstream of the rhaBAD operon at bases-32 to-81 relative to the transcription initiation site to increase rhaBAD expression [1]. In addition, the rhaSR-rhaBAD intergenic region contains a CRP binding site at position-92.5 (CRP 1) relative to the transcription initiation site of the rhaBAD operon and a CRP binding site [1] at positions-92.5 (CRP 2), -115.5 (CRP 3), and-116.5 (CRP 4) relative to the transcription initiation site of the rhaSR operon. Cyclic AMP receptor protein (CRP) regulates expression of more than 100 promoters in escherichia coli.

DNA constructs comprising DNA sequences encoding RhaS, rhaR and rhaBAD promoters are known in the art. US8138324 discloses pTACO and pLEMO derived plasmids (i.e. DNA constructs) comprising DNA sequences encoding RhaS, rhaR and rhaBAD promoter. However, US8138324 does not mention the use of host cells with rhamnose metabolic disorders.

DNA constructs based on pRha derived plasmids comprising DNA sequences encoding RhaS, rhaR and rhaBAD promoter are also known in the art, e.g.from Giacalone et al [5] or Hjelm et al [2]. For example, giacalone et al describe plasmids pRha A and pRha A and Hjelm et al disclose plasmid pRha67K.

Although DNA constructs comprising DNA sequences encoding RhaS, rhaR and rhaBAD promoters are known in the art, many challenges remain, especially in the industrial production of recombinant proteins (particularly monoclonal antibodies or fragments thereof). The main challenges are:

(i) The host cell is stressed, which results in damage to cellular macromolecules (e.g., cell membranes, proteins, and nucleic acids);

(ii) Poor growth of host cells;

(iii) The activity of the produced recombinant protein is poor; and/or

(Iv) The yield of the obtained recombinant protein is low.

Thus, there is a need for improved DNA constructs, as well as host cells and methods suitable for efficient production of recombinant proteins (e.g., monoclonal antibodies or fragments thereof) in high yields.

In particular, there is a need for improved DNA constructs and host cells for efficient production of cetuximab (Certolizumab), a humanized Fab' fragment (from the IgG 1 subtype) of an anti-Tumor Necrosis Factor (TNF) monoclonal antibody with TNF- α affinity. The conjugation of cetuximab with about 40kDa polyethylene glycol (PEG) resulted in cetuximab polyethylene glycol (Certolizumab pegol) which was prepared by UCBThe drugs sold are administered by subcutaneous injection for the treatment of crohn's disease, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis.

For example, EP1287140 and US7012135 disclose DNA constructs for producing cetuximab. However, these DNA constructs contain an unequalized Translation Initiation Region (TIR) and appear to lack the nucleotide sequence encoding the PelB signal peptide, and thus are not the best option for producing cetuximab in high yield.

Typically, in the generation of recombinant expression vectors (e.g., those disclosed in EP1287140 and US 7012135), TIR is formed by fusing the 5' utr (i.e., the untranslated region upstream of the ATG start codon) from the expression vector to the coding sequence of the signal. Each time a different signal peptide is used, a different TIR is generated. Such TIR are referred to as non-evolved because they are formed by specific gene fusions and are not synthetically evolved TIR as described in the present invention and in US10696963 and WO 21158163.

Patents such as US6828121 and EP1341899 relate to host cells for the production of various types of antibodies and antibody fragments, such as humanized Fab' fragments. Specific examples of some antibodies that may be produced by these host cells are anti-IgE, anti-IgG, anti-Her-2, anti-CD 11a, anti-CD 18, anti-CD 20 and anti-VEGF. Examples of host cells disclosed in US6828121 and EP1341899 are e.coli strains deficient in chromosomal DegP and Prc encoding proteases DegP and Prc, respectively, which carry a mutated spr gene, wherein the mutated spr gene product is characterized by a tryptophan to arginine at position 148. However, neither US6828121 nor EP1341899 mention (a) host cell mutations associated with rhamnose metabolism, and (b) the production of specific Fab' fragments (e.g. cetuzumab).

International patent application WO21158163 relates to DNA constructs containing synthetically evolved TIR for modulating the properties of signal peptides in recombinant protein production. WO21158163 clearly shows that synthetically evolved TIR has technical advantages over non-evolved TIR. However, WO21158163 does not mention synthetically evolved TIR specifically developed for optimal expression of cetuximab.

WO21158163 also relates to nucleotide sequences for expressing Pelb signal peptides. However, WO21158163 does not mention nucleotide sequences specifically developed for optimal expression of cetuximab.

Thus, there is a need to optimize DNA constructs, host cells, TIR, and signal peptides to express recombinant proteins (e.g., cetuzumab).

Object of the Invention

It is an object of the present invention to provide the beneficial technical effects of DNA constructs.

It is another object of the present invention to provide the beneficial technical effects of TIR.

It is a further object of the present invention to provide beneficial technical effects of the host cell.

It is another object of the present invention to provide beneficial technical effects of the signal peptide nucleotide sequence.

It is another object of the present invention to provide an advantageous method for efficiently producing recombinant proteins.

Disclosure of Invention

The objects of the invention have been achieved by any one or more aspects of the invention disclosed below.

The first aspect of the invention relates to a DNA construct suitable for expressing cetuximab in a host cell, wherein the cetuximab comprises: (i) a light chain comprising the amino acid sequence of SEQ ID 3; (ii) A heavy chain comprising the amino acid sequence of SEQ ID 4,

Wherein the DNA construct comprises a nucleotide sequence encoding cetuximab, wherein the DNA construct further comprises at least one nucleotide sequence encoding a signal peptide operably linked in the direction of transcription to a nucleotide sequence encoding a light chain of cetuximab and/or a heavy chain of cetuximab, wherein the DNA construct further comprises a nucleotide sequence encoding:

the presence of a promoter which,

The RhaR transcriptional activator is selected from the group consisting of,

The RhaS transcriptional activator is selected from the group consisting of,

The marker of resistance to antibiotics,

At least one terminator, and

-An origin of replication.

In a preferred embodiment, the DNA construct is characterized in that:

the presence of a promoter which,

The RhaR transcriptional activator is selected from the group consisting of,

The RhaS transcriptional activator is selected from the group consisting of,

The marker of resistance to antibiotics,

A promoter operably linked to a nucleotide sequence encoding an antibiotic resistance marker,

At least one terminator, and

-An origin of replication.

In a preferred embodiment, the DNA construct is characterized in that:

the rhaBAD promoter is used in the expression of,

The RhaR transcriptional activator is selected from the group consisting of,

The RhaS transcriptional activator is selected from the group consisting of,

The marker of resistance to antibiotics,

The rrnB T1 terminator,

-RrnB T2 terminator, and

-A pMB1 origin of replication.

In a preferred embodiment, the DNA construct is characterized in that:

The antibiotic resistance marker is a kanamycin resistance marker, preferably a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12 or a sequence having at least 90% sequence identity thereto;

the promoter operably linked to a nucleotide sequence encoding an antibiotic resistance marker is an AmpR promoter, preferably an AmpR promoter comprising the nucleotide sequence of SEQ ID 13 or a sequence having at least 90% sequence identity thereto;

the rrnB T1 terminator comprises the nucleotide sequence of SEQ ID 14 or a sequence having at least 90% sequence identity thereto;

The rrnB T2 terminator comprises the nucleotide sequence of SEQ ID 15 or a sequence having at least 90% sequence identity thereto; and/or

The pMB1 origin of replication comprises the nucleotide sequence of SEQ ID 16 or a sequence having at least 90% sequence identity thereto.

In a preferred embodiment, the DNA construct is characterized in that:

-the antibiotic resistance marker is a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12;

the promoter operably linked to the nucleotide sequence encoding the antibiotic resistance marker is an AmpR promoter comprising the nucleotide sequence of SEQ ID 13;

The rrnB T1 terminator comprises the nucleotide sequence of SEQ ID 14;

The rrnB T2 terminator comprises the nucleotide sequence of SEQ ID 15; and/or

The pMB1 origin of replication comprises the nucleotide sequence of SEQ ID 16.

In a preferred embodiment, the DNA construct is characterized in that:

The rhaBAD promoter comprises the nucleotide sequence of SEQ ID 8 or a sequence having at least 90% sequence identity thereto;

-RhaR transcriptional activator comprises the nucleotide sequence of SEQ ID 9 or a sequence having at least 90% sequence identity thereto;

-RhaS transcriptional activator comprises the nucleotide sequence of SEQ ID 11 or a sequence having at least 90% sequence identity thereto;

-the antibiotic resistance marker is a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12 or a sequence having at least 90% sequence identity thereto;

the AmpR promoter comprises the nucleotide sequence of SEQ ID 13 or a sequence having at least 90% sequence identity thereto;

The rrnB T2 terminator comprises the nucleotide sequence of SEQ ID 15 or a sequence having at least 90% sequence identity thereto; and

In a preferred embodiment, the DNA construct is characterized in that:

The rhaBAD promoter comprises the nucleotide sequence of SEQ ID 8;

-RhaR transcriptional activator comprises the nucleotide sequence of SEQ ID 9;

-RhaS transcriptional activator comprises the nucleotide sequence of SEQ ID 11;

the AmpR promoter comprises the nucleotide sequence of SEQ ID 13;

The rrnB T1 terminator comprises the nucleotide sequence of SEQ ID 14;

The rrnB T2 terminator comprises the nucleotide sequence of SEQ ID 15; and

The pMB1 origin of replication comprises the nucleotide sequence of SEQ ID 16.

In preferred embodiments, the DNA construct may comprise one or more restriction sites that can be cut by restriction enzymes such as EcoRI、NdeI、NotI、XhoI、PspXI、PaeR71、BbsI、StyI、AvrII、BanI、Acc65I、KpnI、Eco53kI、SacI、BamHI、XbaI、SalI、AccI、PstI、SbfI、SphI and/or HindIII.

In a preferred embodiment, the DNA construct further comprises a nucleotide sequence encoding said recombinant protein operably linked to a rhaBAD promoter, wherein said recombinant protein is a monoclonal antibody or fragment thereof, preferably said recombinant protein is cetuzumab. More preferably, the recombinant protein is cetuzumab comprising (i) a light chain comprising the amino acid sequence of SEQ ID 3, and/or (ii) a heavy chain comprising the amino acid sequence of SEQ ID 4.

In preferred embodiments, the DNA construct comprises a nucleotide sequence encoding a recombinant protein operably linked to a rhaBAD promoter comprising (i) a nucleotide sequence encoding a light chain of cetuzumab comprising the sequence of SEQ ID 5 or a sequence having at least 90% sequence identity thereto, and/or (ii) a nucleotide sequence encoding a heavy chain of cetuzumab comprising the sequence of SEQ ID 6 or a sequence having at least 90% sequence identity thereto; preferably, the nucleotide sequence encoding the recombinant protein comprises (i) a nucleotide sequence encoding a cetuximab light chain comprising the sequence of SEQ ID 5, and/or (ii) a nucleotide sequence encoding a cetuximab heavy chain comprising the sequence of SEQ ID 6.

In one embodiment, the DNA construct further comprises a nucleotide sequence encoding a recombinant protein operably linked to the rhaBAD promoter, comprising at least one nucleotide sequence encoding a signal peptide operably linked to one or both of the nucleotide sequences of SEQ ID 5 and SEQ ID 6 in the direction of transcription, preferably the signal peptide is a PelB (pectin lyase B) signal peptide.

The nucleotide sequence encoding the PelB signal peptide, which is operably linked to the nucleotide sequence of the light chain of cetuzumab in the direction of transcription, is referred to herein as PelB1. The nucleotide sequence of PelB1 comprises the sequence of SEQ ID 18 or a sequence having at least 90% sequence identity thereto.

The nucleotide sequence encoding the PelB signal peptide operably linked to the nucleotide sequence of the cetuximab heavy chain in the direction of transcription is referred to as PelB2 in the present invention. The nucleotide sequence of PelB2 comprises the sequence of SEQ ID 19 or a sequence having at least 90% sequence identity thereto.

The resulting PelB signal peptide comprises the amino acid sequence of SEQ ID 7 [6]: MKYLLPTAAAGLLLLAAQPAMA.

In one embodiment, the DNA construct comprises TIR with the nucleotide sequence of SEQ ID 20, wherein the sequence of SEQ ID 20 comprises at least the first 9 nucleotides of the nucleotide sequence of PelB1, i.e. the first 9 nucleotides of SEQ ID 18. This particular TIR is also referred to herein as TIR-LC.

In one embodiment, the DNA construct comprises TIR with the sequence of SEQ ID 21, wherein the sequence of SEQ ID 21 comprises at least the first 9 nucleotides of the nucleotide sequence of PelB2, i.e. the first 9 nucleotides of SEQ ID 19. This particular TIR is also referred to herein as TIR-HC.

In a preferred embodiment, the DNA construct comprises the sequence of SEQ ID 17 or a sequence having at least 90% sequence identity thereto, preferably comprising the sequence of SEQ ID 17.

The second aspect of the invention relates to a DNA construct for expressing a recombinant protein, wherein the DNA construct comprises:

-at least one of the nucleotide sequences of SEQ ID nos 20 and 21, wherein the nucleotide sequences of SEQ ID nos 20 and 21 are TIR sequences; and

-A nucleotide sequence encoding a signal peptide;

Wherein the nucleotide sequences of SEQ ID Nos 20 and 21 comprise at least the first 9 nucleotides of the signal peptide coding sequence.

In one embodiment, the nucleotide sequence encoding the signal peptide is operably linked to:

-a first nucleotide sequence encoding an antibody light chain; and/or

-A second nucleotide sequence encoding a heavy chain of the antibody.

In one embodiment, the DNA construct comprises a Shine-Dalgarno sequence. The Shine-Dalgarno sequence is located upstream of the ATG start codon of the nucleotide sequence encoding the signal peptide. In one embodiment, the Shine-Dalgarno sequence is located upstream of an ATG start codon encoding a nucleotide sequence of a signal peptide operably linked to the light chain and/or heavy chain of an antibody. In one embodiment, the Shine-Dalgarno sequence comprises nucleotide sequences AGGAGGAA and/or GAGGAGAA in the direction of transcription. Preferably AGGAGGAA is located upstream of the nucleotide sequence encoding the light chain of the antibody. Preferably GAGGAGAA is located upstream of the nucleotide sequence encoding the heavy chain of the antibody. More preferably AGGAGGAA is located upstream of the TIR-LC. More preferably GAGGAGAA is located upstream of the TIR-HC.

In one embodiment, a first nucleotide sequence encoding a signal peptide (e.g., pelB 1) is operably linked to a first nucleotide sequence encoding an antibody light chain.

In one embodiment, a second nucleotide sequence encoding a signal peptide (e.g., pelB 2) is operably linked to a second nucleotide sequence encoding an antibody heavy chain.

In one embodiment, the first and second nucleotide sequences encoding the light and heavy chains, respectively, of the antibody encode the amino acid sequences of SEQ ID No 3 and SEQ ID No 4, respectively.

In one embodiment, the first and second nucleotide sequences encoding the light and heavy chains, respectively, of an antibody comprise the nucleotide sequences of SEQ ID No 5 and SEQ ID No 6, respectively.

The third aspect of the present invention relates to a DNA construct for expressing a signal peptide, wherein the DNA construct comprises a nucleotide sequence encoding a PelB signal peptide, wherein the nucleotide sequence encoding the PelB signal peptide comprises at least one of the nucleotide sequences of SEQ ID No 18 and SEQ ID No 19. In one embodiment, the DNA construct comprises the nucleotide sequences of SEQ ID No 18 and SEQ ID No 19.

A fourth aspect of the invention relates to a DNA construct comprising a nucleotide sequence encoding an amino acid sequence, wherein the amino acid sequence comprises:

a. Amino acid sequence of cetuzumab;

b. a first signal peptide of the amino acid sequence of SEQ ID No 7 fused to the N-terminus of the light chain amino acid sequence of cetuximab; and

C. A second signal peptide of the amino acid sequence of SEQ ID NO 7 fused to the N-terminus of the heavy chain amino acid sequence of cetuximab; and

In one embodiment, the nucleotide sequences encoding the first and second signal peptides comprise the nucleotide sequences of SEQ ID No 18 and SEQ ID No 19, respectively.

A fifth aspect of the invention relates to an expression vector comprising any of the DNA constructs of the first, second, third and/or fourth aspects of the invention.

A sixth aspect of the invention relates to a host cell, characterized in that the chromosome comprises:

a. Mutations in the nucleotide sequence encoding RhaB that disable rhamnose metabolism;

Mutations in the DegP gene that result in (i) expression of the DegP protease and/or (ii) a failure of the DegP protease's activity;

mutations in the Prc gene that result in (i) expression of Prc protease and/or (ii) failure of Prc protease activity;

mutation in the spr gene.

In one embodiment, the mutation is selected from the group consisting of a frameshift, a deletion, a substitution, and an insertion.

In one embodiment, the mutation in the nucleotide sequence encoding RhaB that disrupts rhamnose metabolism is a frameshift mutation in the nucleotide sequence encoding RhaB.

In one embodiment, the mutation in the degP gene is a degP deletion.

In one embodiment, the mutation in the prc gene is a prc deletion.

In one embodiment, the mutation in the spr gene is a sprW R mutation, characterized in that the substitution in the spr gene results in a change in tryptophan to arginine at position 148.

In one embodiment, the host cell is characterized in that the chromosome comprises:

Mutations in the DegP gene that result in failure of expression of DegP protease;

Mutations in the Prc gene that result in failure of Prc protease expression; and

Mutation in the spr gene.

In one embodiment, the host cell is a bacterial cell, more preferably E.coli, most preferably E.coli W3110.

In one embodiment, the host cell is E.coli W3110 comprising a chromosome comprising a frameshift mutation in the nucleotide sequence encoding RhaB. This particular host cell is referred to herein as E.coli W3110rhaBfs and XB17.

In one embodiment, the host cell is E.coli W3110 rhaBfs further comprising a chromosome containing the degP deletion. This particular host cell is referred to herein as E.coli W3110 rhaBfs ΔDegP and XB83.

In one embodiment, the host cell is E.coli W3110 rhaBfs DeltaDegP further comprising a chromosome containing the prc deletion. This particular host cell is referred to herein as E.coli W3110 rhaBfs DeltadegP Deltaprc and XB152.

In one embodiment, the host cell is E.coli W3110rhaBfs ΔdegP Δprc further comprising a chromosome containing the sprW R mutation. This particular host cell is referred to herein as E.coli W3110rhaBfs ΔDegPΔ prc sprW148R and XB166.

A seventh aspect of the invention relates to a host cell according to the sixth aspect of the invention comprising a DNA construct according to the first, second, third and/or fourth aspects of the invention.

An eighth aspect of the invention relates to a method for producing a recombinant protein comprising the step of exposing a host cell of the seventh aspect of the invention to rhamnose, thereby inducing the expression of said recombinant protein. In a preferred embodiment, the method further comprises the step of recovering the recombinant protein from the bacterial host cell; and optionally further comprising one or more steps of purifying the recovered recombinant protein, preferably by one or more chromatographic steps.

A ninth aspect of the invention relates to a method for producing a recombinant protein comprising the step of introducing a DNA construct according to the first, second, third and/or fourth aspect of the invention into a host cell according to the sixth aspect of the invention.

In one embodiment, the method further comprises the step of exposing the host cell to rhamnose, thereby inducing expression of the recombinant protein.

In one embodiment, the method further comprises the step of recovering the recombinant protein from the host cell; and optionally further comprising one or more steps of purifying the recovered recombinant protein, preferably by one or more chromatographic steps.

In one embodiment, the method further comprises the step of derivatizing the purified recombinant protein, preferably with a polyethylene glycol moiety, more preferably with a polyethylene glycol moiety of about 40 kDa.

A tenth aspect of the invention relates to a recombinant protein obtainable by the method of the ninth aspect of the invention. The recombinant protein is preferably an antibody or fragment thereof, more preferably a Fab' fragment antibody, most preferably cetuximab.

An eleventh aspect of the invention relates to a cetuximab antibiotic analogue obtainable by the method of the ninth aspect of the invention. Preferably, the cetuximab antibiotic analogue comprises a polyethylene glycol moiety, for example a polyethylene glycol moiety of about 40 kDa. In order to preserve the molecular structure of the cetuximab antibiotic, the cetuximab antibiotic is disclosed herein as a method-defined product. The biosimilar is highly similar to the reference product, i.e., cetuximab anti-biosimilar is polyethylene glycol of cetuximab produced by sponsors of the reference productHas highly similar molecular structure and bioactivity. Furthermore, the biosimilar drug is not clinically different from the reference product, and the clinical trial performed on the biosimilar drug evaluates pharmacokinetics and immunogenicity. However, the minor structural differences between the cetuximab antibiotic of the invention and the cetuximab polyethylene glycol of the reference product sponsor are due in part to the method of the ninth aspect of the invention (as well as the eighth aspect of the invention).

A twelfth aspect of the invention relates to a cetuximab antibiotic analogue or a derivative thereof for use as a medicament, preferably for the treatment of crohn's disease, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis. The derivative preferably comprises a polyethylene glycol moiety, for example a polyethylene glycol moiety of about 40 kDa. One embodiment of the invention relates to a method of treating a disease by using a cetuximab antibiotic analogue. The disease may be Crohn's disease, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis.

A thirteenth aspect of the invention relates to a method for producing a signal peptide comprising the step of introducing a DNA construct according to the first, second, third and/or fourth aspect of the invention into a host cell according to the sixth aspect of the invention.

In some embodiments, one or more of SEQ ID 1-21 of the various aspects of the invention described above (and embodiments thereof) may be substituted with a sequence having at least 90% sequence identity thereto. The term "sequence identity" as used herein is used in reference to amino acid or nucleotide sequences, and sequence identity is over the entire length of a particular sequence. Thus, a sequence may be a sequence that is at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a specified amino acid or nucleotide sequence. Thus, such sequences of the invention include single or multiple nucleotide or amino acid changes (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level, preferred sequences having the sequence identity defined above contain up to 5, for example 1,2, 3, 4 or 5, preferably 1,2 or 3, more preferably 1 or 2, altered amino acids in the sequences of the invention.

Drawings

Plasmid map of FIGS. 1-KTXHIS

The nucleotide sequence of the Multiple Cloning Site (MCS) of FIGS. 2-KTXHIS

FIG. 3-KTXHIS-plasmid map of Cert-PelB1-LC-PelB2-HC

FIG. 4-example of TIR library selection and isolation of evolved TIR therefrom

FIG. 5 Western blot analysis of Medium Components

FIG. 6-Western blot analysis of total fractions

FIG. 7-NanoDrop ^TM andChromatography was performed to detect the yield and titer differences between expression system XB62 (containing XB17 host cells with expression vector D37 with non-evolving TIR) and XB102 (containing XB17 host cells with expression vector E83 with synthetically evolving TIR for modulating cetuximab heavy chain expression).

FIG. 8-comparative expression analysis by periplasmic extraction followed by affinity-HPLC: e83 expression vector expressed in XB17 host cell and E83 vector expressed in XB166 host cell

FIG. 9-comparative expression analysis by periplasmic extraction followed by affinity-HPLC: e83 expression vector expressed in XB166 host cell and E111 vector expressed in XB166 host cell

Detailed Description

Particular embodiments of the invention relate to DNA constructs for expressing antibodies, wherein the DNA constructs comprise improved TIR of SEQ ID 20:

TTGCTCATGAAGTAT

another embodiment relates to a DNA construct for expressing an antibody, wherein the DNA construct comprises the improved TIR of SEQ ID 21:

TGTTAAATGAAGTAT

TIR of SEQ ID 20 and SEQ ID 21 may be contained in the same DNA construct. An example of such an embodiment is that TIR of SEQ ID 20 is located upstream of the nucleotide sequence of the light chain expressing the antibody or fragment thereof (e.g., cetuzumab), while TIR of SEQ ID 21 is located upstream of the nucleotide sequence of the heavy chain expressing the antibody or fragment thereof (e.g., cetuzumab).

Particular embodiments of the present invention relate to improved nucleotide sequences of SEQ ID 18 encoding a PelB signal peptide operably linked to nucleotide sequences encoding an antibody chain:

ATGAAGTATCTTCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCA CAGCCTGCAATGGCA

Another embodiment relates to an improved nucleotide sequence of SEQ ID 19 encoding a PelB signal peptide operably linked to a nucleotide sequence encoding an antibody chain:

ATGAAGTATCTGTTGCCGACTGCTGCAGCGGGACTGCTGCTGTTAGCGGCA CAACCGGCGATGGCG

The PelB nucleotide sequences of SEQ ID 18 and SEQ ID 19 may be contained in the same DNA construct. An example of such a DNA construct is the PelB nucleotide sequence of SEQ ID 18 operably linked to a nucleotide sequence encoding the light chain of an antibody or fragment thereof (e.g., cetuzumab), while the PelB nucleotide sequence of SEQ ID 19 is operably linked to a nucleotide sequence encoding the heavy chain of an antibody or fragment thereof (e.g., cetuzumab).

However, in other embodiments, the sequences of SEQ ID 18-21 described above may be contained in the same DNA construct. In such embodiments, the TIR nucleotide sequences of SEQ ID No 20 and SEQ ID No 21 will comprise at least the first 9 nucleotides of the signal peptide nucleotide sequences of SEQ ID No 18 and SEQ ID No 19.

Other embodiments of the invention may relate to regulating the production of recombinant proteins (e.g., cetuzumab) based on the L-rhamnose rhaBAD promoter. In other words, cetuximab can be produced by:

a. cloning a nucleotide sequence encoding cetuzumab into a DNA construct such that the nucleotide sequence is operably linked to the rhaBAD promoter, and

B. the resulting nucleotide sequence is introduced into a bacterial host cell comprising a chromosome comprising a mutation or modification that disrupts rhamnose metabolism.

In one embodiment, the nucleotide sequence of the rhaBAD promoter comprises the sequence of SEQ ID 8 (wherein the sequence is referred to in FIGS. 1 and 3 "rhaBAD")：CACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCA ATGGCCCATTTTCTTGTCAGTAACGAGAAGGTCGCGAATCCAGGCGCTTTTTAG ACTGGTCGTA.

The DNA construct may comprise a nucleotide sequence encoding a RhaR transcriptional activator. In an embodiment of the invention, the nucleotide sequence of the RhaR transcriptional activator comprises the sequence of SEQ ID 9 (wherein the sequence is referred to as "rhaR" in fig. 1 and 3):

ATGGCTTTCTGCAATAACGCGAATCTTCTCAACGTATTTGTACGCCATATTGCGAATAATCAACTTCGTTCTCTGGCCGAGGTAGCCACGGTGGCGCATCAGTTAAAACTTCTCAAAGATGATTTTTTTGCCAGCGACCAGCAGGCAGTCGCTGTGGCTGACCGTTATCCGCAAGATGTCTTTGCTGAACATACACATGATTTTTGTGAGCTGGTGATTGTCTGGCGCGGTAATGGCCTGCATGTACTCAACGATCGCCCTTATCGCATTACCCGTGGCGATCTCTTTTACATTCATGCTGATGATAAACACTCCTACGCTTCCGTTAACGATCTGGTTTTGCAGAATATTATTTATTGCCCGGAGCGTCTGAAGCTGAATCTTGACTGGCAGGGGGCGATTCCGGGATTTAACGCCAGCGCAGGGCAACCACACTGGCGCTTAGGTAGCATGGGGATGGCGCAGGCGCGGCAGGTTATTGGTCAGCTTGAGCATGAAAGTAGTCAGCATGTGCCGTTTGCTAACGAAATGGCTGAGTTGCTGTTCGGGCAGTTGGTGATGTTGCTGAATCGCCATCGTTACACCAGTGATTCGTTGCCGCCAACATCCAGCGAAACGTTGCTGGATAAGCTGATTACCCGGCTGGCGGCTAGCCTGAAAAGTCCCTTTGCGCTGGATAAATTTTGTGATGAGGCATCGTGCAGTGAGCGCGTTTTGCGTCAGCAATTTCGCCAGCAGACTGGAATGACCATCAATCAATATCTGCGACAGGTCAGAGTGTGTCATGCGCAATATCTTCTCCAGCATAGCCGCCTGTTAATCAGTGATATTTCGACCGAATGTGGCTTTGAAGATAGTAACTATTTTTCGGTGGTGTTTACCCGGGAAACCGGGATGACGCCCAGCCAGTGGCGTCATCTCAATTCGCAGAAAGAT.

The DNA construct may also comprise a nucleotide sequence encoding an extension (extension) of the RhaR transcriptional activator in frame with RhaR due to a lost stop codon. In an embodiment of the invention, the extended nucleotide sequence of the RhaR transcriptional activator comprises the sequence of seq id 10 (wherein the sequence is referred to as "extended rhaR" in fig. 1 and 3):

AGACGAAAGGGCCTCGTGATACGCCTATTTTTATAG.

The DNA construct may comprise a nucleotide sequence encoding a RhaS transcriptional activator. In an embodiment of the invention, the nucleotide sequence of the RhaS transcriptional activator comprises the sequence of SEQ ID 11 (wherein the sequence is referred to in figures 1 and 3 "rhaS")：ATGACCGTATTACATAGTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAACCCCGGCTCCCGCAGGCGGATTTTCCTGAACATCATCATGATTTTCATGAAATTGTGATTGTCGAACATGGCACGGGTATTCATGTGTTTAATGGGCAGCCCTATACCATCACCGGTGGCACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATGAACATACCGATAATCTGTGTCTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCCGGGCTGAATCAGTTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAGCGTATTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTACCCTCGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGCAGTTTGCAGGAGAACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTGGAGGACCATTTTGCCGATGAGGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCACTGCGTACGCTACATCGGCAGCTTAAGCAGCAAACGGGACTGACGCCTCAGCGATACCTGAACCGCCTGCGACTGATGAAAGCCCGACATCTGCTACGCCACAGCGAGGCCAGCGTTACTGACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTCGACGCTTTTTCGCCGAGAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGCAATAA.

The DNA construct may comprise a nucleotide sequence encoding an "antibiotic resistance marker" or a "selectable marker". Such markers are DNA fragments containing genes whose products confer resistance to antibiotics (e.g., chloramphenicol, ampicillin, gentamicin, streptomycin, tetracycline, kanamycin, neomycin) or the ability to grow on selective media (e.g., ura (uracil), leu (leucine), trp (tryptophan), his (histidine)). Typically, the plasmid contains an antibiotic resistance marker to force the bacterial cell to maintain the plasmid. In embodiments of the invention, the DNA construct may comprise the nucleotide sequence of a kanamycin resistance marker. In a specific embodiment of the present invention, the nucleotide sequence conferring kanamycin resistance comprises the sequence of SEQ ID 12 (wherein the sequence is referred to as "KanR" in FIGS. 1 and 3)

ATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA.

The DNA construct may comprise a nucleotide sequence encoding a promoter operably linked to a nucleic acid sequence encoding an antibiotic resistance marker. Such promoters may increase the expression of the antibiotic resistance markers discussed in the preceding paragraphs. In an embodiment of the invention, the ampicillin resistance promoter is an AmpR promoter which promotes not only the expression of the ampicillin resistance marker but also the expression of the kanamycin resistance marker. In a specific embodiment of the invention, the nucleic acid sequence of the AmpR promoter comprises the sequence of SEQ ID 13 (wherein, this sequence is referred to as "AmpR promoter" in fig. 1 and 3):

CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT

The DNA construct may comprise nucleotide sequences encoding a rrnB T1 terminator and a rrnB T2 terminator. The rrnB T1 and T2 terminators are both effective transcription terminators in isolated form, but when used together, the rrnB T1 and T2 terminators can terminate transcription more effectively.

In one embodiment, the nucleotide sequence of the rrnB T1 terminator comprises the sequence of SEQ ID 14:

CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGT TGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT

In one embodiment, the nucleotide sequence of the rrnB T2 terminator comprises the sequence of SEQ ID 15:

AGAAGGCCATCCTGACGGATGGCCTTTT

The DNA construct may further comprise an origin of replication, which is a specific nucleotide sequence that initiates DNA replication. DNA replication may proceed bi-directionally or uni-directionally from this point. Some commonly used origins of replication are ColE1, pMB1, pSC101, R6K, pBR, R6K, p15A and pUC. In an embodiment of the invention, the origin of replication is pMB1 or a derivative thereof. In a specific embodiment of the invention, the nucleic acid sequence of pMB1 comprises the sequence of SEQ ID 16:

TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA.

in one embodiment, the DNA construct is an expression vector comprising a nucleotide sequence encoding one or more of the following:

the rhaBAD promoter is used in the expression of,

The RhaR transcriptional activator is selected from the group consisting of,

The RhaS transcriptional activator is selected from the group consisting of,

The marker of resistance to antibiotics,

A promoter operably linked to a nucleic acid encoding an antibiotic resistance marker,

At least one terminator, and

-An origin of replication.

In one embodiment, the expression vector comprises a nucleotide sequence encoding:

the rhaBAD promoter is used in the expression of,

The RhaR transcriptional activator is selected from the group consisting of,

The RhaS transcriptional activator is selected from the group consisting of,

The marker of resistance to antibiotics,

A promoter operably linked to a nucleic acid sequence encoding an antibiotic resistance marker,

The rrnB T1 terminator,

-RrnB T2 terminator, and

-An origin of replication.

the rhaBAD promoter is used in the expression of,

The RhaR transcriptional activator is selected from the group consisting of,

The RhaS transcriptional activator is selected from the group consisting of,

The kanamycin resistance marker,

The sequence of the AmpR promoter,

The rrnB T1 terminator,

-RrnB T2 terminator, and

-A pMB1 origin of replication.

rhaBAD promoter comprising the nucleotide sequence of SEQ ID 8,

RhaR transcriptional activator comprising the nucleotide sequence of SEQ ID 9,

RhaS transcriptional activator comprising the nucleotide sequence of SEQ ID 11,

Kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12,

An AmpR promoter comprising the nucleotide sequence of SEQ ID 13,

RrnB T1 terminator comprising the nucleotide sequence of SEQ ID 14,

-RrnB T2 terminator comprising the nucleotide sequence of SEQ ID 15, and

-A pMB1 origin of replication comprising the nucleotide sequence of SEQ ID 16.

The nucleotide sequence encoding the recombinant protein to be cloned into the above-described DNA construct may comprise a nucleic acid encoding a monoclonal antibody or fragment thereof, preferably cetuzumab. The nucleic acid encoding cetuximab comprises nucleic acids encoding the light and heavy chains of cetuximab.

In one embodiment, the nucleotide sequence encoding the light chain of cetuximab comprises the sequence of SEQ ID 5:

gatattcagatgactcagagcccaagttcgctgagcgcttctgttggcgatcgtgtgaccattacatgcaaagcctcacagaacgttggtaccaatgtcgcctggtatcagcagaaacctggaaaagcgcccaaagcgctcatctactcagcgagcttcctgtattcaggcgtgccgtatcgctttagcggctctggttccggtacagactttaccctcacgatttcgtccttacaaccggaagatttcgccacgtactattgccagcaatacaacatctatccgctgacctttggacaaggcaccaaagtggagatcaaacgcactgttgctgcaccgagtgtgttcatctttccaccgtctgatgagcagctgaagtctggtacagcaagtgttgtgtgtctgctgaacaacttctatccgcgtgaagctaaagtacagtggaaagtcgacaatgccttgcaatccgggaatagccaggaaagcgtgactgaacaggacagcaaggattcgacctacagtctgagcagtaccttaaccttgtcgaaagcggattacgagaaacacaaggtctatgcctgtgaagtcacgcaTCAAGGCCTGTCATCGCCTGTTACTAAATCATTTAATAGAGGAGAATGTTAA

In a specific embodiment of the invention, the nucleotide sequence encoding the heavy chain of cetuximab comprises the sequence of SEQ ID 6:

gaagtgcagcttgtggagtctggaggtggcttagtccagccaggtggttccctgcgcttgtcctgtgcagcgagcgggtatgtAttcacagattatggcatgaactgggttcggcaagcaccaggcaaaggcctcgaatggatggggtggatcaacacgtatattggggaaccgatttatgcggatagcgtcaaaggtcgcttcacgttcagtctggataccagcaaatcaaccgcgtatctccagatgaatagcctccgtgctgaagatactgccgtgtactactgtgcgcgtggttatcgcagttatgcgatggattactggggccaaggcaccttagtcaccgttagttctgcctccaccaaaggcccatcagtgtttccgctggccccttcgtctaaatcgacgagtggtggcacagccgcactgggatgcctggtcaaagactactttcccgaacctgtaaccgtaagctggaatagtggtgctttgacctcaggcgtgcatacgtttccggctgtcctgcagtcatccggtctgtactcgctttcgagcgttgttactgtaccctctagctccctgggcacccagacgtacatctgcaatgtgaaccataagccgtcgaacaccaaagtggacaagaaagttgagccgaaaagctgcgacaaaacgcacacatgtgccgccTAA

In one embodiment, the nucleic acid encoding the light and heavy chains of cetuximab further comprises a nucleotide sequence encoding a signal peptide operably linked to one or both of the nucleotide sequences encoding the heavy and light chains of cetuximab. The signal peptide is preferably selected from the group consisting of MalE, ompA, phoA, dsbA and Pelb. The nucleic acid sequence encoding the signal peptide is preferably a nucleotide sequence encoding a PelB signal peptide.

In one embodiment, the nucleotide sequence encoding the PelB signal peptide is operably linked to a nucleotide sequence encoding a cetuzumab light chain, which comprises the sequence of SEQ ID 18 and is also referred to herein as PelB signal sequence 1 (see fig. 3), abbreviated PelB1:

ATGAAGTATCTtCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCA CAGCCTGCAATGGCA

In one embodiment, the nucleotide sequence encoding the PelB signal peptide is operably linked to a nucleotide sequence encoding a cetuximab heavy chain, which comprises the sequence of SEQ ID 19 and is also referred to herein as PelB signal sequence 2 (see fig. 3), abbreviated PelB2:

ATGAAGTATCTGTTGCCGACTGCTGCAGCGGGACTGCTGCTGTTAGCGGCA CAACCGGCGATGGCG

in one embodiment, the DNA construct comprises the improved TIR of SEQ ID 20:

TTGCTCATGAAGTAT

in one embodiment, the DNA construct comprises the improved TIR of SEQ ID 21:

TGTTAAATGAAGTAT

In one embodiment, the DNA construct comprises TIR of nucleotide sequences SEQ ID No 20 and SEQ ID No 21, wherein these TIR nucleotide sequences will comprise at least the first 9 nucleotides of the signal peptide nucleotide sequences of SEQ ID 18 and SEQ ID 19, respectively.

In a specific embodiment of the invention, the DNA construct comprises the nucleotide sequence of SEQ ID 17, also referred to herein as KTXHIS-Cert-PelB1-LC-PelB2-HC. The DNA construct is preferably an expression vector.

The bacterial host cells used to produce the recombinant proteins comprise chromosomes with mutations or modifications that disable rhamnose metabolism. The bacterial host cell may be an E.coli cell. In a preferred embodiment, the bacterial host cell is an E.coli K-12 cell, more preferably the bacterial host cell is an E.coli W3110 cell. Failure of rhamnose metabolism is achieved by a mutation in the nucleotide sequence encoding RhaB that inactivates RhaB. Or by using a bacterial host cell having a chromosome with a deletion of the nucleotide sequence encoding RhaB; for example, this may be achieved by deleting the nucleotide sequence encoding RhaB. Preferably, the chromosome of the bacterial host cell comprises a nucleic acid sequence encoding RhaT, i.e. the RhaT gene is intact.

In a specific embodiment of the invention, the bacterial host cell is E.coli W3110 rhaBfs ΔDegPΔ prc sprW148R.

In particular embodiments of the invention, the bacterial host cell E.coli W3110 rhaBfs ΔDegPΔ prc sprW148R comprises a KTXHIS-Cert-PelB1-LC-PelB2-HC expression vector and is used in a method for expressing cetuzumab, which method can be used to produce cetuzumab antibiotic biosimilar.

The biosimilar is highly similar to the reference product, i.e., the cetuximab biosimilar has the cetuximab polyethylene glycol produced by the sponsor of the reference product (i.e., the original research drug manufacturer)Highly similar molecular structure and function (i.e., bioactivity). Furthermore, the biosimilar drug is not clinically different from the reference product, and the clinical trial performed on the biosimilar drug evaluates pharmacokinetics and immunogenicity. Most importantly, biological analogues: a) meets the approval criteria of the medical institution, (b) is produced in a factory licensed by the medical institution, (c) is continuously tracked as part of post-market supervision to continuously ensure security (as shown in https:// www.fda.gov/media/108905/download).

The invention may be illustrated by the disclosures of examples 1 to 9 in the following non-limiting examples section.

It is to be understood that these examples relate to XB166 host cells and KTXHIS-Cert-PelB1-LC-PelB2-HC expression vectors, and their use in combination, although the preferred embodiments of the invention are shown for illustration only. From the above disclosed embodiments of the invention and the following examples, it will be apparent to those skilled in the art that various changes and modifications can be made to the invention to adapt it to various types of therapeutic antibodies and immunoglobulins without departing from the spirit and scope of the invention. Thus, various modifications of the present invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. An example of such a modification is that one or more of the above SEQ ID 1-21 may be substituted with a sequence having at least 90% sequence identity thereto.

Examples

Example 1 relates to the construction of XB166 host cells, also referred to herein as E.coli W3110rhaBfs ΔDegP.DELTA. prc sprW R.

Example 2 relates to the construction of KTXHIS-Cert-PelB1-LC-PelB2-HC expression vectors.

Example 3 relates to the light and heavy chains of cetuximab expressed by the expression vectors disclosed in example 2.

Example 4 shows that cetuximab produced by the invention is phage-free.

Example 5 relates to TIR library selection and isolation of evolving TIR.

Example 6 involves western blot analysis of medium component expression-results demonstrating that E83 expression vectors comprising synthetically evolved TIR for modulating cetuximab heavy chain expression produce the highest level of cetuximab.

Example 7 involves western blot analysis of total component expression-results demonstrating that E83 expression vectors comprising synthetically evolved TIR for modulating cetuximab heavy chain expression produce the highest level of cetuximab.

Example 8 relates toAndChromatography, performed to detect the yield and titer differences between expression system XB62 (XB 17 host cells containing D37 expression vector with unexplored TIR and wild-type nucleotide sequence of PelB signal peptide upstream of the respective nucleotide sequences of light and heavy chains of cetuzumab) and XB102 (XB 17 host cells containing expression vector E83 with synthetically engineered TIR for modulating cetuzumab heavy chain expression) -the results indicated that the yield and titer of cetuzumab produced by expression system XB102 containing expression vector E83 containing synthetically engineered TIR for modulating cetuzumab heavy chain expression was highest.

Example 9 relates to a comparison of the amount of cetuximab expressed in host cells XB17 and XB 166-the results indicate that the use of XB166 host cells (e.coli W3110 rhaBfs ΔdegpΔ prc sprW 148R) in combination with E111 (KTXHIS-Cert-Pelb-LC-Pelb 2-HC) yields the highest relative yields of cetuximab.

EXAMPLE 1 Strain development

The XB166 host cell is an E.coli W3310 derivative, genetically engineered for the production of recombinant proteins, such as antibodies and antibody fragments. In addition, XB166 host cells were designed as strains of the rhamnose-induced system in which the nucleotide sequence encoding the recombinant protein of interest was cloned into the KTXHIS plasmid and expressed under the control of the rhamnose-induced promoter.

The XB166 host cells were developed from the parent escherichia coli strain W3110, from the university of jerusalem (nikken in the united states) escherichia coli gene library center (CGSC), catalog No.: 4474: genotype F-, lambda-, IN (rrnD-rrnE) 1, rph-1.

XB166 was tailored for efficient induction from the rhamnose-dependent promoter by inactivation of the rhaB gene [7]. Furthermore, as described in detail in the following subsections, three genomic modifications are presented that can be used to generate antibody fragments [8].

In summary, methods of developing XB166 host cells involve modifications listed below, which will be discussed in detail in the following subsections:

a) RhaB frameshift mutation;

b) degP deletion;

c) prc deletion; and

D) sprW148R mutation.

Step (a) -construction of RhaB frameshift mutant.

The parent E.coli strain W3110 was engineered to produce a derivative with a frame shift mutation of the rhaB chromosomal copy, rendering it unusable as a carbon source. For this purpose, cells were genetically engineered using the gene replacement plasmid pMAK705-rhaBfs [9].

The engineered strain was phenotyped to verify that rhamnose could no longer be used as a carbon source. In addition, chromosomal fragments containing rhaB frameshifts were PCR amplified and the PCR products sequenced to confirm the correct insertion of both bases (see underlined CG bases in SEQ ID NO 22 disclosed below):

tgtggcagcaactgattcagcccggcgagaaactgaaatcgatccggcgagcgatacagcacattggtcagacacagattatcggtatgttcatacagatgccgatcatgatcgcgtacgaaacagaccgtgccaccggtgatggtatagggctgcccattaaacacatgaatacccgtgccatgttcgacaatcacaatttcatgaaaatcatgatgatgttcaggaaaatccgcctgcgggagccggggttctatcgccacggacgcgttaccagacggaaaaaaatccacactatgtaatacggtcatactggcctcctgatgtcgtcaacacggcgaaatagtaatcacgaggtcaggttcttaccttaaattttcgacggaaaaccacgtaaaaaacgtcgatttttcaagatacagcgtgaattttcaggaaatgcggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcgaattcaggcgctttttagactggtcgtaatgaaattcagcaggatcacattatgacctttcgcaattgtgtcgccgtcgatctcggcgcatccagtgggcgcgtgatgctggcgcgttacgagcgtgaatgccgcagcctgacgctgcgcgaaatccatcgttttaacaatgggctgcatagtcagaacggctatgtcacctgggatgtggatagcctGgaaagtgccattcgccttggattaaacaaggtgtgcgaggaagggattcgtatcgCGatagcattgggattgatacctggggcgtggactttgtgctgctcgaccaacagggtcagcgtgtgggcctgcccgttgcttatcgcgatagccgcaccaatggcctaatggcgcaggcacaacaacaactcggcaaacgcgatatttatcaacgtagcggcatccagtttctgcccttcaatacgctttatcagttgcgtgcgctgacggagcaacaacctgaacttattccacacattgctcacgctctgctgatgccggattacttcagttatcgcctgaccggcaagatgaactgggaatataccaacgccacgaccacgcaactggtcaatatcaatagcgacgactgggacgagtcgctactggcgtggagcggggccaacaaagcctggtttggtcgcccgacgcatccgggtaatgtcataggtcactggatttgcccgcagggtaatgagattccagtggtcgccgttgccagccatgataccgccagcgcggttatcgcctcgccgttaaacggctcacgtgctgcttatctctcttctggcacctggtcattgatgggcttcgaaagccagacgccatttaccaatgacacggcactggcagccaacatcaccaatgaaggcggggcggaaggtcgctatcgggtgctgaaaaatattatgggcttatggctgcttcagcgagtgcttcaggagcagcaaatcaacgatcttccggcgcttatctccgcgacacaggcacttccggcttgccgcttcattatcaatcccaatgacgatcgct

Individual colonies were picked and cultured in LB vegitone to prepare glycerol stocks. The resulting strain E.coli W3110 rhaBf was designated XB17 as the starting strain for the following genetic modification.

Step (b) -degP deletion

The XB17 (e.coli W3110 rhaBfs) strain was further engineered to have three key modifications (DEGP PRC SPR) so that a "triple mutant" host strain could be created, which allows for high level accumulation of recombinant antibody fragments due to reduced proteolytic degradation of the light chain in the periplasm [8].

For this, a genomic copy of the gene encoding the periplasmic serine endoprotease DegP was knocked out using the gene replacement plasmid pMAK705-sacB-DegP (in which a fragment homologous to the region upstream of DegP was fused to a fragment homologous to the region downstream of DegP). To avoid polar effects, the gene substitution cassette was designed to retain the degP start codon and the last 7 degP codons. The gene replacement plasmid also carries sacB gene for counter selection near the temperature sensitive replication origin, thereby promoting plasmid elimination after strain construction (plasmid curing) [10].

Deletion of the chromosomal degP gene can be confirmed by PCR amplification of the degP "scar" region and sequencing of the PCR product (see SEQ ID 23 below, where the underlined codons are the degP start codons and the bold codons are the last 7 codons of the degP gene):

atataaaaatgtcgctgtaaaacatgtgtttagccatccagatgtcgagcggcttgaattgcagggctatcgggtcattagcggattattagagatttatcgtcctttattaagcctgtcgttatcagactttactgaactggtagaaaaagaacgggtgaaacgtttccctattgaatcgcgcttattccacaaactctcgacgcgccatcggctggcctatgtcgaggctgtcagtaaattaccgtcagattctcctgagtttccgctatgggaatattattaccgttgccgcctgctgcaggattatatcagcggtatgaccgacctctatgcgtgggatgaataccgacgtctgatggccgtagaacaataaccaggcttttgtaaagacgaacaataaatttttaccttttgcagaaactttagttcggaacttcaggctataaaacgaatctgaagaacacagcaattttgcgttatctgttaatcgagactgaaatacATGatctacctgttaatgcagTAAtctccctcaaccccttcctgaaaacgggaaggggttctccttacaatctgtgaacttcaccacaactccatacatcttcatcatcctttaggcatttgcacaatgccgtacgttacgtacttccttatgctaagccgtgcataacggaggacttatggctggctggcatcttgataccaaaatggcgcaggatatcgtggcacgtaccatgcgcatcatcgataccaatatcaacgtaatggatgcccgtgggcgaattatcggcagcggcgatcgtgagcgtattggtgaattgcacgaaggtgcattgctggtactttcacagggacgagtcgtcgatatcgatgacgcggtagcacgtcatctgcacggtgtgcggcaggggattaatctaccgttacggctggaaggtgaaattgtcggcgtaattggcctgacaggtgaaccagagaatctgcgtaaatatggcgaactggtctgcatgacggc

single colonies of the engineering strain were picked and grown in LB vegitone to prepare glycerol stocks. The resulting strain E.coli W3110 rhaBfs ΔDegP was designated XB83 as the starting strain for the following genetic modification.

Step (c) -prc deletion

Similar to the degP deletion described above, the prc gene was knocked out using the gene replacement plasmid pMAK705-sacB-prc using E.coli W3110 rhaBfs ΔDegP (XB 83) as the starting strain. Deletion of the chromosomal prc gene can be confirmed by PCR amplification of the prc scar region and sequencing of the PCR product (see SEQ ID 24 below, where the underlined codons are the prc start codon and the bold codons are the last 7 codons of the prc gene):

tttacggtgttaaacccggcgcaacgcgtgtcgatcttgacggcaacccatgcggtgagctggacgagcaacatgtagagcatgctcgcaagcagcttgaagaagcgaaagcgcgtgttcaggcacagcgtgctgaacagcaagcgaaaaaacgcgaagctgccgcaactgctggtgagaaagaagacgcaccgcgccgcgaacgcaagccacgtccgactacgccacgccgcaaagaaggcgctgaacgtaaacctcgtgcgcaaaagccggtagagaaagcgccaaaaacagtaaaagcacctcgcgaagaacagcacaccccggtttctgacatttcagctctgactgtcggacaagccctgaaggtgaaagcgggtcaaaacgcgatggatgccaccgtattagaaatcaccaaagacggcgtccgcgtccagctgaattcgggtatgtctttgattgtgcgcgcagaacacctggtgttctgaaacggaggccgggccaggcATGcaacccgctcccgtcaagTAAtatcaatcaggcacaagaaattgtgcctgattttttaacagcgacaagatgccgtaaatcagatgctacaaaatgtaaagttgtgtctttctggtgacttacgcactatccagacttgaaaatagtcgcgtaacccatacgatgtgggtatcgcatattgcgttttgttaaactgaggtaaaaagaaaattatgatgcgaatcgcgctcttcctgctaacgaacctggccgtaatggtcgttttcgggctggtactgagcctgacagggatacagtcgagcagcgttcaggggctgatgatcatggccttgctgttcggttttggtggttccttcgtttcgcttctgatgtccaaatggatggcattacgatctgttggcggggaagtgatcgagcaaccgcgtaacgaaagggaacgttggctggtcaatactgtagcaacccaggctcgtcaggcggggatcgctatgccgcaagtggctatctacc

Single colonies of the engineering strain were picked and grown in LB vegitone to prepare glycerol stocks. The resulting strain E.coli W3110 rhaBfs DeltagP Deltaprc was designated herein as XB152 as the starting strain for the final genetic engineering step.

Step (d) -sprW R

The final step in engineering the expression host is to complement the triple mutant genotype by introducing the sprW R mutation that results in an amino acid substitution in the spr gene. While degP and prc deletions are expected to produce strains with reduced proteolytic degradation of the antibody fragment light chain, spr mutations are described as producing higher amounts of recombinant protein [8].

Genomic copies of the spr gene were engineered using E.coli W3110 rhaBfs ΔDegP Δprc (XB 152) as the starting strain using the gene replacement plasmid pMAK705-sacB-sprW148R containing the mutated spr fragment [10]. The chromosomal fragment containing sprW R mutation was PCR amplified and the PCR product sequenced to confirm the correct replacement of the genomic spr gene by the mutant allele (see SEQ ID 25 below; T to A mutation highlighted in bold):

aacaaacaacatggtcaaatctcaaccgattttgagatatatcttgcgcgggattcccgcgattgcagtagcggttctgctttctgcatgtagtgcaaataacaccgcaaagaatatgcatcctgagacacgtgcagtgggtagtgaaacatcatcactgcaagcttctcaggatgaatttgaaaacctggttcgtaatgtcgacgtaaaatcgcgaattatggatcagtatgctgactggaaaggcgtacgttatcgtctgggcggcagcactaaaaaaggtatcgattgttctggtttcgtacagcgtacattccgtgagcaatttggcttagaacttccgcgttcgacttacgaacagcaggaaatgggtaaatctgtttcccgcagtaatttgcgtacgggtgatttagttctgttccgtgccggttcaacgggacgccatgtcggtatttatatcggcaacaatcagtttgtccatgcttccaccagcagtggtgttattatttccagcatgaatgaaccgtacAggaagaagcgttacaacgaagcacgccgggttctcagccgcagctaataaaccgtttggatgcaatcccttggctatcctgacgagttaactgaaagcactgcttaggcagtgcttttttgttttcattcatcagagaaaatgatgtttccgcgtcttgatccaggctatagtccggtcattgttatcttttaaatgttgtcgtaatttcaggaaattaacggaatcatgttcatacgcgctcccaattttggacgtaagctcctgcttacctgcattgttgcaggcgtaatgattgcgatactggtgagttgccttcagtttttagtggcctggcataagcacgaagtcaaatacgacacactgattaccgacgtacaaaagtatctcgatacctattttgccgacctgaaatccactactgaccggctccagccgctgaccttagatacctgccagcaagctaaccccgaactgaccgcccgcgcagcgtttagcatgaatgtccgaacgtttgtgctggtgaaagataaaa

Single colonies of the engineering strain were picked and grown in LB vegitone to prepare glycerol stocks. The resulting strain E.coli W3110 rhaBfs ΔDegP.DELTA. prc sprW148R was designated XB166.

Example 8 and figure 8 show and discuss in detail the beneficial technical effects of XB 166.

Examples 2-KTXHIS-Cert-PelB1-LC-PelB2-HC

The first construct of cetuzumab was cloned into KTXHIS plasmid using the signal peptide OmpA-LC, pelB-HC (gene synthesis) via EcoRI and HindIII sites. The signal peptide was then exchanged from the first construct in E.coli using homologous recombination of the PCR fragments. I have made several versions of PelBLC and PelBHC in KTXHIS vectors in which the codons are different even though the amino acid sequence of PelB is the same. In the old, I did not determine which starting plasmid Kiavash was used.

A parent construct for expression of cetuzumab was made with the signal peptides OmpA (upstream of the nucleotide sequence encoding the light chain) and PelB (upstream of the nucleotide sequence encoding the heavy chain) and cloned into the KTXHIS plasmid (see SEQ ID 1) via EcoRI and HindIII sites (see SEQ ID 2). Then, by using homologous recombination of the PCR fragment in E.coli, the existing signal peptide nucleotide sequence was replaced with the signal peptide nucleotide sequences of SEQ ID 18 and 19 in the parent construct, resulting in expression vector D37. After the synthetic construction of two TIR that regulate the expression of the cetuximab light and heavy chains (as explained in examples 5 to 10 below), an expression vector comprising SEQ ID 17 was generated.

Expression plasmid KTXHIS has been described in European patent application EP 20201096.3. The resulting nucleotide sequence comprises the nucleotide sequence of SEQ ID 17, designated KTXHIS-Cert-PelB1-LC-PelB2-HC in the present invention. The plasmid map of KTXHIS-Cert-PelB1-LC-PelB2-HC is shown in FIG. 3:

GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACTAGGCTTCCGCGCCCTCATCCGAAAGGGCGTATTCATATATGCGGTGTtAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTATCTTTCTGCGAATTGAGATGACGCCACTGGCTGGGCGTCATCCCGGTTTCCCGGGTAAACACCACCGAAAAATAGTTACTATCTTCAAAGCCACATTCGGTCGAAATATCACTGATTAACAGGCGGCTATGCTGGAGAAGATATTGCGCATGACACACTCTGACCTGTCGCAGATATTGATTGATGGTCATTCCAGTCTGCTGGCGAAATTGCTGACGCAAAACGCGCTCACTGCACGATGCCTCATCACAAAATTTATCCAGCGCAAAGGGACTTTTCAGGCTAGCCGCCAGCCGGGTAATCAGCTTATCCAGCAACGTTTCGCTGGATGTTGGCGGCAACGAATCACTGGTGTAACGATGGCGATTCAGCAACATCACCAACTGCCCGAACAGCAACTCAGCCATTTCGTTAGCAAACGGCACATGCTGACTACTTTCATGCTCAAGCTGACCAATAACCTGCCGCGCCTGCGCCATCCCCATGCTACCTAAGCGCCAGTGTGGTTGCCCTGCGCTGGCGTTAAATCCCGGAATCGCCCCCTGCCAGTCAAGATTCAGCTTCAGACGCTCCGGGCAATAAATAATATTCTGCAAAACCAGATCGTTAACGGAAGCGTAGGAGTGTTTATCATCAGCATGAATGTAAAAGAGATCGCCACGGGTAATGCGATAAGGGCGATCGTTGAGTACATGCAGGCCATTACCGCGCCAGACAATCACCAGCTCACAAAAATCATGTGTATGTTCAGCAAAGACATCTTGCGGATAACGGTCAGCCACAGCGACTGCCTGCTGGTCGCTGGCAAAAAAATCATCTTTGAGAAGTTTTAACTGATGCGCCACCGTGGCTACCTCGGCCAGAGAACGAAGTTGATTATTCGCAATATGGCGTACAAATACGTTGAGAAGATTCGCGTTATTGCAGAAAGCCATCCCGTCCCTGGCGAATATCACGCGGTGACCAGTTAAACTCTCGGCGAAAAAGCGTCGAAAAGTGGTTACTGTCGCTGAATCCACAGCGATAGGCGATGTCAGTAACGCTGGCCTCGCTGTGGCGTAGCAGATGTCGGGCTTTCATCAGTCGCAGGCGGTTCAGGTATCGCTGAGGCGTCAGTCCCGTTTGCTGCTTAAGCTGCCGATGTAGCGTACGCAGTGAAAGAGAAAATTGATCCGCCACGGCATCCCAATTCACCTCATCGGCAAAATGGTCCTCCAGCCAGGCCAGAAGCAAGTTGAGACGTGATGCGCTGTTTTCCAGGTTCTCCTGCAAACTGCTTTTACGCAGCAAGAGCAGTAATTGCATAAACAAGATCTCGCGACTGGCGGTCGAGGGTAAATCATTTTCCCCTTCCTGCTGTTCCATCTGTGCAACCAGCTGTCGCACCTGCTGCAATACGCTGTGGTTAACGCGCCAGTGAGACGGATACTGCCCATCCAGCTCTTGTGGCAGCAACTGATTCAGCCCGGCGAGAAACTGAAATCGATCCGGCGAGCGATACAGCACATTGGTCAGACACAGATTATCGGTATGTTCATACAGATGCCGATCATGATCGCGTACGAAACAGACCGTGCCACCGGTGATGGTATAGGGCTGCCCATTAAACACATGAATACCCGTGCCATGTTCGACAATCACAATTTCATGAAAATCATGATGATGTTCAGGAAAATCCGCCTGCGGGAGCCGGGGTTCTATCGCCACGGACGCGTTACCAGACGGAAAAAAATCCACACTATGTAATACGGTCATACTGGCCTCCTGATGTCGTCAACACGGCGAAATAGTAATCACGAGGTCAGGTTCTTACCTTAAATTTTCGACGGAAAACCACGTAAAAAACGTCGATTTTTCAAGATACAGCGTGAATTTTCAGGAAATGCGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCACGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCTTGTCAGTAACGAGAAGGTCGCGAATCCAGGCGCTTTTTAGACTGGTCGTAATGAAATTCAGGAGGAAtTgctcATGAAGTATCTtCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCAATGGCAgatattcagatgactcagagcccaagttcgctgagcgcttctgttggcgatcgtgtgaccattacatgcaaagcctcacagaacgttggtaccaatgtcgcctggtatcagcagaaacctggaaaagcgcccaaagcgctcatctactcagcgagcttcctgtattcaggcgtgccgtatcgctttagcggctctggttccggtacagactttaccctcacgatttcgtccttacaaccggaagatttcgccacgtactattgccagcaatacaacatctatccgctgacctttggacaaggcaccaaagtggagatcaaacgcactgttgctgcaccgagtgtgttcatctttccaccgtctgatgagcagctgaagtctggtacagcaagtgttgtgtgtctgctgaacaacttctatccgcgtgaagctaaagtacagtggaaagtcgacaatgccttgcaatccgggaatagccaggaaagcgtgactgaacaggacagcaaggattcgacctacagtctgagcagtaccttaaccttgtcgaaagcggattacgagaaacacaaggtctatgcctgtgaagtcacgcaTCAAGGCCTGTCATCGCCTGTTACTAAATCATTTAATAGAGGAGAATGTTAAATGAAGTATCTGTTGCCGACTGCTGCAGCGGGACTGCTGCTGTTAGCGGCACAACCGGCGATGGCGgaagtgcagcttgtggagtctggaggtggcttagtccagccaggtggttccctgcgcttgtcctgtgcagcgagcgggtatgtAttcacagattatggcatgaactgggttcggcaagcaccaggcaaaggcctcgaatggatggggtggatcaacacgtatattggggaaccgatttatgcggatagcgtcaaaggtcgcttcacgttcagtctggataccagcaaatcaaccgcgtatctccagatgaatagcctccgtgctgaagatactgccgtgtactactgtgcgcgtggttatcgcagttatgcgatggattactggggccaaggcaccttagtcaccgttagttctgcctccaccaaaggcccatcagtgtttccgctggccccttcgtctaaatcgacgagtggtggcacagccgcactgggatgcctggtcaaagactactttcccgaacctgtaaccgtaagctggaatagtggtgctttgacctcaggcgtgcatacgtttccggctgtcctgcagtcatccggtctgtactcgctttcgagcgttgttactgtaccctctagctccctgggcacccagacgtacatctgcaatgtgaaccataagccgtcgaacaccaaagtggacaagaaagttgagccgaaaagctgcgacaaaacgcacacatgtgccgccTAATAAaagctt

EXAMPLE 3 recombinant protein-cetuximab

Expression vector KTXHIS-Cert-PelB1-LC-PelB2-HC expressed cetuzumab light chain amino acid sequence comprising the sequence of SEQ ID 3 ：DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSGVPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Expression vector KTXHIS-cert-PelB1-LC-PelB2-HC expressed cetuzumab heavy chain amino acid sequence comprising the sequence of SEQ ID 4 ：EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYIGEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCAA

Furthermore, the resulting PelB signal peptide comprises the amino acid sequence [6] of SEQ ID 3: MKYLLPTAAAGLLLLAAQPAMA A

EXAMPLE 4 cell Bank

XB166 was converted with KTXHIS-Cert-Pelb1-LC-Pelb 2-HC.

RCB was prepared by picking individual colonies and culturing overnight in Defined Bioreactor Media (DBM). The next day, 20% (final concentration) glycerol was aseptically added and the culture was aliquoted and stored at-80 ℃.

All materials used to make RCBs are of non-animal and non-human origin.

EXAMPLE 5 selection of TIR library and isolation of evolved TIR

By using the methods disclosed in US10696963 and Mirzadeh et al 2015[11] and briefly described in this example, synthetic evolution was used to select TIRs that are more compatible with host cell ribosomes (see also WO21158163 for synthetic evolution of TIRs). A TIR library was created, designed as follows: six nucleotides immediately upstream of the ATG start codon are completely random, six nucleotides immediately downstream of the ATG start codon are random, with only synonymous codon changes. Theoretically, each TIR library contains >18000 expression plasmids with different TIR. In this experiment, an expression plasmid comprising expression cassette PelBss-cetuximab-LC-PelBss-cetuximab-HC (where "ss" is an abbreviation for signal sequence) was fused to β -lactamase for the TIR library generation. For each cetuximab chain in a separate experiment, the TIR library was transformed into bacteria and plated onto LB agar plates containing fixed amounts of rhamnose and increasing concentrations of ampicillin (fig. 4). Colonies resistant to high concentrations of ampicillin (upper panel/row in fig. 4) or colonies visually larger than the corresponding colonies from plates of the non-evolved expression cassette (lower panel/row in fig. 4) were isolated from the TIR library plates and TIR was sequenced. The identified TIR (i.e., synthetically evolved TIR) was then reverse engineered into a plasmid that did not contain β -lactamase to assess its effect on cetuximab expression at a larger fermentation scale.

A specific example of TIR produced by using the synthetic evolution method described above is referred to herein as TIR-LC and has SEQ ID 20, which regulates the expression of the light chain of cetuximab. The TIR-LC of SEQ ID 20 is the TIR contained in the E111 expression vector discussed in example 10.

Example 6-inFermentation cylinder (i.e. the)Mini bioreactor system) to induce western blot analysis of the medium components after 20 hours of expression.

Cetuximab is expressed by using:

i. An expression vector designated D36 comprising a codon-optimized version of the wild-type PelB signal peptide nucleotide sequence (SEQ ID 19) upstream of the cetuzumab heavy chain nucleotide sequence, and

Expression vectors E81, E82 and E83, which differ from the D36 vector in that each of the E81, E82 and E83 expression vectors comprises a synthetically engineered modified TIR responsible for modulating the expression of the cetuximab heavy chain (i.e., TIR upstream of the cetuximab heavy chain nucleotide sequence).

The synthetic engineered TIR (for modulating cetuximab heavy chain expression) contained in the E83 expression vector has the nucleotide sequence of SEQ ID 21. The nucleotide sequences of TIRs upstream of the heavy chains of E81 and E82 (and other expression vectors that have been studied, developed and tested) are not shown in the present invention. The objective of synthetic engineering of TIR upstream of the heavy chain nucleotide sequence was to test the effect of nucleotide substitutions on cetuximab expression.

A volume of 100 μl of each sample containing cetuximab was centrifuged at 14000X g min, then the supernatant was separated and added water to a total of 100 μl. Next, 100. Mu.l of 2 Xsample buffer was added to the sample, which was then boiled at 95℃for 5 minutes, then an equal volume was added to each well, and separated by 12% SDS-PAGE. Protein levels were observed by immunoblotting with cetuximab antisera. The results are shown in fig. 5, which are, in order from left to right: d36, E81, E82 and E83 expression vectors.

As shown in the western blot in fig. 5, the E83 expression vector produced cetuzumab (represented as fragment antibody Fab' in the figure) at the basal portion of the culture at the highest level. Fab' dimers and blots of free Light (LC) and Heavy (HC) chains were also present in the samples.

Example 7-inWestern blot analysis of all (Medium and cell) fractions after 20 hours of induced expression in fermentors

Cetuximab was expressed using D36, E81, E82, E83 expression vectors.

Mu.l of each sample was added to 100. Mu.l of 2 Xsample buffer, which was then boiled at 95℃for 5 minutes, equal volumes were added to each well, and separated by 12% SDS-PAGE. Protein levels were observed by immunoblotting with cetuximab antisera. The results are shown in fig. 6, which are, in order from left to right: d36, E81, E82 and E83 expression vectors.

As shown in the western blot in fig. 6, the E83 expression vector (containing synthetically evolved TIR for modulating cetuximab heavy chain expression) produced the highest level of cetuximab (denoted as fragment antibody Fab' in the figure).

Example 8-AndChromatography method

The cetuximab is expressed by using an XB17 host cell (e.coli W3110 rhaBfs) by using (i) a D37 expression vector having an unevended TIR and a wild-type nucleotide sequence of the PelB signal peptide upstream of the respective nucleotide sequences of the light and heavy chains of cetuximab, and (ii) an expression vector E83 containing a synthetically engineered TIR for modulating cetuximab heavy chain expression.

At the position ofAfter 20 hours of expression in the fermentor, cetuximab was purified using frozen clarified lysates from expression systems XB102 and XB 62. The clarified lysate was prepared by the following steps: (1) 2 homogenizations at about 800 to 900 bar, (2) centrifugation, and (3) filtration of the supernatant with a 0.45PES (polyethersulfone) filter. More specifically, 9.5mL of clarified lysate was loaded into the column at each purificationThe chromatography system was attached to a CaptureSelect ^TM CH1-XL column (affinity resin selective for CH1 domain) and 28mL of the eluted phase containing the majority of the assembled cetuximab was collected each time.

By usingThe micro UV-VIS spectrophotometer measured the protein concentration in the collected elution pool at a wavelength of 280nm with an extinction coefficient set to 1.6 (see fig. 7 for the result, column titled "Nanodrop"). Then, the total amount of protein in the elution pool can be calculated by multiplying 28mL of the eluent by the measured concentration. The total amount of protein in the collected elution pool is then divided by the sample volume loaded onto the column to calculate the yield of the target protein in the clarified lysate, which is compared between the two batches.

Furthermore, these two runs were also evaluated by calculating the protein content throughout the elution and stripping phases using Unicorn internal evaluation software (AKTA system), the extinction coefficient was set to 1.6 (see fig. 7 for the results, titledIs a column of (b). Titer values were 2/3 of the yield values because a portion of the harvested cell mass was lost during clarification prior to column purification.

Summarized in FIG. 7AndThe analysis results clearly show that the XB102 expression system comprising expression vector E83 containing the synthetically engineered TIR (for modulating cetuximab heavy chain expression) produced the highest yield and titer of cetuximab (compared to expression vector D37 lacking the synthetically engineered TIR for modulating cetuximab heavy chain expression).

Example 9 comparison of cetuximab expression in host cells XB17 and XB166

When using an E83 expression vector (which comprises a synthetically engineered TIR for modulating cetuximab heavy chain expression), the expression level of cetuximab was analyzed in the following host cells:

-XB17 (E.coli W3110 rhaBfs), and

XB166 (E.coli W3110 rhaBfs ΔDegP.DELTA. prc sprW 148R).

At the position ofAfter 20 hours of induction of expression in the fermenter, 1ml of the sample was centrifuged at 13500rpm for 20 minutes at 4 ℃. The pellet and media components were separated and the pellet resuspended with 0.5ml 100mM Tris HCl/10mM EDTA (pH 7.4). Next, the heavy suspension was vortexed well and then incubated at 60 ℃ for 16 hours. The precipitated sample was then clarified by centrifugation at 13500rpm for 20 minutes at 4℃and the supernatant (extracted periplasmic sample) was collected and treated with DNase (final concentration 0.02 mg/ml). The samples were filtered using a low protein binding syringe filter (0.2 μm, spartan 13, ge Healthcare). After periplasmic extraction, the samples were centrifuged at 14000X g min and then 20 μl of supernatant was analyzed directly using affinity column (CH 1-XL) -HPLC. The protein concentration was compared to a standard curve using purified cetuximab at known concentrations.

The results shown in fig. 8 demonstrate that the use of XB166 host cells produced higher relative yields of cetuximab when compared to the amount of expression in host cell XB 17.

Example 10-comparison of the expression of cetuximab in host cell XB166 by using E83 and E111 expression vectors

Because of the beneficial effects of the E83 expression vectors described above (see examples 6-9), the E83 expression vectors were templated for synthetic evolved light chain TIR (according to the general method described in example 5). The resulting optimally performing vector contains synthetically evolved TIR (TIR-LC) upstream of the light chain of SEQ ID 20. The vector is referred to herein as E111.

In other words, the E111 expression vector comprises:

-synthetic evolved TIR (TIR-LC) of SEQ ID 20 for modulating expression of a cetuximab light chain; and

Synthetic engineered TIR (TIR-HC) of SEQ ID 21 for modulating cetuzumab heavy chain expression

This example is similar to example 9, but differs in that the differences in cetuximab expression are compared as follows:

an XB166 host cell comprising an E83 expression vector (synthetic engineered TIR with SEQ ID NO 21 for modulating only the heavy chain of cetuzumab), wherein the combination of host cell and vector is referred to as XB174 in fig. 9; and

-An XB166 host cell comprising expression vector E111 with SEQ ID NO 20 and SEQ ID NO 21 synthetically constructed for regulating the expression of the light and heavy chains of cetuzumab, respectively.

The results shown in fig. 9 demonstrate that the relative yield of cetuximab produced by expression vector E111 is higher when compared to E83. In other words, an E111 expression vector comprising:

-synthetically engineered TIR for modulating heavy chains; and

TIR for regulating synthetic evolution of light chains,

The resulting cetuximab yields are higher when compared to E83 without synthetic evolved TIR for modulating light chain expression.

Reference material

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Claims

1. A DNA construct for expressing a recombinant protein, wherein the DNA construct comprises:

-at least one of the nucleotide sequences of SEQ ID No 20 and SEQ ID No 21, wherein the nucleotide sequences of SEQ ID No 20 and SEQ ID No 21 are TIR sequences; and

-A nucleotide sequence encoding a signal peptide; and

Wherein the nucleotide sequences of SEQ ID No 20 and SEQ ID No 21 comprise at least the first 9 nucleotides of the signal peptide coding sequence.

2. The DNA construct of claim 1, wherein the TIR sequence is transcribed as an RNA motif that is the start site for protein translation in an mRNA transcript.

3. The DNA construct of claim 1 or 2, wherein when the DNA construct comprises TIR of the nucleotide sequence of SEQ ID No 20, the nucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID No 18.

4. A DNA construct according to any one of claims 1 to 3, wherein when the DNA construct comprises TIR of the nucleotide sequence of SEQ ID No 21, the nucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID No 19.

5. The DNA construct of any one of claims 1 to 4, wherein the DNA construct comprises the nucleotide sequences of SEQ ID No 20 and SEQ ID No 21, wherein the DNA construct comprises two nucleotide sequences encoding a signal peptide, wherein the first nucleotide sequence encoding a signal peptide comprises the nucleotide sequence of SEQ ID No 18, wherein the second nucleotide sequence encoding a signal peptide comprises the nucleotide sequence of SEQ ID No 19, wherein the nucleotide sequence of SEQ ID No 20 comprises at least the first 9 nucleotides of the nucleotide sequence of SEQ ID No 18, wherein the nucleotide sequence of SEQ ID No 21 comprises at least the first 9 nucleotides of the nucleotide sequence of SEQ ID No 19.

6. The DNA construct according to any one of claims 1 to 5, wherein the DNA construct comprises a nucleotide sequence encoding the recombinant protein, wherein the recombinant protein is preferably an antibody, wherein the antibody is most preferably a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a fragment of any of the antibodies.

7. The DNA construct of any one of claims 1 to 6, wherein the nucleotide sequence encoding a signal peptide is operably linked to the nucleotide sequence encoding a recombinant protein.

8. The DNA construct according to any one of claims 1 to 7, wherein the nucleotide sequence encoding the recombinant protein comprises:

-a first nucleic acid sequence encoding an antibody light chain; and

-A second nucleic acid sequence encoding an antibody heavy chain.

9. The DNA construct of claim 8, wherein the nucleotide sequence encoding a signal peptide is operably linked to:

-a first nucleotide sequence encoding an antibody light chain; and/or

-A second nucleotide sequence encoding a heavy chain of the antibody.

10. The DNA construct according to any one of claims 1 to 9, wherein the DNA construct comprises a Shine-Dalgarno sequence, preferably the Shine-Dalgarno sequence is located upstream of the ATG start codon of the nucleotide sequence encoding the signal peptide, more preferably the Shine-Dalgarno sequence is located upstream of the ATG start codon of the nucleotide sequence encoding the signal peptide operably linked to the antibody light chain.

11. The DNA construct according to any one of claims 8 to 10, wherein the first nucleotide sequence encoding an antibody light chain encodes the amino acid sequence of SEQ ID No 3.

12. The DNA construct according to any one of claims 8 to 11, wherein the second nucleotide sequence encoding the antibody heavy chain encodes the amino acid sequence of SEQ ID No 4.

13. The DNA construct according to any one of claims 8 to 12, wherein the first and second nucleotide sequences encoding the antibody light and heavy chains, respectively, encode the amino acid sequences of SEQ ID No 3 and SEQ ID No 4, respectively.

14. The DNA construct according to any one of claims 8 to 13, wherein the first nucleotide sequence encoding an antibody light chain comprises the nucleotide sequence of SEQ ID No 5.

15. The DNA construct according to any one of claims 8 to 14, wherein the second nucleotide sequence encoding an antibody heavy chain comprises the nucleotide sequence of SEQ ID No 6.

16. The DNA construct according to any one of claims 8 to 15, wherein the first and second nucleotide sequences encoding the antibody light and heavy chains, respectively, comprise the nucleotide sequences of SEQ ID No 5 and SEQ ID No 6, respectively.

17. An expression vector comprising the DNA construct of any one of claims 1 to 16.

18. A host cell comprising the DNA construct of any one of claims 1 to 16 or comprising the expression vector of claim 17, wherein the host cell is a bacterial cell, more preferably e.coli (e.coli), most preferably e.coli comprising a chromosome comprising a mutation or modification that disrupts rhamnose metabolism.

19. The host cell of claim 18, wherein the host cell comprises any one of (i) a chromosome containing a mutation in the nucleotide sequence of the RhaB gene that inactivates RhaB, or (ii) a chromosome in which the nucleotide sequence encoding RhaB is deleted.

20. The host cell according to claim 18 or 19, wherein the host cell is e.coli W3110, preferably comprising a chromosome comprising a frameshift mutation in the nucleotide sequence encoding RhaB.

21. The host cell according to any one of claims 18 to 20, wherein the host cell comprises a chromosome comprising:

a. Frame shift mutations in the nucleotide sequence encoding RhaB;

degP deletion;

prc deletion; and

The sprw148r mutation.

22. The host cell according to any one of claims 18 to 21, wherein the host cell is e.coli W3110 rhaBfs ΔdegpΔ prc sprW148R.

23. An RNA expressed from the DNA construct of any one of claims 1 to 16.

24. A method of expressing a recombinant protein comprising using the host cell of any one of claims 18 to 22.

25. The method of claim 24, further comprising the step of recovering recombinant protein from the host cell; optionally further comprising one or more steps for purifying the recovered recombinant protein, preferably by one or more chromatographic steps.

26. A recombinant protein obtainable by the method of claim 24 or 25.

27. A DNA construct for expressing cetuximab in a host cell, wherein cetuximab comprises (i) a light chain comprising the amino acid sequence of SEQ ID 3 and/or (ii) a heavy chain comprising the amino acid sequence of SEQ ID 4,

Wherein the DNA construct comprises a nucleotide sequence encoding cetuximab, wherein the DNA construct further comprises at least one nucleotide sequence encoding a signal peptide operably linked in the direction of transcription to a nucleotide sequence encoding the light chain of cetuximab and/or the heavy chain of cetuximab,

Wherein the DNA construct further comprises a nucleotide sequence encoding:

the rhaBAD promoter is used in the expression of,

The RhaR transcriptional activator is selected from the group consisting of,

The RhaS transcriptional activator is selected from the group consisting of,

The marker of resistance to antibiotics,

At least one terminator, and

-An origin of replication.

28. The DNA construct of claim 27, wherein the DNA construct further comprises a nucleotide sequence encoding a promoter operably linked to a nucleotide sequence encoding an antibiotic resistance marker.

29. The DNA construct according to claim 28, wherein the at least one terminator comprises two terminators, preferably the terminator comprises a rrnB T1 terminator and a rrnB T2 terminator.

30. The DNA construct of claim 29, wherein the origin of replication comprises a pMB1 origin of replication.

31. The DNA construct of claim 30, wherein,

-The antibiotic resistance marker is a kanamycin resistance marker, preferably a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12 or a sequence having at least 90% sequence identity thereto;

-the promoter operably linked to a nucleotide sequence encoding an antibiotic resistance marker is an AmpR promoter, preferably an AmpR promoter comprising the nucleotide sequence of SEQ ID 13 or a sequence having at least 90% sequence identity thereto;

-the rrnB T1 terminator comprises the nucleotide sequence of SEQ ID 14 or a sequence having at least 90% sequence identity thereto;

-the rrnB T2 terminator comprises the nucleotide sequence of SEQ ID 15 or a sequence having at least 90% sequence identity thereto; and/or

-The pMB1 origin of replication comprises the nucleotide sequence of SEQ ID 16 or a sequence having at least 90% sequence identity thereto.

32. The DNA construct of claim 31, wherein,

-the promoter operably linked to a nucleotide sequence encoding an antibiotic resistance marker is an AmpR promoter comprising the nucleotide sequence of SEQ ID 13;

-the rrnB T1 terminator comprises the nucleotide sequence of SEQ ID 14;

-the rrnB T2 terminator comprises the nucleotide sequence of SEQ ID 15; and/or

-The pMB1 origin of replication comprises the nucleotide sequence of SEQ ID 16.

33. The DNA construct of claim 31 or 32, wherein,

-The rhaBAD promoter comprises the nucleotide sequence of SEQ ID 8 or a sequence having at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 8;

-the RhaR transcriptional activator comprises the nucleotide sequence of SEQ ID 9 or a sequence having at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 9;

-the RhaS transcriptional activator comprises the nucleotide sequence of SEQ ID 11 or a sequence having at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 11;

-the antibiotic resistance marker is a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12 or a sequence having at least 90% sequence identity thereto, preferably a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12;

-the AmpR promoter comprises the nucleotide sequence of SEQ ID 13 or a sequence having at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 13;

-the rrnB T1 terminator comprises the nucleotide sequence of SEQ ID 14 or a sequence having at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 14;

-the rrnB T2 terminator comprises the nucleotide sequence of SEQ ID 15 or a sequence having at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 15;

-the pMB1 origin of replication comprises the nucleotide sequence of SEQ ID 16 or a sequence having at least 90% sequence identity thereto, preferably the nucleotide sequence of SEQ ID 16.

34. The DNA construct of any one of claims 27 to 33, wherein the nucleotide sequence encoding a recombinant protein is operably linked to a rhaBAD promoter.

35. The DNA construct of any one of claims 27 to 34, wherein the nucleotide sequence encoding cetuximab comprises (i) a nucleotide sequence encoding a light chain of cetuximab comprising a sequence of SEQ ID 5 or a sequence having at least 90% sequence identity thereto, and/or (ii) a nucleotide sequence encoding a heavy chain of cetuximab comprising a sequence of SEQ ID 6 or a sequence having at least 90% sequence identity thereto.

36. The DNA construct of any one of claims 27 to 34, wherein the nucleotide sequence encoding cetuximab comprises (i) a nucleotide sequence encoding a light chain of cetuximab comprising a sequence of SEQ ID 5, and/or (ii) a nucleotide sequence encoding a heavy chain of cetuximab comprising a sequence of SEQ ID 6.

37. The DNA construct of claim 36, wherein the nucleotide sequence encoding the signal peptide is operably linked to one or both of the nucleotide sequences of SEQ ID 5 and SEQ ID 6 in the direction of transcription.

38. The DNA construct according to any of claims 27 to 37, wherein the signal peptide is selected from the group consisting of MalE, ompA, phoA, dsbA and Pelb, preferably the signal peptide is Pelb.

39. The DNA construct according to any one of claims 27 to 38, wherein the DNA construct comprises at least one of the nucleotide sequences of SEQ ID nos 20 and 21, wherein the nucleotide sequences of SEQ ID nos 20 and 21 are TIR sequences, and wherein the nucleotide sequences of SEQ ID nos 20 and 21 comprise at least the first 9 nucleotides of the signal peptide coding sequence.

40. The DNA construct according to any one of claims 27 to 39, wherein the signal peptide is PelB, wherein the nucleotide sequence encoding PelB signal peptide operably linked to a light chain in the direction of transcription comprises the sequence of SEQ ID 18 or a sequence having at least 90% sequence identity thereto.

41. The DNA construct of claim 40, wherein the nucleotide sequence encoding the PelB signal peptide operably linked to the light chain in the direction of transcription comprises the sequence of SEQ ID 18.

42. The DNA construct according to claim 40 or 41, wherein the DNA construct comprises TIR of nucleotide sequence SEQ ID No 20, wherein the nucleotide sequence of SEQ ID No 20 comprises at least the first 9 nucleotides of nucleotide sequence SEQ ID 18 encoding a PelB signal peptide.

43. The DNA construct according to any one of claims 27 to 42, wherein the signal peptide is PelB, wherein the nucleotide sequence encoding PelB signal peptide operably linked to a heavy chain in the direction of transcription comprises the sequence of SEQ ID 19 or a sequence having at least 90% sequence identity thereto.

44. The DNA construct of claim 43, wherein the nucleotide sequence encoding the PelB signal peptide operably linked to the heavy chain in the direction of transcription comprises the sequence of SEQ ID 19.

45. The DNA construct according to claim 43 or 44, wherein the DNA construct comprises TIR of nucleotide sequence SEQ ID No 21, wherein the nucleotide sequence of SEQ ID No 21 comprises at least the first 9 nucleotides of nucleotide sequence SEQ ID 19 encoding a PelB signal peptide.

46. The DNA construct according to any of claims 27 to 45, wherein the DNA construct comprises the sequence of SEQ ID 17 or a sequence having at least 90% sequence identity thereto, preferably comprising the sequence of SEQ ID 17.

47. An expression vector comprising the DNA construct of any one of claims 27 to 46.

48. A host cell containing the DNA construct of any one of claims 27 to 46 or containing the expression vector of claim 47, wherein the host cell is a bacterial cell, preferably e.coli (e.coli), most preferably e.coli comprising a chromosome containing a mutation or modification that deregulates rhamnose metabolism.

49. The host cell of claim 48, wherein the host cell comprises any one of (i) a chromosome comprising a mutation in the nucleotide sequence of the RhaB gene that inactivates RhaB, or (ii) a chromosome in which the nucleotide sequence encoding RhaB is deleted.

50. The host cell according to claim 48 or 49, wherein the host cell is an e.coli W3110 cell, preferably comprising a chromosome comprising a frameshift mutation in the nucleotide sequence encoding RhaB.

51. The host cell of any one of claims 22 to 24, wherein the host cell comprises a chromosome comprising:

a. Frame shift mutations in the nucleotide sequence encoding RhaB;

degP deletion;

prc deletion; and

The sprw148r mutation.

52. The host cell of any one of claims 22 to 25, wherein the host cell is e.coli W3110 rhaBfs ΔdegpΔ prc sprW148R.

53. An RNA expressed from the DNA construct of any one of claims 27 to 46.

54. A method of expressing cetuzumab comprising using the host cell of any one of claims 48-52.

55. The method of claim 54, further comprising the step of recovering cetuximab from the host cell; optionally further comprising one or more steps of purifying the recovered cetuximab, preferably by one or more chromatographic steps.

56. Cetuximab obtained by the method according to claim 54 or claim 55.

57. The cetuximab antibiotic biological analog obtained by the method of claim 54 or 55.

58. The cetuximab antibiotic analogue or derivative thereof of claim 57 for use as a medicament, preferably the derivative comprises a polyethylene glycol moiety, more preferably the derivative comprises a polyethylene glycol moiety of about 40 kDa.

59. The cetuzumab anti-biological analog of claim 58 for use in the treatment of crohn's disease, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis.

60. A DNA construct for expressing a signal peptide, wherein the DNA construct comprises a nucleotide sequence encoding a PelB signal peptide, wherein the nucleotide sequence encoding the PelB signal peptide comprises at least one of the nucleotide sequences of SEQ ID No18 and SEQ ID No 19.

61. The DNA construct of claim 60, wherein the DNA construct comprises the nucleotide sequences of SEQ ID No 18 and SEQ ID No 19.

62. The DNA construct of claim 60, wherein the DNA construct comprises the nucleotide sequence of SEQ ID No 18.

63. The DNA construct of claim 60, wherein the DNA construct comprises the nucleotide sequence of SEQ ID No 19.

64. An expression vector comprising the DNA construct of any one of claims 60 to 63.

65. A host cell comprising the DNA construct of any one of claims 60 to 63 or comprising the expression vector of claim 64, wherein the host cell is preferably a bacterial cell, more preferably e.coli (e.coli), most preferably e.coli comprising a chromosome comprising a mutation or modification that disrupts rhamnose metabolism.

66. The host cell of claim 65, wherein the host cell comprises any one of (i) a chromosome comprising a mutation in the nucleotide sequence of the RhaB gene that inactivates RhaB, or (ii) a chromosome in which the nucleotide sequence encoding RhaB is deleted.

67. The host cell according to claim 65 or 66, wherein the host cell is e.coli W3110, preferably comprising a chromosome comprising a frameshift mutation in the nucleotide sequence encoding RhaB.

68. The host cell according to any one of claims 65 to 67, wherein the host cell comprises a chromosome comprising:

a. Frame shift mutations in the nucleotide sequence encoding RhaB;

degP deletion;

prc deletion; and

The sprw148r mutation.

69. The host cell of any one of claims 65 to 68, wherein the host cell is escherichia coli W3110 rhaBfs ΔdegpΔ prc sprW148R.

70. An RNA expressed from the DNA construct of any one of claims 60 to 63.

71. A method of expressing a signal peptide comprising using the host cell of any one of claims 65 to 69.

72. The PelB signal peptide obtained by the method of claim 71, wherein the PelB signal peptide comprises the amino acid sequence of SEQ ID No 7.

73. A DNA construct comprising a nucleotide sequence encoding an amino acid sequence, wherein the amino acid sequence comprises:

a. Amino acid sequence of cetuzumab;

C. A second signal peptide of the amino acid sequence of SEQ ID NO 7 fused to the N-terminus of the heavy chain amino acid sequence of cetuximab.

74. The DNA construct of claim 73, wherein the nucleotide sequence encoding the first signal peptide comprises the nucleotide sequence of SEQ ID No 18.

75. The DNA construct of claim 73, wherein the nucleotide sequence encoding the second signal peptide comprises the nucleotide sequence of SEQ ID No 19.

76. An expression vector comprising the DNA construct of any one of claims 73 to 75.

77. A host cell comprising the DNA construct of any one of claims 73 to 75 or comprising the expression vector of claim 76, wherein the host cell is preferably a bacterial cell, more preferably e.coli (e.coli), most preferably e.coli comprising a chromosome comprising a mutation or modification that disrupts rhamnose metabolism.

78. The host cell of claim 77, wherein said host cell comprises any one of (i) a chromosome comprising a mutation in the nucleotide sequence of the RhaB gene that inactivates RhaB, or (ii) a chromosome in which the nucleotide sequence encoding RhaB is deleted.

79. The host cell according to claim 77 or 78, wherein the host cell is e.coli W3110, preferably comprising a chromosome comprising a frameshift mutation in the nucleotide sequence encoding RhaB.

80. The host cell according to any one of claims 77 to 79, wherein the host cell comprises a chromosome comprising:

a. Frame shift mutations in the nucleotide sequence encoding RhaB;

degP deletion;

prc deletion; and

The sprw148r mutation.

81. The host cell according to any one of claims 77 to 80, wherein the host cell is e.coli W3110 rhaBfs ΔdegpΔ prc sprW148R.

82. An RNA expressed from the DNA construct of any one of claims 73 to 75.

83. A method of expressing a recombinant protein comprising using the host cell of any one of claims 77 to 81.

84. The method of claim 83, further comprising the step of recovering recombinant protein from the bacterial host cell; optionally further comprising one or more steps for purifying the recovered recombinant protein, preferably by one or more chromatographic steps.

85. Cetuximab obtained by the method of claim 83 or 84.

86. A cetuximab antibiotic biological analog obtained by the method of claim 83 or 84.

87. The cetuximab antibiotic analogue or derivative thereof for use as a medicament according to claim 86, preferably the derivative comprises a polyethylene glycol moiety, more preferably the derivative comprises a polyethylene glycol moiety of about 40 kDa.

88. The cetuzumab antibiotic analog of claim 86 or 87, for use in the treatment of crohn's disease, rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis.

89. A host cell suitable for expressing a recombinant protein, wherein the host cell is characterized by a chromosome comprising:

mutation in the spr gene.

90. The host cell according to claim 89, wherein the mutation is selected from the group consisting of a frameshift, a deletion, a substitution, and an insertion.

91. The host cell of claim 89 or 90 wherein the mutation in the nucleotide sequence encoding RhaB that disrupts rhamnose metabolism is a frameshift mutation in the nucleotide sequence encoding RhaB.

92. The host cell according to any one of claims 89 to 91, wherein the mutation in the degP gene is a degP deletion.

93. The host cell of any one of claims 89-92 wherein the mutation in the prc gene is a prc deletion.

94. The host cell of any one of claims 89 to 93, wherein the mutation in the spr gene is a sprW R mutation, characterized in that the substitution in the spr gene results in a change in tryptophan to arginine at position 148.

95. The host cell of any one of claims 89 to 94, comprising,

A. The mutation in the nucleotide sequence encoding RhaB that disrupts rhamnose metabolism is a frameshift mutation in the nucleotide sequence encoding RhaB;

degP deletion;

prc deletion; and

The sprw148r mutation.

96. The host cell according to any one of claims 89 to 95, wherein the host cell is a bacterial cell, more preferably e.

97. The host cell of any one of claims 89 to 96, wherein the host cell is e.coli W3110 rhaBfs ΔdegpΔ prc sprW148R.

98. The host cell according to any one of claims 89 to 97, wherein the host cell is transformed with a DNA construct encoding a recombinant protein, wherein the recombinant protein is preferably an antibody, wherein the antibody is most preferably a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a fragment of any of the antibodies.

99. A method of expressing a recombinant protein comprising the steps of:

-transforming the host cell of any one of claims 89 to 98 with a DNA construct encoding a recombinant protein;

-exposing the resulting transformed host cell to rhamnose, thereby inducing the expression of the recombinant protein; and

-Recovering recombinant protein from the host cell; optionally further comprising one or more steps for purifying the recovered recombinant protein, preferably by one or more chromatographic steps.

100. The method of claim 99, wherein the recombinant protein is an antibody, wherein the antibody is preferably a monoclonal antibody, a polyclonal antibody, a chimeric antibody or a fragment of any of the antibodies, most preferably a Fab' fragment, most preferably cetuzumab.