The modular vector (MODVEC) system: a platform for constructing next-generation expression vectors

Abstract

The present invention is directed to a method for optimizing the expression level of a multi-chain protein comprising at least two different polypeptide chains. The production of multi-chain proteins is a major challenge, especially when pairing heavy and light chains of antibodies in a multispecific format, due to the pairing / folding of novel quaternary structures composed of multiple polypeptide chains. Critical to the efficient and proper assembly of multiple polypeptide chain molecules is the expression of the different chains in the correct ratio within the cell. The present invention addresses this problem.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 317,949, filed March 8, 2022, the entirety of which is incorporated herein by reference.

[0002] Incorporation by Reference The Sequence Listing, in XML format, identifiable by the following file properties, is incorporated by reference in its entirety: Filename: A-2671-WO01-SEC_Sequence_Listing.XML, File Size: 8,686 bytes, Creation Date: March 6, 2023.

[0003] The present invention relates to a method for preparing a standardized expression cassette, and also to a method for recombining such a standardized expression cassette in a host cell in vivo. [Background technology]

[0004] Multispecific antibodies and multispecific antibody-like constructs have several attractive features for developers of therapeutic molecules. The clinical potential of multispecific antibodies that simultaneously target multiple targets, such as bispecific and trispecific antibodies, holds great promise in targeting complex diseases. However, the generation of these molecules poses significant challenges, especially when pairing the heavy and light chains of the antibody, due to the pairing / folding of novel quaternary structures composed of multiple polypeptide chains upon transfection into a single cell. Critical to the efficient and proper assembly of multiple polypeptide chain molecules is the expression of the different chains in the correct ratio within the cell. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention addresses this problem using a modular vector (ModVec) system for rapid construction of diverse expression vectors and vector libraries for recombinant protein production. ModVec enables rapid, high-throughput construction of both simple and complex vector designs, allowing optimization of expression vectors for individual molecules, for specific large molecule modalities, and / or for different expression hosts.

[0006] One major challenge in the production of multi-chain molecules is the variability in product quality due to the inconsistent expression ratio of the chains, which is both molecule-specific and specific to the development process of the cell host containing the expression vector elements. Currently, there are no methods for high-throughput determination of the optimal expression ratio of molecule-specific chains. The ModVec vector library provides a platform for rapid determination of the best expression configuration for individual multi-chain molecules. ModVec can be used in more generalized applications for assembling large complex DNA molecules for purposes other than expression vectors for antibody therapeutics. [Means for solving the problem]

[0007] In one aspect, the present invention provides a method for producing a. providing a polynucleotide sequence encoding at least two different polypeptides, said polynucleotide sequence being flanked on both the 5' and 3' sides by a cleavage site followed by a recognition site for a Type IIS restriction endonuclease; b. providing two or more sets of element arrays, each set of element arrays comprising together at least one functional expression cassette; each element sequence is flanked on both the 5' and 3' sides by a cleavage site followed by a recognition site for a type IIS restriction endonuclease; The recognition and cleavage sites for the type IIS restriction endonuclease are selected so that each set of element sequences can be assembled into a functional expression cassette; providing two or more sets of element arrays; c. providing at least one backbone entry vector, the backbone entry vector comprising a plasmid containing at least one cleavage site followed by a recognition site for a Type IIS restriction endonuclease and sequences for maintaining the vector in a bacterial cell; d. Assembling polynucleotide sequences encoding at least two different polypeptides with two or more sets of element sequences to generate functional expression cassettes in a backbone entry vector using a method based on the use of type IIS restriction enzyme digestion and ligation via cleavage sites and overhangs to obtain a ligated vector comprising at least two functional expression cassettes capable of expressing at least two different polypeptide chains; e. transfecting a mammalian cell with the ligated vector; f. comparing the level of the desired product produced with the level of the impurity of each of the at least two polypeptides; g. determining an optimal expression ratio of the two polypeptides for the transfected mammalian cells and identifying a transfected mammalian cell having an optimal expression ratio of at least two polypeptide chains; The present invention is directed to a method for expressing a multi-chain protein comprising at least two distinct polypeptide chains, comprising:

[0008] In one embodiment, the method comprises: h. Further comprising the step of identifying a configuration of vector elements that provides an optimal expression ratio of the at least two polypeptide chains.

[0009] In one aspect, the present invention is directed to a vector comprising an arrangement of vector elements that provides an optimal expression ratio of at least two polypeptide chains.

[0010] In one embodiment, the element sequence comprises at least two promoter sequences and at least two polyA sequences.

[0011] In one embodiment, the level of the desired product produced is measured by a method selected from the group consisting of reducing or non-reducing cation exchange chromatography, mass spectrometry, any other chromatographic separation method, or a combination thereof.

[0012] In one embodiment, none of the Type IIS restriction endonuclease cleavage sites produces a 4 nucleotide 5' overhang selected from the group consisting of GTAA, TCCA, and CACA upon cleavage with the Type IIS restriction endonuclease.

[0013] In one embodiment, the type IIS restriction endonuclease cleavage site is Selected from the group consisting of AGGT, AGTA, ATCA, CAGT, CCAT, GAAT, GAGG, GGCA, GGTC, TAGC, TCTT, GGAG and CCAC.

[0014] In one embodiment, the mammalian cell is selected from the group consisting of CHO cells, CHOK1 cells, DXB-11, DG-44, COS-7, HEK293, BHK, TM4, CV1, VERO-76, HELA, MDCK, BRL3A, W138, Hep G2, MMT060562, TRI cells, MRC5 cells, FS4 cells, and mammalian myeloma cells.

[0015] In one embodiment, the optimal expression ratio of the at least two polypeptides is selected from the group consisting of 1:1, 1:2 and 1:3.

[0016] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain and a second antibody light chain, wherein the first antibody heavy chain in combination with the first antibody light chain binds a first antigen or epitope and the second antibody heavy chain in combination with the second antibody light chain binds a second antigen or epitope, and the optimal expression ratio of the first antibody heavy chain to the first antibody light chain to the second antibody heavy chain to the second antibody light chain is 1:1:1:1.

[0017] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a second antibody heavy chain and a common antibody light chain, wherein the first antibody heavy chain is combined with the common antibody light chain to bind a first antigen or epitope and the second antibody heavy chain is combined with the common antibody light chain to bind a second antigen or epitope, and the optimal expression ratio of the first antibody heavy chain to the second antibody heavy chain to the common antibody light chain is 1:1:2.

[0018] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a first antibody light chain, a modified antibody heavy chain and a second antibody light chain; The modified antibody heavy chain comprises, from N-terminus to C-terminus, the following group: VH-CH1-binding domain (BD)-hinge-CH2-CH3, BD-VH-CH1-hinge-CH2-CH3, and VH-CH1-hinge-CH2-CH3-BD, and The BD is selected from either a single chain Fv (scFv) or a single chain Fab (scFab); the antibody heavy chain combines with a first antibody light chain to bind a first antigen or epitope, the VH of the modified antibody heavy chain combines with a second antibody light chain to bind a second antigen or epitope, and the BD binds a third antigen or epitope; and The optimal expression ratio of the first antibody heavy chain to the first antibody light chain to the modified antibody heavy chain to the second antibody light chain is 1:1:1:1.

[0019] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a modified antibody heavy chain and a generic antibody light chain, The modified antibody heavy chain comprises, from N-terminus to C-terminus, the following group: VH-CH1-binding domain (BD)-hinge-CH2-CH3, BD-VH-CH1-hinge-CH2-CH3, and VH-CH1-hinge-CH2-CH3-BD, and The BD is selected from either a single chain Fv (scFv) or a single chain Fab (scFab); The antibody heavy chain combines with a common antibody light chain to bind a first antigen or epitope, the VH of the modified antibody heavy chain combines with a common antibody light chain to bind a first antigen or epitope, and the BD binds a second antigen or epitope; and The optimal expression ratio of the first antibody heavy chain, the modified antibody heavy chain and the common antibody light chain is 1:1:2. [Brief description of the drawings]

[0020] [Figure 1A]("ModVec") represents a modular vector platform for high-throughput vector engineering. Fig. 1A) Exemplary sequences of DNA modules. Each module (indicated by N and shaded gray) is flanked by Golden Gate adaptors (boxed regions) containing recognition sites for BsmBI (highlighted in bold). BsmBI cleavage sites are labeled with ^ and overhangs generated by BsmI digestion are underlined. Fig. 1B) Assembly of expression vectors is achieved by a Golden Gate reaction using BsmBI with predefined complementary overhangs and flanking vector elements, plus the expression vector backbone. Fig. 1C) Schematic of the 11-piece (including vector backbone) ModVec assembly of 11 kb. ITR: inverted terminal repeats of piggyBac transposon. CDS: coding sequence. Fig. 1D) Using ModVec, all 19 constructs were successfully assembled in a single round of cloning. 96% of the colonies (109 out of 114) had correctly assembled constructs. Figure 1E) Schematic of one-tube vector library assembly. Figure 1F) Efficient one-tube vector library construction using ModVec. 69 out of 72 construct candidates were obtained by evaluating 350 colonies. [Figure 1B]("ModVec") represents a modular vector platform for high-throughput vector engineering. Fig. 1A) Exemplary sequences of DNA modules. Each module (indicated by N and shaded gray) is flanked by Golden Gate adaptors (boxed regions) containing recognition sites for BsmBI (highlighted in bold). BsmBI cleavage sites are labeled with ^ and overhangs generated by BsmI digestion are underlined. Fig. 1B) Assembly of expression vectors is achieved by a Golden Gate reaction using BsmBI with predefined complementary overhangs and flanking vector elements, plus the expression vector backbone. Fig. 1C) Schematic of the 11-piece (including vector backbone) ModVec assembly of 11 kb. ITR: inverted terminal repeats of piggyBac transposon. CDS: coding sequence. Fig. 1D) Using ModVec, all 19 constructs were successfully assembled in a single round of cloning. 96% of the colonies (109 out of 114) had correctly assembled constructs. Figure 1E) Schematic of one-tube vector library assembly. Figure 1F) Efficient one-tube vector library construction using ModVec. 69 out of 72 construct candidates were obtained by evaluating 350 colonies. [Figure 1C]("ModVec") represents a modular vector platform for high-throughput vector engineering. Fig. 1A) Exemplary sequences of DNA modules. Each module (indicated by N and shaded gray) is flanked by Golden Gate adaptors (boxed regions) containing recognition sites for BsmBI (highlighted in bold). BsmBI cleavage sites are labeled with ^ and overhangs generated by BsmI digestion are underlined. Fig. 1B) Assembly of expression vectors is achieved by a Golden Gate reaction using BsmBI with predefined complementary overhangs and flanking vector elements, plus the expression vector backbone. Fig. 1C) Schematic of the 11-piece (including vector backbone) ModVec assembly of 11 kb. ITR: inverted terminal repeats of piggyBac transposon. CDS: coding sequence. Fig. 1D) Using ModVec, all 19 constructs were successfully assembled in a single round of cloning. 96% of the colonies (109 out of 114) had correctly assembled constructs. Figure 1E) Schematic of one-tube vector library assembly. Figure 1F) Efficient one-tube vector library construction using ModVec. 69 out of 72 construct candidates were obtained by evaluating 350 colonies. [Figure 1D]("ModVec") represents a modular vector platform for high-throughput vector engineering. Fig. 1A) Exemplary sequences of DNA modules. Each module (indicated by N and shaded gray) is flanked by Golden Gate adaptors (boxed regions) containing recognition sites for BsmBI (highlighted in bold). BsmBI cleavage sites are labeled with ^ and overhangs generated by BsmI digestion are underlined. Fig. 1B) Assembly of expression vectors is achieved by a Golden Gate reaction using BsmBI with predefined complementary overhangs and flanking vector elements, plus the expression vector backbone. Fig. 1C) Schematic of the 11-piece (including vector backbone) ModVec assembly of 11 kb. ITR: inverted terminal repeats of piggyBac transposon. CDS: coding sequence. Fig. 1D) Using ModVec, all 19 constructs were successfully assembled in a single round of cloning. 96% of the colonies (109 out of 114) had correctly assembled constructs. Figure 1E) Schematic of one-tube vector library assembly. Figure 1F) Efficient one-tube vector library construction using ModVec. 69 out of 72 construct candidates were obtained by evaluating 350 colonies. [Figure 1E]("ModVec") represents a modular vector platform for high-throughput vector engineering. Fig. 1A) Exemplary sequences of DNA modules. Each module (indicated by N and shaded gray) is flanked by Golden Gate adaptors (boxed regions) containing recognition sites for BsmBI (highlighted in bold). BsmBI cleavage sites are labeled with ^ and overhangs generated by BsmI digestion are underlined. Fig. 1B) Assembly of expression vectors is achieved by a Golden Gate reaction using BsmBI with predefined complementary overhangs and flanking vector elements, plus the expression vector backbone. Fig. 1C) Schematic of the 11-piece (including vector backbone) ModVec assembly of 11 kb. ITR: inverted terminal repeats of piggyBac transposon. CDS: coding sequence. Fig. 1D) Using ModVec, all 19 constructs were successfully assembled in a single round of cloning. 96% of the colonies (109 out of 114) had correctly assembled constructs. Figure 1E) Schematic of one-tube vector library assembly. Figure 1F) Efficient one-tube vector library construction using ModVec. 69 out of 72 construct candidates were obtained by evaluating 350 colonies. [Figure 1F]("ModVec") represents a modular vector platform for high-throughput vector engineering. Fig. 1A) Exemplary sequences of DNA modules. Each module (indicated by N and shaded gray) is flanked by Golden Gate adaptors (boxed regions) containing recognition sites for BsmBI (highlighted in bold). BsmBI cleavage sites are labeled with ^ and overhangs generated by BsmI digestion are underlined. Fig. 1B) Assembly of expression vectors is achieved by a Golden Gate reaction using BsmBI with predefined complementary overhangs and flanking vector elements, plus the expression vector backbone. Fig. 1C) Schematic of the 11-piece (including vector backbone) ModVec assembly of 11 kb. ITR: inverted terminal repeats of piggyBac transposon. CDS: coding sequence. Fig. 1D) Using ModVec, all 19 constructs were successfully assembled in a single round of cloning. 96% of the colonies (109 out of 114) had correctly assembled constructs. Figure 1E) Schematic of one-tube vector library assembly. Figure 1F) Efficient one-tube vector library construction using ModVec. 69 out of 72 construct candidates were obtained by evaluating 350 colonies. [Figure 2A] It is shown that the choice of promoter significantly impacts manufacturability. Figure 2A) Schematic of vector construction. Both LC and HC genes were under the control of the same promoter (either promoter 1 or promoter 2). The influence of the promoter on the manufacturability of standard mAb A, bsAb B and bsAb C is shown in Figure 2B, Figure 2C and Figure 2D, respectively. [Figure 2B] It is shown that the choice of promoter significantly impacts manufacturability. Figure 2A) Schematic of vector construction. Both LC and HC genes were under the control of the same promoter (either promoter 1 or promoter 2). The influence of the promoter on the manufacturability of standard mAb A, bsAb B and bsAb C is shown in Figure 2B, Figure 2C and Figure 2D, respectively. [Figure 2C]It is shown that the choice of promoter significantly impacts manufacturability. Figure 2A) Schematic of vector construction. Both LC and HC genes were under the control of the same promoter (either promoter 1 or promoter 2). The influence of the promoter on the manufacturability of standard mAb A, bsAb B and bsAb C is shown in Figure 2B, Figure 2C and Figure 2D, respectively. [Figure 2D] It is shown that the choice of promoter significantly impacts manufacturability. Figure 2A) Schematic of vector construction. Both LC and HC genes were under the control of the same promoter (either promoter 1 or promoter 2). The influence of the promoter on the manufacturability of standard mAb A, bsAb B and bsAb C is shown in Figure 2B, Figure 2C and Figure 2D, respectively. [Figure 3A] Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 3B]Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 3C] Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 3D]Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 3E] Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 3F]Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 3G] Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 3H]Figure 3 shows the improvement of productivity of four-chain hetero-IgG using vector construction library screening. Figure 3A) Schematic of vector construction for three scaled-up pools. Figure 3B) LC1:LC2 ratios of ProA purified samples from pools 905, 910 and 911 before scale-up for further analysis. Figure 3C) ProA yields of scaled-up pools 905, 910 and 911. Figure 3D) Cation exchange purification recovery of the three pools. Figure 3E) Final yield of correctly assembled hetero-IgG-D after cation exchange. Cation exchange chromatograms of ProA purified samples from pools 905, 910 and 911 are shown in Figure 3F, Figure 3G and Figure 3H, respectively. The identity of each peak was determined by mass spectrometry. [Figure 4A]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 4B]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 4C]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 4D]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 4E]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 4F]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 4G]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 4H]Figure 4A) Schematic of vector construct library design. Three different construct groups were tested: single LC, dual LC A and dual LC B. In each construct, three different promoters were used to control the transcription of the polypeptide chain: promoter 1, promoter 2 and promoter 3. Figure 4B) ProA yield of all vector constructs. The average ProA yield of vector constructs in dual LC A, dual LC B and single LC groups was 117.2±43.4mg / L, 112.6±39.2mg / L and 40.7±20.73mg / L, respectively. Values ​​are expressed as mean±standard deviation. Figure 4C) Product quality of ProA purified samples from all vector constructs. The average nrMCE%MP of vector constructs in dual LC A, dual LC B and single LC groups was 75.6±14.9%, 72.4±16.6% and 71.7±21.6%, respectively. FIG. 4D) Final yield of correctly assembled tsAb E for all vector constructs. The average final yields of vector constructs in the duplex LC A, duplex LC B and single LC groups were 88.7±33.6mg / L, 81.4±35.6mg / L and 28.5±16.8mg / L, respectively. FIG. 4E) ProA yields of stable pools generated from defined and optimal constructs using standard cell line development protocols. The optimal pool had a 124% increase in ProA yield compared to the defined pool. FIG. 4F) Product quality of ProA samples derived from defined and optimal pools was comparable. FIG. 4G) Final yield of tsAb E was 114mg / L from the defined pool and 257mg / L from the optimal pool. The optimal pool had a 125% increase in final yield compared to the defined pool. FIG. 4H) Schematic of optimal vector constructs identified by library screening and our platform vector constructs. [Figure 5A]Vector construct library screening for difficult to express heterodimer IgG. Figure 5A) Schematic of vector constructs used. Expression of heterodimer IgG D was effected by co-transfection of two bicistronic vectors containing genes for all four polypeptide chains. Config 1 vector has LC and HC genes from the same mAb in the same bicistronic vector. Config 2 vector has LC and HC genes in separate bicistronic vectors. Figure 5B) ProA yield of all stable CHO cell pools recovered. 29 pools out of 44 pools were recovered. Figure 5C) LC1:LC2 ratio of ProA purified samples from all pools. Pools marked with an asterisk were scaled up for further analysis. Figure 5D) Product quality of all pools measured by non-reduced MCE main peak and SEC main peak. [Figure 5B] Vector construct library screening for difficult to express heterodimer IgG. Figure 5A) Schematic of vector constructs used. Expression of heterodimer IgG D was effected by co-transfection of two bicistronic vectors containing genes for all four polypeptide chains. Config 1 vector has LC and HC genes from the same mAb in the same bicistronic vector. Config 2 vector has LC and HC genes in separate bicistronic vectors. Figure 5B) ProA yield of all stable CHO cell pools recovered. 29 pools out of 44 pools were recovered. Figure 5C) LC1:LC2 ratio of ProA purified samples from all pools. Pools marked with an asterisk were scaled up for further analysis. Figure 5D) Product quality of all pools measured by non-reduced MCE main peak and SEC main peak. [Figure 5C]Vector construct library screening for difficult to express heterodimer IgG. Figure 5A) Schematic of vector constructs used. Expression of heterodimer IgG D was effected by co-transfection of two bicistronic vectors containing genes for all four polypeptide chains. Config 1 vector has LC and HC genes from the same mAb in the same bicistronic vector. Config 2 vector has LC and HC genes in separate bicistronic vectors. Figure 5B) ProA yield of all stable CHO cell pools recovered. 29 pools out of 44 pools were recovered. Figure 5C) LC1:LC2 ratio of ProA purified samples from all pools. Pools marked with an asterisk were scaled up for further analysis. Figure 5D) Product quality of all pools measured by non-reduced MCE main peak and SEC main peak. [Figure 5D] Vector construct library screening for difficult to express heterodimer IgG. Figure 5A) Schematic of vector constructs used. Expression of heterodimer IgG D was effected by co-transfection of two bicistronic vectors containing genes for all four polypeptide chains. Config 1 vector has LC and HC genes from the same mAb in the same bicistronic vector. Config 2 vector has LC and HC genes in separate bicistronic vectors. Figure 5B) ProA yield of all stable CHO cell pools recovered. 29 pools out of 44 pools were recovered. Figure 5C) LC1:LC2 ratio of ProA purified samples from all pools. Pools marked with an asterisk were scaled up for further analysis. Figure 5D) Product quality of all pools measured by non-reduced MCE main peak and SEC main peak. [Figure 6A]It is shown that gene location in an expression vector can significantly affect manufacturability. Figure 6A) Schematic of DA2 and DB2 constructs. Both constructs used promoter 2 to drive expression of all polypeptide chains. Figure 6B) Pool DA2 had higher ProA yields than pool DB2. Figure 6C) Product quality of ProA purified samples from pool DA2 and pool DB2 was comparable. Figure 6D) Pool DA2 had higher final yields of tsAb E than pool DB2. Figure 6E) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DA2. For the ProA purified samples, the relative peak areas were 10.1 for half mAb1 and 7.4 for half mAb2. Figure 6F) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DB2. The relative peak areas were 5.8 for half mAb1 and 9.8 for half mAb2. FIG. 6G) The half mAb1:half mAb2 relative peak ratio for the ProA-purified sample from pool DA2 was 1.4, whereas for the ProA-purified sample from pool DB2 this value was 0.6. [Figure 6B]It is shown that gene location in an expression vector can significantly affect manufacturability. Figure 6A) Schematic of DA2 and DB2 constructs. Both constructs used promoter 2 to drive expression of all polypeptide chains. Figure 6B) Pool DA2 had higher ProA yields than pool DB2. Figure 6C) Product quality of ProA purified samples from pool DA2 and pool DB2 was comparable. Figure 6D) Pool DA2 had higher final yields of tsAb E than pool DB2. Figure 6E) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DA2. For the ProA purified samples, the relative peak areas were 10.1 for half mAb1 and 7.4 for half mAb2. Figure 6F) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DB2. The relative peak areas were 5.8 for half mAb1 and 9.8 for half mAb2. FIG. 6G) The half mAb1:half mAb2 relative peak ratio for the ProA-purified sample from pool DA2 was 1.4, whereas for the ProA-purified sample from pool DB2 this value was 0.6. [Figure 6C]It is shown that gene location in an expression vector can significantly affect manufacturability. Figure 6A) Schematic of DA2 and DB2 constructs. Both constructs used promoter 2 to drive expression of all polypeptide chains. Figure 6B) Pool DA2 had higher ProA yields than pool DB2. Figure 6C) Product quality of ProA purified samples from pool DA2 and pool DB2 was comparable. Figure 6D) Pool DA2 had higher final yields of tsAb E than pool DB2. Figure 6E) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DA2. For the ProA purified samples, the relative peak areas were 10.1 for half mAb1 and 7.4 for half mAb2. Figure 6F) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DB2. The relative peak areas were 5.8 for half mAb1 and 9.8 for half mAb2. FIG. 6G) The half mAb1:half mAb2 relative peak ratio for the ProA-purified sample from pool DA2 was 1.4, whereas for the ProA-purified sample from pool DB2 this value was 0.6. [Figure 6D]It is shown that gene location in an expression vector can significantly affect manufacturability. Figure 6A) Schematic of DA2 and DB2 constructs. Both constructs used promoter 2 to drive expression of all polypeptide chains. Figure 6B) Pool DA2 had higher ProA yields than pool DB2. Figure 6C) Product quality of ProA purified samples from pool DA2 and pool DB2 was comparable. Figure 6D) Pool DA2 had higher final yields of tsAb E than pool DB2. Figure 6E) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DA2. For the ProA purified samples, the relative peak areas were 10.1 for half mAb1 and 7.4 for half mAb2. Figure 6F) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DB2. The relative peak areas were 5.8 for half mAb1 and 9.8 for half mAb2. FIG. 6G) The half mAb1:half mAb2 relative peak ratio for the ProA-purified sample from pool DA2 was 1.4, whereas for the ProA-purified sample from pool DB2 this value was 0.6. [Figure 6E]It is shown that gene location in an expression vector can significantly affect manufacturability. Figure 6A) Schematic of DA2 and DB2 constructs. Both constructs used promoter 2 to drive expression of all polypeptide chains. Figure 6B) Pool DA2 had higher ProA yields than pool DB2. Figure 6C) Product quality of ProA purified samples from pool DA2 and pool DB2 was comparable. Figure 6D) Pool DA2 had higher final yields of tsAb E than pool DB2. Figure 6E) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DA2. For the ProA purified samples, the relative peak areas were 10.1 for half mAb1 and 7.4 for half mAb2. Figure 6F) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DB2. The relative peak areas were 5.8 for half mAb1 and 9.8 for half mAb2. FIG. 6G) The half mAb1:half mAb2 relative peak ratio for the ProA-purified sample from pool DA2 was 1.4, whereas for the ProA-purified sample from pool DB2 this value was 0.6. [Figure 6F]It is shown that gene location in an expression vector can significantly affect manufacturability. Figure 6A) Schematic of DA2 and DB2 constructs. Both constructs used promoter 2 to drive expression of all polypeptide chains. Figure 6B) Pool DA2 had higher ProA yields than pool DB2. Figure 6C) Product quality of ProA purified samples from pool DA2 and pool DB2 was comparable. Figure 6D) Pool DA2 had higher final yields of tsAb E than pool DB2. Figure 6E) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DA2. For the ProA purified samples, the relative peak areas were 10.1 for half mAb1 and 7.4 for half mAb2. Figure 6F) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DB2. The relative peak areas were 5.8 for half mAb1 and 9.8 for half mAb2. FIG. 6G) The half mAb1:half mAb2 relative peak ratio for the ProA-purified sample from pool DA2 was 1.4, whereas for the ProA-purified sample from pool DB2 this value was 0.6. [Figure 6G]It is shown that gene location in an expression vector can significantly affect manufacturability. Figure 6A) Schematic of DA2 and DB2 constructs. Both constructs used promoter 2 to drive expression of all polypeptide chains. Figure 6B) Pool DA2 had higher ProA yields than pool DB2. Figure 6C) Product quality of ProA purified samples from pool DA2 and pool DB2 was comparable. Figure 6D) Pool DA2 had higher final yields of tsAb E than pool DB2. Figure 6E) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DA2. For the ProA purified samples, the relative peak areas were 10.1 for half mAb1 and 7.4 for half mAb2. Figure 6F) Electropherograms (non-reduced microcapillary electrophoresis) of ProA purified samples from pool DB2. The relative peak areas were 5.8 for half mAb1 and 9.8 for half mAb2. FIG. 6G) The half mAb1:half mAb2 relative peak ratio for the ProA-purified sample from pool DA2 was 1.4, whereas for the ProA-purified sample from pool DB2 this value was 0.6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The method of the present invention allows the production of a desired expression cassette from each set of element sequences by assembling a nucleic acid fragment construct via single-stranded overhangs formed at both ends of the nucleic acid fragment using a type II restriction endonuclease. In the present invention, a type II restriction enzyme may be used. A type II restriction endonuclease recognition site is a recognition site for a restriction endonuclease that recognizes double-stranded DNA and cleaves the double-stranded DNA at a cleavage site that is outside the recognition site of the double-stranded DNA. A type II restriction endonuclease cleaves so that an overhang of 3 to 6 nucleotides is produced, depending on the particular type II restriction endonuclease. Typically, an enzyme that produces an overhang of 4 nucleotides may be used in the method of the present invention. However, it is also possible to use a type II endonuclease that produces a longer single-stranded overhang. The range of nucleotides that forms an overhang upon cleavage is referred to herein as the cleavage site. Because the nucleotide at the cleavage site is not part of the recognition site, it can be chosen as desired without destroying the cleavage activity of the Type II restriction endonuclease.

[0022] Any type II restriction enzyme that provides sufficient "sticky" ends for efficient ligation at the cleavage site can be used to practice the invention. A selection of such enzymes is described on the REBASE web page (rebase.neb.com / cgi-bin / asymmlist) and in the review by Szybalsky et al. (1991, Gene, 100:13-26). Most preferred are the following type II restriction endonucleases: Bsal, Bbsl, BsmBI, Sapl, BspMI, Aarl, Esp3l, Bpil and Hgal. Many of the listed restriction endonucleases are available from New England Biolabs. Sources of these enzymes can also be found on the REBASE web page mentioned above. Type II restriction enzymes with asymmetric recognition sites (such as those shown on this web page) that have a cutting site outside of the recognition site and provide an overhang of at least 3, preferably 4 or more nucleotide residues upon cutting (e.g., BsmBI, BN736I, BpuAI, VpaK321, SfaNI, etc.) can be used in the present invention. In one embodiment, the type II restriction endonuclease is BsmBI.

[0023] It is recommended that the recognition site contains at least 4, more preferably at least 6 or more base pairs, to minimize the possibility that such a recognition site will be found in the sequence portion of interest. Type II restriction nucleases (e.g., SfaNI) with a 5 bp recognition site can also be used. Type II restriction endonucleases that produce 4 nt single-stranded overhangs at each end of the digested fragment can generate ends that can theoretically have 256 different sequences. Type II restriction enzymes have been designed with even longer recognition sites, for example, containing 10 or more base pairs. In one embodiment, the recognition site is 5'-CGTCTC-3'. In one embodiment, when cleaved by an IIS restriction endonuclease, the 5' overhang is 4 nucleotides long, but is not selected from the group consisting of GTAA, TCCA, and CACA. In one embodiment, when cleaved by the IIS restriction endonuclease, the 5' overhang is 4 nucleotides in length and is selected from the group consisting of AGGT, AGTA, ATCA, CAGT, CCAT, GAAT, GAGG, GGCA, GGTC, TAGC, TCTT, GGAG, and CCAC.

[0024] Examples of ligases to be used in the present invention include T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, all of which are commercially available from New England Biolabs.

[0025] In one aspect, the present invention provides a method for producing a. providing a polynucleotide sequence encoding at least two different polypeptides, said polynucleotide sequence being flanked on both the 5' and 3' sides by a cleavage site followed by a recognition site for a Type IIS restriction endonuclease; b. providing two or more sets of element arrays, each set of element arrays comprising together at least one functional expression cassette; each element sequence is flanked on both the 5' and 3' sides by a cleavage site followed by a recognition site for a type IIS restriction endonuclease; The recognition and cleavage sites for the type IIS restriction endonuclease are selected so that each set of element sequences can be assembled into a functional expression cassette; providing two or more sets of element arrays; c. providing at least one backbone entry vector, the backbone entry vector comprising a plasmid containing at least one cleavage site followed by a recognition site for a Type IIS restriction endonuclease and sequences for maintaining the vector in a bacterial cell; d. Assembling polynucleotide sequences encoding at least two different polypeptides with two or more sets of element sequences to generate functional expression cassettes in a backbone entry vector using a method based on the use of type IIS restriction enzyme digestion and ligation via cleavage sites and overhangs to obtain a ligated vector comprising at least two functional expression cassettes capable of expressing at least two different polypeptide chains; e. transfecting a mammalian cell with the ligated vector; f. comparing the level of the desired product produced with the level of the impurity of each of the at least two polypeptides; g. determining an optimal expression ratio of the two polypeptides for the transfected mammalian cells and identifying a transfected mammalian cell having an optimal expression ratio of at least two polypeptide chains; The present invention is directed to a method for expressing a multi-chain protein comprising at least two distinct polypeptide chains, comprising:

[0026] In one embodiment, the method comprises: h. Further comprising the step of identifying a configuration of vector elements that provides an optimal expression ratio of the at least two polypeptide chains.

[0027] In one aspect, the present invention is directed to a vector comprising an arrangement of vector elements that provides an optimal expression ratio of at least two polypeptide chains.

[0028] Each set of element sequences can typically be assembled as an expression cassette. Expression cassette in the context of the present invention is intended to mean a nucleic acid sequence that directs the cellular machinery to make RNA and protein. Typically, an expression cassette will include a coding sequence and a sequence that controls the expression of the coding sequence. Typically, an expression cassette may include at least a promoter, an open reading frame, and a termination sequence. Other element sequences include control sequences, insulator sequences, barcode DNA sequences, primer sequences, promoter sequences, polyA sequences, and IRES sequences, as well as mammalian selectable marker sequences. In one embodiment, the element sequence includes at least two promoter sequences and at least two polyA sequences.

[0029] The term "control sequence" is defined herein to include all components that are necessary or advantageous for the production of an mRNA or a polypeptide either in vitro or in a host cell. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader sequence, a Shine-Dalgarno sequence, an optimal translation initiation sequence (described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, a pre-pro-peptide sequence, a promoter, a signal sequence, and a transcription termination signal. At a minimum, control sequences typically include a promoter and a transcription termination signal (termination or termination signal). Translation initiation and termination signals may also typically be present. Control sequences may be optimized for their particular purpose.

[0030] The term "promoter" is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to a precise downstream transcription start site of a nucleic acid sequence encoding a biological compound to initiate transcription. RNA polymerase efficiently catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region. The term "promoter" is also understood to include the 5'-non-coding region (between the promoter and the translation start) for post-transcriptional translation into mRNA, cis-acting transcriptional regulatory elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors.

[0031] The method of the present invention is typically carried out such that the elements of the expression cassette are assembled in an operably linked backbone entry vector. The terms "operably linked" or "operably linked" and the like are defined herein as a configuration in which a control sequence is appropriately positioned relative to the coding sequence of a DNA sequence such that the control sequence directs the production of an mRNA or polypeptide.

[0032] An "insulator sequence" or "insulator" is a nucleic acid segment that reduces or eliminates transcription from adjacent regions from affecting the nucleic acid segment with which the insulator is involved. Insulators are preferably placed upstream of other regulatory sequences and / or downstream of a gene. Insulators are preferably placed between different genes, transcription units, or gene domains to reduce or prevent interference of adjacent expressed sequences.

[0033] An "enhancer sequence" or "enhancer" functions to increase transcription from a promoter that is adjacent to the enhancer. Enhancers can function in either orientation, both upstream and downstream of a gene.

[0034] A "barcode DNA sequence" or "barcode" can be used to identify a nucleic acid molecule, for example, when sequencing can reveal a particular barcode bound to a nucleic acid molecule of interest. In some cases, when at least a portion of the barcode is found in a sequence-specific event, for example, when at least a portion of the barcode can participate in a ligation or extension reaction, the sequence-specific event can be used to identify the nucleic acid molecule. Thus, the barcode can allow for the identification, selection, or amplification of the DNA molecule bound to the barcode.

[0035] "IRES" or "internal ribosome entry site" refers to a site that allows for sufficient internal ribosome entry to initiate translation in an assay for cap-independent translation, such as the bicistronic reporter assay described herein. The presence of an IRES allows for cap-independent translation of a sequence encoding an associated protein that would not be translated in its absence.

[0036] A "selectable marker gene" or "selectable marker" encodes a protein necessary for the survival and growth of a host cell grown in a selective medium. Typical selectable marker genes encode (a) a protein that confers resistance to antibiotics or other toxins, such as ampicillin, tetracycline or kanamycin, on the prokaryotic host cell, (b) a protein that complements an auxotrophic deficiency of the cell, or (c) a protein that supplies a vital nutrient that is not available from a complex or defined medium. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

[0037] Other selectable genes may be used to amplify the expressed gene. Amplification is the process by which genes required for the production of a protein critical for growth or cell survival are repeated in tandem in the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoterless thymidine kinase gene. The mammalian cell transformant is placed under selection pressure in which the transformant is only adapted to survive due to the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions of successively increasing concentrations of the selection agent in the medium, thereby leading to the amplification of both the selectable gene and the DNA encoding another gene, such as one or more components of the multi-chain protein construct described herein. As a result, large amounts of polypeptides are synthesized from the amplified DNA.

[0038] Thus, an element in the context of the present invention is any component of an expression cassette. A set of elements is a group of elements that can be combined to produce an expression cassette. The method of the present invention requires the provision of two sets of element sequences. This means that sufficient elements should be provided so that at least two different expression cassettes can be obtained. This implies that at least two different species of at least one element must be provided. That is, for the purposes of the present invention, one promoter is combined with two ORFs and two termination signals to constitute two sets of element sequences.

[0039] In the methods of the invention, typically at least two of the sets of element sequences include a promoter element, an open reading frame element and a termination signal element.

[0040] In the methods according to the invention, one or more of the sets of elements may include "partial" element sequences, such as UTRs, signal peptides and split open reading frames.

[0041] Each set of element sequences is provided in a form such that the set can be assembled into a functional expression cassette in a backbone entry vector. Typically, each element is then flanked on both sides by a cleavage site followed by a recognition site for a type II restriction endonuclease, and the recognition site and cleavage site for the type II restriction endonuclease are selected such that each set of element sequences can be assembled into a functional expression cassette. Thus, each element sequence and adjacent sequences typically includes, in order from one end to the other, a type II restriction endonuclease recognition site, its cleavage site, the element sequence, a type II restriction endonuclease cleavage site, its recognition site.

[0042] Thus, each set of elements is prepared or provided in a suitable vector with recognition sites and standardized cleavage sites (preferably 4-bp) for type II restriction endonucleases selected such that after assembly using a one-pot method such as Golden gate cloning, a functional expression cassette is formed.

[0043] A set of backbone entry vectors are prepared or provided that contain left and right connector sequences suitable for assembly using sequence homology.

[0044] A subset of element sequences are selected along with a backbone (bbn) entry vector, which can be assembled, for example using Golden Gate cloning, into a functional expression cassette contained within the backbone entry vector.

[0045] Exemplary host cells include prokaryotic, yeast or higher eukaryotic cells. Prokaryotic host cells include eubacteria, such as gram-negative or gram-positive microorganisms, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescens, and the like. marcescens, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, and Streptomyces. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.However, Pichia, e.g. P. pastoris, Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, Schwanniomyces, e.g. Schwanniomyces occidentalis, Several other genera, species, and strains are generally available and useful herein, including A. occidentalis, and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger.

[0046] Host cells for the expression of glycosylated antigen-binding proteins can be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many strains and variants of baculoviruses have been identified, as well as corresponding insect permissive host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. Various virus strains for transfecting such cells are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

[0047] Vertebrate host cells are also suitable hosts, and the recombinant production of antigen binding proteins from such cells is routine. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980), monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651), human embryonic kidney lines (293 cells or 293 cells subcloned for growth in suspension culture) (Graham et al., J. Gen Virol. 36:59, 1977), baby hamster kidney cells (BHK, ATCC CCL10), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL2), dog kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocellular carcinoma cells (Hep G2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982), MRC5 cells or FS4 cells, mammalian myeloma cells, and several other cell lines. In certain embodiments, cell lines may be selected by determining which cell lines have high expression levels of the multi-chain protein of the invention and constitutively produce it, hi another embodiment, cell lines derived from the B cell lineage may be selected that do not produce their own antibodies but have the ability to produce and secrete heterologous antibodies.In some embodiments, CHO cells are host cells for expressing the multi-chain proteins of the present invention.

[0048] Host cells are transformed or transfected with the above nucleic acids or vectors for the production of multi-chain proteins and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. In addition, novel vectors and transfected cell lines with multiple copies of transcription units separated by selectable markers are particularly useful for expressing antigen binding proteins. Thus, the present invention also provides a method for preparing the multi-chain proteins described herein, comprising culturing a host cell containing one or more expression vectors described herein in a culture medium under conditions that allow expression of the multi-chain protein encoded by the one or more expression vectors, and recovering the multi-chain protein from the culture medium.

[0049] The host cells used to produce the antigen binding proteins of the invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44, 1979; Barnes et al., Anal. Biochem. 102:255, 1980, U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S. Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, or U.S. Pat. No. 5,122,469, WO 90103430, WO 87 / 00195, or U.S. Reissue Pat. No. 30,985 can be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug Gentamycin™), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary nutritional supplements known to those of skill in the art may also be included at appropriate concentrations. Culture conditions such as temperature and pH will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0050] When the host cells are cultured, the multi-chain protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antigen-binding protein is produced intracellularly, as a first step, particulate debris (host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Bispecific antigen-binding proteins can be purified, for example, using hydroxyapatite chromatography, cation exchange chromatography or anion exchange chromatography, or affinity chromatography using the antigen of interest or protein A or protein G as affinity ligands. Protein A can be used to purify proteins, including polypeptides based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13, 1983). Protein G is recommended for all mouse isotypes and human gamma 3 (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. If the protein contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the particular multichain protein to be recovered, other techniques for protein purification are also possible, such as ethanol precipitation, reversed-phase HPLC, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0051] As used herein, the term "antibody" refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (each of about 25 kDa) and two heavy chain polypeptides (each of about 50-70 kDa). The term "light chain" or "immunoglobulin light chain" refers to a polypeptide comprising, from the amino terminus to the carboxyl terminus, a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL). The immunoglobulin light chain constant domain (CL) can be kappa (κ) or lambda (λ). The term "heavy chain" or "immunoglobulin heavy chain" refers to a polypeptide comprising, from the amino terminus to the carboxyl terminus, a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and optionally, immunoglobulin heavy chain constant domain 4 (CH4). Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α) and epsilon (ε) and define the antibody isotype as IgM, IgD, IgG, IgA and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, i.e., IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA and IgD antibodies have three domains (CH1, CH2 and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3 and CH4). Immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. Antibody chains are linked via interpolypeptide disulfide bonds between the CL and CH1 domains (i.e., between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0052] In human antibodies, CH1 refers to the region having the amino acid sequence at positions 118-215 of the EU index. A highly flexible amino acid region called the "hinge region" is located between CH1 and CH2. CH2 represents the region having the amino acid sequence at positions 231-340 of the EU index, and CH3 represents the region having the amino acid sequence at positions 341-446 of the EU index.

[0053] "CL" represents the constant region of the light chain. In the case of the κ chain of a human antibody, CL represents the region having the amino acid sequence at positions 108 to 214 of the EU index. In the case of the λ chain, CL represents the region having the amino acid sequence at positions 108 to 215 of the EU index.

[0054] Both the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), and the AHo numbering scheme (Honegger A. and Plueckthun AJ Mol Biol. 2001 Jun 8;309(3):657-70) can be used in the present invention. The amino acid positions of a given antibody as well as the complementarity determining regions (CDRs) and framework regions (FRs) can be identified using either system. For example, EU heavy chain positions 39, 44, 183, 356, 357, 360, 370, 392, 399, and 409 are identical to AHo heavy chain positions 46, 51, 230, 484, 485, 491, 501, 528, 535, and 551, respectively.

[0055] In one embodiment, the optimal expression ratio of the at least two polypeptides is selected from the group consisting of 1:1, 1:2 and 1:3.

[0056] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain and a second antibody light chain, wherein the first antibody heavy chain in combination with the first antibody light chain binds a first antigen or epitope and the second antibody heavy chain in combination with the second antibody light chain binds a second antigen or epitope, and the optimal expression ratio of the first antibody heavy chain to the first antibody light chain to the second antibody heavy chain to the second antibody light chain is 1:1:1:1.

[0057] In one embodiment, the multi-chain protein comprises a first antibody heavy chain, a second antibody heavy chain and a common antibody light chain, wherein the first antibody heavy chain is combined with the common antibody light chain to bind a first antigen or epitope and the second antibody heavy chain is combined with the common antibody light chain to bind a second antigen or epitope, and the optimal expression ratio of the first antibody heavy chain to the second antibody heavy chain to the common antibody light chain is 1:1:2.

[0058] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a first antibody light chain, a modified antibody heavy chain and a second antibody light chain; The modified antibody heavy chain comprises, from N-terminus to C-terminus, the following group: VH-CH1-binding domain (BD)-hinge-CH2-CH3, BD-VH-CH1-hinge-CH2-CH3, and VH-CH1-hinge-CH2-CH3-BD, and The BD is selected from either a single chain Fv (scFv) or a single chain Fab (scFab); the antibody heavy chain combines with a first antibody light chain to bind a first antigen or epitope, the VH of the modified antibody heavy chain combines with a second antibody light chain to bind a second antigen or epitope, and the BD binds a third antigen or epitope; and The optimal expression ratio of the first antibody heavy chain to the first antibody light chain to the modified antibody heavy chain to the second antibody light chain is 1:1:1:1.

[0059] In one embodiment, the multi-chain protein comprises an antibody heavy chain, a modified antibody heavy chain and a generic antibody light chain, The modified antibody heavy chain comprises, from N-terminus to C-terminus, the following group: VH-CH1-binding domain (BD)-hinge-CH2-CH3, BD-VH-CH1-hinge-CH2-CH3, and VH-CH1-hinge-CH2-CH3-BD, and The BD is selected from either a single chain Fv (scFv) or a single chain Fab (scFab); The antibody heavy chain combines with a common antibody light chain to bind a first antigen or epitope, the VH of the modified antibody heavy chain combines with a common antibody light chain to bind the first antigen or epitope, and the BD binds a second antigen or epitope; and The optimal expression ratio of the first antibody heavy chain, the modified antibody heavy chain and the common antibody light chain is 1:1:2.

[0060] A "binding domain" or "BD" typically contains an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not have to contain both. An Fd fragment, for example, has two VH regions and often retains some antigen-binding function of an intact antigen-binding domain. Further examples of formats of antibody fragments, antibody variants or binding domains include (1) Fab fragments, which are monovalent fragments having VL, VH, CL and CH1 domains, (2) F(ab')2 fragments, which are bivalent fragments having two Fab fragments linked by disulfide bridges at the hinge region, (3) Fd fragments having two VH domains and a CH1 domain, (4) Fv fragments having the VL and VH domains of one arm of an antibody, (5) dAb fragments having a VH domain (Ward et al., (1989) Nature 341:544-546), (6) isolated complementarity determining regions (CDRs), and (7) single chain Fvs (scFvs), the latter being preferred (e.g., from scFV libraries). Cation exchange chromatography ("CEX") is a form of ion exchange chromatography (IEX) and is used to separate molecules based on their net surface charge. Cation exchange chromatography, more specifically, uses a negatively charged ion exchange resin that has an affinity for molecules with a net positive surface charge. Cation exchange chromatography is used for both preparative and analytical purposes and can separate a wide range of molecules, from amino acids and nucleotides to large proteins. Herein, we focus on preparative cation exchange chromatography of proteins. CEX can be performed under reducing and non-reducing conditions.

[0061] Mass spectrometry ("MS") is an analytical technique used to measure the mass-to-charge ratio of ions, the results of which are represented as a mass spectrum, i.e., a plot of intensity as a function of mass-to-charge ratio.

[0062] The disclosure of each reference mentioned herein is incorporated herein by reference in its entirety.

[0063] The present invention is further illustrated by the following examples. EXAMPLES

[0064] ModVec is designed to be extremely flexible using a modular "slot-based" approach, allowing one or more expression cassettes to be assembled in a variety of possible configurations while allowing combinatorial exploration of sequence diversity within each slot. The Modvec system is divided into multiple slots, each of which can contain a DNA sequence with its own Golden Gate overhang. The DNA sequences can be vector elements, including those required for vector replication (ColE Ori, pMB1 Ori...), antibiotic resistance (Amp, Kan, Crm...), viral infection (LTR), transposition (ITR), episomal replication (OriP), targeted integration (loxP, Frt, attB / P), and elements for mammalian gene expression (e.g., promoters, enhancers, internal ribosome entry sites (IRES), self-processing viral 2A peptides, polyA signals, regulatory sequences, insulator sequences such as MAR and UCOE).

[0065] As a proof of principle, ModVec was first utilized to assemble complex mammalian expression vectors. Two ModVec backbones were generated, including pMVP5, which can be used in common mammalian cell lines used in research (HEK, CHO), and a vector backbone specifically designed to be compatible with the CHO cell line manufactured by Amgen. These vector backbones contain only the minimal vector elements required for replication and antibiotic selection in E. coli for plasmid maintenance.

[0066] In our first experiment, we tested the ModVec concept by assembling a vector with functional DNA sequences in all slots, a 14-slot (including the vector backbone), 8 kb DNA assembly. It contained three ECs, EC1 and EC2 were bicistronic, and EC3 was monocistronic. It had three different promoters to drive the expression of five coding sequences. It has been shown that using a circular plasmid as the entry point for the GG reaction improves the efficiency, especially for multi-piece golden gate assemblies (Potapov, Ong et al. 2018). Therefore, vector elements that were expected to be frequently reused within each slot were first cloned into the pGG_Cm_BsaI vector using GG with BsaI to generate a "parts" vector that was confirmed by Sanger sequencing. In this process, we assembled a "parts library" of vector elements such as promoters, IRES, polyA, regulatory sequences, insulators, etc., with overhangs suitable for ModVec, in a format that is easy to store and easy to replenish when needed. Using a combination of circular "parts" and synthesized linear DNA, a 20ul GG reaction was used with 1ul of each vector element (at concentrations between 40-70ng / ul) for the initial 14-slot assembly. pMVP5 was used as the vector backbone. The GG reaction was transformed into E. coli and 32 colonies were picked for Sanger sequencing. Although we were successful in obtaining an 8kB DNA vector design, the efficiency of correct assembly was only 3.125% (1 out of 32 correct colonies picked). Internal and external data (Kanigowska, Shen et al. 2016) indicated that GG efficiency could be increased with smaller reaction volumes (small-scale GG). Next, reaction volumes were reduced to 2ul, 1ul and 0.5ul and 14-slot assemblies were performed using the ECHO acoustic liquid handler, which allows for accurate liquid transfer even in these extremely small volumes. These reactions were proportionally scaled down from the initial 20ul reaction.Small-scale GG increased the efficiency of this 14-piece ModVec assembly to 16% (13 of 78 correct colonies).

[0067] Both successful and failed initial PoC ModVec assemblies were closely analyzed and in this context, it was determined that a specific set of 11GG overhangs was always correctly ligated. We then tested whether the efficiency of ModVec could be improved by using only this set of overhangs. ModVec was used to assemble 10 slot assemblies with these optimized overhangs in a 1ul GG reaction. Over 95% of the clones were found to contain correctly assembled vectors (548 of 576 correct colonies).

[0068] The 95% efficiency of assembly obtained under the optimized ModVec design and conditions allows one-tube construction of expression vector libraries. Functionally similar vector elements that had identical overhangs together were mixed to create a "slot library". Two different promoters driving 12 CDSs in EC1 were combined with three different promoters driving selectable marker expression EC3, including a bridge to exclude EC2, resulting in 72 different expression vectors by combinatorial assembly. After transformation, 350 colonies were picked, generating a total of 312 contigs after sequencing verification. These 312 contigs matched 69 of the 72 vector candidates, demonstrating low sequence-dependent bias in Modvec assembly and showing that library strategies are feasible for this system.

[0069] Materials and Methods: Vector construction All DNA fragments, including vector elements and antibody LC and HC, were synthesized by Twist Bioscience or PCR amplified from existing templates and cloned into the vector backbone using the standard Golden Gate assembly protocol (Engler et al., 2008). To generate partial vectors, each Golden Gate reaction contained 2 μL of DNA fragment (5 ng / μL) and 2 μL of pGG vector (20 ng / μL), 5 μM ATP (Thermo Fisher, R0441), 1 μL of FastDigest buffer (Thermo Fisher, B64) with 0.5 μL of T4 ligase (Thermo Fisher, EL0014) and 0.5 μL of BsaI (Thermo Fisher, ER0291), and 3 μL of H2O. Golden Gate reactions were performed in a thermal cycler for 15 cycles of 2 min at 37°C and 3 min at 16°C. A final 5 min incubation was performed at 85°C to inactivate all enzymes. The miniaturized Golden Gate reactions and one-pot vector library cloning reactions were set up using an ECHO525 liquid handler (Labcyte) to perform nanoliter-scale liquid transfers. The volumes of each DNA fragment and vector backbone were scaled down proportionally for the miniaturized Golden Gate reactions. For ModVec assembly, 15 μL of Golden Gate reaction mixture containing 2 μL of pGG partial vector (40-70 ng / μL), 2 μL of expression vector backbone (50 ng / μL), 1.5 μL of FastDigest buffer (Thermo Fisher, B64) with 5 μM ATP (Thermo Fisher, R0441), 0.75 μL of T4 ligase (Thermo Fisher, EL0014) and 0.75 μL of BsmBI (Thermo Fisher, FD0454), and a variable amount of H2O to 15 μL were mixed using a TECAN liquid handler. For ModVec assembly, for each Golden Gate cycle, the incubation at 37°C was extended to 3 min, the incubation at 16°C was extended to 5 min, and the number of cycles was increased to 20.Plasmid DNA was prepared using a Qiagen miniprep Kit (Qiagen, 27104). After sequencing confirmation, plasmid DNAs encoding HC and LC were mixed in a defined ratio.

[0070] Cell culture and protein expression To generate stable cell pools with random genomic integration, 25 μg of DNA was electroporated into proprietary suspension Chinese Hamster Ovary cells (CHO) using the Bio-Rad Gene Pulser® Xcell Electroporation System. After electroporation, the entire transfectant was plated in proprietary harvest medium. Viable cell density and viability were monitored using a VI-CELL® counter (Beckman Coulter) and medium was changed every 2-3 days until harvest. Harvest was defined as >85% viability by VI-CELL®. Harvested cells were used to seed 50 mL fed-batch products into shake flasks, which were harvested after 10 days. To generate stable cell pools using piggyBac transposase-based expression vectors, 4 µg of DNA was used to inoculate our own suspension CHO cells with glutamine synthetase knockout (CHO GS KO) at 4 × 10 ng / ml with Lipofectamine LTX (Thermo Fisher, 11668030). 6 Transfections were performed using viable cells / mL. Transfected cells were transferred to selection medium with methionine sulfoximine (MSX) 48-72 hours after transfection. Harvested cells were used to culture 4 mL of fed-batch product in 24-well culture blocks at 1 × 10 6Cells were seeded at 10000000 cells / mL and harvested after 10 days. During production, viable cell density and viability were monitored using a VI-CELL® counter (Beckman Coulter) and medium was changed on days 3, 6, and 8. On day 10, viable cell density and viability were measured and conditioned medium from these production batches was used to determine titers by ForteBio OCTET® Red with Protein A biosensor.

[0071] High-throughput protein purification The KingFisher® Flex system (Thermo Fisher) was used with magnetic ProA beads (GE Life Sciences) to purify proteins as previously described (Gong et al., 2021). Briefly, 1 day before harvest, 100 μL of magnetic ProA beads were added to 4 mL of CHO GS KO culture. The beads were collected and subjected to Kingfisher purification using a 24-probe magnetic head. After washing three times with phosphate-buffered saline and two times with Milli-Q water, the proteins were eluted with 500 μL of 100 mM sodium acetate (pH 3.6) for 10 min and then neutralized by adding 10 μL of 3 M Tris (pH 11.0).

[0072] Large-scale ProA purification followed by cation exchange chromatography Protein expressed in 50 mL CHO cultures was purified using ProA affinity capture (1 mL HiTrap MabSelect SuRe, GE Life Sciences, catalog no. GE11-0034-93) as previously described (Gong et al., 2021; Yoo et al., 2014) and eluted with 100 mM sodium acetate, pH 3.6, followed immediately by buffer exchange into 10 mM sodium acetate, 150 mM NaCl, pH 5.2, using a 5 mL HiTrap desalting column (GE Life Sciences, catalog no. GE17-1408-01).

[0073] Cation exchange chromatography was performed as previously described (Gong et al., 2021). Briefly, 1.5–1.8 mL of ProA purified sample was diluted with 20 mL of 20 mM MES (pH 6.2) and loaded onto a 1 mL cation exchange column (SP-HP HiTrap, GE Life Sciences, catalog no. GE29-0513-24) at 1 mL / min. After washing the column with 8 column volumes of the same buffer at 1 mL / min, the protein was eluted with a linear gradient of 0–400 mM NaCl over 40 column volumes at 0.4 ml / min. Fractions with ≥90% purity as measured by size exclusion chromatography (SEC) were pooled and their concentrations were measured using a Multiscan GO microplate reader (Thermo Fisher) as previously described (Winters et al., 2015). The final pooled samples were analyzed by SEC non-reducing microcapillary electrophoresis (nrMCE) and liquid chromatography-mass spectrometry (LC-MS).

[0074] Non-reduced microcapillary electrophoresis and size-exclusion chromatography Product quality of the purified material was analyzed using reducing and non-reducing microcapillary electrophoresis and SEC as previously described (Gong et al., 2021; Guo et al., 2021). For nrMCE, 6 μL of protein was mixed with 21 μL of sample buffer (8.4 mM TrisHCl pH 7.0, 7.98% glycerol, 2.38 mM EDTA, 2.8% SDS and 2.4 mM iodoacetamide), heated at 85°C for 10 min, and then analyzed using a Caliper LabChip GXII Touch instrument (PerkinElmer). For analytical SEC, protein samples were analyzed on an Acquity® HPLC instrument (Waters) using a BEH column (200 Å, 1.7 micron, 4.7 × 300 mm) with 100 mM sodium phosphate (pH 6.9), 50 mM NaCl, 7.5% ethanol as the running buffer at a flow rate of 0.45 mL / min.

[0075] Liquid Chromatography Mass Spectrometry and RapidFire Mass Spectrometry Intact non-reduced LC-MS was performed to determine the MW of the purified samples as previously reported (Campuzano et al., 2019; Spahr et al., 2018). A 30 μL aliquot of 1% trifluoroacetic acid (TFA) was added to 30 μL of each purified sample. Approximately 15 μg of each sample was then injected into an Agilent 1290 UPLC with the column eluate directly coupled to an Agilent 6224 electrospray time-of-flight (ESI-TOF) mass spectrometer (Agilent Technologies). Chromatographic separation utilized a Zorbax® RRHD300SB-C8 2.1×50 mm, 1.8 μm particle size, ultra-performance liquid chromatography (UPLC) column (Agilent Technologies). The column was heated to 70° C. with a flow rate of 0.5 mL / min. The following chromatographic solvents were used: aqueous solvent "A" (0.1% TFA in H2O) and organic solvent "B" (0.1% TFA in 90% n-propanol). The gradient used was isocratic at 80% A / 20% B for 4 min, followed by 28% A / 72% B for 2 min, 10% A / 90% B for 0.5 min, and finally 5% A / 95% B for 0.5 min. The MS method scanned m / z [1000-7000] and acquired 0.7 spectra / sec. The acquired spectra were summed and then deconvoluted using either the Agilent Mass Hunter Qualitative Analysis software (version B.07.00) or the Intact Program module from Protein Metrics (PMI Intact).

[0076] The RapidFire-MS system was used for samples from the high throughput vector assembly library screen. An equal volume of 0.1% w / w formic acid was added to 50uL of the supplied sample and 20uL of this solution was injected into the RapidFire-MS for analysis. The SPE cartridge was a 4uL PLRP 1000Å cartridge / column. The mobile phase was 10% n-propanol with 0.1% formic acid and 90% n-propanol with 0.1% formic acid. All data was processed using PMI Intact.

[0077] result The Modular Vector (ModVec) platform for high-throughput assembly of complex vectors To enable high-throughput vector engineering, we developed a GG assembly-based mammalian modular vector (ModVec) system to construct diverse expression vectors and vector libraries for recombinant protein production. ModVec enables high-throughput construction of both simple and complex vector designs, and aids in the optimization of expression vectors for individual molecules, specific large molecule modalities, and / or for different expression hosts.

[0078] ModVec is designed to be extremely flexible, allowing the assembly of one or more expression cassettes in a variety of possible configurations while combinatorial exploration of sequence diversity within each module. Each module contains a DNA sequence or a library of DNA sequences with carefully designed GG overhangs (Figure 1B). These DNA sequences can be vector elements commonly used for recombinant expression, such as sequences required for vector replication (origin of replication) and antibiotic resistance, elements required for viral infection, transposition, episomal replication, targeted integration, or elements commonly used for mammalian gene expression. These DNA sequences can be flanked by GG adapters (boxed regions in Figure 1A) containing, for example, BsmBI restriction sites, and can be generated by DNA synthesis or PCR amplification. A set of complementary, non-promiscuous 4-bp overhangs flanking BsmBI restriction sites was designed to allow seamless unidirectional assembly of DNA elements into rational expression cassettes (Figure 1B).

[0079] The ModVec concept was first tested by assembling an 11 kb, 11 module bicistronic construct in a 15 μL GG reaction (Figure 1C). 19 constructs were assembled and 6 colonies for each construct were inoculated for sequence verification. All 19 bicistronic constructs were successfully assembled in a single round of cloning. More than 95% (109 / 114) of the clones contained correctly assembled vectors (Figure 1D). These results demonstrate that ModVec is an efficient method for the assembly of large, complex pieces of DNA, provided that the sets of GG overhangs are compatible (i.e., non-promiscuous with each other). The 95% efficiency of correct assembly obtained with optimized ModVec enables one-tube construction of expression vector libraries. Functionally similar vector elements with identical overhangs were mixed together to create a "module library" (Figure 1E). As a demonstration of a single-tube vector engineering library, two different promoters driving 12 coding sequences (CDS1–CDS12) were combined with three different promoters driving CDS13 expression, and combinatorial assembly resulted in 72 different expression vectors. A miniaturized GG reaction was set up using an ECHO liquid handler. Miniaturized GG reactions have been shown to improve GG assembly efficiency (Kanigowska et al., 2016). 80 nL of each of the 25 parts was mixed in a 2.5 μL GG assembly reaction. After 30 cycles of GG assembly, the entire volume was transformed and plated. After overnight incubation, approximately 1000 colonies were grown on agar plates, of which 350 were inoculated into liquid cultures for sequence verification by Sanger sequencing. A total of 312 contigs were generated, matching 69 of the 72 vector candidates (Figure 1F).

[0080] These results demonstrate that ModVec is an efficient cloning platform for HT assembly of complex expression vector and expression construct libraries. Thus, it is now possible to improve bsAb productivity and product quality by high-throughput vector construct screening to identify vector constructs with balanced expression levels of polypeptide chains in bsAbs. We next used the ModVec cloning system to explore different transcriptional regulation strategies to improve the productivity and product quality of complex multi-chain multispecific antibodies recombinantly produced in mammalian cells.

[0081] Effect of promoters on the productivity of double-chain monoclonal antibodies (mAbs) and symmetric bispecific antibodies (bsAbs) The effect of promoters on the productivity of symmetric double-stranded molecules A, B, and C was tested (Figures 2B-2D). Molecule A is a reference monoclonal antibody, and molecules B and C are symmetric bispecific antibodies. A bicistronic vector was constructed in which the LC and HC genes of each molecule were driven by the same promoter (either promoter 1 or promoter 2) (Figure 2A). Both promoter 1 and promoter 2 are derived from the huCMV promoter, but promoter 2 is stronger than promoter 1. For efficient genome integration and rapid generation of stable cell pools, piggyBac transposase-based expression vectors were used. Internal observations suggested that piggyBac transposase-based expression vectors have extremely high reproducibility, so one stable Chinese hamster ovary (CHO) cell pool was generated for each construct. Regardless of the type of molecule, pools transfected with constructs using Promoter 2 had higher yields after protein A (ProA) affinity chromatography than pools transfected with constructs using Promoter 1 (Figures 2B-2D). ProA yields for molecule A were 403 mg / L for pools transfected with constructs using Promoter 1 and 521 mg / L for pools transfected with constructs using Promoter 2, a 30% increase in yield by switching from Promoter 1 to Promoter 2. More dramatic increases were observed for molecules B and C, where switching from Promoter 1 to Promoter 2 increased ProA yields by 83% and 100%, respectively. Cell recovery and growth characteristics were also comparable between pools transfected with each construct. For these three symmetric molecules, varying the promoter strength of both HC and LC genes together did not significantly affect the product quality as measured by the percentage of the major species in size exclusion chromatography (SEC % main peak, Figures 2B-2D, middle panels). As a result, an increase of 28%, 122% and 96% in the final yield of molecules A, B and C was realized by using promoter 2.More dramatic increases in productivity were observed for molecules that initially gave lower yields (molecules B and C). The results indicate that promoter strength modulation is an effective approach to improve mAb and bsAb productivity and that the approach may be most efficiently applied to difficult-to-express proteins.

[0082] The vector engineering strategy was then extended to improve both the productivity and product quality of a more complex four-chain bsAb, hetero-IgG.

[0083] Improving product quality and productivity of heterologous IgG by chain balancing Hetero-IgG is the most common bispecific form due to its antibody-like structure (Labrijn et al., 2019), but its production is also more challenging than mAbs due to the potential for multiproduct impurities due to inaccurate LC-HC and HC-HC pairing (Brinkmann & Kontermann, 2017). Many protein engineering strategies, such as charge pair mutation (CPM) (Dillon et al., 2017; Gunasekaran et al., 2010), knobs-into-holes (KiH) (Ridgway et al., 1996), strand-exchange engineered domain (SEED) (Davis et al., 2010) and common light chain (Krah et al., 2017; Shiraiwa et al., 2019), have been used to favor correct chain pairing and prevent incorrect chain pairing, thereby improving the manufacturability of hetero-IgG. However, differences in the expression levels of each polypeptide chain comprising hetero-IgG in mammalian cells may lead to the formation of product-related impurities and reduce the yield of accurate bsAb. For example, in stable CHO pools of heterodimer IgG-D molecules transfected with our defined expression vectors, we observed up to 60-fold higher production of LC1 relative to LC2, with similarly abundant mispaired species with two copies of LC1 (Guo et al., 2021). We hypothesized that the yield of correctly paired heterodimer IgG relative to mispaired product-related impurities could be improved by vector engineering to balance the expression of each polypeptide chain closer to a 1:1:1:1 ratio. Informed by our success with double-stranded molecules, we employed a vector engineering approach to balance the expression of the polypeptide chains using promoters of different strengths.

[0084] Eleven vector constructs of heterodimer IgG-D were constructed using ModVec. Each vector construct contained two bicistronic vectors co-transfected, in which the two polypeptide CDSs were driven by the same or different promoters (promoter 1, promoter 2 and promoter 3, FIG. 5A). In some vector constructs, the positions of the LC or HC coding sequences were swapped within the bicistronic cassette (FIG. 5A construct 2). Four transfections for each vector construct were performed in CHO cells to generate a total of 44 pools. Stable pools were generated by random genomic integration with metabolic selection. Pools derived from different vector constructs had different recovery rates; that is, of the 44 transfected pools, 15 could not be recovered. Pools that failed to be recovered tended to have been transfected with constructs in which LC1 / LC2 and HC1 / HC2 were in the same vector (FIG. 5A construct 2). In addition to differences in recovery of stable CHO pools, vector-assembled libraries showed a wide range in yield after ProA affinity purification (Figure 5B), product quality as measured by LC1:LC2 ratio (Figure 5C), and percentage of major species peak (%MP) in nonreducing microcapillary electrophoresis (nrMCE) and size-exclusion chromatography (SEC) (Figure 5D).

[0085] Three pools were selected for scale-up production and further analysis. Two pools had LC1:LC2 ratios close to the ideal value of 1 (Figure 3B, pools 910 and 911), and one pool had a biased LC1:LC2 ratio of >5 (Figure 3B, pool 905). The vector constructs used to generate these stable pools are illustrated in Figure 3A. Conditioned media from these three scaled-up pools were purified by ProA affinity capture followed by cation exchange (CEX) using Sepharose SP resin. All three pools had similar ProA yields (Figure 3C). Recovery of the major hetero-IgG-D species from pool 905 after cation exchange was 12%, while pools 910 and 911 had recoveries of 46% and 40%, respectively (Figure 3D). As a result, the yields of correctly assembled hetero-IgG-D after cation exchange were higher for pools 910 and 911 (115 and 75 mg / mL, respectively) than for pool 905 (34 mg / L) (Figure 3E).

[0086] In addition to differing relative levels of production of the major species, pools transfected with different vector constructs also differed in the profile of product-associated impurities as shown in cation exchange chromatograms (Figures 3F-3H). The major species in the ProA-purified sample of pool 905 was identified by mass spectrometry as a mispaired heterotetramer with two copies of LC1 (2×LC1, i.e., LC1:HC1:LC1:HC2, major peak in Figure 3F), with correctly assembled heterodimeric IgG-D being the minor species (minor peak in Figure 3F). This explains the lower purification yield and recovery of pool 905 (Figures 3D-3E) and is consistent with the high LC1:LC2 ratio (Figure 3B). This also indicates that the expression levels of LC1 and LC2 were unbalanced when promoter 1 was used to drive expression of both LCs. No antibody-like homodimers were observed from pool 905, likely due to HC1:HC2 mispairing. The main issue with the CHO pool transfected with this vector construct was LC-HC mispairing. When the promoters driving both LC1 and LC2 were switched to Promoter 2 (pool 910, Figure 3A), 2xLC1 became the minor species and correctly assembled hetero-IgG-D became the majority species in the ProA purified material (Figure 3G), which increased the recovery of the major species after cation exchange (Figures 3D-3E). It is interesting that changing both LC promoters to Promoter 2 balances the expression of LC1 and LC2. We previously reported that LC2 is poorly secreted from mammalian cells (Guo et al., 2021). Switching both LC promoters to Promoter 2 may have had a greater impact on the "poorly expressed" LC2, resulting in a more balanced LC1:LC2 ratio. When more than 10 replicates of the CHO pools were transfected with the promoter arrangement used to generate pool 905, consistently high LC1:LC2 ratios (5-60) and high levels of the 2×LC1 species were observed.Furthermore, when the promoter of LC1 was changed to promoter 3, whose transcription strength is intermediate between promoter 1 and promoter 2, in pool 911, and the promoter of LC2 was not changed (promoter 2), correctly assembled heterodimeric IgG-D was still the major species. However, the major impurity changed to half antibody 1 (LC1:HC1) instead of 2×LC1 (Figure 3H). The change in the major impurity from 2×LC1 in pools 905 and 910 to half antibody 1 in pool 911 may be due to the change in the LC gene promoter affecting the expression of both the LC and HC genes from the bicistronic cassette.

[0087] High-throughput vector engineering of asymmetric, three-chain heterodimeric IgG-based trispecific molecules We next tested whether we could create a highly productive vector for the challenging three-chain asymmetric trispecific antibody E (tsAb E). In transient transfection studies, tsAb E was produced at titers that were 5-10 times lower than the levels expected for mAbs. Inspired by the successful screening of 11 vector configurations for heterodimeric IgG, we decided to apply vector engineering approaches to improve the productivity of tsAb E, but included a larger number of vector configurations in the library design. It was speculated that testing more strategies for regulating polypeptide chain expression would increase the chances of finding a configuration with an optimal stoichiometric ratio for all three polypeptide chains of asymmetric tsAb E. In addition to promoters of different strengths, we varied the gene dosage (LC gene copy number) and the cistron arrangement of the LC and HC genes in the library design. The final library contained 189 vector configurations, including 27 monocistronic vectors with a single copy of the LC gene (single LC), 81 bicistronic vectors with two copies of the LC gene in the first cistron of each vector (double LC A), and 81 bicistronic vectors with two copies of the LC gene in the second cistron of each vector (double LC B) (Figure 4A). Three promoters (promoter 1, promoter 2, and promoter 3) were used to drive the expression of each polypeptide chain in a combinatorial fashion. Thus, in this vector library design, the genes in the bicistronic vectors can be optionally driven by the same or different promoters.

[0088] A proprietary high-throughput expression system that relies on transposase-mediated integration with metabolic selection was used to transfect CHO cells in 24-well culture plates. One pool was generated for each vector configuration. All pools had comparable recovery and growth profiles (data not shown), which is typical for this expression system. As shown in Figure 4B, there were orders of magnitude differences in productivity of this tsAb depending on the vector configuration. ProA yields for all pools ranged from 20 mg / L to 220 mg / L, clearly demonstrating the ability of vector configuration to affect yield. Pools transfected with the dual LC A configuration (117.2±43.4mg / L) or dual LC B configuration (112.6±39.2mg / L) had higher mean ProA yields than pools transfected with the single LC configuration (40.7±20.73mg / L) (Student's t-test, dual LC A vs single LC p<0.0001, dual LC B vs single LC p<0.0001, values ​​shown as mean±s.d.). The dual LC A and dual LC B pools had comparable ProA yields (Student's t-test, dual LC A vs dual LC B p=0.48). A preliminary assessment of product quality was performed by performing non-reducing microcapillary electrophoresis (nrMCE) on each ProA pool. The average percentage of the major species peak (%MP) among pools generated from these three vector configurations was comparable (Figure 4C, duplex LC A MP: 75.6 ± 14.9%, duplex LC B MP: 72.4 ± 16.6%, single LC MP: 71.7 ± 21.6%, duplex LC A vs. duplex LC B p = 0.19, duplex LC A vs. single LC p = 0.34, duplex LC B vs. single LC p = 0.88, Student's t test). As a result, as shown in Figure 4D, the average yield of correctly assembled tsAb E was higher for pools generated from the duplex LC A configuration (88.7 ± 33.6 mg / L) or the duplex LC B configuration (81.4 ± 35.6 mg / L) than for pools generated from the single LC configuration (28.5 ± 16.8 mg / L).The yield of correct species was comparable between pools generated from dual LC A and dual LC B (dual LC A vs dual LC B p=0.48, dual LC A vs single LC p<0.0001, dual LC B vs single LC p<0.0001, Student's t-test). These results indicate the impact of LC gene copy number on the productivity of complex therapeutic molecules. This is consistent with previous findings that gene dosage, determined by gene copy number, can greatly affect productivity and that excess LC is generally beneficial for productivity (Gonzalez et al., 2002).

[0089] Since the performance of the double LC A and double LC B groups was comparable, the performance of the individual configurations of these two groups was compared to understand the effect of gene position on productivity and product quality. Configuration DA2 (double LC A configuration with promoter 2 driving the expression of all polypeptide chains) and configuration DB2 (double LC B configuration with promoter 2 driving the expression of all polypeptide chains) were selected for further analysis (Figure 6A). In DA2, the LC gene was in the first cistron and the HC gene was in the second cistron, while in DB2 the positions of the LC and HC genes were swapped. As shown in Figure 6B, the ProA yield of pool DA2 was 155 mg / L. Swapping the positions of the LC and HC genes by placing the HC gene in the first cistron and the LC gene in the second cistron reduced the overall productivity. The ProA yield of pool DB2 was 113 mg / L. This is consistent with the effect of gene position in multicistronic expression vectors, where the expression levels of genes downstream of the first gene are generally lower compared to the expression level of the first gene (Eszterhas et al., 2002; Patel et al., 2021). In this case, lowering the expression of LC genes and increasing the expression of HC genes resulted in an overall decrease in productivity, again consistent with the expectation that high levels of expression of LC are beneficial for overall productivity (Gonzalez et al., 2002). Product quality, as measured by nrMCE MP and preMP, was comparable between ProA samples purified from pools DA2 and DB2 (nrMCE MP, 80% for DA2 and 81% for DB2; nrMCE preMP, 18% for DA2 and 19% for DB2, Figure 6C). Mass spectrometry confirmed that the nrMCE MP was the correctly assembled tsAb E and the nrMCE pre-MP was half mAb1 (LC:HC1) and half mAb2 (LC:HC2) (Figures 6E and 6F).

[0090] Although the levels of nrMCE pre-MPs were comparable between ProA samples from pools DA2 and DB2, the relative abundances of half mAb1 and half mAb2 differed (Figures 6E and 6F). In the nrMCE electropherogram of the ProA-purified sample from pool DA2, the relative peak area of ​​half mAb1 was 10.1 and half mAb2 was 7.4 (Figure 6E). For the ProA-purified sample from pool DB2, the relative peak area of ​​half mAb1 was 5.8 and half mAb2 was 9.8 (Figure 6F). The relative peaks of half mAb1:half mAb2 were calculated. The results are shown in Figure 6G. The relative peak ratio of half mAb1:half mAb2 was 1.4 for the ProA-purified sample from pool DA2 and 0.6 for the ProA-purified sample from pool DB2. These results further indicate that the location of the LC or HC genes in the expression vector can significantly affect both the productivity of the major species and the profile of impurities produced from CHO cells. These results also support our hypothesis that combining promoters with different strengths, gene copy numbers, and gene locations is an effective approach to modulate the expression levels of polypeptide chains, and may increase the possibility of identifying vector configurations that yield high productivity and high quality of product for multi-chain protein therapeutics.

[0091] To progress to cell line development (CLD) of tsAb E, we selected the best vector configurations from the vector library screen and compared these vectors to a defined platform vector (depicted in Figure 4H). The defined vector configuration was a bicistronic vector with the LC gene in the first cistron and HC1 in the second cistron, combined with a monocistronic vector containing HC2, using promoter 1 to drive expression of all polypeptide chains. In contrast, the optimal configuration identified from the vector library consisted of two bicistronic vectors, each of which expressed a copy of the LC gene in the first cistron and either HC1 or HC2 in the second cistron, with promoter 2 driving expression of both LC genes and promoter 1 driving expression of the HC1 and HC2 genes (Figure 4H). Stable CHO pools generated from the defined constructs produced tsAb E with a ProA yield of 119 mg / L, whereas the optimal construct from the vector library produced 267 mg / L after ProA, a 124% increase (Figure 4E). Product quality as measured by nrMCE was comparable between ProA-purified samples from both pools (Figure 4F). As a result, the final yield of correctly assembled tsAb E was 114 mg / L for the defined pool and 257 mg / L for the optimal pool (Figure 4G). The optimal vector constructs identified by vector construct library screening allowed a 125% increase in productivity of this promising, difficult-to-express tsAb therapeutic over our defined vector constructs.

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j.chroma.2014.04.014

Claims

1. a. providing a polynucleotide sequence encoding at least two different polypeptides, said polynucleotide sequence flanked on both the 5' and 3' sides by a cleavage site followed by a recognition site for a Type IIS restriction endonuclease; b. providing two or more sets of element arrays, each set of element arrays comprising together at least one functional expression cassette; each element sequence is flanked on both the 5' and 3' sides by a cleavage site followed by a recognition site for a type IIS restriction endonuclease; The recognition and cleavage sites of the type IIS restriction endonuclease are selected such that each set of element sequences can be assembled into a functional expression cassette; preparing two or more sets of element arrays; c. providing at least one backbone entry vector, said backbone entry vector comprising a plasmid comprising at least one cleavage site followed by a recognition site for a Type IIS restriction endonuclease and a sequence for maintaining said vector in a bacterial cell; d. Assembling the polynucleotide sequences encoding the at least two different polypeptides with the two or more sets of element sequences to generate functional expression cassettes in the backbone entry vector using a method based on the use of type IIS restriction enzyme digestion and ligation via cleavage sites and overhangs to obtain a ligated vector comprising at least two functional expression cassettes capable of expressing the at least two different polypeptide chains; e. transfecting a mammalian cell with the ligated vector; f. comparing the level of the desired product produced with the level of the impurity of each of said at least two polypeptides; g. determining an optimal expression ratio of the two polypeptides for the transfected mammalian cells and identifying a transfected mammalian cell having an optimal expression ratio of the at least two polypeptide chains; 2. A method for expressing a multi-chain protein comprising at least two different polypeptide chains, comprising:

2. h) The method of claim 1, further comprising identifying a vector element arrangement that provides an optimal expression ratio of said at least two polypeptide chains.

3. The method according to claim 1 or 2, wherein the element sequence is selected from the group consisting of a control sequence, an insulator sequence, a barcode DNA sequence, a primer sequence, a promoter sequence, a polyA sequence and an IRES sequence, and a mammalian selectable marker sequence.

4. The method of claim 3 , wherein the element sequence comprises at least two promoter sequences and at least two polyA sequences.

5. 5. The method of any one of claims 1 to 4, wherein the level of the preferred product produced is measured by a method selected from the group consisting of reducing or non-reducing cation exchange chromatography, mass spectrometry, or a combination thereof.

6. 6. The method of any one of claims 1 to 5, wherein none of the Type IIS restriction endonuclease cleavage sites produces a 4 nucleotide 5' overhang selected from the group consisting of GTAA, TCCA and CACA upon cleavage with the Type IIS restriction endonuclease.

7. The type IIS restriction endonuclease cleavage site is 7. The method of any one of claims 1 to 6, wherein the amino acid sequence is selected from the group consisting of AGGT, AGTA, ATCA, CAGT, CCAT, GAAT, GAGG, GGCA, GGTC, TAGC, TCTT, GGAG and CCAC.

8. 8. The method of any one of claims 1 to 7, wherein the mammalian cell is selected from the group consisting of CHO cells, CHOK1 cells, DXB-11, DG-44, COS-7, HEK293, BHK, TM4, CV1, VERO-76, HELA, MDCK, BRL3A, W138, Hep G2, MMT060562, TRI cells, MRC5 cells, FS4 cells and mammalian myeloma cells.

9. The method according to any one of claims 1 to 8, wherein the optimal expression ratio of the at least two polypeptides is selected from the group consisting of 1:1, 1:2 and 1:

3.

10. 2. The method of claim 1, wherein the multi-chain protein comprises a first antibody heavy chain, a first antibody light chain, a second antibody heavy chain and a second antibody light chain, wherein the first antibody heavy chain in combination with the first antibody light chain binds a first antigen or epitope and the second antibody heavy chain in combination with the second antibody light chain binds a second antigen or epitope, and wherein the optimal expression ratio of the first antibody heavy chain, the first antibody light chain, the second antibody heavy chain and the second antibody light chain is 1:1:1:

1.

11. 2. The method of claim 1, wherein the multi-chain protein comprises a first antibody heavy chain, a second antibody heavy chain and a common antibody light chain, the first antibody heavy chain in combination with the common antibody light chain binds a first antigen or epitope and the second antibody heavy chain in combination with the common antibody light chain binds a second antigen or epitope, and the optimal expression ratio of the first antibody heavy chain, the second antibody heavy chain and the common antibody light chain is 1:1:

2.

12. the multi-chain protein comprises an antibody heavy chain, a first antibody light chain, a modified antibody heavy chain and a second antibody light chain; The modified antibody heavy chain comprises, from N-terminus to C-terminus, the following group: a. VH-CH1-binding domain (BD)-hinge-CH2-CH3; b. BD-VH-CH1-hinge-CH2-CH3, and c. VH-CH1-hinge-CH2-CH3-BD; and the BD is selected from either a single chain Fv (scFv) or a single chain Fab (scFab); the antibody heavy chain in combination with the first antibody light chain binds a first antigen or epitope, the VH of the modified antibody heavy chain in combination with the second antibody light chain binds a second antigen or epitope, and the BD binds a third antigen or epitope; and the optimal expression ratio of the first antibody heavy chain, the first antibody light chain, the modified antibody heavy chain and the second antibody light chain is 1:1:1:1; The method of claim 1.

13. The multi-chain protein includes an antibody heavy chain, a modified antibody heavy chain and a general antibody light chain, The modified antibody heavy chain comprises, from N-terminus to C-terminus, the following group: a. VH-CH1-binding domain (BD)-hinge-CH2-CH3; b. BD-VH-CH1-hinge-CH2-CH3, and c. VH-CH1-hinge-CH2-CH3-BD; and the BD is selected from either a single chain Fv (scFv) or a single chain Fab (scFab); the antibody heavy chain in combination with the generic antibody light chain binds a first antigen or epitope, the VH of the modified antibody heavy chain in combination with the generic antibody light chain binds the first antigen or epitope, and the BD binds a second antigen or epitope; and the optimal expression ratio of the first antibody heavy chain, the modified antibody heavy chain and the common antibody light chain is 1:1:2; The method of claim 1.

14. A vector comprising the arrangement of vector elements according to claim 2, step h.

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