WO2018229767A1 - Methods for improving cell protein production yields - Google Patents

Abstract

The present invention is directed to a cell comprising: a polynucleotide sequence encoding an onco-KIT, or an analogue thereof having at least 80% homology to the onco-KIT, and an exogenous polynucleotide encoding a peptide of interest. Further provided is a method for improving protein production yields.

Description

METHODS FOR IMPROVING CELL PROTEIN PRODUCTION YIELDS

CROSS REFERENCE

[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/518,659 filed June 13, 2017, the contents of which are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[002] The present invention is in the field of cell biology and biomanufacturing.

BACKGROUND OF THE INVENTION

[003] The manufacturing of glycoproteins for therpeutic applications is performed in bioreactors, which can reach a capacity of a few tons. The ability to grow cells in constant conditions of temperature, pH, available nutrients and dissolved oxygen is a formidable task that gets more complicated as the bioreactor size increases. Because fluctuations in growth conditions influence all steps of the biosynthetic pathway, and thus influence the quantity and the quality of the product, it is important to use cells that exhibit stability with respect to their genetic material, growth and metabolic properties.

[004] Growth and survival of most cell types is supported by growth factor stimulations, which signal through tyrosine kinase receptors (RTK). RTK stimulation activates signaling pathways critical for progression and survival. Deprivation of RTK signaling that occurs for example upon serum starvation leads in most cell types to cell cycle arrest, senescence and apoptosis.

[005] The c-KIT RTK plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity, and resistance to kinase inhibitors. Importantly, this onco-KIT mutant when introduced into various cell types is arrested in the endoplasmic reticulum in a constitutively active form.

[006] In contrast to standard tissue culture, as used in research which utilizes serum- supplemented media that provides the required growth factors, the media used for biomanufacturing is chemically defined and must not contain any animal products. Thus, aside from specific growth factors that are recombinantly available, such as insulin, cells are mostly grown in the absence of RTK stimulation. Although cells that are used for biomanufacturing grow rapidly in an optimized chemically-defined medium and maintain high viability, incidents of stress, whether due to technical malfunctions or owing to the biological drug itself, may fail production to reach the desired quantity or quality, with certain batches even require disposal.

SUMMARY OF THE INVENTION

[007] The present invention is directed to cells comprising a mutated KIT tyrosine kinase receptor and compositions comprising same. The invention further provides a method comprising expression of a mutated KIT tyrosine kinase receptor in a cell for improving protein production yields.

[008] According to one aspect, there is provided a cell comprising: (i) a polynucleotide sequence encoding SEQ ID NO: 1, or an analogue thereof having at least 80% homology to the SEQ ID NO: 1, and (ii) an exogenous polynucleotide encoding a peptide of interest. In some embodiments, the polynucleotide sequence encoding SEQ ID NO: 1 is an exogenous polynucleotide.

[009] In some embodiments, the cell is a eukaryotic cell having increased peptide production efficacy compared to control. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is selected from a Chinese hamster ovary cell (CHO) or a human embryonic kidney cell 293 (HEK293).

[010] In some embodiments, the peptide is a glycopeptide. In some embodiments, the cell comprises a glycopeptide translated from the exogenous polynucleotide. In some embodiments, the glycopeptide has human-like glycans. In some embodiments, the glycopeptide comprises a polypeptide selected from the group consisting of: an antibody, an immunogen, and a growth factor. In some embodiments, the glycopeptide comprises a signal peptide sequence.

[011] In some embodiments, a composition comprising the cell and a carrier is provided. In some embodiments, the carrier is a chemically defined animal serum-free medium.

[012] According to another aspect, there is provided a method for improving protein production yields, the method comprising: culturing a cell comprising an exogenous polynucleotide sequence encoding SEQ ID NO: 1, or an analogue thereof having at least 80% homology to the SEQ ID NO: 1. [013] In some embodiments, improving protein production yields is improving global protein production. In some embodiments, improving protein production yields is of a protein of interest.

[014] In some embodiments, the cell has increased peptide production efficacy compared to control. In some embodiments, the method further comprises a step of isolating the secreted glycopeptide.

[015] In some embodiments, the culturing comprises growing of the cell in a medium. In some embodiments, the medium is a chemically defined serum-free medium.

[016] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[017] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[018] Figs. 1A-1C demonstrate the expression of onco-KIT enhances protein translation in a serum- supplemented DMEM medium. (A) is a graph showing a typical flow cytometry analysis of onco-KIT and hKIT expressing CHO-Kl/GPF-Fc compared to parental untransfected cells. On the right is vertical bar graph depicting a quantification of intracellular GFP-Fc levels indicative for transgene expression. Shown is the average of three independent analyses + SD. GFP-Fc expression was found to be significantly elevated in cells expressing onco-KIT (p<0.05). (B) is a vertical bar graph showing qPCR analysis of mRNA levels of GFP-Fc relative to RPLP0 (a housekeeping gene) in CHO-K1 or in cells that express either onco-KIT or hKIT. (C) comprises images of immunoblots of puromycin-labeled proteins and the assessment of KIT and mTOR activity (P-S6, P-4EBP1) in cells that expressed KIT variants.

[019] Figs. 2A-2B demonstrate translation-promoting effects of onco-KIT are maintained in chemically-defined serum-free medium. (A) comprises a graph showing a typical flow cytometry analysis of intracellular GFP-Fc of the various cell lines following the adaptation to growth in chemically-defined serum-free medium (upper panel). Quantification of fluorescence levels is shown as the average of three independent measurements + SD (lower panel). GFP-Fc expression was significantly elevated in cells expressing onco-KIT (p<0.05). (B) comprises images of immunoblots of puromycin-labeled proteins and the assessment of KIT and mTOR activity (P-S6, P- 4EBP1) in cells that expressed KIT variants.

[020] Figs. 3A-3D demonstrate onco-KIT promotes cell proliferation and mitochondrial activity. (A and B) are graphs showing proliferation analysis for four days in serum- supplemented DMEM medium or in chemically defined serum-free medium, respectively. (C and D) are vertical bar graphs of MTT analysis (representing cell viability and mitochondrial activity) which was performed at the end of the proliferation experiment for cells growing in serum-supplemented DMEM or chemically-defined serum-free medium, respectively. Shown is the average of three independent measurements + SD. For both conditions, MTT readouts were significantly elevated for cells expressing the onco-KIT (*p<0.05).

[021] Figs. 4A-4C demonstrate onco-KIT improves cellular resilience to serum deprivation. (A) comprises micrographs of cells plated at an equal density and documented for up to four days in serum-deprived DMEM medium (lOOx resolution). (B) is a vertical bar graph summarizing the percentage of viable cells and (C) is a vertical bar graph summarizing the viable cells' density (millions/well), both as were recorded on the fourth day. Shown is the average of three independent measurements + SD. Expression of onco-KIT significantly improved both parameters (*p<0.05).

[022] Figs. 5A-5B demonstrate onco-KIT protects cells from hypoxia stress. (A) is a vertical bar graph demonstrating percentages of viable cells as measured 6 hours after hypoxia initiation in serum- supplemented DMEM (black) and Fusion medium (gray). (B) is a vertical bar graph demonstrating the fluorescence intensity of the supernatants of the indicated samples at the end of the hypoxia stress. Shown is the average of three independent measurements + SD. Expression of onco-KIT significantly improved secretion (*p<0.05).

[023] Figs. 6A-6C demonstrate onco-KIT enhances unfolded protein response (UPR) activity following ER stress. (A and B) are expression profiling images comprising western blot analysis of P-IRE1 (top) and RT-PCR of XBP1 splicing (bottom) in CHO- Kl and KIT mutant expressing cell-lines, after thapsigargin or tunicamycin induced ER stress, respectively. (C) is a vertical bar graph demonstrating quantitative analysis of ERdj4 mRNA levels, as indicative for XBP-ls activity in thapsigargin (TG)-mediated ER stress. Shown are results of a typical experiment out of three.

[024] Figs. 7A-7C demonstrate expression of onco-KIT improves protein secretion. (A) is a vertical bar graph demonstrating fluorescence analysis of cell culture media following cells incubation with cycloheximide for 2 and 4 hours. (B) is a graph demonstrating cell viability and (C) is a corresponding graph of the fluorescence intensity of the cell culture media following a seven-day secretion assay in chemically- defined serum-free medium.

[025] Figs. 8A-8B are illustrations of the PI3 K/AKT/mTOR pathway. (A) is an illustration depicting activation in a ligand-dependent fashion in a serum-supplemented medium. (B) is an illustration of onco-KIT mediated constitutive activation in a ligand- independent manner in serum-free chemically-defined medium. In both scenarios, cells demonstrate proliferation and survival, increase in protein synthesis and stress resilience improvement.

DETAILED DESCRIPTION OF THE INVENTION

[026] The present invention is directed to cells comprising a mutated KIT tyrosine kinase receptor, compositions comprising same and methods for improving protein production yields.

[027] The present invention is based, in part, on the finding that cells expressing a mutated KIT provide had significantly elevated levels of protein production yields, both of total protein and/or of a protein of interest which was transected into the cells, such as a glycopeptide.

Protein biomanufacturing

[028] In some embodiments, the invention is directed to improving protein production yields, the method comprises culturing a cell comprising SEQ ID NO: 1, or an analogue thereof having at least 80% homology to SEQ ID NO: 1. In some embodiments, the cell further comprises an exogenous polynucleotide encoding a peptide of interest.

[029] In some embodiments, the invention is directed to improving protein production yields of a peptide of interest, the method comprises culturing a cell comprising SEQ ID NO: 1, or an analogue thereof having at least 80% homology to SEQ ID NO: 1, and an exogenous polynucleotide encoding a peptide of interest, thereby improving protein production yields of the peptide of interest.

[030] In another embodiment, the method of the present invention further improves culture performance of a cell of the invention compared to control. In some embodiments, a cell performance comprises global protein synthesis. In some embodiments, a cell performance comprises expression of a peptide of interest. In some embodiments, a cell performance comprises cell proliferation. In some embodiments, a cell performance comprises stress resistance. In one embodiment, stress comprises oxygen stress, including but not limited to hypoxia, or ER stress. In some embodiments, a cell of the invention has performance level equal to or greater than the performance level of a control cell. Methods for determining cell performance as defined above are well known to one of ordinary skill in the art. Non-limiting examples include, but are not limited to, qPCR, western blotting, proliferation assays, metabolism assays (such as MTT) and flow cytometry, all as described herein below.

[031] In some embodiments, a method of the present invention comprises culturing a cell in a medium. In some embodiments, a cell is grown in the medium. In some embodiments, medium is a cell culture medium suitable for growth and maintenance of a cell having increased peptide production yields. In one embodiment, cell culture medium is optimized for cell growth. In some embodiments, cell culture medium is optimized for protein synthesis. In some embodiments, "cell culture medium" refers to any liquid medium which enables cells proliferation. Growth media are known in the art and can be selected depending of the type of cell to be grown. In some embodiments, cell of the invention is cultured under effective conditions, which allow for increased yield of production from the cultured cell. Non-limiting example for increased yield include, but not limited to, increased gene expression, protein production and secretion, molecule biosynthesis, proliferation, stress resistance and others. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit for increased production yield. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce a peptide of interest of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, growth medium of the present invention is chemically defined so as to not include any animal-derived molecule or compound, such as animal serum. In some embodiments, a cell of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a mammalian cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.

[032] In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art. In some embodiments, medium of the present invention is chemically defined. In some embodiments, a chemically defined medium is devoid of animal products. In some embodiments, a medium devoid of animal products comprises a serum free medium. In some embodiments, a chemically defined serum free medium is optionally supplemented with hormones, including but not limited to insulin.

[033] In some embodiments, depending on the vector and host system used for production, resultant polypeptides of the present invention either remain within the recombinant cell, secreted into the fermentation medium or secreted into a space between two cellular membranes. In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is affected.

[034] In one embodiment, the phrase "recovering the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.

[035] In one embodiment, polypeptides of the present invention are purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

[036] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography; e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854-15859 (1990)].

[037] In one embodiment, the polypeptide of the present invention is retrieved in "substantially pure" form that allows for the effective use of the protein in subsequent applications, such as for therapy or diagnosis.

[038] As used herein, the term "substantially pure" describes a peptide/polypeptide or other material which has been separated from its native contaminants. Typically, a monomeric peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide backbone. Minor variants or chemical modifications typically share the same peptide sequence. A substantially pure peptide can comprise over about 85 to 90% of a peptide sample, and can be over 95% pure, over 97% pure, or over about 99% pure. Purity can be measured on a polyacrylamide gel, with homogeneity determined by staining. Alternatively, for certain purposes high resolution may be necessary and HPLC or a similar means for purification can be used. For most purposes, a simple chromatography column or polyacrylamide gel can be used to determine purity.

[039] The term "purified" does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition. A peptide is in the "purified" state after purification of the starting material or of the natural material by at least one order of magnitude, 2 or 3, or 4 or 5 orders of magnitude.

[040] In one embodiment, the polypeptides of the present invention are substantially free of naturally-associated host cell components. The term "substantially free of naturally- associated host cell components" describes a peptide or other material which is separated from the native contaminants which accompany it in its natural host cell state. Thus, a peptide which is chemically synthesized or synthesized in a cellular system different from the host cell from which it naturally originates will be free from its naturally-associated host cell components.

Cells

[041] In some embodiments, a cell of the disclosed invention has increased peptide production efficacy compared to control. In some embodiments, the term "increased peptide production efficacy" as used herein refers to an endogenous polypeptide or an exogenous polynucleotide encoding a protein of interest. In some embodiments, a cell having increased production efficacy of a peptide comprises increased mRNA transcription levels, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased mRNA translation rates, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased translated peptide levels, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased stability of a translated peptide, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased levels of properly folded translated peptide, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased level of properly post-translationally modified translated peptide, compared to a control cell. In some embodiments, a cell having increased production efficacy of a peptide comprises increased secretion levels of a translated peptide, compared to a control cell.

[042] In some embodiments, the terms "increasing" and "improving" used herein are interchangeable and denote values compared to a control. In some embodiments, increasing is by at least 5%, by at least 10%, by at least 20%, by at least 30%, by at least 50%, by at least 60%, by at least 75%, by at least 80%, by at least 90%, by at least 95%, or by at least 100%, compared to control. In some embodiments, increasing is by 1-5%, 3-8%, 7-12%, 10-15%, 13-20%, 18-25%, 22-30%, 26-35%, 33-45%, 40-55%, 50-65%, 60-75%, 70-85%, 80-90%, 90-99%, or 95-100%, compared to control. In some embodiments, increasing is by at least 2-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, compared to control. Each possibility represents a separate embodiment of the present invention.

[043] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to multiply or express the polynucleotide or polypeptide of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the polypeptide coding sequence.

[044] In some embodiments, non-bacterial expression systems are used. In some embodiments, cells of the invention include mammalian cells to express the polypeptide of the present invention. In some embodiments, mammalian cells are derived of human origin. In some embodiments, mammalian cells are derived of murine origin. Non- limiting examples for mammalian cells include, but are not limited to 3T3-L1, 4T1, 9L, A172, A20, A253, A2780, A2780ADR, A2780cis, A431, A549, AHL-1, ALC, B 16, B53, BCP-1, BEAS-2B, bEnd.3, BHK-21, BOSC23, BT-20, BxPC3, C2C12, C3H- 10T1/2, C6, Caco-2, Cal-27, Calu-3, CGR8, CHO, CML Tl, CMT12, COR-L23, COR- L23/5010, COR-L23/CPR, COR-L23/R23-, COS-7, COV-434, CT26, D17, DAOY, DH82, DU145, DuCaP, E14Tg2a, EL4, EM-2, EM-3, EMT6/AR10.0, FM3, GL261, H1299, HaCaT, HCA2, HEK 293, HEK 293T, HeLa, Hepalclc7, Hep G2, HL-60, HT- 1080, HT-29, J558L, Jurkat, JY, K562, KBM-7, KCL-22, KG1, Ku812, KYO-1, L1210, L243, LNCaP, MA- 104, MA2.1, MC-38, MCF-7, MCF-IOA, MDA-MB-231, MDA- MB-361, MDA-MB-468, MDCK II, MG63, MOR/0.2R, Mono-Mac-6, MRC-5, MTD- 1A, MyEnd, NCI-H69, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI- H69/LX4, Neuro-2a, NIH-3T3, NALM-1, NK-92, NTERA-2, NW-145, OK, OPCN, P3X63Ag8, PC12, PC-3, Peer, PNT1A, PNT2, Pt K2, Raji, rbl-1, RenCa, RIN-5F, RMA-S, SaOS-2, SH-SY5Y, SiHa, SK-BR-3, SK-OV-3, SK-N-SH, T2, T-47D, T84, T98G, THP-1, U20S, U373, U87, U937, VCaP, Vero, VG-1, WM39, WT-49, YAC-1, YAR and others. In some embodiments, the expression vector is used to express polynucleotides of the present invention in mammalian cells.

[045] In some embodiments, in bacterial systems of the present invention, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[046] In some embodiments, polynucleotides of the present invention are prepared using PCR techniques as described in Example 1, or any other method or procedure known to one skilled in the art. In some embodiments, the procedure involves the ligation of two different DNA sequences (See, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[047] In one embodiment, polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant polypeptide. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

[048] In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.

[049] In one embodiment, the expression vector of the present invention may further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES).

[050] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+), pGL3, pZeoSV2(+), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMTl, pNMT41, pNMT81, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[051] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[052] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression of the polynucleotides of the present invention. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[053] Various methods can be used to introduce the expression vector of the present invention into cells, in some embodiments, cells introduced with any exogenous polynucleotide, as abovementioned are termed herein "transformed cells" or "recombinant cells". Methods for introducing polynucleotide vectors are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Bio techniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[054] A person with skill in the art will appreciate that the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture.

[055] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[056] In some embodiments, the invention is directed to a composition comprising a cell having increased protein production yield and comprising SEQ ID NO: 1, or an analogue thereof having at least 80% homology to SEQ ID NO: 1, an exogenous polynucleotide encoding a peptide of interest; and a carrier or diluent. Polypeptides and polynucleotides

[057] In some embodiments, the disclosed invention is directed to a cell comprising: (i) a polynucleotide sequence encoding a mutated c-KIT tyrosine kinase receptor (onco- KIT), and (ii) an exogenous polynucleotide encoding a peptide of interest.

[058] Wild type (WT) and mutated (Onco) KIT transcripts and proteins of the invention are specified herein below (Table 1).

Table 1 - Polynucleotides and polypeptides of the invention

[059] In some embodiments, an onco-KIT of the invention comprises or consists of the amino acid sequence as set forth in SEQ ID NO: 1, or an analogue thereof having at least 80% sequence identity thereto. In another embodiment, an onco-KIT protein (SEQ ID NO: 1) has a ligand-independent phosphorylation activity as described herein. In another embodiment, an onco-KIT protein is a mutant of SEQ ID NO: 9 having ligand- independent KIT protein activity as described herein. In another embodiment, an onco- KIT protein (SEQ ID NO: 2) is a fragment of SEQ ID NO: 1 having a ligand-independent phosphorylation activity. In another embodiment, an onco-KIT protein is a mutant of SEQ ID NO: 9 or an analogue thereof having at least 80% sequence identity thereto and having an Aspartic acid residue at position 816 substituted by a Valine (D816V). [060] In another embodiment, an onco-KIT protein of the invention is encoded by a DNA sequence which comprises or consists the nucleic acid sequence as set forth in SEQ ID Nos.: 3-4 or an analogue thereof having at least 75% sequence identity thereto.

[061 ] In some embodiments, an onco-KIT of the invention is a murine onco-KIT which comprises or consists the amino acid sequence as set forth in SEQ ID NO: 5, or an analogue thereof having at least 80% sequence identity thereto. In another embodiment, a murine onco-KIT protein (SEQ ID NO: 5) has a ligand-independent phosphorylation activity as described herein. In another embodiment, a murine onco-KIT protein is a mutant of SEQ ID NO: 10 having ligand-independent KIT protein activity as described herein. In another embodiment, a murine onco-KIT protein (SEQ ID NO: 6) is a fragment of SEQ ID NO: 5 having a ligand-independent phosphorylation activity. In another embodiment, a murine onco-KIT protein is a mutant of SEQ ID NO: 10 or an analogue thereof having at least 80% sequence identity thereto and having an Aspartic acid residue at position 818 substituted by a Tyrosine (D818Y).

[062] In another embodiment, a murine onco-KIT protein of the invention is encoded by a DNA sequence which comprises or consists the nucleic acid sequence as set forth in SEQ ID Nos.: 7-8 or an analogue thereof having at least 75% sequence identity thereto.

[063] As used herein, the term "analogue" includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.

[064] As used herein, the phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite function as specified herein. [065] In one embodiment, the chimeras and/or peptides of the invention encompass variant thereof. As used herein, the term "variant" refers to a polypeptide or nucleotide sequence which comprises a modification of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively. In some embodiments, the modifications are substitution, deletion, and/or insertion of one or more amino acids or nucleotides as compared to another polypeptide or polynucleotide, respectively. In some embodiments, the changes may be of minor nature, such as conservative amino acid substitutions or for nucleotide sequence resulting in conservative amino acid substitutions that do not significantly affect the activity of the polypeptide. In some embodiments, the changes may be substitution of an amino acid molecule, resulting in an addition of a glycosylation site, thereby increasing glycosylation of the polypeptide.

[066] The invention further encompasses a polynucleotide sequence comprising a nucleic acid encoding a peptide of interest. In some embodiments, a peptide of interest in an exogenous peptide. In some embodiments, polynucleotide encoding a peptide of interest is transformed into a cell of the invention. Non-limiting examples for methods of transforming exogenous polynucleotide molecule into a cell of the disclosed invention include, but not limited to: membrane permeabilization, electroporation, viral transformation, transfection, among others, all of which are known to a person of ordinary skill in the art.

[067] In some embodiments, the peptide of interest comprises a signal peptide sequence for secretion. The term "signal peptide sequence" refers to an approximately 16-40 amino acid stretch present on the amino -terminus of a protein which directs the nascent protein to the periplasm (prokaryotic cells) or permits the secretion of the protein (eukaryotic cells). The signal peptide can be cleaved from the protein once the protein has been directed to its desired location (i.e., periplasm, secretory granule, etc.). The terms "signal peptide," "signal peptide sequence" and "leader sequence peptide" are used interchangeably in the art.

[068] As defined herein, a control cell is any cell having a native KIT activity. As used herein, a native KIT activity comprises ligand dependent phosphorylation activity. In some embodiments, a control cell comprises SEQ ID Nos.: 9 or 10. [069] The term "endogenous" is used to refer to a polypeptide that is naturally expressed or produced by a cell, a tissue or an organism. The term "exogenous" is used to refer to any polynucleotide or polypeptide that originate outside of the organism of concern or study and is transformed into the cell.

[070] The term "chimeric" is used to refer to a polypeptide formed by the joining of two or more peptides through a peptide bond formed between the amino terminus of one peptide and the carboxyl terminus of another peptide. The chimeric polypeptide may be expressed as a single polypeptide fusion protein from a nucleic acid sequence encoding the single contiguous conjugate.

[071] In some embodiments, the polypeptide of interest of the disclosed invention is a glycosylated polypeptide. The terms "glycosylated polypeptide" and "glycopeptide" are interchangeable. In another embodiment, a glycopeptide of the present invention requires glycosylation for rendering activity. The term "glycosylation" used herein, refers to the attachment of oligosaccharides (carbohydrates containing two or more simple sugars linked together e.g. from two to about twelve simple sugars linked together) to the polypeptide. The oligosaccharide side chains are linked to the backbone of the polypeptide through either N- or O-linkages. "N-linked glycosylation" refers to the attachment of the carbohydrate moiety to an asparagine (i.e., N) residue in a glycoprotein chain.

[072] In some embodiments, a glycopeptide of the invention is selected from the group consisting of: antibodies, growth factors, immunogens, enzymes, coagulation or anticoagulation proteins.

[073] In some embodiments, the polynucleotide of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis -regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing tissue- specific expression of the polypeptide of the present invention. In some embodiments, the cis- regulatory sequence is suitable for directing inducible expression of the polypeptide of the present invention.

[074] The term "polynucleotide" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide. In one embodiment, a polynucleotide refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

[075] In one embodiment, "complementary polynucleotide sequence" refers to a sequence, which results from reverse transcription of messenger RNA using a reverse transcriptase or any other RNA dependent DNA polymerase. In one embodiment, the sequence can be subsequently amplified in vivo or in vitro using a DNA polymerase.

[076] In one embodiment, "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence can include some exonal sequences required to encode the polypeptide of the present invention, as well as some intronic sequences interposing there between. In one embodiment, the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. In one embodiment, intronic sequences include cis acting expression regulatory elements.

Polypeptide sequences

[077] In some embodiments, SEQ ID NO: 1 comprises or consists of the acid sequence as set forth:

MRG ARG A WDFLC VLLLLLRVQTGS S QPS VSPGEPS PPS IHPGKS DLIVR VGDEI

RLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSI

YVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKD

LRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPV

VS VS KAS YLLREGEEFT VTCTIKD VS S S V YS TWKRENS QTKLQEK YNS WHHG

DFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMI

NTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIR

YVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGM

LQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSID

SSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVA

GMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFP

RNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREAL

MSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFIC S KQEDH AE A ALYKNLLHS KES S C S DSTNE YMDMKPG VS Y V VPTKADKRRS VR IGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAAR NILLTHGRITKICDFGLARVIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFES D VWS YGIFLWELFS LGS S PYPGMP VDS KFYKMIKEGFRMLS PEH AP AEM YDIM KTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRI NSVGSTASSS QPLLVHDD V .

[078] In some embodiments, SEQ ID NO: 2 comprises or consists of the acid sequence as set forth:

TLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYID

PTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVK

MLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYG

DLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVS

YVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAF

LASKNCIDRDLAARNILLTHGRITKICDFGLARVIKNDSNYVVKGNARLPVKW

M APES IFNCVYTFESD VWS YGIFLWELFS LGS SPYPGMPVDS KFYKMIKEGFR

MLS PEH AP AEM YDIMKTC WD ADPLKRPTFKQIVQLIEKQIS ES TNHI YS NLANC

SPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV.

[079] In some embodiments, SEQ ID NO: 9 comprises or consists of the acid sequence as set forth:

MRG ARG A WDFLC VLLLLLRVQTGS S QPS VSPGEPS PPS IHPGKS DLIVR VGDEI

RLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSI

YVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKD

LRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPV

VS VS KAS YLLREGEEFT VTCTIKD VS S S V YS TWKRENS QTKLQEK YNS WHHG

DFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMI

NTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIR

YVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGM

LQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSID

SSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVA

GMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFP

RNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREAL

MSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFIC

S KQEDH AE A ALYKNLLHS KES S C S DSTNE YMDMKPG VS YV VPTKADKRRS VR IGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAAR NILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFES D VWS YGIFLWELFS LGS S PYPGMP VDS KFYKMIKEGFRMLS PEH AP AEM YDIM KTC WD ADPLKRPTFKQIVQLIEKQIS ES TNHI YS NLANCS PNRQKP V VDHS VRI NSVGSTASSS QPLLVHDD V .

[080] In some embodiments, SEQ ID NO: 5 comprises or consists of the acid sequence as set forth:

MRGARGAWDLLCVLLVLLRGQTATSQPSASPGEPSPPSIHPAQSELIVEAGDTL

S LTCIDPDFVRWTFKTYFNEM VENKKNEWIQEKAEATRTGTYTCS NSNGLTS S

IYVFVRDPAKLFLVGLPLFGKEDSDALVRCPLTDPQVSNYSLIECDGKSLPTDL

TFVPNPKAGITIKNVKRAYHRLCVRCAAQRDGTWLHSDKFTLKVRAAIKAIPV

VS VPETS HLLKKGDTFT V VCTIKD VS TS VNS MWLKMNPQPQHIAQ VKHNS WH

RGDFNYERQETLTISSARVDDSGVFMCYANNTFGSANVTTTLKVVEKGFINISP

VKNTTVFVTDGENVDLVVEYEAYPKPEHQQWIYMNRTSANKGKDYVKSDNK

S NIR Y VNQLRLTRLKGTEGGT YTFLVS NS D AS AS VTFN V Y VNTKPEILT YDRLI

NGMLQCVAEGFPEPTIDWYFCTGAEQRCTTPVSPVDVQVQNVSVSPFGKLVV

QSSIDSSVFRHNGTVECKASNDVGKSSAFFNFAFKGNNKEQIQAHTLFTPLLIG

FVVAAGAMGIIVMVLTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYD

HKWEFPRNRLS FGKTLG AG AFGKV VE AT A YGLIKS D A AMT V A VKMLKPS AH

LTEREALMSELKVLSYLGNHMNIVNLLGACTVGGPTLVITEYCCYGDLLNFLR

RKRDSFIFSKQEEQAEAALYKNLLHSTEPSCDSSNEYMDMKPGVSYVVPTKTD

KRRSARIDSYIERDVTPAIMEDDELALDLDDLLSFSYQVAKGMAFLASKNCIHR

DLAARNILLTHGRITKICDFGLARYIRNDSNYVVKGNARLPVKWMAPESIFSCV

YTFES D VWS YGIFLWELFS LGS S PYPGMP VDS KFYKMIKEGFRMVS PEH AP AE

MYDVMKTCWDADPLKRPTFKQVVQLIEKQISDSTKHIYSNLANCNPNPENPV

V VDHS VR VNS VGS S AS S TQPLLVHED A.

[081] In some embodiments, SEQ ID NO: 6 comprises or consists of the acid sequence as set forth:

TLFTPLLIGFVVAAGAMGIIVMVLTYKYLQKPMYEVQWKVVEEINGNNYVYI

DPTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVK

MLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTVGGPTLVITEYCCY

GDLLNFLRRKRDSFIFSKQEEQAEAALYKNLLHSTEPSCDSSNEYMDMKPGVS

YVVPTKTDKRRSARIDSYIERDVTPAIMEDDELALDLDDLLSFSYQVAKGMAF LASKNCIHRDLAARNILLTHGRITKICDFGLARYIRNDSNYVVKGNARLPVKW MAPESIFSCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRM VSPEHAPAEMYDVMKTCWDADPLKRPTFKQVVQLIEKQISDSTKHIYSNLANC NPNPENP V V VDHS VRVNS VGS S AS S TQPLLVHED A .

[082] In some embodiments, SEQ ID NO: 10 comprises or consists of the acid sequence as set forth:

MRGARGAWDLLCVLLVLLRGQTATSQPSASPGEPSPPSIHPAQSELIVEAGDTL

S LTCIDPDFVRWTFKTYFNEM VENKKNEWIQEKAEATRTGTYTCS NSNGLTS S

IYVFVRDPAKLFLVGLPLFGKEDSDALVRCPLTDPQVSNYSLIECDGKSLPTDL

TFVPNPKAGITIKNVKRAYHRLCVRCAAQRDGTWLHSDKFTLKVRAAIKAIPV

VS VPETS HLLKKGDTFT V VCTIKD VS TS VNS MWLKMNPQPQHIAQ VKHNS WH

RGDFNYERQETLTISSARVDDSGVFMCYANNTFGSANVTTTLKVVEKGFINISP

VKNTTVFVTDGENVDLVVEYEAYPKPEHQQWIYMNRTSANKGKDYVKSDNK

S NIR Y VNQLRLTRLKGTEGGT YTFLVS NS D AS AS VTFN VY VNTKPEILT YDRLI

NGMLQCVAEGFPEPTIDWYFCTGAEQRCTTPVSPVDVQVQNVSVSPFGKLVV

QSSIDSSVFRHNGTVECKASNDVGKSSAFFNFAFKGNNKEQIQAHTLFTPLLIG

FVVAAGAMGIIVMVLTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYD

HKWEFPRNRLS FGKTLG AG AFGKV VE AT A YGLIKS D A AMT V A VKMLKPS AH

LTEREALMSELKVLSYLGNHMNIVNLLGACTVGGPTLVITEYCCYGDLLNFLR

RKRDSFIFSKQEEQAEAALYKNLLHSTEPSCDSSNEYMDMKPGVSYVVPTKTD

KRRSARIDSYIERDVTPAIMEDDELALDLDDLLSFSYQVAKGMAFLASKNCIHR

DLAARNILLTHGRITKICDFGLARDIRNDSNYVVKGNARLPVKWMAPESIFSCV

YTFES D VWS YGIFLWELFS LGS S P YPGMP VDS KF YKMIKEGFRM VS PEH APAE

MYDVMKTCWDADPLKRPTFKQVVQLIEKQISDSTKHIYSNLANCNPNPENPV

V VDHS VRVNS VGS S AS S TQPLLVHED A.

[083] In the discussion unless otherwise stated, adjectives such as "substantially" and "about" modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word "or" in the specification and claims is considered to be the inclusive "or" rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins. [084] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms "a," "an" and "at least one" are used interchangeably in this application.

[085] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[086] In the description and claims of the present application, each of the verbs, "comprise," "include" and "have" and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[087] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[088] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[089] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub -combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

EXAMPLES

[090] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Materials and Methods

Cell lines and pharmacological treatments

[091] CHO-K1 (ATCC) cells were cultured in Dulbecco's Modified Eagle medium (Biological Industries, Israel): Nutrient Mixture F-12 (F-12 DMEM, Sigma- Aldrich) supplemented with 10% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine (Biological Industries, Israel), 1% penicillin- streptomycin solution (Biological Industries, Israel), and 1 mM sodium pyruvate (Biological Industries, Israel) at 37 °C under 5% C0₂. Chemically-induced ER stress was performed by treating cells with either thapsigargin or tunicamycin (purchased from Fermentek, Israel), at a concentration of 2.5 μg/mL for 8 hours. Compounds were dissolved in DMSO to 2.5 mg/mL stock solution.

Generation of stable transgene expressing CHO-K1 cells

[092] GFP-Fc was cloned into pcDNA3.1(+) between EcoRI and EcoRV restriction sites. In approximately 70% confluence, CHO-K1 cells were transfected using TransIT^® 2020 (3 μΐ_^ of transfection reagent^g of DNA). The transfected cells were recovered for 24 hours followed by FACS sorting of GFP positive cells. Following one week of culturing, sorting was preformed, and the process was then repeated three more times until a stable pool of GFP-positive cells was obtained. From this bulk, single-cell cloning was performed by limiting dilution. The expanded clones were validated for GFP-Fc expression by flow cytometry and immunoblotting, and the same clone was used for further engineering (termed 'CHO-Kl/GFP-Fc').

[093] Onco-KIT and hKIT were cloned into pcDNA 3. l/Hygro⁽⁺⁾ mammalian expression vector between Notl and Nhel restriction sites for stable expression in CHO- Kl/GFP-Fc cells. KIT variants were separately transfected into the same clone of the CHO-Kl/GFP-Fc cells following two weeks of selection with Hygromycin B (A.G. Scientific, Inc.) at a concentration of 500 μg/mL. KIT expression was verified by immunoblotting .

Western blotting

[094] Cells were either trypsinized or directly harvested, centrifuged at 3,000 rpm for 5 min, and washed twice in PBS. For cell lysis, radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors was added in a volume about four times the volume of the cells' pellet, then vibrated for 20 min at 4 °C. Lysates were cleared by centrifugation in 14,000 rpm for 30 min at 4 °C. Five times (5x) reduced Laemmli sample buffer was added, boiled for 5 min at 95 °C, and loaded on SDS-PAGE. Blotting into PVDF membranes was performed using Biorad PowerPac™. Blots were blocked in 10% skim milk in TBST buffer for 1 hour at room temperature. Primary antibodies were rabbit anti-phospho S6 Ribosomal Protein (Ser240/244) (cell signaling #2215), c-Kit (D13A2) (cell signaling #3074), Phospho-c-Kit (Tyr719) (cell signaling #3391), Phospho-4E-BPl (Thr37/46) (236B4) (cell signaling #2855), Anti-IREl (phospho S724) Rabbit [EPR5253] (abeam ab 124945), mouse anti-puromycin Antibody, clone 12D10 (Millipore #MABE343). For GFP and p97 polyclonal antibodies were used. Secondary HRP-conjugated goat anti-rabbit and anti-mouse (Jackson Immunoresearch, West Grove, PA) were used. Blots were developed in Bio-Rad ChemiDoc™ XR and analyzed using Image Lab™ software.

Puromycin labeling for protein synthesis evaluation

[095] Cells were harvested, centrifuged and resuspended in 3 ml of fresh medium. A portion of 1 ml was analyzed for cell counts and the remaining 2 ml were pulsed for 10 min by adding 10 μΐ of puromycin solution (Millipore, Cat. No. 508838, 10 mg/ml). Following the addition of puromycin, cells were immediately incubated at 37 °C under 300 rpm shaking and ice-cold PBS was added to a volume of 40 ml to terminate the protein labeling. Cells were washed twice with cold PBS and 10 x 10⁶ cells, according to the count analysis performed, were lysed in 200 μΐ of lx reduced Laemmli sample buffer preheated to 70 °C. Equal volumes were loaded on 12% SDS-PAGE and following blotting, probed with an anti-puromycin antibody.

Flow cytometry

[096] Cells were harvested, centrifuged and washed twice in PBS, filtered through a 100 μΜ strainer directly to FACS tubes. Analyses were performed with Cytoflex FACS using the CytExpert software.

qPCR and RT-PCR

[097] Total RNA was isolated using TRI-reagent (Bio-Rad). cDNA was synthesized using 1 μg RNA with qScript^™ cDNA Synthesis KIT (Quanta Biosciences) according to the manufacturer's instructions. Bio-Rad iTaq™ universal SYBR^® Green Supermix was used for quantitative Real-Time PCR analyses. hRPLPO was used as an endogenous housekeeping gene for PCR quantification. Analysis was performed on CFX Connect™ Real-Time PCR Detection System (Bio-Rad) using the Bio-Rad CFX manager 3.1 software. The following primers were used: GFP-Fc: forward- 5'- TGAAGTTC ATCTGC ACC ACCG-3 ' (SEQ ID NO: 11), reverse - 5'- AGTCGTGCTGCTTC ATGTGGT-3 ' (SEQ ID NO: 12); for RPLP0 forward: 5'- CCAACTACTTCCTTAAGATCATCCAACT-'3 (SEQ ID NO: 13), reverse: 5'- ACATGCGGATCTGCTGCA-'3 (SEQ ID NO: 14). RT-PCR was used for the detection of XBP1 mRNA splicing using 5x Red Load Taq (LAROVA) with CHO-XBP1 splicing primers: forward- 5 '-CCTTGT AATTGAGAACC AGG-3 ' (SEQ ID NO: 15), reverse - 5'-CCAAAAGGATATCAGACTCGG-3' (SEQ ID NO: 16). ERdj4: forward-5- GGTGTGCCAAAATCGGCATC-3' (SEQ ID NO: 17), reverse: 5'- GCACTGTGTCCAAGTGTATCA-3' (SEQ ID NO: 18).

Cell proliferation kinetics evaluation [098] The same number of cells was plated (DMEM) or suspended (Fusion) in 24-well plates. On each day of the experiment three wells were harvested and cell counting was performed using a hemocytometer and trypan blue exclusion to discriminate live and dead cells. The mean of three wells was recorded as cell number per well or cells per ml medium.

Analysis of mitochondrial activity by MTT assay

[099] An equal number of cells were seeded in 96-well plates in phenol-red free DMEM medium or in Fusion CD. MTT assay was performed according to the supplier's instructions (Vybrant® MTT Cell Proliferation Assay Kit, ThermoFisher).

Hypoxia stress

[0100] A day prior to the experiment, an equal number of cells was either plated or seeded in 6 cm plates. Plates were subjected to hypoxia stress for 6 hours using a nitrogen chamber. At the end of the treatment, equal volumes of each sample were used to measure the cell counts and fluorescence in the supernatants.

Growth and productivity assessments

[0101] Cells were adapted to shaking conditions at 120 rpm in EX-CELL CD CHO Fusion medium for five passages using shake flasks. Then, cells were seeded at a density of 0.5 x 10⁶ cells/mL in the chemically-defined medium and examined for growth kinetics, viability and secretion capacity for the duration of seven days. On each day of the experiment a small portion was taken for cell counts. Samples were then centrifuged, and supernatants were collected and stored at -80 °C till the end of the experiment. Productivity was evaluated by the measurement of the fluorescence intensity of the supernatants using a Cytation 3 plate reader.

EXAMPLE 1

Onco-KIT expression enhances protein translation in serum-supplemented medium

[0102] As proof-of-concept for assessing the effects of KIT on protein expression and secretion the inventors generated a CHO-K1 single-cell clone that stably expresses an Ig fusion composed of a GFP domain, preceded by a signal peptide, fused to the constant region of a human IgGl, termed GFP-Fc. These cells were further engineered to express either the wild type (WT) hKIT or SEQ ID NO: 2 (also known in the art to comprise a D816V substitution) in a stable fashion under the selection of hygromycin. Flow cytometry analysis of the intracellular levels of GFP-Fc indicated a significantly higher expression in CHO-Kl cells that co-expressed onco-KIT compared to parental cells or hKIT (Fig. 1A). When the three cell lines were compared, levels of the GFP-Fc encoding mPvNA were of similar values (Fig. IB). The discrepancy between the mRNA levels and the measured fluorescence indicated a regulation at the post-transcription level. To examine whether protein translation is affected by the expression of the different KIT variants the inventors pulsed the cells for a short time with puromycin and performed immunoblotting with an anti-puromycin antibody that resolves only the newly synthesized proteins. Expression of onco-KIT, but not hKIT, elevated the global protein translation (Fig. 1C, top panel).

[0103] When expressed in cells, KIT yields two major polypeptides that are distinct in their glycan types and are readily separated on SDS-PAGE. The heavier one, which contains complex N-linked carbohydrate modifications and resides at the cell surface, and a lighter one that express the high mannose N-linked glycans, which is found in the ER. When expressed in CHO cells, the WT hKIT allele was mostly expressed in the heavier form, while the onco-KIT was mostly expressed in the lighter form, corresponding to its typical intracellular localization. Analysis of phosphorylated KIT status indicated that both KIT variants, when cultured in the presence of serum, were active (Fig. 1C). KIT activates the PI3K pathway, which subsequently activates the mammalian target of rapamycin (mTOR) pathway. mTOR, when activated, increases protein translation by multiple pathways. To assess the activation of the mTOR pathway the inventors measured the levels of phosphorylated 4-EBP1 and ribosomal S6 proteins by immunoblotting. As both of these targets are phosphorylated in an mTOR-dependent fashion, an elevation in the activity of mTOR in the onco-KIT expressing cells was indicated (Fig. 1C).

EXAMPLE 2

Onco-KIT translation-promoting effects is maintained in chemically defined serum-free medium

[0104] To further assess the ability of onco-KIT to improve performance under conditions more relevant to biomanufacturing, the inventors adapted the cells for growth in the chemically-defined animal-free medium, EX-CELL® CD CHO Fusion. Examination of the intracellular GFP-Fc levels by flow cytometry displayed a higher expression for cells expressing the onco-KIT than those expressing hKIT and the CHO- Kl controls (Fig. 2A). Translation analysis indicated that the onco-KIT expressing cells increased the levels of protein synthesis using the puromycin-pulse method (Fig. 2B, upper panel). This was consistent with the analysis of phospho KIT levels, which was detected only for the onco-KIT expressing cells and not for the hKIT expressing cells. Thus, onco-KIT signals in the ligand-free chemically-defined medium, while hKIT is inactive. Accordingly, higher phosphorylation levels of S6 and 4EBP1 were observed for the onco-KIT expressing cells indicating the hyperactivation of mTOR. The inventors concluded that in a chemically-defined medium, hKIT-expressing cells behave as CHO-Kl, while onco-KIT maintains a strong signal that promotes translation and mTOR activation in a manner that promotes transgene expression (Fig. 2B).

EXAMPLE 3

Onco-KIT promotes cell proliferation and mitochondrial activity

[0105] Owing to the fact that onco-KIT operates as a driver mutation for several types of tumors, the inventors investigated whether its expression in CHO would promote proliferation. The inventors followed the proliferation kinetics following the seeding of an equal number of cells. Whether adhered to the plastic as in serum-supplemented DMEM or whether in suspension, as for EX-CELL® CD CHO Fusion, onco-KIT endowed cells with faster proliferation rates (Fig. 3A and 3B). In addition, to assess the viability of the cells at day 4 of the proliferation assay, the inventors performed an MTT assay (Fig. 3C and 3D), which correlated to the cell numbers. These data indicated that expression of onco-KIT in CHO cells promoted their proliferation capacity and maintained cell viability.

EXAMPLE 4

Onco-KIT improves resilience to conditions of serum deprivation

[0106] Onco-KIT triggered a strong RTK signal in the absence of the ligand. To test whether the signal was sufficient to compensate for the growth factor stimulation, the inventors followed cell number and viability under serum deprivation conditions. While the parental CHO-Kl and the hKIT expressing CHO-Kl cells showed clear morphological signs of apoptosis after 4 days without the serum, the onco-KIT expressing cells continued to proliferate and reached 100% confluence (Fig. 4A). Total cell numbers were three times more than the controls and the onco-KIT cells maintained higher viability (Fig. 4B and 4C). The continuing proliferation of onco-KIT transduced CHO-Kl cells, even in the absence of serum, indicated that the ligand-independent signal emitted by onco-KIT can replace the growth supporting elements that are provided by the serum.

EXAMPLE 5

Onco-KIT protects cells from hypoxia stress

[0107] To determine whether onco-KITs can provide protection to hypoxia stress, an equal number of cells were seeded in serum- supplemented DMEM or in chemically- defined medium. Cells were then subjected to 6-hour hypoxia treatment using a nitrogen chamber. Primarily, for the cells cultured in EX-CELL® CD CHO Fusion, the inventors observed improved viability (Fig. 5A). Analysis of the supernatants indicated that expression of onco-KIT conferred elevated levels of GFP-Fc, suggesting that onco-KIT supported secretion under hypoxic conditions (Fig. 5B).

EXAMPLE 6

Onco-KIT augments UPR activity under ER stress conditions

[0108] For reasons that are not fully understood, onco-KIT is mainly found in the ER and its trafficking to the cell surface can be expedited by manipulating its phosphorylation status. The difference in the intracellular localization urged the inventors to investigate whether onco-KIT has any effect on ER function in stressful conditions. Under basal culturing conditions, the stable expression of onco-KIT or hKIT did not result in a strong unfolded protein response (UPR) as assessed by the expression of P-IRE1 and the splicing of XBP1, a hallmark of the UPR. When ER stress was provoked by thapsigargin, which reduces calcium concentrations in the ER, or tunicamycin, which inhibits N-linked glycosylation, the presence of onco-KIT enhanced the UPR. This was evident by the increase in phosphorylated IREl and enhancement of XBP1 mRNA splicing (Fig. 6A and B). Accordingly, ERdj4, a target of XBPls, was induced to a higher level in the onco-KIT expressing cells than the hKIT (Fig. 6C). Thus, onco-KIT may have improved the ability to sustain UPR signaling under ER stress conditions.

EXAMPLE 7

Onco-KIT improves transgene protein secretion

[0109] To assess whether the higher translation and transgene expression conferred by onco-KIT was also manifested in higher productivity, cells were treated with the protein synthesis inhibitor cycloheximide for a short time and the fluorescence in the medium was monitored. In a manner that correlated with the expression level, supernatants of the cells that expressed onco-KIT contained higher levels of GFP-Fc (Fig. 7A). the inventors then subjected the cells to a 7-day batch secretion assay in EX-CELL® CD CHO Fusion in shake flasks. Viable cell densities were significantly higher in the cells that expressed onco-KIT compared to CHO-K1 and cells with hKIT (Fig. 7B). Fluorescent intensities of the supernatants, which are indicative to the overall secreted GPF-Fc, showed that onco-KIT enhanced overall productivity starting from day 4 until the final day of the experiment, reaching more than a two-fold increase in the total fluorescence intensity (Fig. 7C). This correlated to the viability of the cells. While under the current experimental conditions the viability of CHO-K1 and hKIT expression cells was compromised at day 4 and onwards, probably owing to the depletion of nutrients, onco- KIT expressing cells endured the batch conditions much better, hence allowing secretion to commence and titers to rise till the end of the experiment. The inventors concluded that expression of onco-KIT can serve as a useful strategy to improve cell robustness and overall productivity for biomanufacturing.

[0110] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

1. A cell comprising:

(i) a polynucleotide sequence encoding SEQ ID NO: 1, or an analogue thereof having at least 80% homology to said SEQ ID NO: 1, and

(ii) an exogenous polynucleotide encoding a peptide of interest.

2. The cell of claim 1, wherein said polynucleotide sequence encoding SEQ ID NO: 1 is an exogenous polynucleotide.

3. The cell of any one of claims 1 or 2, wherein said cell is a eukaryotic cell having increased peptide production efficacy compared to control.

4. The cell of claim 3, wherein said eukaryotic cell is a mammalian cell.

5. The cell of claim 4, wherein said mammalian cell is selected from a Chinese hamster ovary cell (CHO) or a human embryonic kidney cell 293 (HEK293).

6. The cell of any one of claims 1 to 5, wherein said peptide is a glycopeptide.

7. The cell of claim 6, wherein said glycopeptide has human-like glycans.

8. The cell of claim 7, wherein said glycopeptide is selected from the group consisting of: an antibody, an immunogen, and a growth factor.

9. The cell of claim 8, wherein said glycopeptide comprises a signal peptide sequence.

10. A composition comprising the cell of any one of claims 1 to 9, and a carrier.

11. The composition of claim 10, wherein said carrier is a chemically defined animal serum-free medium.

12. A method for improving protein production yields, the method comprising: culturing a cell comprising an exogenous polynucleotide sequence encoding SEQ ID NO: 1, or an analogue thereof having at least 80% homology to said SEQ ID NO: 1.

13. The method of claim 12, wherein said cell further comprises an exogenous polynucleotide encoding a peptide of interest.

14. The method of claim 13, wherein said cell has increased peptide production efficacy compared to control.

15. The method of claim 14, wherein said peptide is a glycopeptide.

16. The method of claim 15, wherein said glycopeptide has human-like glycans.

17. The method of any one of claims 12 to 16, wherein said cell is a eukaryotic cell.

18. The method of claim 17, wherein said eukaryotic cell is a mammalian cell.

19. The method of claim 18, wherein said mammalian cell is selected from a Chinese hamster ovary cell (CHO) or a human embryonic kidney cell 293 (HEK293).

20. The method of claim 19, wherein said glycopeptide is selected from the group consisting of: an antibody, an immunogen, and a growth factor.

21. The method of any one of claims 15 to 20, wherein said glycopeptide comprises a signal peptide sequence.

22. The method of claim 21, further comprising a step of isolating said secreted glycopeptide.

23. The method of any one of claims 12 to 22, wherein said culturing comprises growing said cell in a medium

24. The method of claim 23, wherein said medium is a chemically defined serum- free medium.