Modified cells for producing a recombinant product of interest

Abstract

The present disclosure relates to cells (e.g., Chinese Hamster Ovary (CHO) cells) that have been modified to reduce or eliminate expression of specific endogenous cellular proteins, and methods of using such cells for the production of recombinant products of interest, such as recombinant proteins, recombinant viral particles, or recombinant viral vectors. These modifications were specifically selected to generate engineered cells with desired traits in several key areas, including improved cell culture performance (e.g., increased viability and product titer).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 191,781, filed May 21, 2021, the contents of which are incorporated by reference in their entirety and to which priority is claimed.

[0002] The present disclosure relates to cells (e.g., Chinese Hamster Ovary (CHO) cells) that have been modified to reduce or eliminate expression of specific endogenous proteins, and methods of using such cells for the production of recombinant products of interest, such as recombinant proteins, recombinant viral particles, or recombinant viral vectors. These modifications generate engineered cells with unexpected synergistic traits in key areas, including improved viability and increased product titers. [Background technology]

[0003] Rapid advances in cell biology and immunology have driven the demand for developing novel therapeutic recombinant products, such as recombinant proteins, recombinant viral particles and recombinant viral vectors, for various diseases, including cancer, cardiovascular and metabolic diseases. These biopharmaceutical candidates are generally produced by commercially available cell lines capable of expressing the product of interest. For example, CHO cells have been widely adapted to produce monoclonal antibodies.

[0004] The expression of certain proteins by cells is detrimental to cell culture performance. For example, proteins that promote apoptosis can reduce the viability and productivity of the culture. Furthermore, during the production of biopharmaceuticals, the expression of recombinant products can impose a high proteostatic burden that induces cellular adaptation. For example, cells often utilize the unfolded protein response (UPR) to mitigate the increased proteostatic burden. However, the UPR can ultimately result in a decrease in overall protein translation, negatively impacting the titer achieved for the recombinant product of interest.

[0005] Thus, there is a need in the art for more efficient methods, modified cells and compositions for producing recombinant products of interest, such as recombinant proteins, recombinant viral particles or recombinant viral vectors, where the modified cells expressing the recombinant products of interest exhibit improved attributes related to cell viability and titers associated with the production of the product of interest. Such improved cells can be achieved by modifying the genome of the cells (i.e., cell line engineering). Summary of the Invention

[0006] In certain embodiments, the present disclosure relates to modified cells, wherein the cells have been modified to reduce or eliminate expression of two or more endogenous proteins compared to expression of the endogenous proteins in unmodified cells, and wherein (a) one or more of the endogenous proteins whose expression is reduced or eliminated promotes apoptosis of the modified cells in cell culture, and (b) one or more of the endogenous proteins whose expression is reduced or eliminated modulates the unfolded protein response (UPR).

[0007] In certain embodiments, the disclosure relates to modified cells, wherein the cells have been modified to reduce or eliminate expression of two or more endogenous proteins compared to expression of the endogenous proteins in a non-modified cell, wherein one or more endogenous proteins are selected from the group of endogenous proteins consisting of BCL2 binding X, apoptosis regulator (BAX) and BCL2 antagonist / killer 1 (BAK), and one of the endogenous proteins is protein kinase R-like ER kinase (PERK).

[0008] In certain embodiments, the present disclosure relates to modified cells in which expression of BAX, BAK and PERK is reduced or eliminated.

[0009] In certain embodiments, the disclosure relates to the modified cell, wherein the modified cell is engineered to express a recombinant product of interest. In certain embodiments, the disclosure relates to the modified cell, wherein the modified cell is generated from a recombinant cell expressing a recombinant product of interest. In certain embodiments, the disclosure relates to the modified cell, wherein one or more endogenous proteins have no detectable expression. In certain embodiments, the disclosure relates to the modified cell, wherein the recombinant product of interest comprises a viral vector. In certain embodiments, the disclosure relates to the modified cell, wherein the recombinant product of interest comprises a viral particle. In certain embodiments, the disclosure relates to the modified cell, wherein the recombinant product of interest comprises a recombinant protein. In certain embodiments, the disclosure relates to the modified cell, wherein the recombinant protein is an antibody or an antibody fusion protein, or an antigen-binding fragment thereof. In certain embodiments, the disclosure relates to the modified cell, wherein the antibody is a multispecific antibody, or an antigen-binding fragment thereof. In certain embodiments, the disclosure relates to the modified cell, wherein 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 present disclosure relates to the modified cells described above, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody. In certain embodiments, the present disclosure relates to the modified cells described above, wherein the antibody is a monoclonal antibody. In certain embodiments, the present disclosure relates to the modified cells described above, wherein the recombinant product of interest is encoded by an exogenous nucleic acid sequence integrated into the cell genome at one or more target locations.

[0010] In certain embodiments, the present disclosure relates to the modified cells described above, wherein the modified cells do not express detectable BAX, BAK, and PERK. In certain embodiments, the present disclosure relates to the modified cells described above, wherein the modified cells express reduced levels of BAX, BAK, and PERK.

[0011] In certain embodiments, the present disclosure relates to the modified cell described above, wherein the modified cell is a modified animal cell. In certain embodiments, the modified animal cell is a modified Sf9 cell, a CHO cell, a HEK 293 cell, a HEK-293T cell, a BHK cell, an A549 cell, or a HeLa cell.

[0012] In certain embodiments, the present disclosure relates to compositions comprising the modified cells described above.

[0013] In certain embodiments, the present disclosure relates to a method of producing a recombinant product of interest comprising: (a) culturing the modified cells described above; and (b) recovering the recombinant product of interest from the culture medium or the modified cells, wherein the modified cells expressing the recombinant product of interest exhibit reduced or eliminated expression of BAX, BAK and PERK.

[0014] In certain embodiments, the disclosure relates to methods of producing modified cells comprising: (a) applying nuclease-assisted and / or nucleic acid targeting BAX, BAK, and PERK to cells to reduce or eliminate expression of the endogenous genes; and (b) selecting modified cells in which expression of the endogenous genes is reduced or eliminated compared to unmodified cells.

[0015] In certain embodiments of the above method for producing modified cells, the modification is performed before the introduction of an exogenous nucleic acid encoding a recombinant product of interest or after the introduction of an exogenous nucleic acid encoding a recombinant product of interest. In certain embodiments, the nuclease-assisted gene targeting system is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN or meganuclease. In certain embodiments, the reduction of gene expression is mediated by RNA silencing. In certain embodiments, the RNA silencing is selected from the group consisting of siRNA gene targeting and knockdown, shRNA gene targeting and knockdown, and miRNA gene targeting and knockdown.

[0016] In certain embodiments, the recombinant product of interest is encoded by a nucleic acid sequence. In certain embodiments, the nucleic acid sequence is integrated into the cellular genome of the modified cell at one or more target positions. In certain embodiments, the recombinant product of interest expressed by the modified cell is encoded by a nucleic acid sequence that is randomly integrated into the cellular genome of the modified cell.

[0017] In certain embodiments, the recombinant product of interest comprises a viral vector. In certain embodiments, the recombinant product of interest comprises a viral particle. In certain embodiments, the recombinant product of interest comprises a recombinant protein. In certain embodiments, the recombinant protein is an antibody or an antibody fusion protein, or an antigen-binding fragment thereof. In certain embodiments, 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. In certain embodiments, the antibody is a monoclonal antibody.

[0018] Certain embodiments of the above methods include purifying the product of interest, harvesting the product of interest, and / or formulating the product of interest.

[0019] In certain embodiments of the above method, the modified cell is a modified animal cell. In certain embodiments, the modified animal cell is a modified Sf9 cell, a CHO cell, a HEK 293 cell, a HEK 293T cell, a BHK cell, an A549 cell or a HeLa cell.

[0020] In certain embodiments, the present disclosure relates to modified cells that have a higher specific productivity than the corresponding isolated animal cells that contain polynucleotides and functional copies of each of the wild-type Bax, Bak and PERK genes.

[0021] In certain embodiments, the present disclosure relates to modified cells that are more resistant to apoptosis than corresponding isolated animal cells that contain functional copies of each of the Bax, Bak and PERK genes.

[0022] In certain embodiments, the present disclosure relates to modified cells for use in fed-batch, perfusion, process intensification, semi-continuous perfusion, or continuous perfusion cell culture processes. [Brief description of the drawings]

[0023] [Figure 1A] PDGFRa is downregulated by UPR activation. Figure 1A shows that PDGFRa protein and mRNA levels were downregulated when mAb1-expressing CHO cells were grown at pH 7.07. [Figure 1B] FIG. 1B shows that PDGFRa protein and mRNA levels were each downregulated when mAb1-expressing CHO cells were grown at pH 7.07. [Figure 1C] FIG. 1C shows Western blot analysis of two mAb1-expressing host cell lines, CHO DG44 and CHO-K1, treated with chemical UPR inducers: tunicamycin and DTT. [Figure 1D] FIG. 1D shows qPCR analysis of PDGFRa mRNA levels in both host cell lines from FIG. 1C treated with tunicamycin and DTT. [Figure 1E] Figure 1E shows Western blot analysis of mAb1-expressing CHO-K1 cells treated with tunicamycin to activate the UPR in the presence of a UPR pathway-specific inhibitor. RT-PCR panel of XBP-1 shows IRE1 alpha RNase activation. [Figure 1F] FIG. 1F shows qPCR analysis of PDGFRa mRNA levels in CHO-K1 cells treated with tunicamycin in the presence of a UPR pathway-specific inhibitor. [Figure 1G]Figure 1G shows Western blot analysis of WT and PERK KO empty host CHO-K1 (clone 9) cell lines treated with tunicamycin and a PERK inhibitor. [Figure 2A] Figure 2A shows Western blot analysis of mAb1-expressing CHO-K1 cells treated with thapsigargin to activate the UPR in the presence of various UPR pathway-specific inhibitors. RT-PCR panel of XBP-1 shows IRE1 alpha RNase activation. [Figure 2B] FIG. 2B shows Western blot analysis of empty host CHO-K1 cells treated with tunicamycin to activate the UPR in the presence of various UPR pathway-specific inhibitors. [Figure 2C] FIG. 2C shows qPCR analysis of PDGFRa mRNA levels in CHO-K1 cells treated with thapsigargin in the presence of various UPR pathway-specific inhibitors. [Fig. 2D-E] Figure 2D shows a Western blot analysis of Cas9-sgRNA against the PERK gene, with sgRNA against luciferase as a control. Figure 2E shows a Western blot analysis of empty host CHO-K1 single cell clones after using Cas9 to knock out PERK. Clone 9 was used in Figure 1G. [Figure 3A-B] PDGFRa signaling is important for cell proliferation, e.g., CHO cell proliferation, and growth factor signaling is intact following PDGFRa inhibition. Figure 3A is a schematic of PDGFRa and insulin receptor (IR) signaling upstream of protein synthesis, cell cycle progression and cell proliferation. Thick arrows indicate high intensity activation by the respective receptors. Figure 3B shows the VCC and viability % of empty CHO-K1 host cells after 4 days in the presence or absence of PDGFRa inhibitors and / or insulin in the seed train medium. [Figure 3C] Figure 3C shows Western blot analysis of empty host CHO-K1 cells (from Figure 3B) after 4 days in the presence or absence of PDGFRa inhibitors and / or insulin in the seed train medium. [Figure 3D] FIG. 3D shows the relative IVCC, % viability, relative potency and relative Qp of mAb2-expressing CHO-K1 cells in the presence of PDGFRa inhibitors and / or insulin during production at day 12. [Figure 4A] FIG. 4A shows viable cell count (VCC) and % viability of empty host CHO-K1 cells after 4 days in the presence of increasing concentrations of PDGFRa inhibitors in the seed train medium. [Figure 4B] FIG. 4B shows Western blot analysis of empty host CHO-K1 cells after 4 days in the presence of increasing concentrations of PDGFRa inhibitors in the seed train medium. [Figure 4C] FIG. 4C shows Western blot analysis mAb2-expressing CHO-K1 cells when produced in the presence or absence of a 10 μM concentration of PERK inhibitor. [Figure 4D] FIG. 4D shows qPCR analysis of downstream targets of the PERK branch of UPR, CHOP and GADD34 during production of mAb2-expressing CHO-K1 cells in the presence or absence of a PERK inhibitor. [Figure 5A] PDGFRa levels are stabilized during production in PERK KO cell lines. Figure 5A shows Western blot analysis of mAb2-expressing CHO-K1 single cell clones after using CRISPR-Cas9 to knock out PERK. [Figure 5B] FIG. 5B shows the relative IVCC, % viability, relative titer and relative Qp of mAb2-expressing CHO-K1 PERK KO cells at day 14. [Figure 5C] FIG. 5C shows Western blot analysis of the production of mAb2-expressing CHO-K1 WT and PERK KO cells. [Figure 6A] PERK and Bax / Bak TKO synergistically increase bioprocessing outcomes. Figure 6A shows Western blot analysis of mAb3-expressing CHO-K1 single cell clones in seed trains after using Cas9 to knock out PERK. [Figure 6B]Overall titers of various mAb3 expressing CHO-K1 hosts across different bioprocesses, namely lean production medium, rich production medium, and enriched process using rich production medium (shown in FIG. 6B). [Figure 6C] Relative Qp of different mAb3 expressing CHO-K1 hosts across different bioprocesses, namely lean production medium, rich production medium and enriched process using rich production medium (shown in Figure 6C). [Figure 6D] FIG. 6D shows Western blot analysis of various mAb3-expressing CHO-K1 hosts in rich production medium. [Figure 6E-1] FIG. 6E shows qPCR analysis of heavy and light chain mRNA levels in lean and rich production media. [Figure 6E-2] FIG. 6E shows qPCR analysis of heavy and light chain mRNA levels in lean and rich production media. [Figure 7A] FIG. 7A shows bioprocess results of a 6-day production of the mAb3 expression pool in either a Bax / Bak DKO background or a PERK / Bax / Bak TKO background showing relative titer, Qp and IVCC. [Figure 7B] FIG. 7B shows bioprocess results of 14-day production of Fab1 expression pools in either WT, PERK KO, Bax / Bak DKO or PERK / Bax / Bak TKO backgrounds showing relative titer, Qp and IVCC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present disclosure relates to cells (e.g., Chinese Hamster Ovary (CHO) cells) that have been modified to reduce or eliminate expression of specific endogenous proteins, and methods of using such modified cells for the production of recombinant products of interest, such as recombinant proteins, recombinant viral particles, or recombinant viral vectors. These modifications generate engineered cells that have desirable traits in several key areas, including improved viability and increased product titers.

[0025] One commonly used method for the expression of heterologous molecules, such as monoclonal antibodies, is by a fed-batch process, in which cells are initially seeded and then fed in batches over the course of the production period. Various factors during the production process can lead to poor production and product quality outcomes. One factor is increased proteostatic stress due to the synthesis of large amounts of heterologous molecules. The endoplasmic reticulum (ER) can adapt to this stress by activating the unfolded protein response (UPR). The UPR is typically triggered when the ER reaches its maximum protein folding capacity and is unable to keep up with the increased demands for protein synthesis and folding. The UPR has three identified ER transmembrane proteins that sense the accumulation of unfolded polypeptides and respond by activating signaling pathways that promote ER homeostasis by expanding the ER, increasing chaperone production, and decreasing overall protein translation. If these processes are unable to alleviate proteostatic stress, sustained UPR activation can result in apoptotic cell death. Under normal conditions, these UPR sensors are bound by ER chaperones called immunoglobulin-binding proteins (BiPs) in the ER lumen, which keeps them inactive. Upon accumulation of unfolded proteins in the ER lumen, BiPs dissociate from these sensors and assist in folding of unfolded or misfolded proteins to reduce ER proteostasis stress, thereby allowing activation of UPR receptors.

[0026] The three UPR sensors are inositol-requiring enzyme 1 (IRE1), protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The IRE1 branch of the UPR is the most conserved, with IRE1 functioning as both a kinase and a ribonuclease (RNase). IRE1 is involved in non-conventional splicing of XBP1 (X-box binding protein 1) mRNA transcripts. This splicing produces a short form of XBP1, a transcription factor that regulates UPR target genes to increase the protein folding capacity of the ER. These target genes expand and modify the ER by regulating lipid biosynthetic enzymes and ERAD (ER-associated degradation) components. Another branch of the UPR, PERK (protein kinase R-like ER kinase), is a kinase but lacks ribonuclease activity. When activated, PERK reduces global mRNA translation by phosphorylating eIF2alpha, thereby decreasing its activity. At the same time, a subset of mRNAs, containing short open reading frames, undergo selective protein translation. One of these mRNAs encodes activating transcription factor 4 (ATF4), which regulates the expression of another transcription factor called C / EBP homologous protein (CHOP), which in turn regulates components of the apoptotic pathway, including Bim and death receptor 5 (DR5). Moderate activation of PERK functions cytoprotectively through CHOP, whereas extended PERK activation leads to cell death. Finally, the ATF6 branch of the UPR involves the transport and proteolytic activation of ATF6 from the ER to the Golgi apparatus. During protein degradation, ATF6 functions as a transcription factor that helps increase ER capacity by increasing the production of ER proteins involved in protein folding chaperones, such as BiP and GRP94, as well as foldases such as protein disulfide isomerase. The three branches of the UPR work together to alleviate proteostasis stress by simultaneously decreasing the overall protein folding load and increasing the protein folding capacity of the ER.

[0027] The present disclosure is based, at least in part, on the discovery that knocking out PERK in Bax / Bak double knockout (DKO) antibody-expressing cell lines demonstrated increased viability and proliferation, and surprisingly also demonstrated a synergistic increase in specific productivity and titer compared to control cell lines.

[0028] For clarity, and not for limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections: 5.1 Definitions; 5.2 Reduction or elimination of expression of endogenous proteins; 5.3 Cells containing gene-specific modifications; 5.4 Cell culture methods; 5.5 Production of the recombinant product of interest; and 5.6 Exemplary Non-Limiting Embodiments

[0029] 5.1. definition The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure and in the specific context in which each term is used. Particular terms are discussed below or elsewhere herein to provide additional guidance to the practitioner describing the compositions and methods of the present disclosure, and how to make and use them.

[0030] As used herein, in the claims and / or specification, when used in conjunction with the term "comprising," the use of the words "a" or "an" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more."

[0031] As used herein, the terms "comprise(s) / include(s)," "having / has," "can," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0032] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, as is customary in the art. Alternatively, "about" can mean within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0033] The terms "cell culture medium" and "culture medium" refer to a nutrient solution used to grow mammalian cells that typically provides at least one component from one or more of the following categories: 1) A source of energy, usually in the form of carbohydrates such as glucose; 2) all essential amino acids, usually a basic set of 20 amino acids plus cysteine; 3) Vitamins and / or other organic compounds required in low concentrations; 4) free fatty acids; and 5) Trace Elements (Trace elements are defined as inorganic compounds or naturally occurring elements that are typically required in very low concentrations, usually in the micromolar range).

[0034] The nutrient solution can optionally be supplemented with one or more components from any of the following categories: 1) Hormones and other growth factors such as insulin, transferrin and epidermal growth factor; 2) Salts and buffers, such as calcium, magnesium and phosphate; 3) nucleosides and bases, such as adenosine, thymidine and hypoxanthine; and 4) Protein and tissue hydrolysates.

[0035] "Culturing" a cell means contacting the cell with a cell culture medium under conditions suitable for the survival and / or growth and / or proliferation of the cell.

[0036] By "batch culture" is meant a culture medium in which all components for cell culture, including cells and all culture nutrients, are supplied to the culture bioreactor at the start of the culture process.

[0037] As used herein, "fed-batch culture cell culture" refers to a batch culture in which cells and medium are initially fed into a culture bioreactor, additional culture nutrients are fed to the culture continuously or in discrete increments during the culture process, and cells and / or products are or are not harvested periodically prior to the end of the culture.

[0038] A "perfusion culture", sometimes referred to as a continuous culture, is a culture in which the cells are maintained in the culture, e.g., by filtration, encapsulation, anchoring to microcarriers, etc., and medium is introduced and removed from the culture bioreactor continuously, in stages, or intermittently (or any combination of these).

[0039] As used herein, the term "cell" refers to animal cells, mammalian cells, cultured cells, host cells, recombinant cells, and recombinant host cells. Such cells are generally cell lines obtained or derived from animals, e.g., mammalian, tissues, that are capable of growth and survival when placed in medium containing the appropriate nutrients and / or growth factors.

[0040] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid can be subsequently introduced to create recombinant cells. These host cells may also be modified (i.e., engineered) to alter or delete the expression of certain endogenous host cell proteins. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny need not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell are included herein. Introduction of exogenous nucleic acid into these host cells (e.g., by transfection) creates recombinant cells derived from the original "host cell", "host cell line" or "host cell line". The terms "host cell", "host cell line" and "host cell culture" may also refer to such recombinant cells and their progeny.

[0041] The terms "recombinant cell," "recombinant cell line," and "recombinant cell culture" are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid has been introduced to allow expression of a recombinant product of interest. The recombinant product expressed by such cells may be a recombinant protein, a recombinant viral particle, or a recombinant viral vector.

[0042] The term "animal host cell" or "animal cell" refers to a cell line derived from an animal that can grow and survive when placed in either monolayer or suspension culture in a medium containing appropriate nutrients and growth factors. In addition to the mammalian cells described below, examples of suitable animal host cells within the context of the present disclosure may include, but are not limited to, invertebrate cells and non-mammalian vertebrate (e.g., bird, reptile and amphibian) cells. Examples of invertebrate cells include the following insect cells: Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. See, e.g., Luckow et al., Bio / Technology, 6:47-55 (1988); Miller et al., in Genetic Engineering, Setlow, JK et al., eds., Vol. 8 (Plenum Publishing, 1986). pp. 277-279; and Maeda et al., Nature, 315:592-594 (1985).

[0043] The term "mammalian host cell" or "mammalian cell" refers to a cell line derived from a mammal capable of growth and survival when placed in either monolayer or suspension culture in a medium containing appropriate nutrients and growth factors. The growth factors required for a particular cell line can be readily determined empirically without undue experimentation, for example, as described in Mammalian Cell Culture (Mather, JP ed., Plenum Press, NY 1984), and Barnes and Sato, (1980) Cell, 22:649. Typically, the cells are capable of expressing and secreting large amounts of a particular protein of interest, e.g., a glycoprotein, into the medium.Examples of suitable mammalian host cells within the context of the present disclosure include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dp12.CHO cells (European Patent No. 307,247 published March 15, 1989); CHO-K1 (ATCC, CCL-61); monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); 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 tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NYAcad. Sci., 383:44-68 1982); MRC 5 cells; FS4 cells; and human hepatoma line (Hep G2). In certain embodiments, mammalian cells include Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 1980); dp12.CHO cells (European Patent No. 307,247 published March 15, 1989).

[0044] The "growth phase" of a cell culture refers to a period of exponential cell growth (log phase) during which cells generally divide rapidly. The length of time that cells are maintained in the growth phase can vary based on, for example, the type of cell, the growth rate of the cells, and / or the conditions of the medium. In certain embodiments, during this phase, cells are cultured under conditions that maximize cell growth for a period of time (usually 1-4 days). The determination of the growth cycle of the host cell is determined for the particular host cell, as contemplated without undue experimentation. "Duration and conditions that maximize cell growth" and the like refers to culture conditions in which cell growth and division are determined to be optimal for a particular cell line. In certain embodiments, during the growth phase, cells are cultured in a nutrient medium containing necessary additives, typically in a humidified, controlled atmosphere at about 30-40° C., such that optimal growth for the particular cell line is achieved. In certain embodiments, cells are maintained in the growth phase for a period of about 1-4 days, usually 2-3 days.

[0045] "Production phase" of cell culture refers to the period during which cell growth is / remains stagnant. Logarithmic cell growth typically declines before or during this phase and is taken over by protein production. During the production phase, logarithmic cell growth ceases and protein production predominates. During this period, the medium is generally replenished to support the continuous production of protein and to obtain the desired glycoprotein product. Fed-batch and / or perfusion cell culture processes replenish the cell medium or provide fresh medium during this phase to achieve and / or maintain the desired cell density, viability, and / or titer of recombinant protein product. The production phase can be carried out on a large scale.

[0046] As used herein with respect to the activity of a protein, the term "activity" refers to any activity of the protein, including, but not limited to, enzymatic activity, ligand binding, drug transport, ion transport, protein localization, receptor binding, and / or structural activity. Such activities can be modulated, e.g., reduced or eliminated, by reducing or eliminating the expression of the protein, thereby reducing or eliminating the presence of the protein. Such activities can also be modulated, e.g., reduced or eliminated, by modifying the nucleic acid sequence encoding the protein, such that the resulting modified protein exhibits reduced or eliminated activity compared to the wild-type protein.

[0047] The term "expression" or "expressing" is used herein to mean transcription and translation occurring in a host cell. The degree of expression of a product gene in a host cell can be measured based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by the product gene produced by the cell. For example, the mRNA transcribed from the product gene is preferably quantified by Northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). The protein encoded by the product gene can be quantified either by assaying the biological activity of the protein or by using an assay unrelated to such activity, such as Western blot or radioimmunoassay, using an antibody capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0048] As used herein, "polypeptide" generally refers to peptides and proteins having more than about 10 amino acids. The polypeptides may be homologous to the host cell or, preferably, exogenous, meaning heterologous or foreign to the host cell being utilized, such as human proteins produced by Chinese hamster ovary cells, or yeast polypeptides produced by mammalian cells. In certain embodiments, mammalian polypeptides (polypeptides originally derived from mammalian organisms) are used, more preferably those that are directly secreted into the medium.

[0049] The term "protein" refers to a sequence of amino acids with sufficient chain length to generate higher order tertiary and / or quaternary structure. This is to distinguish it from "peptides" or other low molecular weight drugs that do not have such structures. Proteins herein typically have a molecular weight of at least about 15-20 kD, preferably at least about 20 kD. Examples of proteins encompassed by the definition herein include host cell proteins, as well as all mammalian proteins, particularly therapeutic and diagnostic proteins such as therapeutic and diagnostic antibodies, and general proteins containing one or more disulfide bonds, including multi-chain polypeptides containing one or more inter- and / or intra-chain disulfide bonds.

[0050] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies (e.g., antibodies consisting of a single heavy chain sequence and a single light chain sequence (including multimers of such pairings)), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0051] As used herein, an "antibody fragment," "antigen-binding portion" (or simply "antibody portion") of an antibody, or an "antigen-binding fragment" of an antibody refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0052] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular origin or species, and the remainder of the heavy and / or light chain is derived from a different origin or species.

[0053] The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is an IgG1 isotype. In certain embodiments, the antibody is an IgG2 isotype. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0054] As used herein, the term "titer" refers to the total amount of recombinantly expressed antibody produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of antibody per milliliter or liter of medium (mg / mL or mg / L). In certain embodiments, titer is expressed in grams of antibody per liter of medium (g / L). Titer can be expressed or evaluated in terms of a relative measurement, such as the percentage increase in titer compared to obtaining protein product under different medium conditions.

[0055] The terms "nucleic acid", "nucleic acid molecule" or "polynucleotide" include any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, whereby the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is usually represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein can include naturally occurring or non-naturally occurring nucleotides. Examples of non-natural nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for directly expressing the antibodies of the present disclosure in vitro and / or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can 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 antibodies in vivo (see, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0056] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0057] A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or a human cell, or to an antibody derived from a non-human source that utilizes the human antibody repertoire, or to sequences encoding other human antibodies. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0058] A "humanized" antibody refers to a chimeric antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDRs corresponding to the CDRs of a non-human antibody and all or substantially all of the FRs corresponding to the FRs of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0059] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain, e.g., the "complementarity determining regions" (CDRs), which are hypervariable in sequence and determine antigen-binding specificity.

[0060] Generally, an antibody comprises six CDRs: three in the VH (CDR-H1, CDR-H2, CDR-H3) and three in the VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include the following: (a) hypervariable loops located 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 located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) Antigenic contacts present 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)).

[0061] Unless otherwise specified, CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the designations of CDRs can be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0062] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, a cytotoxic agent.

[0063] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical and / or bind the same epitope, except for possible variant antibodies (which may, for example, include naturally occurring mutations or arise during the production of the monoclonal antibody preparation, and such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which usually contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies according to the subject matter of the present disclosure can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and such methods and other exemplary methods of producing monoclonal antibodies are described herein.

[0064] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three complementarity determining regions (CDR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0065] As used herein, the term "cell density" refers to the number of cells in a given volume of medium. In certain embodiments, a high cell density is desirable in that it results in higher protein production capacity. Cell density can be monitored by any technique known in the art, including but not limited to extracting a sample from the medium and analyzing the cells under a microscope, using a commercially available cell counting device, or using a suitable commercially available probe introduced into the bioreactor itself (or into the loop through which the medium and suspended cells pass back to the bioreactor).

[0066] As used herein, the term "recombinant protein" generally refers to peptides and proteins, including antibodies, encoded by nucleic acid that is "heterologous," i.e., foreign to the host cell being utilized, e.g., nucleic acid encoding a human antibody, that is introduced into a non-human host cell.

[0067] As used herein, the term "recombinant viral particle" refers generally to a viral particle that may be naturally occurring or that may be produced by recombining exogenous nucleic acid for use in vaccine production.

[0068] As used herein, the term "recombinant viral vector" generally refers to viral vectors that have been modified to express exogenous viral elements for use in, e.g., gene therapy, including, but not limited to, recombinant vectors based on adeno-associated virus (AAV), herpes simplex virus (HSV), retroviruses, poxviruses, and lentiviruses.

[0069] 5.2. Reducing or eliminating expression of endogenous proteins In certain embodiments, the present disclosure relates to modified cells, such as CHO cells, in which the expression of one or more endogenous proteins is reduced or eliminated.For example, but not limited to, methods for reducing or eliminating endogenous protein expression in modified cells include (1) modifying the gene encoding the endogenous protein or its components, for example, by introducing deletion, insertion, substitution or combinations thereof into the gene, (2) reducing or eliminating the transcription and / or stability of the mRNA encoding the endogenous protein or its components, and (3) reducing or eliminating the translation of the mRNA encoding the endogenous protein or its components.In certain embodiments, the reduction or elimination of protein expression is obtained by targeted genome editing.For example, genome editing based on CRISPR / Cas9 can be used to modify one or more target genes, resulting in the reduction or elimination of the expression of the gene or genes targeted for editing.

[0070] In certain embodiments, one or more of the endogenous cellular proteins targeted for expression reduction or elimination are selected based on their role in promoting apoptosis.Apoptosis can reduce the viability and productivity of culture, so reducing or eliminating the expression of such proteins can have a positive effect on the viability and productivity of culture.For example, but not limited to, the cellular proteins selected based on their role in promoting apoptosis are BCL2 binding X, apoptosis regulator (BAX) or BCL2 antagonist / killer 1 (BAK).

[0071] In certain embodiments, the modified cells of the present disclosure exhibit reduced or eliminated expression of BAX. In certain embodiments, BAX, as used herein, refers to eukaryotic BAX cellular protein, e.g., CHO BAX cellular protein (Entrez Gene ID: 100689032; GenBank ID: EF104643.1) and functional variants thereof. In certain embodiments, functional variants of BAX, as used herein, encompass BAX sequence variants that have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the wild-type BAX sequence of the modified cells used to generate the recombinant product of interest.

[0072] In certain embodiments, the modified cells of the present disclosure exhibit reduced or eliminated expression of BAK. In certain embodiments, BAK, as used herein, refers to eukaryotic BAK cell protein, such as CHO BAK cell protein (GenBank ID: EF104644.1), and functional variants thereof. In certain embodiments, functional variants of BAK, as used herein, encompass BAK sequence variants that have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the wild-type BAK sequence of the modified cells used to produce the recombinant product of interest.

[0073] In certain embodiments, the modified cells of the present disclosure exhibit reduced or eliminated expression of BAX and BAK.

[0074] In certain embodiments, one or more of the endogenous cellular proteins targeted for expression reduction or elimination are selected based on their role in regulating unfolded protein response (UPR).For example, but not limited to, the cellular proteins selected based on their role in regulating UPR are inositol-requiring enzyme 1 (IRE1), protein kinase R-like ER kinase (PERK) or activating transcription factor 6 (ATF6).In certain embodiments, the modified cells of the present disclosure show the expression reduction or elimination of PERK.In certain embodiments, PERK, as used herein, refers to eukaryotic PERK cellular proteins, such as CHO PERK cellular proteins (gene ID: 100765343; GenBank: EGW03658.1; and isoforms NCBI reference sequence: XP_027285344.2 and NCBI reference sequence: XP_016831844.1) and their functional variants. In certain embodiments, a functional variant of PERK, as used herein, encompasses a PERK sequence variant having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the wild-type PERK sequence of the modified cell used to produce the recombinant product of interest.

[0075] In certain embodiments, the modified cells of the present disclosure exhibit reduced or eliminated expression of the following endogenous proteins: BAX, BAK, and PERK.

[0076] In certain embodiments, the cells of the present disclosure are modified to reduce or eliminate the expression of one or more endogenous cellular proteins compared to the expression of the endogenous cellular proteins in unmodified, i.e., "reference" cells. In certain embodiments, the reference cells are cells in which the expression of one or more particular endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, is not reduced or eliminated. In certain embodiments, the reference cells are cells that contain at least one or both wild-type alleles of genes encoding BAX, BAK and / or PERK. For example, and not by way of limitation, the reference cells are cells that have both wild-type alleles of genes encoding BAX, BAK and / or PERK. In certain embodiments, the reference cells are WT cells. In certain embodiments, the modification to reduce or eliminate the expression of one or more endogenous cellular proteins is performed prior to the introduction of an exogenous nucleic acid encoding a recombinant product of interest. In certain embodiments, the modification to reduce or eliminate the expression of one or more endogenous cellular proteins is performed after the introduction of an exogenous nucleic acid encoding a recombinant product of interest.

[0077] In certain embodiments, expression of one or more endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in a cell modified to reduce or eliminate expression of the endogenous protein 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 corresponding endogenous protein expression in a reference cell, e.g., a WT cell. In certain embodiments, expression of one or more endogenous proteins in a cell modified to reduce or eliminate expression of the endogenous protein 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 corresponding endogenous protein expression in a reference cell, e.g., a WT cell.

[0078] In certain embodiments, expression of one or more endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in a cell modified to reduce or eliminate expression of the endogenous protein 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 corresponding endogenous protein expression in a reference cell, e.g., a WT cell. In certain embodiments, expression of one or more endogenous proteins in a cell modified to reduce or eliminate expression of the endogenous protein 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 corresponding endogenous protein expression in a reference cell, e.g., a WT cell.

[0079] In certain embodiments, the expression of one or more specific endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in cells modified to reduce or eliminate expression of endogenous proteins 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 corresponding endogenous protein expression in a reference cell, e.g., a WT cell. In certain embodiments, the expression of one or more endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in cells modified to reduce or eliminate expression of endogenous proteins is about 40% or less of the corresponding endogenous protein expression in a reference cell, e.g., a WT cell. In certain embodiments, expression of one or more endogenous proteins in a cell modified to reduce or eliminate expression of the endogenous protein 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 corresponding endogenous protein expression in a reference cell, e.g., a WT cell.

[0080] In certain embodiments, expression of one or more endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in a cell modified to reduce or eliminate expression of the endogenous protein is between about 1% and about 90%, between about 10% and about 90%, between about 20% and about 90%, between about 25% and about 90%, between about 30% and about 90%, between about 35% and about 90%, between about 40% and about 90%, between about 40% and about 90%, between about 50% and about 90%, between about 50% and about 90%, between about 60% and about 90%, between about 60% and about 90%, between about 70% and about 90%, between about 80% and about 90%, between about 80% and about 90%, between about 90% and about 90%, between about 10% and about 90%, between about 15 ... 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 7 0%, 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%, and about 5% to about 40%.In certain embodiments, expression of one or more endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in a cell modified to reduce or eliminate expression of the endogenous protein is between about 1% and about 90%, between about 10% and about 90%, between about 20% and about 90%, between about 25% and about 90%, between about 30% and about 90%, between about 35% and about 90%, between about 40% and about 90%, between about 40% and about 90%, between about 50% and about 90%, between about 50% and about 90%, between about 60% and about 90%, between about 60% and about 90%, between about 70% and about 90%, between about 80% and about 90%, between about 80% and about 90%, between about 90% and about 90%, between about 10% and about 90%, between about 15 ... 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 7 0%, 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%, and about 5% to about 40%.

[0081] In certain embodiments, expression of one or more endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in a cell modified to reduce or eliminate expression of the endogenous protein is about 5% to about 40% of the corresponding endogenous protein expression in a reference cell, e.g., a WT cell.

[0082] In certain embodiments, the expression levels of one or more endogenous proteins, e.g., BAX, BAK and / or PERK polypeptides, in various reference cells (e.g., cells that contain at least one or both wild-type alleles of the corresponding genes) may vary.

[0083] In certain embodiments, a genetic engineering system is used to reduce or eliminate the expression of one or more specific endogenous proteins (e.g., BAX, BAK and / or PERK expression). A variety of genetic engineering systems known in the art can be used for the methods disclosed herein. Non-limiting examples of such systems include the use of CRISPR / Cas systems, zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, and other tools for reducing or eliminating protein expression by gene silencing, such as small interfering RNA (siRNA), short hairpin RNA (shRNA) and microRNA (miRNA). Any CRISPR / Cas system known in the art, including conventional, improved or modified Cas systems, and other bacterial-based genome excision tools, such as Cpf-1, can be used with the methods disclosed herein.

[0084] In certain embodiments, one or more genes, for example, a portion of a gene encoding an endogenous protein such as a BAX, BAK and / or PERK polypeptide, are deleted to reduce or eliminate the expression of the corresponding endogenous protein in the cell. 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 gene are 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 genes are deleted.In a specific embodiment, 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 ...0% to about 90%, about 85% to about 90%, about 2% to about 80%, about 10% to about 80%, about 20% to about 90%, about 30% to about 90%, about 40% % 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 4 0%, about 20% to about 40%, about 30% to about 40%, about 35% to about 40%, about 2% to about 30%, about 10% to about 30%, about 20% to about 30%, about 25% to about 30%, about 2% to about 20%, about 5% to about 20%, about 10% to about 20%, about 15% to about 20%, about 2% to about 10%, about 5% to about 10%, or about 2% to about 5% is deleted.

[0085] In certain embodiments, at least one exon of the gene encoding a BAX, BAK and / or PERK polypeptide is at least partially deleted in the cell.As used herein, "partially deleted" refers to, for example, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 20 ...40%, at least about 40%, at least about 40%, at least about 40%, % or less, approx. 5% or less, approx. 10% or less, approx. 15% or less, approx. 20% or less, approx. 25% or less, approx. 30% or less, approx. 35% or less, approx. 40% or less, approx. 45% or less, approx. 50% or less, approx. % or less, approx. 90% or less, approx. 95% or less, approx. 2% to approx. 90%, approx. 10% to approx. 90%, approx. 20% to approx. 90%, approx. 25% to approx. 90%, approx. 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% is deleted.

[0086] In certain non-limiting embodiments, the CRISPR / Cas9 system is used to reduce or eliminate the expression of one or more endogenous proteins in a cell, such as BAX, BAK and / or PERK polypeptides. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains an RNA to guide the crRNA to the correct part of the host DNA, along with a region that is used by Cas9 and binds to tracrRNA (which is generally in the form of a hairpin loop) that forms an active complex with Cas9), and transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9). The terms "guide RNA" and "gRNA" refer to any nucleic acid that facilitates the specific association (or "targeting") of an RNA-guided nuclease, such as Cas9, to a target sequence, such as a genomic or episomal sequence in a cell. gRNAs can be unimolecular (comprising a single RNA molecule, or called chimeric) or modular (comprising multiple, typically two separate RNA molecules, e.g., crRNA and tracrRNA, that are usually associated with each other by duplexing).

[0087] The CRISPR / Cas9 strategy can transfect cells using vectors. Guide RNA (gRNA) can be designed for each application, as it is the sequence that identifies and directly binds to the target DNA in the cell using Cas9. Multiple crRNAs and tracrRNAs can be packaged together to form a single-stranded guide RNA (sgRNA). The sgRNA can be combined with the Cas9 gene and incorporated into a vector for transfection into cells.

[0088] In certain embodiments, the CRISPR / Cas9 system for use in reducing or eliminating the expression of one or more endogenous proteins, such as BAX, BAK and / or PERK polypeptides, comprises a Cas9 molecule and one or more gRNAs that comprise a targeting domain that is complementary to the target sequence of the gene that codes for the endogenous protein or its components.In certain embodiments, the target gene is the region of the gene that codes for the endogenous protein, such as BAX, BAK and / or PERK polypeptide.The target sequence can be any exon or intron region within the gene.

[0089] 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) that includes the Cas9 protein complexed with one or more gRNAs, for example, by electroporation (see, for example, DeWitt et al., Methods 121-122:9-15 (2017) for additional methods of delivering RNPs to cells). In certain embodiments, administering the CRISPR / Cas9 system to the cell reduces or eliminates the expression of endogenous proteins, for example, BAX, BAK and / or PERK polypeptides.

[0090] In certain embodiments, the genetic engineering system is a ZFN system for reducing or eliminating the expression of one or more specific endogenous proteins in cells, such as BAX, BAK and / or PERK polypeptides. ZFN can act as a restriction enzyme, which is created by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing zinc finger nucleases to target desired sequences in genomes. The DNA binding domain of each ZFN typically contains 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 specificity. The most common cleavage domain of ZFN is the non-specific cleavage domain derived from type II restriction endonuclease FokI. ZFNs regulate protein expression by creating double-stranded breaks (DSBs) within target DNA sequences, which are repaired by non-homologous end joining (NHEJ) in the absence of a homologous template. Such repairs can result in deletions or insertions of base pairs, generating frameshifts and preventing the production of harmful proteins (Durai et al., Nucleic Acids Res.; 33(18):5978-90(2005)). Multiple pairs of ZFNs can also be used to completely remove entire large segments of genomic sequences (Lee et al., Genome Res.; 20(1):81-9(2010)).

[0091] In certain embodiments, the genetic engineering system is a TALEN system to reduce or eliminate the expression of one or more specific endogenous proteins in a cell, such as BAX, BAK and / or PERK polypeptides. TALENs are restriction enzymes that can be engineered to cleave specific sequences in DNA. The TALEN system operates on a principle similar to ZFNs. TALENs are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of a repeating motif of 33-34 amino acids with two variable positions that strongly recognize specific nucleotides. By assembling an array of these TALEs, the TALE DNA binding domain can be engineered to bind to a desired DNA sequence, thereby directing a nuclease to cleave at a specific location in the genome (Boch et al., Nature Biotechnology; 29(2):135-6 (2011)). In certain embodiments, the target gene encodes BAX, BAK and / or PERK.

[0092] In certain embodiments, the expression of one or more specific endogenous proteins, such as BAX, BAK and / or PERK polypeptides, can be reduced or eliminated using oligonucleotides having complementary sequences to corresponding nucleic acids (e.g., 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 BAX, BAK and / or PERK nucleic acid sequences, and the homology of the portion to the corresponding 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 percent. In certain non-limiting embodiments, the complementary portion can comprise at least 10 nucleotides, or at least 15 nucleotides, or at least 20 nucleotides, or at least 25 nucleotides, or at least 30 nucleotides, and the antisense nucleic acid, shRNA, mRNA, or siRNA molecule can be up to 15, or up to 20, or up to 25, or up to 30, or up to 35, or up to 40, or up to 45, or up to 50, or up to 75, or up to 100 nucleotides in length. The antisense nucleic acid, shRNA, mRNA, or siRNA molecule can comprise DNA or atypical or non-naturally occurring residues, such as, but not limited to, phosphorothioate residues.

[0093] The genetic engineering system disclosed herein can be delivered to cells using viral vectors, such as retroviral vectors, such as gamma retroviral vectors, and lentiviral vectors.If the capsid protein is functional to infect human cells, the combination of retroviral vectors and suitable packaging lines is suitable.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). Non-amphotropic particles, such as particles pseudotyped with VSVG, RD114, or GALV envelopes and any others known in the art, are also suitable.Possible methods of transduction also include direct co-culture of cells with producer cells, for example by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culture with viral supernatant alone or concentrated vector stocks, with or without appropriate growth factors and polycations, for example by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0094] Other transduction virus vectors can be used to modify the cells disclosed herein.In certain embodiments, the selected vector shows high infection efficiency and 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.USA94:10319,1997). Other viral vectors that can be used include, for example, adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, vaccinia virus, bovine papilloma virus, or herpes viruses, such as Epstein-Barr virus (see, e.g., 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; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).Retroviral vectors have been particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0095] Non-viral techniques can also be used to genetically engineer the cells disclosed herein. For example, by administering nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989) or by microinjection under surgical conditions (Wolff et al., Science Nucleic acid molecules can be introduced into cells by viral vectors (V. et al., 247:1465,1990). Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for delivering nucleic acid molecules into cells. Transplantation of normal genes into diseased tissues of subjects can also be achieved by transferring normal nucleic acids into ex vivo cultivable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into target tissues or systemically.

[0096] 5.3 Cells containing gene-specific modifications In one aspect, the present disclosure relates to a cell or composition comprising one or more cells, e.g., animal cells, in which expression of one or more endogenous proteins is reduced or eliminated. In certain embodiments, the cell has reduced or eliminated expression of BAX, BAK and / or PERK polypeptides.

[0097] As used herein, elimination of expression refers to elimination of the expression of a particular endogenous protein in a cell, such as BAX, BAK and / or PERK polypeptide, when compared with a reference cell.As used herein, reduction of expression refers to the reduction of the expression of an endogenous protein in a cell, such as BAX, BAK and / or PERK polypeptide, when compared with a reference cell.

[0098] Non-limiting examples of cells useful in connection with the subject matter of the present disclosure include invertebrate cells and non-mammalian vertebrate (e.g., avian, reptile and amphibian) cells, such as Spodoptera frugiperda cells, Aedes aegypti cells, Aedes albopictus cells, Drosophila melanogaster cells and Bombyx mori cells, or mammalian cells, such as CHO cells (e.g., DHFR CHO cells), dp12.CHO cells, CHO-K1 (ATCC, CCL-61), SV40 transformed monkey kidney CV1 line (e.g., COS-7 ATCC CRL-1651), human embryonic kidney lines (e.g., HEK 293 or HEK 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (e.g., BHK, ATCC ... cells (e.g., BHK, ATCC CCL-61), SV40 transformed monkey kidney CV1 line (e.g., COS-7 ATCC CRL-16 10), mouse Sertoli cells (e.g., TM4), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), 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 tumor (e.g., MMT 060562, ATCC CCL 51), TRI cells, MRC 5 cells, FS4 cells, human liver cancer lines (e.g., Hep G2), myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0). In certain embodiments, the cells are CHO cells. Additional non-limiting examples of CHO cells include CHO K1SV cells, CHO DG44 cells, CHO DUKXB-11 cells, CHOK1S cells, and CHO K1M cells.

[0099] In certain embodiments, the cells disclosed herein express a recombinant product of interest. In certain embodiments, the recombinant product of interest is a recombinant protein. In certain embodiments, the recombinant product of interest is a monoclonal antibody. Additional non-limiting examples of recombinant products of interest are described in Section 5.5.

[0100] In certain embodiments, the cells disclosed herein can be used to produce commercially useful amounts of a recombinant product of interest. In certain embodiments, the cells disclosed herein facilitate the production of commercially useful amounts of a recombinant product of interest, at least in part, through higher productivity and higher titer and increased / expanded viability than reference cells, e.g., WT cells.

[0101] In certain embodiments, the cells disclosed herein may contain a nucleic acid encoding a recombinant product of interest. In certain embodiments, the nucleic acid may be present in one or more vectors, such as expression vectors. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors can replicate autonomously in the cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the genome of the cell upon introduction into the cell, thereby replicating along with the cellular genome. In addition, certain vectors, expression vectors, can induce the expression of genes to which they are operably linked. In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids (vectors). Further non-limiting examples of expression vectors for use in the present disclosure include viral vectors that serve equivalent functional roles (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0102] In certain embodiments, the nucleic acid encoding the recombinant product of interest can be introduced into the cells disclosed herein. In certain embodiments, the introduction of the nucleic acid into the cells can be performed by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In certain embodiments, the cells are eukaryotic cells, such as Chinese Hamster Ovary (CHO) cells. In certain embodiments, the cells are lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0103] In certain embodiments, a nucleic acid encoding a recombinant product of interest may be randomly integrated into a host cell genome ("random integration" or "RI"). For example, and not by way of limitation, a nucleic acid encoding a recombinant product of interest may be randomly integrated into the genome of a cell that has also been modified to reduce or eliminate expression of one or more specific endogenous proteins, such as BAX, BAK and / or PERK polypeptides.

[0104] In certain embodiments, a nucleic acid encoding a recombinant product of interest may be integrated into a host cell genome in a targeted manner ("targeted integration" or "TI" as described in detail herein). For example, but not limited to, a nucleic acid encoding a recombinant product of interest may be integrated into a genome of a cell that has been modified to reduce or eliminate expression of one or more specific endogenous proteins, such as BAX, BAK and / or PERK polypeptides. In certain embodiments, the use of TI host cells for the introduction of a nucleic acid encoding a recombinant product of interest results in robust and stable cell culture performance and reduces the risk of sequence variants in the resulting recombinant product of interest. TI host cells, and strategies for their use, are described in detail in U.S. Patent Application Publication No. 20210002669, the entire contents of which are incorporated by reference.

[0105] In certain embodiments using targeted integration, exogenous nucleotide sequence is integrated into the site of specific locus of genome of TI host cell.In certain embodiments, the locus where exogenous nucleotide sequence is integrated is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to the sequence selected from Contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0106] In certain embodiments, the nucleotide sequence immediately 5' of the integrated exogenous sequence is selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 5' of the integrated exogenous sequence is nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 69303-79768 of NW_003615063. NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1.

[0107] In certain embodiments, the nucleotide sequence immediately 3' of the integrated exogenous sequence is selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence immediately 3' of the integrated exogenous sequence is selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 79769-100059 of NW_003615063. NW_006882936.1, nucleotides 2662055-2701768 of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

[0108] In certain embodiments, the integrated exogenous sequence is flanked 5' by a nucleotide sequence selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the integrated exogenous sequence is flanked 3' by a nucleotide sequence selected from the group consisting of nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1, and sequences at least 50% homologous thereto.In certain embodiments, the nucleotide sequence adjacent to the 5' end of the integrated exogenous nucleotide sequence is selected from the group consisting of nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, and nucleotides 69303-79768 of NW_003615 NW_006882936.1, nucleotides 2650443-2662054 of NW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1. In certain embodiments, the nucleotide sequence 3' adjacent to the integrated exogenous nucleotide sequence is selected from the group consisting of nucleotides 45270 to 45490 of NW_006874047.1, nucleotides 207912 to 792374 of NW_006884592.1, nucleotides 491910 to 667813 of NW_006881296.1, nucleotides 79769 to 100059 of NW_0036150 NW_006882936.1, nucleotides 2662055 to 2701768 of NW_006882936.1, and nucleotides 97706 to 105117 of NW_003615411.1.

[0109] In certain embodiments, the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of Contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1, and sequences at least 50% homologous thereto. In certain embodiments, the nucleotide sequence operably linked to the exogenous nucleotide sequence is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or at least about 99.9% homologous to a sequence selected from Contigs NW_006874047.1, NW_006884592.1, NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, and NW_003615411.1.

[0110] In certain embodiments, the nucleic acid encoding the product of interest can be integrated into the host cell genome using transposase-type integration.Transposase-based integration technology is disclosed in, for example, 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, the entire contents of which are incorporated herein by reference.

[0111] In certain embodiments, the nucleic acid encoding the recombinant product of interest may be randomly integrated into the host cell genome ("random integration" or "RI"). In certain embodiments, random integration may be mediated by any method or system known in the art. In certain embodiments, random integration is mediated by the MaxCyte STX® Electroporation System.

[0112] In certain embodiments, targeted integration can be combined with random integration. In certain embodiments, targeted integration can be followed by random integration. In certain embodiments, random integration can be followed by targeted integration. For example, but not limited to, a nucleic acid encoding a desired recombination product can be randomly integrated into the genome of a cell in which the expression of one or more specific endogenous proteins, such as BAX, BAK and / or PERK, is regulated to be reduced or eliminated, and a nucleic acid encoding the same desired recombination product can be integrated into the genome of the cell in a targeted manner.

[0113] In certain embodiments, the cells disclosed herein comprise one or more altered genes. In certain embodiments, the alterations to the genes reduce or eliminate the expression of endogenous proteins. In certain embodiments, the cells disclosed herein comprise one or more altered BAX, BAK and / or PERK genes. In certain embodiments, the subsequent transcription of the altered BAX, BAK and / or PERK genes encodes the endogenous proteins whose expression is reduced or eliminated. In certain embodiments, the one or more altered genes are altered by disruption of the coding region. In certain embodiments, the alterations to the genes comprise biallelic alterations. In certain embodiments, the genetic alteration comprises a deletion of 1 or more base pairs, 2 or more base pairs, 3 or more base pairs, 4 or more base pairs, 5 or more base pairs, 6 or more base pairs, 7 or more base pairs, 8 or more base pairs, 9 or more base pairs, 10 or more base pairs, 11 or more base pairs, 12 or more base pairs, 13 or more base pairs, 14 or more base pairs, 15 or more base pairs, 16 or more base pairs, 17 or more base pairs, 18 or more base pairs, 19 or more base pairs, or 20 or more base pairs.

[0114] In certain embodiments, the present disclosure relates to modified cells, or compositions comprising one or more modified cells, where the modified cells, or compositions comprising one or more modified cells, exhibit one or more of the following characteristics: 1) the modified cells exhibit improved cell culture performance compared to similar cells lacking the modification; and 2) the modified cells exhibit improved viability compared to similar cells lacking the modification.

[0115] In certain embodiments, the present disclosure relates to cells, or compositions comprising one or more of the cells, having all of the following characteristics: 1) the modified cells exhibit improved cell culture performance compared to similar cells lacking the modification; and 2) the modified cells exhibit improved viability compared to similar cells lacking the modification.

[0116] In certain embodiments, the modified cells of the present disclosure exhibit improved cell culture performance compared to similar cells lacking the modification. In certain embodiments, the modified cells of the present disclosure exhibit improved cell culture performance due to i) increased / expanded viability and healthier mitochondria for metabolism, and / or ii) higher productivity and higher titer. In certain embodiments, the modified cells of the present disclosure exhibit increased / expanded viability and healthier mitochondria for metabolism due to reduced or eliminated expression of BAX, BAK and / or PERK. In certain embodiments, the modified cells of the present disclosure exhibit increased / expanded viability and healthier mitochondria for metabolism due to reduced or eliminated expression of BAX. In certain embodiments, the modified cells of the present disclosure exhibit increased / expanded viability and healthier mitochondria for metabolism due to reduced or eliminated expression of BAK. In certain embodiments, the modified cells of the present disclosure exhibit higher productivity and higher titer due to reduced or eliminated expression of PERK. In certain embodiments, the modified cells of the present disclosure exhibit increased / expanded viability and improved cell culture performance due to the reduction or elimination of expression of BAX, BAK and / or PERK.

[0117] In certain embodiments, the present disclosure relates to modified cells or compositions comprising one or more TI cells that exhibit improved cell culture performance. In certain embodiments, the TI cells of the present disclosure exhibit reduced or eliminated expression of BAX, BAK and PERK. In certain embodiments, the TI cells of the present disclosure exhibit reduced or eliminated expression of BAX. In certain embodiments, the TI cells of the present disclosure exhibit reduced or eliminated expression of BAK. In certain embodiments, the TI cells of the present disclosure exhibit reduced or eliminated expression of PERK.

[0118] In certain embodiments, the cell is a cell line. In certain embodiments, the cell is a cell line that has been cultured for a number of generations. In certain embodiments, the cell is a primary cell.

[0119] In certain embodiments, expression of a polypeptide of interest is stable if the expression level is maintained at a particular level and increases or decreases by less than 20% over 10, 20, 30, 50, 100, 200 or 300 generations. In certain embodiments, expression of a polypeptide of interest is stable if the culture can be maintained without any selection. In certain embodiments, expression of a polypeptide of interest is high if the polypeptide product of the gene of interest reaches about 1 g / L, about 2 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 10 g / L, about 12 g / L, about 14 g / L or about 16 g / L.

[0120] The exogenous nucleotide or vector of interest can be introduced into the host cell by conventional cell biology methods, including but not limited to transfection, transduction, electroporation or injection. In certain embodiments, the exogenous nucleotide or vector of interest is introduced into the host cell by chemical-based transfection methods, including lipid-based transfection methods, calcium phosphate-based transfection methods, cationic polymer-based transfection methods, or nanoparticle-based transfection methods. In certain embodiments, the exogenous nucleotide of interest is introduced into the host cell by virus-mediated transduction, including but not limited to lentivirus, retrovirus, adenovirus, or adeno-associated virus-mediated transduction. In certain embodiments, the exogenous nucleotide or vector of interest is introduced into the host cell by gene gun-mediated injection. In certain embodiments, both DNA and RNA molecules are introduced into the host cell using the methods described herein.

[0121] 5.4. Cell culture method In one aspect, the present disclosure provides a method for producing a recombinant product of interest, comprising culturing the modified cells disclosed herein. Culture conditions suitable for mammalian cells known in the art can be used to culture the modified cells disclosed herein (J.Immunol.Methods(1983)56:221-234) or can be easily determined by those skilled in the art (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, BD, eds. Oxford University Press, New York(1992)).

[0122] Cell cultures can be prepared in a medium suitable for the particular cells to be cultured. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma) are exemplary nutrient solutions. In addition, any of the media described in Ham and Wallace, (1979) Meth. Enz., 58:44; Barnes and Sato, (1980) Anal. Biochem., 102:255; U.S. Patent No. 4,767,704; U.S. Patent No. 4,657,866; U.S. Patent No. 4,927,762; U.S. Patent No. 5,122,469 or U.S. Patent No. 4,560,655; International Publication No. WO 90 / 03430; and International Publication No. WO 87 / 00195, the entire disclosures of which are incorporated herein by reference, can be used as culture media. Any of these media may be supplemented as necessary with hormones and / or other growth factors (insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (e.g., adenosine and thymidine), antibiotics (e.g., gentamycin / gentamicin), trace elements (usually defined as inorganic compounds present in final concentrations in the micromolar range), lipids (linoleic acid or other fatty acids) and suitable salts thereof, and glucose, or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[0123] In certain embodiments, the cells modified to reduce and / or eliminate the activity of a particular endogenous protein are CHO cells. Any suitable medium can be used to culture the CHO cells of the present disclosure. In certain embodiments, a suitable medium for culturing CHO cells may contain basal medium components such as a formulation based on DMEM / HAM F-12, optionally containing glycine, hypoxanthine and thymidine; recombinant human insulin, hydrolyzed peptone, e.g., Primatone HS or Primatone RL (Sheffield, England) or equivalent; cytoprotectants, e.g., Pluronic F68 or equivalent pluronic polyol; gentamicin; and trace elements (for the composition of DMEM and HAM F12 media, see Culture Media Formulations in the American Type Culture Collection Catalog of Cell Lines and Hybridomas, 6th Edition, 1988, pp. 346-349) (media formulations as described in U.S. Pat. No. 5,122,469 are particularly suitable).

[0124] In certain embodiments, the cells modified to reduce and / or eliminate the expression of a particular endogenous protein, e.g., BAX, BAK and / or PERK polypeptide, are cells expressing a recombinant product. The recombinant product may be produced by growing cells expressing the recombinant product of interest under various cell culture conditions. For example, cell culture procedures for large-scale or small-scale production of recombinant products may be useful within the context of the present disclosure. Procedures may be used, including but not limited to, fluidized bed bioreactors, hollow fiber bioreactors, roller bottle cultures, shake flask cultures, or stirred tank bioreactor systems (with or without microcarriers in the latter two systems), and may alternatively be operated in batch, fed-batch, or continuous modes.

[0125] In certain embodiments, the cell culture of the present disclosure is carried out in a stirred tank bioreactor system and uses a fed-batch procedure. In fed-batch culture, cells and culture medium are initially fed to the culture vessel, and additional culture nutrients are fed to the culture continuously or in individual increments during the culture, with or without periodic cell and / or product harvesting before the end of the culture. Fed-batch culture can include, for example, semi-continuous fed-batch culture, in which the entire culture (including cells and medium) is periodically removed and replaced with fresh medium. Fed-batch culture is distinguished from simple batch culture, in which all components for cell culture (including cells and all culture nutrients) are fed to the culture vessel at the beginning of the culture process. Fed-batch culture can further be distinguished from perfusion culture, insofar as the supernatant is not removed from the culture vessel during the process (in perfusion culture, the cells are kept in the culture, for example, by filtration, encapsulation, anchoring to microcarriers, and medium is continuously or intermittently introduced and removed from the culture vessel).

[0126] In certain embodiments, the cells of the culture can be grown according to any scheme or routine that may be suitable for the particular cells and the particular production plan being contemplated. Thus, the present disclosure contemplates single-step or multi-step culture procedures. In single-step culture, the cells are inoculated into a culture environment, and the process of the present disclosure is used during a single production phase of the cell culture. Alternatively, multi-step culture is contemplated. In multi-step culture, the cells can be cultured in multiple steps or stages. For example, the cells can be grown in a first step or growth phase medium. In this stage, the cells, optionally removed from storage, are seeded in a medium suitable for promoting growth and high viability. The cells can be maintained in the growth phase for a suitable period of time by adding fresh medium to the cell culture.

[0127] In certain embodiments, flow or continuous cell culture conditions are devised to enhance the growth of mammalian cells in the growth phase of the cell culture. In the growth phase, the cells are grown under conditions and for a period of time maximized for growth. Culture conditions, such as temperature, pH, dissolved oxygen (dO2), etc., will be used for a particular host and will be apparent to one of skill in the art. Generally, pH is adjusted to a level between about 6.5 and 7.5 using either an acid (e.g., CO2) or a base (e.g., Na2CO3 or NaOH). A suitable temperature range for the culture of mammalian cells, such as CHO cells, is between about 30°C and 38°C, with a suitable dO2 of 5-90% of air saturation.

[0128] At certain stages, the cells can be used to seed a production phase or step of a cell culture, or the production phase or step can be continuous with a seeding or growth phase or step, as described above.

[0129] In certain embodiments, the culture method described in the present disclosure may further include harvesting the recombinant product from the cell culture, for example, from the production phase of the cell culture. In certain embodiments, the recombinant product produced by the cell culture method of the present disclosure may be harvested from a third bioreactor, for example, a production bioreactor. For example, but not limited to, the disclosed method may include harvesting the recombinant product at the completion of the production phase of the cell culture. Alternatively or additionally, the recombinant product may be harvested prior to the completion of the production phase. In certain embodiments, the recombinant product may be harvested from the cell culture when a certain cell density is achieved. For example, but not limited to, the cell density may be about 2.0×10 6 cells prior to harvest. 7 cells / mL ~ approx. 5.0×10 7 It may be cells / mL.

[0130] In certain embodiments, recovering the product from the cell culture may include one or more of centrifugation, filtration, sonication, flocculation and cell removal techniques.

[0131] In certain embodiments, the recombinant product of interest may be secreted from the cells or may be a membrane-bound, cytosolic or nuclear protein. In certain embodiments, a soluble form of the recombinant product may be purified from conditioned cell culture medium, and a membrane-bound form of the recombinant product may be purified by preparing a total membrane fraction from the expressing cells and extracting the membranes with a non-ionic detergent such as TRITON® X-100 (EMD Biosciences, San Diego, Calif.). In certain embodiments, a cytosolic or nuclear protein may be prepared by lysing the cells (e.g., by mechanical force, sonication and / or detergent), removing the cell membrane fraction by centrifugation, and retaining the supernatant.

[0132] 5.5 Production of the desired recombinant product The cells and / or methods of the disclosure can be used to produce any recombinant product of interest that can be expressed by the cells disclosed herein.

[0133] 5.5.1 Viral particles and viral vector products In certain embodiments, the cells and / or methods of the present disclosure can be used to produce viral particles or viral vectors. In certain embodiments, the methods of the present disclosure can be used to produce viral particles. In certain embodiments, the methods of the present disclosure can be used to produce viral vectors. In certain embodiments, the methods of the present disclosure can be used to express viral polypeptides. Non-limiting examples of such polypeptides include viral proteins, viral structural (Cap) proteins, viral packaging (Rep) proteins, AAV capsid proteins and viral helper proteins. In certain embodiments, the viral polypeptide is an AAV viral polypeptide.

[0134] In certain embodiments, cells useful in connection with the production of viral particles or viral vectors include, but are not limited to, human embryonic kidney lines (e.g., HEK 293 or HEK 293 cells subcloned for growth in suspension culture), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g., Hep G2, HB 8065), human hepatoma lines (e.g., Hep G2), myeloma cell lines (e.g., Y0, NS0 and Sp2 / 0), monkey kidney CV1 line transformed by SV40 (e.g., COS-7 ATCC CRL-1651), 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 ... ovarian cancer cells (e.g., ATCC CCL 10), mouse Sertoli cells (e.g., TM4), mouse Sertoli cells (e.g., TM4), mouse Sertoli cells (e.g., TM4), mouse Sertoli cells (e.g., TM4), mouse CRL-1587), dog kidney cells (e.g., MDCK, ATCC CCL 34), buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442), mouse mammary tumor (e.g., MMT 060562, ATCC CCL 51), TRI cells, MRC 5 cells, and FS4 cells. In certain embodiments, the cells are CHO cells. Additional non-limiting examples of CHO cells include CHO K1SV cells, CHO DG44 cells, CHO DUKXB-11 cells, CHOK1S cells, and CHO K1M cells.

[0135] In certain embodiments, examples of genes of interest that may be carried by the viral particles produced by the methods described herein include, for example, mammalian polypeptides such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; leptin; clotting factors, such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulants, such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, such as urokinase or human urinary or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; tumor necrosis factor receptors, such as death receptor 5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL); B cell maturation antigen (BCMA); B lymphocyte stimulatory factor (BLyS); proliferation-inducing ligand (APRIL); enkephalinase; RANTES (regulated upon activation and normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, e.g., human serum albumin; Müllerian inhibitory factor; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; microbial proteins, e.g., beta-lactamase; DNase; IgE; cytotoxic T lymphocyte-associated antigen (CTLA ), such as CTLA-4; inhibin; activin; platelet-derived endothelial cell growth factor (PD-ECGF); vascular endothelial growth factor family proteins (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and P1GF); platelet-derived growth factor (PDGF) family proteins (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D and dimers thereof); fibroblast growth factor (FGF) family, such as aFGF, bFGF, FGF4, and FGF9; epidermal growth factor (EGF);Hormone or growth factor receptors, such as VEGF receptors (e.g., VEGFR1, VEGFR2, and VEGFR3), epidermal growth factor (EGF) receptors (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptors), platelet-derived growth factor (PDGF) receptors (e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptors; TIE ligands (angiopoietin, ANGPT1, ANGPT2), angiopoietin receptors, such as TIE1 and TIE2; protein A or D; rheumatoid factor; neurotrophic factors, such as For example, bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors, e.g., NGF-b; transforming growth factors (TGF), TGF-alpha and TGF-beta, e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factors I and II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factors I and II (IGF-I and IGF-II); Threonine-like growth factor binding proteins (IGFBPs); CD proteins, such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; bone morphogenetic factors; immunotoxins; bone morphogenetic proteins (BMPs); chemokines, such as CXCL12 and CXCR4; interferons, such as interferon-alpha, interferon-beta and interferon-gamma; colony stimulating factors (CSFs), such as M-CSF, GM-CSF and G-CSF; cytokines, such as interleukins (ILs), such as IL-1 to IL-2. -10;midkine;superoxide dismutase;T cell receptor;surface membrane protein;decay-accelerating factor;viral antigens, e.g., parts of the AIDS envelope;transport proteins;homing receptors;addressins;regulatory proteins;integrins, e.g., CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM;ephrins;Bv8;delta-like ligand 4 (DLL4);Del-1;BMP9;BMP10;follistatin;hepatocyte growth factor (HGF) / scatter factor (SF);Alk1;Robo4;ESM1;perlecan;EGF-like domain multiple 7 (EGFL7); CTGF and members of its family; thrombospondins, e.g., thrombospondin 1 and thrombospondin 2; collagens, e.g., collagen IV and collagen XVIII; neuropilins, e.g., NRP1 and NRP2; pleiotrophin (PTN); progranulin; proliferin; Notch proteins, e.g., Notch1 and Notch4; semaphorins, e.g., Sema3A, Sema3C, and Sema3F; tumor associated antigens, e.g., CA125 (ovarian cancer antigen); immunoadhesins; and fragments and / or variants of any of the above-mentioned polypeptides, as well as antibodies, including antibody fragments, that bind to one or more proteins, including, for example, any of the above-mentioned proteins;

[0136] In some embodiments, the gene of interest carried by the viral particles produced by the mammalian cells of the present disclosure includes, but is not limited to, 8MPI, 8MP2, 8MP38 (GDF1O), 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, FGF1 O 1A, IL 1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL1 0, IL 11, IL 12A, IL 12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B, IL 18, IL 19, 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 (4-1 BB ligand), TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGF D), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, ILThe present invention may encode a protein that binds to or interacts with any protein, including cytokines, cytokine-related proteins and cytokine receptors selected from the group consisting of: 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.

[0137] INSL4;IRAK1;IRTA2 (immunoglobulin superfamily receptor translocation associated 2);ERAK2;ITGA1;ITGA2;ITGA3;ITGA6 (a6 integrin);ITGAV;ITGB3;ITGB4 (b4 integrin);α4β7 and αEβ7 integrin heterodimer;JAG1;JAK1;JAK3;JUN;K6HF;KAI1;KDR;KITLG;KLF5 (GC Box BP);KLF6;KLKIO;KLK12;KLK13;KLK14;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KRT19 (keratin 19);KRT2A;KHTHB6 (hair-specific H-type keratin);LAMAS;LEP (leptin);LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49,GPR67);Lingo-p75;Lingo-Troy;LPS;LTA (TNF-b );LTB;LTB4R(GPR16);LTB4R2;LTBR;LY64(Lymphocyte antigen 64(RP105), type I membrane protein of the leucine-rich repeat (LRR) family);Ly6E(Lymphocyte antigen 6 complex, locus E;Ly67, RIG-E, SCA-2, TSA-1);Ly6G6D(Lymphocyte antigen 6 complex, locus G6D;Ly6-D,MEGT1);LY6K(Lymphocyte antigen 6 complex, locus K;LY6K;HSJ0013 48;FLJ35226;MACMARCKS;MAG or OMgp;MAP2K7 (c-Jun);MDK;MDP;MIB1;midkine;MEF;MIP-2;MKI67;(Ki-67);MMP2;MMP9;MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin);MS4A1;MSG783 (RNF124, hypothetical protein FLJ20315);MSMB;MT3 (metallothionectin-111);MTSS1;MU C1 (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; OX40; P2RX7; P2X5 (P2X ligand-gated ion receptor channel 5);PAP;PART1;PATE;PAWR;PCA3;PCNA;PD-L1;PD-L2;PD-1;POGFA;POGFB;PECAM1;PF4(CXCL4);PGF;PGR;phosphacan;PIAS2;PIK3CG;PLA U(uPA);PLG;PLXDC1;PMEL17(silver homolog;SILV;D12S53E;PMEL17;SI;SIL);PPBP(CXCL7);PPID;PRI;PRKCQ;PRKDI;PRL;PROC;PROK2;PSAP;PSCA hlg(2700050C12Rik, C530008O16Rik, 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 monocyte-activating cytokine);SDF2;Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, semaphorin 5b Hlog, sema domain, 7 thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B); SERPINA1; SERPINA3; SERP1NB5 (maspin); SERPINE1 (PAI-1); SERPDMF1; SHBG; SLA2; SLC2A2;SLC33A1;SLC43A1;SLIT2;SPPI;SPRR1B (Sprl);ST6GAL1;STABI;STAT6;STEAP (prostate transmembrane epithelial antigen of 6);STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, prostate transmembrane epithelial antigen 2 of 6, transmembrane prostate protein of 6);TB4R2;TBX2 1;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 two follistatin-like domains 1; tomoregulin-1);TMEM46 (shisa homolog 2);TNF;TNF-a;TNFAEP2 (B94);TNFAIP3;TNFRSFIIA;TNFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6 (Fas);TNFRSF7;TNFRSF8;TNFRSF9;T NFSF10(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(4-1 BB ligand);TOLLIP;Toll-like receptor;TOP2A(Topoisomerase Ea);TP53;TPM1;TPM2;TRADD;TMEM118(Ring finger protein, transmembrane 2;RNFT2;FLJ14627);TRAF1;TRAF2;TRAF3;TRAF4;TRAF5;TRAF6;TREM1;TREM2;may bind to or interact with 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 / or ZFPM2.

[0138] Many other viral components and / or other genes of interest may be packaged by mammalian cells in accordance with the present disclosure, and the above list is not meant to be limiting.

[0139] 5.5.2 Recombinant Protein Products In certain embodiments, the cells and / or methods of the present disclosure may be used to produce recombinant proteins, e.g., recombinant mammalian proteins. Non-limiting examples of such recombinant proteins include hormones, receptors, fusion proteins, including antibody fusion proteins (e.g., antibody-cytokine fusion proteins), regulatory factors, growth factors, complement system factors, enzymes, coagulation factors, anticoagulant factors, kinases, cytokines, CD proteins, interleukins, therapeutic proteins, diagnostic proteins, and antibodies. The cells and / or methods of the present disclosure are not specific to the molecule being produced, e.g., antibodies.

[0140] In certain embodiments, the methods of the present disclosure can be used to produce antibodies, including therapeutic and diagnostic antibodies, or 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 consisting of a single heavy chain sequence and a single light chain sequence (including multimers of such pairings)), multispecific antibodies, and antigen-binding fragments thereof. For example, but not limited to, multispecific antibodies can be bispecific antibodies, dual epitope antibodies, T-cell-dependent bispecific antibodies (TDBs), dual acting FAbs (DAFs), or antigen-binding fragments thereof.

[0141] 5.5.2.1 Multispecific antibodies In certain embodiments, the antibodies produced by the cells and methods provided herein are multispecific antibodies, e.g., bispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has at least two different moieties, i.e., binding specificities for different epitopes of different antigens (i.e., bispecific), or different epitopes of the same antigen (i.e., dual epitopes). In certain embodiments, a multispecific antibody has three or more binding specificities. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments as described herein.

[0142] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (see, e.g., WO 2009 / 089004), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992), and WO 2011 / 034605), using common light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605), and using diabody technology to create bispecific antibody fragments (see, e.g., WO 98 / 50431). al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147:60 (1991).

[0143] Engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs, are also included herein (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Further non-limiting examples of multispecific antibodies with 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 acting FAbs" or "DAFs" (see, for example, U.S. Patent No. 2008 / 0069820 and WO 2015 / 095539).

[0144] Multispecific antibodies can also be provided in asymmetric form with domain crossover in one or more binding arms of 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 complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). In certain embodiments, multispecific antibodies comprise cross-Fab fragments. The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which either the variable or constant regions of the heavy and light chains have been exchanged. A cross-Fab fragment comprises a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1) and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to induce correct Fab pairing. See, for example, WO 2016 / 172485.

[0145] A variety of additional molecular formats for multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0146] In certain embodiments, a particular type of multispecific antibody also included herein is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell, e.g., a tumor cell, and to an activation-invariant component of the T cell receptor (TCR) complex, such as CD3, to retarget the T cell to kill the target cell.

[0147] Additional non-limiting examples of bispecific antibody formats that may be useful for this purpose include, but are not limited to, so-called "BiTE" (bispecific T cell engager) molecules in which two scFv molecules are fused by a flexible linker (see, e.g., WO 2004 / 106381, WO 2005 / 061547, WO 2007 / 042261 and WO 2008 / 119567; Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot. Eng. 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies ("TandAbs"; Kipriyanov et al., J Mol Biol 1999, 2010 ... 293, 41-56 (1999); "DART" (dual affinity retargeting) molecules based on the diabody format but featuring a C-terminal disulfide bridge for further stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), as well as the so-called triomabs, which are all-hybrid mouse / rat IgG molecules (reviewed in Seimetz et al., Cancer Treat. Rev. 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO 2013 / 026833, WO 2013 / 026839, WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0148] 5.5.2.2 Antibody fragments In certain embodiments, the antibody produced by the cells and methods provided herein is an antibody fragment. For example, but not limited to, the antibody fragment is a Fab fragment, a Fab' fragment, a Fab'-SH fragment or a F(ab')2 fragment, in particular a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each of which contains the variable domains of the heavy and light chains (VH and VL, respectively) as well as the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain. Thus, a "Fab fragment" is an antibody fragment having a light chain containing a VL domain and a CL domain, and a heavy chain fragment containing a VH domain and a CH1 domain. A "Fab' fragment" differs from a Fab fragment by the addition of residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab')2 fragment that contains two antigen binding sites (two Fab fragments) and a portion of the Fc region. See U.S. Patent No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that comprise salvage receptor binding epitope residues and have increased in vivo half-lives.

[0149] In certain embodiments, the antibody fragment is a diabody, triabody, or tetrabody. A "diabody" is an antibody fragment that has two antigen binding sites, which may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat.Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

[0150] In a further embodiment, the antibody fragment is a single chain Fab fragment. A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, said antibody domains and said linker having one of the following orders from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized via a natural disulfide bond between the CL and CH1 domains. In addition, these single-chain Fab fragments may be further stabilized by the insertion of cysteine ​​residues (e.g., position 44 of the variable heavy chain and position 100 of the variable light chain according to the Kabat numbering) to generate interchain disulfide bonds.

[0151] In another embodiment, the antibody fragment is a single chain variable fragment (scFv). A "single chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy (VH) and light (VL) chains of an antibody connected by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids, usually rich in glycine for flexibility and serine or threonine for solubility, which can connect either the N-terminus of VH to the C-terminus of VL, or vice versa. The protein retains the specificity of the original antibody, even though the constant regions have been removed and the linker introduced. For a review of scFv fragments, see, e.g., Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185 and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0152] In another embodiment, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0153] In certain aspects, the antibody fusion protein produced by the cells and methods provided herein is an antibody-cytokine fusion protein. While such antibody-cytokine fusion proteins can include full-length antibodies, the antibody of the antibody-cytokine fusion protein is, in certain embodiments, an antibody fragment, such as a single-chain variable fragment (scFv), a diabody, a Fab fragment, or a small immune protein (SIP). In certain embodiments, the cytokine can be fused to the N-terminus or C-terminus of the antibody. In certain embodiments, the cytokine of the antibody-cytokine fusion protein is composed of multiple subunits. In certain embodiments, the cytokine subunits are the same (homomeric). In certain embodiments, the cytokine subunits are different (heteromeric). In certain embodiments, the cytokine subunits are fused to the same antibody. In certain embodiments, the cytokine subunits are fused to different antibodies. For a review of antibody-cytokine fusion proteins, see, for example, Murer et al., N Biotechnol., 52:42-53 (2019).

[0154] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of an intact antibody.

[0155] 5.5.2.3 Chimeric and Humanized Antibodies In certain aspects, the antibody produced by the cells and methods provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. The chimeric antibody comprises an antigen-binding fragment thereof.

[0156] In certain aspects, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody also comprises at least a portion of a human constant region. In certain embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0157] Humanized antibodies and methods for making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. No. 5,821,337, U.S. Pat. No. 7,527,791, U.S. Pat. No. 6,982,321, and U.S. Pat. No. 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach to FR shuffling).

[0158] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al. al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0159] 5.5.2.4 Human antibodies In certain embodiments, the antibody produced by the cells and methods provided herein is a human antibody.Human antibodies can be produced using various techniques known in the art.Human antibodies are generally described in van Dijk and van de Winkel, Curr.Opin.Pharmacol.5:368-74(2001) and Lonberg, Curr.Opin.Immunol.20:450-459(2008).

[0160] Human antibodies can be prepared by administering immunogens to transgenic animals that have been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin loci that replace endogenous immunoglobulin loci or are extrachromosomal or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat.Biotech.23:1117-1125(2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0161] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, 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 Boerner et al., J.Immunol., 147:86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc.Natl.Acad.Sci.USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0162] 5.5.2.5 Target molecule Non-limiting examples of molecules that can be targeted by antibodies produced by the cells and methods disclosed herein include soluble serum proteins and their receptors, as well as other membrane-bound proteins (e.g., adhesins). In certain embodiments, the antibodies produced by the cells and methods disclosed herein target 8MPI, 8MP2, 8MP38 (GDFIO), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (αFGF), FGF2 (βFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF1 (HST-2), FGF2 (HST-2), FGF3 (HST-2), FGF4 (HST-2), FGF5 (HST-2), FGF6 (HST-2), FGF7 (KGF), FGF9, FGF1 (HST-2), FGF2 ... 0, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2, IFNA4, IFNA5, IFN A6, 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 (APO3L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HG F(VEGFD), VEGF, VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, IL10RA, IL10RB, IL The cytokines may bind to one, two or more cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of 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.

[0163] In certain embodiments, antibodies produced by the cells and methods disclosed herein are selected from the group consisting of CCLI(1-309), CCL2 (MCP-1 / MCAF), CCL3 (MIP-Iα), CCL4 (MIP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin), CCL13 (MCP-4), CCL15 (MIP-Iδ), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC / Exodus-2), CCL22 (MDC / STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2 / Eotaxin-2), CCL25 (CCL26), CCL27 (CCL28), CCL29 (CCL29), CCL30 (CCL31), CCL32 (CCL33), CCL34 (CCL35), CCL35 (CCL36), CCL37 (CCL38), CCL39 (CCL39), CCL40 (CCL40), CCL41 (CCL41), CCL42 (CCL42), CCL43 (CCL43), CCL44 (CCL44), CCL45 (CCL45), CCL46 (CCL46), CCL47 (CCL47), CCL48 (CCL48), CCL49 (CCL49), CCL50 (CCL50), CCL51 (CCL51), CCL52 (CCL52), CCL53 (CCL53), CCL54 (CCL54), CCL55 (CCL55), CCL56 (CCL57), CCL57 (CCL57), CCL58 (CCL59), CCL59 (CCL59), CCL CL25(TECK), CCL26(eotaxin-3), CCL27(CTACK / ILC), CCL28, CXCLI(GROI), CXCL2(GR02), CXCL 3(GR03), CXCL5(ENA-78), CXCL6(GCP-2), CXCL9(MIG), CXCL10(IP10), CXCL11(1-TAC), CXCL1 2(SDFI), CXCL13, CXCL14, CXCL16, PF4(CXCL4), PPBP(CXCL7), CX3CL1(SCYDI), SCYEI, nhotactin), XCL2 (SCM-Iβ), BLRI (MDR15), CCBP2 (D6 / JAB61), CCRI (CKRI / HM145), CCR2 (mcp-IRB IRA), CCR3(CKR3 / CMKBR3), CCR4, CCR5(CMKBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBII), CCR8(CMKBR8 / TER1 / CKR-L1), CCR9(GPR-9-6), CCRL1(VSHK1), CCRL2(L-CCR), XCR1(GPR5 / CCXCR1), CMKLR1, CMKOR1(R DC1), CX3CR1(V28), CXCR4, GPR2(CCR10), GPR31, GPR81(FKSG80), CXCR3(GPR9 / CKR-L2), CXCR6(TYMSTR / STRL33 / Bonzo), HM74, IL8RA(IL8Rα), IL8RB(I L8Rβ), 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.

[0164] In certain embodiments, antibodies (e.g., multispecific antibodies, such as bispecific antibodies) produced by the methods disclosed herein are capable of binding to one or more target molecules selected from the following: 0772P (CA125, MUC16) (i.e., ovarian cancer antigen), ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1; ADORA2A; aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2; amyloid beta; ANGPTL; ANGPT2; ANGPTL3; A NGPTL4;ANPEP;APC;APOC1;AR;ASLG659;ASPHD1 (Aspartate beta-hydroxylase domain containing 1;LOC253982);AZGP1 (Zinc-a-glycoprotein);B7.1;B7.2;BAD;BAFF-R (B cell activating factor receptor,BLyS receptor 3,BR3;BAG1;BAI1;BCL2;BCL6;BDNF;BLNK;BLRI (MDR15);BMP1;BMP2;BMP3B (GDF10);BMP4;BMP6;BMP8;BMPR1A;BMPR1B (Bone morphogenetic protein receptor -IB type);BMPR2;BPAG1(plectin);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(MT P-2);SLC;Exodus-2;CCL22(MDC / STC-1);CCL23(MPIF-1);CCL24(MPIF-2 / Eotaxin-2);CCL25(TECK);CCL26(Eotaxin-3);CCL27(CTACK / ILC);CCL28;CC L3(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 alpha, 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;C DK7;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;COL 18A1;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's lymphoma receptor 1, G protein-coupled receptor);CXCR6(TYMSTR / STRL33 / Bonzo);CYB5;CYC1;CYSLTR1;DAB2IP;DES;DKFZp451J0118;DNCLI;DPP4;E16(LAT1, SLC7A5);E2F1;ECGF1;EDG1;EFNA1;EFNA3 ;EFNB2;EGF;EGFR;ELAC2;ENG;ENO1;ENO2;ENO3;EPHB4;EphB2R;EPO;ERBB2 (Her-2);EREG;ERK8;ESR1;ESR2;ETBR (endothelin type B receptor);F3 (TF);FADD;FasL;FASN;FCER1A;FCER2;FCGR3A;FcRH1 (Fc receptor-like protein 1);FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain-containing phosphatase anchor protein 1a), SPAP1B, SPAP1C);FGF;FGF1(α FG F5;FGF6(HST-2);FGF7(KGF);FGF8;FGF9;FGFR;FGFR3;FIGF(VEGFD);FELl(EPSILON);FILl(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 alpha 1;GFRA1;GDNFR;GDNFRA;RETL1;TRNR1;RET1L;GDNFR-alpha 1;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 (C10); GRP; GSN (gelsolin); GSTP1; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIF1A; HOP1; histamine and histamine receptor; HLA-A; HLA-DOB (beta 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; IFN-cancer Ma;DFNW1;IGBP1;IGF1;IGF1R;IGF2;IGFBP2;IGFBP3;IGFBP6;IL-l;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;IL1A;IL1B;ILIF10;IL1F5;IL1F6;IL1F7;IL1F8;IL 1F9;IL1HY1;IL1R1;IL1R2;IL1RAP;IL1RAPL1;IL1RAPL2;IL1RL1;IL1RL2, ILIRN;IL2;IL20;IL20Rα;IL21 R;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;IRAK1;IRTA2 (immunoglobulin superfamily receptor translocation associated 2);ERAK2;ITGA1;ITGA2;ITGA3;ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); α4β7 and αEβ7 integrin heterodimers; JAG1; JAK1; JAK3; JUN; K6HF; KAI1; KDR; KITLG; KLF5 (GC Box BP);KLF6;KLKIO;KLK12;KLK13;KLK14;KLK15;KLK3;KLK4;KLK5;KLK6;KLK9;KRT1;KRT19 (keratin 19);KRT2A;KHTHB6 (hair-specific H-type keratin);LAMAS;LEP (leptin);LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49,GPR67);Lingo-p75;Lingo-Troy;LPS;LTA (TNF-b);LTB;LTB4R (GPR16 );LTB4R2;LTBR;LY64 (lymphocyte antigen 64 (RP105), a type I membrane protein of the leucine-rich repeat (LRR) family);Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);Ly6G6D (lymphocyte antigen 6 complex, locus G6D; Ly6-D,MEGT1);LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348; FLJ35226);MACMARCKS;MAG or OMgp; MAP2K7 (c-Jun); MDK; MDP; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2; MMP9; MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin); MS4A1; MSG783 (RNF124, hypothetical protein FLJ20315); MSMB; MT3 (metallothionectin-111); MTSS1; MUC1 (mucin); MYC; MY088; Napi3b (also known as NaPi2b) (NAPI-3B, NPT IIb, 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;OX40; P2RX7;P2X5 (P2X ligand-gated ion channel 5;PAP;PART1;PATE;PAWR;PCA3;PCNA;PD-L1;PD-L2;PD-1;POGFA;POGFB;PECAM1;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, C530008O16Rik, RIKEN cDNA 2700050C12 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 monocyte-activating cytokine);SDF2;Sema 5b(FLJ10372,KIAA1445,Mm.42015,SEMA5B,SEMAG,semaphorin 5b Hlog, sema domain, 7 thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin)5B);SERPINA1;SERPINA3;SERP1NB5 (maspin);SERPINE1 (PAI-1);SERPDMF1;SHBG;SLA2;SLC2A2;SLC33A1;SLC43A1;SLIT2;SPPI;SPRR1B (Sprl);ST6GAL1;STABI;STAT6;STEAP (six prostate transmembrane epithelial antigens);STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer-associated gene 1, prostate cancer-associated protein 1, six prostate transmembrane epithelial antigens 2, six prostate transmembrane 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 two follistatin-like domains 1; tomoregulin-1);TMEM46 (shisa homolog 2);TNF;TNF-a;TNFAEP2 (B 94);TNFAIP3;TNFRSFIIA;TNFRSF1A;TNFRSF1B;TNFRSF21;TNFRSF5;TNFRSF6(Fas);TNFRSF7;TNFRSF8;TNFRSF9;TNFSF10(TRAIL);TNFSF11(TRANCE);TNFSF12(AP03L);TNFSF1 3(April);TNFSF13B;TNFSF14(HVEM-L);TNFSF15(VEGI);TNFSF18;TNFSF4(OX40 ligand);TNFSF5(CD40 ligand);TNFSF6(FasL);TNFSF7(CD27 ligand);TNFSFS(CD30 ligand);TNFSF9(4-1 BB ligand);TOLLIP;Toll-like receptor;TOP2A (Topoisomerase Ea);TP53;TPM1;TPM2;TRADD;TMEM118 (Ring finger protein, transmembrane 2;RNFT2;FLJ14627);TRAF1;TRAF2;TRAF3;TRAF4;TRAF5;TRAF6;TREM1;TREM2;TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, Transient receptor potential cation channel, subfamily M, member 4);TRPC6;TSLP;TWEAK;Tyrosinase (TYR;OCAIA;OCA1A;Tyrosinase;SHEP3);VEGF;VEGFB;VEGFC;Versican;VHL C5;VLA-4;XCL1 (Lymphotactin);XCL2 (SCM-1b);XCRI (GPR5 / CCXCRI);YY1; and ZFPM2. ;

[0165] In certain embodiments, the antibodies produced by the cells and methods disclosed herein are directed to CD proteins, such as 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, CD79β, IGb (immunoglobulin-related beta), B29); CD200 members of the ErbB receptor family, such as the EGF receptor, the HER2 receptor, the HER3 receptor, or the HER4 receptor. cell adhesion molecules, e.g., LFA-1, Mac1, p150.95, VLA-4, ICAM-1, VCAM, alpha4 / beta7 integrin, and alphav / beta3 integrin, e.g., either the alpha or beta subunits thereof (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); growth factors, e.g., VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (alpha IFN); TNF alpha, interleukins, e.g., IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL 17 It can bind to AF, IL-1S, IL-13R alpha 1, IL13R alpha 2, IL-4R, IL-5R, IL-9R, IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F protein, protein C, etc.

[0166] In certain embodiments, the cells and methods provided herein can be used to produce antibodies (or multispecific antibodies, e.g., bispecific antibodies) that specifically bind to complement protein C5 (e.g., anti-C5 agonist antibodies that specifically bind to human C5). In certain embodiments, the anti-C5 antibody comprises one, two, three, four, five, or six CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of SSYYMA (SEQ ID NO: 1); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of AIFTGSGAEYKAEWAKG (SEQ ID NO: 26); (c) a heavy chain variable region CDR3 comprising the amino acid sequence of DAGYDYPTHAMHY (SEQ ID NO: 27); (d) a light chain variable region CDR1 comprising the amino acid sequence of RASQGISSSLA (SEQ ID NO: 28); (e) a light chain variable region CDR2 comprising the amino acid sequence of GASETES (SEQ ID NO: 29); and (f) a light chain variable region CDR3 comprising the amino acid sequence of QNTKVGSSYGNT (SEQ ID NO: 30). For example, in certain embodiments, the anti-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 (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 domain CDR3 comprising the amino acid sequence of QNTKVGSSYGNT (SEQ ID NO: 30). The sequences of CDR1, CDR2 and CDR3 of the heavy chain variable domain and CDR1, CDR2 and CDR3 of the light chain variable domain are disclosed in US 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 US 2016 / 0176954).

[0167] In certain embodiments, the anti-C5 antibody comprises a VH sequence and a VL sequence. QVQLVESGGG LVQPGRSLRL SCAASGFTVH SSYYMAWVRQ APGKGLEWVG AIFTGSGAEY KAEWAKGRVT ISKDTSKNQV VLTMTNMDPV DTATYYCASD AGYDYPTHAM HYWGQGTLVT VSS (SEQ ID NO: 31), respectively and DIQMTQSPSS LSASVGDRVT ITCRASQGIS SSLAWYQQKP GKAPKLLIYG ASETESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQN TKVGSSYGNT FGGGTKVEIK (SEQ ID NO: 32) (including post-translational modifications of these sequences). The above VH and VL sequences are disclosed in US 2016 / 0176954 as SEQ ID NO: 106 and SEQ ID NO: 111, respectively. (See Tables 7 and 8 of US Patent Application Publication No. 2016 / 0176954). In a specific embodiment, the anti-C5 antibody is 305L015 (see US 2016 / 0176954).

[0168] In certain embodiments, the antibody produced by the methods disclosed herein can bind to OX40 (e.g., an anti-OX40 agonist antibody that specifically binds to human OX40). In certain embodiments, the anti-OX40 antibody comprises one, two, three, four, five or six CDRs selected from: (a) heavy chain variable region CDR1 comprising the amino acid sequence of DSYMS (SEQ ID NO:2); (b) heavy chain variable region CDR2 comprising the amino acid sequence of DMYPDNGDSSYNQKFRE (SEQ ID NO:3); (c) heavy chain variable region CDR3 comprising the amino acid sequence of APRWYFSV (SEQ ID NO:4); (d) light chain variable region CDR1 comprising the amino acid sequence of RASQDISNYLN (SEQ ID NO:5); (e) light chain variable region CDR2 comprising the amino acid sequence of YTSRLRS (SEQ ID NO:6); and (f) 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 comprises a VH sequence and a VL sequence. EVQLVQSGAE VKKPGASVKV SCKASGYTFT DSYMSWVRQA PGQGLEWIGD MYPDNGDSSY NQKFRERVTI TRDTSTSTAY LELSSLRSED TAVYYCVLAP RWYFSVWGQG TLVTVSS (SEQ ID NO: 8), respectively. and DIQMTQSPSS LSASVGDRVT ITCRASQDIS NYLNWYQQKP GKAPKLLIYY TSRLRSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GHTLPPTFGQ GTKVEIK (SEQ ID NO: 9) (including post-translational modifications of these sequences).

[0169] In certain embodiments, the anti-OX40 antibody comprises one, two, three, four, five or six CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of NYLIE (SEQ ID NO: 10); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of VINPGSGDTYYSEKFKG (SEQ ID NO: 11); (c) a heavy chain variable region CDR3 comprising the amino acid sequence of DRLDY (SEQ ID NO: 12); (d) a light chain variable region CDR1 comprising the amino acid sequence of HASQDISSYIV (SEQ ID NO: 13); (e) a light chain variable region CDR2 comprising the amino acid sequence of HGTNLED (SEQ ID NO: 14); and (f) a light chain variable region CDR3 comprising the amino acid sequence of VHYAQFPYT (SEQ ID NO: 15). For example, in certain embodiments, the anti-OX40 antibody comprises a heavy chain variable domain (VH) sequence comprising one, two or three CDRs selected from: (a) a heavy chain variable region CDR1 comprising the amino acid sequence of NYLIE (SEQ ID NO: 10); (b) a heavy chain variable region CDR2 comprising the amino acid sequence of VINPGSGDTYYSEKFKG (SEQ ID NO: 11); and (c) a heavy chain variable region CDR3 comprising the amino acid sequence of DRLDY (SEQ ID NO: 12); and / or a light chain variable domain (VL) sequence comprising one, two or three CDRs selected from: (a) a light chain variable region CDR1 comprising the amino acid sequence of HASQDISSYIV (SEQ ID NO: 13); (b) a light chain variable region CDR2 comprising the amino acid sequence of HGTNLED (SEQ ID NO: 14); and (c) a light chain variable region CDR3 comprising the amino acid sequence of VHYAQFPYT (SEQ ID NO: 15). In certain embodiments, the anti-OX40 antibody comprises a VH sequence and a VL sequence. EVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWIGV INPGSGDTYY SEKFKGRVTI TRDTSTSTAY LELSSLRSED TAVYYCARDR LDYWGQGTLV TVSS (SEQ ID NO: 16), respectively. and DIQMTQSPSS LSASVGDRVT ITCHASQDIS SYIVWYQQKP GKAPKLLIYH GTNLEDGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCVH YAQFPYTFGQ GTKVEIK (SEQ ID NO: 17) (including post-translational modifications of these sequences).

[0170] Further details regarding anti-OX40 antibodies are disclosed in WO 2015 / 153513, which is incorporated by reference in its entirety.

[0171] In certain embodiments, antibodies produced by the cells and methods disclosed herein can bind to influenza virus B hemagglutinin, i.e., "fluB" (e.g., antibodies that bind in vitro and / or in vivo to hemagglutinin from the Yamagata lineage of influenza B virus, to hemagglutinin from the Victoria lineage of influenza B virus, to hemagglutinin from an ancestral lineage of influenza B virus, or to hemagglutinin from the Yamagata lineage, Victoria lineage and ancestral lineage of influenza B virus). Further details regarding anti-FluB antibodies are described in WO 2015 / 148806, which is incorporated herein by reference in its entirety.

[0172] In certain embodiments, the antibodies produced by the cells and methods disclosed herein are capable of binding to at least one target selected from the group consisting of low density lipoprotein receptor-related protein (LRP)-1 or LRP-8 or transferrin receptor, and beta-secretase (BACE1 or BACE2), alpha-secretase, gamma-secretase, tau secretase, amyloid precursor protein (APP), death receptor 6 (DR6), amyloid beta peptide, alpha-synuclein, parkin, huntingtin, p75 NTR, CD40, and caspase-6.

[0173] In certain embodiments, the antibody produced by the cells and methods disclosed herein is a human IgG2 antibody against CD40. In certain embodiments, the anti-CD40 antibody is RG7876.

[0174] In certain embodiments, the cells and methods of the present disclosure can be used to produce a polypeptide. For example, but not limited to, the polypeptide is a targeted immunocytokine. In certain embodiments, the targeted immunocytokine is a CEA-IL2v immunocytokine. In certain embodiments, the CEA-IL2v immunocytokine is RG7813. In certain embodiments, the targeted immunocytokine is a FAP-IL2v immunocytokine. In certain embodiments, the FAP-IL2v immunocytokine is RG7461.

[0175] In certain embodiments, a multispecific antibody (such as a bispecific antibody) produced by the cells or methods provided herein can bind to CEA and at least one additional target molecule. In certain embodiments, a multispecific antibody (such as a bispecific antibody) produced according to the methods provided herein can bind to a tumor-targeting cytokine and at least one additional target molecule. In certain embodiments, a multispecific antibody (such as a bispecific antibody) produced according to the methods provided herein is fused to IL2v (i.e., an interleukin 2 variant) and binds to an IL1-based immune cytokine and at least one additional target molecule. In certain embodiments, a multispecific antibody (such as a bispecific antibody) produced according to the methods provided herein is a T cell bispecific antibody (i.e., a bispecific T cell engager or BiTE).

[0176] In certain embodiments, multispecific antibodies (such as bispecific antibodies) produced according to the methods provided herein are directed to any of the following: IL-1 alpha and IL-1 beta, 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-1 beta; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-12 and TWEAK, IL-13 and 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;CD138 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,TNF alpha and TGF-beta,TNF alpha and IL-1 beta;TNF alpha and IL-2, TNF alpha and IL-3, TNF alpha and IL-4, TNF alpha and IL-5, TNF alpha and IL-6, TNF alpha and IL-8, TNF alpha and IL-9, TNF alpha and IL-10, TNF alpha and IL-11, TNF alpha and IL-12, TNF alpha and IL-13, TNF alpha and IL-14, TNF alpha and IL-15, TNF alpha and IL-16, TNF alpha and IL-17, TNF alpha and IL-18, TNF a alpha and IL-19, TNF alpha and IL-20, TNF alpha and IL-23, TNF alpha and IFN alpha, TNF alpha and CD4, TNF alpha and VEGF, TNF alpha and MIF, TNF alpha and ICAM-1, TNF alpha and PGE4, TNF alpha and PEG2, TNF alpha and RANK ligand, TNF alpha and Te38, TNF alpha and BAFF, TNF alpha and CD22, TNF alpha and CTLA-4, TNF alpha 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, IL4 and CD40L, TNFR1 and IL-1 R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A;CTLA-4 and BTN02;IGF1 and IGF2;IGF1 / 2 and Erb2B;MAG and RGM A;NgR and RGM A;It is capable of binding to at least two target molecules selected from NogoA and RGM A; OMGp and RGM A; POL-1 and CTLA-4; and RGM A and RGM B.

[0177] In certain embodiments, a multispecific antibody (such as a bispecific antibody) generated according to the methods provided herein is an anti-CEA / anti-CD3 bispecific antibody. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody is RG7802. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody comprises the amino acid sequence set forth in SEQ ID NOs: 18-21, as shown below: DIQMTQSPSS LSASVGDRVT ITCKASAAVG TYVAWYQQKP GKAPKLLIYS ASYRKRGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCHQ YYTYPLFTFG QGTKLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC (SEQ ID NO: 18) QAVVTQEPSL TVSPGGTVTL TCGSSTGAVT TSNYANWVQE KPGQAFRGLI GGTNKRAPGT PARFSGSLLG GKAALTLSGA QPEDEAEYYC ALWYSNLWVF GGGTKLTVLS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSC (SEQ ID NO: 19) QVQLVQSGAE VKKPGASVKV SCKASGYTFT EFGMNWVRQA PGQGLEWWMGW INTKTGEATY VEEFKGRVTF TTDTSTSTAY MELRSLRSDD TAVYYCARWD FAYYVEAMDY WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDGGGGS GGGGSEVQLL ESGGGLVQPG GSLRLSCAAS GFTFSTYAMN WVRQAPGKGL EWVSRIRSKY NNYATYYADS VKGRFTISRD DSKNTLYLQM NSLRAEDTAV YYCVRHGNFG NSYVSWFAYW GQGTLVTVSS ASVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGECDKT HTCPPCPAPE AAGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALGAPIE KTISKAKGQP REPQVYTLPP CRDELTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK (SEQ ID NO: 20) QVQLVQSGAE VKKPGASVKV SCKASGYTFT EFGMNWVRQA PGQGLEWMG WINTKTGEATY VEEFKGRVTF TTDTSTSTAY MELRSLRSDD TAVYYCARWD FAYYVEAMD YWGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTS GVHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKKVE PKSCDKTHT CPPCPAPEAAG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVH NAKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALGAPIEKTI SKAKGQPRE PQVCTLPPSRD ELTKNQVSLS CAVKGFYPSD ​​IAVEWESNGQ PENNYKTTPP VLDSDGSFF LVSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K (SEQ ID NO: 21)

[0178] Further details regarding anti-CEA / anti-CD3 bispecific antibodies are provided in WO 2014 / 121712, which is incorporated herein by reference in its entirety.

[0179] In certain embodiments, a multispecific antibody (e.g., bispecific antibody) made by the cells and methods disclosed herein is an anti-VEGF / anti-angiopoietin bispecific antibody. In certain embodiments, the anti-VEGF / anti-angiopoietin bispecific antibody is a crossmab. In certain embodiments, the anti-VEGF / anti-angiopoietin bispecific antibody is RG7716. In certain embodiments, the anti-CEA / anti-CD3 bispecific antibody comprises the amino acid sequence set forth in SEQ ID NOs: 22-25, as shown below: EVQLVESGGG LVQPGGSLRL SCAASGYDFT HYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP YYYGTSHWYF DVWGQGTLVT VSSASTKGPS VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMAS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLAQDWL NGKEYKCKVS NKALGAPIEK TISKAKGQPR EPQVYTLPPC RDELTKNQVS LWCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN AYTQKSLSLS PGK (SEQ ID NO: 22) QVQLVQSGAE VKKPGASVKV SCKASGYTFT GYYMHWVRQA PGQGLEWWMGW INPNSGGTNY AQKFQGRVTM TRDTSISTAY MELSRLRSDD TAVYYCARSP NPYYYDSSGY YYPGAFDIWG QGTMVTVSSA SVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGECDKTH TCPPCPAPEA AGGPSVFLFP PKPKDTLMAS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLAQDWL NGKEYKCKVS NKALGAPIEK TISKAKGQPR EPQVCTLPS RDELTKNQVS LSCAVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLVSKLTVDK SRWQQGNVFS CSVMHEALHN AYTQKSLSLS PGK (SEQ ID NO: 23) DIQLTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC (SEQ ID NO: 24) SYVLTQPPSV SVAPGQTARI TCGGNNIGSK SVHWYQQKPG QAPVLVVYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG DEADYYCQVW DSSSDHWVFG GGTKLTVLSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSC (SEQ ID NO: 25)

[0180] In certain embodiments, a multispecific antibody (e.g., a bispecific antibody) generated by the methods disclosed herein is an anti-Ang2 / anti-VEGF bispecific antibody. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is RG7221. In certain embodiments, the anti-Ang2 / anti-VEGF bispecific antibody is CAS number 1448221-05-3.

[0181] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens to generate antibodies. In the case of transmembrane molecules such as receptors, fragments of these (e.g., extracellular domains of receptors) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells can be derived from natural sources (e.g., cancer cell lines) or can be cells that have been transformed by recombinant techniques to express transmembrane molecules. Other antigens and forms thereof useful for preparing antibodies will be apparent to those skilled in the art.

[0182] In certain embodiments, the polypeptides (e.g., antibodies) produced by the cells and methods disclosed herein can be linked to further conjugate to chemical molecules such as dyes or cytotoxic agents, e.g., chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes (i.e., radioconjugates). Immunoconjugates including antibodies or bispecific antibodies produced using the methods described herein can contain a cytotoxic agent conjugated to the constant region of only one of the heavy chains or only one of the light chains.

[0183] 5.5.2.6 Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein, such as those set forth in Section 5.5.5, are contemplated. For example, it may be desirable to change 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 from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics (e.g., antigen binding).

[0184] 5.5.2.6.1 Substitution, insertion and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions" and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. TIFF2024521107000002.tif159170

[0185] Amino acids can be classified according to general 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 affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0186] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0187] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing will have modified (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will have substantially retained specific biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for a specific biological activity (e.g., binding affinity).

[0188] Modifications (e.g., substitutions) can be made in the CDRs, for example to improve antibody affinity. Such modifications can be made to CDR "hot spots", i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or to residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting secondary libraries 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 embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, where several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0189] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may, for example, be outside the antigen contact residues within the CDRs. In the particular variant VH and VL sequences provided above, each CDR is either unmodified or contains no more than one, two, or three amino acid substitutions.

[0190] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted or removed as candidates for substitution. The variants may be screened to determine whether they have the desired properties.

[0191] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody.

[0192] 5.5.2.6.2 Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0193] If the antibody comprises an Fc region, the oligosaccharides attached to the Fc region can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region by N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNac), galactose, and sialic acid, as well as fucose attached to the GlcNac of the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present disclosure can be modified to generate antibody variants with specific improved properties.

[0194] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures that lack fucose attached (directly or indirectly) to the Fc region. Such nonfucosylated oligosaccharides (also called "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc of the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO 2006 / 082515. Asn297 refers to an asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located upstream or downstream of position 297, i.e., about ±3 amino acids between positions 294 and 300, due to minor sequence variations of the antibody. Such antibodies with an increased percentage of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding, and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108 and 2004 / 0093621.

[0195] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or eliminated activity of GDP-fucose synthesis or transporter proteins (see, e.g., U.S. Patent No. 2004259150, U.S. Patent No. 2005031613, U.S. Patent No. 2004132140, U.S. Patent No. 2004110282).

[0196] In further embodiments, the antibody variants are provided with bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999), Ferrara et al., Biotechn Bioeng 93, 851-861 (2006), WO 99 / 54342, WO 2004 / 065540, and WO 2003 / 011878.

[0197] Also provided is an antibody variant that has at least one galactose residue in the oligosaccharide attached to Fc region.Such an antibody variant can have improved CDC function.Such an antibody variant is described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0198] 5.5.2.6.3 Fc Region Variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0199] In certain aspects, the present disclosure contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications where antibody half-life in vivo is important, yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest 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 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo, for example in animal models such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assay can be carried out to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929).

[0200] Antibodies with reduced effector function include those containing substitutions at one or more of residues 238, 265, 269, 270, 297, 327 and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0201] Certain antibody variants have been described with improved or diminished binding to FcRs. (See, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0202] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0203] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., Fc region positions 234 and 235 (residues in EU numbering). 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 a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831). In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in the Fc region derived from a human IgG1 Fc region.

[0204] In some embodiments, modifications are made to the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0205] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve 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, e.g., a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0206] The Fc region residues important for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434 and H435 (EU index numbering) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310 and H435 were found to be important for the interaction of human Fc with mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435 and Y436 are important for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671) reported and investigated various mutant forms of residues 248-259, 301-317, 376-382, and 424-437.

[0207] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., substitutions at positions 253 and / or 310 and / or 435 (residues of EU numbering) of the Fc region. 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 the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0208] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions 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 the Fc region derived from human IgG1 Fc region. (See, for example, WO2014 / 177460A1).

[0209] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., substitutions 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 the Fc region derived from a human IgG1 Fc region. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0210] The C-terminus of the heavy chain of the antibody as reported herein may be a complete C-terminus terminating in amino acid residue PGK. The C-terminus of the heavy chain may be a truncated C-terminus with one or two of the C-terminal amino acid residues removed. In one preferred embodiment, the C-terminus of the heavy chain is PG terminating in a truncated C-terminus. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, amino acid positions of the EU index numbering). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, amino acid position of the EU index numbering).

[0211] 5.5.2.6.4 Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, such as THIOMAB™ antibodies, in which one or more residues of an antibody are replaced by cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0212] 5.5.2.6.5 Antibody derivatives In certain aspects, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. Suitable moieties for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, prolypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing due to its stability in water. The 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 or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative is to be used for therapy under defined conditions, etc.

[0213] 5.5.2.7 Immunoconjugates The present disclosure also provides immunoconjugates comprising an antibody disclosed herein conjugated (chemically linked) to one or more therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of microbial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope.

[0214] In one embodiment, the immunoconjugate is an antibody drug conjugate (ADC) in which an antibody is conjugated to one or more of the above-mentioned therapeutic agents. The antibody is typically connected to one or more therapeutic agents using a linker. A review of ADC technology, including examples of therapeutic agents and drugs and linkers, is provided in Pharmacol Review 68:3-19 (2016).

[0215] In another embodiment, the immunoconjugate comprises an antibody that binds to an enzymatically active toxin or fragment thereof as described herein, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aethiops curcas protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitgellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0216] In another embodiment, the immunoconjugate comprises the antibody described herein conjugated to a radioactive atom to form a radioconjugate.Various radioisotopes are available for the production of radioconjugates.Examples include At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu radioisotopes.When the radioconjugate is used for detection, it can contain a radioactive atom for scintigraphy studies, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0217] Conjugates of antibodies and cytotoxic drugs can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0218] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linking reagents, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0219] 5.6 Exemplary Non-Limiting Embodiments A. A modified cell, wherein the cell is modified to reduce or eliminate expression of two or more endogenous proteins compared to expression of the endogenous proteins in a non-modified cell; (a) one or more of the endogenous proteins whose expression is reduced or eliminated promotes apoptosis of the modified cells in cell culture; (b) Modified cells, in which one or more of the endogenous proteins whose expression is reduced or eliminated regulate the unfolded protein response (UPR).

[0220] B. A modified cell, wherein the cell is modified to reduce or eliminate expression of two or more endogenous proteins compared to expression of the endogenous proteins in a non-modified cell, wherein one or more endogenous proteins are selected from the group of endogenous proteins consisting of BCL2 binding X, apoptosis regulator (BAX) and BCL2 antagonist / killer 1 (BAK), and one of the endogenous proteins is protein kinase R-like ER kinase (PERK).

[0221] B1. Modified cells of B in which expression of BAX, BAK and PERK is reduced or eliminated.

[0222] B2. A modified cell of any one of A-B1, engineered to express a recombinant product of interest.

[0223] B3. A modified cell of any one of A-B1, generated from a recombinant cell expressing a recombinant product of interest.

[0224] B4. Modified cells of B2 or B3, in which one or more endogenous proteins have no detectable expression.

[0225] B5. A modified cell of B2 or B3, in which the recombinant product of interest comprises a viral vector.

[0226] B6. Modified cells of B2 or B3, in which the recombinant product of interest comprises a viral particle.

[0227] B7. The modified cells of B2 or B3, wherein the recombinant product of interest comprises a recombinant protein.

[0228] B8. The modified cell of B7, wherein the recombinant protein is an antibody or an antibody fusion protein or an antigen-binding fragment thereof.

[0229] B9. The modified cell of B8, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

[0230] B10. A modified version of B8, in which the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

[0231] B11. The modified cell of any one of B8 to B10, wherein the antibody is a chimeric antibody, a human antibody or a humanized antibody.

[0232] B12. The modified cell of any one of B8 to B11, wherein the antibody is a monoclonal antibody.

[0233] B13. The modified cell of B2 or B3, wherein the desired recombinant product is encoded by an exogenous nucleic acid sequence integrated into the cell genome at one or more targeted locations.

[0234] B14. Modified cells of any one of A-B13 that do not express detectable BAX, BAK and PERK.

[0235] B15. Modified cells of any one of A-B13, expressing reduced levels of BAX, BAK and PERK.

[0236] B16. The modified cell of any one of A to B15, which is a modified animal cell.

[0237] B17. A modified animal cell of B16, which is a modified Sf9 cell, a CHO cell, a HEK 293 cell, a HEK-293T cell, a BHK cell, an A549 cell or a HeLa cell.

[0238] B18. A composition comprising any one of the modified cells of A-B17.

[0239] B19. (a) culturing any one of the modified cells of A to B17; and (b) recovering the recombinant product of interest from the culture medium or the modified cells. 1. A method for producing a recombinant product of interest, comprising: The method, wherein the modified cells expressing the recombinant product of interest exhibit reduced or eliminated expression of BAX, BAK and PERK.

[0240] C. (a) applying nuclease assistance and / or nucleic acids targeting BAX, BAK and PERK to cells to reduce or eliminate expression of said endogenous genes; and (b) selecting modified cells in which expression of the endogenous gene is reduced or eliminated compared to unmodified cells. A method for producing a modified cell, comprising:

[0241] C1. The method according to C, wherein the modification is performed before introduction of an exogenous nucleic acid encoding a recombinant product of interest or after introduction of an exogenous nucleic acid encoding a recombinant product of interest.

[0242] C2. The method according to C or C1, wherein the nuclease-assisted gene targeting system is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN or meganuclease.

[0243] C3. A method according to C or C1, wherein the reduction in gene expression is mediated by RNA silencing.

[0244] C4. The method according to C3, wherein the RNA silencing is selected from the group consisting of siRNA gene targeting and knockdown, shRNA gene targeting and knockdown, and miRNA gene targeting and knockdown.

[0245] C5. The method of any one of B19 and C1-C4, wherein the recombinant product of interest is encoded by a nucleic acid sequence.

[0246] C6. The method of any one of B19 and C1-C5, wherein the nucleic acid sequence is integrated into the cellular genome of the modified cell at one or more target locations.

[0247] C7. The method of any one of B19 and C1-C5, wherein the recombinant product of interest expressed by the modified cell is encoded by a nucleic acid sequence randomly integrated into the cellular genome of the modified cell.

[0248] C8. The method of any one of B19 and C1-C7, wherein the recombinant product of interest comprises a viral vector.

[0249] C9. The method of any one of B19 and C1-C7, wherein the recombinant product of interest comprises a viral particle.

[0250] C10. The method of any one of B19 and C1-C7, wherein the recombinant product of interest comprises a recombinant protein.

[0251] C11. The method of C10, wherein the recombinant protein is an antibody or an antibody fusion protein or an antigen-binding fragment thereof.

[0252] C12. The method of C11, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

[0253] C13. The method of C11, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

[0254] C14. The method of any one of C11-C13, wherein the antibody is a chimeric antibody, a human antibody, or a humanized antibody.

[0255] C15. The method of any one of C11-C13, wherein the antibody is a monoclonal antibody.

[0256] C16. The method of any one of B19-C15, including purifying the product of interest, harvesting the product of interest, and / or formulating the product of interest.

[0257] C17. The method of C16, wherein the modified cell is a modified animal cell.

[0258] C18. The method of any one of B19-C17, wherein the modified animal cell is a modified Sf9 cell, a CHO cell, a HEK 293 cell, a HEK 293T cell, a BHK cell, an A549 cell, or a HeLa cell.

[0259] C19. The modified cell or method of any of A-C18, having a higher specific productivity than a corresponding isolated animal cell containing a polynucleotide and a functional copy of each of the wild-type Bax, Bak and PERK genes.

[0260] C20. The modified cell or method of any of A-C19, which is more resistant to apoptosis than a corresponding isolated animal cell that contains functional copies of each of the Bax, Bak and PERK genes.

[0261] C21. The modified cell or method of any of A-C20 for use in a fed-batch, perfusion, process intensification, semi-continuous perfusion, or continuous perfusion cell culture process. EXAMPLES

[0262] The following examples are merely illustrative of the subject matter disclosed herein and should not be construed as limiting in any way.

[0263] material and method Vector constructs, cell culture conditions and production Expression of both heavy chain (HC) and light chain (LC) as two separate units was driven by their respective cytomegalovirus (CMV) promoter and regulator elements. Plasmids encoded dihydrofolate reductase (DHFR) or puromycin as selection markers driven by simian virus (SV) 40 early promoter and enhancer elements. The SV40 late polyadenylation (polyA) signal sequence was used in the 3' region of the HC and LC DNA. Cells were cultured in proprietary serum-free DMEM / F12-based medium in a 50 mL tube roller shaking at 150 rpm, 37 °C and 5% CO2, and 4 × 10 5 The cells were passaged every 3 to 4 days at a seeding density of 1000 cells / mL (Hu, et al., 2013).

[0264] Fed-batch production cultures were performed with proprietary chemically defined media using various vessels (tube spinner and AMBR15) with bolus feeding on days 3, 7 and 10 as previously mentioned (Hsu, Aulakh, Traul, & Yuk, 2012). Anti-cell clumping agents were used in all cultures during the production assay to prevent cell clumping due to release of DNA from dead cells. Lean or rich production media were used to culture low (1–2 × 10 6 cells / mL) or high (10 × 10 6Cells were seeded at a seeding density of 1000 x 1000 cells / mL. On day 3, the culture temperature was shifted from 37°C to 35°C. Titers were determined using Protein A affinity chromatography with UV detection. Viability and viable cell counts were determined using a Vi-Cell XR instrument (Beckman Coulter item number 383721).

[0265] CRISPR / Cas9-mediated disruption of PERK (EIF2AK3) The sgRNA primer sequences were as follows: PERK sgRNA 1:5'AGTCACGGCGGGCACTCGCG PERK sgRNA 2:5'TACGGCCGAAGTGACCGTGG PERK sgRNA 3:5'GCGTGACTCATGTTCGCCAG Luciferase sgRNA: 5'ATCCTGTCCCTAGTGGCCC

[0266] Five million cells were washed and suspended in Buffer R (Neon 100uL Kit Catalog:MPK10025 Invitrogen). Five micrograms of Cas9:sgRNA RNP complex was added to the cell culture mixture. Cells were electroporated using 3x10ms pulses at 1,620V. Transfected cells were cultured for 3 days followed by single cell cloning via limiting dilution. Pools and single cell clones were screened for PERK knockout by Western blot analysis.

[0267] RT-PCR analysis to detect IRE1 alpha RNase activity CHO-XBP1s forward primer: 5'CCTTGTAATTGAGAACCAGG CHO-XBP1s reverse primer: 5'CCAAAAGGATATCAGACTCGG

[0268] Power SYBR Green RNA-to CT-1 Step Kit and protocol used from Applied Biosystems (#4389986).

[0269] Immunoblotting and reagents One and a half million cells were lysed in 1x NP40 buffer (10 mM Tris, pH 8.0, 0.5% NP40, 150 mM NaCl, 10 mM DTT, and 5 mM MgCl2) containing a protease inhibitor cocktail (Roche EDTA-free mini-tablet cocktail) for 20 min on ice. Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4–12% Tris glycine) and transferred to nitrocellulose membranes. After blocking with 5% milk in Tris-buffered saline (TBS)-0.1% Tween buffer, the membranes were blotted with the respective antibodies. Blots were visualized using HRP-conjugated anti-rabbit antibodies and SuperSignal West Dura Extended Duration Substrate. The following inhibitors were used: ATF6i (10 μM Ceapin-A7 (Gallagher, et al., 2016)), PERKi (10 μM Compound 39 (Axten, et al., 2012)), IRE1i6 (10 μM 4u8c (Cross, et al., 2012)), IRE1i9 (10 μM in-house / Genentech), PDGFRi (5–20 μM Abcam, AG-1296). The following antibodies were used: anti-PDGFRa (Cell Signaling Technology (CST), D1E1E), rabbit anti-BiP (C50B12, Cell Signaling Technology, 3177), rabbit anti-PERK (CST, C33E10), mouse anti-β-actin-HRP (AC-15) (Abcam, ab49900), rabbit anti-phospho-Akt (Ser473) (CST, D9E), rabbit anti-Akt (CST, 5G3), rabbit cleaved caspase 3 (CST, asp175), goat anti-human IgG-HRP (MP Biomedicals, 0855252), rabbit IRE1a (CST, 14C10), mouse anti-phospho-IRE1, mouse anti-XBP1, rabbit anti-Bax (Abcam, ab32503), rabbit anti-Bak (CST, D4E4), donkey anti-rabbit-HRP (Jackson ImmunoResearch Laboratories, Inc., 711-035-152), rabbit anti-sod2 (CST, D3X8F).

[0270] Example 1: UPR activation attenuates PDGFRa transcription and downregulates its expression An interesting phenomenon was previously described where activation of the UPR during seed train culture, triggered by relatively low pH conditions in a particular CHO cell line, negatively impacted culture growth in production medium at the target pH (Tung, et al., 2018). When exposed to low pH conditions, high intracellular BiP levels were detected in this cell line, which correlated with a low growth profile during production, and poor bioprocess outcomes (Tung, et al., 2018). To better understand the mechanisms underlying the decline in production culture growth under low vs. high pH conditions, seed train cultures were maintained under high and low pH conditions and subjected to proteomic analysis by mass spectrometry. A significant decrease in the expression of PDGFRa protein levels was observed under low pH conditions (Figure 1A), which was associated with transcriptional attenuation of the PDGFRa gene (Figure 1B). Since high intracellular BiP levels indicate UPR activation, we decided to investigate the potential correlation between UPR levels and reduced PDGFRa levels in CHO cells. Tunicamycin (Tun, a strong UPR inducer) and DTT (a weak UPR inducer) were used to chemically induce the UPR in seed train cultures of two antibody-expressing (mAb1) CHO host strains, CHO DG44 and CHO-K1. Under optimal pH conditions, with the strong UPR inducer (Tun), fully functional PDGFRa levels were reduced at both protein and mRNA levels in both CHO host backgrounds (Figure 1C and Figure 1D). Note that BiP levels, as an indicator of UPR activation, increased accordingly in response to strong and weak UPR chemical inducers (Figure 1C). The low molecular weight PDGFRa protein band observed upon tunicamycin treatment represents the non-glycosylated form of this protein, since tunicamycin treatment inhibits protein glycosylation (Figure 1C).

[0271] To further analyze which branch of the UPR is involved in regulating PDGFRa levels, strong UPR inducers (tunicamycin and thapsigargin) were used to induce the UPR in CHO-K1 cells treated with specific inhibitors for the ATF6, PERK or IRE1a branches of the UPR pathway (Figure 1E, 1F and Figure 2A, 2B and 2C). These data revealed that inhibition of the PERK branch of the UPR pathway rescued the downregulation of PDGFRa at the protein level (Figure 1E) and mRNA level (Figure 1F) without affecting the activation of other branches of the UPR, as evidenced by increased levels of intracellular BiP protein and XBP-1 RNA processing in tunicamycin-treated (Figure 1E) and thapsigargin-treated (Figure 2A) cultures. Downregulation of PDGFRa via activation of the PERK branch of the UPR pathway occurred in both antibody-expressing cells (Figure 1E and Figure 2A) and empty host cells (Figure 2B). The slightly lower molecular weight of the PERK protein observed in the presence of the PERK inhibitor is likely due to covalent modification of PERK by this specific inhibitor (Figure 2B).

[0272] Additionally, we designed and tested sgRNAs to knock out the PERK gene in CHO-K1 cells using CRISPR-Cas9 (Figure 2D), and single-cell cloned the transfected pool (sgPERK#2) with the best knockout phenotype to isolate empty CHO-K1 host cell lines that do not express PERK protein (Figure 2E). These empty CHO-K1 PERK KO host cell lines were assessed for growth, transfection rate, recovery in selective medium, and culture performance to identify PERK KO host cell lines with overall culture performance comparable to wild-type (WT) CHO-K1 hosts. Empty WT and empty PERK KO host cell lines (clone 9, Figure 2E) were then treated with or without tunicamycin and a PERK inhibitor to evaluate PDGFRa regulation upon UPR induction (Figure 1G). Compared to WT controls, PDGFRa expression was not downregulated upon UPR induction, and addition of a PERK inhibitor did not further stabilize PDGFRa expression in PERK KO hosts ( Fig. 1G ).

[0273] This investigation on one of our antibody-expressing cell lines, mAb1 CHO DG44, revealed that transcriptional downregulation, and therefore reduced expression of PDGFRa protein, was likely responsible for poor growth outcomes during production when cells were derived from seed train cultures exposed to low pH (Figure 1A and Figure 1B). It was previously shown that this poor growth outcome correlated with increased intracellular BiP levels, indicative of UPR activation (Tung, et al., 2018). When the UPR was chemically induced, PDGFRa protein levels were also reduced due to transcriptional downregulation, a phenomenon that could be reversed by chemical inhibition of the PERK branch of the UPR pathway, suggesting that PERK activation mediates PDGFRa downregulation (Figure 1C, Figure 1D, Figure 1E, Figure 1F and Figure 2A, Figure 2B and Figure 2C). This was further confirmed when chemical induction of the UPR on a PERK KO cell line did not result in downregulation of PDGFRa expression (Figure 1G).

[0274] Example 2: The PDGFRa signaling pathway is important for CHO culture proliferation and functions in parallel with the insulin signaling pathway It was previously shown that UPR-induced poor proliferation profiles correlate with reduced PDGFRa levels (Figure 1A and Figure 1B) (Tung, et al., 2018). Although PDGFRa and insulin signaling pathways have overlapping downstream targets (Figure 3A), insulin signaling negatively regulates PDGFRa signaling (Cirri, et al., 2005). To test the importance of the PDGFRa signaling pathway in CHO cell proliferation, we cultured empty host CHO-K1 cells in the presence of various concentrations of PDGFRa inhibitors, which reduced cell proliferation by approximately 50% at 20 μM concentrations (Figure 3B) due to attenuation of the Akt signaling pathway (Figure 3C). Addition of insulin to CHO cultures treated with PDGFRa inhibitors partially rescued cell proliferation (Figure 3B) and increased Akt phosphorylation, and thus its activation, compared to untreated cultures (Figure 3C). These findings confirmed that the PDGFRa and insulin signaling pathways indeed have overlapping downstream targets in CHO cells, and that the Akt signaling pathway remains intact in the presence of PDGFRa inhibitors (Figure 3B and Figure 3C). PDGFRa signaling is also important for CHO production culture growth, as in an antibody-expressing (mAb2) CHO cell line, inhibition on day 3 of fed-batch production markedly reduced cell growth without affecting cell viability (Figure 3D). Similar to seed train cultures (Figure 3B), the addition of insulin on day 3 of the production cultures partially rescued the observed cell growth inhibition (Figure 3D).

[0275] In our CHO cells cultured in a chemically defined medium without growth factors, the PDGFRa signaling pathway proved to be important for cell proliferation (Figure 4A and Figure 4B), suggesting that our CHO cells secrete PDGFRa ligands or that the PDGFRa signaling pathway is constitutively active in these cells. Addition of insulin to the culture medium partially rescued cell proliferation when PDGFRa signaling was inhibited, and both PDGFRa and insulin receptor (IR) have partially overlapping signaling pathways (Figure 3A), implying that PDGFRa inhibitors are specific and do not affect downstream signaling (Figure 3B and Figure 3C).

[0276] In production cultures, we also observed downregulation of PDGFRa by the PERK branch of the UPR, and the decline in PDGFRa levels toward the end of the culture period coincided with even higher levels of PERK activity, as evidenced by a sharp increase in the mRNA levels of its downstream target proteins (Figures 4C and 4D). Chemical inhibition of PERK prevented the increased transcription of its downstream targets and also stabilized PDGFRa levels during production (Figures 4C and 4D).

[0277] Example 3: Activation of the PERK branch of the UPR attenuates PDGFRa signaling, reduces specific productivity and promotes viability of producing cultures The mAb2-expressing CHO-K1 cell line was used to monitor the correlation between PERK activation and downregulation of PDGFRa expression in production cultures in the absence (control) or presence (added on day 3 of production) of a PERK inhibitor. The downregulation of PDGFRa observed on days 13 and 14 of production culture (Figure 4C, left panel) correlated with an increase in the mRNA levels of the CHOP and GADD34 genes, which are downstream targets of PERK (Marciniak, et al., 2004), indicating activation of the PERK signaling pathway (Figure 4D). Addition of a PERK inhibitor blocked PERK signaling (without increasing the mRNA levels of CHOP and GADD34) and prevented the downregulation of PDGFRa expression (Figure 4C, right panel, and Figure 4D). Because the use of PERK inhibitors is cost-prohibitive and their potential off-target activity on cultured cells cannot be completely ruled out, we decided to generate a PERK KO mAb2-expressing CHO-K1 cell line to directly investigate the role of this signaling pathway in PDGFRa downregulation and production culture performance.

[0278] Using CRISPR-Cas9 technology, the PERK gene was knocked out in the mAb2-expressing CHO-K1 cell line, and after single cell cloning, the derived PERK KO cell line (Figure 5A, underlined clone) with a comparable growth profile to the parental cell line was evaluated in production cultures (Figure 5B and Figure 5C). Although the PERK KO cell lines showed an overall reduction in growth and viability compared to the parental cell line (Figure 5B), the PERK KO cell lines all had higher specific productivity and, for the most part, higher titers compared to the WT parental cell line (Figure 5B). Western blot analysis of these cell lines during production confirmed that PDGFRa levels were stabilized in the PERK KO cell lines compared to the WT parental cell line, which showed a reduction in PDGFRa expression levels toward the end of production (Figure 5C). Although higher levels of intracellular BiP protein in the PERK KO cell line indicated increased UPR activation (Figure 5C), the observed decrease in cell proliferation and viability (Figure 5B) correlated with increased caspase-3 cleavage, implying activation of the apoptotic pathway towards the end of the production culture (Figure 5C). Antibodies expressed by WT or PERK KO cell lines had comparable product quality.

[0279] These findings confirmed that activation of the PERK branch of the UPR downregulated expression of PDGFRa in both seed train and production cultures. Interestingly, PERK KO cultures exhibited lower overall viability and proliferation, but higher titers and specific productivity during production (Figure 5B). Increased intracellular BiP levels and higher levels of caspase-3 cleavage in these cultures, indicating activation of the UPR and apoptotic pathways, respectively, correlated with reduced viability of the cultures (Figure 5C). High levels of specific productivity during production trigger cell apoptosis, and premature PERK activation likely attenuates cell apoptosis by simply reducing the specific productivity of these cells.

[0280] Example 4: Knockout of PERK in a Bax / Bak double knockout CHO cell line dramatically increased specific productivity and titer by enhancing transgene transcription and attenuating apoptotic cell death Because PERK KO clones showed higher levels of apoptosis during production (Figure 5C), we knocked out the PERK gene in mAb3-expressing WT cell lines or in the mAb3-expressing pool of Bax / Bak double knockout (DKO) cell lines (Figure 6A). Bax / Bak are proteins that act at the mitochondria to initiate apoptotic cell death (Taylor, Cullen, & Martin, 2008), and deleting these genes may render the cell lines more resistant to apoptosis and improve viability and productivity during long-term production processes compared to WT CHO cell lines (Misaghi, Qu, Snowden, Chang, & Snedcor, 2013). After single-cell sorting, we compared the PERK / Bax / Bak triple knockout (TKO) clones (Figure 6A) to controls (WT, PERK KO, and Bax / Bak DKO pool) across three different production platforms: 1) using lean production medium, 2) using rich production medium, and 3) using rich production medium in an enriched process. The TKO clones demonstrated good bioprocess results, showing higher titers and relative specific productivity than the control (Figures 6B and 6C and Table 2), while maintaining comparable product quality attributes across all production platforms (Table 3). Similar production platforms testing PERK / Bax / Bak TKO pools and clones clearly reveal that deletion of the PERK gene results in higher specific productive CHO cells expressing antibody (mAb3) or Fab (Fab1) (Figures 7A, 7B and Table 4). These data suggest that the observed increase in specific productivity of Bax / Bak / PERK TKO CHO cells is not clone- or product-specific, but rather a general phenomenon. Table 2. Bioprocess results of mAb3-expressing CHO-K1 TKO cells across different bioprocesses. TIFF2024521107000003.tif113170Table 3. Product quality of mAb3-expressing CHO-K1 TKO cells across different bioprocesses. TIFF2024521107000004.tif49170Table 4. Bioprocessing results of Bax / Bak DKO and PERK / Bax / Bak TKO single cell clones. TIFF2024521107000005.tif57170

[0281] Western blot analysis revealed that PERK / Bax / Bak TKO clones had higher intracellular levels of antibody heavy and light chains in seed trains (Figure 6A) and production medium (Figure 6D) than the parental line. Furthermore, the TKO clones showed stabilized PDGFRa expression during production and no caspase-3 cleavage, indicating inhibition of the apoptotic pathway (Figure 6D) than the parental line. Interestingly, PERK / Bax / Bak TKO clones had higher levels of IRE1a, phospho-IRE1a, and significantly higher levels of spliced ​​XBP-1 transcription factor, indicating that these cells experience increased protein translation and proteostasis stress during production (Figure 6D). TKO clones also showed higher levels of Sod2 protein, implying activation of the reactive oxygen species (ROS) pathway (Figure 6D). These findings suggest that activation of the PERK branch of the UPR during production cumulatively reduces proteostasis stress by decreasing protein translation, as well as attenuation of the IRE1a and ROS pathways, thereby mitigating apoptotic cell death in production cultures. Further investigation of these production processes linked increased specific productivity to increased mRNA levels of heavy and light chain transcripts in TKO clones compared to parental cell lines (Figure 6E). This suggests that the PERK branch of the UPR reduces transgene transcription from the CMV promoter during production, either directly or indirectly, via attenuation of IRE1a or PDGFRa signaling.

[0282] As mentioned above, PERK was knocked out in antibody expressing Bax / Bak double knockout cell lines to prevent apoptosis due to increased specific productivity in PERK KO cell lines (Figure 6A). Surprisingly, the synergistic effect in the producing TKO clones resulted in higher overall titers (up to 8g / L) and relative specific productivity than the parental lines, while having comparable IVCC and viability (Figure 6B, Figure 6C and Table 2). This synergistic effect can be explained by the increased IRE1a signaling caused by the deletion of PERK, which has been shown to attenuate the IRE1a branch of the UPR (Chang, et al., 2018), along with the prolongation of IRE1a activity due to the attenuation of the apoptotic signaling pathway by the deletion of Bax and Bak genes. In our producing TKO clones, we observed increased and prolonged IRE1a signaling, indicated by increased phosphorylation of IRE1a and the increased presence of its downstream target, spliced ​​XBP-1 (Figure 6D). XBP-1 has been shown to transiently improve bioprocessing outcomes (Rajendra, Hougland, Schmitt, & Barnard, 2015), and the observed increase in antibody transcript levels (Figure 6E) suggests that activation of the PERK branch of the UPR attenuates transgene transcription from the CMV promoter, or that PDGFRa and / or IRE1a signaling plays a role in enhancing transcription from the CMV promoter, either directly or through their downstream targets. However, the exact mechanisms and interactions between these signaling pathways remain to be determined.

[0283] The findings presented in this disclosure suggest that chronic activation of the UPR in antibody-expressing CHO cells may cause poor growth, primarily through the PERK pathway, which downregulates PDGFRa levels. The UPR in these cells is primarily caused by proteostasis stress in the ER, which can be caused by many different factors ranging from cell culture parameters to the amino acid sequence and composition of the expressed proteins. This is thought to be a way to promote adaptive growth when protein production, and therefore the load on the ER, is increased. Slowing down cell growth and metabolism by regulating PDGFRa levels can allow more time for ER expansion, which is also regulated by the PERK pathway. Knocking out the PERK pathway may allow cells to grow, but it may also result in apoptosis, as the cells cannot adapt to the additional stress imposed by high rates of specific productivity and protein synthesis. To avoid this, knocking out the PERK pathway in conjunction with deletion of components of the apoptosis pathway (Bax / Bak genes) achieves high rates of specific productivity and increased cell viability. Therefore, this disclosure proposes that knocking out PERK in mammalian protein-expressing host cell lines with attenuated apoptotic pathways may significantly increase specific productivity and therefore culture titers.

[0284] The contents of all figures and all references, patents and published patent applications, and deposit numbers cited throughout this application are hereby expressly incorporated by reference.

Claims

1. A modified cell, wherein the cell is modified to reduce or eliminate expression of two or more endogenous proteins compared to expression of the endogenous proteins in a non-modified cell; (a) one or more of the endogenous proteins whose expression is reduced or eliminated promotes apoptosis of the modified cells in cell culture; (b) A modified cell, in which one or more of the endogenous proteins whose expression is reduced or eliminated regulates the unfolded protein response (UPR).

2. A modified cell, wherein the cell has been modified to reduce or eliminate expression of two or more endogenous proteins compared to expression of the endogenous proteins in a non-modified cell, wherein one or more endogenous proteins are selected from the group of endogenous proteins consisting of BCL2 binding X, apoptosis regulator (BAX) and BCL2 antagonist / killer 1 (BAK), and one of the endogenous proteins is protein kinase R-like ER kinase (PERK).

3. The modified cell of claim 2, wherein expression of BAX, BAK and PERK is reduced or eliminated.

4. 2. The modified cell of claim 1 , engineered to express a recombinant product of interest.

5. The modified cell of claim 1 , produced from a recombinant cell expressing a recombinant product of interest.

6. The modified cell of claim 4 , wherein one or more endogenous proteins have no detectable expression.

7. The modified cell of claim 4 , wherein the recombinant product of interest comprises a viral vector.

8. The modified cell of claim 4 , wherein the desired recombinant product comprises a viral particle.

9. The modified cell of claim 4 , wherein the recombinant product of interest comprises a recombinant protein.

10. 10. The modified cell of claim 9, wherein the recombinant protein is an antibody or an antibody fusion protein or an antigen-binding fragment thereof.

11. The modified cell of claim 10 , wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

12. The modified cell of claim 10 , wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

13. The modified cell of claim 10 , wherein the antibody is a chimeric antibody, a human antibody or a humanized antibody.

14. The modified cell of claim 10 , wherein the antibody is a monoclonal antibody.

15. The modified cell of claim 4 , wherein the desired recombinant product is encoded by an exogenous nucleic acid sequence integrated into the cell genome at one or more target locations.

16. The modified cell of claim 1 , which does not express detectable BAX, BAK, or PERK.

17. The modified cell of claim 1 , which expresses reduced levels of BAX, BAK and PERK.

18. The modified cell of claim 1 which is a modified animal cell.

19. The modified cell of claim 18, wherein the modified animal cell is a modified Sf9 cell, a CHO cell, a HEK 293 cell, a HEK-293T cell, a BHK cell, an A549 cell or a HeLa cell.

20. A composition comprising the modified cells of claim 1.

21. (a) culturing the modified cell of claim 1; and (b) recovering the recombinant product of interest from the culture medium or the modified cells.

1. A method for producing a recombinant product of interest, comprising: The method, wherein the modified cells expressing the recombinant product of interest exhibit reduced or eliminated expression of BAX, BAK and PERK.

22. (a) applying nuclease assistance and / or nucleic acids targeting BAX, BAK and PERK to cells to reduce or eliminate expression of endogenous genes; and (b) selecting modified cells in which expression of the endogenous gene is reduced or eliminated compared to unmodified cells; A method for producing a modified cell, comprising:

23. 23. The method of claim 22, wherein the modification is performed prior to introduction of an exogenous nucleic acid encoding a recombinant product of interest or after introduction of an exogenous nucleic acid encoding a recombinant product of interest.

24. 23. The method of claim 22, wherein the nuclease-assisted gene targeting system is selected from the group consisting of CRISPR / Cas9, CRISPR / Cpf1, zinc finger nuclease, TALEN or meganuclease.

25. 23. The method of claim 22, wherein the reduction in gene expression is mediated by RNA silencing.

26. 26. The method of claim 25, wherein the RNA silencing is selected from the group consisting of siRNA gene targeting and knockdown, shRNA gene targeting and knockdown, and miRNA gene targeting and knockdown.

27. 22. The method of claim 21 , wherein the desired recombinant product is encoded by a nucleic acid sequence.

28. 22. The method of claim 21, wherein the nucleic acid sequence is integrated into the cellular genome of the modified cell at one or more target locations.

29. 22. The method of claim 21, wherein the desired recombinant product expressed by the modified cell is encoded by a nucleic acid sequence that is randomly integrated into the cellular genome of the modified cell.

30. 22. The method of claim 21 , wherein the recombinant product of interest comprises a viral vector.

31. 22. The method of claim 21 , wherein the recombinant product of interest comprises a viral particle.

32. 22. The method of claim 21 , wherein the recombinant product of interest comprises a recombinant protein.

33. 33. The method of claim 32, wherein the recombinant protein is an antibody or an antibody fusion protein or an antigen-binding fragment thereof.

34. 34. The method of claim 33, wherein the antibody is a multispecific antibody or an antigen-binding fragment thereof.

35. 34. The method of claim 33, wherein the antibody consists of a single heavy chain sequence and a single light chain sequence, or an antigen-binding fragment thereof.

36. 34. The method of claim 33, wherein the antibody is a chimeric antibody, a human antibody or a humanized antibody.

37. 34. The method of claim 33, wherein the antibody is a monoclonal antibody.

38. 22. The method of claim 21, comprising purifying the product of interest, harvesting the product of interest, and / or formulating the product of interest.

39. 39. The method of claim 38, wherein the modified cell is a modified animal cell.

40. 22. The method of claim 21, wherein the modified animal cell is a modified Sf9 cell, a CHO cell, a HEK 293 cell, a HEK 293T cell, a BHK cell, an A549 cell or a HeLa cell.

41. 2. The modified cell of claim 1, having a higher specific productivity than a corresponding isolated animal cell that contains a polynucleotide and a functional copy of each of the wild-type Bax, Bak and PERK genes.

42. The modified cell of claim 1 , which is more resistant to apoptosis than a corresponding isolated animal cell that contains functional copies of each of the Bax, Bak and PERK genes.

43. 10. The modified cell of claim 1 for use in a fed-batch, perfusion, process intensification, semi-continuous perfusion, or continuous perfusion cell culture process.

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