U.S. patent application number 13/967977 was filed with the patent office on 2014-02-20 for chimeric anti-ricin antibody.
This patent application is currently assigned to LFB BIOTECHNOLOGIES. The applicant listed for this patent is ETAT FRANCAIS REPRESENTE PAR LE DELEGUE GENERAL POUR L'ARMEMENT, LFB BIOTECHNOLOGIES. Invention is credited to Alexandre FONTAYNE, Philippe THULLIER.
Application Number | 20140050722 13/967977 |
Document ID | / |
Family ID | 42227649 |
Filed Date | 2014-02-20 |
United States Patent
Application |
20140050722 |
Kind Code |
A1 |
THULLIER; Philippe ; et
al. |
February 20, 2014 |
CHIMERIC ANTI-RICIN ANTIBODY
Abstract
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.
Inventors: |
THULLIER; Philippe; (Bernin,
FR) ; FONTAYNE; Alexandre; (La Madeleine,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LFB BIOTECHNOLOGIES
ETAT FRANCAIS REPRESENTE PAR LE DELEGUE GENERAL POUR
L'ARMEMENT |
Courtaboeuf Cedex
Bagneux |
|
FR
FR |
|
|
Assignee: |
LFB BIOTECHNOLOGIES
Courtaboeuf Cedex
FR
ETAT FRANCAIS REPRESENTE PAR LE DELEGUE GENERAL POUR
L'ARMEMENT
Bagneux
FR
|
Family ID: |
42227649 |
Appl. No.: |
13/967977 |
Filed: |
August 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13508108 |
Jun 27, 2012 |
8535668 |
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PCT/FR2010/052375 |
Nov 4, 2010 |
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13967977 |
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Current U.S.
Class: |
424/133.1 ;
424/93.2; 435/320.1; 435/7.4; 514/44R; 530/387.3; 536/23.53 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 43/00 20180101; C07K 2317/24 20130101; C07K 16/16 20130101;
G01N 33/573 20130101; C07K 16/40 20130101; C07K 2317/21
20130101 |
Class at
Publication: |
424/133.1 ;
530/387.3; 536/23.53; 435/320.1; 514/44.R; 424/93.2; 435/7.4 |
International
Class: |
C07K 16/40 20060101
C07K016/40; G01N 33/573 20060101 G01N033/573 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 4, 2009 |
FR |
09/57786 |
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.
Description
FIELD OF THE INVENTION
[0001] This invention concerns a chimeric antibody directed against
Ricin.
BACKGROUND OF THE INVENTION
[0002] Ricin 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.
[0003] As a toxin, ricin is extremely toxic. However, its toxicity
varies according to the means by which it penetrates the
organism.
[0004] When ricin is absorbed through digestion, it is largely
destroyed by proteolytic digestive enzymes but its perlingual
absorption may increase the quantity absorbed.
[0005] In contrast, when ricin is inhaled (pulmonary route) or
administered via the parenteral route its toxicity is multiplied
1000-fold.
[0006] 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.
[0007] After an apparent improvement, infection may have a fatal
outcome.
[0008] In humans, the dose of ricin estimated to be lethal is
between 1 and 10 .mu.g/kg.
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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).
[0015] 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.
[0016] 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 INVENTION
[0017] Thus, the object of the invention is to provide
ricin-neutralising antibodies.
[0018] The invention is also intended to provide the means of
producing these ricin-specific antibodies.
[0019] Another object of the invention concerns the use of these
antibodies as medication or as decontamination agents in response
to ricin contamination.
[0020] The invention concerns a chimeric monoclonal antibody
against ricin, in which the light chain and the heavy chain are
such that: [0021] the constant region of the light chain
essentially comprises the constant region of the light chain of a
human immunoglobulin; [0022] the constant region of the heavy chain
essentially comprises the constant region of the heavy chain of a
human immunoglobulin; [0023] the variable region of the light chain
includes the variable region of the light chain of a macaque
immunoglobulin; and [0024] 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.
[0025] 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 INVENTION
[0026] Under the invention, the term "antibody" refers to an
immunoglobulin, an oligomeric protein comprising 4 chains that
contribute to acquired immune response.
[0027] 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.
[0028] 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:
[0029] the base of the Y corresponds to the constant region Fc,
which is recognised by the Fc receptors and complement; and [0030]
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 (V.sub.L) and a constant region (C.sub.L). Each
heavy chain comprises a variable region (V.sub.H) and a constant
region composed of three constant domains C.sub.H1, C.sub.H2 and
C.sub.H3. The domains C.sub.H2 and C.sub.H3 make up the area
Fc.
[0031] The structure of an antibody is depicted diagrammatically in
FIG. 1.
[0032] 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.
[0033] The pearl-necklace structure of a variable region of a light
or heavy chain of an antibody is depicted in FIG. 2.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] "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.
[0038] "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.
[0039] "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.
[0040] "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.
[0041] 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.
[0042] Thus, the definition of the monoclonal antibody under the
invention covers both: [0043] The precursor of the chimeric
human/macaque antibody as defined above; and [0044] The chimeric
human/macaque antibody as defined above.
[0045] 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.
[0046] 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.
[0047] 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
[0048] 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%.
[0049] 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.
[0050] Consequently, the monoclonal antibodies under the invention
neutralise ricin toxicity.
[0051] 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.
[0052] This test measures the capacity of the antibodies under the
invention to protect J774A.1 cells put in contact with ricin
against death.
[0053] In short, J774A.1 cells (ATCC-LGC, Molsheim, France) are
cultured at a density of 14,000 cells/well (200 .mu.l/well) in a
culture dish and cultivated at 37.degree. C. with 5% CO.sub.2 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).
[0054] 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].
[0055] In short, the luciferase messenger RNA translation assay is
as follows:
[0056] 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.RTM., amino acids and luciferase messenger
RNA is then added into each well. The reaction lasts for 1 hr 30
mins.
[0057] 5 .mu.L of the reaction is then sampled and added to 45
.mu.L 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 .dbd.(CPS processed/control CPS).times.100)).
[0058] Another beneficial method of producing the invention
concerns a monoclonal antibody defined above, wherein: [0059] 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 [0060] 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.
[0061] 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.
[0062] 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 (.kappa.) type.
[0063] Under another beneficial method of production, the invention
concerns a monoclonal antibody against ricin as described above,
wherein: [0064] The light chain of the said monoclonal antibody
includes at least the complementarity-determining regions
(CDR.sub.Lm) of the variable region of a macaque immunoglobulin
light chain; and [0065] The heavy chain of the said monoclonal
antibody includes at least the complementarity-determining regions
(CDR.sub.Hm) of the variable region of a macaque immunoglobulin
heavy chain.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] Another beneficial method of producing the invention
concerns a monoclonal antibody as defined above, wherein: [0070]
The variable region of the light chain of the said monoclonal
antibody includes or comprises the following SEQ ID No 2
sequence:
TABLE-US-00001 [0070] (SEQ ID NO 2)
Nter-ELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPK
LLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYP
LTFGGGTKVEIK-Cter;
and [0071] The variable region of the heavy chain of the said
monoclonal antibody includes or comprises the following SEQ ID No 4
sequence:
TABLE-US-00002 [0071] (SEQ ID NO 4)
Nter-QVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGL
EWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYF
CARDDIVVSRIFDDWGQGVLVTVSS-Cter.
[0072] Another beneficial method of producing the invention
concerns a monoclonal antibody as defined above, wherein: [0073]
The variable region of the light chain of the said monoclonal
antibody comprises the following SEQ ID No 2 sequence:
TABLE-US-00003 [0073] (SEQ ID NO 2)
Nter-ELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPK
LLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYP
LTFGGGTKVEIK-Cter;
and [0074] The variable region of the heavy chain of the said
monoclonal antibody comprises the following SEQ ID NO 4
sequence:
TABLE-US-00004 [0074] (SEQ ID NO 4)
Nter-QVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGL
EWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYF
CARDDIVVSRIFDDWGQGVLVTVSS-Cter.
REV8
[0075] Another beneficial method of producing the invention
concerns a monoclonal antibody as defined above, including: [0076]
A light chain including or consisting of the following SEQ ID No 6
sequence:
TABLE-US-00005 [0076]
Nter-ELQMTQSPSSLSASVGDRVTITCRASQSIRSYLAWYQQKPGKAPK
LLIYDAAHLQSGVPSRFSGSGSGTEFSLTISSLQPEDFAVYYCQQRNSYP
LTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK
VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE
VTHQGLSSPVTKSFNRGEC-Cter;
and [0077] A heavy chain including or consisting of the following
SEQ ID No 8 sequence:
TABLE-US-00006 [0077]
Nter-QVQLVESGGGLVKPGGSLRLSCAASGFTFTDYYMDWVRQAPGKGL
EWVSRISPGGDVTWYADSVKGRFTISRDNAQNTLYLQMNSLRAEDTAVYF
CARDDIVVSRIFDDWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAAL
GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS
LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK-Cter.
REV7
[0078] The invention also concerns a monoclonal antibody against
ricin, as defined above, wherein: [0079] The light chain of the
said monoclonal antibody includes the leader region of the variable
region of a human immunoglobulin light chain (LV.sub.Lh), [0080]
The heavy chain of the said monoclonal antibody includes the leader
region of the variable region of a human immunoglobulin heavy chain
(LV.sub.Hh).
[0081] Another beneficial method of producing the invention
concerns a monoclonal antibody against ricin, as defined above,
wherein: [0082] 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 [0083] 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.
[0084] 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.
[0085] In addition, the invention concerns a precursor of an
aforementioned monoclonal antibody, wherein: [0086] The leader
region LV.sub.Lh includes or consists of the following SEQ ID No 17
sequence:
TABLE-US-00007 [0086] Nter-MDMRVPAQLLGLLLLWLPGARC-Cter;
[0087] The leader region LV.sub.Hh includes or consists of the
following SEQ ID No 18 sequence:
TABLE-US-00008 [0087] Nter-MKHLWFFLLLVAAPRWVLS- Cter . . .
[0088] Under another preferred means of production, the invention
concerns a precursor of the monoclonal antibody described above,
wherein: [0089] The variable region of the light chain of the said
monoclonal antibody includes or consists of the following SEQ ID No
10 sequence:
TABLE-US-00009 [0089]
Nter-MDMRVPAQLLGLLLLWLPGARCELQMTQSPSSLSASVGDRVTITC
RASQSIRSYLAWYQQKPGKAPKLLIYDAAHLQSGVPSRFSGSGSGTEFSL
TISSLQPEDFAVYYCQQRNSYPLTFGGGTKVEIK-Cter;
and [0090] The variable region of the heavy chain of the said
monoclonal antibody includes or consists of the following SEQ ID No
12 sequence:
TABLE-US-00010 [0090]
Nter-mkhlwfflllvaaprwvlsQVQLVESGGGLVKPGGSLRLSCAASG
FTFTDYYMDWVRQAPGKGLEWVSRISPGGDVTWYADSVKGRFTISRDNAQ
NTLYLQMNSLRAEDTAVYFCARDDIVVSRIFDDWGQGVLVTVSS-Cter.
[0091] 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.
[0092] Under one beneficial method of production, the invention
concerns a monoclonal antibody defined above, including: [0093] A
light chain consisting of the following SEQ ID No 14 sequence:
TABLE-US-00011 [0093]
Nter-MDMRVPAQLLGLLLLWLPGARCELQMTQSPSSLSASVGDRVTITC
RASQSIRSYLAWYQQKPGKAPKLLIYDAAHLQSGVPSRFSGSGSGTEFSL
TISSLQPEDFAVYYCQQRNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDE
QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-Cter;
and [0094] A heavy chain consisting of the following SEQ ID No 16
sequence:
TABLE-US-00012 [0094]
Nter-MKHLWFFLLLVAAPRWVLSQVQLVESGGGLVKPGGSLRLSCAASG
FTFTDYYMDWVRQAPGKGLEWVSRISPGGDVTWYADSVKGRFTISRDNAQ
NTLYLQMNSLRAEDTAVYFCARDDIVVSRIFDDWGQGVLVTVSSASTKGP
SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT
HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV
SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK-Cter
[0095] 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.
[0096] 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.
[0097] The invention also concerns a monoclonal antibody fragment
as defined above, the said fragment being a Fab or F(ab)'2
fragment.
[0098] scFv fragments are excluded from the invention.
[0099] Thus, Fab and F(ab)'2 fragments are constituted of: [0100]
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; [0101] For F(ab)'2, of the combination of the two
Fabs described above via a disulphide bridge.
[0102] 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
[0103] 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.
[0104] 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.
[0105] 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.
[0106] 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.
[0107] Another beneficial method of producing the invention
concerns a nucleic acid as defined above, including: [0108] 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 [0109] 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.
[0110] 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.
[0111] 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.
[0112] Another beneficial method of producing the invention
concerns a nucleic acid as defined above, including: [0113] A
nucleic acid including or constituted of a sequence selected from
among the nucleic acids SEQ ID No 1, 5, 9 and 13; and [0114] A
nucleic acid including or constituted of a sequence selected from
among the nucleic acids SEQ ID No 3, 7, 11 and 15.
[0115] Another beneficial method of producing the invention
concerns a nucleic acid as defined above, including: [0116] A
nucleic acid including or constituted of the sequence SEQ ID No 5
or 13; and [0117] A nucleic acid including or constituted of the
sequence SEQ ID No 7 or 15.
[0118] Another beneficial method of producing the invention
concerns a nucleic acid as defined above, including: [0119] A
nucleic acid including or constituted of the sequence SEQ ID No 13;
and [0120] A nucleic acid including or constituted of the sequence
SEQ ID No 15.
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] Another beneficial method of producing the invention
concerns an expression vector, as above, including: [0132] 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 [0133] 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.
[0134] Another beneficial method of producing the invention
concerns an expression vector, as above, including: [0135] 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 [0136] 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.
[0137] 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.
[0138] 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.
[0139] Another means of producing the invention concerns an
expression vector defined above, including at least one antibiotic
resistance gene.
[0140] 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.
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 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'.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] The cells "consisting of at least one vector "correspond to
the cells in which at least one expression vector mentioned above
has been introduced.
[0150] 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.
[0151] 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.
[0152] 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 . . . .
[0153] 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.
[0154] 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.
[0155] The favourable cell line of the invention is line YB2/0
available at the ATCC under number CRL 1662.
[0156] In another beneficial method, the invention concerns a cell
or a cell line consisting of an expression vector defined above,
permitting the expression of: [0157] a monoclonal antibody
previously defined [0158] or of a light chain of a monoclonal
antibody such as that previously described [0159] or of a heavy
chain of a monoclonal antibody such as that previously
described
[0160] In another beneficial method, the invention concerns a cell
or a cell line obtained by the cloning of an aforementioned
cell.
[0161] 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.
[0162] The general principal of cellular cloning is the limit
dilution of cells.
[0163] 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: [0164] present an
apoptosis of below 25%, and [0165] secrete at least cloning [0166]
20 .mu.g/ml of monoclonal antibody previously defined
[0167] 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 .mu.g/ml of monoclonal antibody. The measure of the
quantity of antibody is easily achievable by the experts, using
simple protein dosage techniques.
[0168] 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: [0169] it presents an
apoptosis of below 25% [0170] it is stable throughout cellular
divisions, and [0171] it secretes at least 14 .mu.g/ml of
monoclonal antibody previously defined.
[0172] 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.
[0173] A further aspect of the invention concerns the
pharmaceutical composition, in particular vaccinal, comprising at
least [0174] one monoclonal antibody defined above, or [0175] one
nucleic acid defined above, or [0176] one expression vector defined
above, or [0177] one fragment of the said monoclonal antibody
defined above, combined with a vehicle pharmaceutically
acceptable.
[0178] 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.
[0179] The dosage of the active substance depends in particular on
the mode of administration, and can be easily determined by an
expert.
[0180] "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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] The favourable formulation for the pharmaceutical
composition of the invention is an aerosol comprising [0185] a
monoclonal antibody defined above, or [0186] a nucleic acid defined
above, or [0187] an expression vector defined above, or [0188] a
fragment of the said monoclonal antibody defined above, combined
with an excipient, with or without a propulsion agent.
[0189] 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).
[0190] 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.
[0191] 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 .mu.m and preferably smaller than 9 .mu.m, or
smaller than 5 .mu.m. 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.
[0192] 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).
[0193] The invention also includes the utilisation of at least:
[0194] one monoclonal antibody defined above, or [0195] one
fragment of the said monoclonal antibody defined above, [0196] one
nucleic acid defined above, or [0197] one expression vector defined
above, or [0198] one cell defined above, for the preparation of a
drug for the treatment or prevention of a pathology linked to ricin
contamination.
[0199] 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.
[0200] 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 . . . .
[0201] 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:
[0202] placing the said sample in contact with at least one
monoclonal antibody as previously defined, and [0203] 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.
[0204] The monoclonal antibodies of the invention can therefore be
utilised to detect the presence of ricin in a biological
sample.
[0205] 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).
[0206] The invention also concerns an in vitro decontamination
procedure, for a sample vulnerable to contamination by ricin,
particularly a biological sample, which involves: [0207] the
placement of the said sample in contact with at least one of the
above monoclonal antibodies, and [0208] the elimination from the
said sample of the immune complexes formed between ricin and the
said monoclonal antibody.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] One beneficial method of producing the invention concerns a
procedure for the preparation of an aforementioned anti-ricin
monoclonal antibody, which involves: [0213] a. the transformation
of a cell, particularly that of a mammal, specifically the YB2/0
line, with at least one vector defined above; [0214] b. the
selection of the transformed cells; [0215] 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.
[0216] 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.
[0217] 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 .mu.g/mL are
selected.
[0218] One beneficial method of producing the invention concerns a
procedure defined above, in which the cells selected in Step C are
cloned.
[0219] One beneficial method of producing the invention concerns a
procedure defined above, in which the cells selected in Step C are
re-cloned for: [0220] their resistance to at least one antibiotic,
and [0221] their capacity to produce at least 11 .mu.g/mL of the
said monoclonal antibody.
[0222] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0223] FIG. 1 corresponds to a schematic representation of an
antibody. The black parts correspond to the constant regions of the
heavy chains, the dark grey parts correspond to the constant region
of the light chain, the parts in light grey correspond to the
variable region of the heavy chain, and the white parts correspond
to the variable region of the light chain. --S--S-- represents the
disulphide bridges between two cysteines. The CDR regions and
structures are indicated by arrows. The Fab and Fc fragments are
also represented.
[0224] FIG. 2 corresponds to a schematic pearl-necklace
representation of the amino acid sequence of a variable section of
the light chain or heavy chain of immunoglobulin. The black circles
correspond to the amino acids forming the structured regions, and
the grey circles correspond to the amino acids representing the
CDRs.
[0225] FIG. 3 corresponds to a schematic representation of the bond
between the leader peptide of the variable section of the heavy
chain of a human immunoglobulin and the variable section of the
heavy chain of a macaque immunoglobulin. The oligonucleotides which
have served as PCRs are drawn on the schematic. The unique
restriction sites are also indicated.
[0226] FIG. 4 corresponds to a schematic representation of the bond
between the leader peptide of the variable region of the heavy
chain of a human immunoglobulin and the variable region of the
light chain of macaque immunoglobulin. The oligonucleotides which
have served as PCRs are drawn on the schematic. The unique
restriction sites are also indicated.
[0227] FIG. 5 corresponds to the schematic representation of the
intermediate cloning vector H622-26 containing the `double hybrid`
heavy chain, in which the human leader is bound to the variable
region of the macaque heavy chain (VH 43RCA), which is itself bound
to the constant region of the human immunoglobulin (CH T125).
[0228] 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.
[0229] FIG. 6 corresponds to the schematic representation of the
final cloning vector HK622-26 (SEQ ID No. 21) containing the
`double hybrid` heavy chain in which the human leader is bound to
the variable region of the macaque heavy chain (VH 43RCA), which is
itself bound to the constant region of the human immunoglobulin (CH
T125), and the `double hybrid` light chain in which the human
leader is bound to the variable region of the macaque light chain
(VK 43RCA), which itself is bound to the constant region of the
human immunoglobulin (CK T125).
[0230] 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.
[0231] FIG. 7 corresponds to a 2% agarose gel on which the PCR
products allowing the detection of the mycoplasms have been
separated.
[0232] 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.
[0233] 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.
[0234] 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.
[0235] FIG. 8 represents in graph form the stability of antibody
production for the three cloids EE9, GG3 and IF2. The grey bars
represent measurements taken during the first dosage, and the black
bars represent measurements taken during the second dosage.
[0236] The abscissa or x-axis represents time, in days, and the
ordinate or y-axis represents the quantity of antibodies produced
in ng/mL.
[0237] FIG. 9 corresponds to a 2% agarose gel on which the PCR
products allowing the detection of the mycoplasms have been
separated.
[0238] 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.
[0239] 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.
[0240] FIG. 10 represents in graph form the stability of antibody
production for the clones EE9-2G10, EE9-5D8, EE9-5G7, GG3-1G9,
GG3-2G4, IF2-1C7, IF2-2D8 and IF2-2E9 over 8 days. The grey bars
represent measurements taken during the first dosage, and the black
bars represent measurements taken during the second dosage.
[0241] The abscissa or x-axis represents time, in days, and the
ordinate or y-axis represents the quantity of antibodies produced
in ng/mL.
[0242] FIG. 11 represents the cumulative Kaplan-Meier curve for
mice having received 50 .mu.g of ricin by pulmonary instillation as
well as a control human IgG (curve with triangles), or the antibody
according to the invention (43RCA), 6 hours (curve with squares),
22 hours (curve with diamonds) or 24 hours (dashed curve with
circles) after the instillation of the ricin.
[0243] FIG. 12 represents the cumulative Kaplan-Meier survival
curve for mice having received 50 .mu.g of ricin by pulmonary
instillation as well as 20 .mu.g of control human IgG (curve with
triangles), or the antibody according to the invention (43RCA) at a
concentration of 20, 10, 5 and 1 .mu.g;
[0244] FIG. 13 represents the cumulative Kaplan-Meier survival
curve for mice having received 50 .mu.g of ricin by pulmonary
instillation as well as 150 .mu.g of the antibody according to the
invention (43RCA), 44 hours (curve with squares), 54 hours (curve
with diamonds) after the instillation of the ricin. A control (the
curve with triangles) indicates the survival rate of mice treated
with the ricin and a non-relevant immunoglobulin.
EXAMPLES
Example 1
Construction and Sequencing of an HK622-26 Expression Vector for
the Expression of Antibodies According to the Invention
[0245] The expression vector HK622-26 (SEQ ID No 21) was
constructed for the expression of the chimeric macaque-human (IgG)
anti-ricin monoclonal antibody.
[0246] 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.
[0247] 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 Assembly
[0248] The VH43RCA region corresponds to the chimerization of:
[0249] 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:
TABLE-US-00013 [0249] (SEQ ID NO 19)
5'-ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATG
GGTCCTGTCC-3',
and [0250] 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.
[0251] The chimerization is performed in the following manner:
[0252] the leader VH sequence (SEQ ID No 19) is amplified by PCR
using the following initiator pair:
TABLE-US-00014 [0252] VH1-Ricin sense initiator: (SEQ ID NO 22)
5'-CTCAGTGCTAGCGCCGCCACCATGAAACATCTGTGGT-3' VH2-Ricin antisense
initiator: (SEQ ID NO 23) 5'-CCAGCTGCACCTGGGACAGGACCCAT-3'
starting from a plasmidic HK558-12 DNA matrix.
[0253] The PCR reaction is performed according to the following
protocol: [0254] denaturation: 5 min. at 95.degree. C. [0255]
denaturation: 0.5 min. at 95.degree. C..right brkt-bot. [0256]
hybridisation: 0.5 min at 50.degree. C.} 10 repetitions [0257]
elongation: 0.5 min. at 72.degree. C..right brkt-bot. [0258]
elongation: 10 min. at 72.degree. C.
[0259] 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).
[0260] the macaque VH sequence (SEQ ID No 3) is amplified by PCR
using the following initiator pair:
TABLE-US-00015 [0260] VH3-Ricin sense initiator: (SEQ ID NO 24)
5'-ATGGGTCCTGTCCCAGGTGCAGCTGG-3' VH4-Ricin antisense initiator:
(SEQ ID NO 25) 5'-ACCGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCA-3',
starting with a plasmidic phAi14 DNA matrix.
[0261] The PCR reaction is performed according to the following
protocol: [0262] denaturation: 5 min. at 95.degree. C. [0263]
denaturation: 0.5 min. at 95.degree. C..right brkt-bot. [0264]
hybridisation: 0.5 min at 50.degree. C.} 10 repetitions [0265]
elongation: 0.5 min. at 72.degree. C..right brkt-bot. [0266]
elongation: 10 min. at 72.degree. C.
[0267] 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).
[0268] 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.
[0269] The PCR reaction is performed according to the following
protocol: [0270] denaturation: 5 min. at 95.degree. C. [0271]
denaturation: 0.5 min. at 95.degree. C..right brkt-bot. [0272]
hybridisation: 0.5 min at 50.degree. C.} 15 repetitions [0273]
elongation: 1 min. at 72.degree. C..right brkt-bot. [0274]
elongation: 10 min. at 72.degree. C.
[0275] FIG. 3 shows the VH43RCA region with the different initiator
pairs having served in the preparation of the final chimerical VH
fragment (amplicon 3) of 461 bp.
B-Synthesis of the VK43RCA Region by PCR Assembly
[0276] The VK43RCA region corresponds to the chimerization of:
[0277] 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:
TABLE-US-00016 [0277] (SEQ ID NO 17)
5'-ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTG
GCTCCCAGGTGCCAGATGT-3',
and [0278] 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.
[0279] The chimerization is performed in the following manner:
[0280] the leader VK sequence (SEQ ID No 17) is amplified by PCR
using the following initiator pair:
TABLE-US-00017 [0280] VK1-Ricin sense initiator: (SEQ ID NO 26)
5'-CTCAGTACTAGTGCCGCCACCATGGACATGAGGGTCCCCG-3' VK2-Ricin antisense
initiator: (SEQ ID NO 27) 5'-TGTCATCTGGAGCTCACATCTGGCACCTGG-3'
starting from a plasmidic HK558-12 DNA matrix.
[0281] The PCR reaction is performed according to the following
protocol: [0282] denaturation: 5 min. at 95.degree. C. [0283]
denaturation: 0.5 min. at 95.degree. C..right brkt-bot. [0284]
hybridisation: 0.5 min at 50.degree. C.} 10 repetitions [0285]
elongation: 0.5 min. at 72.degree. C..right brkt-bot. [0286]
elongation: 10 min. at 72.degree. C.
[0287] 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).
[0288] the macaque VK sequence (SEQ ID No 1) is amplified by PCR
using the following initiator pair:
TABLE-US-00018 [0288] VK3-Ricin sense initiator: (SEQ ID NO 28)
5'-CCAGGTGCCAGATGTGAGCTCCAGATGACA-3' VK4-Ricin antisense initiator:
(SEQ ID NO 29) 5'-TGAAGACACTTGGTGCAGCCACAGTTCGTTTGATCTCCACCTTGGTC
C-3'
starting from a plasmidic phAi14 DNA matrix.
[0289] The PCR reaction is performed according to the following
protocol: [0290] denaturation: 5 min. at 95.degree. C. [0291]
denaturation: 0.5 min. at 95.degree. C..right brkt-bot. [0292]
hybridisation: 0.5 min at 50.degree. C.} 15 repetitions [0293]
elongation: 1 min. at 72.degree. C..right brkt-bot. [0294]
elongation: 10 min. at 72.degree. C.
[0295] 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).
[0296] 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.
[0297] The PCR reaction is performed according to the following
protocol: [0298] denaturation: 5 min. at 95.degree. C. [0299]
denaturation: 0.5 min. at 95.degree. C..right brkt-bot. [0300]
hybridisation: 0.5 min at 50.degree. C.} 15 repetitions [0301]
elongation: 1 min. at 72.degree. C..right brkt-bot. [0302]
elongation: 10 min. at 72.degree. C.
[0303] FIG. 4 shows the VK43RCA region with the different initiator
pairs having served in the preparation of the final chimeric VH
fragment (amplicon 3') of 436 bp.
C-Construction of Vector H622-26
[0304] 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.
[0305] 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.
[0306] The digestion fragments are then purified, and a
vector/amplicon 3 mixture is subjected to a cohesive ligation.
[0307] The products of the ligation are then used to transform
bacteria, and the transformers are selected on an LB medium with an
ampicillin complement.
[0308] 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.
[0309] 6 positive clones in PCR were selected for a screening via
enzymatic digestion: [0310] by Nhe I+Apa I control digestion
(verification of the insert and junctions): The expected profile
consists of 2 strips, 447+9917 bp, and [0311] by Hind III digestion
(verification of the entire vector): The expected profile consists
of 4 strips, 3759+2894+2559+1152 bp.
[0312] All of the clones are positive, and the H622-26 clone, whose
restriction map is represented in FIG. 5, is chosen for the second
step.
2--Amplicon 3' and the vector H622-26 are subjected to a double
digestion by the restriction enzymes Spe I-Dra III.
[0313] The digestion fragments are then purified, and a
vector/amplicon 3' mixture is subjected to a cohesive ligation.
[0314] The products of the ligation are then used to transform
bacteria, and the transformers are selected on an LB medium with an
ampicillin complement.
[0315] 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.
[0316] 6 positive clones in PCR were selected for a screening via
enzymatic digestion: [0317] by Spe I+Dra III control digestion
(verification of the insert and junctions): The expected profile
consists of 2 strips, 420+10344 bp, and [0318] by Hind III
digestion (verification of the entire vector): The expected profile
consists of 4 strips, 3759+2894+2559+1151 bp.
[0319] 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 FIG. 6.
[0320] 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 Transfection
[0321] This study has the aim of obtaining clones producing
anti-ricin in the YB2/0 line by direct double transfection.
[0322] The YB2/0 cells were maintained (by re-treatment with
1.times.10.sup.5 cell/ml twice per week) in light of transfection
at 10.sup.5 cells/ml in an EMS medium, 5% SVF.
[0323] 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.
Transfection
[0324] The cells are transfected according to the following
protocol:
4 cuvettes containing 500 .mu.L of cells are prepared in the
following manner: [0325] Cuvettes 1 and 2: 42.8 .mu.g of vector
HK622-26/EcoRV [0326] Positive control cuvette: 25.2 .mu.g of
vector H416-24, linearised; [0327] 23.2 .mu.g of vector H416-23,
linearised; [0328] Negative control cuvette: no vectors.
[0329] The cells are then subjected to electrophoresis at a voltage
of 230 V and a capacitance of 960 .mu.F for 17.9 ms.
[0330] 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: [0331] Cuvettes
1 and 2 (per cuvette): 1 plate, 24 wells, at 25,000 cells/well
[0332] 5 plates, 96 wells, at 2500 cells/well [0333] 1 plate, 96
wells, at 100 cells/well [0334] T+Control cuvette: 1 plate, 96
wells, at 2500 cells/well [0335] 1 plate, 96 wells, at 100
cells/well [0336] T-Control cuvette: 1 plate, 96 wells, at 2500
cells/well [0337] 1 plate, 96 wells, at 100 cells/well
[0338] The cells are then cultivated in the presence of antibiotic
for 4 weeks.
[0339] For the 96 well plates, the medium is changed twice per
week.
[0340] The rest of the transformed cells are spread over 6 wells
for each of cuvettes 1 and 2.
Selection of Transformers
[0341] Evaluation of the Production of Antibodies
[0342] 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.
[0343] In total 6 pools were tested, and the mean concentration of
monoclonal antibodies was measured in .mu.g/mL. The cellular
density and cellular viability were also determined.
[0344] The results obtained are presented in Table 1 below:
TABLE-US-00019 TABLE 1 Viability and dosage of antibody production
from the various pools. Antibody Cellular Density Viability
Concentration Cuvette Pool (10.sup.6 cells/ml) (%) (.mu.g/ml) 1 1
0.62 82 5.6 2 0.52 74 5.1 3 0.44 88 4.2 2 1 0.62 84 3.6 2 0.72 86
5.8 3 0.52 72 6
[0345] We observe that the two transfections allow the production
of anti-ricin monoclonal antibodies.
[0346] First Clonal Selection
[0347] After 22 days of culture in a selective medium, the
appearance of clones resistant to G418 and to methotrexate was
evaluated.
[0348] The results of the clones appearing are described in Table 2
below:
TABLE-US-00020 TABLE 2 Number of clones having emerged. Number of
Number of wells wells Number of where where cells clones clones
Cuvette seeded Number emerged emerged/ No. Selection per well of
P96 (total) P96 1 RPMI + 5% SVF 2500 5 34 6.8 dialysed + G418 0.5
g/L + 25 nM MTX 2 RPMI + 5% SVF 2500 5 42 8.4 dialysed + G418 0.5
g/L + 25 nM MTX T+ RPMI + 5% SVF 2500 1 1 1 dialysed + G418 0.5 g/L
+ 25 nM MTX
P96=96-well plates
[0349] 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).
[0350] The cells which had been grown in the wells are considered
as not being pure clones, as 2500 cells per well were seeded.
[0351] For each of the pseudo clones, or cloids, the quantity of
immunoglobulin secreted was measured.
[0352] The results of the measurements from the best 24 clones are
shown in Table 3 below:
TABLE-US-00021 TABLE 3 quantities of antibodies produced for the
different cloids tested. Name Average of the of the 1 IgG ng/ml 2
IgG ng/mL 3 IgG ng/mL 3 IgG ng/mL cloid quantity quantity quantity
quantities measured BA1 4800 5862 5843 5502 CB8 1700 1978 2253 1977
CE10 1100 1283 1413 1265 DH6 700 1405 2809 1638 EC2 12300 13153
21402 15618 EC9 9300 10638 11428 10455 ED9 12300 13734 15564 13866
EE9 22000 21738 24161 22633 EG11 16800 15716 18330 16949 GF11 6100
5529 6125 5918 GF2 11000 11715 13187 11967 GG3 27000 27371 28737
27703 HC9 5200 6386 7151 6246 HE7 8000 11015 13036 10684 HE8 1400
1492 1764 1552 IA1 10000 11779 12322 11367 IA6 6300 4978 7044 6107
IA7 3800 4887 5493 4727 IF2 26000 29707 31385 29031 JB3 16500 14736
17530 16255 JB9 6600 6973 7970 7181 JE6 8200 8626 9557 8794 KB6 400
899 1165 821 KC9 9900 7996 8832 8909
[0353] Of the 24 cloids tested, 10 had a monoclonal antibody
production of more than 8.9 .mu.g/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.degree. C. in liquid nitrogen.
[0354] 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 (FIG. 7).
[0355] All the clones had a satisfactory survival rate (>85%)
and no clone was contaminated by mycoplasma.
TABLE-US-00022 TABLE 4 viability of the cloids Cloid Viability (%)
GF2 89 EE9 97 IF2 89 EG11 89 KC9 85 EC2 95 ED9 95 GG3 100 IA1 100
JB3 100
[0356] The 3 cloids (EE9, CG3 and IF2) whose production of
antibodies was highest (more than 22 .mu.g/mL) were selected for
further cell cloning.
[0357] The stability of the monoclonal antibody production was also
tested for each of the cloids selected.
[0358] 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:
TABLE-US-00023 TABLE 5 antibody production over time. Name Name
Name of the Quantity Quantity of the Quantity Quantity of the
Quantity Quantity cloid Week 1 (ng/ml) 2 ng/mL cloid Week 1 (ng/ml)
2 ng/mL cloid Week 1 (ng/ml) 2 ng/mL EE9 1 13800 18415 GG3 1 14600
19131 IF2 1 12700 19743.5 EE9 2 17000 22901.5 GG3 2 12500 18022 IF2
2 16300 21009.5 EE9 3 17500 25188.5 GG3 3 9500 13153.5 IF2 3 12800
15733.5 EE9 4 14400 23695 GG3 4 7200 9257.5 IF2 4 7800 12186.5 EE9
5 16100 23878.5 GG3 5 5800 7270.5 IF2 5 10700 11822.5 EE9 6 15000
19890.5 GG3 6 3000 4433.5 IF2 6 7100 10581.5
[0359] The stability of the cloids over time is also represented in
graphic form in FIG. 8.
[0360] Of the 3 cloids selected, the GG3 cloid saw its production
of antibodies go down very significantly during the various
stages.
[0361] On the basis of this information, the cloids selected were
recloned in order to achieve "pure" clones.
[0362] Second Clonal Selection
[0363] A second cloning of the cloids EE9, CG3 and IF2 was
conducted in DMEM 5% dialysed SVF medium. 5.times.96 well plates,
with 40 cells per well, were seeded in a DMEM 5% dialysed SVF
medium. There was no clone for this cloning.
[0364] A second cloning was carried out in a DMEM and EMS 5%
dialysed SVF medium.
[0365] 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 4.5.10.sup.5 cellules/mL on the eve of
cloning.
[0366] 5.times.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.
TABLE-US-00024 TABLE 6 Table 6: number of clones which emerged
after cloning the 3 cloids, as a function of the culture medium.
Number of clones appearing Medium Cloids DMEM EMS IF2 22 1 GG3 30 0
EE9 2 25
[0367] Two siftings using ELISA allowed the 10 best clones to be
selected from the 80 cloid clones which emerged.
[0368] 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:
TABLE-US-00025 TABLE 7 quantities of antibodies produced for the
different cloids tested. Quantity 1 Quantity 2 Average E-IgG
Culture Stage ng/mL ng/mL ng/mL EE9-5G7 17 536 21 002 19 269
EE9-5D8 14 572 14 647 14 610 EE9-2G10 14 901 15 642 15 272 EE9-2F8
4 198 5 433 4 816 EE9-4B1 2 637 6 475 4 556 EE9-1B6 3 773 4 865 4
319 EE9-1G5 3 571 2 807 3 189 EE9-3G4 1 715 2 255 1 985 EE9-3G2 793
1 544 1 543 EE9-4H3 <1600 1 031 1 031 EE9-4G9 <1600 910 910
GG3-2G4 21227 26174 23 701 GG3-1G9 15816 18720 17 268 GG3-2G2 11445
10988 11 217 GG3-1D8 10 631 9 854 10 243 GG3-5G11 6251 7283 6 767
GG3-1F3 5 553 6 343 5 948 GG3-2E7 6782 8059 7 421 GG3-4F3 6060 5568
5 814 GG3-1F7 3 340 5 158 4 249 GG3-2B8 3898 5048 4 473 GG3-4B9
1785 2933 2 359 IF2-2E9 33267 42379 37 823 IF2-2D8 28917 32700 30
809 IF2-IC7 15770 18316 17 043 IF2-ID5 11990 13224 12 607 IF2-2F7
8386 8024 8 205 IF2-4B7 4688 5222 4 955 IF2-3D11 3147 6179 4 663
IF2-3B6 <1600 975 975 IF2-2E10 <1600 1234 1 234 IF2-2C4
<1600 761 761
[0369] 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 .mu.g/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 .mu.g/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 .mu.g/mL
monoclonal antibody.
[0370] 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 (FIG. 9).
[0371] All the clones had a satisfactory survival rate (>70%)
and no clone was contaminated by mycoplasma.
TABLE-US-00026 TABLE 7 viability of the clones selected Viability
Viability Viability Clone (%) Clone (%) Clone (%) GG3-2G4 78
IF2-2F7 79 EE9-2F8 80 GG3-2G2 83 IF2-1C7 75 EE9-4B1 84 GG3-1D8 86
IF2-2D8 90 EE9-5D8 85 GG3-1G9 89 IF2-2E9 71 EE9-2G10 90 GG3-5G11 78
IF2-1D5 95 EE9-5G7 93
[0372] Finally 8 clones were selected, based on the following
criteria: [0373] their production of monoclonal antibodies was
.gtoreq.14 .mu.g/mL [0374] their viability was .gtoreq.70%
[0375] The clones selected were thus the following: EE9-5G7,
EE9-5D8, EE9-2G10, GG3-2G4, GG3-1G9, IF2-2E9, IF2-2D8 and
IF2-1C7.
[0376] Stability of the Clones
[0377] The aim of these experiments is to verify that the various
clones secrete substantially the same quantity of monoclonal
antibody through the different stages.
[0378] 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.
[0379] The production of monoclonal antibodies was evaluated in two
independent ELISA assays and the results are shown in Table 8
below:
TABLE-US-00027 TABLE 8 antibody production over time. Name of the
Number of Average Average Name of the Number of Average Average
clones weeks ng/mL .mu.g/mL clones weeks ng/mL .mu.g/mL EE9-2G10 1
29592 30 EE9-5D8 1 0 0 2 4577 5 2 17072 17 3 9019 9 3 21419 21 4
20163 20 4 18034 18 5 10908 11 5 13317 13 6 10965 11 6 15010 15 7
7848 8 7 14607 15 8 5191 5 8 8543 9 EE9-5G7 1 31003 31 GG3-1G9 1
22030 22 2 17087 17 2 18223 18 3 21407 21 3 20848 21 4 15508 16 4
26141 26 5 12454 12 5 29579 30 6 14053 14 6 31577 32 7 13466 13 7
39374 39 8 12404 12 8 60901 61 GG3-2G4 1 23966 24 IF2-1C7 1 18266
18 2 22002 22 2 16667 17 3 23668 24 3 18738 19 4 23469 23 4 10165
10 5 32319 32 5 7717 8 6 32727 33 6 6852 7 7 41491 41 7 8158 8 8
47648 48 8 8675 9 IF2-2D8 1 39692 40 IF2-2E9 1 29219 29 2 41974 42
2 30861 31 3 33515 34 3 33623 34 4 30602 31 4 28549 29 5 29631 30 5
27918 28 6 31373 31 6 36222 36 7 35701 36 7 34909 35 8 35279 35 8
38246 38
[0380] The stability of the cloids over time is also represented in
graphic form in FIG. 10.
[0381] 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
Invention
[0382] The 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.
[0383] 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.
[0384] 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.
[0385] The cells were unfrozen and seeded to 2.10.sup.5 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.
[0386] The progress of culture and roller production is shown in
Table 9 for clone IF2-2D8 and in Table 10 for clone IF2-2E9.
TABLE-US-00028 TABLE 9 Follow-up of the culture and roller
production of clone IF2-2D8 Subculture Volume of Concentration
Viability density Type of medium Date (.times.10.sup.6/ml) (%)
(.times.10E6/ml) container (ml) Flask number Medium J0 Change of
medium T75 30 2 DMEM J3 -10 ml +10 ml medium T75 30 2 DMEM J7 0.64
86 0.1 T75 30 1 DMEM J10 1.1 95 0.1 T75 30 1 DMEM J14 1.38 96 0.1
T75 30 1 DMEM T75 30 1 RPMI T75 30 1 EMS J17 0.9 100 0.2 T175 100 1
RPMI 0.88 98 0.2 T175 100 1 EMS J20 1.48 97 0.3 Roller 500 1 EMS
1.12 100 0.3 Roller 400 1 RPMI J22 0.8 95 0.44 Roller 900
Production EMS Start 0.74 95 0.32 Roller 900 Production RPMI Start
J24 2.12 93 Roller 900 EMS 0.92 96 Roller 900 RPMI J27 Viability
<50% Stop Roller EMS Viability <50% Stop Roller RPMI
TABLE-US-00029 TABLE 10 Progress of culture and roller production
of clone IF2-2E9 Cell Concentration Viability density Type of
Volume of Date (.times.10.sup.6/ml) (%) (.times.1E6/ml) container
medium (ml) Flask Number Medium J0 0.56 82 0.1 T75 30 1 DMEM J3
0.76 95 0.1 T75 30 1 DMEM J7 1.4 91 0.1 T75 30 1 DMEM 1 EMS 1 RPMI
J10 0.68 100 0.1 T75 30 1 RPMI 0.9 100 0.1 T75 30 1 EMS J14 0.98 98
0.2 T175 100 1 RPMI 1.2 91 0.2 T175 100 1 EMS J17 1.26 95 0.25
Roller 500 1 RPMI 1.12 96 0.22 Roller 500 1 EMS J20 1.74 92 0.9
Roller 900 Production Start RPMI 1.84 98 0.9 Roller 900 Production
Start EMS J22 1.54 90 Roller 900 1 RPMI 2.1 88 Roller 900 1 EMS J24
Viability <50% Stop roller RPMI Viability <50% Stop roller
EMS
[0387] The supernatants for these rollers were quantified by ELISA
(Table 11), in order to measure the quantity of antibody
produced.
TABLE-US-00030 TABLE 11 Doses of roller supernatants. Concentration
Clones Medium (.mu.g/ml) IF2-2D8 EMS 39 IF2-2D8 RPMI 32 IF2-2E9 EMS
47 IF2-2E9 RPMI 34
[0388] The supernatants were filtered through a 0.22 .mu.m filter
and the antibodies were purified by affinity chromatography on a
HiTrap protein A FF column.
[0389] Purification assessment of the anti-ricin antibodies IF2-2D8
and IF2-2E9
TABLE-US-00031 Culture Antibodies supernatant purified Name of
Culture IgG Vol. IgG Yield clone medium Vol. (ml) (mg) (ml) (mg)
(%) IF2-2D8 EMS 1094 27.7 57.2 21.1 76.2 IF2-2D8 RPMI 1096 21.7
11.6 16.1 74.2 IF2-2E9 EMS 1095 38 17.2 32.8 86.3 IF2-2E9 RPMI 1095
24.3 24.3 22 90.5
[0390] The concentrates allowed between 16 and 32.8 mg purified
antibody to be obtained for each of the clones.
[0391] 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
[0392] Production and purification of a batch of 500 mg anti-ricin
antibody. [0393] 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.
Example 4
In Vitro Test of Ricin Neutralisation by the Human/Macaque Chimeric
Monoclonal Antibody--Survival Test
[0394] 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.
[0395] Briefly, J774A.1 cells (ATCC-LGC, Molsheim, France) were
seeded at a density of 14.000 cells/well (200 .mu.l/well), in a
culture plate and cultivated at 37.degree. C. in the presence of 5%
CO.sub.2 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 Test
[0396] Another 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.RTM., amino acids
of the luciferase messenger RNA is then added to each well. The
reaction is achieved over 1.5 hours.
[0397] 5 .mu.L of the reaction are then taken and added to 45 .mu.L
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 .dbd.(CPS treated/CPS control).times.100)).
[0398] In the two tests (Example 4 and Example 5), the comparison
between the 50% protecting dose of scFv (1 .mu.g/ml) and that of
the IgG (0.5 .mu.g/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 Antibody
[0399] In 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 .mu.g/kg by weight of
ricin and 50 .mu.g anti-ricin antibody, following an adjustment of
the protocol described by Ezzell et al. (Ezzell, et al. 1984.
Infect. Immun 45:761-767)
[0400] 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.
[0401] 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 Ricin
[0402] The 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.
[0403] 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 .mu.g/mL). After each dilution of the
antibody, the chip was regenerated with 1.5 glycin (Biacore) at a
flow of 10 .mu.l/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 Survival
[0404] BALb/c mice (20-22 g) were exposed to ricin by pulmonary
instillation at the level of 16 .mu.g/kg body weight and survival
was checked 10 days after administration of the ricin.
[0405] 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 .mu.g. 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.
[0406] 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
FIG. 11)
[0407] The mice which had received the 43RCA antibody 6 hours after
administration of the ricin also revealed 100% survival (n=7)
[0408] 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%
[0409] 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.
TABLE-US-00032 Ratio wet weight/dry weight of the lungs SD Saline
Solution 4.47 0.31 Ricin 6.56 0.655 Ricin + 43RCA IgG 5.36
0.607
Example 9
Administration of the Human/Macaque Chimeric Monoclonal Antibody to
Mice by Instillation and Determination of Minimum Doses
[0410] BALb/c mice were exposed to ricin by pulmonary instillation
at a level of 16 .mu.g/kg body weight and received an
administration of 43RCA 6 hours afterwards, as 1, 5, 10, 20
.mu.g.
[0411] FIG. 12 shows that whatever the dose administered, 100%
survival rate is observed in the mice. The 43RCA antibody proved to
be therapeutically effective from 1 .mu.g upwards when it is
administered 6 hours after intoxication by ricin.
[0412] BALb/c mice were exposed to ricin by pulmonary instillation
at a level of 16 .mu.g/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.
[0413] As shown in FIG. 13, the mice which had received the ricin
and an IgG control all died on Day 4. The mice which had received
the 43RCA antibody 44 hours after the pulmonary administration of
ricin had a survival rate of 83% (n=12). The mice which had
received the 43RCA antibody 54 hours after the pulmonary
administration of ricin had a survival rate of 75% (n=8). The 43RCA
antibody therefore has a therapeutic effect even when it is
administered more than two days after intoxication by ricin.
Example 10
Administration of the Human/Macaque Chimeric Monoclonal Antibody by
Aerosol
[0414] 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).
[0415] 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.
[0416] 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.)
[0417] The diffusion conditions for the aerosols and their recovery
remained constant throughout the study. The aerosol concentration
(.mu.g/L) was calculated and the dose of ricin inhale [sic]
(.mu.g/mouse) was estimated by using the Guyton formula.
Sequence CWU 1
1
291321DNAMacaca fascicularisCDS(1)..(321) 1gag ctc cag atg aca cag
tct cca tcc tcc ctg tct gca tct gta gga 48Glu Leu Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc
atc act tgt cgg gca agt cag agc att aga agt tat 96Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Tyr 20 25 30 tta gcc
tgg tat cag cag aaa cca ggg aaa gcc cct aag ctc ctg atc 144Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
tat gat gca gcc cat ttg caa agt ggg gtc cca tca agg ttc agc ggc
192Tyr Asp Ala Ala His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 agt gga tct ggg aca gaa ttc agt ctc acc atc agc agc ctg
caa cct 240Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 gaa gat ttt gca gtt tat tac tgt caa cag cgt aat
agt tat cct ctg 288Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn
Ser Tyr Pro Leu 85 90 95 act ttc ggc gga ggg acc aag gtg gag atc
aaa 321Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
2107PRTMacaca fascicularis 2Glu Leu Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Arg Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ala
His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Ser Tyr Pro Leu 85
90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
3360DNAMacaca fascicularisCDS(1)..(360) 3cag gtg cag ctg gtg gag
tcg ggg ggc ggc ttg gta aag cct ggg ggg 48Gln Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 tcc ctg aga ctc
tcc tgc gca gcc tcc gga ttc acc ttc act gac tac 96Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 tac atg
gac tgg gtc cgc cag gct cca ggg aag ggg ctg gag tgg gtc 144Tyr Met
Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
tca cgt att agc cct ggt ggt gat gtc aca tgg tac gca gac tcc gtg
192Ser Arg Ile Ser Pro Gly Gly Asp Val Thr Trp Tyr Ala Asp Ser Val
50 55 60 aag ggc aga ttt acc atc tcc aga gac aac gcc cag aac aca
ctg tat 240Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr
Leu Tyr 65 70 75 80 ctt caa atg aac agc ctg aga gct gag gac acg gct
gtc tat ttc tgt 288Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Phe Cys 85 90 95 gcg aga gat gat ata gtg gtg tcc aga att
ttt gat gac tgg ggc cag 336Ala Arg Asp Asp Ile Val Val Ser Arg Ile
Phe Asp Asp Trp Gly Gln 100 105 110 gga gtc ctg gtc acc gtc tcc tca
360Gly Val Leu Val Thr Val Ser Ser 115 120 4120PRTMacaca
fascicularis 4Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asp Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Ser Pro Gly Gly
Asp Val Thr Trp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala
Arg Asp Asp Ile Val Val Ser Arg Ile Phe Asp Asp Trp Gly Gln 100 105
110 Gly Val Leu Val Thr Val Ser Ser 115 120 5642DNAArtificial
sequenceHybrid human/macaque 5gag ctc cag atg aca cag tct cca tcc
tcc ctg tct gca tct gta gga 48Glu Leu Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly 1 5 10 15 gac aga gtc acc atc act tgt
cgg gca agt cag agc att aga agt tat 96Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Arg Ser Tyr 20 25 30 tta gcc tgg tat cag
cag aaa cca ggg aaa gcc cct aag ctc ctg atc 144Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 tat gat gca
gcc cat ttg caa agt ggg gtc cca tca agg ttc agc ggc 192Tyr Asp Ala
Ala His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 agt
gga tct ggg aca gaa ttc agt ctc acc atc agc agc ctg caa cct 240Ser
Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80 gaa gat ttt gca gtt tat tac tgt caa cag cgt aat agt tat cct
ctg 288Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Ser Tyr Pro
Leu 85 90 95 act ttc ggc gga ggg acc aag gtg gag atc aaa cga act
gtg gct gca 336Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr
Val Ala Ala 100 105 110 cca agt gtc ttc atc ttc ccg cca tct gat gag
cag ttg aaa tct gga 384Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly 115 120 125 act gcc tct gtt gtg tgc ctg ctg aat
aac ttc tat ccc aga gag gcc 432Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gta cag tgg aag gtg gat
aac gcc ctc caa tcg ggt aac tcc cag 480Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gag agt gtc aca
gag cag gac agc aag gac agc acc tac agc ctc agc 528Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agc acc
ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc
624Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 ttc aac agg gga gag tgt 642Phe Asn Arg Gly Glu Cys 210
6214PRTArtificial sequenceSynthetic Construct 6Glu Leu Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Arg Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Asp Ala Ala His Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn
Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 71350DNAArtificial
sequenceHybrid human/macaque 7cag gtg cag ctg gtg gag tcg ggg ggc
ggc ttg gta aag cct ggg ggg 48Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgc gca
gcc tcc gga ttc acc ttc act gac tac 96Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 tac atg gac tgg gtc
cgc cag gct cca ggg aag ggg ctg gag tgg gtc 144Tyr Met Asp Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 tca cgt att
agc cct ggt ggt gat gtc aca tgg tac gca gac tcc gtg 192Ser Arg Ile
Ser Pro Gly Gly Asp Val Thr Trp Tyr Ala Asp Ser Val 50 55 60 aag
ggc aga ttt acc atc tcc aga gac aac gcc cag aac aca ctg tat 240Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr 65 70
75 80 ctt caa atg aac agc ctg aga gct gag gac acg gct gtc tat ttc
tgt 288Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe
Cys 85 90 95 gcg aga gat gat ata gtg gtg tcc aga att ttt gat gac
tgg ggc cag 336Ala Arg Asp Asp Ile Val Val Ser Arg Ile Phe Asp Asp
Trp Gly Gln 100 105 110 gga gtc ctg gtc acc gtc tcc tca gcc tcc acc
aag ggc cca tcg gtc 384Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val 115 120 125 ttc ccc ctg gca ccc tcc tcc aag agc
acc tct ggg ggc aca gcg gcc 432Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala 130 135 140 ctg ggc tgc ctg gtc aag gac
tac ttc ccc gaa ccg gtg acg gtg tcg 480Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 tgg aac tca ggc
gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc 528Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 cta cag
tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc 576Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190
tcc agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag
624Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 ccc agc aac acc aag gtg gac aag aaa gtt gag ccc aaa tct
tgt gac 672Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220 aaa act cac aca tgc cca ccg tgc cca gca cct gaa
ctc ctg ggg gga 720Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 ccg tca gtc ttc ctc ttc ccc cca aaa
ccc aag gac acc ctc atg atc 768Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 tcc cgg acc cct gag gtc aca
tgc gtg gtg gtg gac gtg agc cac gaa 816Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 gac cct gag gtc aag
ttc aac tgg tac gtg gac ggc gtg gag gtg cat 864Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 aat gcc aag
aca aag ccg cgg gag gag cag tac aac agc acg tac cgt 912Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 gtg
gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag 960Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc
gag 1008Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca
cag gtg tac 1056Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 acc ctg ccc cca tcc cgg gat gag ctg acc aag
aac cag gtc agc ctg 1104Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu 355 360 365 acc tgc ctg gtc aaa ggc ttc tat ccc
agc gac atc gcc gtg gag tgg 1152Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380 gag agc aat ggg cag ccg gag
aac aac tac aag acc acg cct ccc gtg 1200Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 ctg gac tcc gac
ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac 1248Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 aag agc
agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat 1296Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
gag gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg
1344Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445 ggt aaa 1350Gly Lys 450 8450PRTArtificial
sequenceSynthetic Construct 8Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asp Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile
Ser Pro Gly Gly Asp Val Thr Trp Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe
Cys 85 90 95 Ala Arg Asp Asp Ile Val Val Ser Arg Ile Phe Asp Asp
Trp Gly Gln 100 105 110 Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195
200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly Gly 225 230 235 240 Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275
280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395
400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445 Gly Lys 450 9387DNAMacaca
fascicularisCDS(1)..(387) 9atg gac atg agg gtc ccc gct cag ctc ctg
ggg ctt ctg ctg ctc tgg 48Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp 1 5 10 15 ctc cca ggt gcc aga tgt gag ctc
cag atg aca cag tct cca tcc tcc 96Leu Pro Gly Ala Arg Cys Glu Leu
Gln Met Thr Gln Ser Pro Ser Ser 20 25 30 ctg tct gca tct gta gga
gac aga gtc acc atc act tgt cgg gca agt 144Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 35 40 45 cag agc att aga
agt tat tta gcc tgg tat cag cag aaa cca ggg aaa 192Gln Ser Ile Arg
Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 gcc cct
aag ctc ctg atc tat gat gca gcc cat ttg caa agt ggg gtc 240Ala Pro
Lys Leu Leu Ile Tyr Asp Ala Ala His Leu Gln Ser Gly Val 65 70 75 80
cca tca agg ttc agc ggc agt gga tct ggg aca gaa ttc agt ctc acc
288Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr
85 90 95 atc agc agc ctg caa cct gaa gat ttt gca gtt tat tac tgt
caa cag 336Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln 100 105 110 cgt aat agt tat cct ctg act ttc ggc gga ggg acc
aag gtg gag atc 384Arg Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile 115 120 125 aaa 387Lys 10129PRTMacaca fascicularis
10Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 Leu Pro Gly Ala Arg Cys Glu Leu Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser 35 40 45 Gln Ser Ile Arg Ser Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile Tyr Asp Ala
Ala His Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Ser Leu Thr 85 90 95 Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 100 105 110 Arg Asn Ser
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys
11417DNAMacaca fascicularisCDS(1)..(417) 11atg aaa cat ctg tgg ttc
ttc ctt ctc ctg gtg gca gct ccc aga tgg 48Met Lys His Leu Trp Phe
Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 gtc ctg tcc cag
gtg cag ctg gtg gag tcg ggg ggc ggc ttg gta aag 96Val Leu Ser Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 cct ggg
ggg tcc ctg aga ctc tcc tgc gca gcc tcc gga ttc acc ttc 144Pro Gly
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45
act gac tac tac atg gac tgg gtc cgc cag gct cca ggg aag ggg ctg
192Thr Asp Tyr Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60 gag tgg gtc tca cgt att agc cct ggt ggt gat gtc aca tgg
tac gca 240Glu Trp Val Ser Arg Ile Ser Pro Gly Gly Asp Val Thr Trp
Tyr Ala 65 70 75 80 gac tcc gtg aag ggc aga ttt acc atc tcc aga gac
aac gcc cag aac 288Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Gln Asn 85 90 95 aca ctg tat ctt caa atg aac agc ctg aga
gct gag gac acg gct gtc 336Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110 tat ttc tgt gcg aga gat gat ata
gtg gtg tcc aga att ttt gat gac 384Tyr Phe Cys Ala Arg Asp Asp Ile
Val Val Ser Arg Ile Phe Asp Asp 115 120 125 tgg ggc cag gga gtc ctg
gtc acc gtc tcc tca 417Trp Gly Gln Gly Val Leu Val Thr Val Ser Ser
130 135 12139PRTMacaca fascicularis 12Met Lys His Leu Trp Phe Phe
Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30 Pro Gly Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Thr
Asp Tyr Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55
60 Glu Trp Val Ser Arg Ile Ser Pro Gly Gly Asp Val Thr Trp Tyr Ala
65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Gln Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val 100 105 110 Tyr Phe Cys Ala Arg Asp Asp Ile Val Val
Ser Arg Ile Phe Asp Asp 115 120 125 Trp Gly Gln Gly Val Leu Val Thr
Val Ser Ser 130 135 13708DNAArtificial sequenceHybrid human/macaque
13atg gac atg agg gtc ccc gct cag ctc ctg ggg ctt ctg ctg ctc tgg
48Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1
5 10 15 ctc cca ggt gcc aga tgt gag ctc cag atg aca cag tct cca tcc
tcc 96Leu Pro Gly Ala Arg Cys Glu Leu Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30 ctg tct gca tct gta gga gac aga gtc acc atc act tgt
cgg gca agt 144Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser 35 40 45 cag agc att aga agt tat tta gcc tgg tat cag
cag aaa cca ggg aaa 192Gln Ser Ile Arg Ser Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Lys 50 55 60 gcc cct aag ctc ctg atc tat gat gca
gcc cat ttg caa agt ggg gtc 240Ala Pro Lys Leu Leu Ile Tyr Asp Ala
Ala His Leu Gln Ser Gly Val 65 70 75 80 cca tca agg ttc agc ggc agt
gga tct ggg aca gaa ttc agt ctc acc 288Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Glu Phe Ser Leu Thr 85 90 95 atc agc agc ctg caa
cct gaa gat ttt gca gtt tat tac tgt caa cag 336Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 100 105 110 cgt aat agt
tat cct ctg act ttc ggc gga ggg acc aag gtg gag atc 384Arg Asn Ser
Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 aaa
cga act gtg gct gca cca agt gtc ttc atc ttc ccg cca tct gat 432Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135
140 gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg ctg aat aac
480Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
145 150 155 160 ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat
aac gcc ctc 528Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu 165 170 175 caa tcg ggt aac tcc cag gag agt gtc aca gag
cag gac agc aag gac 576Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 180 185 190 agc acc tac agc ctc agc agc acc ctg
acg ctg agc aaa gca gac tac 624Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 195 200 205 gag aaa cac aaa gtc tac gcc
tgc gaa gtc acc cat cag ggc ctg agc 672Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 tcg ccc gtc aca aag
agc ttc aac agg gga gag tgt 708Ser Pro Val Thr Lys Ser Phe Asn Arg
Gly Glu Cys 225 230 235 14236PRTArtificial sequenceSynthetic
Construct 14Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu
Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Glu Leu Gln Met Thr Gln
Ser Pro Ser Ser 20 25 30 Leu Ser Ala Ser Val Gly Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser 35 40 45 Gln Ser Ile Arg Ser Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60 Ala Pro Lys Leu Leu Ile
Tyr Asp Ala Ala His Leu Gln Ser Gly Val 65 70 75 80 Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Glu Phe Ser Leu Thr 85 90 95 Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln 100 105 110
Arg Asn Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115
120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235
151407DNAArtificial sequenceHybrid human/macaque 15atg aaa cat ctg
tgg ttc ttc ctt ctc ctg gtg gca gct ccc aga tgg 48Met Lys His Leu
Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 gtc ctg
tcc cag gtg cag ctg gtg gag tcg ggg ggc ggc ttg gta aag 96Val Leu
Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys 20 25 30
cct ggg ggg tcc ctg aga ctc tcc tgc gca gcc tcc gga ttc acc ttc
144Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45 act gac tac tac atg gac tgg gtc cgc cag gct cca ggg aag
ggg ctg 192Thr Asp Tyr Tyr Met Asp Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu 50 55 60 gag tgg gtc tca cgt att agc cct ggt ggt gat gtc
aca tgg tac gca 240Glu Trp Val Ser Arg Ile Ser Pro Gly Gly Asp Val
Thr Trp Tyr Ala 65 70 75 80 gac tcc gtg aag ggc aga ttt acc atc tcc
aga gac aac gcc cag aac 288Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Gln Asn 85 90 95 aca ctg tat ctt caa atg aac agc
ctg aga gct gag gac acg gct gtc 336Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat ttc tgt gcg aga gat
gat ata gtg gtg tcc aga att ttt gat gac 384Tyr Phe Cys Ala Arg Asp
Asp Ile Val Val Ser Arg Ile Phe Asp Asp 115 120 125 tgg ggc cag gga
gtc ctg gtc acc gtc tcc tca gcc tcc acc aag ggc 432Trp Gly Gln Gly
Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 cca tcg
gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc 480Pro Ser
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155
160 aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg gtg
528Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
165 170 175 acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac
acc ttc 576Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe 180 185 190 ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc
agc agc gtg gtg 624Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val 195 200 205 acc gtg ccc tcc agc agc ttg ggc acc cag
acc tac atc tgc aac gtg 672Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val 210 215 220 aat cac aag ccc agc aac acc aag
gtg gac aag aaa gtt gag ccc aaa 720Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 tct tgt gac aaa act
cac aca tgc cca ccg tgc cca gca cct gaa ctc 768Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 ctg ggg gga
ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc 816Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 ctc
atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg 864Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280
285 agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg
912Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
290 295 300 gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac
aac agc 960Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser 305 310 315 320 acg tac cgt gtg gtc agc gtc ctc acc gtc ctg
cac cag gac tgg ctg 1008Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 325 330 335 aat ggc aag gag tac aag tgc aag gtc
tcc aac aaa gcc ctc cca gcc 1056Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 340 345 350 ccc atc gag aaa acc atc tcc
aaa gcc aaa ggg cag ccc cga gaa cca 1104Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360 365 cag gtg tac acc ctg
ccc cca tcc cgg gat gag ctg acc aag aac cag 1152Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc
1200Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
385 390 395 400 gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac
aag acc acg 1248Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr 405 410 415 cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc
ctc tac agc aag ctc 1296Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu 420 425 430 acc gtg gac aag agc agg tgg cag cag
ggg aac gtc ttc tca tgc tcc 1344Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser 435 440 445 gtg atg cat gag gct ctg cac
aac cac tac acg cag aag agc ctc tcc 1392Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 ctg tct ccg ggt aaa
1407Leu Ser Pro Gly Lys 465 16469PRTArtificial sequenceSynthetic
Construct 16Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro
Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Lys 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe 35 40 45 Thr Asp Tyr Tyr Met Asp Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Arg Ile
Ser Pro Gly Gly Asp Val Thr Trp Tyr Ala 65 70 75 80 Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn 85 90 95 Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110
Tyr Phe Cys Ala Arg Asp Asp Ile Val Val Ser Arg Ile Phe Asp Asp 115
120 125 Trp Gly Gln Gly Val Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 210 215 220 Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235
240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val 275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325 330 335 Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 340 345 350 Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 355 360
365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445 Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450 455 460 Leu Ser Pro
Gly Lys 465 1757DNAArtificial Sequenceleader sequence 17atg aaa cat
ctg tgg ttc ttc ctt ctc ctg gtg gca gct ccc aga tgg 48Met Lys His
Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 gtc
ctg tcc 57Val Leu Ser 1819PRTArtificial SequenceSynthetic Construct
18Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1
5 10 15 Val Leu Ser 1966DNAArtificial Sequenceleader sequence 19atg
gac atg agg gtc ccc gct cag ctc ctg ggg ctt ctg ctg ctc tgg 48Met
Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10
15 ctc cca ggt gcc aga tgt 66Leu Pro Gly Ala Arg Cys 20
2022PRTArtificial SequenceSynthetic Construct 20Met Asp Met Arg Val
Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly
Ala Arg Cys 20 2110763DNAArtificial sequenceExpression vector
21gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
60gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
120aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt
agggttaggc 180gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
tatctgaggg gactagggtg 240tgtttaggcg aaaagcgggg cttcggttgt
acgcggttag gagtcccctc aggatatagt 300agtttcgctt ttgcataggg
agggggaaat gtagtcttat gcaatactct tgtagtcttg 360caacatggta
acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg
420ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
agacgggtct 480gacatggatt ggacgaacca ctgaattccg cattgcagag
atattgtatt taagtgccta 540gctcgataca ataaacgcca tttgaccatt
caccacattg gtgtgcacct ccaagcttgg 600taccgagctc ggatccacta
gagcagaagt tggtcgtgag gcactgggca ggtaagtatc 660aaggttacaa
gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga
720ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc
ctttctctcc 780acaggtgtcc actcccagtt caattacagc tcttgctagc
gccgccacca tgaaacatct 840gtggttcttc cttctcctgg tggcagctcc
cagatgggtc ctgtcccagg tgcagctggt 900ggagtcgggg ggcggcttgg
taaagcctgg ggggtccctg agactctcct gcgcagcctc 960cggattcacc
ttcactgact actacatgga ctgggtccgc caggctccag ggaaggggct
1020ggagtgggtc tcacgtatta gccctggtgg tgatgtcaca tggtacgcag
actccgtgaa 1080gggcagattt accatctcca gagacaacgc ccagaacaca
ctgtatcttc aaatgaacag 1140cctgagagct gaggacacgg ctgtctattt
ctgtgcgaga gatgatatag tggtgtccag 1200aatttttgat gactggggcc
agggagtcct ggtcaccgtc tcctcagcct ccaccaaggg 1260cccatcggtc
ttccccctgg caccctcctc caagagcacc tctgggggca cagcggccct
1320gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcgtgga
actcaggcgc 1380cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag
tcctcaggac tctactccct 1440cagcagcgtg gtgaccgtgc cctccagcag
cttgggcacc cagacctaca tctgcaacgt 1500gaatcacaag cccagcaaca
ccaaggtgga caagaaagtt gagcccaaat cttgtgacaa 1560aactcacaca
tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct
1620cttcccccca aaacccaagg acaccctcat gatctcccgg acccctgagg
tcacatgcgt 1680ggtggtggac gtgagccacg aagaccctga ggtcaagttc
aactggtacg tggacggcgt 1740ggaggtgcat aatgccaaga caaagccgcg
ggaggagcag tacaacagca cgtaccgtgt 1800ggtcagcgtc ctcaccgtcc
tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa 1860ggtctccaac
aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca
1920gccccgagaa ccacaggtgt acaccctgcc cccatcccgg gatgagctga
ccaagaacca 1980ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc
gacatcgccg tggagtggga 2040gagcaatggg cagccggaga acaactacaa
gaccacgcct cccgtgctgg actccgacgg 2100ctccttcttc ctctacagca
agctcaccgt ggacaagagc aggtggcagc aggggaacgt 2160cttctcatgc
tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc
2220cctgtctccg ggtaaatgat agggcgcgcc gctagagagg atcccgggtg
gcatccctgt 2280gacccctccc cagtgcctct cctggccctg gaagttgcca
ctccagtgcc caccagcctt 2340gtcctaataa aattaagttg catcattttg
tctgactagg tgtccttcta taatattatg 2400gggtggaggg gggtggtatg
gagcaagggg caagttggga agacaacctg tagggcctgc 2460ggggtctatt
gggaaccaag ctggagtgca gtggcacaat cttggctcac tgcaatctcc
2520gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
attccaggca 2580tgcatgacca ggctcagcta atttttgttt ttttggtaga
gacggggttt caccatattg 2640gccaggctgg tctccaactc ctaatctcag
gtgatctacc caccttggcc tcccaaattg 2700ctgggattac aggcgtgaac
cactgctccc ttccctgtcc ttctgatttt aaaataacta 2760taccagcagg
aggacgtcca gacacagcat aggctacctg gccatgccca accggtggga
2820catttgagtt gcttgcttgg cactgtcctc tcatgcgttg ggtccactca
gtagatgcct 2880gttcatatgc tcgagatctc ccgatcccct atggtgcact
ctcagtacaa tctgctctga 2940tgccgcatag ttaagccagt atctgctccc
tgcttgtgtg ttggaggtcg ctgagtagtg 3000cgcgagcaaa atttaagcta
caacaaggca aggcttgacc gacaattgca tgaagaatct 3060gcttagggtt
aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgtatctg
3120aggggactag ggtgtgttta ggcgaaaagc ggggcttcgg ttgtacgcgg
ttaggagtcc 3180cctcaggata tagtagtttc gcttttgcat agggaggggg
aaatgtagtc ttatgcaata 3240ctcttgtagt cttgcaacat ggtaacgatg
agttagcaac atgccttaca aggagagaaa 3300aagcaccgtg catgccgatt
ggtggaagta aggtggtacg atcgtgcctt attaggaagg 3360caacagacgg
gtctgacatg gattggacga accactgaat tccgcattgc agagatattg
3420tatttaagtg cctagctcga tacaataaac gccatttgac cattcaccac
attggtgtgc 3480acctccaagc ttggtaccga gctcggatcc actagagcag
aagttggtcg tgaggcactg 3540ggcaggtaag tatcaaggtt acaagacagg
tttaaggaga ccaatagaaa ctgggcttgt 3600cgagacagag aagactcttg
cgtttctgat aggcacctat tggtcttact gacatccact 3660ttgcctttct
ctccacaggt gtccactccc agttcaatta cagctcttac tagtgccgcc
3720accatggaca tgagggtccc cgctcagctc ctggggcttc tgctgctctg
gctcccaggt 3780gccagatgtg agctccagat gacacagtct ccatcctccc
tgtctgcatc tgtaggagac 3840agagtcacca tcacttgtcg ggcaagtcag
agcattagaa gttatttagc ctggtatcag 3900cagaaaccag ggaaagcccc
taagctcctg atctatgatg cagcccattt gcaaagtggg 3960gtcccatcaa
ggttcagcgg cagtggatct gggacagaat tcagtctcac catcagcagc
4020ctgcaacctg aagattttgc agtttattac tgtcaacagc gtaatagtta
tcctctgact 4080ttcggcggag ggaccaaggt ggagatcaaa cgaactgtgg
ctgcaccaag tgtcttcatc 4140ttcccgccat ctgatgagca gttgaaatct
ggaactgcct ctgttgtgtg cctgctgaat 4200aacttctatc ccagagaggc
caaagtacag tggaaggtgg ataacgccct ccaatcgggt 4260aactcccagg
agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc
4320accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg
cgaagtcacc 4380catcagggcc tgagctcgcc cgtcacaaag agcttcaaca
ggggagagtg ttagtgaact 4440ctagagctcg ctgatcagcc tcgactgtgc
cttctagttg ccagccatct gttgtttgcc 4500cctcccccgt gccttccttg
accctggaag gtgccactcc cactgtcctt tcctaataaa 4560atgaggaaat
tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
4620ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
gacatatgat 4680ttaaatactg gggctcgact gtggaatgtg tgtcagttag
ggtgtggaaa gtccccaggc 4740tccccagcag gcagaagtat gcaaagcatg
catctcaatt agtcagcaac caggtgtgga 4800aagtccccag gctccccagc
aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 4860accatagtcc
cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
4920tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
cgcctcggcc 4980tctgagctat tccagaagta gtgaggaggc ttttttggag
gcctaggctt ttgcaaaaag 5040cttggggggg gggacagctc agggctgcga
tttcgcgcca aacttgacgg caatcctagc 5100gtgaaggctg gtaggatttt
atccccgctg ccatcatggt tcgaccattg aactgcatcg 5160tcgccgtgtc
ccaaaatatg gggattggca agaacggaga cctaccctgg cctccgctca
5220ggaacgagtt caagtacttc caaagaatga ccacaacctc ttcagtggaa
ggtaaacaga 5280atctggtgat tatgggtagg aaaacctggt tctccattcc
tgagaagaat cgacctttaa 5340aggacagaat taatatagtt ctcagtagag
aactcaaaga accaccacga ggagctcatt 5400ttcttgccaa aagtttggat
gatgccttaa gacttattga acaaccggaa ttggcaagta 5460aagtagacat
ggtttggata gtcggaggca gttctgttta ccaggaagcc atgaatcaac
5520caggccacct cagactcttt gtgacaagga tcatgcagga atttgaaagt
gacacgtttt 5580tcccagaaat tgatttgggg aaatataaac ttctcccaga
atacccaggc gtcctctctg 5640aggtccagga ggaaaaaggc atcaagtata
agtttgaagt ctacgagaag aaagactaac 5700aggaagatgc tttcaagttc
tctgctcccc tcctaaagct atgcattttt ataagaccat 5760gggacttttg
ctggctttag atcgatcttt gtgaaggaac cttacttctg tggtgtgaca
5820taattggaca aactacctac agagatttaa agctctaagg taaatataaa
atttttaagt 5880gtataatgtg ttaaactact gattctaatt gtttgtgtat
tttagattcc aacctatgga 5940actgatgaat gggagcagtg gtggaatgcc
tttaatgagg aaaacctgtt ttgctcagaa 6000gaaatgccat ctagtgatga
tgaggctact gctgactctc aacattctac tcctccaaaa 6060aagaagagaa
aggtagaaga ccccaaggac tttccttcag aattgctaag ttttttgagt
6120catgctgtgt ttagtaatag aactcttgct tgctttgcta tttacaccac
aaaggaaaaa 6180gctgcactgc tatacaagaa aattatggaa aaatattctg
taacctttat aagtaggcat 6240aacagttata atcataacat actgtttttt
cttactccac acaggcatag agtgtctgct 6300attaataact atgctcaaaa
attgtgtacc tttagctttt taatttgtaa aggggttaat 6360aaggaatatt
tgatgtatag tgccttgact agagatcata atcagccata ccacatttgt
6420agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
aacataaaat 6480gaatgcaatt gttgttgtta acttgtttat tgcagcttat
aatggttaca aataaagcaa 6540tagcatcaca aatttcacaa ataaagcatt
tttttcactg cattctagtt gtggtttgtc 6600caaactcatc aatgtatctt
atcatgtctg gatccgcgta tggtgcactc tcagtacaat 6660ctgctctgat
gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
6720ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
tctccgggag 6780ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
gcgagacgaa agggcctcgt 6840gatacgccta tttttatagg ttaatgtcat
gataataatg gtttcttaga gatctcgagg 6900gtgggccatc gccctgatag
acggtttttc gccctttgac gttggagtcc acgttcttta 6960atagtggact
cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
7020atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
atttaacaaa 7080aatttaacgc gaattttaac aaaatattaa cgcttacaat
ttcctgatgc ggtattttct 7140ccttacgcat ctgtgcggta tttcacaccg
catacgcgga tctgcgcagc accatggcct 7200gaaataacct ctgaaagagg
aacttggtta ggtaccttct gaggcggaaa gaaccagctg 7260tggaatgtgt
gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
7320caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
ctccccagca 7380ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
ccatagtccc gcccctaact 7440ccgcccatcc cgcccctaac tccgcccagt
tccgcccatt ctccgcccca tggctgacta 7500atttttttta tttatgcaga
ggccgaggcc gcctcggcct ctgagctatt ccagaagtag 7560tgaggaggct
tttttggagg cctaggcttt tgcaaaaagc ttgattcttc tgacacaaca
7620gtctcgaact taaggctaga gccaccatga ttgaacaaga tggattgcac
gcaggttctc 7680cggccgcttg ggtggagagg ctattcggct atgactgggc
acaacagaca atcggctgct 7740ctgatgccgc cgtgttccgg ctgtcagcgc
aggggcgccc ggttcttttt gtcaagaccg 7800acctgtccgg tgccctgaat
gaactgcagg acgaggcagc gcggctatcg tggctggcca 7860cgacgggcgt
tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
7920tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
cctgccgaga 7980aagtatccat catggctgat gcaatgcggc ggctgcatac
gcttgatccg gctacctgcc 8040cattcgacca ccaagcgaaa catcgcatcg
agcgagcacg tactcggatg gaagccggtc 8100ttgtcgatca ggatgatctg
gacgaagagc atcaggggct cgcgccagcc gaactgttcg 8160ccaggctcaa
ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
8220gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
tgtggccggc 8280tgggtgtggc ggaccgctat caggacatag cgttggctac
ccgtgatatt gctgaagagc 8340ttggcggcga atgggctgac cgcttcctcg
tgctttacgg tatcgccgct cccgattcgc 8400agcgcatcgc cttctatcgc
cttcttgacg agttcttctg agcgggactc tggggttcga 8460aatgaccgac
caagcgacgc ccaacctgcc atcacgatgg ccgcaataaa atatctttat
8520tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat agcgataagg
atcgatcctc 8580tagctagagt cgatcgacct gcagggatcc gcgtatggtg
cactctcagt acaatctgct 8640ctgatgccgc atagttaagc cagccccgac
acccgccaac acccgctgac gcgccctgac 8700gggcttgtct gctcccggca
tccgcttaca gacaagctgt gaccgtctcc gggagctgca 8760tgtgtcagag
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
8820gcctattttt ataggttaat gtcatgataa taatggtttc ttagagatct
tagatccagt 8880tgaactgcag ggaaatccgg acgtgtatac cagtttaaac
atgttaatta agtcgacgcg 8940gccgcaggtg gcacttttcg gggaaatgtg
cgcggaaccc ctatttgttt atttttctaa 9000atacattcaa atatgtatcc
gctcatgaga caataaccct gataaatgct tcaataatat 9060tgaaaaagga
agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
9120gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
agatgctgaa 9180gatcagttgg gtgcacgagt gggttacatc gaactggatc
tcaacagcgg taagatcctt 9240gagagttttc gccccgaaga acgttttcca
atgatgagca cttttaaagt tctgctatgt 9300ggcgcggtga tatcccgtat
tgacgccggg caagagcaac tcggtcgccg catacactat 9360tctcagaatg
acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
9420acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
ggccaactta 9480cttctgacaa cgatcggagg accgaaggag ctaaccgctt
ttttgcacaa catgggggat 9540catgtaactc gccttgatcg ttgggaaccg
gagctgaatg aagccatacc aaacgacgag 9600cgtgacacca cgatgcctgt
agcaatggca acaacgttgc gcaaactatt aactggcgaa 9660ctacttactc
tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
9720ggaccacttc tgcgctcggc ccttccggct ggctggttta
ttgctgataa atctggagcc 9780ggtgagcgtg ggtctcgcgg tatcattgca
gcactggggc cagatggtaa gccctcccgt 9840atcgtagtta tctacacgac
ggggagtcag gcaactatgg atgaacgaaa tagacagatc 9900gctgagatag
gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
9960atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
gaagatcctt 10020tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
cgttccactg agcgtcagac 10080cccgtagaaa agatcaaagg atcttcttga
gatccttttt ttctgcgcgt aatctgctgc 10140ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt tgccggatca agagctacca 10200actctttttc
cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
10260gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
atacctcgct 10320ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
agtcgtgtct taccgggttg 10380gactcaagac gatagttacc ggataaggcg
cagcggtcgg gctgaacggg gggttcgtgc 10440acacagccca gcttggagcg
aacgacctac accgaactga gatacctaca gcgtgagcta 10500tgagaaagcg
ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
10560gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
tctttatagt 10620cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
tgtgatgctc gtcagggggg 10680cggagcctat ggaaaaacgc cagcaacgcg
gcctttttac ggttcctggc cttttgctgg 10740ccttttgctc acatggctcg aca
107632237DNAArtificial SequenceVH1-Ricin sense initiator
22ctcagtgcta gcgccgccac catgaaacat ctgtggt 372326DNAArtificial
SequenceVH2-Ricin antisense initiator 23ccagctgcac ctgggacagg
acccat 262426DNAArtificial SequenceVH3-Ricin sense initiator
24atgggtcctg tcccaggtgc agctgg 262540DNAArtificial
SequenceVH4-Ricin antisense initiator 25accgatgggc ccttggtgga
ggctgaggag acggtgacca 402640DNAArtificial SequenceVK1-Ricin sense
initiator 26ctcagtacta gtgccgccac catggacatg agggtccccg
402730DNAArtificial SequenceVK2-Ricin antisense initiator
27tgtcatctgg agctcacatc tggcacctgg 302830DNAArtificial
SequenceVK3-Ricin sense initiator 28ccaggtgcca gatgtgagct
ccagatgaca 302948DNAArtificial SequenceVK4-Ricin antisense
initiator 29tgaagacact tggtgcagcc acagttcgtt tgatctccac cttggtcc
48
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