Method for producing a two-chain protein in a prokaryotic host cell

Abstract

Provided herein are methods and host cells for producing polypeptides comprising two chains, such as antibodies, half antibodies, antibody fragments, or one-armed antibodies. The methods and host cells enable production of the two-chain polypeptide using expression of polynucleotides encoding the polypeptide chains from extrachromosomal polynucleotide(s) and expression of one or more chaperone proteins (e.g., peptidyl-prolyl isomerase and / or protein disulfide oxidoreductase) from the host cell chromosome using non-natural combination(s) of promoters and translation units encoding the chaperone proteins.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 755,915, filed November 5, 2018, which is incorporated by reference in its entirety. Submission of sequence listing via ASCII text file

[0002] The contents of the following ASCII text file submission are incorporated herein by reference in their entirety: Sequence Listing Computer Readable Form (CRF) (Filename: 146392044040SEQLIST.TXT, Recorded: October 28, 2019, Size: 6KB).

[0003] The present disclosure relates to methods for producing recombinant polypeptides such as antibodies (e.g., bispecific antibodies, half antibodies, one-armed antibodies, antibody fragments, etc.), and to prokaryotic host cells that may find use in said methods. [Background technology]

[0004] Recombinant protein production in prokaryotic host cells has been the source of many important therapeutic drugs since the production of human insulin in E. coli in 1978. As molecular biology tools and knowledge develop, the complexity of recombinant therapeutics also increases. The production of these recombinant proteins requires that the products exhibit properties such as proper translation, folding, assembly, disulfide bonds, and transport to the periplasm. It is known that the expression of many recombinant proteins, especially those with disulfide bonds (e.g., two-chain proteins, including but not limited to antibodies and antibody fragments), leads to the formation of inclusion bodies in prokaryotic host cells (Spadiut et al., Trends in Biotechnology, 32:54, 2014). Thus, there is a need for expression systems and processes for the recombinant production of properly folded and assembled two-chain proteins in prokaryotic host cells on an industrial scale.

[0005] Monoclonal antibodies represent one of the fastest growing types of recombinant therapeutic drugs, and many monoclonal antibodies have already been approved or are under review for the treatment of various diseases (Nelson et al., Nature Review Drug Discovery, 9:767, 2010). Conventional monoclonal antibodies bind to a single target antigen. In many diseases, it may be advantageous to employ antibodies that bind to two or more target antigens, i.e., multispecific antibodies. Such antibodies can be employed in combinatorial approaches to multiple therapeutic targets (see, for example, Bostrom et al., Science, 323:1610, 2009, and Wu et al., Nature Biotechnology, 25:1290, 2007). For example, bispecific antibodies can be produced that simultaneously bind to epitopes expressed on the surface of cancer cells and epitopes expressed on T cells to induce T cell-mediated killing of tumor cells (Shalaby et al., Clinical Immunology, 74:185, 1995). Other monoclonal antibody formats, such as antibody fragments and one-armed antibodies, have also been used (see, e.g., Merchant et al., Proc. Natl. Acad. Sci. 110:E2987-E2996, 2013).

[0006] The use of antibodies in the clinic requires the ability to produce two-chain proteins in industrially necessary quantities. Vector components that improve recombinant protein production in prokaryotic host cells have been described (see, for example, Schlapschy et al., Protein Engineering, Design and Selection, 19:385, 2006, and Simmons et al., Journal of Immunological Methods, 263:133, 2002), and in particular the expression of chaperone protein(s) has been used to increase antibody titers. However, these chaperone protein(s) are typically expressed from a plasmid in the host cell. This means that for every new recombinant protein to be expressed, significant time and expense must be spent to construct a unique expression plasmid encoding both the recombinant product and the chaperone(s) and to tune their expression (e.g., by testing various promoters and / or translation initiation regions). This also requires the use of larger plasmid sizes to accommodate the coding sequence(s) and the associated regulatory elements for the chaperone protein(s). Plasmid expression also typically results in higher expression levels of the chaperone protein(s) (as the plasmid can be present at at least 10-15 copies per cell), and in some cases, plasmid expression requires additional purification steps to remove the chaperone protein from the recombinant product titer.

[0007] All references cited herein, including patent applications, patent publications, and UniProtKB / Swiss-Prot accession numbers, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0008] There remains a need for an optimal method for efficient production of recombinant two-chain proteins on a laboratory scale. In particular, the integration of the translation unit(s) encoding the chaperone protein into the prokaryotic host cell chromosome and / or the integration of a non-native promoter to drive the expression of the native chaperone protein allows a single host cell to be used to express a variety of recombinant protein products and simplify the plasmid manipulation and protein purification protocols required for production.

[0009] To meet these and other needs, prokaryotic host cells and methods for using same to produce two-chain polypeptides are provided herein. Advantageously, these host cells and methods allow for more efficient production of two-chain polypeptides, for example, without the need for up-front time and cost to optimize chaperone expression plasmids or downstream purification steps to remove chaperone proteins.

[0010] In one aspect, provided herein is a method of producing a polypeptide comprising two chains in a prokaryotic host cell comprising a host cell chromosome, the method comprising: (a) culturing a host cell to express the two chains of the polypeptide in a culture medium under conditions suitable for expression of the two chains of the polypeptide, whereby, upon expression, the two chains fold and assemble to form a biologically active polypeptide in the host cell, the host cell comprising: (1) a first polynucleotide comprising a first translation unit encoding a first chain of the polypeptide; (2) a second polynucleotide comprising a second translation unit encoding a second chain of the polypeptide, wherein the first and second polynucleotides are covalently linked to one another; (b) forming a third polynucleotide comprising a third translation unit that is part of an extrachromosomal polynucleotide selected from the group consisting of peptidyl-prolyl isomerase and protein disulfide oxidoreductase, the third translation unit being part of a host cell chromosome, the third translation unit being integrated into the host cell chromosome and in operable combination with a promoter that drives transcription of the third translation unit, the combination of the third translation unit and the promoter being non-native to the host cell chromosome; and (b) recovering the biologically active polypeptide from the host cell.

[0011] In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is a Pho promoter that drives transcription of the third translation unit when phosphate in the culture medium is depleted. In some embodiments, the inducible promoter is an IPTG-inducible promoter that drives transcription of the third translation unit when isopropyl β-D-thiogalactoside (IPTG) is present in the culture medium. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a CP25 promoter. In some embodiments, the third translation unit is native to the host cell chromosome. In some embodiments, the third translation unit is non-native to the host cell chromosome. In some embodiments, the chaperone protein is a peptidyl-prolyl isomerase. In some embodiments, the peptidyl-prolyl isomerase is an FkpA protein. In some embodiments, the FkpA is E. coli FkpA. In some embodiments, the chaperone protein is a protein disulfide oxidoreductase. In some embodiments, the protein disulfide oxidoreductase is a DsbC protein. In some embodiments, the protein disulfide oxidoreductase is E. coli DsbC. In some embodiments, the protein disulfide oxidoreductase is a DsbA protein. In some embodiments, the protein disulfide oxidoreductase is E. coli DsbA.

[0012] In another aspect, provided herein is a method of producing a polypeptide comprising two chains in a prokaryotic host cell comprising a host cell chromosome, the method comprising: (a) culturing a host cell to express the two chains of the polypeptide in a culture medium under conditions suitable for expression of the two chains of the polypeptide, whereby upon expression, the two chains fold and assemble to form a biologically active polypeptide in the host cell, the host cell comprising: (1) a first polynucleotide comprising a first translation unit encoding a first chain of the polypeptide, (2) a second polynucleotide comprising a second translation unit encoding a second chain of the polypeptide, wherein the first and second polynucleotides are part of one or more extrachromosomal polynucleotides; (3) a third polynucleotide comprising a third translation unit encoding a protein disulfide oxidoreductase, wherein the third polynucleotide comprises a second translation unit encoding a protein disulfide oxidoreductase, (b) forming a third polynucleotide, wherein a third translation unit is part of the host cell chromosome, the third translation unit being integrated into the host cell chromosome and in operable combination with a first promoter that drives transcription of the third translation unit, the combination of the third translation unit and the first promoter being non-native to the host cell chromosome; and (4) a fourth polynucleotide comprising a fourth translation unit encoding a peptidyl-prolyl isomerase, the fourth translation unit being part of the host cell chromosome, the fourth translation unit being integrated into the host cell chromosome and in operable combination with a second promoter that drives transcription of the fourth translation unit, the combination of the fourth translation unit and the second promoter being non-native to the host cell chromosome; and (b) recovering the biologically active polypeptide from the host cell.

[0013] In some embodiments, the first and second promoters are both inducible promoters. In some embodiments, the first and second promoters are both Pho promoters that drive transcription of the third and fourth translation units, respectively, when phosphate in the culture medium is depleted. In some embodiments, one of the first and second promoters is an inducible promoter, and the other of the first and second promoters is a constitutive promoter. In some embodiments, the first promoter is a Pho promoter that drives transcription of the third translation unit when phosphate in the culture medium is depleted, and the second promoter is a CP25 promoter. In some embodiments, the second promoter is an inducible promoter, and the first promoter is a constitutive promoter. In some embodiments, one or both of the third translation unit and the fourth translation unit are native to the host cell chromosome. In some embodiments, both the third translation unit and the fourth translation unit are native to the host cell chromosome. In some embodiments, one or both of the third translation unit and the fourth translation unit are non-native to the host cell chromosome. In some embodiments, the protein disulfide oxidoreductase is a DsbC protein. In some embodiments, the protein disulfide oxidoreductase is E. coli DsbC. In some embodiments, the peptidyl-prolyl isomerase is a FkpA protein. In some embodiments, the FkpA is E. coli FkpA. In some embodiments, the protein disulfide oxidoreductase is E. coli DsbC, the first promoter is a Pho promoter that drives transcription of the third translation unit when phosphate is depleted in the culture medium, the peptidyl-prolyl isomerase is E. coli FkpA, and the second promoter is a CP25 promoter.In some embodiments, the protein disulfide oxidoreductase is E. coli DsbC, the first promoter is a Pho promoter that drives transcription of a third translation unit when phosphate in the culture medium is depleted, and the peptidyl-prolyl isomerase is E. coli FkpA, and the second promoter is a Pho promoter that drives transcription of a fourth translation unit when phosphate in the culture medium is depleted. In some embodiments, the host cell further comprises (5) a fifth polynucleotide comprising a fifth translation unit encoding a second protein disulfide oxidoreductase, the fifth translation unit being part of the host cell chromosome, the fifth translation unit being integrated into the host cell chromosome and in operable combination with a third promoter that drives transcription of the fifth translation unit, and the combination of the fifth translation unit and the third promoter being non-native to the host cell chromosome. In some embodiments, the second protein disulfide oxidoreductase is a DsbA protein. In some embodiments, the second protein disulfide oxidoreductase is E. coli DsbA. In some embodiments, the third promoter is an inducible promoter. In some embodiments, the third promoter is an IPTG-inducible promoter that drives transcription of the fifth translation unit when isopropyl β-D-thiogalactoside (IPTG) is present in the culture medium. In some embodiments, the fifth translation unit is native to the host cell chromosome. In some embodiments, the fifth translation unit is non-native to the host cell chromosome.In some embodiments, the first protein disulfide oxidoreductase is E. coli DsbC, the first promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter that drives transcription of the third translation unit when IPTG is present in the culture medium, the peptidyl-prolyl isomerase is E. coli FkpA, the second promoter is a CP25 promoter, the second protein disulfide oxidoreductase is E. coli DsbA, and the third promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter that drives transcription of the fifth translation unit when IPTG is present in the culture medium. In some embodiments, the host cell further comprises (6) a sixth polynucleotide comprising a sixth translation unit encoding a third chain of the polypeptide, the sixth polynucleotide being part of one or more extrachromosomal polynucleotides, such that upon expression, the three chains fold and assemble to form a biologically active polypeptide in the host cell. In some embodiments, the first translation unit encodes an immunoglobulin heavy chain, the second translation unit encodes an immunoglobulin light chain, and the sixth translation unit encodes an immunoglobulin Fc fragment, and the three chains fold and assemble to form a biologically active monovalent antibody. In some embodiments, the monovalent antibody can specifically bind to an antigen.

[0014] In some embodiments of any of the above embodiments, the first and second polynucleotides are both part of a single extrachromosomal expression vector. In some embodiments, the extrachromosomal expression vector further comprises a polynucleotide encoding a selectable marker that promotes resistance to a selection agent, and the host cell is cultured under conditions suitable for expression of the selectable marker, and the culture medium further comprises the selection agent. In some embodiments, the extrachromosomal expression vector further comprises an origin of replication suitable for replicating the extrachromosomal expression vector in a prokaryotic host cell. In some embodiments, the two chains of the polypeptide are linked to each other by at least one disulfide bond. In some embodiments, the polypeptide is a heterodimeric monomer. In some embodiments, the polypeptide is a half antibody, the first chain and the second chain comprising an immunoglobulin heavy chain and an immunoglobulin light chain. In some embodiments, the half antibody is capable of specifically binding to an antigen. In some embodiments, the polypeptide is a secreted protein. In some embodiments, the secreted protein is recovered from the periplasm of the host cell. In some embodiments, the prokaryotic host cell is a gram-negative bacterium. In some embodiments, the gram-negative bacterium is E. coli. In some embodiments, the E. coli is of a strain that lacks endogenous protease activity. In some embodiments, the E. coli is of a strain that has a degpS210A mutation. In some embodiments, the E. coli is of a strain that has enhanced LacI production or activity. In some embodiments, the E. coli is of a strain that has a lacI Q In some embodiments, the E. coli is a strain having the mutations ΔfhuA ΔphoA ilvG2096 (IlvG+, Valr) Δprc spr43H1 ΔmanA lacI Q This is from the ΔompT ΔmenE742 degPS210A strain.

[0015] In another aspect, provided herein is a method of producing a bispecific antibody comprising a first half antibody capable of binding to a first antigen and a second half antibody capable of binding to a second antigen, the method comprising: producing a first half antibody according to the method of any one of the above embodiments, wherein a first translation unit encodes a heavy chain of the first half antibody and a second translation unit encodes a light chain of the first half antibody, the first half antibody comprising at least one knob-forming mutation; and producing a second half antibody according to the method of any one of the above embodiments, wherein a first translation unit encodes a heavy chain of the second half antibody and a second translation unit encodes a light chain of the second half antibody, the second half antibody comprising at least one hole-forming mutation; and combining the first half antibody with the second half antibody under reducing conditions to generate the bispecific antibody.

[0016] In some embodiments, the first antigen and the second antigen are different antigens. In some embodiments, the method further comprises adding a reducing agent to achieve reducing conditions. In some embodiments, the reducing agent is glutathione.

[0017] In another aspect, provided herein is a host cell (e.g., a prokaryotic host cell) comprising a host cell chromosome, the prokaryotic host cell comprising: (1) a first polynucleotide comprising a first translational unit encoding a peptidyl-prolyl isomerase, the first translational unit being part of the host cell chromosome, the first translational unit being integrated into the host cell chromosome and in operable combination with a first promoter driving transcription of the first translational unit, the combination of the first translational unit and the first promoter being integrated into the host cell chromosome; and (2) a second polynucleotide comprising a second translational unit encoding a protein disulfide oxidoreductase, wherein the second translational unit is part of the host cell chromosome, the second translational unit being integrated into the host cell chromosome and in operable combination with a second promoter that drives transcription of the second translational unit, wherein the combination of the second translational unit and the second promoter is non-native to the host cell chromosome.

[0018] In some embodiments, one or both of the first translation unit and the second translation unit are native to the prokaryotic host cell chromosome. In some embodiments, both the first translation unit and the second translation unit are native to the prokaryotic host cell chromosome. In some embodiments, one or both of the first translation unit and the second translation unit are non-native to the prokaryotic host cell chromosome. In some embodiments, the first promoter is a first inducible promoter. In some embodiments, the first inducible promoter is a Pho promoter. In some embodiments, the first inducible promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter. In some embodiments, the first promoter is a first constitutive promoter. In some embodiments, the first constitutive promoter is a CP25 promoter. In some embodiments, the second promoter is a second inducible promoter. In some embodiments, the second inducible promoter is a Pho promoter. In some embodiments, the second inducible promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter. In some embodiments, the second promoter is a second constitutive promoter. In some embodiments, the second constitutive promoter is a CP25 promoter. In some embodiments, the peptidyl-prolyl isomerase is an FkpA protein. In some embodiments, the FkpA is E. coli FkpA. In some embodiments, the protein disulfide oxidoreductase is a DsbC protein. In some embodiments, the protein disulfide oxidoreductase is E. coli DsbC. In some embodiments, the peptidyl-prolyl isomerase is an FkpA protein, the first promoter is a CP25 promoter, the protein disulfide oxidoreductase is a DsbC protein, and the second promoter is a Pho promoter.In some embodiments, the peptidyl-prolyl isomerase is an FkpA protein, the first promoter is a Pho promoter, the protein disulfide oxidoreductase is a DsbC protein, and the second promoter is a Pho promoter. In some embodiments, the host cell further comprises (3) a third polynucleotide comprising a third translation unit encoding a second protein disulfide oxidoreductase, the third translation unit being part of the host cell chromosome, the third translation unit being integrated into the host cell chromosome and in operable combination with a third promoter driving transcription of the third translation unit, and the combination of the third translation unit and the third promoter being non-native to the host cell chromosome. In some embodiments, the second protein disulfide oxidoreductase is a DsbA protein. In some embodiments, the second protein disulfide oxidoreductase is E. coli DsbA. In some embodiments, the third promoter is a third inducible promoter. In some embodiments, the third inducible promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter. In some embodiments, the peptidyl-prolyl isomerase is an FkpA protein, the first promoter is a CP25 promoter, the first protein disulfide oxidoreductase is a DsbC protein, the second promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter, the second protein disulfide oxidoreductase is a DsbA protein, and the third promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter. In some embodiments, the prokaryotic host cell is a gram-negative bacterium. In some embodiments, the gram-negative bacterium is Escherichia coli. In some embodiments, the E. coli is of a strain that lacks endogenous protease activity. In some embodiments, the E. coli is a strain with a degpS210A mutation. In some embodiments, the E. coli is of a strain with enhanced LacI production or activity. In some embodiments, the E. coli is of a strain with enhanced LacI production or activity. QIn some embodiments, the E. coli is a strain having the mutations ΔfhuA ΔphoA ilvG2096 (IlvG+, Valr) Δprc spr43H1 ΔmanA lacI Q This is from the ΔompT ΔmenE742 degPS210A strain.

[0019] In some embodiments, the host cell further comprises an extrachromosomal expression vector comprising (a) a first extrachromosomal polynucleotide comprising a first extrachromosomal translation unit encoding a first chain of a two-chain polypeptide, and (b) a second extrachromosomal polynucleotide comprising a second extrachromosomal translation unit encoding a second chain of the two-chain polypeptide, whereby, upon expression, the two chains fold and assemble to form a biologically active two-chain polypeptide in the host cell. In some embodiments, the extrachromosomal expression vector further comprises an origin of replication suitable for replicating the extrachromosomal expression vector in a prokaryotic host cell. In some embodiments, the extrachromosomal expression vector further comprises a polynucleotide encoding a selectable marker that promotes resistance to a selection agent. In some embodiments, the two chains of the two-chain polypeptide are linked to each other by at least one disulfide bond. In some embodiments, the two-chain polypeptide is a heterodimeric monomer. In some embodiments, the polypeptide is a half antibody, where the first chain and the second chain comprise an immunoglobulin heavy chain and an immunoglobulin light chain. In some embodiments, the half antibody is capable of specifically binding to an antigen. In some embodiments, the two-chain polypeptide is a secreted protein. In some embodiments, the secreted protein is recovered from the periplasm of the host cell. In some embodiments, the extrachromosomal expression vector further comprises a third extrachromosomal polynucleotide comprising a third extrachromosomal translation unit encoding a third chain of the two-chain polypeptide, whereby, upon expression, the three chains fold and assemble to form a biologically active polypeptide in the host cell. In some embodiments, the first extrachromosomal translation unit encodes an immunoglobulin heavy chain, the second extrachromosomal translation unit encodes an immunoglobulin light chain, and the third extrachromosomal translation unit encodes an immunoglobulin Fc fragment, and the three chains fold and assemble to form a biologically active monovalent antibody. In some embodiments, the monovalent antibody can specifically bind to an antigen.

[0020] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present disclosure. These and other aspects of the present disclosure will be apparent to those skilled in the art. These and other embodiments of the present disclosure are further described in the following detailed description. [Brief description of the drawings]

[0021] [Figure 1] Plasmid maps of an expression vector (MD156, left) used to overexpress the same chaperone protein and two-chain protein product (in this case, antibody fragment heavy and light chains, "HC" and "LC", respectively) are shown compared to an expression vector (CS392, right) for expression of the two-chain protein product in a host cell with chromosomal overexpression of the chaperone protein. Vector sizes are provided in base pairs bp.

[0022] [Diagram 2] Titers (black) and FkpA expression levels (grey) of strains with the indicated promoter-fkpA pairings are shown compared to strains with plasmid-based expression of FkpA.

[0023] [Figure 3A-3C] Relative chaperone expression levels (fold over native expression levels) of DsbA (FIG. 3A), DsbC (FIG. 3B), or FkpA (FIG. 3C) in the indicated strains in shake flasks are shown. Sh.Fl. represents shake flask culture, + represents positive control (plasmid chaperone expression), - represents negative control (no chaperone expression), and Sh.Fl.(-) refers to native expression levels.

[0024] [Figure 4A-4C]A indicates the relative chaperone expression levels (fold over native expression levels) of DsbA (FIG. 4A), DsbC (FIG. 4B), or FkpA (FIG. 4C) in the indicated strains from a 10 L fermenter. + represents the positive control (plasmid chaperone expression), - represents the negative control (no chaperone expression), and Ambr(-) refers to the native expression level.

[0025] [Diagram 5] indicates the xIL13 titer (g / L) produced by strains harboring the indicated chromosomally engineered plasmid and native chaperone locus pairs. 67A6 / MD157 refers to strains without chromosomal engineering and harboring the MD157 plasmid expressing the indicated chaperone under the indicated promoter (see FIG. 1 for a diagram of the MD157 plasmid).

[0026] [Figure 6A-6B] The optical density (OD, FIG. 6A) and osmolality (FIG. 6B) over time of cultures of the indicated strain / plasmid combinations producing xIL13 are shown.

[0027] [Figure 7A-7C] Shown are xIL13 titers (g / L, FIG. 7A), DsbC concentrations (FIG. 7B), and FkpA concentrations (FIG. 7C) produced by the indicated strains over time.

[0028] [Figure 8A-8B] The optical density (OD, FIG. 8A) and osmolality (FIG. 8B) over time of cultures of the indicated strain / plasmid combinations producing AF2 are shown.

[0029] [Figure 9] AF2 titers (g / L) produced by the indicated strains are shown over time.

[0030] [Figure 10A-10B] The optical density (OD, FIG. 10A) and osmolality (FIG. 10B) over time of cultures of the indicated strain / plasmid combinations producing MetMAb are shown.

[0031] [Figure 11] MetMAb titers (g / L) produced by the indicated strains are shown over time.

[0032] [Figure 12A-12B] The optical density (OD, FIG. 12A) and osmolality (FIG. 12B) over time of cultures of the indicated strain / plasmid combinations producing anti-VEGF antibody fragments are shown.

[0033] [Figure 13] Anti-VEGF antibody fragment titers (g / L) produced by the indicated strains are shown over time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present disclosure provides host cells (e.g., prokaryotic host cells) incorporating a non-native promoter:chaperone protein combination(s) suitable for large-scale production of recombinant two-chain protein products, as well as related methods. The examples provided herein demonstrate that prokaryotic host cells expressing chaperone proteins from host cell chromosomes produce titers comparable to plasmid-based chaperone expression. These results were consistent across multiple antibody formats, such as half antibodies, one-arm antibodies, and antibody fragments, and required little or no additional process development. Importantly, the data presented herein show that chaperone expression from host cell chromosomes, rather than plasmids, results in lower chaperone expression levels (potentially eliminating the need for further downstream purification to remove chaperone proteins from the product) yet comparable or higher product titers. These results demonstrate that, compared to using host cells expressing the chaperone protein(s) from a plasmid, the host cells and / or methods of the present disclosure can be used to produce products at least as efficiently on an industrial scale without the need for up-front time and cost to optimize the chaperone expression plasmid or downstream purification steps to remove the chaperone protein.

[0035] In one aspect, provided herein is a method of producing a polypeptide comprising two chains in a prokaryotic host cell comprising a host cell chromosome, the method comprising culturing the host cell in a culture medium under conditions suitable for expression of the two chains of the polypeptide to express the two chains of the polypeptide, whereby, upon expression, the two chains fold and assemble to form a biologically active polypeptide in the host cell, and (b) recovering the biologically active polypeptide from the host cell, the host cell comprising (1) a first polynucleotide comprising a first translation unit encoding a first chain of the polypeptide, (2) a second translation unit encoding a second chain of the polypeptide, and (3) a third polynucleotide comprising a third translation unit encoding a chaperone protein selected from the group consisting of peptidyl-prolyl isomerase and protein disulfide oxidoreductase, wherein the third translation unit is part of the host cell chromosome, the third translation unit is integrated into the host cell chromosome and is in operable combination with a promoter that drives transcription of the third translation unit, and the combination of the third translation unit and the promoter is non-native to the host cell chromosome.

[0036] In another aspect, provided herein is a method of producing a polypeptide comprising two chains in a prokaryotic host cell comprising a host cell chromosome, the method comprising culturing the host cell to express the two chains of the polypeptide in a culture medium under conditions suitable for expression of the two chains of the polypeptide, whereby, upon expression, the two chains fold and assemble to form a biologically active polypeptide in the host cell, and (b) recovering the biologically active polypeptide from the host cell, the host cell comprising: (1) a first polynucleotide comprising a first translation unit encoding a first chain of the polypeptide, (2) a second polynucleotide comprising a second translation unit encoding a second chain of the polypeptide, wherein the first and second polynucleotides are part of one or more extrachromosomal polynucleotides, (3) a protein disulfide oxynucleotide, and (4) a protein disulfide oxynucleotide. and (4) a fourth polynucleotide comprising a fourth translation unit encoding a peptidyl-prolyl isomerase, wherein the fourth translation unit is part of the host cell chromosome, the third translation unit is integrated into the host cell chromosome and in operable combination with a first promoter that drives transcription of the third translation unit, the combination of the third translation unit and the first promoter being non-native to the host cell chromosome.

[0037] In another aspect, provided herein is a prokaryotic host cell comprising a host cell chromosome, the prokaryotic host cell comprising: (1) a first polynucleotide comprising a first translational unit encoding a peptidyl-prolyl isomerase, the first translational unit being part of the host cell chromosome, the first translational unit being integrated into the host cell chromosome and in operable combination with a first promoter driving transcription of the first translational unit, the combination of the first translational unit and the first promoter being non-native to the host cell chromosome; and (2) a second polynucleotide comprising a second translational unit encoding a protein disulfide oxidoreductase, wherein the second translational unit is part of the host cell chromosome, the second translational unit is integrated into the host cell chromosome and is in operable combination with a second promoter that drives transcription of the second translational unit, and the combination of the second translational unit and the second promoter is non-native to the host cell chromosome. I. Definition

[0038] Before describing the present disclosure in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0039] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[0040] The term "about" as used herein refers to a normal error range for each value, as would be readily understood by one of ordinary skill in the art. Reference herein to a value or parameter of "about" includes (and describes) embodiments directed to the value or parameter itself. The term "about" as used herein with respect to a value encompasses up to 90%-110% of that value (e.g., a relative translation strength of the first and second TIR of about 1.0 to about 3.0 refers to a relative translation strength in the range between 0.9 and 3.3).

[0041] It is to be understood that aspects and embodiments of the present disclosure described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.

[0042] As used herein, the term "polypeptide comprising two chains" (the terms "two-chain protein" and "two-chain polypeptide" may also be used interchangeably herein) is intended to refer to any polypeptide that contains two or more separate polypeptide chains. In some embodiments, a two-chain protein may include a macromolecular complex of two or more polypeptides linked together through one or more intermolecular linkages, including but not limited to disulfide bonds. In some embodiments, a two-chain protein may include a single polypeptide having amino acid sequences belonging to two separate polypeptide chains (e.g., an antibody heavy chain and an antibody light chain) linked by a polypeptide linker. In this case, the two-chain protein physically represents a single chain, but the two or more portions of the single chain may behave functionally as if they were two separate protein chains. For example, a single-chain antibody may include a functional heavy chain and a functional light chain, which are joined by a polypeptide linker but nevertheless fold and assemble as if they were separate polypeptides associated only by intermolecular linkages (e.g., one or more disulfide bonds).

[0043] The terms "native" and "non-native" as used herein with respect to one or more genetic elements (e.g., promoters, translation units, or combinations thereof) are intended to refer to the genomic context of the genetic elements in a host cell chromosome as they occur in nature. For example, a translation unit is "native" with respect to a host cell or host cell chromosome if the translation unit naturally occurs in the genome of the host cell, and is "non-native" if the translation unit does not naturally occur in the genome of the host cell. A promoter is "native" with respect to a host cell or host cell chromosome if the promoter naturally occurs in the genome of the host cell, and is "non-native" if the promoter does not naturally occur in the genome of the host cell. An operable combination of a promoter and a translation unit is "non-native" if the promoter does not naturally occur in the genome of the host cell in the same operable linkage as the translation unit, or vice versa. For example, a promoter:translation unit combination is "non-native" with respect to a host cell or host cell chromosome when one or both of the promoter and translation unit do not naturally occur in the host cell genome, when a promoter is present in the host cell genome operably linked to a translation unit with which it is not operably combined in the naturally occurring host cell genome (even if the same promoter sequence is naturally present elsewhere in the host cell genome), or when a translation unit is present in the host cell genome operably linked to a promoter with which it is not operably combined in the naturally occurring host cell genome (even if the same translation unit is naturally present elsewhere in the host cell genome).

[0044] The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome. Certain vectors can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "recombinant vectors"). In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector.

[0045] The term "cistron," as used herein, is intended to refer to a genetic element broadly equivalent to a translation unit that includes a nucleotide sequence encoding a polypeptide chain and adjacent control regions. A "cistron" can include, for example, one or more open reading frames, a translation initiation region (TIR, as defined herein below), a signal sequence, and a termination region.

[0046] A "polycistronic" expression vector refers to a single vector that contains and expresses multiple cistrons under the regulatory control of one single promoter. A common example of a polycistronic vector is a "bicistronic" vector that contains and expresses two different polypeptides under the control of one promoter. During the expression of a bicistronic or polycistronic vector, the multiple genes are first transcribed as a single transcription unit and then translated separately.

[0047] "Transcription unit" refers to a polynucleotide that is transcribed as a single RNA transcript. "Translation unit" refers to a polynucleotide that encodes a polypeptide and produces the polypeptide when translated. As described above, a polycistronic polynucleotide may contain a single transcription unit with multiple translation units.

[0048] A "separate cistron" expression vector according to the present disclosure refers to a single vector that contains at least two separate promoter-cistron pairs, where each cistron is under the control of its own promoter. Upon expression of a separate cistron expression vector, both the transcription and translation processes of the different genes are separate and independent.

[0049] As used herein, "chaperone protein" refers to any protein that aids in the folding or assembly of other macromolecules, including, but not limited to, two-chain proteins. In general, chaperone proteins may act to facilitate protein folding or assembly by many different mechanisms. For example, chaperone proteins may facilitate protein folding and / or assembly, catalyze the formation of intrachain disulfide bonds, promote protein unfolding and / or degradation (e.g., of aggregated or misfolded proteins or multiprotein complexes), prevent aggregation, assist in protein degradation, etc.

[0050] "Secretory signal sequence" or "signal sequence" refers to a nucleic acid sequence encoding a short signal peptide that can be used to guide a newly synthesized protein of interest through a cell membrane, usually the inner membrane or both the inner and outer membranes of prokaryotes. In this way, the protein of interest, such as an immunoglobulin light or heavy chain polypeptide, is secreted into the periplasm or into the culture medium of a prokaryotic host cell. The signal peptides encoded by the secretory signal sequence may be endogenous to the host cell or they may be exogenous, including the signal peptide native to the expressed polypeptide. Secretory signal sequences are typically present at the amino terminus of the expressed polypeptide and are typically enzymatically removed between biosynthesis and secretion of the polypeptide from the cytoplasm. Thus, signal peptides are usually not present in the mature protein product.

[0051] "Operably linked" refers to the juxtaposition of two or more components, the components so described being in a relationship that allows them to function in their intended manner. For example, a promoter is operably linked to a coding sequence or translation unit if it acts in cis to control or regulate the transcription of the linked sequence. In general, "operably linked" DNA sequences are not necessarily contiguous, but are contiguous and in reading frame, as is the case when joining two protein coding regions or in the case of secretory leaders. However, an operably linked promoter is generally located upstream of a coding sequence or translation unit, but is not necessarily contiguous with it. An operably linked enhancer may be located upstream, within, or downstream of a coding sequence / translation unit at a substantial distance from the promoter. Linking is accomplished by recombinant methods known in the art, for example, using PCR methods, by annealing, or by ligation at convenient restriction enzyme sites. If no convenient restriction enzyme sites exist, synthetic oligonucleotide adaptors or linkers are used according to the convention.

[0052] As used herein, "regulatory element" refers to a nucleotide sequence present in cis that is essential for the transcription and translation of a polynucleotide encoding a heterologous polypeptide into a polypeptide.Transcriptional regulatory elements usually include the promoter 5' of the gene sequence to be expressed, the transcriptional initiation and termination sites, and the polyadenylation signal sequence.The term "transcriptional initiation site" refers to the nucleic acid in the construct that corresponds to the first nucleic acid that is incorporated into the primary transcription product, i.e., mRNA precursor, and the transcriptional initiation site may overlap with the promoter sequence.

[0053] "Promoter" refers to a polynucleotide sequence that controls the transcription of a gene or sequence to which it is operably linked. A promoter contains signals for binding RNA polymerase and initiating transcription. The promoter used is functional in the cell type of the host cell in which the expression of the selected sequence is intended. Numerous promoters, including constitutive, inducible, and repressible promoters from a variety of different sources, are well known in the art (and identified in databases such as GenBank) and are available as or within cloned polynucleotides (e.g., from depositories such as ATCC and other commercial or personal sources). For inducible promoters, the activity of the promoter increases or decreases in response to a signal, such as the presence of IPTG or phosphate starvation.

[0054] The term "host cell" (or "recombinant host cell"), as used herein, is intended to refer to a cell that has been genetically modified or can be genetically modified by the introduction of an exogenous or non-natural polynucleotide, such as a recombinant plasmid or vector. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0055] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. A "pharmaceutical acceptable" excipient (vehicle, additive) is one that can be reasonably administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0056] A "subject" or "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0057] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0058] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. The contaminating components of its natural environment are substances that would interfere with the antibody's experimental, diagnostic, or therapeutic use, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight, in some embodiments, greater than 99% by weight, for example, as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example, using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, for example, using Coomassie blue or silver staining. An isolated antibody includes an in situ antibody within a recombinant cell, since at least one component of the antibody's natural environment will be absent. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0059] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by several constant domains. Each light chain has a variable domain (V L ) at its other end and a constant domain, in which the light chain constant domain aligns with the first constant domain of the heavy chain and the light chain variable domain aligns with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0060] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portions of the immunoglobulin, the variable domains that contain the antigen binding sites. The constant domains are the C H 1. C H 2, and C H It contains three domains (collectively, CH) of the light chain, as well as a CHL (or CL) domain of the light chain.

[0061] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain is H The variable domain of the light chain may be referred to as "V L These domains are generally the most variable parts of the antibody and contain the antigen binding sites.

[0062] The term "variable" refers to the fact that certain parts of the variable domains vary widely in sequence between antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed throughout the variable domains of antibodies. The variability is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions that predominantly adopt a beta-sheet configuration connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs within each chain are held in close proximity to each other by the FR regions and, together with the HVRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md, 1991). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as involvement of the antibody in antibody-dependent cellular toxicity.

[0063] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0064] The term IgG "isotype" or "subclass" as used herein means any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[0065] Depending on the amino acid sequence of the constant domain of the heavy chain, antibodies (immunoglobulins) can be assigned to different classes. The five main classes of immunoglobulins are IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunit structures and three-dimensional organization of the various classes of immunoglobulins are well known and are generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th Edition (WB Saunders, Co., 2000). An antibody can be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[0066] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody in a substantially intact form, rather than an antibody fragment as defined below. These terms specifically refer to an antibody having a heavy chain that contains an Fc region.

[0067] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[0068] An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen-binding region thereof. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0069] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0070] "Fv" is the smallest antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight non-covalent association. In a single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in the two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0071] Fab fragments contain heavy and light chain variable domains, and also contain a light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0072] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, and these domains are present in a single polypeptide chain. In general, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form a desired structure for antigen binding. For a review of scFvs, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, Eds. (Springer-Verlag, New York, 1994), pp. 269-315.

[0073] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, these domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are described more fully, for example, in EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9, 129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0074] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogenous antibodies, e.g., the individual antibodies that make up the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the characteristic of the antibody that is not a mixture of separate antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence may be further modified, e.g., to improve affinity to the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and an antibody that includes a modified target-binding sequence is also a monoclonal antibody of the present disclosure. In contrast to polyclonal antibody preparations, which usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are usually uncontaminated by other immunoglobulins.

[0075] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present disclosure may be produced using a variety of techniques including, for example, expression in prokaryotic host cells, the hybridoma method (see, e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Antibodies (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellowes, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004)). ), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993), Jakobovits et al., Nature 362:255-258 (1993), Bruggemann et al., Year in Immunol.7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-813 (1994), Fishwild et al., Nature Biotechnol. 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0076] The monoclonal antibodies herein specifically include "chimeric antibodies" in which a portion of the heavy and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, for example, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies, whose antigen-binding regions are derived from, for example, antibodies produced by immunizing macaque monkeys with an antigen of interest.

[0077] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient or donor antibody. These modifications can further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence. A humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op.Struct.Biol.2:593-596 (1992). Also see, e.g., Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol.1:105-115 (1998); Harris, Biochem.Soc.Transactions23:1035-1038 (1995); Hurle and Gross, Curr.Op.Biotech.5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0078] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. The methods described in Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991), Marks et al., J. Mol. Biol. 222:581 (1991), Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985), Boerner et al., J. Immunol. 147:(1)86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology), which have been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled. Also see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B cell hybridoma technology.

[0079] A "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Typically, species-dependent antibodies bind "specifically" to a human antigen (e.g., about 1×10 -7 M or less, preferably about 1×10 -8 M or less, preferably about 1×10 -9The species-dependent antibody has a binding affinity for a homolog of the antigen from a second non-human mammalian species that is at least about 50 times, or at least about 500 times, or at least about 1000 times weaker than the binding affinity for the human antigen, the binding affinity (Kd) value being equal to or less than M. The species-dependent antibody can be any of the various types of antibodies defined above, but is preferably a humanized or human antibody.

[0080] The term "hypervariable region", "HVR", or "HV" as used herein refers to a region of an antibody variable domain that is sequence hypervariable and / or forms structurally defined loops. Generally, an antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 show the highest diversity of the six HVRs, and H3 in particular is thought to play a unique role in conferring excellent specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, Methods in Molecular Biology 248:1-25 (ed. Lo, Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies, consisting only of heavy chains, are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0081] Several HVR descriptions are used and encompassed herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of available complex crystal structures. Residues from each of these HVRs are shown below. TIFF0007620546000001.tif78170

[0082] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al. (see above) for each of these definitions.

[0083] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0084] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al. (see above). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or an insertion into, the FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (such as residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues may be determined by alignment of the homologous regions of the sequence of the antibody with the "standard" Kabat numbered sequence for a given antibody.

[0085] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[0086] The term "linear antibody" refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific. II.Host cells

[0087] Provided herein are host cells (e.g., prokaryotic host cells) having a host cell chromosome that includes a translational unit encoding at least one chaperone protein (e.g., a peptidyl-prolyl isomerase or a protein disulfide oxidoreductase), the translational unit being in operative combination or linked to a promoter (that is also a part of the host cell chromosome) that drives transcription of the translational unit, such that the combination of the promoter and translational unit is non-native to the host cell or host cell chromosome.

[0088] In some embodiments, a host cell chromosome comprises (1) a first polynucleotide comprising a first translational unit encoding a peptidyl-prolyl isomerase, (2) a second polynucleotide comprising a second translational unit encoding a protein disulfide oxidoreductase, the first and second translational units being part of the host cell chromosome and in operable combination or linked with a first and second (respective) promoter (also part of the host cell chromosome) driving the transcription of the first and second translational units, respectively. In some embodiments, the combination of the first translational unit and the first promoter and / or the combination of the second translational unit and the second promoter is non-native to the host cell chromosome. For example, one or both of the promoters may be non-native to the host cell chromosome, one or both of the translational units may be non-native to the host cell chromosome, or one or both of the translational units may be native to the host cell chromosome but operably combined with a promoter in a combination that is non-native to the host cell chromosome.

[0089] In some embodiments, the host cell further comprises one or more extrachromosomal polynucleotides encoding two or more chains of the two-chain polypeptide of the present disclosure. For example, in some embodiments, the host cell comprises (1) a first polynucleotide comprising a first translation unit encoding a first chain of the two-chain polypeptide of the present disclosure, (2) a second polynucleotide comprising a second translation unit encoding a second chain of the two-chain polypeptide of the present disclosure, and (3) a third polynucleotide comprising a third translation unit encoding a chaperone protein (e.g., peptidyl-prolyl isomerase or protein disulfide oxidoreductase), in operable combination with a promoter that drives transcription of the third translation unit. In some embodiments, the combination of the third translation unit and the promoter is non-native to the host cell chromosome. In some embodiments, the first and second polynucleotides (i.e., encoding the first and second translation units, respectively) are part of one or more extrachromosomal polynucleotides (e.g., plasmids), and the third polynucleotide (and associated promoter) is part of the host cell chromosome.

[0090] In some embodiments, the host cell comprises: (1) a first polynucleotide comprising a first translational unit encoding a first chain of a two-chain polypeptide of the present disclosure; (2) a second polynucleotide comprising a second translational unit encoding a second chain of a two-chain polypeptide of the present disclosure; (3) a third polynucleotide comprising a third translational unit encoding a protein disulfide oxidoreductase, the third polynucleotide being in operable combination with a promoter that drives transcription of the third translational unit; and (4) a fourth polynucleotide comprising a fourth translational unit encoding a peptidyl-prolyl isomerase, the fourth polynucleotide being in operable combination with a promoter that drives transcription of the fourth translational unit. In some embodiments, the combination of the third translational unit with its associated promoter and / or the combination of the fourth translational unit with its associated promoter is non-native to the host cell chromosome. In some embodiments, the first and second polynucleotides (i.e., encoding the first and second translation units, respectively) are part of one or more extrachromosomal polynucleotides (e.g., plasmids), and the third and fourth polynucleotides (and associated promoters) are part of a host cell chromosome.

[0091] In some embodiments, the host cell comprises: (1) a first polynucleotide comprising a first translational unit encoding a first chain of a two-chain polypeptide of the present disclosure; (2) a second polynucleotide comprising a second translational unit encoding a second chain of a two-chain polypeptide of the present disclosure; (3) a third polynucleotide comprising a third translational unit encoding a protein disulfide oxidoreductase, the third polynucleotide being in operable combination with a first promoter that drives transcription of the third translational unit; and (4) a fourth polynucleotide comprising a fourth translational unit encoding a protein disulfide oxidoreductase, the fourth polynucleotide being in operable combination with a second promoter that drives transcription of the fourth translational unit. In some embodiments, the combination of the third translational unit with the first promoter and / or the combination of the fourth translational unit with the second promoter is non-native to the host cell chromosome. In some embodiments, the first and second polynucleotides (i.e., encoding the first and second translation units, respectively) are part of one or more extrachromosomal polynucleotides (e.g., plasmids), and the third and fourth polynucleotides (and associated promoters) are part of a host cell chromosome.

[0092] In some embodiments, the host cell comprises: (1) a first polynucleotide comprising a first translation unit encoding a first chain of a two-chain polypeptide of the disclosure; (2) a second polynucleotide comprising a second translation unit encoding a second chain of a two-chain polypeptide of the disclosure; (3) a third polynucleotide comprising a third translation unit encoding a protein disulfide oxidoreductase, the third polynucleotide being in operable combination with a first promoter that drives transcription of the third translation unit; (4) a fourth polynucleotide comprising a fourth translation unit encoding a peptidyl-prolyl isomerase, the fourth polynucleotide being in operable combination with a second promoter that drives transcription of the fourth translation unit; and (5) a fifth polynucleotide comprising a fifth translation unit encoding a protein disulfide oxidoreductase, the fifth polynucleotide being in operable combination with a third promoter that drives transcription of the fifth translation unit. In some embodiments, the combination of the third translational unit and the first promoter, the combination of the fourth translational unit and the second promoter, and / or the combination of the fifth translational unit and the third promoter are non-native to the host cell chromosome. In some embodiments, the first and second polynucleotides (i.e., encoding the first and second translational units, respectively) are part of one or more extrachromosomal polynucleotides (e.g., plasmids), and the third, fourth, and fifth polynucleotides (and associated promoters) are part of the host cell chromosome.

[0093] In some embodiments, a polynucleotide or translation unit of the present disclosure that encodes a chaperone protein (e.g., a peptidyl-prolyl isomerase or a protein disulfide oxidoreductase) is native to a host cell chromosome. For example, a polynucleotide or translation unit that encodes a chaperone protein can be a native chaperone protein gene or locus. In some embodiments, a promoter is inserted into the host cell genome (e.g., by insertion into or replacement of one or more native regulatory sequences or genetic elements) in operable combination with a native chaperone protein gene or locus, generating a promoter:translation unit combination that is non-native to the host cell chromosome.

[0094] In other embodiments, a polynucleotide or translational unit of the disclosure that encodes a chaperone protein (e.g., a peptidyl-prolyl isomerase or a protein disulfide oxidoreductase) is non-native to the host cell (e.g., chromosomally integrated into the host cell).

[0095] In some embodiments, a host cell chromosome of the present disclosure may include one or more native translation units encoding chaperone proteins of the present disclosure and one or more non-native translation units encoding chaperone proteins of the present disclosure. In some embodiments, a host cell chromosome of the present disclosure may include multiple non-native translation units encoding chaperone proteins of the present disclosure. Furthermore, many of the host cells of the present disclosure are known to include host cell chromosomes encoding multiple chaperone proteins (e.g., FkpA, DsbA, and DsbC in E. coli). In some embodiments, one or more native translation units encoding chaperone proteins of the present disclosure are operably combined with a promoter of the present disclosure in a combination that is non-native to the host cell or host cell chromosome.

[0096] Methods for introducing the polynucleotide or translation unit of the present disclosure into a host cell (e.g., a prokaryotic host cell) are known in the art. An exemplary method, allelic exchange, is described in more detail below. Advantageously, the allelic exchange method does not leave a "scar" on the host cell genome. Other methods include, but are not limited to, those described in Datsenko, KA and Wanner, BL (2000) Proc. Natl. Acad. Sci. 97:6640-6645.

[0097] In some embodiments of any of the above embodiments, the host cell further comprises a translation unit encoding a third chain of the two-chain polypeptide of the present disclosure. In some embodiments, the translation unit is part of an extrachromosomal polynucleotide encoding the first and / or second chain of the two-chain polypeptide. For example, in some embodiments, the two-chain polypeptide is a one-arm antibody comprising, for example, an immunoglobulin heavy chain, an immunoglobulin light chain, and an immunoglobulin Fc fragment, assembled to form a biologically active monovalent antibody (e.g., a monovalent antibody capable of specifically binding to an antigen). Chaperone Proteins

[0098] Certain aspects of the present disclosure relate to chaperone proteins. Chaperone proteins can refer to any protein that aids in the folding or assembly of other macromolecules, including but not limited to two-chain proteins. Examples of chaperone proteins can include, but are not limited to, peptidyl-prolyl isomerase, protein disulfide oxidoreductase, and heat shock proteins (such as Hsp60, Hsp70, Hsp90, and Hsp100 proteins). Chaperone proteins can also aid in transporting proteins through membranes, for example, translocation of polypeptide chains through plasma membranes or endoplasmic reticulum membranes.

[0099] In some embodiments, the chaperone protein may be a peptidyl-prolyl isomerase. Peptidyl-prolyl isomerase (the terms "prolyl isomerase", "rotamase" and "PPiase" may be used interchangeably herein) may refer to any enzyme that catalyzes the interconversion of cis and trans isomers of proline or prolyl-iminopeptide bonds. The EC number for this reaction is EC5.2.1.8. Any protein known or predicted to catalyze the reaction represented by this EC number may be a peptidyl-prolyl isomerase of the present disclosure. Peptidyl-prolyl isomerase activity may also be represented by the GO term ID GO:0003755. Any protein known or predicted to possess the molecular function represented by this GO term ID may be a peptidyl-prolyl isomerase of the present disclosure.

[0100] Peptidyl-prolyl isomerase activity is known in the art to promote protein folding and assembly. In some embodiments, peptidyl-prolyl isomerase can aid in protein folding and assembly by converting trans-prolyl bonds to cis-prolyl bonds for proteins whose properly folded structure contains cis-prolyl bonds. Some peptidyl-prolyl isomerases are also known to improve the folding and assembly of proteins that lack cis-prolyl bonds (Bothmann H and Pluckthun A 2000 J.Biol.Chem.275:17100). In some embodiments, peptidyl-prolyl isomerase can aid in protein folding and assembly of proteins that lack cis-prolyl bonds. Thus, while peptidyl-prolyl isomerase activity can serve as a functional characteristic to identify chaperone proteins that are useful in the methods described herein, the utility of a peptidyl-prolyl isomerase is not necessarily limited to its catalytic activity per se.

[0101] In some embodiments, the peptidyl-prolyl isomerase is FkpA protein. In some embodiments, the FkpA protein is E. coli FkpA. E. coli FkpA can refer to any polypeptide encoded by the fkpA gene in any strain or isolate of bacteria belonging to the E. coli species. In some embodiments, E. coli FkpA refers to the protein encoded by the fkpA gene represented by EcoGene accession number EG12900. In some embodiments, E. coli FkpA refers to the protein having the sequence represented by NCBI RefSeq accession number NP_417806.

[0102] Other FkpA proteins are known in the art. Examples of FkpA proteins include, but are not limited to, S. boydii peptidyl-prolyl isomerase (NCBI RefSeq No. WP_000838252), C. youngae peptidyl-prolyl isomerase (NCBI RefSeq No. WP_006687366), K. oxytoca peptidyl-prolyl isomerase (NCBI RefSeq No. WP_004125943), S. enterica peptidyl-prolyl isomerase (NCBI RefSeq No. WP_000838233), K. pneumoniae peptidyl-prolyl isomerase (NCBI RefSeq No. WP_019704642), S. cerevisiae FPR3p (NCBI RefSeq No. NP_013637), M. musculus Fkpb1a (NCBI RefSeq No. NP_013638), and K. oxytoca peptidyl-prolyl isomerase (NCBI RefSeq No. WP_004125943). RefSeq No. NP_032045), M. musculus Fkpb2 (NCBI RefSeq No. NP_032046), H. sapiens FKBP2 (NCBI RefSeq No. NP_001128680), and D. melanogaster CG14715 (NCBI RefSeq No. NP_650101). In some embodiments, the FkpA protein of the present disclosure has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to E. coli FkpA.

[0103] In some embodiments, the chaperone protein may be a protein disulfide oxidoreductase. Protein disulfide oxidoreductase (the terms "protein disulfide isomerase" and "thiol-disulfide isomerase" may be used interchangeably herein) may refer to any enzyme that catalyzes the rearrangement of disulfide bonds in proteins. For example, protein disulfide oxidoreductase may catalyze the oxidation of cysteine ​​to form disulfide bonds in proteins. Protein disulfide oxidoreductase may also catalyze the isomerization of unpaired disulfide bonds in proteins. The EC number for this reaction is EC5.3.4.1. Any protein known or predicted to catalyze the reaction represented by this EC number may be a protein disulfide oxidoreductase of the present disclosure. Protein disulfide oxidoreductase activity may also be represented by the GO term ID GO:0015035. Any protein known or predicted to possess the molecular function represented by this GO term ID can be a protein disulfide oxidoreductase of the present disclosure.

[0104] Protein disulfide oxidoreductase activity is known in the art to promote protein folding and assembly.For example, protein disulfide oxidoreductase activity promotes the formation of appropriate intramolecular and intermolecular disulfide bonds during protein folding and assembly.In particular, protein disulfide oxidoreductase activity is important for proteins with disulfide bonds expressed in the periplasm of prokaryotic cells.

[0105] In some embodiments, the protein disulfide oxidoreductase is DsbA protein. In some embodiments, the DsbA protein is E. coli DsbA. E. coli DsbA can refer to any polypeptide encoded by dsbA gene in any strain or isolate of bacteria belonging to the E. coli species. In some embodiments, E. coli DsbA refers to the protein encoded by dsbA gene represented by EcoGene accession number EG11297. In some embodiments, E. coli DsbA refers to the protein having the sequence represented by NCBI RefSeq accession number NP_418297.

[0106] Other DsbA proteins are known in the art. Examples of DsbA proteins may include, but are not limited to, S. flexneri thiol-disulfide isomerase (NCBI RefSeq No. WP_000725335), S. dysenteriae thiol-disulfide isomerase (NCBI RefSeq No. WP_000725348), C. youngae thiol-disulfide isomerase (NCBI RefSeq No. WP_006686108), and S. enterica thiol-disulfide isomerase (NCBI RefSeq No. WP_023240584). In some embodiments, a DsbA protein of the disclosure has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to E. coli DsbA.

[0107] In some embodiments, the protein disulfide oxidoreductase is DsbC protein. In some embodiments, the DsbC protein is E. coli DsbC. E. coli DsbC can refer to any polypeptide encoded by dsbC gene in any strain or isolate of bacteria belonging to the E. coli species. In some embodiments, E. coli DsbC refers to the protein encoded by dsbC gene represented by EcoGene accession number EG11070. In some embodiments, E. coli DsbC refers to the protein having the sequence represented by NCBI RefSeq accession number NP_417369.

[0108] Other DsbC proteins are known in the art. Examples of DsbC proteins may include, but are not limited to, S.sonnei protein disulfide isomerase (NCBI RefSeq No. WP_000715206), S.dysenteriae protein disulfide isomerase (NCBI RefSeq No. WP_000715209), E.fergusonii protein disulfide isomerase (NCBI RefSeq No. WP_000715225), S.bongori thiol:disulfide exchange protein DsbC (NCBI RefSeq No. WP_020845161), and S.enterica protein disulfide isomerase DsbC (NCBI RefSeq No. WP_023183515). In some embodiments, a DsbC protein of the disclosure has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to E. coli DsbC.

[0109] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In one embodiment, the length of the reference sequence aligned for comparison purposes is at least 50%, typically at least 75%, and even more typically at least 80%, 85%, 90%, 95%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").

[0110] The percent identity between two sequences is a function of the number of identical positions shared by sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences.For sequence comparison, typically, one sequence serves as a reference sequence, and is compared with the test sequence.When using sequence comparison algorithm, the test sequence and the reference sequence are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.When comparing two sequences for identity, the sequences do not need to be contiguous, but any gap can have a penalty that can reduce the overall percent identity.For blastn, the default parameters are gap clearance penalty of 5 and gap extension penalty of 2.For blastp, the default parameters are gap clearance penalty of 11 and gap extension penalty of 1.

[0111] A "comparison window," as used herein, includes reference to any one segment of a number of contiguous positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, within which a sequence is compared to a reference sequence of the same number of contiguous positions after optimally aligning the two sequences. Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed using known algorithms (e.g., by the local homology algorithm of Smith and Waterman, Adv Appl Math, 2:482, 1981, by the homology alignment algorithm of Needleman and Wunsch, J Mol Biol, 48:443, 1970, by the search for similarity method of Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444, 1988, by computerized implementations of the algorithms FASTDB (Intelligenetics), BLAST (National Center for Biomedical Information), GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI)), or by manual alignment and visual inspection.

[0112] A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the FASTA algorithm (Pearson and Lipman, Proc Natl Acad Sci USA, 85:2444, 1988, and Pearson, Methods Enzymol, 266:227-258, 1996). To calculate percent identity, the preferred parameters used in FASTA alignment of DNA sequences are optimized: BL50 matrix 15:-5, k-tuple=2, joining penalty=40, optimization=28, gap penalty-12, gap length penalty=-2, and width=16.

[0113] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms (Altschul et al., Nuc Acids Res, 25:3389-3402, 1977 and Altschul et al., J Mol Biol, 215:403-410, 1990, respectively). Using the parameters described herein, BLAST and BLAST 2.0 are used to determine percent sequence identity for the nucleic acids and proteins of the present disclosure. Software for performing BLAST analyses is publicly available via the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short-length words W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in database sequences. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds to initiate searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score decreases by an amount X from its maximum performance value, when the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proc Natl Acad Sci USA, 89:10915, 1989) alignment (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0114] BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul, Proc Natl Acad Sci USA, 90:5873-5787, 1993). One measure of similarity provided by BLAST algorithm is the minimum total probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum total probability when comparing the test nucleic acid with the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0115] Another example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from a group of related sequences using progressive pairwise alignments to show the relationships and percent sequence identity. It also plots a tree or dendogram showing the cluster relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method (Feng and Doolittle, J Mol Evol, 35:351-360, 1987) that uses a method similar to that published (Higgins and Sharp, CABIOS 5:151-153, 1989). The program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple alignment procedure starts with a pairwise alignment of the two most similar sequences, generating a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is run by indicating the specific sequence and its amino acid or nucleotide coordinates for the region of sequence comparison, as well as indicating the program parameters. PILEUP is used to compare the reference sequence with other test sequences to determine the sequence identity percent relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, for example, version 7.0 (Devereaux et al., Nuc Acids Res, 12:387-395, 1984).

[0116] Another preferred example of an algorithm suitable for multiple DNA and amino acid sequence alignment is the CLUSTALW program (Thompson et al., Nucl Acids. Res, 22:4673-4680, 1994). CLUSTALW performs multiple pairwise comparisons between sequences and organizes them into multiple alignments based on homology. Gap opening and gap extension penalties were 10 and 0.05, respectively. For amino acid alignments, the BLOSUM algorithm can be used as the protein weighting matrix (Henikoff and Henikoff, Proc Natl Acad Sci USA, 89:10915-10919, 1992). Promoter

[0117] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and operably linked to the nucleic acid encoding the antibody. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the antibody. As mentioned above, the promoter can be inserted into a host cell chromosome in operable combination with a translation unit (e.g., a natural translation unit, such as one encoding a chaperone protein of the present disclosure) to generate a promoter:translation unit combination that is non-native to the host cell or host cell chromosome.

[0118] In some embodiments, the promoter of the present disclosure is an inducible promoter. The activity of an inducible promoter increases or decreases in response to a signal. For example, an inducible promoter may promote transcription in response to the presence of a signal, such as IPTG. An inducible promoter may promote transcription in response to the absence of a signal, such as phosphate. In any of these scenarios, the amount of transcription may or may not be proportional to the amount or lack of signal. Numerous examples of inducible promoters suitable for prokaryotic host cells are known in the art. These include lac, tac, trc, trp, pho, recA, tetA, nar, phage P L , cspA, T7, and P BAD In some embodiments, multiple copies of an inducible promoter are used to coordinately drive the expression of separate translation units encoding chaperone proteins such as DsbC and FkpA.

[0119] In some embodiments, the inducible promoter is an IPTG-inducible promoter. An IPTG-inducible promoter can refer to any polynucleotide sequence that promotes transcription in a manner that responds to isopropyl β-D-1-thiogalactopyranoside (IPTG) or any other lactose derivative (e.g., allolactose) that can promote transcription from the lac operon. Many examples of IPTG-inducible promoters are known in the art, including but not limited to tac (e.g., tacI, tacII, etc.) promoter, lac promoter, and their derivatives (e.g., lacUV5, taclac, etc.).

[0120] In some embodiments, the inducible promoter is a pho promoter that drives the transcription of the translation unit when phosphate in the culture medium is depleted. The pho promoter can refer to any polynucleotide sequence that promotes transcription in a manner that responds to extracellular phosphate (e.g., inorganic phosphate). For example, the phosphate (Pho) regulon in E. coli contains protein components that sense extracellular phosphate and regulate the expression of a number of downstream genes through the Pho promoter in response to phosphate levels (see Hsieh YJ and Wanner BL 2010 Curr.Opin.Microbiol.13(2):198 for a more detailed description). When bacteria are grown in a culture medium, it is known that the expression of this Pho regulon is repressed when phosphate (e.g., inorganic phosphate, Pi) is available in the medium and induced when phosphate is depleted. One non-limiting example of a pho promoter used in the methods described herein is the E. coli phoA promoter. This promoter is widely known and used in the art to regulate recombinant protein expression in prokaryotic host cells in a manner dependent on the phosphate concentration in the cell culture medium (for a more detailed description see Lubke C et al. 1995 Enzyme Microb. Technol. 17(10):923).

[0121] In some embodiments, the promoter of the present disclosure is a constitutive promoter. The activity of a constitutive promoter is believed to maintain a constant level of gene expression regardless of variations in the conditions under which the host cell is grown (e.g., nutrient conditions, cell density, etc.). For example, the activity of a constitutive promoter may depend on the availability of RNA polymerase rather than the activity or expression of one or more transcription factors. In some embodiments, the promoter is a synthetic promoter or a non-naturally occurring promoter. Exemplary constitutive promoters suitable for a range of prokaryotic host cells are described, for example, in Jensen PR, Hammer K. Appl Environ Microbiol 1998, 64:82-87. In some embodiments, the constitutive promoter is the CP25 promoter.

[0122] As described herein, a host cell chromosome of the present disclosure can include multiple non-natural combinations of promoters operably linked or combined with a translation unit encoding a chaperone protein of the present disclosure. Different types of promoters can be inserted into a host cell chromosome in any number or combination. For example, in some embodiments, a host cell chromosome of the present disclosure includes an inducible promoter (e.g., operably combined with a translation unit encoding a chaperone protein of the present disclosure) and a constitutive promoter (e.g., operably combined with a different translation unit encoding a chaperone protein of the present disclosure). For example, in some embodiments, a host cell chromosome of the present disclosure includes an inducible promoter of the present disclosure operably combined with a translation unit encoding a chaperone protein of the present disclosure, and a constitutive promoter of the present disclosure operably linked with a different translation unit encoding a chaperone protein of the present disclosure, both of which promoter:translation unit combinations are non-native to the host cell or host cell chromosome.

[0123] In some embodiments, a host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding a chaperone protein of the present disclosure, and a CP25 promoter of the present disclosure operably linked to a different translation unit encoding a chaperone protein of the present disclosure. In some embodiments, a host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding a protein disulfide oxidoreductase of the present disclosure, and a CP25 promoter of the present disclosure operably linked to a translation unit encoding a peptidyl-prolyl isomerase of the present disclosure. In some embodiments, a host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding DsbC, and a CP25 promoter of the present disclosure operably linked to a translation unit encoding FkpA. In some embodiments, the host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding E. coli DsbC, and a CP25 promoter of the present disclosure operably linked to a translation unit encoding E. coli FkpA. In some embodiments, the host cell is E. coli, and the translation units encoding DsbC and FkpA are native. In some embodiments, the host cell further comprises one or more extrachromosomal polynucleotides comprising two or more translation units encoding two or more polypeptide chains of a two-chain polypeptide of the present disclosure.

[0124] In some embodiments, a host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding a chaperone protein of the present disclosure, and a Pho promoter of the present disclosure operably linked to a different translation unit encoding a chaperone protein of the present disclosure. In some embodiments, a host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding a protein disulfide oxidoreductase of the present disclosure, and a Pho promoter of the present disclosure operably linked to a translation unit encoding a peptidyl-prolyl isomerase of the present disclosure. In some embodiments, a host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding DsbC, and a Pho promoter of the present disclosure operably linked to a translation unit encoding FkpA. In some embodiments, a host cell chromosome of the present disclosure comprises a Pho promoter of the present disclosure operably linked to a translation unit encoding E. coli DsbC, and a Pho promoter of the present disclosure operably linked to a translation unit encoding E. coli FkpA. In some embodiments, the host cell is E. coli and the translation units encoding DsbC and FkpA are native. In some embodiments, the host cell further comprises one or more extrachromosomal polynucleotides comprising two or more translation units encoding two or more polypeptide chains of the two-chain polypeptide of the present disclosure.

[0125] In some embodiments, a host cell chromosome of the present disclosure comprises a tac promoter of the present disclosure operably linked to a translation unit encoding a chaperone protein of the present disclosure, a tac promoter of the present disclosure operably linked to a second translation unit encoding a chaperone protein of the present disclosure, and a CP25 promoter of the present disclosure operably linked to a third translation unit encoding a chaperone protein of the present disclosure. In some embodiments, a host cell chromosome of the present disclosure comprises a tac promoter of the present disclosure operably linked to a translation unit encoding a protein disulfide oxidoreductase of the present disclosure, a tac promoter of the present disclosure operably linked to a second translation unit encoding a protein disulfide oxidoreductase of the present disclosure, and a CP25 promoter of the present disclosure operably linked to a third translation unit encoding a peptidyl-prolyl isomerase of the present disclosure. In some embodiments, the host cell chromosome of the present disclosure comprises a tac promoter of the present disclosure operably linked to a translation unit encoding DsbC, a tac promoter of the present disclosure operably linked to a translation unit encoding DsbA, and a CP25 promoter of the present disclosure operably linked to a translation unit encoding FkpA. In some embodiments, the host cell chromosome of the present disclosure comprises a tac promoter of the present disclosure operably linked to a translation unit encoding E. coli DsbC, a tac promoter of the present disclosure operably linked to a translation unit encoding E. coli DsbA, and a CP25 promoter of the present disclosure operably linked to a translation unit encoding E. coli FkpA. In some embodiments, the host cell is E. coli, and the translation units encoding DsbC, DsbA, and FkpA are native. In some embodiments, the host cell further comprises one or more extrachromosomal polynucleotides comprising two or more translation units encoding two or more polypeptide chains of the two-chain polypeptide of the present disclosure. Extrachromosomal polynucleotides and expression vectors

[0126] In some embodiments, the host cell comprises (1) a first polynucleotide comprising a first translation unit encoding a first chain of a two-chain polypeptide of the present disclosure, and (2) a second polynucleotide comprising a second translation unit encoding a second chain of the two-chain polypeptide. In some embodiments, the first and second polynucleotides are part of one or more extrachromosomal polynucleotides. In some embodiments, the first and second polynucleotides are part of the same extrachromosomal polynucleotide. In some embodiments, the extrachromosomal polynucleotide(s) further comprises a third translation unit encoding a third chain of the two-chain polypeptide. In some embodiments, the extrachromosomal polynucleotide(s) comprises one or more expression vectors or plasmids.

[0127] In some embodiments, the first translation unit encoding the first chain of the two-chain polypeptide and the second translation unit encoding the second chain of the two-chain polypeptide are part of a single extrachromosomal polynucleotide (e.g., a plasmid or other expression vector). In some embodiments, the first translation unit encoding the first chain of the two-chain polypeptide and the second translation unit encoding the second chain of the two-chain polypeptide are expressed from separate extrachromosomal polynucleotides (e.g., a plasmid or other expression vector).

[0128] In some embodiments, the extrachromosomal polynucleotide(s) further comprises a selectable marker (e.g., a translation unit encoding a selectable marker protein). A selectable marker may refer to any polynucleotide that encodes a protein that promotes survival of a host cell when the cell is exposed to any condition used for selection, i.e., selectively increasing the abundance of a cell(s) carrying the selectable marker compared to the abundance of a cell(s) lacking the selectable marker. Typical selectable markers encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply essential nutrients that cannot be obtained from complex media (e.g., a gene encoding D-alanine racemase for Bacilli). Numerous selectable markers and corresponding selection agents with a single antibiotic are known in the art. For example, without limitation, many selectable markers and corresponding antibiotics are described and cited in Jang CW and Magnuson T 2013 PLoS ONE 8(2):e57075. In some embodiments, a selectable marker can refer to a gene (e.g., a gene expressed from a plasmid) that complements a gene deletion present in the genome of a host cell. In these examples, when cells are selected (i.e., grown under conditions that require the activity of the gene deleted from the host genome), the copy of the gene provided by the plasmid complements the defect in the host genome, thereby selecting the cell(s) that carry the exogenous complementing gene. Such genes can include auxotrophic markers or genes required to produce a particular nutrient that is lacking in the cell culture medium, examples of which are further described herein. Some exemplary selectable markers and antibiotics are further described herein.

[0129] In some embodiments, the selectable marker promotes resistance to a selection agent and the culture medium includes the selection agent to cause the host cells to retain the polynucleotide. In some embodiments, the selection agent is an antibiotic. One example of a selection scheme utilizes a drug that stops the growth of the host cells. Cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and therefore survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0130] Another selection scheme uses prokaryotic host cells with chromosomal deletions that remove genes whose gene products are essential for growth in a particular culture medium. In these examples, cells that are successfully transformed with a heterologous gene that complements the chromosomal deletion of the host cell will survive when grown in the particular culture medium. Examples of genes useful in this scheme can include auxotrophic marker genes or other genes that are required to produce essential nutrients when the host cell is grown in the particular culture medium.

[0131] In some embodiments, the extrachromosomal polynucleotide(s) further comprises a suitable origin of replication for replicating the extrachromosomal expression vector in prokaryotic host cells.Generally, in cloning vectors, this sequence allows the vector to replicate independently of host chromosomal DNA, and includes origin of replication or autonomously replicating sequence.Such sequences are well known for various prokaryotic host cells.For example, the origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria.

[0132] Expression vectors used in prokaryotic host cells may also contain sequences required for the termination of transcription and stabilization of mRNA. In prokaryotic cells, termination factors may include Rho-dependent or Rho-independent termination factors. An example of a termination factor useful in prokaryotic host cells includes, but is not limited to, λt0 termination factor (Scholtissek and Grosse, Nucleic Acids Res.15:3185, 1987).

[0133] The antibody of the present disclosure can be recombinantly produced not only directly but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected is preferably one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. In the case of a prokaryotic host cell that does not recognize and process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from the group of, for example, alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. Recombinant Polypeptides

[0134] Certain aspects of the present disclosure relate to methods for producing two-chain polypeptides. Advantageously, the methods described herein can be useful for facilitating the expression, folding, and assembly of many different types of proteins, particularly those with disulfide bonds, such as two-chain proteins as described above. Certain two-chain proteins are described below, but the methods described herein are not limited to these specific embodiments. As used herein, two-chain proteins can include proteins that contain more than two separate polypeptide chains. Many embodiments described herein relate to two-chain proteins that include two polypeptide chains, but two-chain proteins having more than two polypeptide chains (e.g., three or more polypeptides) are contemplated and may be produced by the methods described herein. As described above, two-chain proteins consisting of a single polypeptide chain that associates with what would otherwise be two separate polypeptide chains (e.g., single-chain antibodies, single-chain variable fragments, etc.) are also contemplated and may be produced by the methods described herein.

[0135] In some embodiments, the two chains of the two-chain polypeptide of the present disclosure are linked together by at least one disulfide bond. Disulfide bond may refer to any covalent bond linking two thiol groups. Disulfide bonds in polypeptides typically form between thiol groups of cysteine ​​residues. It is known in the art that polypeptide disulfide bonds are important for the folding and assembly of many polypeptides, such as the two-chain proteins of the present disclosure. Polypeptide disulfide bonds may include disulfide bonds between cysteine ​​residues in a single polypeptide chain (i.e., intramolecular or intrachain disulfide bonds). Polypeptide disulfide bonds may also include disulfide bonds between cysteine ​​residues found on separate polypeptide chains (i.e., intermolecular or interchain disulfide bonds). Thus, in some embodiments, the two chains of the two-chain polypeptide are linked together by at least one disulfide bond.

[0136] It is known in the art that disulfide bonds are important for the folding and assembly of antibodies and antibody fragments. Different antibody isotopes, and different subclasses within an isotope, are known to possess different patterns of disulfide bonds. For example, an IgG antibody may contain 12 intrachain disulfide bonds, one interchain disulfide bond between each light chain and its corresponding heavy chain, and 2-11 interchain disulfide bonds between heavy chains, depending on the particular IgG subclass (for a more detailed description, see Liu H and May K 2012 MAbs.4(1):17). IgM (see, e.g., Wiersma EJ and Shulman MJ 1995 J. Immunol. 154(10):5265), IgE (see, e.g., Helm BA et al. 1991 Eur. J. Immunol. 21(6):1543), IgA (see, e.g., Chintalacharuvu KR et al. 2002 J. Immunol. 169(9):5072), and IgD (see, e.g., Shin SU et al. 1992 Hum. Antibodies Hybridomas 3(2):65) are also known to form disulfide bonds during folding and assembly.

[0137] In some embodiments, the two-chain polypeptide of the present disclosure is heterologous to the host cell. As used herein, a heterologous polypeptide when used in relation to a host cell can refer to any polypeptide that is not endogenously expressed in the host cell (i.e., when the host cell is isolated from nature). A heterologous polypeptide can also refer to a polypeptide that can be endogenously expressed by the host cell, but is expressed under different regulation than when the host cell is isolated from nature. Examples of different regulation can include, but are not limited to, different amounts of expression, expression in response to different stimuli, or any other altered amount of expression, such as by using a heterologous promoter, such as an inducible promoter.

[0138] In some embodiments, the two-chain polypeptide of the present disclosure is a monomer of a heterodimer. As used herein, a heterodimer may refer to any polypeptide complex that contains two separate polypeptides or polypeptide complexes that are operably linked. A non-limiting example of a heterodimer is a bispecific or bivalent antibody (i.e., a light chain-heavy chain pair that is operably linked) that is composed of two separate antibody monomers. In this example, folding and assembly of a first heavy chain-light chain pair that recognizes a first antigen produces a first antibody monomer. Folding and assembly of a second heavy chain-light chain pair that recognizes a second antigen produces a second antibody monomer. These monomers can be assembled by any means known in the art (described in more detail below with respect to bispecific antibodies) to form a heterodimer. For further details regarding examples of heterodimeric antibody formation, see Ridgway JBB et al. 1996 Protein Eng.9(7):617.

[0139] In some embodiments, the two-chain polypeptide of the present disclosure is a monovalent antibody, in which the first and second chains correspond to immunoglobulin heavy and light chains.As used herein, a monovalent antibody may refer to any polypeptide complex made of an antibody heavy chain and an antibody light chain that are operably linked together to form a heavy-light chain pair, in which the heavy-light chain pair is not operably linked to a second heavy-light chain pair.The term "half antibody (hAb)" may be used interchangeably herein.

[0140] In some embodiments, the monovalent antibody of the present disclosure can specifically bind to an antigen. As used herein, the terms "bind", "specifically bind", or "specific for" refer to a measurable and reproducible interaction, such as binding, between a target (i.e., an antibody that determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules). For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or for longer than it binds to other targets. In one embodiment, the extent to which an antibody binds to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, or 0.1 nM or less. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0141] In some embodiments, the two-chain polypeptide of the present disclosure is a secreted protein. As used herein, a secreted protein may refer to any protein that is secreted by a host cell into the host cell periplasm or extracellular environment. A secreted protein may be a protein that is endogenously secreted by a host cell, or a secreted protein may be a protein that is not endogenously secreted by a host cell but is modified in a manner to facilitate its secretion. For example, the presence of a signal sequence, typically found at the N-terminus of a polypeptide, may direct the polypeptide into the secretory pathway for secretion. A number of signal sequences are known in the art and may be useful to facilitate the secretion of secreted proteins or to enable the secretion of proteins that are not naturally secreted by a host cell. See, for example, Picken et al., Infect.Immun.42:269-275 (1983), Simmons and Yansura, Nature Biotechnology 14:629-634 (1996), and Humphreys DP et al. 2000 Protein Expr.Purif.20(2):252. One non-limiting example of a signal sequence is the heat-stable enterotoxin II (STII) signal sequence.

[0142] In some embodiments, the secreted protein of the present disclosure is recovered from the periplasm of host cell. Periplasm is known in the art to refer to the space between the inner or cytoplasmic membrane and the outer membrane of gram-negative bacterial cells. Without wishing to be bound by theory, it is believed that the periplasm is an oxidizing environment suitable for the formation of disulfide bonds. Therefore, it may be advantageous to localize a polypeptide (e.g., a two-chain protein of the present disclosure) that has disulfide bonds as part of its properly folded and assembled structure in the periplasm (for more detailed explanation, see Schlapschy M et al. 2006 Protein Eng.Des.Sel.19(8):385).

[0143] Numerous methods for recovering periplasmic proteins are known in the art. One non-limiting example of large-scale purification of periplasmic proteins is described in European Patent EP1356052 B1 (see, for example, Example 4). Periplasmic proteins can be recovered by extracting periplasmic fraction from spheroplast preparation (see, for example, Schlapschy M et al. 2006 Protein Eng.Des.Sel.19(8):385). Once periplasmic extract is produced, periplasmic proteins can be purified by any standard protein purification technique known in the art, such as affinity purification, chromatography, etc. host cell

[0144] Certain embodiments of the present disclosure relate to prokaryotic host cells.Prokaryotic organisms suitable for cloning or expressing DNA in the vectors herein include eubacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, and Bacilli, such as B.subtilis and B.licheniformis (e.g. B.licheniformis41P, disclosed in DD266,710, published April 12, 1989), Pseudomonas, such as P.aeruginosa and P.aeruginosa. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative rather than limiting.

[0145] In some embodiments, the prokaryotic host cell is a gram-negative bacterium. Gram-negative bacteria refers to any bacterium that contains an outer membrane surrounding a peptidoglycan layer that is detected by gram staining. Many gram-negative bacterial host cells are known in the art. For example, gram-negative bacteria are known to include Proteobacteria, such as, but not limited to, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Zetaproteobacteria, Epsilonproteobacteria, Deltaproteobacteria, and Acidobacteria, Cyanobacteria, and Spirochetes. Well-known gram-negative bacteria may include species of genera such as Escherichia, Salmonella, Shigella, Pseudomonas, Heliobacter, Legionella, Neisseria, and Klebsiella.

[0146] In some embodiments, the Gram-negative bacteria of the present disclosure is Escherichia coli. As used herein, Escherichia coli may refer to any strain or isolate of bacteria belonging to the species Escherichia coli. Escherichia coli may include naturally occurring strains or strains that have been genetically modified, such as by mutation or transformation with a plasmid as described herein.

[0147] In some embodiments, the E. coli of the present disclosure is of a strain lacking endogenous protease activity. Without wishing to be bound by theory, it is believed that a strain lacking endogenous protease activity may allow for improved production of recombinant proteins, such as the periplasmic proteins of the present disclosure, since some endogenous proteases have activity against recombinantly expressed substrates (see Baneyx F and Georgiu G 1990 J. Bacteriol. 172(1):491 for one such example). A strain lacking endogenous protease activity may include a strain in which a gene encoding an endogenous protease is mutated, deleted, or otherwise inactivated. Examples of such genes may include, but are not limited to, degP, prc, and ompT. Methods for introducing mutations into a wide variety of prokaryotic host cells (e.g., to engineer a strain lacking endogenous protease activity) are well known in the art. For example, Snyder L et al. 2013 Molecular Genetics of Bacteria 第4版 See ASM Press. In certain embodiments, the E. coli of the present disclosure is of a strain carrying a degpS210A mutation.

[0148] In some embodiments, the E. coli of the present disclosure is of a strain having enhanced LacI production or activity. An exemplary LacI protein sequence is represented by UniProt KB Accession No. P03023. In certain embodiments, the E. coli is of a strain having enhanced LacI production or activity. Q A strain carrying a mutation (see, for example, Muller-Hill, B. et al. (1968) Proc. Natl. Acad. Sci. 59:1259-1264) that is known to result in overproduction of the LacI repressor of the lac operon.

[0149] In certain embodiments, the E. coli of the present disclosure is ΔfhuA ΔphoA ilvG2096 (IlvG+, Valr) Δprc spr43H1 ΔmanA lacI Q This is from the ΔompT ΔmenE742 degPS210A strain. Antibodies and antibody fragments

[0150] The two-chain protein described herein can be prepared by any suitable technique known in the art.One exemplary class of two-chain protein is antibody.As described below, antibody is prepared using the technique available in the art for producing antibody, the exemplary method of which is described in more detail in the following section.Those skilled in the art will recognize that many of the methods described below can be applied to two-chain proteins other than antibodies.

[0151] The antibody is directed against an antigen of interest (for example, and without limitation, PD-L1 (such as human PD-L1), HER2, or CD3 (such as human CD3), IL13, IL4, VEGFC, VEGFA, and VEGF). Preferably, the antigen is a biologically important polypeptide, such that administration of the antibody to a mammal suffering from a disorder can provide a therapeutic benefit to the mammal.

[0152] In some embodiments, the antibody of the present disclosure is directed against interleukin-13 (herein referred to as IL-13 or IL13). For example, the antibody can be a monovalent antibody or "half antibody" against IL13, a complete antibody comprising two monovalent heavy-light chain pairs against IL13 (e.g., two identical monovalent heavy-light chain pairs, two monovalent heavy-light chain pairs each comprising different HVRs or CDRs that recognize the same epitope of IL13, or two monovalent heavy-light chain pairs each comprising different HVRs or CDRs that recognize non-overlapping or partially overlapping epitopes of IL13), or a bispecific antibody comprising a heavy-light chain pair against IL13 and a heavy-light chain pair against a different antigen.

[0153] Examples of IL13 polypeptides are known in the art. In some embodiments, the IL13 polypeptide is a human IL13 polypeptide. In some embodiments, the IL13 polypeptide is a precursor form of IL13. A non-limiting example of a precursor form of an IL13 polypeptide is the human IL13 precursor as represented by Swiss-Prot Accession No. P35225.2. In some embodiments, the IL13 polypeptide has the sequence: Contains MALLLTTVIA LTCLGGFASP GPVPPSTALRELIEEL VNITQNQKAP LCNGSMVWSI NLTAGMYCAA LESLINVSGC SAIEKTQRML SGFCPHKVSA GQFSSLHVRD TKIEVAQFVK DLLLHLKKLF REGRFN (sequence number 1).

[0154] In other embodiments, the IL13 is a mature form of IL13 (e.g., lacking a signal sequence). In some embodiments, the IL13 polypeptide has the sequence: Contains SPGPVPPSTALR ELIEELVNIT QNQKAPLCNG SMVWSINLTA GMYCAALESL INVSGCSAIE KTQRMLSGFC PHKVSAGQFS SLHVRDTKIE VAQFVKDLLL HLKKLFREGR FN (SEQ ID NO: 2).

[0155] In some embodiments, provided herein is an anti-IL13 antibody comprising a heavy chain variable domain and a light chain variable domain, (a) (a) the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to AYSVN (SEQ ID NO:5), MIWGDGKIVYNSALKS (SEQ ID NO:6), and DGYYPYAMDN (SEQ ID NO:7), respectively; and / or (b) (b) the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity to RASKSVDSYGNSFMH (SEQ ID NO: 8), LASNLES (SEQ ID NO: 9), and QQNNEDPRT (SEQ ID NO: 10), respectively. In certain embodiments, sequence identity is at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% compared to a reference sequence.

[0156] In some embodiments, the anti-IL13 antibody comprises a heavy chain variable domain sequence of SEQ ID NO: 3 and / or a light chain variable domain sequence of SEQ ID NO: 4. In yet further embodiments, an isolated anti-IL13 antibody is provided comprising a heavy chain sequence and / or a light chain sequence, (a) the heavy chain variable domain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the following reference heavy chain sequence: (b) EVTLRESGPALVKPTQTLTLTCTVSGFSLSAYSVNWIRQPPGKALEWLAMIWGDGKIVYNSALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCAGDGYYPYAMDNWGQGSLVTVSS (SEQ ID NO: 3) (c)(b) the light chain variable domain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the following reference light chain sequence: (d) DIVLTQSPDSLSVSLGERATINCRASKSVDSYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPRTFGGGTKVEIKR (SEQ ID NO: 4).

[0157] In some embodiments, the antibody of the present disclosure is directed against interleukin-33 (herein referred to as IL-33 or IL33). For example, the antibody can be a monovalent antibody or "half antibody" against IL33, a complete antibody comprising two monovalent heavy-light chain pairs against IL33 (e.g., two identical monovalent heavy-light chain pairs, two monovalent heavy-light chain pairs each comprising different HVRs or CDRs that recognize the same epitope of IL33, or two monovalent heavy-light chain pairs each comprising different HVRs or CDRs that recognize non-overlapping or partially overlapping epitopes of IL33), or a bispecific antibody comprising a heavy-light chain pair against IL33 and a heavy-light chain pair against a different antigen.

[0158] Various isoforms of IL33 are known.For example, human IL33 isoforms include, but are not limited to, those represented by NCBI RefSeq accession numbers AOZ26495, ADR77828, AAH47085, NP_254274, NP_001186569, NP_001300977, NP_001340731, NP_001300975, NP_001300976 and XP_06870774.

[0159] In one aspect, a multispecific antibody is provided, comprising a first monovalent or half antibody and a second monovalent or half antibody, wherein the first half antibody comprises a first VH / VL unit that binds IL-33 and the second half antibody comprises a second VH / VL unit that binds IL-13.

[0160] HVR and variable domain sequences of exemplary anti-IL33 antibodies (including anti-IL33 / anti-IL13 bispecific antibodies) can be found, for example, in WO2016077381.

[0161] In some embodiments, the CH3 and / or CH2 domains of the antibodies of the disclosure are derived from an IgG (e.g., an IgG1 subtype, an IgG2 subtype, an IgG2A subtype, an IgG2B subtype, an IgG3 subtype, or an IgG4 subtype). In some embodiments, the CH3 and / or CH2 domains of the antibodies of the disclosure may comprise one or more knob- or hole-forming mutations (such as, for example, those listed in Table 2 below).

[0162] In certain embodiments, the CH3 and / or CH2 domains of the antibodies of the present disclosure are derived from the IgG4 subtype. In some embodiments, the IgG4 CH3 and / or CH2 domains of the antibodies of the present disclosure may contain one or more additional mutations, including but not limited to the S228P mutation (EU numbering).

[0163] In some embodiments, the antibody of the present disclosure is an antibody fragment, as discussed in more detail below. As used herein, an antibody fragment refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen that the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabody, linear antibody, single-chain antibody molecule (e.g., scFv), and multispecific antibody formed from antibody fragments.

[0164] In some embodiments, the antibody of the present disclosure is a one-arm antibody. In some embodiments, the one-arm antibody comprises an immunoglobulin heavy chain, an immunoglobulin light chain, and an immunoglobulin Fc fragment, and the three chains fold and assemble to form a biologically active monovalent antibody. For a description of an exemplary and non-limiting one-arm antibody Onartuzumab (e.g., MetMAb), see, for example, Merchant, M. et al. (2013) Proc. Natl. Acad. Sci. 110:E 2987-E2996. Antibody properties

[0165] In certain embodiments, the antibodies provided herein have a concentration of 1 μM or less, 150 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of 1 M.

[0166] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with a Fab version of the antibody of interest and its antigen, as illustrated by the following assay: The solution binding affinity of the Fab for the antigen is determined by the minimum concentration of ( 125 I) Fab is equilibrated with labeled antigen, and then bound antigen is measured by capturing it on a plate coated with anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol., 293:865-881 (1999)). To establish the conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / mL of capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23° C.). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I] antigen is mixed with serial dilutions of the Fab of interest. The Fab of interest is then incubated overnight, although incubation may continue for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to the capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μL / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes on a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that results in 20% or less of maximum binding is selected for use in the competitive binding assay.

[0167] According to another embodiment, Kd is measured using a surface plasmon resonance assay using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25° C. with an immobilized antigen CM5 chip at about 10 response units (RU). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM-500 nM) in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) are injected at a flow rate of approximately 25 μL / min at 25° C. Association rates (kon) and dissociation rates (koff) are calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen et al., J. Mol. Biol., 293:865-881 (1999).If the association rate by the surface plasmon resonance assay described above exceeds 106 M-1 s-1, the association rate can be determined using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations as measured in a spectrometer such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer with stirred cuvette (ThermoSpectronic). (i) Antigen preparation

[0168] In some cases, soluble antigens or fragments thereof conjugated to other molecules can be used as immunogens to generate antibodies. For transmembrane molecules such as receptors, fragments of these (e.g., extracellular domains of receptors) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells can be from natural sources (e.g., cancer cell lines) or can be cells transformed by recombinant techniques to express transmembrane molecules. Other antigens and forms thereof useful for preparing antibodies will be apparent to those skilled in the art. (ii) Certain antibody-based methods

[0169] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR, where R and R 1are different alkyl groups) to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

[0170] Animals are immunized against the antigen, immunogenic conjugate or derivative, for example, by combining the protein or conjugate (100 μg or 5 μg for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. The animals are boosted until the titer plateaus. Preferably, the animals are boosted with a conjugate of the same antigen, but conjugated to a different protein and / or with a different cross-linking reagent. Conjugates can also be made in recombinant cell culture as protein fusions. Agglutinating agents such as alum are also suitably used to enhance the immune response.

[0171] The monoclonal antibodies of the present disclosure can be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975) and further described, for example, in Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988), for human-human hybridomas, Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), and Ni, Xiandai Mianyixue 26(4):265-268 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826, for the production of monoclonal human native IgM antibodies from hybridoma cell lines. Human hybridoma technology (trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005), and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Once the desired monoclonal antibodies are isolated from the hybridomas, the polynucleotides encoding them can be subcloned into a prokaryotic expression vector and expressed in a prokaryotic host cell to produce the antibody by any of the methods described herein. (iii) Library-derived antibodies

[0172] Antibodies of the present disclosure can be isolated by screening combinatorial libraries for antibodies with the desired activity(ies). A variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics, such as, for example, the method described in Example 3. Additional methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology, 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and described in, for example, McCafferty et al., Nature, 348:552-554; Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology, 248:161-175 (Lo, eds., Human Press, Totowa, NJ, 2001). Press, Totowa, NJ, 2003), Sidhu et al., J. Mol. Biol., 338(2):299-310(2004), Lee et al., J. Mol. Biol., 340(5):1073-1093(2004), Fellouse, Proc. Natl. Acad. Sci. USA, 101(34):12467-12472(2004), and Lee et al., J. Immunol. Methods, 284(1-2):119-132(2004).

[0173] In certain phage display methods, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages as described by Winter et al., Ann. Rev. Immunol. 12:433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) without immunization to provide a single source of antibodies against a wide range of non-self and even self antigens, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol. 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0174] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments. (iv) Chimeric, Humanized, and Human Antibodies

[0175] In certain embodiments, the antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc.Natl.Acad.Sci.USA, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. The chimeric antibody comprises an antigen-binding fragment thereof.

[0176] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, in which the HVR, e.g., CDR (or a portion thereof) is derived from a non-human antibody, and the FR (or a portion thereof) is derived from a human antibody sequence. A humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0177] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and described, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (description of SDR (a-CDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) ( "Resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (description of "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (description of a "guided selection" approach to FR shuffling).

[0178] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol., 151:2296 (1993)), framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992) and Presta et al., J. Immunol., 151:2623 (1999)). 3)), human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., 13:1619-1633 (2008)), and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem., 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem., 271:22611-22618 (1996)).

[0179] In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr.Opin.Pharmacol.5:368-74 (2001) and Lonberg, Curr.Opin.Immunol.20:450-459 (2008). Human antibodies can be produced, for example and without limitation, by expression from a prokaryotic expression vector in a prokaryotic host cell by any of the methods described herein.

[0180] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below. (v)Antibody fragment

[0181] Antibody fragments can be produced by conventional means such as enzyme digestion or recombinant techniques. In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may provide improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al. (2003) Nat.Med.9:129-134.

[0182] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained via proteolysis of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, allowing for facile large-scale production of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and increased in vivo half-life are described in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO93 / 16185, U.S. Pat. Nos. 5,571,894, and 5,587,458. Fv and scFv are the only species with intact binding sites that lack constant regions, and therefore they may be suitable for reducing non-specific binding during in vivo use. scFv fusion proteins can be constructed to provide fusion of effector proteins at either the amino or carboxy terminus of the scFv. See Antibody Engineering, edited by Borrebaeck, supra. An antibody fragment may also be a "linear antibody," for example, as described in U.S. Patent No. 5,641,870. Such linear antibodies may be monospecific or bispecific. (vi) Multispecific antibodies

[0183] A multispecific antibody has binding specificities for at least two different epitopes, which are usually derived from different antigens. Such molecules usually bind only two different epitopes (i.e., bispecific antibodies, BsAbs), but antibodies with additional specificities, such as trispecific antibodies, are encompassed by this term as used herein. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0184] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of this correct molecule, usually performed by an affinity chromatography step, is somewhat cumbersome and the product yield is low. Similar procedures are described in WO93 / 08829, and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0185] One approach known in the art for making bispecific antibodies is the "knob-into-hole" or "projection-into-cavity" approach (see, e.g., U.S. Pat. No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each contain an interface. An interface of one immunoglobulin polypeptide interacts with a corresponding interface of the other immunoglobulin polypeptide, thereby allowing the association of the two immunoglobulin polypeptides. These interfaces can be engineered such that a "knob" or "projection" (these terms may be used interchangeably herein) located at the interface of one immunoglobulin polypeptide corresponds to a "hole" or "cavity" (these terms may be used interchangeably herein) located at the interface of the other immunoglobulin polypeptide. In some embodiments, the holes are of the same or similar size as the knobs and are suitably positioned such that a knob of one interface is positionable within a corresponding hole of the other interface when the two interfaces interact. Without wishing to be bound by theory, it is believed that this stabilizes the heteromultimer and makes it more favorable for formation of heteromultimers than other species, e.g., homomultimers. In some embodiments, this approach can be used to promote heteromultimerization of two different immunoglobulin polypeptides to generate bispecific antibodies comprising two immunoglobulin polypeptides with binding specificities for different epitopes.

[0186] In some embodiments, knobs can be constructed by replacing small amino acid side chains with larger side chains. In some embodiments, holes can be constructed by replacing large amino acid side chains with smaller side chains. The knobs or holes can be present in the original interface or can be synthetically introduced. For example, knobs or holes can be synthetically introduced by modifying the nucleic acid sequence encoding the interface and replacing at least one "original" amino acid residue with at least one "import" amino acid residue. Methods for modifying nucleic acid sequences can include standard molecular biology techniques well known in the art. The side chain volumes of various amino acid residues are shown in the table below. In some embodiments, the original residues have small side chain volumes (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and the imported residues to form the knobs are naturally occurring amino acids and can include arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residue has a large side chain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan) and the imported residue to form the hole is a naturally occurring amino acid, which may include alanine, serine, threonine, and valine. TIFF0007620546000002.tif164170 a Molecular weights of amino acids are minus the molecular weight of water. Values ​​from Handbook of Chemistry and Physics, 43rd ed., Cleveland, Chemical Rubber Publishing Co., 1961. b Values ​​from AAZamyatnin, Prog. Biophys. Mol. Biol. 24:107-123, 1972. c Values ​​from C. Chothia, J. Mol. Biol. 105:1-14, 1975. Accessible surface area is defined in Figures 6-20 of this reference.

[0187] In some embodiments, the original residues for forming the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques for obtaining the three-dimensional structure known in the art may include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of an immunoglobulin constant domain. In these embodiments, the CH3 / CH3 interface of human IgG1 involves 16 residues on each domain located on four antiparallel β-strands. Without wishing to be bound by theory, the mutated residues are preferably located on the two central antiparallel β-strands to minimize the risk that the knob may be accommodated by the surrounding solvent rather than the compensatory hole in the partner CH3 domain. In some embodiments, the mutations that form the corresponding knob and hole in the two immunoglobulin polypeptides correspond to one or more pairs provided in the table below. TIFF0007620546000003.tif56170 Mutations are represented by the original residue, followed by its position using the Kabat numbering system, then the imported residue (all residues are shown in single letter amino acid code). Multiple mutations are separated by colons.

[0188] In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain that comprises one or more amino acid substitutions listed in Table 2 above. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide that comprises a CH3 domain that comprises one or more amino acid substitutions listed in the left column of Table 2, and a second immunoglobulin polypeptide that comprises a CH3 domain that comprises one or more corresponding amino acid substitutions listed in the right column of Table 2. As a non-limiting example of a knob-and-hole forming pair, in some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide that comprises a CH3 domain that comprises a T366W mutation, and a second immunoglobulin polypeptide that comprises a CH3 domain that comprises T366S, L368A, and Y407V mutations.

[0189] Each half antibody can have either a knob (protrusion) or a hole (cavity) engineered into the heavy chain, as described in U.S. Pat. No. 7,642,228. Briefly, CH3 knob mutants can be generated first. A library of CH3 hole mutants can then be created by randomizing residues 366, 368, and 407, which are adjacent to the knob on the partner CH3 domain. In certain embodiments, the knob mutation includes T366W, and the hole mutation includes T366S, L368A, and Y407V in an IgG1 or IgG4 backbone. Equivalent mutations in other immunoglobulin isotypes can be made by those skilled in the art. Furthermore, those skilled in the art will readily understand that it is preferred that the two half antibodies used in a bispecific antibody are of the same isotype.

[0190] Exemplary, non-limiting techniques for producing multispecific (eg, bispecific) antibodies are provided in Section III.

[0191] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al., J. Immunol. 147:60 (1991).

[0192] In some embodiments, the two-chain protein is part of a multispecific or bispecific antibody, which contains two or more monovalent antibodies of the present disclosure.

[0193] In some embodiments, the first antigen-binding domain of the bispecific antibody comprises one or more heavy chain constant domains, the one or more heavy chain constant domains being selected from a first CH1 (CH11) domain, a first CH2 (CH21) domain, a first CH3 (CH31) domain, and the second antigen-binding domain of the bispecific antibody comprises one or more heavy chain constant domains, the one or more heavy chain constant domains being selected from a second CH1 (CH12) domain, a second CH2 (CH22) domain, and a second CH3 (CH32) domain. In some embodiments, at least one of the one or more heavy chain constant domains of the first antigen-binding domain is paired with another heavy chain constant domain of the second antigen-binding domain. In some embodiments, the CH31 and CH32 domains each comprise a protrusion or cavity, and the protrusion or cavity in the CH31 domain is positionable in the cavity or protrusion in the CH32 domain, respectively. In some embodiments, the CH31 and CH32 domains meet at the interface between the protrusion and the cavity. An exemplary set of amino acid substitutions in the CH31 and CH32 domains is shown in Table 2 herein. In some embodiments, the CH21 and CH22 domains each comprise a protrusion or a cavity, and the protrusion or cavity in the CH21 domain can be positioned in the cavity or protrusion in the CH22 domain, respectively. In some embodiments, the CH21 and CH22 domains meet at the interface between the protrusion and the cavity. In some embodiments, the CH31 and / or CH32 domains of IgG contain one or more amino acid substitutions at residues selected from the group consisting of 347, 349, 350, 351, 366, 368, 370, 392, 394, 395, 398, 399, 405, 407, and 409 according to the amino acid numbering shown in Figure 5 of U.S. Pat. No. 8,216,805. In some embodiments, the protrusion comprises one or more introduced residues selected from the group consisting of arginine (R) residues, phenylalanine (F) residues, tyrosine (Y) residues, and tryptophan (W) residues.In some embodiments, the cavity comprises one or more introduced residues selected from the group consisting of an alanine (A) residue, a serine (S) residue, a threonine (T) residue, and a valine (V) residue. In some embodiments, the CH3 and / or CH2 domains are derived from an IgG (e.g., an IgG1 subtype, an IgG2 subtype, an IgG2A subtype, an IgG2B subtype, an IgG3 subtype, or an IgG4 subtype). In some embodiments, one CH3 domain of the bispecific antibody comprises the amino acid substitution T366Y and the other CH3 domain comprises the amino acid substitution Y407T. In some embodiments, one CH3 domain comprises the amino acid substitution T366W and the other CH3 domain comprises the amino acid substitution Y407A. In some embodiments, one CH3 domain comprises the amino acid substitution F405A and the other CH3 domain comprises the amino acid substitution T394W. In some embodiments, one CH3 domain comprises amino acid substitutions T366Y and F405A, and the other CH3 domain comprises amino acid substitutions T394W and Y407T. In some embodiments, one CH3 domain comprises amino acid substitutions T366W and F405W, and the other CH3 domain comprises amino acid substitutions T394S and Y407A. In some embodiments, one CH3 domain comprises amino acid substitutions F405W and Y407A, and the other CH3 domain comprises amino acid substitutions T366W and T394S. In some embodiments, one CH3 domain comprises amino acid substitution F405W, and the other CH3 domain comprises amino acid substitution T394S. Mutations are represented by the original residue, followed by the position using the Kabat numbering system, followed by the imported residue. See also numbering in Figure 5 of U.S. Pat. No. 8,216,805. (vii) single domain antibody

[0194] In some embodiments, the antibody of the present disclosure is a single domain antibody. A single domain antibody is a single polypeptide chain that includes all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass., see, e.g., U.S. Patent No. 6,248,516 B1). In one embodiment, a single domain antibody consists of all or a portion of the heavy chain variable domain of an antibody. (viii) antibody variants

[0195] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion, and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid alterations may be introduced into the amino acid sequence of the subject antibodies when that sequence is generated. (ix) Substitution, insertion, and deletion variants

[0196] In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. The sites of interest for substitutional mutagenesis include HVR and FR. Conservative substitutions are shown in Table 1 under the heading of "conservative substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions" and are as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product is screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF0007620546000004.tif175170

[0197] Amino acids can be grouped according to common side chain properties. a. Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile, b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln, c. Acidic: Asp, Glu, d. Basic: His, Lys, Arg, e. Residues that affect chain orientation: Gly, Pro, f. Aromatics: Trp, Tyr, Phe.

[0198] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[0199] One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have a modification (e.g., improvement) in certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have certain biological properties of the parent antibody substantially retained. An exemplary substitution variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0200] Modifications (e.g., substitutions) can be made to the HVRs, for example, to improve antibody affinity. Such modifications may be made in "hot spots" of the HVRs, i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves HVR-directed approaches, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0201] In certain embodiments, substitution, insertion, or deletion may occur within one or more HVRs, as long as such modification does not substantially reduce the ability of antibody to bind to antigen.For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs.Such modifications may be outside HVR "hot spots" or SDRs.In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unmodified or contains no more than one, two, or three amino acid substitutions.

[0202] A useful method for identifying residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of the antigen-antibody complex to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they have the desired properties.

[0203] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the N- or C-terminal fusion of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody. (x) Fc domain variant

[0204] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0205] In certain embodiments, the present disclosure contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications in which antibody half-life in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus potentially lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only Fc(RIII, whereas monocytes express F(RI, Fc(RII, and Fc(RIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest include those described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7063 (1986)). Acad.Sci.USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J.Exp.Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model, for example, as disclosed in Clynes et al., Proc.Nat'l Acad.Sci.USA, 95:652-656 (1998). A C1q binding assay can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)); FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0206] Antibodies with reduced effector function include antibodies with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0207] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0208] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at Fc region positions 298, 333, and / or 334 (EU numbering of residues). In an exemplary embodiment, the antibody comprises the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

[0209] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 164:4178-4184 (2000).

[0210] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding to FcRn. Such Fc variants include variants with substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution at Fc region residue 434 (U.S. Patent No. 7,371,826)). See also Duncan and Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351 for other examples of Fc region variants. (xi) Antibody derivatives

[0211] The antibodies of the present disclosure may be further modified to include additional non-proteinaceous moieties that are known in the art and readily available. In certain embodiments, the moieties suitable for derivatization of the antibody are water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in a given treatment under defined conditions, etc. III. Production Method

[0212] Provided herein is a method for producing a polypeptide comprising two chains (e.g., a two-chain polypeptide) in a prokaryotic host cell of the present disclosure.In some embodiments, the method includes culturing a host cell of the present disclosure in a culture medium under conditions suitable for the expression of the two chains of the polypeptide to express the two chains of the polypeptide, whereby, upon expression, the two chains fold and assemble to form a biologically active polypeptide in the host cell, and recovering the biologically active polypeptide from the host cell.

[0213] Any of the host cells of the present disclosure (e.g., as described in Section II) can find use in the methods of the present disclosure. For example, in some embodiments, the host cell comprises one or more extrachromosomal polynucleotides of the present disclosure and one or more translation units of the present disclosure (e.g., operably linked to a promoter and present on a host cell chromosome in a non-natural combination). In some embodiments, the host cell comprises a first polynucleotide (part of the extrachromosomal polynucleotide) comprising a first translation unit encoding a first chain of a polypeptide, a second polynucleotide (part of the extrachromosomal polynucleotide) comprising a second translation unit encoding a second chain of a polypeptide, and a third polynucleotide (part of the host cell chromosome) comprising a third translation unit encoding a chaperone protein (e.g., a peptidyl-prolyl isomerase or protein disulfide oxidoreductase of the present disclosure), the third polynucleotide being in operable combination with a promoter of the present disclosure in a non-natural combination. In some embodiments, the host cell comprises a first polynucleotide (part of an extrachromosomal polynucleotide) comprising a first translation unit encoding a first strand of a polypeptide, a second polynucleotide (part of an extrachromosomal polynucleotide) comprising a second translation unit encoding a second strand of a polypeptide, a third polynucleotide (part of a host cell chromosome) comprising a third translation unit encoding a protein disulfide oxidoreductase of the present disclosure, in operable combination with a promoter of the present disclosure in a non-natural combination, and a fourth polynucleotide (part of a host cell chromosome) comprising a fourth translation unit encoding a peptidyl-prolyl isomerase of the present disclosure, in operable combination with a promoter of the present disclosure in a non-natural combination.In some embodiments, the host cell comprises a first polynucleotide (part of an extrachromosomal polynucleotide) comprising a first translation unit encoding a first chain of a polypeptide, a second polynucleotide (part of an extrachromosomal polynucleotide) comprising a second translation unit encoding a second chain of a polypeptide, a third polynucleotide (part of a host cell chromosome) comprising a third translation unit encoding a protein disulfide oxidoreductase of the present disclosure, in operable combination with a promoter of the present disclosure in a non-natural combination, a fourth polynucleotide (part of a host cell chromosome) comprising a fourth translation unit encoding a peptidyl-prolyl isomerase of the present disclosure, in operable combination with a promoter of the present disclosure in a non-natural combination, and a fifth translation unit encoding a second protein disulfide oxidoreductase of the present disclosure, in operable combination with a promoter of the present disclosure in a non-natural combination. In some embodiments of any of the above embodiments, the host cell further comprises a translation unit (part of the extrachromosomal polynucleotide) encoding a third chain of the two-chain protein.

[0214] In some embodiments, the host cell is cultured to express two chains of the polypeptide, and upon expression, the two chains are folded and assembled to form a biologically active polypeptide in the host cell. As used herein, folding and assembly of the two chains can refer to any or all steps that facilitate the final fit of the appropriate three-dimensional two-chain protein conformation, two-chain protein assembly, or both. Folding and assembly can refer to folding and assembly of each chain into its appropriate conformation and fold, or to folding and assembly of the complex created by the intermolecular linkage of the two protein chains. Similarly, each chain can be folded and assembled to form a biologically active polypeptide, or the complex created by the intermolecular linkage of the two protein chains can generally be folded and assembled to form a biologically active polypeptide.

[0215] Biologically active polypeptide can refer to any polypeptide that can perform the function attributed to the polypeptide.The function of biologically active polypeptide includes, but is not limited to, proper folding or assembly, binding or other interaction with another macromolecule, and enzymatic activity.For illustrative purposes, biologically active antibody can refer to an antibody that can perform at least one function attributed to the antibody, including, but not limited to, binding to an epitope or possessing the characteristics of antibody Fc region, as described in more detail herein.

[0216] Antibodies can be produced using recombinant methods. For recombinant production of anti-antigen antibodies, nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. DNA encoding the antibody can be easily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains). Many vectors are available. Components of a vector generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence (e.g., as described above). Production of multispecific (e.g., bispecific) antibodies

[0217] Certain aspects of the present disclosure relate to methods of producing bispecific antibodies (e.g., comprising a first half antibody capable of binding to a first antigen and a second half antibody capable of binding to a second antigen, the first and second antigens being optionally different). In some embodiments, the method comprises producing a first half antibody as described herein, the first half antibody comprising a heavy chain and a light chain encoded by a translation unit (e.g., a portion of one or more extrachromosomal polynucleotides) of the present disclosure, and producing a second half antibody as described herein, the second half antibody comprising a heavy chain and a light chain encoded by a translation unit (e.g., a portion of one or more extrachromosomal polynucleotides) of the present disclosure. In some embodiments, one of the first and second half antibodies comprises at least one knob-forming mutation of the present disclosure, and the other of the first and second half antibodies comprises at least one hole-forming mutation of the present disclosure. In some embodiments, the method further comprises combining the first half antibody with a second half antibody under reducing conditions to generate a bispecific antibody. Exemplary methods for half antibody production and bispecific antibody assembly are provided below.

[0218] Polynucleotides encoding modified immunoglobulin polypeptides with one or more corresponding knob or hole forming mutations can be expressed and purified using standard recombinant techniques and cell systems known in the art. See, for example, U.S. Patent Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228, 7,695,936, 8,216,805, U.S. Patent Application Publication No. 2013 / 0089553, and Spiess et al., Nature Biotechnology 31:753-758, 2013. Modified immunoglobulin polypeptides may be produced using prokaryotic host cells such as E. coli. Immunoglobulin polypeptides with corresponding knobs and holes can be expressed in host cells in co-culture and purified together as heteromultimers, or expressed in monoculture, purified separately, and assembled in vitro. In some embodiments, two strains of bacterial host cells, one expressing an immunoglobulin polypeptide with a knob and the other expressing an immunoglobulin polypeptide with a hole, are co-cultured using standard bacterial culture techniques known in the art. In some embodiments, the two strains can be mixed in a specific ratio, for example, to achieve equal expression levels under culture. In some embodiments, the two strains can be mixed in a ratio of 50:50, 60:40, or 70:30. After the polypeptides are expressed, the cells can be lysed together and the proteins can be extracted. Standard techniques known in the art that allow for the measurement of the abundance of homomultimeric versus heteromultimeric species can include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide can be expressed separately, recovered, and assembled together in vitro using standard recombinant techniques. As described in more detail below, assembly can be accomplished, for example, by purifying each modified immunoglobulin polypeptide, mixing and incubating them together in equal masses, reducing disulfides (e.g., by treatment with dithiothreitol), concentrating, and reoxidizing the polypeptides.The bispecific antibodies formed can be purified using standard techniques, including cation exchange chromatography, and can be measured using standard techniques, including size exclusion chromatography. For a more detailed description of these methods, see Speiss et al., Nat Biotechnol 31:753-8, 2013.

[0219] Half antibodies containing either the knob or hole mutations are produced in separate cultures by expressing the heavy and light chain constructs in bacterial host cells (e.g., E. coli). Each half antibody can be purified separately by Protein A affinity chromatography. Clarified cell extracts from the knob and hole half antibodies can be purified by HiTrap MABSELECT SURE™ columns. Protein A purified half antibodies with different specificities can be assembled to form bispecific antibodies in an in vitro redox reaction in the presence of a reducing agent.

[0220] Any suitable method can be used to prepare the desired reducing conditions. For example, the desired reducing conditions can be prepared by adding a reducing agent / reducing agent to the reaction (such as the assembly mixture of the present invention). Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), thioglycolic acid, ascorbic acid, thiolacetic acid, glutathione (GSH), β-mercaptoethylamine, cysteine / cystine, GSH / glutathione disulfide (GSSG), cysteamine / cystamine, glycylcysteine, and β-mercaptoethanol, preferably GSH. In certain particular embodiments, the reducing agent is a weak reducing agent, including, but not limited to, GSH, β-mercaptoethylamine, cysteine / cystine, GSH / GSSG, cysteamine / cystamine, glycylcysteine, and β-mercaptoethanol, preferably GSH. In certain preferred embodiments, the reducing agent is GSH. In certain embodiments, the reducing agent is not DTT. It is within the ability of one of ordinary skill in the art to select an appropriate reducing agent at an appropriate concentration and under appropriate experimental conditions to achieve the desired reducing conditions in the reaction. For example, o 10 mM L-reduced glutathione in a solution with a bispecific antibody protein concentration of 10 g / L at C results in a starting redox potential of about -400 mV. For example, glutathione added to the assembly mixture creates a weakly reducing condition that favors knob-into-hole bispecific assembly. Other reducing agents of a similar class, such as BMEA (β-mercaptoethylamine), may have a similar effect. See WO2013 / 055958, which is incorporated herein by reference in its entirety. The reducing conditions of the reaction can be estimated and measured using any suitable method known in the art. For example, it can be measured using a resazurin indicator (color change from blue to colorless under reducing conditions). For more accurate measurements, a redox potential meter (such as an ORP electrode from BROADLEY JAMES®) can be used.

[0221] In certain particular embodiments, the reducing conditions are weak reducing conditions. The term "weak reducing agent" or "weak reducing conditions" as used herein refers to a reducing agent or reducing conditions prepared by a reducing agent having a negative oxidation potential at 25°C. The oxidation potential of the reducing agent is preferably between -50 and -600 mV, between -100 and -600 mV, between -200 and -600 mV, between -100 and -500 mV, between -150 and -300 mV, more preferably between about -300 and -500 mV, and most preferably about -400 mV, when the pH is between 7 and 9 and the temperature is between 15°C and 39°C. A person skilled in the art will be able to select an appropriate reducing agent to prepare the desired reducing conditions. A person skilled in the art will recognize that a strong reducing agent, i.e., one having a more negative oxidation potential than the above-mentioned reducing agents at the same concentration, pH, and temperature, may be used at a lower concentration. In a preferred embodiment, the protein can form disulfide bonds in the presence of a reducing agent when incubated under the above conditions. Examples of weak reducing agents include, but are not limited to, glutathione, β-mercaptoethylamine, cystine / cysteine, GSH / GSSG, cysteamine / cystamine, glycylcysteine, and β-mercaptoethanol. In certain embodiments, the oxidation potential of a 200X molar ratio of GSH:antibody can be used as a reference point for weak reducing conditions that can be expected to assemble efficiently using other reducing agents.

[0222] The glutathione concentration can be expressed as a molar concentration, or a molar ratio or molar excess relative to the amount of half antibodies present in the assembly mixture. The use of a target molar ratio of reducing agent control to protein concentration in the assembly mixture, which prevents over-reduction or under-reduction as a result of variable protein concentrations. In certain other embodiments, the reducing agent is added to the assembly mixture in a molar excess of 2-600X, 2-200X, 2-300X, 2-400X, 2-500X, 2-20X, 2-8X, 20-50X, 50-600X, 50-200X, or 100-300X, preferably 50-400X, more preferably 100-300X, and most preferably 200X, relative to the total amount of half antibodies. In certain embodiments, the assembly mixture has a pH between 7 and 9, preferably pH 8.5.

[0223] In certain embodiments, the culture of the first half antibody and the second half antibody can be combined, and then dissolved in the combined culture.The combination of the released first half antibody and the second half antibody can form a bispecific antibody under reducing conditions.See WO2011 / 133886, the entirety of which is incorporated herein by reference.

[0224] According to a different approach, an antibody variable domain (antibody-antigen binding site) with the desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion is preferably with an immunoglobulin heavy chain constant domain, including at least a part of the hinge, CH2, and CH3 regions. It is typical to have the first heavy chain constant region (CH1), containing the site required for light chain binding, present in at least one of these fusions. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments. However, in cases where expressing at least two polypeptide chains in equal ratios provides high yields, or where the ratio is not particularly important, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

[0225] In one embodiment of this approach, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate separation of the desired bispecific compound from undesired immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy separation method. This approach is disclosed in WO94 / 04690. For further details of the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

[0226] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. One interface is the C HThe method comprises at least a portion of the three domains. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of a second antibody molecule by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products such as homodimers.

[0227] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and to treat HIV infection (WO91 / 00360, WO92 / 200373, and EP03089). Heteroconjugate antibodies may be produced using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0228] Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0229] Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli that can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0230] Various techniques have also been described for producing and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptide from the Fos and Jun proteins was linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. The fragments consist of a light chain variable domain (V) connected to a light chain variable domain (V) by a linker that is too short to allow pairing between the two domains on the same chain. L ) connected to a heavy chain variable domain (V H ) in a given fragment. H and V L The domain is a complementary V L Domains and V H The Fv domains are paired together to form two antigen-binding sites. Another strategy for making bispecific antibody fragments has also been reported by the use of single-chain Fv (sFv) dimers. See Gruber et al., J. Immunol, 152:5368 (1994).

[0231] Another technique for making bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, the single polypeptide chain may comprise a variable heavy chain (V H ) and variable light chain (V LThe single polypeptide comprises two single chain Fv (scFv) fragments each having a CD3 domain and a CD4 domain, the domains being separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. The single polypeptide further comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, such that cells of two different cell types are brought into close proximity or tethered when each scFv engages its cognate epitope. One particular embodiment of this approach comprises an scFv that recognizes a cell surface antigen expressed by an immune cell, e.g., a CD3 polypeptide on a T cell, linked to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0232] Since it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic expression system known in the art. However, specific purification techniques (see, for example, EP1691833) may be necessary to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, the solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using a gradient of imidazole concentration. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a gradient of sodium chloride concentration. Finally, the eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species. Selection and transformation of host cells

[0233] Two-chain proteins such as full length or half antibodies, antibody fusion proteins, one-arm antibodies, and antibody fragments can be produced in bacteria, especially when glycosylation and Fc effector functions are not required, for example, when therapeutic antibodies are conjugated to cytotoxic agents (e.g., toxins) that are effective in destroying tumor cells alone. Full length antibodies have a better half-life in circulation. Production in E. coli is faster and more cost-effective. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Pat. No. 5,648,237 (Carter et al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523 (Simmons et al.), which describe translation initiation regions (TIRs) and signal sequences to optimize expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the E. coli cell paste in a soluble fraction and purified, for example, by a protein A or G column depending on the isotype. Final purification can be performed, for example, similar to the process for purifying antibodies expressed in CHO cells.

[0234] Host cells are transformed with the expression or cloning vectors of the present disclosure for production of the two-chain protein and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Host cell culture

[0235] The host cells of the present disclosure may be cultured in a variety of media. As used herein, "culture medium" refers to any composition or liquid that supports the growth of the bacteria of the present disclosure. A suitable culture medium may be liquid or solid and may contain any nutrients, salts, buffers, elements, and other compounds that support cell growth and survival. Typical nutrients of the culture medium may include nitrogen, carbon, amino acids, carbohydrates, trace elements, vitamins, and mineral sources. These nutrients may be added as individual components (as in defined culture media) or as components of a complex extract (e.g., yeast extract). The culture medium may be nutrient-rich to support rapid growth or nutrient-minimal to support slower growth. The culture medium may also contain any agent (e.g., antibiotics) used to inhibit the growth of or kill contaminating organisms. The culture medium may also contain any compound used to control the activity of an inducible promoter or enzyme (as an example, IPTG may be included to induce the expression of any polynucleotide controlled by the lac operon or a functionally similar promoter). Numerous examples of suitable culture media are well known in the art and include, but are not limited to, M9 medium, Lysogeny Broth (LB), Terrific Broth (TB), NZY broth, SOB broth, and YT broth.

[0236] Any of these media may be supplemented as necessary with salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, antimycotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), glucose, and / or a suitable energy source. Typical components found in prokaryotic cell culture media include yeast extract, salts (e.g., NaCl), tryptone, buffers (e.g., phosphate buffer), glycerol, and the like. Any other necessary supplements may also be included at appropriate concentrations known to those of skill in the art. Culture conditions such as temperature and pH will be those already used with the prokaryotic host cell selected for expression and will be apparent to those of skill in the art. Purification of biologically active polypeptides

[0237] Certain aspects of the present disclosure relate to recovering biologically active polypeptides from host cells. Typically, recovering (the terms "purifying" or "purification" may be used interchangeably herein) biologically active polypeptides of the present disclosure involves isolating the polypeptide from the host cell (or from the cell culture medium, if the polypeptide is excreted into the medium), and purifying the polypeptide from other associated macromolecules, such as cell debris and other polypeptides. Numerous techniques for purifying various proteins from various host cell compartments are known in the art (see, for example, Evans, Jr., TC and Xu MQ (eds.) Heterologous Gene Expression in E. coli (2011) Methods in Molecular Biology, Vol. 705, Humana Press). Exemplary techniques are described below, which are included for illustrative purposes to aid the understanding of those skilled in the art, and are in no way meant to be limiting.

[0238] When using recombinant techniques, two-chain proteins such as secretory proteins can be produced intracellularly, produced in the periplasmic space, or directly secreted into the medium. When secretory proteins are produced intracellularly, the first step is to remove particulate debris, which may be host cells or lysed fragments, for example, by centrifugation or ultrafiltration.

[0239] In some embodiments, secreted proteins are recovered from the periplasm of host cells. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating secreted proteins secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If secreted proteins are excreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors, such as PMSF, may be included in any of the foregoing steps to inhibit protein degradation, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0240] Secreted protein compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography typically being one of the preferred purification steps. With respect to antibodies, the suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. H If it contains three domains, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatographic fractionation, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered. Those skilled in the art will recognize that many of these techniques useful for antibody recovery can be easily adapted to recover other two-chain proteins, such as secreted proteins. EXAMPLES

[0241] The present disclosure will be more fully understood by referring to the following examples. However, these should not be interpreted as limiting the scope of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in consideration thereof will be suggested to those skilled in the art, but they should be included within the spirit and scope of the present application and the scope of the appended claims. Example 1: Engineering an E. coli strain with chromosomally integrated promoters controlling expression of the chaperones DsbA, DsbC, and FkpA

[0242] Overexpression of the chaperones DsbA, DsbC, and FkpA from plasmids can improve antibody-based product production in bacterial cultures. However, expression of these chaperones from plasmids has several drawbacks. For example, such an approach requires the development and adjustment of an expression plasmid for each new product. Large plasmid size may also result in lower product titers in some cases. Furthermore, plasmids are typically present at 10-15 copies per cell, resulting in high levels of overexpression that may require downstream purification step(s) to remove the chaperone protein (e.g., FkpA) from the product. In some cases, the same product titers could be achieved with lower expression levels of one or more of the chaperones.

[0243] Here, the native promoters of dsbA, dsbC, and fkpA in the E. coli genome were replaced with a non-native combination of promoters, phoA, tac, and CP25, to generate engineered strains that chromosomally overexpress the chaperones. These strains were examined for use in the production of two half antibodies (anti-IL13 half antibodies, referred to herein as "xIL13" and AF2), a one-arm antibody (MetMAb), and an antibody Fab fragment (an anti-VEGF antibody fragment). method Vector construction

[0244] Vectors expressing the chaperones and either xIL13 (MD157) or AF2 (MD341) were constructed as described in WO2016073791 (see, e.g., paragraphs 278 and 279, 281-284, 285-288). Stock manipulation

[0245] The phoA (see Wanner BL (1990) Colloqium Mosbach, Mol. Basis Bact. Metab. p. 41), tac (see de Boer HA et al. (1983) PNAS 80, pp. 21-5), and CP25 (see Jensen PR and Hammer K. (1998) Appl. Environ. Microbiol. 64, pp. 82-87) promoters were integrated into the E. coli genome to replace the native promoters of dsbA, dsbC, and fkpA. These promoter modifications were achieved via allelic exchange (see, e.g., Bass, S. et al. (1996) J. Bacteriol. 178:1154-1161 and Innes, D. et al. (2001) Microbiology 147:1887-1896).

[0246] Briefly, a pS1080-based suicide vector was constructed using NEBuilder® HiFi DNA Assembly Master Mix (Gibson Assembly) to contain the promoter of interest, flanked on both sides by 500 base pairs of homologous sequences matching the desired insertion region in the E. coli genome. After sequencing for confirmation, the bacteriophages M13 and P1 were used to infect the 48C8 strain with the plasmid sequence and transduce the introduced sequence into the strain of interest (see, e.g., Nakashima, N. and Miyazaki, K. (2014) Int. J. Mol. Sci. 15:2773-2793). The promoter obtained from the source plasmid replaced the intergenic region upstream of the chaperone. This replaced the native promoters of dsbA, dsbC, and fkpA in the E. coli genome with the phoA, tac, and / or CP25 promoters from the pS1080 suicide vector. Modifications to the lacI gene were also made to further enhance expression from the tac promoter. Vial lots of the resulting strains were produced and stored at -80°C. Shake flask culture and fermenter process

[0247] The engineered strains (see Table B) were cultivated in standard shake flask cultures (Sh.Fl.) and 10 liter fermenters (10L). 10 liter fermentations were performed as described in WO2016073791 (see, e.g., paragraphs 289-292). Strains 67A6 and 64B4, containing only the chaperones expressed from their native promoters, were used as negative controls (i.e., Sh.Fl.(-) and Ambr(-)). As positive controls, these strains were transformed with plasmids expressing DsbA, DsbC, and FkpA (see Table A) (i.e., Sh.Fl.(+), Ambr(+), and 10L(+)). Electrophoresis, Western blot, and HPLC analysis

[0248] The relative concentrations of DsbA, DsbC, and FkpA were measured by Western blot. The concentrations of xIL13, AF2, MetMAb, and anti-VEGF antibody fragments were measured by reversed-phase HPLC. Electrophoresis, Western blot, and HPLC analysis methods were performed as described in WO2016073791 (see, e.g., paragraphs 293-299). result

[0249] Vectors were constructed to overexpress the antibody-based products xIL13, AF2, MetMAb, and anti-VEGF antibody fragments in E. coli. A representative plasmid map of the vector expressing xIL13 is shown in FIG. 1. Each vector contains genes encoding xIL13, AF2, MetMAb, or anti-VEGF antibody fragments, and either (1) no chaperone genes (e.g., FIG. 1, right), or (2) a combination of dsbA, dsbC, and fkpA (e.g., FIG. 1, left), as detailed in Table A below. Strains containing these vectors can be used as positive controls when assessing protein expression in engineered strains. TIFF0007620546000005.tif60170- indicates that the indicated chaperone gene is absent.

[0250] In addition, 17 strains were constructed for overexpression of one or more of the periplasmic chaperones DsbC, DsbA, and FkpA by replacing the native promoters of these chaperones in the E. coli genome with the tac, phoA, and CP25 promoters. The lacI background of strains was also constructed by deleting lacI (ΔlacI and ΔlacI::kan), inserting a wild-type copy of lacI (lacI + or lacIWT), or the original lacI of the parent strain. Q The mutation was engineered to further enhance expression from the tac promoter by leaving it intact. Since the lacI gene product represses expression from the tac promoter, deletion of lacI results in higher expression from the tac promoter. QThe mutations result in increased transcription levels of lacI and stronger repression of the tac promoter (see Calos (1978) Nature 274, 762-765). Furthermore, the tac promoter is inducible with IPTG. Table B lists the engineered strains and indicates the engineered promoter modifications in addition to the lacI gene modifications. TIFF0007620546000006.tif141170--Dashes indicate no modification to the native promoter sequence. a The genotype of the parent strain (67A6) was lacI. q Has a mutation. Example 2: Overexpression of FkpA under the control of a chromosomally integrated promoter

[0251] Overexpression of FkpA was compared between strains engineered as described in Example 1 with an integrated promoter overexpressing native FkpA compared to strains carrying a plasmid expressing FkpA.

[0252] To assess the ability of FkpA to be chromosomally overexpressed, the native promoter of FkpA was replaced with the tac, phoA, or CP25 promoters according to the methods of Example 1. Modifications to the lacI promoter were also made to further enhance the strength of the tac promoter. Strains were grown in 10 liter fermentors and assessed for FkpA production by Western blot.

[0253] Chromosomal expression driven by the CP25 promoter showed the highest level of FkpA expression, followed by expression driven by the phoA promoter (Figure 2). As expected, the tac promoter showed the lowest level of FkpA expression, with the ΔlacI background showing higher FkpA expression than the lacIWT background.

[0254] Thus, chromosome engineering resulted in a range of FkpA expression compared with a single high level of expression using a plasmid-expressed fkpA locus. These results demonstrate that expression of FkpA can be controlled and enhanced by chromosomal overexpression. Example 3: Chromosomal expression of chaperones under the control of the tac, phoA, and CP25 promoters in shake flask cultures

[0255] To assess chaperone expression in strains with promoter modifications of DsbA, DsbC, and FkpA, cultures were grown in shake flasks and expression of these chaperones was measured using Western blot.

[0256] A range of expression levels was observed (Fig. 3A-C). For DsbA, expression from the CP25 promoter was strongest, exceeding the levels of expression seen in the positive controls (Sh.Fl.(+) and 10L(+), DsbA expressed from a plasmid) (Fig. 3A). The next highest levels of DsbA were produced by phoA, followed by tac, and IPTG induction of the tac promoter increased the expression levels of DsbA, as expected. Expression from the tac promoter alone was most likely due to the repressible lacI promoter in this strain. Q Due to background, it was lower than the negative control (Sh.Fl.(-), no exogenous chaperone expression). Similarly, DsbC expression was higher with the phoA promoter compared to the tac promoter, and IPTG induction of the tac promoter resulted in increased DsbC expression (Figure 3B). DsbC expression from the tac promoter alone was also lower than the negative control (Sh.Fl.(-)).

[0257] Interestingly, FkpA expression was slightly different among the engineered strains when compared to the DsbA and DsbC expression results (Figure 3C). The phoA promoter drove the highest expression of FkpA, followed by expression from the CP25 promoter. Similar to DsbA and DsbC, the tac promoter drove the lowest level of expression. In general, the expression levels of FkpA were very high compared to the native level (Sh.Fl.(-)), indicating that the native FkpA promoter is a weaker promoter compared to the native DsbA and DsbC promoters.

[0258] Taken together, these results demonstrate that expression of all three chaperones, DsbA, DsbC, and FkpA, can be similarly regulated and enhanced via chromosomal overexpression. Example 4: Chromosomal expression of chaperones under the control of the tac, phoA, and CP25 promoters in ambr250 fermenter cultures

[0259] To test whether chromosomal overexpression of chaperones could be transferred from shake flask cultures to larger fermenter cultures, the engineered strains were grown in ambr250 high cell density fermenters according to the method of Example 1, and chaperone expression was measured by Western blot.

[0260] As with shake flask cultures, a range of expression levels was observed (Figures 4A-C). For DsbA and DsbC, only the tac promoter with different lacI backgrounds was tested. For both DsbA and DsbC, the lacI Q The tac promoter with a lacIWT background gave the lowest level of expression, while the IPTG-inducible tac promoter with a lacIWT background had the highest expression as seen in shake flask cultures (Figures 4A and 4B). Unlike FkpA expression in shake flasks, the CP25 promoter had the highest FkpA expression in the ambR250 bioreactor when compared to the phoA and tac promoters (Figure 4C).

[0261] Taken together, these data indicate that the expression pattern of chromosomally expressed chaperones can be successfully scaled up from shake flasks to 10 L high cell density fermenter processes. Example 5: Expression of xIL13 half antibodies in engineered lines

[0262] The effect of chaperone overexpression in the host strain (see Table B) on the production of the half antibody xIL13 was tested.

[0263] The host strains were transformed with plasmid CS392 expressing xIL13. Host strain 67A6, which does not contain chromosomally overexpressed chaperones, was transformed with plasmid MD157 expressing xIL13 together with DsbA, DsbC, and FkpA (Figure 1). 67A6 / MD157 was used as a positive control. All strains were grown in 10 L fermentors as described in Example 1, and xIL13 production was assessed by reverse-phase HPLC titer assay.

[0264] The top three xIL13 producing strains (69E1, 69F8, and 69F4) produced equal or higher titers of xIL13 compared to the positive control, where xIL13 was expressed from a plasmid (Figure 5). The highest expression from the 69E1 strain contained DsbC and FkpA driven by the phoA and CP25 promoters, respectively. These results also showed that xIL13 titers were not significantly affected by the expression of DsbA.

[0265] Therefore, chromosomal engineering, such as the three engineered strains 69E1, 69F8, and 69F4, can be used as an alternative to co-expressing chaperones with xIL13 on a plasmid, resulting in comparable xIL13 titers. Example 6: Expression of chaperones and xIL13 half antibodies in engineered 69E1, 69F4, and 69F8 lines

[0266] To further explore the use of 69E1, 69F4 and 69F8 strains in antibody-based product production, these strains were more thoroughly evaluated for expression of xIL13 and chaperones over a 72-hour period. The three engineered strains described in Example 5 were transformed with plasmid CS392 to express xIL13 as described in Examples 1 and 5. The 67A6 strain, as a positive control, was transformed with plasmid MD157 to express xIL13 and chaperones as described in Example 1. All strains were grown in 10 L fermentors and xIL13 concentrations were assessed by reverse-phase HPLC titer assay as described in Example 1.

[0267] Optical density (Figure 6A) and osmolality (Figure 6B) were measured during fermentation and no significant differences were found between the experimental and plasmid control strains.

[0268] For all three experimental strains, xIL13 expression was similar or slightly higher than the positive control, with the 69E1 and 69F8 strains having the highest expression levels at 72 hours (Figure 7A). DsbC levels in the engineered strain fermenters were lower compared to the control process for most fermenters (Figure 7B). This was expected since the plasmid-based process has a much higher copy number than the process where the chaperone is expressed from the chromosome. DsbC levels between the 69E1 and 69F8 strain fermenters were similar, which was expected since both use the phoA promoter for expression. Interestingly, their levels were similar to the plasmid expression levels towards the end of the fermentation. DsbC was expressed from the phoA promoter in the 69E1 and 69F8 strains. Therefore, the promoter was not induced until 18 hours into fermentation due to the presence of phosphate, and levels were low in the first part of the fermentation. Phosphate is completely consumed in about 18 hours, after which the promoter is fully induced, resulting in more DsbC expression. Since 69F4 uses a leaky tac promoter and is independent of the phosphate level in the medium, DsbC levels in 69F4 fermenters were initially higher compared to fermenters of 69E1 and 69F8 strains. These results also indicate that the phoA promoter is a stronger promoter compared to the tac promoter, similar to the expression results obtained in shake flask cultures.

[0269] The 69E1 and 69F4 strains express FkpA under the CP25 promoter, while 69F8 expresses FkpA under the phoA promoter. The strong constitutive CP25 promoter resulted in high levels of FkpA comparable to the plasmid levels (FkpA under the phoA promoter) (Figure 7C). The 69F8 strain accumulated FkpA at lower levels compared to the plasmid-based process, which may be due to differences in copy number (approximately 15 for the plasmid and 1 for the chromosome) despite the use of the phoA promoter in both cases to drive FkpA expression. However, the titers between the phase I plasmid-based process and the 69F8 strain process (Figure 7A) were similar, suggesting that additional FkpA may not be required. Due to the strain imposed on purification development to reduce FkpA levels in the final pool, reduced FkpA levels together with the ability to achieve high titers could be considered an advantage of the 69F8 strain for the xIL13 process.

[0270] These data suggest that expression of xIL13 does not necessarily require high levels of DsbC and FkpA compared to the plasmid control: all three strains were able to produce equal or higher titers of xIL13, even though the strains generally expressed lower levels of the chaperones (although this feature is advantageous in that it eliminates the need for further purification to remove FkpA). Example 7: Expression of AF2 half antibodies in engineered 69E1, 69F4, and 69F8 strains

[0271] The ability of strains 69E1, 69F4, and 69F8 to produce the half antibody AF2 was evaluated. Strains 69E1, 69F4, and 69F8 were transformed with plasmid ERD046 to express AF2, and strain 67A6, as a positive control, was transformed with plasmid MD341 to express AF2 and chaperones, as described in Example 1. Strains were grown in 10 L fermentors, as described in Example 1, and AF2 concentrations were evaluated at various time points over 72 hours.

[0272] Optical density (Figure 8A) and osmolality (Figure 8B) were measured during fermentation. No significant differences were found between the experimental and plasmid control strains. Expression of AF2 was highest in strain 69E1, exceeding the titer seen in the plasmid control (Figure 9). Strains 69F4 and 69F8 had slightly lower but similar titers of AF2 compared to the control.

[0273] These data demonstrate that all three strains, especially strain 69E1, can be used to produce AF2, instead of expressing the chaperone on a plasmid. Example 8: Expression of MetMAb1 armed antibodies in engineered 69E1, 69F4, and 69F8 strains

[0274] The ability of strains 69E1, 69F4, and 69F8 to produce the one-armed antibody MetMAb was evaluated. These three strains were transformed with plasmid p186 to express MetMAb, and strain 64B4, as a positive control, was transformed with plasmid pOA5D5.3630 to express MetMAb and chaperone, as described in Example 1. Strains were grown in 10 L fermentors as described in Example 1, and MetMAb concentrations were evaluated at various time points over 72 hours.

[0275] Optical density (Figure 10A) and osmolality (Figure 10B) were measured during fermentation and no significant differences were found between the experimental and plasmid control strains.

[0276] In the control process, DsbA and DsbC were expressed from plasmids and FkpA was not used. Fermenters using all three strains had similar or higher titers when compared to the control process (Figure 11). The fermenter using the 69E1 strain had an approximately two-fold increase in titer compared to the control process. Surprisingly, the fermenter using the 69F4 strain did not accumulate a similar titer to the 69E1 strain. Without wishing to be bound by theory, this may be due to suboptimal levels of DsbC expressed under the tac promoter in the 69F4 strain compared to the phoA promoter (a stronger promoter) in the 69E1 strain. The fermenter using the 69E8 strain had a similar titer to the control process. Without wishing to be bound by theory, this may be due to suboptimal levels of FkpA. Example 9: Expression of anti-VEGF antibody fragments in engineered 69E1 strains

[0277] The ability of the 69E1 strain to produce anti-VEGF Fab fragments was evaluated. The 69E1 strain and the control strain 67A6 were transformed with plasmid HSK117 to express anti-VEGF antibody fragments as described in Example 1. The strains were grown in 10 L fermentors as described in Example 1, and anti-VEGF antibody fragment concentrations were evaluated at various time points over 72 hours.

[0278] During fermentation, optical density (Figure 12A) and osmolality (Figure 12B) were measured. The 69E1 strain showed a slightly higher optical density than the control at 72 hours (Figure 12A). Expression of the anti-VEGF antibody fragment in the 69E1 strain also exceeded that of the plasmid control at all time points tested (Figure 13).

[0279] These data demonstrate that strains with chromosomal chaperone overexpression can be used to produce higher titers of anti-VEGF antibody fragments compared to strains using plasmid-based chaperone overexpression.

[0280] Taken together, the results of Examples 5-9 demonstrate that chromosomal overexpression of chaperones has the potential to produce titers comparable to plasmid-based chaperone expression. Several molecular formats were tested, including the bispecific half-antibodies xIL13 and AF2, the one-arm antibody MetMAb, and an anti-VEGF Fab fragment. Fermentations using the three engineered strains 69E1, 68F8, and 69F4 had comparable or higher titers compared to the control process with little or no additional process development. Additionally, in one case (AF2 in the 69F8 host), no additional development was undertaken in the case above the control, so it is possible that additional process development efforts could be undertaken for each molecule to further drive titers beyond the levels observed here. In addition to achieving high titers, these strains do not require additional plasmid cloning efforts and provide a quick and easy method to evaluate chaperone expression. In some cases (e.g., the 69F8 strain in the xIL13 process), similar titers were obtained with lower levels of FkpA. This is desirable for downstream purification since no additional column purification is required for clearance of FkpA.

Claims

1. 1. A method for producing a polypeptide comprising two chains in a prokaryotic host cell comprising a host cell chromosome, the method comprising the steps of: (a) culturing a host cell in a culture medium under conditions suitable for expression of the two chains of the polypeptide to express the two chains of the polypeptide, whereby, upon expression, the two chains fold and assemble to form a biologically active polypeptide in the host cell; The host cell comprises (1) a first polynucleotide comprising a first translation unit encoding a first chain of the polypeptide; (2) a second polynucleotide comprising a second translation unit encoding a second strand of the polypeptide, wherein the first and second polynucleotides are part of one or more extrachromosomal polynucleotides; (3) a third polynucleotide comprising a third translational unit encoding a first protein disulfide oxidoreductase, said third translational unit being part of and native to said host cell chromosome, said third translational unit being in operable combination with a first promoter that is integrated into said host cell chromosome and drives transcription of said third translational unit resulting in chromosomal overexpression of a chaperone protein, and wherein the combination of said third translational unit and said first promoter is non-native to said host cell chromosome; and (4) A fourth polynucleotide comprising a fourth translation unit encoding a peptidyl-prolyl isomerase, wherein the fourth translation unit is part of the host cell chromosome and is native to the host cell chromosome, the fourth translation unit being in operable combination with a second promoter that is integrated into the host cell chromosome and drives transcription of the fourth translation unit resulting in chromosomal overexpression of a chaperone protein, and the combination of the fourth translation unit and the second promoter being non-native to the host cell chromosome. Forming, (b) recovering the biologically active polypeptide from the host cell. Including, one or both of the third translation unit and the fourth translation unit are native to a host cell chromosome; the first promoter is an inducible promoter and the second promoter is an inducible promoter or a constitutive promoter; The first promoter is a Pho promoter, and the second promoter is a Pho promoter or a CP25 promoter; the first protein disulfide oxidoreductase is an E. coli DsbC protein and the peptidyl-prolyl isomerase is an E. coli FkpA protein; method.

2. 2. The method of claim 1, wherein the first and second promoters are both Pho promoters that drive transcription of the third and fourth translation units, respectively, when phosphate in the culture medium is depleted.

3. 2. The method of claim 1, wherein the first promoter is an inducible promoter and the second promoter is a constitutive promoter.

4. 2. The method of claim 1, wherein the first promoter is a Pho promoter that drives transcription of the third translation unit when phosphate in the culture medium is depleted, and the second promoter is a CP25 promoter.

5. 2. The method of claim 1, wherein the first protein disulfide oxidoreductase is E. coli DsbC, the first promoter is a Pho promoter that drives transcription of the third translation unit when phosphate in the culture medium is depleted, the peptidyl-prolyl isomerase is E. coli FkpA, and the second promoter is a CP25 promoter.

6. 2. The method of claim 1, wherein the first protein disulfide oxidoreductase is E. coli DsbC, the first promoter is a Pho promoter that drives transcription of the third translation unit when the culture medium is depleted of phosphate, the peptidyl-prolyl isomerase is E. coli FkpA, and the second promoter is a Pho promoter that drives transcription of the fourth translation unit when the culture medium is depleted of phosphate.

7. A method for producing a polypeptide comprising two chains in a prokaryotic host cell comprising a host cell chromosome, the method comprising: (a) culturing a host cell in a culture medium under conditions suitable for expression of the two chains of the polypeptide to express the two chains of the polypeptide, whereby, upon expression, the two chains fold and assemble to form a biologically active polypeptide in the host cell; The host cell comprises (1) a first polynucleotide comprising a first translation unit encoding a first chain of the polypeptide; (2) a second polynucleotide comprising a second translation unit encoding a second strand of the polypeptide, wherein the first and second polynucleotides are part of one or more extrachromosomal polynucleotides; (3) a third polynucleotide comprising a third translational unit encoding a first protein disulfide oxidoreductase, said third translational unit being part of and native to said host cell chromosome, said third translational unit being in operable combination with a first promoter that is integrated into said host cell chromosome and drives transcription of said third translational unit resulting in chromosomal overexpression of a chaperone protein, and the combination of said third translational unit and said first promoter being non-native to said host cell chromosome; (4) a fourth polynucleotide comprising a fourth translational unit encoding a peptidyl-prolyl isomerase, wherein the fourth translational unit is part of and native to the host cell chromosome, the fourth translational unit being in operable combination with a second promoter that is integrated into the host cell chromosome and drives transcription of the fourth translational unit resulting in chromosomal overexpression of a chaperone protein, the combination of the fourth translational unit and the second promoter being non-native to the host cell chromosome; and (5) A fifth polynucleotide comprising a fifth translational unit encoding a second protein disulfide oxidoreductase, the fifth translational unit being part of a host cell chromosome, the fifth translational unit being integrated into the host cell chromosome and in operable combination with a third promoter driving transcription of the fifth translational unit, the combination of the fifth translational unit and the third promoter being non-native to the host cell chromosome. Including, Forming, (b) recovering the biologically active polypeptide from the host cell. Including, one or both of the third translation unit and the fourth translation unit are native to a host cell chromosome; the first promoter is an inducible promoter and the second promoter is an inducible promoter or a constitutive promoter; the first protein disulfide oxidoreductase is E. coli DsbC, the first promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter that drives transcription of the third translation unit when IPTG is present in the culture medium, and the peptidyl-prolyl isomerase is E. coli FkpA; the second promoter is a CP25 promoter, the second protein disulfide oxidoreductase is E. coli DsbA, and The method of claim 1, wherein the third promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter that drives transcription of the fifth translation unit when IPTG is present in the culture medium.

8. 8. The method of any one of claims 1 to 7, wherein the host cell further comprises (6) a sixth polynucleotide comprising a sixth translation unit encoding a third strand of the polypeptide, the sixth polynucleotide being part of the one or more extrachromosomal polynucleotides, whereby upon expression, the three chains fold and assemble to form a biologically active polypeptide in the host cell.

9. 9. The method of claim 8, wherein the first translation unit encodes an immunoglobulin heavy chain, the second translation unit encodes an immunoglobulin light chain, and the sixth translation unit encodes an immunoglobulin Fc fragment, and the three chains fold and assemble to form a biologically active monovalent antibody.

10. 9. The method of any one of claims 1 to 8, wherein the polypeptide is a half antibody and the first chain and the second chain comprise a heavy immunoglobulin chain and an immunoglobulin light chain.

11. 1. A method for producing a bispecific antibody comprising a first half antibody capable of binding to a first antigen and a second half antibody capable of binding to a second antigen, comprising: Producing the first half antibody according to the method of claim 10, wherein the first translation unit encodes a heavy chain of the first half antibody and the second translation unit encodes a light chain of the first half antibody, the first half antibody comprising at least one knob-forming mutation; Producing the second half antibody according to the method of claim 10, wherein the first translation unit encodes the heavy chain of the second half antibody and the second translation unit encodes the light chain of the second half antibody, and the second half antibody comprises at least one hole-forming mutation; combining said first half antibody with said second half antibody under reducing conditions to produce said bispecific antibody.

12. The method of claim 11 , wherein the first antigen and the second antigen are different antigens.

13. 13. The method of claim 11 or claim 12, further comprising the step of adding a reducing agent to achieve said reducing conditions.

14. The method of claim 13, wherein the reducing agent is glutathione.

15. 1. A prokaryotic host cell comprising a host cell chromosome, (1) a first polynucleotide comprising a first translational unit encoding a peptidyl-prolyl isomerase, said first translational unit being part of said host cell chromosome, said first translational unit being integrated into said host cell chromosome and in operable combination with a first promoter that drives transcription of said first translational unit, and wherein the combination of said first translational unit and said first promoter is non-native to said host cell chromosome; and (2) a second polynucleotide comprising a second translational unit encoding a first protein disulfide oxidoreductase, said second translational unit being part of said host cell chromosome and being native to said host cell chromosome, said second translational unit being in operable combination with a second promoter that is integrated into said host cell chromosome and drives transcription of said second translational unit resulting in chromosomal overexpression of the first protein disulfide oxidoreductase, and the combination of said second translational unit and said second promoter being non-native to said host cell chromosome; one or both of the first translational unit and the second translational unit are native to a host cell chromosome; A prokaryotic host cell, wherein the peptidyl-prolyl isomerase is an E. coli FkpA protein, the first promoter is a CP25 promoter or a Pho promoter, the first protein disulfide oxidoreductase is an E. coli DsbC protein, and the second promoter is a Pho promoter.

16. 16. The prokaryotic host cell of claim 15, wherein the peptidyl-prolyl isomerase is the E. coli FkpA protein, the first promoter is the CP25 promoter, the first protein disulfide oxidoreductase is the E. coli DsbC protein, and the second promoter is the Pho promoter.

17. 16. The prokaryotic host cell of claim 15, wherein the peptidyl-prolyl isomerase is an E. coli FkpA protein, the first promoter is a Pho promoter, the first protein disulfide oxidoreductase is an E. coli DsbC protein, and the second promoter is a Pho promoter.

18. A prokaryotic host cell comprising a host cell chromosome, 1) a first polynucleotide comprising a first translational unit encoding a peptidyl-prolyl isomerase, said first translational unit being part of said host cell chromosome, said first translational unit being integrated into said host cell chromosome and in operable combination with a first promoter driving transcription of said first translational unit, said combination of said first translational unit and said first promoter being non-native to said host cell chromosome; (2) a second polynucleotide comprising a second translational unit encoding a first protein disulfide oxidoreductase, said second translational unit being part of and native to said host cell chromosome, said second translational unit being in operable combination with a second promoter that is integrated into said host cell chromosome and drives transcription of said second translational unit resulting in chromosomal overexpression of the first protein disulfide oxidoreductase, and wherein the combination of said second translational unit and said second promoter is non-native to said host cell chromosome; and (3) A third polynucleotide comprising a third translational unit encoding a second protein disulfide oxidoreductase, wherein the third translational unit is part of the host cell chromosome, the third translational unit is integrated into the host cell chromosome and is in operable combination with a third promoter that drives transcription of the third translational unit, and the combination of the third translational unit and the third promoter is non-native to the host cell chromosome. Including, one or both of the first translational unit and the second translational unit are native to a host cell chromosome; 2. A prokaryotic host cell, wherein the peptidyl-prolyl isomerase is an E. coli FkpA protein, the first promoter is a CP25 promoter, the first protein disulfide oxidoreductase is an E. coli DsbC protein, the second promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter, the second protein disulfide oxidoreductase is a DsbA protein, and the third promoter is an isopropyl β-D-thiogalactoside (IPTG) inducible promoter.

19. (i) lacI Ｑ or (ii) ΔfhuA ΔphoA ilvG2096 (IlvG+, Valr) Δprc spr43H1 ΔmanA lacI Ｑ ΔompT ΔmenE742 degPS210A strain of E. coli; A prokaryotic host cell according to any one of claims 15 to 18.

20. (a) a first extrachromosomal polynucleotide comprising a first extrachromosomal translation unit encoding a first chain of a two-chain polypeptide; and (b) a second extrachromosomal polynucleotide comprising a second extrachromosomal translation unit encoding a second chain of the two-chain polypeptide.

20. The prokaryotic host cell of any one of claims 15 to 19, further comprising an extrachromosomal expression vector comprising:

Images