WO2019097290A1

WO2019097290A1 - Enhancement of biologics production in cell culture systems by fertilin-derived peptides

Info

Publication number: WO2019097290A1
Application number: PCT/IB2018/001424
Authority: WO
Inventors: Cécile Bauche; Renaud Vaillant; Julien DEROYER; Aurélie PERRIER
Original assignee: Aratinga.Bio
Priority date: 2017-11-15
Filing date: 2018-11-15
Publication date: 2019-05-23

Abstract

Methods and compositions are provided for obtaining increased biological agent production, such as viral vectors, from eukaryotic cells transfected with plasmids or from mammalian cells that produce biological agents without transfection. The methods include culturing cells during one or more phases of the production of a biological agent in the presence of an analog or derivative of fertilin, such as a cyclic peptide including at least a portion of a fertilin β binding site sequence. The methods and compositions increase the metabolism of the cells and the proliferation or production rate of the biological agent. The methods and compositions can be used to reduce the cost of producing a biological agent, such as a viral vector or a vaccine.

Description

TITLE

Enhancement of Biologies Production in Cell Culture Systems

by Fertilin-Derived Peptides

BACKGROUND

Retroviral vectors, particularly lentiviral vectors, are suitable delivery vehicles for the introduction of a variety of genes of interest into the genomic DNA of a broad range of target cells. The ability of retroviral vectors to deliver a non-rearranged, single copy gene into a broad range of mammalian somatic cells makes retroviral vectors well suited for a myriad of medical applications.

However, the production of replication-defective lentiviral vectors for large-scale clinical use is challenging. Lentiviral vectors can be produced by co-transfecting 293 T human embryonic kidney (HEK) cells with several different plasmid constructs. The process is costly, cumbersome and time-consuming. To produce large batches of lentiviral vector, the process requires days or weeks of cell growth before the quantity of cells is suitable for transfection with plasmids. At least another day of cell growth after transfection is required, after which the culture medium is removed and replaced with a fresh one, followed by one or more days until the first harvest (Tiscornia G., et al. Production and Purification of Lentiviral Vectors. Nature Protocols. Vol. 1, No. 1, pp. 241-245, 2006). It is often possible to replace the culture medium and have a second harvest that is pooled with the first one to increase the yield of production at the end of the downstream purification and concentration process, but typically, at the time of the first harvest the cells are already exhausted and undergoing apoptosis, such that a second harvest, if possible, gives low yield of production, and the consistency must be mastered. Exhaustion of the cells increases the presence of contaminants, resulting in an increase in residual proteins and cell DNA, and debris which may compromise its quality and the ability to purify/concentrate the lentiviral vectors.

Vector harvest is thus currently limited by mammalian cell survival. After days of intense replication and translation of exogenous material, many cells are either dead or exhausted. Dead cells cannot be removed during vector production as the vectors are present in the cell culture supernatant. The energy requirements of cultured cells producing biological agents such as viral vectors are high, and current methods, even if using rich culture media and good oxygenation of the cells, fail to provide long term energy at the levels necessary for cells to produce a biological agent for a long time. There is a need for methods of producing biological agents that increase the length of time during which producing cells are capable of producing a biological agent before cell exhaustion or death. There is also a need for methods to limit the cell death during the time of the biological agent production so as to reduce the contaminant levels in the harvest batch. This would lead to batches of the biological agent having higher purity at the end of the purification/concentration steps, and better fulfilling regulatory guidelines for the human use. Such methods also would provide a higher concentration factor during purification/concentration steps, resulting in a more concentrated product within regulatory guidelines for human use, allowing injection of higher doses in humans. There is also a need to accelerate cell growth before the transfection step in order to reduce the time and cost of biological agent production, and to allow for faster harvests.

SUMMARY

The present technology provides a method for obtaining increased biological agent production from eukaryotic cells transfected with plasmids or from mammalian cells that produce biological agents without transfection.

One aspect of the technology is a method that includes culturing cells, before and after starting the production of a biological agent, in the presence of a cyclic peptide, the cyclic peptide including at least a portion of a fertilin b binding site sequence. Preferably, the fertilin b binding sequence is from human fertilin b. Even more preferably, the binding sequence includes the human tripeptide binding site, FEE.

In some embodiments, the peptide includes at least part of any one of SEQ ID NOS: 1 - 7. Preferably, the peptide includes at least part of SEQ ID NO: l . In some embodiments, the mammalian cells are derived from human embryonic kidney cells. In some embodiments, the mammalian cells are immortalized human embryonic kidney (HEK) cells 293. In certain embodiments, the mammalian cells have been transfected with two or more plasmids.

In some embodiments, the peptide is cyclized by means of a covalent bond. In preferred embodiments, the covalent bond is a disulfide bond. Preferably, the disulfide bond is a bond between two cysteine residues in the peptide. In some embodiments, the method increases the proliferation rate of the biological agent-producing cells in culture. In some embodiments, the method decreases the rate of apoptosis of the cultured cells. In some embodiments, the cells are capable of producing the biological agent for longer periods of time before undergoing exhaustion or apoptosis when compared to cells cultured without the cyclic peptide. In some embodiments, the method increases cellular energy metabolism or energy level. In some embodiments, the method increases mitochondrial activity in the cultured cells. In some embodiments, the method increases ATP production and/or ATP level in the cultured cells.

In some embodiments the cyclic peptide is added to the culture medium in which the cells are cultured. In some embodiments, the cyclic peptide is added to the cell culture medium when beginning cell expansion. In other embodiments, the cyclic peptide is added throughout the process of biological agent production by the cultured cells. In some embodiments, cells are continuously cultured in the presence of cyclic peptide. In some embodiments, the cyclic peptide is added intermittently to the cells or to the culture medium containing cells. In some embodiments, the cyclic peptide is added to the culture medium at a concentration in the range from about 1 mM to about 1000 mM, such as from about 10 pM to about 300 pM, or such as about 100 pM.

The technology also can be summarized with the following list of embodiments.

1. A method of increasing production and/or purity of a biological agent by cultured eukaryotic cells, and/or decreasing the time and/or cost of cell expansion required for said production, the method comprising culturing the cells in the presence of a cyclic peptide, wherein the cyclic peptide has the following formula:

wherein X represents any amino acid which can be different or identical for each position, and m+n is less than or equal to 5, whereby production of the biological agent is increased, and/or the level of impurities reduced, and/or the time and/or cost of cell expansion is reduced, compared to culturing the cells in the absence of the cyclic peptide.

2. The method of embodiment 1, wherein the biological agent is selected from proteins, nucleic acids, antibodies, vaccines, and viral vectors.

3. The method of embodiment 1 or 2, wherein the eukaryotic cells are mammalian cells.

4. The method of embodiment 3, wherein the mammalian cells are human.

5. The method of embodiment 4, wherein the human cells are human embryonic kidney cells and wherein the biological agent is a viral vector. 6. The method of any of the preceding embodiments, wherein the cyclic peptide comprises at least three consecutive amino acids of any of SEQ ID NOS: 1-7.

7. The method of any of the preceding embodiments, wherein the cyclic peptide is cyclized by means of a covalent bond.

8. The method of embodiment 7, wherein the covalent bond is a disulfide bond.

9. The method of any of the preceding embodiments, wherein the biological agent is a viral vector.

10. The method of embodiment 9, wherein the viral vector is a retroviral vector.

11. The method of the embodiment 10, wherein the viral vector is a lentiviral vector.

12. The method of any of the preceding embodiments, wherein the peptide increases energy metabolism in the cultured cells.

13. The method of any of the preceding embodiments, wherein the cyclic peptide is added to the culture medium at the beginning of cell expansion and reduces the time required for cell expansion.

14. The method of any of the preceding embodiments, wherein the cyclic peptide increases the purity of the produced biological agent.

15. The method of any of the preceding embodiments, wherein the cyclic peptide increases the quantity and/or concentration of the produced biological agent.

16. The method of any of the preceding embodiments, wherein the cyclic peptide reduces the cost of producing the biological agent.

17. The method of any of the preceding embodiments, wherein the cyclic peptide reduces apoptosis of the cultured cells.

18. The method of any of the preceding embodiments, wherein the cyclic peptide is added to the culture medium at a concentration from about 10 mM to about 300 pM.

19. The method of embodiment 18, wherein the cyclic peptide is added to the culture medium at a concentration of about 100 pM.

20. The method of any of the preceding embodiments, wherein the method is for production of a viral vector, and wherein the cyclic peptide is added to the culture medium during a seed, stimulation, or transfection phase of said production.

21. The method of embodiment 20, wherein the viral vector production conditions are suboptimal in the absence of the cyclic peptide.

22. The method of any of the preceding embodiments, wherein the biological agent is a therapeutic or diagnostic agent for use in humans or animals.

23. A cell culture medium comprising a cyclic peptide having the following formula:

wherein X represents any amino acid which can be different or identical for each position, and m+n is less than or equal to 5.

24. The cell culture medium of embodiment 23, further comprising one or more cells for the production of a biological agent.

25. The cell culture medium of embodiment 24, further comprising said biological agent.

26. A kit comprising the cell culture medium of embodiment 23 and one or more cells or plasmids for the production of a biological agent.

27. A kit comprising (i) one or more cells or plasmids for the production of a biological agent, and (ii) a cyclic peptide having the following formula:

28. ETse of the cell culture medium of any of embodiments 23-25, or the kit of any of embodiments 26-27, for the production of a biological agent.

29. The cell culture medium of any of embodiments 23-25, or the kit of any of

embodiments 26-27, for use in the production of a biological agent.

30. ETse of a cyclic peptide having the following formula:

wherein X represents any amino acid which can be different or identical for each position, and m+n is less than or equal to 5, in the production of a biological agent by one or more mammalian cells in culture.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the concentration dependence of stimulation of the metabolism of HEK293T cells by a fertilin analog using PRESTOBLUE reagent. See Example 1 for details.

Figures 2A-2B show the effects of fertilin analog on lentiviral vector production added at the indicated stages of a lentiviral vector production protocol. The results were obtained at 24 hours (Fig. 2A) or 48 hours (Fig. 2B) following stimulation of vector production. Cells were seeded at 2.1 x 10⁵ cells/well. See Example 2 for details.

Figures 3 A-3B show the effects of fertilin analog on lentiviral vector production added at the indicated stages of a lentiviral vector production protocol. The results were obtained at 24 hours (Fig. 3A) or 48 hours (Fig. 3B) following stimulation of vector production. Cells were seeded at 1.05 x 10⁵ cells/well. See Example 2 for details.

Figures 4A-4B show the effects of fertilin analog on lentiviral vector production added at the indicated stages of a lentiviral vector production protocol. The results were obtained at 24 hours (Fig. 4A) or 48 hours (Fig. 4B) following stimulation of vector production. Cells were seeded at 0.525 x 10⁵ cells/well. See Example 2 for details.

DETAILED DESCRIPTION

The present technology provides a method of enhancing the production or quality of a biological agent produced by eukaryotic cells, including cells transfected with plasmids or non- transfected cells that produce the biological agent. The eukaryotic cells can be mammalian cells, including human cells, and the biological agent can be a protein, such as a recombinant protein, including an antibody, or it can be a nucleic acid, a vaccine, or a viral vector. The method includes culturing the biological agent-producing cells in the presence of a cyclic peptide derived from the protein fertilin b. Preferably, the fertilin b is human fertilin b.

Lentiviral vectors are normally produced by co-transfecting human embryonic kidney (HEK) cells with several different plasmid constructs. However, this process typically takes several days, and the yield of the viral harvest is often low or contaminant levels too high. The method of the present technology increases viral vector production and/or quality and/or yield. While not intending to limit the technology to any particular mechanism, it is believed that the addition of a cyclic fertilin-derived peptide to the culture medium increases the energy available for cells to survive, grow, and support the production of viral vector. The increased energy level may result from increased ATP production or more efficient use of ATP in the cultured mammalian cell. By enhancing cellular energy supplies, the cyclic peptide allows the cells to synthesize viral genetic material and/or proteins at a higher rate and/or for a longer period of time. Cell apoptosis is delayed or avoided, allowing for higher viral vector yields or the reduction of contaminant levels. The peptide may also accelerate cellular growth rate, thus allowing faster and less expensive production for higher vector yields within a given time frame when compared to the production of the vector in the absence of the peptide. Fertilin b (previously PH-30, also known as ADAM2) is a cell adhesion molecule on the surface of mammalian sperm, which is believed to bind to integrin receptors on the plasma membrane of oocytes, and thus play a crucial role in fertilization. Human fertilin has a putative binding site which is the tripeptide FEE (phenylalanine, glutamic acid, glutamic acid). Fertilin b is a member of the molecular family known as ADAMs (a disintegrin and a metalloprotease domain) or MDC (metalloprotease-disintegrin-cysteine rich). ADAM proteins have a specific domain structure, which includes a signal sequence, prodomain, metalloprotease domain, disintegrin-like domain, cysteine-rich domain, epidermal growth factor (EGF)-like domain, and a transmembrane segment with a short cytoplasmic tail. The disintegrin-like domain has homology to snake venom ligands for integrins, molecules important for cell adhesion. Hence, fertilin b is believed to interact with and/or bind to receptor molecules involved in cell adhesion, such as integrins, cadherins, and immunoglobulin receptors, among others.

Integrins are receptors involved in the cell-extracellular matrix and cell-cell interactions. Integrins are heterodimers, consisting of a (alpha) and b (beta) subunits. In mammals, there are eighteen a and eight b subunits. The a and b subunits each penetrate the plasma membrane and possess small cytoplasmic domains. There are several types of integrins, and a cell may have several types on its surface. Integrins work alongside other receptors such as cadherins, the immunoglobulin superfamily cell adhesion molecules, selectins and syndecans to mediate cell-cell and cell-matrix interaction.

Bomsel et al. (US 9,238,807 and US 8,883,472) found that peptides containing a portion of the fertilin b binding site sequence can increase the fusiogenic capacity of mammalian gametes when added to a culture medium containing the gametes. The peptide is cyclized and comprises at least the tripeptide (FEE) essential for binding to its receptor.

The present inventors have discovered that fertilin b, or peptides containing the cyclic FEE tripeptide (FEEc) of fertilin b or homologous sequences, have the ability to increase the production and/or yield of viral vectors from cultured cells.

FEEc is contained in the disintegrin loop of fertilin beta. This tripeptide differs according to species. However, the organization of the disintegrin loop is highly conserved among species. The amino acid sequences for different mammalian species are shown in Table 1. The tripeptide binding site in each species is underlined. TABLE 1

Human fertilin b

CLFMSKERMCRPSFEECDLPEYCNGSSASC SEQ ID NO: 1

Mouse fertilin b

CKLKRKGE V CRL AODECD VTE Y CN GT SE V C SEQ ID NO:2

Guinea pig fertilin b

CEFKTKGE V CRE S TDECDLPE Y CN GS S G AC SEQ ID NO: 3

Monkey fertilin b

CLFMS QERV CRP SFDE CDLPE Y CN GT S AS C SEQ ID NO:4

Rabbit fertilin b

CTFKEKGQ S CRPP VGECDLFE Y CN GT S ALC SEQ ID NO: 5

Rat fertilin b

CNLKAKGELCRPANOECDVTEYCNGTSEVC SEQ ID NO: 6

A consensus sequence for the disintegrin loop can be deduced from Table 1 :

(SEQ ID NO: 7)

C-X2-(F/L)-(K/M/I)-X₅-(K/R/Q)-(G/E)-X8-X9-C-R-Xi2-Xi3-TriPept-C-D-(L/V)-X₂o-E-Y-C-

N-(G/E)-(T/S)-S-(A/E/G)-X₂₉-C

The X groups represent, independently of one another, an amino acid and“TriPepf’ is the tripeptide essential for binding of fertilin to integrin. Preferably, X₈ is a charged amino acid. More particularly, it is selected from the group consisting of E, R and Q. Preferably, X12 is selected from the group consisting of P, L, E, and G. Preferably, X13 is a small and uncharged amino acid, more particularly it is selected from the group consisting of S, A, P, and T. Preferably, X29 is a small and uncharged amino acid, more particularly selected from the group consisting of S, A, and V.

The cyclic peptide can be cyclized by any method known to those skilled in the art.

The peptide can be cyclized by means of a covalent bond between the main chain and the main chain, between the main chain and a side chain, or between a side chain and another side chain. The covalent bond can be a disulfide, amide or thioether bond. For example, the peptide can be cyclized by a peptide bond between the N-terminal residue and the C-terminal residue, or with amino or carboxylic groups of the side chains of the residues.

Preferably, the peptide is cyclized by means of two cysteine residues, more particularly by means of a disulfide bridge between said two cysteine residues. The cysteine residues must be located in such a way as to permit cyclization of the peptide. The cysteine residues can be located in such a way that, after cyclization, the peptide has a peptide tail, or does not have a tail. Preferably, said cysteine residues are located at the ends of the peptide, and there is no tail after cylization.

The cyclic peptide according to the method of technology can be described by the following formula:

C-Xm-TriPept-Xn-C (SEQ ID NO: 9) wherein X represents any amino acid, m and n are integers ranging from 0 to 14. Amino acids labeled X are independent of one another and can represent, within the same molecule, amino acids which are the same or different. Preferably, when m or n is equal to 0, the other is at least 1. Preferably, m+n is less than 10, preferably less than or equal to 5. In a preferred embodiment, m+n is equal to 3. Preferably, the tripeptide has the sequence X-(Q/D/E)-E or X- (D/E)-E. For example, the tripeptide can be selected from the group consisting of (Q-D-E), (F- E-E), (T-D-E), (V-G-E), (F-D-E), (T-D-E), (N-Q-E), and (L-D-E). In a preferred embodiment, the tripeptide is (F-E-E).

The cysteine residues involved in peptide cyclization can be naturally located in the disintegrin loop or can be introduced into the peptide sequence. The disintegrin loops are rich in cysteine, and cysteine residues are conserved at positions 1, 10, 17, 23 and 30 of the loops. Thus, the peptides can be cyclized by means of a disulfide bridge selected from the group consisting of: C1-C17, C1-C23, C1-C30, C10-C17, C10-C23, and C10-C30. Preferably, the peptides are cyclized by means of a disulfide bridge selected from C10-C17 and C10-C23. The cysteine residues also can be introduced into the peptide to be cyclized.

The cyclic peptide may also contain other domains of fertilin b, such as the signal sequence, prodomain, metalloprotease domain, disintegrin-like domain, cysteine rich domain, epidermal growth factor (EGF)-like domain and a transmembrane segment, in addition to, or instead of, the disintegrin loop.

The amino acids of the cyclic peptide according to the technology can be natural or non-natural. A non-natural amino acid can be an analogue or derivative of a natural amino acid. For example, a non-natural amino acid can have a longer, shorter or different side chain containing suitable functional groups. The amino acids can be L or D stereoisomers or a mixture thereof. In addition, the peptide bonds can be modified to make them resistant to proteolysis. For example, at least one (— CO— NH— ) peptide bond can be replaced by a divalent bond selected in the group consisting of (— CFF— NH— ), (— NH— CO— ), (— CH_2— O— ), (— CH_2— S— ), (— CH_2— CH_2— ), (— CO— CH_2— ), (— CHOH— CH_2— ), (— N=N— ), and (— CH=CH— ).

The residues of the sequences described hereinabove can vary in a conservative manner, meaning that the variant residue displays similar physico-chemical characteristics. Steric hindrance, polarity, hydrophobicity or charge are among the physico-chemical characteristics taken into account.

Consequently, the technology also relates to variants and/or derivatives of said cyclic peptides and to the use thereof, particularly in order to modulate ATP production and/or use by the cell. Said variants and derivatives conserve the binding capacity to integrins and/or to modulate (particularly increase) ATP production in a mammalian cell, and/or to increase viral vector production from cultured mammalian cells transfected with viral plasmids.

The technology also relates to a multimer of the cyclic peptide. This polymerization of the cyclic peptide can be achieved by any method known to those skilled in the art. Preferably, the cyclic peptide is coupled with a carrier molecule allowing the peptide to polymerize. The bond between the cyclic peptide and the carrier molecule can be covalent or noncovalent. The methods by which to attach the cyclic peptide to the carrier molecule are well known to those skilled in the art and comprise amine chemistry, carbodiimide coupling of carboxyl and amino derivatives, activation of cyanogen bromide, N-hydroxysuccinimide, epoxide, sulfhydryl, or hydrazide. The bond between the carrier molecule and the cyclic peptides can be direct or indirect. When it is indirect, it can take place through a linker. Said linker can play a role of spacer which avoids interference of the carrier molecule on the properties of the cyclic peptide. Said linker can be a peptide. The cyclic peptide must be attached to the carrier molecule in such a way as to maintain the accessibility of the tripeptide. The number of cyclic peptides comprised in the multimer is preferably comprised between 2 and 1000. For example, the polymerization can be accomplished by means of a biotin/streptavidin conjugate which allows a tetramer of the cyclic peptide to be prepared, whereby each cyclic peptide is bound to a biotin and four biotins can bind to a streptavidin molecule.

Moreover, several cyclic peptides according to the technology can be immobilized on a solid support. Non-limiting examples of the solid support are agarose, glass, cellulose resins, silica resins, polystyrene, and polyacrylamide. The solid support can be modified with functional groups allowing fixation of the cyclic peptides, for example by means of carboxyl, amino, sulfhydryl, hydroxyl and/or carbohydrate groups contained in said peptides.

The technology concerns a method including culturing cells in the presence of a cyclic peptide, a multimer of cyclic peptides or a culture medium composition including said cyclic peptides in order to enhance biological agent production or quality by eukaryotic cells. The peptide may increase cell growth rate and thus increase viral vector production by reducing the time necessary for vector production. The peptide may accelerate the proliferation rate of the cultured producing cells. The peptide may increase the number of viable producing cells in culture. The peptide may maintain producing cells in culture viable for a longer period of time. The peptide may enhance biological agent production by increasing the yield of production, and/or it may improve quality or purity of the biological agent by reducing the presence of contaminating materials, such as proteins or nucleic acids from dead or dying cells in the culture.

In particular, the cyclic peptides according to the technology can be used to enhance any laboratory or industrial production involving eukaryotic cells, such as the production of viral vectors by mammalian cells. The mammalian cells can be, for example, HEK cells that have been transfected with one or more viral plasmids in order to produce viral vectors. The cyclic peptide can be added to the culture medium in which the mammalian cells are cultured. The cyclic peptide can be added to the cell culture medium at the beginning of cells expansion. The cells can be continuously cultured in the presence of cyclic peptide. The cyclic peptide can be added intermittently to the cells or to the culture medium containing cells. The cyclic peptide can be added to the culture medium at a concentration in the range from about 1 mM to about 1000 mM, such as from about 10 pM to about 300 pM, or such as about 30, 50, 60, 70, 80, 90, 100, 110, 120, or 150 pM.

EXAMPLES

Example 1. Effect of Fertilin on Cell Viability.

The effect of a fertilin peptide analog added to the culture medium at different stages of lentiviral vector production was determined using HEK293T cells as described below. Effect of Fertilin on Cell Viability

The HEK293T cell line was grown and maintained at 37°C, 5% C0₂ in DMEM medium (with red phenol and glutamax, GIBCO #61965-026) containing 10 % fetal bovine serum (FBS), New Zealand Sourced (HyClone, #SH30406.02) and 1% penicillin/streptomycin (PS) (GIBCO #15140-122). The cells were seeded at a density of 25000 cells/well in 96-well plates 4 hours before stimulation with fertilin. The fertilin analog used for stimulation was the cyclic peptide having the sequence MB-CSFEEC-COOH (SEQ ID NO:8), wherein a disulfide bond connects the cysteine residues at positions 1 and 6. Fertilin stimulation was performed by removing the culture medium and adding 90 pL diluted fertilin analog at the indicated concentrations to the cultured cells and incubating at 37°C. The next day, 10 pL PRESTOBLEIE cell viability reagent (Invitrogen A13261) was added to each well. Cells were incubated for 1 to 3 hours at 37°C prior to quantifying the fluorescence signal (VARIOSCAN LEIX, ThermoScientific). The results are shown in Fig. 1, which shows that cell metabolism was stimulated in a dose-dependent manner by the fertilin analog.

Example 2 Effect of Fertilin on Lentiviral Vector Production.

Lentiviral Vector Production

HEK293T cells were seeded at 2.1E+05 cells/well, 1.05E+05 cells/well, and 0.53E+05 cells/well in 24-well plates (Costar, #3526) in 300 pL per well of DMEM as described above and incubated at 37°C under humidified 5% C0₂. The day after seeding, the transient transfection was performed.

Transfer vector plasmid (pARA-CMV-GFP), packaging plasmid (pARA-Pack), and envelope plasmid (pENVl) were diluted with sterile distilled water (GIBCO 10977035) and mixed with 1M CaCL. Details of the plasmids are as follows:

pARA-CMV-GFP

Kanamycin resistant plasmid encoding the provirus (non-pathogenic and non- replicative recombinant proviral DNA derived from HIV-l, strain NL4-3), in which an expression cassette is cloned. The insert contains the antigenic transgene, the promoter for transgene expression and sequences added to increase the transgene expression and to allow the lentiviral vector to transduce all cell types including non-mitotic ones. The transgene is the gene encoding the Green Fluorescent Protein (GFP). The promoter is the human ubiquitin promoter, devoid of any enhancer sequence, which promotes gene expression at a high level in a ubiquitous manner. Non coding sequences and expression signals include: (i) the Long Terminal Repeat sequences (LTR) with the whole cis-active elements for the 5’LTR (U3-R- U5) and the deleted one for the 3’LTR, hence lacking the promoter region; (ii) (AU3-R-U5), for transcription and integration mechanisms; (iii) the encapsidation sequences (SD and 5’Gag); (iv) the central PolyPurine Tract/Central Termination Site for nuclear translocation of the vectors; (v) Bovine Growth Hormone polyadenylation signal to stabilize the RNA.

pARA-Pack

Kanamycin resistant plasmid coding for the structural lentiviral proteins (GAG, POL, TAT and REV) used in trans for the encapsidation of the lentiviral provirus. Coding sequences are a polycistronic gene gag-pol-tat-rev, coding for the structural (Matrix MA, Capsid CA and Nucleocapside NC), enzymatic (Protease PR, Integrase IN and Reverse Transcriptase RT) and regulatory (TAT and REV) proteins. Non-coding sequences and expression signals include (i) minimal promoter from cytomegalovirus (CMV) for transcription initiation; (ii) polyadenylation signal from the insulin gene for the transcription termination; and (iii) HIV-l Rev responsive Element (RRE) participating in nuclear export of the packaging RNA.

pENVl

Kanamycin resistant plasmid coding for glycoproteins G from the Vesicular Stomatitis Virus (VSV-G) Indiana strain, used for pseudotyping of the lentiviral vectors. The VSV-G genes were codon optimized for expression in human cells, and the gene was cloned into pVAXl plasmid (Invitrogen, life technologies), The coding sequence is codon optimized VSV- G gene for optimal expression in human cells. Non-coding sequences and expression signals include: (i) minimal promoter from cytomegalovirus (CMV) for transcription initiation and (ii) BGH polyadenylation signal.

The DNA/CaCh mixture was added to 2X HEPES buffered saline (HBS) (0.28 M NaCl, 0.05 M HEPES and 1.5 mM Na₂HP0₄; optimal pH range, 7.00-7.28) and incubated at room temperature for 30 min. Subsequently, 37 pL of the transfection mixture was applied to culture plates and incubated at 37°C in a 5% C0₂ humidified atmosphere.

The day after transfection, lentiviral vector production was stimulated by FBS removal. The medium was replaced by 300 pL of DMEM w/o red phenol (HyClone, # SH30284.02) and cells were incubated at 37°C under humidified 5% CO2. After 24h of production, 100 pL of supernatant (Vector Harvest 1) were transferred to a well of a 96-well plate. After a short centrifugation to eliminate the cell debris, 90 pL were transferred into a well of a new 96 well- plate and stored at <-70°C. After 48h of production, 200 pL of supernatant (Vector Harvest 2) were transferred to 96-well plate. After a short centrifugation in order to eliminate cell debris, 180 pL were transferred into a well of a new 96-well plate and stored at <-70°C. Effect of Fertilin on Lentiviral Vector Production

10 pL of fertilin analog (SEQ ID NO: 8) solution or a pH control solution (containing acetic acid to mimic the pH change induced by the fertilin solution) were added as follows. Seven fertilin concentrations (50, 100, 150, 172, 200, 343 and 686 mM) were tested as well as three acidic pH conditions (0.01, 0.02 and 0.04 volume % final concentration of acetic acid). The fertilin or pH control solutions were added at different steps of the production flowchart:

4h after seeding

4h after transfection

At stimulation step

4h after seeding and 4h after transfection

4h after seeding and at stimulation

4h after transfection and at stimulation

4h after seeding, 4h after transfection and at stimulation

Titration of Lentiviral Vectors

Titration was perfomed by flow cytometry as follows. HEK293T cells (8x 10⁵ cells/well) were seeded in 24-well plates and incubated at 37°C in a 5% C0₂ humidified atmosphere. After 4 h, the culture medium was removed and 300 pL of fresh DMEM containing 10% FBS, 1% PS and 0.5-30 pL viral supernatant were added to each well. After 2 h, each well was supplemented with 0.5 mL fresh culture medium. Three days after infection, the HEK293T cells in each well were trypsinized, fixed (BD CellFIX solution #340181) and the number of fluorescence-positive cells was determined using flow cytometry (AttuneNXT; Invitrogen, Inc.).

The following formula was used to convert the percentage of eGFP-expressing cells for a specific dilution into TU:

TU/ml = (% of cells expressing eGFP/100) ^x total number ofHEK293T cells at time of infection/volume of virus stock added (mL).

Data Presentation and Statistical Analysis

Three independent experiments were performed for each condition. The means are represented with standard deviations, and all significant differences were evaluated using an ANOVA test. Significantly different results are shown in the figures with p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***). RESULTS

The results for GFP expression obtained in transduced cells as a function of fertilin analog concentration and stage of production at which fertilin was introduced are shown in Figs. 2A, 2B, 3A, 3B, 4A, and 4B; the different figures reflect different seeding densities and different time to harvest after stimulation (24h or 48h). All data are shown as normalized to the control condition (absence of fertilin).

High Cell Density

A cell density of 2.1 x 10⁵ cells/well was used for the experiments shown in Figs. 2A (cells harvested 24h after stimulation) and 2B (48h after stimulation). Each figure shows results using different fertilin analog concentrations and for fertilin added at the indicated step of the protocol.

With this cell density and harvest at 24h after stimulation, vector production was optimal, and not surprisingly there was little meaningful stimulation of vector production by fertilin regardless of where it was added in the production protocol. An increase of production was seen at low fertilin analog concentrations under 171.5 mM during the transfection and seed + transfection conditions. As no effect was observed during the seed step, and as no significant cumulative effect was observed when fertilin was added during seed + transfection, seed + stimulation steps, the effect observed was likely related to the addition of a low concentration of fertilin during the transfection step. When added during the stimulation and transfection steps, a negative effect was observed, probably due to the increase of product concentration.

At 48h after stimulation, increases of vector production to 130% were observed when fertilin was added at low concentrations during seeding + transfection.

Intermediate Cell Density

A cell density of 1.05 x 10⁵ cells/well was used for the experiments shown in Figs. 3 A (cells harvested 24h after stimulation) and 3B (48h after stimulation).

With vector harvest at 24h after stimulation, an increase of production was observed at low fertilin concentrations (<100 pM) during the seed and stimulation steps. A cumulative effect was observed at low concentrations during the seed + stimulation steps. A clear deleterious effect was observed when fertilin was added at high concentrations during the stimulation and seed + stimulation + transfection steps. Harvesting at 48h revealed increased production at low fertilin concentrations (<100 mM) during the seed step. A deleterious effect of high concentrations of fertilin was observed when it was added during most steps of the production.

Overall, fertilin addition stimulated vector production under these non-optimal production conditions.

Low Cell Density

A cell density of 0.525 x 10⁵ cells/well was used for the experiments shown in Figs. 4A (cells harvested 24h after stimulation) and 4B (48h after stimulation).

With harvest performed at 24h after stimulation, there was a higher increase (150%) at almost all steps of production, using low fertilin concentrations. A deleterious effect of fertilin was observed at higher fertilin concentrations.

When harvest was performed at 48h after stimulation, the transfection and transfection + stimulation portions of the protocol showed an increase in vector production if fertilin was added at high concentrations.

Overall, fertilin showed a significant increase of production at almost all steps of the process using the low cell density.

The present application claims the benefit of U.S. Provisional Appl. No. 62/586,244, filed 15 November 2017, which is hereby incorporated by reference in its entirety.

As used herein, "consisting essentially of' allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of elements of a device, can be exchanged with "consisting essentially of' or "consisting of'.

Claims

CLAIMS What is claimed is:

1. A method of increasing production and/or purity of a biological agent by cultured eukaryotic cells, and/or decreasing the cost of cell expansion required for said production, the method comprising culturing the cells in the presence of a cyclic peptide, wherein the cyclic peptide has the following formula:

wherein X represents any amino acid which can be different or identical for each position, and m+n is less than or equal to 5, whereby production of the biological agent is increased and/or the level of impurities reduced, and/or the time and/or cost of cell expansion is reduced, compared to culturing the cells in the absence of the cyclic peptide.

2. The method of claim 1, wherein the biological agent is selected from proteins, nucleic acids, antibodies, vaccines, and viral vectors.

3. The method of claim 1, wherein the eukaryotic cells are mammalian cells.

4. The method of claim 3, wherein the mammalian cells are human.

5. The method of claim 4, wherein the human cells are human embryonic kidney cells and wherein the biological agent is a viral vector.

6. The method of claim 1, wherein the cyclic peptide comprises at least three

consecutive amino acids of any of SEQ ID NOS: 1-7.

7. The method of claim 1, wherein the cyclic peptide is cyclized by means of a covalent bond.

8 The method of claim 7, wherein the covalent bond is a disulfide bond.

9. The method of claim 1, wherein the biological agent is a viral vector.

10. The method of claim 9, wherein the viral vector is a retroviral vector.

11. The method of the claim 10, wherein the viral vector is a lentiviral vector.

12. The method of claim 1, wherein the peptide increases energy metabolism in the cultured cells.

13. The method of claim 1, wherein the cyclic peptide is added to the culture medium at the beginning of cell expansion and reduces the time required for cell expansion.

14. The method of claim 1, wherein the cyclic peptide increases the purity of the produced biological agent.

15. The method of claim 1, wherein the cyclic peptide increases the quantity and/or concentration of the produced biological agent.

16. The method of claim 1, wherein the cyclic peptide reduces the cost of producing the biological agent.

17. The method of claim 1, wherein the cyclic peptide reduces apoptosis of the cultured cells.

18. The method of claim 1, wherein the cyclic peptide is added to the culture medium at a concentration from about 10 mM to about 300 pM.

19. The method of claim 18, wherein the cyclic peptide is added to the culture medium at a concentration of about 100 pM.

20. The method of claim 1, wherein the method is for production of a viral vector, and wherein the cyclic peptide is added to the culture medium during a seed, stimulation, or transfection phase of said production.

21. The method of claim 20, wherein the viral vector production conditions are suboptimal in the absence of the cyclic peptide.

22. The method of claim 1, wherein the biological agent is a therapeutic or diagnostic agent for use in humans or animals.

24. The cell culture medium of claim 23, further comprising one or more cells for the production of a biological agent.

25. The cell culture medium of claim 24, further comprising said biological agent.

26. A kit comprising the cell culture medium of claim 23 and one or more cells or plasmids for the production of a biological agent.