Modulation of cell growth and viability using nucleoside transport inhibitors, inhibitors of adenosine uptake, adenosine kinase inhibitors and inhibitors of extracellular synthesis

Abstract

Provided is a cell culture medium for the production of a product of interest using a fed-batch process wherein the cell culture medium comprises an extracellular adenosine modulator comprising one or more of a nucleoside transport inhibitor, an inhibitor of adenosine uptake, an adenosine kinase inhibitor or an inhibitor of extracellular synthesis of adenosine.

Description

MODULATION OF CELL GROWTH AND VIABILITY USING NUCLEOSIDETRANSPORT INHIBITORS, INHIBITORS OF ADENOSINE UPTAKE, ADENOSINEKINASE INHIBITORS AND INHIBITORS OF EXTRACELLULAR SYNTHESISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 563,673 filed March 11, 2024, the entire contents of which are incorporated by reference herein.FIELD

[0002] This disclosure relates generally to methods of using nucleoside transport inhibitors, inhibitors of adenosine uptake, adenosine kinase inhibitors and / or inhibitors of extracellular synthesis.BACKGROUND

[0003] Chinese Hamster ovary (CHO) cell lines are the most widely used mammalian cell lines for large-scale production of therapeutic proteins, particularly antibodies. This cell line can adapt to grow in suspension cultures using serum-free and chemically defined media, and it has gene amplification systems that allow for an increase in productivity. Lai 2013.

[0004] Fed batch cultures are a preferred mode of protein production because they are shorter and operationally easier compared to continuous manufacturing. However, unlike continuous manufacturing where inhibitory by-products can be perfused out with spent media allowing cells to achieve very' high peak Viable Cell Densities (VCD), fed batch culture growth is stalled as metabolic by-products keep accumulating in the culture.

[0005] Adenosine is a purine nucleoside that has been shown to control cell growth by its ability to increase cyclic adenosine 5 '-monophosphate AMP (cAMP) levels, resulting in inhibition of cychn-dependent kinases involved in cell cycle checkpoint regulation. Du 2005. It also has been shown to positively influence recombinant protein production by' inducing growth arrest in CHO. Chong 2009, Carvalhal 2003. Intracellular adenosine is produced from 5'- adenosine monophosphate (5'-AMP) by the action of the enzyme 5'-nucleotidase. It is degraded via deamination to inosine by adenosine deaminase (ADA). Kaczmarek 1996, Resta 1998. In the extracellular space, adenosine is mainly produced through adenosine 5'-triphosphate (ATP) dephosphorylation by two ectonucleotidases — ectonucleoside-triphosphate diphosphohydrolase 1 (CD39) and ecto-5-nucleotidase (CD73). CD39 hydrolyzes ATP and ADP to AMP and CD73 converts AMP into adenosine. Latini 2001, Zimmermann 2000.

[0006] Additionally, adenosine can trigger downstream signaling pathways in the cell either through G protein-coupled receptors (GPCR), namely the Al, A2A, A2B and A3ARs or can also be transported out of the cell to the extracellular space and vice versa by different proteins including specific bi-directional nucleoside transporters which are further divided into Equilibrative Nucleoside Transporters (ENTs) and Concentrative Nucleoside Transporters (CNTs). Latini 2001, Fredholm 2011. Different cell lines have different expression levels of these receptors and transporters which govern the mode of adenosine uptake. Naes 2020, Penny cooke 2001. Inhibitors of transporters increase the extracellular concentrations of adenosine and are useful clinically to treat certain cardiovascular complications. Chakrabarti 2008.

[0007] Even though production yields of recombinant therapeutic antibodies in CHO cell lines have increased considerably over the years, the ever-increasing demand for drugs and the possibility to bring down production costs leaves opportunities for further improvement.SUMMARY

[0008] The present disclosure provides a cell culture medium for the production of a product using a fed-batch process wherein the cell culture medium comprises an extracellular adenosine modulator comprising one or more of a nucleoside transport inhibitor, an inhibitor of adenosine uptake, an adenosine kinase inhibitor, or an inhibitor of extracellular synthesis of adenosine.

[0009] In one aspect, the present disclosure provides a fed-batch cultivation process comprising: a) seeding a bioreactor, b) adding nutrients to the bioreactor, c) adding to the bioreactor an extracellular adenosine modulator comprising one or more of a nucleoside transport inhibitor, an inhibitor of adenosine uptake, an adenosine kinase inhibitor, or an inhibitor of extracellular synthesis of adenosine; and d) obtaining a product.

[0010] The summan' of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A and Figure IB show viable cell density (VCD), viability, titer and product quality attributes in fed-batch cultures supplemented with dilazep dihydrochloride (DZD) orDZD + PSB 12379. In this figure, PSB 12379 is referred to as PSB. The cell line used was derived from a CH0-K1 cell line.

[0012] Figure 2 shows a batch experiment showing the impact of different types of inhibitors on cell growth arrest caused by adenosine addition. DP refers to dipyridamole, DCPCX refers to dipropylcyclopentylxanthine, and NBMPR refers to nitrobenzylthioinosine.

[0013] Figure 3 shows an ANOVA analysis demonstrating the impact of DZD and PSB 12379 individually and in combination on VCD in batch culture. In this figure, PSB 12379 is referred to as PSB.

[0014] Figure 4 shows metabolomics data demonstrating the accumulation of extracellular adenosine in cultures treated with inhibitors. In this figure, PSB 12379 is referred to as PSB.DETAILED DESCRIPTION

[0015] Adenosine accumulates in typical fed-batch cultures. Extracellular adenosine is involved in initiation of transition from exponential phase to stationary phase in fed-batch cultures, and potentially also is involved in inducing apoptosis. Blocking the effect of extracellular adenosine can increase overall cell density in fed-batch cultures thereby improving productivity.

[0016] The present disclosure is directed to a cell culture medium for the production of a product using a fed-batch process wherein the cell culture medium comprises an extracellular adenosine modulator comprising one or more of a nucleoside transport inhibitor, an inhibitor of adenosine uptake, an adenosine kinase inhibitor and / or an inhibitor of extracellular synthesis of adenosine.

[0017] The present disclosure is also directed to a fed-batch cultivation process comprising: a) seeding a bioreactor, b) adding nutrients to the bioreactor, c) adding to the bioreactor an extracellular adenosine modulator comprising one or more of a nucleoside transport inhibitor, an inhibitor of adenosine uptake, an adenosine kinase inhibitor, and an inhibitor of extracellular synthesis of adenosine; and d) obtaining a product.

[0018] In an embodiment, the extracellular adenosine modulator is the nucleoside transport inhibitor. In subembodiments, the nucleoside transport inhibitor is selected from: dilazep dihydrochloride (DZD), dipyridamole (DP), nitrobenzylthioinosine (NBMPR). and midazolam.

[0019] In another embodiment, the extracellular adenosine modulator is the inhibitor of extracellular synthesis of adenosine. In subembodiments, the inhibitor of extracellular synthesis of adenosine is a ectonucleotidase (CD73) inhibitor. In further subembodiments, the ectonucleotidase (CD73) inhibitor is PSB 12379.

[0020] In an embodiment, the cell culture medium comprises the nucleoside transport inhibitor and the inhibitor of extracellular synthesis of adenosine. In subembodiments, the cell culture medium comprises dilazep dihydrochloride and PSB 12379.

[0021] In an embodiment, the inhibitor of adenosine uptake is cilostazol.

[0022] In an embodiment, the adenosine kinase inhibitor which is 5-iodotubericidin.

[0023] In an embodiment, the product is selected from the group consisting of: a protein, a recombinant protein related product, a bacteria, an enzy me, and a choloroplast.

[0024] In an embodiment, the cell culture medium further comprises a cell line which is a mammalian cell line.

[0025] In an embodiment, the cell line is a Chinese Hamster ovary cell line.

[0026] In an embodiment, the extracellular adenosine modulator is present at a concentration of about 0.5 pM to about 5 pM. In a subembodiment, the extracellular adenosine modulator is present at a concentration of about 0.1 pM to about 1 pM.

[0027] In an embodiment, the extracellular adenosine modulator increases overall cell density by at least 150% compared to overall cell density of a corresponding fed-batch culture not comprising the extracellular adenosine modulator.

[0028] In an embodiment, the extracellular adenosine modulator increases overall productivity by at least 100%. at least 150% or at least 200% compared to overall cell productivity of a corresponding fed-batch culture not comprising the extracellular adenosine modulator.

[0029] In an embodiment, the first extracellular adenosine modulator is added to the bioreactor prior to adding nutrients. In an embodiment, the first extracellular adenosine modulator is added to the bioreactor prior to seeding the bioreactor.

[0030] In an embodiment, the nucleoside transport inhibitor is publicly known, or otherwise known by a skilled artisan, as of the effective filing date of this application. In an embodiment, the inhibitor of adenosine uptake is publicly known, or otherwise known by a skilled artisan, as of the effective filing date of this application. In an embodiment, the adenosine kinase inhibitor is publicly known, or otherwise known by a skilled artisan, as of the effective filing date of this application. In an embodiment, the inhibitor of extracellular synthesis of adenosine is publicly known or otherwise known by a skilled artisan, as of the effective filing date of this application.Definitions

[0031] Listed below are definitions of various terms used herein. These definitions apply' to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0032] 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. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry', and peptide chemistry are those well-known and commonly employed in the art.

[0033] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0034] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0035] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0036] As used herein, the term “comprising” may include the embodiments “consisting of' and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having.” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0037] The term “batch” or “batch culture” as used herein refers to a method of culturing cells in which all the components that will ultimately be used in culturing the cells, including the medium as well as the cells themselves, are provided at the beginning of the culturing process. A batch culture is ty pically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0038] The term “fed batch” or “fed-batch” as used herein refers to a method of culturing cells in which additional components are provided to the culture at some time subsequent to the beginning of the culture process. The provided components typically comprise nutritional supplements for the cells that have been depleted during the culturing process. A fed-batchculture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified.

[0039] The tenn ‘‘perfusion’" as used herein refers to a method of culturing cells in which additional fresh medium is provided, either continuously over some period of time or intermittently over some period of time, to the culture (subsequent to the beginning of the culture process), and simultaneously spent medium is removed. The fresh medium typically provides nutritional supplements for the cells that have been depleted during the culturing process. Polypeptide product, which may be present in the spent medium, is optionally purified. Perfusion also allows for removal of cellular waste products from the cell culture.

[0040] The term “viability” means the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.

[0041] “Viable cell density ” or “VCD” refers to the number of live cells in a given volume of culture medium, as determined by standard viability' assays (such as trypan blue dye exclusion method).

[0042] The terms “integrated viable cell density”, “integral viable cell concentration”, “IVCC", or “IVCD” are used interchangeably and mean the average density of viable cells over the course of the culture multiplied by the amount of time the culture has run.

[0043] The term “titer” means the total amount of a polypeptide or protein produced by a cell culture in a given amount of medium volume. Titer can be expressed in units of milligrams or micrograms of polypeptide or protein per milliliter (or other measure of volume) of medium. “Cumulative titer” is the titer produced by the cells during the course of the culture, and can be determined, for example, by measuring daily titers and using those values to calculate the cumulative titer.

[0044] PSB 12379 is an ectonucleotidase (CD73) inhibitor having the following structure:

[0045] PSB 1115 is a highly selective, water-soluble, human A2B adenosine receptor antagonist having the following structure:EXAMPLE

[0046] The following example is meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.Example 1: Modulation of adenosine uptake in fed-batch culture.Materials and methodsCell line and seed train

[0047] Recombinant glutamine synthetase knockout Chinese Hamster Ovary cells (GS-CHO) were used to produce mAb in this example. The seed train included vial thaws and culture in vessels of increasing volume to obtain enough cells for each experiment. Culture vessels included shake flasks (Coming) and AMBR 15 and AMBR250 vessels (Sartorius).Bioreactor operations

[0048] Bioreactors were seeded at different cell densities listed in each experiment. Batch and fed batch experiments were completed in Shake flasks (Coming) or ambr250® or ambrl5 bioreactors and mock perfusion was completed in deep well plates (DWP). For all culture conditions: temperature was 37°C, CO2 w as maintained at 5%, shake flasks were spinning at 100 RPM and DWP at 300RPM in incubators (Kuhner AG, Basel, Switzerland). Culture durations were 7 days for a batch experiment, 14 days for fed batch, 5 days for mock perfusion. Cell counts and viability were measured every day. For fed batch culture, cells were seeded at 0.5 million cells / mL in 60 mL of Dynamis (Gibco) in shake flask, and cells were fed daily after day 3 with 2.5% volume of Cell Boost 7A(Cytiva) and 0.25% volume of Cell Boost 7b(Cytiva). Glucose was maintained at 5 g / L throughout the experiment. For mock perfusion the culture was centrifuged at 1000 rpm for 5 min (Beckman Coulter, Brea. CA) to pellet the cells, then 2 mL of medium was removed from each well and then replenished with 2 mL of fresh medium every 24 hours as described in Madabhushi et al. Biotechnol. Prog. 2022; 38(4):e3262. Spent medium was further analyzed for key metabolites, titer and quality attributes.Experimental treatments

[0049] In the experiments in which nucleosides were added, nucleosides were supplemented into cultures on day 3 at the experimental concentrations described. For adenosine inhibitor studies, cells were either treated with (a) 2.5pM DZD daily on day 3 to day 7 of fed batch culture, (b) with combination of 500nM PSB 12379 and 2.5pM DZD as single treatment on day 3, or (c) with combination of 500nM PSB 12379 and 2.5pM DZD daily over day 3 to day 7 of fed batch culture. Mock perfusion media contained nucleosides at the concentrations described. In adenosine inhibitor studies no further nucleosides were added.Cell counting and metabolites

[0050] Viable cell density (VCD) and viability were measured using the trypan blue exclusion method on a Cedex Hi-Res cell counter (Roche Diagnostics GmbH, Mannheim, Germany). Key metabolites were measured using a BioProfile FLEX2 (Nova Biomedical, Waltham, MA).Downstream process

[0051] IgG titer was measured using ProA columns via HPLC.Flow Cytometry

[0052] Flow Cytometry was completed using a Merck Millipore Guava easyCyte 5HT Flow Cytometer HT. Cell cycle analysis was completed using the Guava cell cycle reagent kit from Luminex using manufacturer protocols with an exception that ethanol fixation was completed for 3 days at 4" C. Cells were fixed at lxlOA6 cells / mL.Adenosine measurement

[0053] Adenosine was directly measured using the abeam Adenosine Assay Kit (Fluorometric) using manufacturer protocols on a Molecular Devices Spectra Max 5. Samples were diluted 1 / 10 to 1 / 20 and were frozen until thawing to sample for the assay.ResultsAdenosine and other purine nucleosides caused CHO cell growth arrest, increase in average diameter and 1.4-2 fold improvement in recombinant mAb productivity.

[0054] CHO cell line (Cell line A) expressing a model monoclonal antibody was cultured in media supplemented with different concentrations of adenosine (final concentration on day 3 of 0.05-lmM). An adenosine concentration dependent increase in average diameter of the cells was observed with corresponding cell growth arrest. The titer of the recombinant antibody was measured on day 7 of the batch culture. While a minor increase in overall product titer was observed at higher concentrations of adenosine, the specific productivity of the mAb increased by > 2-fold at 0.25mM, 0.5mM and ImM concentrations. These results are consistent with previously observed findings of increases in specific productivity with adenosine treatment.

[0055] Mock perfusion cultures were also performed in 24-well deep well plates where cells were seeded at 20e6cells / mL, and complete media was exchanged daily to simulate perfusion cultures. Similar to the batch experiment, concentration dependent cell growth arrest, increase in diameter, and specific productivity’ were observed. Additionally, multiple cell lines expressing different recombinant mAbs also showed a similar effect with adenosine supplementation in batch and mock perfusion studies, although the fold increase in specific productivity ranged from 1.5 to 2 fold. Increase in diameter and increase in specific productivity for CHO cells is usually associated with cell cycle arrest in G0 / G1 phase. However, based on cell cy cle analysis, adenosine supplementation seemed to decrease % of cells in G0 / G1 phase by 10% with a corresponding increase in cells in S phase.

[0056] Next, it was tested whether the increase in antibody specific productivity’ was also observed with other nucleoside supplementation. In this regard, batch experiment yvas performed in media supplemented with one of the following nucleosides: adenosine, guanosine, inosine, 5’- methyl uridine or cytidine. Cell groyvth arrest, an increase in average diameter, and an increase in specific productivity were observed primarily with purine nucleosides (adenosine, guanosine and inosine) and not with pyrimidines.

[0057] In the batch experiment, although the specific productivity increased by up to tyvo-fold in the presence of adenosine or guanosine, the overall product titer was low er due to lower cumulative cell density’ observed for these conditions.Perfusion media supplemented with adenosine increases overall mAh productivity in high cell density perfusion processes.

[0058] For commercial production of recombinant proteins, one of the primary goals is to increase overall mAb productivities to improve efficiency and production costs. While the supplementation of adenosine dramatically increases cell specific productivity, the cell growth arrest impacts the cumulative cell densities in the culture which therefore could impact the overall recombinant protein amount in the process. Perfusion cell culture processes, especially intensified processes, are increasingly being adopted for biomanufacturing wherein high cell densities (~100-150e6 cells / mL) are maintained in the reactors by continuous exchange of spent media with fresh media. Once the target high cell density’ is reached, the goal is to transition cellular metabolism from proliferation to recombinant protein production to maximize the overall productivity of the process.

[0059] It was tested whether adenosine can be applied to achieve increase in overall mAb productivity in perfusion processes when it is supplemented in media after targeting high cell density is reached. In this regard, cells w ere seeded at 0.5e6 cells / mL in ambr250 perfusion bioreactors on Day 0 in a perfusion media without added adenosine. Continuous media exchangewas initiated on day 3 at a rate of 0.5 vessel volumes per day (VVD) which was increased to 1 VVD of Day 5 followed by 2 VVD starting Day 7. Once target VCD of 100e6 cells / mL was reached on Day 14, perfusion media was changed to media containing ImM adenosine. Cell growth, titer and metabolite profdes and product quality profiles were determined.

[0060] The results demonstrated that the overall cell grow th and metabolite profiles was comparable between control and ImM adenosine supplemented perfusion cultures. The overall permeate titer increased by more than 25% with adenosine supplementation. The bleed strategy to maintain target cell density in ambr250 perfusion systems was determined based on manual sampling and theoretical growth rate projections. For the adenosine supplemented conditions, an overestimation of bleed resulted in drop in viable cell density on Day 17 which resulted in a dip in product titers. This recovered after a couple of days when cell densities reached back target VCDs and the higher overall productivity with adenosine was consistently observed. Additionally, the product quality was within acceptable variability between control and adenosine supplemented cultures.

[0061] A biphasic perfusion media approach wherein adenosine supplementation in perfusion media after target cell density7is achieved can increase overall process productivity by 25%. Effect of increased specific productivity with adenosine supplementation is only observed in exponentially growing cell culture phase.

[0062] Since adenosine supplementation significantly improved perfusion culture productivities when supplemented after target VCD was reached, it was evaluated whether supplementing adenosine in traditional fed-batch processes could provide similar benefits. In this regard, different concentrations of adenosine (0.25mM. 0.5mM or ImM) were added either on Day 1 (early exponential phase) or on Day 6 (when peak VCD was achieved) to fed-batch cultures in ambrI5 bioreactors.

[0063] The cell density , viability and productivity profiles for the different conditions tested in fed-batch were determined. As expected, when adenosine was supplemented on Day 1, a concentration dependent reduction in cell growth was observed. Titer and cell specific productivities of the Day 1 supplemented conditions were higher than controls on Day 7 and Day 8 of the cultures. However, as the cumulative cell densities increased, the difference with day 1 supplementation was not observed towards the end of the cultures.

[0064] In contrast to the expected observations with Day 1 supplementation of adenosine, adenosine supplementation on Day 6 did not show this behavior. Cell densities were slightly negatively impacted by adenosine supplementation although viabilities as measured by try pan blue exclusion method were similar. Further analysis showed that adenosine supplementation cancause increases in percentage of cells in early apoptosis. No significant impact on titer or specific productivities were observed.

[0065] Overall, the increase in cell specific productivity and growth arrest were observed consistently for multiple cell lines when adenosine was supplemented in early phase of batch culture, mock perfusion studies, perfusion cultures and in early phase of fed-batch cultures. However, adenosine supplementation at later phase of fed-batch culture did not show any meaningful effect indicating that either adenosine has already accumulated at sufficient concentrations at the later phases or the mechanisms or pathways through which adenosine influences cellular metabolism and cell cycle are already in play during the later phase of fed- batch cultures.Adenosine transport is mediated through the equilibrative nucleoside transporters in CHO cell cultures.

[0066] Adenosine is essential for the proper functioning of every cell in the body. Adenosine is synthesized in most mammalian cells via the dephosphorylation of AMP through nucleotidases. A portion of adenosine comes from the metabolism of methionine. Adenosine is released in the extracellular spaces via an equilibrative nucleoside transporter (ENT). In the extracellular spaces, adenosine is formed by the dephosphorylation of ATP and 5'AMP via CD39 and CD73, respectively. Adenosine activates four G-coupled membrane receptors, named AIR, A2AR, A2BR, and A3R. The intra- and extra-cellular levels of adenosine are tightly regulated through its production from ADP and ATP, transport through specific nucleoside transporters, and catabolism. Not much is known about the biochemical pathways in CHO cells that contribute to the effects detailed above with adenosine supplementation.

[0067] Using transcriptomics, the temporal dynamics of gene expression of ENTs, CNTs and GPCRs w ere evaluated for different CHO cell lines in representative fed batch processes. The FPKM Log2 transcript levels of the nucleoside transporters and GPCRs w ere determined over the course of fed batch cultures for three different cell lines. The genes that seem to be expressed are the equilibrative nucleoside transporters (slc29al, slc29a2, slc29a3) and concentrative nucleoside transporter, slc28a3.

[0068] To evaluate whether the effect of adenosine is mediated by its uptake from extracellular space through ENTs, batch cultures were run where adenosine was supplemented in the presence or absence of inhibitors or antagonists for either GPCRs (DP CPX for adorAl, SCH58261 for adorA2a, PSB11 15 for adorA2b and MRS 1334 for adorA3) or nucleoside transporters (dipyridimole or DP and dilazep dihydrochloride). Also evaluated w ere nitrobenzylthioinosine (NBPMR), cilostazol, midazolam, and 5-iodotubericidin. The percentage rescue of viable cell density due to adenosine was calculated when each of the inhibitors was supplemented to thecultures (Figure 2). Specifically, cells were seeded at 0.5 million cells / mL in deep-well plate and treated with nucleosides on day 3 and cultured for a total period of 7 days. The percentage rescue was calculated by dividing the rescue in VCD that was observed by inhibitor treatment in the presence of adenosine compared to no treatment, over VCDs in culture that naturally accumulate adenosine. Here PSB1115, MRS1334 and SCH58261 are GPCR antagonists; DZD and DP are nucleoside transport inhibitors. A positive number represents higher VCD upon treatment with drug and adenosine when compared to cells treated with adenosine. As demonstrated in Figure 2, DP, DZD, NBPMR, cilostazol, midazolam, and iodotubericidin ere able to rescue the suppressed cell growth effect of adenosine; whereas there was no rescue effect seen with the antagonists for GPCRs. For example, DP and DZD were able to rescue the suppressed cell grow th effect of adenosine by about 30%. This indicates that adenosine from the extracellular environment is taken up through the nucleoside transporters (primarily ENTs) and consequently imparts the effects on cell growth and recombinant protein production.Production and secretion of adenosine by CHO cells over the culture duration can initiate the transition of cells from exponential to stationary phase.

[0069] In fed-batch cultures, cells transition from exponentially growing phase to stationary7phase. Constant exchange of media and removal of spent media supports growth of cells in perfusion cultures to very high cell densities depending on the perfusion rate. It is not well understood whether in fed-batch cultures, the transition is triggered by nutrient depletion or metabolic imbalances or due to multiple inhibitory byproducts that could accumulate in culture. Without being bound by theory7, one of the possible reasons w hy adenosine supplementation at later phase of fed-batch culture does not show any meaningful effect could be that it already accumulates in the culture. It was demonstrated that adenosine accumulates to significant amounts in the cell culture supernatant in later stages of fed-batch cultures. Additionally, Figure 4 provides metabolomics data showing an increased accumulation of extracellular adenosine in a culture treated with nucleoside inhibitors. Spent cell medium was collected from days 5, 7 and 10 of fed batch culture where culture was treated w ith PSB 12379 and DZD on day 3 to day 7. Fold changes were calculated by determining relative abundance of adenosine in spent media to abundance of adenosine in fresh media.

[0070] Since it was identified that adenosine uptake through ENTs is primarily how adenosine’s effect on CHO cells is manifested, the impact of the inhibitors of ENTs and CD73 inhibitor in batch cultures in the absence of any additional adenosine supplementation was tested. As seen in Figure 3, blocking ENTs significantly increased the overall cell density on Day 3 of the batch culture by 10%. This effect was also observed when PSB 12379 was added to the cultures. This indicates that adenosine is being synthesized extracellularly by the action of CD73ectonuleosidase. Additionally, a synergistic effect was seen by the addition of both PSB 12379 and DZD in further increasing the cell densities in batch culture by 20% compared to control conditions.

[0071] The batch cultures can typically only be run for shorter durations due to nutrient limitations impacting cell growth. In order to further determine whether the transition of cells from exponential to stationary phase in CHO cell cultures is potentially initiated by uptake of adenosine followed by downstream effects, fed-batch cultures were performed in the presence of ENT inhibitors, without any supplementation of adenosine.

[0072] CHO cells expressing mAbl were seeded in shake flasks at 0.5e6 cells / mL and cultured using a standard fed-batch process with daily supplementation of feeds. To some cultures, DZD was supplemented daily at a concentration of 2.5pM from Day 3 to Day 7. As depicted in Figures 1A and IB, supplementation of DZD resulted in a 50% increase in peak VCD compared to control with no DZD supplementation. In order to investigate whether adenosine is synthesized extracellularly and whether the function of ectonucleosidase contributes to this, cultures were supplemented with 500nM PSB 12379 in addition to 2.5pM DZD. As shown in Figures 1 A and IB, there w as a synergistic effect of PSB 12379 and DZD beyond just the single supplementation of DZD. This suggests that adenosine is synthesized by CHO cells extracellularly as well through the action of CD73. This adenosine then is taken up by the cells via ENTs which then modulates cellular metabolism and cell cycle which subsequently leads to transition of cells from exponential to stationary' phase.

[0073] Surprisingly, the overall mAb productivities increased 1.5-2 fold with supplementation of either DZD alone or a combination of PSB 12379 and DZD. This was due to the overall higher cumulative cell densities observed under these treatment conditions when compared to control. Slc29al expression is highly regulated and inversely correlates with peak viable cell densities under different process conditions.

[0074] The data shown using ENT inhibitors demonstrated that inhibition of ENTs (such as slc29al which is one of the most abundant nucleoside transporters in CHO cells) can increase the peak cell densities in CHO cell fed batch cultures. This indicates that adenosine production and uptake potentially plays a role in transition of cells from exponential to stationary' phase in fed batch. Usually, for the same process (media-feeds and process parameters), different cell lines can show different growth behaviors and different peak VCDs. Moreover, for the same cell line, media and cell culture process parameters such as pH and culture temperature can also impact growth rate and peak VCDs achieved.

[0075] While multiple mechanisms have been proposed in the literature for impact of these different factors on cellular metabolism or cell cycle, there has been no studies on the potential roles of nucleoside transporters and linkage to downstream regulation in CHO cells.

[0076] The temporal dynamics of slc29al expression were analyzed for different cell lines and compared to the viable cell density profiles observed in fed batch processes. The results showed that for three different CHO cell lines expressing mAbs, there is consistently a greater than two fold reduction in the expression of slc29al transcripts over culture duration. Coincidently, viable cell density also reaches its peak for all three cell lines as slc29al expression is downregulated. However, it is common to observe differences in cell growth profiles between different host cell lines, different recombinant protein expressing cell lines as well as clone to clone variability which could be driven by various regulatory' mechanisms and genetic backgrounds. To limit the degree of variability, it was tested whether different cell growth profiles can be achieved for a given cell line by varying just the process parameters. It was also evaluated whether there is any correlation between slc29al expression patterns and peak viable cell density achieved.

[0077] In order to accomplish a phenotype of different peak VCDs, design of experiments (DDEs) were conducted with seeding density7, pH, grow th temperature, temperature of downshift and dissolved oxygen in media (DO) for a given cell line where each parameter was varied at three different levels. 36 different fed batch cultures were run at these range of process conditions based on an I-optimal design and the VCD profiles were determined. Peak VCDs ranging from 17 - 32 million cells / mL were achieved. The histogram distribution of peak cell densities observed for different process conditions in the DOE were determined. Transcriptomics analysis was performed early (day 3) and around the time when peak VCD (Day 5) was achieved. The FPKM Log2 expression levels of slc29al for different process conditions were determined. Different degrees of downregulation of slc29al w ere observed for different conditions (up to 4 fold downregulation). There was an inverse correlation between slc29al expression levels on day 5 and peak VCD achieved by the conditions.

[0078] This aligns with the observations w i th inhibitors of ENT transporters wherein inhibition of ENT transporters increased peak VCD. Transcriptomics data with cultures attaining different peak VCDs indicate that different process conditions can also impact expression levels of slc29al which can then further regulate cell proliferation.DiscussionApplication of adenosine (or other purine nucleosides) in biphasic perfusion media.

[0079] While supplementation of CHO cell cultures with adenosine increased specific productivity' of recombinant proteins, it also caused cell growth arrest. In order to increase the overall productivity of biomanufacturing processes, the strategy' of adenosine supplementationwas applied in perfusion cultures once target cell density was achieved to drive cellular processes from growth phase to production phase. Supplementing perfusion media with ImM adenosine after target VCD was reached increased the overall productivity of the process by 25%. Similar positive improvement in overall productivity of perfusion processes was also observed with guanosine and inosine supplementation.

[0080] When a similar strategy of supplementing adenosine in fed-batch cultures was evaluated (by supplementing cultures once peak VCD was reached), no effect on overall productivities was observed. Interestingly, adenosine seemed to increase specific productivity (qP) of cells that were still in their exponentially growth phase such as in batch cultures, mock perfusion, early phase of fed batch processes or in perfusion cultures. The culture environment in bioprocesses is quite complex. In mammalian cell systems, there is a dramatic interplay between cellular growth, metabolism, signaling pathways, protein production and product uality regulation. The discrepancy in the effect of adenosine between exponential phase and stationary phase of cultures could be caused by various factors including: a) adenosine is one of the byproducts produced over time in a typical fed batch culture and the downstream pathways regulated by adenosine are already ongoing and therefore further supplementation with adenosine has no significant effect; b) other inhibitory byproducts in culture already exist in culture and prevent the effect of adenosine; or c) nutrient depletion or nutrient imbalance can trigger cellular stress and cell growth arrest or a combination of these factors.

[0081] Adenosine is a key signaling molecule that has been shown to affect various physiological functions in central nervous system, cardiovascular and immunity'. Without being bound by theory, it is hypothesized that adenosine gets naturally produced by CHO cells over time in fed batch cultures which can be taken up by the cells and subsequently regulate the transition of cells from exponential phase to stationary phase. In perfusion cultures, since the spent media (containing inhibitory byproducts / regulatory molecules such as adenosine) is constantly exchanged with fresh perfusion media, cells continue to grow to higher cell densities.

[0082] Using ENT inhibitors such as DZD and DP and not GPCR antagonists, the growth inhibitory effects of adenosine supplementation w ere reduced in batch cultures indicating the primary' uptake of adenosine in CHO cell cultures is mediated by ENTs (Slc29al being the primary transporter). Interestingly in batch cultures, 10% higher peak VCDs were observed in the presence of ENT inhibitors without any added adenosine. The effect was further improved w ith a combination of CD73 inhibitor and ENT inhibitor indicating that at least some of the adenosine is synthesized extracellularly by the action of ectonucleosidases.

[0083] Supplementation of normal fed batch cultures with just the ENT inhibitors or a combination of ENT and CD73 inhibitors increased the number of cells continuing to growexponentially and thus doubled the peak VCDs achieved as well as resulted in a twofold increase in recombinant protein productivity.

[0084] These observations suggest that adenosine uptake through ENTs (such as Slc29al) regulates transition of CHO cells in fed batch from exponential to stationary phase.

[0085] It was also observed that different process conditions can differentially regulate the expression levels of Slc29al. An inverse correlation in the expression levels of slc29al and peak VCDs in fed batch cultures were observed. This indicates that inhibition or downregulation of Slc29al can improve peak VCDs in fed batch cultures. It has been demonstrated that process conditions such as pH and temperature of downshift can also influence expression levels of transporters that regulate nutrient uptake which can subsequently influence regulation of cell growth and metabolism.

[0086] These cellular mechanisms can be manipulated or leveraged to further improve productivities of biologies manufacturing processes. Provided is the use of ENT and CD73 inhibitors in fed batch cultures to increase recombinant protein productivity by up to twofold. Also provided is the use of biphasic perfusion media strategy by supplementing adenosine or purine nucleosides in the media during the production phase of the process.

[0087] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.References:

[0088] Throughout this application various publications are referenced, most typically by the last name of the first author and the year of publication. Full citations for these publications are set forth below.Lai, T.; Yang, Y.; Ng, S. K., Advances in Mammalian cell line development technologies for recombinant protein production. Pharmaceuticals (Basel) 2013, 6 (5), 579-603.Du, Z.; Treiber, D.; McCarter, J. D.; Fomina-Yadlin, D.; Saleem, R. A.; McCoy, R. E.; Zhang, Y.; Tharmalingam, T.; Leith, M.; Follstad, B. D.; Dell, B.; Grisim, B.; Zupke, C.; Heath, C.; Morris, A. E.; Reddy, P., Use of a small molecule cell cycle inhibitor to control cell grow th and improve specific productivity and product quality of recombinant proteins in CHO cell cultures. Biotechnol Bioeng 2015, 112 (1), 141-55.Chong, W. P.; Sim, L. C.; Wong, K. T.; Yap, M. G., Enhanced IFNgamma production in adenosine-treated CHO cells: a mechanistic study. Biotechnol Prog 2009, 25 (3), 866-73.CarvalhaL A. V.; Santos, S. S.; Calado, J.; Haury, M.; Carrondo, M. J., Cell growth arrest by nucleotides, nucleosides and bases as a tool for improved production of recombinant proteins. Biotechnol Prog 2003, 19 (1), 69-83.Kaczmarek, E.: Koziak, K.; Sevigny, J.; Siegel, J. B.; Anrather, J.; Beaudoin. A. R.;Bach, F. H.; Robson, S. C., Identification and characterization of CD39 / vascular ATP diphosphohydrolase. J Biol Chem 1996, 271 (51), 33116-22.Resta, R.; Yamashita, Y.; Thompson, L. F., Ecto-cnz me and signaling functions of lymphocyte CD73. Immunol Rev 1998, 161. 95-109.Latini, S.; Pedata, F.. Adenosine in the central nervous system: release mechanisms and extracellular concentrations. J Neurochem 2001, 79 (3), 463-84.Zimmermann, H., Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 2000, 362 (4-5), 299-309.Fredholm, B. B.; AP. I. J.; Jacobson. K. A.; Linden, J.; Muller, C. E., International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors— an update. Pharmacol Rev 2011, 63 (1), 1-34.Naes, S. M.; Ab-Rahim, S.; Mazlan, M.; Abdul Rahman, A., Equilibrative Nucleoside Transporter 2: Properties and Physiological Roles. Biomed Res Int 2020, 2020, 5197626.Pennycooke. M.: Chaudary, N.; Shuralyova. 1.; Zhang, Y.; Coe. 1. R.. Differential expression of human nucleoside transporters in normal and tumor tissue. Biochem Biophys Res Commun 2001, 280 (3), 951-9.Chakrabarti, S.; Freedman, J. E., DP, cerebrovascular disease, and the vasculature. Vascul Pharmacol 2008, 48 (4-6). 143-9.

[0089] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A cell culture medium for the production of a product using a fed-batch process wherein the cell culture medium comprises an extracellular adenosine modulator comprising one or more of a nucleoside transport inhibitor, an inhibitor of adenosine uptake, an adenosine kinase inhibitor or an inhibitor of extracellular synthesis of adenosine.

2. The cell culture medium of claim 1, wherein the nucleoside transport inhibitor is: dilazep dihydrochloride, dipyridamole, nitrobenzylthioinosine (NBMPR), or midazolam.

3. The cell culture medium of claim 1 or 2, wherein the inhibitor of extracellular synthesis of adenosine is an ectonucleotidase (CD73) inhibitor.

4. The cell culture medium of claim 3, wherein the ectonucleotidase (CD73) inhibitor is PSB 12379.

5. The cell culture medium of any one of claims 1-4. wherein the cell culture comprises the nucleoside transport inhibitor and the inhibitor of extracellular synthesis of adenosine.

6. The cell culture medium of any one of claims 1-5, wherein the inhibitor of adenosine uptake is cilostazol.

7. The cell culture medium of any one of claims 1-6, wherein the adenosine kinase inhibitor is 5-iodotubericidin.

8. The cell culture medium of any one of claims 1-7. wherein the product is selected from the group consisting of: a protein, a recombinant protein related product, a bacteria, an enzyme, and a choloroplast.

9. The cell culture medium of any one of claims 1-8, further comprising a cell line which is a mammalian cell line.

10. The cell culture medium of claim 9, wherein the cell line is a Chinese Hamster ovary’ cell line.

11. The cell culture medium of any one of claims 1-10, wherein the extracellular adenosine modulator is present at a concentration of 0.5 pM- 5 pM.

12. The cell culture medium of claim 11, wherein the extracellular adenosine modulator is present at a concentration of 0. 1 pM -1 pM.

13. The cell culture medium of any one of claims 1-12, wherein the extracellular adenosine modulator increases overall cell density by at least 150% compared to overall cell density of a corresponding fed-batch culture not comprising the extracellular adenosine modulator.

14. The cell culture medium of any one of claims 1-13, wherein the extracellular adenosine modulator increases overall productivity by at least 100%, at least 150% or at least 200% compared to overall cell productivity of a corresponding fed-batch culture not comprising the extracellular adenosine modulator.

15. A fed-batch cultivation process comprising:(a) seeding a bioreactor,(b) adding nutrients to the bioreactor,(c) adding to the bioreactor an extracellular adenosine modulator comprising one or more of a nucleoside transport inhibitor, an inhibitor of adenosine uptake, an adenosine kinase inhibitor, or an inhibitor of extracellular synthesis of adenosine; and(d) obtaining a product.

16. The process of claim 15, wherein a first extracellular adenosine modulator is added to bioreactor prior to adding nutrients or prior to seeding the bioreactor.

17. The process of claim 15 or 16, wherein the nucleoside transport inhibitor is dilazep dihydrochloride, dipyridamole, nitrobenzylthioinosine (NBMPR), or midazolam.

18. The process of any one of claims 15-17, wherein the inhibitor of extracellular synthesis of adenosine is an ectonucleotidase (CD73) inhibitor.

19. The process of claim 18, wherein the ectonucleotidase (CD73) inhibitor is PSB 12379.

20. The process of claim any one of claims 15-19, wherein the extracellular adenosine modulator comprises the nucleoside transport inhibitor and the inhibitor of extracellular synthesis of adenosine.

21. The process of claim any one of claims 15-20, wherein the inhibitor of adenosine uptake is cilostazol.

22. The process of claim any one of claims 15-21. wherein the adenosine kinase inhibitor is 5-iodotubericidin.

23. The process of any one of claims 15-22, wherein the bioreactor comprises a cell line which is a mammalian cell line or a Chinese Hamster ovary cell line.

24. The process of any one of claims 15-23, wherein the product is selected from the group consisting of: a protein, a recombinant protein related product, a bacteria, an enzyme, and a choloroplast.

25. The process of any one of claims 15-24. wherein the extracellular adenosine modulator is present at a concentration of about 0.5 pM to about 5 pM or about 0. 1 pM to about 1 pM.

26. The process of any one of claims 15-25, wherein the extracellular adenosine modulator increases overall cell density by at least 150% compared to overall cell density of a corresponding fed-batch culture not comprising the extracellular adenosine modulator.

27. The process of any one of claims 15-26, wherein the extracellular adenosine modulator increases overall productivity of the process by at least 100%, at least 150% or at least 200% compared to overall cell productivity of a corresponding fed-batch culture process not comprising the extracellular adenosine modulator.

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