Chimeric Anti-ricin antibody

a technology of ricin and anti-ricin, which is applied in the direction of immunological disorders, drug compositions, peptides, etc., can solve the problems of ricin being extremely toxic, affecting the anti-ricin effect, so as to achieve the effect of conserving the resistance of anti-ricin properties and being easy to determin

Inactive Publication Date: 2014-02-20
LABE FR DU FRACTIONNEMENT & DES BIOTECH SA +1
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  • Abstract
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  • Claims
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AI Technical Summary

Benefits of technology

[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 μg / 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:
[0171]it secretes at least 14 μg / 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
[0177]one fragment of the said monoclonal antibody defined above,combined with a vehicle pharmaceutically acceptable.

Problems solved by technology

As a toxin, ricin is extremely toxic.
However, its toxicity varies according to the means by which it penetrates the organism.
Absorption via inhalation causes weakness, fever, dizziness, dyspnoea, coughing, pulmonary oedemas and pain in the limbs.
After an apparent improvement, infection may have a fatal outcome.
However, no ricin-specific therapy is currently available.
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.
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).

Method used

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Examples

Experimental program
Comparison scheme
Effect test

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 Asse...

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 1×105 cell / ml twice per week) in light of transfection at 105 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 μL of cells are prepared in the following manner:[0325]Cuvettes 1 and 2: 42.8 μg of vector HK622-26 / EcoRV[0326]Positive control cuvette: 25.2 μg of vector H416-24, linearised;[0327]23.2 μg of vector H416-23, linearised;[0328]Negative control cuv...

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...

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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.

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 bu...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/40G01N33/573
CPCG01N33/573C07K16/40C07K16/16C07K2317/21C07K2317/24A61P37/04A61P43/00
Inventor THULLIER, PHILIPPEFONTAYNE, ALEXANDRE
Owner LABE FR DU FRACTIONNEMENT & DES BIOTECH SA
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