EP1179048A2 - CULTURES OF INSECT CELLS WHICH REPRODUCE AT 37 oC, METHOD FOR OBTAINING SAID CULTURES AND THEIR USES - Google Patents

Abstract

The invention relates to a method for obtaining insect cells, especially S. frugiperda cells, which are able to reproduce at 37 °C, and to the use of said cells for producing recombinant proteins.

Description

 CROPS OF INSECT CELLS MULTIPLYING AT 37 ° C, PROCESS FOR OBTAINING SAME, AND USES THEREOF.

The present invention relates to cultures of insecre cells capable of growing at 37 ° C., and allowing the multiplication of baculoviruses, as well as to the use of these cell lines for the expression of genes of interest cloned into baculoviruses.

The expression, in insect cell cultures, of genes of interest cloned into vectors derived from baculovirus, is one of the most widely used methods of producing recombinant proteins today.

The optimum temperature for insect cells in culture is between 26 and 28 ° C. At temperatures above 30 ° C, their growth slows considerably, and they die quickly if kept at temperatures above 35 ° C.

In the case of Spodoptera frugiperda cells in culture, it was found that a short-term thermal shock (30 min.) At 42 ° C did not reduce their viability [CLEM and MILLER, Mol. Cell. Biol. , 14,

5212-5222, (1994)]. However, when these cells are kept at suboptimal temperatures for a longer period of time, their growth is affected; HARA et al. [Biosc. Biotech. Biochem. , 57 (6), 996-997,, (1993)] report, for example, that no growth of Sf21 cell cultures was observed at 37 ° C. When these cells are infected with a recombinant baculovirus expressing the β-galactosidase gene under the control of the polyhedrin promoter, the expression of this gene is maximum for temperatures of the order of

30 ° C, decreases considerably at 33 ° C, and becomes zero at

35 ° C. The production of recombinant proteins in baculovirus / insect cell systems is therefore conventionally carried out at temperatures approaching 27 ° C. This can have drawbacks, particularly when it is possible to express certain proteins which are naturally synthesized at a higher temperature (for example proteins from warm-blooded animals, and in particular from mammals); indeed, it is known that temperature can have a direct influence on the folding of polypeptide chains, and that for certain proteins, a variation of a few degrees C in the temperature at which they are synthesized can modify their activity significantly . In addition, it is known that the lipid composition of the membranes is different at 27 ° C. and at 37 ° C., which is important for the environment of certain proteins and in particular membrane proteins.

This is why it is desirable to be able to maintain in culture and to multiply insect cells allowing the multiplication of baculoviruses and the expression of genes of interest cloned into them, at a temperature as close as possible to body temperature. mammals.

The Japanese application in the name of NORINSUISANSHO KAJU SKIKEN published on 04/19/91 under the number J03094074 mentions the obtaining of a line of cells derived from larvae of Λgrot-is segetum, and capable of surviving at 37 ° C. These cells are obtained by carrying out a random mutagenesis by ultraviolet rays, then a selection of the mutants by acclimatization to alternating temperatures (30 ° C for 2 to 5 days, 25 ° C for 4 to 7 days, 33 ° C for 2 to 5 days, 25 ° C for 5 to 7 days, 35 ° C for 2 to 5 days, 25 ° C for 5 to 7 days, 37 ° C for 2 to 5 days, 25 ° C for 4 to 7 days) . The cells thus obtained allow, when cultivated at temperatures of 33 to 37 ° C, the multiplication of baculovirus patnogenes for insects of the genus Agrot-is. However, demand J03094674 gives no indication on the st ^'aoïlité of these cell lines, or their ability to multiply indefinitely at 37 ° C, or the possibility of using them as host cells for the expression of recombinant genes clones in baculoviruses.

The inventors have now found that, surprisingly, cells of the Sf9 and Sf21 lines of Spodop tera frugiperda (which are conventionally used for the production of recombinant baculoviruses), could when placed under certain conditions, give rise to cultures capable to grow and multiply normally and indefinitely at 37 ° C and perfectly suitable for the multiplication of oaculovirus and the expression in them of active recombinant proteins.

The subject of the present invention is a process for obtaining cultures of insect cells, and in particular of pidoptera, capable of multiplying at 37 ° C; this process comprises the following stages: a) culturing said insect cells at a temperature of approximately 27 ± 2 ° C; b) increasing the culture temperature to about 37 ± 1 ° C, c) maintaining said cells in culture at this temperature for about 4 to 6 days; d) recovering the cells from step c) and re-cultivating them at a temperature of approximately 37 ± 1 ° C.

The temperature increase in step b) can be carried out in a single step, by raising the temperature of the culture directly from 28 ° C to 37 ° C.

It can also be carried out in several stages, for example a first temperature rise of 27 a

30 ° C approximately, followed by maintaining at the temperature of 30 ° C for 2 to 3 days, the recovery of the cells, their cultivation at 30 ° C, the elevation of the temperature of this culture from approximately 30 to 33 ° C, followed by maintaining at the temperature of 33 ° C for 2 to 3 days, the recovery of the cells, their cultivation at 33 ° C., the elevation of the temperature of this culture from approximately 33 to 35 ° C., followed by maintaining at the temperature of 35 ° C. for 2 to 3 days, the recovery of the cells, their cultivation at 35 ° C, and the raising of the temperature of this culture from 35 to 37 ° C approximately. Each of the steps for raising the temperature of the culture can be carried out simply by passing from an oven maintained at the lowest temperature to an oven maintained at the highest temperature.

Unlike the process described in Japanese application J03094674, the process according to the invention does not require any mutagenesis step.

Insect cells which can advantageously be used, in accordance with the invention, for obtaining cultures of cells capable of multiplying at 37 ° C. are in particular cells of Spodoptera frugiperda, for example those of the line Sf21 or of the Sf9 line. The culture media conventionally used for the culture of insect cells are suitable for implementing the process according to the invention, and for maintaining the cultures resulting therefrom; for example, in the case of the Sf9 or Sf21 lines, one of them can use one of the media described by O'REILLY et al. [Baculovirus

Expression Vectors: A Laboratory Manual; Freeman -and

Cie, New York, (1992)], such as GRACE medium, IPL-41 medium, TC-100 medium, etc. or any other medium. The present invention also relates to cultures of insect cells which are susceptible of being obtained by a process in accordance with the invention, as defined above, and the lines derived from these cultures. These lines will be referred to below as “HT lines”, and designated by the name of the original line, followed by the letters “HT”. For example, or will denote: “Sf9-HT” and “Sf21-HT” the lines respectively derived, according to the invention, from the Sf9 and Sf21 cells.

The lines thus denoted differ from the mother lines from which they originate by their number of chromosomes, which is 2 times higher (endodiploidy). They can be maintained in culture at 37 ° C, and multiply indefinitely at this temperature; they can also be stored in frozen form, and can be returned to culture directly at 37 ° C. They retain the ability to grow and multiply at 27 ° C (although slower than the cells from which they originate), as well as the ability to support thawing. The cells in accordance with the invention are permissive for the same baculoviruses as the mother cells from which they are derived. In cell cultures according to the invention, these baculoviruses multiply at 37 ° C. and produce infectious particles.

The inventors have further found that genes of interest inserted into recombinant baculoviruses can be expressed at 37 ° C. in cell cultures according to the invention, and make it possible to obtain functional proteins. The cell cultures according to the invention can therefore be used, in the same way as cultures of the cells from which they are derived, to express genes of interest cloned in baculoviruses. The present invention relates to a method for expressing at least one recombinant protein in insect cells, which proceeds is characterized in that said recomoining protein is expressed in the cells of a culture according to the invention.

According to a preferred embodiment of the method according to the invention, said recombinant protein is expressed at a temperature above 35 ° C, preferably between 35 and 39 ° C.

All expression vectors used in haoïtuellement lines Sf21 and Sf9 ^• can also be used for the expression of genes of interest in Sf21-HT cell cultures and Sf9-HT according to the invention. It is in particular possible to use vectors in which the gene of interest to be expressed is placed under the control of a very early promoter, such as the IE-1 promoter, of an early promoter, such as the 39K promoter, of a late promoter, such as the gp67 promoter or that of the capsid protein, or of a very late promoter, such as that of the polyhedron or of the P10 protein. Cellular promoters capable of functioning at 37 ° C. can also be used, such as, for example, the Actme 3 promoter from Bombyx mon

(silkworm) or mammalian cell promoters.

It is also possible to use vectors of the “multi-recombinant” type, in which 2 or more genes of interest are placed under αe 2 control or more promoters. In αe cell cultures in accordance with the invention derived from the Sf9-HT monoclonal line, the very early, early or late promoters appear to be more active at 37 ° C. than the very late promoters (polyedrine or P10); we can therefore choose the promoter used according to the level of expression that we wish to obtain.

For the expression of genes of interest in cell cultures in accordance with the invention, it is also possible to use vectors derived from baculovirus, such as those described for example in Application EP 0 638 647 to the names of the NATIONAL INSTITUTE OF AGRONOMIC RESEARCH and the NATIONAL CENTER OF SCIENTIFIC RESEARCH.

The present invention will be better understood with the aid of the additional description which follows, which refers to examples of obtaining cell cultures in accordance with the invention, and of using these cultures for the expression of αe genes. clones in oaculoviruses. EXAMPLE 1 OBTAINING CULTURES OF CELLS MULTIPLYING AT 37 ° C

The cell lines used are on the one hand, the lines Sf21 (IPL3-SF-21) and Sf9 (ATCC CRL 1711) originating from the Lepidoptera Spodoptera frugiperda, and on the other hand, by way of comparison, the line S2 originating from the fly Drosophila melanogas ter, the line C3 1225 from the Aedes albopi ctus mosquito, and the line Mb, from the Lepidoptera Mamestra brassicae.

The cells of the lepidopteran lines Sf21, Sf9, and Mb, and αe the line Drosophila S2 are subcultured in TC-100 medium (LIFE TECHNOLOGIES, INC.), Supplemented with 5% fetal calf serum.

The mosquito cells CB 1225 are maintained in the medium of MITSUHASHI-MARAMOROSCH [Contπb. Boyce Thompson Inst., 22, 435-460, (1964)]. Cultures of Sf9 or Sf21 cells, as well as cultures of Mb, S2, and CB 1225 cells are placed in an oven at 28 ° C; the temperature is gradually increased over four days, up to 37 ° C. The cells are kept at this temperature and their growth is monitored daily.

After a week at 37 ° C, only cells from Sf9 and Sf21 cultures are able to survive. Cells from the Mb, S2 and CB 1225 lines stop growing from a temperature of 30 ° C, and die from 35 ° C. Samples of the cultures from the Sf9 and Sf21 lines are diluted in fresh medium at a rate of ^{1/5 th} , and returned to culture at 37 ° C. The stable lines obtained after several passages in culture under these conditions are called Sf9-HT and Sf21-HT.

The growth of the Sf9-HT line at 37 ° C and 28 ° C was compared with that of the Sf-9 mother line at 28 ° C. The growth curves, which are represented in FIG. 1, show that at 28 ° C, the Sf9-HT (O) cells grow slower (doubling time 33.6 hours) than the Sf9 (D) cells (time doubling 26.4 hours). In contrast, Sf9-HT cells at 37 ° C (Δ) grow faster (doubling time 19.2 hours) than Sf9 cells at 28 ° C. The stationary phase is reached more quickly (4 days) and corresponds to a lower cell density (1.2.10 ⁶ cells) in the case of Sf9-HT cells than in that of Sf9 cells (8 days, and 2.7.10 ⁶ cells ). Examination of Sf9-HT cells under a microscope shows that they have a normal morphology; however their average diameter is larger than that of Sf9 cells. Analysis by flow cytometry shows that the DNA content of Sf9-HT cells is 2 times greater than that of Sf9 cells.

The comparative analysis of the membrane lipids of Sf9 and Sf9-HT cells was carried out. The results of this analysis are presented in Table 1 below.

In the Sf9-HT line, there is an increase in sphingomyeline and phosphatidylinositol, and a decrease in phosphatidylcholine and phosphatidylethanolamine, which leads to an increase in the sphingomyelin / phosphatidylcholine ratio. There is also an overall increase in the percentage of saturated fatty acids, mainly due to an increase in palmitic acid.

EXAMPLE 2 MULTIPLICATION OF THE BACULOVIRUS ACMNPV AND EXPRESSION OF THE PROMOTERS IN SF9-HT CELLS CULTIVATED AT 37 ° C

Cultures of the Sf9-HT line were infected with the baculovirus AcMNPV, at 28 ° C. or at 37 ° C., according to the following protocol:

The Sf9-HT cells are taken up in the TC-100 medium used in Example 1, without fetal calf serum and distributed in new dishes. One hour later, when the cells adhere to the bottom of the dishes of culture, the culture medium is eliminated and the viral inoculum, produced on Sf9 cells cultured at 28 ° C., is added at the rate of 10 infectious viral units per cell. After one hour of incubation at room temperature, complete medium is added, and the cultures are placed at 28 ° C., or at 37 ° C. The viral cycle is observed by electron microscopy.

72 hours after infection, viral particles present in large numbers in the nucleus, as well as polyhedra and are observed in Sf9-HT cells cultured at 28 ° C. fibrillar structures corresponding to the P10 protein. In the cells to

37 ° C, on the other hand, one clearly distinguishes only the intranuclear viral particles. In both cases, the Sf9-HT cells secrete infectious viral particles in the culture medium; in the case of infected cells grown at

37 ° C, the titer in infectious viral particles produced is 10 times lower than at 28 ° C. When viral particles secreted by infected Sf9-HT cells cultured at 37 ° C., are used as an inoculum for a new cycle of viral infection in Sf9-HT cells cultured at

28 ° C, we observe in the latter the formation of polyhedra and fibrillar structures; in addition, the titer in infectious viral particles is identical to that observed with the viral inoculum produced on Sf9 cells cultured at 28 ° C.

Figure 2 shows the evolution of the viral titer in SF9-HT and Sf9 cells during the different passages, from the same initial inoculum

(10 infectious viral units per cell) obtained on Sf9 cells cultured at 28 ° C:

(^) initial inoculum obtained on Sf9 cells cultured at 28 ° C; passage on Sf9 cells. at 28 ° C, then 4 successive passages at 28 ° C with the inoculum of the preceding passage obtained at 28 ° C;

(A) initial inoculum maintained on Sf9 cells cultured at 28 ° C; passage through Sf9-HT cells at 37 ° C., then successive passages at 37 ° C. with the inoculum of the preceding passage obtained at 37 ° C.,

(Ξ) initial inoculum obtained on Sf9 cells cultured at 28 ° C, passage through Sf9-HT cells cultured at 28 ° C, then 4 successive passages at 28 ° C with the inoculum with the preceding passage obtained at 28 ° C;

(M) (control) initial inoculum obtained on Sf9 cells cultured at 28 ° C; passage on Sf9 cells cultured at 37 ° C. then 4 successive passages at 37 ° C. with the inoculum of the preceding passage. In this case, no viral replication is observed.

When viral particles secreted by infected Sf9-HT cells cultivated at 37 ° C., are used as an inoculum for a new cycle of viral infection in Sf9-HT or Sf9 cells cultured at 28 ° C. formation of polyhedra and fibrillar structures; in addition, the titer in infectious viral particles is identical to that observed with the viral inoculum produced on Sf9 cells cultured at 28 ° C. The expression profile of the baculovirus genes infecting these cell cultures is different at 28 ° C. and at 37 ° C., as shown in FIGS. 3a to 3f, which illustrate the comparison of the expression kinetics in Sf9 to 28 cells. ° C (§ ^ i), and at 37 ° C (≡≡) or in Sf9-HT cells at 28 ° C {££ and at 37 ° C

(), messenger RNAs corresponding to very early proteins (IE1), early (39K), late (gp67 and vp39) or very late (polyedπne and P10).

On the abscissa, the culture time after infection (hours); on the ordinate, the quantity of RNA (expressed in cpm) for the genes of the proteins IE1 (3a), 39K (3b), gp67 (3c), vp39 (3d), P10 (3e), and polyhedrin (ph) (3f), at 28 ° C and 37 ° C. These mRNAs are extracted and assayed using specific ³² P-labeled cRNA probes on residues C, obtained using the RIBOPROBE PROMEGA KIT.

In the case of Sf9 cells: at 28 ° C, 24 hours after infection, the messenger RNAs mainly present are those of the gp61, iel, vp39 and 39k genes; from 48 hours after infection, the most expressed messenger RNAs are those of very late genes (polyhedrin and P10); at 37 ° C. no expression of the mRNA is observed, with the exception of a weak expression of the 39k mRNA.

In the case of Sf9-HT cells infected at 37 ° C., the expression of the iel, 39K, gp67 and Vp39 genes is reduced, with only 6, 20, 20 and 30% of expression at 24 h post-infection, by compared to what is observed at 28 ° C with Sf9 cells. As for the expression of the very late polyhedrin and PI C genes, it is only 15 to 20% (at 24 hours).

Although the production of the mRNAs of the polyhedrin and P10 genes appears to be low in relative values (especially at 48 h), they are however of the same order of magnitude as that which is observed for the iel and 39K genes.

In fact, the probes used to measure the synthesis of the RNAs expressed under the control of the PI O and iel promoters are of comparable size (iel: 278bp / P10 233bp). However, their content of residues C is however lower for P10 (70 for iel and 44 for P10). Likewise, the polyhedrin and 39K probes are of comparable size (513bp for the 39K probe and 460 for the polyhedrin probe), but the content of residues C is 104 for the 39K probe and 125 for the polyhedrin probe, respectively). Thus, at 24 hours post-infection, the iel, PI O and polyhedrin genes are transcribed with the same efficiency; and the 39K gene is best expressed under these conditions. EXAMPLE 3 MULTIPLICATION OF BACULOVIRUS ACMNPV IN SF21-HT CELLS CULTIVATED AT 37 ° C

A culture of Sf21-HT cells capable of multiplying at 37 ° C. was infected with the baculovirus AcMNPV according to the protocol indicated in Example 2 above. The viral cycle was observed by electron microscopy in cells cultured at 27 ° C, and those cultured at 37 ° C.

72 hours after infection, the appearance of the infected Sf21-HT cells cultivated at 27 ° C. is identical to that of the Sf9-HT cells cultivated under the same conditions, described in Example 2. The major part of the Sf21- cells HT infected cultured at 37 ° C also has the same appearance as that of Sf9-HT cells cultured under the same conditions. However, the presence of polyhedra is also observed in approximately 1 to 2% of the Sf21-HT cells.

EXAMPLE 4 EXPRESSION IN SF9-HT CELLS CULTIVATED AT 37 ° C OF A HETEROLOGOUS GENE CLONED IN A BACULOVIRUS.

The cZ gene coding for β-galactosidase was placed under the control of the promoter of the P10 protein gene, or else under the control of the promoter of the polyhedrin gene, in recombinant baculoviruses derived from the AcMNPV virus.

Cultures of Sf9-HT cells capable of multiplying at 37 ° C. were infected with one or other of these recombinant baculoviruses, according to the same protocol as that indicated above for the baculovirus AcMNPV.

After 3 days of culture at 37 ° C., the β-galactosidase activity is revealed in the cells, using as chromogenic substrate, X-Gal (5-bromo-4-chloro-3- mdoyl-β-D-galactopyranoside) and the cultures are observed under a microscope.

In the case of the promoter of the PIO protein gene, a large proportion of the cells (approximately 95%) have a blue coloration, indicating the expression of active β-galactosidase.

In the case of the polyhedrin gene promoter, approximately 5% of the cells have a blue color. - Immunoglobulms

A recombinant baculovirus expressing the heavy and light chains of a human immunoglobulin [POUL et ai. , Eur. J. Immunol., 25, 2005-2009, (1995)] was used to infect a culture of cells according to the invention. After 4 days of culture at 37 ° C., cultures are recovered in the supernatant up to 2 mg / l of functional antibodies (ie approximately 2 mg of antibody for 5.10 ⁸ cells).

Claims

1) Process for obtaining cultures of insect cells capable of multiplying at 37 ° C., characterized in that it comprises the following steps: a) culturing said insect cells at a temperature of approximately 27 ± 2 ° C; b) increasing the culture temperature to about 37 ± 1 ° C, c) maintaining said cells in culture at this temperature for 4 to 6 days; d) recovering the cells from step c) and re-cultivating them at a temperature of approximately 37 ± 1 ° C. 2) Method according to claim 1, characterized in that the cells cultured are Spodoptera frugiperda cells. 3) Method according to any one of claims 1 or 2 characterized in that said cells of Spodoptera frugiperda belong to the line Sf9 or to the line Sf21. 4) Culture of insect cells, characterized in that it is capable of being obtained by the method according to any one of claims 1 to 3. 5) Method for culturing insect cells, characterized in that cells from a culture according to claim 4 are cultured at 37 ° C. 6) A method of expression of a recombinant protein in insect cells, which method is characterized in that said recombinant protein is expressed in the cells of a culture according to claim 4. 7) Method according to claim 6, characterized in that said recombinant protein is expressed at a temperature above 35 ° C, preferably between 35 and 39 ° C. 8) Use of the Sf9 and Sf21 cell lines of Spodoptera frugiperda, for obtaining cell cultures capable of multiplying at 37 ° C.