Method for producing full-length antibodies using Escherichia coli

Abstract

This invention provides a method for producing full-length antibodies in Escherichia coli at a yield of approximately 200 mg / L or more. [Solution] A method for producing a full-length antibody comprising a heavy chain (HC) and a light chain (LC), comprising culturing Escherichia coli cells expressing the coding sequence of the HC and the coding sequence of the LC in a culture medium under conditions acceptable for producing the HC and the LC, wherein the full-length antibody is produced in an amount of at least about 200 mg per liter of the culture medium, or the weight percentage of the produced full-length antibody relative to the weight of the cell pellet produced from the Escherichia coli cells is in the range of 0.05% to 20%.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority and interest to U.S. Provisional Application No. 63 / 018,436, filed on 30 April 2020, which is incorporated in its entirety by reference for all purposes.

[0002] Sequence List This application includes a sequence listing submitted electronically in ASCII format, the entirety of which is incorporated by reference into this disclosure. A copy of the said ASCII file, created on 29 April 2021, is named 091200-1246043-006810WO_SL.txt and has a size of 81,355 bytes. [Background technology]

[0003] Full-length antibodies play a major role in treating various human diseases. As the demand for antibody therapy increases, the development of host systems for enhanced and cheaper production of full-length antibodies has become increasingly important. Almost all therapeutic antibodies approved to date are produced primarily in mammalian hosts. These production methods have drawbacks such as high manufacturing costs and long culture times. Alternative production systems, such as bacteria and yeast, have been investigated. However, these systems have their own limitations. For example, the proteins produced in these systems often require refolding and secretion through several biological membranes and lack the disulfide bonds essential for maintaining the three-dimensional shape of the antibody. Furthermore, ensuring that the heavy and light chains of the same antibody are expressed in the appropriate ratio to produce the heterotetramer form of the full-length antibody in maximum yield remains a goal. Therefore, there is still a need to produce full-length antibodies in high yield to meet the demand for commercial production. [Overview of the project]

[0004] This disclosure provides a method for producing full-length antibodies comprising heavy chains (HC) and light chains (LC) in high yield. The method comprises culturing Escherichia coli cells expressing the coding sequences of the HC and the LC under conditions acceptable for producing the HC and the LC, wherein the full-length antibody can be produced in an amount of at least 200 mg / L. In some embodiments, the full-length antibody is produced in a wet weight percentage in the range of 0.05% to 20%, 0.05% to 10%, 0.05% to 5%, or 0.05% to 1% based on the weight of a wet cell pellet produced from the Escherichia coli cells in a culture. In some embodiments, at least a portion of the Escherichia coli cells contain oxidative cytoplasm. In some embodiments, the method comprises transforming the Escherichia coli strain with a plasmid. In some embodiments, the HC and the LC are produced in the cytoplasm of the Escherichia coli. In some embodiments, the method further comprises assembling the produced HC and the LC under non-reductive conditions to form a full-length antibody. In some embodiments, the molar ratio of the HC produced from the E. coli to the LC produced is about 1:1 to about 1:3.

[0005] In some embodiments, the expression of HC, LC, or both is controlled by a promoter, for example, a T7 promoter, or a promoter having a promoter strength substantially similar to that of the T7 promoter. In some embodiments, the plasmid comprises a bicistronic operon, the bicistronic operon comprising a coding sequence for HC and a coding sequence for LC. The bicistronic operon may also comprise a promoter that drives the expression of both HC and LC, the promoter being a T7 promoter, or a promoter having a promoter strength substantially similar to that of the T7 promoter. In some embodiments, the bicistronic operon comprises a T7 terminator. In some embodiments, the plasmid encoding HC and LC comprises a first monocistronic operon for HC and a second monocistronic operon for LC. In some embodiments, the first operon comprises a ribosome binding site comprising a sequence selected from any of SEQ ID NOs: 18-20, and the second monocistronic operon comprises a ribosome binding site comprising the sequence of SEQ ID NO: 17. In some embodiments, the first monocistrone operon or the second monocistrone operon each independently includes a T7 promoter or a promoter having a promoter strength substantially similar to that of the T7 promoter. In some embodiments, the first monocistrone operon or the second monocistrone operon each independently includes a T5 promoter or a promoter having a promoter strength substantially similar to that of the T5 promoter. In some embodiments, the first monocistrone operon and the second monocistrone operon include the same promoter. In some embodiments, the first monocistrone operon and the second monocistrone operon include different promoters.

[0006] The E. coli cell may include a first ribosome binding site for HC translation and a second ribosome binding site for LC translation, the first and second ribosome binding sites being selected such that the molar ratio of HC to LC produced from the E. coli is in the range of 1:1 to 1:3. In some embodiments, the first ribosome binding site includes a sequence selected from the group consisting of SEQ ID NOs: 17 to 19, and the second ribosome binding site includes a sequence selected from the group consisting of SEQ ID NOs: 20 to 23. In some embodiments, the first ribosome binding site includes the sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0007] In some embodiments, the HC and / or LC of the full-length antibody contain at least one non-natural amino acid. For example, the non-natural amino acid may be paramethylazide-L-phenylalanine (pAMF), AEK, or pAcF. For example, the coding sequence of the HC and / or LC may be modified to have at least one non-natural amino acid codon, where the non-natural amino acid codon does not result in the incorporation of one of the 20 natural amino acids. The at least one non-natural amino acid may be introduced by charging a tRNA containing an anticodon complementary to the at least one non-natural amino acid codon, for example, the amber codon (TAG). In some embodiments, the codon adjacent to the 3' end of at least one of the non-natural amino acids is codon-optimized to promote the expression of the HC or LC.

[0008] In certain embodiments, the full-length antibodies that can be produced using this method are B10 antibody, H01 antibody, 7219 antibody, anti-PD1 antibody, anti-HER2 antibody (e.g., trastuzumab), anti-Tim3 antibody, or anti-LAG3 antibody. In some embodiments, the coding sequence of the HC of the B10 antibody includes a mutation to SEQ ID NO: 1, resulting in the substitution of a natural amino acid codon with a non-natural amino acid codon, where the natural amino acid to be substituted is one or more amino acids selected from F404, Y180, and F241. The non-natural amino acid can be introduced into the HC or LC by charging a tRNA complementary to the non-natural amino acid codon. In some embodiments, the coding sequence of the LC of the B10 antibody or the anti-HER2 antibody includes a mutation to SEQ ID NO: 2, resulting in the substitution of a natural amino acid codon with a non-natural amino acid codon, where the natural amino acid is K42 or E161.

[0009] In some embodiments, a method for producing a full-length FolRa-B10 IgG antibody comprises transforming an E. coli host strain with a construct expressing FolRa-B10 IgG, where the S181 codon of the HC coding sequence is AGC or AGT.

[0010] In some embodiments, the method further includes covalently linking a warhead moiety to the non-natural amino acid on the HC or LC of the full-length antibody via a linker. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows a comparison of titers in cell lysates from Shuffle and Snuggle strains co-expressing heavy chain (HC) and light chain (LC) for FoRa B10 wild-type (WT) IgG, demonstrating that additional mutations present in Snuggle, including, for example, the deletion of the trxA gene and the presence of the wild-type ahpC gene, are essential for high levels of IgG synthesis.

[0012] [Figure 2] Figure 2 shows a comparison of titers in cell lysates for FolRa-B10 IgG produced using 0, 2, or 4 pAMF non-natural amino acids and the indicated S181 codon. "DAR0" represents B10 IgG without non-natural amino acids.

[0013] [Figure 3] Figure 3 shows a comparison of FolRa-B10 IgG assemblies produced using 0, 2, or 4 pAMF non-natural amino acids after overnight reassembly with FlowerPlate®, as measured by a Caliper bioanalyzer under non-reductive conditions.

[0014] [Figure 4] Figure 4 shows the expression of B10-F404pAcF and B10-F404AEK in and without the presence of appropriate non-natural amino acids in the culture medium. The low titer in the absence of non-natural amino acids (NNAAs) indicates that NNAAs are required for amber suppression in the production of full-length IgG.

[0015] [Figure 5] Figure 5 shows the results of the deconvoluted LCMS spectrum of the Fc fragment of B10 F404pAMF conjugated to the DBCO-meitansine drug linker SC236. The main species observed has a mass of 25257.97 Da, compared to the theoretical mass of the conjugate, which is 25258.04 Da. No species were observed around the theoretical mass of the unconjugated Fc fragment, 23974.14 Da. The calculated conjugation efficiency of this conjugate is 99.997%.

[0016] [Figure 6]Figure 6 shows the LCMS results of purified Trastuzumab LC K42pAcPhe E161pAcPhe run in positive ionization mode. There is one clean peak corresponding to the mass expected for the 2×pAcPhe LC product.

[0017] [Figure 7] Figure 7 shows the LCMS results of purified Trastuzumab LC K42pAcPhe E161pAcPhe after oxime ligation with PEG8-alkoxyamine run in positive ionization mode. The conjugation efficiency was calculated using the peaks at 23692 AMU, 24073 AMU, and 24455 AMU. These correspond to the expected masses of the 0PEG8 product, 1PEG8 product, and 2PEG8 product, respectively. A) shows the LC before oxime ligation and B) shows the LC after oxime ligation. The calculated conjugation efficiency was approximately 98%.

[0018] [Figure 8A] Figures 8A - 8I show exemplary unnatural amino acids that can be used in the methods disclosed in the present disclosure. [Figure 8B] Figures 8A - 8I show exemplary unnatural amino acids that can be used in the methods disclosed in the present disclosure. [Figure 8C] Figures 8A - 8I show exemplary unnatural amino acids that can be used in the methods disclosed in the present disclosure. [Figure 8D] Figures 8A - 8I show exemplary unnatural amino acids that can be used in the methods disclosed in the present disclosure. [Figure 8E] Figures 8A - 8I show exemplary unnatural amino acids that can be used in the methods disclosed in the present disclosure. [Figure 8F] Figures 8A - 8I show exemplary unnatural amino acids that can be used in the methods disclosed in the present disclosure. [Figure 8G] Figures 8A - 8I show exemplary unnatural amino acids that can be used in the methods disclosed in the present disclosure. [Figure 8H]Figures 8A–8I show exemplary non-natural amino acids that can be used in the manner disclosed herein. [Figure 8I] Figures 8A–8I show exemplary non-natural amino acids that can be used in the manner disclosed herein. [Modes for carrying out the invention]

[0019] I. Introduction This disclosure provides a method for producing full-length antibodies by expressing both the heavy chain (HC) and light chain (LC) of an antibody in E. coli cells. The method of the present invention utilizes a combination of features that enables the production of full-length antibodies in high yields, for example, at protein concentrations exceeding 200 mg per liter of culture medium.

[0020] E. coli cells typically possess oxidative cytoplasm, which facilitates the formation of disulfide bonds necessary to maintain the three-dimensional structure and stability of antibodies. The method uses a strong promoter to drive the transcription of HC, LC, or both. In some embodiments, the promoter is a T7 promoter or a T5 promoter. In some cases, the promoter strength is substantially similar to that of a T7 promoter or a T5 promoter. In some cases, the method uses an optimized ribosome binding site to ensure efficient translation and / or assembly of the antibody's HC and LC.

[0021] The inventors have found that maintaining proportional expression of heavy chains (HC) and light chains (LC), i.e., an optimal ratio of HC to LC expression levels, can increase the yield of properly assembled full-length antibodies. For example, it is desirable to maintain the molar ratio of HC to LC produced from E. coli cultures within an appropriate range, e.g., 1:1 to 1:3, 1:1 to 1:2.5, or 1:1 to 1:2. In some methods, maintaining HC and LC expression within the optimal range ratio is achieved by using one or two promoters that can control the transcription levels of HC and LC. In some methods, proportional expression of HC and LC in E. coli cultures is achieved by controlling the translation efficiency of HC and LC using modified ribosome binding sites. Exemplary transbosomal binding sequences are given herein.

[0022] In some methods, at least one of the HC coding sequence and the LC coding sequence is modified to include at least one non-natural amino acid codon (e.g., the amber codon), provided that this non-natural amino acid codon does not result in the incorporation of one of the 20 natural amino acids. These non-natural amino acids can function as potential reactive groups for site-specific binding to one or more biologically active adducts.

[0023] This disclosure incorporates, by reference, the entirety of International Application PCT / US2019 / 060345.

[0024] II. Definition Unless otherwise defined, technical and scientific terms used in this disclosure have the same meanings as those generally understood by those skilled in the art. See, for example, Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th ed. 2007), Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989), and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons (Hoboken, NY 1995).

[0025] When used in this disclosure, the singular forms “a,” “and,” and “the” also encompass multiple referents unless the context explicitly indicates otherwise. For example, “a cell” encompasses multiple such cells, and “the protein” encompasses one or more proteins and their equivalents known to those skilled in the art, and so on. All technical and scientific terms used in this disclosure have the same meaning as commonly understood to those skilled in the art in which the invention pertains, unless explicitly indicated otherwise.

[0026] The term "approximately" indicates a range of - / +10% of the reference value. For example, "approximately 10" means 10 - / +10 × 10% = 9 to 11.

[0027] Unless otherwise specified, the terms "yield" or "titer" refer to the amount of antibody produced (including the total amount of HC and LC) relative to the volume of culture medium. For example, 200 mg / L means that 200 mg of antibody is produced per liter of culture medium.

[0028] The term "E. coli strain" refers to a subtype of E. coli, whose cells(s) possess a specific biological morphology and share a specific genetic makeup. The term "E. coli strain with oxidative cytoplasm" refers to a strain in which some or all of the cells derived from that strain each possess oxidative cytoplasm.

[0029] The term "amino acid" refers to natural and non-natural amino acids, as well as amino acids that function in a similar manner to natural amino acids, such as proline, amino acid analogs, and amino acid mimetics.

[0030] The terms “natural amino acids” or “naturally encoded amino acids” refer to protein-constituting amino acids known to those skilled in the art. These include 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as the less common pyrrolidine and selenocysteine. Naturally encoded amino acids include 22 post-translational variants of natural amino acids, such as prenylated amino acids, isoprenylated amino acids, myristoylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids.

[0031] The term "non-natural amino acids" refers to amino acids that are not protein-forming amino acids or their post-translational modification variants. In particular, this term refers to amino acids that are not one of the 20 common amino acids, pyrrolidine, selenocysteine, or their post-translational modification variants.

[0032] The terms "aminoacylation" or "aminoacylate" refer to the complete process of adding an aminoacyl group to a compound, which results in the charging of the correct amino acid onto tRNA. In the present invention, the aminoacylated or aminoacylated tRNA is the tRNA that is charged with an amino acid, and the aminoacylated or aminoacylated amino acid is the amino acid that is charged to the tRNA molecule.

[0033] The terms "aminoacyl-tRNA synthetase," "tRNA synthetase," "synthetase," "aaRS," or "RS" refer to enzymes that catalyze covalent linkage between amino acids and tRNA molecules. This results in aminoacylated tRNA molecules, which are tRNA molecules containing each amino acid linked via ester bonds.

[0034] In the context of tRNA, the term "charged" refers to the aminoacylation of tRNA by an amino acid, which may be natural or unnatural. Aminoacylation allows ribosomes to incorporate the amino acid into the polypeptide translated from mRNA.

[0035] The term "biologically active adduct" refers to a chemical substance, molecule, or reagent that can perform a function in a cell or organism. For example, such functions may include cell proliferation, apoptosis, post-translational modification (e.g., phosphorylation), activation or deactivation of cell signaling, cell death, or cell labeling.

[0036] In the context of protein translation, the term "selective incorporation" refers to the incorporation or introduction of specific amino acids (e.g., specific non-natural amino acids) into a protein sequence at a desired, predetermined amino acid position without interfering with the desired function of the protein.

[0037] The term "preferential aminoacylation" refers to the preference of a tRNA synthase for aminoacyling (charging) a particular tRNA molecule with a given amino acid molecule compared to other amino acid molecules. In other words, a tRNA synthase can selectively aminoacylate non-natural amino acids (nnAAs) over natural amino acids. For example, a tRNA synthase can aminoacylate a particular nnAA with a frequency of more than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) compared to any other or all other natural amino acids.

[0038] The term "natural amino acids" refers to any of the 20 amino acids encoded by the genetic code, including arginine (Arg, R), histidine (His, H), lysine (Lys, K), aspartic acid (Asp, D), glutamic acid (Glu, E), serine (Ser, S), threonine (Thr, T), asparagine (Asn, N), glutamine (Gln, Q), cysteine ​​(Cys, G), glycine (Gly, G), proline (Pro, P), alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V), which are precursors of proteins.

[0039] In this disclosure, amino acids may be represented by commonly known three-letter symbols or single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by commonly accepted single-letter codes.

[0040] The terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded forms. Unless otherwise specified, the terms encompass nucleic acids including known analogs of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to native nucleotides. Unless otherwise specified, a particular nucleic acid sequence implicitly includes not only the explicitly stated sequence but also its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. Specifically, degenerate codon substitution can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The terms nucleic acid or polynucleotide are used interchangeably with genes, cDNA, and mRNA encoded by genes.

[0041] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably in this disclosure and refer to polymers of amino acid residues. These terms apply to natural and non-natural amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetic to corresponding natural amino acids. As used in this disclosure, these terms encompass amino acid chains of any length in which amino acid residues are linked by covalent peptide bonds, including full-length proteins and cleaved proteins.

[0042] The term "amino acid position substitution" refers to a change in an amino acid residue at a specific position in the amino acid sequence of a protein. For example, the term "X20Y" refers to the substitution of the wild-type (reference) amino acid X at amino acid position 20 of a protein with amino acid Y.

[0043] The term "suppression codon" refers to a nucleotide triplet that is introduced into a polynucleotide at a predetermined position and recognized by a specific tRNA capable of recognizing a stop codon (e.g., an amber stop codon, an ochre stop codon, or an opal stop codon), thereby allowing translation to read through the codon and produce a protein, and thus suppressing the stop codon.

[0044] The term “antibody” refers to a protein functionally defined as a binding protein and structurally defined as containing an amino acid sequence that is recognized by those skilled in the art as originating from the framework region of an immunoglobulin-coding gene of an animal producing the antibody. An antibody may consist of one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a wide variety of immunoglobulin variable region genes. The light chain is classified as kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, defining the classes of immunoglobulins IgG, IgM, IgA, IgD, and IgE, respectively.

[0045] The term "full-length antibody" is used interchangeably with "full-length IgG" and refers to an immunoglobulin (antibody) structural unit containing a tetramer, each composed of two identical polypeptide chain pairs, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region consisting of approximately 100-110 or more amino acids, primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively.

[0046] Antibodies exist either as untreated (intact) immunoglobulins or as numerous well-characterized fragments produced by digestion using various peptidases. For example, pepsin digests antibodies under the disulfide bond of the hinge region to produce F(ab)'2, which is itself a dimer of Fab, a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 can be reduced under mild conditions, which breaks the disulfide bond of the hinge region, thereby converting the (Fab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). While various antibody fragments are defined by the digestion of untreated (intact) antibodies, those skilled in the art will understand that such Fab' fragments may be synthesized de novo, either chemically or by utilizing recombinant DNA methods. Therefore, as used in this disclosure, the term antibody also encompasses antibody fragments produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods. Antibodies also include single-chain antibodies (antibodies existing as a single polypeptide chain) and single-chain Fv antibodies (sFv or scFv) in which a variable heavy chain and a variable light chain are linked (directly or via a peptide linker) to form a continuous polypeptide. Single-chain Fv antibodies are covalently linked VH-VL heterodimers, which may be expressed from nucleic acids containing VH-coding sequences and VL-coding sequences that are directly linked or linked by a peptide-coding linker. (Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883.) Although VH and VL are linked to each other as a single polypeptide chain, the VH and VL domains associate non-covalently. The first functional antibody molecule expressed on the surface of filamentous phages was single-stranded Fv(scFv), but alternative expression strategies have also been successful.For example, a Fab molecule can be displayed on a phage if one of the chains (heavy or light) is fused to a g3 capsid protein and the other (complementary) chain is exported to the periplasm as a soluble molecule. The two chains can encode on the same or different replicons. The key point is that the two antibody chains of each Fab molecule assemble post-translation, and the dimer is incorporated into the phage particle via binding of one of the chains to g3p (see, for example, U.S. Patent No. 5,733,743). Numerous other structures that convert scFv antibodies, as well as naturally assembling but chemically distinct light and heavy polypeptide chains from the V region of the antibody, into molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen-binding site, are known to those skilled in the art (see, for example, U.S. Patents Nos. 5,091,513, 5,132,405, and 4,956,778). Antibodies include all those displayed on phages (e.g., scFv, Fv, Fab, and disulfide-bonded Fv (Reiter et al. (1995) Protein Eng. 8: 1323-1331)). Antibodies may also include diantibodies, miniantibodies, and scFv-Fc fusions.

[0047] The term "reductase" refers to any enzyme capable of reducing thioredoxin reductase (TrxB), glutathione or glutathione reductase (Gor), or any other enzyme capable of reducing thioredoxin or glutaredoxin components.

[0048] The term "thioredoxin" encompasses thioredoxin 1 (TrxA) and thioredoxin 2 (TrxC), as described in Rietsch and Beckwith (1998) Ann. Rev. Genet. 32: 163. Thioredoxins are small proteins characterized by the presence of the motif Cys-Xaa-Xaa-Cys (where Xaa represents any amino acid) in their active site. Thioredoxins are re-reduced by thioredoxin reductase (encoded by the trxB gene) and NADPH. In trxB mutants, thioredoxins accumulate in their oxidized form. TrxA is encoded by the trxA gene, and TrxB is encoded by the trxB gene.

[0049] The term "gor" refers to the glutathione oxidoreductase gene, while the term "GOR" refers to glutathione oxidoreductase itself.

[0050] "DsbC" is a protein encoded by the gene dsbC, which catalyzes disulfide bond isomerization. The DsbC null mutant has defects in the folding of proteins containing multiple disulfide bonds.

[0051] The term "glutathione" refers to γ-L-glutamyl-L-cysteinyl-glycine (GSH), a highly conserved low molecular weight thiol found in many organisms, including cyanobacteria, proteobacteria, several strains of Gram-positive bacteria, and all eukaryotes with mitochondria and chloroplasts. Glutathione is synthesized by the action of two enzymes: glutamate cysteine ​​ligase (gshA) and glutathione synthase (gshB). Glutamate cysteine ​​ligase catalyzes the reaction between glutamate and cysteine ​​to form γ-glutamylcysteine, which is then conjugated to glycine by glutathione synthase to form GSH.

[0052] A nucleic acid can be said to be "operably linked" to another nucleic acid sequence if it is positioned to have a functional relationship with that sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory reader, it means that they are contiguous and in the reading phase. Linking is achieved by ligation at a convenient restriction enzyme site. If such a site does not exist, a synthetic oligonucleotide adapter or linker is used according to conventional methods.

[0053] AhpC is one of two subunits of the alkyl hydroperoxide reductase AhpCF. The other subunit of AhpCF is the flavonoid AhpF (Tarataglia et al, J. Biol. Chem., Volume 265, 10535-10540, 1990 and Smillie et al, Genbank submission NCBL gi; 216542, 1993). These two proteins work together. AhpF uses NADH or NADPH as an electron donor to AhpC, and AhpC reduces physiological lipid peroxides such as linoleic acid hydroperoxide, as well as thymine hydroperoxide and non-physiological alkyl hydroperoxides, to their respective non-toxic alcohol forms. This enzyme complex (or system) removes oxygen and its derivatives. AhpC has been shown to act as a specific alkyl hydroperoxide scavenging enzyme to protect against damage from oxygen radicals, but it has also been shown that the removal of reactive nitrogen intermediates occurs. AhpF is associated with a thioredoxin reductase that has an extended additional N-terminal fragment essential for the specific reduction of AhpC.

[0054] When used to describe proteins, the term "cytosol" refers to the fact that proteins reside in the cytosol of a cell.

[0055] A "heterogeneous protein or polypeptide" refers to a protein or polypeptide that is not normally produced within a host cell. Heterogeneous polypeptides may originate from the same species and type as the host cell, provided they are expressed from nucleic acids introduced into the host cell.

[0056] An "exogenous polypeptide" refers to a polypeptide that is not naturally produced within a cell but is expressed or present within the cell after being introduced into it.

[0057] A "null mutation" refers to a gene mutation that results in a non-functional gene. A null mutation may result in a complete absence of the production of the associated gene product, or in the production of a product that does not function properly.

[0058] The term "protein disulfide isomerase" is used interchangeably with the terms "disulfide isomerase" or "PDI" and refers to an enzyme that catalyzes the formation and isomerization of disulfide bonds. In vitro data have shown that PDI is involved in the catalytic activity of disulfide bond formation and rearrangement. (Creighton et al. (1980) J. Mol. Biol. 142:43, Feedman et al. (1989) Biochem. Soc. Symp. 5:167, and Bardwell and Beckwith (1993) Cell 74:899. Yeast mutants in PDI have been shown to have a defect in the formation of disulfide bonds in carboxypeptidase Y (LaMantia and Lennarz (1993) Cell 74:899). The use of PDI for heterologous protein expression in host cells is further described in PCT applications with international publications 93 / 25676 and 94 / 08012, and European publication 509,841.

[0059] The term "prolyl isomerase," used interchangeably with "peptidyl prolyl isomerase" or "PPlase," refers to an enzyme found in both prokaryotes and eukaryotes that interconverts the cis and trans isomers of the peptide bond with the amino acid proline. Proteins possessing prolyl isomerase activity include, but are not limited to, cyclophyllin (e.g., accession number Q13427), FKBP (e.g., accession number Q02790), palburin (e.g., accession number Q9Y237), Tig (e.g., accession number P0A850), SlyD (e.g., accession number P0A9K9), and yCpr6 (e.g., accession number S000004206).

[0060] The term “deaggregase” refers to a protein chaperone that assists in the deaggregation and / or solubilization of a target protein, which is produced, for example, in a sterile translation system. Such chaperones are particularly useful at high concentrations because their mechanism of action is stoichiometric rather than catalytic, and they are thought to function by stabilizing the hydrophobic patch of a newly synthesized protein while it is folding. Non-exclusive examples of deaggregases include Skp (e.g., accession number P0AEU7), GroEL (e.g., accession number P0A6F5), GroES (e.g., accession number P0A6F9), DnaK (e.g., accession number P0A6Y8), DnaJ (e.g., accession number P08622), or GrpE (e.g., accession number P09372).

[0061] When a protein is described as being in a "reduced state," it means that the protein has more electrons than its oxidized state.

[0062] The term "oxidative cytoplasm" refers to the cytosol of a cell in which the substrate is more readily oxidized than reduced.

[0063] The term "thioredoxin reductase activity" refers to the ability of thioredoxin reductase (TRXB) to maintain thioredoxin 1 in a reduced state.

[0064] The term "thioredoxin 1 activity" refers to the ability of thioredoxin 1 (TRXA) to maintain ribonucleotide reductase in a reduced state.

[0065] The term "peroxireductase activity" refers to the ability of AhpC to reduce physiological lipid oxides.

[0066] The term "glutathione reductase activity" refers to the ability to catalyze the reduction of glutathione disulfide (GSSG) to sulfhydryl glutathione (GSH). For example, glutathione reductase (GOR) possesses glutathione reductase activity.

[0067] The terms “recombinant” or “recombinantly” refer to biomolecules, such as genes or proteins, that (1) have been removed from their natural environment, (2) the gene does not contain all or part of the naturally occurring polynucleotides, (3) are operably ligated to polynucleotides that are not naturally bound, or (4) are not naturally occurring. The term “recombinant” may be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, and proteins and / or mRNA encoded by such nucleic acids.

[0068] The term "culture titer" refers to the concentration of protein in the culture medium. For example, a culture titer of "136 mg / L" means 136 mg of protein per liter of cell culture medium.

[0069] The term "lysate titer" refers to the concentration of protein in cell lysate. For example, a lysate titer of "0.655 g / L" means 0.655 g of protein per liter of cell lysate.

[0070] When the term "weight percentage" is used to express the titer or yield of protein produced from a cell culture, it refers to the wet weight percentage, i.e., the ratio of the weight of protein (e.g., IgG) to the weight of the cell pellet (undried) from which the protein is extracted. The cell pellet is collected from a cell culture expressing protein and lysed using standard techniques (e.g., as described in Example 6) to release the protein. As an illustrative example, if 100 g of cell pellet is lysed to produce 1 liter of lysate, which is determined to contain 50 mg of IgG, then the weight percentage of IgG is 50 mg / 100 g = 0.05%.

[0071] III. Host Cells - Escherichia coli Strains with Oxidative Cytoplasm The Escherichia coli strains in this disclosure may be any Escherichia coli strain known to those skilled in the art. In some embodiments, the Escherichia coli strain is strain A(K-12), strain B, strain C, or strain D. In some embodiments, at least some Escherichia coli cells of the strain each contain oxidative cytoplasm capable of forming and maintaining disulfide bonds in antibodies. The oxidation state of the cytosol is typically assessed by measuring the redox potential of the cytoplasm of the Escherichia coli cells. Methods for measuring the redox state of cells are well known and are described, for example, in Gilbert et al. (1990) Adv. Enzymol. Rel. Areas Mol. Biol. 63:69, Holmgren and Fgestedt (1982) J. Biol. Chem. 257: 6926, and Hwang et al. (1992) Science 257: 1496. In some cases, the cytosol of an E. coli strain has a redox potential in the range of -400mV to -100mV, for example, -350mV to -150mV, -300mV to -200mV, or -291mV to -250mV.

[0072] Genetic engineering of E. coli strains with oxidative cytoplasm An E. coli strain having oxidative cytoplasm may exhibit reduced or absent expression or activity of at least one reductase compared to the corresponding wild-type E. coli strain. The reductase may be one or more reductases selected from the group consisting of thioredoxin reductase, glutathione reductase, and glutathione. In some methods, the E. coli strain contains a null mutation in at least one of the reductases disclosed above. In some embodiments, the E. coli strain lacks activity of thioredoxin reductase encoded by trxB, lacks activity of thioredoxin 1 encoded by trxA, and / or lacks activity of glutathione reductase encoded by gor. In some embodiments, the E. coli strain expresses a mutant AhpC protein, which has glutathione reductase activity. In one exemplary embodiment, the E. coli strain is a Snuggle strain containing null mutations in trxA, trxB, and gor, and lacking thioredoxin reductase activity (TrxB), thioredoxin 1 activity (TrxA), and glutathione reductase activity (GOR). Furthermore, the Snuggle strain overexpresses DsbC lacking its own signal sequence, and is a variant of the ahpC gene encoding an enzyme lacking peroxireductase activity but possessing glutathione reductase activity (ahpC * ) is overexpressed. Methods for confirming that the expression of these enzymes is reduced are well known, for example, as disclosed below.

[0073] A method for introducing mutations into E. coli. In some embodiments, gene modification, such as the knockout of trxA and trxB, can be performed by site-directed recombination. Site-directed recombination uses an enzyme that has both endonuclease and ligase activity, which recognizes a specific portion of a DNA sequence and replaces it with any other corresponding DNA sequence. See Yang W. and Mizuuchi K., Structure, 1997, Vol. 5, 1401-1406(9). Site-directed recombination systems are well known in the art and include, for example, the Int / att system from bacteriophages, the Cre / LoxP system from PI bacteriophages, and the FLP-FRT system from yeast.

[0074] Non-limiting examples of methods for introducing site-directed recombination into various proteins disclosed herein include the Cre / Lox recombination system and the Flp / Frt recombination system. Both systems are well known in the art. For example, site-specific integration into bacterial chromosomes has been reported (e.g., Sauer et al., Proc. Natl. Acad. Sci. 85. 5166-5170 (1988), Fukushige et al., Proc. Natl. Acad. Sci., 89. 7905-7907 (1992), Baubonis et al., Nucleic Acids Research. 21, 2025-2029 (1993), Hasan et al., Gene, 150. 51-56 (1994), Golic et al., Cell. 5_9, 499-509 (1989), Sauer, Mol. Cell. Biolo. 1_, 2087-2096 (1987), Sauer et al., Methods: Companion to Methods in Enzymol. 4.). 143-149 (1992), Sauer et al., The New Biologist. 2., 441-449 (1990), Sauer et al., Nucleic Acids Res.. 17. 147-161 (1989), Qin et al., Proc. Natl. Acad. Sci. 91. 1706-1710 (1994), Orban et al., Proc. Natl. Acad. Sci., 89, 6861-6865 (1992)). Specific deletions and rearrangements of chromosome sequences, as well as the excision of foreign DNA as plasmids from lambda vectors, are also known (e.g., Barinaga, Science. 265, 27-28 (1994); Sauer, Methods in Enzymol. 225. 890-900 (1993); Sauer et al., Gene, 70. 331-341 (1988); See Brunelli et al., Yeast, 1309-1318 (1993); Invitrogen (San Diego, CA) 1995 Catalog, 35; Clontech (Palo Alto, CA) 1995 / 1996 Catalog, 187-188). Cloning schemes have been developed so that recombination reconstructs or inactivates functional transcription units by deletion or inversion of sequences between recombination sites (see, for example, Odell et al., Plant Phvsiol.. 106. 447-458 (1994), Gu et al., Cell. 73. 1155-1164 (1993), Lakso et al., Proc. Natl. Acad. Sci.. 89. 6232-6236 (1992), Fiering et al., Proc. Natl. Acad. Sci. 90. 8469-8473 (1973), O'Gorman et al., Science. 251, 1351-55 (1991), Jung et al., Science, 259, 984-987 (1993)).

[0075] The genes encoding Cre recombinase or Flp recombinase may be provided in trans under the control of a constitutive promoter, an inductive promoter, or a developmentally regulated promoter, or purified recombinase may be introduced (see, for example, Baubonis et al., supra; Dang et al., Develop. Genet. 13, 367-375 (1992), Chou et al., Genetics. 131. 643-653 (1992), Morris et al., Nucleic Acids Res. 19. 5895-5900 (1991)).

[0076] In some embodiments, the genomic manipulations disclosed herein are performed using a site-specific recombination protocol from Kirill A. Datsenko and Barry L. Wanner Proc Natl Acad Sci US A. 2000 Jun 6; 97(12): 6640-6645. In one embodiment, knockout of a gene, e.g., trxA, can be performed as follows: A PCR amplicon is constructed containing an antibiotic resistance gene flanked on both sides by two FRT sites and homology extensions (H1 and H2) homologous to the two ends of the gene to be knocked out. After transforming cells with this PCR product, the gene to be knocked out is replaced by the antibiotic resistance gene by Red-mediated recombination at these adjacent homologous regions. After selection, the resistance gene can be removed using a helper plasmid expressing FLP recombinase. FLP recombinase acts on the FRT (FLP recognition target) site of the direct repeat adjacent to the resistance gene. Since Red and FLP helper plasmids are temperature-sensitive replicas, they can be simply removed (cured) by growth at 37°C. Knock-in of genes such as dsbC can be performed by standard molecular cloning techniques well known to those skilled in the art.

[0077] In some embodiments, gene inactivation, e.g., deletion, is performed using a CRISPR / Cas system. The CRISPR / Cas system removes a gene using a Cas protein and at least one or two ribonucleic acids that can direct the Cas protein to a sequence in the target gene, e.g., in gor. Methods of using the CRISPR / Cas system to eliminate gene expression are well known, for example, described in U.S. Patent Application Publication 2014 / 0170753, the disclosure of which is incorporated in its entirety by reference.

[0078] Further methods for knocking out target genes include, but are not limited to, homologous recombination techniques, effector nuclease transcriptional activation (activator-like effector nucleases (TALENs)), and zinc finger nucleases (ZFNs). These methods are also well known in the art.

[0079] Confirmation of modified reductase expression Various methods can be used to measure the protein expression levels of various modified genes in E. coli and / or to confirm whether the genes have been knocked out or knocked in. For example, gene expression can be measured by conventional Northern blotting for quantifying mRNA transcription. Various labels may be used, the most common being radioisotopes. However, other techniques may be used, such as using biotin-modified nucleotides for introduction into polynucleotides. The biotin then functions as a site for binding to avidin or antibody, which may be labeled with a wide variety of labels such as radionuclides, fluorescent substances, enzymes, etc.

[0080] In some embodiments, expressed proteins can be purified and quantified using gel electrophoresis (e.g., PAGE), Western blotting, or capillary electrophoresis (e.g., Caliper's LabChip). Protein synthesis in cell-free translation reactions is typically carried out using radiolabeled amino acids. 35 S-labeled methionine or 14 The incorporation of 1C-labeled leucine may also be monitored. Radiolabeled proteins can be visualized and quantified for molecular size by autoradiography after electrophoresis, or isolated by immunoprecipitation. Incorporation of recombinant His tag is Ni 2+ This provides another purification method: purification by affinity column chromatography. Protein production from the expression system can be measured as the yield of soluble protein, or using assays for enzyme activity or binding activity.

[0081] In some embodiments, if the protein to be quantified has a defined biological activity, such as enzymatic activity (e.g., alkaline phosphatase) or growth inhibitory activity, the expression of the protein of interest can be confirmed by assaying the activity of the protein after incubation with a suitable substrate.

[0082] Measurement of enzyme activity In some embodiments, mutations introduced into one or more genes, e.g., trxA, do not invalidate protein or mRNA expression but result in mutains that lack the activity of the corresponding wild-type protein, e.g., thioredoxin activity of TrxA. Knocking out a gene does not necessarily require complete loss of its activity, and therefore, for the purposes of this disclosure, it will be understood by those skilled in the art that lack of activity means that the mutant protein ("mutein") loses 85-100% of the activity of the control protein, e.g., the wild-type protein. Various mutains produced can be tested to confirm that they lack the activity of the wild-type protein. For example, each of the coding sequences of mutains can be expressed separately in a host strain, and these mutains can be purified and tested for their activity as described below.

[0083] Confirmation of loss of thioredoxin reductase activity In one embodiment, thioredoxin reductase activity can be measured by its activity in reducing 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) in the presence of NADPH. This reaction is typically initiated by mixing DTNB with thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH. An increase in absorbance at 412 nm is observed over time. Enzyme activity can be defined as the rate of increase in absorbance. Embodiments for detecting thioredoxin reductase activity are disclosed in U.S. Patent No. 8,592,468, the relevant portion of which is incorporated herein by reference.

[0084] Confirmation of loss of thioredoxin activity Methods for measuring thioredoxin activity are also well known. In one embodiment, this assay is an insulin precipitation assay, as described, for example, in Sung-Jong Jeon et al., European Journal of Biochemistry, Vol. 269, No. 22. Thioredoxin is known to have activity as an insulin disulfide reductase, and the decrease in insulin disulfide bonds can be measured by the increase in turbidity due to the precipitation of free insulin B chains. In an example for explanation, the standard assay mixture contained 0.1 M potassium phosphate (pH 7.0), 1 mM EDTA, and 0.13 mM bovine insulin, either in the absence or presence of recombinant protein, and the reaction was started by adding 1 mM dithiothreitol. The increase in absorbance at 650 nm was monitored at 30°C.

[0085] Confirmation of loss of glutathione reductase activity AhpC * The loss of glutathione reductase activity of the protein or the glutathione reductase activity of mutaine GOR can also be monitored. In some embodiments, glutathione reductase activity is measured by its own activity in reducing cysteine. For example, in the presence of a cofactor, a candidate protein, such as AhpC * Alternatively, cysteine ​​was incubated with a reducing solution containing mutaine GOR. Preferably, the cofactor is a coenzyme. Preferably, the cofactor is nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH). This reaction reduces cystine to cysteine. A schematic reaction is as follows: CYS - CYS + 2GSH → 2CYS + GSSG GSSG+NADPH→2GSH+NADP+H The aforementioned activity can be measured by measuring cysteine ​​production. In one specific example, the glutathione reductase activity for cysteine ​​reduction is described in International Publication No. 2018 / 114576.

[0086] Confirmation of loss of AhpC peroxireductase activity AhpC * The lack of peroxireductase activity in this protein can be confirmed by incubating it with an organic hydroperoxide or hydrogen peroxide in the presence of NADH. Functional peroxireductase converts these substrates to water via an NADH-dependent mechanism. The lack of evidence for such conversion indicates a lack of peroxireductase activity.

[0087] Oxidative maintenance of the cytoplasm by inhibiting reductase activity. Alternatively, the cytoplasmic redox potential of E. coli cells can be regulated by exposing the cells to an agent that inhibits the activity of one or more intracellular reductases. The cytoplasmic redox potential can be adjusted to an oxidative state that promotes disulfide bond formation and protein stability. Agents that can inhibit one or more of the above-mentioned reductases are well known, such as gold thioglucose, gold thiomalate, or copper(II) sulfate (CuSO4).

[0088] Suitable reductase inhibitors can also be identified by screening a library of compounds using in vitro enzyme assays. The activity of the target reductase can be measured and compared in the presence or absence of the candidate compound, and if the candidate compound shows the ability to reduce reductase activity in the assay, the candidate compound can be selected as a reductase inhibitor.

[0089] Further characteristics of E. coli strains that can promote antibody yield In some embodiments, the E. coli strain further expresses a prokaryotic cytoplasmic disulfide isomerase, which facilitates protein folding and can further improve antibody yield. In one embodiment, the disulfide isomerase is DsbC. In one embodiment, the disulfide isomerase is yeast protein disulfide isomerase (yPDI), as described, for example, in Groff et al., MAbs 6(3): 671-678 (2014). In some embodiments, the E. coli strain further expresses a disulfide oxidase to facilitate disulfide formation and enhance IgG assembly. In one embodiment, the disulfide oxidase is DsbA. In one embodiment, the disulfide reductase is Qsox. In some embodiments, the protein isomerase is prolyl isomerase. Suitable prolyl isomerases may include, but are not limited to, FkpA, cyclophylline, FKBP, palbrin, or slyD, deaggregase skP or groEL / groES, danK, dnaJ, or grpE.

[0090] Confirmation that the cytosol of the E. coli strain is in an oxidized state. Whether an E. coli strain has oxidative cytoplasm can be determined by measuring the cytoplasmic redox potential, as disclosed above. Alternatively, the oxidative state of the cytoplasm can be determined by evaluating the biological activity of a test protein having one or more disulfide bonds, such as LC, which is difficult to express in wild-type E. coli strains. Preferred test polypeptides or proteins are those normally secreted by cells or are membrane proteins. In some cases, these polypeptides are modified by deletions or mutations in signal sequences so that the protein is not exported outside the cell's cytoplasm.

[0091] As an illustrative example, the coding sequence for the LC of the B10 antibody (SEQ ID NO: 2) can be engineered into an expression cassette under a suitable promoter and transformed into a modified E. coli strain. The soluble protein fraction containing the LC is measured. A suitable E. coli strain can express at least 1 mg / 100 mL of LC in its soluble form. Methods for preparing bacterial lysates and measuring the protein expression levels (e.g., LC expression) in the lysates are well known. In some embodiments, lysates can be produced by treating E. coli cells with a lysis agent. Cytoplasmic proteins can be released by treating the lysate with enzymes such as benzonase or oval lysozyme. The insoluble protein fraction can be separated from the soluble fraction, for example, by centrifugation. The soluble protein fraction (containing the LC) can be collected and analyzed by SDS-PAGE. The amount of LC protein in the soluble protein fraction can then be quantified, for example, by densitometry.

[0092] IV. Antibodies of Focus The methods provided in this disclosure can be used to recombinantly produce any full-length antibody in E. coli cells. Because antibodies contain disulfide bonds, the systems of this disclosure are beneficial in preventing antibody degradation and increasing yield. Using the methods of this disclosure, any antibody can be produced in yields of approximately 200 mg / L or more, approximately 250 mg / L or more, approximately 500 mg / L or more, approximately 750 mg / L or more, or approximately 1000 mg / L or more, expressed as the amount of protein per liter of culture medium. In some embodiments, the antibody is selected from the group consisting of B10 antibody, H01 antibody, 7219 antibody (anti-CD74 antibody), anti-PD1 antibody, anti-Tim3 antibody, anti-HER2 antibody (e.g., trastuzumab), and anti-LAG3 antibody. B10 and H01 are different antibodies, but both target the folate receptor. Polynucleotides encoding the HC and LC of an antibody can be introduced into host cells to express the HC and LC, and then they can be assembled to form a full-length antibody. Table 1 lists non-specific examples of antibodies. [Table 1]

[0093] V. Promoter The promoters that can be used in the methods of the present invention to drive the transcription of HC or LC may be any suitable promoter sequence suitable for Escherichia coli. Such promoters may include mutant promoters, truncated promoters, and hybrid promoters, and may be obtained from polynucleotides encoding extracellular or intracellular polypeptides that may be endogenous (native) or heterologous (exogenous) to the cell. The promoters used in this disclosure may be constitutive promoters or inducible promoters.

[0094] In some embodiments, the promoter is a constitutive promoter. The promoter may have substantially the same promoter strength as T7, i.e., the promoter strength is at least 60%, at least 70%, or at least 80% of the T7 promoter strength. In some embodiments, the T7 promoter includes or consists of the sequence of SEQ ID NO: 19. In some embodiments, the promoter may exhibit a strength in the range of 50% to 200%, e.g., 80% to 150% or 90% to 140%, of the T7 promoter strength. Non-limiting examples of promoters suitable for driving HC and LC transcription in E. coli cells include the T3 promoter, SP6 promoter, pBad, XylA, or PhoA promoter.

[0095] The strengths of two promoters can be compared by comparing the transcription levels of the same gene product initiated by the two promoters. For example, host cells containing an expression construct with the promoter of interest ("test host cells") and control host cells containing a control expression construct (containing a reference promoter, e.g., the T7 promoter) can be grown in culture under replicate conditions. Total RNA from the host cells and controls can be extracted and measured at 260 nm absorbance. Then, cDNA can be synthesized from equal amounts of total RNA from the test host cells and control host cells. RT-PCR can be performed to amplify the cDNA corresponding to the transcripts produced from the promoters. An exemplary method is described in De Mey et al. ("Promoter knock-in: a novel rational method for the fine tuning of genes", BMC Biotechnol 2010 Mar 24; 10:26).

[0096] In some embodiments, the promoters driving the expression of HC and LC are the same, for example, both being T7 promoters. In some cases, HC and LC are directed by a single promoter, for example, a single T7 promoter in a bicistronic operon, as disclosed below. In some embodiments, the promoter for HC and the promoter for LC are different promoters. In some embodiments, the promoter intensities of HC and LC are substantially the same. In some embodiments, the promoter intensities of HC and LC are different.

[0097] VI. Vectors The polynucleotides encoding the LC and HC of an antibody can be inserted into one or two replicable vectors for expression in E. coli. Many vectors are available for this purpose, and those skilled in the art can easily select a suitable vector for use in the methods disclosed herein. In addition to the gene of interest and a promoter that drives the expression of said gene, the vector typically includes one or more of a signal sequence, an origin of replication, or one or more marker genes.

[0098] In some embodiments, the bicistronic vector uses a promoter for transcribing both the HC coding sequence and the LC coding sequence. In some embodiments, the HC coding sequence is proximal to the promoter. In some embodiments, the LC coding sequence is proximal to the promoter. Any promoter among those disclosed herein may be used in the bicistronic vector. In some embodiments, the promoter is a T7 promoter. In some embodiments, the bicistronic vector further includes a transcription terminator such as a T7 terminator. The HC coding sequence and the LC coding sequence may be 15 to 30 nucleotides, for example 20 to 24 nucleotides, apart from each other; that is, the two nearest nucleotides, one on the HC and the other on the LC, are 15 to 30 nucleotides, for example 20 to 24 nucleotides apart. In some embodiments, the HC coding sequence and the LC coding sequence are separated by a nucleotide sequence containing a sequence encoding a ribosome binding site.

[0099] In some embodiments, plasmids encoding HC and LC include two monocistronic operons, one for HC production and the other for LC production. In some embodiments, the two monocistronic operons share the same promoter, e.g., a T7 promoter or a T5 promoter. In some embodiments, the two monocistronic operons include different promoters; that is, the promoter of the monocistronic operon for LC and the promoter of the monocistronic operon for HC have different sequences and / or different promoter activities. Any of the promoters disclosed herein can be used in a vector containing the two monocistronic operons. In some embodiments, each monocistronic operon further includes a transcriptional terminator, such as a T7 terminator.

[0100] In some embodiments, the translation of HC is regulated by a first ribosome binding site, and the translation of LC is regulated by a second ribosome binding site. The ribosome binding sites are independently selected to ensure that the expression levels of HC and LC are in the desired ratio, for example, that the molar ratio of HC to LC is in the range of 1:1 to 1:3, e.g., about 2.5. This technique can increase the yield of full-length antibodies formed by HC and LC produced in E. coli cultures. The ratio of HC to LC can be determined based on the amount of HC and LC produced in the cell lysate obtained from the E. coli culture. The amounts of HC and LC can be measured using any method suitable for protein quantification. In one exemplary embodiment, the E. coli culture is recovered and lyzed to prepare a cell lysate. Optionally, the cell lysate is purified, for example, by chromatography. The cell lysate or purified cell lysate is treated with a reducing agent (e.g., DTT) to separate HC and LC. Next, this reduced cell lysate sample can be analyzed on an SDS-PAGE gel, where HC and LC are separated and resolved according to their size. Then, the gel is blotted with an antibody that recognizes HC and / or LC. The signal associated with the binding of the antibody to HC or LC can be quantified. This signal is proportional to the amount of HC or LC produced in the culture.

[0101] In some embodiments, the first ribosome binding site or the second ribosome binding site includes SEQ ID NO: 28. In some embodiments, the second ribosome binding site includes SEQ ID NO: 29. In some embodiments, the first ribosome binding site includes SEQ ID NO: 28, and the second ribosome binding site includes SEQ ID NO: 29. In some embodiments, the first ribosome binding site includes a sequence selected from the group consisting of SEQ ID NOs: 17-19, and the second ribosome binding site includes a sequence selected from the group consisting of SEQ ID NOs: 20-23; see Example 13, Tables 2 and 3. In some embodiments, the first ribosome binding site includes a sequence selected from the group consisting of SEQ ID NOs: 20-22, and the second ribosome binding site includes SEQ ID NOs: 17-19 (e.g., SEQ ID NO: 17); see Example 24. In some embodiments, the second ribosome binding site includes SEQ ID NO: 21. In some embodiments, the second ribosome binding site includes SEQ ID NO: 22. In some embodiments, the first ribosome binding site includes SEQ ID NO: 17 or SEQ ID NO: 18. As long as the expression levels of HC and LC can be maintained at the desired ratio described above, any sequence disclosed for the first ribosome binding site can be used as the second ribosome binding site.

[0102] In some embodiments, the antibody is selected from the group consisting of SP7219 IgG, B10 IgG, H01 IgG, αPD1 IgG, αTim3 IgG, αLAG3 IgG, and their variants.

[0103] VII. Antibodies containing unnatural amino acids The antibodies produced using the above method may contain at least one non-natural amino acid. In some embodiments, the non-natural amino acid is incorporated into the HC or LC at a specific site in the protein. These non-natural amino acids typically have bio-orthogonal reactive chemical side chains that can be used for conjugation with various biologically active adducts. See below. This method can introduce additional functionality to the antibody, such as fluorescent or radiolabeling, photoactivatable markers, PEGs that modify pharmacokinetics, or chemotherapeutic agents.

[0104] The one or more non-natural amino acids may be located at selected site-specific locations in the heavy chain, light chain, or both of the antibody. These site-specific locations may be in any domain of the antibody, such as any variable domain and any constant domain. The number of non-natural amino acids present in the LC, HC, or both may vary, ranging from 1 to 10, for example, 2 to 8, 3 to 6, 5 to 10, or 3 to 7.

[0105] Heavy chains containing one or more unnatural amino acids In certain embodiments, the antibodies provided in this disclosure contain one or more non-natural amino acids that substitute for a native amino acid at a position selected from the group consisting of HC-F404, HC-K121, HC-Y180, HC-F241, HC-221, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC-R19, HC-Y52, or HC-S70 in the heavy chain residue. In these notations, HC represents a heavy chain residue.

[0106] Light chains containing one or more unnatural amino acids In certain embodiments, the antibodies provided in this disclosure include one or more non-natural amino acids that substitute for native amino acids at positions selected from LC-K42, LC-E161, LC-T22, LC-S7, LC-N152, LC-K42, LC-E161, or LC-D170, respectively. In these notations, LC represents a light chain residue.

[0107] The antibodies disclosed herein may also contain non-natural amino acids at any combination of the above-mentioned positions in HC and LC.

[0108] Exemplary antibodies containing non-natural amino acids In one exemplary embodiment, the antibody is a B10 antibody containing an HC having one or more mutations selected from the positions of HC-F404, HC-Y180, and HC-F241 relative to SEQ ID NO: 1, and the position of LC-K42 relative to SEQ ID NO: 5. The mutations cause one or more amino acid codons at these positions to be replaced by non-natural amino acid codons. In one embodiment, the non-natural amino acid codon is an amber codon. In some embodiments, the B10 antibody contains an HC having a sequence selected from the group consisting of SEQ ID NOs: 3, 4, 6, and 7, and / or an LC having the sequence of SEQ ID NO: 5. In another exemplary embodiment, the antibody is a trastuzumab containing an LC in which at least one of LC-E42 and LC-E161 is replaced by a non-natural amino acid.

[0109] Non-natural amino acids Suitable non-natural amino acids that can be incorporated into antibodies include, for example, those described in U.S. Patents 10,179,909, 9,938,516, 9,682,934, 10,596,270, and 10,610,571, the entire contents of which are incorporated by reference into this disclosure.

[0110] The non-natural amino acids may contain reactive groups useful for forming covalent bonds to linkers or biologically active adducts (also known as payloads), as described below. In certain embodiments, the reactive groups are selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazide, semicarbazide, sulfanyl, azide, and alkynyl. The non-natural amino acids may be L-amino acids, D-amino acids, or racemic amino acids. In certain embodiments, the non-natural amino acids described herein include D-forms and racemic forms of natural amino acids.

[0111] In a particular embodiment, the non-natural amino acid relates to one of the following formulas. [ka]

[0112] In the above formula, the wavy line indicates a bond that connects to the rest of the antibody polypeptide chain. These non-natural amino acids can be incorporated into the polypeptide chain in the same way that natural amino acids are incorporated into the same polypeptide chain. In certain embodiments, the non-natural amino acids are incorporated into the polypeptide chain via amide bonds, as shown in the formula. In the above formula, R represents any functional group without restriction, as long as the amino acid residue is not identical to a natural amino acid residue. In certain embodiments, R may be a hydrophobic group, a hydrophilic group, a polar group, an acidic group, a basic group, a chelating group, a reactive group, a therapeutic moiety, or a labeling moiety. In the above formula, each L represents a linker (e.g., a divalent linker), which will be further described below.

[0113] In some embodiments, the non-naturally encoded amino acids include side-chain functional groups that react efficiently and selectively with functional groups not found in the 20 common amino acids (including, but not limited to, azide groups, ketone groups, aldehyde groups, and aminooxy groups) to form stable conjugates. For example, an antigen-binding polypeptide containing a non-naturally encoded amino acid with an azide functional group can be reacted with a polymer (including, but not limited to, poly(ethylene glycol)) or with a second polypeptide containing an alkyne moiety to form a stable conjugate by selective reaction between the azide and alkyne functional groups to form a huisgen[3+2] cycloaddition product.

[0114] Examples of non-naturally encoded amino acids that are suitable for use in the present invention and useful for reaction with water-soluble polymers include, but are not limited to, those having carbonyl reactive groups, aminooxy reactive groups, hydrazine reactive groups, hydrazide reactive groups, semicarbazide reactive groups, azide reactive groups, and alkyne reactive groups. In some embodiments, the non-naturally encoded amino acids include a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids include those in which the naturally occurring N- or O-bond between the amino acid and the sugar is replaced by a covalent linkage not commonly found in nature, such as alkenes, oximes, thioethers, amides, etc. Examples of such amino acids include sugars not commonly found in natural proteins, such as 2-deoxyglucose and 2-deoxygalactose.

[0115] Many of the non-natural amino acids suitable for use in the present invention are generally available, for example, from Sigma (USA) or Aldrich (Milwaukee, Wisconsin, USA). Those not commercially available may be optionally synthesized as indicated in this disclosure, as indicated in various publications, or using standard methods known to those skilled in the art. For organic synthesis techniques, see, for example, *Organic Chemistry by Fessendon and Fessendon* (1982, Second Edition, Willard Grant Press, Boston Mass.); and also, as incorporated by reference in this disclosure, U.S. Patent Publication Nos. 2003 / 0082575 and 2003 / 0108885, *Advanced Organic Chemistry by March* (Third Edition, 1985, Wiley and Sons, New York), and *Advanced Organic Chemistry by Carey and Sundberg* (Third Edition, Parts A and B, 1990, Plenum Press, New York). Other publications describing the synthesis of non-natural amino acids include, for example, International Publication No. 2002 / 085923 titled "In vivo incorporation of Non-natural Amino Acids," Matsoukas et al., (1995) J. Med. Chem., 38, 4660-4669, King, FE & Kidd, DAA (1949) A New Synthesis of Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates. J. Chem. Soc., 3315-3319, and Friedman, OM & Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates for Anti-Tumor Agents.J. Am. Chem. Soc. 81, 3750-3752, Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine). J. Org. Chem. 53,1167-1170, Azoulay, M., Vilmont, M. & Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur. J. Med. Chem. 26, 201-5, Koskinen, A. M. P. & Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino Acid Analogues. J. Org. Chem. 54, 1859-1866, Christie, B. D. & Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates from L-Asparagine. Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem. 1989:1859-1866, Barton et al., (1987) Synthesis of Novel a-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis of L- and D-α-Amino-Adipic Acids, L-a-aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron Lett. 43:4297-4308, and Subasinghe et al.See also (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Also see U.S. Patent Application No. 10 / 744,899, filed December 22, 2003, and U.S. Patent Application No. 60 / 435,821, filed December 22, 2002, both titled "Protein Arrays".

[0116] Many non-natural amino acids are based on natural amino acids such as tyrosine, glutamine, phenylalanine, etc., and are suitable for use in the present invention. Tyrosine analogs include, but are not limited to, para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine. These substituted tyrosines include, but are not limited to, keto groups (including, but not limited to, acetyl groups), benzoyl groups, amino groups, hydrazine, hydroxylamine, thiol groups, carboxyl groups, isopropyl groups, methyl groups, C6-C20 linear or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, nitro groups, alkynyl groups, etc. Furthermore, multiple-substituted aryl rings are also within the scope of consideration. Glutamine analogs suitable for use in the present invention include, but are not limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclic derivatives, and amide-substituted glutamine derivatives. Examples of phenylalanine analogs suitable for use in the present invention include, but are not limited to, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine. Substituents include, but are not limited to, hydroxyl groups, methoxy groups, methyl groups, allyl groups, aldehydes, azides, iodines, bromos, keto groups (including, but not limited to, acetyl groups), benzoyl groups, alkynyl groups, and the like.Specific examples of non-natural amino acids suitable for use in the present invention include azidoethoxycarbonyllysine (AEK), p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, and p-azidoethoxycarbonyllysine. Examples of non-natural amino acids suitable for use in the present invention include, but are not limited to, do-L-phenylalanine, p-azidomethyl-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine. Examples of structures of various non-natural amino acids suitable for use in the present invention are presented, for example, in International Publication No. 2002 / 085923, titled "In vivo incorporation of non-natural amino acids." For additional methionine analogs, see Kiick et al., (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation, PNAS 99:19-24.

[0117] Specific examples of useful non-natural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc-β-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-methyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine. Further useful examples include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine.

[0118] In certain embodiments, the non-natural amino acid is selected from p-acetylphenylalanine, p-ethynylphenylalanine, p-propargyloxyphenylalanine, p-azidomethylphenylalanine, and p-azidophenylalanine. In one embodiment, the non-natural amino acid is p-azidophenylalanine. It is known to those skilled in the art that this amino acid residue promotes a hysgen[3+2] cycloaddition reaction (a so-called "click" chemistry reaction) with, for example, a compound having an alkynyl group. This reaction allows those skilled in the art to easily and rapidly conjugate the antibody at a site-specific location of the non-natural amino acid.

[0119] In certain embodiments, the first reactant is an alkynyl moiety (including, but not limited to, the moiety in the unnatural amino acid p-propargyloxyphenylalanine, where the propargyl group may also be referred to as the acetylene moiety), and the second reactant is an azide moiety, and [3+2] cycloaddition chemistry can be used. In certain embodiments, the first reactant is an azide moiety (including, but not limited to, the moiety in the unnatural amino acid p-azido-L-phenylalanine), and the second reactant is an alkynyl moiety.

[0120] The non-natural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) At least one functional group on the side chain of the non-natural amino acid is orthogonal to the chemical reactivity of 20 genetically encoded common amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), or at least one feature and / or activity and / or reactivity that is orthogonal to the chemical reactivity of natural amino acids present in polypeptides containing the non-natural amino acid; (2) The introduced non-natural amino acid is genetically encoded (3) Non-natural amino acids that are substantially chemically inert to the 20 common amino acids, are stably incorporated into the polypeptide, preferably with the same stability as the natural amino acids, or the incorporation occurs under typical physiological conditions, more preferably such incorporation can occur via an in vivo system, and (4) the non-natural amino acids include an oxime functional group, or a functional group convertible to an oxime group by reaction with a reagent, preferably such conversion is made under conditions that do not destroy the biological properties of the polypeptide containing the non-natural amino acid (not to mention, unless such destruction of biological properties is for the purpose of modification / conversion), or such conversion can occur under aqueous conditions with a pH of about 4 to about 8, or the reaction site on the non-natural amino acid is an electrophilic site. Any number of non-natural amino acids can be introduced into the polypeptide. Non-natural amino acids may also include protected or masked oximes, or protected or masked groups convertible to an oxime group after deprotection of the protected group or demasking of the masked group.Non-natural amino acids may also contain protected or masked carbonyl or dicarbonyl groups, which can be converted to carbonyl or dicarbonyl groups after deprotection of the protected group or demasking of the masked group, thereby making them available for reaction with hydroxylamines or oximes to form oxime groups.

[0121] In further embodiments, non-natural amino acids that can be used in the methods and compositions of the present disclosure include, but are not limited to, amino acids containing photoactivatable crosslinkers, spin-labeled amino acids, fluorescent amino acids, metal-linked amino acids, metal-containing amino acids, radioactive amino acids, amino acids having novel functional groups, amino acids that interact covalently or acovalently with other molecules, photocaged and / or photoisomerizable amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids such as sugar-substituted serine, other carbohydrate-modified amino acids, keto-containing amino acids, aldehyde-containing amino acids, amino acids containing polyethylene glycol or other polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids having elongated side chains compared to natural amino acids (including, but not limited to, polyethers or long-chain hydrocarbons (containing about 5 or about 10 carbon atoms)), carbon-linked sugar-containing amino acids, redox-active amino acids, aminothio acid-containing amino acids, and amino acids containing one or more toxic moieties.

[0122] In some embodiments, non-natural amino acids include a sugar moiety. Examples of such amino acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine, N-acetyl-L-glucosaminyl-L-asparagine, and O-mannosaminyl-L-serine. Examples of such amino acids include cases where the naturally occurring N- or O-bond between the amino acid and the sugar is replaced by a covalent linkage not commonly found in nature, such as alkenes, oximes, thioethers, amides, etc. Examples of such amino acids also include sugars not commonly found in natural proteins, such as 2-deoxyglucose, 2-deoxygalactose, etc.

[0123] In certain embodiments, the non-natural amino acid is selected from the group of non-natural amino acids shown in Figures 8A to 8D. Such non-natural amino acids may be in the form of salts, or incorporated into non-natural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and may be optionally post-translationally modified.

[0124] In some embodiments, the non-natural amino acid is paramethylazido-L-phenylalanine (pAMF), azidoethoxycarbonyllysine (AEK), or p-acetyl-L-phenylalanine (pAcF). In some embodiments, the non-natural amino acid is (S)-2-amino-3-(5-(6-methyl-1,2,4,5-tetrazin-3-ylamino)pyridine-3-yl)propanoic acid.

[0125] Incorporation of non-natural amino acids Methods for incorporating non-natural amino acids are well known and are described, for example, in U.S. Patents 10,610,571, 9,988,619, and 9,938,516, which are incorporated in their entirety by reference in this disclosure.

[0126] In one method, the coding sequence of HC or LC is modified to include at least one non-natural amino acid codon. The non-natural amino acid codon is a codon that does not result in the incorporation of any of the 20 natural amino acids. In some embodiments, the non-natural amino acid codon is an amber, opal, or ochre stop codon that, instead of terminating translation, is repurposed to be charged onto its cognate tRNA by a tRNA synthetase.

[0127] Non-natural amino acids can be loaded onto tRNA by tRNA synthetases that preferentially acetylate non-natural amino acids compared to any of the 20 natural amino acids. Such tRNA synthetases are known; for example, U.S. Patent No. 9,938,516 discloses a tRNA synthetase that selectively incorporates the non-natural amino acid paramethylazido-L-phenylalanine (pAMF). Other tRNA synthetases capable of selectively incorporating other non-natural amino acids, such as azidoethoxycarbonyllysine (AEK) or p-acetyl-L-phenylalanine (pAcF), are also known; see, for example, Chen et al., Angew Chem Int Ed Engl. 2009; 48(22):4052-5 (doi: 10.1002 / anie.200900683) and Li et al., Proc Natl Acad Sci USA, 2003 Jan 7;100(1):56-61. The entire contents of the aforementioned publications are incorporated herein by reference. Further exemplary non-natural amino acids that can be incorporated into the antibodies disclosed herein include aralkyl, heterocyclyl, and heteroaralkyl, as well as lysine derivative non-natural amino acids. In some embodiments, such non-natural amino acids include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocyclic groups. In some embodiments, such amino acids include azide, tetrazine, or other chemical groups that can be conjugated to a coupling partner such as a water-soluble moiety.

[0128] tRNA synthetases capable of selectively incorporating non-natural amino acids may also be obtained by genetically modifying wild-type tRNA synthetases to produce mutant tRNA synthetases. Each of these mutant tRNA synthetases can then be tested for its activity in selectively incorporating non-natural amino acids using a reporter gene containing a desired non-natural amino acid codon. The activity of mutant tRNA synthetase variants in the presence of non-natural amino acids (e.g., pAMF) compared to 20 common natural amino acids can be measured by detecting the presence or absence of the reporter protein. One exemplary method for producing mutant tRNA synthetases for incorporating non-natural amino acids is disclosed in U.S. Patent No. 9,938,516, the entirety of which is incorporated by reference in this disclosure.

[0129] In some embodiments, the non-natural amino acid codon is a synthetic codon, and the non-natural amino acid is incorporated into the antibody using an orthogonal synthetase / tRNA pair. The orthogonal synthetase may be a modified version of any of the natural amino acid synthetases. For example, the orthogonal synthetase may be a proline synthetase, a modified serine synthetase, a modified tryptophan synthetase, or a modified phosphoserine synthetase. The orthogonal tRNA may also be a modified version of any of the natural amino acid tRNAs. For example, the orthogonal tRNA may be modified alanine tRNA, modified arginine tRNA, modified aspartate tRNA, modified cysteine ​​tRNA, modified glutamine tRNA, modified glutamate tRNA, modified alanin lysine, modified histidine tRNA, modified leucine tRNA, modified isoleucine tRNA, modified lysine tRNA, modified methionine tRNA, modified phenylalanine tRNA, modified proline tRNA, modified serine tRNA, modified threonine tRNA, or modified tryptophan tRNA. In some embodiments, it may be modified tyrosine tRNA, modified valine tRNA, or modified phosphoserine tRNA.

[0130] Codon optimization Codon optimization can be used to increase the translation rate of HC or LC, or to produce recombinant RNA transcripts with desirable properties such as a longer half-life, compared to transcripts produced using unoptimized sequences. HC coding sequences or LC coding sequences can be optimized to maximize expression efficiency in E. coli. In particular, when expressing HC or LC polynucleotides containing non-natural amino acid codons in E. coli, bases adjacent to the non-natural amino acid codons can affect the mRNA three-dimensional structure at the P site of the ribosome, potentially impacting the integration efficiency of the non-natural amino acid. Therefore, it is desirable to optimize the codons of amino acids adjacent to the non-natural amino acid codons to maximize the yield of antibodies containing non-natural amino acids. In some embodiments, codons adjacent to the 3' side of the non-natural amino acid codon are optimized to maximize expression. The optimal codon can be selected by comparing the yields of HC (or LC) produced from expressing HC (or LC) coding sequences containing different codons for the same amino acid located adjacent to the 3' side of the non-natural amino acid codon, and selecting the coding sequence that yields the highest yield. In one illustrative example, the HC coding sequence of the B10 antibody has an amber codon at position 180 instead of a tyrosine (Y) codon. Position 181, adjacent to the 3' side of the amber codon, is serine. HC coding sequences with a serine codon AGC or AGT at that position exhibited a higher titer than HC coding sequences with a serine codon TCG at the same position. See Example 11 and Figure 2.

[0131] VIII. Method Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meanings as those generally understood by those skilled in the art to which the invention pertains. For definitions and terms in the art, practitioners should refer, in particular, to Green, MR, and Sambrook, J., eds., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012) and Ausubel, FM, et al., Current Protocols in Molecular Biology (Supplement 99), John Wiley & Sons, New York (2012) (these are incorporated by reference into this disclosure). Standard methods are also shown in Bindereif, Schon, & Westhof (2005) Handbook of RNA Biochemistry, Wiley-VCH, Weinheim, Germany, which describes detailed methods for RNA manipulation and analysis and is incorporated by reference into this disclosure. Examples of sufficient explanations to direct those skilled in the art through appropriate molecular techniques for generating recombinant nucleic acids and numerous cloning exercises can be found, incorporated by reference, in Green, MR, and Sambrook, J., (ibid.), Ausubel, FM, et al., (ibid.), Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology (Volume 152 Academic Press, Inc., San Diego, Calif. 1987), and PCR Protocols: A Guide to Methods and Applications (Academic Press, San Diego, Calif. 1990).

[0132] PCR amplification methods are well known in the art and are described, for example, in Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif., 1990. The amplification reaction system typically includes the DNA to be amplified, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), a reaction buffer, and magnesium. Typically, the desired number of thermal cycles is 1 to 25. Methods for primer design and optimization of PCR conditions are well known in the art and can be found in Ausubel et al., Short Protocols in Molecular Biology, 5 th This can be found in standard molecular biology textbooks such as Edition, Wiley, 2002 and Innis et al., PCR Protocols, Academic Press, 1990. Computer programs can help design primers with the required specificity and optimal amplification characteristics (e.g., Oligo Version 5.0 (National Biosciences)). In some embodiments, PCR primers may further include a recognition site for a restriction endonuclease to facilitate the insertion of the amplified DNA fragment into a specific restriction enzyme site in the vector. When adding a restriction enzyme site to the 5' end of a PCR primer, it is preferable to include several extra 5' bases (e.g., two or three) to allow for more efficient enzymatic cleavage. In some embodiments, PCR primers may also include an RNA polymerase promoter site, e.g., T7 or SP6, to allow for subsequent in vitro transcription. Methods for in vitro transcription are well known to those skilled in the art (see, for example, Van Gelder et al., Proc. Natl. Acad. Sci. USA 87:1663-1667, 1990 and Eberwine et al., Proc. Natl. Acad. Sci. USA 89:3010-3014, 1992).

[0133] Where antibodies described in this disclosure are indicated by name, it is understood that this includes antibodies having similar functions and similar amino acid sequences in their HC and LC. For example, the name "B10 antibody" includes wild-type antibodies having the HC sequence of SEQ ID NO: 1 and the LC sequence of SEQ ID NO: 2, as well as antibodies having HC and / or light chains with amino acid sequences similar to SEQ ID NO: 1 or SEQ ID NO: 2. Sequence similarity may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Protein sequence identity is measured using the BLASTP program with a default word length of 3, expected value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992).

[0134] Transformation Vectors containing HC coding sequences and LC coding sequences may be transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass the wide variety of techniques commonly used for introducing exogenous DNA into E. coli cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. E. coli cells transformed with the vector can be cultured under appropriate growth conditions to enable HC and LC expression. Cells are harvested, and cell lysates are prepared using standard techniques.

[0135] HC and LC assemblies Cell lysates prepared from E. coli cultures can be adjusted to conditions that allow two LCs and two HCs to assemble into a tetramer of full-length antibodies, which involves dimerization of the two heavy chains and linkage of the heavy and light chains by disulfide bond formation. Typically, the cell lysate is adjusted to a suitable pH (e.g., pH 8.0) and incubated for an extended time with shaking to allow disulfide bond formation and assembly of full-length antibodies. In an example for explanation, the cell lysate is adjusted to pH 8.0 with 100 mM Tris·HCl by incubation on a FlowerPlate (m2p-labs) at 25°C for 16 hours with shaking at 650 rpm.

[0136] Purification of full-length antibodies Assembled IgG was purified from cell lysates using standard affinity chromatography. Various methods for protein purification, chromatography, electrophoresis, centrifugation, and crystallization are described in Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Methods for cell-free synthesis are described in Spirin & Swartz (2008) Cell-free Protein Synthesis, Wiley-VCH, Weinheim, Germany. The degree of HC and LC assembly in antibodies can be measured using methods well known in the art, for example, under non-reducible conditions using a Caliper bioanalyzer (Perkin Elmer, Richmond, California).

[0137] Titer / Yield The titer of the assembled full-length antibody can be measured using standard methods, such as SDS-PAGE, Western blotting, chromatography (e.g., analytical size exclusion chromatography, liquid chromatography, and HPLC), immune-based assays, and enzyme-linked immunosorbent assay (ELISA), but is not limited thereto. An example for illustration is shown in Example 22.

[0138] In some embodiments, particularly when E. coli cells are grown at high density for IgG production, e.g., when the 600 OD is in the range of 50 - 150, the titer of the full-length antibody produced using the methods disclosed herein (expressed as the amount of antibody per liter of medium) is greater than about 200 mg / L, greater than about 250 mg / L, greater than about 350 mg / L, greater than about 400 mg / L, greater than about 450 mg / L, greater than about 500 mg / L, greater than about 550 mg / L, greater than about 600 mg / L, greater than about 650 mg / L, greater than about 700 mg / L, greater than about 750 mg / L, greater than about 800 mg / L, greater than about 850 mg / L, greater than about 900 mg / L, greater than about 950 mg / L, greater than about 1000 mg / L, or more. In another embodiment, the protein of interest is produced at a concentration (amount or level) greater than about 1000 mg / L, e.g., greater than about $1100 mg / L$, greater than about 1200 mg / L, greater than about 1300 mg / L, greater than about 1400 mg / L, greater than about 1500 mg / L, greater than about 1600 mg / L, greater than about 1700 mg / L, greater than about 1800 mg / L, greater than about 1900 mg / L, greater than about 2000 mg / L, or more. Typically, high-density growth is carried out in a bioreactor with a volume of 0.5 liters to 5 liters, and an example thereof is shown in Example 6. However, it is contemplated that the methods described in the present disclosure may be performed on a larger scale, e.g., culture volumes of 500 L or more, 1000 L or more, 2000 L or more, 2500 L or more, 3000 L or more, 5000 L or more, 8000 L or more, 10,000 L or more, or more.

[0139] In some embodiments, the titer of the full-length antibody produced using the methods disclosed herein is a wet weight percentage in the range of 0.05–20%, 0.05–10%, 0.05–5%, 0.05–4%, 0.05–3%, 0.05–2%, 0.05–1%, or 0.05–0.5% relative to the weight of the wet cell pellet produced from E. coli cells in culture. For the lower end of this range, the titer of the full-length antibody produced is a wet weight percentage of at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, or at least 0.6% relative to the weight of E. coli cells in culture.

[0140] IX. Antibody-drug conjugates Antibodies produced using the methods disclosed herein can be conjugated to a biologically active adduct (also known as a payload) using chemical reactions such as click chemistry. In some cases, the antibody contains one or more non-natural amino acids at specific sites in the protein sequence, and the biologically active adduct can be conjugated to these non-natural amino acids. If an antibody has non-natural amino acids at a desired amino acid position, the biologically active adduct can be conjugated to the non-natural amino acids using chemical reactions such as click chemistry. For example, a pAMF-containing antibody produced using the methods disclosed herein can be purified by standard procedures. The purified protein is then subjected to a click chemistry reaction (e.g., a copper(I)-catalyzed azide-alkyne 1,3-cycloaddition reaction or a copper-free catalyzed azide-alkyne 1,3-cycloaddition reaction) to directly conjugate the biologically active adduct to the pAMF residue.

[0141] Examples of biologically active adducts for use in the present invention include, but are not limited to, small molecules, oligonucleotides, peptides, amino acids, nucleic acids, sugars, oligosaccharides, polymers, synthetic polymers, chelating agents, fluorophores, chromophores, other detectable agents, drug moieties, cytotoxic agents, detectable agents, and the like.

[0142] Detailed descriptions of click chemistry reactions for the conjugation of biologically active adducts can be found, for example, in Baskin et al., Proc. Natl. Acad. Sci., 2007, 104: 16793-16797, Kim et al., Curr. Opin. Chem. Biol., 2013, 14:412-419, and Bundy and Swartz, Bioconjug. Chem., 2010, 21(2):255-263.

[0143] In click chemistry reactions, alkynes are activated for [3+2] cycloaddition with azides. A biologically active adduct containing a strained alkyne (e.g., cyclooctin or a variant thereof) (e.g., linked to the alkyne) can undergo a strain-promoted alkyne-azide cycloaddition with the non-natural amino acid paramethylazide-L-phenylalanine on the protein of interest, thereby conjugating the biologically active adduct to the protein at the amino acid position of the non-natural amino acid. A preferred strained alkyne reagent is the reagent DBCO shown below. [ka]

[0144] Linker In certain embodiments, the antibody may be linked to a biologically active adduct using one or more linkers that can react with the antibody's amino acids (e.g., non-natural amino acids) and the biologically active adduct. The term “linker” is used to refer to a group or bond that is typically formed as a result of a chemical reaction, and is typically a covalent bond. The linkers used in this disclosure may be any linker that is obvious to those skilled in the art. In certain embodiments, the linker is any divalent or polyvalent linker known to those skilled in the art. Useful divalent linkers include alkylenes, substituted alkylenes, heteroalkylenes, substituted heteroalkylenes, arylenes, substituted arylenes, heteroarylenes, and substituted heteroarylenes. In certain embodiments, the linker is a C1-10 alkylene or a C1-10 heteroalkylene. Suitable linkers are also disclosed in U.S. Patent No. 10,596,270, the entirety of which is incorporated by reference in this disclosure.

[0145] The conjugated proteins of interest can be purified according to standard methods known in the art to obtain substantially pure polypeptides, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatofocusing, and size exclusion), electrophoresis procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, *Protein Purification*, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

[0146] The conjugated protein of interest can be quantified according to standard methods known in the art, which include, but are not limited to, mass spectrometry (e.g., ESI-TOF mass spectrometry and tandem mass spectrometry), microfluidic electrophoresis, gel electrophoresis, Western blotting, immunoassays (e.g., ELISA), and other assays for evaluating the activity of the conjugated protein.

[0147] X. Use Antibodies produced by the present invention, including those incorporating non-natural amino acids, can be used for one or more of the following purposes or effects: inhibiting the proliferation, infection, or function of an infectious agent, or killing the infectious agent (such infectious agents include, without limitation, bacteria, viruses, fungi, and other parasites); affecting (suppressing or enhancing) physical characteristics (such physical characteristics include, without limitation, height, weight, hair color, eye color, skin, fat-to-lean ratio, or other tissue pigmentation, or the size or shape of organs or body parts (e.g., breast enlargement or reduction, changes in form or shape, etc.)); affecting biorhythms or circadian cycles or rhythms; affecting the reproductive capacity of male or female subjects; affecting the metabolism, catabolism, anabolism, processing, utilization, storage, or excretion of dietary fats, lipids, proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional factors or components; and affecting behavioral characteristics (such as appetite, libido, stress, recognition). Including cognitive impairment, depression (including depressive disorders), and violent behavior; providing analgesic or other pain-relieving effects; promoting the differentiation and proliferation of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting enzyme deficiencies and treating deficiency-related diseases; treating hyperproliferative disorders (e.g., psoriasis); immunoglobulin-like activity (such as the ability to bind to antigens or complement); and the ability to act as an antigen in a vaccine composition and induce an immune response against a protein or another material or entity that cross-reacts with such protein.

[0148] The antibodies produced by the present invention can be used for any purpose known to those skilled in the art. Preferred uses include medical applications, including diagnostic, prophylactic, and therapeutic applications. For example, the antibodies can be prepared for topical administration or other types of administration. Thus, the proteins produced by the present invention can be solubilized or suspended in a pharmacologically acceptable solution to form a pharmaceutical composition for administration to a subject. Appropriate buffers and methods for administering pharmaceutical compositions for medical purposes are further described below. It will be understood by those skilled in the art that the medical compositions can be administered to non-human subjects, for example, for veterinary purposes.

[0149] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be suggested to those skilled in the art, which are understood to be within the spirit and scope of this application and the attached claims. All publications, sequence accession numbers, patents, and patent applications referenced herein are incorporated in their entirety by reference for all purposes.

[0150] Embodiment This disclosure includes the following exemplary embodiments. Embodiment 1. A method for producing a full-length antibody containing heavy chains (HC) and light chains (LC), This includes culturing E. coli cells expressing the coding sequences of the HC and the LC in a culture medium under conditions acceptable for producing the HC and the LC, The full-length antibody is produced in an amount of at least about 200 mg per liter of the culture medium, or The weight percentage of the full-length antibodies produced relative to the weight of the cell pellet derived from the aforementioned E. coli cells is in the range of 0.05% to 20%. The aforementioned method.

[0151] Embodiment 2. The method according to Embodiment 1, wherein at least a portion of the Escherichia coli cells contain oxidative cytoplasm.

[0152] Embodiment 3. A method according to any one of the above embodiments, comprising transforming the plasmid into the Escherichia coli strain.

[0153] Embodiment 4. The method according to any one of the above embodiments, wherein the HC and LC are produced in the cytoplasm of the Escherichia coli.

[0154] Embodiment 5. The method according to any one of the above embodiments, further comprising assembling the produced HC and LC under non-reductive conditions to form a full-length antibody.

[0155] Embodiment 6. The method according to any one of the above embodiments, wherein the molar ratio of the HC produced from the Escherichia coli to the LC produced is about 1:1 to about 1:3.

[0156] Embodiment 7. The expression of HC, LC, or both is controlled by a promoter. The promoter is a T7 promoter, or a promoter having a promoter strength substantially similar to that of a T7 promoter, or The promoter is a T5 promoter, or a promoter having a promoter strength substantially similar to that of a T5 promoter. The method according to any one of claims 1 to 6.

[0157] Embodiment 8. The plasmid comprises a bisistronic operon, The bisistronic operon includes the coding sequence of HC and the coding sequence of LC, or The plasmid comprises a first monocistronic operon for the HC and a second monocistronic operon for the LC. A method according to any one of the embodiments described above.

[0158] Embodiment 9. The bisistronic operon includes a promoter that drives the expression of both the HC and the LC, The promoter is a T7 promoter, or a promoter having a promoter strength substantially similar to that of a T7 promoter, or The first monocistrone operon or the second monocistrone operon includes a T7 promoter. The method described in Embodiment 8.

[0159] Embodiment 10. The bisistron operon includes a T7 terminator, or The first monocistronic operon or the second monocistronic operon includes a T7 terminator. A method according to any one of the embodiments described above.

[0160] Embodiment 11. The method according to any one of claims 1 to 10, wherein the Escherichia coli cell includes a first ribosome binding site for the translation of the HC and a second ribosome binding site for the translation of the LC, and the first ribosome binding site and the second ribosome binding site are selected such that the molar ratio of HC and LC produced from the Escherichia coli is in the range of 1:1 to 1:3.

[0161] Embodiment 12. The method according to Embodiment 11, wherein the first ribosome binding site is transcribed from a DNA sequence selected from the group consisting of SEQ ID NOs: 17 to 19, and the second ribosome binding site is transcribed from a DNA sequence selected from the group consisting of SEQ ID NOs: 20 to 23.

[0162] Embodiment 13. The method according to either Embodiment 11 or Embodiment 12, wherein the first ribosome binding site has the sequence of SEQ ID NO: 17 or SEQ ID NO: 18.

[0163] Embodiment 14. The method according to any one of the above embodiments, wherein the HC and / or LC of the full-length antibody comprises at least one non-natural amino acid.

[0164] Embodiment 15. The method according to Embodiment 14, wherein the coding sequence of HC and / or the coding sequence of LC is modified to have at least one non-natural amino acid codon, and the non-natural amino acid codon does not result in the incorporation of any natural amino acid.

[0165] Embodiment 16. The method according to Embodiment 14, wherein the at least one non-natural amino acid is introduced by loading (charging) it onto a tRNA containing an anticodon complementary to the at least one non-natural amino acid codon.

[0166] Embodiment 17. The method according to any one of Embodiments 14 to 16, wherein the at least one non-natural amino acid codon is the amber codon TAG.

[0167] Embodiment 18. The method according to any one of Embodiments 14 to 16, wherein the at least one non-natural amino acid is paramethylazide-L-phenylalanine (pAMF), AEK, or pAcF.

[0168] Embodiment 19. The method according to any one of claims 15 to 18, wherein at least one codon adjacent to the 3' side of the non-natural amino acid is codon-optimized.

[0169] Embodiment 20. The method according to any one of the above embodiments, wherein the full-length antibody is B10 antibody, H01 antibody, 7219 antibody, anti-PD1 antibody, anti-Tim3 antibody, anti-LAG3 antibody, or anti-Her2 antibody.

[0170] Embodiment 21. The coding sequence of the HC of the B10 antibody contains a mutation relative to SEQ ID NO: 1, and as a result of the mutation, a natural amino acid codon is replaced with a non-natural amino acid codon. The aforementioned natural amino acid is one or more amino acids selected from F404, Y180, and F241, and The method according to Embodiment 20, wherein a non-natural amino acid is introduced into the HC by loading (charging) a tRNA complementary to the non-natural amino acid codon.

[0171] Embodiment 22. The method according to any one of the above embodiments, wherein the coding sequence of the LC of the B10 antibody or the coding sequence of the LC of the trastuzumab contains at least one mutation relative to SEQ ID NO: 2, the at least one mutation substituted a natural amino acid codon with a non-natural amino acid codon, and the natural amino acid is K42 or E161.

[0172] Embodiment 23. The method according to any one of the above embodiments, further comprising covalently linking the warhead portion to the non-natural amino acid on the HC or LC of the full-length antibody via a linker.

[0173] Embodiment 24. The method according to Embodiment 23, wherein the warhead is SC236 or aminooxy-PEG8-methane. [Examples]

[0174] The following examples are provided to illustrate the invention described in the claims and are not intended to limit the invention.

[0175] Example 1. Production of IgG expression plasmid in Snuggle IgG production requires the simultaneous synthesis of heavy chain (HC) polypeptides and light chain (LC) polypeptides. To construct the IgG expression plasmid pJ411-HC-LC B10, the HC and LC genes were cloned into a single bicistronic operon having a T7 promoter and T7 terminator. This bicistronic operon contains SEQ ID NO: 35. This plasmid has a high-copy pUC replication origin and contains kanamycin-selectable markers. The plasmid sequence was confirmed by cloning. Both HC and LC had independent ribosome binding sites. To optimize the HC to LC ratio, mutations were introduced into the ribosome binding sites of either gene. The parent plasmids PJ411, PJ411, PJ401, and PJ434, used to construct various expression plasmids in the examples, were purchased from ATUM, Inc. (Newark, California). The same expression plasmid pJ411-HC-LC B10 was used in Examples 1 to 6.

[0176] For the purposes of this disclosure and the examples, “αFolRa B10” and “B10 WT” are used interchangeably. In some cases, additional nomenclature is added to indicate the promoter used in the construct. For example, “B10 WT-T7p” represents a B10 wild-type (WT) construct using the T7 promoter.

[0177] Example 2. Example of wild-type (WT) IgG expression in a shaking flask. The E. coli strain expressing αFolR IgG B10 was prepared by transforming Snuggle, described in international patent application PCT / US2019 / 060345, with plasmid pJ411-HC-LC B10. This strain was grown overnight at 37°C in terrific broth (TB) containing 50 μg / mL kanamycin. In the morning, it was diluted 1:50 in fresh TB + kanamycin (Kan) and OD was prepared. 600The cells were grown at 37°C until the concentration reached 1.5. At this point, the expression of the HC and LC genes was induced by adding arabinose to a final concentration of 0.2%. The temperature was adjusted to 25°C, and protein expression continued for 16 hours. The cells were harvested by centrifugation at 6000×g for 10 minutes.

[0178] Example 3. Analysis of in vivo IgG expression in a shaking flask Cells were collected by centrifugation at 6000×g for 10 minutes as described in Example 2, and then frozen. The thawed cells were lyseed by sonication in modified S30 buffer (10 mM Tris·HCl pH 8.2, 2 mM magnesium acetate, 60 mM potassium acetate, and 0.1 mg / L egg white lysozyme) at 10 mL / g wet cell weight (wcw), and debris was removed by centrifugation at 19,000×g for 30 minutes. IgG was purified from the cell lysates using standard protein A affinity chromatography, and antibody concentrations were measured by UV / Vis spectroscopy.

[0179] Example 4. Comparison of in vivo IgG expression in oxidative cytoplasmic lines Shuffle and Snuggle Snuggle and Shuffle strains (NEB, Cambridge, Massachusetts) were transformed with plasmid pJ411-HC-LC B10. The differences in the genetic makeup of Suggle and Shuffle are described in international patent application PCT / US2019 / 060345. In short, Shuffle strain lacks thioredoxin reductase activity (TRXB) and glutathione reductase activity (GOR). Furthermore, Shuffle strain has a DsbC gene lacking a signal sequence and a variant of the ahpC gene encoding an enzyme that lacks peroxyreductase activity but possesses glutathione reductase activity (ahpC *) overexpresses and . The Snuggle strain has been further modified (based on Shuffle) to also lack thioredoxin 1 activity (TrxA) compared to Shuffle. As a result, the Snuggle strain contains null mutations in trxA, trxB, and gor, and therefore lacks thioredoxin reductase activity (TrxB), thioredoxin 1 activity (TrxA), and glutathione reductase activity (GOR). The Snuggle strain also overexpresses DsbC without a signal sequence, and a variant of the ahpC gene (ahpC) encoding an enzyme that lacks peroxyreductase activity but has glutathione reductase activity. * ) is also overexpressed.

[0180] Cells from both strains were grown overnight at 37°C in Terrific Broth (TB) containing 50 μg / mL kanamycin. In the morning, both cultures were diluted 1:50 in fresh TB+Kanamycin, and both strains were given their final OD (Oral Dissociation). 600 Cells were grown at 37°C until the concentration reached 1.5. IgG expression was induced in SBDG419 by adding a final concentration of 0.2% arabinose. IgG expression in Shuffle cells was induced by adding a final concentration of 1 mM IPTG. The temperature was adjusted to 25°C, and protein expression continued for 16 hours.

[0181] Figure 1 shows a comparison of the titers of cell lysates from Shuffle and Snuggle strains co-expressing HC and LC of FoRa B10 wild-type (WT) IgG, suggesting that the additional mutations present in Snuggle, as described above, contribute to high levels of IgG synthesis.

[0182] Example 5. Cell bank strain for in vivo production of wild-type (WT) IgG in Escherichia coli. Each transformed culture of the wild-type (WT) IgG strain described herein (i.e., pJ411-HC-LC B10) was streaked onto an LB plate containing 50 μg / mL kanamycin and incubated in a 30°C incubator for colony development. Single colonies were then picked and inoculated into a 50 mL bioreactor tube filled with 10 mL of I17-SF shaking flask medium (Tables 8 and 9 describe the components of the I17-SF shaking flask medium). The tube was then incubated in a 25 mm orbital diameter shaker at 30°C and 250 rpm for 15–24 hours. When the OD595 nm of the culture reached 2–4, the cells were subcultured in I17-SF shaking flask medium at a seeding density of 10%–25% (v / v). The flask was incubated in a 25 mm orbital diameter shaker at 37°C and 250 rpm. When the OD595nm reached 2-3, the cells were mixed with 80% pre-sterilized glycerol to a concentration of 20% (v / v), aliquoted into 1 mL cryovials, flash-frozen with liquid nitrogen, and then placed in a -80°C freezer for long-term storage.

[0183] Example 6. Expression and cell lysis of wild-type (WT) IgG produced in E. coli by high-density fermentation. The fermentation process was initiated by taking a 1 mL vial from the cell bank and inoculating it into a shaking flask containing I17-SF Shaking Flask Limited Medium containing 50 μg / mL kanamycin at a seeding density of approximately 3% (v / v). When the OD595 nm reached 2–4, the flask culture was used to inoculate a 500 mL bioreactor at a seeding density of 1.5% (v / v) in batch medium, which consisted of 30 μg / mL kanamycin in deionized water, 0.1% (v / v) P2000 defoamer, and 1.2% (v / v) 10×I17 medium (Tables 5, 6, and 7 list the components of the 10×I17 medium). The bioreactor temperature, dissolved oxygen, and pH settings were 37°C, 30%, and 7, respectively. Once the cells had grown to an OD595 nm of 2–5 in the batch phase, they were inoculated for 0.20 h-1 The fed-batch phase was initiated by supplying 10 × I¹⁷ medium at an exponential rate. After the 10-hour fed-batch phase, the temperature of the bioreactor was reduced to 25°C, and the exponential supply rate was reduced to 0.02h -1 The concentration was reduced to 1. After 1 hour, the induction phase was initiated by adding L-arabinose to a target concentration of 4 g / L based on the initial volume of the bioreactor. The induction phase lasted 24 to 48 hours before harvesting. At the end of fermentation, the culture was harvested and centrifuged in a floor centrifuge at 18,592 × g and 2°C to 8°C for 15 minutes. The supernatant was discarded, the cell pellet was resuspended, washed with S30 buffer at a concentration of 16.67% (w / w) (Table 10 lists the components of S30 buffer), and centrifuged again under the same conditions as used in the initial harvesting step. After washing, the supernatant was discarded, and the cells were resuspended with S30-5 buffer at a concentration of 16.67% (w / w) (Table 11 lists the components of S30-5 buffer). Next, the cell resuspension was passed through an Avestin homogenizer (EmulsiFlex-C5) at 17,000 Psi to disrupt the cells and produce crude lysate. This crude lysate was further clarified by centrifugation at 18,000-20,000 × g and 2°C-8°C for 30 minutes using a floor centrifuge. The supernatant (clarified lysate) was collected, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0184] Example 7. Plasmid preparation for pAMF-containing IgG expression in Snuggle The production of IgG containing non-natural amino acids (NNAAs) requires the simultaneous synthesis of heavy chain (HC) polypeptides and light chain (LC) polypeptides, where the HC and / or LC contain NNAAs. To construct the plasmid pJ411-HC F404TAG-LC B10 for the expression of antibody B10 HC F404 pAMF, the genes for HC and LC were cloned into a single bicistronic operon having a T7 promoter and T7 terminator. The codon of HC residue F404 was mutated to TAG to specify the location of the NNAA. This plasmid has a high-copy pUC replication origin and contains a gene for kanamycin resistance. The plasmid sequence was confirmed by cloning. Both HC and LC had independent ribosome binding sites. To optimize the ratio of HC to LC, mutations were made in the ribosome binding site of either gene. pJ411-HC F404TAG-LC B10 is identical to pJ411-HC-LC B10 except for the mutation of the F404 codon to TAG.

[0185] Simultaneous translation of pAMF requires the expression of an amber suppressor tRNA that is orthogonal to the existing aminoacyl-tRNA synthetase (AAtRS), as well as an orthogonal AAtRS that specifically recognizes the amber suppressor tRNA and pAMF NNAA. These pAMF AAtRS and tRNA genes were cloned into vector pJ434 as a bicistronic operon, downstream of the inducible T7 promoter and the constitutive promoter Pc0. This vector contains a p15A replication origin and a β-lactamase selection marker that confers carbenicillin resistance. Both the replication origin and the marker are compatible with pJ411. The plasmid sequence was confirmed by cloning.

[0186] Example 8. Example of pAMF IgG expression in a shaking flask. An E. coli strain expressing αFolR IgG with pAMF at residue 404 was prepared by co-transforming Snuggle strain SBDG419 with plasmids pJ411-HC F404TAG-LC B10 and pJ434 Pc0 pAMF RS-tRNA. This strain was grown overnight at 37°C in terrific broth (TB) containing 50 μg / mL kanamycin and 100 μg / mL carbenicillin. In the morning, this was diluted 1:50 in fresh TB + Kana / Carbenicillin (Carb) and OD 600 The cells were grown at 37°C until the ratio reached 1.5. At this point, T7-driven transcription of the HC, LC, AATRS, and tRNA genes was induced by adding a final concentration of 0.2% arabinose and 2 mM pAMF. The temperature was adjusted to 25°C, and protein expression continued for 16 hours. Cells were harvested by centrifugation at 6000×g for 10 minutes.

[0187] Example 9. Cell bank strain for in vivo production of pAMF-IgG in E. coli The transformed culture of the nnAA IgG strain described in Example 8 was streaked onto an LB plate containing 50 μg / mL kanamycin and 100 μg / mL carbenicillin, and incubated in a 30°C incubator for colony development. Single colonies were then picked and inoculated into a 50 mL bioreactor tube filled with 10 mL of I17-SF shaking flask medium containing 100 μg / mL carbenicillin (see Tables 8 and 9 for the components of I17-SF shaking flask medium). The tube was then incubated in a 25 mm orbital diameter shaker at 30°C and 250 rpm for 15–24 hours. Once the OD595 nm of the culture reached 2–4, the cells were subcultured in I17-SF shaking flask medium at a seeding density of 10%–25% (v / v). The flask was incubated in a 25 mm orbital diameter shaker at 37°C and 250 rpm. When the OD595nm reached 2-3, the cells were mixed with 80% pre-sterilized glycerol to a concentration of 20% (v / v), aliquoted into 1 mL cryovials, flash-frozen in liquid nitrogen, and then placed in a -80°C freezer for long-term storage.

[0188] Example 10. Expression and cell lysis of pAMF-IgG produced in E. coli by high-density fermentation. The fermentation process was initiated by taking a 1 mL vial from the cell bank described in Example 9 and inoculating it into a shaking flask containing I17-SF shaking flask medium with 100 μg / mL carbenicillin at a seeding density of approximately 3% (v / v). When the OD595 nm reached 2-4, the flask culture was used to inoculate a 500 mL bioreactor with batch medium at a seeding density of 1.5% (v / v), which consisted of 30 μg / mL kanamycin, 100 μg / mL carbenicillin, 0.1% (v / v) P2000 defoamer, and 1.2% (v / v) 10×I17 medium in deionized water (Tables 5, 6, and 7 list the components of the 10×I17 medium). The bioreactor temperature, dissolved oxygen, and pH settings were 37°C, 30%, and 7, respectively. Once the aforementioned cells have proliferated to an OD595nm of 2-5 in the batch phase, 0.20h -1 The fed-batch phase was initiated by supplying 10 × I¹⁷ medium at an exponential rate. After the 10-hour fed-batch phase, the temperature of the bioreactor was reduced to 25°C, and the exponential supply rate was reduced to 0.02h -1The concentration was reduced to [value missing]. After 1 hour, the induction phase was initiated by adding pAMF to a target concentration of 2 mM based on the culture volume of the bioreactor before induction, and L-arabinose to a target concentration of 4 g / L based on the initial volume of the bioreactor. The induction phase took 24 to 48 hours before harvesting. At the end of fermentation, the culture was harvested and centrifuged in a floor centrifuge at 18,592 × g and 2°C to 8°C for 15 minutes. The supernatant was discarded, the cell pellet was resuspended, washed with S30 buffer at a concentration of 16.67% (w / w) (see Table 10 for further information on the components of S30 buffer), and centrifuged again under the same conditions used in the initial harvesting step. After washing, the supernatant was discarded, and the cells were resuspended with S30-5 buffer at a concentration of 16.67% (w / w) (see Table 11 for information on the components of S30-5 buffer). Next, the cell resuspension was passed through an Avestin homogenizer (EmulsiFlex-C5) at 17,000 Psi to disrupt the cells and produce crude lysate. This crude lysate was further clarified by centrifugation at 18,000–20,000 × g and 2°C–8°C for 30 minutes using a floor centrifuge. The supernatant (clarified lysate) was collected, aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.

[0189] Example 11. Optimization of the 3' codon of the TAG for the expression of NNAA-containing IgG. To optimize the S181 codon for the production of B10 Y180 pAMF-containing IgG, the HC Y180TAG gene with the Ser codon AGC or AGT at the Ser181 position was produced. The HC and LC genes were cloned into pJ411 using a single bicistronic operon with a T7 promoter and T7 terminator. This plasmid, pJ411-HC Y180TAG-LC B10, has a high-copy pUC replication origin and contains the gene for kanamycin resistance. The plasmid sequence was confirmed by cloning. Both HC and LC had independent ribosome binding sites with sequences of SEQ ID NO: 17 and SEQ ID NO: 20, respectively. pJ411-HC Y180TAG-LC B10 is identical to pJ411-HC-LC B10 except for the mutation of the Y180 codon to TAG.

[0190] The SBDG419 strain was co-transformed with a codon-optimized B10 expression plasmid and pJ434 containing pAMF RS and tRNA, and IgG containing pAMF was expressed in the same manner as in Example 8.

[0191] Figure 2 shows a comparison of the titer in cell lysates for FolRa-B10 IgG produced using 0, 2, or 4 pAMF non-natural amino acids and the indicated S181 codon. As shown in Figure 2, the HC Y180TAG gene produced with Ser codons AGC and AGT at the Ser181 position shows a higher titer than that with TCG at the same position. This indicates that optimizing the 3' S181 codon can substantially improve the titer.

[0192] Example 12. Reassembly of IgG produced in vivo. IgG reassembly was performed by incubating cell lysates, adjusted to pH 8.0 with 100 mM Tris·HCl, in a FlowerPlate (m2p-labs) at 25°C with shaking at 650 rpm for 16 hours. The reassembled IgG was purified from the cell lysates using standard affinity chromatography, and the assembly was measured using a Caliper bioanalyzer under non-reductive conditions.

[0193] Figure 3 shows a comparison of FolRa-B10 IgG assemblies produced with 0, 2, or 4 pAMF non-natural amino acids after overnight reassembly with FlowerPlate, as measured by a Caliper bioanalyzer under non-reductive conditions.

[0194] Example 13. Screening of ribosome binding site variants for optimal IgG expression in E. coli. Plasmids containing both HC and LC ribosome-binding site (RBS) variants for SP7219 IgG were co-transformed into SBDG419 with pJ434 Pc0 pAMF RS-tRNA. Cells were grown overnight at 37°C in terrific broth (TB) containing 50 μg / mL kanamycin and 100 μg / mL carbenicillin. In the morning, these were diluted 1:50 in fresh TB+Kan / Carb and OD (Oxygen-Drug). 600Cells were grown at 37°C until the ratio reached 1.5. At this point, T7-driven transcription of the HC, LC, AATRNAS, and tRNA genes was induced by adding a final concentration of 0.2% arabinose, and 2 mM pAMF was added to the culture. The temperature was adjusted to 25°C, and protein expression continued for 16 hours. Cells were harvested by centrifugation at 6000×g for 10 minutes. Cells were lyse by sonication in 10 mL / g (wet cell weight (wcw)) of modified S30 buffer (10 mM Tris·HCl pH 8.2, 2 mM magnesium acetate, and 60 mM potassium acetate), and debris was removed by centrifugation at 19,000×g for 30 minutes. IgG was purified from the cell lysate using standard affinity chromatography, and its concentration was measured by UV / Vis spectroscopy. [Table 2] [Table 3]

[0195] The ribosome-binding sequence for heavy chain translation can be transcribed from the DNA sequence SEQ ID NO: 28:AX1GAGX2T (where X1 is A or G, or X2 is A or G). The ribosome-binding sequence for light chain translation can be transcribed from the DNA sequence SEQ ID NO: 29:AX1X2AX3AT (where X1 is G or A, X2 is G or A, or X3 is G or T).

[0196] Example 14. Shaking flask expression of IgG containing pAcF and AEK non-natural amino acids in Escherichia coli. SBDG419 strains were co-transformed with either pJ434 Pc0 pAcF RS-tRNA or pJ434 Pc0 Mm PylK RS-Mb PylK tRNA using plasmids encoding B10 F404TAG IgG. Azidoethoxycarbonyllysine (AEK) is a substrate for wild-type (WT) pyrrolidine synthase. These strains were grown overnight at 37°C in terrific broth (TB) containing 50 μg / mL kanamycin and 100 μg / mL carbenicillin. In the morning, these were diluted 1:50 in fresh TB + Kana / Carb and OD (Oxygen Extract). 600 Cells were grown at 37°C until the ratio reached 1.5. At this point, T7-driven transcription of the HC gene, LC gene, AATRNAS gene, and tRNA gene was induced by adding 0.2% arabinose, and 2 mM pAcF or AEK was added to the culture as appropriate. The temperature was adjusted to 25°C, and protein expression continued for 16 hours. Cells were harvested by centrifugation at 6000×g for 10 minutes. Cells were lysed by sonication in 10 mL / g (wcw) modified S30 buffer (10 mM Tris·HCl pH 8.2, 2 mM magnesium acetate, and 60 mM potassium acetate), and debris was removed by centrifugation at 19,000×g for 30 minutes. IgG was purified from the cell lysate using standard affinity chromatography, and its concentration was measured by UV / Vis spectroscopy.

[0197] Figure 4 shows the expression of B10-F404pAcF and B10-F404AEK (both possessing the HC ribosome binding sequence of SEQ ID NO: 17 and the LC ribosome binding sequence of SEQ ID NO: 20) in and without the presence of appropriate non-natural amino acids in the culture medium. The low titer in the absence of NNAA indicates that NNAA is required for amber suppression in the production of full-length IgG. Example 15 Summary of IgG titers in untreated (intact) E. coli Tables 4 and 5 summarize the titer results of IgG produced from E. coli in a shaking flask or bioreactor under the conditions described in the above examples. The volume of the bioreactor shown in Table 4 was 0.5 liters. [Table 4-1] [Table 4-2] [Table 4-3]

[0198] Purified A280 titers for NNAA IgG expressed in Snuggle cells co-transformed with plasmids encoding the listed IgGs and plasmids encoding tRNA / AAtRS for specific NNAAs. All NNAAs were used at 2 mM, except for the third entry in the table, which was expressed in the case of 0 mM pAMF. The very low titer of this sample indicates that NNAAs are required for amber suppression and full-length IgG production. [Table 5]

[0199] A280 titer of purified NNAA IgG expressed in Snuggle cells co-transformed with plasmids encoding the listed IgGs and plasmids encoding specific NNAA tRNA / AAtRS. pAMF was used at 2 mM. Example 16: Culture medium and components for high-density E. coli IgG fermentation [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

[0200] Example 17. Conjugation of nnAA-containing mAb Click chemistry bioconjugation reactions were performed on B10-F404TAG IgG expressed in vivo, containing the non-natural amino acids pAMF, AEK, and pAcF. Azide nnAA-containing IgG, B10 F404pAMF, and B10 F404AEK were conjugated to DBCO-maytan sine linker warheads (SC236) via strain-accelerated alkyne-azide cycloaddition (SPAAC) "click" chemistry as follows: 1 mg / mL of IgG was reacted with a 10-fold molar excess of SC236 in PBS at 22°C for 16 hours. B10 F404pAcF IgG was conjugated to aminooxy-PEG8-methane via oxim ligation as follows. 13 mg / mL of IgG was reacted with 40-fold molar excess aminooxy-PEG8-methane in 100 mM sodium acetate (pH 4.5) at 30°C for 72 hours. The IgG was digested with IdeS protease and reduced with DTT, and the degree of conjugation reaction was measured by LC-MS (Agilent Technologies 6520 Accurate-Mass Q-TOF LC / MS).

[0201] Figure 5 shows the deconvoluted LCMS spectrum of the Fc fragment of B10 F404pAMF conjugated to the DBCO-meitansine drug linker SC236. While the theoretical mass of the conjugate is 25258.04 Da, the observed main species has a mass of 25257.97 Da. No species were observed around the theoretical mass of the unconjugated Fc fragment, 23974.14 Da. The calculated conjugation efficiency of this conjugate is 100%. [Table 13]

[0202] Example 18. Plasmid construction for expression of NNAA-containing LC in Snuggle The production of LC containing non-natural amino acids (NNAAs) requires the synthesis of a light chain with simultaneous incorporation of NNAAs. To produce a plasmid for expressing LC with NNAAs at positions K42 and E161 (pJ411-trastuzumab LC K42TAG E161TAG), the LC gene was cloned into an operon with a T7 promoter and T7 terminator. The NNAA positions were specified by mutating the codons at LC residues K42 and E161 to TAGs. This plasmid has a high-copy pUC replication origin and contains genes for kanamycin resistance. The plasmid sequence was confirmed by cloning.

[0203] Simultaneous translation of NNAA uptake requires the expression of an amber suppressor tRNA that is orthogonal to the existing aminoacyl-tRNA synthetase (AAtRS), as well as an orthogonal AAtRS that specifically recognizes the amber suppressor tRNA and pAMF NNAA. These NNAA AAtRS and tRNA genes were cloned into vector pJ434 as a bicistronic operon, downstream of the inducible T7 promoter and the constitutive promoter Pc0. This vector contains a p15A replication origin and a β-lactamase selection marker that confers carbenicillin resistance. Both the replication origin and the marker are compatible with pJ411. The plasmid sequence was confirmed by cloning.

[0204] Example 19. Example of p-Ac-Phe LC expression in a shaking flask. E. coli strains expressing trastuzumab LC with pAcPhe at residues K42 and E161 were prepared by co-transforming Snuggle strain SBDG419 with plasmid pJ411-trastuzumab LC K42TAG E161TAG and pJ434 Pc0 pAcPhe RS-tRNA. This strain was grown overnight at 37°C in terrific broth (TB) containing 50 μg / mL kanamycin and 100 μg / mL carbenicillin. In the morning, this was diluted 1:50 in fresh TB+Kan / Carb and OD was used. 600The cells were grown at 37°C until the ratio reached 1.5. At this point, T7-driven transcription of the HC, LC, AATRS, and tRNA genes was induced by adding 0.2% arabinose and 2 mM pAcPhe to the culture. The temperature was adjusted to 25°C, and protein expression continued for 16 hours. Cells were harvested by centrifugation at 6000×g for 10 minutes.

[0205] Example 20. Analysis of p-Ac-Phe LC expression Cells were frozen overnight at -80°C, thawed, and resuspended in B-PER bacterial protein extraction reagent containing 0.01 mg / mL lysosomes and 1 μL of benzonase nuclease per 1 mL of B-PER™ solution to lyse the E. coli cells and hydrolyze the nucleic acids. After incubation at room temperature for 10 minutes, the cells were completely lyzed and the viscosity due to nucleic acid polymers decreased. At this point, the lysate was clarified by centrifugation at 24,000 × g for 10 minutes. The LC-containing supernatant was set aside for analysis. After reduction with 10 mM DTT, the samples were analyzed by PAGE gel.

[0206] Example 21. Purification and conjugation of p-Ac-PHE LC Following expression in a shaking flask, trastuzumab LC K42 pAcPhe E161pAcPhe was purified from the lysate using standard proL chromatography. After purification, 143 mg of purified LC was recovered. LC-MS of untreated (intact) LC (Figure 6) showed that all recovered proteins were full-length and there was no evidence of misuptake. This means that the synthetase had good fidelity at both TAG sites, and the cleaved proteins had no affinity for proL. The purified K42pAcPhe E161pAcPhe LC was conjugated. LC-MS of the conjugated LC (Figure 7) showed a drug load of 1.96 molecules per LC, indicating a conjugation efficiency of 98%. [Table 14]

[0207] Example 22. Quantitative determination of protein production To measure the amount of HC and LC present in cell lysates, which were reduced with 10 mM DTT and separated from LC by treatment with 10 mM DTT, the protein concentrations of HC and LC were measured using a Sally Sue automated Western blot (Protein Simple, San Jose, California) according to the manufacturer's instructions for the 12-230 kDa separation module (SM-S0001, Protein Simple). The reduced cell lysates were diluted 1:20 in 0.1× sample buffer. Anti-human IgG(H+L) Biotin-SP (109-065-003, Jackson ImmunoResearch) was used as the primary antibody, diluted 1:150 in Antibody Diluent 2. Standard samples were prepared using serial dilutions of purified H01 antibody in 0.1× sample buffer.

[0208] Example 23. Production of IgG using an alternative promoter. IgG production requires the simultaneous synthesis of heavy chain (HC) and light chain (LC) polypeptides. To construct the IgG expression plasmid pJ401-LC-HC B10, the HC and LC genes were cloned into a single bicistronic operon with a T5 promoter (T5p) (SEQ ID NO: 23) that recruits E. coli RNA polymerase for transcription. This plasmid has a high-copy pUC replication origin and contains kanamycin-selectable markers. The plasmid sequence was confirmed by cloning. Both HC and LC had independent ribosome-binding sites.

[0209] An Escherichia coli strain for expressing B10 using this plasmid was prepared by transforming Snuggle described in International Application No. PCT / US2019 / 060345 with plasmid pJ401-LC-HC B10. This strain was grown overnight at 37 °C in terrific broth (TB) containing 50 μg / mL kanamycin. In the morning, this was diluted 1:50 into fresh TB+Kan and grown at 37 °C until the OD600 reached 1.5. At this point, the expression of the HC gene and the LC gene was induced by adding IPTG to a final concentration of 1 mM. The temperature was adjusted to 25 °C and protein expression continued for 16 hours. The cells were harvested by centrifugation at 6000g for 10 minutes, lysed, and then IgG expression was analyzed in the manner described above. The results are shown in Table 15, which means that the expression of B10 IgG by the T5 promoter ("B10 WT-T5p") is consistent with other high-yield IgGs in shake flasks. The conventional expression using the T7 promoter (T7p) ("B10 WT-T7p") is shown for comparison. [Table 15] Based on the titer in shake flasks, it is expected that the same strain of B10 WT-T5p will achieve more than 200 mg / L of B10 IgG, or even more than 1 g / L of B10 IgG, when produced in a high-density fermentation process such as a bioreactor. This is because previous tests, such as Example 15, Table 4, have shown that switching from shake flasks to high-density fermentation can usually increase antibody production by more than fivefold.

[0210] Example 24. Production of IgG Using Alternative Operon Configurations IgG production requires the simultaneous synthesis of heavy chain (HC) and light chain (LC) polypeptides. Plasmid pJ411 B10 MO was cloned to produce IgG using separate operons for HC and LC. In this vector, the HC gene and the LC gene were cloned into monocistronic complete operons containing a T7 promoter, LC or HC gene, and then a T7 terminator, respectively. This plasmid has a high-copy pUC replication origin and contains kanamycin-selectable markers. Plasmid sequences were confirmed by cloning. Both HC and LC had independent ribosome binding sites. Mutations were introduced within the HC ribosome binding site to optimize the HC-LC ratio. Three distinct plasmids, B10-MO-SDs, B10-MO-SDm, and B10-MO-SDw, were constructed, each containing strong, moderate, and weak HC ribosome binding sites, including SEQ ID NOs. 20, 21, and 22, respectively. Previous studies have shown that more LC expression is required than HC expression, so LC used the same strong ribosome binding site (SEQ ID NO: 17) for all constructs.

[0211] E. coli strains expressing B10 using these plasmids were prepared by transforming Snuggle, described in international patent application PCT / US2019 / 060345, with plasmids B10-MO-SDs (SEQ ID NO: 36), B10-MO-SDm (SEQ ID NO: 37), and B10-MO-SDw (SEQ ID NO: 38). These strains were grown overnight at 37°C in terrific broth (TB) containing 50 μg / mL kanamycin. In the morning, this was diluted 1:50 in fresh TB+Kan and grown at 37°C until the OD600 reached 1.5. At this point, HC and LC gene expression was induced by adding arabinose to a final concentration of 0.2%. The temperature was adjusted to 25°C, and protein expression continued for 16 hours. Cells were harvested by centrifugation at 6000 g for 10 minutes, lysed, and then IgG expression was analyzed using the method described above. The results are shown in Table 16, which demonstrates that high expression of B10 IgG using a separate operon can be achieved with appropriate SD tuning and HC:LC ratio. The yield for the best construct from this series was consistent with other high-yielding IgG in a shaking flask. For comparison, conventional expression ("B10 WT") using the bisistronic operon described in Example 1 is shown. [Table 16] Previous studies, such as Example 15, Table 4, have shown that switching from a shaking flask to high-density fermentation can typically increase antibody production by more than five times compared to the titer in the shaking flask. Therefore, the results in this example demonstrate that the same strain of B10-MO-SDw can produce B10 IgG levels exceeding 200 mg / L, or even exceeding 1 g / L, when cultured in a high-density fermentation setting such as a bioreactor.

[0212] Abbreviations in this application OD595nm = Optical density measured at a wavelength of 595nm RPM = revolutions per minute (v / v%) = Volume of solute / Volume of solution (%) (w / w%) = weight of solute / weight of solution (%) Qs = Quantum satis (add the amount necessary to achieve the desired result) SD = Shine Dalgarno sequence, ribosome binding site, or RBS SDs or RBSs = Strong ribosome binding sites SDm or RBSm = moderate strength ribosome binding site SDw or RBSw = weak ribosome binding site

[0213] Example sequence Protein sequences of LC and HC of IgG expressed in E. coli * indicates the position of non-natural amino acids (NNAAs). Sequence ID 1 (WT B10 HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNTTTKSIHWVRQAPGKGLEWVGEIYPRDGITDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGWHWRSGYSYYLD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0214] Sequence ID No. 2 (WT B10 LC or H01 LC or trastuzumab LC) MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0215] Sequence ID 3 (B10 F404TAG HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNTTTKSIHWVRQAPGKGLEWVGEIYPRDGITDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGWHWRSGYSYYLD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS*FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0216] Sequence ID 4 (B10 Y180TAG HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNTTTKSIHWVRQAPGKGLEWVGEIYPRDGITDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGWHWRSGYSYYLD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL*SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0217] Sequence ID 5 (B10 K42TAG LC) MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPG*APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0218] Sequence ID 6 (B10 Y180TAG F404TAG HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNTTTKSIHWVRQAPGKGLEWVGEIYPRDGITDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGWHWRSGYSYYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL*SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS*FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0219] Sequence number 7 (B10 241TAG HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNTTTKSIHWVRQAPGKGLEWVGEIYPRDGITDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGGWHWRSGYSYYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV*LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0220] Sequence number 8 (H01 Y180TAG F404TAG HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNIRTQSIHWVRQAPGKGLEWIGDIFPIDGITDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSWSWPSGMDYYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL*SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS*FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0221] Sequence number 9 (7219 LC) MDIQMTQSPSSVSASVGDRVTITCRASQGIGSWLAWYQQKPGKAPKLLIYAADRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYHTYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0222] Sequence number 10 (7219 F404TAG HC) MQVQLVESGGVVQPGRSLRLSCAASGFNFSDYGMHWVRQAPGKGLEWVAVIWYDGSISYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGTVEHGAVYGTDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS*FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0223] SEQ ID NO:11(αPD1 HC) MEVQLVQSGAEVKKPGASVKVSCKASGYTFDSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTNTAYMELRSRSDDTAVYYCARDVDYGTGSGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCEVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0224] Sequence ID 12 (αPD1 LC) MSYELTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVMVIYKDTERPSGIPERFSGSSSGTKVTLTISGVQAEDEADYYCQSADNSITYRVFGGGTKVT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0225] Sequence ID 13 (αTim3 HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNIDRYYIHWVRQAPGKGLEWVAGITPVRGYTEYADSVKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGYVYRMWDSYDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCEVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0226] Sequence ID 14 (αTim3 LC) MDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0227] Sequence ID 15 (αLAG3 HC) MQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSYKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREEAPENWDYALDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCEVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0228] Sequence ID 16 (αLAG3 LC) MEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGRSPFSFGPGTKVDI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0229] Sequence ID 17 (HC-RBS1; Consensus RBS) AGGAGGT Sequence ID No. 18 (HC-RBS2; first variant) AAGAGAT Sequence ID 19 (HC-RBS3; second variant) AAAAGAT Sequence ID 20 (LC-RBS1; standard LC RBS) AGGAGAT Sequence ID 21 (LC-RBS2; first variant) AAGAGAT Sequence ID 22 (LC-RBS3; double mutant) AAAAGAT Sequence ID 23 (LC-RBS4; triple mutant) AAAATAT

[0230] Sequence ID 24 (H01 HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNIRTQSIHWVRQAPGKGLEWIGDIFPIDGITDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGSWSWPSGMDYYLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0231] Sequence number 25 (7219 HC) MQVQLVESGGGVVQPGRSLRLSCAASGFNFSDYGMHWVRQAPGKGLEWVAVIWYDGSISYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGTVEHGAVYGTDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0232] Sequence ID 26 (T7 promoter) TAATACGACTCACTATAGGG Sequence ID 27 (Trastuzumab HC) MEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0233] Sequence ID 28 (ribosome binding sequence, e.g., ribosome binding sequence for heavy chains) AX1GAGX2T (where X1 is A or G, and X2 is A or G) Sequence ID 29 (ribosome binding sequence, e.g., ribosome binding sequence for light chains) AX1X2AX3AT (where X1 is G or A, X2 is G or A, and X3 is G or T) Sequence ID 30 (T5 promoter sequence) TAATTGTGAGCGGATAACAATTACGAGCTTCATGCACAGTGAAATCATGAAAAATTTATTTGCTTTGTGAGCGGATAACAATTATAATA

[0234] Sequence ID 31 (including underlined Sequence ID 17) [ka] Sequence ID 32 (including sequence ID 20, which is underlined) [ka] Sequence ID 33 (including sequence ID 21, which is underlined) [ka] Sequence ID 34 (including sequence ID 22, which is underlined) [ka]

[0235] Sequence ID 35 (operon sequence for B10 WT; single underline (Sequence ID 31) codes for LC's 5'UTR, double underline (Sequence ID 32) codes for HC's 5'UTR) [ka]

[0236] Sequence ID 36 (operon sequence for B10-MO-SDs; single underline (Sequence ID 31) codes for LC's 5'UTR, double underline (Sequence ID 32) codes for HC's 5'UTR) [ka]

[0237] Sequence ID 37 (operon sequence for B10-MO-SDm; single underline (sequence ID 31) codes for LC's 5'UTR, double underline (sequence ID 33) codes for HC's 5'UTR) [ka]

[0238] Sequence ID 38 (operon sequence for B10-MO-SDw; single underline (sequence ID 31) codes for LC's 5'UTR, double underline (sequence ID 34) codes for HC's 5'UTR) [ka]

Claims

1. A method for producing a full-length antibody containing heavy chains (HC) and light chains (LC), This includes culturing E. coli cells expressing the coding sequences of the HC and the LC in a culture medium under conditions acceptable for producing the HC and the LC, The full-length antibody is produced in an amount of at least about 200 mg per liter of the culture medium, or The weight percentage of the full-length antibodies produced relative to the weight of the cell pellet generated from the E. coli cells is in the range of 0.05% to 20%. The aforementioned method.

2. The method according to claim 1, wherein at least a portion of the Escherichia coli cells contain oxidative cytoplasm.

3. The method according to claim 1, comprising transforming the plasmid into the Escherichia coli strain.

4. The method according to claim 1 or claim 3, wherein the HC and LC are produced in the cytoplasm of the E. coli.

5. The method according to claim 1, further comprising assembling the produced HC and LC under non-reductive conditions to form a full-length antibody.

6. The method according to claim 1 or claim 5, wherein the molar ratio of the HC produced from the E. coli to the LC produced is about 1:1 to about 1:

3.

7. The expression of HC, LC, or both is controlled by a promoter. The promoter is a T7 promoter, or a promoter having a promoter strength substantially similar to that of a T7 promoter, or The promoter is a T5 promoter, or a promoter having a promoter strength substantially similar to that of a T5 promoter. The method according to any one of claims 1 to 6.

8. The plasmid comprises a bisistronic operon, The bisistron operon includes the coding sequence of HC and the coding sequence of LC, or The plasmid comprises a first monocistronic operon for the HC and a second monocistronic operon for the LC. The method according to any one of claims 1 to 6.

9. The bisistronic operon includes a promoter that drives the expression of both the HC and the LC, The promoter is a T7 promoter, or a promoter having a promoter strength substantially similar to that of a T7 promoter, or The first monocistrone operon or the second monocistrone operon includes a T7 promoter. The method according to claim 8.

10. The bisistron operon includes a T7 terminator, or The first monocistrone operon or the second monocistrone operon includes a T7 terminator. The method according to claim 9.

11. The method according to any one of claims 1 to 10, wherein the Escherichia coli cell includes a first ribosome binding site for the translation of the HC and a second ribosome binding site for the translation of the LC, and the first ribosome binding site and the second ribosome binding site are selected such that the molar ratio of the HC and LC produced from the Escherichia coli is in the range of 1:1 to 1:

3.

12. The method according to claim 11, wherein the first ribosome binding site is transcribed from a DNA sequence selected from the group consisting of SEQ ID NOs: 17 to 19, and the second ribosome binding site is transcribed from a DNA sequence selected from the group consisting of SEQ ID NOs: 20 to 23.

13. The method according to claim 12, wherein the first ribosome binding site has the sequence of SEQ ID NO: 17 or SEQ ID NO:

18.

14. The method according to claim 1, wherein the HC and / or LC of the full-length antibody comprises at least one non-natural amino acid.

15. The method according to claim 14, wherein the coding sequence of HC and / or the coding sequence of LC is modified to have at least one non-natural amino acid codon, and the non-natural amino acid codon does not result in the incorporation of any natural amino acid.

16. The method according to claim 14, wherein the at least one non-natural amino acid is introduced by loading (charging) it onto a tRNA containing an anticodon complementary to the at least one non-natural amino acid codon.

17. The method according to claim 16, wherein the at least one non-natural amino acid codon is an amber codon TAG.

18. The method according to claim 16, wherein the at least one non-natural amino acid is paramethylazide-L-phenylalanine (pAMF), AEK, or pAcF.

19. The method according to any one of claims 15 to 18, wherein the codon adjacent to the 3' side of at least one of the non-natural amino acids is codon-optimized.

20. The method according to claim 1, wherein the full-length antibody is B10 antibody, H01 antibody, 7219 antibody, anti-PD1 antibody, anti-Tim3 antibody, anti-LAG3 antibody, or anti-Her2 antibody.

21. The coding sequence of HC of the B10 antibody contains a mutation relative to SEQ ID NO: 1, and as a result of the mutation, a natural amino acid codon is replaced with a non-natural amino acid codon. The aforementioned natural amino acid is one or more amino acids selected from F404, Y180, and F241, and The method according to claim 20, wherein a non-natural amino acid is introduced into the HC by loading (charging) a tRNA complementary to the non-natural amino acid codon.

22. The method according to claim 20 or claim 21, wherein the coding sequence of the LC of the B10 antibody or the coding sequence of the LC of the trastuzumab includes at least one mutation relative to SEQ ID NO: 2, the at least one mutation causing a natural amino acid codon to be replaced by a non-natural amino acid codon, the natural amino acid being K42 or E161.

23. The method according to claim 14, further comprising covalently linking the warhead portion to the non-natural amino acid on the HC or LC of the full-length antibody via a linker.

24. The method according to claim 23, wherein the warhead is SC236 or aminooxy-PEG8-methane.

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