Patent Application
20200263144
The invention relates to a method for producing a recombinant baculovirus of which the genome comprises one or more transgene(s) each encoding a protein maturation enzyme and at least two transgenes each encoding a polypeptide of interest, baculoviruses or recombinant baculovirus genomes obtained by this method, sets of homologous recombination elements, cells comprising a recombinant baculovirus or a recombinant baculovirus genome as well as the use of baculoviruses or recombinant baculovirus genomes for the production of polypeptides of interest.
Baculoviruses are a family of rod-shaped viruses, specific to arthropods, which comprises four genera (Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus) encompassing 49 species. Baculoviruses are not capable of replicating in the cells of mammals or other vertebrae.
The baculovirus genome is constituted of a double-stranded DNA molecule, circular, of a size comprised between 80 and 180 kpb. The baculovirus genome associates with highly basic proteins of 605 kDa, within a nucleocapsid with helicoidal symmetry, which contains a capsid protein of 39 kDa. The size of the genome determines the length of the nucleocapsid.
The nucleocapsid is next enclosed in a lipoprotein envelope to form the viral particle or virion. These structures may be covered with a crystalline or polyhedral matrix essentially constituted of a single protein (polyhedrin) of around 30 kDa. Polyhedrons are large structures of which the size varies from 1 to 15 μm diameter with an outer polysaccharide envelope which confers additional protection.
Baculoviruses, of which the genome has been modified genetically, are used in biotechnology for the production of recombinant proteins (i.e. recombinant baculoviruses). After penetration in an insect cell, these recombinant baculoviruses are going to use the machinery of the insect cell to produce the recombinant protein.
Recombinant baculoviruses are obtained by inserting one or more genes originating from other species (for example from human beings, other vertebrates, bacteria and viruses) into the genome of a parent baculovirus. These genes are placed under the control of a viral or cellular promoter (for example the promoter of the polyhedrin gene) to generate a recombinant baculovirus genome. The promoter enables the transcription of the foreign gene into messenger RNA which in its turn is translated into protein in the insect cell infected by the recombinant baculovirus. The advantage of using this system is that the level of production of the recombinant protein in insect cells infected by the recombinant baculovirus may be very important. The recombinant protein may next be purified from infected cells if the protein is intracellular or instead from the culture medium if the protein is secreted. The baculovirus expression system is widely used in industry and in research laboratories. In addition to the important productivity of the baculovirus expression system, this system is also highly appreciated because it makes it possible to produce biologically active recombinant proteins. Indeed, insect cells generally make it possible to obtain appropriate post-translational modifications.
However, certain proteins require post-translational modifications that insect cells are not capable of carrying out. These post-translational modifications being normally carried out by maturation enzymes of specific proteins.
For example, the majority of human glycoproteins have a so-called complex glycosylation, they are in general sialylated (
Thus, if it is wished to produced highly cytotoxic antibodies, antibodies capable of inducing high ADCC and CDC activities, for example to destroy specifically a tumour, the ideal is to produce galactosylated antibodies. On the other hand, an antibody that will be used simply as a ligand, a ligand of a cellular receptor for example to induce apoptosis, or instead to carry out imaging by specifically marking a tissue, it will be desirable to use an antibody incapable of inducing cellular cytotoxicity, in this case a sialylated antibody will be the most suitable.
Numerous studies on the N-glycosylation potential of insect cells very clearly show that if the addition of glycans is specific, that is to say that it is always carried out on asparagine residues identical to those which are glycosylated naturally on the original protein expressed by the tissue (for example the Asn297 of the antibodies), the structure of glycans is different, they are shorter and there are no complex glycans (
With the aim of obtaining correctly matured proteins of interest, it is thus important to complete the enzymatic maturation potential of the insect cell.
Baculoviruses comprising genes encoding protein maturation enzymes have already been described.
The article of Palmberger et al. (2012) relates to a recombinant baculovirus comprising in its genome sequences encoding for two glycosylation enzymes in a same locus. This baculovirus is used for the production of the antibodies 3D6 anti-gp41 of HIV. Two genes encoding the heavy and light chains of the antibodies were inserted into the genome of this baculovirus. These recombinant baculoviruses are generated from a bacmid (infectious in insect cells) in a bacterium. The construction method uses the Cre/Lox system and Tn7 transposition for an iterative integration of the different genes (genes of interest and protein maturation genes). A selection of the bacteria on different antibiotics is then necessary to isolate the infectious recombinant bacmids that will next be introduced, in the form of DNA, into insect cells to produce recombinant baculoviruses.
The article of Chang et al. (2003) discloses a recombinant baculovirus expressing both a polypeptide of interest (human α1-antitrypsin) and a series of glycosyltransferases in a single locus. The recombination is carried out in insect cells, with a non-infectious linearized viral DNA. The repair of this DNA consecutive to the homologous recombination with the transfer vectors makes it possible to reconstruct a circular and thus infectious viral DNA.
The methods for constructing these baculovirus are based on steps of iterative integrations of genes of interest and protein maturation genes bringing into play conventional integration systems.
In both cases, for each integrated transgene, a step of selection is necessary either (i) in the bacterium, a selection based on the presence of antibiotic resistance gene adjacent to the transgene, or (ii) in the insect cell, each recombination step necessitates the presence, in the viral DNA, of a new unique site for specific cleavage of a restriction enzyme to be able again to linearize the viral DNA and to perform a new integration. The repair of this DNA consecutive to homologous recombination with the transfer vectors makes it possible to reconstruct a circular and thus infectious viral DNA.
Thus, there still exists a need to develop methods that are easy to implement and which make it possible to produce recombinant proteins of interest, notably proteins comprising several distinct sub-units, such as antibodies in baculovirus expression systems and which may notably be developed on an industrial scale and which are capable of inducing an appropriate maturation of the protein of interest.
It is in particular necessary, in order to produce certain recombinant proteins of interest, composed of several peptide sub-units, to have a baculovirus expression system in which several transgenes, each encoding a sub-unit, may be integrated easily and preferably in a single step.
Yet, to be able to integrate several transgenes in a baculovirus, the methods known until now have necessitated:
In order to eliminate the step of selection of recombinant baculoviruses having integrated with success a transgene of interest, a method has been proposed based on the joint use:
This method is notably described in the patent application WO 01/12829 and in the article of Possee et al., 2008.
However, this method only makes it possible to integrate one or two transgenes of interest (head to tail), in a single locus. The operation must be repeated several times to integrate a third or a fourth transgene in another locus, bound to another gene essential for replication which will have to be non-functional.
In addition, the integration of a transgene upstream or downstream of a gene essential for replication as described by Possee is capable of generating viruses of which the replication could be altered, and thus viruses replicating insufficiently, that is to say having in the culture supernatant a much lower content of infectious viral particles (PFU/ml) than a wild virus.
On the basis of this finding, the Applicant has developed a method that is particularly efficient and easy to implement for preparing homogeneous and stable recombinant baculoviruses, and which makes it possible to envisage a development at the industrial level for the production of recombinant proteins, for example multimeric proteins comprising several distinct sub-units.
In a first aspect the invention relates to a method for producing a recombinant baculovirus of which the genome comprises one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest, said method comprising the steps of:
a) Preparing, in an insect cell, a baculovirus genome capable of replicating which comprises one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest, by homologous recombination between:
In a second aspect the invention relates to a recombinant baculovirus or a recombinant baculovirus genome comprising:
said spacer of nucleic acid sequence is constituted of 0 to 600 pb, preferably from 1 to 600 pairs of bases,
In a third aspect the invention relates to a set of homologous recombination elements comprising:
n being an integer at least equal to 2.
In a fourth aspect the invention relates to a cell comprising a recombinant baculovirus or a recombinant baculovirus genome according to the invention or a set of homologous recombination elements according to the invention.
In a fifth aspect the invention relates to the use of a recombinant baculovirus or a recombinant baculovirus genome according to the invention or a cell according to the invention for the production of n polypeptides of interest.
Within the context of the present invention the expression “baculovirus” is taken to designate a rod-shaped virus specific to arthropods. A baculovirus generally comprises a nucleocapsid enclosing a baculovirus genome. Examples of baculoviruses are BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV and TeNPV.
Within the context of the present invention the expression “baculovirus genome” is taken to designate the whole of the genetic material of a baculovirus, comprising in particular all the encoding and non-encoding nucleotide sequences of a baculovirus.
Within the context of the present invention the expression “replication deficient baculovirus genome” is taken to designate a baculovirus genome in which at least two genes essential for viral replication have been either deleted (entirely or partially) or mutated in such a way that the baculovirus genome has lost its ability to replicate in an insect cell. For example, the gene essential for viral replication no longer expresses itself or it is transcribed then translated into a non-functional protein. Thus, the genes deleted (entirely or partially) or mutated are called “non-functional genes essential for viral replications”. The viral replication deficient baculovirus genomes are produced from parent baculovirus genomes using molecular biology techniques well known to those skilled in the art, and notably enabling the insertion and/or the deletion of nucleotide sequences in the parent baculovirus genome. Preferably, the replication deficient baculovirus viral genome comprises at least one nucleotide sequence which allows it to replicate within a bacterial cell. The nucleotide sequences enabling replication within a bacterial cell are not transgenes of interest in the sense of the invention. An example of bacterial replication element is the nucleotide sequence “Mini-F”. Such replication elements are well known in the prior art. The bacterial cell may be Escherichia coli. A baculovirus genome which comprises a nucleotide sequence which makes it possible to replicate within a bacterial cell is known by the denomination “Bacmid”. Preferably, the replication deficient baculovirus genome also comprises one or more nucleotide sequences encoding one or more selection markers enabling to select or to identify the bacterial cells transfected by the replication deficient baculovirus genome. The selection nucleotide sequences are not transgenes of interest in the sense of the invention. It may be for example an ampicillin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, a zeocin resistance gene and/or a tetracycline resistance gene.
Within the context of the present invention the expression “recombinant baculovirus genome” is taken to designate a baculovirus genome which comprises one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest. The term “recombinant baculovirus genome” according to the invention corresponds to the baculovirus genome obtained by the implementation of the method according to the invention, that is to say the baculovirus genome obtained by homologous recombination between the replication deficient baculovirus genome and n transfer vectors.
Within the context of the present invention the expression “recombinant baculovirus” is taken to designate a baculovirus of which the genome is a recombinant baculovirus genome, that is to say a baculovirus of which the genome comprises one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest. The recombinant baculovirus may be produced after replication of the recombinant baculovirus genome in an insect cell. The recombinant baculovirus is capable of infecting insect cells. Preferably, the recombinant baculovirus according to the invention is infectious for an insect cell.
“Gene” is taken to designate a nucleotide sequence capable of being transcribed then translated into polypeptide, for example into protein. One then speaks of gene encoding a polypeptide.
Within the context of the present invention “transgene” is taken to designate a gene which is not naturally present in the genome of a baculovirus. It may be for example a gene of human origin, a gene of animal origin, a gene of plant origin, a gene of viral origin or a gene of bacterial origin. Within the context of the invention, the transgene is either a “transgene encoding a protein maturation enzyme”, or a “transgene encoding a polypeptide of interest”.
“Distinct transgenes” is taken to designate transgenes not having the same nucleotide sequence.
A transgene in the sense of the present invention is placed under the control of appropriate elements for its expression in the insect cell. “Appropriate elements” is taken to designate the set of elements necessary for its transcription into messenger RNA (RNAm) and for the translation of RNAm into polypeptide. Among the elements necessary for transcription, the promoter assumes particular importance. It may be a constituent promoter or a regulatable promoter and it may be of baculoviral origin or of arthropod origin (e.g. of insect origin). The important point is that the chosen promoter is suited for the expression of the transgene in the insect cell. Generally speaking, a promoter in use in the present invention may be modified so as to contain regulatory sequences. As examples of promoters it is possible to cite the promoter polyhedrin, the promoter P10, synthetic promoters derived from polyhedrin and P10 promoters, the promoter IE1 of the baculovirus CfMNPV, the promoter IE1 of the baculovirus LdMNPV, the promoter gp64 of the baculovirus OpMNPV, the promoter IE1 of the shrimp virus WSSV (white spot syndrome virus), the promoter P9 of the densovirus of Junonia coenia (JcDNV), the cellular promoter A3 (actin 3) of the silkworm Bombyx mori. In a particular embodiment, one or more transgenes is placed under the control of a synthetic promoter derived from the wild promoter P10 (SEQ ID NO: 1), preferably the synthetic promoter P10S1A (SEQ ID NO: 2) or P10S1B (SEQ ID NO: 3).
“Expression cassette” is taken to designate a nucleotide sequence generally constituted of one or more genes and suitable elements for its/their expression, for example a transgene and the suitable elements for its expression in the insect cell.
“Protein maturation enzyme” is taken to designate an enzyme involved in the maturation of proteins. In particular, the maturation operated by a protein maturation enzyme leads to the production of a stable protein and/or a protein having all or part of its biological activity. For example, the protein maturation enzyme may act at the level of the peptide sequence of a protein (for example by cleavage), at the folding level (this is the case for example of chaperone proteins), at the glycosylation level, or at the level of any other post-translational modification such as for example phosphorylation or methylation. The protein maturation enzyme may be a signal peptidase, a furin, a proprotein convertase, a glycosyltransferase, a glycosidase, a chaperone protein, an isomerase disulphide, an acyltransferase, a methyltransferase, a hydroxylase, a transglutaminase, a farnesyltransferase, a geranylgeranyltransferase, a N-myristoyltransferase, a palmityltransferase, a protein kinase, a phosphatase, a transpeptidase, a carboxylase and/or a ubiquitin ligase.
“Glycosyltransferase” is taken to designate an enzyme capable of catalysing the transfer of a monosaccharide, from an activated sugar (donor), generally by a phosphate, to an acceptor molecule (usually an alcohol or an amine). The transfer acceptor may also be a peptide residue, usually serine, threonine or more rarely tyrosine, hydroxylysine and hydroxyproline during O-glycosylations (O-mannose, O-fucose, O-GalNAc, O-GlcNAc, O-galactose and O-glucose), or an asparagine during N-glycosylation. An activated mannose may also be transferred onto a tryptophan to form a C-mannosyl tryptophan. The glycosyltransferase may be selected from N-acetylglucosaminyltransferases I, II, III, IV, V, VB, VI and IX, a galactosyltransferase, for example a beta-1,4-galactosyltransferase, for example selected from beta-1,4-galactosyltransferase 1, 2, 3, 4, 5, 6 and 7, CMP-NeuAc synthase, NeuAc synthase, protein-O-mannosyltransferases 1 and 2, protein-O-fucosyltransferases 1 and 2, protein-O-glucosyltransferase 1, protein-O-GlcNAc transferase, GalNAc transferase, fucosyltransferases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 (FUT1 á FUT11) and sialyltransferases, for example α2,3 sialyltransferase and α2,6 sialyltransferase.
“Polypeptide” is taken to designate a chain of amino acids bound by peptide bonds. For example, a polypeptide may be a protein, a protein sub-unit, a protein fragment or simply a chain of amino acids. Generally, a polypeptide is formed of at least 10 amino acids.
“Polypeptide of interest” or “recombinant polypeptide of interest” is taken to designate a polypeptide encoded by a transgene. For example, the polypeptide of interest may be a sub-unit of a multimeric protein. Advantageously, it is a polypeptide of therapeutic and/or diagnostic interest, that is to say a polypeptide that may be used in therapy or in diagnostics.
“Distinct polypeptides of interest” is taken to designate polypeptides not having the same sequence of amino acids.
“Distinct sub-units” is taken to designate sub-units of a multimeric protein not having the same sequence of amino acids. Thus, a “protein comprising n distinct sub-units” is a protein which comprises n sub-units each having a specific sequence of amino acids which are bound together by non-covalent bonds and/or covalent bonds.
“Multimeric protein” is taken to designate a protein comprising several sub-units. A multimeric protein may comprise several identical sub-units (homomultimeric protein) or several distinct sub-units (heteromultimeric protein).
“Protein complex” is taken to designate an assembly constituted of several proteins having a functional or structural bond with each other, for example a “Virus-Like-Particle” (VLP) or a multienzymatic complex.
According to the present invention, the expression “replication” or “viral replication” is understood to extend to both the replication of the baculovirus genome and the baculovirus.
Given that the replication of the baculovirus genome in an insect cell is essential for the replication of the baculovirus in the insect cell. Thus, a gene essential for viral replication is taken to designate a gene essential for replication of the baculovirus in the insect cell. The replication of the baculovirus in the insect cell enables the generation of infectious baculovirus. Thus, the method of the invention makes it possible to generate an infectious recombinant baculovirus in a cell, notably an insect cell.
Within the context of the present invention the expression “homologous recombination” is taken to designate the exchange of genetic information between two different nucleotide sequences, necessitating the presence of homologous sequences between two different nucleotide sequences.
The term “antibody” is used herein in the widest sense and encompasses various antibody structures widely described in the literature, including, but without being limited thereto, antibodies whatever their origin, monoclonal antibodies, polyclonal antibodies and fragments of antibodies as long as they exhibit the desired activity (for example bond to the antigen). It may be a mono-specific or multi-specific antibody, for example bi-specific. The antibodies may be an IgA, IgD, IgE, IgG or an IgM. Examples of fragments of antibodies include, but without being limited thereto, fragments Fv, Fab, Fab′, F(ab′)2; diabodies; scFv/Fc; antibodies of camelid type (for example the VHH); single chain antibody molecules (for example scFv).
The invention relates to a method for producing a recombinant baculovirus of which the genome comprises one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest, said method comprises the steps of:
a) Preparing, in an insect cell, a baculovirus genome capable of replicating which comprises one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest, by homologous recombination between:
b) Generating a recombinant baculovirus in an insect cell which comprises the recombinant baculovirus genome obtained at step a).
The n transgenes each encoding a polypeptide of interest are borne by the n transfer vectors which recombine with the replication deficient baculovirus viral genome in which n genes essential for viral replication are non-functional and which comprises one or more transgene(s) each encoding a protein maturation enzyme. After recombination, the n transgenes each encoding a polypeptide of interest are integrated in the genome of the recombinant baculovirus.
The recombination takes place in an insect cell between (a1) a replication deficient baculovirus genome in which n genes essential for viral replication are non-functional and which comprises one or more transgene(s) each encoding a protein maturation enzyme and (a2) the n transfer vectors.
Advantageously, recombination within the context of the present invention takes place in a single step in the insect cell, and this is so whatever the number n of transgenes to integrate. That is to say that the recombination of the n transgenes each encoding a polypeptide of interest with the replication deficient baculovirus genome takes place simultaneously or quasi-simultaneously in the insect cell. This simultaneous recombination is one of the main advantages of the method according to the invention because it makes it possible to produce the desired recombinant baculovirus genome rapidly and in a single step.
In a particular embodiment, the replication deficient baculovirus genome is obtained from a baculovirus genome selected from or derived from the genome of BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV or TeNPV, preferably AcMNPV.
In a preferred embodiment, the replication deficient baculovirus genome implemented in the method is in circular form. Thus, the method according to the invention does not necessitate the linearization of the replication deficient genome.
The transfer vectors may also contain one or more nucleotide sequences that allow them to replicate within a bacterial cell. They may also contain genes encoding a selection marker enabling to select or to identify bacterial cells transformed with a transfer vector.
One of the main advantages of the method of the invention is that the ability of the replication deficient viral baculovirus genome to replicate is restored by recombination with the n transfer vectors. Indeed, each of the n transfer vectors encode, in addition to one of the n transgenes encoding a polypeptide of interest, a nucleotide sequence enabling to restore the function of one of the n non-functional genes essential for viral replications. Thus, only a recombination with the set of n transfer vectors makes it possible to restore the replication of the replication deficient baculovirus genome. Thus, the method of the invention guarantees that only the genomes of recombinant baculoviruses containing the n transgenes encoding a polypeptide of interest are able to generate infectious recombinant baculoviruses. This method avoids having to use expensive, time consuming tests to identify the recombinant baculoviruses containing the n transgenes encoding a polypeptide of interest.
In a preferred embodiment, the genes essential for viral replication are chosen from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147) and lef-2 (ORF6). These genes are preferred because they are adjacent to a gene not essential for viral replication. Thus, in a preferred embodiment, in the replication deficient baculovirus viral genome, the n non-functional genes essential for viral replication are each adjacent to a gene not essential for viral replication. As detailed throughout the present application, the n transgenes each recombine at the locus of a gene not essential for viral replication adjacent to a gene essential for viral replication that is non-functional. Given that the gene not essential for viral replication is not essential for the replication of the baculovirus genome, recombination does not affect the capacity of the recombinant baculovirus genome to replicate.
The n transgenes are integrated either within a gene not essential for viral replication or in an intergenic zone, between 2 non-essential genes, or instead upstream or downstream of a gene essential for viral replication.
When the transgene is integrated upstream or downstream of an essential gene (construction of BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS-5T3 (or BacSia3) described in example 12, with the integration of the gene ST3 downstream of orf51), the baculovirus thus generated, although viable, may however have an altered replication and thus relatively low viral titres.
According to a preferred embodiment of the invention, the n transgenes are integrated within n genes not essential for viral replication, which makes it possible to obtain baculoviruses much more stable in the course of replication cycles, and thus to obtain sufficient viral titres to envisage industrial production.
Examples of integration of a transgene within a gene not essential for viral replication are described in the examples, notably in example 9, with the integration of the gene fur in the genes Chit/Cath, in example 10, with the integration of the gene β1,4GalT in the gene egt, in example 12 with the integration of the genes NeuAc synthase and CMP-NeuAc synthase in the gene iap2 and in example 14 relative to the cloning of the transgene α2,6-sialyltransferase I (ST6GalI) in ORF119 (PIF1) of BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS.
In the sense of the present invention, a non-functional gene essential for viral replication is adjacent to a gene not essential for viral replication when the two genes succeed each other or partially overlap on the genome of the baculovirus, preferably no other gene is comprised between the non-functional gene essential for viral replication and the gene not essential for viral replication. Advantageously, the two aforementioned genes are separated by a spacer nucleotide sequence, for example a non-encoding spacer nucleotide sequence. In particular, the spacer nucleotide sequence has a length ranging from 1 pb to 600 pb. It is also possible that no separating nucleotide sequence (i.e. 0 pb) separates the two aforementioned genes, that is to say that the two aforementioned genes are placed side by side on the genome of the baculovirus or partially overlap. Alternatively, the spacer nucleotide sequence may comprise a gene not essential for viral replication.
The Applicant has noticed that the choice of non-functional genes essential for viral replications adjacent to genes not essential for viral replication was particularly advantageous and made it possible to obtain homogeneous homologous recombination and thus to obtain homogeneous recombinant baculovirus genomes. As explained elsewhere, only a perfect recombination of the n vectors makes it possible to obtain a recombinant baculovirus genome capable of replicating in an insect cell. Thus, the genomes of the recombinant baculoviruses prepared by the method according to the invention are homogeneous to more than 90%, advantageously to more than 95%, preferably to more than 99% and in a completely preferred manner around 100%, for example the recombinant baculovirus genomes prepared by the method according to the invention are all identical.
In a particular embodiment, the gene not essential for viral replication is selected from Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 (ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37 (ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 (ORF96), he65 (ORF105), ORF108, ORF110, cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), ORF5.
Advantageously, the gene essential for viral replication/gene not essential for viral replication couple is selected from the couples listed in table 1 below:
In an advantageous embodiment, the n nucleotide sequences enabling to restore the function of the n non-functional genes essential for viral replications each recombine with a non-functional gene essential for viral replication such as listed in table 1; whereas the n transgenes encoding a polypeptide of interest each recombine at the locus of a gene not essential for viral replication, said non-essential gene being the gene adjacent to said essential gene of which the function was restored during this step of homologous recombination, such as presented in table 1.
Thus, when the essential gene of which the function is restored is the gene 1629 (ORF9), the transgene encoding for a peptide of interest will be integrated in the non-essential gene Ph (ORF5); when the essential gene of which the function is restored is the gene Pk1 (ORF10), the transgene encoding for a peptide of interest will be integrated in the non-essential gene ORF11, and so on for each couple of adjacent genes listed in table 1.
The method according to the invention implements a mechanism of homologous intermolecular recombination. Generally speaking, the mechanism of homologous recombination consists in the exchange of homologous nucleotide sequences between the replication deficient baculovirus genome and the n transfer vectors. These nucleotide sequences may be identical or substantially homologous.
In a particularly advantageous embodiment, the transfer vectors comprise, on either side of the expression cassette of the transgene encoding a polypeptide of interest, flanking sequences homologous to the replication deficient baculovirus genome. The degree of homology of the flanking sequences with the corresponding part of the replication deficient baculovirus genome may be variable but must be sufficient to enable intermolecular recombination. For the purposes of the present invention, it is preferable that it is greater than 70%, advantageously greater than 80%, preferably greater than 90% and in an entirely preferred manner around 100%, preferably identical. In addition, a short region of homology may be sufficient to enable intermolecular recombination, that is to say at least 10 consecutive nucleotides (or pairs of bases) common between the flanking sequences and their homologous sequences in the replication deficient baculovirus genome. Within the context of the present invention, the length of the flanking sequences may range from 10 pb (i.e. 10 pairs of bases) to 10 kb (i.e. 10,000 pairs of bases), advantageously from 100 pb to 6 kb, preferably from 200 pb to 6 kb and, in an entirely preferred manner from 400 pb to 6 kb. Thus, the genetic material located between the flanking sequences of the n transfer vectors replaces the genetic material located between the two sequences homologous to the flanking sequences of the replication deficient baculovirus genome. This intermolecular exchange makes it possible to obtain a recombinant baculovirus genome capable of generating an infectious recombinant baculovirus in the insect cell.
According to the invention, the set of nucleotide sequences i) (i.e. the nucleotide sequences enabling to restore the function of the n non-functional genes essential for viral replications) of the n transfer vectors are capable of restoring the replication of the replication deficient baculovirus genome. Indeed, intermolecular exchange makes it possible to restore the function of the n non-functional genes essential for viral replications. In other words, the restauration of the function of the n non-functional genes essential for viral replications occurs when the homologous recombination takes place correctly. This comes from the fact that the n transfer vectors each comprise a nucleotide sequence enabling to restore the function of one of the n non-functional genes essential for viral replications.
For example, when n=2, a replication deficient baculovirus genome in which two genes essential for viral replication are non-functional recombine with two transfer vectors which each comprise a nucleotide sequence enabling to restore the function of one of the two non-functional genes essential for viral replication. Which means that recombination with the first transfer vector makes it possible to restore the function of a first non-functional gene essential for viral replication and recombination with the second transfer vector makes it possible to restore the function of the second non-functional gene essential for viral replication. Thus, only the recombination of two transfer vectors with the replication deficient baculovirus genome makes it possible to restore the function of the two non-functional genes essential for viral replications and thus to restore the replication of the replication deficient baculovirus genome. This restoration of the function of the two essential genes makes it possible to obtain a recombinant baculovirus genome capable of generating infectious recombinant baculoviruses in the insect cell.
Thus, according to the method of the invention, recombination with the n transfer vectors, or multi-recombination, is necessary to restore the replication of the replication deficient baculovirus genome.
In a surprising manner, the inventors have demonstrated that multi-recombination can be done in a single step, simultaneously, in the insect cell. This is particularly advantageous since the replication deficient baculovirus genome and the n transfer vectors may be introduced at the same time in the insect cell, that is to say that the replication deficient baculovirus genome and the n transfer vectors are introduced simultaneously in the insect cell, in other words the replication deficient baculovirus genome and the n transfer vectors are introduced in a single step in the insect cell, and this is so whatever the number n of transfer vectors. Multi-recombination in a single step makes it possible to obtain easily and rapidly homogeneous recombinant baculovirus genomes.
The method according to the invention makes it possible to produce a recombinant baculovirus which comprises n transgenes each encoding a polypeptide of interest. The set of n polypeptides of interest may form, for example, a protein comprising several sub-units. The set of n polypeptides of interest may also be the constituent proteins of a protein complex, for example a VLP. The set of n polypeptides of interest are produced by an insect cell infected by the recombinant baculovirus which comprises the n transgenes each encoding a polypeptide of interest.
Thus, in a particular embodiment, the n polypeptides of interest form several distinct proteins of interest. It may involve several distinct proteins of interest comprising a single polypeptide chain, several distinct proteins of interest comprising several identical sub-units and/or several distinct proteins of interest comprising several distinct sub-units. The number of distinct proteins of interest formed by the n polypeptides of interest will be equal to or less than n. For example, three polypeptides of interest (n=3) may form (i) a first protein of interest comprising a single polypeptide chain and a second protein of interest comprising two distinct sub-units, (ii) three distinct proteins of interest each comprising a single polypeptide chain, (iii) a first protein of interest comprising several identical sub-units and a second protein of interest comprising two distinct sub-units, or (iv) three distinct proteins of interest each comprising several identical sub-units.
In another particular embodiment, the n polypeptides of interest form a single protein. In this embodiment, the protein then comprises n distinct sub-units, each of the sub-units being one of the n polypeptides of interest.
The method according to the invention is thus particularly advantageous for preparing a recombinant baculovirus which comprises transgenes encoding a protein of interest comprising several distinct sub-units, for example a protein of interest which is only active when it comprises all the sub-units. The sub-units being generally bound together by non-covalent bonds (e.g. hydrophobic bonds) and/or covalent bonds (e.g. disulphide bridges between two cysteines). The method of the invention is thus particularly advantageous for preparing a recombinant baculovirus which comprises transgenes encoding a multimeric protein, for example an antibody or an antibody fragment.
The number of transfer vectors will depend on the desired number of distinct polypeptides of interest that it is wished to produce. For example, two transfer vectors will be used for a protein comprising two distinct sub-units, three transfer vectors will be used for a protein comprising three distinct sub-units, etc. Advantageously, each transfer vector comprises a transgene encoding a polypeptide of interest different from the transgenes encoding the other polypeptides of interest comprised in the other transfer vectors. In a particular embodiment, a transfer vector may comprise more than one transgene, for example two transgenes, each encoding a polypeptide of interest. In this particular embodiment, the transgenes may be present in a same locus, preferably at the most two transgenes per locus.
Advantageously, n is an integer ranging from 2 to 31, for example ranging from 2 to 10. For example, for a protein of interest comprising several distinct sub-units, the value of n will correspond to the number of distinct sub-units of said protein of interest.
In a particular embodiment, n=2. In this case, it is possible to distinguish the following implementations:
In a particular embodiment, n=3. In this case, it is possible to distinguish the following implementations:
For this particular embodiment, the baculovirus BacMid3 described in example 3, of which the genes essential for replication 1629, DNApol and gp64 are non-functional, could be used for the simultaneous integration of these three transgenes.
In a particular embodiment, n=4. In this case, it is possible to distinguish the following implementations:
Advantageously, the recombinant baculovirus produced by the implementation of the method of the invention does not comprise a nucleic acid sequence which enables it to replicate within a bacterial cell. Optionally, the nucleic acid sequence that makes it possible to replicate the replication deficient baculovirus genome within a bacterial cell may be eliminated during the step of homologous recombination in the insect cell.
Step a) is carried out after introduction into the insect cell of the transfer vectors and the replication deficient baculovirus genome. This introduction may be carried out with techniques widely described in the prior art. It is possible to cite notably the calcium phosphate technique, the DEAE dextran technique, electroporation, methods based on osmotic shock, microinjection or methods based on the use of liposomes, preferably lipofection. The method according to the invention is particularly advantageous because it makes it possible to introduce the n transfer vectors and the replication deficient baculovirus genome in a single step into the insect cell. The quantities of replication deficient baculovirus genome and transfer vectors introduced into the insect cell may vary. It is preferred to employ a quantity 5 times greater of each of the n transfer vectors with respect to the quantity of replication deficient baculovirus genome. The replication deficient baculovirus genome is advantageously introduced into the insect cell in circular form, that is to say without having been linearized beforehand. Linearization is unnecessary since the baculovirus genome is replication deficient, even in circular form since it comprises non-functional genes essential for viral replications. The absence of linearization step is one of the major advantages of the method of the invention.
As detailed above, the protein maturation enzyme may be selected from a peptidase signal, a furin, a proprotein convertase, a glycosyltransferase, a glycosidase, a protein chaperone, a disulphide isomerase, an acyltransferase, a methyltransferase, a hydroxylase, a transglutaminase, a farnesyltransferase, a geranylgeranyl-transferase, a N-myristoyltransferase, a palmityltransferase, a protein kinase, a phosphatase, a transpeptidase, a carboxylase or a ubiquitin ligase.
The choice of the protein maturation enzyme(s) will depend on the polypeptides of interest, notably the type of maturation that the polypeptides of interest will have to undergo. For example, when the polypeptides of interest form the sub-units of a glycosylated protein of interest, for example an antibody, the maturation enzyme(s) may be one or more glycosyltransferase(s) enabling to obtain the desired glycosylation. Those skilled in the art could easily select suitable glycosyltransferase(s) as a function of the desired glycosylation.
For example, the glycosyltransferase(s) may be chosen from N-acetylglucosaminyltransferase I, II, II, IV, V; VB, VI, IX, a galactosyltransferase (e.g. a beta-1,4-galactosyltransferase, for example selected from beta-1,4-galactosyltransferase 1, 2, 3, 4, 5, 6 and 7), CMP-NeuAc synthase, NeuAc synthase, a sialyltransferase (e.g. α2,3 sialyltransferase or α2,6 sialyltransferase), protein-O-mannosyltransferases 1 and 2, protein-O-fucosyltransferases 1 and 2, protein-O-glucosyltransferase 1, protein-O-GlcNAc transferase, GalNAc transferase, fucosyltransferases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 (FUT1 to FUT11).
In a particular embodiment, the glycosyltransferase is one or more glycosyltransferase(s) chosen from N-acetylglucosaminyltransferase II, a beta-1,4-galactosyltransferase and a sialyltransferase.
Step a1
In a particular embodiment, the replication deficient baculovirus genome of step a1) is prepared, in a bacterial cell, by homologous recombination between:
i) a replication deficient baculovirus genome in which n genes essential for viral replication are non-functional, and
ii) one or more nucleotide sequence(s) each comprising one or more transgene(s) each encoding a protein maturation enzyme.
Thus, in this particular embodiment, the method of the invention comprises the steps of:
a) Preparing, in an insect cell, a second baculovirus genome capable of replicating which comprises one or more transgene(s) each encoding a protein maturation enzyme and n transgenes each encoding a polypeptide of interest (recombinant baculovirus genome), by homologous recombination between:
In this embodiment, a first recombination takes place in a bacterial cell between (a′1) a replication deficient baculovirus genome in which n genes essential for viral replication are non-functional and (a′2) one or more nucleotide sequence(s) each comprising one or more transgene(s) each encoding a protein maturation enzyme.
Preferably, the transgene(s) encoding a protein maturation enzyme each recombine at the locus of a gene not essential for viral replication, preferably at the locus of a gene not essential for viral replication non-adjacent to a non-functional gene essential for viral replication. The gene not essential for viral replication is advantageously selected from ptp (ORF1), ctx (ORF3), ORF4, ORF7, odv-e26 (ORF16), ORF17, ORF18, ORF19, ARIF-1 ORF20-21, pif2 (ORF22), protein F (ORF23), iap1 (ORF27), lef6 (ORF28), ORF29, ORF30, sod (ORF31), fgf (ORF32), gta (ORF42), ORF43, ORF44, ORF45, odv-e66 (ORF46), ORF47, ORF56, ORF57, chaB-like (ORF58/59), chaB-like (ORF60), mtase (ORF69), hcf-1 (ORF70), iap2 (ORF71), ORF86, ORF87, ORF111, ORF114, pif3 (ORF115), ORF116, ORF117, pif1 (ORF119), ORF120, ORF121, ORF122, pk2 (ORF123), ORF124, lef7 (ORF125), chitinase (ORF126), gp16 (ORF130), p35 (ORF135), p26 (ORF136), p10 (ORF137), p74 (ORF138), ORF149, ORF150, ie2 (ORF151), pe38 (ORF153) and ORF154.
In this particular embodiment, the bacterial cell is a bacterium which supports the replication of baculovirus genomes containing a mini-F origin of replication. The bacterial cell is preferably E. coli, notably DH10B or EL350.
Step b) consists in generating a recombinant baculovirus in an insect cell which comprises the recombinant baculovirus genome obtained at step a). For example, it may be the insect cell of step a).
Advantageously, the insect cell is cultured in suitable conditions so that it expresses the recombinant baculovirus, notably in a culture medium suited to the growth of the cells. The culture medium may contain a serum of animal origin or may be a culture medium without serum.
Advantageously, the insect cell is selected from Sf9, Sf21, Tn5-b14, lepidoptera cell lines sensitive to baculovirus AcMNPV, lines Sf21, the “High Five” line, preferably it is Sf9.
The recombinant baculovirus thus generated may be used to infect other insect cells. These insect cells infected by the recombinant baculovirus may then each produce transgenes. This production of each of the transgenes thus makes it possible to obtain the n polypeptides of interest having been matured by the protein maturation enzyme(s).
In a particular embodiment, the recombinant baculovirus may be used to infect eukaryotic cells. It has in fact been shown that the baculovirus could infect eukaryotic cells.
The method according to the invention thus makes it possible to produce easily and rapidly a recombinant baculovirus whose genome comprises one or more transgene(s) each encoding a protein maturation enzyme and n distinct transgenes each encoding a distinct polypeptide of interest.
Recombinant Baculovirus
The present invention also aims to protect a recombinant baculovirus or recombinant baculovirus genome comprising:
a) one or more transgene(s) each encoding a protein maturation enzyme, and
b) n nucleotide sequences of formula (I):
[transgene encoding a polypeptide of interest]-[spacer nucleotide sequence]-[gene essential for functional viral replication] (I),
said spacer nucleic acid sequence is constituted of 0 to 600 pb, preferably 1 to 600 pairs of bases,
said gene essential for functional viral replication is selected from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), Vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), lef-2 (ORF6);
n being an integer at least equal to 2.
Advantageously, the recombinant baculovirus or the recombinant baculovirus genome according to the invention does not comprise a nucleic acid sequence which enables it to replicate within a bacterial cell. It has in fact been demonstrated that the absence of such a sequence makes it possible to increase the stability of the recombinant baculovirus, compared to a recombinant baculovirus comprising such a sequence (Piljmann et al. (2003) Journal of General Virology).
Advantageously, the recombinant baculovirus or recombinant baculovirus genome according to the invention does not comprise n genes not essential for viral replication chosen from Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 (ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37 (ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 (ORF96), he65 (ORF105), ORF108, ORF110, cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), ORF5. Advantageously, one of the n genes not essential for viral replication not comprised in the recombinant baculovirus or recombinant baculovirus genome is the gene encoding cathepsin because it has been shown that cathepsin may have a deleterious effect on the polypeptides of interest produced.
According to the invention, n is an integer at least equal to 2, for example an integer ranging from 2 to 30, for example ranging from 2 to 10, and more specifically being equal to 2, 3 or 4, as detailed in the section “Method for preparing the recombinant baculovirus” above.
According to a particular embodiment of the invention, n is greater than or equal to 3.
The present invention also aims to protect a recombinant baculovirus or a recombinant baculovirus genome, capable of being obtained by the production method according to the invention, comprising:
a) one or more transgene(s) each encoding a protein maturation enzyme, and
b) n nucleotide sequences of formula (I):
[transgene encoding a polypeptide of interest]-[spacer nucleotide sequence]-[gene essential for functional viral replication] (I),
said spacer nucleic acid sequence is constituted of 0 to 600 pb, preferably 1 to 600 pairs of bases,
said gene essential for functional viral replication is selected from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), Vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), lef-2 (ORF6);
n being an integer at least equal to 2.
Advantageously, the n nucleotide sequences of formula (I) are spread out over the whole the genome of the recombinant baculovirus, which makes it possible to improve the stability thereof. The n nucleotide sequences of formula (I) are thus sufficiently spaced apart on the genome of the baculovirus. Advantageously, each of the n nucleotide sequences of formula (I) is spaced apart by at least 500 nucleotides with respect to another of the n nucleotide sequences of formula (I). Obtaining a recombinant baculovirus or a recombinant baculovirus genome with n nucleotide sequences of formula (I) spread out over the whole the genome does not present any particular difficulty for those skilled in the art since the distribution on the genome will be linked to the “essential gene/non-essential gene” couples that will be chosen.
Advantageously, and this is inherent in the implementation of the preparation method according to the invention, the n nucleotide sequences of formula (I) are not duplicated on the genome of the recombinant baculovirus. A decrease in the stability of the recombinant baculovirus when the sequences are duplicated on the genome (data not presented) has in fact been demonstrated.
The present invention also aims to protect a set of homologous recombination elements comprising:
a) a replication deficient baculovirus genome in which n genes essential for viral replication are non-functional and which comprises one or more transgene(s) each encoding a protein maturation enzyme;
b) n transfer vectors each comprising:
Advantageously, the non-functional genes essential for viral replications are each adjacent to a gene not essential for viral replication, as described in the section “Method for preparing the recombinant baculovirus” above.
In a particular embodiment, the transfer vectors comprise, on either side of the expression cassette of the transgene, flanking sequences homologous to the replication deficient baculovirus genome. Advantageously, the flanking sequences of each of the transfer vectors are homologous to all or part of said non-functional gene essential for viral replication and to all or part of said gene not essential for viral replication. Advantageously, the flanking sequences have a length that may range from 10 pb (i.e. 10 pairs of bases) to 10 kb (i.e. 10,000 pairs of bases), advantageously ranging from 100 pb to 6 kb, preferably ranging from 200 pb to 6 kb and, in an entirely preferred manner ranging from 400 pb to 6 kb. The flanking sequences are described in detail in the section “Method for preparing the recombinant baculovirus” above.
Advantageously, n is an integer ranging from 2 to 31, for example ranging from 2 to 10, as detailed in the section “Method for preparing the recombinant baculovirus” above.
The present invention also aims to protect a cell comprising a recombinant baculovirus or a recombinant baculovirus genome according to the invention, or a cell comprising a set of homologous recombination elements according to the invention.
In a particular embodiment, the cell is an insect cell, preferably selected from Sf9, Sf21, Tn5-b14, lepidoptera cell lines sensitive to baculovirus AcMNPV, lines Sf21, preferably Sf9, as described in greater detail in the section “Method for preparing the recombinant baculovirus” above.
The present invention also targets the use of a recombinant baculovirus or a recombinant baculovirus genome according to the invention or a cell according to the invention for the production of n transgenes each encoding a polypeptide of interest.
The production of recombinant polypeptides of interest from a baculovirus is well described in the prior art and may easily be implemented by techniques well known to those skilled in the art.
“Mono-Recombinant” Baculovirus
In a particular embodiment, the method of the invention may be used to produce a “mono-recombinant” baculovirus, that is to say a baculovirus comprising a single transgene encoding a polypeptide of interest.
In this particular embodiment, the method for producing a recombinant baculovirus of which the genome comprises one or more transgene(s) each encoding a protein maturation enzyme and a transgene encoding a polypeptide of interest, comprises the steps of:
Caption:
ORF: open reading frame
Polyhedrin or PH: baculovirus gene encoding polyhedrin: gene not essential for viral replication.
ORF603: baculovirus gene encoding the protein 603, non-essential gene.
ORF1629: baculovirus gene encoding the protein 1629: gene essential for viral replication
pVT: plasmid transfer vector.
Mini-F: origin of bacterial replication.
KanR: bacterial expression cassette expressing the kanamycin resistance gene.
AmpR: bacterial expression cassette expressing the ampicillin resistance gene.
Recombination fragment: fragment of DNA containing the expression cassette to integrate in the target DNA. This fragment has flanking regions on either side of the expression cassette to be able to target specifically the region that will undergo homologous recombination via Red Recombinase.
Caption:
gp37: baculovirus gene encoding glycoprotein gp37, gene not essential for viral replication. gp37 Δ252 aa: gene gp37 deleted from the region encoding the 252 N-terminal amino acids.
DNAPol: baculovirus gene encoding viral DNA polymerase, gene essential for viral replication.
DNAPol⋅Δ466 aa: dna pol gene deleted from the region encoding the 466 C-terminal amino acids.
HygroR: bacterial expression cassette expressing the hygromycin resistance gene.
Recombination fragment: fragment of DNA containing the expression cassette to integrate in the target DNA. This fragment has flanking regions on either side of the expression cassette to be able to target specifically the region that will undergo homologous recombination via Red Recombinase.
Caption:
Chit: baculovirus gene encoding chitinase, gene not essential for viral replication.
Cath: baculovirus gene encoding viral cathepsin, gene not essential for viral replication.
gp64: baculovirus gene encoding viral glycoprotein gp64, gene essential for viral replication.
gp64⋅Δ188 aa: gene gp64 deleted from the region encoding the 188 C-terminal amino acids.
ZeoR: bacterial expression cassette expressing the zeocin resistance gene.
Recombination fragment: fragment of DNA containing the expression cassette to integrate in the target DNA. This fragment has flanking regions on either side of the expression cassette to be able to target specifically the region that will undergo homologous recombination via Red Recombinase.
Caption:
pVT: plasmid transfer vector.
gp37: baculovirus gene encoding the glycoprotein gp37, gene not essential for viral replication.
DNAPol: baculovirus gene encoding viral DNA polymerase, gene essential for viral replication.
DNAPol.Δ466 aa: dna pol gene deleted from the region encoding the 466 C-terminal amino acids.
Exogenous gene: transgene of interest.
P: viral or cellular promoter which controls the expression of the transgene.
HygroR: bacterial expression cassette expressing the hygromycin resistance gene.
Caption:
pVT/PH: polyhedrin transfer vector plasmid.
PH: all or part of the baculovirus gene encoding polyhedrin, gene not essential for viral replication.
ORF603: baculovirus gene encoding protein 603, non-essential gene.
ORF1629: baculovirus gene encoding protein 1629, gene essential for viral replication.
Exogenous gene: transgene of interest.
P: viral or cellular promoter which controls the expression of the transgene.
KanR: bacterial expression cassette expressing the kanamycin resistance gene.
Mini-F: origin of bacterial replication.
Caption:
pVT: plasmid transfer vector.
pVT/gp37-Cγ1: plasmid transfer vector specific to the heavy chain of an immunoglobulin.
Cγ1: DNAc encoding the constant domain γ1 of a human immunoglobulin.
VH: DNAc encoding the variable domain of the heavy chain of an immunoglobulin.
DNAPol: baculovirus gene encoding viral DNA polymerase, gene essential for viral replication.
DNAPolΔ466 aa: DNAPol gene deleted from the region encoding the 466 C-terminal amino acids.
P: viral or cellular promoter which controls the expression of the transgene.
HygroR: bacterial expression cassette expressing the hygromycin resistance gene.
PS: DNAc encoding a signal sequence (secretion of the heavy chain).
Caption:
ORF603: baculovirus gene encoding protein 603.
ORF1629: baculovirus gene encoding protein 1629: gene essential for viral replication.
Mini-F: origin of bacterial replication.
pVT: plasmid transfer vector.
pVT/PH-Cκ: plasmid transfer vector specific to the light chain of an immunoglobulin.
Cκ: DNAc encoding the kappa constant domain of a human immunoglobulin.
VL: DNAc encoding the variable domain of the light chain of an immunoglobulin.
P: viral or cellular promoter which controls the expression of the transgene.
KanR: bacterial expression cassette expressing the kanamycin resistance gene.
PS: DNAc encoding a signal sequence (secretion of the light chain).
Caption:
pVT Chit/Cath: plasmid transfer vector capable of recombining at the locus of the region comprising the 2 non-essential genes, ChiA encoding chitinase and Cath encoding cathepsin. gp64Δ188 aa: gp64 gene, essential gene, deleted from the region encoding the 188 C-terminal amino acids.
Exogenous gene: transgene of interest.
P: viral or cellular promoter which controls the expression of the transgene.
ZeoR: bacterial expression cassette expressing the zeocin resistance gene.
Caption:
A: analysis of the organisation of the genomes of 3 independent recombinant baculoviruses expressing the antibodies 13B8II. These baculoviruses were isolated from a single transfection experiment. The hybridizations carried out respectively with a probe specific to the constant region of the kappa light chain: probe Cκ and a probe specific to the constant region of the heavy chain gamma 1: probe Cγ1 demonstrates a correct and identical organisation of the 3 recombinant viruses.
B. Analysis by electrophoresis in polyacrylamide gel (SDS, 2-mercaptoethanol) and silver staining of the purified recombinant antibodies. The antibodies secreted in the culture medium of cells infected with the recombinant baculovirus were purified on a Protein A Sepharose column.
PC1: Plasmid control, plasmid containing the gene of the kappa light chain,
PC2: Plasmid control, plasmid containing the gene of the heavy chain γ1, R1-3, recombinant baculovirus 1, 2 and 3,
MW: size marker.
Caption:
M: gene of the flu virus encoding the matrix protein.
HA: gene of the flu virus encoding hemagglutinin.
NA: gene of the flu virus encoding neuraminidase.
bp: size of the DNA fragments expressed in pairs of bases.
Caption:
A: Diagrammatical representation of the structure of the bispecific antibodies. L1: light chain of the antibodies 1; L2, light chain of the antibodies 2.
B: Purified bispecific antibodies, analysed by electrophoresis on polyacrylamide gel. The proteins were revealed by silver staining. (1) electrophoresis under reducing conditions (SDS, 2-mercaptoethanol). (2) electrophoresis under non-reducing conditions. H: heavy chain of the antibodies, L: light chain of the antibodies, H2L4: composition of the bispecific antibodies: 2 fused heavy chains bound by 4 disulphide bridges at the level of 2 hinge regions+4 light chains (2 chains L1+2 chains L2) paired in a specific manner to the corresponding regions VH1-CH1 and VH2-CH1.
Two expression cassettes were successively inserted into the region IG35/36 cloned beforehand in a plasmid pUC (i) a viral expression cassette, composed of an early viral promoter (see Table 2) and the gene encoding GNT-II and (ii) a bacterial expression cassette controlling the zeocin resistance gene (ZeoR). A “recombination fragment” containing the 2 cassettes was generated by digestion of the above plasmid by 2 restriction endonucleases.
The latter was introduced into the bacterium EL350/BacMid2 by electroporation. A homologous recombination took place via Red Recombinase between the flanking regions of the recombination fragment and the DNA of BacMid2 enabling the integration of the 2 expression cassettes. The recombinant bacteria thus obtained were selected with zeocin then the gene encoding the resistance to this antibiotic was eliminated from the DNA of the bacmid by simple digestion/reparation/religation. The bacmid obtained, called BacMid2-GNTII, was re-introduced by electroporation into a bacterium EL350 (EL350/BacMid2-GNT-II).
A and B: Control of the genomic organisation of BacMids BacFur and BacGal-Fur. A. The DNA of the 2 bacmids was digested by EcoRI, the fragments generated were separated on agarose gel at 1% then stained with ethidium bromide. B. The DNA was transferred onto a nylon membrane according to the Southern technique. The membranes were incubated with a probe specific to the gene fur expressed by the Sf9 cell.
Caption:
A, Agarose gel stained with ethidium bromide, Well 1: restriction profile EcoRI of BacMid2Gal-Fur, Well 2: Restriction profile EcoRI of BacMid2-Fur. B. Southern blot, Well 1: restriction profile EcoRI of BacMid2Gal-Fur, Well 2: restriction profile EcoRI of BacMid2-Fur.
C and D: two recombinant viruses co-expressing the polyprotein Pr55Gag and gp160 of the virus HIV-1 were constructed one from BacMid2 and the other from BacMid2-Fur. The Sf9 cells were infected for 48 hours with the different viruses and the proteins secreted in the culture supernatant were concentrated or not with a solution of “Retro Concentin™ Virus Precipitation (SBI, reference RV100A-1)” then deposited on a polyacrylamide gel at 10% under denaturing and reducing conditions then analysed by Western blot. C. The proteins were revealed with an anti-gp120 antibody (reference Ab21179, Abcam). D. The proteins were revealed with an anti-Pr55Gag antibody (reference. 63917, Abcam).
Caption:
BACWT: wild baculovirus, BACgp160/Gag/Fur: triple-recombinant baculovirus expressing gp160 and polyprotein Pr55Gag of HIV-1 as well as the furin of the cell Sf9 BACgp160/Gag: double-recombinant baculovirus expressing gp160 and polyprotein Pr55Gag of HIV-1, BACgp120: mono-recombinant baculovirus expressing gp120 of HIV-1, BACGag: mono-recombinant baculovirus expressing polyprotein Pr55Gag of HIV-1.
A. Generation of a double-recombinant baculovirus genome.
The 2 transgenes of interest are cloned in their respective transfer vector, pVT/PH targeting the couple GNE/GE PH/1629 and pVT/gp37 targeting the couple GNE/GE gp37/DNAPol. The Sf9 cells are transfected with 2 pVT and DNA of BacMid2-Gal. During homologous recombination, the 2 transgenes of interest are integrated in the genome of BacMid2-Gal whereas simultaneously the non-functional genes 1629 and dnapol borne by BacMid2-Gal are replaced by a functional copy. These events will have for consequence the elimination of the origin of bacterial replication and the generation of an infectious recombinant baculovirus genome. Recombinant baculoviruses are then produced and secreted in the culture medium, then cloned by the phage plaque assay method.
B. Generation of a mono-recombinant baculovirus genome.
The gene of interest is cloned in the transfer vector pVT/gp37. The Sf9 cells are transfected with pVT/gp37 comprising a transgene, pVT/PH not containing transgene and DNA of BacMid2-Gal. During homologous recombination, there is repair of the bacmid in the 2 loci and thus generation of infectious baculovirus.
Caption:
GE: Essential gene
GNE: Non-essential gene
pVT/PH: Transfer vector which targets the couple GNE/GE PH/1629
pVT/gp37: Transfer vector which targets the couple GNE/GE gp37/DNAPol
DNA PolNF: Gene encoding non-functional viral DNA polymerase
DNA PolF: Gene encoding functional viral DNA polymerase
1629NF: Gene encoding the non-functional protein 1629
1629F: Gene encoding the functional protein 1629
β1,4 GalT: β1,4 galactosyltransferase
GNT-II: N acetylglucosaminyltransferase II
KanR: kanamycin resistance gene
mini-F: origin of bacterial replication
PH: polyhedrin gene
Caption:
GE: Essential gene
GNE: Non-essential gene
pVT/PH: Transfer vector which targets the couple GNE/GE PH/1629
pVT/gp37: transfer vector which targets the couple GNE/GE gp37/DNAPol
DNA PolNF: Gene encoding non-functional viral DNA polymerase
DNA PolF: Gene encoding functional viral DNA polymerase
1629NF: Gene encoding the non-functional protein 1629
1629F: Gene encoding the functional protein 1629
β1,4 GalT: β1,4 galactosyltransferase
GNT-II: N acetylglucosaminyltransferase II
KanR: kanamycin resistance gene
mini-F: origin of bacterial replication
PH: polyhedrin gene
Caption:
A and B. Western blot. A: the membrane was incubated with a sheep anti-human IgG whole antibody peroxidase conjugated (reference NA933V, GE Healthcare). B: The membrane was incubated with a sheep anti-mouse IgG whole antibody peroxidase conjugated (reference NA931V, GE Healthcare).
A. Well 1: Fetuin (61-68 kDa), supplied in the “Dig Glycan Differentiation” kit from Roche, this α2,3 and α2,6 sialylated protein constitutes the positive control for analyses with lectin blot whether for SNA, MAA or diCBMA. According to the supplier, the molar mass (*) of this protein varies between 68 and 61 kDa, Well 2: Recombinant antibodies 13B8II/BacGal, Well 3: Recombinant antibody 13B8II/BacMan,
B. Well 1: Recombinant mouse antibody/BacGal, Well 2: Recombinant mouse antibody/BacMan,
C. Lectin blot. The membrane was incubated in the presence of RCA120 conjugated with biotin. The presence of lectin was revealed as described in example 17.
Well: Fetuin (61-68 kDa) Well 2: Recombinant mouse antibody/BacMan, Well 3: Recombinant mouse antibody/BacGal, Well 4: Recombinant antibody 13B8II/BacMan, Well 5: Recombinant antibody 13B8II/BacGal.
Caption:
GE: Essential gene
GNE: Non-essential gene
pVT/PH: Transfer vector which targets the couple GNE/GE PH/1629
pVT/gp37: Transfer vector which targets the couple GNE/GE gp37/DNAPol
DNA PolNF: Gene encoding non-functional viral DNA polymerase
DNA PolF: Gene encoding functional viral DNA polymerase
1629NF: Gene encoding the non-functional protein 1629
1629F: Gene encoding the functional protein 1629
β1,4 GalT: β1,4 galactosyltransferase
GNT-II: N acetylglucosaminyltransferase II
α2,3 ST: α2,3 sialyltransferase
α2,6 ST: α2,6 sialyltransferase
KanR: kanamycin resistance gene
mini-F: origin of bacterial replication
PH: polyhedrin gene
Caption:
a: Analysis of the electrophoretic profile of digestion by EcoRI of 2 clones of BacMid2Sia6-II (1 and 2) in comparison with BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS (T) shows that the integration of the cassette ST6GalI in the region Pif1 generates 2 fragments EcoRI of 3122pb and 6138pb.
b: The hybridisation carried out with a probe specific to ST6GalI makes it possible to verify the marking of the 2 fragments EcoRI of 3122pb and 6138pb and the control plasmid (PC), plasmid containing the ST6GalI gene and demonstrates a correct and identical organisation of the 2 BacMids obtained.
MW: Smart Ladder (Eurogentec)
A. Western blot. The membrane was incubated with the antibodies anti-gp64 AcV5 (reference SC65499, Santa Cruz Biotechnology)
B and C: Lectin blot. B. The membrane was incubated in the presence of SNA, lectin which specifically recognises α2,6 bound sialic acids (C) the lectin di-CBM40 which recognises α2,3 bound sialic acids and to a lesser extent α2,6 bound sialic acids.
Caption:
Fet: Fetuin
Mq: Molecular weight markers
ST3: Virus generated from the bacmid BacSia3
ST6: Virus generated from the bacmid BacSia6
ST3/6: Virus generated from the bacmid BacSia3/6
Man: Virus generated from BacMid2
Caption:
Well 1, the protein X was produced after infection of the Sf9 cells with a recombinant baculovirus generated from BacMid2. Well 2, the protein X was produced after infection of the Sf9 cells with a recombinant baculovirus generated from BacSia6. Well 3, commercially available Protein X produced in CHO cells. The presence of lectin was revealed as described in example 17. MW: Molecular weight marker (Pre-stained marker, Biolabs reference P7706).
A and B: Analysis of the protein VSVg.
A. Western blot, after electrophoresis in polyacrylamide gel then transfer onto a membrane, the proteins were incubated in the presence of a specific antibody directed against VSVg (mouse antibodies peroxidase conjugated, reference A5977 Sigma).
Caption:
Well 1, Fetuin, Well 2, and 3, Sf9 cells infected with a recombinant baculovirus expressing the protein VSVg generated from BacSia6 (Well 2) or Bacmid2 (Well 3).
B. Lectin blot. The proteins transferred onto a nitrocellulose membrane were placed in the presence of lectin SNA (Sambucus nigra agglutinin) specific to residues of α2,6 bound sialic acids. The presence of lectin was revealed as described in example 17.
C and D. Analysis of the viral protein gp64.
We also verified that the recombinant baculovirus expressing sialylated VSVg was also bearing a sialylated gp64. To do so, the baculoviruses secreted in the culture supernatant were sedimented then taken up by a lysis buffer to be analysed by Western blot then by lectin blot. C. Western blot, gp64 was revealed with a specific antibody (antibodies anti-gp64 AcV5, reference SC65499, Santa Cruz Biotechnology). D. Lectin blot. In the presence of lectin SNA as described in example 17.
Caption:
Well 1, Fetuin, Wells 2, and 3, particles of baculovirus prepared from the culture supernatants of cells infected with the recombinant baculovirus expressing protein VSVg and generated from BacMid2-Sia6 (Well 2) or a Bacmid2 (Well 3).
Examples 1 to 3 are relative to the construction of replication deficient baculoviruses, in which 1, 2 or 3 genes respectively are non-functional.
Examples 4 and 5 describe the generation of recombinant baculoviruses having integrated 2 or 3 transgenes, respectively.
Examples 6 to 8 are relative to the use of these recombinant baculoviruses having integrated 2 or 3 transgenes for the production of proteins of interest.
Examples 9 to 15 describe the construction of recombinant baculoviruses comprising transgenes encoding for protein maturation enzymes.
Examples 16 to 19 demonstrate that proteins of interest produced thanks to the baculoviruses of examples 9 to 15 have satisfactory maturation and/or glycosylation.
BacMid1 has the deletion of a gene essential for viral replication, the gene 1629.
1. Integration of the Origin of Bacterial Replication in a Genome of the Baculovirus
This operation is carried out in the insect cell.
The origin of bacterial replication Mini-F was introduced into the polyhedrin locus of the baculovirus genome AcMNPV by homologous recombination in insect Sf9 cells (Spodoptera frugiperda). To do so, the cells were transfected with (i) a transfer vector PH (pVT/Mini-F-KanR) in which the sequence of the gene ph was replaced by a fragment of DNA bearing the Mini-F+a bacterial expression cassette conferring kanamycin resistance (KanR), and (ii) a baculovirus genome AcMNPV (baculovirus isolated from the lepidoptera Autographa californica). The baculovirus generated were purified by the phage plaque assay technique then characterised in order to confirm that they had indeed integrated the Mini-F and the expression cassette KanR. A baculovirus was selected and was next transferred into the bacterium E. coli EL350, thus generating a first BacMid (BacMid0, non-deficient for viral replication in insect cells).
1. Deletion of the Essential Gene 1629
A bacterial expression cassette conferring ampicillin resistance (AmpR) and having on 5′ and 3′ the restriction site MauBI—site absent from the baculovirus genome AcMNPV —was integrated downstream of the bacterial expression cassette KanR by homologous recombination in the bacterium E. coli EL350. In the course of this recombination, a fragment of DNA encoding the 27 C-terminal amino acids of the protein 1629 was deleted, making the protein 1629 non-functional (BacMid0/ampR). The ampicillin resistance gene was next eliminated after digestion by MauBI then religation, thus generating BacMid1. The genome of the baculovirus (i.e. BacMid1) is then deficient for replication in insect cells, because a gene essential for viral replication (i.e. the gene encoding the protein 1629) is non-functional. The bacteria containing BacMid1 are called hereafter “bacteria E. coli EL350/BacMid1.”.
BacMid2 exhibits the deletion of 2 genes essential for viral replication, the gene 1629 and the gene encoding viral DNA polymerase (DNAPol). From BacMid1, the deletion of the gene DNAPol was carried out in the bacteria E. coli EL350/BacMid1 after electroporation of a recombination fragment of 4222 bp in which a part of the genes encoding gp37 (252 amino acids) and DNAPol (466 C-terminal amino acids) was deleted and replaced by a bacterial expression cassette enabling the production of hygromycin B phosphotransferase (HygroR) thus conferring hygromycin resistance (HygroR). The HygroR gene was placed under the control of the bacterial promoter EM7 (derived from the commercially available vector pSelect-Hygro-mcs, Invitrogen), the terminator glms was introduced downstream of the HygroR gene (Gay N. J. et al. Biochem J., 1986, 234, 111-117). The bacteria containing BacMid2 (E. coli EL350/BacMid2) were selected for their hygromycin resistance. The baculovirus genome (i.e. BacMid2) is deficient for replication in insect cells, because two genes essential for viral replication (i.e. the gene encoding the protein 1629 and the gene encoding DNAPol) are non-functional.
Note: It is possible to use BacMid2 to produce a single protein (see Example 4). It suffices to have two transfer vectors, one providing the transgene and all or part of the deleted essential gene 1 and the other providing the wild gene corresponding to the deleted essential gene 2. The two deleted genes are repaired during homologous recombination.
BacMid3 has the deletion of 3 essential genes, 1629, DNAPol and gp64.
From BacMid2, the deletion of gene gp64 was carried out in the bacteria E. coli EL350/BacMid2 after electroporation of a recombination fragment of 3260pb in which the totality of the gene of the cathepsin plus 779 bp of the sequence encoding 259 amino acids of chitinase and a part of the gene of gp64, deletion of 566 bp encoding 188 amino acids, was replaced by a bacterial expression cassette conferring zeocin resistance (ZeoR) (Drocourt et al., Nucleic Acids Research, vol. 18no 13, 1990). The ZeoR gene derived from the commercially available plasmid pCR®-Blunt (Invitrogen) was placed under the control of the bacterial promoter T5N25, derived from the phage T5 (Gentz and Sward, J. Bacteriology, vol. 164 no 1, 1985) and followed by the transcription terminator rrnBT1 (E. coli ribosomal RNA operon T1 terminator) (Kwon et al., J Biol. Chem., vol 274 no 41, 1999). The bacteria containing BacMid3 (E. coli EL350/BacMid3) were selected for their zeocin resistance. The baculovirus genome (i.e. BacMid3) is deficient for replication in insect cells, because three genes essential for viral replication (i.e. the gene encoding protein 1629 and the gene encoding DNAPol and the gene encoding gp64) are non-functional.
A transfer vector pVT/gp37 was constructed to be able to generate recombinant baculoviruses expressing 2 transgenes. To do so, the fragment EcoRI F of the baculovirus genome AcMNPV containing the gene gp37 and the gene DNAPol was cloned in a bacterial plasmid pUC, thus generating pUC/gp37.
This plasmid was next modified in the following manner: a large part of the gene encoding gp37 was deleted (724pb), the ATG initiator was mutated and replaced by two unique restriction sites XbaI and AvrII enabling the integration of a transgene under control of the natural promoter of gp37. These modifications thus led to the transfer vector pVT/gp37 being obtained.
The Sf9 cells were transfected by lipofection with the transfer vectors pVT/PH and pVT/gp37 loaded with the transgenes and DNA of BacMid2. The viruses generated after homologous recombination were cloned by the phage plaque assay method. The production of the recombinant protein was verified by a suitable method (e.g. for example ELISA, Western blot, enzymatic assay). The genome of the recombinant viruses was verified by Southern blot and the sequence of the transgene integrated in the viral genome was verified by sequencing after PCR amplification.
The genomes of recombinant baculoviruses generated after homologous recombination between BacMid2 and the transfer vectors no longer express gp37 (protein not essential for viral replication).
In order that the viral DNA is repaired in the 2 loci of BacMid2, a second recombination must take place with a transfer vector PH loaded or not with a transgene. In all cases, the DNA of the baculovirus genome will be repaired and thus infectious.
It will also be possible to use pVT/PH containing a wild sequence, that is to say containing the wild expression cassette (non-modified) leading to the production of polyhedrin. The pVT/PH could also be “empty” that is to say not contain transgene or polyhedrin gene.
In the same way it will be possible to integrate the transgene in the locus PH. In this case, a pVT/gp37 not deleted (functional non-essential gene) or deleted totally or partially such as described in
Direction of transcription of the gene
Caption:
Sequence of the gene in bold type
ATG initiator underlined
Polylinker XbaI/AvrII/BamHI in boxed section
The nucleic sequence illustrated above is the sequence SEQ ID NO: 16
Expression of the Heavy Chain of an Antibody.
Construction of a specific pVT/gp37, pVT/gp37-Cγ1
This transfer vector contains the following expression cassette:
Expression of the Light Chain of an Antibody.
Construction of a specific pVT/PH, pVT/PH-CL.
This transfer vector contains the following expression cassette:
A transfer vector, pVT/Chit-Cath, was constructed to be able to generate genomes of recombinant baculoviruses expressing 3 transgenes.
The fragment BstXI-XbaI derived from the regions EcoRI E and H of the baculovirus AcMNPV was cloned in a plasmid pUC. A deletion EcoNI-EcoRI of 1175 pb makes it possible to inactivate the genes encoding chitinase, non-essential, and cathepsin, also non-essential. The addition of a site XbaI between the sites EcoNI and EcoRI makes it possible to integrate a transgene. These modifications thus led to the transfer vector pVT/Chit-Cath being obtained.
The Sf9 cells are transfected by lipofection with the transfer vectors pVT/PH, pVT/gp37 and pVT/chitCath loaded with the transgenes and the DNA of BacMid3. The viruses generated during homologous recombination were cloned by the phage plaque assay method. The production of the recombinant protein was controlled by a suitable method, ELISA, Western blot, enzymatic assay, etc., the genome of the recombinant viruses was controlled by Southern blot and the sequence of the transgene was controlled after PCR amplification.
The DNAc encoding the regions VH and VL of the antibodies were integrated respectively in the transfer vectors pVT/PH-C; and pVT/gp37-Cγ1. Recombinant baculoviruses were generated after homologous recombination between 2 pVT and the DNA of BacMid2 of example 4:
The DNAc encoding the region VL of the antibodies was introduced into pVTPH/Ck which recombines with the region PH/1629 of BacMid2,
The DNAc encoding the region VH of the antibodies was cloned in pVT/gp37-Cγ1 which recombines with the region gp37 of BacMid2.
The Sf9 cells were transfected by lipofection with BacMid2 and the 2 transfer vectors obtained in example 4 then incubated for 4 days at 28° C. The culture supernatants were collected and the recombinant baculoviruses generated and secreted in the culture medium were cloned by the phage plaque assay technique.
The organisation of the genome of the recombinant baculoviruses was controlled by Southern blot (see
Production of flu VLP
To produce these VLPs, the 3 genes of the flu virus, M, HA and NA, were co-expressed.
These 3 genes were integrated in the three transfer vectors necessary to recombine with the BacMid3 of example 5: The gene M was introduced into the transfer vector pVT/PH as described in
The gene HA was introduced into the transfer vector pVT/gp37 as described in
The gene NA was introduced into the transfer vector pVT/Chit/Cath as described in
The Sf9 cells were transfected by lipofection with BacMid3 and the 3 transfer vectors obtained above, then incubated for 4 days at 28° C. The recombinant baculoviruses generated then secreted in the culture supernatant were cloned by the phage plaque assay method.
The organisation of the genomes of recombinant baculoviruses was controlled by Southern blot (see
The bispecific antibody constructed according to the international application WO 2013/005194 is constituted of a heavy chain composed of the domains VH+CH1+CH2+CH3 of an antibody 1, N-terminal fused to the domains VH+CH1 of an antibody 2. Mutations introduced at the interface of the regions CL and CH1 of the antibodies 1 favour the correct pairings between the domains VL1 and VL2 of the light chains L1 and L2 which are produced separately and the corresponding domains VH1 and VH2. The production of this antibody necessitates the simultaneous production and in equal quantity of 3 chains, the fused heavy chain, the light chain L1 and the light chain L2.
The DNAc encoding the light chain L1 was introduced into the transfer vector pVT/PH as described in
The DNAc encoding the light chain L2 was introduced into the transfer vector pVT/gp37 as described in
The DNAc encoding the fused heavy chain was introduced into the transfer vector pVT/Chit-Cath as described in
The general principle that was used to introduce into the bacmids the genes enabling to optimise post translational modifications of the proteins (BacMid2/MPT (MPT: Post-Translational Modification) is described in
Caption of table 2: the genes involved in the elaboration of the post-translational modifications (example: glycosylation, endoproteolytic cleavage) were inserted into non-essential genes/regions of the bacmids. Except in the case of the over-expression of cellular furin which is produced under the control of a strong late promoter P1051, the promoters used to control the expression of these genes are early so as to produce these enzymes before the biosynthesis of the proteins of interest that will be expressed under the control of late promoters.
BacMid2-Fur was constructed from the BacMid2 obtained in example 2. The gene encoding furin of the lepidoptera cell Sf9 (fur) was cloned downstream of a synthetic late promoter P1051 of sequence:
The gene fur was integrated in the chitinase-cathepsin locus. The transfer vector, pVT/Chit-Cath of which the construction is described in example 5 was used. The expression cassette comprising the gene fur under control of the synthetic promoter P1051 was introduced at the unique site XbaI of the pVT/Chit-Cath (position 106160 in the genome of the baculovirus), to give the plasmid pVT/Chit-Cath-Fur. The gene fur was cloned in the same direction as the inactivated cathepsin gene.
A bacterial expression cassette “zeocin resistance (ZeoR)” composed as follows: [Bacterial promoter T5N25-ZeoR-terminator rmBT1] containing a site Bsu36I on either side was cloned at the site EcoRI of pVT/Chit-Cath-Fur, to give the plasmid pVT/Chit-Cath-Fur-ZeoR. This second cassette enables the expression of the gene ZeoR and thus confers on the bacterium bearing this plasmid zeocin resistance.
The recombination fragment of 5927 pb was prepared after digestion of the plasmid pVT/Chit-Cath-Fur-ZéoR by BglII thus generating flanking regions for the homologous recombination of 652 pb and 704 pb on either side of the fragment. After electroporation in the bacterium EL350/BacMid2, the bacteria were selected on zeocin. As described in
BacMid2/Fur was thus obtained. The genomes of these new BacMids were controlled by Southern (
BacMid2-Gal was constructed from the BacMid2 obtained in example 2. The DNAc encoding 2 glycosyltransferases missing in lepidoptera cells and necessary for the biosynthesis of galactosylated glycans, human N-acetylglucosaminyltransferase II (GNT-II) (EC 2.4.1.143, Accession no NM_002408.3) and bovine β 1,4 galactosyltransferase (β1,4GalT) (EC 2.4.1.38, Accession no NM_177512.2) were introduced into non-essential genes or regions of BacMid2 by homologous recombination. In order that the enzymatic activities of β1,4GalT and GNT-II are expressed before the synthesis of the transgene(s) of interest encoding a polypeptide of interest, the transgenes encoding GNT-II and β1,4GalT were cloned downstream of early viral promoters such as described in table 2.
The addition of transgenes encoding respectively GNT-II and β1,4GalT was carried out in an iterative manner in BacMid2.
Insertion of the transgene GAIT-II
This transgene was introduced in position 29226 of the viral genome by homologous recombination between orf35 (v-ubi) and orf36 (39k) designated intergenic region IG35/36. In order to be able to insert the expression cassette in the genome of BacMid, the unique sites for cloning XbaI (italics) and Bsu36I (underlined) were integrated by PCR in the region IG35/36 with the following primers:
The PCR fragment obtained of 861 bp was cloned in a plasmid pGEM®Teasy and controlled by sequencing, to give the plasmid pGEM-IG35/36.
Two expression cassettes were introduced into the above plasmid pGEM-IG35/36.
A viral expression cassette, composed as follows [Promoter P9 of the densovirus—JcNDV—transgene encoding GNTII—stop TkpA] was inserted at the level of the site XbaI, to give the plasmid pGEM-IG35/36-GNTII. The Promoter P9 of the densovirus JcNDV is described in Shirk P D, Bossin H, Furlong R B, Gillett J L. Regulation of Junonia coenia densovirus P9 promoter expression. Insect Mol Biol. 2007 October; 16(5):623-33. Epub 2007 Aug. 22.
The bacterial expression cassette “zeocin resistance” (ZeoR) (obtained from the commercially available plasmid pCR®Blunt, InVitrogen) composed as follows: [Bacterial promoter T5N25-ZeoR-terminator rrnBT1] was cloned at the level of the site Bsu36I. This second cassette enables the expression of the ZeoR gene and thus will confer on the bacterium bearing it zeocin resistance, to give the plasmid pGEM-IG35/36-GNTII-ZeoR. The bacterial promoter T5N25 is described in Gentz R, Bujard H. Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. J Bacteriol. 1985 October; 164(1):70-7. The transcription terminator rrnBT1 is described in Kwon Y S, Kang C. Bipartite modular structure of intrinsic, RNA hairpin-independent termination signal for phage RNA polymerases. J Biol Chem. 1999 Oct. 8; 274(41):29149-55.
The recombination fragment of 3493 bp<IG35/36-GNTII-ZeoR> obtained after digestion by EcoRI of the plasmid generated above pGEM-IG35/36-GNTII-ZeoR and having flanking regions for the homologous recombination of 420 bp and 428 pb on either side of the recombination fragment, was electrophoresed in the bacteria EL350/BacMid2. The bacteria containing BacMid2/GNTII-ZeoR (E. coli EL350/BacMid2/GNTII-ZeoR) were selected for their kanamycin, hygromycin and zeocin resistance. The DNA of 3 clones of Bacmid2/GNTII-ZeoR selected was extracted then the GNT-II and ZeoR genes inserted into the region IG35/36 were controlled by PCR then sequencing.
The bacterial expression cassette flanked on either side of a site Bsu36I was next eliminated by simple digestion by Bsu36I, repair of the ends of the DNA with the DNA polymerase of Klenow then ligation of the plasmid on itself. It should be noted that the “repaired” sequence Bsu36I [5′ CCTNATNAGG 3′] was conserved in Bacmid2/GNTII thus generated after ligation of the plasmid. This sequence is thus present in the recombinant baculovirus and it may constitute a specific signature.
The transgene encoding GNTII was cloned in the same direction as the gene 39K.
BacMid2/GNTII was thus obtained then controlled as described above before being used for the insertion of the gene encoding β1,4GalT.
Insertion of the gene β1,4GalT
The transgene encoding β1,4GalT was integrated in the locus of the non-essential gene egt (Ecdysteroid glycosyltransferase, ORF15, position in the genome position 11426-12946 of the viral genome AcMNPV) of BacMid2/GNTII according to the general principle described above. The fragment PstI-BamHI of 5110 bp (position 9999 to 15110 in the viral genome of AcMNPV) containing the gene egt, was cloned beforehand in a plasmid pUC to give the plasmid pUC-EGT. Then, the viral expression cassette comprising the DNAc encoding bovine ß1,4GalT under control of the promoter gp67 of OpMNPV was introduced into the gene egt by insertion (inactivation of the gene by insertion) at the unique site XbaI (position 12782 in the genome of the baculovirus) present in the sequence encoding the gene egt, to give the plasmid pUC-EGT-GalT. The transgene encoding ß1,4GT was cloned in the same direction as the gene egt.
An adaptor NsiI-Bsu36I-NsiI was next inserted in the site NsiI situated downstream of the gene ß1,4GalT, which enabled the introduction of the bacterial expression cassette ZéoR in Bsu36I generating the plasmid pUC-EGT-GalT-ZéoR.
The recombination fragment of 3128 bp was prepared after digestion of the above plasmid pUC-EGT-GalT-ZéoR by SnaBI-NruI thus generating flanking regions for the homologous recombination of 474 bp and 866 pb on either side of the fragment. After electroporation in the bacterium EL350/BacMid2-GNTII, the bacteria were selected on zeocin. As previously, the bacterial expression cassette ZéoR was eliminated by digestion Bsu36I, repair then ligation.
BacMid2/GNTII/ß1,4GalT (also called BacMid2-Gal or BacGal) was thus obtained. The genome of BacMid2-Gal was controlled by Southern then sequencing of all the integrated genes.
BacMid2-Gal-Fur was constructed as described for BacMid2-Fur (Example 9).
The bacteria EL350/BacMid2-Gal were electrophoresed with the recombination fragment of 5927 pb described in example 9, then selected on zeocin. As previously, the bacterial expression cassette ZéoR was eliminated by digestion Bsu36I, repair then ligation. BacMid2Gal-Fur was thus obtained. The genome of the bacmid was controlled by Southern (
The transgenes encoding human CMPNeuAc synthase (CMPNeuAc synthase or CMPNeuAcS) (EC 2.7.7.43, accession no NM_018686.5), human NeuAc synthase (NeuAc Synthase or NeuAcS) (EC 2.5.1.56, accession no AF257466) and human α2,3 sialyltransferase (ST3), ST3GalIV (EC 2.4.99.4, accession no X74570) were inserted into BacMid2/GNTII-ß1,4GT in an iterative manner according to the general principle described in
Cloning of the Two Transgenes Encoding Respectively NeuAc Synthase and CMP NeuAc Synthase in Locus Iap2 of BacMid2-Gal.
The Applicant chose to clone these two enzymes head to tail under the control of very early promoters, the promoter IE1 (immediate-early 1) of the baculovirus of Choristoreura fumiferana for the control of the expression of the gene CMP NeuAc synthase and that of the baculovirus of Lymantria dispar for the control of the expression of the gene NeuAc synthase (see Table 2)
The region comprising the gene iap2 (ORF71) of the baculovirus AcMNPV (position in the genome 61016-61765) was amplified beforehand by double PCR with the following primers:
These successive PCRs also made it possible (i) to integrate the unique sites Bsu36I (underlined above) and XbaI (double underlined above) and (ii) to delete a large part of the sequence encoding iap2, deletion of 335 bp/112 amino acids. The amplified fragment of 896 bp was cloned in a plasmid pGEM® Teasy, to give the plasmid pGEM-IAP2.
A viral expression cassette, composed as follows [Stop SV40-CMPNeuAc Synthase-Promoter IE1Cf-Promoter IE1Ld-NeuAc Synthase] was inserted at the site XbaI of pGEM-IAP2, to give the plasmid pGEM-iap2-CMPNeuAcS-NeuAcS.
The bacterial expression cassette “zeocin resistance” (ZeoR) composed as follows: [Bacterial promoter T5N25-ZeoR-terminator rrnBT1] was cloned at site Bsu36I of pGEM-iap2-CMPNeuAcS-NeuAcS, to give the plasmid pGEM-iap2-CMPNeuAcS-NeuAcS-ZeoR. This second cassette enables the expression of the ZeoR gene and thus confers on the bacterium bearing this plasmid zeocin resistance.
The recombination fragment [CMPNeuAc-NeuAcS-ZeoR] of 4548 pb was prepared after digestion of the plasmid pGEM-iap2-CMPNeuAcS-NeuAcS-ZeoR by the restriction endonuclease NotI which generates flanking regions for the homologous recombination of 486 bp and 396pb on either side of the fragment. The bacteria EL350/BacMid2-GNTII-ß1,4GT were electrophoresed with the recombination fragment thus generating BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS-ZéoR. As previously, the zeocin resistance cassette was eliminated after digestion by Bsu36I, repair then religation. BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS was thus obtained.
Cloning of the Transgene α2,3-Sialyltransferase IV (ST3GalIV) in the Intergenic Region Comprised Between orf51 and orf52 (IG51/52) of BacMid2-GNTII-B1,4GT-CMPNeuAcS-NeuAcS.
This region located in the fragment EcoRI N of the baculovirus AcMNPV was isolated after amplification by double PCR with the following primers:
During these successive PCRs there is addition of 2 unique sites Bsu36I (underlined above) and XbaI (double underlined above) in the intergenic region ORF51/ORF52 which will enable integration of the expression cassette ST3 at site XbaI in position 44298 in the viral genome. The fragment obtained of 922 bp was cloned in a pGEM® T easy (Promega), to give the plasmid pGEM-IG51/52.
As for the other enzymes, ST3GalIV must be present in the cells before the glycoproteins of interest are expressed. We chose the promoter IE1 of the shrimp virus WSSV (White Spot Syndrome Virus) identified as being a functional cell promoter Sf9 of “immediate-early” type (See Table 2)(Liu et al., Virology, 2005; Liu et al. J of virology, 2007; Gao et al., J. Biotechnology, 2007).
A viral expression cassette, composed as follows [promoter WSSV-5T3] was inserted at site XbaI of pGEM-IG51/52, to give the plasmid pGEM-IG51/52-5T3. ST3GalIV was cloned in the opposite direction of orf51.
The bacterial expression cassette “zeocin resistance (ZeoR)” composed as follows: [Bacterial promoter T5N25-ZeoR-terminator rrnBT1] was cloned at site Bsu36I of pGEM-ORF51-ST3, to give the plasmid pGEM-IG51/52-ST3-ZeoR. This second cassette enables the expression of the ZeoR gene and thus confers on the bacterium bearing this plasmid zeocin resistance.
A recombination fragment [ST3GalIV-ZeoR] of 2589 pb was generated from pGEM-IG51/52-ST3-ZeoR by digestion by the restriction endonuclease NotI. This digestion generated flanking regions for the homologous recombination of 498 bp and 411pb on either side of the expression cassette. Homologous recombination was carried out in the bacterium EL350/BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS, generating BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS-ST3 (or BacSia3). The genome of BacSia3 was controlled by Southern then sequencing of all the integrated genes.
Cloning of the Transgene α2,6-Sialyltransferase I (ST6GalI) in the Intergenic Region orf51/orf52 (IG51/52) of BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS.
The method for cloning the gene encoding human α2,6 sialyltransferase, ST6GalI (EC 2.4.99.1, accession no X17247) is similar to that described in example 12 for the transgene encoding ST3GalIV, summarised as follows:
a. Cloning of the Transgene α2,6-Sialyltransferase I (ST6GalI) in ORF119 (PIF1) of BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS.
ORF119 (position in the genome 100699-102291), encoding PIF1, protein not essential for viral replication, is located in the fragment EcoRI E of the baculovirus AcMNPV. A fragment on either side of the gene was obtained after amplification by double PCR with the following primers:
These successive PCRs made it possible to eliminate the totality of ORF119, to integrate the unique sites Bsu36I (underlined above) and XbaI (double underlined above). The site XbaI will enable the integration of the expression cassette ST6 in position 100697 in the viral genome. The fragment obtained of 998 bp was cloned in a pGEM® Teasy (Promega), to give the plasmid pGEM-PIF1.
The viral expression cassette, described in example 12 [promoter WSSV-5T6] was inserted at site XbaI of pGEM-PIF1, to give the plasmid pGEM-PIF1-ST6. ST6GalI was cloned in the direction of pif1.
The bacterial expression cassette “zeocin resistance (ZeoR)” composed as follows: [Bacterial promoter T5N25-ZeoR-terminator rrnBT1] was cloned at site Bsu36I of pGEM-PIF1-ST6, to give the plasmid pGEM-PIF1-ST6-ZeoR. This second cassette enables the expression of the ZeoR gene and thus confers on the bacterium bearing this plasmid zeocin resistance.
A recombination fragment [ST6GalI-ZeoR] of 2903 pb was generated from pGEM-PIF1-ST6-ZeoR by digestion by the restriction endonuclease NotI. This digestion generated flanking regions for the homologous recombination of 498 bp and 411pb on either side of the expression cassette. The homologous recombination was carried out in the bacterium EL350/BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS, generating BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS-ST6-II (or BacSia6-II). The genome of BacSia6-II was controlled by Southern (
Head to Tail Cloning of the Transgenes ST6GalI and ST3GalIV in the Intergenic Region orf51/orf52 (IG51/52) in BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS.
The cloning method is similar to that described in Examples 12 and 13 for the transgene encoding ST3GalIV and the transgene encoding ST6GalI, summarised as follows:
A recombination fragment [ST3/ST6-ZeoR] of 5008 pb was generated from pGEM-IG51/52-ST3/ST6-ZeoR by digestion by the restriction endonuclease NotI. This digestion generated flanking regions for the homologous recombination of 490 bp and 426pb on either side of the expression cassette. The homologous recombination was carried out in the bacterium EL350/BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS, to give BacMid2-GNTII-ß1,4GT-CMPNeuAcS-NeuAcS-ST3/ST6 (or BacSia3/6). The genome of BacSia3/6 was controlled by Southern then sequencing of all the integrated genes.
HIV1 gp160 must undergo a step of maturation in order that the virus is infectious and that the surface glycoproteins of the virus are organised into trimers. These structures are today considered as essential for the formation of epitopes of interest necessary for the elaboration of a vaccine against HIV-1. This maturation is carried out by cellular furin which is going to cleave gp160 into gp120+gp41. The production of gp160 in a recombinant form in general leads to a partially matured form and does so whatever the expression system.
In order to obtain a completely matured gp160 we integrated the gene encoding furin of the Sf9 cell in the viral genome under the control of a very active promoter, a promoter P10-like called P1051 and constructed BacMid2-Fur (Example 9).
From this bacmid, we constructed a double-recombinant virus expressing 2 proteins of HIV1, polyprotein Pr55Gag and gp160. The production of these 2 proteins leads to the secretion in the culture medium of virus-like-particles (VLP). In this experiment we concentrated (noted C and NC for non-concentrated) the VLP secreted with a solution of “Retro Concentin™ Virus Precipitation” (SBI, reference RV100A-1). The different samples obtained after infection with a wild virus BACWT or multi-recombinant viruses expressing gp160 and Pr55Gag (BAC/gp160/Gag), gp160 and Pr55Gag and furin (BAC/gp160/Gag) or mono-recombinant viruses like the virus BAC gp120 which expresses uniquely soluble gp120 and the virus BACGag which expresses uniquely the polyprotein Pr55Gag (BACGag) were analysed by Western blot with an antibody anti-gp120, Panel A (goat polyclonal antibodies directed against gp120 of HIV1, reference Ab21179, Abcam) or an antibody anti-Gag, Panel B (Anti-p55+p24+p17, Reference Ab63917, Abcam).
As is shown in
A terminal N-galactosylation being a characteristic of glycosylation of Asn297 situated in the constant domain of the IgG (
BacMid2-Gal was used like BacMid2 described previously (Examples 4 and 6) for the generation in a single step of a double recombinant baculovirus (
Insertion of Transgenes Encoding the Heavy and Light Chains of an Antibody.
1. Principle of the Generation of Recombinant Baculoviruses.
DNAc encoding the variable regions VH and VL of the antibodies of interest were inserted into specific baculovirus transfer vectors (pVT) of the heavy and light chains of the antibodies, pVT/gp37-H (for cloning the variable region of the heavy chain) (
2. Construction of a Recombinant Virus Expressing a Galactosylated Antibody.
Recombinant viruses expressing the same antibodies were produced from BacMid2 and BacMid2-Gal. The antibodies produced after infection of Sf9 cells with the recombinant virus derived from BacMid2 serves as control since they have an “insect” glycosylation, that is to say glycan motifs of paucimannosidic type and to a lesser proportion of oligomannosidic type (
Cloning in the Transfer Vectors.
The fragments of DNAc encoding the variable regions of the heavy and light chains of the antibodies of interest were cloned in the respective transfer vectors (pVT/gp37-H and pVT/PH-L). In the course of this cloning, the complete genes encoding the 2 chains of antibodies are reconstituted (
Generation and Cloning of the Recombinant Baculoviruses.
The Sf9 cells were transfected by lipofection with the loaded pVT/gp37-H and pVT/PH-L and DNA of Bacmid2-Gal or BacMid2 (
Control of the Genomic Organisation of the Recombinant Viruses.
Several baculoviral clones were selected, amplified and their genome extracted to be analysed by Southern blot. The genes encoding the heavy and light chains inserted into the baculoviral genome were also amplified by PCR then sequenced.
Production and Purification of the Recombinant Antibodies.
Sf9 cells suited to the growth in serum free medium were infected at a level of 3 PFU/cell. After 3 days of infection, the culture supernatant was collected and deposited on a Protein A Sepharose column (GE-Healthcare). The quality of the antibodies was verified after migration in polyacrylamide gel and silver staining.
Analysis of Glycosylation by Lectin Blot.
Principle of lectin blot: lectins are molecules that attach themselves specifically on glycan motifs. It is thus very simple to demonstrate the presence of a particular glycan bound to a protein after electrophoresis in polyacrylamide gel, transfer onto a nitrocellulose membrane and incubation of the membrane with a biotin conjugated lectin (example biotinylated lectin RCA120, reference B1085, Vector Laboratories) or with digoxigenin (example the lectins of the
“DIG Glycan Differentiation Kit”, reference 11210238001, Roche). The presence of lectins is then detected indirectly thanks to an antibody directed against biotin or the digoxigenin itself peroxidase conjugated or with alkaline phosphatase. The presence of these enzymes is then detected thanks to their enzymatic activity which will generate either a brown red precipitate for peroxidase or a blue coloration for alkaline phosphatase.
Experimentation
The production of antibodies was controlled by Western blot. The proteins were separated by electrophoresis on a polyacrylamide gel at 10% in the presence of SDS and 2-mercaptoethanol then transferred onto a nitrocellulose membrane (Protran™ 0.45 μm NC, GE Healthcare). The transfer of the proteins was verified after ponceau red staining. The membrane was incubated with (
Analysis by lectin blot (
3. Results
Human (
These experiments clearly demonstrate that the BacGal virus is capable of complementing Sf9 cells to be able to produce galactosylated glycoproteins.
1. Construction of a Recombinant Baculovirus Expressing its Alpha 2,3 Sialylated Glycoprotein Envelope Gp64.
The activity of BacSia3 was controlled using as model protein the surface glycoprotein of the virus, gp64. Glycoprotein gp64 is the major glycoprotein of the baculovirus, it is involved in all the first steps of infection. It is located on the surface of the virus. It has been shown that this glycoprotein is capable of being galactosylated and sialylated (Jarvis et al. 1995). To do so, a recombinant virus was obtained by homologous recombination between BacMidSia3 and the empty transfer vectors pVTPH and pVT/gp37. The presence of α2,3 sialyl motifs was demonstrated thanks to a lectin blot carried out with lectin di-CBM40 described in the article (Ribeiro et al., 2016).
Generation and Cloning of the Recombinant Baculoviruses.
The Sf9 cells were transfected by lipofection with empty pVTPH and pVT/gp37 and DNA of Bacmid2 (control) or BacMid-Sia3 obtained in example 12 and according to the principle of
Analysis of Glycosylation by Lectin Blot.
The lectin used in this example was biotinylated di-CBM40. The protocol that was used is similar to that which is described in example 17. After saturation, the membrane was incubated with diCBM40-Biotinylated lectin diluted 1/200 (5.7 μg/ml) in TBS-T or with SNA-Dig lectin (Roche, Kit DIG Glycan Differentiation Kit) diluted 1/1000 in TBS-T. The revelation of the membranes was carried out as described in example 17. The presence of gp64 was controlled by Western blot in the presence of an anti-gp64 antibody (monoclonal mice antibodies AcV5 reference SC65499, Santa Cruz Biotechnology).
2. Results
As shown in
These experiments clearly demonstrate that the virus BACSia3 is capable of complementing Sf9 cells to be able to produce α2,3 sialylated glycoproteins.
1. Construction of a Recombinant Baculovirus Expressing a Recombinant Alpha 2,6 Sialylated Protein.
The activity of BacSia6 was controlled using as model protein Glycoprotein G Vesicular Stomatitis virus, VSVg, the protein X and glycoprotein gp64 of the baculovirus.
Generation and Cloning of the Recombinant Baculo Viruses.
The DNAc fragment encoding the protein of interest was cloned in pVTPH according to the general principle described in
Production of Recombinant Proteins
The proteins were produced as described in example 18.
Analysis of Glycosylation by Lectin Blot.
The presence of recombinant proteins was controlled by Western blot. After transfer of the proteins, the nitrocellulose membranes were incubated in the presence of different specific antibodies, anti-VSVg (mice monoclonal antibodies peroxidase conjugated, reference A5977, Sigma), anti-gp64 (mice monoclonal antibodies AcV5 reference SC65499, Santa Cruz Biotechnology). Revelation was carried out either directly as described in example 16 when the antibody is directly peroxidase conjugated or after incubation with a secondary antibody peroxidase conjugated (rabbit anti-mouse IgG serum peroxidase conjugated, reference A9044). The peroxidase was revealed by chemiluminescence with the ECL SuperSignal® West Pico Chemiluminescent Substrate system (reference 34077, Thermo Scientific).
The lectin that was used in this example was SNA (Sambuscus nigra agglutinin) which recognises α2,6 bound sialic acids. SNA was revealed as described in example 17.
2. Results
a. Glycosylation of Protein X Expressed by the Recombinant Baculovirus Generated from BacSia6
As shown by Western lot,
Analysis by lectin Blot,
b. Glycosylation of VSVg Expressed by the Recombinant Baculovirus Generated from BacSia6.
VSVg being a membranal protein, we analysed the pellets of the infected cells. As shown in
Conversely, analysis by lectin blot with SNA (revelation protocol described in example 17) showed an intense marking of protein VSVg uniquely when it is expressed from the baculovirus derived from BacSia6 (
c. Glycosylation of Gp64 of the Recombinant Baculovirus Generated from BacSia6.
We also verified that the recombinant virus expressing sialylated VSVg (see above) was also bearing a sialylated gp64. To do so, the baculoviruses secreted in the culture supernatant were sedimented (35,000 rpm for 60 minutes, Beckman Optima LE-80K centrifuge, TI-70-1 rotor) then taken up by a lysis buffer to be analysed by Western blot then lectin blot. As previously, fetuin was used as positive marker for SNA (
These experiments clearly demonstrate that the BacSia6 virus is capable of complementing Sf9 cells to be able produce α2,6 sialylated glycoproteins.
We analysed the genome of BacMidSia6II by Southern blot to control its genomic organisation.
As shown in
1. Construction of a Recombinant Baculovirus Expressing a Recombinant Alpha 2,3 and Alpha 2,6 Sialylated Protein.
The activity of BacSia3-6 was controlled using as model protein the surface glycoprotein of the virus, gp64. To do so, a recombinant virus was obtained by homologous recombination between BacMidSia3/6 and the empty transfer vectors pVTPH and pVT/gp37. The presence of α2,3 sialyl- and α2,6 sialyl-motifs was analysed by lectin blot carried out in the presence of lectin di-CBM40 which recognises α2,3 sialic acids and to a lesser extent α2,6 bound sialic acids and SNA which only recognises α2,6 bound sialic acids and not those which are α2,3 bound.
Generation and Cloning of Recombinant Baculoviruses.
Sf9 cells were transfected by lipofection with empty pVTPH and pVT/gp37 and DNA of Bacmid2 (control) or BacMid-Sia3/6 obtained in example 15. After 7 days of infection at 28° C., the viruses secreted in the culture supernatant were cloned by the phage plaque assay technique. Four viral clones were selected, amplified and their genome extracted to be analysed by Southern blot. The gene inserted in the viral genome was amplified by PCR then sequenced.
Production of Recombinant Proteins
The proteins were produced as described in example 17.
Analysis of Glycosylation by Lectin Blot
The analysis protocols were identical to those described in Examples 20, 21 and 22.
2. Results
The gp64 produced by the GalSia3-6 virus is the only one which is recognised both by SNA (
These experiments clearly demonstrate that the BacSia3-6 virus is capable of complementing Sf9 cells to be able to produce α2,3 and α2,6 sialylated glycoproteins.
WO 01/12829
WO 2013/005194
| Number | Date | Country | Kind |
|---|---|---|---|
| 1760068 | Oct 2017 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/FR2018/052652 | 10/25/2018 | WO | 00 |
