Regulation of lactogenic activity in mammalian cells

Abstract

Methods, cells, and compositions for producing a product of interest, such as a recombinant protein, are provided. The present invention provides mammalian cells with reduced or eliminated lactogenic activity, wherein expression of a pyruvate kinase muscle (PKM) polypeptide isoform is knocked down or knocked out, the PKM polypeptide isoform comprising the PKM-1 polypeptide isoform. Also provided are methods and compositions for reducing or eliminating lactogenic activity in cells, as well as compositions comprising cells with reduced or eliminated lactogenic activity, and methods of using the same.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 649,963, filed March 29, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on March 28, 2019, is named 00B206_0785_SL.txt and is 444,511 bytes in size.

[0003] 1. Field of the invention The present disclosure relates to methods and compositions for producing a product of interest, such as a recombinant protein. In particular, the disclosure relates to mammalian cells expressing a product of interest, where the cells (e.g., Chinese Hamster Ovary (CHO) cells) have regulated lactogenic activity. The disclosure also relates to methods and compositions for regulating pyruvate kinase muscle (PKM) expression (e.g., PKM-1 expression) in mammalian cells to reduce or eliminate lactogenic activity in the cells, as well as compositions comprising one or more cells with reduced or eliminated lactogenic activity, and methods of use thereof. [Background technology]

[0004] 2.Background Chinese hamster ovary (CHO) cells are widely used in the production of therapeutic proteins for clinical use due to their capacity for proper protein folding, assembly, and post-translational processing. Normally, like other immortalized cell lines, CHO cells tend to consume glucose and produce lactate through aerobic glycolysis, a process known as the Warburg effect (Warburg, 1956, Science 123(3191):309-14). Accumulation of lactate in the production medium can have adverse effects on cell growth, viability, and productivity. Such lactogenic behavior (i.e., lactate-producing behavior) of CHO cells during the production process can cause a decrease in viability and productivity, and may alter the quality of the therapeutic protein produced.

[0005] Several approaches targeting process conditions have been developed to mitigate lactate production in lactogenic CHO cell lines. For example, optimizing the amount of copper has been shown to be effective in preventing lactogenic behavior in some CHO cell lines (Luo et al., 2012, Biotechnol. Bioeng. 109(1):146-56; Xu et al., 2016, Bioprocess Biosyst. Eng. 39(11):1689-702). Another approach involving nutrient supply control (high-end pH-controlled glucose delivery, or HIPDOG), triggered by increasing the pH in the medium, has been shown to be effective in reducing or eliminating lactate accumulation in large-scale CHO culture media (Gagnon et al., 2011, Biotechnol. Bioeng. 108(6):1328-37). However, these approaches are limited because the former is not applicable to all lactogenic CHO cell lines and the latter may complicate the large-scale manufacturing process, and furthermore, these approaches do not target the mechanisms underlying the lactogenic behavior in CHO cells.

[0006] Therefore, there is a need in the art for techniques to reduce lactate production in cell culture. Summary of the Invention

[0007] 3. Overview The present disclosure relates to methods, cells, and compositions for producing a product of interest, such as a recombinant protein. In particular, the methods, cells, and compositions described herein include improved mammalian cells, such as Chinese Hamster Ovary (CHO) cells, that express the product of interest and have regulated lactogenic activity. The methods and compositions described herein control the lactogenic activity of mammalian cells, thereby reducing or eliminating the underlying effects associated with lactogenic activity, such as reduced viability and productivity of the mammalian cells, and altered quality of the product of interest produced.

[0008] The disclosure further relates to methods and compositions for reducing or eliminating lactogenic activity in cells by regulating pyruvate kinase myoglobin (PKM) expression (e.g., PKM-1 expression) in mammalian cells, as well as cells with reduced or eliminated lactogenic activity and methods of use thereof.

[0009] In one aspect, the disclosure relates to mammalian cells having reduced or eliminated lactogenic activity, in which expression of one or more isoforms of pyruvate kinase muscle (PKM) polypeptide is knocked down or knocked out. In certain embodiments, the knocked out or knocked down PKM polypeptide isoform is a PKM-1 polypeptide isoform. In certain embodiments, the knocked out or knocked down PKM polypeptide isoform is both a PKM-1 polypeptide isoform and a PKM-2 polypeptide isoform. In certain embodiments, the lactogenic activity of the mammalian cells is less than about 50%, such as less than about 20%, of the lactogenic activity of a reference cell. In certain embodiments, the reference cell is a cell comprising one or more wild type alleles of the PKM gene. For example, both alleles of the PKM gene are wild type or unmodified. In certain embodiments, the lactogenic activity of the mammalian cells is measured on day 14 or day 15 of the production phase. In certain embodiments, the mammalian cells produce less than about 1.0 g / L, or less than about 2.0 g / L, of lactate during the production phase, e.g., less than about 1.0 g / L, or less than about 2.0 g / L, of lactate in shake flasks during the production phase. In certain embodiments, the mammalian cells produce less than about 2.0 g / L of lactate in a bioreactor during the production phase. The present disclosure provides a mammalian cell comprising an allele of the PKM gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-41, or comprising the nucleotide sequences set forth in SEQ ID NOs: 37 and 38. The present disclosure further provides compositions comprising one or more cells, e.g., mammalian cells, disclosed herein.

[0010] In certain embodiments, the mammalian cell comprises a nucleic acid sequence encoding a product of interest. In certain embodiments, the nucleic acid sequence is integrated in the cellular genome of the mammalian cell at a targeted location. Alternatively and / or in addition, the nucleic acid encoding the product of interest is randomly integrated into the cellular genome of the mammalian cell. In certain embodiments, the mammalian cell is a CHO cell.

[0011] In certain embodiments, the product of interest comprises a protein, e.g., a recombinant protein. In certain embodiments, the product of interest comprises an antibody or an antigen-binding fragment thereof. For example, but not limited to, the antibody is a multispecific antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof. In certain embodiments, the antibody is a chimeric antibody, a human antibody, or a humanized antibody, and / or a monoclonal antibody.

[0012] In another aspect, the disclosure relates to a method for reducing or eliminating lactogenic activity in a cell. In certain embodiments, the method comprises knocking down or knocking out expression of a pyruvate kinase muscular (PKM) polypeptide isoform. In certain embodiments, the method comprises administering to the cell a genetic modification system that knocks down or knocks out expression of a pyruvate kinase muscular (PKM) polypeptide isoform. In certain embodiments, the genetic modification system is selected from the group consisting of a CRISPR / Cas system, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, and combinations thereof. In certain embodiments, the method results in a cell having a lactogenic activity that is less than about 50%, such as less than about 20%, of the lactogenic activity of a reference cell. In certain embodiments, the reference cell is a cell that comprises one or more wild-type alleles of the PKM gene. For example, both alleles of the PKM gene are wild-type or unmodified. In certain embodiments, the lactogenic activity of the cell is measured on day 14 or day 15 of the production phase. In certain embodiments, the cells produce less than about 1.0 g / L or less than about 2.0 g / L of lactate during the production phase, e.g., less than about 1.0 g / L or less than about 2.0 g / L of lactate in shake flasks during the production phase. In certain embodiments, the cells produce less than about 2.0 g / L of lactate in a bioreactor during the production phase.

[0013] In certain non-limiting embodiments, the genetic engineering system for use in the present disclosure is a CRISPR / Cas9 system comprising a Cas9 molecule and one or more guide RNAs (gRNAs) comprising a targeting domain complementary to a target sequence of the PKM gene. In certain embodiments, the target sequence is selected from the group consisting of a portion of the PKM gene, a 5' intron region adjacent to exon 9 of the PKM gene, a 3' intron region adjacent to exon 9 of the PKM gene, a 3' intron region adjacent to exon 10 of the PKM gene, a region within exon 1 of the PKM gene, a region within exon 2 of the PKM gene, a region within exon 12 of the PKM gene, and combinations thereof. In certain embodiments, the one or more gRNAs comprise a first gRNA comprising a target sequence complementary to the 5' intron region adjacent to exon 9 of the PKM gene, and a second gRNA comprising a targeting domain complementary to the 3' intron region adjacent to exon 9 of the PKM gene. In certain embodiments, the one or more gRNAs include a first gRNA that includes a target sequence complementary to a region in exon 2 of the PKM gene, and a second gRNA that includes a target domain complementary to a region in exon 12 of the PKM gene. For example, but not limited to, the one or more gRNAs include a sequence selected from the group consisting of SEQ ID NOs: 33-34 and 42-43, and combinations thereof. In certain embodiments, expression of a PKM polypeptide isoform is knocked out or knocked down, eliminating lactogenic activity of the cell. In certain embodiments, the PKM polypeptide isoform is a PKM-1 polypeptide isoform, or a combination of PKM-1 and PKM-2 polypeptide isoforms.

[0014] In certain embodiments, the genetic engineering system for use in the present disclosure is a zinc finger nuclease (ZFN) system or a transcription activator-like effector nuclease (TALEN) system. In certain non-limiting embodiments, the genetic engineering system comprises an RNA selected from the group consisting of short hairpin RNA (shRNA), small interfering RNA (siRNA), and microRNA (miRNA), where the RNA is complementary to the mRNA expressed by the PKM gene. In certain embodiments, the mRNA expressed by the PKM gene encodes a PKM-1 polypeptide isoform. In certain embodiments, the expression of the PKM-1 polypeptide isoform is knocked down to reduce the lactogenic activity of the cell. In certain embodiments, the genetic engineering system further comprises a second RNA selected from the group consisting of shRNA, siRNA, and microRNA miRNA, where the second RNA is complementary to a portion of the mRNA expressed by the PKM gene that encodes the PKM-2 polypeptide isoform. In certain embodiments, expression of the PKM-1 and PKM-2 polypeptide isoforms is knocked out or knocked down, reducing the lactogenic activity of the cells.

[0015] In a further aspect, the disclosure provides a method for producing a product of interest, e.g., from a cell disclosed herein. For example, the method for producing a product of interest includes culturing mammalian cells having reduced or eliminated lactogenic activity to produce the product of interest. In certain embodiments, the disclosure provides a method for culturing a population of mammalian cells expressing a product of interest, wherein the mammalian cells have reduced or eliminated lactogenic activity. In certain embodiments, the reduced or eliminated lactogenic activity is brought about by knocking down or knocking out expression of a pyruvate kinase muscle (PKM) polypeptide isoform in the mammalian cells. In certain embodiments, the PKM polypeptide isoform is a PKM-1 polypeptide isoform. In certain embodiments, expression of a PKM-2 polypeptide isoform is also knocked down or knocked out. In certain embodiments, the method can also include isolating the product of interest from the cell culture. [Brief description of the drawings]

[0016] [Fig. 1A-1F] The amount of lactate in mAb-1 cell line directly correlates with the amount of PKM-1. Cell viability (Fig. 1A), titer at day 14 (Fig. 1B), and lactate amount (Fig. 1C) in mAb-1 cell line under low lactate and high lactate process. (Fig. 1D) PKM-1 and PKM-2 Western blot analysis of mAb-1 cell lysates at different days during production under low lactate and high lactate process. Actin was used as a loading control. Quantification of relative PKM-1 (Fig. 1E) and PKM-2 (Fig. 1F) amounts normalized to actin. [Fig. 2A-2F]Lactate content of mAb-2 cell line directly correlates with the amount of PKM-1. Cell viability (Fig. 2A), titer at day 14 (Fig. 2B), and lactate content (Fig. 2C) in mAb-2 cell line under low lactate and high lactate process. (Fig. 2D) PKM-1 and PKM-2 Western blot analysis of mAb-2 cell lysates at different days during production under low lactate and high lactate process. Actin was used as a loading control. Quantification of relative PKM-1 (Fig. 2E) and PKM-2 (Fig. 2F) content normalized to actin. [Figure 3A-3E] Although CHO cells do not express PKL / R enzymes, the amount of these enzymes does not correlate with the amount of lactate in the production culture. (Figure 3A) Western blot analysis of intracellular expression of PKL / R proteins in two different CHO host cell lines (K1 and DHFR- / -) and human 293 cells. Actin is used as a loading control. (Figures 3B and 3C) Intracellular protein amounts of PKL / R in mAb-1 (Figure 3B) and mAb-2 (Figure 3C) cell lines at different days during the high-lactate and low-lactate manufacturing process. (Figures 3D and 3E) Quantification of the relative amounts of PKL / R normalized to actin in mAb-1 (Figure 3D) and mAb-2 (Figure 3E) cell lines. [Figure 4A-4C]The cell lines are knocked out by generating PKM-1. (Figure 4A) Schematic of exons 8-10 of the PKM gene and gRNA and screening primers used to evaluate the deletion of exon-9 in mAb-2 cell lines. Two gRNAs flanking exon 9 of the PKM gene were co-transfected with the Cas9 transgene to induce the deletion of exon 9. Transfected cells were cloned single cells. PCR primers flanking the deletion region were used to screen for cell lines with successful deletion of exon-9 in the PKM allele. (Figure 4B) Wild-type (WT) mAb-2 cells and cell lines in which the deletion of exon-9 was targeted via gRNA and Cas9 transgene were analyzed by screening PCR primers. The upper band represents the WT PKM allele, while the lower band represents the PKM allele with the deletion of exon 9. KO: knockout cell line, HET: heterozygous cell line. (FIG. 4C) Sequence comparison of the PKM WT allele versus the exon-9 KO allele in the targeted cell lines. Note that the KO-15 cell line has a larger deletion in the 5′ region of exon-9 in the targeted PKM allele than was intended (by 122 bp). [Figure 5A-5H] Turning off or reducing expression of PKM-1 prevented lactogenic behavior in mAb-2 cell lines, even under high lactate processes. Viable cell counts (FIG. 5A), cell viability (FIG. 5B), day 14 titer (FIG. 5C), day 14 specific productivity (FIG. 5D), and lactate amount (FIG. 5E) of WT and PKM-1 HET and KO mAb-2 cell lines during a 14-day batch-fed production assay using an AMBR15 bioreactor. Process controls were the same as for the high lactate process in FIG. 2. (FIG. 5F) Western blot assay of cell lysates of the indicated cell lines for PKM-1 and PKM-2 proteins. Actin is used as a loading control. (FIGS. 5G and 5H) Quality of antibody products, including aggregation rate (FIG. 5G) and percentage of charge variants (FIG. 5H), of mAb-2 from the indicated cell lines. [Figures 6A-6G]Lactogenic behavior is prevented or reduced in PKM-1 KO or HET mAb-2 cell lines by using the high lactate process, regardless of cell age or post-thaw. Viable cell count (FIG. 6A), cell viability (FIG. 6B), day 14 titer (FIG. 6C), day 14 specific productivity (FIG. 6D), and lactate content (FIG. 6E) for the indicated cell lines at young cell ages in a 14-day batch-fed production assay using an AMBR15 bioreactor. (FIGS. 6F and 6G) ​​Antibody product quality, including aggregation rate (FIG. 6F) and percentage of charge variants (FIG. 6G) for mAb-2 from the indicated cell lines. Process controls were the same as in FIG. 5. Error bars represent standard error of four independent experiments. [Figure 7] MAb-3 producing pools derived from PKM and PKM-1 KO host cell lines had comparable or high Qp and titer (in shake flask production) but poor growth. [Figure 8A-8B] MAb-3 producing pools derived from PKM and PKM-1 KO host cell lines produced less lactate (FIG. 8A) but consumed more glucose (FIG. 8B) in shake flask production. [Figure 9A] MAb-3 producing pools derived from PKM and PKM-1 KO host cell lines exhibited different amino acid synthesis / consumption rates in shake flask production. [Figure 9B] The bars and pathways enclosed in rectangles indicate that the PKM KO host cell line accumulated 3-phosphoglycerate. [Figure 9C] Bars and pathways enclosed in rectangles indicate that the WT host cell line accumulated pyruvate. [Figure 9D] The bars and pathways enclosed in rectangles indicate that the PKM-1 KO host cell line accumulated the TCA cycle product α-ketoglutarate (a-KG). [Figure 9E] The bars and pathways enclosed in rectangles indicate that the PKM-1 KO host cell line accumulated the TCA cycle product oxaloacetate. [Figure 10]The host cell consumption or production rates of glucose, lactate, and amino acids. [Figure 11] MAb-3 producing pools derived from the PKM and PKM-1 KO host cell lines exhibited distinct glycosylation profiles in shake flask production, with reduced galactosylation in the PKM KO host and slightly reduced fucosylation in the PKM KO and PKM-1 KO host cell lines. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 5. Detailed Description For purposes of clarity, and not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections: 5.1 Definitions; 5.2 Regulation of PKM expression; 5.3 Cells with reduced or eliminated lactogenic activity; 5.4 Cell culture methods; 5.5 Products; and 5.6 Illustrative Embodiments.

[0018] 5.1 Definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which each term is used. Particular terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner describing the compositions and methods of the present disclosure, and how to make and use them.

[0019] As used herein, in the claims and / or specification, when used in conjunction with the term "comprising," the use of the words "a" or "an" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0020] As used herein, the terms "comprise(s) / include(s)," "having / has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0021] As used herein, the term "lactogenic behavior" or "lactogenic activity" refers to the lactogenic activity of a cell, for example by consuming glucose to produce lactate by aerobic glycolysis. In certain embodiments, the lactogenic activity of a cell can be measured by the amount of lactate accumulated in the cell culture medium.

[0022] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on the limitations of the method, i.e., the measurement system, by which the value is measured or determined. For example, "about" can mean within three or more than three standard deviations per one practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. The terms "cell culture medium" and "culture medium" refer to a nutrient solution used to grow mammalian cells that typically provides at least one component from one or more of the following categories: 1) A source of energy, usually in the form of carbohydrates such as glucose; 2) All the essential amino acids, and usually a basic group of 20 amino acids plus cysteine; 3) Vitamins and / or other organic compounds required in low concentrations; 4) Free fatty acids; and 5) Trace Elements (Trace elements are defined as inorganic compounds or naturally occurring elements that are typically required in very low concentrations, usually in the micromolar range).

[0023] The nutrient solution can optionally be supplemented with one or more components from any of the following categories: 1) hormones and other growth factors, such as insulin, transferrin, and epidermal growth factor; 2) Salts and buffers, such as calcium, magnesium, and phosphate; 3) nucleosides and bases, such as adenosine, thymidine, and hypoxanthine; and 4) Protein and tissue hydrolysates.

[0024] "Culturing" a cell means contacting the cell with a cell culture medium under conditions suitable for the survival and / or growth and / or proliferation of the cell.

[0025] By "batch culture" is meant a culture medium in which all components for cell culture, including cells and all culture nutrients, are supplied to the culture bioreactor at the start of the culture process.

[0026] As used herein, "fed-batch cell culture" refers to a batch culture in which cells and medium are initially fed into a culture bioreactor, additional culture nutrients are fed to the culture either continuously or in discrete increments during the culture process, and cells and / or product may or may not be harvested periodically prior to the end of the culture.

[0027] A "perfusion culture", sometimes referred to as a continuous culture, is a culture in which the cells are maintained in the culture, e.g., by filtration, encapsulation, anchoring to microcarriers, etc., and medium is introduced and removed from the culture bioreactor continuously, in stages, or intermittently (or any combination of these).

[0028] As used herein, the term "cell" refers to animal cells, mammalian cells, cultured cells, host cells, recombinant cells, and recombinant host cells. Such cells are generally cell lines obtained from or derived from mammalian tissues that are capable of proliferation and viability when placed in medium containing the appropriate nutrients and / or growth factors.

[0029] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the host cell regardless of the number of passages. The progeny need not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the invention.

[0030] The term "mammalian host cell" or "mammalian cell" refers to a cell line derived from a mammal capable of growth and survival when placed in either monolayer or suspension culture in a medium containing appropriate nutrients and growth factors. The growth factors required for a particular cell line can be readily determined empirically without undue experimentation, for example, as described in Mammalian Cell Culture (Mather, JP ed., Plenum Press, NY 1984), and Barnes and Sato, (1980) Cell, 22:649. Typically, the cells are capable of expressing and secreting large amounts of a particular protein of interest, e.g., a glycoprotein, into the medium.Examples of suitable mammalian host cells within the context of the present disclosure include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dp12.CHO cells (European Patent No. 307,247 published March 15, 1989); CHO-K1 (ATCC, CCL-61); monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci., 383:44-68 1982); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). In certain embodiments, mammalian cells include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dp12.CHO cells (European Patent No. 307,247 published March 15, 1989).

[0031] The term "peptone" within the context of this disclosure is meant to refer to a media supplement that is essentially hydrolyzed animal protein. The source of this protein can be animal by-products from slaughtered horses, purified gelatin, or plant material. The protein is typically hydrolyzed using acid, heat, or various enzyme preparations.

[0032] The "growth phase" of a cell culture refers to a period of exponential cell growth (log phase) during which cells generally divide rapidly. The length of time that cells are maintained in the growth phase can vary based on, for example, the type of cell, the growth rate of the cells, and / or the conditions of the medium. In certain embodiments, during this phase, cells are cultured under conditions that maximize cell growth for a period of time (usually 1-4 days). The determination of the growth cycle of the host cell is determined for the particular host cell, as contemplated without undue experimentation. "Duration and conditions that maximize cell growth" and the like refers to culture conditions in which cell growth and division are determined to be optimal for a particular cell line. In certain embodiments, during the growth phase, cells are cultured in a nutrient medium containing necessary additives, typically in a humidified, controlled atmosphere at about 30-40° C., such that optimal growth for the particular cell line is achieved. In certain embodiments, cells are maintained in the growth phase for a period of about 1-4 days, usually 2-3 days.

[0033] The "transition phase" of a cell culture refers to the period during which the culture conditions of the production phase are engaged. During the transition phase, environmental factors such as the temperature of the cell culture medium, the osmolality of the medium, etc., are shifted from growth conditions to production conditions.

[0034] "Production phase" of cell culture refers to the period during which cell growth is / remains stagnant. Exponential cell growth typically declines before or during this phase and is taken over by protein production. During the production phase, exponential cell growth ceases and protein production predominates. During this period, the medium is generally replenished to support the continuous production of protein and to obtain the desired glycoprotein product. Fed-batch and / or perfusion cell culture processes replenish the cell culture medium or supply fresh medium during this phase to achieve and / or maintain the desired cell density, viability, and / or titer of recombinant protein product. The production phase can be carried out on a large scale.

[0035] The term "expression" or "expressing" is used herein to mean transcription and translation occurring in a host cell. The degree of expression of a product gene in a host cell can be measured based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell. For example, the mRNA transcribed from the product gene is preferably quantified by Northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). The protein encoded by the product gene can be quantified either by assaying the biological activity of the protein or by using an assay independent of such activity, such as Western blot or radioimmunoassay, using an antibody capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0036] As used herein, "polypeptide" generally refers to peptides and proteins having more than about 10 amino acids. The polypeptide can be homologous to the host cell, or, preferably, exogenous, meaning that it is heterologous, i.e., foreign, to the host cell utilized, e.g., a human protein produced by a Chinese hamster ovary cell, or a yeast polypeptide produced by a mammalian cell. In certain embodiments, mammalian polypeptides (polypeptides originally derived from a mammalian organism), more preferably polypeptides that are directly secreted into the medium, are used.

[0037] The term "protein" is meant to refer to a sequence of amino acids whose chain length is sufficient to produce higher tertiary and / or quaternary structures. This is to distinguish it from "peptides" or other low molecular weight drugs that do not have such structures. Typically, proteins herein have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD. Examples of proteins encompassed by the definition herein include all mammalian proteins, particularly therapeutic and diagnostic proteins such as therapeutic and diagnostic antibodies, as well as general proteins that contain one or more disulfide bonds, including multi-chain polypeptides that contain one or more inter- and / or intra-chain disulfide bonds.

[0038] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence (including multimers of such pairs)), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0039] As used herein, an "antibody fragment," "antigen-binding portion" (or simply "antibody portion") of an antibody, or an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0040] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0041] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, an antibody is an IgG1 isotype. In certain embodiments, an antibody is an IgG2 isotype. The heavy chain constant domains that correspond to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0042] As used herein, the term "titer" refers to the total amount of recombinantly expressed antibody produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of antibody per milliliter or liter of medium (mg / mL or mg / L). In certain embodiments, titer is expressed in grams of antibody per liter of medium (g / L). Titer can be expressed or evaluated in terms of relative measurements, such as the percentage increase in titer compared to obtaining protein product under different medium conditions.

[0043] The terms "nucleic acid", "nucleic acid molecule" or "polynucleotide" include any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the base sequence, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. Here, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases, including derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for directly expressing the antibodies of the present disclosure in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, the mRNA may be chemically modified to increase the stability of the RNA vector and / or expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356, or European Patent No. 2 101 823 B1).

[0044] As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0045] The term "hybridoma" refers to a hybrid cell line produced by the fusion of an immunogenic immortalized cell line with an antibody-producing cell. The term encompasses the progeny of a heterohybrid myeloma fusion resulting from the fusion of a human cell with a mouse myeloma cell line, followed by a plasma cell commonly known as a trioma cell line. In addition, the term is meant to include any immortalized hybrid cell line that produces antibodies, such as quadromas. See, e.g., Milstein et al., Nature, 537:3053 (1983).

[0046] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody derived from a non-human source produced by a human or human cell, or utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0047] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises nearly all of at least one, usually two, variable domains in all or nearly all of the CDRs corresponding to the variable domains of a non-human antibody and all or nearly all of the FRs corresponding to the variable domains of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0048] As used herein, the term "hypervariable region" or "HVR" refers to the regions of an antibody variable domain that are hypervariable in sequence and determine antigen-binding specificity, e.g., each of the "complementarity determining regions" (CDRs). Generally, an antibody comprises six CDRs, three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include: (a) the hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) the CDRs located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996));

[0049] Unless otherwise indicated, CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the designations of CDRs can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0050] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0051] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or arising during the preparation of monoclonal antibodies, such mutations usually being present in small amounts). In comparison to a polyclonal antibody preparation, which typically includes different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the subject matter of the present disclosure can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or a portion of the human immunoglobulin loci, and such methods and other exemplary methods of producing monoclonal antibodies are described herein.

[0052] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of a natural antibody generally have a similar structure, and each domain contains four conserved framework regions (FR) and three complementarity determining regions (CDR). See, for example, Kindt et al., Kuby Immunology, 6 th, WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Moreover, antibodies that bind to a specific antigen may be isolated by using a VH or VL domain from an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0053] As used herein, the term "cell density" refers to the number of cells in a given volume of medium. In certain embodiments, a high cell density is desirable in that it results in higher protein production capacity. Cell density can be monitored by any technique known in the art, including but not limited to extracting a sample from the medium and analyzing the cells under a microscope, using a commercially available cell counting device, or using a suitable commercially available probe introduced into the bioreactor itself (or into the loop through which the medium and suspended cells pass back to the bioreactor).

[0054] As used herein, the term "seeding" refers to the addition or inoculation of growing cells into a medium at the beginning of the production phase. Further, as used herein, the term "seed train" refers to the continuous passaging of cells in a volume of medium of about 20 L or less to maintain a cell line.

[0055] As used herein, the term "recombinant cell" refers to a cell that has some genetic modification relative to the original parent cell from which it was derived. Such genetic modification can be the result of the introduction of a heterologous gene to express a gene product, such as a recombinant protein.

[0056] As used herein, the term "recombinant protein" generally refers to peptides and proteins, including antibodies, that are "heterologous," i.e., foreign to the antibody produced by the host cell utilized, e.g., CHO cells.

[0057] As used herein, "PKM polypeptide" refers to a polypeptide encoded by a PKM gene. PKM polypeptides include the PKM-1 and / or PKM-2 polypeptide isoforms.

[0058] 5.2 Regulation of PKM expression Glycolysis is the process of glucose metabolism used to generate energy by mammalian cells, such as CHO cells. Glycolysis can occur in high or low flux states. The cellular response to glucose levels and the switch between these flux states can vary depending on the cell line and the amount and combination of isoenzymes present in the glycolytic pathway (Mulukutla et al., 2014, PLoS One 9(6):e98756). Under normal culture conditions, CHO cells, like other immortalized cell lines, tend to consume glucose and produce lactate through aerobic glycolysis, a process known as the Warburg effect (Warburg, 1956, Science 123(3191):309-14). Accumulation of lactate in the production medium can adversely affect cell growth, viability, and productivity. Therefore, the control of cellular energy flux and metabolism can be leveraged to avoid the undesirable consequences caused by lactate accumulation during the cell culture production phase (Mulukutla et al., 2010, Trends Biotechnol. 28(9):476-84; Luo et al., 2012, Biotechnol. Bioeng. 109(1):146-56; Ahn and Antoniewicz, 2012, Biotechnol. J. 7(1):61-74).

[0059] The final step of the glycolysis process involves the conversion of phosphoenolpyruvate (PEP) to pyruvate, mediated by the pyruvate kinase (PK) enzyme. Four different PK isoforms have been identified: PK liver (PKL), PK erythroid (PKR), PK muscle 1 (PKM-1), and PK muscle 2 (PKM-2). The PK enzyme is expressed by two different genes: PKLR and PKM. Alternative exon splicing results in the different PK isoforms. The presence of both exons 1 and 2 in the mRNA transcript from the PKLR gene results in the expression of the PKR protein, whereas an mRNA transcript starting in exon 2 results in the expression of the PKL protein. Alternative splicing of exons 9 or 10 in the PKM gene transcript results in PKM-1 or PKM-2, respectively. Specifically, PKM-1 includes exon 9 and excludes exon 10 of the PAM gene, and PKM-2 includes exon 10 and excludes exon 9 of the PKM gene. Tissue-specific promoters, transcription factors, and alternative splicing control the expression of these isoforms in different tissues and cell lines (Chaneton and Gottlieb, 2012, Trends. Biochem. Sci. 37(8):309-16; Israelsen and Vander Heiden, 2015, Semin Cell Dev Biol 43:43-51; Mazurek, 2011, Int. J. Biochem. Cell Biol 43(7):969-80; Harada et al., 1978, Biochim. Biophys. Acta. 524(2):327-39; Noguchi et al., 1986, J. Biol. Chem. 261(29):13807-12; Noguchi et al., 1987, J. Biol. Chem. 262(29):14366-71).

[0060] PKM-2 has been extensively studied due to its central role in cancer cell metabolism and tumor growth, characterized by high glucose consumption and lactate production. While the PKM-1 enzyme is constitutively active, PKM-2 activity is regulated by oligomerization, substrate binding, and post-translational modifications (Christofk et al., 2008, Nature 452(7184):230-3; Chaneton and Gottlieb, 2012, Trends Biochem. Sci. 37(8):309-16; Israelsen and Vander Heiden, 2015, Semin. Cell Dev. Biol 43:43-51). For example, fructose 1,6-bisphosphate (FBP) reversibly binds to PKM-2 and promotes tetramerization and therefore activation of PKM-2 (Ashizawa et al., 1991, J. Biol. Chem. 266(25):16842-6; Dombrauckas et al., 2005, Biochemistry 44(27):9417-29). Phosphorylation of PKM-2 at tyrosine 105 or binding of PKM-2 to other phosphotyrosine proteins inactivates PKM-2 by blocking FBP binding and preventing PKM-2 tetramerization (Christofk et al., 2008, Nature 452(7184):181-6; Hitosugi et al., 2009, Sci. Signal 2(97):ra73). However, increasing levels of glycolysis promote acetylation of PKM-2 at lysine 305 as part of a metabolic feedback loop, targeting PKM-2 for degradation by chaperone-mediated autophagy (Lv et al., 2011, Mol. Cell 42(6):719-30) (Macintyre and Rathmell, 2011, Mol. Cell 42(6):713-4). Furthermore, PKM-2 dimers have been shown to utilize PEP as a phosphate donor and localize to the nucleus where they act as protein kinases, phosphorylating STAT3 and activating MEK5, promoting cell proliferation (Gao et al., 2012, Mol. Cell 45(5):598-609).

[0061] According to one aspect, the present disclosure relates to a method of controlling lactogenic activity of a mammalian cell by controlling PKM expression, e.g., PKM polypeptide expression, in the cell. For example, a method of controlling lactogenic activity of a mammalian cell includes, but is not limited to, knocking out or knocking down PKM polypeptide expression in the cell. In certain embodiments, the expression of PKM-1 is knocked down or knocked out. In certain embodiments, the expression of PKM-2 is knocked down or knocked out. In certain embodiments, the expression of both PKM-1 and PKM-2 is knocked down or knocked out. As used herein, knocked out expression refers to removing the expression of a PKM polypeptide, e.g., PKM-1 polypeptide and / or PKM-2 polypeptide, in a cell when compared to a reference cell. As used herein, knocked down expression refers to reducing the expression of a PKM polypeptide, e.g., PKM-1 polypeptide and / or PKM-2 polypeptide, in a cell when compared to a reference cell.

[0062] In certain embodiments, the reference cell is a cell in which expression of a PKM polypeptide, such as PKM-1 and / or PKM-2, is unregulated, e.g., unreduced. In certain embodiments, the reference cell is a cell that contains at least one or both wild-type alleles of a PKM gene. For example, but not limited to, the reference cell is a cell that has both wild-type PKM alleles. In certain embodiments, the reference cell is a WT CHO cell.

[0063] In certain embodiments, expression of a PKM polypeptide, e.g., PKM-1 and / or PKM-2, in a cell engineered to knock down expression of a PKM polypeptide is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the PKM polypeptide expression in a reference cell, e.g., a WT CHO cell. In certain embodiments, expression of PKM-1 polypeptide in a cell modified to knock down expression of PKM-1 polypeptide is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the PKM-1 polypeptide expression in a reference cell, e.g., a WT CHO cell.

[0064] In certain embodiments, expression of a PKM polypeptide, e.g., PKM-1 and / or PKM-2, in a cell engineered to knock down expression of a PKM polypeptide is at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% of the expression of a PKM polypeptide in a reference cell, e.g., a WT CHO cell. In certain embodiments, expression of a PKM-1 polypeptide in a cell modified to knock down expression of a PKM-1 polypeptide is at least about 90%, at least about 80%, at least about 70%, at least about 60%, at least about 50%, at least about 40%, at least about 30%, at least about 20%, at least about 10%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% of the expression of a PKM-1 polypeptide in a reference cell, e.g., a WT CHO cell.

[0065] In certain embodiments, expression of a PKM polypeptide, e.g., PKM-1 and / or PKM-2, in a cell engineered to knock down expression of a PKM polypeptide is about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of the PKM polypeptide expression in a reference cell, e.g., a WT CHO cell. In certain embodiments, expression of a PKM polypeptide, e.g., PKM-1 and / or PKM-2, in a cell engineered to knock down expression of a PKM polypeptide is about 40% or less of the PKM polypeptide expression in a reference cell, e.g., a WT CHO cell. In certain embodiments, expression of PKM-1 polypeptide in a cell in which expression of the PKM-1 polypeptide has been knocked down is about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1% or less of the PKM-1 polypeptide expression in a reference cell, e.g., a WT CHO cell.

[0066] In certain embodiments, expression of a PKM polypeptide, e.g., PKM-1 and / or PKM-2, in a cell modified to knock down expression of a PKM polypeptide is about 1% to about 90%, about 10% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 85% to about 90%, about 1% to about 80%, about 10% to about 80%, about 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 75% to 80%, 1% to 70%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, 50% to 70%, 60% to 70%, 65% to 70%, 1 % to about 60%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, about 55% to about 60%, about 1% to about 50%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 45% to about 50%, about 1% to about 40%, about 10% to about 40%, about 20% to about 40%, about 30% to about 40%, about 35% to about 40%, about 1% to about 30%, about 10% to about 30%, about 20% to about 30%, about 25% to about 30%, about 1% to about 20%, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 1% to about 10%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, and about 5% to about 40%.In certain embodiments, the expression of PKM-1 polypeptide in a cell in which expression of the PKM-1 polypeptide has been knocked down is about 1% to about 90%, about 10% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 85% to about 90%, about 1% to about 80%, about 10% to about 80%, about 20% to 80%, 30% to 80%, 40% to 80%, 50% to 80%, 60% to 80%, 70% to 80%, 75% to 80%, 1% to 70%, 10% to 70%, 20% to 70%, 30% to 70%, 40% to 70%, 50% to 70%, 60% to 70%, 65% to 70%, 1 % to about 60%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, about 55% to about 60%, about 1% to about 50%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 45% to about 50%, about 1% to about 40%, about 10% to about 40%, about 20% to about 40%, about 30% to about 40%, about 35% to about 40%, about 1% to about 30%, about 10% to about 30%, about 20% to about 30%, about 25% to about 30%, about 1% to about 20%, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 1% to about 10%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, and about 5% to about 40%.

[0067] In certain embodiments, the expression of PKM polypeptides, such as PKM-1 and / or PKM-2, in cells engineered to knock down expression of PKM polypeptides is about 5% to about 40% of the expression of PKM polypeptides in reference cells, such as WT CHO cells. In certain embodiments, the expression of PKM-1 polypeptides in cells engineered to knock down expression of PKM-1 polypeptides is about 5% to about 40% of the expression of PKM-1 polypeptides in reference cells, such as WT CHO cells. The expression levels of PKM polypeptides, such as PKM-1 and / or PKM-2, in different reference cells (e.g., cells that contain at least one or both wild-type alleles of the PKM genes) can vary. For example, seed train cells, or low lactate producing cells, can produce small amounts of PKM-1, while high lactate producing cells can produce large amounts of PKM-1.

[0068] Alternative splicing of exons 9 or 10 in the PKM gene transcript gives rise to PKM-1 or PKM-2, respectively. The knocked-down or knocked-out PKM gene can be a PKM gene of human origin. In certain embodiments, the PKM gene can be from a non-human, such as a rhesus monkey, dog, green monkey, chicken, cow, pig, mouse, Chinese hamster, rat, or rabbit.

[0069] In certain embodiments, the knocked down or knocked out PKM gene can be a Chinese hamster PKM gene. In certain embodiments, the Chinese hamster PKM gene sequence has NCBI reference sequence ID NW_003613709.1 (range: 200602..223561) (SEQ ID NO: 44). In certain embodiments, the PKM gene sequence differs from the NW_003613709.1 sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In certain embodiments, the PKM gene sequence differs by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more from the sequence set forth in SEQ ID NO:44.

[0070] Further non-limiting examples of PKM genes include human PKM genes (e.g., NC_000015.10 (range 72199029..72231624) (SEQ ID NO: 45)), rhesus monkey PKM genes (e.g., NC_027899.1 (range 49211766..49245983) (SEQ ID NO: 46)), green monkey PKM genes (e.g., NC_023667.1 (range 11224332..11255538) (SEQ ID NO: 47)), canine PKM genes (e.g., NC_006612.3 (range 35712853..35737643) (SEQ ID NO: 48)), mouse PKM genes (e.g., NC_000075.6 (range 59656368..59679375) (SEQ ID NO: 49)), rat PKM gene (e.g., NC_005107.4 (range 64480963..64502957) (SEQ ID NO: 50)), rabbit PKM gene (e.g., NC_013685.1 (range 304096..317843) (SEQ ID NO: 51)), chicken PKM gene (e.g., NC_006097.4 (range 1506428..1523684) (SEQ ID NO: 52)), porcine PKM gene (e.g., NC_010449.5 (range 60971807..61032780) (SEQ ID NO: 53)), and bovine PKM gene (e.g., AC_000167.1 (range 18965981..18992644) (SEQ ID NO: 54)). In certain embodiments, the PKM gene sequence differs by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides from any one of the sequences set forth in SEQ ID NOs: 45-54. In certain embodiments, the PKM gene sequence differs by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more from any one of the sequences set forth in SEQ ID NOs: 45-54.

[0071] One skilled in the art will know that different mammalian cells, even those derived from the same species, such as two different CHO host cell lines, may not share the same PKM gene sequence. Small differences in the sequence of the PKM gene, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide differences, may be present in two mammalian cells derived from the same species.

[0072] 5.2.1 Methods for controlling PKM expression In certain embodiments, a genetic engineering system is used to control (e.g., knock down or knock out) the expression of a PKM polypeptide (e.g., PKM-1 expression). A variety of genetic engineering systems known in the art can be used for the methods disclosed herein. Non-limiting examples of such systems include the use of CRISPR / Cas systems, zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, and other tools for protein knockdown by gene silencing, such as small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). Any CRISPR / Cas system known in the art, including conventional, improved, or modified Cas systems, and other bacterial-based genome excision tools, such as Cpf-1, can be used with the methods disclosed herein.

[0073] Any PKM inhibitor known in the art can be used with the methods disclosed herein to regulate PKM activity and thus the lactogenic activity of the cells disclosed herein. Non-limiting examples of PKM inhibitors include sodium monofluorophosphate, L-phenylalanine, creatine phosphate, Ca 2+ , fluorophosphate, and pyridoxal 5'-phosphate.

[0074] In certain embodiments, a portion of the PKM gene is deleted to control, e.g., knock down or knock out, expression of a PKM polypeptide, in certain embodiments, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% of the PKM gene is deleted. In certain embodiments, about 2% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, about 30% or less, about 35% or less, about 40% or less, about 45% or less, about 50% or less, about 55% or less, about 60% or less, about 65% or less, about 70% or less, about 75% or less, about 80% or less, about 85% or less, or about 90% or less of the PKM gene is deleted.In certain embodiments, the concentration of the serotonin-containing compound is about 2% to about 90%, about 10% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, about 85% to about 90%, about 2% to about 80%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 75% to about 80%, about 2% to about 70%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 60% to about 70%, about 65% to about 70%, about 2% ~60%, ~10%~60%, ~20%~60%, ~30%~60%, ~40%~60%, ~50%~60%, ~55%~60%, ~2%~50%, ~10%~50%, ~20%~50%, ~30%~50%, ~40%~50%, ~45%~50%, ~2%~40%, ~10%~40%, ~2%~5 ... 0% to about 40%, about 30% to about 40%, about 35% to about 40%, about 2% to about 30%, about 10% to about 30%, about 20% to about 30%, about 25% to about 30%, about 2% to about 20%, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 2% to about 10%, about 5% to about 10%, or about 2% to about 5% of PKM genes are deleted.

[0075] In certain embodiments, at least one exon of the PKM gene is at least partially deleted. As used herein, "partially deleted" refers to, for example, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 142%, at least about 146%, at least about 147%, at least about 148%, at least about 149%, at least about 150%, 2% or less, approx. 5% or less, approx. 10% or less, approx. 15% or less, approx. 20% or less, approx. 25% or less, approx. 30% or less, approx. 35% or less, approx. 40% or less, approx. 45% or less, approx. 5% or less, about 90% or less, about 95% or less, about 2% to about 90%, about 10% to about 90%, about 20% to about 90%, about 25% to about 90%, about 30% to approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, approximately 80% to approximately 90%, approximately 85% to about 90%, about 2% to about 80%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 60% to about 80%, about 70% to about 80%, about 75% to about 80%, about 2% to about 70%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, about 60% to about 70%, about 65% to about 70%, about 2% to about 60%, about 10% to about 60%, about 20% to about 60%, about 30% to about 60%, about 40% to about 60%, about 50% to about 60%, about This means that 55% to about 60%, about 2% to about 50%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, about 40% to about 50%, about 45% to about 50%, about 2% to about 40%, about 10% to about 40%, about 20% to about 40%, about 30% to about 40%, about 35% to about 40%, about 2% to about 30%, about 10% to about 30%, about 20% to about 30%, about 25% to about 30%, about 2% to about 20%, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 2% to about 10%, about 5% to about 10%, or about 2% to about 5% is deleted.For example, but not limited to, exon 9 of the PKM gene can be at least partially or completely deleted. In certain embodiments, exon 10 of the PKM gene can be at least partially or completely deleted. In certain embodiments, exons 9 and 10 of the PKM gene can be at least partially or completely deleted. In certain embodiments, the region encompassing exons 1 to 12 is at least partially or completely deleted.

[0076] In certain non-limiting embodiments, the CRISPR / Cas9 system is used to control the expression of PKM polypeptides. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains an RNA to guide the crRNA to the correct part of the host DNA, along with a region that is used by Cas9 to bind to tracrRNA (which is generally in the form of a hairpin loop) that forms an active complex with Cas9), and transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9). The terms "guide RNA" and "gRNA" refer to any nucleic acid that facilitates the specific association (i.e., "targeting") of an RNA-guided nuclease, such as Cas9, to a target sequence, such as a genomic or episomal sequence in a cell. The gRNA can be unimolecular (comprising a single-stranded RNA molecule, alternatively called a chimera) or modular (comprising, for example, two or more, typically two, separate RNA molecules, e.g., crRNA and tracrRNA, that are usually associated with each other by duplexing). The CRISPR / Cas9 method can use vectors for transfecting mammalian cells. Guide RNA (gRNA) can be designed for each application, since it is the sequence that uses Cas9 to identify and directly bind to the target DNA in the cell. Multiple crRNAs and tracrRNAs can be packaged together to form a single-stranded guide RNA (sgRNA). The sgRNA can be combined with the Cas9 gene and incorporated into a vector for transfection into cells.

[0077] In certain embodiments, CRISPR / Cas9 for use in controlling expression of one or more PKM polypeptides comprises a Cas9 molecule and one or more gRNAs comprising a targeting domain that is complementary to a target sequence of a PKM gene. In certain embodiments, the target gene is a region of the PKM gene. The target sequence can be any exon or intron region within the PKM gene. For example, the target is to eliminate or reduce expression of PKM1 and / or PKM2 polypeptides. In certain embodiments, the target sequence can be the 5' region adjacent to exon 1, a region within exon 1, a 5' region adjacent to exon 2, a region within exon 2, a 5' region adjacent to exon 9, a 3' region adjacent to exon 9, a 3' region adjacent to exon 10, a region within exon 12, and / or a 3' region adjacent to exon 12 of the PKM gene. For example, the target sequence is selected from the group consisting of, but is not limited to, the 5' intron region adjacent to exon 9 of the PKM gene, the 3' intron region adjacent to exon 9 of the PKM gene, the 3' intron region adjacent to exon 10 of the PKM gene, and combinations thereof.

[0078] In certain embodiments, both the 5' intron region adjacent to exon 9 of the PKM gene and the 3' intron region adjacent to exon 9 of the PKM gene are targeted using the CRISPR / Cas9 system disclosed herein. For example, the CRISPR / Cas9 system includes, but is not limited to, a Cas9 molecule, a gRNA targeting the 5' intron region adjacent to exon 9 of the PKM gene, and a gRNA targeting the 3' intron region adjacent to exon 9 of the PKM gene to generate cells in which PKM-1 is knocked down or knocked out. In certain embodiments, the gRNA targeting the 5' intron region adjacent to exon 9 of the PKM gene comprises the sequence set forth in SEQ ID NO: 33. In certain embodiments, the gRNA targeting the 3' intron region adjacent to exon 9 of the PKM gene comprises the sequence set forth in SEQ ID NO: 34.

[0079] In certain embodiments, the target sequence can be a 5' region adjacent to exon 1, a region within exon 1, a 5' region adjacent to exon 2, a region within exon 2, a region within exon 12, a 3' region adjacent to exon 12, or a combination thereof. For example, but not limited to, the CRISPR / Cas9 system of the present disclosure can include a Cas9 molecule, a gRNA targeting a region within exon 1 of the PKM gene, and a gRNA targeting a region within exon 12 of the PKM gene, e.g., for generating cells in which both PKM-1 and PKM-2 are knocked down or knocked out. In certain embodiments, the CRISPR / Cas9 system of the present disclosure can include a Cas9 molecule, a gRNA targeting a region within exon 2 of the PKM gene, and a gRNA targeting a region within exon 12 of the PKM gene, e.g., for generating cells in which both PKM-1 and PKM-2 are knocked down or knocked out. In certain embodiments, the gRNA targeting a region within exon 2 of the PKM gene comprises the sequence set forth in SEQ ID NO:42. In a particular embodiment, the gRNA targeting a region within exon 12 of the PKM gene comprises the sequence set forth in SEQ ID NO:43.

[0080] In certain embodiments, the gRNA is administered to the cell in a single vector and the Cas9 molecule is administered to the cell in a second vector. In certain embodiments, the gRNA and the Cas9 molecule are administered to the cell in a single vector. Alternatively, each of the gRNA and the Cas9 molecule can be administered by a separate vector. In certain embodiments, the CRISPR / Cas9 system can be delivered to the cell as a ribonucleoprotein complex (RNP), comprising the Cas9 protein complexed with one or more gRNAs, e.g., by electroporation (see, e.g., DeWitt et al., Methods 121-122:9-15 (2017) for further methods of delivering RNPs to cells). In certain embodiments, administering the CRISPR / Cas9 system to the cell results in knockout or knockdown of expression of the PKM-1 polypeptide. In certain embodiments, administering the CRISPR / Cas9 system to the cell results in knockout or knockdown of expression of both the PKM-1 and PKM-2 polypeptides.

[0081] In a particular embodiment, the genetic engineering system is a ZFN system for controlling the expression of PKM polypeptide in mammalian cells. ZFNs can act as restriction enzymes, which are created by combining zinc finger DNA binding domains with DNA cleavage domains. Zinc finger domains can be engineered to target specific DNA sequences, allowing zinc finger nucleases to target desired sequences in the genome. The DNA binding domain of an individual ZFN typically contains multiple, individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating new zinc finger domains is to combine small zinc finger "modules" with known specificity. The most common cleavage domain of ZFNs is the non-specific cleavage domain derived from the type II restriction endonuclease FokI. ZFNs regulate protein expression by making double-strand breaks (DSBs) in the target DNA sequence, which are repaired by non-homologous end joining (NHEJ) in the absence of a homologous template. Such repair can result in the deletion or insertion of base pairs, creating a frameshift and preventing the production of harmful proteins (Durai et al., Nucleic Acids Res.; 33(18):5978-90(2005)). Multiple ZFN pairs can also be used to completely remove entire large segments of genomic sequences (Lee et al., Genome Res.; 20(1):81-9(2010)). In certain embodiments, the target gene is a portion of the PKM gene. In certain embodiments, the target sequence is exon 9 of the PKM gene. In certain embodiments, the target sequence is exon 9 and exon 10 of the PKM gene.

[0082] In certain embodiments, the genetic engineering system is a TALEN system for controlling expression of a PKM polypeptide in mammalian cells. TALENs are restriction enzymes that can be modified to cleave specific sequences in DNA. The TALEN system operates on a principle similar to ZFNs. TALENs are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) consist of a 33-34 amino acid repeat motif with two positions that strongly recognize specific nucleotides. By assembling an array of these TALEs, the TALE DNA binding domain can be modified to bind to a desired DNA sequence, thereby guiding a nuclease to cleave at a specific location in the genome (Boch et al., Nature Biotechnology; 29(2):135-6 (2011)). In certain embodiments, the target gene is part of the PKM gene. In certain embodiments, the target sequence is exon 9 of the PKM gene. In certain embodiments, the target sequence is exon 9 and exon 10 of the PKM gene.

[0083] In certain embodiments, expression of a PKM polypeptide can be knocked down using an oligonucleotide having a complementary sequence to a PKM nucleic acid (e.g., PKM mRNA, PKM-1 mRNA, or PKM-2 mRNA). Non-limiting examples of such oligonucleotides include small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). In certain embodiments, such oligonucleotides can be homologous to at least a portion of a PKM nucleic acid sequence, such as a PKM, PKM-1, or PKM-2 nucleic acid sequence, where the homology of the portion to the PKM nucleic acid sequence is at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%. In certain non-limiting embodiments, the complementary portion can comprise at least 10 nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, and the antisense nucleic acid, shRNA, mRNA, or siRNA molecule can be up to 15, or up to 20, or up to 25, or up to 30, or up to 35, or up to 40, or up to 45, or up to 50, or up to 75, or up to 100 nucleotides in length. Antisense nucleic acid, shRNA, mRNA, or siRNA molecules can comprise DNA or atypical or non-naturally occurring residues, such as, but not limited to, phosphorothioate residues.

[0084] The genetic recombination system disclosed herein can be delivered to mammalian cells using viral vectors, e.g., retroviral vectors, such as gamma retroviral vectors, and lentiviral vectors. If the capsid protein is functional to infect human cells, a combination of a retroviral vector and an appropriate packaging line is suitable. A variety of amphotropic virus producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114, or GALV envelopes, and any others known in the art, are also suitable. Possible transduction methods also include direct co-culturing of cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or with viral supernatant alone or concentrated vector stocks, with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0085] Other transducing viral vectors can be used to modify the mammalian cells disclosed herein. In certain embodiments, the selected vector exhibits high efficiency of infection, as well as stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263 267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, for example, Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al, BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cometta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).Retroviral vectors in particular have been developed and used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0086] Non-viral approaches can also be used for the genetic modification of mammalian cells as disclosed herein. For example, administration of nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989) or microinjection under surgical conditions (Wolff et al., Science Nucleic acid molecules can be introduced into mammalian cells by the method of the present invention (247:1465, 1990). Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially useful for delivering nucleic acid molecules into cells. Transplantation of a conventional gene into an infected tissue of a subject can also be performed by transferring the conventional nucleic acid into a cell type capable of ex vivo culture (e.g., autologous or heterologous primary cells or their progeny) and then injecting the cells (or their progeny) into the target tissue or by systemic injection.

[0087] 5.3 Cells with reduced or eliminated lactogenic activity In one aspect, the disclosure relates to cells or compositions comprising one or more cells, e.g., mammalian cells, with reduced or eliminated lactogenic activity, and methods of use thereof, in which expression of a PKM polypeptide (e.g., expression of PKM-1) has been knocked down or knocked out in a cell, resulting in reduced or eliminated lactogenic activity of the cell. Non-limiting examples of cells include CHO cells (e.g., DHFR CHO cells), dp12.CHO cells CHO-K1 (ATCC, CCL-61), monkey kidney CV1 transformed with SV40 (e.g., COS-7 ATCC CRL-1651), human embryonic kidney lines (e.g., 293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), mouse mammary carcinoma (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human hepatoma lines (e.g., Hep G2), myeloma cell lines (e.g., Y0, NS0 and Sp2 / 0). In certain embodiments, the cell is a CHO cell. Further non-limiting examples of CHO host cells include CHO K1SV cells, CHO DG44 cells, CHO DUKXB-11 cells, CHOK1S cells, and CHO KIM cells. In certain embodiments, only one allele of the PKM gene is modified in the cells of the present disclosure. In certain embodiments, both alleles of the PKM gene are modified. In certain embodiments, the cells of the present disclosure comprise at least one allele of the PKM gene comprising a sequence selected from the group consisting of SEQ ID NOs: 39-41, or comprise the nucleotide sequence set forth in SEQ ID NOs: 37 and 38 (see FIG. 4C).For example, the cells of the disclosure include, but are not limited to, an allele of the PKM gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-41, or comprising the nucleotide sequences set forth in SEQ ID NOs: 37 and 38. In certain embodiments, the cells of the disclosure include at least one allele of the PKM gene comprising a sequence selected from the group consisting of SEQ ID NOs: 39-41. In certain embodiments, the cells are CHO cells. Non-limiting examples of cells with reduced or eliminated lactogenic activity include HET-3, HET-18, KO-2, and KO-15, as disclosed herein.

[0088] In certain embodiments, expression of a PKM polypeptide (e.g., PKM-1 and / or PKM-2) is knocked out, resulting in the elimination of lactogenic activity of the cell compared to a reference cell. In certain embodiments, expression of a PKM polypeptide is knocked out in a cell, resulting in the reduction of lactogenic activity of the cell compared to a reference cell. In certain embodiments, the reference cell is a cell in which expression of a PKM polypeptide is not regulated. In certain embodiments, the reference cell is a cell with at least one wild-type PKM allele. In certain embodiments, the reference cell is a cell with both wild-type PKM alleles. In certain embodiments, the reference cell is a WT CHO cell.

[0089] In certain embodiments, the lactogenic activity of the cells is indicated by the lactate concentration in the cell culture medium during the culture period of the cells. In certain embodiments, the lactogenic activity of the cells is indicated by the lactate concentration in the cell culture medium on the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 25th, 30th, 35th, 40th, 45th, 50th, 55th, 60th, 65th, 70th, or 80th day of the culture period. In certain embodiments, the lactogenic activity of the cells is indicated by the lactate concentration in the cell culture medium after more than 80 days from the start of the culture. In certain embodiments, the lactogenic activity of the cells disclosed herein is indicated by the lactate concentration in the cell culture medium during the production phase, e.g., on day 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the production phase of the cell culture process. In certain embodiments, the lactate concentration is measured on day 6, 7, 10, 11, 14, and / or 15 of the production phase. In certain embodiments, the lactate concentration is measured on day 7, 10, and / or 14 of the production phase.

[0090] Any method known in the art for measuring the lactogenic activity of cells and / or measuring lactate production by cells in culture medium can be used with the subject matter disclosed herein. Non-limiting exemplary methods include those disclosed in TeSlaa and Teitell, Methods Enzymol (2014) 542:91-114, and Lehman et al., Med Sci Sports Exerc (1991) 23(8):935-8, which are incorporated by reference in their entirety. For example, but not limited to, such techniques include using a commercial extracellular lactate kit, using an extracellular bioanalyzer, measuring the extracellular acidification rate (ECAR), for example using a Seahorse XF analyzer, measuring the activity of rate-limiting glycolytic enzymes, and measuring lactate production using a tracer.

[0091] In certain embodiments, the lactogenic activity of a cell having reduced lactogenic activity (e.g., by regulating the PKM gene in the cell to knock down or knock out the PKM polypeptide) is about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% of the lactogenic activity of a reference cell. In certain embodiments, the lactogenic activity of a cell having reduced lactogenic activity (e.g., by regulating the PKM gene in the cell to knock down or knock out the PKM polypeptide) is less than about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% of the lactogenic activity of a reference cell. In certain embodiments, the lactogenic activity of the cells is less than about 50% of the lactogenic activity of the reference cells, e.g., when observed at day 14 or day 15 of the production phase of the cell culture. In certain embodiments, the lactogenic activity of the cells is less than about 20% of the lactogenic activity of the reference cells, e.g., when observed at day 14 or day 15 of the production phase of the cell culture. In certain embodiments, the lactogenic activity of the cells is less than about 10% of the lactogenic activity of the reference cells, e.g., when observed at day 14 or day 15 of the production phase of the cell culture. In certain embodiments, the reference cells are cells that contain at least one, or both, wild-type alleles of the PKM genes.

[0092] In certain embodiments, the lactate concentration in cell culture medium produced by the cells of the disclosure is less than about 15 g / L, less than about 14 g / L, less than about 13 g / L, less than about 12 g / L, less than about 101 g / L, less than about 10 g / L, less than about 9 g / L, less than about 8 g / L, less than about 7 g / L, less than about 6 g / L, less than about 5 g / L, less than about 4 g / L, less than about 3 g / L, less than about 2 g / L, less than about 1 g / L, or less than about 0.5 g / L, for example, during the production phase of the cell culture (e.g., on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, or day 20 of the production phase). In certain embodiments, the lactate concentration in the cell culture medium produced by the cells of the present disclosure is less than about 5 g / L, less than about 2 g / L, or less than about 1 g / L. In certain embodiments, the lactate concentration in the cell culture medium produced by the cells of the present disclosure is less than about 5 g / L, less than about 2 g / L, or less than about 1 g / L on day 7, day 10, day 14, or day 15 of the production phase of the cell culture. In certain embodiments, the lactate concentration in the cell culture medium produced by the cells of the present disclosure is less than about 1 g / L, or less than about 2 g / L during the production phase of the shake flask culture. In certain embodiments, the lactate concentration in the cell culture medium produced by the cells of the present disclosure is less than about 2 g / L during the production phase of the cell culture in a bioreactor.

[0093] In certain embodiments, cells with reduced or eliminated lactogenic activity disclosed herein (e.g., cells generated by knocking out or knocking down expression of a PKM polypeptide) have a titer and / or specific productivity comparable to cells with normal lactogenic activity (e.g., wild-type cells). In certain embodiments, the difference in titer and / or specific productivity between cells with reduced or eliminated lactogenic activity and cells with normal lactogenic activity is less than about 1%, less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, or less than about 30% of cells with normal lactogenic activity (e.g., reference cells). In certain embodiments, cells with reduced or eliminated lactogenic activity have a higher titer and / or specific productivity than cells with normal lactogenic activity. In certain embodiments, the titer of cells with reduced or eliminated lactogenic activity is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% greater than the titer of cells with normal lactogenic activity. In certain embodiments, the titer of cells with reduced or eliminated lactogenic activity is more than about 50% greater than the titer of cells with normal lactogenic activity. In certain embodiments, the specific productivity (Qp) of cells with reduced or eliminated lactogenic activity is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90% greater than the specific productivity of cells with normal lactogenic activity. In certain embodiments, the specific productivity of cells with reduced or eliminated lactogenic activity is more than about 90% greater than the specific productivity of cells with normal lactogenic activity.

[0094] Lactate concentration in cell culture medium can be measured by a chemistry analyzer or lactate assay kit. Non-limiting examples of chemistry analyzers include Bioprofile 400 (Nova Biomedical), Piccolo Xpress Chemistry Analyzer, Excel-Semi-automated Chemistry Analyzer, Indiko Clinical and Specialty Chemistry System, and ACE Axcel® Clinical Chemistry System. Non-limiting examples of lactate assay kits include L-Lactate Assay Kit (Colorimetric) (ab65331, Abeam), BioVision Lactate Colorimetric / Fluorometric Assay Kit, PicoProbe™ Lactate Fluorometric Assay Kit, Lactate Colorimetric Assay Kit II, Cell Biolabs Lactate Assay Kits.

[0095] In certain embodiments, the cells disclosed herein express a product of interest. In certain embodiments, the product of interest is a recombinant protein. In certain embodiments, the product of interest is a monoclonal antibody. Further non-limiting examples of products of interest are described in Section 5.5. In certain embodiments, the cells disclosed herein can be used to produce commercially useful quantities of the product of interest.

[0096] In certain embodiments, the cells disclosed herein can include a nucleic acid encoding a product of interest. In certain embodiments, the nucleic acid can be present in one or more vectors, such as expression vectors. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) are introduced into the genome of the host cell, thereby replicating along with the host genome. Furthermore, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operatively linked. In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids (vectors). Further non-limiting examples of expression vectors for use in the present disclosure include viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses) that serve equivalent functional roles.

[0097] In certain embodiments, a nucleic acid encoding a product of interest can be introduced into a host cell disclosed herein. In certain embodiments, the introduction of the nucleic acid into the cell can be performed by transfection, electroporation, microinjection, infection with a nucleic acid sequence-containing viral or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In certain embodiments, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0098] In certain embodiments, a nucleic acid encoding a product of interest can be randomly integrated into a host cell genome ("random integration" or "RI"). For example, and not by way of limitation, a nucleic acid encoding a product of interest can be randomly integrated into the genome of a cell that has been engineered to knock down or knock out expression of a PKM polypeptide (e.g., PKM-1).

[0099] In certain embodiments, a nucleic acid encoding a product of interest can be integrated into a host cell genome in a targeted manner ("targeted integration" or "TI"). For example, but not limited to, a nucleic acid encoding a product of interest can be integrated into the genome of a controlled cell to knock down or knock out expression of a PKM polypeptide (e.g., PKM-1) in a targeted manner. An "integration site" includes a sequence in a host cell genome where an exogenous nucleotide sequence is inserted. In certain embodiments, an integration site is between two adjacent nucleotides in a host cell genome. In certain embodiments, an integration site includes a stretch of nucleotide sequence. In certain embodiments, an integration site is located within a specific locus in the genome of a TI host cell. In certain embodiments, an integration site is within an endogenous gene of a TI host cell. Any integration site known in the art can be controlled and used with the presently disclosed subject matter. Targeted integration can be mediated by methods and systems known in the art. For example, but not limited to, the methods and systems disclosed in International Application No. PCT / US18 / 067070, filed December 21, 2018, the entire contents of which are incorporated herein, can be used for targeted integration.

[0100] In certain embodiments, the nucleic acid encoding the product of interest can be integrated into the host cell genome using transposase-based integration, as disclosed, for example, in Trubitsyna et al., Nucleic Acids Res. 45(10):e89 (2017), Li et al., PNAS 110(25):E2279-E2287 (2013), and WO 2004 / 009792, the entireties of which are incorporated herein by reference.

[0101] In certain embodiments, the nucleic acid encoding the product of interest can be randomly integrated into the host cell genome ("random integration" or "RI"). In certain embodiments, random integration can be mediated by any method or system known in the art. In certain embodiments, random integration is mediated by the MaxCyte STX® electroporation system.

[0102] In certain embodiments, targeted integration can be combined with random integration. In certain embodiments, targeted integration can be followed by random integration. In certain embodiments, random integration can be followed by targeted integration. For example, but not limited to, a nucleic acid encoding a product of interest can be randomly integrated into the genome of a cell that has been controlled to knock down or knock out the expression of a PKM polypeptide, such as PKM-1, and a nucleic acid encoding the same product of interest can be integrated into the genome of the cell in a targeted manner.

[0103] In certain embodiments, the host cell is a RI host cell. In certain embodiments, the host cell is a TI host cell.

[0104] 5.4 Cell culture method In one aspect, the present disclosure provides a method for producing a product of interest, comprising culturing the cells disclosed herein. Culture conditions suitable for mammalian cells known in the art can be used for culturing the cells described herein (J. Immunol. Methods (1983) 56:221-234) or can be readily determined by one of skill in the art (see, e.g., Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, BD, eds. Oxford University Press, New York (1992)).

[0105] Mammalian cells can be prepared in a medium suitable for the particular cells being cultured. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are exemplary nutrient solutions. In addition, any of the media disclosed in Ham and Wallace, (1979) Meth. Enz., 58:44; Barnes and Sato, (1980) Anal. Biochem., 102:255; U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 5,122,469, or 4,560,655; WO 90 / 03430; and WO 87 / 00195 (the disclosures of which are all incorporated herein by reference) can be used as the culture medium. Any of these media may be supplemented as necessary with hormones and / or other growth factors (insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics (e.g., gentamycin / gentamicin), trace elements (usually defined as inorganic compounds present in final concentrations in the micromolar range), lipids (linoleic acid or other fatty acids) and suitable salts thereof, and glucose, or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[0106] In certain embodiments, the mammalian cells modified to reduce and / or eliminate expression of a PKM polypeptide are CHO cells. Any suitable medium can be used to culture the CHO cells. In certain embodiments, media suitable for culturing CHO cells may contain basal medium components, such as DMEM / HAM F-12 based formulations (for the composition of DMEM and HAM F12 media, see American Type Culture Collection Catalogue of Cell Lines and Hybridomas, Sixth Edition, 1988, pp. 346-349) with altered concentrations of several components, such as amino acids, salts, sugars, and vitamins, optionally containing glycine, hypoxanthine, and thymidine; hydrolyzed peptone, such as recombinant human insulin, Primatone HS or Primatone RL (Sheffield, England), or equivalent; cytoprotectants, such as Pluronic F68 or an equivalent pluronic polyol; gentamicin; and trace elements (formulations of media described in U.S. Pat. No. 5,122,469 are particularly suitable).

[0107] In certain embodiments, the mammalian cells modified to reduce and / or eliminate expression of a PKM polypeptide are cells expressing a recombinant protein. Recombinant proteins can be produced by growing cells expressing a product of interest under a variety of cell culture conditions. For example, cell culture procedures for large-scale or small-scale production of proteins are potentially useful in the context of the present disclosure. Procedures can be used, including but not limited to fluidized bed bioreactors, hollow fiber bioreactors, roller bottle cultures, shake flask cultures, or stirred tank bioreactor systems (with or without microcarriers in the latter two systems), and can alternatively be operated in batch, fed-batch, or continuous modes.

[0108] In certain embodiments, the cell culture of the present disclosure is carried out in a stirred tank bioreactor system and employs a fed-batch procedure. In a fed-batch culture, the mammalian host cells and medium are initially fed to the culture vessel, additional culture nutrients are fed to the culture vessel continuously or incrementally during the culture, and the cells and / or product may or may not be harvested periodically before the end of the culture. Fed-batch cultures can include, for example, semi-continuous fed-batch cultures, in which the entire culture medium (including cells and medium) is periodically removed and replaced with fresh medium. Fed-batch cultures are distinct from simple batch cultures, in which all components for cell culture (including cells and all culture nutrients) are fed to the culture vessel at the beginning of the culture process. Fed-batch cultures can further be distinguished from perfusion cultures insofar as supernatant is not removed from the culture vessel during the process (in perfusion cultures, the cells are maintained in the culture medium, e.g., by filtration, encapsulation, anchoring to microcarriers, and medium is continuously or intermittently introduced and removed from the culture vessel).

[0109] In certain embodiments, the cells in the culture can be propagated according to any scheme or routine that may be suitable for the particular host cell and the particular production contemplated. Thus, the present disclosure contemplates single-step or multi-step culture procedures. In a single-step culture, the host cells are seeded in a culture medium environment and the process of the present disclosure is used during one production phase of the cell culture. Alternatively, multi-step culture is contemplated. In a multi-step culture, the cells can be cultured in multiple steps or stages. For example, the cells can be grown in a first step or growth phase culture medium. At this stage, the cells, optionally removed from storage, are seeded in a medium suitable to promote growth and high viability. The cells can be maintained in the growth phase for a suitable period of time by adding fresh medium to the host cell culture.

[0110] In certain embodiments, flow or continuous cell culture conditions are devised to enhance the growth of mammalian cells in the growth phase of the cell culture. In the growth phase, the cells are grown under conditions and for a period of time maximized for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dO2), etc., will be used for a particular host and will be apparent to one of skill in the art. Generally, pH is adjusted to a level of about 6.5-7.5 using either an acid (e.g., CO2) or a base (e.g., Na2CO3 or NaOH). A suitable temperature range for culturing mammalian cells, such as CHO cells, is about 30-38°C, and a suitable dO2 is 5-90% of air saturation.

[0111] At a particular stage, the cells can be used to seed a production phase or step of a cell culture, or the production phase or step can be continuous with a seeding or growth phase or step, as described above.

[0112] In certain embodiments, the culture methods described herein can further include harvesting a product from the cell culture medium, for example, during the production phase of the cell culture. In certain embodiments, the product produced by the cell culture methods of the present disclosure can be harvested from a third bioreactor, for example, a production bioreactor. For example, but not limited to, the disclosed methods can include harvesting the product upon completion of the production phase of the cell culture. Alternatively, or in addition, the product can be harvested prior to completion of the production phase. In certain embodiments, the product can be harvested from the cell culture medium once a certain cell density is achieved. For example, but not limited to, the cell density can be about 2.0×10 6 cells / mL or more prior to harvest. 7 cells / mL ~ approx. 5.0×10 7 It can be in cells / mL.

[0113] In certain embodiments, recovering the product from the cell culture may include one or more of centrifugation, filtration, sonication, flocculation, and cell removal techniques.

[0114] In certain embodiments, the product of interest can be secreted from the host cells, or the product of interest can be a membrane-bound, cytosolic, or nuclear protein. In certain embodiments, a soluble form of the polypeptide can be purified from cell culture medium, and a membrane-bound form of the polypeptide can be purified by preparing a total membrane fraction from the expressing cells and extracting the membranes with a non-ionic detergent such as TRITON® X-100 (EMD Biosciences, San Diego, Calif.). In certain embodiments, a cytosolic or nuclear protein can be prepared by lysing the host cells (e.g., by mechanical force, sonication, and / or detergent), removing the cellular membrane fraction by centrifugation, and retaining the supernatant.

[0115] 5.5 Products The cells and / or methods of the present disclosure can be used to produce any product of interest expressible by the cells disclosed herein. In certain embodiments, the cells and / or methods of the present disclosure can be used to produce polypeptides, such as mammalian polypeptides. Non-limiting examples of such polypeptides include hormones, receptors, fusion proteins, regulators, growth factors, complement system factors, enzymes, clotting factors, anticoagulants, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. The cells and / or methods of the present disclosure are not specific to the molecule, e.g., the antibody, that is produced.

[0116] In certain embodiments, the methods of the present disclosure can be used to generate antibodies, including therapeutic and diagnostic antibodies, or antigen-binding fragments thereof. In certain embodiments, the antibodies generated by the cells and methods of the present disclosure can be, but are not limited to, monospecific antibodies (e.g., antibodies comprising such paired multimers consisting of a single heavy chain sequence and a single light chain sequence), multispecific antibodies, and antigen-binding fragments thereof. For example, but not limited to, multispecific antibodies can be bispecific antibodies, dual epitope antibodies, T-cell-dependent bispecific antibodies (TDBs), dual acting FAbs (DAFs), or antigen-binding fragments thereof.

[0117] 5.5.1 Multispecific antibodies In certain embodiments, the antibodies produced by the cells and methods provided herein are multispecific antibodies, e.g., bispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has at least two different moieties, i.e., binding specificities for different epitopes of different antigens (i.e., bispecific), or different epitopes of the same antigen (i.e., dual epitopes). In certain embodiments, a multispecific antibody has three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments as described herein.

[0118] Techniques for making multispecific antibodies include, but are not limited to, recombinant coexpression of two immunoglobulin heavy chain-light chains with different specificities (see Milstein and Cuello, Nature 305:537 (1983)) and "knobs-in-holes" recombination (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be produced using techniques such as the modification of electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)); production of bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992), and WO 2011 / 034605); use of general light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); use of "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and by the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and by the preparation of trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0119] Modified antibodies, such as "Octopus antibodies" or DVD-Igs, having three or more antigen binding sites are also included herein (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Further non-limiting examples of multispecific antibodies having three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Trispecific antibodies or antigen-binding fragments thereof also include "dual acting FAbs" or "DAFs" (see, for example, U.S. Patent Application Publication No. 2008 / 0069820 and WO 2015 / 095539).

[0120] Multispecific antibodies can also be provided in asymmetric form with domain crossovers in one or more binding arms with the same antigen specificity, i.e. by swapping VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In certain embodiments, multispecific antibodies comprise cross-Fab fragments. The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. A cross-Fab fragment comprises a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, for example, WO 2016 / 172485.

[0121] Various additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0122] In certain embodiments, a particular type of multispecific antibody, also included herein, is a bispecific antibody designed to simultaneously bind to a surface antigen and to an activated and invariant component of the T cell receptor (TCR) complex, such as CD3, on a target cell, e.g., a tumor cell, to retarget the T cell and kill the target cell.

[0123] Further non-limiting examples of bispecific antibody formats that may be useful for this purpose include so-called "BiTEs" (bispecific T cell engagers), in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261, and WO 2008 / 119567; Nagorsen and Baeuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot. Eng. 9, 299-305 (1996)) and derivatives thereof, such as tandem bispecific antibodies ("TandAbs"; Kipriyanov et al., J Mol Biol 1999, 2010 ... 293, 41-56 (1999); "DART" (dual affinity retargeting) molecules based on the diabody format but featuring a C-terminal disulfide bridge for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)); and the so-called triomabs, which are all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat. Rev. 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) el203498.

[0124] 5.5.2 Antibody fragments In certain embodiments, the antibody produced by the cells and methods provided herein is an antibody fragment. For example, but not limited to, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, in particular a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each of which contains heavy and light chain variable domains (VH and VL, respectively), as well as a constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). The term "Fab fragment" therefore refers to an antibody fragment that contains a light chain containing a VL domain and a CL domain, and a heavy chain fragment containing a VH domain and a CH1 domain. A "Fab' fragment" differs from a Fab fragment by the addition of residues at the carboxy terminus of the CH1 domain that contain one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear a thiol group. Pepsin treatment yields an F(ab')2 fragment that contains two antigen binding sites (two Fab fragments) and a portion of the Fc region. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0125] In certain embodiments, the antibody fragment is a diabody, triabody, or tetrabody. A "diabody" is an antibody fragment having two antigen binding sites, which may be bivalent or bispecific. See, for example, European Patent Application Publication No. 404,097, International Publication No. WO 1993 / 01161, Hudson et al., Nat Med. 9, 129-134 (2003), and Hollinger et al., Proc Natl Acad Sci. USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0126] In a further embodiment, the antibody fragment is a single chain Fab fragment. A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized by a native disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab fragments may be further stabilized by the insertion of cysteine ​​residues (eg, at position 44 of the variable heavy chain and position 100 of the variable light chain, according to the Kabat numbering) to generate interchain disulfide bonds.

[0127] In another embodiment, the antibody fragment is a single chain variable fragment (scFv). A "single chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light (VL) chains of an antibody connected by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, which can connect either the N-terminus of VH to the C-terminus of VL, or vice versa. The protein can retain the specificity of the original antibody, even though the constant regions have been removed and the linker has been introduced. For a review of scFv fragments, see, e.g., Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458.

[0128] In another embodiment, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0129] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of an intact antibody.

[0130] 5.5.3 Chimeric and humanized antibodies In certain embodiments, the antibody produced by the cells and methods provided herein is a chimeric antibody. Certain chimeric antibodies are disclosed, for example, in U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0131] In certain aspects, the chimeric antibody is a humanized antibody. Generally, humanization of a non-human antibody reduces its immunogenicity to humans, but maintains the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody, and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally also comprises at least a portion of a human constant region. In certain embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0132] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (discussing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface reconstruction"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR reconstruction"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach for FR construction).

[0133] Human framework regions for use in humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from human antibody consensus sequences of particular subtypes of heavy or light chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Immunol. 272:10678-10684 (1997)). et al., J. Biol. Chem. 271:22611-22618 (1996).

[0134] 5.5.4 Human antibodies In certain embodiments, the antibodies produced by the cells and methods provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0135] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce fully human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or is present extrachromosomally or randomly introduced into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are typically inactive. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining different human constant regions.

[0136] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J.Immunol., 147:86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562, 2006. Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0137] 5.5.5 Target molecules Non-limiting examples of molecules that can be targeted by antibodies produced by the cells and methods disclosed herein include soluble serum proteins and their receptors, as well as other membrane-bound proteins (e.g., attachment factors). In certain embodiments, antibodies produced by the cells and methods disclosed herein target 8MPI, 8MP2, 8MP38 (GDFIO), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF18, FGF19, FGF20, FGF21, FGF22, FGF23, FGF24, FGF25, FGF26, FGF27, FGF28, FGF29, FGF30, FGF31, FGF32, FGF33, FGF34, FGF35, FGF36, FGF37, FGF38, FGF39, FGF40, FGF41, FGF42, FGF43, FGF44, FGF45, FGF46, FGF47, FGF48, FGF4 ...1, FGF42, FGF43, FGF44, FGF45, FGF46, FGF47, FGF48, FGF49, FGF49, FGF41, FGF42, FGF43, FGF44, FGF45, FGF46, FGF47, FGF4 , FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1(EP SELON), FEL1(ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B , IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA(TNF- β), LTB, TNF (TNF-α), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand) Gund), TNFSF10(TRAIL), TNFSF11(TRANCE), TNFSF12(AP03L), TNFSF13(April), TNFSF13B, TNFSF14(HVEM-L), TNFSF15(VEGI), TNFSF18 , HGF(VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB,The present invention can bind to one, two or more cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of IL9R, IL10RA, IL10RB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.k.

[0138] In certain embodiments, the antibodies produced by the cells and methods disclosed herein are selected from the group consisting of CCLI(1-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP-Iα), CCL4 (MIP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Iδ), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC / Exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / Eotaxin-2), CCL25 (CCL26), CCL27 (CCL28), CCL29 (CCL29), CCL30 (CCL31), CCL32 (CCL33), CCL34 (CCL35), CCL35 (CCL36), CCL37 (CCL38), CCL39 (CCL39), CCL40 (CCL40), CCL41 (CCL41), CCL42 (CCL42), CCL43 (CCL43), CCL44 (CCL44), CCL45 (CCL45), CCL46 (CCL46), CCL47 (CCL47), CCL48 (CCL48), CCL49 (CCL49), CCL50 (CCL50), CCL51 (CCL51), CCL52 (CCL52), CCL53 (CCL53), CCL54 (CCL54), CCL55 (CCL55), CCL56 (CCL55), CCL57 (CCL57), CCL58 (CCL58), CCL59 (CCL59), C CL25(TECK), CCL26(eotaxin-3), CCL27(CTACK / ILC), CCL28, CXCLI(GROI), CXCL2(GR02), CXCL 3(GR03), CXCL5(ENA-78), CXCL6(GCP-2), CXCL9(MIG), CXCL10(IP10), CXCL11(1-TAC), CXCL1 2(SDFI), CXCL13, CXCL14, CXCL16, PF4(CXCL4), PPBP(CXCL7), CX3CL1(SCYDI), SCYEI, Nhotactin), XCL2 (SCM-Iβ), BLRI (MDR15), CCBP2 (D6 / JAB61), CCRI (CKRI / HM145), CCR2 (mcp-IRB IRA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBII), CCR8(CMKBR8 / TER1 / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), XCR1(GPR5 / CCXCR1), CMKLR1, CMKOR1( RDC1), CX3CR1(V28), CXCR4, GPR2(CCR10), GPR31, GPR81(FKSG80), CXCR3(GPR9 / CKR-L2), CXCR6(TYMSTR / STRL33 / Bonzo), HM74, IL8RA(IL8Rα), IL8RB( IL8Rβ), LTB4R (GPR16), TCP10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5, C5R1, CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HDF1, HDF1α, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL.

[0139] In certain embodiments, the antibodies (e.g., multispecific antibodies, such as bispecific antibodies) generated by the methods disclosed herein are capable of binding to one or more target molecules selected from the following: 0772P (CA125, MUC16) (i.e., ovarian cancer antigen), ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; amyloid beta; ANGPTL; ANGPT2; ANGPTL3; ANGPT L4;ANPEP;APC;APOC1;AR;ASLG659;ASPHD1(aspartate beta-hydroxylase domain containing 1;LOC253982);AZGP1(zinc-a-glycoprotein);B7.1;B7.2;BAD;BAFF-R(B cell-activating factor receptor, BLyS receptor 3,BR3;BAG1;BAI1;BCL2;BCL6;BDNF;BLNK;BLRI(MDR15);BMP1;BMP2;BMP3B(GDF10);BMP4;BMP6;BMP8;BMPR1A;BMPR1B(type IB bone morphogenetic protein receptor); BMPR2;BPAG1(plectin);BRCA1;Brevican;C19orf10(IL27w);C3;C4A;C5;C5R1;CANT1;CASP1;CASP4;CAV1;CCBP2(D6 / JAB61);CCL1(1-309) CCL13(MCP-4);CCL15(MIP1δ);CCL16(HCC-4);CCL17(TARC);CCL18(PARC);CCL19(MIP-3β);CCL2(MCP-1);MCAF;CCL20(MIP-3α);CCL21(MTP- 2);SLC;exodus-2;CCL22(MDC / STC-1);CCL23(MPIF-1);CCL24(MPIF-2 / eotaxin-2);CCL25(TECK);CCL26(eotaxin-3);CCL27(CTACK / ILC);CCL28;CCL 3(MTP-Iα);CCL4(MDP-Iβ);CCL5(RANTES);CCL7(MCP-3);CCL8(mcp-2);CCNA1;CCNA2;CCND1;CCNE1;CCNE2;CCR1(CKRI / HM145);CCR2(mcp-IRβ / RA);CCR3(CKR / CMKBR3);CCR4;CCR5(CMKBR5 / ChemR13);CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6);CCR7(CKBR7 / EBI1);CCR8(CMKBR8 / TER1 / CKR-L1);CCR9(GPR-9-6);CCRL1(VSHK1);CCRL2(L-CCR);CD164;CD19;CD1C;CD20;CD200;CD22(B-cell receptor CD22-B isoform);CD24;CD28;CD3;CD37;CD38;CD3E;CD3G;CD3Z ;CD4;CD40;CD40L;CD44;CD45RB;CD52;CD69;CD72;CD74;CD79A (CD79α, immunoglobulin-related α, B cell-specific protein);CD79B;CDS;CD80;CD81;CD83;CD86;CDH1 (E-cadherin);CDH10;CDH12;CDH13;CDH18;CDH19;CDH20;CDH5;CDH7;CDH8;CDH9;CDK2;CDK3;CDK4;CDK5;CDK6;CDK7;CDK9;CDKN1A (p21 / WAF1 / Cip1);CDKN1B ( p27 / Kip1;CDKN1C;CDKN2A (P16INK4a);CDKN2B;CDKN2C;CDKN3;CEBPB;CER1;CHGA;CHGB;chitinase;CHST10;CKLFSF2;CKLFSF3;CKLFSF4;CKLFSF5;CKLFSF6;CKLFSF7;CKLFSF8;CLDN3;CLDN7 (claudin-7);CLL-1 (CLEC12A, MICL, and DCAL2);CLN3;CLU (clusterin);CMKLR1;CMKOR1 (RDC1);CNR1;COL18A1;COL1A 1;COL4A3;COL6A1;Complement factor D;CR2;CRP;CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor);CSFI (M-CSF);CSF2 (GM-CSF);CSF3 (GCSF);CTLA4;CTNNB1 (b-catenin);CTSB (cathepsin B);CX3CL1 (SCYDI);CX3CR1 (V28);CXCL1 (GRO1);CXCL10 (IP-10);CXCL11 (I-TAC / IP-9);CXCL12 (SDF1);CXCL13;CXCL14;CXCL16;CXCL2(GRO2);CXCL3(GRO3);CXCL5(ENA-78 / LIX);CXCL6(GCP-2);CXCL9(MIG);CXCR3(GPR9 / CKR-L2);CXCR4;CXCR5(Burkitt's lymphoma receptor 1, G protein-coupled receptor);CXCR6(TYMSTR / STRL33 / Bonzo);CYB5;CYC1;CYSLTR1;DAB2IP;DES;DKFZp451J0118;DNCLI;DPP4;E16(LAT1, SLC7A5);E2F1;ECGF1;EDG1;EFNA1;EFNA 3;EFNB2;EGF;EGFR;ELAC2;ENG;ENO1;ENO2;ENO3;EPHB4;EphB2R;EPO;ERBB2 (Her-2);EREG;ERK8;ESR1;ESR2;ETBR (endothelin type B receptor);F3 (TF);FADD;FasL;FASN;FCER1A;FCER2;FCGR3A;FcRH1 (Fc receptor-like protein 1);FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C);FGF;FGF 1(αFGF);FGF10;FGF11;FGF12;FGF12B;FGF13;FGF14;FGF16;FGF17;FGF18;FGF19;FGF2(bFGF);FGF20;FGF21;FGF22;FGF23;FGF3(int-2);FGF4(HST );FGF5;FGF6(HST-2);FGF7(KGF);FGF8;FGF9;FGFR;FGFR3;FIGF(VEGFD);FEL1(EPSILON);FIL1(ZETA);FLJ12584;FLJ25530;FLRTI(fibronectin);FLT 1;FOS;FOSL1(FRA-1);FY(DARC);GABRP(GABAa);GAGEB1;GAGEC1;GALNAC4S-6ST;GATA3;GDF5;GDNF-Ra1(GDNF family receptor alpha 1;GFRA1;GDNFR;GDNFRA;RETL1;TRNR1;RET1L;GDNFR-α1;GFR-ALPHA-1);GEDA;GFI1;GGT1;GM-CSF;GNASI;GNRHI;GPR2(CCR10);GPR19(G protein-coupled receptor 19;Mm.4787);GPR31;GPR44;GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12); GPR81 (FKSG80); GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856; D15Ertd747e); GRCCIO (CIO); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HOP1; histamine and histamine receptor; HLA-A; HLA-DOB (MHC class II beta subunit of molecule (Ia antigen);HLA-DRA;HM74;HMOXI;HUMCYT2A;ICEBERG;ICOSL;1D2;IFN-a;IFNA1;IFNA2;IFNA4;IFNA5;IFNA6;IFNA7;IFNB1;IFNγ;DFNW1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2;IGFBP3;IGFBP6;IL-1;IL10;IL10RA;IL10RB; IL11;IL11RA;IL-12;IL12A;IL12B;IL12RB1;IL12RB2;IL13;IL13RA1;IL13RA2;IL14;IL15;IL15RA;IL16;IL17;I L17B;IL17C;IL17R;IL18;IL18BP;IL18R1;IL18RAP;IL19;ILIA;IL1B;ILIF10;IL1F5;IL1F6;IL1F7;IL1F8;IL1F9 ;IL1HY1;IL1R1;IL1R2;IL1RAP;IL1RAPL1;IL1RAPL2;IL1RL1;IL1RL2, ILIRN;IL2;IL20;IL20Rα;IL21R;IL22;IL- 22c;IL22R;IL22RA2;IL23;IL24;IL25;IL26;IL27;IL28A;IL28B;IL29;IL2RA;IL2RB;IL2RG;IL3;IL30;IL3RA;IL 4;IL4R;IL5;IL5RA;IL6;IL6R;IL6ST (glycoprotein 130);Influenza A;Influenza B;EL7;EL7R;EL8;IL8RA;DL8RB;IL8RB;DL9;DL9R;DLK;INHA;INHBA;INSL3;INSL4;IRAKI;IRTA2 (immunoglobulin superfamily receptor translocation associated 2);ERAK2;ITGA1;ITGA2;ITGA3;ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); α4β7 and αEβ7 integrin heterodimers; JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP);KLF6;KLKIO;KLK12;KLK13;KLK14;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KRT19 (keratin 19);KRT2A;KHTHB6 (hair-specific H-type keratin);LAMAS;LEP (leptin);LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49, GPR67);Lingo-p75;Lingo-Troy;LPS;LTA (TNF-b);LTB;LTB4R (GPR16);LTB4R2;LTBR;LY64 (lymphocyte antigen 64 (RP105), a member of the leucine-rich repeat (LRR) family I type 6 membrane protein); Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-l); Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226); MACMARCKS; MAG or OMgp; MAP2K7 (c-Jun); MDK; MDP; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2; MMP9; MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin); MS4A1; MSG783 (RNF124, hypothetical protein FLJ20315); MSMB; MT3 (metallothionectin-111); MTSS1; MUC1 (mucin); MYC; MY088; Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), member 2, type II sodium-dependent phosphate transporter 3b); NCA; NCK2; Neurocan; NFKB1; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1 (NM23A); NOX5; NPPB; NR0B1; NR0B2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4; NR112;NR113;NR2C1;NR2C2;NR2E1;NR2E3;NR2F1;NR2F2;NR2F6;NR3C1;NR3C2;NR4A1;NR4A2;NR4A3;NR5A1;NR5A2;NR6A1;NRP1;NRP2;NT5E;NTN4;ODZI;OPRD1;0X40;P2RX7;P2X5(purinergic; receptor P2X ligand-gated ion channel 5);PAP;PARTI;PATE;PAWR;PC A3;PCNA;PD-L1;PD-L2;PD-1;POGFA;POGFB;PEC AMI;PF4(CXCL4);PGF;PGR;phosphacan;PIAS2;PIK3CG;PLAU(uPA);PLG;PLXDC1;PMEL17(silver homolog;SILV;D12S53E;PMEL17;SI;SIL);PPBP(CXCL7);PPID;PRI;PRKCQ;PRKDI;PRL;PROC;PROK2;PSAP;PSCA hlg(2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene);PTAFR;PTEN;PTGS2(COX-2);PTN;RAC2(p21 Rac2);RARB;RET(ret prototypic oncogene;MEN2A;HSCR1;MEN2B;MTC1;PTC;CDHF12;Hs.168114;RET51;RET-ELE1);RGSI;RGS13;RGS3;RNF110(ZNF144);ROBO2;S100A2;SCGB1D2(lipophilin B);SCGB2A1(mammaglobin 2);SCGB2A2(mammaglobin 1);SCYEI(endothelial mononuclear cell activating cytokine);SDF2;Serna 5b(FLJ10372,KIAA1445,Mm.42015,SEMA5B,SEMAG,semaphorin 5b Hlog, sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM), and short cytoplasmic domain (semaphorin) 5B);SERPINA1;SERPINA3;SERP1NB5 (maspin);SERPINEl (PAI-l);SERPDMF1;SHBG;SLA2;SLC2A2;SLC33A1;SLC43A1;SLIT2;SPPI;SPRR1B (Sprl);ST6GAL1;STABI;STAT6;STEAP (six, transmembrane epithelial antigen of prostate);STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six, transmembrane epithelial antigen of prostate 2, six transmembrane prostate protein);TB4R2;TBX21;TCPIO;TOGFI;TEK;TENB2 (putative transmembrane proteoglycan);TGFA;TGFBI;TGFB1II;TGFB2;TGFB3;TGFBI;TGFBRI;TGFBR2;TGFBR3;THIL;THBSI (thrombospondin-1);THBS2;THBS4;THPO;TIE (Tie-1);TMP3;tissue factor;TLR1;TLR2;TLR3;TLR4;TLR5;TLR6;TLR7;TLR8;TLR9;TLR10;TMEFF1 (transmembrane protein with EGF-like and follistatin-like domains 1; tomoregulin-1);TMEM46 (sca homolog 2);TNF;TNF-a;TNFAEP2 (B94);TNF AIP3;TNFRSFIIA;TNFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSF10(TRAIL);TNFSF11(TRANCE);TNFSF12(AP03L);TNFSF13(Ap ril);TNFSF13B;TNFSF14(HVEM-L);TNFSF15(VEGI);TNFSF18;TNFSF4(OX40 ligand);TNFSF5(CD40 ligand);TNFSF6(FasL);TNFSF7(CD27 ligand);TNFSFS(CD30 ligand);TNFSF9(4-1 BB ligand);TOLLIP;Toll-like receptor;TOP2A (Topoisomerase Ea);TP53;TPM1;TPM2;TRADD;TMEM118 (RING finger protein, transmembrane 2;RNFT2;FLJ14627);TRAF1;TRAF2;TRAF3;TRAF4;TRAF5;TRAF6;TREM1;TREM2;TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subfamily M, member 4);TRPC6;TSLP;TWEAK;tyrosinase (TYR;OCAIA;OCA1A;tyrosinase;SHEP3);VEGF;VEGFB;VEGFC;versican;VHL C5;VLA-4;XCL1 (lymphotactin);XCL2 (SCM-1b);XCRI (GPR5 / CCXCRI);YY1;and ZFPM2. ;

[0140] In certain embodiments, the antibodies produced by the cells and methods disclosed herein are directed to any of the following: CD3, CD4, CD5, CD16, CD19, CD20, CD21 (CR2 (complement receptor 2) or C3DR (C3d / Epstein-Barr virus receptor), or Hs.73792); CD33; CD34; CD64; CD72 (B cell differentiation antigen CD72, Lyb-2); CD79b (CD79B, CD79P, IGb (immunoglobulin-related beta), B29); CD200 members of the ErbB receptor family, such as EGF receptor, HER2, HER3, or HER4 receptor; LFA-1, Mac1, pl50.95, VLA-4, ICAM-1, VCAM, α4 / β7 receptor, or any of the following: Cell adhesion molecules such as tegrin and αv / β3 integrin (including either the α or β subunits thereof) (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); VEGF-A, VEGF-C; tissue factor (TF); growth factors such as α interferon (αIFN); interleukins such as IL-1β, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-17AF, IL-1S, IL-13Rα1, IL13Rα2, IL-4R, IL-5R, IL-9R, IgE, TNFα; blood group antigens, flk2 / flt3 receptors; obesity (OB) receptor; mpl receptor; CTLA-4; CD proteins such as RANKL, RANK, RSV F protein, and protein C.

[0141] In certain embodiments, the cells and methods provided herein can be used to produce antibodies (multispecific antibodies, such as bispecific antibodies) that specifically bind to complement protein C5 (e.g., anti-C5 agonist antibodies that specifically bind to human C5). In certain embodiments, the anti-C5 antibody comprises one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SSYYMA (SEQ ID NO: 1); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of AIFTGSGAEYKAEWAKG (SEQ ID NO: 26); (c) a heavy chain variable region CDR3 comprising the amino acid sequence of DAGYDYPTHAMHY (SEQ ID NO: 27); (d) a light chain variable region CDR1 comprising the amino acid sequence of RASQGISSSLA (SEQ ID NO: 28); (e) a light chain variable region CDR2 comprising the amino acid sequence of GASETES (SEQ ID NO: 29); and (f) a light chain variable region CDR3 comprising the amino acid sequence of QNTKVGSSYGNT (SEQ ID NO: 30). For example, in certain embodiments, the anti-05 antibody comprises a heavy chain variable domain (VH) sequence comprising one, two, or three CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SSYYMA (SEQ ID NO: 1); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of AIFTGSGAEYKAEWAKG (SEQ ID NO: 26); (c) a heavy chain variable region CDR3 comprising the amino acid sequence of DAGYDYPTHAMHY (SEQ ID NO: 27); and / or a light chain variable domain (VL) sequence comprising one, two, or three CDRs selected from: (d) a light chain variable region CDR1 comprising the amino acid sequence of RASQGISSSLA (SEQ ID NO: 28); (e) a light chain variable region CDR2 comprising the amino acid sequence of GASETES (SEQ ID NO: 29); and (f) a light chain variable region CDR3 comprising the amino acid sequence of QNTKVGSSYGNT (SEQ ID NO: 30). The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region and CDR1, CDR2, and CDR3 of the light chain variable region are disclosed in U.S. Patent Application Publication No. 2016 / 0176954 as SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 125, respectively. (See Tables 7 and 8 in U.S. Patent Application Publication No. 2016 / 0176954.)

[0142] In certain embodiments, the anti-C5 antibody has the VH and VL sequences QVQLVESGGG LVQPGRSLRL SCAASGFTVH SSYYMAWVRQ APGKGLEWVG AIFTGSGAEY KAEWAKGRVT ISKDTSKNQV VLTMTNMDPV DTATYYCASD AGYDYPTHAM HYWGQGTLVT VSS (SEQ ID NO: 31), respectively. and DIQMTQSPSS LSASVGDRVT ITCRASQGIS SSLAWYQQKP GKAPKLLIYG ASETESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQN TKVGSSYGNT FGGGTKVEIK (SEQ ID NO:32), including post-translational modifications of these sequences. The VH and VL sequences are disclosed in US Patent Application Publication No. 2016 / 0176954 as SEQ ID NO:106 and SEQ ID NO:111, respectively. (See Tables 7 and 8 of US Patent Application Publication No. 2016 / 0176954.) In a particular embodiment, the anti-C5 antibody is 305L015 (See US Patent Application Publication No. 2016 / 0176954).

[0143] In certain embodiments, the antibody produced by the methods disclosed herein can bind to OX40 (e.g., an anti-OX40 agonist antibody that specifically binds to human OX40). In certain embodiments, the anti-OX40 antibody comprises one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of DSYMS (SEQ ID NO: 2); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of DMYPDNGDSSYNQKFRE (SEQ ID NO: 3); (c) a heavy chain variable region CDR3 comprising the amino acid sequence of APRWYFSV (SEQ ID NO: 4); (d) a light chain variable region CDR1 comprising the amino acid sequence of RASQDISNYLN (SEQ ID NO: 5); (e) a light chain variable region CDR2 comprising the amino acid sequence of YTSRLRS (SEQ ID NO: 6); and (f) a light chain variable region CDR3 comprising the amino acid sequence of QQGHTLPPT (SEQ ID NO: 7). For example, in certain embodiments, the anti-OX40 antibody comprises a heavy chain variable domain (VH) sequence comprising one, two, or three CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of DSYMS (SEQ ID NO: 2); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of DMYPDNGDSSYNQKFRE (SEQ ID NO: 3); and (c) a heavy chain variable region CDR3 comprising the amino acid sequence of APRWYFSV (SEQ ID NO: 4); and / or a light chain variable domain (VL) sequence comprising one, two, or three CDRs selected from: (a) a light chain variable region CDR1 comprising the amino acid sequence of RASQDISNYLN (SEQ ID NO: 5); (b) a light chain variable region CDR2 comprising the amino acid sequence of YTSRLRS (SEQ ID NO: 6); and (c) a light chain variable region CDR3 comprising the amino acid sequence of QQGHTLPPT (SEQ ID NO: 7). In certain embodiments, the anti-OX40 antibody has the VH and VL sequences EVQLVQSGAE VKKPGASVKV SCKASGYTFT DSYMSWVRQA PGQGLEWIGD MYPDNGDSSY NQKFRERVTI TRDTSTSTAY LELSSLRSED TAVYYCVLAP RWYFSVWGQG TLVTVSS (SEQ ID NO: 8), respectively. and DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLRSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GHTLPPTFGQ GTKVEIK (SEQ ID NO: 9) (including post-translational modifications of these sequences).

[0144] In certain embodiments, the anti-OX40 antibody comprises one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of NYLIE (SEQ ID NO: 10); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of VINPGSGDTYYSEKFKG (SEQ ID NO: 11); (c) a heavy chain variable region CDR3 comprising the amino acid sequence of DRLDY (SEQ ID NO: 12); (d) a light chain variable region CDR1 comprising the amino acid sequence of HASQDISSYIV (SEQ ID NO: 13); (e) a light chain variable region CDR2 comprising the amino acid sequence of HGTNLED (SEQ ID NO: 14); and (f) a light chain variable region CDR3 comprising the amino acid sequence of VHYAQFPYT (SEQ ID NO: 15). For example, in certain embodiments, the anti-OX40 antibody comprises a heavy chain variable domain (VH) sequence comprising one, two, or three CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of NYLIE (SEQ ID NO: 10); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of VINPGSGDTYYSEKFKG (SEQ ID NO: 11); and (c) a heavy chain variable region CDR3 comprising the amino acid sequence of DRLDY (SEQ ID NO: 12); and / or a light chain variable domain (VL) sequence comprising one, two, or three CDRs selected from: (a) a light chain variable region CDR1 comprising the amino acid sequence of HASQDISSYIV (SEQ ID NO: 13); (b) a light chain variable region comprising the amino acid sequence of HGTNLED (SEQ ID NO: 14); and (c) a light chain variable region CDR3 comprising the amino acid sequence of VHYAQFPYT (SEQ ID NO: 15). In certain embodiments, the anti-OX40 antibody has the VH and VL sequences EVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWIGV INPGSGDTYY SEKFKGRVTI TRDTSTSTAY LELSSLRSED TAVYYCARDR LDYWGQGTLV TVSS (SEQ ID NO: 16), respectively. and DIQMTQSPSS LSASVGDRVT ITCHASQDIS SYIVWYQQKP GKAPKLLIYH GTNLEDGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCVH YAQFPYTFGQ GTKVEIK (SEQ ID NO: 17) (including post-translational modifications of these sequences).

[0145] Further details regarding anti-OX40 antibodies are disclosed in WO 2015 / 153513, which is incorporated by reference in its entirety.

[0146] In certain embodiments, antibodies produced by the cells and methods disclosed herein can bind to influenza virus B hemagglutinin, or "fluB" (e.g., antibodies that bind to hemagglutinin from the Yamagata lineage of influenza B virus in vitro and / or in vivo, that bind to hemagglutinin from the Victoria lineage of influenza B virus, that bind to hemagglutinin from the ancestral lineage of influenza B virus, or that bind to hemagglutinin from the Yamagata lineage, Victoria lineage, and ancestral lineage of influenza B virus). Further details regarding anti-FluB antibodies are disclosed in WO 2015 / 148806, which is incorporated by reference in its entirety.

[0147] In certain embodiments, the antibodies produced by the cells and methods disclosed herein are capable of binding to at least one target selected from the group consisting of low density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor, and beta-secretase (BACE1 or BACE2), alpha-secretase, gamma-secretase, tau secretase, amyloid precursor protein (APP), death receptor 6 (DR6), amyloid beta peptide, alpha-synuclein, parkin, huntingtin, p75 NTR, CD40, and caspase-6.

[0148] In certain embodiments, the antibody produced by the cells and methods disclosed herein is a human IgG2 antibody against CD40. In certain embodiments, the anti-CD40 antibody is RG7876.

[0149] In certain embodiments, the cells and methods of the present disclosure can be used to produce a polypeptide. For example, but not limited to, the polypeptide is a targeted immunocytokine. In certain embodiments, the targeted immunocytokine is a CEA-IL2v immunocytokine. In certain embodiments, the CEA-IL2v immunocytokine is RG7813. In certain embodiments, the targeted immunocytokine is a FAP-IL2v immunocytokine. In certain embodiments, the FAP-IL2v immunocytokine is RG7461.

[0150] In certain embodiments, a multispecific antibody (such as a bispecific antibody) made by the cells or methods provided herein can bind to CEA and at least one additional target molecule. In certain embodiments, a multispecific antibody (such as a bispecific antibody) made according to the methods provided herein can bind to a tumor-targeting cytokine and at least one additional target molecule. In certain embodiments, a multispecific antibody (such as a bispecific antibody) made according to the methods provided herein is fused to IL2v (i.e., an interleukin 2 variant) and binds to an IL-1 system immune cytokine and at least one additional target molecule. In certain embodiments, a multispecific antibody (such as a bispecific antibody) made according to the methods provided herein is a T cell bispecific antibody (i.e., a bispecific T cell engager or BiTE).

[0151] In certain embodiments, multispecific antibodies (such as bispecific antibodies) generated according to the methods provided herein are directed to any of the following: IL-1α and IL-1β, IL-12 and IL-1S; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-1β; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-12 and TWEAK, IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAMS, IL-13 and PED2, IL17A and IL17F, CEA and CD3, CD3 and CD19, CD138 and CD20; CD 138 and CD40; CD19 and CD20; CD20 and CD3; CD3S and CD13S; CD3S and CD20; CD3S and CD40; CD40 and CD20; CD-S and IL-6; CD20 and BR3, TNFα and TGF-β, TNFα and IL-1β;TNFα and IL-2, TNFα and IL-3, TNFα and IL-4, TNFα and IL-5, TNFα and IL6, TNFα and IL8, TNFα and IL-9, TNFα and IL-10, TNFα and IL-11, TNFα and IL-12, TNFα and IL-13, TNFα and IL-14, TNFα and IL-15, TNFα and IL-16, TNFα and IL-17, TNFα and IL-18, TNF α and IL-19, TNFα and IL-20, TNFα and IL-23, TNFα and IFNα, TNFα and CD4, TNFα and VEGF, TNFα and MIF, TNFα and ICAM-1, TNFα and PGE4, TNFα and PEG2, TNFα and RANK ligand, TNFα and Te38, TNFα and BAFF, TNFα and CD22, TNFα and CTLA-4, TNFα and GP130, TNF a and IL-12p40, VEGF and angiopoietin, VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGFA and ANG2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, EGFR and MET, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR (HER1) and HER2, EGFR and HER3, EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L, JL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTN02; IGF1 and IGF2; IGF 1 / 2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; POL-1 and CTLA-4; and RGM A and RGM B.

[0152] In certain embodiments, a multispecific antibody (e.g., a bispecific antibody) generated according to the methods provided herein is an anti-CEA / anti-CD3 bispecific antibody. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody is RG7802. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody comprises the amino acid sequence set forth in SEQ ID NOs: 18-21, as shown below: TIFF0007641263000001.tif39170TIFF0007641263000002.tif34170TIFF0007641263000003.tif92170TIFF0007641263000004.tif66170

[0153] Further details regarding anti-CEA / anti-CD3 bispecific antibodies are provided in WO 2014 / 121712, which is incorporated by reference in its entirety.

[0154] In certain embodiments, the multispecific antibody (e.g., bispecific antibody) produced by the cells and methods disclosed herein is an anti-VEGF / anti-angiopoietin bispecific antibody. In certain embodiments, the anti-VEGF / anti-angiopoietin bispecific antibody is a crossmab. In certain embodiments, the anti-VEGF / anti-angiopoietin bispecific antibody is RG7716. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody comprises the amino acid sequence set forth in SEQ ID NOs: 22-25, as shown below: TIFF0007641263000005.tif67170TIFF0007641263000006.tif71170TIFF0007641263000007.tif40170TIFF0007641263000008.tif34170

[0155] In certain embodiments, the multispecific antibody (e.g., bispecific antibody) produced by the methods disclosed herein is an anti-Ang2 / anti-VEGF bispecific antibody. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is RG7221. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is CAS number 1448221-05-3.

[0156] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens to generate antibodies. In the case of transmembrane molecules such as receptors, fragments of these (e.g., extracellular domains of receptors) can be used as immunogens. Alternatively, cells expressing the transmembrane molecule can be used as immunogens. Such cells can be derived from natural sources (e.g., cancer cell lines) or can be cells transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for the preparation of antibodies will be apparent to those skilled in the art.

[0157] In certain embodiments, the polypeptides (e.g., antibodies) produced by the cells and methods disclosed herein can be further conjugated by linkage to a chemical molecule such as a dye or a cytotoxic agent, e.g., a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of microbial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Immunoconjugates, including antibodies or bispecific antibodies, produced using the methods described herein can contain a cytotoxic agent conjugated to the constant region of only one of the heavy chains or only one of the light chains.

[0158] 5.5.6 Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein, e.g., those set forth in Section 5.5.5, are contemplated. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by incorporating appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0159] 5.5.6.1 Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions" and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1]

[0160] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro, (6) Aromatic: Trp, Tyr, Phe.

[0161] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0162] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., improvement) in a certain biological property (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have a certain biological property of the parent antibody substantially retained. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibody is displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0163] Modifications (e.g., substitutions) can be made to CDRs, for example, to improve the affinity of the antibody. Such modifications can be made within CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing and then reselecting from a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0164] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may, for example, be outside of the antigen contact residues within the CDRs. In the above specific variant VH and VL sequences, each CDR is either unmodified or has no more than one, two, or three amino acid substitutions.

[0165] A useful method for identifying antibody residues or regions that may be targeted for mutation is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. The variants may be screened to determine whether they have the desired properties.

[0166] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the N- or C-terminal fusion of the antibody to an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

[0167] 5.5.6.2 Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0168] If the antibody comprises an Fc region, the oligosaccharides attached to the Fc region can be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present disclosure can be modified to generate antibody variants with specific improved properties.

[0169] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures that lack fucose linkages (direct or indirect) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc at the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the endogenous or parent antibody. For example, the proportion of nonfucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as determined, for example, by MALDI-TOF mass spectrometry as described in WO 2006 / 082515. By Asn297 is meant the asparagine residue located at about position 297 (EU numbering of Fc region residues) of the Fc region, although Asn297 can also be located about ±3 upstream or downstream of amino acid position 297, i.e., at positions 294-300, due to minor sequence variations in the antibody. Such antibodies having an increased percentage of nonfucosylated oligosaccharides in the Fc region can have improved FcγRIIIa receptor binding and / or improved effector function, in particular improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621.

[0170] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as FUT8 knockout CHO cells of the α-1,6-fucosyltransferase gene (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or abolished activity of GDP-fucose synthesis or transporter proteins (see, e.g., U.S. Patent Application Publication Nos. 2004259150, 2005031613, 2004132140, and 2004110282).

[0171] In further embodiments, antibody variants are provided with bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0172] Also provided is an antibody variant having at least one galactose residue in the oligosaccharide attached to the Fc region. Such an antibody variant can have improved CDC function. Such an antibody variant is described, for example, in WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0173] 5.5.6.3 Fc Region Variants In certain aspects, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0174] In certain embodiments, the present disclosure contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important, yet certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII FcR expression in hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al, Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, ADCC activity of the molecule of interest can be assessed in vivo in an animal model, e.g., as disclosed in Clynes et al. Proc. Nat'l. Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., the C1q and C3c binding ELISAs described in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life measurements can also be performed using methods known in the art (see, e.g., Petkova, S B. et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929A1).

[0175] Antibodies with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 have been substituted with alanine (U.S. Patent No. 7,332,581).

[0176] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0177] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering residues) of the Fc region.

[0178] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., Fc region positions 234 and 235 (EU numbering residues). In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831.) In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.

[0179] In some embodiments, changes are made within the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0180] Antibodies with increased half-lives and improved binding to fetal Fc receptors (FcRn) that play a role in transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substituents that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall' Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0181] The Fc region residues critical for mouse Fc mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall' Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index numbering) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are critical for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671), various mutations of residues 248-259, 301-317, 376-382, and 424-437 have been reported and investigated.

[0182] The antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at Fc region positions 253, and / or 310, and / or 435 (EU numbering residues). In a particular embodiment, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in an Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0183] The antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310, and / or 433, and / or 436 (EU numbering residues) of the Fc region. In a particular embodiment, the antibody variant comprises an Fc region having amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2014 / 177460A1.)

[0184] The antibody variants include Fc regions with one or more amino acid substitutions that increase FcRn binding, e.g., mutations at Fc region positions 252, and / or 254, and / or 256 (EU numbering residues). In certain embodiments, the antibody variants include Fc regions with amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from a human IgG1 Fc region. See also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260; 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0185] The C-terminus of the heavy chain of an antibody as reported herein may be a complete C-terminus terminating in amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two of the C-terminal amino acid residues have been removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus terminating in PG. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, amino acid positions of the EU index numbering). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, amino acid position of the EU index numbering).

[0186] 5.5.6.4 Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, such as THIOMAB™ antibodies, in which one or more residues of the antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody, which can be used to couple the antibody to other moieties, such as drug moieties, or linker / drug moieties, and to generate immunoconjugates as further described herein. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0187] 5.5.6.5 Antibody derivatives In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing due to its stability in water. The polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0188] 5.5.7 Immunoconjugates The present disclosure also provides immunoconjugates comprising an antibody disclosed herein conjugated (chemically linked) to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of microbial, fungal, plant or animal origin, or fragments thereof), or a radioisotope.

[0189] In one embodiment, the immunoconjugate is an antibody drug conjugate (ADC) in which an antibody is conjugated to one or more of the aforementioned therapeutic agents. The antibody is typically connected to one or more of the therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents and agents and linkers, is provided in Pharmacol Review 68:3-19 (2016).

[0190] In another embodiment, the immunoconjugate comprises an antibody that binds to an enzymatically active toxin or fragment thereof as described herein, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aetophora curcas protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitgellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0191] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When a radioconjugate is used for detection, it can contain a radioactive atom for scintigraphy studies, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0192] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0193] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with crosslinker reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0194] 5.6 Illustrative Embodiments A. In certain non-limiting embodiments, the presently disclosed subject matter provides mammalian cells with reduced or eliminated lactogenic activity in which expression of a pyruvate kinase muscle (PKM) polypeptide isoform is knocked down or knocked out, the PKM polypeptide isoform comprising a PKM-1 polypeptide isoform.

[0195] A1. The mammalian cell as described above in A, wherein expression of a PKM-2 polypeptide isoform is knocked down or knocked out.

[0196] A2. The mammalian cell according to A or A1, wherein the cell is a CHO cell.

[0197] A3. The mammalian cell as described in any one of A to A2, comprising a nucleic acid sequence encoding a product of interest.

[0198] A4. The mammalian cell as described above in A3, wherein the product of interest comprises a protein.

[0199] A5. The mammalian cell as described in A3 or A4 above, wherein the product of interest comprises a recombinant protein.

[0200] A6. The mammalian cell according to any one of A3 to A5, wherein the desired product comprises an antibody or an antigen-binding fragment thereof.

[0201] A7. The mammalian cell as described in A6, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

[0202] A8. The mammalian cell described in A6, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

[0203] A9. The mammalian cell according to any one of A6 to A8, wherein the antibody comprises a chimeric antibody, a human antibody, or a humanized antibody.

[0204] A10. The mammalian cell according to any one of A6 to A9, wherein the antibody comprises a monoclonal antibody.

[0205] A11. The mammalian cell according to any one of A3 to A10, wherein the nucleic acid sequence is integrated into the cellular genome of the mammalian cell at a targeted location.

[0206] A12. The mammalian cell as described in A11, further comprising a nucleic acid encoding a product of interest randomly integrated into the cellular genome of the mammalian cell.

[0207] A13. The mammalian cell according to any one of A to A12, wherein the lactogenic activity of the mammalian cell is less than about 50% of the lactogenic activity of a reference cell.

[0208] A14. The mammalian cell as described above in A13, wherein the lactogenic activity of the mammalian cell is less than about 20% of the lactogenic activity of a reference cell.

[0209] A15. The mammalian cell according to A13 or A14, wherein the reference cell is a cell comprising a wild-type allele of the PKM gene.

[0210] A16. The mammalian cell according to any one of A to A15, wherein the lactogenic activity of the mammalian cell is measured on the 14th or 15th day of the production phase.

[0211] A17. The mammalian cell according to any one of A-A16, wherein the mammalian cell produces less than about 2.0 g / L of lactate during the production phase.

[0212] A18. The mammalian cell according to any one of A-A16, wherein the mammalian cell produces less than about 2.0 g / L of lactate in shake flasks during the production phase.

[0213] A19. The mammalian cell according to any one of A-A16, wherein the mammalian cell produces less than about 2.0 g / L of lactate in a bioreactor during the production phase.

[0214] B. In certain non-limiting embodiments, the presently disclosed subject matter provides a mammalian cell comprising an allele of the PKM gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-41, or the nucleotide sequences set forth in SEQ ID NOs: 37 and 38.

[0215] C. In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising any of the foregoing mammalian cells described in A-A19.

[0216] D. In certain non-limiting embodiments, the presently disclosed subject matter provides methods of reducing or eliminating lactogenic activity in a cell, comprising knocking down or knocking out expression of a pyruvate kinase muscle (PKM) polypeptide isoform.

[0217] E. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of reducing or eliminating lactogenic activity in a cell comprising administering to the cell a genetic modification system, wherein the genetic modification system knocks down or knocks out expression of a pyruvate kinase muscle (PKM) polypeptide isoform.

[0218] E1. The method described in E, wherein the genetic modification system is selected from the group consisting of a CRISPR / Cas system, a zinc finger nuclease (ZFN) system, a transcription activator-like effector nuclease (TALEN) system, and combinations thereof.

[0219] E2. The method described in E or E1, wherein the genetic modification system is a CRISPR / Cas9 system.

[0220] E3. CRISPR / Cas9 system (a) a Cas9 molecule, and (b) one or more guide RNAs (gRNAs) that include a targeting sequence complementary to a target sequence within the PKM gene; The aforementioned method described in E2, comprising:

[0221] E4. The aforementioned method of E3, wherein the target sequence is selected from the group consisting of a portion of the PKM gene, a region within exon 1, a 5' region adjacent to exon 2, a region within exon 2, a 5' intron region adjacent to exon 9 of the PKM gene, a 3' intron region adjacent to exon 9 of the PKM gene, a 3' intron region adjacent to exon 10 of the PKM gene, a region within exon 1 of the PKM gene, a region within exon 12 of the PKM gene, and combinations thereof.

[0222] E5. The aforementioned method described in any one of E3 to E4, wherein one or more gRNAs comprise a sequence selected from the group consisting of SEQ ID NOs: 33 to 34, and 42 to 43, and combinations thereof.

[0223] E6. The aforementioned method of E3 or E4, wherein the one or more gRNAs comprise: (1) a first gRNA comprising a target sequence complementary to a 5' intron region adjacent to exon 9 of the PKM gene; and (2) a second gRNA comprising a target domain complementary to a 3' intron region adjacent to exon 9 of the PKM gene.

[0224] E7. The aforementioned method described in any one of E3 to E6, wherein one or more gRNAs comprise a sequence selected from the group consisting of SEQ ID NOs: 33 to 34, and combinations thereof.

[0225] E8. The aforementioned method of E3 or E4, wherein the one or more gRNAs comprise: (1) a first gRNA comprising a target sequence complementary to a region within exon 2 of the PKM gene; and (2) a second gRNA comprising a target domain complementary to a region within exon 12 of the PKM gene.

[0226] E9. The aforementioned method described in any one of E3 to E5, and E8, wherein one or more gRNAs comprise a sequence selected from the group consisting of SEQ ID NOs: 42 to 43, and combinations thereof.

[0227] E10. The method of any one of D and E-E9, wherein expression of a PKM polypeptide isoform is knocked out and lactogenic activity of the cell is eliminated or reduced compared to the lactogenic activity of a reference cell.

[0228] E11. The aforementioned method of any one of D and E-E9, wherein expression of a PKM polypeptide isoform is knocked down and lactogenic activity of the cells is reduced compared to the lactogenic activity of a reference cell.

[0229] E12. The aforementioned method of E10 or E11, wherein the lactogenic activity of the cells is less than about 50% of the lactogenic activity of the reference cells.

[0230] E13. The aforementioned method of E10 or E11, wherein the lactogenic activity of the cells is less than about 20% of the lactogenic activity of the reference cells.

[0231] E14. The aforementioned method according to any one of D and E-E13, wherein the lactogenic activity of the cells is measured on day 14 or 15 of the production phase.

[0232] E15. The aforementioned method of any one of D and E-E14, wherein the cells produce less than about 2.0 g / L lactate during the production phase.

[0233] E16. The aforementioned method of any one of D and E-E14, wherein the cells produce less than about 2.0 g / L lactate in shake flasks during the production phase.

[0234] E17. The aforementioned method of any one of D and E-E14, wherein the cells produce less than about 2.0 g / L of lactate in the bioreactor during the production phase.

[0235] E18. The aforementioned method according to any one of E11 to E17, wherein the reference cell is a cell comprising a wild type allele of the PKM gene.

[0236] E19. The aforementioned method of any one of D and E-E18, wherein the PKM polypeptide isoform is a PKM-1 polypeptide isoform.

[0237] E20. The method of any one of D and E-E19, wherein the PKM polypeptide isoforms are a PKM-1 polypeptide isoform and a PKM-2 polypeptide isoform.

[0238] E21. The method described in E, wherein the genetic modification system comprises RNA selected from the group consisting of short hairpin RNA (shRNA), small interfering RNA (siRNA), and microRNA (miRNA), and the RNA is complementary to a portion of the mRNA expressed by the PKM gene.

[0239] E22. The aforementioned method of E21, wherein the mRNA expressed by the PKM gene encodes a PKM-1 polypeptide isoform.

[0240] E23. The aforementioned method of E22, wherein expression of a PKM-1 polypeptide isoform is knocked out or knocked down, and lactogenic activity of the cell is reduced compared to lactogenic activity of a reference cell.

[0241] E24. The method of any one of E21 to E23, wherein the genetic modification system further comprises a second RNA selected from the group consisting of shRNA, siRNA, and microRNA miRNA, and the second RNA is complementary to a portion of the mRNA expressed by the PKM gene encoding a PKM-2 polypeptide isoform.

[0242] E25. The aforementioned method according to E24, wherein expression of PKM-1 and PKM-2 polypeptide isoforms is knocked out or knocked down, thereby reducing lactogenic activity of the cells.

[0243] E26. The method described in E, wherein the genetic modification system is a zinc finger nuclease (ZFN) system or a transcription activator-like effector nuclease (TALEN) system.

[0244] E27. The method according to any one of D and E-E26, wherein the cell is a mammalian cell.

[0245] E28. The aforementioned method according to E27, wherein the mammalian cell is a CHO cell.

[0246] E29. The method according to any one of D and E-E28, wherein the cell expresses a product of interest.

[0247] E30. The method according to E29, wherein the product of interest expressed by the cell is encoded by a nucleic acid sequence.

[0248] E31. The method as described in E30, wherein the nucleic acid sequence is integrated into the cellular genome of the cell at a targeted location.

[0249] E32. The aforementioned method of any one of E26 to E31, wherein the product of interest expressed by the cell is further encoded by a nucleic acid sequence randomly integrated into the cellular genome of the mammalian cell.

[0250] E33. The method as described above in E26 to E31, wherein the product of interest comprises a protein.

[0251] E34. The method of E33, wherein the desired product comprises a recombinant protein.

[0252] E35. The method of any one of E26 to E33, wherein the desired product comprises an antibody or antigen-binding fragment thereof.

[0253] E36. The aforementioned method of E35, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

[0254] E37. The aforementioned method of E35, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

[0255] E38. The aforementioned method according to any one of E35 to E37, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0256] E39. The aforementioned method described in any one of E35 to E38, wherein the antibody is a monoclonal antibody.

[0257] F. In certain non-limiting embodiments, the presently disclosed subject matter provides a method of making a product of interest comprising culturing a mammalian cell that expresses the product of interest, wherein the mammalian cell expresses the product of interest and has reduced or eliminated lactogenic activity.

[0258] G. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for culturing a population of mammalian cells expressing a product of interest, wherein the mammalian cells have reduced or eliminated lactogenic activity.

[0259] G1. The method according to F or G, wherein the reduction or elimination of lactogenic activity is caused by knocking out or knocking down expression of pyruvate kinase muscle (PKM) polypeptide isoform in mammalian cells.

[0260] G2. The method as described above in G1, wherein the PKM polypeptide isoform is a PKM-1 polypeptide isoform.

[0261] G3. The method according to G1, wherein the PKM polypeptide isoform is a PKM-1 polypeptide isoform or a PKM-2 polypeptide isoform.

[0262] G4. The method of any one of F and G-G3, wherein the lactogenic activity of the mammalian cells is less than about 50% of the lactogenic activity of the reference cells.

[0263] G5. The method of any one of F and G-G3, wherein the lactogenic activity of the mammalian cells is less than about 20% of the lactogenic activity of the reference cells.

[0264] G6. The method according to any one of F and G-G5, wherein the lactogenic activity of the mammalian cells is measured on day 14 or 15 of the production phase.

[0265] G7. The aforementioned method of any one of F and G-G6, wherein the mammalian cells produce less than about 2.0 g / L of lactate during the production phase.

[0266] G8. The aforementioned method of any one of F and G-G6, wherein the mammalian cells produce less than about 2.0 g / L of lactate in the percolation flask during the production phase.

[0267] G9. The aforementioned method of any one of F and G-G6, wherein the mammalian cells produce less than about 2.0 g / L of lactate in the bioreactor during the production phase.

[0268] G10. The aforementioned method of any one of F and G-G9, wherein the reference cell is a cell containing at least one or both wild-type alleles of the PKM gene.

[0269] G11. The method according to any one of F and G to G10, wherein the mammalian cell is a CHO cell.

[0270] G12. The method according to any one of F and G-G11, wherein the product of interest expressed by the mammalian cell is encoded by a nucleic acid sequence.

[0271] G13. The method as described in G12, wherein the nucleic acid sequence is integrated into the cellular genome of the mammalian cell at a targeted location.

[0272] G14. The method according to any one of F and G-G13, wherein the product of interest expressed by the cell is further encoded by a nucleic acid sequence randomly integrated into the cellular genome of the mammalian cell.

[0273] G15. The method according to any one of F and G-G14, wherein the desired product comprises a protein.

[0274] G16. The method according to any one of F and G-G15, wherein the desired product comprises a recombinant protein.

[0275] G17. The method according to any one of F and G-G16, wherein the desired product comprises an antibody or an antigen-binding fragment thereof.

[0276] G18. The aforementioned method described in G17, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

[0277] G19. The method described in G17, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

[0278] G20. The method according to any one of G17 to G19, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0279] G21. The method according to any one of G17 to G20, wherein the antibody is a monoclonal antibody.

[0280] G22. The method according to any one of F and G-G21, further comprising recovering the desired product. EXAMPLES

[0281] The following examples are merely illustrative of the subject matter disclosed herein and should not be construed as limiting in any way.

[0282] Example 1: PKM-1 expression drives lactogenic behavior in CHO cell lines, causing poor viability and productivity During the process of cell line development (CLD), two lactogenic cell lines expressing different antibody molecules were identified. The lactogenic behavior of these cell lines can be differentially mitigated through optimization of nutrient supply or culture pH, depending on the cell line. Analysis of various proteins involved in the glycolysis pathway revealed a direct correlation between pyruvate kinase myosin-1 (PKM-1) isoforms and lactogenic behavior.

[0283] CRISPR / Cas9 was used for targeted deletion of exon-9 to knock out PKM-1 expression. Knocking out PKM-1 expression completely abolished lactogenic behavior in two selected cell lines, eliminating the need for mitigation methods. One cell line was identified in which expression of both PKM-1 and PKM-2 genes was completely abolished without any adverse effects on growth and viability. Further analysis of the parental and CHO cell lines revealed that they all expressed PKL and PKR versions of pyruvate kinase, which, without being bound by theory, allowed the cells to tolerate the complete lack of PKM expression. The unique mitigation methods used to control the lactogenic behavior of these cell lines shifted metabolic pathways and altered the patterns of PKM-1 transcription and expression to prevent or delay lactogenic behavior. The PKM gene is completely knocked out in order to analyze the behavior of the resulting cell lines in culture and in bioreactors.

[0284] In summary, the present disclosure depicts a direct correlation between lactogenic behavior and abundance of PKM-1 in production cultures. Furthermore, ablation of PKM-1 expression permitted and reversed lactogenic behavior in selected CHO cell lines. Thus, permanent deletion of PKM-1, or the entire PKM gene for this issue, in CHO cells can be beneficial in reducing the occurrence of lactogenic behavior during production in bioreactors.

[0285] Materials and Methods cell line Cell lines secreting recombinant monoclonal antibodies mAb-1 or mAb-2 were derived from CHO-K1 hosts utilizing the glutamine synthetase (GS) selection marker. Seed trains were maintained in proprietary DMEM / F12-based medium containing methionine sulfoximine (MSX) as the selection agent at 150 rpm, 37°C, and 5% CO2. Cells were passaged every 3 or 4 days.

[0286] 2L bioreactor production Production in 2 L bioreactors was carried out in glass stirred tank bioreactors (Applikon, Foster City, CA) with a Finesse controller (Applikon, Foster City, CA). All production media utilized one seed train phase (N-1) in the bioreactor for 3 days followed by a production phase (N) lasting 14 days. The N-1 seed media was inoculated at an operating volume of 1.5-1.7 L for a target packed cell volume (PCV) of 0.17%. These were operated at 37°C, a pH setpoint of 7.0, a dissolved oxygen (DO) setpoint of 30%, and agitation at 275 rpm. After 3 days, cells were transferred and inoculated into the production (N) bioreactors at a volume of 1.4-1.7 L at a target PCV of 0.25%. All bioreactors were operated at 37°C with a temperature shift to 35°C for 72 hours. Cultures were operated at a deadband of 0.03, pH 7.00, using CO2 as an acid control and 1M sodium carbonate as a base control. The only exception to this pH manipulation was the pH level of mAb-2, which was operated at a constant pH of 6.80, with the same deadband of 0.03. All cultures were set to operate at 350 rpm and 30% DO, with DO maintained constant using a combination of air and oxygen gas flows. For the control process culture of mAb-1, the feed occurred on day 3 (72 hours) of production, at which point the total volume of the culture was 20%. For the improved feed process, the feed occurred on days 3 (72 hours) and 6 (144 hours), at 15% of the total volume of the culture for each feed.

[0287] AMBR15 operation Production cultures in an AMBR15 (Sartorius, Goettingen Germany) system were operated at set points of 37° C. temperature, 30% DO, pH 7.0, and agitation speed of 1400 rpm. The N-1 seed train was run for 4 days, and then the production (N) stage was inoculated at 1 million cells / mL in a total volume of 13 mL, at a temperature of 37° C., 30% DO, pH 7.0, and agitation speed of 1400 rpm. For these cultures, a scale-down process in a 2 L bioreactor was carried out.

[0288] Offline sample analysis Supernatant and cell pellet samples were collected for assay or Western blotting analysis using a Vi-Cell XR (Beckman Coulter) for viable cell concentration (VCC) and viability, and for pO2, pH, pCO2, Na + Samples were analyzed using a Bioprofile 400 (Nova Biomedical) for arginine, glucose, and lactate. All samples from the 2L and AMBR bioreactors were analyzed on the BioProfile 400 for several minutes after sampling to minimize gas evolution. The same Vi-Cell XR, BioProfile 400, and osmometer (Model 2020, Advanced Instruments) were used for all samples to remove instrument-to-instrument variability. Antibody titers were measured using high pressure liquid chromatography (HPLC) with a protein A column. Quality assays of antibody production were performed using cell culture supernatant samples purified by a PhyTip (PhyNexus, San Jose, CA) protein A column. Antibody molecular size distribution was analyzed by size exclusion chromatography (SEC). Protein charge heterogeneity was measured using imaging capillary isoelectric focusing (icIEF), and all charge-heterogeneous samples were pretreated with carboxypeptidase B. Quality assays for all protein products were developed in-house and detailed procedures have been published (Hopp et al., 2009, Biotechnol. Prog. 25(5):1427-32).

[0289] Immunoblotting The cell pellet was lysed in cell lysis buffer (10 mM Tris pH 8.0, 0.5% NP40, 150 mM NaCl, 5 mM MgCl2 with protease inhibitors) and incubated on ice for 20 minutes. Samples were then spun down at 13,000 rpm for 10 minutes and the supernatant transferred to a new tube and protein concentration was measured using a Nanodrop 2000 (Thermo Scientific, Wilmington DE) using absorbance at 280 nm. 15 μL of lysate was combined with 5 μL of 4x running buffer (400 μL 2x Invitrogen loading buffer + 400 μL 50% glycerol + 200 μL 20% SDS + 100 μL β-mercaptoethanol) and heated at 90°C for 5 minutes. Equal amounts of protein were then loaded onto 12-well 4-20% Tris-glycine gels and run at 150V for 1.5 hours using SDS running buffer. Proteins were then transferred to nitrocellulose membranes using a Thermo Fisher iBlot2. After washing with 1xTBST buffer, blots were blocked in 5% milk solution for a minimum of 1 hour and then incubated overnight with primary antibody. Blots were then washed and incubated in secondary antibody for a minimum of 1 hour and washed again with TBST buffer. Blots were imaged using ECL reagent followed by a Bio-Rad imaging instrument (Bio-Rad, Hercules, CA).

[0290] PKM-1 knockout To knock out PKM-1, guide RNAs (gRNAs) targeting both the 5' and 3' intronic regions flanking exon 9 of the PKM gene were cloned into a gRNA expression vector from Genentech. This construct was co-transfected with a Cas9 expression plasmid into mAb-2 expressing cells. Transfected cells were single cell cloned and deletion of exon 9 was confirmed by genomic DNA PCR. The following gRNA oligos were found to be most effective for deleting exon-9 (PKM-1), and a pool targeted with these oligos was used to isolate PKM-1 knockout mAb-2 cell lines: 5'gRNA: GTCCTTTGGGCAGAGACAG (SEQ ID NO: 33) 3'gRNA: GACCAGAGTACTCCCTCGT (SEQ ID NO: 34) Genomic DNA PCR primer sequences: 5'-Primer: CCAGATTTGGTGAGGACGAT (SEQ ID NO: 35) 3'-Primer: AGCTGTGTTGTGAGGCATTG (SEQ ID NO: 36)

[0291] result Increased amounts of PKM-1 during production correlate with the lactogenic behavior of CHO cells A culture seed train of a cell line expressing mAb-1 was used to set up production broth in a bioreactor using standard or enhanced feeding. Standard feeding induced lactogenic behavior in the mAb-1 cell line, while enhanced feeding attenuated lactogenic behavior. High lactate conditions induced a decrease in cell viability after 10 days in the production broth (Figure 1A) and reduced titers on day 14 compared to the low lactate condition (Figure 1B). Although both culture conditions had significant amounts of lactate by day 7, the high lactate condition showed a significant increase in lactate accumulation in the production broth from day 7, reaching an upper 15 g / L by day 14. For the low lactate condition, a 3-4 day delay was observed before lactate tended to be higher (days 12-14), only reaching 8 g / L by day 14 (Figure 1C).

[0292] To identify enzymes that may correlate with the observed lactogenic behavior, Western blot analysis was performed on various proteins involved in cell growth signaling and glycolysis pathways. Since cell viability dropped sharply after 10 days in high lactate conditions, this set of samples from days 0, 2, 7, and 10 were compared with samples from days 0, 7, 10, and 14 in low lactate conditions (Figures 1D-1F). Among all the different proteins analyzed (data not shown), only PKM-1 abundance showed a direct correlation with the lactogenic behavior of mAb-1 expressing cell lines. Figures 1D-1F show the abundance of PKM-1 and PKM-2 proteins in cell pellets. The expression abundance of PKM-1 in high lactate samples started to increase divergently after 7 days in production culture compared to low lactate samples. Interestingly, even under low lactate conditions, the increase in PKM-1 abundance towards the end of the production culture (Fig. 1E, days 10–14) trended together with high lactate amounts during the culture (Fig. 1C), so that the increase in PKM-1 abundance correlated closely with lactate accumulation, whereas the abundance of PKM-2 correlated inversely with the lactogenic behavior in the mAb-1-expressing cell line (Fig. 1F).

[0293] Similar bioreactor experiments were performed for mAb-2 expressing cell lines. Such cell lines were cultured in production media with conditions promoting lactogenic or non-lactogenic behavior. Cell viability of the mAb-2 cell line was comparable between high and low lactate conditions by days 13 and 14 of production culture (Figure 2A). This resulted in a decrease in titer of about 25% in high lactate conditions relative to low lactate conditions (Figure 2B). Throughout the production process, lactate accumulation in the medium was lower in the low lactate control group of the mAb-2 cell line experiments. However, under high lactate conditions, the amount of lactate began to diverge from day 7, reaching an upper 15 g / L by day 14 of production culture (Figure 2C). Western blot analysis revealed that the amount of PKM-1 protein began to increase in the high lactate samples compared to the low lactate samples at approximately day 7 of production and increased linearly until the end of the production culture (Figures 2D and 2E). This increase in PKM-1 amount correlated closely with the increase in accumulated lactate (Figures 2C and 2E). Similar to mAb-1, the overall amount of PKM-2 in the low lactate mAb-2 samples was greater than that in the high lactate samples, as shown in Figures 2D (bottom panel) and 2F. Taken together, these data suggest a direct correlation between lactogenic behavior and PKM-1 amount in cells expressing mAb-1 and mAb-2 in these cells.

[0294] PKL / R proteins are expressed in CHO cells but do not correlate with lactogenic behavior. To assess whether other forms of PK (besides PKM) are expressed in the proprietary CHO host cells, we analyzed the expression of PKL and PKR (PKL / R) proteins in two different CHO host cell lines (CHO-K1 and DHFR- / -) as well as in a human (HEK293) cell line. Both CHO cell lines, as well as the control HEK293 cell line, showed expression of the PKL / R enzyme (Figure 3A), indicating that all isoforms of the PK gene (including PKM-1, PKM-2, and PKL / R) are expressed in the selected CHO host. We next assessed whether expression of PKL / R was differentially regulated when comparing high and low lactate conditions in mAb-1 and / or mAb-2 expressing cells. The expression levels of PKL / R were comparable in high / low lactate conditions in both mAb-1 and mAb-2 cell lines (when protein amounts were normalized to actin as an internal control), indicating no correlation with lactogenic behavior in these cell lines (Figures 3B-3D). The low amount of PKL / R in the mAb-2 high lactate samples at days 7 and 10 in Figure 3C was due to low overall protein loading, reflected by the low amount of actin (as a loading control) in the same lane. Besides PK isoforms, the expression of several different proteins involved in cell growth signaling and glycolysis pathways was also analyzed. However, it was observed that there was no clear correlation between the expression of proteins and the lactogenic behavior observed in mAb-1 or mAb-2 expressing cell lines (data not shown). The fact that different PK isoforms were expressed in the selected CHO host cells suggested that these cell lines may be able to tolerate targeted deletion of one or more isoforms of PK. The lactogenic behavior of the mAb-1 and mAb-2 cell lines observed during the production phase and the direct correlation with PKM-1 abundance made this enzyme a good candidate for targeted deletion.

[0295] CRISPR / Cas9-mediated targeted deletion of exon-9 in the PKM gene The PKM gene is responsible for the expression of both PKM-1 and PKM-2 enzymes, which differ only by alternatively including exon 9 or exon 10, respectively. The selected CHO cell lines were not sequenced or annotated, so no information was available on PKM allele type, gene copy number, or sequence. Therefore, to eliminate expression of the PKM-1 gene, guide RNA constructs were designed targeting the intronic region adjacent to exon 9 using the CHO sequence available in public databases (Kent et al. 2002, Genome Res. 12(6):996-1006). A 5' screening PCR primer was designed to match the intronic region between exons 8 and 9, and a 3' screening PCR primer was designed against the matched exon 10 (Figure 4A). Sequence information of the exons was obtained based on amplification and sequencing of PKM cDNA from mRNA of the selected CHO host. mAb-2 expressing cell lines were transfected with Cas9 and the various gRNA constructs. After the initial screening, the pool showing the most efficient exon 9 targeting was passed through single cell cloning. Twenty cell lines from this pool were screened for targeted deletion of exon-9, and two heterozygous (HET-3 and HET-18) and two knockout (KO-2 and KO-15) cell lines were identified for which the deletion of the targeted allele was fully confirmed by PCR and sequencing (Figures 4B and 4C). Cultures of these cell lines were then expanded for further evaluation in bioreactors. Sequencing of the targeted allele confirmed the deletion of exon 9 in all selected cell lines (Figure 4C). Three cell lines (KO-2, HET-3, and HET-18) had the exact deletion of exon 9 at the 5' and 3' gRNA targeting sites (Figure 4C). For the KO-15 cell line, the PCR size of the targeted allele was smaller than that observed in the other cell lines (Figure 4B). Sequencing analysis confirmed that 122 base pairs (bp) upstream of the 5' gRNA were deleted in the KO-15 cell line and partially replaced with an unrelated insertion (Figure 4C and data not shown).Deletions in this region may affect the integrity of the intron between exons 8 and 9. Regardless, sequencing analysis showed that exon 9 was completely deleted in all selected cell lines.

[0296] Blocking or reducing PKM-1 expression prevented or reduced, respectively, lactogenic behavior in the mAb-2 cell line. Four mAb-2 cell lines, all with deletion of some (HET) or all (KO) alleles, and deletion of exon-9 in the WT mAb-2 line, were sourced and set up in AMBR bioreactors for 14 days. Growth and viability of the PKM-1 KO, HET and WT cell lines are shown in Figures 5A and 5B. The PKM-1 HET or mAb-2 lines had relatively similar titers and specific productivity to that of the WT mAb-2 cell line (Figures 5C and 5D). The PKM-1 KO (KO-2 and KO-15) and one of the HET mAb-2 cell lines (HET-3) had significantly less lactate when cultured under control (lactogenic) conditions compared to the WT mAb-2 cell line (Figure 5E). On the other hand, the HET-18 strain exhibited similar lactogenic behavior compared to WT mAb-2 up to day 10. After 10 days, the rate of lactate accumulation in the WT mAb-2 strain increased and by day 14 was approximately double that of the HET-18 strain (Figure 5E).

[0297] Western blot analysis of these cell lines showed that KO-2 and KO-15 lines completely lost expression of PKM-1, and HET-3 cell line had low PKM-1 expression compared to HET-18 cell line (Figure 5F). The fact that the lactogenic behavior of PKM-1 HET and KO cell lines directly correlated with PKM-1 protein expression in some cell lines suggested that the observed changes in lactogenic behavior were not solely due to other clonal differences. Since the band corresponding to the full-length WT allele could have been amplified from genomic DNA of HET cell lines (Figure 4B), possible explanations for these observations include, but are not limited to, differential expression of PKM-1 from different CHO PKM alleles; targeting of the WT PKM allele with only one gRNA construct resulting in disruption of proper exon splicing; inversion of the gRNA targeting region, or 4 homogenization mediated by allelic recombination.

[0298] The amount of PKM-2 was low for the WT mAb-2 cell line (the relative difference was considered with the caveat that the actin loading control was also low in this sample), but was high in the HET-3, HET-18, and KO-2 cell lines (Figure 5F). Interestingly, the KO-15 mAb-2 cell line completely lacked expression of PKM-2, in addition to PKM-1 (Figure 5F). This is likely due to a larger deletion observed upstream of the 5' gRNA targeting region of the PKM allele, possibly affecting the functionality of the intronic region between exons 8 and 9 (Figure 4C). This suggested that the selected CHO cells could tolerate the complete absence of both PKM-1 and PKM-2 enzymes, possibly due to their ability to express PKL / R enzymes (Figure 3A).

[0299] Product quality attributes such as large molecular weight (aggregates) and charge variant species were investigated between WT and PKM-1 KO and HET mAb2 expressing cell lines. Although no significant differences in product aggregation were observed between WT, PKM-1 KO and HET cell lines (Figure 5G), some variation was observed in the mAb-2 product charge variant (Figure 5H) profile. There was no clear trend or correlation between the amount of lactate and product quality attributes (Figures 5G and 5H), so the observed differences may be due to normal variations that may occur during cell line derivation. Additionally, the media and feeds used in these experiments were optimized for the WT mAb-2 cell line, not the PKM-1 KO or HET cell lines. Altering the amount of PKM may affect the overall metabolic profile of these cell lines, so fine tuning of media and feeds may be required to investigate the full effect and impact on product quality attributes.

[0300] Since it is important to analyze the behavior of any cell line after cell storage and thawing, PKM-1 KO and HET mAb-2 cell lines were stored and then thawed and kept in culture for 2.5 weeks along with the WT cell line as a control. These cells were then used as a source to set up quadruple production media for each cell line to evaluate their post-thaw performance. The data showed that the growth, viability, titer, specific productivity, and lactogenic profile of PKM-1 KO and HET cell lines were comparable before and after cell storage (Figures 5A-5D and 6A-6D). As previously observed (data not shown), fresh, thawed WT mAb-2 cells were less lactogenic (10 g / L, Figure 6E) versus older cells (15 g / L, Figure 5E). However, this behavior was unique to the WT mAb-2 cell line, as cell age had no significant effect on the lactogenic behavior of the mAb-2 PKM-1 KO and HET cell lines (Figures 5E and 6E). Note that for all cell lines, a small accumulation of lactate was observed in the medium until day 7 of production culture, after which the PKM-1 KO and HET-3 cell lines consumed lactate, while a lactate-dissipating profile was observed for both the WT and HET-18 cell lines (Figure 6E). Although all young cell lines (after thawing) had a relatively low acidic peak rate (Figure 6G) compared to the older cell lines (Figure 5G), a feature likely related to cell age, there were no major differences in product quality between lactogenic (WT or PKM-1 HET-18) and non-lactogenic (PKM-1 KO or HET-3) cell lines (Figures 6F-6G).

[0301] Consideration Lactogenic behavior in CHO cell cultures can result in culture acidification and high osmolality as a result of adding base to adjust culture pH, negatively affecting viability, VCC, and final production titer (Li et al., 2010, MAbs 2(5):466-79). Lactate production occurs via aerobic glycolysis, a behavior commonly known as the Warburg effect (Warburg, 1956, Science 123(3191):309-14), and is observed in many cultured and cancer cells. These cells utilize the glycolytic process to produce energy and regulate this process in response to different energy and metabolic fluxes (Mulukutla et al., 2014, PLoS One 9(6):e98756; Mulukutla et al. 2010, Trends Biotechnol. 28(9):476-84; Luo et al., 2012, Biotechnol. Bioeng. 109(1):146-56; Ahn and Antoniewicz, 2012, Biotechnol. J. 7(l):61-74). Although some lactogenic behavior can be controlled by process modifications (Luo et al., 2012, Biotechnol. Bioeng. 109(1):146-56; Gagnon et al., 2011, Biotechnol. Bioeng. 108(6):1328-37), these approaches did not reveal the underlying causes of the observed behavior. To better understand lactogenic behavior in the CHO host, we identified two lactogenic cell lines expressing mAb-1 or mAb-2, whose lactogenic behavior could be tuned by altering either the feeding method or the pH of the culture medium. These independently derived cell lines were utilized as tools in analyzing a panel of proteins and enzymes involved in cell growth signaling or glycolysis pathways to delineate possible correlations with lactogenic behavior. These findings revealed a direct correlation between PKM-1 expression and lactogenic behavior in both mAb-1 and mAb-2 cell lines (FIGS. 1D-1F and 2D-2F).

[0302] The amount of PKM-1 detected in both mAb-1 and mAb-2 production cultures was low at early time points during production and increased in a manner that correlated with the increasing amount of lactate in the cultures, whereas the amount of PKM-2 correlated inversely with the lactogenic behavior in these cell lines (Figures 1D-1F and 2D-2F). This may be due to the function of proteins involved in alternative splicing of the PKM-1 or PKM-2 transcripts from the PKM genes (Chaneton and Gottlieb, 2012, Trends Biochem. Sci. 37(8):309-16; Israelsen and Vander Heiden, 2015, Semin. Cell Dev. Biol. 43:43-51; Mazurek, 2011, Int. J. Biochem. Cell Biol. 43(7):969-80; Harada et al., 1978, Biochim. Biophys. Acta. 524(2):327-39; Noguchi et al., 1986, J. Biol. Chem. 261(29):13807-12; Noguchi et al., 1987, J. Biol. Chem. 262(29):14366-71). Of the two isoforms of the PKM gene, PKM-2 has been reported to function as a central switch to alter metabolic pathways, allowing cancer cells to survive and grow under physiologically unfavorable conditions, such as within the tumor environment (Christofk et al., 2008, Nature 452(7184):230-3; Chaneton and Gottlieb, 2012, Trends Biochem. Sci. 37(8):309-16; Israelsen and Vander Heiden, 2015, Semin. Cell Dev. Biol. 43:43-51). On the other hand, the PKM-1 isoform is constitutively active once expressed. Without wishing to be bound by theory, the correlation between the amount of PKM-1 and the rate of lactogenic behavior in the production cultures may be due to the uncontrollable conversion of PEP to pyruvate by this enzyme, followed by the LDH-mediated conversion of pyruvate to lactate.

[0303] CRISPR mediated deletion of exon-9, and therefore PKM-1, in mAb-2 expressing cells resulted in a complete reversal of the lactogenic behavior of PKM-1 KO cells under conditions where WT mAb-2 expressing cells exhibit lactogenic behavior (Figure 5E). The two PKM-1 HET mAb-2 cell lines behaved differently. The HET-3 KO cell line produced very little lactate (slightly more than the PKM-1 KO cell line), while the HET-18 cell line was lactogenic and produced approximately 2 / 3 of the lactate of WT mAb-2 cells (Figure 5E). A direct correlation between the lactogenic behavior of these PKM-1 HET cell lines and the higher amount of PKM-1 in these cells was also observed (Figure 5F). The difference in PKM-1 abundance observed in HET-18 compared to the HET-3 cell line may be due to differential expression of PKM-1 from different PKM alleles, or partial targeting of the PKM allele by the gRNA construct in the HET-3 cell line, or inversion of the targeted region within the targeted PKM allele.

[0304] A PKM-1 knockout cell line (KO-15) (Figure 5F) was identified that is unable to express PKM-2 protein. The cell line has a 122 bp deletion and insertion of random bases in the intron upstream of the 5' gRNA targeting region (Figure 3C). The KO-15 cell line can also tolerate the loss of both PKM-1 and PKM-2 enzymes because CHO cell lines also express PKL / R (Figure 3A), thus compensating for the lack of PKM expression. Expression of PKL / R protein in WT and all PKM-1 HET and KO cell lines was confirmed by Western blot analysis (data not shown). Nevertheless, the absence or reduction of PKM-1 expression resulted in lower lactate production in all PKM-1 KO or HET cell lines, respectively, without affecting titer or specific productivity (Figures 5C-5D and 6C-6D). The absence or weakening of lactogenic behavior in these cell lines was reproducible and consistent with the young cell ages post-thaw and among all replicates (Figures 5A-5E and 6A-6E).

[0305] Controlling lactogenic behavior during production culture is important to obtain optimal titers and product quality from manufacturing runs. Therefore, knocking out PKM-1 expression from the host can be advantageous in manipulating the lactogenic behavior of CHO cells. Because PKL / R enzymes can compensate for the loss of PKM, it is important to confirm and monitor the expression of other PK enzymes in CHO cells before targeting the entire PKM-1 or PKM gene for deficiency. Knocking out the entire PKM gene can be tolerated in CHO hosts, and PKM KO CHO hosts can express the antibody or product of interest with titers and product quality comparable to those of the WT host. These PKM KO hosts can have reduced lactogenic behavior and better growth profiles than the WT host, as observed in the KO-2 and KO-15 cell lines (Figures 5A and 6A).

[0306] Example 2: Production of mAb-3 in PKM knockout and PKM-1 knockout CHO cell lines The PKM or PKM-1 gene was knocked out in the CHO-K1M cell line to generate one PKM KO and four PKM-1 KO host cell lines. A transgene encoding mAb-3 was introduced into the WT CHO-K1M, PKM KO, and PKM-1 KO host cell lines by targeted integration to generate three WT pools expressing mAb-3 from the same host cell line, three PKM KO pools expressing mAb-3 from the same host cell line, and four PKM-1 KO pools expressing mAb-3 from four different host cell lines.

[0307] The following experiments were performed as disclosed in Example 1, except that the gRNAs for generating PKM KO hosts were different and are as follows: The gRNAs used to generate PKM-1 KO host cells were the same as those used in Example 1. The gRNA for generating PKM KO host cells had the following sequence: 5'-gRNA: CCCATCACGGCCCGCAACAC (SEQ ID NO: 42, targets a region within exon 2 of the PKM gene) 3'-gRNA: CTTCTTCAAGACGGGGGATG (SEQ ID NO: 43, targeting a region within exon 12 of the PKM gene)

[0308] In shake flask production, MAb-3 producing pools derived from PKM and PKM-1 KO host cell lines had comparable or higher Qp and titer than the WT pool, but had lower growth (represented by integrated viable cell concentration (IVCC)) (Figure 7). In shake flask production, MAb-3 producing pools derived from PKM and PKM1 KO host cell lines also produced less lactate than the WT host cell line (Figure 8A) but consumed more glucose (Figure 8B). As shown in Figure 8A, the WT host cells produced approximately 1.0 g / L lactate by day 15 of production in shake flasks. During shake flask production, lactate concentrations of 1-2.5 g / L are considered high. In contrast, by day 15, PKM-1 KO host cells produced very little lactate, and PKM KO host cells produced less than 0.2 g / L lactate, resulting in a reduction in lactate production of over 80% when PKM-1, or both PKM-1 and PKM-2, were knocked out.

[0309] In shake flask production, MAb-3 production pools derived from the PKM / PKM-1 KO host cell lines had different amino acid synthesis / consumption rates (Figures 9A and 10). For example, the PKM KO host cell line accumulated 3-phosphoglycerate, resulting in increased production of serine and glycine compared to the WT and PKM-1 host cells (Figure 9B). The WT host cell line accumulated pyruvate compared to the PKM KO and PKM-1 host cells (Figure 9C). Without being limited to a particular theory, accumulation of pyruvate in the WT host cells may result in accumulation of lactate and increased production of alanine (Figure 10). The PKM-1 KO host cell line accumulated TCA cycle products such as oxaloacetate (leading to accumulation of aspartate and asparagine (Figure 9D)) and α-ketoglutarate (a-KG) (leading to accumulation of glutamate, arginine, and glutamine (Figure 9E)). Without being limited to a particular theory, these results suggest that PKM-1 and PKM-2 may function to control the amount of pyruvate produced, such that pyruvate preferentially enters the TCA cycle. A summary of the above findings is shown in Figure 10. Based on these data, the cell culture medium and conditions used to culture PKM-1 and PKM KO cells can be adjusted to compensate for the amount of glucose consumed and / or the amount of amino acids consumed or synthesized.

[0310] MAb-3 production pools derived from PKM / PKM1 KO host cell lines had distinct glycosylation profiles in shake flask production. Galactosylation was reduced in the PKM KO host, and fucosylation was slightly reduced in the PKM KO and PKM-1 KO host cell lines (Figure 11). Without being limited to a particular theory, these changes in glycosylation likely do not affect the activity of the antibodies generated.

[0311] The contents of all figures and all references, patents and published patent applications, and deposit numbers cited throughout this application are hereby expressly incorporated by reference.

Claims

1. A Chinese hamster ovary (CHO) cell having reduced or eliminated lactogenic activity, wherein expression of a pyruvate kinase muscle (PKM) polypeptide isoform is knocked down or knocked out, and the PKM polypeptide isoform comprises a PKM-1 polypeptide isoform.

2. The CHO cell of claim 1, wherein expression of a PKM-2 polypeptide isoform is knocked down or knocked out.

3. 3. The CHO cell of claim 1 or 2, comprising a nucleic acid sequence encoding a product of interest.

4. 4. The CHO cell of claim 3, wherein the product of interest comprises a protein.

5. 5. The CHO cell of claim 3 or 4, wherein the product of interest comprises a recombinant protein.

6. The CHO cell of any one of claims 3 to 5, wherein the product of interest comprises an antibody or an antigen-binding fragment thereof.

7. The CHO cell of claim 6, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

8. The CHO cell of claim 6, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

9. The CHO cell of any one of claims 6 to 8, wherein the antibody comprises a chimeric antibody, a human antibody, or a humanized antibody.

10. The CHO cell of any one of claims 6 to 9, wherein the antibody comprises a monoclonal antibody.

11. The CHO cell of any one of claims 3 to 10, wherein the nucleic acid sequence is integrated into the cellular genome of the CHO cell at a targeted location.

12. 12. The CHO cell of claim 11, further comprising a nucleic acid encoding said product of interest randomly integrated into the cellular genome of said CHO cell.

13. The CHO cell of any one of claims 1 to 12, wherein the lactogenic activity of the CHO cell is less than about 50% of the lactogenic activity of a reference cell.

14. 14. The CHO cell of claim 13, wherein the lactogenic activity of the CHO cell is less than about 20% of the lactogenic activity of a reference cell.

15. The CHO cell of claim 13 or 14, wherein the reference cell is a cell containing a wild-type allele of the PKM gene.

16. The CHO cell of any one of claims 1 to 15, wherein the lactogenic activity of the CHO cell is measured on day 14 or 15 of the production phase.

17. 17. The CHO cell of any one of claims 1-16, wherein the CHO cell produces less than about 2.0 g / L of lactate during a production phase.

18. 17. The CHO cell of any one of claims 1-16, wherein the CHO cell produces less than about 2.0 g / L lactate in shake flasks during the production phase.

19. 18. The CHO cells of any one of claims 1-17, wherein the CHO cells produce less than about 2.0 g / L of lactate in a bioreactor during a production phase.

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