CHIMERIC ANTI-RICIN ANTIBODY
A chimeric monoclonal antibody targeted to ricin is presented. The light chain and heavy chain constant regions are respectively made up of the light chain and heavy chain constant regions of human immunoglobulin, and the light chain and heavy chain variable regions respectively include the light chain and heavy chain variable regions of macaque immunoglobulin. The antibody does not substantially induce any immune response against chimeric antibodies.
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This invention concerns a chimeric antibody directed against Ricin.
BACKGROUND OF THE INVENTIONRicin is a toxalbumin produced by a shrub belonging to the Euphorbiaceae family, the Castor Oil Plant (Ricinus communis). Ricin is a highly toxic glycoprotein with a molecular weight of 66 kDa formed by two polypeptide chains A and B connected together by a disulphide bridge. Chain B allows the toxin to attach itself to the cell wall while Chain B, which is responsible for its toxic properties, is capable of inhibiting protein synthesis by inhibiting 28S ribosomal RNA, causing cell death. It is present in the castor oil seed in concentrations of between 1% and 10%. It may be extracted from incompletely purified castor oil.
As a toxin, ricin is extremely toxic. However, its toxicity varies according to the means by which it penetrates the organism.
When ricin is absorbed through digestion, it is largely destroyed by proteolytic digestive enzymes but its perlingual absorption may increase the quantity absorbed.
In contrast, when ricin is inhaled (pulmonary route) or administered via the parenteral route its toxicity is multiplied 1000-fold.
Symptoms are fairly non-specific and vary according to the route by which the ricin is absorbed. They generally become evident within a period of 2 to 24 hours and rarely take longer than 2 days to appear. Absorption through ingestion causes vomiting, feelings of faintness, abdominal pain, bloody diarrhoea (stools resembling rice water), a painful need to defecate or urinate (anuria), dehydration, drowsiness, muscle weakness, cramps, vasomotor paralysis and tachycardia. Absorption via inhalation causes weakness, fever, dizziness, dyspnoea, coughing, pulmonary oedemas and pain in the limbs.
After an apparent improvement, infection may have a fatal outcome.
In humans, the dose of ricin estimated to be lethal is between 1 and 10 μg/kg.
In view of these varied symptoms and the danger caused by ricin at a very low dose, there is a real need for protection against ricin contamination, including in response to its potential use in the context of bioterrorist attacks.
A rapid diagnostic test for ricin poisoning via the pulmonary route has recently been developed (Guglielmo-Viret et al. 2007). After ricin exposure, the following antidotes may be used: sugar analogues to prevent the ricin from connecting to its target, or catalytic subunit inhibitors such as Azidothymidine.
Vaccination might constitute another ricin poisoning treatment strategy. For example, antibodies have been developed that are designed to interfer with the connection that the anthrax toxin makes with cell surface receptors or to inhibit the assembly of the toxin. However, no ricin-specific therapy is currently available.
Wang et al. (Wang et al., 2007, Biotechnol Lett 29: 1811-1816) recently developed a human-mouse chimeric antibody against ricin. However, although this first-generation chimeric antibody is ricin-specific, it is capable of generating a Human Anti-Chimeric Antibody (HACA) immune response and of inducing low patient tolerance.
The international application WO 2009/053637 describes single-chain Fv (scFv) fragments from constant regions of macaque antibodies that are capable of effectively neutralising ricin, as well as humanized or super-humanized scFv fragments.
However, such fragments are small in size, have a very short half-life, are rapidly eliminated by the kidneys and are incapable of providing long-term protection. Furthermore, given the lack of a constant region, these fragments are relatively ineffective at stimulating the immune system (recruitment of immune effectors).
Thus, there is a real need to provide ricin-specific antibodies that are stable after administration and that possess a very high toxin neutralization rate.
It is also important to provide antibodies that do not contribute to a HACA-type immune response in the host when they are administered.
SUMMARY OF THE INVENTIONThus, the object of the invention is to provide ricin-neutralising antibodies.
The invention is also intended to provide the means of producing these ricin-specific antibodies.
Another object of the invention concerns the use of these antibodies as medication or as decontamination agents in response to ricin contamination.
The invention concerns a chimeric monoclonal antibody against ricin, in which the light chain and the heavy chain are such that:
-
- the constant region of the light chain essentially comprises the constant region of the light chain of a human immunoglobulin;
- the constant region of the heavy chain essentially comprises the constant region of the heavy chain of a human immunoglobulin;
- the variable region of the light chain includes the variable region of the light chain of a macaque immunoglobulin; and
- the variable region of the heavy chain includes the variable region of the heavy chain of a macaque immunoglobulin;
the said monoclonal antibody not substantially inducing a Human Anti-Chimeric Antibody immune response.
The invention relies on the discovery made by the Inventors that chimeric macaque/human antibodies under the invention display improved ricin neutralisation, compared to the antibodies or antibody fragments used previously, and that the said antibodies are similar to human antibodies and therefore should not cause a Human Anti-Chimeric Antibody immune response.
DETAILED DESCRIPTION OF THE INVENTIONUnder the invention, the term “antibody” refers to an immunoglobulin, an oligomeric protein comprising 4 chains that contribute to acquired immune response.
Immunoglobulins are familiar to professionals and they consist of a combination of two dimers each comprising a heavy chain and a light chain. The oligomeric complex is assembled through the connection of a light chain and a heavy chain via a disulphide bridge between two cysteines, the two heavy chains in turn being connected to one another by two disulphide bridges.
Each heavy chain and each light chain comprises a constant region and a variable region. The assembly of the constituent chains of an antibody allow for the definition of a characteristically Y-shaped three-dimensional structure, where:
-
- the base of the Y corresponds to the constant region Fc, which is recognised by the Fc receptors and complement; and
- the ends of the arms of the Y correspond to the corresponding assembly of the variable regions of the light chain and of the heavy chain.
More specifically, each light chain comprises a variable region (VL) and a constant region (CL). Each heavy chain comprises a variable region (VH) and a constant region composed of three constant domains CH1, CH2 and CH3. The domains CH2 and CH3 make up the area Fc.
The structure of an antibody is depicted diagrammatically in
The variable region of the light chain comprises three antigen recognition determining domains (CDRs) surrounded by four structural domains. The three-dimensional folding of the variable region is such that the 3 CDRs are exposed on the same side of the protein and allow for the formation of a special structure to recognise a specific antigen.
The pearl-necklace structure of a variable region of a light or heavy chain of an antibody is depicted in
The antibodies described in the invention are isolated and purified and they are different from natural antibodies as they are chimeric. These antibodies are mature, meaning that they possess a three-dimensional ad hoc structure allowing them to recognise the antigen and they possess all the post-translational modifications essential to their antigen recognition.
They are monoclonal antibodies, meaning that they only recognise a single antigenic determinant in ricin, unlike polyclonal antibodies, which correspond to a mixture of monoclonal antibodies and can therefore recognise multiple antigenic determinants within a single protein.
For the purposes of the invention, “chimeric monoclonal antibody” is defined as an isolated antibody in which the sequence of each constituent light chain and/or heavy chain includes or consists of a hybrid sequence derived from at least two different animals. More specifically, the chimeric antibodies in the invention are human/macaque hybrids, meaning that a region of the sequence of the light chains and heavy chains derives from the sequence of a macaque immunoglobulin while the rest of the sequence of those heavy chains and light chains derives from the sequence of one, or potentially several human immunoglobulins.
“The constant region of the light chain essentially comprises the constant region of the light chain of a human immunoglobulin” means that the constant region of the light chain may comprise the sequence of the constant region of a human immunoglobulin light chain, but may also comprise a sequence corresponding to the fusion of several sequences from several constant regions of several human immunoglobulins. In other words, the constant region of the light chain may comprise a sequence corresponding to a mosaic of sequences from constant regions of light chains, provided that this mosaic sequence reconstitutes a sequence of a constant region of a light chain.
“The constant region of the heavy chain essentially comprises the constant region of the heavy chain of a human immunoglobulin” means that the constant region of the heavy chain may comprise the sequence of the constant region of a human immunoglobulin heavy chain, but may also comprise a sequence corresponding to the fusion of several sequences from several constant regions of several human immunoglobulins. In other words, the constant region of the heavy chain may comprise a sequence corresponding to a mosaic of sequences from constant regions of heavy chains, provided that this mosaic sequence reconstitutes a sequence of a constant region of a heavy chain.
“The variable region of the light chain includes the variable region of the light chain of a macaque immunoglobulin” means that the sequence of the variable region of the light chain corresponds to the sequence of the variable region of a macaque immunoglobulin light chain. This variable region of the light chain may be merged into its N-terminal region, in the C-terminal region of a sequence allowing for the excretion of the antibody. This sequence allowing for the excretion of the antibody is called the signal peptide or leader sequence.
“The variable region of the heavy chain includes the variable region of the heavy chain of a macaque immunoglobulin” means that the sequence of the variable region of the heavy chain corresponds to the sequence of the variable region of a macaque immunoglobulin heavy chain.
This variable region of the heavy chain may be merged into its N-terminal region, in the C-terminal region of a sequence allowing for the excretion of the antibody.
Thus, the definition of the monoclonal antibody under the invention covers both:
-
- The precursor of the chimeric human/macaque antibody as defined above; and
- The chimeric human/macaque antibody as defined above.
Within the cell that produces the monoclonal antibody covered by the invention, the said monoclonal antibody against ricin is produced in the form of a precursor. Thus, in the N-terminal region of the light chain and heavy chain, this precursor possesses a leader sequence or signal peptide. This precursor therefore undergoes various stages of maturation, and in particular its leader sequences are cleaved so as to allow the antibody to be secreted in the extra-cellular environment. The secreted antibody is therefore a mature antibody.
The monoclonal antibodies under the invention “do not substantially induce a Human Anti-Chimeric Antibody immune response”. This means that when the monoclonal antibodies under the invention are administered to an individual, including a human being, the immune system of the said individual is not substantially stimulated or undergoes little stimulation, and so the said individual does not produce any antibodies against the antibodies covered by the invention. One beneficial method of producing the invention concerns a monoclonal antibody as defined above that is capable of neutralising ricin in vitro and in vivo, specifically yielding a ricin neutralisation rate higher than the neutralisation rate of an scFv fragment against ricin.
The Inventors have demonstrated that the antibody according to the invention is capable of inhibiting ricin more effectively and at a lower dose than a single-chain fragment of an immunoglobulin (scFv), including a chimeric human/macaque scFv fragment as defined under the invention
One beneficial method of producing the invention concerns a monoclonal antibody as defined above that is capable of neutralising ricin, specifically yielding a ricin neutralisation rate of at least 40%, 50%, 60%, 70% and preferably at least 80%.
The monoclonal antibodies under the invention are “capable of neutralising ricin”. This means that the monoclonal antibodies according to the invention are capable of preventing the action of ricin, or in other words of inhibiting the toxicity of ricin on subunit 28S of ribosomes. This inhibition is due to ricin sequestration or to masking of the ricin domain or domains responsible for its toxicity.
Consequently, the monoclonal antibodies under the invention neutralise ricin toxicity.
The antibody neutralisation activity may be measured with the assistance of the routine protocol in regular use among professionals. Among these tests, we can cite the neutralisation test based on cell survival.
This test measures the capacity of the antibodies under the invention to protect J774A.1 cells put in contact with ricin against death.
In short, J774A.1 cells (ATCC-LGC, Molsheim, France) are cultured at a density of 14,000 cells/well (200 μl/well) in a culture dish and cultivated at 37° C. with 5% CO2 for 24 hours in some DMEM to which 10% foetal calf serum is added. The antibodies under the invention are incubated with 480 ng/ml of ricin or with control serum (irrelevant antibodies) for 1 hour. The mixture is then added to the cells. 24 hours later, cell viability is measured via techniques known to professionals (Trypan blue exclusion, Cytotox (Promega), apoptosis measurement etc). Each test is corrected in relation to cell controls that are “100% viable” (no ricin and no antibodies) and “0% viable” (ricin without antibodies).
Another test can be used to measure the neutralising activity of the monoclonal antibodies of the invention, which involves measuring the inhibition of protein translation. For this, one measures the translation of a marker protein (e.g. luciferase translation) under an acellular in vitro translation system [Hale M L Pharmacol Toxicol 2001, 88(5):255-260].
In short, the luciferase messenger RNA translation assay is as follows:
The monoclonal antibodies are deposited in some Phosphate Buffered Saline (PBS) in 96-well dishes and ricin is added at a final concentration of 4 mM. Rabbit reticulocyte lysate complemented with RNAsin®, amino acids and luciferase messenger RNA is then added into each well. The reaction lasts for 1 hr 30 mins.
5 μL of the reaction is then sampled and added to 45 μL of reaction buffer allowing luciferase activity to be detected (Luciferase assay reagent, Promega, Inc.). Light emission (luminescence) is measured in counts per second (CPS) using a Victor multi-plate reader (PerkinElmer Wallac, Boston Mass.). The data is expressed as a percentage in relation to the control (% control ═(CPS processed/control CPS)×100)).
Another beneficial method of producing the invention concerns a monoclonal antibody defined above, wherein:
-
- The constant region of the light chain includes or consists of the constant region of the light chain of a kappa type human immunoglobulin; and
- The constant region of the heavy chain includes or consists of the constant region of the heavy chain of a type IgG1 human immunoglobulin;
the said light chain of a kappa type human immunoglobulin and the said heavy chain of a type IgG1 human immunoglobulin being, in particular, the constant region of a light chain and the constant region of a heavy chain of a human immunoglobulin obtained through immunisation with the Rhesus D antigen.
Thus, the monoclonal antibody under the invention is not only capable of neutralising ricin via its variable regions, but also of facilitating ricin degradation by promoting its degradation by macrophages.
More specifically, the constant regions of the light chains and heavy chains of the monoclonal antibody under the invention derive from the IgG1s of an individual immunised with a Rhesus D antigen. Under another beneficial method of producing the invention, the monoclonal antibody described above and below possesses a light chain and, in particular, a constant region of the said light chain of the kappa (κ) type.
Under another beneficial method of production, the invention concerns a monoclonal antibody against ricin as described above, wherein:
-
- The light chain of the said monoclonal antibody includes at least the complementarity-determining regions (CDRLm) of the variable region of a macaque immunoglobulin light chain; and
- The heavy chain of the said monoclonal antibody includes at least the complementarity-determining regions (CDRHm) of the variable region of a macaque immunoglobulin heavy chain.
The variable region of the light chain of the monoclonal antibody under the invention therefore includes at least the 3 CDRs of a macaque immunoglobulin, while the remainder of the variable region of the light chain may come from a macaque or from any other mammalian species, including humans.
The variable region of the heavy chain of the monoclonal antibody under the invention therefore includes at least the 3 CDRs of a macaque immunoglobulin, while the remainder of the variable region of the heavy chain may come from a macaque or from any other mammalian species, including humans.
When the variable region of the light chain of the monoclonal antibody only possesses the CDRs of the light chain of a macaque immunoglobulin, and the variable region of the heavy chain only possesses CDRs from the heavy chain of a macaque immunoglobulin, and all the remainder of the two variable regions derives from a human immunoglobulin, the antibody is then described as a humanized chimeric antibody.
Another beneficial method of producing the invention concerns a monoclonal antibody as defined above, wherein:
-
- The variable region of the light chain of the said monoclonal antibody includes or comprises the following SEQ ID No 2 sequence:
and
-
- The variable region of the heavy chain of the said monoclonal antibody includes or comprises the following SEQ ID No 4 sequence:
Another beneficial method of producing the invention concerns a monoclonal antibody as defined above, wherein:
-
- The variable region of the light chain of the said monoclonal antibody comprises the following SEQ ID No 2 sequence:
and
-
- The variable region of the heavy chain of the said monoclonal antibody comprises the following SEQ ID NO 4 sequence:
Another beneficial method of producing the invention concerns a monoclonal antibody as defined above, including:
-
- A light chain including or consisting of the following SEQ ID No 6 sequence:
and
-
- A heavy chain including or consisting of the following SEQ ID No 8 sequence:
The invention also concerns a monoclonal antibody against ricin, as defined above, wherein:
-
- The light chain of the said monoclonal antibody includes the leader region of the variable region of a human immunoglobulin light chain (LVLh),
- The heavy chain of the said monoclonal antibody includes the leader region of the variable region of a human immunoglobulin heavy chain (LVHh).
Another beneficial method of producing the invention concerns a monoclonal antibody against ricin, as defined above, wherein:
-
- The N-terminal region of the variable region of the light chain includes a signal sequence, particularly the leader region of the variable region of the light chain of a second immunoglobulin, specifically a human one; and
- The N-terminal region of the variable region of the heavy chain includes a signal sequence, particularly the leader region of the variable region of the heavy chain of a third immunoglobulin, specifically a human one;
whereby the second and third immunoglobulins may be identical or different.
As stated above, the leader region or signal peptide, which is located in the N-terminal region of the variable region of the light chain and of the heavy chain, corresponds to a protein secretion sequence. During protein synthesis of the light chain and heavy chain, the said leader sequence means that the protein undergoing synthesis must remain in the light of the Rough Endoplasmic Reticulum (RER). This sequence is then eliminated from the mature light chain and from the mature heavy chain, so that the mature monoclonal antibody capable of interacting with ricin no longer possesses this sequence.
In addition, the invention concerns a precursor of an aforementioned monoclonal antibody, wherein:
-
- The leader region LVLh includes or consists of the following SEQ ID No 17 sequence:
-
- The leader region LVHh includes or consists of the following SEQ ID No 18 sequence:
Under another preferred means of production, the invention concerns a precursor of the monoclonal antibody described above, wherein:
-
- The variable region of the light chain of the said monoclonal antibody includes or consists of the following SEQ ID No 10 sequence:
and
-
- The variable region of the heavy chain of the said monoclonal antibody includes or consists of the following SEQ ID No 12 sequence:
In the aforementioned sequences (the sequences SEQ ID No 10 and SEQ ID No 12) those amino acids underlined whose “single-letter” coding symbol is in lower case correspond to the leader sequence amino acids. Those amino acids whose “single-letter” coding symbol is in upper case correspond to the variable region.
Under one beneficial method of production, the invention concerns a monoclonal antibody defined above, including:
-
- A light chain consisting of the following SEQ ID No 14 sequence:
and
-
- A heavy chain consisting of the following SEQ ID No 16 sequence:
The invention also concerns the light chain of the monoclonal antibody defined above, specifically including the sequence SEQ ID No 6, and more particularly consisting of the sequence SEQ ID No 6 or SEQ ID No 14.
The invention also concerns the heavy chain defined above, specifically including the sequence SEQ ID No 8, and more particularly consisting of the sequence SEQ ID No 8 or SEQ ID No 16.
The invention also concerns a monoclonal antibody fragment as defined above, the said fragment being a Fab or F(ab)′2 fragment.
scFv fragments are excluded from the invention.
Thus, Fab and F(ab)′2 fragments are constituted of:
-
- For Fab: a light chain including a constant region of a human immunoglobulin light chain and a variable region of a macaque immunoglobulin, and a heavy chain comprising a constant region of a human immunoglobulin heavy chain and a variable region of a macaque immunoglobulin;
- For F(ab)′2, of the combination of the two Fabs described above via a disulphide bridge.
The object of the invention is also a nucleic acid including a sequence coding the light chain of the monoclonal antibody defined above, and specifically including the sequence SEQ ID No 1 or 5
Thus, under the invention the nucleic acid sequences are such that the sequence SEQ ID No 1 codes for the protein SEQ ID No 2, SEQ ID No 3 codes for the protein SEQ ID No 4, SEQ ID No 5 codes for the protein SEQ ID No 6, SEQ ID No 7 codes for the protein SEQ ID No 8, SEQ ID No 9 codes for the protein SEQ ID No 10, SEQ ID No 11 codes for the protein SEQ ID No 12, SEQ ID No 13 codes for the protein SEQ ID No 14 and SEQ ID No 15 codes for the protein SEQ ID No 16.
The object of the invention is also a nucleic acid including or constituted of a sequence coding the light chain of the monoclonal antibody defined above, and specifically including the sequence SEQ ID No 9 or 13.
Another beneficial method of production concerns a nucleic acid including or constituted of a sequence coding the heavy chain of the monoclonal antibody as defined above, specifically including the sequence SEQ ID 7.
Another beneficial method of production concerns a nucleic acid including or constituted of a sequence coding the heavy chain of the monoclonal antibody as defined above, specifically including the sequence SEQ ID 15.
Another beneficial method of producing the invention concerns a nucleic acid as defined above, including:
-
- A nucleic acid, coding the light chain of the monoclonal antibody defined above, including or constituted of a sequence selected from among the nucleic acids SEQ ID No 1, 5, 9 and 13; and/or
- A nucleic acid, coding the heavy chain of the monoclonal antibody defined above, including or constituted of a sequence selected from among the nucleic acids SEQ ID No 3, 7, 11 and 15.
Another beneficial method of producing the invention concerns a nucleic acid including or constituted of a sequence coding the light chain of the monoclonal antibody as defined above, specifically including or constituted of any one of the sequences SEQ ID No 1, 5, 9 or 13.
Another beneficial method of producing the invention concerns a nucleic acid including a sequence coding the heavy chain of the monoclonal antibody defined above, specifically including or constituted of any one of the sequences SEQ ID No 3, 7, 11 or 15.
Another beneficial method of producing the invention concerns a nucleic acid as defined above, including:
-
- A nucleic acid including or constituted of a sequence selected from among the nucleic acids SEQ ID No 1, 5, 9 and 13; and
- A nucleic acid including or constituted of a sequence selected from among the nucleic acids SEQ ID No 3, 7, 11 and 15.
Another beneficial method of producing the invention concerns a nucleic acid as defined above, including:
-
- A nucleic acid including or constituted of the sequence SEQ ID No 5 or 13; and
- A nucleic acid including or constituted of the sequence SEQ ID No 7 or 15.
Another beneficial method of producing the invention concerns a nucleic acid as defined above, including:
-
- A nucleic acid including or constituted of the sequence SEQ ID No 13; and
- A nucleic acid including or constituted of the sequence SEQ ID No 15.
Under another beneficial method of production, the invention concerns a nucleic acid including a sequence coding the light chain of the monoclonal antibody defined above and including a sequence coding the heavy chain of the monoclonal antibody defined above.
In other words, the aforementioned sequence includes, within a single molecule, or more specifically in the same strand, a sequence coding the light chain of the monoclonal antibody defined above followed by a sequence coding the heavy chain of the monoclonal antibody defined above. This also means that the aforementioned sequence includes, within a single molecule, a sequence coding the heavy chain of the monoclonal antibody defined above followed by a sequence coding the heavy chain of the monoclonal antibody defined above.
The invention also concerns an expression vector including at least one nucleic acid defined above, the said nucleic acid being under the control of elements allowing its expression.
Under the invention, “expression vector” is defined to mean a molecule of DNA that possesses elements allowing for its replication (duplication) in at least one living organism. In particular, these elements allowing for replication originate from the replication of yeast or bacteria, or from elements controlling the replication of a virus.
In particular, according to the invention, the vectors are plasmids, phages, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), modified genomes of replicative or integrative viruses etc.
These are known as “expression” vectors because they possess nucleotide sequences that allow expression, namely the transcription into RNA of the nucleotide sequences that they control. Under the invention, the said nucleic acid sequence contained in the said vector is placed “under the control of elements allowing its expression”. This means that the said expression vector possesses at least one transcription initiation sequence such as a virus promoter like the early promoter of the Simian Virus SV40 or of the Cytomegalovirus (CMV), or sequences promoting Rous Sarcoma Virus (RSV), and in particular a sequence or promoter including a TATA box. Furthermore, the said vector also possesses at least one transcription termination sequence, and in particular a polyadenylation sequence deriving from a mammalian, or more specifically a human gene.
Other sequences allowing for the regulation or modulation of the expression of the nucleotide sequence contained in the said vector may be added to those sequences indispensable to the expression of the said sequence. A non-exhaustive list includes: introns from mammalian, and particularly human genes, enhancement-type transcription regulation sequences (“enhancers”) or sequences transcribed but not translated from mammalian, and particularly human genes.
One beneficial method of producing the invention concerns an expression vector as defined above, including at least one nucleic acid selected from among those nucleic acids including the following sequences SEQ ID No 1, 3, 5, 7, 9, 11, 13 and 15.
Another beneficial method of producing the invention concerns a combination comprising two expression vectors;
the first expression vector including a nucleic acid that includes a sequence selected from among the nucleic acids SEQ ID No 1, 5, 9 and 13; and
the second expression vector including a nucleic acid that includes a sequence selected from among the nucleic acids SEQ ID No 3, 7, 11 and 15.
Another beneficial method of producing the invention concerns a combination comprising two expression vectors, as above, wherein:
The first expression vector includes a nucleic acid that includes the sequence SEQ ID No 13; and
The second expression vector includes a nucleic acid that includes the sequence SEQ ID No 15.
Another beneficial method of producing the invention concerns an expression vector, as above, including:
-
- A first nucleic acid selected from among the nucleic acids from the following sequences: SEQ ID No 1, 5, 9 and 13, the said first nucleic acid being under the control of elements allowing its expression; and
- A second nucleic acid selected from among the nucleic acids from the following sequences: SEQ ID No 3, 7, 11 and 15, the said second nucleic acid being under the control of elements allowing its expression.
Another beneficial method of producing the invention concerns an expression vector, as above, including:
-
- A first nucleic acid including or constituted of the sequence SEQ ID No 13, the said first nucleic acid being under the control of elements allowing its expression; and
- A second nucleic acid including or constituted of the sequence SEQ ID No 15, the said second nucleic acid being under the control of elements allowing its expression.
This expression vector therefore includes two nucleic acid sequences, as above, and more specifically it includes a nucleic acid sequence coding the light chain of the monoclonal antibody defined above, and a nucleic acid sequence coding the heavy chain of the monoclonal antibody defined above.
By preference, the said expression vector contains a first element allowing the expression of the nucleic acid sequence coding the light chain of the monoclonal antibody defined above and a second element allowing the expression of the nucleic acid sequence coding the heavy chain of the monoclonal antibody defined above, the said first and the said second element allowing the expression of the said nucleic acid sequences being identical or different, and preferably identical. In particular, these control elements are the long terminal repeat (LTR) sequences of the RSV virus.
Another means of producing the invention concerns an expression vector defined above, including at least one antibiotic resistance gene.
Under the invention, “at least one [ . . . ] resistance gene” is defined to mean that the said expression vector may contain 1 or 2, or 3 or 4 or 5 or 6 antibiotic resistance genes.
Under the invention, “antibiotic resistance gene” is defined to mean a gene whose expression output exerts a cytostatic (growth inhibiting) or cytolytic (cell death) effect on cells. In particular, the antibiotics concerned by the invention have an effect on prokaryotic cells, but may also have an effect on eukaryotic cells, whether these are yeasts, plants, insects, amphibians or mammals.
More specifically, the aforementioned expression vector possesses an antibiotic resistance gene specific to prokaryotic cells and at least one or preferably 2 antibiotic resistance genes specific to eukaryotic cells.
The following can be cited as antibiotics specific to prokaryotic cells: Ampicillin, Tetracycline and its derivatives, Hygromycin, Kanamycin etc. The following can be cited as antibiotics specific to eukaryotic cells: G418, Geneticin (G418 salts), Puromycin, Methotrexate, Blasticidin etc.
More particularly, a mode of embodiment of the invention concerns an expression vector such as previously defined, comprising or consisting of the sequence SEQ ID No 21.
The transcriptional units (TU) of interest coding for the heavy chain and the light chain are cloned under the form of cDNA and under the dependence of the RSV promoter. This promoter corresponds to the LTR (Long Terminal Repeat) of the Rous sarcoma virus which contains an enhancer element in its region 5′.
An artificial intron optimised for alternate splicing is cloned immediately in region 3′ of the promoter and is composed of a donor sequence in 5′ isolated from the human beta-globin and in region 3′ of the acceptor sequence derived from the variable gene of the immunoglobulin heavy chain. The TU's of interest end by sequences of polyadenylation derived from the growth hormone gene (GH) of human origin (hGH) for the heavy chain and bovine (bGH) for the light chain. This difference of origin in the choice of polyA is carried out with the aim of limiting the combinations between the genes of interest. This promoter association LTRRSV, chimeric intron, cDNA and polyA sequence has been selected because it confers a high transcriptional and translational activity in the cell line YB2/0.
In addition to the UT's of interest the expression vector contains several UT's resistant to some chemical molecules:
Bla gene: This gene (named Amp in the vector restriction maps) expresses the enzyme beta-lactamase in the bacteria (prokaryotic promoter) and confers a resistance to ampicillin.
Neo gene: This gene codes for the nptII enzyme (neomycin-phosphotransferase II) under the control of the SV40 promoter and confers a resistance to various antibiotics such as neomycin, kanamycin or G418 to the transfected mammalian cells expressing this gene.
Dhfr gene: This gene codes for the DHFR enzyme (DiHydroFolate Reductase) under the control of the SV40 promoter and confers a resistance to methotrexate (MTX). This process can be used to carry out gene amplification by increasing the concentration of MTX resulting from the increase in production of antibodies by the transfected cells.
The invention also seeks to have a cell, or several cells, or a cell line consisting of at least one expression vector such as previously defined.
The cells “consisting of at least one vector “correspond to the cells in which at least one expression vector mentioned above has been introduced.
Experts in this area of work know perfectly the techniques of molecular biology which allow the introduction of a DNA sequence or an expression vector to the interior of a cell, and notably with reference for example to (Sambrook, J et al. in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989). Thereafter the term “transfection” will be commonly used to describe the action of the introduction of a vector in a cell.
For example, the techniques of calcium phosphate transfection, by the use of lipidic particles or “lipofectants”, or by techniques which allow the generation of holes in the cellular membrane by means of an electric shock (electroporation). This list is not exhaustive.
The cells or cellular lines utilised in the invention are cells from prokaryotes or eukaryotes such as bacteria, yeast or other mushrooms, insect cells, amphibian cells, mammalian cells and notably rodents, human cells . . . .
The cell is distinguished from the cellular line by the fact that the cellular line is a cellular population in an established culture, that is to say it has acquired the characteristics which allow their proliferation in vitro, and notably a characteristic of immortalisation.
A beneficial method of the invention concerns a cell or a cell line previously mentioned, presenting a substantially reduced fucosylation activity compared with a normal cell, the aforementioned cell or cell line being notably a mammalian cell, and in particular the YB2/0 line.
The favourable cell line of the invention is line YB2/0 available at the ATCC under number CRL 1662.
In another beneficial method, the invention concerns a cell or a cell line consisting of an expression vector defined above, permitting the expression of:
-
- a monoclonal antibody previously defined
- or of a light chain of a monoclonal antibody such as that previously described
- or of a heavy chain of a monoclonal antibody such as that previously described
In another beneficial method, the invention concerns a cell or a cell line obtained by the cloning of an aforementioned cell.
The techniques of cellular cloning are largely known to experts in this field, and are based on the principle of the isolation of cells from a cellular population in order that each individual cell generates daughter cells (or clones) isolated from the daughter cells from the division of other cells of the population.
The general principal of cellular cloning is the limit dilution of cells.
Also, another beneficial method of the invention concerns a cell or a cell line from the aforementioned cloning, the said cells or cell line characterised by the fact that they:
-
- present an apoptosis of below 25%, and
- secrete at least cloning
- 20 μg/ml of monoclonal antibody previously defined
The measure of apoptosis, or programmed cell death, is made by techniques routinely employed by experts in this field which involve the evaluation of at least one of the stages characteristic of apoptosis: modification of the plasma membrane, modification of the proteins from the Caspase family, modification of the factors of transactions and fragmentation of DNA. Among the cells or cell lines obtained by cloning, the advantage of the invention is that the cells conserved are only those which produce a significant quantity of monoclonal antibody, and notably those which produce at least 20 μg/ml of monoclonal antibody. The measure of the quantity of antibody is easily achievable by the experts, using simple protein dosage techniques.
Another embodiment of the invention concerns a cell or a cell line obtained by the cloning of the aforementioned cell, notably characterised by the fact that:
-
- it presents an apoptosis of below 25%
- it is stable throughout cellular divisions, and
- it secretes at least 14 μg/ml of monoclonal antibody previously defined.
The notion of cellular stability implies that the cells from the cloning of cells cloned from cells containing at least one vector permitting the expression of a monoclonal antibody in accordance with the invention are capable during the different divisions of conserving their properties of resistance to antibiotics and of producing the monoclonal antibodies.
A further aspect of the invention concerns the pharmaceutical composition, in particular vaccinal, comprising at least
-
- one monoclonal antibody defined above, or
- one nucleic acid defined above, or
- one expression vector defined above, or
- one fragment of the said monoclonal antibody defined above,
combined with a vehicle pharmaceutically acceptable.
Advantageously, the invention involves a pharmaceutical composition, in particular vaccinal, consisting of at least one monoclonal antibody defined above, combined with an acceptable pharmaceutical vehicle.
The dosage of the active substance depends in particular on the mode of administration, and can be easily determined by an expert.
“A pharmaceutically acceptable vehicle” refers to a non-toxic material which is compatible with a biological system such as a cell, a cellular culture, a tissue or an organism.
An effective therapeutic quantity can vary between 0.01 mg/kg and 50 mg/kg, preferably between 0.1 mg/kg and 20 mg/kg, and more preferably between 0.1 mg/kg and 2 mg/kg, in one or several daily administrations, over one or several days.
The pharmaceutical composition of the product can be administered intravenously, notably by injection or by gradual drip, subcutaneously, by systemic route, locally by means of infiltration, by bone, or by respiratory or pulmonary route by means of aerosol.
The preparations for parenteral administration can include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, or injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcohol/water solutions, emulsions or suspensions.
The favourable formulation for the pharmaceutical composition of the invention is an aerosol comprising
-
- a monoclonal antibody defined above, or
- a nucleic acid defined above, or
- an expression vector defined above, or
- a fragment of the said monoclonal antibody defined above,
combined with an excipient, with or without a propulsion agent.
In one embodiment of the product, the aerosol presents in the form of a liquid containing the anti-ricin antibody and an excipient. Usually the excipient is alcohol, but any other excipient known by the experts could be utilised within the framework of the invention. A liquid form aerosol can be linked to a gas propellant such as chlorofluorocarbon (CFC) or hydro fluorocarbon (HFA).
The aerosol in liquid form can also be made up of lipidic micro particles and an excipient. In this case the excipients can be chosen from synthetic dipalmitoylphosphatidylcholine (DPPC), lactose or hydroxyethylamidon (HES). The micro particles are then administered with the aid of an insufflator.
In another embodiment of the invention the aerosol presents in powder form. The powder is composed of particles of sizes of between 1 and 10 μm and preferably smaller than 9 μm, or smaller than 5 μm. As a non-exhaustive guide, the following methods can be used to obtain a dry powder: pulverisation together with desiccation by freezing or crystallisation by ultrasound, controlled precipitation.
The administration of the aerosol will be carried out according to whether it presents in liquid or solid form with the aid of a nebuliser which can be pneumatic, ultrasonic or sieved or with the aid of a metered dose inhaler (pressurised liquid, mechanic, electrodynamic, thermic) for liquid formulations or with the aid of an inhaler for solid formulations. (Reychler G., Dessanges J F and Vecellio L, Respiratory Journal, 2007; 24:1013-1023).
The invention also includes the utilisation of at least:
-
- one monoclonal antibody defined above, or
- one fragment of the said monoclonal antibody defined above,
- one nucleic acid defined above, or
- one expression vector defined above, or
- one cell defined above,
for the preparation of a drug for the treatment or prevention of a pathology linked to ricin contamination.
By treatment, we mean the method of treatment for a manifested pathology, whose symptoms are visible. By prevention, we mean the method of stopping the said pathology from manifesting.
Pathologies associated with ricin correspond to symptoms linked to ricin contamination, in particular diarrhoea, changes in electrolytes, dehydration, swelling, and respiratory, hepatic and renal problems . . . .
Equally the invention aims to achieve in vitro dosage of ricin in a biological sample taken from an individual susceptible to ricin contamination, the aforementioned method comprising:
-
- placing the said sample in contact with at least one monoclonal antibody as previously defined, and
- the determination of the presence or absence of ricin in the said sample by the detection of the formulation of an eventual immune complex between ricin and the said monoclonal antibody.
The monoclonal antibodies of the invention can therefore be utilised to detect the presence of ricin in a biological sample.
In particular, the said antibodies can be utilised for the implementation of detection techniques known by experts such as ELISA, RIE, immunoprecipitation or immunolabelling. (Western blot).
The invention also concerns an in vitro decontamination procedure, for a sample vulnerable to contamination by ricin, particularly a biological sample, which involves:
-
- the placement of the said sample in contact with at least one of the above monoclonal antibodies, and
- the elimination from the said sample of the immune complexes formed between ricin and the said monoclonal antibody.
A means of implementing the said decontamination procedure consists, for example, in the use of the invention's monoclonal antibodies, upon which magnetic spheres are grafted to the constant region of the said antibody.
Once the aforementioned antibodies, coupled with magnetic spheres, are placed in contact with the sample to be decontaminated, the ricin is eliminated from the sample by capturing the antibodies according to the invention, with magnets.
Another means to implement the said decontamination procedure consists, for example, in the use of monoclonal antibodies that have been immobilised on a column on which Staphylococcus aureus protein A or G is present. The sample to decontaminate is then passed through the column in order to capture, on the antibodies, according to the immobilised invention, any ricin which may be contained in the said sample.
One beneficial method of producing the invention concerns a procedure for the preparation of an aforementioned anti-ricin monoclonal antibody, which involves:
-
- a. the transformation of a cell, particularly that of a mammal, specifically the YB2/0 line, with at least one vector defined above;
- b. the selection of the transformed cells;
- c. the evaluation of the production of the said antibody by the clones selected in the preceding stage, by determining the presence or absence of the formation of any immune complexes between the ricin and the said monoclonal antibody.
One beneficial method of producing the invention concerns a procedure defined above, in which the selection of transformed cells is achieved by determining their resistance to at least one antibiotic.
One beneficial method of producing the invention concerns a procedure defined above, in which the cells producing a quantity of the said monoclonal antibody greater than 22 μg/mL are selected.
One beneficial method of producing the invention concerns a procedure defined above, in which the cells selected in Step C are cloned.
One beneficial method of producing the invention concerns a procedure defined above, in which the cells selected in Step C are re-cloned for:
-
- their resistance to at least one antibiotic, and
- their capacity to produce at least 11 μg/mL of the said monoclonal antibody.
The invention is better illustrated by the following examples and figures. The examples below aim to clarify the object of the invention, and to illustrate beneficial methods of realising it, but in no way limit the scope of the invention.
The different regulation elements (promoters, chimeric introns, polyadenylation sites, etc.), as well as the genes for resistance to antibiotics and the origins of replication, are also represented.
The different regulation elements (promoters, chimeric introns, polyadenylation sites, etc.), as well as the genes for resistance to antibiotics and the origins of replication, are also represented.
Bands 3 and 22 correspond to the migration of the molecular weight marker; bands 1 and 20 correspond to the migration of the PCR products made from a negative control, and bands 2 and 21 correspond to the migration of PCR products made from a positive control.
Bands 4, 6, 8, 10, 12, 14, 16, 18, 23, and 25 correspond to the migration of the PCR products made respectively from the cloid bases GG3, IF2, EE9, EG11, JB3, ED9, EC2, GF2, 1A1 and KC9.
Bands 5, 7, 9, 11, 13, 15, 17, 19, 24, and 26 correspond to the migration of the PCR products made respectively from the cloid bases GG3, IF2, EE9, EG11, JB3, ED9, EC2, GF2, 1A1 and KC9.
The abscissa or x-axis represents time, in days, and the ordinate or y-axis represents the quantity of antibodies produced in ng/mL.
Bands 4, 17, 30 and 33 correspond to the migration of the molecular weight markers; bands 3 and 31 correspond to the migration of the PCR products made from a negative control, and bands 2 and 32 correspond to the migration of PCR products made from a positive control. Bands 5, 7, 9, 11, 13, 15, 18, 20, 22, 24, 26 and 28 correspond to the migration of the PCR products made respectively from the clone bases GG9-4B2, GG9-2F8, GG9-5D8, GG9-2G10, IF2-2F2, IF2-2E3, GG3-2G4, GG3-EC2, GG3-1G9, GG3-2G2, GG3-5G11, GG3-1D8 and EE9-5G7.
Bands 6, 8, 10, 12, 14, 16, 19, 21, 23, 25, 27 and 29 correspond to the migration of the PCR products made respectively from the clone bases GG9-4B2, GG9-2F8, GG9-5D8, GG9-2G10, IF2-2F2, IF2-2E3, GG3-2G4, GG3-EC2, GG3-1G9, GG3-2G2, GG3-5G11, GG3-1D8 and EE9-5G7.
The abscissa or x-axis represents time, in days, and the ordinate or y-axis represents the quantity of antibodies produced in ng/mL.
The expression vector HK622-26 (SEQ ID No 21) was constructed for the expression of the chimeric macaque-human (IgG) anti-ricin monoclonal antibody.
The HK622-26 vector was constructed from the CHK622-05 vector by ‘double chimerization,’ meaning the addition by PCR of assemblies from human leader regions, and by a cloning addition of the constant CK and CH human regions to the VH and VK macaque variable sequences.
The variable heavy and light chain VH and VK regions are extracted from a coding vector for an anti-ricin ScFv, ScFv43RCA/H2-V116, and are introduced into the ‘generic’ CHK622-08 vector, after adding leader sequences to ensure a good synthesis of the chimerical antibody. The CK and CH constant regions are of human origin, and are derived from clone T125-A2, directed against the Rhesus D antigen.
A—Synthesis of the VH43RCA Region by PCR AssemblyThe VH43RCA region corresponds to the chimerization of:
-
- the leader VH sequence of the human M29812 sequence (in groups VH4, VH4-59), contained in the vector HK588-12, where the said leader VH sequence has been constituted in the following manner:
and
-
- the macaque VH anti-ricin region contained in the plasmid phAi14 containing the fragment ScFv43RCA/H2-V116, where the said VH macaque sequence has been constituted from the SEQ ID No 3 sequence.
The chimerization is performed in the following manner:
-
- the leader VH sequence (SEQ ID No 19) is amplified by PCR using the following initiator pair:
starting from a plasmidic HK558-12 DNA matrix.
The PCR reaction is performed according to the following protocol:
-
- denaturation: 5 min. at 95° C.
- denaturation: 0.5 min. at 95° C.┐
- hybridisation: 0.5 min at 50° C.} 10 repetitions
- elongation: 0.5 min. at 72° C.┘
- elongation: 10 min. at 72° C.
This pair of initiators allows the creation of an amplicon (amplicon 1) of 91 bases containing the site Nhe I (GCTAGC), the Kozak sequence (bold) and the human leader sequence SEQ ID No 17).
-
- the macaque VH sequence (SEQ ID No 3) is amplified by PCR using the following initiator pair:
starting with a plasmidic phAi14 DNA matrix.
The PCR reaction is performed according to the following protocol:
-
- denaturation: 5 min. at 95° C.
- denaturation: 0.5 min. at 95° C.┐
- hybridisation: 0.5 min at 50° C.} 10 repetitions
- elongation: 0.5 min. at 72° C.┘
- elongation: 10 min. at 72° C.
This initiator pair allows the creation of an amplicon (amplicon 2) of 396 bases containing the macaque VH sequence and the site Apa I (GGGCCCC).
Amplicons 1 and 2, previously obtained, are then combined to give the final amplicon 3 by PCR assembly using the aforementioned VH1 and VH4 initiators.
The PCR reaction is performed according to the following protocol:
-
- denaturation: 5 min. at 95° C.
- denaturation: 0.5 min. at 95° C.┐
- hybridisation: 0.5 min at 50° C.} 15 repetitions
- elongation: 1 min. at 72° C.┘
- elongation: 10 min. at 72° C.
The VK43RCA region corresponds to the chimerization of:
-
- the leader VK sequence of the human Z0006 sequence (in groups VK1, VK1-13), contained in the vector HK588-12, where the said leader VK sequence has been constituted in the following manner:
and
-
- the macaque VK anti-ricin region contained in the plasmid phAi14 containing the fragment ScFv43RCA/H2-V116, where the said VH macaque sequence has been constituted from the SEQ ID No 1 sequence.
The chimerization is performed in the following manner:
-
- the leader VK sequence (SEQ ID No 17) is amplified by PCR using the following initiator pair:
starting from a plasmidic HK558-12 DNA matrix.
The PCR reaction is performed according to the following protocol:
-
- denaturation: 5 min. at 95° C.
- denaturation: 0.5 min. at 95° C.┐
- hybridisation: 0.5 min at 50° C.} 10 repetitions
- elongation: 0.5 min. at 72° C.┘
- elongation: 10 min. at 72° C.
This pair of initiators allows the creation of an amplicon (amplicon 1′) of 102 bases containing the site Spe I (ACTAGT), the Kozak sequence (bold) and the human leader sequence (SEQ ID No 19).
-
- the macaque VK sequence (SEQ ID No 1) is amplified by PCR using the following initiator pair:
starting from a plasmidic phAi14 DNA matrix.
The PCR reaction is performed according to the following protocol:
-
- denaturation: 5 min. at 95° C.
- denaturation: 0.5 min. at 95° C.┐
- hybridisation: 0.5 min at 50° C.} 15 repetitions
- elongation: 1 min. at 72° C.┘
- elongation: 10 min. at 72° C.
This initiator pair allows the creation of an amplicon (amplicon 2′) of 364 bases containing the macaque VK sequence and the Dra III site (CACTTGGTG).
Amplicons 1′ and 2′, previously obtained, are then combined to give the final amplicon 3′ by PCR assembly using the aforementioned VK1 and VK4 initiators.
The PCR reaction is performed according to the following protocol:
-
- denaturation: 5 min. at 95° C.
- denaturation: 0.5 min. at 95° C.┐
- hybridisation: 0.5 min at 50° C.} 15 repetitions
- elongation: 1 min. at 72° C.┘
- elongation: 10 min. at 72° C.
Amplicons 3 and 3′ are introduced into the ‘generic’ vector CHK622-08 containing the constant CH heavy and CK light chain sequences from the IgG1 of the clone T125-A2 directed against the Rhesus D antigen.
The cloning of amplicons 3 and 3′ is performed sequentially in the following way:
1—Amplicon 3 and the vector CHK622-08 are subjected to a double digestion by the restriction enzymes Nhe I−Apa I.
The digestion fragments are then purified, and a vector/amplicon 3 mixture is subjected to a cohesive ligation.
The products of the ligation are then used to transform bacteria, and the transformers are selected on an LB medium with an ampicillin complement.
The resistant clones are then tested by PCR by using the initiators SPRSVBIS and GSP2ANP: a fragment of 783 bp is expected for the positive clones.
6 positive clones in PCR were selected for a screening via enzymatic digestion:
-
- by Nhe I+Apa I control digestion (verification of the insert and junctions): The expected profile consists of 2 strips, 447+9917 bp, and
- by Hind III digestion (verification of the entire vector): The expected profile consists of 4 strips, 3759+2894+2559+1152 bp.
All of the clones are positive, and the H622-26 clone, whose restriction map is represented in
2—Amplicon 3′ and the vector H622-26 are subjected to a double digestion by the restriction enzymes Spe I−Dra III.
The digestion fragments are then purified, and a vector/amplicon 3′ mixture is subjected to a cohesive ligation.
The products of the ligation are then used to transform bacteria, and the transformers are selected on an LB medium with an ampicillin complement.
The resistant clones are then tested by PCR by using the initiators 5′1PLC and CK4: a fragment of 539 bp is expected for the positive clones.
6 positive clones in PCR were selected for a screening via enzymatic digestion:
-
- by Spe I+Dra III control digestion (verification of the insert and junctions): The expected profile consists of 2 strips, 420+10344 bp, and
- by Hind III digestion (verification of the entire vector): The expected profile consists of 4 strips, 3759+2894+2559+1151 bp.
All of the clones are positive. After sequencing, only 4 clones possess a correct sequence. Clone 2 was chosen, and corresponds to vector H622-26. The restriction map of the vector H622-26 is represented in
The vector is thus ready for the transformation of cells.
Example 2 Obtaining Anti-Ricin-Producing Clones in the YB2/0 Line by Direct Double TransfectionThis study has the aim of obtaining clones producing anti-ricin in the YB2/0 line by direct double transfection.
The YB2/0 cells were maintained (by re-treatment with 1×105 cell/ml twice per week) in light of transfection at 105 cells/ml in an EMS medium, 5% SVF.
The vectors utilised for the transfection are as follows: HK622-26/EcoRV, H416-24 (T+) et K416-23 (T+). HK622-26/EcoRV signifies that the vector obtained in example 1 was linearised by digestion with the Eco RV enzyme in order to promote its integration into the transformed cells.
TransfectionThe cells are transfected according to the following protocol:
4 cuvettes containing 500 μL of cells are prepared in the following manner:
-
- Cuvettes 1 and 2: 42.8 μg of vector HK622-26/EcoRV
- Positive control cuvette: 25.2 μg of vector H416-24, linearised;
- 23.2 μg of vector H416-23, linearised;
- Negative control cuvette: no vectors.
The cells are then subjected to electrophoresis at a voltage of 230 V and a capacitance of 960 μF for 17.9 ms.
Once the electrophoresis is complete, the cells are carefully removed from the cuvettes and placed in a selective medium, RPMI, 5% dialysed SVF, 1 g/L geneticin for the P24 (25,000 cells/ml) and P96 at 100 cells/well (geneticin being applied only at J+3, once the resistance gene has been expressed) and in RPMI, 5% dialysed SVF, 0.5 g/L geneticin, 50 nM methotrexate (MTX) and seeded in culturing dishes in the following manner:
-
- Cuvettes 1 and 2 (per cuvette): 1 plate, 24 wells, at 25,000 cells/well
- 5 plates, 96 wells, at 2500 cells/well
- 1 plate, 96 wells, at 100 cells/well
- T+Control cuvette: 1 plate, 96 wells, at 2500 cells/well
- 1 plate, 96 wells, at 100 cells/well
- T−Control cuvette: 1 plate, 96 wells, at 2500 cells/well
- 1 plate, 96 wells, at 100 cells/well
- Cuvettes 1 and 2 (per cuvette): 1 plate, 24 wells, at 25,000 cells/well
The cells are then cultivated in the presence of antibiotic for 4 weeks.
For the 96 well plates, the medium is changed twice per week.
The rest of the transformed cells are spread over 6 wells for each of cuvettes 1 and 2.
Selection of TransformersEvaluation of the Production of Antibodies
In an initial step, the mean rate of antibody production is evaluated over 3 pools from each of the transfections of cuvettes 1 and 2, after 13 days in a selective culture medium.
In total 6 pools were tested, and the mean concentration of monoclonal antibodies was measured in μg/mL. The cellular density and cellular viability were also determined.
The results obtained are presented in Table 1 below:
We observe that the two transfections allow the production of anti-ricin monoclonal antibodies.
First Clonal Selection
After 22 days of culture in a selective medium, the appearance of clones resistant to G418 and to methotrexate was evaluated.
The results of the clones appearing are described in Table 2 below:
P96=96-well plates
In total 76 double resistant (G418 and methotrexate) pseudo-clones (wells) emerged following the double selection, for a transfection efficiency of 7.9% (76 positive wells for 960 seeded wells).
The cells which had been grown in the wells are considered as not being pure clones, as 2500 cells per well were seeded.
For each of the pseudo clones, or cloids, the quantity of immunoglobulin secreted was measured.
The results of the measurements from the best 24 clones are shown in Table 3 below:
Of the 24 cloids tested, 10 had a monoclonal antibody production of more than 8.9 μg/mL. All these cloids (EC2, ED9, EE9, EG11, GF2, CG3, IA1, IF2, JB3 and KC9) were deep frozen in a medium without antibiotic, in the presence of 10% DMSO, and kept at −195° C. in liquid nitrogen.
A cell viability test was conducted for each of these 10 cloids before freezing (Table 4) and they were screened for the possible presence of mycoplasma (
All the clones had a satisfactory survival rate (>85%) and no clone was contaminated by mycoplasma.
The 3 cloids (EE9, CG3 and IF2) whose production of antibodies was highest (more than 22 μg/mL) were selected for further cell cloning.
The stability of the monoclonal antibody production was also tested for each of the cloids selected.
The three cloids EE9, GG3 and IF2 were kept for 6 weeks by cascade dilution in 24 well plates in a DMEM medium+5% dialysed SVF and subcultured every 7 days. The production of monoclonal antibodies was evaluated in two independent ELISA assays and the results are shown in Table 5 below:
The stability of the cloids over time is also represented in graphic form in
Of the 3 cloids selected, the GG3 cloid saw its production of antibodies go down very significantly during the various stages.
On the basis of this information, the cloids selected were recloned in order to achieve “pure” clones.
Second Clonal Selection
A second cloning of the cloids EE9, CG3 and IF2 was conducted in DMEM 5% dialysed SVF medium. 5×96 well plates, with 40 cells per well, were seeded in a DMEM 5% dialysed SVF medium. There was no clone for this cloning.
A second cloning was carried out in a DMEM and EMS 5% dialysed SVF medium.
The cells were unfrozen in a RPMI 5% dialysed SVF medium then passed into an EMS 5% dialysed SVF and DMEM 5% SVF medium. The cells were subcultured to 4.5.105 cellules/mL on the eve of cloning.
5×96 well plates, with 40 cells per well, were seeded per cloid and per medium, namely a total of 30 plates, or 2880 wells. The numbers of clones which appeared are shown in Table 6.
Two siftings using ELISA allowed the 10 best clones to be selected from the 80 cloid clones which emerged.
The ELISA results from the 11 clones produced from cloid EE9, the 11 clones produced from cloid GG3 and the 10 clones produced from cloid IF2, are shown in Table 7 below:
5 clones were kept for each cloid. The 5 clones produced from cloid EE9 (EE9-5G7,EE9-5D8, EE9-2F8, EE9-4B1 and EE8-2G10) on average secrete more than 4.5 μg/mL monoclonal antibodies, the 5 clones from the cloid GG3 (GG3-2G4, GG3-1G9, GG3-2G2, GG3-1D8 and GG3-5G11) on average secrete more than 6.5 μg/mL monoclonal antibody and the 5 clones from the cloid IF2 (IF2-2E9, IF2-2D8, IF2-1C7, IF2-1D5 and IF2-2F7) secrete more than 8 μg/mL monoclonal antibody.
A cell viability test was conducted for each of these 15 cloids before freezing (Table 7) and they were screened for the possible presence of mycoplasma (
All the clones had a satisfactory survival rate (>70%) and no clone was contaminated by mycoplasma.
Finally 8 clones were selected, based on the following criteria:
-
- their production of monoclonal antibodies was ≧14 μg/mL
- their viability was ≧70%
The clones selected were thus the following: EE9-5G7, EE9-5D8, EE9-2G10, GG3-2G4, GG3-1G9, IF2-2E9, IF2-2D8 and IF2-1C7.
Stability of the Clones
The aim of these experiments is to verify that the various clones secrete substantially the same quantity of monoclonal antibody through the different stages.
The eight clones selected were kept for 8 weeks by cascade dilution in 24 well plates and subcultured every 7 days. The clones from cloid EE9 were kept in a EMS+5% dialysed SVF medium and the clones from cloids GG3 and IF2 in a DMEM=5% dialysed SVF medium.
The production of monoclonal antibodies was evaluated in two independent ELISA assays and the results are shown in Table 8 below:
The stability of the cloids over time is also represented in graphic form in
In the end, 3 clones were selected for their stability of monoclonal antibody production: clone EE9-5G7, clone IF2-2D8 and clone IF2-2E9.
Example 3 Production of Antibodies in Roller Bottles According to the InventionThe EE9-5G7 clone was selected for the production of 500 mg of antibodies on the basis of the dosages of maximum static pseudo production which established its level of production as 19 mg/L and also based on the results of the stability study conducted on the parental EE9 cloid, whose production level was stable for six weeks.
On the basis of these data, clone EE9-5G7 was amplified in such a way as to achieve two successive productions of 30 roller bottles in batch mode. Production was stopped when the cell viability dropped below 50%. As the supernatant measured at the end of production was only 12 mg/L, namely 648 mg antibody produced. This supernatant was measured, centrifuged, filtered before being concentrated 15 times. The concentrate was purified by affinity chromatography and this allowed 468 mg purified antibody to be obtained.
The production of anti-ricin monoclonal antibody by clones IF2-2D8 and IF2-2E9 was achieved by roller bottle production in an RPMI and EMS medium.
The cells were unfrozen and seeded to 2.105 cells/ml in a DMEM, 5% SVF medium depleted in Ig, 0.5 g/l geneticine. The viability from unfreezing was 91% for clone IF2-2D8 and 93% for clone IF2-2E9.
The progress of culture and roller production is shown in Table 9 for clone IF2-2D8 and in Table 10 for clone IF2-2E9.
The supernatants for these rollers were quantified by ELISA (Table 11), in order to measure the quantity of antibody produced.
The supernatants were filtered through a 0.22 μm filter and the antibodies were purified by affinity chromatography on a HiTrap protein A FF column.
Purification assessment of the anti-ricin antibodies IF2-2D8 and IF2-2E9
The concentrates allowed between 16 and 32.8 mg purified antibody to be obtained for each of the clones.
Clones IF2-2D8 and IF2-2E9 proved to be stable during the different stages (unlike their parental cloid) produce [sic] twice as many monoclonal antibodies as clone EE9-5G7. The physico-chemical characterisation of the antibodies showed that it is clone IF2-2E9 which produced the antibody most like the antibody produced by clone EE9-5G7. For this reason, it was clone IF2-2E9 which was chosen for the next productions of anti-ricin antibodies
Production and purification of a batch of 500 mg anti-ricin antibody.
-
- The culture supernatant for clone IF2-2E9 was concentrated, filtered, then the antibodies were purified in a first stage by affinity chromatography on Sepharose protein A followed by two stages of ion exchange. The quantity of antibody purified is 642 mg.
This test measures the capacity of the antibodies forming the subject of the invention to protect J774A.1 cells which have come into contact with Ricin from dying.
Briefly, J774A.1 cells (ATCC-LGC, Molsheim, France) were seeded at a density of 14.000 cells/well (200 μl/well), in a culture plate and cultivated at 37° C. in the presence of 5% CO2 for 24 hours in a DMEM medium complemented by 10% fcetal vole serum. The antibodies forming the subject of the invention were incubated with 480 ng/ml ricin or with control serum (antibodies not relevant) for one hour. The mixture was then added to the cells. 24 hours afterwards, cell viability was measured by techniques known to the expert in the field (Trypan Blue Exclusion Test, Cytox (Promega), measurement of apoptosis . . . ). Each test was corrected in relation to the “100% cell viability” controls (no ricin and no antibodies) and “0%” viability (ricin without antibodies).
Example 5 In Vitro Test of Ricin Neutralisation by the Human/Macaque Chimeric Monoclonal Antibody—Translation Inhibition TestAnother test may be used for measuring the neutralising activity of the monoclonal antibodies forming the subject of the invention, this means measuring protein translation inhibition. To do this, the translation of a marker protein is measured, for example the translation of luciferase, in an acellular in vitro translation system [Hale M L Pharmacol Toxicol 2001, 88(5):255-260]. Briefly, the test for measuring the translation of the luciferase messenger RNA is the following: The monoclonal antibodies are placed in 96 well plates in phosphate buffered saline (PBS) and ricin is added at a final concentration of 4 mM. Rabbit reticulocyte lysate complemented by RNAsin®, amino acids of the luciferase messenger RNA is then added to each well. The reaction is achieved over 1.5 hours.
5 μL of the reaction are then taken and added to 45 μL of rectional buffer solution, allowing luciferase activity to be detected (Luciferase assay reagent, Promega, Inc.). The light emitted (luminescence) is measured in counts per second (CPS) with the aid of a Victor multi-plate reader (PerkinElmer Wallac, Boston Mass.). The data are expressed as a percentage compared to a control (% control ═(CPS treated/CPS control)×100)).
In the two tests (Example 4 and Example 5), the comparison between the 50% protecting dose of scFv (1 μg/ml) and that of the IgG (0.5 μg/ml) shows a net benefit of the expression in the form of IgG.
Example 6 In Vivo Test of Ricin Neutralisation by the Human/Macaque Chimeric Monoclonal AntibodyIn order to test the neutralising effect of the anti-ricin antibody in vivo, Fisher rats (Charles River Laboratories L′Arbresle, France) received a dose of 16 μg/kg by weight of ricin and 50 μg anti-ricin antibody, following an adjustment of the protocol described by Ezzell et al. (Ezzell, et al. 1984. Infect. Immun 45:761-767)
The tests were carried out by measuring the viability of the animals as a function of the doses of ricin and of anti-ricin antibodies injected.
As a control, the viability of the rats treated solely with the ricin (positive test) and the rats treated solely with the anti-ricin antibody (negative test) was also evaluated.
Example 7 Affinity Assay of the Human/Macaque Chimeric Monoclonal Antibody for RicinThe affinity assay of the anti-ricin antibody for its antigen, that is to say ricin, was measured by surface plasmon resonance by means of a BIAcore X instrument (Biacore, Uppsala, Sweden). The ricin was immobilised on a CMS chip (Biacore), by means of an amine coupling, in accordance with the manufacturer's instructions.
For each measurement, a minimum of six dilutions, in an HBS-EP (Biacore) buffer of anti-ricin antibody was tested for 900s (dilutions from 10 to 0.1 μg/mL). After each dilution of the antibody, the chip was regenerated with 1.5 glycin (Biacore) at a flow of 10 μl/min for 30 s. The affinity constants were calculating by using the method described by Karlsson et al. (Karlsson et al. 1991, J. Immunol. Methods 145:229-240) and checked using internal tests as described by Schuck, et al. (Schuck, P., and A. P. Minton. 1996. Anal. Biochem. 240:262-272).
Example 8 Administration of the Human/Macaque Chimeric Monoclonal Antibody to Mice by Instillation and Determination of SurvivalBALb/c mice (20-22 g) were exposed to ricin by pulmonary instillation at the level of 16 μg/kg body weight and survival was checked 10 days after administration of the ricin.
The anti-chain A IgG 43RCA type of ricin antibody or a human IgG control were administered by pulmonary instillation at a level of 50 μg. The protective effect of the anti-chain A IgG 43RCA type of ricin antibody was measured by the percentage of mice which survived and by the ratio of wet weight over dry weight in the lungs, taking account of the fact that ricin induces pulmonary oedema.
The mice exposed to the ricin who had received the human control IgG were all dead by Day 3 3 (0% survival) (n=4). 100% survival was observed in the mice which had received the ricin and the 43RCA antibody in the same injection (n=2, not represented in
The mice which had received the 43RCA antibody 6 hours after administration of the ricin also revealed 100% survival (n=7)
Among the mice which had received the 43RCA antibody 22 hours after the administration of the ricin (n=7), 2 died on day 4, 1 died on Day 8 and the other 4 mice survived. The survival percentage is equal to 57.1% when the antibody is administered 22 hours after the administration of the ricin by the pulmonary route. Among the mice which had received the 43RCA antibody 24 hours after the administration of the ricin (n=11), 2 died on day 4, 2 died on Day 6. On Day 10, 7 of the 11 mice had survived. The survival rate is equal to 63.6%
The ratio of wet weight to dry weight of the lungs was measured 3 days after administration of the ricin to the mice, whether or not they received the 43RCA antibody. As shown in the table below, the ratio goes down if the mice receive the ricin pre-mixed with the 43RCA antibody, demonstrating that this 43RCA antibody neutralises the toxicity of the toxin and limits oedema of the lungs.
BALb/c mice were exposed to ricin by pulmonary instillation at a level of 16 μg/kg body weight and received an administration of 43RCA 6 hours afterwards, as 1, 5, 10, 20 μg.
BALb/c mice were exposed to ricin by pulmonary instillation at a level of 16 μg/kg body weight and survival was checked for 10 days after administration of the ricin. The anti-chain A IgG 43RCA type of ricin antibody or a human IgG control were administered by pulmonary instillation at a level of 150 mg.
As shown in
The mice were exposed to aerosols containing ricin and the anti-ricin antibody obtained by a Collison nebuliser (mass median aerodynamic diameter [MMAD]=1.2 urn).
The concentrations of ricin and antibody were identical for each exposure (ricin=1 mg/ml, antibody=14 mg/ml); the aerosol concentration being determined by the exposure time.
The aerosols were continually measured during exposure by means of a glass impact tester (AGI). The concentration of protein collected in the impact tester was measured by using the BCA micro protein assay kit (Pierce Co., Rockford, Ill., U.S.A.)
The diffusion conditions for the aerosols and their recovery remained constant throughout the study. The aerosol concentration (μg/L) was calculated and the dose of ricin inhale [sic] (μg/mouse) was estimated by using the Guyton formula.
Claims
1. A chimeric anti-ricin monoclonal antibody, wherein:
- the constant region of the light chain essentially comprises the constant region of the light chain of a human immunoglobulin;
- the constant region of the heavy chain essentially comprises the constant region of the heavy chain of a human immunoglobulin;
- the variable region of the light chain comprises the variable region of the light chain of a macaque immunoglobulin;
- the variable region of the heavy chain comprises the variable region of the heavy chain of a macaque immunoglobulin; and
- the monoclonal antibody does not substantially induce an immune response against chimeric antibodies.
2. The antibody according to claim 1, wherein the antibody is capable of neutralizing ricin.
3. The antibody according to claim 1, wherein the antibody is capable of neutralizing ricin and has a ricin neutralization rate that is higher than the neutralization rate of a fragment of scFv targeted at ricin.
4. The antibody according to claim 1, wherein:
- the constant region of the light chain comprises the constant region of the light chain of a kappa type human immunoglobulin; and
- the constant region of the heavy chain comprises the constant region of the heavy chain of a type IgG1 human immunoglobulin.
5. The antibody according to claim 4, wherein the constant region of the light chain and the constant region of the heavy chain are obtained by immunization with a Rhesus D antigen.
6. The antibody according to claim 4, wherein the constant region of the light chain and the constant region of the heavy chain are obtained from the T125-A2 clone.
7. A Fab or F(ab)′2 fragment of the monoclonal antibody according to claim 1.
8. Nucleic acid coding for the light chain of the monoclonal antibody according to claim 1.
9. The nucleic acid according to claim 8, comprising SEQ ID NO: 1, 5, 9 or 13.
10. Nucleic acid coding for the heavy chain of the monoclonal antibody according to claim 1.
11. The nucleic acid according to claim 8, comprising SEQ ID NO: 3, 7, 11 or 15.
12. Nucleic acid coding for the light chain and coding for the heavy chain of the monoclonal antibody according to claim 1.
13. An expression vector, comprising nucleic acid coding for the light chain, coding for the heavy chain, or coding for the light chain and the heavy chain of the monoclonal antibody according to claim 1, said nucleic acid being under control of elements that permit expression of the nucleic acid.
14. A pharmaceutical composition, comprising at least one of:
- the monoclonal antibody according to claim 1;
- a Fab or F(ab)′2 fragment of said monoclonal antibody;
- a nucleic acid coding for the light chain of said monoclonal antibody, coding for the heavy chain of said monoclonal antibody, or coding for the light chain and the heavy chain of said monoclonal antibody; or
- a vector comprising said nucleic acid,
- in association with a pharmaceutically acceptable vehicle.
15. A pharmaceutical composition, comprising at least one monoclonal antibody according to claim 1, in association with a pharmaceutically acceptable vehicle.
16. A method for treating or preventing a pathology associated with ricin contamination in a subject, comprising administering to the subject at least one of:
- the monoclonal antibody according to claim 1;
- a Fab or F(ab)′2 fragment of said monoclonal antibody;
- a nucleic acid coding for the light chain of said monoclonal antibody, coding for the heavy chain of said monoclonal antibody, or coding for the light chain and the heavy chain of said monoclonal antibody;
- a vector comprising said nucleic acid; or
- a cell comprising said monoclonal antibody, said fragment, said nucleic acid, or said vector.
17. A method of measuring ricin in vitro, in a biological sample from an individual who may have been contaminated by ricin, the method comprising:
- bringing the sample into contact with at least one monoclonal antibody according to claim 1; and
- determining the presence or absence of ricin in said sample by detecting the formation of an immune complex between the ricin and said monoclonal antibody.
18. A process for decontaminating in vitro, a sample that may have been contaminated by ricin, the process comprising:
- bringing the sample into contact with at least one monoclonal antibody according to claim 1, and
- removing from said sample any immune complexes formed between the ricin and said monoclonal antibody.
19. A process for preparing the antibody according to claim 1, comprising:
- transforming a cell with a vector comprising a nucleic acid coding for the light chain of said monoclonal antibody, coding for the heavy chain of said monoclonal antibody, or coding for the light chain of said monoclonal antibody and the heavy chain of said monoclonal antibody;
- selecting the transformed cells; and
- assessing the production of said antibody from the selected cells, by determining the presence or absence of the formation of an immune complex between ricin and said monoclonal antibody.
20. The process according to claim 19, wherein the cell is of the cell line YB2/0.
Type: Application
Filed: Aug 15, 2013
Publication Date: Feb 20, 2014
Applicants: LFB BIOTECHNOLOGIES (Courtaboeuf Cedex), ETAT FRANCAIS REPRESENTE PAR LE DELEGUE GENERAL POUR L'ARMEMENT (Bagneux)
Inventors: Philippe THULLIER (Bernin), Alexandre FONTAYNE (La Madeleine)
Application Number: 13/967,977
International Classification: C07K 16/40 (20060101); G01N 33/573 (20060101);
