Model system and biomarkers to prevent lactate runaway

Abstract

The invention provides a model system for identifying biomarkers of lactate runaway, as well as a panel of biomarkers identified as being associated with lactate runaway. Also provided are methods of detecting or modulating the biomarkers at any stage of cell culture, and or upstream process development stages to control lactate runaway in cell culture.

Description

Attorney Docket No. JB 16944 WOPCT1MODEL SYSTEM AND BIOMARKERS TO PREVENT LACTATE RUNAWAYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 694,065, filed September 12, 2024, which is hereby incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0002] Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. However high levels of lactate have clear negative impacts on cell culture processes and can particularly have a negative impact on bioreactor cell cultures. Cells in some bioreactor cultures may enter a state of lactate accumulation which causes lactate runaway in the bioreactor. The increase in the lactate concentration is responsible for a decrease in growth and productivity.

[0003] Thus there remains a need in the art for compositions and methods that can be used to reduce or prevent lactate accumulation in cell culture systems. The present disclosure meets this unmet need.SUMMARY OF THE INVENTION

[0004] The invention provides a model system for identifying biomarkers of lactate runaway, as well as a set of biomarkers identified as being associated with lactate runaway. Also provided are methods of detecting or modulating the biomarkers to control lactate runaway in cell culture.

[0005] In some embodiments, the invention relates to a model system comprising high-lactate producing cell lines for use in methods of identifying biomarkers associated with an increased risk of lactate runaway and engineering targets for controlling lactate runaway. In some embodiments, the model system further comprises at least one low-lactate producing cell line.

[0006] In some embodiments, the invention relates to a method of identifying biomarkers associated with an increased risk of lactate runaway, engineering targets for preventing or controlling lactate runaway or a combination thereof, the method comprising performing at least one omics assay comprising a high-throughput screening method to measure biological molecules in a systematic way.Attorney Docket No. JB 16944 WOPCT1

[0007] In some embodiments, at least one omics assay comprises a genomic assay, a proteomic assay, a transcriptomic assay, a metabolomic assay, a lipidomic assay, an epigenomic assay, ribosequencing, or any combination thereof. In some embodiments, at least one omics assay is a spatial omics assay, a temporal omics assay, or an activity-based omics assay.

[0008] In some embodiments, the invention relates to a method of identifying a cell as having increased risk of lactate runaway, the method comprising: a) detecting the level or localization of at least one of hexokinase-2(HK2), asparagine synthetase (ASNS), Mitochondrial Ribosome Protein S7 (MRPS7), Mitochondrial Ribosome Protein S5 (MRPS5), Mitochondrial Ribosome Protein S33 (MRPS33), Mitochondrial Ribosome Protein S27 (MRPS27), Mitochondrial Ribosome Protein S18C (MRPS18C), Mitochondrial Ribosome Protein S10B (MRPS10B), Mitochondrial Ribosome Protein Sil (MRPS11), Mitochondrial Ribosome Protein L9 (MRPL9), Mitochondrial Ribosome Protein L58 (MRPL58), Mitochondrial Ribosome Protein L52 (MRPL52), Mitochondrial Ribosome Protein L49 (MRPL49), Mitochondrial Ribosome Protein L48 (MRPL48), Mitochondrial Ribosome Protein L46 (MRPL46), Mitochondrial Ribosome Protein L45 (MRPL45), Mitochondrial Ribosome Protein L41 (MRPL41), Mitochondrial Ribosome Protein L40 (MRPL40), Mitochondrial Ribosome Protein L4 (MRPL4), Mitochondrial Ribosome Protein L37 (MRPL37), Mitochondrial Ribosome Protein L35 (MRPL35), Mitochondrial Ribosome Protein L30 (MRPL30), Mitochondrial Ribosome Protein L3 (MRPL3), Mitochondrial Ribosome Protein L24 (MRPL24), Mitochondrial Ribosome Protein L23 (MRPL23), Mitochondrial Ribosome Protein L19 (MRPL19), Mitochondrial Ribosome Protein L18 (MRPL18), Mitochondrial Ribosome Protein L16 (MRPL16), Mitochondrial Ribosome Protein L15 (MRPL15), Mitochondrial Ribosome Protein LI 4 (MRPL14), Mitochondrial Ribosome Protein Lil (MRPL11), mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS2), Fatty Acid Desaturase 3 (FADS3), Malic Enzyme 1 (MEI), atlastin-1 (ALT1), ATP-dependent 6- phosphofructokinase type C (PFKP), LIM Homeobox 9 (LHX9), pyruvate dehydrogenase phosphatase 1 (PDP1), pyruvate dehydrogenase phosphatase regulatory subunit (PDPR), acylglycerol kinase (AGK), tumor protein 53 (P53), cyclin-dependent kinase inhibitor 1 A (P21 / CDKN1A), cyclin-dependent kinase inhibitor IB (P27 / CDKN1B), Bcl-2-associated X protein (BAX), Bcl-2 homologous antagonist killer (BAK), MAPK Activated Protein Kinase 3 (MAPKAPK3 / MK3), RUN Domain Containing 1 (RUNDC1), dynamin-l-like protein (Dnmll / DPRl), Septin2 (SEPT2), Sirtuin 5 (SIRT5), Sirtuin 7 (SIRT7), Hermansky-PudlakAttorney Docket No. JB 16944 WOPCT1 syndrome 1 (HPS1), ATPase Family AAA Domain Containing 1 (ATAD1), Mitogen- Activated Protein Kinase 9 (MAPK9 / JNK2), and N-myc downstream regulated gene 1 (NDRG1) in the cell; b) comparing the level or localization of the biomarker to a comparator control level or localization of the biomarker; and c) identifying the cell as having an increased risk of lactate runaway when the cell or cell culture has an elevated level of expression one or more of HK2, MEI, PFKP, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, or MAPKAPK3 (MK3), a decreased level of expression at least one of ASNS, MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPE58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11, HMGCS2, FADS3, AET1, PDP1, PDPR or NDRG1, or altered localization of MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPE9, MRPE58, MRPE52, MRPE49, MRPE48, MRPE46, MRPE45, MRPE41, MRPE40, MRPE4, MRPE37, MRPE35, MRPE30, MRPE3, MRPE24, MRPE23, MRPE19, MRPE18, MRPE16, MRPE15, MRPE14, MRPL11, HMGCS2, PFKP, P21 / CDKN1A, EHX9, AGK, P53, RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, or MAPK9 (JNK2) as compared to the comparator control.

[0009] In some embodiments, the comparator control comprises a statistically significant cut-off determined based on the normal level of the biomarker in a cell line or culture of the same type as the cell. In some embodiments, the method further comprises a step of depleting the cell from a cell culture. In some embodiments, the method further comprises removing a cell culture comprising the cell from a production pipeline.

[0010] In some embodiments, the method further comprises a step of contacting the cell with an agent to prevent or control lactate runaway, wherein the agent is an agonist or activator of ASNS, HMGCS2, FADS3, AET1, EHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or an antagonist or inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).

[0011] In some embodiments, the invention relates to a method of treating a cell culture to prevent or control lactate runaway, the method comprising contacting a cell culture with at least one agent to prevent or control lactate runaway, wherein the agent is selected from the group consisting of an agonist or activator of ASNS, HMGCS2, FADS3, AET1, EHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or an antagonistAttorney Docket No. JB 16944 WOPCT1 or inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).

[0012] In some embodiments, the invention relates to an engineered cell comprising a modification to prevent or control lactate runaway, the cell comprising at least one modification selected from the group consisting of insertion of a nucleic acid construct for expression of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or a modification to prevent expression of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).

[0013] In some embodiments, the engineered cell comprises a genetic modification to delete or disrupt expression of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following detailed description of preferred examples of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the examples shown in the drawings.

[0015] Figure 1 depicts in-bioreactor performance trends of a model system to study lactate runaway (lactate, cell growth, and extracellular titer).

[0016] Figure 2A and Figure 2B depict the metabolic characterization methods to study lactate runaway in the model system.

[0017] Figure 3 depicts an in-silico characterization of lactate runaway.

[0018] Figure 4 depicts the proteomics characterization of lactate runaway.

[0019] Figure 5 depicts data demonstrating that the control cells (EE) and lactate runaway cells (HL) have distinct proteomic signatures in the 10 L bioreactor.

[0020] Figure 6 depicts data demonstrating the spatial proteomics characterization of a model system to study lactate runaway.

[0021] Figure 7 depicts data demonstrating that control cells (LL) and lactate runaway cells (HL) have distinct proteomic signatures in the growth media.

[0022] Figure 8 depicts data demonstrating that control (LL) and lactate runaway cells (HL) have distinct proteomic signatures in the growth media.Attorney Docket No. JB 16944 WOPCT1

[0023] Figure 9 depicts data demonstrating the intra and extra cellular lactate levels in two bioreactor systems (10L and 250mL).

[0024] Figure 10 depicts data demonstrating the multi-omics findings on the LDH complex.

[0025] Figure 11 depicts proteomics data demonstrating the high lactate clones display markers of cell cycle arrest and senescence in the 10 L bioreactor.

[0026] Figure 12 depicts proteomics data demonstrating the gene ontology of lactate runaway nuclear proteomics.

[0027] Figure 13 depicts proteomics data demonstrating that P53 is enriched in the nuclei of clones that are predisposed to lactate runaway.

[0028] Figure 14 depicts proteomics data demonstrating the upregulation of P53 targets in lactate runaway clones.

[0029] Figure 15 depicts proteomics data demonstrating that P53 activating factors (Sirtuins, MAPK and JNK) are primed in lactate runaway clones.

[0030] Figure 16 depicts proteomics data demonstrating that cell death factors are primed in lactate runaway clones.

[0031] Figure 17 depicts proteomics data demonstrating that mitochondrial ribosomes are decreased in the 10 L bioreactor in lactate runaway.

[0032] Figure 18 depicts proteomics data demonstrating that mitochondrially encoded genes decrease in the 10 L bioreactor during lactate runaway.

[0033] Figure 19 depicts proteomics data demonstrating that mitochondrial ribosome proteins are localized to the cytosol for clones that are predisposed to lactate runaway.

[0034] Figure 20 provides proteomics evidence for mitochondrial fission in clones that are predisposed to lactate runaway.

[0035] Figure 21 depicts proteomics data demonstrating that Electron Transport Chain (ETC) proteins are enriched in the cytosol of cells that are predisposed to lactate runaway.

[0036] Figure 22 depicts data demonstrating that NAD+ recycling is disrupted in lactate runaway cells.

[0037] Figure 23 depicts metabolomics data from monitoring glycolytic & TCA cycle intermediates.

[0038] Figure 24 depicts metabolomics data demonstrating that the concentration of TCA cycle intermediates are decreased during lactate runaway.Attorney Docket No. JB 16944 WOPCT1

[0039] Figure 25 depicts proteomics data demonstrating that mitochondrial ribosomal proteins are downregulated in 10 L bioreactor during lactate runaway and mislocalized to the cytosol.

[0040] Figure 26 depicts proteomics data demonstrating that HMGCS2 is downregulated in 10 L bioreactor during runaway lactate and mislocalized to the cytosol and has lower predicted flux.

[0041] Figure 27 depicts metabolomics data demonstrating that FADS3 has higher flux with proteomic data demonstrating downregulated in 10 L bioreactors during lactate runaway.

[0042] Figure 28 depicts computational modeling of metabolomics data demonstrating that MEI has increased predicted flux and proteomics data demonstrating upregulation in 10 L bioreactor.

[0043] Figure 29 depicts computational modeling of metabolomics data demonstrating that ALT1 has lower predicted flux with differing directions and is downregulated in 10 L bioreactor.

[0044] Figure 30 depicts proteomics data demonstrating that HK2 is upregulated in 10 L bioreactor and mis-localized to the mitochondria during lactate runaway.

[0045] Figure 31 depicts proteomics data demonstrating that PFKP is upregulated in WCL and translocated to the cytosol during runaway lactate.

[0046] Figure 32 depicts spatial proteomics data demonstrating that LHX9 is mislocalized from nucleus to cytosol during lactate runaway.

[0047] Figure 33 depicts proteomics data demonstrating that ASNS is downregulated in 10 L bioreactor while metabolomics data shows concomitant underutilization of aspartic acid

[0048] Figure 34 depicts proteomics data demonstrating that PDP1 is downregulated in 10 L bioreactor during lactate runaway.

[0049] Figure 35 depicts proteomics data demonstrating that PDPR is downregulated in 10 L bioreactors during lactate runaway.

[0050] Figure 36 depicts spatial proteomics data demonstrating that AGK is mislocalized away from mitochondria.

[0051] Figure 37 depicts spatial proteomics data demonstrating that P53 is mislocalized away from cytosol

[0052] Figure 38 depicts proteomics data demonstrating that P21 / CDKN1A is upregulated in 10 L proteomics and translocated to nucleus during runaway lactate.Attorney Docket No. JB 16944 WOPCT1

[0053] Figure 39 depicts proteomics data demonstrating that P27 / CDKN1B is upregulated in 10 L bioreactor during lactate runaway.

[0054] Figure 40 depicts proteomics data demonstrating that BAX is upregulated in 10 L bioreactor during lactate runaway.

[0055] Figure 41 depicts proteomics data demonstrating that BAK is upregulated in 10 L bioreactor during lactate runaway.

[0056] Figure 42 depicts proteomics data demonstrating that MAPKAPK3 (MK3) is upregulated in 10 L bioreactor during lactate runaway.

[0057] Figure 43 depicts spatial proteomics data demonstrating that RUNDC1 is mislocalized away from nucleus during lactate runaway.

[0058] Figure 44 depicts spatial proteomics data demonstrating that Dnmll (DPR1) is localized to mitochondria during lactate runaway.

[0059] Figure 45 depicts spatial proteomics data demonstrating that SEPTIN2 is localized to mitochondria during lactate runaway.

[0060] Figure 46 depicts spatial proteomics data demonstrating that SIRT5 is localized away from mitochondria during lactate runaway.

[0061] Figure 47 depicts spatial proteomics data demonstrating that SIRT7 is localized to nucleus during lactate runaway.

[0062] Figure 48 depicts spatial proteomics data demonstrating that HPS 1 is mislocalized away from nucleus during lactate runaway.

[0063] Figure 49 depicts spatial proteomics data demonstrating that AT ADI is overexpressed in mitochondria during lactate runaway.

[0064] Figure 50 depicts proteomics data demonstrating that MAPK9 (JNK2) is localized to nucleus during lactate runaway.

[0065] Figure 51 depicts proteomics data demonstrating that NDRG1 is downregulated in 10 L bioreactor and spatial proteomics.

[0066] Figure 52 depicts data demonstrating that the proteomic signature, higher levels of HK2 which were previously seen (Figure 30), was maintained in several high lactate cell lines (TNFR2, EMP2xVB17 and IL- 11).

[0067] Figure 53 depicts the workflow for Seahorse inhibitor validation studies performed with VDAC inhibitors.

[0068] Figure 54 depicts data demonstrating that high lactate (HL) clones show lower oxygen consumption rate (OCR) and basal respiration.Attorney Docket No. JB 16944 WOPCT1

[0069] Figure 55 demonstrates the maximal oxygen consumption rate attained by adding the uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) is lower in HL clones.

[0070] Figure 56 depicts data demonstrating the spare respiratory capacity which, the difference between maximal and basal respiration, indicates the capability of the cell to respond to an energetic demand as well as how closely the cell is respiring to its theoretical maximum.

[0071] Figure 57 depicts data demonstrating that specific HK2 inhibition via VDAC binding domain peptide at lOpM (inhibitory concentration (IC), IC50) rescues OCR of TNFR2 HL clone while inhibition of both HK1 and HK2 with 2-Deoxy-D-glucose fails to rescue OCR.

[0072] Figure 58 depicts data demonstrating that specific HK2 inhibition via VDAC binding domain peptide at lOpM rescues maximal respiration of TNFR2 HL clone while2-Deoxy-D-glucose, which inhibits both HK1 and HK2, at all doses does not rescue maximal respiration of TNFR2 HL clone. 2 -deoxy glucose (2DG) fails to get metabolized in glycolysis and inhibits HK1 and HK2.

[0073] Figure 59A depicts data demonstrating the development of an artificial lactate regulation challenge.

[0074] Figure 59B depicts data demonstrating high and low lactate clones will be evaluated using the artificial lactate regulation challenge.

[0075] Figure 60 depicts data demonstrating the viable cell density (VCD) and viability of high and low lactate clones in the artificial lactate regulation challenge.

[0076] Figure 61 depicts data demonstrating the glucose and lactate levels in high and low lactate clones in the artificial lactate regulation challenge.

[0077] Figure 62 depicts data demonstrating the cell specific lactate production in high and low lactate clones in the artificial lactate regulation challenge.

[0078] Figure 63 depicts data demonstrating the basal and maximal respiration rates are not different in HK2 knockouts of both high and low lactate clones.

[0079] Figure 64 depicts data demonstrating lower proton leak, and increased coupling efficiency in HK2 knockout HL clone. Lower proton leak and increased coupling efficiency in HK2 knockout cells indicates less wasteful respiration resulting from leaking of protons across mitochondrial membrane and increased coupling to ATP synthesis.

[0080] Figure 65 depicts a diagram of the role of ASNS in converting aspartic acid and glutamine to asparagine and glutamate.Attorney Docket No. JB 16944 WOPCT1

[0081] Figure 66 depicts data demonstrating that the proteomic signature, low levels of ASNS which was previously seen (Figure 33), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL-11).

[0082] Figure 67 depicts data demonstrating the levels of asparagine in high and low lactate clones in media and intra-cellular.

[0083] Figure 68 depicts data demonstrating the levels of glutamine in high and low lactate clones.

[0084] Figure 69 depicts the levels of glutamate in high and low lactate clones.

[0085] Figure 70 depicts data demonstrating that ASNS inhibition reduces maximal respiration of LL clones with specific ASNS inhibitor AMA at 5, 10 and 50 pM (0.05%, 0.1% and 0.5%) and with specific and potent ASNS inhibitor ATF4-in2 at 1 pM, 5 pM and 10 pM. A significant drop in OCR is observed at 1 pM with a specific and potent ATF4 inhibitor (inhibitor #2), with IC50= 50nM.

[0086] Figure 71 depicts data demonstrating that ATP coupled respiration is reduced upon specific ASNS inhibition with AMA at 5, 10 and 50 pM (0.05%, 0.1% and 0.5%) and ATF4-in2 at 1 pM, 5 pM and 10 pM.

[0087] Figure 72 depicts data demonstrating that the proteomic signature, high levels of MAPKAPK3 (MK3) which was previously seen (Figure 42), was maintained in the IL-11 high lactate cell line.

[0088] Figure 73 depicts data demonstrating that the proteomic signature, high levels of BAK which was previously seen (Figure 41), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL-11).

[0089] Figure 74 depicts data demonstrating that the proteomic signature, low levels of NDRG1 which was previously seen (Figure 51), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL- 11).

[0090] Figure 75 depicts data demonstrating that the proteomic signature, low levels of HMGCS2 which was previously seen (Figure 26), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL- 11).

[0091] Figure 76 depicts data demonstrating that the proteomic signature, low levels of PDPR which was previously seen (Figure 35), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL-11).

[0092] Figure 77 depicts data demonstrating that the proteomic signature, high levels of MEI which was previously seen (Figure 28), was variable when additional high lactate cell lines (TNFR2, EMR2xVB17 and IL- 11) were evaluated.Attorney Docket No. JB 16944 WOPCT1

[0093] Figure 78 depicts data demonstrating that the proteomic signature, low levels of FADS3 which was previously seen (Figure 27), was variable when additional high lactate cell lines (TNFR2, EMR2xVB17 and IL- 11) were evaluated.

[0094] Figure 79 depicts data demonstrating that the proteomic signature, low levels of SEPTIN-2 which was previously seen (Figure 44), was variable when additional high lactate cell lines (TNFR2, EMR2xVB17 and IL-11) were evaluated.DETAILED DESCRIPTION

[0095] The present disclosure is based, in part, on the development of an assay and model system for high-lactate production including a high-lactate cell and the use of the assay or model system to identify markers that can be used to screen for high lactate cells or can be targeted for inhibition or overexpression to reduce or prevent lactate runaway.

[0096] Therefore, some examples of this disclosure include model systems comprising high-lactate producing cell lines, low-lactate producing cell lines, or a combination of high- and low-lactate producing cell lines for comparative analyses. In some embodiments, the model system is used for a multi-omics analysis to identify biomarkers associated with lactate production levels.

[0097] Some examples of this disclosure include biomarkers identified as being associated with an increased risk of high lactate production and methods of use thereof for screening for high-lactate producing cells using a biomarker or biomarker panel identified as being associated with a risk of high lactate production. In some embodiments, the screening method includes removing a culture comprising high-lactate producing cells from a production pipeline prior to the cell culture being cultured in a bioreactor.

[0098] Some examples of this disclosure include methods of modifying the lactate production of a cell comprising modulating one or more biomarker identified as being associated with a risk of high lactate production. In some embodiments, the method includes genetically modifying at least one cell from a cell culture prior to the cell culture being cultured in a bioreactor.Definitions

[0099] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of some examples of this disclosure.Attorney Docket No. JB 16944 WOPCT1

[0100] As used herein, each of the following terms has the meaning associated with it in this section.

[0101] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical objects of the article. By way of example, “an element” means one element or more than one element.

[0102] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

[0103] The term “bioreactor” as used herein refers to any vessel useful for the growth of a cell culture. The bioreactor can be of any size so long as it is useful for the culturing of cells. In certain embodiments, such cells are mammalian cells. Typically, the bioreactor will be at least 1 liter and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000, 24,000, 36,000 liters or more, or any volume in between, however in some embodiments the bioreactor is a microbioreactor, a nano bioreactor or a microfluidic bioreactor having a volume of less than 1 liter. The internal conditions of the bioreactor, including, but not limited to pH and temperature, are optionally controlled during the culturing period. The bioreactor can be composed of any material that is suitable for holding mammalian cell cultures suspended in media under the culture conditions of the present invention, including glass, plastic or metal. The term “production bioreactor” as used herein refers to the final bioreactor used in the production of the antibody of interest. The volume of the production bioreactor is typically at least 500 liters and may be 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. One of ordinary skill in the art will be aware of and will be able to choose suitable bioreactors for use in practicing the present invention.

[0104] As used herein, the terms “culture”, “culturing”, “cultured”, and “cell culture” refer to a cell population that is suspended in a medium under conditions suitable to survival and / or growth of the cell population. As will be clear from context to those of ordinary skill in the art, these terms as used herein also refer to the combination comprising the cell population and the medium in which the population is suspended. Cell culture includes, e.g., cells grown by batch, fed-batch or perfusion cell culture methods and the like. In certain embodiments, the cell culture is a mammalian cell culture.

[0105] As used herein, the terms “chemically defined medium” or “chemically defined media” refer to a synthetic growth medium in which the identity and concentration ofAttorney Docket No. JB 16944 WOPCT1 all the components are known. Chemically defined media do not contain bacterial, yeast, animal, or plant extracts, animal serum or plasma although they may or may not include individual plant or animal-derived components (e.g., proteins, polypeptides, etc). Chemically defined media may contain inorganic salts such as phosphates, sulfates, and the like needed to support growth. The carbon source is defined, and is usually a sugar such as glucose, lactose, galactose, and the like, or other compounds such as glycerol, lactate, acetate, and the like. While certain chemically defined media also use phosphate salts as a buffer, other buffers may be employed such as citrate, triethanolamine, and the like. As used herein, a chemically defined medium contains no more than micromolar amounts of any metal ion. Examples of commercially available chemically defined mediums include, but are not limited to, ThermoFisher's CD Hybridoma Medium and CD Hybridoma AGT™ Medium, various Dulbecco's Modified Eagle's (DME) mediums (Sigma-Aldrich Co; SAFC Biosciences, Inc), Ham's Nutrient Mixture (Sigma-Aldrich Co; SAFC Biosciences, Inc), combinations thereof, and the like. Methods of preparing chemically defined mediums are known in the art, for example in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007 / 077217, and U.S. Patent Application Publication Nos. 2008 / 0009040 and 2007 / 0212770.

[0106] Ranges: throughout this disclosure, various examples of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Description

[0107] In one embodiment, the invention relates to cell lines for use in methods of identifying biomarkers associated with an increased level of lactate production, mitochondrial dysfunction, or a combination thereof, and the use of the identified biomarkers to detect or prevent lactate runaway.Model System with High-Eactate Expressing CellsAttorney Docket No. JB 16944 WOPCT1

[0108] In some embodiments, the present invention provides an assay and model system comprising a high-lactate producing cell line. In one embodiment, the invention relates to methods of using the model systems of the present invention to identify biomarkers useful for identifying a cell as having a risk of increased lactate production, mitochondrial dysfunction, or a combination thereof. The consistently high-lactate production of the cell line allows for the use of the model system for performing multi-omics analysis, including, but not limited to, genomic, epigenomic, transcriptomic, ribosequencing and proteomic analyses, as well as other pertinent molecular assays to study cell response and lactate production. In some embodiments, the model system can be used to evaluate the effect of libraries of compounds on lactate production, mitochondrial dysfunction, or a combination thereof.

[0109] In certain examples, the model system comprises a cultured cell that has consistent high lactate levels. In some embodiments, the cultured cell is a high lactate producing Chinese hamster ovary (CHO) cell.

[0110] In certain examples, the model system further comprises a cultured cell that has consistent low lactate levels. In some embodiments, the cultured cell is a low lactate producing CHO cell.

[0111] In some embodiments, the invention provides methods of performing one or more omics analyses of the cell line having consistent high lactate levels, consistent low lactate levels or both, to identify biomarkers or engineering targets for controlling or preventing lactate runaway or lactate runaway. Exemplary omics analyses that can be performed include, but are not limited to, genomic, transcriptomic, proteomic, metabolomic, lipidomic, ribosequencing and epigenomic analyses.

[0112] In some embodiments, one or more omics analysis is a spatial omics analysis. Exemplary spatial omics assays include, but are not limited to, spatial proteomics, spatial transcriptomics, or spatial epigenomics.

[0113] In some embodiments, one or more omics analysis is a temporal omics analysis. Exemplary temporal omics assays include, but are not limited to, temporal proteomics, temporal transcriptomics, or temporal epigenomics.

[0114] In some embodiments, one or more omics analysis is an activity-based omics assay. Exemplary activity-based omics assays include, but are not limited to, metabolomics.

[0115] In some embodiments, two or more omics analysis may be combined in a multi-omics approach to identify biomarkers or engineering targets for controlling or preventing lactate runaway or lactate runaway.Attorney Docket No. JB 16944 WOPCT1Biomarkers of increased lactate production or mitochondrial dysfunction

[0116] The present invention relates to the discovery that the level of expression or expression pattern of particular biomarkers were identified as biomarkers of cells with high lactate production and therefore cells which present a risk of lactate runaway. In some embodiments, one or more biomarkers associated with lactate production, mitochondrial dysfunction, or a combination thereof are up-regulated, or expressed at a higher than normal level. In other embodiments, one or more biomarkers associated with lactate production, mitochondrial dysfunction, or a combination thereof are down-regulated, or expressed at a lower than normal level. In other embodiments, one or more biomarkers associated with lactate production, mitochondrial dysfunction, or a combination thereof are mislocalized, or differentially enriched, as compared to the localization or enrichment of the biomarker in normal cells. Thus, the invention relates to compositions and methods useful for monitoring lactate production, mitochondrial dysfunction, or a combination thereof, in cells as well as for identifying cells at risk of lactate runaway, based upon the expression level or expression pattern of one or more biomarkers that is associated with lactate production, mitochondrial dysfunction, or a combination thereof.

[0117] In an exemplary embodiment, at least one biomarker associated with increased lactate production, mitochondrial dysfunction, or a combination thereof, is: MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11, HMGCS2, FADS3, MEI, ALT1, HK2, PFKP, LHX9, ASNS, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), or NDRG1 or any combination thereof. In some embodiments, the invention relates to a screening assay of a cell or cell culture to determine whether the cell or cell culture has an elevated level of expression one or more of HK2, MEI, PFKP, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, or MAPKAPK3 (MK3), and identifying the cell or cell culture as having an increased risk of lactate runaway based on the increased level of the biomarker. In some embodiments, the invention relates to a screening assay of a cell or cell culture to determine whether the cell or cell culture has a decreased level of expression at least one of ASNS, Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40,Attorney Docket No. JB 16944 WOPCT1MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, FADS3, ALT1, PDP1, PDPR or NDRG1, and identifying the cell or cell culture as having an increased risk of lactate runaway based on the decreased level of the biomarker.

[0118] In some embodiments, the invention relates to a screening assay of a cell or cell culture to determine whether the cell or cell culture has spatial differential enrichment or mislocalization of at least one of Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, PFKP, P21 / CDKN1A, LHX9, AGK, P53, RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, or MAPK9 (JNK2), and identifying the cell or cell culture as having an increased risk of lactate runaway based on the altered cellular localization of the biomarker. In some embodiments, Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, AGK, LHX9 RUNDC1, or SIRT5 is enriched in the cytosol. In some embodiments, P21 / CDKN1A, SIRT7, HPS1, or MAPK9 (JNK2) is enriched in the nucleus. In some embodiments, Dnmll (DPR1), SEPTIN2, or ATAD1 is enriched in the mitochondria.

[0119] In some embodiments, the invention provides a method of identifying a cell as having increased risk of lactate runaway, the method comprising detecting the level or localization of at least one biomarker selected from the group consisting of HK2, Mitochondrial Ribosome Proteins, HMGCS2, FADS3, MEI, ALT1, PFKP, LHX9, ASNS, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), and NDRG1 in the cell; comparing the level or localization of the biomarker to a comparator control level or localization of the biomarker; and identifying the cell as having increased risk of lactate runaway when the cell or cell culture has an elevated level of expression one or more of HK2, MEI, PFKP, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, or MAPKAPK3 (MK3), a decreased level of expression at least one of ASNS, Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4,Attorney Docket No. JB 16944 WOPCT1MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, FADS3, ALT1, PDP1, PDPR or NDRG1, or altered localization of Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, PFKP, P21 / CDKN1A, LHX9, AGK, P53, RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, or MAPK9 (JNK2) as compared to the comparator control.

[0120] In some embodiments, the level of one or more or HK2, MEI, PFKP, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, or MAPKAPK3 (MK3) is determined to be increased when the level of one or more of the markers of the invention is increased by at least 1%, by at least 2%, by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100%, when compared to with a comparator control.

[0121] In various embodiments, the level of one or more of markers of the invention in the cell is compared with the level of a corresponding biomarker in a comparator. Nonlimiting examples of comparators include, but are not limited to, a negative control, a positive control, an expected normal background value of the marker, a historical normal background value of the marker, an expected normal background value in a cell population that the cell is a member of, or a historical normal background value of a cell population that the cell is a member of. In one embodiment, the comparator control comprises a statistically significant cut-off determined based on the normal level of the biomarker in a cell line or culture.

[0122] In some embodiments, the level of one or more or ASNS, Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, FADS3, ALT1, PDP1, PDPR or NDRG1 is determined to be decreased when the level of one or more of the markers of the invention is decreased by at least 1%, by at least 2%, by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 100%, when compared to with a comparator control.

[0123] The level of one or more of the markers of the invention can be determined by assessing the amount of polypeptide of one or more of the biomarkers of the invention in theAttorney Docket No. JB 16944 WOPCT1 cell or cell culture, the amount of mRNA of one or more of the biomarkers of the invention in the cell or cell culture, the amount of enzymatic activity of one or more of the biomarkers of the invention in the cell or cell culture, or a combination thereof.

[0124] In some embodiments, a combination of two or more of the biomarkers associated with lactate production, mitochondrial dysfunction, or a combination thereof is identified as a cellular fingerprint. The fingerprint may be a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than 50 specific biomarkers (e.g., the increase or decrease in the abundance, localization, turnover rate or post-translational modification of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more than 50 specific proteins.)

[0125] In some embodiments, the cellular fingerprint can be used to identify cells as having a high risk of lactate runaway, based upon the expression level or expression pattern of one or more biomarkers that is associated with lactate production, mitochondrial dysfunction, or a combination thereof. Cells identified as having a high risk of lactate runaway can be excluded growth in a bioreactor, cell line engineering, continuation through a bioproduct production pipeline, or any combination thereof. In some embodiments, the cellular fingerprint can be used to identify cells as having a low risk of lactate runaway, based upon the expression level or expression pattern of one or more biomarkers that is associated with lactate production, mitochondrial dysfunction, or a combination thereof. Cells identified as having a low risk of lactate runaway can be selected for growth in a bioreactor, for cell line engineering, for continuation through a bioproduct production pipeline, or any combination thereof.

[0126] In some embodiments, one or more biomarkers are up-regulated, or expressed at a higher-than-normal level in a cell having a higher probability of lactate runaway in a bioreactor. In some embodiments, one or more biomarkers are down-regulated, or expressed at a lower-than-normal level in a cell having a higher probability of lactate runaway in a bioreactor.

[0127] In various embodiments, the level of one or more of markers of the invention in the cell is compared with the level of a corresponding biomarker in a comparator. Nonlimiting examples of comparators include, but are not limited to, a negative control, a positive control, an expected normal background value of the marker, a historical normal background value of the marker, an expected normal background value in a cell population that the cell is a member of, or a historical normal background value of a cell population that the cell is aAttorney Docket No. JB 16944 WOPCT1 member of. In one embodiment, the comparator control comprises a statistically significant cut-off determined based on the normal level of the biomarker in a cell line or culture.Engineering Targets for Controlling Lactate Production or Mitochondrial Dysfunction

[0128] In some embodiments, one or more of the biomarkers associated with lactate production, mitochondrial dysfunction, or a combination thereof serve as engineering targets for controlling the level of lactate production. Therefore, in some embodiments, the invention relates to methods of modulating the level or activity of at least one biomarker associated with lactate production, mitochondrial dysfunction, or a combination thereof. In some embodiments the invention relates to methods of increasing the expression level or activity of at least one of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1 or any combination thereof. In some embodiments the invention relates to methods of decreasing the expression level or activity of at least one of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2) or any combination thereof.

[0129] In other embodiments, one or more biomarkers associated with lactate production, mitochondrial dysfunction, or a combination thereof are down-regulated, or expressed at a lower-than-normal level. Thus, the invention relates to compositions and methods useful for monitoring lactate production, mitochondrial dysfunction, or a combination thereof, in cells as wells as for identifying cells at risk of lactate runaway, based upon the expression level or expression pattern of one or more biomarkers that is associated with lactate production, mitochondrial dysfunction, or a combination thereof.

[0130] “Inhibitors,” “activators,” and “modulators” of the markers are used to refer to activating, inhibitory, or modulating molecules that can increase or decrease the expression or activity of a protein or biomarker. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of a biomarker. “Activators” are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate activity of a biomarker, e.g., agonists. Inhibitors, activators, or modulators also include genetically modified versions of the biomarkers, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, RNAi, microRNA, and siRNA molecules, small organic molecules and the like.Attorney Docket No. JB 16944 WOPCT1

[0131] Accordingly, the invention provides methods of modulating a biomarker using an activator (e.g., agonist) or inhibitor (e.g., antagonist) of at least one of HK2, ASNS, Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPE58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPE11), HMGCS2, FADS3, MEI, ALT1, PFKP, LHX9, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), or NDRG1. In some embodiments, the agent to modulate a biomarker comprises an activator of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1. In one embodiment, the activator of at least one of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or any combination thereof, includes but is not limited to a small molecule, a chemical compound, a protein, a peptide, a peptidomemetic, a nucleic acid, and the like.

[0132] In some embodiments, the agent to modulate a biomarker comprises an inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2). In one embodiment, the inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2) or any combination thereof includes, but is not limited to, an antibody or a fragment thereof, a peptide, a nucleic acid, small molecule, a chemical compound, and the like. In some embodiments, the inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2) is specific for HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2). For example, in one embodiment, the inhibitor specifically inhibits HK2, but not hexokinase 1 (HK1).Exemplary HK2 inhibitors include, but are not limited to, inhibitors of VDAC (voltage-dependent anion selective channel), 2-Deoxy-D-glucose (2-DG), Clotrimazole (CEZ), CyclosporinA (CsA), and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In one embodiment the HK2 inhibitor comprises 2-DG.Modified CellsAttorney Docket No. JB 16944 WOPCT1

[0133] In some embodiments, the invention provides genetically modified, or engineered, cell lines that have been modified to have increased or decreased expression of at least one of HK2, ASNS, Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, FADS3, MEI, ALT1, PFKP, LHX9, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), or NDRG1.

[0134] In some embodiments, the genetically modified, or engineered, cell line is a knock-in or knock-out cell line. In some embodiments, the modified or engineered, cell line is a cell line for generating the active substance of a biopharmaceutical medicine. In some embodiments, the modified or engineered, cell line is a cell line for generating protein therapeutics including, but not limited to, monoclonal antibodies (mAbs), peptides, recombinant proteins or antigen production.

[0135] As used herein, “knock-in” means a genetic modification that replaces the genetic information encoded at a chromosomal locus in a cell with a different, or heterologous, DNA sequence, wherein the heterologous, DNA sequence encodes a protein for expression in the modified cell. In some embodiments, the heterologous, DNA sequence further comprises a promoter. In some embodiments, the knock-in cell line comprises a heterologous DNA sequence for overexpression of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or any combination thereof.

[0136] In certain embodiments, the modified cell comprises an exogenous nucleic acid molecule (e.g., DNA or RNA) comprising a nucleic acid sequence that encodes ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or any combination thereof.

[0137] In certain aspects, the present invention comprises methods for modifying a cell comprising introducing to the cell a nucleic acid molecule (e.g., DNA or RNA) comprising a nucleic acid sequence that encodes ASNS,HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or any combination thereof. In certain embodiments, the cell being modified is a high-lactate producing cell and wherein modifying the cell results in the decreased production of lactate in the cell. Transduction or transfection of appropriate cells with a recombinant DNA or RNA moleculeAttorney Docket No. JB 16944 WOPCT1 for overexpression of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1 is accomplished by well-known methods. With regard to transduction or transfection of vertebrate cells with vectors containing recombinant DNA, electroporation, cationic lipid or salt treatment methods can be employed (see, for example, Graham et al, (1973) Virology 52, 456-467; Wigler et al., (1979) Proc. Natl. Acad. Sci. USA 76, 1373-1376). With regard to transfection of vertebrate cells with RNA molecules, RNAs can be delivered to a cell as a DNA molecule, from which an RNA molecule is launched, packaged in a viral replicon particle (VRP), packaged in a lipid nanoparticle (LNP) or delivered as a naked modified or unmodified RNA molecule.

[0138] In some embodiments, the LNPs comprise a cationic lipid to encapsulate and / or enhance the delivery of in vitro transcribed (IVT) RNA into a target cell. The cationic lipid can be any lipid species that carries a net positive charge at a selected pH, such as physiological pH. The lipid nanoparticles can be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and PEG-modified lipids. Several cationic lipids have been described in the literature, many of which are commercially available.

[0139] LNPs can be prepared using methods well known in the art. For example, the LNPs can be prepared using ethanol injection or dilution, thin film hydration, freeze-thaw, French press or membrane extrusion, diafiltration, sonication, detergent dialysis, ether infusion, and reverse phase evaporation.

[0140] Successfully transformed cells can be identified by well-known techniques including the selection for a selectable marker. For example, cells resulting from the introduction of a recombinant DNA of the present invention can be cloned to produce single colonies. Cells from those colonies can be harvested, lysed and their DNA content examined for the presence of the recombinant DNA using a method such as that described by Southern, (1975) J. Mol. Biol. 98, 503-517, PCR, mass spectrometry or the level of proteins produced from the cell assayed via an immunological method.

[0141] As used herein, “knock-out” means an alteration in the sequence of a gene that results in a decrease in the function of the gene. In some embodiments, the knock-out is such that the gene expression is undetectable or insignificant. Knock-outs as used herein also include conditional knock-outs, where alteration of the gene can occur upon, for example, exposure of the cell to a substance that promotes gene alteration, introduction of an enzyme that promotes recombination at a gene site, or other method for directing the gene alteration. In some embodiments, the knock-out cell line comprises a mutation or modification thatAttorney Docket No. JB 16944 WOPCT1 decreases expression of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2) or any combination thereof. In certain embodiments, the cell being modified is a high-lactate producing cell and wherein modifying the cell allows for the generation of a cell line with decreased production of lactate.Genome Editing Compositions

[0142] In one embodiment, the invention provides for generating a modified cell through use of a genome editing system. In some embodiments, the modified or engineered cell is for use for generating the active substance of a biopharmaceutical medicine. In some embodiments, the modified or engineered, cell is for use for generating protein therapeutics including, but not limited to, monoclonal antibodies (mAbs), peptides, recombinant proteins or antigen production.

[0143] A series of programmable nuclease-based genome editing technologies have developed (see for example, Hsu et al., Cell 157, Jun. 5, 2014 1262-1278), including, but not limited to, meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effectorbased nucleases (TALENs) and CRISPR-Cas systems (see e.g. Platt et al., Cell 159(2), 440- 455 (2014); Shalem et al., Science 3 84-87 (2014); and Le Cong et al., Science 339, 819 (2013)) or alternative CRISPR systems. Genome editing systems have a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating, repressing, altering methylation, transferring specific moieties) a target polynucleotide in a multiplicity of cell types.

[0144] In one embodiment, a CRISPR-Cas system is used to integrate the reporter constructs of the invention into a host genome. The CRISPR-Cas system can include at least one guide RNA (gRNA) targeted to a target nucleic acid sequence, and a CRISPR-associated (Cas) peptide form a complex to induce insertion of the reporter constructs at the targeted nucleic acid sequence.

[0145] In one embodiment, the target polynucleotide is a DNA molecule. DNA molecules include, but are not limited to, genomic DNA molecules, extrachromosomal DNA molecules, conjugative plasmids, and exogenous DNA molecules. In some embodiments, the target sequence is a gene encoding Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14,Attorney Docket No. JB 16944 WOPCT1MRPL11), HMGCS2, FADS3, MEI, ALT1, HK2, PFKP, LHX9, ASNS, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), or NDRGE In some embodiments, the target sequence is a regulatory region of a gene encoding Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, FADS3, MEI, ALT1, HK2, PFKP, LHX9, ASNS, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), or NDRGE

[0146] In general, “CRISPR-Cas system” or “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (transactivating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

[0147] In some embodiments, the site of reporter integration is determined by the CRISPR-Cas system guide RNA. In general, a “CRISPR-Cas guide RNA” or “guide RNA” refers to an RNA that directs sequence-specific binding of a CRISPR complex to the target sequence. Typically, a guide RNA comprises (i) a guide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and (ii) a trans-activating cr (tracr) mate sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. In some embodiments, a guideAttorney Docket No. JB 16944 WOPCT1 sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.

[0148] In the context of formation of a CRISPR complex, a “target sequence” or “a sequence of a target DNA” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides or DNA / RNA hybrid polynucleotides. In some embodiments, a target sequence is in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast.

[0149] In some embodiments, the CRISPR-Cas domain comprises a Cas protein. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2. Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Mad7, Cas-Clover, and homologs thereof, orthologs thereof, or modified versions thereof. In some embodiments, the Cas protein has DNA or RNA cleavage activity. In some embodiments, the Cas protein directs cleavage of one or both strands of a nucleic acid molecule at the location of a target sequence, such as within the target sequence and / orAttorney Docket No. JB 16944 WOPCT1 within the complement of the target sequence. In some embodiments, the Cas protein directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.High-Lactate Cell Depleted Cell Culture

[0150] In some embodiments, the invention provides a method of depleting a cell culture of cells having high-lactate lactate production, mitochondrial dysfunction, or a combination thereof. The invention also provides a cell culture composition that has been depleted of cells having high-lactate lactate production, mitochondrial dysfunction, or a combination thereof.

[0151] In some embodiments, the cell culture is for use for generating the active substance of a biopharmaceutical medicine. In some embodiments, the cell culture is for use for generating protein therapeutics including, but not limited to, monoclonal antibodies (mAbs), peptides, recombinant proteins or antigen production.

[0152] In some embodiments, the method comprises identifying a cell in a mixed cell culture as expressing at least one biomarker associated with a risk of increased lactate production, mitochondrial dysfunction, or a combination thereof and removing the cell from the mixed cell culture to generate a high-lactate depleted cell culture. Depletion of cells having high-lactate lactate production, mitochondrial dysfunction, or a combination thereof can be performed by any method known in the art, including, but not limited to, positive or negative selection, or FACS.

[0153] In some embodiments, the invention includes a pipeline which comprises a step of identifying cells or cell lines with decreased risk of lactate runaway for growth in a bioreactor. In some embodiments, the pipeline includes a step of determining the level of at least one protein in the cell or cell line which can be used to identify a cell has likely having decreased risk of lactate runaway and therefore having increased production or fitness in a bioreactor. In some embodiments, the pipeline includes a step of determining the level of at least one protein in the cell or cell line which can be used to identify a cell has likely having increased risk of lactate runaway and therefore decreased production or fitness in a bioreactor, and removing or excluding the identified cell or cell line from a bioreactor or a bioproduction pipeline. Therefore, in some embodiments, the invention includes a step of identifying protein levels within a clone which can be used to identify those clones for further expansion and inoculation of a bioreactor for bioproduct production.Attorney Docket No. JB 16944 WOPCT1Lactate Controlled Cell Culture

[0154] In some embodiments, the invention provides a method of treating a cell culture with an agent to prevent lactate runaway, mitochondrial dysfunction, or a combination thereof. The invention also provides a cell culture composition that has been treated to prevent lactate runaway, mitochondrial dysfunction, or a combination thereof.

[0155] In some embodiments, the cell culture is for use for generating the active substance of a biopharmaceutical medicine. In some embodiments, the cell culture is for use for generating protein therapeutics including, but not limited to, monoclonal antibodies (mAbs), peptides, recombinant proteins or antigen production.

[0156] In some embodiments, the method comprises administering an agent to modulate a molecule associated with increased lactate production, mitochondrial dysfunction, or a combination thereof to a cell culture or to cell culture growth medium.

[0157] In some embodiments, the agent to modulate a molecule associated with increased lactate production, mitochondrial dysfunction, or a combination thereof comprises an activator of HMGCS2, FADS3, ALT1, LHX9, ASNS, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1. In some embodiments, the agent to modulate a molecule associated with increased lactate production, mitochondrial dysfunction, or a combination thereof comprises an inhibitor of MEI, HK2, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).EMBODIMENTS

[0158] This invention provides the following non-limiting embodiments.

[0159] 1. A model system comprising a high-lactate producing cell line for use in methods of identifying biomarkers associated with an increased risk of lactate runaway and engineering targets for controlling lactate runaway.

[0160] 2. The model system of embodiment 1, further comprising at least one low-lactate producing cell line.

[0161] 3. A method of identifying biomarkers associated with an increased risk of lactate runaway, engineering targets for preventing or controlling lactate runaway or a combination thereof, the method comprising performing at least one omics assay comprising a high-throughput screening method to measure biological molecules in a systematic way.Attorney Docket No. JB 16944 WOPCT1

[0162] 4. The method of embodiment 3, wherein the at least one omics assay comprises a genomic assay, a proteomic assay, a transcriptomic assay, a metabolomic assay, a lipidomic assay, an epigenomic assay, ribosequencing or any combination thereof.

[0163] 5. The method of embodiment 3 or 4, wherein the at least one omics assay is a spatial omics assay, a temporal omics assay, or an activity-based omics assay.

[0164] 6. A method of identifying a cell as having an increased risk of lactate runaway, the method comprising: a) detecting the level or localization of at least one biomarker selected from the group consisting of Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, FADS3, MEI, ALT1, HK2, PFKP, LHX9, ASNS, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), and NDRG1 in the cell; b) comparing the level or localization of the biomarker to a comparator control level or localization of the biomarker; and c) identifying the cell as having increased risk of lactate runaway when the cell or cell culture has an elevated level of expression one or more of MEI, HK2, PFKP, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, or MAPKAPK3 (MK3), a decreased level of expression at least one of Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, FADS3, ALT1, ASNS, PDP1, PDPR or NDRG1, or altered localization of Mitochondrial Ribosome Proteins (MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11), HMGCS2, PFKP, P21 / CDKN1A, LHX9, AGK, P53, RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, AT ADI, or MAPK9 (JNK2) as compared to the comparator control.Attorney Docket No. JB 16944 WOPCT1

[0165] 7. The method of embodiment 6, wherein the comparator control comprises a statistically significant cut-off determined based on the normal level of the biomarker in a cell line or culture of the same type as the cell.

[0166] 8. The method of embodiment 6 or 7, further comprising depleting the cell from a cell culture.

[0167] 9. The method of any one of embodiments 6 to 8, further comprising removing a cell culture comprising the cell from a production pipeline.

[0168] 10. The method of any one of embodiments 6 to 9, further comprising a contacting the cell with an agent to prevent or control lactate runaway, wherein the agent is an agonist or activator of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or an antagonist or inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2), or a combination thereof.

[0169] 11. A method of treating a cell culture to prevent or control lactate runaway, the method comprising contacting a cell culture with at least one agent to prevent or control lactate runaway, wherein the agent is selected from the group consisting of an agonist or activator of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or an antagonist or inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).

[0170] 12. An engineered cell comprising a modification to prevent or control lactate runaway, the cell comprising at least one modification selected from the group consisting of insertion of a nucleic acid construct for expression of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or a modification to prevent expression of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).

[0171] 13. The engineered cell of embodiment 12, wherein the cell comprises a genetic modification to delete or disrupt expression of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).Attorney Docket No. JB 16944 WOPCT1EXPERIMENTAL EXAMPLES

[0172] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0173] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the examples of this disclosure and practice the claimed methods. The following working examples therefore, point out specific examples of the disclosure, and are not to be construed as limiting in any way the remainder of the invention.Example 1 : Characterization of a Model System to Study Lactate Runaway

[0174] Figure 1 shows bioreactor data (Lactate, cell growth, and extracellular Titer) from high and low lactate-producing cells. Control and lactate runaway cells have distinct signatures in the 10 L bioreactor. Figure 2 a metabolic characterization of a model system to study lactate runaway.

[0175] Control and lactate runaway cells have different rates of synthesis / consumption of metabolites. Figure 3 shows an in-silico characterization of a model system to study lactate runaway.

[0176] Control and lactate runaway cells have distinct proteomic signatures in the 10 L bioreactor (Figure 4 and Figure 5).

[0177] Control and lactate runaway cells have distinct proteomic signatures in the growth media (Figure 6 - Figure 8).

[0178] Intra and extracellular lactate levels from metabolomics are shown in Figure 9.

[0179] Figure 10 shows lactate flux findings and LDHA / LDHD levels.Example 2: Lactate runaway is associated with cell stress and senescence

[0180] High lactate clones display markers of cell cycle arrest and senescence in the 10 L Bioreactor (Figure 11).

[0181] Figure 12 demonstrates the gene ontology of lactate runaway nuclear proteomics. These data suggest that HL clones are predisposed to cellular senescence, and the control clones display hallmarks of cell growth.Attorney Docket No. JB 16944 WOPCT1

[0182] P53 is enriched in clones that are predisposed to lactate runaway (Figure 13).P53 is enriched in its active site (nucleus) where it acts as a transcription factor. Upregulation of P53 Targets in lactate runaway Clones (Figure 14) indicates that P53 is active in the HL clones. P53 Activating Factors (Sirtuins, MAPK and JNK) are primed in lactate runaway Clones (Figure 15).

[0183] Cell Death Markers are elevated at early timepoints of the 10 L bioreactor and in growth media (Figure 16).Example 3: Mitochondrial Dysfunction is Associated with Lactate Runaway

[0184] Mitochondrial ribosomes decrease in the 10 L bioreactor (Figure 17) which indicates decreased mitochondrial function.

[0185] Mitochondrially encoded genes decrease in the 10 L bioreactor during lactate runaway (Figure 18). These are the products of the mitochondrial ribosome. This decrease is a functional outcome of decreased mitochondrial ribosomes.

[0186] Mitochondria ribosome proteins are localized to the cytosol for clones that are predisposed to lactate runaway (Figure 19) which indicates that the mitochondria of HL clones are compromised and have diminished function.

[0187] Mitochondria fission is observed in clones that are predisposed to lactate runaway (Figure 20) which suggests a phenotype of mitochondrial fragmentation and dysfunction in HL clones.

[0188] Electron transport chain (ETC) proteins are enriched in the cytosol of cells that are predisposed to lactate runaway (Figure 21) which suggests that ETC function is disrupted in HL clones, which is linked to NAD+ recycling and tricarboxylic acid (TCA) cycle function.

[0189] NAD+ recycling is disrupted in the lactate runaway cells (Figure 22). The metabolomics data shows lower concentration of NAD+ in high lactate clone across time.

[0190] Figure 23 shows results from monitoring lactate & TCA intermediates. The data suggests that clones have similar pyruvate levels, yet clones with high lactate exhibit low level citate and succinate (TCA intermediate metabolites).

[0191] TCA cycle intermediates are decreased during lactate runaway (Figure 24). TCA cycle intermediates (citrate and succinate) were examined across two cell lines (one producing low and high lactate levels). In the low lactate clone, citrate levels are significantly higher intracellular and extracellular. Succinate levels are lower in high lactate compared to low lactate.Attorney Docket No. JB 16944 WOPCT1Example 4: Biomarkers and Engineering Targets to Detect and Control Lactate Runaway

[0192] Through a combination of proteomics, spatial proteomics and modeling, a set of biomarkers that can be used to detect cells prone to lactate runaway were identified (Table 1). These markers also serve as engineering targets to control lactate runaway. Figures 25 through 51 depict data demonstrating the behaviors of various biomarkers of Table 1. The biomarkers identified are also engineering targets for controlling lactate runaway. Table 2 provides an overview of the engineering actions for controlling or preventing lactate runaway.Table 1: Biomarkers of Lactate RunawayDescription Gene Name Behavior (Relative to control)Downregulated in 10 L and MislocalizedMitochondrial Ribosome Proteins MRP# [M->C] in GMDownregulated in 10 L and MislocalizedHydroxymethylglutaryl-CoA synthase HMGCS2 [M->C] in GMFatty acid desaturase 3 FADS3 Downregulated in 10 LNADP-dependent malic enzyme 1 MEI Upregulated in 10 LAlanine aminotransferase 1 ALT1 Downregulated in 10 LHexokinase 2 HK2 Upregulated in 10 L and in GMUpregulated and mislocalized [M->C] inATP-dependent 6-phosphofructokinase PFK GMLIM / homeobox protein Lhx9 LHX9 Mislocalized in GM [N->C]Asparagine synthetase ASNS Downregulated in 10 L and in GMPyruvate dehydrogenase [acetyl-transferring]]- phosphatase 1, PDP1 Downregulated in 10 LPyruvate dehydrogenase phosphatase regulatory subunit PDPR Downregulated in 10 LAcylglycerol kinase AGK Mislocalized in Grwoth Media [M->C]Cellular tumor antigen p53 P53 Mislocalized in GM [C->N] Upregulated in 10 L and MislocalizedCyclin-dependent kinase inhibitor 1 P21 / CDKN1A [C->N] in GMCyclin-dependent kinase inhibitor IB P27 / CDKN1B Upregulated in 10 LApoptosis regulator BAX BAX Upregulated in 10 LBcl-2 homologous antagonist / killer BAK Upregulated in 10 L MAPKAPK3MAP kinase-activated protein kinase 3 (MK3) Upregulated in 10 LRUN domain-containing protein 1 RUNDC1 Mislocalized [N-C] in GM DnmllDynamin-l-like protein (DPR1) Mislocalized [C->M] in GMSeptin-2 SEPTIN2 Mislocalized [C->M] in GMNAD-dependent protein deacylase sirtuin-5 SIRT5 Mislocalized [M->C] in GMNAD-dependent protein deacylase sirtuin-7 SIRT7 Mislocalized [C->N] in GMBLOC-3 complex member HPS 1 HPS1 Mislocalized [C->N] in GMOuter mitochondrial transmembrane helix translocase AT ADI Mislocalized [C->M] in GMAttorney Docket No. JB 16944 WOPCT1MAPK9Mitogen-activated protein kinase 9 (JNK2) Mislocalized [C->N] in GMN-myc downstream regulated 1 NDRG1 Downregulated in 10 L and in GMGM = growth mediumC = cytosolM = mitochondriaN = nucleus10 L = 10 L bioreactorMRP# = MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11Table 2: Engineering ActionGene Name ActionHMGCS2 OE / Act / KIFADS 3 OE / Act / KIMEI KO / KD / InhibALT1 OE / Act / KIHK2 KO / KD / InhibPFK KO / KD / InhibLHX9 OE / Act / KIASNS OE / Act / KIPDP1 OE / Act / KIPDPR OE / Act / KIAGK OE / Act / KIP53 KO / KD / InhibP21 / CDKN1A KO / KD / InhibP27 / CDKN1B KO / KD / InhibBAX KO / KD / InhibBAK KO / KD / InhibMAPKAPK3 (MK3) KO / KD / InhibRUNDC1 OE / Act / KIDnmll (DPR1) KO / KD / InhibSEPTIN2 KO / KD / InhibSIRT5 OE / Act / KISIRT7 OE / Act / KIHPS1 OE / Act / KIAT ADI KO / KD / InhibMAPK9 (JNK2) KO / KD / InhibNDRG1 OE / Act / KIKD = knockdownKO = knockoutInhib = inhibitAct = activate OE = overexpressKI = knockinExample 5: Confirmation of HK2 as an engineering target

[0193] High lactate clones exhibit a signature of cellular stress, metabolic dysfunction, and mitochondrial toxicity / mitophagy. One of the most promising targets wasAttorney Docket No. JB 16944 WOPCT1Hexokinase 2 (HK2). Validation experiments were performed in ambrl5 which allowed for triplicate samples and higher throughput.

[0194] Figure 52 demonstrates that the proteomic signature, higher levels of HK2 which was previously seen (Figure 30), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL-11).

[0195] HK2 dissociation from the voltage-dependent anion channel (VDAC) in the outer membrane of mitochondria triggers activation of inositol triphosphate receptors leading to release of calcium from the ER, which is taken up by mitochondria. This influx of calcium into mitochondria leads to oligomerization of VDAC, which is known to form a macromolecule size pore in the outer membrane of mitochondria that allows proteins and mitochondrial DNA (mtDNA) to exit the mitochondria, and is often associated with apoptosis and inflammation. Seahorse inhibitor validation studies were performed with VDAC inhibitors (Figure 53). Cells are continuously exposed to the inhibitor before and throughout seahorse evaluation.

[0196] The high lactate (HL) clones show lower basal respiration and oxygen consumption rate (Figure 54). Basal respiration, the oxygen consumption used to meet cellular ATP demand and engage Kreb’s cycle, is much reduced in the high lactate clones, which shows energetic demand of the cell under baseline conditions. The low lactate (LL) clones at three different cell densities show higher oxygen consumption rate (OCR) compared to HL clones.

[0197] Figure 55 demonstrates the maximal oxygen consumption rate attained by adding the uncoupler FCCP. FCCP mimics a physiological “energy demand” by stimulating the respiratory chain to operate at maximum capacity, which causes rapid oxidation of substrates (sugars, fats, and amino acids) to meet this metabolic challenge. The data shows the maximum rate of respiration that the cell can achieve.

[0198] Figure 56 demonstrates the spare respiratory capacity which indicates the capability of the cell to respond to an energetic demand as well as how closely the cell is to respiring to its theoretical maximum. The cell's ability to respond to demand can be an indicator of cell fitness or flexibility. The spare respiratory capacity is reduced in HL clones.

[0199] Figure 57 demonstrates that HK2 inhibition via VDAC binding domain peptide at lOpM rescues OCR of TNFR2 HL clone. Figure 57 depicts data demonstrating that specific HK2 inhibition via VDAC binding domain peptide at lO M (inhibitory concentration (IC), IC50) rescues OCR of TNFR2 HL clone while inhibition of both HK1 and HK2 with 2-Deoxy-D-glucose fails to rescue OCR.Attorney Docket No. JB 16944 WOPCT1

[0200] Figure 58 demonstrates that specific HK2 inhibition via VDAC binding domain peptide at lOpM rescues maximal respiration of TNFR2 HL clone while inhibition of both HK1 and HK2 via 2-Deoxy-D-glucose at all doses does not rescue OCR of TNFR2 HL clone. 2-deoxy glucose (2DG) fails to get metabolized in glycolysis and inhibits HK1 and HK2.

[0201] An artificial lactate regulation challenge was generated. The experimental set up involves a control 20% working volume and a lactate challenge condition 40% working volume Bolus fed batch. The higher working volume results in less aeration and drives an artificial but reproduceable runaway lactate phenotype (Figure 59A). Importantly this is different from the bioreactor set up where high and low lactate clones are split. This condition drives high lactate in all Wild Type (unedited) clones. Three HL HK2 KO cell lines and the parental HL +LL clones were each grown in the different working volume conditions (Figure 59B). In order to track metabolites and feed cells in a way that helps drive the lactate phenotype, samples were taken daily and measured on the nova to inform feeding with complex feed and glucose.

[0202] Viable cell density (VCD) and viability were tracked during the run and cells were fed based on glucose measurements taken on the Novaflex (Figure 60). The edited clones perform similarly in the 20% WV but outperform both parental cell lines in the 40% challenge condition. When measuring lactate, in the 20% condition KO cell lines are markedly lower from the beginning and most remain that way for the duration of the run (Figure 61). In the 40% condition, the HK2 KO lactate remains underneath the predefined 4 g / L cutoff (above this is considered runaway lactate for the reactor runs). This is not true for either the HL or LL parental cell lines (Figure 61). Cell specific lactate production was calculated for the cultures from beginning to peak VCD. Although there is not a marked difference in the 40% working volume (likely because the significant changes in production happen post peak VCD) the 20% condition shows a clear difference in the cell specific lactate production (Figure 62). These trends are reflected in the titer where the 20% WV has a consistent small titer benefit and the 40% working volume is similar across the board (Figure 62).

[0203] HK2 knockout improves coupling efficiency despite overall low basal oxidative respiration. Figure 63 shows that HL clones show lower oxidative phosphorylation (basal and maximal). The HK2 KO clones show lower basal and maximal respiration. Despite the lower basal and maximal respiration, the proportion of oxygen consumed for ATP synthesis during oxidative phosphorylation (coupling efficiency) is increased in HK2 koAttorney Docket No. JB 16944 WOPCT1 clones and proton leak (wasteful OCR without ATP Synthesis) is reduced in HK2 KO (Figure 64).Example 6: Confirmation of ASNS as an engineering target

[0204] ASNS converts aspartic acid and glutamine to asparagine (Figure 65). Studies were designed to evaluate aspartic acid and glutamine content in the spent media of HL and LL clones. Without being bound by theory, it was hypothesized that since ASNS is poorly expressed in HL clones aspartic acid and glutamine levels in spent media will be high because they are not being efficiently used by low ASNS expression.

[0205] Figure 66 demonstrates that the proteomic signature, low levels of ASNS which was previously seen (Figure 33), was maintained in several high lactate cell lines generated from TNFR2, EMR2xVB17 or IL-11 high lactate producing clones.

[0206] Daily timepoints for spent media supernatant samples & pellet samples were submitted for ambr250 and 10K bioreactor for high lactate (HL) and low lactate (LL) clones in duplicates. Samples were processed and acquired on a MSI method for both positive and negative modes to measure amino acids and metabolites. Internal standards [IS] (3- chlorotyrosine & 2,5DHB) were added to each sample. For supernatant, protein was precipitated using acetonitrile and samples were diluted with a factor of 1 lOx. IS were spiked in and samples were acquired. For cell pellets, cells were lysed using BeatBox in lOOpL mixture of 2:2:1 ACN:MeOH:H2O. Samples were spun down at 14K rpm for 10 min at 4°C. Supernatant were moved to a new plate, IS were added and samples were acquired. Data were analyzed using Skyline and exported out as normalized area.

[0207] Figure 67 depicts the levels of asparagine in high and low lactate clones. For both cell lines (high and low lactate), asparagine depletion begins as early as Day 1. In the high lactate clone, asparagine is not consumed at the same rate as in the low lactate clone. This trend is reflected in the media samples. No significant differences are observed in the pellet data.

[0208] Figure 68 depicts the levels of glutamine in high and low lactate clones. Media glutamine levels do not exhibit the same trend in both vessels (10 L and Ambr 250). In the 10 L bioreactors, glutamine levels appear stable, whereas in the ambr 250, they seem to increase starting from Day 8. Monitoring glutamine levels is challenging in these clones, as they are glutamine synthetase (GS) knockouts.

[0209] Figure 69 depicts the levels of glutamate in high and low lactate clones. Glutamate levels might not be a good indicator of what is happening with ASNS. TheAttorney Docket No. JB 16944 WOPCT1Gln / Glu serves as a medium to transfer the NH3 group. Glutamate has multiple ways to be synthesized and balanced.

[0210] Aspartate levels in the media of High Lac clones are high, possibly because it is not being efficiently converted to asparagine due to low ASNS. Glutamine levels in the media of High Lac clones are lower than in LL clone because it is being synthesized by the GS gene in the antibody plasmid and LL clones proliferate more than HL clones. Asparagine is high in spent media of HL clone because of poor proliferation of cells and therefore less utilization.

[0211] Figure 70 demonstrates that ASNS inhibition reduces maximal respiration of LL clones with specific ASNS inhibitor AMA at 5, 10 and 50 pM (0.05%, 0.1% and 0.5%) and with specific and potent ASNS inhibitor ATF4-in2 at 1 pM, 5 pM and 10 pM.

[0212] Figure 71 demonstrates that respiration coupled to ATP synthesis is reduced upon specific ASNS inhibition with AMA at 5, 10 and 50 pM (0.05%, 0.1% and 0.5%) and ATF4-in2 at 1 pM, 5 pM and 10 pM in LL clone.

[0213] Figure 72 demonstrates the proteomic signature, high levels of MAPKAPK3 (MK3) which was previously seen (Figure 42), was maintained in the IL-11 high lactate cell line.

[0214] Figure 73 demonstrates that the proteomic signature, high levels of BAK which was previously seen (Figure 41), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL-11).

[0215] Figure 74 demonstrates that the proteomic signature, low levels of NDRG1 which was previously seen (Figure 51), was maintained in several high lactate cell lines (TNFR2 and IL- 11).

[0216] Figure 75 demonstrates that the proteomic signature, low levels of HMGCS2 which was previously seen (Figure 26), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL-11).

[0217] Figure 76 demonstrates that the proteomic signature, low levels of PDPR which was previously seen (Figure 35), was maintained in several high lactate cell lines (TNFR2, EMR2xVB17 and IL-11).

[0218] Figure 77 demonstrates that the proteomic signature, high levels of MEI which was previously seen (Figure 28), was variable when additional high lactate cell lines (TNFR2 and IL- 11) were evaluated.Attorney Docket No. JB 16944 WOPCT1

[0219] Figure 78 demonstrates that the proteomic signature, low levels of FADS3 which was previously seen (Figure 27), was variable when additional high lactate cell lines (TNFR2, EMR2xVB17 and IL-11) were evaluated.

[0220] Figure 79 demonstrates that the spatial proteomic signature, low levels of SEPTIN-2 in mitochondria which was previously seen (Figure 44), was variable when additional high lactate cell lines (TNFR2, EMR2xVB17 and IL- 11) were evaluated.

[0221] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific examples, it is apparent that other examples and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such examples and equivalent variations.

Claims

1. CLAIMSWhat is claimed is:

1. A model system comprising a high-lactate producing cell line for use in methods of identifying biomarkers associated with an increased risk of lactate runaway and engineering targets for controlling lactate runaway.

2. The model system of claim 1, further comprising at least one low- lactate producing cell line.

3. A method of identifying biomarkers associated with an increased risk of lactate runaway, engineering targets for preventing or controlling lactate runaway or a combination thereof, the method comprising: performing at least one omics assay comprising a high-throughput screening method to measure biological molecules in a systematic way.

4. The method of claim 3, wherein the at least one omics assay comprises a genomic assay, a proteomic assay, a transcriptomic assay, a metabolomic assay, a lipidomic assay, an epigenomic assay, ribosequencing or any combination thereof.

5. The method of claim 3, wherein the at least one omics assay is a spatial omics assay, a temporal omics assay, or an activity-based omics assay.

6. A method of identifying a cell as having an increased risk of lactate runaway, the method comprising: a) detecting the level or localization of at least one biomarker selected from the group consisting of HK2, ASNS, MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPL58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11, HMGCS2, FADS3, MEI, ALT1, PFKP, LHX9, PDP1, PDPR, AGK, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, MAPK9 (JNK2), and NDRG1 in the cell;b) comparing the level or localization of the biomarker to a comparator control level or localization of the biomarker; and c) identifying the cell as having increased risk of lactate runaway when the cell or cell culture has an elevated level of expression one or more of HK2, MEI, PFKP, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, or MAPKAPK3 (MK3), a decreased level of expression at least one of ASNS, MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPL9, MRPE58, MRPL52, MRPL49, MRPL48, MRPL46, MRPL45, MRPL41, MRPL40, MRPL4, MRPL37, MRPL35, MRPL30, MRPL3, MRPL24, MRPL23, MRPL19, MRPL18, MRPL16, MRPL15, MRPL14, MRPL11, HMGCS2, FADS3, AET1, PDP1, PDPR or NDRG1, or altered localization of MRPS7, MRPS5, MRPS33, MPRS27, MRPS18C, MRPS10B, MRPS11, MRPE9, MRPE58, MRPE52, MRPE49, MRPE48, MRPE46, MRPE45, MRPE41, MRPE40, MRPE4, MRPE37, MRPE35, MRPE30, MRPE3, MRPE24, MRPE23, MRPE19, MRPE18, MRPE16, MRPE15, MRPE14, MRPL11, HMGCS2, PFKP, P21 / CDKN1A, EHX9, AGK, P53, RUNDC1, Dnmll (DPR1), SEPTIN2, SIRT5, SIRT7, HPS1, ATAD1, or MAPK9 (JNK2) as compared to the comparator control.

7. The method of claim 6, wherein the comparator control comprises a statistically significant cut-off determined based on the normal level of the biomarker in a cell line or culture of the same type as the cell.

8. The method of claim 6, further comprising depleting the cell from a cell culture.

9. The method of claim 6, further comprising removing a cell culture comprising the cell from a production pipeline.

10. The method of claim 6, further comprising a contacting the cell with an agent to prevent or control lactate runaway, wherein the agent is an antagonist or inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2) or an agonist or activator of ASNS, HMGCS2, FADS3, AET1, EHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1.

11. A method of treating a cell culture to prevent or control lactate runaway, the method comprising contacting a cell culture with at least one agent to prevent or control lactate runaway, wherein the agent is selected from the group consisting of an antagonist or inhibitor of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2) or an agonist or activator of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or a combination thereof.

12. An engineered cell comprising a modification to prevent or control lactate runaway, the cell comprising at least one modification selected from the group consisting of a modification to prevent expression of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2), or insertion of a nucleic acid construct for expression of ASNS, HMGCS2, FADS3, ALT1, LHX9, PDP1, PDPR, AGK, RUNDC1, SIRT5, SIRT7, HPS1, or NDRG1, or a combination thereof.

13. The engineered cell of claim 12, wherein the cell comprises a genetic modification to delete or disrupt expression of HK2, MEI, PFKP, P53, P21 / CDKN1A, P27 / CDKN1B, BAX, BAK, MAPKAPK3 (MK3), Dnmll (DPR1), SEPTIN2, ATAD1, or MAPK9 (JNK2).

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