Methods for improving protein expression

Abstract

The present disclosure relates to a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, the nucleotide sequence of which is deleted from one or two introns in the immunoglobulin heavy chain, the nucleic acid being useful for increasing immunoglobulin expression.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application, filed January 12, 2023, claims the benefit under 35 U.S.C. § 119(e) of the following U.S. Provisional Application No. 63 / 299,303, filed January 13, 2022. Each of the above-listed applications is hereby incorporated by reference in its entirety for all purposes.

[0002] The present disclosure relates to improved methods of expressing a polypeptide of interest. A nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain and an intron deletion is useful for increasing the cell-specific productivity of immunoglobulin. [Background technology]

[0003] DNA is composed of sequences of introns and exons, and introns are removed during mRNA processing by splicing. This process is closely linked to mRNA export through the nuclear pore complex. This export is fundamental for expression and is well documented in the literature. See Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3.

[0004] It has been observed that the presence of an intron in a codon-optimized heavy chain constant region encoded in an expression vector improves the harvest titer compared to the same nucleotide sequence in this region without the presence of an intron. However, a risk associated with using an expression vector containing an intron is that during expression of a protein of interest, such as an antibody or immunoglobulin, intron-retention and mis-splicing events may occur, resulting in unwanted aberrant protein species that need to be removed during purification. This adds extra complexity to the purification process and can lead to batch-to-batch variability if these additional species cannot be removed during downstream processing.

[0005] It has been shown that the use of non-codon-optimized sequences containing heavy chain constant region introns can eliminate the occurrence of splice variants in the heavy chain constant region. It is believed that the nucleotide sequence changes resulting from codon optimization induce hidden splicing to produce these alternative species. It has also been shown that removing each of the introns individually (thereby leaving two of the three introns) results in a surprising improvement in recovery titers.

[0006] Removal of one or two introns from the immunoglobulin heavy chain constant region can reduce the risk of intron retention and mis-splicing events while increasing immunoglobulin expression, which results in the de novo generation of expression vectors and lowers the risk of generating aberrant protein species during the production of antibody- or immunoglobulin-based recombinant proteins. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kohler A. et al., Nature Review Molecular Cell Biology 8:761-773(2007) [Non-Patent Document 2] Bjork, P. et al., Seminars in Cell & Developmental Biology 32:47-54(2014) [Non-Patent Document 3] Reed, R.Current Opinion in Cell Biology,15:326-331(2003) Summary of the Invention [Means for solving the problem]

[0008] The disclosure is generally directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of all introns in the immunoglobulin heavy chain is deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region. In one aspect, the leader intron or the VH-CH1 intron is deleted. In one aspect, the leader intron is deleted. In one aspect, the VH-CH1 intron is deleted.

[0009] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron.

[0010] The present disclosure is also directed to an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of all introns in the immunoglobulin heavy chain is deleted except for intron 1 in the heavy chain constant region.

[0011] In one aspect, the nucleic acid, when expressed with a nucleic acid encoding an immunoglobulin light chain, expresses an immunoglobulin at a higher titer than a nucleic acid that includes all the intron sequences of an immunoglobulin heavy chain. In another aspect, the nucleic acid, when expressed with a nucleic acid encoding an immunoglobulin light chain, expresses an immunoglobulin at a higher titer than a nucleic acid that does not include the intron sequences of an immunoglobulin heavy chain constant region. In another aspect, the nucleic acid, when co-expressed with a nucleic acid encoding an immunoglobulin light chain, does not express an immunoglobulin fragment. In another aspect, the immunoglobulin light chain is a kappa light chain or a lambda light chain. In another aspect, the nucleic acid is codon-optimized. In another aspect, the expressed immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4. In another aspect, the immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin. In another aspect, the nucleic acid is a deoxyribonucleic acid (DNA).

[0012] In another aspect, the disclosure is directed to a vector or expression vector comprising a nucleic acid of the disclosure. In another aspect, the disclosure is directed to a host cell comprising a vector or expression vector of the disclosure. In one aspect, the host cell is a eukaryotic cell, for example, a Chinese Hamster Ovary (CHO) cell.

[0013] The disclosure is also generally directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions in which the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of all introns in the immunoglobulin heavy chain is deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, the host cell expressing the immunoglobulin at the same or higher titers as a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, wherein all introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain. In one embodiment, the leader intron or the VH-CH1 intron is deleted. In one embodiment, the leader intron is deleted. In one embodiment, the VH-CH1 intron is deleted.

[0014] The disclosure is also generally directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions where the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, where the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, and a nucleic acid encoding an immunoglobulin light chain, the host cell expressing immunoglobulins at the same or higher titers as a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, where the nucleic acid encoding the immunoglobulin heavy chain, where all of introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

[0015] The disclosure is also generally directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions where the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, where the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, the host cell expressing immunoglobulins at the same or higher titers as a host cell comprising a nucleic acid ... all of introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

[0016] The disclosure is also directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions in which the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence of all introns in the immunoglobulin heavy chain is deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, the host cell expressing the immunoglobulin at a higher titer than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, wherein none of introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain. In one embodiment, the leader intron or the VH-CH1 intron is deleted. In one embodiment, the leader intron is deleted. In one embodiment, the VH-CH1 intron is deleted.

[0017] The disclosure is also directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions where the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, where the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, and a nucleic acid encoding an immunoglobulin light chain, the host cell expressing immunoglobulins at higher titers compared to a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, where the nucleic acid encoding the immunoglobulin heavy chain, where none of introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

[0018] The disclosure is also directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions where the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, where the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell expresses immunoglobulins at higher titers compared to a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, where the nucleic acid encoding the immunoglobulin heavy chain, where none of introns 1-3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

[0019] The disclosure is also directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions in which the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell does not express an immunoglobulin fragment. In one aspect, the leader intron or the VH-CH1 intron is deleted. In one aspect, the leader intron is deleted. In one aspect, the VH-CH1 intron is deleted.

[0020] The disclosure is also directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions whereby the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, where the nucleotide sequence of all introns in the immunoglobulin heavy chain is deleted except for the leader intron, and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell does not express an immunoglobulin fragment.

[0021] The disclosure is also directed to a method of producing an immunoglobulin comprising culturing a host cell in a medium and under conditions whereby the cell expresses an immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, where the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell does not express an immunoglobulin fragment.

[0022] In one aspect, the immunoglobulin heavy chain encoding nucleic acid is deoxyribonucleic acid (DNA). In another aspect, the immunoglobulin light chain is a kappa light chain or a lambda light chain. In another aspect, the immunoglobulin heavy chain encoding nucleic acid is codon optimized. In another aspect, the expressed immunoglobulin has an IgG1, IgG2, IgG3, or IgG4 isotype. In another aspect, the expressed immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin.

[0023] In one embodiment, the expressed immunoglobulins produced from the pool of clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, or at least 5,000 mg / L. In another embodiment, immunoglobulins produced from the highest expressing clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 6,000 mg / L, at least 7,000 mg / L, at least 8,000 mg / L, at least 9,000 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, or at least 12,000 mg / L.

[0024] In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the eukaryotic cell is a CHO cell. [Brief description of the drawings]

[0025] [Figure 1] Shown is a high-performance size-exclusion chromatograph of MAb1 and a depiction of the immunoglobulin variants produced. [Diagram 2][Figures 2A-2B] Figure 2A shows a schematic diagram of the non-codon-optimized genomic DNA (gDNA) and codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2. Figure 2B shows the immunoglobulin titer (mg / L) on day 14 of MAb2 production using either the gDNA or cDNA nucleotide sequence. [Diagram 3] [Figures 3A-3B] Figure 3A shows schematic diagrams of non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. FIG. 3B shows the immunoglobulin titer (mg / L) on day 13 of MAb2 production using each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. [Figure 4][Figures 4A-4D] Figure 4A shows graphs depicting the mean viable cell count (VCN) (x106 / mL) for each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. FIG. 4B shows a graph depicting the percent cell viability for each of the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon-optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. FIG. 4C shows a graph depicting the integrated viable cell concentration (IVC) (109 cells-day / L) for each of the following constructs: (1) non-codon optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2.FIG. 4D shows a graph depicting the cellular productivity (qP) (pg / cell day) for each of the following constructs: (1) non-codon optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region, (2) codon optimized complementary DNA (cDNA) of the immunoglobulin heavy chain constant region of MAb2, (3) gDNA without intron 1 of the immunoglobulin heavy chain constant region of MAb2, (4) gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2, (5) gDNA without intron 3 of the immunoglobulin heavy chain constant region of MAb2, and (6) gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2. [Diagram 5][Figures 5A-5D] Figure 5A shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (6) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (7) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (8) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (9) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (10) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (11) gDNA with introns 1 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (12) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (13) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (14) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (15) gDNA with introns 2 shows the immunoglobulin titers (mg / L) on day 11 of MAb2 production using gDNA from which intron 2 has been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (6) gDNA from which introns 1 and 2 have been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (7) gDNA from which introns 2 and 3 have been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (8) gDNA that does not have any introns in the immunoglobulin heavy chain constant region of MAb2 (codon optimized). FIG. 5B shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (6) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (7) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (8) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (9) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (10) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (11) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (12) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (13) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (14) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (15) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant Graphs showing the mean viable cell counts (VCN) (×106 / ml) for each of: (5) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (6) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (7) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (8) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (9) MAb3 (positive control).FIG. 5C shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (6) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (7) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (8) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (9) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (10) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (11) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (12) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (13) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (14) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (15) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 1 shows a graph depicting the integrated viable cell concentration (IVC) (109 cell-hour / L) for each of (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb1 (codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (4) gDNA with introns 3 and 4 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (5) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (6) gDNA with introns 2 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (7) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (8) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (9) MAb3 (positive control). FIG. 5D shows the following constructs: (1) gDNA with intron 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (2) gDNA with introns 1 and 2 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (3) gDNA with introns 2 and 3 removed from the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (4) gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2 (non-codon optimized), (5 1 shows a graph depicting the cellular productivity (qP) (pg / cell day) for each of: (1) gDNA in which intron 2 has been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (2) gDNA in which introns 1 and 2 have been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), (3) gDNA in which introns 2 and 3 have been removed from the immunoglobulin heavy chain constant region of MAb2 (codon optimized), and (4) gDNA that does not have any introns in the immunoglobulin heavy chain constant region of MAb2 (codon optimized). [Figure 6][FIGS. 6A-6B] FIG. 6A shows the following constructs: (1) non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region of MAb2; (2) non-codon-optimized gDNA lacking intron 2 of the immunoglobulin heavy chain constant region of MAb2; (3) non-codon-optimized gDNA lacking introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2; (4) non-codon-optimized gDNA lacking introns 1 and 2 of the immunoglobulin heavy chain constant region of MAb2; (5) non-codon-optimized gDNA with intron 3 in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2; (6) non-codon-optimized gDNA with a modified intron 3 nucleotide sequence in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2; (7) Schematic diagrams of non-codon optimized gDNA having intron 1 in place of intron 3 in the immunoglobulin heavy chain constant region of MAb2, (8) non-codon optimized gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2, (9) non-codon optimized genomic DNA (gDNA) having all introns in the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, (10) non-codon optimized gDNA without introns 2 and 3 and wild-type IgG1 in the immunoglobulin heavy chain constant region of MAb2, (11) non-codon optimized gDNA without introns 1 and 2 in the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, and (12) non-codon optimized gDNA of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1.FIG. 6B illustrates the following constructs: (1) non-codon optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region of MAb2; (2) non-codon optimized gDNA without intron 2 of the immunoglobulin heavy chain constant region of MAb2; (3) non-codon optimized gDNA without introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2; (4) non-codon optimized gDNA without introns 1 and 2 of the immunoglobulin heavy chain constant region of MAb2; (5) non-codon optimized gDNA with intron 3 in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2; (6) non-codon optimized gDNA with a modified intron 3 nucleotide sequence in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2; and (7) non-codon optimized gDNA with intron 3 in place of intron 1 of the immunoglobulin heavy chain constant region of MAb2. 1 shows immunoglobulin titers (mg / L) on day 11 of MAb2 production using (1) non-codon optimized gDNA having intron 1 in position of intron 3 of MAb2, (2) non-codon optimized gDNA without any introns in the immunoglobulin heavy chain constant region of MAb2, (3) non-codon optimized genomic DNA (gDNA) having all introns of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, (4) non-codon optimized gDNA without introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, (5) non-codon optimized gDNA without introns 1 and 2 of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1, and (6) non-codon optimized gDNA without introns 1 and 2 of the immunoglobulin heavy chain constant region of MAb2 and wild-type IgG1. [Figure 7] Schematic diagrams of non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant region of MAb2, MAb1, MAb3, and MAb4, non-codon-optimized gDNA without introns 2 and 3 of the immunoglobulin heavy chain constant region of MAb2, MAb1, MAb3, and MAb4, and non-codon-optimized gDNA without any introns of the immunoglobulin heavy chain constant region of MAb2, MAb1, MAb3, and MAb4 are shown. [Figure 8]Shown are immunoglobulin titers (mg / L) on day 11 for non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, non-codon-optimized gDNA lacking introns 2 and 3 of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, and non-codon-optimized gDNA lacking any introns of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4. [Figure 9] Graphs showing the time course of titer levels of non-codon-optimized genomic DNA (gDNA) of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, non-codon-optimized gDNA lacking introns 2 and 3 of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4, and non-codon-optimized gDNA lacking any introns of the immunoglobulin heavy chain constant regions of MAb2, MAb1, MAb3, and MAb4. [Figure 10] 1 shows a schematic representation of the genomic DNA sequence of the immunoglobulin heavy chain constant region. [Figure 11] [FIGS. 11A-11B] Figure 11A shows a schematic diagram of the following constructs of MAb2: (1) non-codon-optimized genomic DNA (gDNA) of immunoglobulin heavy chain, with stars representing (from left to right): (i) leader intron, (ii) VH-CH1 intron, (iii) intron 1 of the heavy chain constant region, (iv) intron 2 of the heavy chain constant region, and (v) intron 3 of the heavy chain constant region; (2) non-codon-optimized gcDNA with (i) leader intron and (ii) VH-CH1 intron; and (3) non-codon-optimized gcDNA with leader intron only. Figure 11B shows a graph of immunoglobulin titers (mg / L) at day 11 for the constructs of Figure 11A. [Figure 12][Figures 12A-12B] Figure 12A shows a schematic diagram of the following constructs MAb1, MAb2, MAb3, and MAb4: (1) non-codon-optimized genomic DNA (gDNA) with all introns of the immunoglobulin heavy chain constant region, (2) non-codon-optimized gDNA with only intron 1 of the immunoglobulin heavy chain constant region; and (3) non-optimized gDNA without any introns of the immunoglobulin heavy chain constant region. Figure 12B shows an agarose gel separating the reverse transcriptase PCR products of the constructs in Figure 12A. [Figure 13] [Figures 13A-13C] Figure 13A shows schematic diagrams of the following constructs of MAb2: (1) non-codon-optimized genomic DNA (gDNA) of an immunoglobulin heavy chain, where the stars represent (from left to right) (i) leader intron, (ii) VH-CH1 intron, (iii) intron 1 of the heavy chain constant region, (iv) intron 2 of the heavy chain constant region, and (v) intron 3 of the heavy chain constant region; (2) non-codon-optimized gDNA with (i) leader intron, (ii) VH-CH1 intron, and (iii) intron 1 of the immunoglobulin heavy chain constant region; (3) non-codon-optimized gcDNA with (i) leader intron and (ii) VH-CH1 intron; (4) non-codon-optimized gcDNA with a leader intron; (5) non-codon-optimized gcDNA without the immunoglobulin heavy chain intron; and (6) non-codon-optimized gDNA with only intron 1 of the immunoglobulin heavy chain. Figure 13B shows a graph of immunoglobulin titer (mg / L) of the constructs of Figure 13A on day 11. Figure 13C shows a graph depicting cellular productivity (qP) (pg / (cell day)) of the constructs of Figure 13A. [Figure 14][Figures 14A-14C] Figure 14A shows schematic diagrams of the following constructs of Mab2: (1) non-codon optimized genomic DNA (gDNA) from an immunoglobulin heavy chain constant region; (2) non-codon optimized gDNA without introns 2 and 3 from an immunoglobulin heavy chain constant region; (3) non-codon optimized gDNA without introns 1 and 2 from an immunoglobulin heavy chain constant region; (4) non-codon optimized gcDNA without introns 1-3 from an immunoglobulin heavy chain constant region; (5) non-codon optimized gDNA with intron 3 at the position of intron 1 in the immunoglobulin heavy chain constant region; (6) non-codon optimized gDNA with a modified intron 3 nucleotide sequence at the position of intron 1 in the immunoglobulin heavy chain constant region; and (7) non-codon optimized gDNA with intron 1 at the position of intron 3 in the immunoglobulin heavy chain constant region. Figure 14B shows agarose gel separation of reverse transcriptase PCR products of the constructs of Figure 14A. FIG. 14C shows a graph of immunoglobulin titers (mg / L) on day 11 for the constructs in FIG. 14A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0027] I. Definition As used herein, the terms "immunoglobulin," "antibody," and "antibodies" are terms of art and may be used interchangeably herein and refer to a molecule or complex of molecules that have at least one antigen-binding site that specifically binds to an antigen.

[0028] Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, human antibodies, humanized antibodies, resurfaced antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fvs (scFvs), camelid antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFvs), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), bispecific antibodies, and multispecific antibodies. In certain aspects, the antibodies described herein refer to polyclonal antibody populations. An antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG 2a or IgG 2b ). In certain aspects, the antibodies described herein are IgG antibodies, or classes thereof (e.g., human IgG1, IgG2, or IgG4), or subclasses thereof. In certain aspects, the antibodies are humanized monoclonal antibodies. In another particular aspect, the antibodies are human monoclonal antibodies, e.g., human monoclonal antibodies that are immunoglobulins. In certain aspects, the antibodies described herein are IgG1, IgG2, or IgG4 antibodies.

[0029] As used herein, the terms "antigen-binding domain," "antigen-binding region," "antigen-binding site," and similar terms refer to the portion of an antibody molecule that contains the amino acid residues that confer specificity to the antibody molecule for an antigen (e.g., the complementarity determining region (CDR)). The antigen-binding region can be derived from any animal species, including rodents (e.g., mice, rats, or hamsters) and humans.

[0030] A "monoclonal" antibody refers to a homogeneous antibody population that is involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to a polyclonal antibody, which typically contains different antibodies directed against different antigenic determinants. The term "monoclonal" antibody encompasses both intact and full-length immunoglobulin molecules, as well as Fab, Fab', F(ab')2, Fv), single chain (scFv), fusion proteins containing an antibody portion, and any other modified immunoglobulin molecule that contains an antigen recognition site. Furthermore, a "monoclonal" antibody refers to an antibody that may be produced in any number of ways, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0031] The term "chimeric" antibody refers to an antibody whose amino acid sequences are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

[0032] The term "humanized" antibody refers to a form of a non-human (e.g., murine) antibody that contains minimal non-human (e.g., murine) sequence. Typically, humanized antibodies are human immunoglobulins in which residues of the complementarity determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity ("CDR-grafted") (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and capacity. The humanized antibody may be further modified by substitution of additional residues within the Fv framework regions and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or potency of the antibody. Generally, a humanized antibody will contain substantially all of at least one, and typically two or three, variable domains, which contain all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. The humanized antibody may also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996).

[0033] The term "resurfaced antibody" or "multiple resurfaced antibodies" refers to a murine antibody that has been redesigned to resemble a human antibody by humanizing only those amino acids available on the surface of the V region of the recombinant FV. Resurfacing a murine monoclonal antibody to reduce its immunogenicity can be beneficial to maintain the avidity of the original monoclonal antibody in the resurfaced form, since the native framework-CDR interactions are preserved.

[0034] The term "human antibody" means an antibody having an amino acid sequence derived from the human immunoglobulin locus, such an antibody being made using any technique known in the art.

[0035] The variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically about the amino-terminal 110-125 amino acids in the mature heavy chain and about 90-115 amino acids in the mature light chain, which vary widely in sequence between antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0036] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the portion of an antibody (e.g., the carboxyl terminal portion of the light and / or heavy chain) that is not directly involved in binding the antibody to an antigen, but may exert various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a conserved amino acid sequence compared to the immunoglobulin variable domain. The "constant region" or "constant domain" of an immunoglobulin may contain a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, or a subset of these domains, such as a CH2 domain and a CH3 domain. In certain aspects provided herein, the immunoglobulin constant region does not contain a CH1 domain. In certain aspects provided herein, the immunoglobulin constant region does not contain a hinge. In certain aspects provided herein, the immunoglobulin constant region contains a CH2 domain and a CH3 domain.

[0037] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from the portion of a source antibody that is responsible for binding to cellular antibody receptors and the C1q component of complement. Fc means "fragment crystallizable" and refers to a fragment of an antibody that readily forms protein crystals. The different protein fragments originally described by protein digestion may define the overall general structure of an immunoglobulin protein. "Fc region" or "Fc domain" contains all or part of the CH2 domain, CH3 domain, and optional hinge. "Fc region" or "Fc domain" may refer to a single polypeptide or two disulfide-bonded polypeptides. For reviews of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc includes variants of the native sequence.

[0038] A "wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequences that are located between and connect the CH1 and CH2 domains (in the case of IgG, IgA, and IgD) or between and connect the CH1 and CH3 domains (in the case of IgE and IgM) found in the heavy chain of a naturally occurring antibody. In certain aspects, the wild-type immunoglobulin hinge region sequence is human and may include a human IgG hinge region. A "modified wild-type immunoglobulin hinge region" or "modified immunoglobulin hinge region" refers to a wild-type immunoglobulin hinge region that is (a) from about 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (b) from about 5 amino acids in length (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) to about 120 amino acids in length (e.g., from about 10 to about 40 amino acids in length, or about 15 "Immunoglobulin hinge region" refers to a portion of a wild-type immunoglobulin hinge region having up to about 30 amino acids in length, or about 15 to about 20 amino acids in length, or about 20 to about 25 amino acids in length, having up to about 30% amino acid changes (e.g., up to about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% amino acid substitutions or deletions, or a combination thereof), and having an IgG core hinge region as disclosed in U.S. Patent Application Publication No. 2013 / 0129723 and U.S. Patent Application Publication No. 2013 / 0095097.

[0039] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant region, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, and include the subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0040] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ), based on the amino acid sequence of the constant region. Light chain amino acid sequences are well known in the art. In certain aspects, the light chain is a human light chain.

[0041] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that are modified, naturally or by intervening, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides that contain, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It will be understood that because the polypeptides of the present invention are based on antibodies, in certain aspects the polypeptides can occur as single chains or associated chains.

[0042] As used herein, the term "nucleic acid", "nucleic acid molecule", or "polynucleotide" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, these terms encompass nucleic acids that contain analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly specified. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene. As used herein, the term "nucleic acid," "nucleic acid molecule," or "polynucleotide" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs, and derivatives, fragments, and homologs thereof.

[0043] The term "intron" as used herein refers to a sequence of nucleotides that is transcribed into RNA and then removed from the RNA, typically by splicing, to make a mature form of RNA, such as an mRNA. Typically, an intron's nucleotide sequence is not incorporated into the mature RNA, and neither the intron sequence nor any portion thereof is typically translated or incorporated into a polypeptide. Splice signal sequences, such as splice donors and splice acceptors, are used by the cell's splicing machinery to remove the intron from the RNA.

[0044] The term "vector," as used herein, refers to a linear or circular nucleic acid that contains a segment of nucleic acid of interest.

[0045] The term "expression vector" as used herein refers to a linear or circular nucleic acid molecule that contains one or more expression units. In addition to one or more expression units, an expression vector may also contain additional nucleic acid segments such as. Expression vectors may generally be derived from plasmid or viral DNA, or may contain elements of both.

[0046] As used herein, the term "host cell" can be any type of cell, e.g., a primary cell, a cultured cell, or a cell from a cell line. In certain aspects, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule, and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell that was transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that may occur in subsequent generations or upon integration of the nucleic acid molecule into the host cell genome.

[0047] The term "viable cell number," as used herein, refers to the number of living (surviving) cells present in culture.

[0048] The term "cell viability," as used herein, refers to the ability of cultured cells to survive under a given set of culture conditions or experimental variations. The term, as used herein, also refers to the proportion of cells that are alive at a particular time in culture relative to the total number of live and dead cells at that time.

[0049] The term "recovery titer" or "titer" as used herein refers to the total amount of expressed polypeptide or immunoglobulin produced in a cell culture divided by a given amount of medium volume.

[0050] The term "qP", as used herein, refers to cell specific productivity and is determined from the total immunoglobulin produced divided by the integral viable cell concentration.

[0051] The term "IVC" as used herein refers to integral viable cell concentration;

number

[0052] An "isolated" polypeptide, antibody, nucleic acid, vector, cell, or composition is a polypeptide, antibody, nucleic acid, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, nucleic acids, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in the form found in nature. In some aspects, an isolated antibody, nucleic acid, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants). In some examples, the material is at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0053] It will be understood that the terms "a" and "an" as used herein refer to "one or more" of the listed components, unless otherwise indicated.

[0054] As used herein, unless otherwise specified or clear from the context, the term "or" is understood to be inclusive. When used herein in phrases such as "A and / or B," the term is intended to include both "A and B," "A or B," "A" and "B." Similarly, when used herein in phrases such as "A, B and / or C," the term is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0055] It should be understood that whenever an embodiment is described herein with the term "comprises", otherwise similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided and are part of the disclosure of this application. In this disclosure, "comprises", "comprising", "containing", "having" and the like may have the meanings they have in U.S. and European patent law and may mean "includes", "including", and the like. "Consisting essentially of" or "consisting essentially of" likewise have the meanings under U.S. and European patent law. It should be understood that as far as U.S. patent law is concerned, the term is open-ended, allowing for the presence of more than what is recited, but excluding prior art aspects, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited. It should also be understood that as far as European patent law is concerned, the use of "consisting essentially of" or "consisting essentially of" means that certain further components may be present, i.e., those that do not substantially affect the essential characteristics of the compound or composition.

[0056] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate a deviation of up to 5% above or below that value or range while remaining within the intended meaning of the stated value or range.

[0057] II. Nucleic Acids Encoding Immunoglobulins Human immunoglobulin G (IgG) contains heavy and light chain polypeptides, which together form an immunoglobulin. Immunoglobulin light chains have a variable light chain, which contains the variable light chain complementarity determining regions (CDRs) that help bind to an epitope. Immunoglobulin light chains also contain a light chain constant region. IgG light chains can be either kappa or lambda light chains.

[0058] Immunoglobulin heavy chains have variable heavy chains, which contain the variable heavy chain region complementarity determining regions (CDRs) that help bind to epitopes. Immunoglobulin heavy chains also contain heavy chain constant regions. In the IgG heavy chain constant region, there are three constant domains (CH1, CH2, and CH3) and a hinge region.

[0059] The nucleotide sequence encoding the human IgG heavy chain constant region contains three introns (see FIG. 10). Intron 1 in the human IgG heavy chain constant region is located between the exon encoding the CH1 region and the exon encoding the hinge region. Intron 2 in the human IgG heavy chain constant region is located between the exon encoding the hinge region and the exon encoding the CH2 region. Intron 3 in the human IgG heavy chain constant region is located between the exon encoding the CH2 region and the exon encoding the CH3 region. Similarly, the nucleotide sequence encoding the human IgG heavy chain region includes a leader intron and an intron between the variable heavy domain (VH) and constant domain 1 (CH1).

[0060] Introns are known to be involved in mRNA export through the nuclear pore complex. For general information, see Kohler A. et al., Nature Review Molecular Cell Biology 8:761-773 (2007); Bjork, P. et al., Seminars in Cell & Developmental Biology 32:47-54 (2014); Reed, R. Current Opinion in Cell Biology, 15:326-331 (2003). Therefore, when preparing nucleic acids for recombinant immunoglobulin production, endogenous introns are usually not excised, because nucleic acids with introns typically increase immunoglobulin production titers compared to the corresponding cDNA versions. See, for example, FIG. 2.

[0061] Immunoglobulin production using nucleic acids containing endogenous introns in human IgG heavy chains increases immunoglobulin titers, but the introns can introduce incorrect splice sites that can result in immunoglobulin fragments or variants, thus reducing the purity of the product. The introduction of immunoglobulin fragments or variants into the immunoglobulin pool can increase the difficulty in purifying these fragments and variants, thus unduly burdening the purification process and contributing to reduced product purity.

[0062] In one aspect, the deletion of one or two intronic nucleotide sequences in the IgG heavy chain constant region reduces the production of immunoglobulin fragments or variants and results in increased immunoglobulin titers when expressed together with the appropriate nucleic acid encoding an IgG light chain.

[0063] In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, the nucleotide sequence of the second and third introns of the immunoglobulin heavy chain constant region being deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, the nucleotide sequence comprising the sequence of the first intron and the sequence of the second and third introns of the immunoglobulin heavy chain constant region being deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, the nucleotide sequence comprising only the first intron of the immunoglobulin heavy chain constant region. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, the nucleotide sequence comprising only the second intron of the immunoglobulin heavy chain constant region. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, the nucleotide sequence comprising only the third intron of the immunoglobulin heavy chain constant region.

[0064] In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of one of the three endogenous introns of the immunoglobulin heavy chain constant region is deleted. In some examples, the first intron is deleted. In some examples, the second intron is deleted. In some examples, the third intron is deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence comprises the sequence of the second and third introns, but the sequence of the first intron of the immunoglobulin heavy chain constant region is deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence comprises the sequence of the first and third introns, but the sequence of the second intron of the immunoglobulin heavy chain constant region is deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence comprises the sequence of the first and second introns, but lacks the sequence of the third intron of an immunoglobulin heavy chain constant region.

[0065] In certain aspects, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequences of two of the three endogenous introns in the immunoglobulin heavy chain constant region are deleted. In some examples, the first and second introns are deleted. In some examples, the first and third introns are deleted. In some examples, the second and third introns are deleted. In certain aspects, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence comprises the sequence of the first intron, but the sequence of the second and third introns of the immunoglobulin heavy chain constant region is deleted. In certain aspects, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence comprises the sequence of the second intron, but the sequence of the first and third introns of the immunoglobulin heavy chain constant region is deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequence includes the sequence of the third intron but lacks the sequences of the first and second introns of an immunoglobulin heavy chain constant region.

[0066] In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the first intron has been deleted, the nucleotide sequence of the second and / or third intron of the immunoglobulin heavy chain constant region has been deleted, and the nucleotide sequence of the second and / or third intron has been replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the second intron has been replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the third intron has been replaced with the nucleotide sequence of the first intron. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the first and second introns has been deleted from the immunoglobulin heavy chain constant region, and the nucleotide sequence of the second intron has been replaced with the nucleotide sequence of the first intron. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the first and third introns have been deleted from the immunoglobulin heavy chain constant region, and the nucleotide sequence of the third intron has been replaced with the nucleotide sequence of the first intron. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the first intron has been deleted, and the nucleotide sequences of the second and third introns of the immunoglobulin heavy chain constant region have been deleted, and the nucleotide sequences of the second and third introns have each been replaced with the nucleotide sequence of the first intron.

[0067] In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the second and / or third intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the second intron is replaced with the nucleotide sequence of the first intron. In some examples, the nucleotide sequence of the third intron is replaced with the nucleotide sequence of the first intron. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the second intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of the first intron, and the sequences of the first and third introns are not deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of the first intron, and the sequences of the first and second introns are not deleted. In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequences of the second and third introns in an immunoglobulin heavy chain constant region are replaced with the nucleotide sequence of the first intron, and the sequence of the first intron is not deleted.

[0068] In certain aspects, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In some examples, the nucleotide sequence of the first intron in the immunoglobulin heavy chain constant region is deleted. In certain aspects, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron comprising approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region, and the sequences of the first and / or second introns are deleted.

[0069] In certain aspects, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the second intron in the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron that comprises approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In some examples, the nucleotide sequence of the first intron in the immunoglobulin heavy chain constant region is deleted. In certain aspects, the present disclosure encompasses nucleic acids comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequence of the second intron in the immunoglobulin heavy chain constant region is replaced with a nucleotide sequence of an intron that comprises approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region, and the sequences of the first and / or third introns are deleted.

[0070] In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region. In one aspect, the leader intron or the VH-CH1 intron is deleted. In one aspect, the leader intron is deleted. In one aspect, the VH-CH1 intron is deleted.

[0071] In certain aspects, the disclosure encompasses an isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron.

[0072] In certain aspects, the disclosure encompasses a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for intron 1 in the heavy chain constant region.

[0073] In any of the above aspects, a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain may also be expressed together with a nucleic acid encoding an immunoglobulin light chain. In some examples, the immunoglobulin light chain is a kappa light chain. In some examples, the immunoglobulin light chain is a lambda light chain.

[0074] In any of the above aspects, the nucleic acid expresses immunoglobulins at higher titers when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain as compared to a nucleic acid containing all intron sequences in the heavy chain constant region.

[0075] In any of the above aspects, the nucleic acid expresses immunoglobulins with higher titers when the nucleic acid is expressed together with a nucleic acid encoding an immunoglobulin light chain compared to a nucleic acid that does not contain an intron sequence in the heavy chain constant region.

[0076] In any of the above aspects, the nucleic acid is not codon optimized. In any of the above aspects, the nucleic acid is codon optimized.

[0077] In any of the above aspects, the expressed immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4.

[0078] In any of the above aspects, the expressed immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin.

[0079] The nucleic acid of the present disclosure can be in the form of RNA or in the form of DNA.DNA includes genomic DNA or synthetic DNA, and can be double-stranded or single-stranded, and if single-stranded, can be coding strand or non-coding (antisense) strand.In some aspects, the nucleic acid is DNA that lacks another endogenous intron.

[0080] In some aspects, the nucleic acid comprises non-naturally occurring nucleotides. In some aspects, the nucleic acid is recombinantly produced.

[0081] In certain aspects, the nucleic acid is isolated.

[0082] III. Cells and Vectors Vectors and cells containing the nucleic acids described herein are also provided.

[0083] In certain aspects, provided herein are cells (e.g., host cells) that contain expression vectors that express (e.g., recombinantly express) nucleic acids described herein encoding immunoglobulins. Provided herein are vectors (e.g., expression vectors) that contain nucleotide sequences encoding immunoglobulin heavy and light chains for recombinant expression in a host cell (e.g., a mammalian host cell). Also provided herein are host cells that contain such vectors for recombinantly expressing nucleic acids that contain nucleotide sequences encoding immunoglobulins.

[0084] Recombinant expression of an immunoglobulin requires an expression vector containing a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4) as described herein. In some aspects, the vector comprises a nucleic acid as described herein that contains a nucleotide sequence encoding an immunoglobulin. In some aspects, the host cell comprises the vector. The disclosure also provides constructs in the form of plasmids, vectors, transcription cassettes, or expression cassettes that comprise a nucleic acid as described herein. In some aspects, the vector is an expression vector, additional nucleotide sequences (e.g., a promoter) to aid in recombinant immunoglobulin production in the host cell.

[0085] The expression vector can be introduced into a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the immunoglobulins described herein. Thus, provided herein is a host cell containing a polynucleotide encoding an immunoglobulin or polypeptide thereof described herein, operably linked to a promoter for expression of such sequence in the host cell.

[0086] In certain aspects, the host cell contains a vector that includes nucleic acid encoding the immunoglobulin heavy and light chain polypeptides of the immunoglobulins described herein, hi certain aspects, the host cell contains a plurality of different vectors that include nucleic acid encoding all of the immunoglobulin polypeptides described herein.

[0087] A vector or combination of vectors can contain nucleic acids encoding two or more polypeptides that interact to form an immunoglobulin as described herein: for example, a first nucleic acid encoding a heavy chain and a second nucleic acid encoding a light chain. When the two polypeptides are encoded by nucleic acids in two separate vectors, these vectors can be transfected into the same host cell.

[0088] A variety of host-expression vector systems may be utilized to express the immunoglobulins or polypeptides thereof (e.g., immunoglobulin constant regions; heavy or light chains) described herein. Such host-expression systems are either vehicles in which a coding sequence of interest may be produced and subsequently purified, or cells which, when transformed or transfected with the appropriate nucleotide coding sequences, are capable of expressing the immunoglobulins or polypeptides thereof described herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., Cauliflower Mosaic Virus, CaMV; Tobacco Mosaic Virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the antibody coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter).

[0089] Once the immunoglobulins described herein or polypeptides thereof (e.g., immunoglobulin constant regions; heavy or light chains) are produced by recombinant expression, they may be purified by any antibody purification method known in the art, such as, for example, chromatography (e.g., ion exchange chromatography, particularly affinity chromatography for a particular antigen followed by protein A, and sizing column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Additionally, the antibodies described herein may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification (e.g., FLAG tags, his tags, or avidin).

[0090] IV. Immunoglobulin Production Immunoglobulins may be produced by any method of immunoglobulin synthesis known in the art, for example, by recombinant expression techniques. The methods described herein use, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the skill of the art. These techniques are described, for example, in the references cited herein and are described in detail in the literature. For example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. al.,Current Protocols in Molecular Biology,John Wiley & Sons(1987 and annual revision);Current Protocols in Immunology,John Wiley & Sons(1987 and annual revision)Gait(ed.)(1984)Oligonucleotide Synthesis:A Practical Approach,IRL Press;Eckstein(ed.)(1991)Oligonucleotides and Analogues:A Practical Approach,IRL Press;Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0091] In some aspects, isolated nucleic acids are provided having a nucleotide sequence encoding any of the immunoglobulin heavy chain constant regions, and optionally, the immunoglobulin heavy chain variable regions and / or the immunoglobulin light chains of the present disclosure. L and / or C H Such nucleic acids may encode an amino acid sequence comprising an immunoglobulin V constant chain, such as an immunoglobulin light constant chain and / or a heavy constant chain. L and / or V H In some aspects, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In some aspects, host cells comprising such nucleic acids are also provided. In some aspects, the host cell comprises (e.g., is transfected with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an immunoglobulin light chain and an amino acid sequence comprising an immunoglobulin heavy chain, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an immunoglobulin light chain, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an immunoglobulin heavy chain.

[0092] In some aspects, host cells containing nucleic acids of the present disclosure encoding immunoglobulins are cultured under conditions suitable for expression of the antibody. For recombinant production of immunoglobulins using nucleic acids of the present disclosure, one or more nucleic acids encoding heavy and / or light chains are isolated and inserted into one or more vectors for further cloning and / or expression in host cells as described herein.

[0093] In certain aspects, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain lacking the nucleotide sequence of the second and third introns of the immunoglobulin heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at a higher titer than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain constant region that includes all intron sequences or none of the intron sequences. In certain aspects, the cell is an isolated cell. In some aspects, the core sequence of the first intron is deleted. In some aspects, the core sequence of the second intron is deleted. In some aspects, the core sequence of the third intron is deleted. In some aspects, the nucleotide sequence of the first and second introns is deleted. In some aspects, the nucleotide sequence of the first and third introns is deleted. In some aspects, the nucleotide sequence of the second intron is replaced with the nucleotide sequence of the first intron. In some aspects, the nucleotide sequence of the third intron is replaced with the nucleotide sequence of the first intron. In some embodiments, the nucleotide sequence of the second intron is replaced with the nucleotide sequence of the first intron. In some embodiments, the nucleotide sequence of the third intron is replaced with the nucleotide sequence of the first intron. In certain embodiments, the nucleotide sequence of the third intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of an intron that comprises approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In certain embodiments, the nucleotide sequence of the second intron in the immunoglobulin heavy chain constant region is replaced with the nucleotide sequence of an intron that comprises approximately the same number of nucleotides as the nucleotide sequence of the first intron of the immunoglobulin heavy chain constant region. In some embodiments, the nucleotide sequence of the first intron in the immunoglobulin heavy chain constant region is also deleted.

[0094] In a particular aspect, the host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at the same or higher titers as a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, in which all introns 1 to 3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain. In one aspect, the leader intron or the VH-CH1 intron is deleted. In one aspect, the leader intron is deleted. In one aspect, the VH-CH1 intron is deleted.

[0095] In a particular aspect, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at the same or higher titers as a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, in which all of introns 1 to 3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

[0096] In a particular aspect, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at the same or higher titers as a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, in which all of introns 1 to 3 of the immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

[0097] In a particular aspect, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at the same or higher titers as a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain and a nucleic acid encoding an immunoglobulin light chain, in which all of introns 1 to 3 of the immunoglobulin heavy chain constant region are present.

[0098] In a particular aspect, the host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, in which the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at a higher titer than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain and a nucleic acid encoding an immunoglobulin light chain in which none of introns 1 to 3 of the immunoglobulin heavy chain constant region is present. In one aspect, the leader intron or the VH-CH1 intron is deleted. In one aspect, the leader intron is deleted. In one aspect, the VH-CH1 intron is deleted.

[0099] In a particular aspect, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at higher titers than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain and a nucleic acid encoding an immunoglobulin light chain, wherein none of introns 1 to 3 of the immunoglobulin heavy chain constant region are present.

[0100] In a particular aspect, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses immunoglobulins at higher titers than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain and a nucleic acid encoding an immunoglobulin light chain in which none of introns 1 to 3 of the immunoglobulin heavy chain constant region are present.

[0101] In certain aspects, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell does not express an immunoglobulin fragment. In one aspect, the leader intron or the VH-CH1 intron is deleted. In one aspect, the leader intron is deleted. In one aspect, the VH-CH1 intron is deleted.

[0102] In certain aspects, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein all intron nucleotide sequences in the immunoglobulin heavy chain are deleted except for the leader intron, and a nucleic acid encoding an immunoglobulin light chain, and the host cell does not express an immunoglobulin fragment.

[0103] In certain aspects, a host cell comprises a nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in the immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region, and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell does not express an immunoglobulin fragment.

[0104] In any of the above aspects, the immunoglobulin light chain is a kappa light chain or a lambda light chain.

[0105] In any of the above aspects, the nucleic acid is codon optimized. In any of the above aspects, the nucleic acid is not codon optimized.

[0106] In any of the above aspects, the immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4.

[0107] In any of the above aspects, the immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin.

[0108] In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovery titer of at least 1,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovery titer of at least 1,500 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovery titer of at least 2,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovery titer of at least 2,500 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovery titer of at least 3,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovery titer of at least 3,500 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovery titer of at least 4,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovered titer of at least 4,500 mg / L. In any of the above embodiments, the immunoglobulin produced from the pool of clones has a recovered titer of at least 5,000 mg / L.

[0109] In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 1,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 1,500 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 2,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 3,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 4,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 5,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 6,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 7,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 8,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 9,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 10,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 11,000 mg / L. In any of the above embodiments, the immunoglobulin produced from the highest expressing clone has a recovered titer of at least 12,000 mg / L.

[0110] In any of the above embodiments, the host cell is a eukaryotic cell. In any of the above embodiments, the eukaryotic cell is a CHO cell.

[0111] In some aspects, exogenous nucleic acid is introduced into the cell.

[0112] In some aspects, the method further comprises purifying the immunoglobulin from the cell or host cell.

[0113] Aspects of the present disclosure may be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies of the present disclosure and methods of using the antibodies of the present disclosure. It will be apparent to those skilled in the art that numerous modifications, both to materials and methods, may be made without departing from the scope of the present disclosure. EXAMPLES

[0114] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light thereof will be suggested to those skilled in the art and are to be included within the spirit and scope of the present application.

[0115] Example 1. Characterization of Misspliced ​​Immunoglobulin Variants Introns are important in several areas, including regulating alternative splicing, enhancing gene expression, and controlling the transport of mRNA from the nucleus. Therefore, nucleic acids used in expression vectors for producing immunoglobulins contain endogenous introns. However, this can result in mis-spliced ​​immunoglobulin variants that can be difficult to purify and / or reduce the recovered titer of the immunoglobulin product.

[0116] Cell culture production of MAb1 resulted in three immunoglobulin variants due to intron splice variants. Expression vectors containing the light and heavy chain sequences of each immunoglobulin, including endogenous intron sequences in the heavy chain constant region, were linearized and transfected into Chinese Hamster Ovary (CHO) cells. Transfection of the linearized expression vector was performed by nucleofection to generate pools of cells. The linearized expression vector was integrated into the CHO genome by random integration. The pool of cells expressing MAb1 was cloned to generate cell lines that produced the clones using the cell line development process appropriate at that time.

[0117] CHO cells were grown and MAb1 was secreted during culture pre-loss. Immunoglobulins were harvested and purified using high performance size exclusion chromatography (HPSEC). For HPSEC fractionation, MAb1 was injected onto a TSK-gel G3000SW×L column (7.8 mm×30 cm; Tosoh Bioscience, King of Prussia, PA, USA) at ambient column temperature. Samples were eluted isocratically with a mobile phase composed of 0.1 M sodium phosphate, 0.1 M sodium sulfate, pH 6.8, at a flow rate of 1.0 mL / min. Fractions collected from multiple injections were pooled and concentrated prior to characterization and analysis. See Harris, C. et al., MABS, 11:1452-1463 (2019).

[0118] As shown in FIG. 1, HPSEC reveals three splice variants, namely, a monomeric immunoglobulin with an extension, and two fragments. The immunoglobulin variant with an extension was confirmed to have an additional lambda light chain constant domain at the C-terminus of the immunoglobulin. This was caused by an alternative heavy chain transcript with an additional lambda light chain constant domain at the C-terminus of the heavy chain. Two fragments were determined to be splice variants associated with intron 2 between the hinge and CH2 regions. Specifically, one fragment is a truncated heavy chain due to an in-frame stop codon, and the other is a mis-splicing event where a frameshift occurs to create a stop codon and results in a truncated heavy chain. Thus, the immunoglobulin fragment variants are produced by introns in the immunoglobulin heavy chain constant region.

[0119] Example 2. Engineering Nucleic Acids to Reduce Immunoglobulin Splice Variants To eliminate the immunoglobulin fragments produced in Example 1, a codon-optimized cDNA nucleotide sequence was made without any introns in the immunoglobulin heavy chain constant region of MAb2. The cDNA version removed intron 2 in the immunoglobulin heavy chain constant region, preventing the production of two fragments.

[0120] However, it is known that introns are required to enhance protein expression in CHO cells. Therefore, CHO cells were transfected by nucleofection with non-codon-optimized gDNA or codon-optimized cDNA to generate pools, grown, and secreted immunoglobulins during a pool fed-batch process in shake flasks. See Figure 2A. Figure 2B shows that the cDNA version of MAb2 produced significantly less immunoglobulin compared to the genomic DNA (gDNA) version, as shown by the recovery titers.

[0121] Since the two immunoglobulin fragment variants result from missplicing in intron 2, individual introns were removed from the immunoglobulin heavy chain constant region and compared to cDNA or gDNA versions without any introns of the immunoglobulin heavy chain constant region. The following constructs were made and tested: (1) gDNA containing all three introns in the heavy chain constant region (non-codon optimized), (2) cDNA (codon optimized), (3) gDNA with intron 1 removed (non-codon optimized), (4) gDNA with intron 2 removed (non-codon optimized), (5) gDNA with intron 3 removed (non-codon optimized), and (6) gDNA with all introns removed (non-codon optimized). See Figure 3A.

[0122] Nucleic acids were prepared and transfected into CHO cells and pooled as described in Example 1. Pools were screened for single cell clones using a Single Cell Printer™ (Cytena), which deposits droplets containing single cells into wells of a 384-well plate. Single cell deposition was confirmed using a Cellavista® plate reader (Synentec). The highest expressing clones were selected for further characterization. The highest clones were expanded and secreted MAb2 for each construct. MAb2 was harvested on day 13. Figure 3B shows that cDNA and gDNA (without intron) had the lowest recovery titers. Meanwhile, gDNA constructs lacking either intron 1, intron 2, or intron 3 had increased recovery titers compared to gDNA, gDNA (without intron), and cDNA constructs containing all three introns.

[0123] Figures 4A-C show that the mean viable cell count (VCN), cell viability, and integral viable cell concentration (IVC) were similar across all constructs, while Figure 4D reveals that the increased titer was due to increased cellular productivity (qP). It was surprising that removing one intron increased the recovered titer, since introns are known to increase immunoglobulin expression.

[0124] Example 3. Manipulation of nucleic acids to increase recovery titers To further determine the importance of each intron to the ability of CHO cells to produce immunoglobulins, the following MAb2 constructs were made: (1) gDNA with intron 2 deleted (non-codon optimized), (2) gDNA with introns 1 and 2 deleted (non-codon optimized), (3) gDNA with introns 2 and 3 deleted (non-codon optimized), and (4) gDNA without any introns (non-codon optimized). To determine the importance of using codon-optimized nucleotide sequences, a set of identical constructs was made except that codon-optimized sequences were used.

[0125] The constructs were transfected into CHO cells as in Example 2. Similarly, during the culturing process in Example 2, the CHO cells were grown to secrete immunoglobulins. On day 11, the immunoglobulin products were harvested and the recovery titers, qP, VCN, and IVC were all determined. Figure 5A reveals that the recovery titers of immunoglobulins produced from gDNA without intron 2 and gDNA without introns 2 and 3 were the highest, regardless of whether the nucleotide sequence was codon-optimized or not. However, gDNA without introns 1 and 2 had similar titer levels to gDNA without any introns. Figures 5B and 5C show that the VCN and IVC for each were almost the same. However, the cell productivity (qP) revealed that gDNA without intron 2 and gDNA without introns 2 and 3 were the most productive. See Figure 5D. This suggests that intron 1 is important for increasing immunoglobulin production.

[0126] To further understand the importance of intron 1 to increased immunoglobulin production, several novel MAb2 constructs were generated. In addition to the non-codon optimized constructs generated above (i.e., gDNA with all introns (non-codon optimized), gDNA without intron 2 (non-codon optimized), gDNA without introns 2 and 3 (non-codon optimized), and gDNA without introns 1 and 2 (non-codon optimized)), the following constructs were generated: (1) gDNA with intron 3 moved to the position of intron 1 and introns 1 and 2 deleted (non-codon optimized), (2) gDNA with intron 3 moved to the position of intron 1, where the nucleotide sequence of intron 3 was modified to enhance the 5' splice donor site by making a single nucleotide change in the intron sequence. (3) gDNA with intron 1 moved to the position of intron 3 and with introns 2 and 3 deleted (non-codon optimized), (4) gDNA without any introns (non-codon optimized), (5) gDNA with wild-type IgG1 and all introns (non-codon optimized), (6) gDNA with wild-type IgG1 and without introns 2 and 3 (non-codon optimized), (7) gDNA with wild-type IgG1 and without introns 1 and 2 (non-codon optimized), and (8) gDNA with wild-type IgG1 and without any introns (non-codon optimized). See Figure 6A.

[0127] These constructs were transfected into CHO cells as described in Example 1. The CHO cells were grown to secrete immunoglobulins during the culture process described in Example 1. Immunoglobulins were harvested on day 11 and the recovery titers were determined. Figure 6B shows that moving intron 1 to the position of intron 3 results in similar recovery titers to gDNA with all introns and gDNA with only intron 1 in the normal position. Figure 6B also confirms that the effect of intron 1 in maintaining similar titers to constructs containing all three introns is not affected by whether the type is wild type IgG1 or half-life extended IgG1.

[0128] Example 4. Manipulation of the nucleic acid in additional immunoglobulins to determine if there is an increase in recovery titer Additional immunoglobulin molecules are tested to determine whether the results seen with MAb2 are specific to that immunoglobulin. Figure 7 shows the various constructs made for MAb2, MAb1, MAb3, and MAb4. For each immunoglobulin, the following constructs were made: (1) gDNA with all introns (non-codon optimized), (2) gDNA with only intron 1 (non-codon optimized), and (3) gDNA without any introns (non-codon optimized). MAb2 and MAb3 contain kappa light chains, and MAb1 and MAb4 contain lambda light chains. The lambda light chains have different introns between the variable and constant light chains and different polyA tails. These constructs were made as previously described in Example 1. These constructs were transfected into CHO cells to generate pools as described in Example 2. The CHO cells were grown to secrete immunoglobulins during the culture process described in Example 2. Immunoglobulins were harvested on day 11 and the recovery titer was determined.

[0129] FIG. 8 shows that the presence of only intron 1 in the heavy chain constant region of MAb2, MAb3, MAb1, and MAb4 resulted in similar immunoglobulin titers compared to the respective constructs with all introns in the heavy chain constant region. In addition, the constructs with only intron 1 increased the titers compared to gDNA without any introns in the immunoglobulin heavy chain constant region. Furthermore, FIG. 9 shows that for MAb2, MAb3, MAb1, and MAb4, the immunoglobulin titers from days 7 to 11 were similar between the constructs with all introns in the heavy chain constant region compared to the constructs with only intron 1 in the heavy chain constant region. For all molecules tested, gDNA without any introns resulted in significantly lower immunoglobulin titers compared to the constructs with all introns in the heavy chain constant region and the constructs with only intron 1.

[0130] These data provide further evidence that intron 1 of the heavy chain constant region is important in maintaining higher immunoglobulin titer levels. Furthermore, the data show that the increase in titer levels is not limited to MAb2. Furthermore, the presence of immunoglobulin kappa or lambda light chains does not affect titer levels.

[0131] Example 5. Manipulation of nucleic acid to replace intron 3 with an intron of the same size as intron 1 To identify why intron 1 may increase the recovered titer, this example will demonstrate that the reason is its size rather than the nucleotide sequence of intron 1 itself. Intron 1 in MAb2 is 391 nucleotides, while intron 3 is 97 nucleotides. The following constructs will be made: (1) MAb2 gDNA (non-codon optimized) with all introns, (2) MAb2 (non-codon optimized) without introns 2 and 3, (3) MAb2 (non-codon optimized) without introns 1 and 2 but with the number of nucleotides in intron 3 increased to approximately the same size as intron 1, (4) MAb2 (non-codon optimized) but with the size of intron 1 reduced to approximately the size of intron 3 and introns 2 and 3 deleted, and (5) MAb2 gDNA (non-codon optimized) without any introns.

[0132] These constructs will be made as previously described in Example 2. These constructs will be transfected into CHO cells as described in Example 2. The CHO cells will be grown and induced to produce immunoglobulins as described in Example 1. The immunoglobulins will be harvested on day 11 and the recovery titers will be determined. The results will show that increasing the size of intron 3 will result in an increase in the recovery titer, making it comparable to MAb2 without introns 2 and 3. However, reducing the size of intron 1 will result in comparable results to MAb2 without introns 1 and 2. This example will reveal that the size of intron 1 is more important than its nucleotide sequence itself.

[0133] Example 6. Engineering of the nucleic acid to remove the VH-CH1 intron increases potency Immunoglobulin heavy chains have five introns: (1) leader intron; (2) VH-CH1 intron; (3) intron 1 of the heavy chain constant region; (4) intron 2 of the heavy chain constant region; and (5) intron 3 of the heavy chain constant region. As shown above, removal of introns 2 and / or 3 of the heavy chain constant region reduces the production of immunoglobulin fragments and increases the titer. Therefore, to determine whether introns other than introns 1-3 of the heavy chain constant region affect antibody titer, the following constructs were made: (1) MAb2 gDNA with all five introns (non-codon optimized); (2) MAbX gcDNA without introns 1-3 of the heavy chain constant region (non-codon optimized); and (3) MAbX gDNA with only the leader intron (non-codon optimized). See FIG. 11A.

[0134] These constructs were made by generating an expression vector, linearizing the vector, and using the vector to transfect CHO cells by nucleofection to generate pools of CHO cells. These constructs were transfected into CHO cells as described in Example 2. The CHO cells were grown to secrete immunoglobulins during the culture process described in Example 1. Immunoglobulins were harvested on day 11 and the harvest titer was determined. Figure 11B showed that construct #2 significantly reduced the titer level compared to the control (construct #1), while construct #3 had a titer level similar to the control construct. This example suggests that the VH-CH1 intron reduces immunoglobulin production.

[0135] Example 7. Intron 1 of the heavy chain constant region relieves VH-CH1 intron retention To determine whether intron 1 of the heavy chain constant region increases immunoglobulin titers by reducing VH-CH1 intron retention, the following constructs were made: (1) MAb1 gDNA with introns 1-3 of the heavy chain constant region (non-optimized); (2) MAb1 gDNA with only intron 1 of the heavy chain constant region (non-optimized); (3) MAb1 gcDNA without an intron of the heavy chain constant region (non-optimized); (4) MAb2 gDNA with introns 1-3 of the heavy chain constant region (non-optimized); (5) MAb2 gDNA with only intron 1 of the heavy chain constant region (non-optimized); (6) MAb1 gcDNA without an intron of the heavy chain constant region (non-optimized); (7) MAb3 gDNA with introns 1-3 of the heavy chain constant region (non-optimized); (8) MAb3 gDNA with only intron 1 of the heavy chain constant region (non-optimized); (9) MAb3 without an intron of the heavy chain constant region. (10) MAb4 gDNA with heavy chain constant region introns 1-3 (non-optimized); (11) MAb4 gDNA with only heavy chain constant region intron 1 (non-optimized), and (12) MAb4 gcDNA without the heavy chain constant region intron (non-optimized). See Figure 12A.

[0136] These constructs were made by generating an expression vector, linearizing the vector, and using the vector to transfect CHO cells by nucleofection to generate pools of CHO cells. RNA was isolated from the CHO cells and cDNA was prepared using standard methods known in the art. Reverse transcriptase PCR was performed with specific primers for the VH and CH1 regions to determine constructs containing the VH-CH1 intron. Agarose gel electrophoresis was performed to separate the PCR products.

[0137] Figure 12B shows that intron 1 of the heavy chain constant region reduces retention of the VH-CH1 intron in four different antibody constructs, but when intron 1 of the heavy chain constant region is removed (i.e., gcDNA construct), the VH-CH1 intron is retained. This example suggests that splice variants reduce titer levels when intron 1 of the heavy chain constant region is absent.

[0138] Example 8. Intron 1 of the heavy chain constant region maintains high generation of immunoglobulin titers Because retention of the VH-CH1 intron reduces titer levels, the following constructs were made to determine whether immunoglobulin titers were altered compared to control gDNA: (1) MAb2 gDNA with all five immunoglobulin heavy chain introns; (2) MAbX with leader intron, VH-CH1, and intron 1 of the heavy chain constant region; (3) MAbX (gcDNA) with only the leader intron and VH-CH1 intron; (4) MAbX with only the leader intron; (5) MAbX without the immunoglobulin heavy chain intron; and (6) MAbX with only intron 1 of the heavy chain constant region. See Figure 13A.

[0139] These constructs were made by generating an expression vector, linearizing the vector, and using the vector to transfect CHO cells by nucleofection to generate pools of CHO cells. The CHO cells were grown to secrete immunoglobulins during the culture process described in Example 1. Immunoglobulins were harvested on day 11 and harvest titers and cell productivity were determined.

[0140] Removal of the VH-CH1 intron from the gcDNA construct increases titer and cellular productivity (construct #3 compared to construct #4). See Figures 13B and 13C. Further removal of the leader intron shows no difference in titer or cellular productivity (construct #4 compared to #5), and intron 1 alone shows no difference in titer or cellular productivity compared to no intron or leader intron alone (#6 compared to #4 or #5). Ibid. This example shows that intron 1 of the heavy chain constant region increases titer levels and cellular productivity by precisely removing the VH-CH1 intron.

[0141] Example 9. Intron 1 of the heavy chain constant region relieves retention of the VH-CH1 intron when moved to a new location in the heavy chain constant region To further understand the importance of intron 1 of the heavy chain constant region in alleviating VH-CH1 intron retention, several new MAb2 constructs were generated. In addition to the non-codon optimized constructs generated above (i.e., gDNA with all introns (non-codon optimized), gDNA without introns 2 and 3 (non-codon optimized), gDNA without introns 1 and 2 (non-codon optimized), and gcDNA without introns 1 to 3 of the heavy chain constant region (non-codon optimized)), the following constructs were generated: (1) gDNA in which intron 3 has been moved to the position of intron 1 and introns 1 and 2 have been deleted (non-codon optimized); (2) gDNA in which intron 3 has been moved to the position of intron 1, the nucleotide sequence of intron 3 has been modified to increase the strength of the 5' splice donor site by changing one nucleotide in the intron sequence, and introns 1 and 2 have been deleted (non-codon optimized); and (3) gDNA in which intron 1 has been moved to the position of intron 3 and introns 2 and 3 have been deleted (non-codon optimized). See FIG. 14A.

[0142] These constructs were made by generating an expression vector, linearizing the vector, and using the vector to transfect CHO cells by nucleofection to generate pools of CHO cells. The CHO cells were grown to secrete immunoglobulins during the culture process described in Example 1. Immunoglobulins were harvested on day 11 and the harvest titer and cell productivity were determined. RNA was isolated from the CHO cells and cDNA was prepared using standard methods known in the art. Reverse transcriptase PCR was performed with specific primers for the VH and CH1 regions to determine the constructs containing the VH-CH1 intron. Agarose gel electrophoresis was performed to separate the PCR products.

[0143] FIG. 14B shows that constructs containing only intron 3 of the heavy chain constant region, constructs in which intron 3 of the heavy chain constant region is moved to the position of intron 1 of the heavy chain constant region, and constructs in which intron 3 of the heavy chain constant region is moved (with a mutation) to the position of intron 1 of the heavy chain constant region did not reduce retention of the VH-CH1 intron, whereas the construct in which intron 1 of the heavy chain constant region is moved to the position of intron 3 resulted in the VH-CH1 intron being properly spliced ​​out. Similarly, the recovered titers of constructs containing only intron 3 of the heavy chain constant region, constructs in which intron 3 of the heavy chain constant region was moved to the position of intron 1 of the heavy chain constant region, and constructs in which intron 3 of the heavy chain constant region was moved (with a mutation) to the intron 1 position of the heavy chain constant region were significantly reduced compared to the control construct (gDNA), whereas the construct in which intron 1 of the heavy chain constant region was moved to the position of intron 3 had recovered titer levels similar to the control construct, suggesting that the position of intron 1 is not important but the sequence of intron 1 is. * * *

[0144] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0145] All references (e.g., publications, or patents, or patent applications) cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication, or patent, or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0146] Other aspects are within the scope of the following claims.

Claims

1. 1. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in said immunoglobulin heavy chain are deleted except for the leader intron, the VH-CH1 intron, and intron 1 in the heavy chain constant region.

2. The nucleic acid of claim 1, wherein the leader intron or the VH-CH1 intron is deleted.

3. The nucleic acid of claim 2, wherein the leader intron is deleted.

4. The nucleic acid of claim 2, wherein the VH-CH1 intron is deleted.

5. 1. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in said immunoglobulin heavy chain are deleted except for the leader intron.

6. An isolated nucleic acid comprising a nucleotide sequence encoding an immunoglobulin heavy chain, wherein the nucleotide sequences of all introns in said immunoglobulin heavy chain are deleted except for intron 1 in the heavy chain constant region.

7. The nucleic acid, when co-expressed with a nucleic acid encoding an immunoglobulin light chain, expressing immunoglobulins at higher titers than nucleic acids that include all intron sequences of the immunoglobulin heavy chain, or expressing immunoglobulins at higher titers than nucleic acids that do not include intron sequences of the immunoglobulin heavy chain constant region; and / or expressing immunoglobulins but not immunoglobulin fragments; The nucleic acid according to any one of claims 1 to 6.

8. The nucleic acid of claim 7 , wherein the immunoglobulin light chain is a kappa light chain or a lambda light chain.

9. The nucleic acid according to any one of claims 1 to 6, wherein the nucleic acid is codon-optimized.

10. 8. The nucleic acid of claim 7, wherein the expressed immunoglobulin has an isotype of IgG1, IgG2, IgG3, or IgG4.

11. The nucleic acid of claim 10 , wherein the immunoglobulin is a human, humanized, chimeric, or resurfaced immunoglobulin.

12. The nucleic acid according to any one of claims 1 to 6, wherein the nucleic acid is deoxyribonucleic acid (DNA).

13. A vector comprising the nucleic acid according to any one of claims 1 to 6.

14. An expression vector comprising the nucleic acid according to any one of claims 1 to 6.

15. A host cell comprising the vector of claim 13.

16. A host cell comprising the expression vector of claim 14.

17. The host cell of claim 15, wherein the host cell is a eukaryotic cell or a Chinese hamster ovary (CHO) cell.

18. The host cell described in claim 16, wherein the host cell is a eukaryotic cell or a CHO cell.

19. 10. A method for producing an immunoglobulin, comprising culturing a host cell in a medium and under conditions in which the cell expresses the immunoglobulin; the host cell comprises the immunoglobulin heavy chain-encoding nucleic acid of any one of claims 1 to 6 and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell expresses the immunoglobulin at the same or a higher titer than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, wherein the nucleic acid encodes an immunoglobulin heavy chain and in which all of introns 1 to 3 of an immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

20. 10. A method for producing an immunoglobulin, comprising culturing a host cell in a medium and under conditions in which the cell expresses the immunoglobulin; the host cell comprises the immunoglobulin heavy chain-encoding nucleic acid of any one of claims 1 to 6 and a nucleic acid encoding an immunoglobulin light chain, wherein the host cell expresses the immunoglobulin at a higher titer than a host cell comprising a nucleic acid encoding an immunoglobulin heavy chain, wherein none of introns 1 to 3 of an immunoglobulin heavy chain constant region are present, and a nucleic acid encoding an immunoglobulin light chain.

21. 10. A method for producing an immunoglobulin, comprising culturing a host cell in a culture medium and under conditions in which the cell expresses the immunoglobulin; the host cell comprises an immunoglobulin heavy chain-encoding nucleic acid of any one of claims 1 to 6 and a nucleic acid encoding an immunoglobulin light chain, and the host cell expresses the immunoglobulin but does not express an immunoglobulin fragment.

22. the nucleic acid encoding the immunoglobulin heavy chain is deoxyribonucleic acid (DNA), and / or the immunoglobulin light chain is a kappa light chain or a lambda light chain, and / or the nucleic acid encoding the immunoglobulin heavy chain is codon-optimized, and / or the expressed immunoglobulin has an IgG1, IgG2, IgG3, or IgG4 isotype, or is a human, humanized, chimeric, or resurfaced immunoglobulin having an IgG1, IgG2, IgG3, or IgG4 isotype; and / or the expressed immunoglobulins produced from the pool of clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, or at least 5,000 mg / L; and / or the immunoglobulins produced from the highest expressing clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 6,000 mg / L, at least 7,000 mg / L, at least 8,000 mg / L, at least 9,000 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, or at least 12,000 mg / L; and / or The host cell is a eukaryotic cell or a CHO cell.

20. The method of claim 19.

23. The nucleic acid encoding the immunoglobulin heavy chain is deoxyribonucleic acid (DNA), and / or the immunoglobulin light chain is a kappa light chain or a lambda light chain, and / or the nucleic acid encoding the immunoglobulin heavy chain is codon-optimized, and / or the expressed immunoglobulin has an IgG1, IgG2, IgG3, or IgG4 isotype, or is a human, humanized, chimeric, or resurfaced immunoglobulin having an IgG1, IgG2, IgG3, or IgG4 isotype; and / or the expressed immunoglobulins produced from the pool of clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, or at least 5,000 mg / L; and / or the immunoglobulins produced from the highest expressing clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 6,000 mg / L, at least 7,000 mg / L, at least 8,000 mg / L, at least 9,000 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, or at least 12,000 mg / L; and / or The host cell is a eukaryotic cell or a CHO cell.

21. The method of claim 20.

24. The nucleic acid encoding the immunoglobulin heavy chain is deoxyribonucleic acid (DNA), and / or the immunoglobulin light chain is a kappa light chain or a lambda light chain, and / or the nucleic acid encoding the immunoglobulin heavy chain is codon-optimized, and / or the expressed immunoglobulin has an IgG1, IgG2, IgG3, or IgG4 isotype, or is a human, humanized, chimeric, or resurfaced immunoglobulin having an IgG1, IgG2, IgG3, or IgG4 isotype; and / or the expressed immunoglobulins produced from the pool of clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 2,500 mg / L, at least 3,000 mg / L, at least 3,500 mg / L, at least 4,000 mg / L, at least 4,500 mg / L, or at least 5,000 mg / L; and / or the immunoglobulins produced from the highest expressing clones have a recovered titer of at least 1,000 mg / L, at least 1,500 mg / L, at least 2,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 6,000 mg / L, at least 7,000 mg / L, at least 8,000 mg / L, at least 9,000 mg / L, at least 10,000 mg / L, at least 11,000 mg / L, or at least 12,000 mg / L; and / or The host cell is a eukaryotic cell or a CHO cell.

22. The method of claim 21.

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