Modulating lactogenic activity in mammalian cells

Abstract

To provide a technique for reducing lactate production in cell culture.SOLUTION: The present invention provides a method for reducing or eliminating lactogenic activity in a cell, the method comprising administering to the cell a genetic engineering system, wherein the genetic engineering system knocks down or knocks out expression of a pyruvate kinase muscle (PKM) polypeptide isoform.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Patent Application No. 62 / 649,963, filed on March 29, 2018, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted in ASCII format via EFS - Web, which is hereby incorporated 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 present disclosure relates to mammalian cells that express a product of interest, wherein the cells (e.g., Chinese hamster ovary (CHO) cells) have a controlled lactogenic activity. The present disclosure also relates to methods and compositions for controlling pyruvate kinase muscle (PKM) expression (e.g., PKM - 1 expression) in mammalian cells to reduce or eliminate lactogenic activity in the cells, and to compositions having one or more cells in which lactogenic activity has been reduced or eliminated, and methods of using the same.

Background Art

[0004] 2. Background Chinese hamster ovary (CHO) cells are widely used in the production of therapeutic proteins for clinical use due to their ability to properly fold, assemble, and post-translationally modify proteins. Typically, 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). The accumulation of lactate in the production medium can have an adverse effect on cell growth, viability, and productivity. Such lactogenic behavior (i.e., lactate-producing behavior) of CHO cells during the manufacturing process can cause a decrease in viability and productivity and may change the quality of the therapeutic protein produced.

[0005] Several approaches targeting processing 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) induced by increasing the pH in the medium has been shown to be effective in reducing or removing lactate accumulation in large-scale CHO media (Gagnon et al., 2011, Biotechnol. Bioeng. 108(6):1328-37). However, the former approach is not applicable to all lactogenic CHO cell lines, and in the latter, the large-scale manufacturing process may be complicated, so these approaches have limitations. Furthermore, these approaches do not target the underlying mechanism of 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. Summary 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 that express the product of interest, such as Chinese hamster ovary (CHO) cells, and have a controlled lactogenic activity. The methods and compositions described herein control the lactogenic activity of mammalian cells by reducing or eliminating underlying effects associated with lactogenic activity, such as a decrease in the viability and productivity of mammalian cells, and a change in the quality of the product of interest produced.

[0008] The present disclosure further relates to methods and compositions for reducing or eliminating lactogenic activity in cells by controlling pyruvate kinase muscle (PKM) expression (e.g., PKM-1 expression) in mammalian cells, as well as cells with reduced or eliminated lactogenic activity, and methods of using the same.

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

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

[0011] In certain embodiments, the product of interest includes a protein, such as a recombinant protein. In certain embodiments, the product of interest includes an antibody or an antigen-binding fragment thereof. For example, without limitation, 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 present disclosure relates to a method for reducing or eliminating lactogenic activity in a cell. In certain embodiments, the method includes knocking down or knocking out the expression of a pyruvate kinase muscle (PKM) polypeptide isoform. In certain embodiments, the method includes administering to the cell a gene recombination system that knocks down or knocks out the expression of a pyruvate kinase muscle (PKM) polypeptide isoform. In certain embodiments, the gene recombination 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 includes 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 the 14th or 15th day of the production period. In certain embodiments, the cell produces less than about 1.0 g / L, or less than about 2.0 g / L, of lactate during the production period, such as less than about 1.0 g / L, or less than about 2.0 g / L, of lactate in a shake flask during the production period. In certain embodiments, the cell produces less than about 2.0 g / L of lactate in a bioreactor during the production period.

[0013] In certain non-limiting embodiments, the gene recombination 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 part of the PKM gene, 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, 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 comprise a first gRNA comprising a target sequence complementary to a region within exon 2 of the PKM gene and a second gRNA comprising a targeting domain complementary to a region within exon 12 of the PKM gene. For example, without limitation, the one or more gRNAs comprise sequences selected from the group consisting of SEQ ID NOs: 33-34 and 42-43, and combinations thereof. In certain embodiments, the expression of the PKM polypeptide isoform is knocked out or knocked down, and the lactogenic activity of the cell is removed. In certain embodiments, the PKM polypeptide isoform is the PKM-1 polypeptide isoform, or a combination of the PKM-1 and PKM-2 polypeptide isoforms.

[0014] In certain embodiments, the gene recombination 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 gene recombination system comprises an RNA selected from the group consisting of short hairpin RNA (shRNA), small interfering RNA (siRNA), and microRNA (miRNA), and 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 and the lactogenic activity of the cells is reduced. In certain embodiments, the gene recombination 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 the PKM-2 polypeptide isoform. In certain embodiments, the expression of the PKM-1 and PKM-2 polypeptide isoforms is knocked out or knocked down and the lactogenic activity of the cells is reduced.

[0015] In a further aspect, the present disclosure provides, for example, a method for producing a product of interest from the cells disclosed herein. For example, the method for producing a product of interest includes culturing mammalian cells with reduced or eliminated lactogenic activity to produce the product of interest. In certain embodiments, the present disclosure provides a method for culturing a population of mammalian cells that express a product of interest, wherein the mammalian cells have reduced or eliminated lactogenic activity. In certain embodiments, the reduction or elimination of lactogenic activity is effected by knocking down or knocking out the expression of the pyruvate kinase muscle (PKM) polypeptide isoform in the mammalian cells. In certain embodiments, the PKM polypeptide isoform is the PKM-1 polypeptide isoform. In certain embodiments, the expression of the 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 medium.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0017] 5. Detailed Description Without limitation and for clarity, the detailed description of the subject matter disclosed herein is divided into the following subsections: 5.1 Definitions; 5.2 Control of PKM Expression; 5.3 Cells with Reduced or Removed Lactogenic Activity; 5.4 Cell Culture Methods; 5.5 Products; and 5.6 Exemplary Embodiments.

[0018] 5.1 Definitions The terms used herein generally have their ordinary meaning in the context of the present disclosure and in the specific context in which each term is used, in the art. Specific terms are discussed below or elsewhere in this specification to provide additional guidance for practitioners regarding the compositions and methods of the present disclosure and their preparation and use.

[0019] As used herein, when the terms "comprising" are used in the claims and / or in the specification, the use of the phrase "a" or "an" can mean "one", which is also consistent with the meaning of "one or more", "at least one", and "one or two or more".

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

[0021] As used herein, the term "lactogenic behavior" or "lactogenic activity" means, for example, the lactate-producing activity of a cell by consuming glucose and producing 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 to some extent on the method by which the value is measured or determined, i.e., the limitations of the measuring system. For example, "about" can mean within three or more standard deviations per single measurement in the art. Alternatively, "about" can mean 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, this term can mean within one order of magnitude, preferably within five-fold, and more preferably within two-fold of a value. The terms "cell culture medium" and "medium" typically mean a nutrient solution used to grow mammalian cells that provides at least one component from one or more of the following categories: 1) An energy source, usually in the form of a carbohydrate such as glucose; 2) All essential amino acids, and usually a basic grouping of 20 amino acids + cysteine; 3) Vitamins and / or other organic compounds required at 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 at 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) Proteins and tissue hydrolysates.

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

[0025] "Batch culture" means a culture in which, at the start of the culture process, all components for cell culture (including cells and all culture nutrients) are supplied to the culture bioreactor.

[0026] As used herein, "fed-batch cell culture" means a batch culture in which cells and medium are initially supplied to the culture bioreactor, and additional culture nutrients are supplied continuously or incrementally to the culture during the culture process, and cells and / or product are periodically withdrawn or not withdrawn before the end of the culture.

[0027] In some cases, the "reflux culture solution", also called the continuous culture solution, is a culture solution in which cells are retained in the culture solution, for example, by filtration, encapsulation, anchoring to a microcarrier, etc., and the medium is introduced continuously, stepwise, or intermittently (or any combination thereof) and removed from the culture bioreactor.

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

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

[0030] The term "mammalian host cell" or "mammalian cell" means a cell line derived from a mammal that is capable of growth and survival when placed in either a monolayer culture or a suspension culture in a medium containing appropriate nutrients and growth factors. The growth factors required for a particular cell line can be readily determined experimentally without undue experimentation, as described, for example, in Mammalian Cell Culture (Mather, J.P. ed., Plenum Press, N.Y. 1984), and Barnes and Sato, (1980) Cell, 22:649. Typically, the cells can express and secrete a large amount of a particular protein of interest, such as a glycoprotein, into the medium.Examples of mammalian host cells suitable 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 on March 15, 1989); CHO-K1 (ATCC, CCL-61); simian kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL 1651); human fetal kidney strain (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 cancer cells (HELA, ATCC CCL 2); dog 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 N.Y. Acad. Sci., 383:44-68 1982); MRC 5 cells; FS4 cells; and human hepatoma strain (Hep G2). In certain embodiments, the 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 on March 15, 1989).

[0031] As used within the context of the present disclosure, the term "peptone" refers to a medium supplement that is essentially a 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 acids, heat, or various enzyme preparations.

[0032] The "growth phase" of a cell culture refers to the period (logarithmic phase) during which cells generally divide rapidly and exponentially. During the growth phase, the length of time the cells are maintained can vary based on, for example, the cell type, the cell growth rate, and / or the state of the medium. In certain embodiments, during this period, the cells are cultured for a certain period (usually 1 - 4 days) under conditions such that cell growth is maximized. The determination of the growth cycle of a host cell is determined for a particular host cell and can be arrived at without undue experimentation. "Period and conditions such that cell growth is maximized" etc. means the culture conditions under which cell growth and division are measured to be optimal for a particular cell line. In certain embodiments, during the growth phase, the cells are cultured in a nutrient medium containing the necessary additives under a humidified and controlled atmosphere at generally about 30 - 40 °C so that optimal growth for a particular cell line is achieved. In certain embodiments, the 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 involved. During the transition phase, environmental factors such as the temperature of the cell culture medium and the osmotic pressure of the medium move from growth conditions to production conditions.

[0034] The "production phase" of cell culture means the period during which cell growth stagnates / continues to stagnate. Logarithmic cell growth typically decreases before or during this phase and is taken over by protein production. During the production phase, logarithmic cell growth ends and protein production becomes dominant. During this period, the medium is generally supplemented to support continuous protein production and obtain the desired glycoprotein product. Fed-batch culture and / or perfusion cell culture processes replenish the cell culture medium or supply fresh medium during this period to achieve and / or maintain the desired cell density, viability, and / or titer of the recombinant protein product. The production phase can be carried out on a large scale.

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

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

[0037] The term "protein" is meant to refer to an amino acid sequence that is long enough to produce a higher order tertiary and / or quaternary structure. This is for the purpose of distinguishing it from "peptides" or other low molecular weight agents that do not have such a structure. Typically, the proteins herein have a molecular weight of at least about 15 - 20 kD, preferably at least about 20 kD. Examples of proteins included in the definitions herein include all mammalian proteins, particularly therapeutic and diagnostic proteins such as therapeutic and diagnostic antibodies, and common proteins containing one or more disulfide bonds, including multichain polypeptides containing one or more interchain and / or intrachain disulfide bonds.

[0038] The term "antibody" is used herein in the broadest sense and includes, but is not limited to, various antibody structures such as 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, provided they exhibit the desired antigen binding activity.

[0039] As used herein, an “antibody fragment,” “antigen-binding portion” (or simply “antibody portion”), or “antigen-binding fragment” of an antibody means a molecule other than an intact antibody that comprises a part of an intact antibody that binds 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 (dAb); and multispecific antibodies formed from antibody fragments. A review of certain antibody fragments can be found in Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0040] The term “chimeric” antibody means 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 main 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, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG2 isotype. The heavy-chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The light chains of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0042] As used herein, the term "titer" means the total amount of recombinantly expressed antibody produced by a cell culture divided by a given volume of media. Titers are typically expressed in units of milligrams of antibody per milliliter or liter of media (mg / mL or mg / L). In certain embodiments, titers are expressed in grams of antibody per liter of media (g / L). Titers can be expressed or evaluated in terms of relative measurements, such as the percentage increase in titer as compared to obtaining a protein product under different media 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 base or a 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. Often, nucleic acid molecules are described by their base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. Here, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), such as complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and hybrid polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition to this, the term nucleic acid molecule includes both sense and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring nucleotides or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides that include derivatized sugars or phosphate backbone linkages or chemically modified residues include modified nucleotide bases. The nucleic acid molecule also includes, for example, DNA molecules and RNA molecules suitable as vectors for directly expressing the antibodies of the present disclosure in vitro and / or in vivo in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into a subject to produce an antibody in vivo (see, for example, Stadler et al, Nature Medicine 2017, published online on June 12, 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 is linked.

[0045] The term "hybridoma" means a hybrid cell line produced by the fusion of an immortalized cell line of an immunogen and an antibody-producing cell. This term encompasses the progeny of heterohybrid myeloma fusions, which are the result of fusing human cells with mouse myeloma cell lines and then with plasma cells, commonly known as trioma cell lines. Furthermore, this term is meant to include any immortalized hybrid cell line that produces an antibody, such as a quadroma, etc. See, for example, Milstein et al., Nature, 537:3053 (1983).

[0046] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody derived from a non-human source that is made by or utilizes a human or human cell, or a human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0047] A "humanized" antibody means a chimeric antibody that contains amino acid residues derived from non-human CDRs and amino acid residues derived from human FRs. In certain embodiments, a humanized antibody contains substantially all or nearly all of the CDRs corresponding to the variable domain of a non-human antibody and substantially all or nearly all of the FRs corresponding to the variable domain of a human antibody, in at least one, usually two, variable domains. A humanized antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody, such as a non-human antibody, means the antibody that has been humanized.

[0048] As used herein, the term "hypervariable region" or "HVR" means each of the regions of an antibody variable domain that are hypervariable within the sequence and determine antigen-binding specificity, such as the "complementarity-determining regions" (CDRs). Generally, an antibody contains six CDRs, three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include (a) 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) CDRs present 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)); are included.

[0049] Unless otherwise indicated, CDRs are determined according to Kabat et al. above. One of ordinary skill in the art will understand that the CDR notations can be determined according to Chothia above, MacCallum above, or any other scientifically approved 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" means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible variant antibodies (e.g., those containing naturally occurring mutations or those occurring during the preparation of the monoclonal antibody and present in minor amounts as such), and / or bind to the same epitope. Each monoclonal antibody in the preparation of monoclonal antibodies is directed against a single determinant (epitope) of the antigen, as compared to the preparation of polyclonal antibodies, which typically include different antibodies directed against different determinants (epitopes). Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a substantially homogeneous population of 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 made by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci, and such methods and other exemplary methods of making monoclonal antibodies are described herein.

[0052] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding of the antibody to antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three complementarity determining regions (CDRs). For example, Kindt et al., Kuby Immunology, 6 th、See W.H.Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Further, an antibody that binds to a particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain from an antibody that binds to that antigen. See, for example, 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" means the number of cells in a given volume of medium. In certain embodiments, a high cell density is desirable in terms of resulting in a 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 commercially available suitable probe introduced into the bioreactor itself (or a loop through which the medium and suspended cells pass and return to the bioreactor).

[0054] As used herein, the term "seeding" means adding or plating growing cells to the medium at the start of the production phase. Further, as used herein, the term "seed train" means continuously passaging cells in a volume of medium of about 20 L or less to maintain the cell line.

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

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

[0057] As used herein, "PKM polypeptide" means a polypeptide encoded by the PKM gene. The PKM polypeptide includes the PKM-1 polypeptide isoform and / or the PKM-2 polypeptide isoform.

[0058] 5.2 Control of PKM Expression Glycolysis is the process of glucose metabolism used to generate energy by mammalian cells, e.g., CHO cells. Glycolysis can occur in a high flux state or a low flux state. The cellular response to glucose levels and the switching between these flux states can vary depending on the cell line and the amount and combination of isozymes present in the glycolytic pathway (Mulukutla et al., 2014, PLoS One 9(6):e98756). Under normal culture conditions, like other immortalized cell lines, CHO cells tend to consume glucose and produce lactate by aerobic glycolysis, a process known as the Warburg effect (Warburg, 1956, Science 123(3191):309-14). The accumulation of lactate in the production medium can have an adverse effect on cell growth, viability, and productivity. Therefore, it is possible to intervene in the control of cellular energy flux and metabolism to avoid the undesirable results caused by the accumulation of lactate during the cell culture production period (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] In the final step of the glycolytic process, the conversion of phosphoenolpyruvate (PEP) to pyruvate is mediated by the pyruvate kinase (PK) enzyme. Four different PK isoforms: PK liver (PKL), PK erythrocyte (PKR), PK muscle 1 (PKM-1), and PK muscle 2 (PKM-2) have been identified. The PK enzyme is expressed by two different genes: PKLR and PKM. Alternative exon splicing results in different PK isoforms. The presence of both exons 1 and 2 in the mRNA transcript from the PKLR gene leads to the expression of the PKR protein, while the mRNA transcript starting with exon 2 results in the expression of the PKL protein. Alternative splicing of exon 9 or 10 in the PKM gene transcript results in PKM-1 or PKM-2, respectively. Specifically, PKM-1 contains exon 9 of the PAM gene and excludes exon 10, and PKM-2 contains exon 10 of the PKM gene and excludes exon 9. 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 because of its central role in cancer cell metabolism and tumor growth, which is 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 the tetramerization and hence 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 the tetramerization of PKM-2 (Christofk et al., 2008, Nature 452(7184):181-6; Hitosugi et al., 2009, Sci. Signal 2(97):ra73). However, increased glycolysis promotes the acetylation of PKM-2 at lysine 305 as part of the 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, the PKM-2 dimer has been shown to localize to the cell nucleus, where it acts as a protein kinase using PEP as a phosphate donor, promoting cell proliferation through phosphorylation of STAT3 and activation of MEK5 (Gao et al., 2012, Mol. Cell 45(5):598-609).

[0061] According to one aspect, the present disclosure relates to a method of controlling the lactogenic activity of mammalian cells by controlling PKM expression in the cells, such as PKM polypeptide expression. For example, methods of controlling the lactogenic activity of mammalian cells include, but are not limited to, knocking out or knocking down PKM polypeptide expression in the cells. 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 means removing the expression of a PKM polypeptide, such as a PKM-1 polypeptide and / or a PKM-2 polypeptide, in a cell when compared to a reference cell. As used herein, knocked down expression means reducing the expression of a PKM polypeptide, such as a PKM-1 polypeptide and / or a PKM-2 polypeptide, in a cell when compared to a reference cell.

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

[0063] In certain embodiments, the expression of PKM polypeptides, such as PKM-1 and / or PKM-2, in cells modified to knockdown the expression of PKM polypeptides 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 of reference cells, such as WT CHO cells. In certain embodiments, the expression of PKM-1 polypeptide in cells modified to knockdown the 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 of reference cells, such as WT CHO cells.

[0064] In certain embodiments, the expression of PKM polypeptides, such as PKM-1 and / or PKM-2, in cells modified to knockdown the expression of PKM polypeptides 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 PKM polypeptide expression of reference cells, such as WT CHO cells. In certain embodiments, the expression of PKM-1 polypeptide in cells modified to knockdown the expression of 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 PKM-1 polypeptide expression of reference cells, such as WT CHO cells.

[0065] In certain embodiments, the expression of PKM polypeptide, such as PKM-1 and / or PKM-2, in cells modified to knockdown the expression of 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 of a reference cell, such as a WT CHO cell. In certain embodiments, the expression of PKM polypeptide, such as PKM-1 and / or PKM-2, in cells modified to knockdown the expression of PKM polypeptide is about 40% or less of the PKM polypeptide expression of a reference cell, such as a WT CHO cell. In certain embodiments, the expression of PKM-1 polypeptide in cells modified to knockdown the expression of PKM-1 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-1 polypeptide expression of a reference cell, such as a WT CHO cell.

[0066] In certain embodiments, the expression of PKM polypeptides, such as PKM-1 and / or PKM-2, in cells modified to knockdown the expression of PKM polypeptides 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 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 1% 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 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%, about 5% to about 40% of the PKM-1 polypeptide expression in reference cells, such as WT CHO cells.In certain embodiments, the expression of the PKM-1 polypeptide in cells engineered to knockdown the expression of the PKM-1 polypeptide is from 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 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 1% 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 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%, about 5% to about 40% of the PKM-1 polypeptide expression in a reference cell, such as a WT CHO cell.

[0067] In certain embodiments, the expression of PKM polypeptides, such as PKM-1 and / or PKM-2, in cells modified to knockdown the expression of PKM polypeptides is about 5% to about 40% of the PKM polypeptide expression of reference cells, such as WT CHO cells. In certain embodiments, the expression of PKM-1 polypeptide in cells engineered to knockdown the expression of PKM-1 polypeptide is about 5% to about 40% of the PKM-1 polypeptide expression of 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 containing at least one or both wild-type alleles of the PKM gene) can vary. For example, seed train cells, or low lactate-producing cells, may produce a small amount of PKM-1, while high lactate-producing cells may produce a large amount of PKM-1.

[0068] Alternative splicing of exon 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 human-derived PKM gene. In certain embodiments, the PKM gene can be derived from a non-human, such as a rhesus monkey, dog, cynomolgus monkey, chicken, cow, pig, mouse, Chinese hamster, rat, or rabbit.

[0069] In certain embodiments, the knocked down or knocked out PKM gene can be the 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 from the sequence set forth in SEQ ID NO: 44 by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more.

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

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

[0072] 5.2.1 Method for Controlling PKM Expression In certain embodiments, a gene recombination system is used to control (e.g., knockdown or knockout) the expression of the PKM polypeptide (e.g., PKM-1 expression). Various gene recombination systems known in the art can be used for the methods disclosed herein. Non-limiting examples of such systems include the CRISPR / Cas system, the zinc finger nuclease (ZFN) system, the transcription activator-like effector nuclease (TALEN) system, and the use of 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 bacteria-based genome excision tools such as Cpf-1, can be used in conjunction with the methods disclosed herein.

[0073] Any PKM inhibitor known in the art can be used in conjunction with the methods disclosed herein to control PKM activity, and thus, the lactogenic activity of the cells disclosed herein can be controlled. 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., knockdown or knockout, the expression of the 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, 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% 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 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% of the PKM gene is deleted.

[0075] In certain embodiments, at least one exon of the PKM gene is at least partially deleted. As used herein, "partially deleted" means, 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%, 2% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less, 70% or less, 75% or less, 80% or less, 85% or less, 90% or less, 95% or less, from about 2% to about 90%, from about 10% to about 90%, from about 20% to about 90%, from about 25% to about 90%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, from about 85% to about 90%, from about 2% to about 80%, from about 10% to about 80%, from about 20% to about 80%, from about 30% to about 80%, from about 40% to about 80%, from about 50% to about 80%, from about 60% to about 80%, from about 70% to about 80%, from about 75% to about 80%, from about 2% to about 70%, from about 10% to about 70%, from about 20% to about 70%, from about 30% to about 70%, from about 40% to about 70%, from about 50% to about 70%, from about 60% to about 70%, from about 65% to about 70%, from about 2% to about 60%, from about 10% to about 60%, from about 20% to about 60%, from about 30% to about 60%, from about 40% to about 60%, from about 50% to about 60%, from about 55% to about 60%, from about 2% to about 50%, from about 10% to about 50%, from about 20% to about 50%, from about 30% to about 50%, from about 40% to about 50%, from about 45% to about 50%, from about 2% to about 40%, from about 10% to about 40%, from about 20% to about 40%, from about 30% to about 40%, from about 35% to about 40%, from about 2% to about 30%, from about 10% to about 30%, from about 20% to about 30%, from about 25% to about 30%, from about 2% to about 20%, from about 5% to about 20%, from about 10% to about 20%, from about 15% to about 20%, from about 2% to about 10%, from about 5% to about 10%, or from about 2% to about 5% of the exon region is deleted.For example, exon 9 of the PKM gene can be at least partially deleted, or completely deleted, but is not limited thereto. In certain embodiments, exon 10 of the PKM gene can be at least partially deleted, or completely deleted. In certain embodiments, exons 9 and 10 of the PKM gene can be at least partially deleted, or completely deleted. In certain embodiments, the region encompassing exons 1-12 is at least partially deleted, or completely deleted.

[0076] In certain non-limiting embodiments, the expression of the PKM polypeptide is controlled using the CRISPR / Cas9 system. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein capable of modifying DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains an RNA for guiding the crRNA to the correct portion of the host DNA, together with a region that binds to a tracrRNA (generally in the form of a hairpin loop) used by Cas9 to form an active complex with Cas9), and trans-activating crRNA (tracrRNA, which binds to the crRNA and forms an active complex with Cas9). The terms "guide RNA" and "gRNA" mean any nucleic acid that promotes the specific association (i.e., "targeting") of a nuclease such as Cas9, guided by the RNA, to a target sequence such as an intracellular genomic or episomal sequence. The gRNA can be a single molecule (including a single-stranded RNA molecule, alternatively called a chimera), or modular (usually two or more, typically two, individual RNA molecules, such as crRNA and tracrRNA, that associate with each other by duplex formation). In the CRISPR / Cas9 method, a vector for transfecting mammalian cells can be used. Since the guide RNA (gRNA) is a sequence for identifying and directly binding to the target DNA in the cell, it can be designed for each application. Multiple crRNAs and tracrRNAs can be packaged together to form a single-stranded guide RNA (sgRNA). For transfection into cells, the sgRNA can be ligated with the Cas9 gene and incorporated into a vector.

[0077] In certain embodiments, the CRISPR / Cas9 used to control the 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 the 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 remove or reduce the expression of the PKM1 and / or PKM2 polypeptide. In certain embodiments, the target sequence is the 5' region adjacent to exon 1 of the PKM gene, a region within exon 1, the 5' region adjacent to exon 2 of the PKM gene, a region within exon 2, the 5' region adjacent to exon 9 of the PKM gene, the 3' region adjacent to exon 9 of the PKM gene, the 3' region adjacent to exon 10 of the PKM gene, a region within exon 12, and / or the 3' region adjacent to exon 12 of the PKM gene. For example, the target sequence is selected from, but not limited to, the group consisting of 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 that targets the 5' intron region adjacent to exon 9 of the PKM gene, and a gRNA that targets the 3' intron region adjacent to exon 9 of the PKM gene for generating cells in which PKM-1 is knocked down or knocked out. In certain embodiments, the gRNA that targets 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 that targets 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, without limitation, the CRISPR / Cas9 system of the present disclosure can include, for example, 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 to generate 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, for example, 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 to generate 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 certain embodiments, 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 a 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 additional methods of delivering RNP to cells). In certain embodiments, administration of the CRISPR / Cas9 system to the cell results in knockout or knockdown of the expression of the PKM-1 polypeptide. In certain embodiments, administration of the CRISPR / Cas9 system to the cell results in knockout or knockdown of the expression of both the PKM-1 and PKM-2 polypeptides.

[0081] In certain embodiments, the gene recombination 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. The zinc finger domains can be modified to target specific DNA sequences, thereby enabling zinc finger nucleases to target desired sequences within the genome. The DNA binding domains of individual ZFNs typically contain multiple, individual zinc finger repeats, each of which can recognize multiple base pairs. The most common method for generating new zinc finger domains is to combine small zinc finger "modules" with known specificities. The most common cleavage domain of ZFNs is a non-specific cleavage domain derived from the type II restriction endonuclease FokI. ZFNs regulate protein expression by making double-stranded breaks (DSBs) within 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 base pair deletions or insertions, 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 large segments of the genomic sequence (Lee et al., Genome Res.; 20(1):81-9(2010)). 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 exons 9 and 10 of the PKM gene.

[0082] In certain embodiments, the gene recombination system is a TALEN system for controlling the expression of PKM polypeptide in mammalian cells. TALEN is a restriction enzyme that can be modified to cleave specific sequences of DNA. The TALEN system operates on a principle similar to that of ZFN. TALEN is generated by combining a transcriptional activator-like effector DNA binding domain with a DNA cleavage domain. The transcriptional activator-like effector (TALE) is composed of 33-34 amino acid repeat motifs with two positions that strongly recognize specific nucleotides. By assembling arrays of these TALEs, the TALE DNA binding domain can be modified to bind to a desired DNA sequence, thereby guiding the 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 the PKM polypeptide can be knocked down using an oligonucleotide having a complementary sequence to the 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 the PKM nucleic acid sequence, e.g., the PKM, PKM-1, or PKM-2 nucleic acid sequence, and the homology of that 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 consist of 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. The antisense nucleic acid, shRNA, mRNA, or siRNA molecule can contain DNA, or non-canonical or non-naturally occurring residues, e.g., phosphorothioate residues, including but not limited to this.

[0084] The gene recombination system disclosed in this specification can be delivered to mammalian cells using viral vectors, such as retroviral vectors such as gamma-retroviral vectors, and lentiviral vectors. When the capsid protein is functional for infecting human cells, a combination of a retroviral vector and an appropriate packaging strain is preferred. Various 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). Pseudotyped particles with non-amphotropic particles, such as VSVG, RD114, or GALV envelope, and any others known in the art are also preferred. Possible transduction methods include, for example, co-culturing cells directly with producer cells by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with only the viral supernatant or with a concentrated vector stock solution with or without appropriate growth factors and polycations by the methods of, for example, Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0085] Other transduction 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, for example, 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. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, or herpes virus, 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 for vectors).Retroviral vectors have been specifically developed and are being 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 recombination of mammalian cells disclosed herein. For example, administration of nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 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 247:1465, 1990) can be used to introduce nucleic acid molecules into mammalian cells. Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also potentially be useful for delivering nucleic acid molecules into cells. Transplanting normal genes into infected tissues of a subject can also be accomplished by transferring normal nucleic acids into cell types 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 systemically injecting them.

[0087] 5.3 Cells with reduced or eliminated lactogenic activity In one aspect, the present disclosure relates to one or more cells in which the lactogenic activity has been reduced or eliminated, such as cells or compositions comprising mammalian cells, and methods of using the same. The expression of the PKM polypeptide (e.g., the expression of PKM-1) has been knocked down or knocked out intracellularly, thereby resulting in a reduction or elimination of the lactogenic activity of the cells. Non-limiting examples of cells include CHO cells (e.g., DHFR CHO cells), CHO-K1 (ATCC, CCL-61) of dp12.CHO cells, monkey kidney CV1 transformed by SV40 (e.g., COS-7 ATCC CRL-1651), human fetal kidney cell 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 cancer cells (e.g., HELA, ATCC CCL 2), dog 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 breast cancer (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, human liver cancer cell 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 sequences set forth in SEQ ID NOs: 37 and 38 (see FIG. 4C).For example, the cells of the present disclosure include an allele of the PKM gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39 to 41, or include the nucleotide sequences set forth in SEQ ID NOs: 37 and 38, but are not limited thereto. In certain embodiments, the cells of the present disclosure include at least one allele of the PKM gene comprising a sequence selected from the group consisting of SEQ ID NOs: 39 to 41. In certain embodiments, the cell is a CHO cell. Non-limiting examples of cells with reduced or eliminated lactogenic activity include HET-3, HET-18, KO-2, and KO-15 disclosed herein.

[0088] In certain embodiments, the expression of the PKM polypeptide (e.g., PKM-1 and / or PKM-2) is knocked out, resulting in the elimination of the lactogenic activity of the cell as compared to a reference cell. In certain embodiments, the expression of the PKM polypeptide is knocked out intracellularly, resulting in a decrease in the lactogenic activity of the cell as compared to a reference cell. In certain embodiments, the reference cell is a cell in which the expression of the PKM polypeptide is not regulated. In certain embodiments, the reference cell is a cell having at least one wild-type PKM allele. In certain embodiments, the reference cell is a cell having 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 cell culture period. 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, during the production period, for example, on the 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th day of the production period of the cell culture process, indicated by the lactate concentration in the cell culture medium. In certain embodiments, the lactate concentration is measured on the 6th, 7th, 10th, 11th, 14th, and / or 15th day of the production period. In certain embodiments, the lactate concentration is measured on the 7th, 10th, and / or 14th day of the production period.

[0090] Any method known in the art for measuring the lactogenic activity of cells and / or for measuring lactate production by cells in a medium can be used in conjunction 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 hereby 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) using, for example, 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 cells with reduced lactogenic activity (e.g., by controlling the PKM gene in the cells to knockdown or knockout 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 reference cells. In certain embodiments, the lactogenic activity of cells with reduced lactogenic activity (e.g., by controlling the PKM gene in the cells to knockdown or knockout 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 reference cells. In certain embodiments, the lactogenic activity of the cells is less than about 50% of the lactogenic activity of reference cells when observed, for example, on the 14th or 15th day of the production phase of cell culture. In certain embodiments, the lactogenic activity of the cells is less than about 20% of the lactogenic activity of reference cells when observed, for example, on the 14th or 15th day of the production phase of cell culture. In certain embodiments, the lactogenic activity of the cells is less than about 10% of the lactogenic activity of reference cells when observed, for example, on the 14th or 15th day of the production phase of cell culture. In certain embodiments, the reference cells are cells containing at least one, or both wild-type alleles of the PKM gene.

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

[0093] In certain embodiments, the cells disclosed herein with reduced or eliminated lactogenic activity (e.g., cells generated by knocking out or knocking down the expression of the PKM polypeptide) have a titer comparable to that of cells with normal lactogenic activity (e.g., wild-type cells) and / or a specific production ability. In certain embodiments, the difference in titer and / or specific production ability 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 the cells with normal lactogenic activity (e.g., reference cells). In certain embodiments, the cells with reduced or eliminated lactogenic activity have a higher titer and / or specific production ability than cells with normal lactogenic activity. In certain embodiments, the titer of the 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% higher than the titer of the cells with normal lactogenic activity. In certain embodiments, the titer of the cells with reduced or eliminated lactogenic activity is greater than about 50% higher than the titer of the cells with normal lactogenic activity. In certain embodiments, the specific production ability (Qp) of the 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% higher than the specific production ability of the cells with normal lactogenic activity. In certain embodiments, the specific production ability of the cells with reduced or eliminated lactogenic activity is greater than about 90% higher than the specific production ability of the cells with normal lactogenic activity.

[0094] The lactate concentration in the cell culture medium can be measured by a chemical analyzer or a lactate assay kit. Non-limiting examples of chemical 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 the product of interest are described in Section 5.5. In certain embodiments, the cells disclosed herein can be used to produce a commercially useful amount of the product of interest.

[0096] In certain embodiments, the cells disclosed herein can contain nucleic acids encoding a product of interest. In certain embodiments, the nucleic acids 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 to which additional DNA segments can be ligated. Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome. Certain vectors can replicate themselves within the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). When introduced into a host cell, other vectors (e.g., non-episomal mammalian vectors) are introduced into the genome of the host cell and replicated along with the host genome. Further, certain vectors, expression vectors, can direct the expression of genes to which they are operably linked. Generally, 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 perform equivalent functional roles.

[0097] In certain embodiments, nucleic acids encoding a product of interest can be introduced into the host cells disclosed herein. In certain embodiments, introduction of the nucleic acids into the cells can be carried out by transfection, electroporation, microinjection, infection with a nucleic acid sequence-containing virus or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. In certain embodiments, the host cells are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0098] In certain embodiments, a nucleic acid encoding a product of interest can be randomly integrated into the host cell genome (“random integration” or “RI”). 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 is controlled to knockdown or knockout the 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 the 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 cell that is controlled to knockdown or knockout the expression of a PKM polypeptide (e.g., PKM-1) in a targeted manner. An “integration site” includes a sequence within the host cell genome into which an exogenous nucleotide sequence is inserted. In certain embodiments, the integration site is between two adjacent nucleotides of the host cell genome. In certain embodiments, the integration site includes an extension of a nucleotide sequence. In certain embodiments, the integration site is located within a specific locus of the genome of a TI host cell. In certain embodiments, the 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 subject matter disclosed herein. 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, a nucleic acid encoding a product of interest can be integrated into the host cell genome using transposase-mediated integration. Transposase-mediated integration is disclosed, for example, in Trubitsyna et al., Nucleic Acids Res. 45(10):e89(2017), Li et al., PNAS 110(25):E2279-E2287(2013), and International Publication No. 2004 / 009792, which are all incorporated herein by reference in their entirety.

[0101] In certain embodiments, a nucleic acid encoding a 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 follow random integration. In certain embodiments, random integration can follow targeted integration. For example, without limitation, a nucleic acid encoding a product of interest can be randomly integrated into the genome of a cell engineered to knockdown or knockout the expression of a PKM polypeptide, such as PKM-1, and the same nucleic acid encoding the product of interest can be integrated into the genome of the cell in a targeted manner.

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

[0104] 5.4 Cell culture methods In one aspect, the present disclosure provides a method for producing a target product, which includes culturing the cells disclosed herein. Culture conditions suitable for mammalian cells, which are known in the art, can be used for culturing the cells described herein (J. Immunol. Methods (1983) 56:221-234), or can be easily measured by the person skilled in the art (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B.D., 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), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle Medium (DMEM, Sigma) are exemplary nutrient solutions. Further, 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 No. 4,767,704; No. 4,657,866; No. 4,927,762; No. 5,122,469, or No. 4,560,655; International Publication Nos. 90 / 03430; and 87 / 00195 (the entire disclosures of which are incorporated herein by reference) can be used as the culture medium. Any of these media can be supplemented, as needed, with hormones and / or other growth factors (such as 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 the micromolar range at final concentration), lipids (linolenic acid or other fatty acids) and their suitable monomers, and glucose, or an equivalent energy source. Any other necessary supplements can also be included at appropriate concentrations known to those of skill in the art.

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

[0107] In certain embodiments, the mammalian cells modified to reduce and / or eliminate the expression of the PKM polypeptide are cells that express a recombinant protein. The recombinant protein can be produced by growing cells that express the product of interest under various cell culture conditions. For example, cell culture procedures for large - scale or small - scale protein production can potentially be 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 culture, shake flask culture, or stirred tank bioreactor systems (in the latter two systems, with or without microcarriers), and alternatively can be operated in batch mode, fed - batch mode, or continuous mode.

[0108] In certain embodiments, the cell culture of the present disclosure is carried out in a stirred tank bioreactor system using a fed-batch culture procedure. In the fed-batch culture medium, mammalian host cells and a medium are first supplied to a culture vessel, and additional culture nutrients are continuously or individually increased and supplied to the culture medium during the culture. Before the end of the culture, cells and / or products are periodically collected or not. Examples of the fed-batch culture medium include a semi-continuous fed-batch culture medium in which all the culture medium (including cells and the medium) is periodically removed and replaced with fresh medium. The fed-batch culture medium is distinguished from a simple batch culture medium in which all the components for cell culture (including cells and all culture nutrients) are supplied to the culture vessel at the start of the culture process. The fed-batch culture medium can further be distinguished from a recycle culture as long as the supernatant is not removed from the culture vessel during the process (in a recycle culture, cells are retained in the culture medium, for example, by filtration, encapsulation, or anchoring to a microcarrier, and the medium is continuously or intermittently introduced and removed from the culture vessel).

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

[0110] In certain embodiments, perfusion culture or continuous cell culture conditions are devised to enhance the growth of mammalian cells during the growth phase of the cell culture. During the growth phase, the cells grow under and for a period of conditions maximized for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dO2), etc., are those used with a particular host and will be apparent to those skilled in the art. Generally, either an acid (e.g., CO2) or a base (e.g., Na2CO3 or NaOH) is used to adjust the pH to a level of about 6.5 - 7.5. 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 the production phase or process of the cell culture. Alternatively, as described above, the production phase or process can be continuous with the seeding or growth phase or process.

[0112] In certain embodiments, the culture method described in the present disclosure can further include, for example, recovering a product from the cell culture during the production phase of the cell culture. In certain embodiments, the product produced by the cell culture method of the present disclosure can be recovered from a third bioreactor, such as a production bioreactor. For example, but not limited to, the disclosed method can include recovering the product at the end of the production phase of the cell culture. Alternatively, or additionally, the product can be recovered before the end of the production phase. In certain embodiments, the product can be recovered from the cell culture when a particular cell density is achieved. For example, but not limited to, the cell density can be about 2.0×10 7 cells / mL to about 5.0×10 7 cells / mL.

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

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

[0115] 5.5 Product Using the cells and / or methods of the present disclosure, any product of interest that can be expressed by the cells disclosed herein can be produced. 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, regulatory factors, growth factors, complement system factors, enzymes, coagulation factors, anticoagulation factors, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. The cells and / or methods of the present disclosure are not specific for molecules, such as the antibodies produced.

[0116] In certain embodiments, the methods of the present disclosure can be used in the production of therapeutic and diagnostic antibodies, or antibodies comprising antigen-binding fragments thereof. In certain embodiments, the antibodies produced by the cells and methods of the present disclosure can be, but are not limited to, monospecific antibodies (e.g., antibodies comprising multimers of such pairs consisting of a single-chain heavy chain sequence and a single-chain light chain sequence), multispecific antibodies, and antigen-binding fragments thereof. For example, but not limited to, multispecific antibodies can be bispecific antibodies, bisepitope antibodies, T cell-dependent bispecific antibodies (TDBs), dual action Fab (DAF), or antigen-binding fragments thereof.

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

[0118] Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305:537 (1983)), and "knob-in-hole" recombination (see, e.g., U.S. Patent No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be generated by modifying the electrostatic steering effect for generating antibody Fc heterodimer molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Patent 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 common light chain technology to avoid light chain mispairing problems (see, e.g., WO 98 / 50431); use of "diabody" technology for generating bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and can be generated, for example, by the preparation of trispecific antibodies as described in Tutt et al. J. Immunol. 147:60 (1991).

[0119] Also included herein are modified antibodies having three or more antigen-binding sites, such as "octopus antibodies" or DVD-Ig (see, e.g., WO 2001 / 77342 and WO 2008 / 024715). Another non-limiting example of a multispecific antibody 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. Bispecific antibodies or antigen-binding fragments thereof also include "dual action FAb" or "DAF" (see, e.g., US 2008 / 0069820 and WO 2015 / 095539).

[0120] Multispecific antibodies can be provided in an asymmetric form having domain crossovers in one or more binding arms having the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253), or entire Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, also see Schaefer et al, PNAS, 108(2011)1187-1191, and Klein at al., MAbs 8(2016)1010-20). In certain embodiments, the multispecific antibody comprises a cross Fab fragment. The terms "cross Fab fragment" or "xFab fragment" or "cross-over Fab fragment" refer 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, e.g., WO 2016 / 172485.

[0121] Various further 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, certain types of multispecific antibodies, also included herein, are bispecific antibodies designed to simultaneously bind to surface antigens on target cells, e.g., tumor cells, and an activated invariant component of the T cell receptor (TCR) complex, such as CD3, to retarget T cells and kill the target cells.

[0123] Further non-limiting examples of bispecific antibody formats that can be useful for this purpose include the so-called "BiTE" (bispecific T cell engager) in which two scFv molecules are fused by a flexible linker (see, for example, International Publication Nos. 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 ("TandAb"; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); "DART" (dual affinity retargeting) molecules (Johnson et al., J Mol Biol 399, 436-449 (2010)) based on the diabody format and characterized by a C-terminal disulfide bridge for further stabilization, and so-called triomabs, which are fully hybrid mouse / rat IgG molecules (identified in Seimetz et al., Cancer Treat. Rev. 36, 458-467 (2010)), but are not limited thereto. Specific T cell bispecific antibody formats included herein are described in International Publication Nos. WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0124] 5.5.2 Antibody Fragments In certain embodiments, the antibodies produced by the cells and methods provided herein are antibody fragments. For example, without limitation, antibody fragments are Fab, Fab’, Fab’-SH, or F(ab’)2 fragments, particularly Fab fragments. Papain digestion of intact antibodies produces two identical antigen-binding fragments, so-called “Fab” fragments, each containing a heavy-chain and a light-chain variable domain (VH and VL, respectively), as well as the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). The term “Fab fragment” thus means an antibody fragment that includes 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 having residues added at the carboxy terminus of the CH1 domain that includes one or more cysteines from the antibody hinge region. Fab’-SH is a Fab’ fragment in which the cysteine residue of the constant domain has a thiol group. Pepsin treatment yields an F(ab’)2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. See U.S. Patent No. 5,869,046 for discussion of Fab and F(ab’)2 that include 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 that may be bivalent or bispecific. See, for example, European Patent Application Publication No. 404,097, International Publication No. 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 tetra-bodies are also described in Hudson et al, Nat. Med. 9:129-134 (2003).

[0126] In a further aspect, 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, and the antibody domains and the linker are in the following order from the N-terminus to the 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, the 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 natural disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab fragments will be further stabilized by the formation of interchain disulfide bonds by the insertion of cysteine residues (e.g., positions 44 of the variable heavy chain and 100 of the variable light chain according to Kabat numbering).

[0127] In another aspect, 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 to 25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, and can connect the N-terminus of VH to the C-terminus of VL or vice versa. This protein can retain the specificity of the original antibody despite the removal of the constant regions and the introduction of a linker. For an overview of scFv fragments, see, for example, Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also International Publication No. 93 / 16185, and U.S. Patent Nos. 5,571,894 and 5,587,458.

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

[0129] Antibody fragments can be made by a variety of techniques, including but not limited to proteolysis of intact antibodies.

[0130] 5.5.3 Chimeric and Humanized Antibodies In certain embodiments, the antibodies produced by the cells and methods provided herein are chimeric antibodies. 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 includes 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-switch" antibody in which the class or subclass has been changed from those of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0131] In certain embodiments, the chimeric antibody is a humanized antibody. Generally, when a non-human antibody is humanized, its immunogenicity in humans is reduced while the specificity and affinity of the parent non-human antibody are maintained. Typically, a humanized antibody includes 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 human antibody sequences. A humanized antibody optionally also includes at least a portion of a human constant region. In certain embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived) to, for example, restore or improve antibody specificity or affinity.

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

[0133] Human framework regions for use in humanization include, but are not limited to, the following: 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 specific 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 (somatic mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J Biol. Chem. 271:22611-22618 (1996)).

[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 an immunogen to a transgenic animal modified to produce a complete human antibody or a complete antibody having a human variable region that responds to antigen administration. Such animals typically contain all or part of a human immunoglobulin locus that replaces the endogenous immunoglobulin locus, is present extrachromosomally, or is randomly introduced into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is typically inactive. For an overview 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 K-M MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions of the complete 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 methods based on hybridomas. 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 produced 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, for example, those described 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 Molecule Non-limiting examples of molecules that can be targeted by the cells and antibodies produced by the methods disclosed herein include soluble serum proteins and their receptors, as well as other membrane-bound proteins (such as adhesion factors). In certain embodiments, the cells and antibodies produced by the methods disclosed herein are 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, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1 (EPSELON), 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), 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,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 can bind to them.

[0138] In certain embodiments, the antibodies produced by the cells and methods disclosed herein are 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(TECK), CCL26(eotaxin-3), CCL27(CTACK / ILC), CCL28, CXCLI(GROI), CXCL2(GR02), CXCL3(GR03), CXCL5(ENA-78), CXCL6(GCP-2), CXCL9(MIG), CXCL10(IP10), CXCL11(1-TAC), CXCL12(SDFI), CXCL13, CXCL14, CXCL16, PF4(CXCL4), PPBP(CXCL7), CX3CL1(SCYDI), SCYEI, XCLI(lymphotactin), 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 / SIt can bind to chemokines, chemokine receptors, or chemokine-related proteins selected from the group consisting of TRL22 / 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) produced by the methods disclosed herein can bind 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 β; ANGPTL; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR; ASLG659; ASPHD1 (aspartate β-hydroxylase domain-containing 1; LOC253982); AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF-R (B cell activation 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 (plecktrin); BRCA1; Brevican; C19orf10 (IL27w); C3; C4A; C5; C5R1; CANT1; CASP1; CASP4; CAV1; CCBP2 (D6 / JAB61); CCL1 (1-309); CCL11 (eotaxin); 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 (TECT); CCL26 (eotaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (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; COL1A1; 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 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; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EphB2R; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2; ETBR (endothelin 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; FGF1 (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); FLT1; FOS; FOSL1 (FRA-1); FY (DARC); GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GDNF-Ra1 (GDNF family receptor α1; 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 receptors; HLA-A; HLA-DOB (β subunit of MHC class II 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; IL17B; 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; IL4; 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-related 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 type H 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), type I membrane protein of the leucine-rich repeat (LRR) family); 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 (metallothionein-III); 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 (Purine-activated; Sex receptor P2X ligand-gated ion channel 5); PAP; PARTI; PATE; PAWR; PCA3; 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 proto-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, Semadomain, 7 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, prostate transmembrane epithelial antigen); STEAP2(HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate cancer associated gene 1, Prostate cancer associated protein 1, Six, prostate transmembrane epithelial antigen 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 (seasahomolog 2); TNF; TNF-a; TNFAEP2 (B94); TNFAIP3; TNFRSFIIA; TNFRSF1A; TNFRSF1B; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE); TNFSF12 (AP03L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6 (FasL); TNFSF7 (CD27 ligand); TNFSFS (CD30 ligand); TNFSF9 (4-1BB 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 can bind to 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 β), B29); CD200 members of the ErbB receptor family such as the EGF receptor, HER2, HER3, or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, pl50.95, VLA-4, ICAM-1, VCAM, α4 / β7 integrin, and αv / β3 integrin (including any of these α or β subunits) (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 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; CD proteins such as RANKL, RANK, RSV F protein, protein C, etc.

[0141] In certain embodiments, using the cells and methods provided herein, antibodies that specifically bind to complement protein C5 (multispecific antibodies such as bispecific antibodies) (e.g., anti-C5 agonist antibodies that specifically bind to human C5) can be produced. 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-C5 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 of U.S. Patent Application Publication No. 2016 / 0176954.)

[0142] In certain embodiments, the anti-C5 antibodies each comprise VH and VL sequences QVQLVESGGG LVQPGRSLRL SCAASGFTVH SSYYMAWVRQ APGKGLEWVG AIFTGSGAEY KAEWAKGRVT ISKDTSKNQV VLTMTNMDPV DTATYYCASD AGYDYPTHAM HYWGQGTLVT VSS (SEQ ID NO: 31), 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 as SEQ ID NO: 106 and SEQ ID NO: 111, respectively, in US Patent Application Publication No. 2016 / 0176954. (See Tables 7 and 8 of US Patent Application Publication No. 2016 / 0176954.) In certain embodiments, the anti-C5 antibody is 305L015 (see US Patent Application Publication No. 2016 / 0176954).

[0143] In certain embodiments, the antibodies produced by the methods disclosed herein are capable of binding to OX40 (e.g., anti-OX40 agonist antibodies that specifically bind 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 VH and VL sequences EVQLVQSGAE VKKPGASVKV SCKASGYTFT DSYMSWVRQA PGQGLEWIGD MYPDNGDSSY NQKFRERVTI TRDTSTSTAY LELSSLRSED TAVYYCVLAP RWYFSVWGQG TLVTVSS (SEQ ID NO: 8), respectively. and It includes 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 1, 2, 3, 4, 5, or 6 CDRs selected from: (a) heavy - chain variable region CDR1 comprising the amino acid sequence of NYLIE (SEQ ID NO: 10); (b) heavy - chain variable region CDR2 comprising the amino acid sequence of VINPGSGDTYYSEKFKG (SEQ ID NO: 11); (c) heavy - chain variable region CDR3 comprising the amino acid sequence of DRLDY (SEQ ID NO: 12); (d) light - chain variable region CDR1 comprising the amino acid sequence of HASQDISSYIV (SEQ ID NO: 13); (e) light - chain variable region CDR2 comprising the amino acid sequence of HGTNLED (SEQ ID NO: 14); and (f) 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 1, 2, or 3 CDRs selected from: (a) heavy - chain variable region CDR1 comprising the amino acid sequence of NYLIE (SEQ ID NO: 10); (b) heavy - chain variable region CDR2 comprising the amino acid sequence of VINPGSGDTYYSEKFKG (SEQ ID NO: 11); and (c) 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 1, 2, or 3 CDRs selected from: (a) light - chain variable region CDR1 comprising the amino acid sequence of HASQDISSYIV (SEQ ID NO: 13); (b) light - chain variable region comprising the amino acid sequence of HGTNLED (SEQ ID NO: 14); and (c) light - chain variable region CDR3 comprising the amino acid sequence of VHYAQFPYT (SEQ ID NO: 15). In certain embodiments, the anti - OX40 antibody comprises, respectively, the VH and VL sequences EVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWIGV INPGSGDTYY SEKFKGRVTI TRDTSTSTAY LELSSLRSED TAVYYCARDR LDYWGQGTLV TVSS (SEQ ID NO: 16) 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 the anti-OX40 antibody are disclosed in International Publication No. WO 2015 / 153513, the entire disclosure of which is incorporated herein by reference.

[0146] In certain embodiments, the antibodies produced by the cells and methods disclosed herein can bind to influenza B virus hemagglutinin, i.e., "fluB" (e.g., bind to hemagglutinin from the Yamagata lineage of influenza B virus, bind to hemagglutinin from the Victoria lineage of influenza B virus, bind to hemagglutinin from the ancestral lineage of influenza B virus, or bind to hemagglutinins from the Yamagata lineage, Victoria lineage, and ancestral lineage of influenza B virus in vitro and / or in vivo). Further details regarding the anti-FluB antibodies are disclosed in International Publication No. WO 2015 / 148806, the entire disclosure of which is incorporated herein by reference.

[0147] In certain embodiments, the antibodies produced by the cells and methods disclosed herein can bind 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 β-secretase (BACE1 or BACE2), α-secretase, γ-secretase, tau-secretase, amyloid precursor protein (APP), cell death receptor 6 (DR6), amyloid β peptide, α-synuclein, parkin, huntingtin, p75 NTR, CD40, and caspase-6.

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

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

[0150] In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced by the cells or methods provided herein can bind to CEA and at least one additional target molecule. In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein can bind to a tumor-targeted cytokine and at least one additional target molecule. In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are fused to IL2v (i.e., interleukin 2 variant) and bind to an IL-1 family immune cytokine and at least one additional target molecule. In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are T cell bispecific antibodies (i.e., bispecific T cell engagers or BiTEs).

[0151] In certain embodiments, the multispecific antibodies (such as bispecific antibodies) made according to the methods provided herein are directed against 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-~; 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;It can bind to at least two target molecules selected from 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, TNFα 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 RGMA; CTLA-4 and BTN02; IGF1 and IGF2; IGF 1 / 2 and Erb2B; MAG and RGMA; NgR and RGMA; NogoA and RGMA; OMGp and RGMA; POL-1 and CTLA-4; and RGMA and RGB.

[0152] In certain embodiments, the multispecific antibodies (such as bispecific antibodies) prepared according to the methods provided herein are anti-CEA / anti-CD3 bispecific antibodies. 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 sequences set forth in SEQ ID NOs: 18-21 shown below: TIFF2025090616000001.tif39170TIFF2025090616000002.tif34170TIFF2025090616000003.tif92170TIFF2025090616000004.tif66170

[0153] Further details regarding the anti-CEA / anti-CD3 bispecific antibody are provided in International Publication No. WO 2014 / 121712, which is hereby incorporated by reference in its entirety.

[0154] In certain embodiments, the multispecific antibodies (such as bispecific antibodies) prepared by the cells and methods disclosed herein are anti-VEGF / anti-angiopoietin bispecific antibodies. 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 sequences set forth in SEQ ID NOs: 22-25 shown below: TIFF2025090616000005.tif67170TIFF2025090616000006.tif71170TIFF2025090616000007.tif40170TIFF2025090616000008.tif34170

[0155] In certain embodiments, the multispecific antibodies (such as bispecific antibodies) produced by the methods disclosed herein are anti-Ang2 / anti-VEGF bispecific antibodies. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is RG7221. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody has the CAS number 1448221-05-3.

[0156] Soluble antigens or fragments thereof optionally conjugated to other molecules can be used as immunogens for generating antibodies. In the case of transmembrane molecules such as receptors, these fragments (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules 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 transmembrane molecules. Other antigens and their forms useful for the preparation of antibodies will be apparent to those skilled in the art.

[0157] In certain embodiments, the polypeptides (such as antibodies) produced by the cells and methods disclosed herein can be further conjugated by binding to chemical molecules such as dyes or cytotoxic agents, e.g., chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., enzyme-active toxins derived from microorganisms, fungi, plants or animals, or fragments thereof), or radioisotopes (i.e., radiolabeled conjugates). Immunoconjugates comprising antibodies or bispecific antibodies prepared using the methods described herein can contain a cytotoxic agent conjugated to the constant region of only one heavy chain or only one light chain.

[0158] 5.5.6 Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein, such as those shown 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 the antibody can be prepared by incorporating appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues within the amino acid sequence of the antibody. Deletions, insertions, and substitutions can be combined arbitrarily to arrive at the final construct, provided that the final construct possesses the desired properties, such as antigen binding.

[0159] 5.5.6.1 Substitution, Insertion, and Deletion Variants In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for mutagenesis by substitution include the CDRs and the FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the product screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. **Table 1**

[0160] Amino acids can be classified 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 would involve exchanging a member of one of these classes for a member of another class.

[0162] Certain types of substituted variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant, which is selected for further study, will have a modification (e.g., an improvement) (e.g., an increase in affinity, a reduction in immunogenicity) in certain biological properties compared to the parent antibody and / or will have certain biological properties of the parent antibody that are substantially retained. Exemplary substituted variants are, for example, affinity matured antibodies that can be conveniently generated using affinity maturation techniques based on phage display such as those described herein. Briefly, one or more CDR residues are mutated, the variant antibody is displayed on a phage, and screened for a particular biological activity (e.g., binding affinity).

[0163] Modifications (e.g., substitutions) can be made to the CDRs, for example, to improve the affinity of an antibody. Such modifications can be made in CDR “hot spots,” i.e., residues encoded by codons that mutate at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within residues that contact the antigen, and the resulting VH or VL variants are tested for binding affinity. Affinity maturation by constructing and then reselecting 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 having the desired affinity. Another method for introducing diversity involves a CDR-directed approach in which some CDR residues (e.g., 4-6 residues at a time) are randomized. The 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 can occur within one or more CDRs so long as such modifications do not substantially reduce the ability of the antibody to bind to its antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made within the CDRs. Such modifications can be, for example, outside of the antigen contact residues within the CDRs. In the particular variant VH and VL sequences described above, each CDR is either unmodified or has one, two, or three or fewer amino acid substitutions.

[0165] A useful method for identifying residues or regions of an antibody that can target mutations, as described by Cunningham and Wells (1989) Science, 244:1081-1085, is called "alanine scanning mutagenesis." In this method, one residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced by a neutral or negatively charged amino acid (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, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they have the desired properties.

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

[0167] 5.5.6.2 Glycosylation Variants In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites to the antibody can be conveniently achieved by modifying the amino acid sequence such that one or more glycosylation sites are created or removed.

[0168] When the antibody contains 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 by N-linkage to Asn297 in the CH2 domain of the Fc region. 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, modifications can be made to the oligosaccharides in the antibodies of the present disclosure to produce antibody variants having certain improved properties.

[0169] In one aspect, provided is an antibody variant having an oligosaccharide structure in which a fucosylated oligosaccharide, i.e., a fucose moiety (either directly or indirectly) bound to the Fc region is lacking. Such a non-fucosylated oligosaccharide (also referred to as an "afucosylated" oligosaccharide) is in particular an N-linked oligosaccharide lacking a fucose residue to which a first GlcNAc is bound to the stem of a biantennary oligosaccharide structure. In one aspect, provided is an antibody variant having an increased ratio of non-fucosylated oligosaccharides in the Fc region as compared to the endogenous or parental antibody. For example, the ratio of non-fucosylated 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 proportion of non-fucosylated oligosaccharides is, for example, the (average) amount of oligosaccharides lacking a fucose residue relative to the total of all oligosaccharides bound to Asn297 (e.g., complex, hybrid, and high mannose structures), measured by the MALDI-TOF mass spectrometry method described in WO 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues), but Asn297 can also be located at positions approximately ±3 upstream or downstream of amino acid position 297, i.e., positions 294 to 300, due to minor sequence variations in the antibody. Such antibodies having an increased ratio of non-fucosylated oligosaccharides in the Fc region can have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621.

[0170] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application Publication No. 2003 / 0157108; and International Publication No. 2004 / 056312, particularly Example 11), and knockout cell lines such as FUT8 knockout CHO cells of the α-1,6-fucosyltransferase gene (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 085107), or cells with reduced or eliminated activity of GDP-fucose synthesis or transporter proteins (see, for example, US Patent Application Publication Nos. 2004259150, 2005031613, 2004132140, 2004110282).

[0171] In a further aspect, the antibody variant is provided, for example, with a bisecting oligosaccharide in which the biantennary oligosaccharide attached to the Fc region of the antibody is 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); International Publication No. 99 / 54342; No. 2004 / 065540, No. 2003 / 011878.

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

[0173] 5.5.6.3 Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies presented herein, thereby creating Fc region variants. The Fc region variants may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0174] In certain embodiments, the present disclosure contemplates antibody variants that, while having some effector functions, but not all, are desirable candidates for applications where the in vivo half-life of an antibody is important while certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduction / absence of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. Primary cells for mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI and FcγRII, and FcγRIII FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991), page 464, Table 3. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a molecule of interest are described in U.S. Patent 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 (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)). Effector cells useful in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in an animal model, as disclosed, for example, 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 lacks CDC activity due to its inability to bind C1q. See, for example, C1q and C3c binding ELISAs described in International Publication Nos. WO 2006 / 029879 and WO 2005 / 100402. A CDC assay can be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. 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, for example, Petkova, S B et al., Int’l. Immunol. 18(12):1759-1769 (2006); International Publication No. WO 2013 / 120929A1).

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

[0176] Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 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, for example, substitutions at positions 298, 333, and / or 334 (residues of EU numbering) 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, for example, positions 234 and 235 (residues of EU numbering) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2012 / 130831.) In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in the Fc region derived from the human IgG1 Fc region.

[0179] In some embodiments, changes occur within the Fc region that result in a change (i.e., either an improvement or a decrease) in C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0180] Antibodies that play a role in transferring maternal IgG to the fetus, with an increased half-life and improved binding to the fetal Fc receptor (FcRn) (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 contain an Fc region having one or more substituents that improve the binding of the Fc region to FcRn. Such Fc variants include those having a substitution 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, for example, those having a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall’ Acqua, W.F., 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, for example, Dall’ Acqua, W.F., 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, J.K., 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, J.K., 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, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671) reported and investigated various mutations at residues 248-259, and 301-317, and 376-382, and 424-437.

[0182] Comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as mutations at Fc region positions 253, and / or 310, and / or 435 (EU numbered residues). In certain embodiments, 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 the human IgG1 Fc region. See, for example, Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0183] It comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as mutations at positions 310, and / or 433, and / or 436 (residues of EU numbering) of the Fc region. In certain embodiments, 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 an Fc region derived from the human IgG1 Fc region. (See, e.g., WO 2014 / 177460A1.)

[0184] It comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, such as mutations at positions 252, and / or 254, and / or 256 (residues of EU numbering) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in an Fc region derived from the human IgG1 Fc region. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; No. 5,624,821; and WO 94 / 29351, which relate to other examples of Fc region variants.

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

[0186] 5.5.6.4 Cysteine-Modified Antibody Variants In certain embodiments, it may be desirable to generate cysteine-modified 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 of the antibody. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties, or linker / drug moieties, to generate immunoconjugates further described herein. Cysteine-modified antibodies can be generated as described, for example, in U.S. Patent Nos. 7,521,541, 8,030,930, 7,855,275, 9,000,130, or WO 2016 / 040856.

[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 derivatizing antibodies include, but are not limited to, water - soluble polymers. Non - limiting examples of water - soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, 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 - vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof, among others. Polyethylene glycol propionaldehyde may be advantageous during production due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and when two or more polymers are attached, they can be the same molecule or different molecules. In general, the number and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is to 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 cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, enzymes active toxins from microorganisms, fungi, plants or animals, or fragments thereof), or radioisotopes.

[0189] In one aspect, 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 drugs and linkers, is described in Pharmacol Review 68:3-19 (2016).

[0190] In another aspect, the immunoconjugate includes an antibody that binds to an enzymatically active toxin or fragments thereof, as described herein. These include, but are not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha sarcin, protein from Sina aburagiri, dianthin protein, protein from Arctium lappa (PAPI, PAPII, and PAP-S), trichosanthin inhibitor, curcin, crotin, sabon inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecene.

[0191] In another aspect, the immunoconjugate includes an antibody as described herein that is conjugated to a radioactive atom to form a radio conjugate. Various radioisotopes are available for the production of radio conjugates. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu. When used for detection of radio conjugates, it can include radioactive atoms for scintigraphy studies, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as again iodine-123, 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 prepared 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-azide 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 the conjugation of radioactive nucleotides to antibodies. See International Publication No. 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug intracellularly. For example, an acid-labile linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker (Chari et al, Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0193] The immunoconjugates or ADCs of the present specification are prepared with cross-linking reagent reagents including, but not limited to, commercially available 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., manufactured by Pierce Biotechnology, Inc., Rockford, IL., U.S.A), and such conjugates prepared with these are explicitly contemplated, but not limited to these.

[0194] 5.6 Exemplary Embodiments A. In certain non-limiting embodiments, the subject matter disclosed herein provides mammalian cells in which the expression of pyruvate kinase muscle (PKM) polypeptide isoforms is knocked down or knocked out, and the PKM polypeptide isoforms include the PKM-1 polypeptide isoform, and the lactogenic activity is reduced or removed.

[0195] A1. The aforementioned mammalian cells according to A, wherein the expression of the PKM-2 polypeptide isoform is knocked down or knocked out.

[0196] A2. The aforementioned mammalian cells according to A or A1, wherein the cells are CHO cells.

[0197] A3. The aforementioned mammalian cells according to any one of A to A2, which contain a nucleic acid sequence encoding the product of interest.

[0198] A4. The aforementioned mammalian cells according to A3, wherein the product of interest contains a protein.

[0199] A5. The aforementioned mammalian cells according to A3 or A4, wherein the product of interest contains a recombinant protein.

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

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

[0202] A8. The mammalian cell according to 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 cell genome of the mammalian cell at a targeted position.

[0206] A12. The mammalian cell according to A11, further comprising a nucleic acid encoding the product of interest randomly integrated into the cell 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 the reference cell.

[0208] A14. The mammalian cell according to A13, wherein the lactogenic activity of the mammalian cell is less than about 20% of the lactogenic activity of the reference cell.

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

[0210] A16. The mammalian cells described above in any one of A to A15, wherein the lactogenic activity of the mammalian cells is measured on the 14th or 15th day of the production period.

[0211] A17. The mammalian cells described above in any one of A to A16, wherein the mammalian cells produce less than about 2.0 g / L of lactate during the production period.

[0212] A18. The mammalian cells described above in any one of A to A16, wherein the mammalian cells produce less than about 2.0 g / L of lactate in a shaking flask during the production period.

[0213] A19. The mammalian cells described above in any one of A to A16, wherein the mammalian cells produce less than about 2.0 g / L of lactate in a bioreactor during the production period.

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

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

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

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

[0218] E1. The method according to E, wherein the gene recombination 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 according to E or E1, wherein the gene recombination system is a CRISPR / Cas9 system.

[0220] E3. The CRISPR / Cas9 system is (a) a Cas9 molecule, and (b) one or more guide RNAs (gRNAs) comprising a targeting sequence complementary to a target sequence within the PKM gene The method according to E2.

[0221] E4. The method according to E3, wherein the target sequence is selected from the group consisting of a part 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 method according to any one of E3 - E4, wherein the one or more gRNAs comprise a sequence selected from the group consisting of SEQ ID NOs: 33 - 34, and 42 - 43, and combinations thereof.

[0223] E6. The above-mentioned method according to E3 or E4, wherein 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 above-mentioned method according to any one of E3 - E6, wherein one or more gRNAs comprise a sequence selected from the group consisting of SEQ ID NOs: 33 - 34, and combinations thereof.

[0225] E8. The above-mentioned method according to E3 or E4, wherein 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 above-mentioned method according to any one of E3 - E5 and E8, wherein one or more gRNAs comprise a sequence selected from the group consisting of SEQ ID NOs: 42 - 43, and combinations thereof.

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

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

[0229] E12. The above-mentioned method according to E10 or E11, wherein the lactogenic activity of the cell is less than about 50% of the lactogenic activity of the reference cell.

[0230] The method according to any one of E10 or E11, wherein the lactogenic activity of the cell is less than about 20% of the lactogenic activity of the reference cell.

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

[0232] E15. The method according to any one of D and E to E14, wherein the cell produces less than about 2.0 g / L of lactate during the production period.

[0233] E16. The method according to any one of D and E to E14, wherein the cell produces less than about 2.0 g / L of lactate in a shake flask during the production period.

[0234] E17. The method according to any one of D and E to E14, wherein the cell produces less than about 2.0 g / L of lactate in a bioreactor during the production period.

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

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

[0237] E20. The method according to any one of D and E to E19, wherein the PKM polypeptide isoform is the PKM-1 polypeptide isoform and the PKM-2 polypeptide isoform.

[0238] E21. The method according to E, wherein the gene recombination system comprises an 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 part of the mRNA expressed by the PKM gene.

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

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

[0241] E24. The method according to any one of E21 to E23, wherein the gene recombination 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 part of the mRNA expressed by the PKM gene encoding the PKM-2 polypeptide isoform.

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

[0243] E26. The method according to E, wherein the gene recombination system is a zinc finger nuclease (ZFN) system or a transcription activator-like effector nuclease (TALEN) system.

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

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

[0246] E29. The method according to D and E to 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 according to E30 above, wherein the nucleic acid sequence is integrated into the cellular genome of the cell at the targeted position.

[0249] E32. The method according to any one of E26 - E31 above, 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 according to E26 - E31 above, wherein the product of interest comprises a protein.

[0251] E34. The method according to E33 above, wherein the product of interest comprises a recombinant protein.

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

[0253] E36. The method according to E35 above, wherein the antibody is a bispecific antibody or an antigen - binding fragment thereof.

[0254] E37. The method according to E35 above, 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 method according to any one of E35 - E37 above, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0256] E39. The method according to any one of E35 - E38 above, wherein the antibody is a monoclonal antibody.

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

[0258] G. In certain non-limiting embodiments, the subject matter disclosed herein provides a method of culturing a population of mammalian cells that express 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 knockout or knockdown of the expression of the pyruvate kinase muscle (PKM) polypeptide isoform in mammalian cells.

[0260] G2. The method according to G1, wherein the PKM polypeptide isoform is the PKM-1 polypeptide isoform.

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

[0262] G4. The method according to any one of F and G to 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 according to any one of F and G to 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 to G5, wherein the lactogenic activity of the mammalian cells is measured on the 14th or 15th day of the production period.

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

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

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

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

[0269] G11. The aforementioned method according to any one of F and G - 10, wherein the mammalian cells are CHO cells.

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

[0271] G13. The aforementioned method according to G12, wherein the nucleic acid sequence is integrated into the cell genome of mammalian cells at a targeted position.

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

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

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

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

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

[0277] G19. The aforementioned method according to 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 aforementioned 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 aforementioned method according to any one of G17 to G20, wherein the antibody is a monoclonal antibody.

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

Examples

[0281] The following examples merely illustrate the subject matter disclosed herein and should not be regarded as limiting in any way.

[0282] Example 1: PKM-1 expression drives lactogenic behavior in CHO cell lines and causes low viability and productivity In 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 reduced differently depending on the cell line, through optimization of nutrient supply or culture pH. By analyzing various proteins involved in the glycolytic pathway, a direct correlation between the pyruvate kinase muscle-1 (PKM-1) isoform and lactogenic behavior was revealed.

[0283] CRISPR / Cas9 was used for targeted deletion of exon-9 to knock out PKM-1 expression. By knocking out PKM-1 expression, the lactogenic behavior in the two selected cell lines completely disappeared, eliminating the need for mitigation methods. When one cell line was identified, the expression of both the PKM-1 and PKM-2 genes completely disappeared without any adverse effects on growth and viability. Further analysis of the parental and CHO cell lines revealed that they all expressed the PKL and PKR versions of pyruvate kinase, which, although not bound by theory, enabled the cells to be resistant to the complete absence of PKM expression. An exclusive mitigation method used to control the lactogenic behavior of these cell lines shifted the metabolic pathway and altered the pattern of PKM-1 transcription and expression to prevent or delay lactogenic behavior. The PKM gene was completely knocked out to analyze the behavior of the obtained cell lines in the culture medium and within the bioreactor.

[0284] In summary, the present disclosure depicts a direct correlation between lactogenic behavior and a high amount of PKM-1 in the production culture medium. Furthermore, by removing PKM-1 expression, the lactogenic behavior in the selected CHO cell lines was tolerated and reversed. Therefore, a permanent deficiency of PKM-1, or the entire PKM gene, regarding this problem in CHO cells can be beneficial in reducing the occurrence of lactogenic behavior during production within the bioreactor.

[0285] Materials and Methods Cell Lines Cell lines secreting recombinant monoclonal antibody mAb-1 or mAb-2 were extracted from CHO-K1 host using the glutamine synthetase (GS) selection marker. The seed train was maintained in a proprietary DMEM / F12-based medium containing methionine sulfoximine (MSX) as the selection agent at an osmotic rate of 150 rpm, 37 °C, and 5% CO2. The cells were passaged every 3 or 4 days.

[0286] Manufacture of 2L bioreactor The manufacture of 2L bioreactors was carried out in a glass stirred tank bioreactor (Applikon, Foster City, CA) with a Finesse controller (Applikon, Foster City, CA). In all production media, a single seeding train stage (N - 1) was utilized within the bioreactor for 3 days, followed by seeding of a 14 - day continuous production stage (N). The N - 1 seeding medium was seeded at an operating volume of 1.5 - 1.7L 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, the cells were transferred and seeded into the production (N) bioreactor at a volume of 1.4 - 1.7L at a target PCV of 0.25%. All bioreactors were operated at 37°C with a temperature shift to 35°C over 72 hours. CO2 was used as the acid control and 1M sodium carbonate as the base control to operate the culture broth at a deadband of 0.03 and pH 7.00. The only exception to this pH operation was the pH level of mAb - 2, which was operated at the same deadband of 0.03 and a constant pH of 6.80. The combination of air and oxygen gas flow was used to maintain DO constant, and all cultures were set to be operated at 350 rpm and 30% DO. 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 broth was 20%. For the improved feed process, for each feed, the feed occurred at 15% of the total volume of the culture broth on day 3 (72 hours) and day 6 (144 hours).

[0287] AMBR15 operation The production culture medium in the AMBR15 (Sartorius, Goettingen, Germany) system was operated at set values of a temperature of 37°C, a DO of 30%, a pH of 7.0, and a stirring speed of 1400 rpm. The N-1 seeding train was carried out for 4 days, and then the production (N) stage was seeded at a temperature of 37°C, a DO of 30%, a pH of 7.0, and a stirring speed of 1400 rpm, with a total volume of 13 mL and a seeding density of 1 million cells / mL. For these culture media, a scale-down process of a 2L bioreactor was carried out.

[0288] Offline sample analysis Supernatant and cell pellet samples were collected to be passed through assays or Western blotting analysis. For viable cell concentration (VCC) and viability, Vi-Cell XR (Beckman Coulter) was used, and for pO2, pH, pCO2, Na + , glucose, and lactate, Bioprofile 400 (Nova Biomedical) was used to analyze the samples. All samples from the 2L and AMBR bioreactors were analyzed with the BioProfile 400 for several minutes after minimizing gas generation by sampling. The same Vi-Cell XR, BioProfile 400, and osmometer (Model 2020, Advanced Instruments) were used for all samples to remove variability between instruments. High performance liquid chromatography (HPLC) equipped with a Protein A column was used to measure antibody titers. A quality assay of the antibody product was carried out using cell culture supernatant samples purified by a PhyTip (PhyNexus, San Jose, CA) Protein A column. The antibody molecular size distribution was analyzed by size exclusion chromatography (SEC). Imaging capillary isoelectric focusing (icIEF) was used to measure the protein charge heterogeneity, and all charge heterogeneous samples were pretreated with carboxypeptidase B. A quality assay for all protein products was developed in-house and the detailed procedure was published (Hopp et al., 2009, Biotechnol. Prog. 25(5):1427-32).

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

[0290] PKM-1 knockout To knockout PKM-1, guide RNAs (gRNAs) targeting both the 5’ and 3’ intron regions adjacent to exon 9 of the PKM gene were cloned into a gRNA expression vector manufactured by Genentech. This construct was co-transfected into mAb-2 expressing cells together with a Cas9 expression plasmid. The transfected cells were single cloned cells, and the deletion of exon 9 was confirmed by genomic DNA PCR. The following gRNA oligos were most effective for the deletion of exon-9 (PKM-1), and a pool targeted by 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) Sequences of genomic DNA PCR primers: 5’-primer: CCAGATTTGGTGAGGACGAT (SEQ ID NO: 35) 3’-primer: AGCTGTGTTGTGAGGCATTG (SEQ ID NO: 36)

[0291] Results The increase in the amount of PKM-1 during production correlates with the lactogenic behavior of CHO cells Using the culture seed train of the cell line expressing mAb-1, the production culture solution was prepared in a bioreactor using standard or enhanced feeding methods. Standard feeding caused lactogenic behavior in the mAb-1 cell line, while the enhanced feeding method reduced lactogenic behavior. Under high lactate conditions, a decrease in cell viability was caused in the production culture solution after 10 days (Figure 1A), and the titer on the 14th day decreased compared to the low lactate conditions (Figure 1B). Although both culture conditions had a considerable amount of lactate until the 7th day, the high lactate condition showed a significant increase in lactate accumulation in the production culture solution from the 7th day, reaching up to 15 g / L above on the 14th day. Regarding the low lactate condition, a delay of 3 - 4 days was observed before the amount of lactate tended to increase (from the 12th day to the 14th day), and it only reached 8 g / L by the 14th day (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 the glycolytic pathway. Under high lactate conditions, since cell viability decreased rapidly after 10 days, this set of samples at 0, 2, 7, and 10 days were compared with samples at 0, 7, 10, and 14 days under low lactate conditions (Figure 1D - 1F). Among all the different proteins analyzed (data not shown), only the amount of PKM-1 showed a direct correlation with the lactogenic behavior of the mAb-1 expressing cell line. Figures 1D - 1F show the amounts of PKM-1 and PKM-2 proteins in the cell pellet. The expression level of PKM-1 in the high lactate samples began to increase divergently after 7 days during production culture compared to the low lactate samples. Interestingly, even under low lactate conditions, the increase in the amount of PKM-1 towards the end of the production culture (Figure 1E, 10th day - 14th day) showed a trend along with the high lactate amount in the culture (Figure 1C), so the increase in the amount of PKM-1 showed a close trend with lactate accumulation. However, the amount of PKM-2 was inversely correlated with the lactogenic behavior in the mAb-1 expressing cell line (Figure 1F).

[0293] Similar bioreactor experiments were performed on the mAb-2 expressing cell line. Such cell lines were cultured in production media having conditions that promoted lactogenic or non-lactogenic behavior. Up to days 13 and 14 of the production culture, the cell viability of the mAb-2 cell line was equivalent between the high lactate and low lactate conditions (Figure 2A). This resulted in a ~25% titer decrease in the high lactate condition relative to the low lactate condition (Figure 2B). Throughout the production process, the accumulation of lactate in the media was low in the low lactate control group of the mAb-2 cell line experiment. However, under the high lactate condition, the amount of lactate began to diverge from day 7 and reached up to 15 g / L by day 14 of the 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 the amount of PKM-1 was closely correlated with the increase in the 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 Figure 2D (bottom) and Figure 2F. Taken together, these data suggest a direct correlation between the lactogenic behavior and the amount of PKM-1 in cells expressing mAb-1 and mAb-2 in these cells.

[0294] The PKL / R protein is expressed in CHO cells but is not correlated with lactogenic behavior. To evaluate whether other forms of PK (other than PKM) are expressed in the proprietary CHO host cells, the expression of PKL and PKR (PKL / R) proteins was analyzed in two different CHO host cell lines (CHO-K1 and DHFR- / -), as well as in a human (HEK293) cell line. Both CHO cell lines, and 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 hosts. Next, it was evaluated whether the expression of PKL / R was differentially regulated when comparing high / low lactate conditions in mAb-1 and / or mAb-2 expressing cells. The expression level of PKL / R was equivalent under high / low lactate conditions in both the mAb-1 and mAb-2 cell lines (when the amount of protein was normalized against actin as an internal control), indicating no correlation with lactogenic behavior in these cell lines (Figures 3B - 3D). The small amount of PKL / R in the mAb-2 high lactate samples on days 7 and 10 in Figure 3C was due to low overall protein loading, as reflected by the low amount of actin (as a loading control) in the same lane. In addition to PK isoforms, the expression of several different proteins involved in cell growth signaling and the glycolytic pathway was also analyzed. However, no clear correlation was observed between protein expression and the lactogenic behavior observed in the 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 suggests that these cell lines may be able to tolerate targeted deletion of one or more isoforms of PK. Based on the direct correlation between the lactogenic behavior of the mAb-1 and mAb-2 cell lines and the amount of PKM-1 observed during production, this enzyme emerged as a good candidate for targeted deletion.

[0295] Targeted deletion of exon-9 in the PKM gene mediated by CRISPR / Cas9 The PKM gene is responsible for the expression of both PKM-1 and PKM-2 enzymes, which differ only in that they alternatively contain exon 9 or exon 10, respectively. Since the selected CHO cell line was not sequenced or annotated, information regarding the PKM genotype, gene copy number, or sequence was not available. Therefore, to eliminate the expression of the PKM-1 gene, a guide RNA construct targeting the intron region adjacent to exon 9 was designed using CHO sequences available in the public database (Kent et al. 2002, Genome Res. 12(6):996-1006). 5’ screening PCR primers were designed to match the intron region between exons 8 and 9, and 3’ screening PCR primers were designed for the corresponding exon 10 (Figure 4A). Based on the amplification and sequencing of PKM cDNA from the mRNA of the selected CHO host, exon sequence information was obtained. The mAb-2 expressing cell line was transfected with Cas9 and various gRNA constructs. After the initial screening, the pool showing the most efficient targeting of exon 9 was passed through single cell cloning. Twenty cell lines from this pool were screened for exon-9 target deletion, and two heterozygous (HET-3 and HET-18), as well as two knockout (KO-2 and KO-15) cell lines, were identified for which the deletion of the targeted allele was completely confirmed by PCR and sequencing (Figures 4B and 4C). The cultures of these cell lines were then expanded for further evaluation in a bioreactor. Sequencing of the targeted allele confirmed the deletion of exon 9 in all of the selected cell lines (Figure 4C). Three cell lines (KO-2, HET-3, and HET-18) had an 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 and partially replaced with an unrelated insertion in the KO-15 cell line (Figure 4C, and data not shown).Defects in this region can affect the integrity of the intron between exons 8 and 9. Regardless, according to the sequencing analysis, exon 9 was completely absent in all selected cell lines.

[0296] By blocking or reducing PKM-1 expression, the lactogenic behavior in the mAb-2 cell line was avoided or reduced, respectively. Using all four mAb-2 cell lines with exon-9 deletion in some (HET) or all (KO) alleles, and the WT mAb-2 strain as a source, a 14-day production culture in an AMBR bioreactor was set up. The growth and viability of PKM-1 KO, HET, and WT cell lines are shown in Figures 5A and 5B. The PKM-1 HET or mAb-2 strain had relatively the same titer and specific production ability as the WT mAb-2 cell line (Figures 5C and 5D). PKM-1 KO (KO-2 and KO-15), and one of the HET mAb-2 cell lines (HET-3) had very little lactate amount when cultured under control (lactogenic) conditions compared to the WT mAb-2 cell line (Figure 5E). On the other hand, the HET-18 strain showed similar lactogenic behavior compared to WT mAb-2 until day 10. After 10 days, the accumulation rate of lactate in the WT mAb-2 strain increased and became approximately twice that of the HET-18 strain by day 14 (Figure 5E).

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

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

[0299] The quality attributes of the product, such as large molecular weight (aggregates) and charge variant species, were investigated among WT and PKM-1 KO and HET mAb2-expressing cell lines. Although no significant difference in the degree of product aggregation was observed among the WT and PKM-1 KO and HET cell lines (Figure 5G), some changes were observed in the mAb-2 product charge variant (Figure 5H) profile. Since there was no clear trend or correlation between the amount of lactate and the quality attributes of the product (Figure 5G and 5H), the observed differences may be due to normal variations that can occur during cell line derivation. Furthermore, the media and feeds used in these experiments were optimized for the WT mAb-2 cell line rather than the PKM-1 KO or HET cell lines. Since changing the amount of PKM may affect the overall metabolic profile of these cell lines, fine-tuned media and feeds may be required to investigate the full effects and impacts on the quality attributes of the product.

[0300] Since it is important to analyze the behavior of all cell lines after cell storage and thawing, PKM-1 KO and HET mAb-2 cell lines were stored and then thawed and maintained in the medium for 2.5 weeks together with the WT cell line as a control. Next, based on these cells, 4 sets of production media were set up for each cell line to evaluate the performance after thawing. The data showed that the growth, viability, titer, specific productivity, and lactogenic profile of the PKM-1 KO and HET cell lines were equivalent before and after cell storage (Figures 5A - 5D and 6A - 6D). As previously observed (data not shown), fresh, thawed WT mAb-2 cells had lower lactogenicity (10 g / L, Figure 6E) compared to old cells (15 g / L, Figure 5E). However, since 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), this behavior was unique to the WT mAb-2 cell line. For all cell lines, it was observed that lactate accumulated gradually in the medium until day 7 of the production culture, and then while the PKM-1 KO and HET-3 cell lines consumed lactate, a lactate disappearance profile was observed in both the WT and HET-18 cell lines (Figure 6E). Note that all young cell lines (after thawing) had a relatively low acidic peak rate compared to old cell lines (Figure 5G) (Figure 6G), which is probably a feature related to cell age, but there was no significant difference 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] Discussion The lactogenic behavior in CHO cell culture medium can cause acidification of the culture medium and high osmolarity as a result of adding bases to regulate the pH of the culture medium, viability that negatively affects, VCC, and the final productivity titer (Li et al., 2010, MAbs 2(5):466 - 79). Lactate production occurs through aerobic glycolysis, a behavior generally known as the Warburg effect (Warburg, 1956, Science 123(3191):309 - 14), and is observed in many cultured cells and cancer cells. These cells utilize the glycolysis process to produce energy and control this process according to various 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 behaviors can be controlled by process modification (Luo et al., 2012, Biotechnol.Bioeng.109(1):146 - 56; Gagnon et al., 2011, Biotechnol.Bioeng.108(6):1328 - 37), these approaches did not clarify the root cause of the observed behavior. To better understand the lactogenic behavior in CHO hosts, two lactogenic cell lines expressing mAb - 1 or mAb - 2 were identified, whose lactogenic behavior can be adjusted by changing either the feeding method or the pH of the culture medium. To depict the possible correlation with lactogenic behavior, these independently derived cell lines were used as tools in analyzing a panel of proteins and enzymes involved in cell growth signaling or the glycolytic pathway. These findings revealed a direct correlation between PKM - 1 expression and lactogenic behavior in both the mAb - 1 and mAb - 2 cell lines (Figures 1D - 1F and 2D - 2F).

[0302] The amount of PKM-1 detected in both the mAb-1 and mAb-2 production cultures was low at the initial time points during production and increased in a manner correlated with the increase in the amount of lactate in the culture. The amount of PKM-2 was inversely correlated with the lactogenic behavior in these cell lines (Figs. 1D–1F and 2D–2F). This may be due to the function of proteins involved in the alternative splicing of PKM-1 or PKM-2 transcripts from the PKM gene (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). Among the two isoforms of the PKM gene, PKM-2 has been reported to function as a central switch for altering metabolic pathways, allowing cancer cells to survive and proliferate under physiologically unfavorable conditions such as within the tumor microenvironment (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 when expressed. Without being bound by theory, the correlation between the amount of PKM-1 in the production culture and the rate of lactogenic behavior may be due to the uncontrolled conversion of PEP to pyruvate by this enzyme and the subsequent conversion of pyruvate to lactate mediated by LDH.

[0303] CRISPR mediated exon-9 deletion and thus 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). Two PKM-1 HET mAb-2 cell lines showed different behaviors. 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 was also observed between the lactogenic behavior of these PKM-1 HET cell lines and the higher amount of PKM-1 in these cells (Figure 5F). The difference in the amount of PKM-1 observed in HET-18 compared to the HET-3 cell line could be due to different 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) that cannot express PKM-2 protein was identified. 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). Since the CHO cell line also expresses PKL / R (Figure 3A), the KO-15 cell line can be resistant to the loss of both PKM-1 and PKM-2 enzymes, thereby compensating for the lack of PKM expression. The 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 lack or decrease of PKM-1 expression resulted in lower lactate production in all PKM-1 KO or HET cell lines without affecting the titer or specific productivity, respectively (Figures 5C-5D and 6C-6D). The absence or attenuation of lactogenic behavior in these cell lines was reproducible and consistent among young cell ages after thawing and all replicates (Figures 5A-5E and 6A-6E).

[0305] Controlling lactogenic behavior during production culture is important for obtaining optimal titers and product quality from manufacturing implementation. Therefore, knocking out PKM-1 expression from the host can be advantageous in dealing with the lactogenic behavior of CHO cells. Since the PKL / R enzyme 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 PKM-1 or the entire PKM gene due to the deficiency. Knocking out the entire PKM gene can be tolerated in the CHO host, and the PKM KO CHO host can express the antibody or product of interest with titers and product quality corresponding to those of the WT host. These PKM KO hosts can reduce lactogenic behavior and have a better growth profile 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 gene or the PKM-1 gene was knocked out in the CHO-K1M cell line to generate one PKM KO host cell line and four PKM-1 KO host cell lines. By targeted integration, the transgene encoding mAb-3 was introduced into the WT CHO-K1M, PKM KO, and PKM-1 KO host cell lines 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 carried out as disclosed in Example 1, except that the gRNAs for generating the PKM KO host were different and are as shown below. The gRNAs used to generate the PKM-1 KO host cells were the same as those used in Example 1. The gRNAs for generating the PKM KO host cells had the following sequences: 5’-gRNA: CCCATCACGGCCCGCAACAC (SEQ ID NO: 42, targeting 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, the MAb-3 producing pools derived from PKM and PKM-1 KO host cell lines had Qp and titers equivalent to, or higher than, those of the WT pool, but had low growth (represented by integrated viable cell concentration (IVCC)) (Figure 7). In shake flask production, the 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 of lactate by day 15 of production in shake flasks. Lactate concentrations of 1 - 2.5 g / L are considered high during shake flask production. In contrast, by day 15, the PKM-1 KO host cells produced very little lactate, the PKM KO host cells produced less than 0.2 g / L of lactate, and knocking out either PKM-1, or both PKM-1 and PKM-2, resulted in a greater than 80% reduction in lactate production.

[0309] In shake flask production, the MAb-3 producing pool 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, the accumulation of pyruvate in the WT host cells may result in the accumulation of lactate and increased production of alanine (Figure 10). The PKM-1 KO host cell line accumulated TCA cycle products such as oxaloacetate (resulting in the accumulation of aspartate and asparagine (Figure 9D)), and α-ketoglutarate (a-KG) (resulting in the accumulation of glutamate, arginine, and glutamine (Figure 9E)). Without being limited to a particular theory, these results suggest that PKM-1 and PKM-2 can function to control the amount of pyruvate products 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 media and conditions used to culture the PKM-1 and PKM KO cells can be adjusted to supplement the amount of glucose consumed and / or the amount of amino acids consumed or synthesized.

[0310] The MAb-3 producing pool derived from the PKM / PKM1 KO host cell lines had different 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 are likely not to affect the activity of the produced antibody.

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

Claims

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

3. The mammalian cell of claim 1 or 2, wherein the cell is a CHO cell.

4. A mammalian cell according to any one of claims 1 to 3, comprising a nucleic acid sequence encoding a product of interest.

5. The mammalian cell of claim 4 , wherein the product of interest comprises a protein.

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

7. The mammalian cell of any one of claims 4 to 6, wherein the product of interest comprises an antibody or an antigen-binding fragment thereof.

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

9. The mammalian cell of claim 7 , wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

10. The mammalian cell of any one of claims 7 to 9, wherein the antibody comprises a chimeric antibody, a human antibody, or a humanized antibody.

11. The mammalian cell of any one of claims 7 to 10, wherein the antibody comprises a monoclonal antibody.

12. The mammalian cell according to any one of claims 4 to 11, wherein the nucleic acid sequence is integrated into the cellular genome of the mammalian cell at a targeted location.

13. 13. The mammalian cell of claim 12, further comprising a nucleic acid encoding the desired product randomly integrated into the cellular genome of the mammalian cell.

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

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

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

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

18. 18. The mammalian cell of any one of claims 1 to 17, wherein the mammalian cell produces less than about 2.0 g / L of lactate during the production phase.

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

20. 18. The mammalian cell of any one of claims 1 to 17, wherein the mammalian cell produces less than about 2.0 g / L of lactate in a bioreactor during a production phase.

21. 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 sequence set forth in SEQ ID NOs: 37 and 38.

22. A composition comprising a mammalian cell according to any one of claims 1 to 21.

23. A method of reducing or eliminating lactogenic activity in a cell comprising knocking down or knocking out expression of a pyruvate kinase muscle (PKM) polypeptide isoform.

24. 1. A method for 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.

25. 25. The method of claim 24, 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.

26. The method of claim 24 or 25, wherein the genetic modification system is a CRISPR / Cas9 system.

27. The CRISPR / Cas9 system (a) a Cas9 molecule; and (b) one or more guide RNAs (gRNAs) comprising a targeting sequence complementary to a target sequence in the PKM gene; 27. The method of claim 26, comprising:

28. 28. The method of claim 27, wherein 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.

29. The one or more gRNAs (i) (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; or (ii) (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.

29. The method of claim 27 or 28, comprising:

30. 30. The method of any one of claims 27 to 29, wherein the one or more gRNAs comprise a sequence selected from the group consisting of SEQ ID NOs: 33-34 and 42-43, and combinations thereof.

31. 31. The method of any one of claims 23 to 30, wherein expression of the PKM polypeptide isoform is knocked out and lactogenic activity of the cell is eliminated or reduced compared to lactogenic activity of a reference cell.

32. 31. The method of any one of claims 23 to 30, wherein expression of the PKM polypeptide isoform is knocked down and lactogenic activity of the cell is reduced compared to lactogenic activity of a reference cell.

33. 33. The method of claim 31 or 32, wherein the lactogenic activity of the cells is less than about 50% of the lactogenic activity of the reference cells.

34. 33. The method of claim 31 or 32, wherein the lactogenic activity of the cells is less than about 20% of the lactogenic activity of the reference cells.

35. The method of any one of claims 23 to 34, wherein the lactogenic activity of the cells is measured on day 14 or 15 of the production phase.

36. 36. The method of any one of claims 23 to 35, wherein the cells produce less than about 2.0 g / L lactate during the production phase.

37. 36. The method of any one of claims 23-35, wherein the cells produce less than about 2.0 g / L lactate in shake flasks during the production phase.

38. 36. The method of any one of claims 23-35, wherein the cells produce less than about 2.0 g / L lactate in a bioreactor during the production phase.

39. The method of any one of claims 31 to 38, wherein the reference cell is a cell comprising a wild-type allele of the PKM gene.

40. The method of any one of claims 23 to 39, wherein the PKM polypeptide isoform is a PKM-1 polypeptide isoform.

41. 40. The method of any one of claims 23 to 39, wherein the PKM polypeptide isoforms are a PKM-1 polypeptide isoform and a PKM-2 polypeptide isoform.

42. 25. The method of claim 24, 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.

43. The method of claim 42, wherein the mRNA expressed by the PKM gene encodes a PKM-1 polypeptide isoform.

44. 44. The method of claim 43, wherein expression of the PKM-1 polypeptide isoform is knocked out or knocked down and the lactogenic activity of the cell is reduced compared to the lactogenic activity of a reference cell.

45. 45. The method of any one of claims 42 to 44, wherein the genetic modification system further comprises a second RNA selected from the group consisting of shRNA, siRNA, and microRNA miRNA, wherein the second RNA is complementary to a portion of an mRNA expressed by the PKM gene encoding a PKM-2 polypeptide isoform.

46. 46. ​​The method of claim 45, wherein expression of the PKM-1 and PKM-2 polypeptide isoforms is knocked out or knocked down, reducing lactogenic activity of the cells.

47. 25. The method of claim 24, wherein the genetic modification system is a zinc finger nuclease (ZFN) system or a transcription activator-like effector nuclease (TALEN) system.

48. The method of any one of claims 23 to 47, wherein the cell is a mammalian cell.

49. 50. The method of claim 49, wherein the mammalian cell is a CHO cell.

50. 50. The method of any one of claims 23 to 49, wherein the cells express a product of interest.

51. 51. The method of claim 50, wherein the product of interest expressed by the cell is encoded by a nucleic acid sequence.

52. 52. The method of claim 51, wherein the nucleic acid sequence is integrated into the cellular genome of the cell at a targeted location.

53. 53. The method of any one of claims 50 to 52, 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.

54. 54. The method of any one of claims 50 to 53, wherein the product of interest comprises a protein.

55. 55. The method of claim 54, wherein the product of interest comprises a recombinant protein.

56. 56. The method of any one of claims 50 to 55, wherein the product of interest comprises an antibody or an antigen-binding fragment thereof.

57. 57. The method of claim 56, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

58. 57. The method of claim 56, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

59. The method of any one of claims 56 to 58, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

60. The method of any one of claims 56 to 59, wherein the antibody is a monoclonal antibody.

61. 1. A method for producing a product of interest, comprising culturing a mammalian cell expressing said product of interest, wherein said mammalian cell expresses said product of interest and has reduced or eliminated lactogenic activity.

62. 23. A method for culturing a population of mammalian cells expressing a product of interest, wherein said mammalian cells have reduced or eliminated lactogenic activity.

63. 63. The method of claim 61 or 62, wherein the reduction or elimination of lactogenic activity occurs by knocking out or knocking down expression of a pyruvate kinase muscle (PKM) polypeptide isoform in the mammalian cell.

64. 64. The method of claim 63, wherein the PKM polypeptide isoform is a PKM-1 polypeptide isoform.

65. 64. The method of claim 63, wherein the PKM polypeptide isoform is a PKM-1 polypeptide isoform or a PKM-2 polypeptide isoform.

66. 66. The method of any one of claims 61 to 65, wherein the lactogenic activity of the mammalian cell is less than about 50% of the lactogenic activity of a reference cell.

67. 66. The method of any one of claims 61 to 65, wherein the lactogenic activity of the mammalian cell is less than about 20% of the lactogenic activity of a reference cell.

68. 68. The method of any one of claims 61 to 67, wherein the lactogenic activity of the mammalian cells is measured on day 14 or 15 of the production phase.

69. 69. The method of any one of claims 61-68, wherein the mammalian cells produce less than about 2.0 g / L lactate during the production phase.

70. 69. The method of any one of claims 61-68, wherein the mammalian cells produce less than about 2.0 g / L lactate in shake flasks during the production phase.

71. 69. The method of any one of claims 61-68, wherein the mammalian cells produce less than about 2.0 g / L of lactate in a bioreactor during the production phase.

72. The method of any one of claims 66 to 71, wherein the reference cell is a cell that comprises at least one or both wild-type alleles of the PKM gene.

73. 73. The method of any one of claims 61 to 72, wherein the mammalian cell is a CHO cell.

74. 74. The method of any one of claims 61 to 73, wherein the product of interest expressed by the mammalian cell is encoded by a nucleic acid sequence.

75. 75. The method of claim 74, wherein the nucleic acid sequence is integrated into the cellular genome of the mammalian cell at a targeted location.

76. 76. The method of any one of claims 61 to 75, 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.

77. 77. The method of any one of claims 61 to 76, wherein the product of interest comprises a protein.

78. 78. The method of any one of claims 61 to 77, wherein the product of interest comprises a recombinant protein.

79. 80. The method of any one of claims 61 to 79, wherein the product of interest comprises an antibody or an antigen-binding fragment thereof.

80. 80. The method of claim 79, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

81. 80. The method of claim 79, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

82. The method of any one of claims 79 to 81, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

83. The method of any one of claims 79 to 82, wherein the antibody is a monoclonal antibody.

84. 84. The method of any one of claims 61 to 83, further comprising recovering the desired product.

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