Method for preparing monoclonal antibodies capable of activating effector cells expressing FCγRIII
View Patent ↗The invention concerns a method for obtaining and selecting monoclonal antibodies by an ADDC-type test, said antibodies capable of activating type III Fcγ receptors and having a particular glycan structure. The inventive anti-D antibodies can be used for preventing Rhesus isoimmunization in Rh negative persons, in particular for haemolytic disease in a new-born baby or for uses such as idiopathic thrombocytopenic purpura (ITP).
1. A method for preparing a monoclonal antibody capable of specifically activating effector cells expressing FcγRIII, wherein the method comprises:
purifying a monoclonal antibody obtained from a clone originated from a cell line selected from hybridomas and animal or human cell lines transfected with a vector comprising a gene encoding said antibody; then
adding the antibody to a reaction mixture comprising:
target cells for said antibody,
effector cells comprising cells expressing FcγRIII and
polyvalent IgGs; then
determining an FcγRIII-type ADCC activity level as a lysis percentage of the target cells then
selecting the monoclonal antibody, which activates the effector cells and causes an FcγRIII-type ADCC activity level greater than 60% and
adding an anti-FcγRIII antibody in the reaction mixture in order to confirm the FcγRIII-type ADCC activity level due to the selected monoclonal antibody.
2. The method of claim 1 , wherein the clone is a clone originating from a heterohybrid cell line obtained by fusion of human B lymphocytes originating from immunized individuals with murine, human or heterohybrid myeloma cells.
3. The method of claim 1 , wherein the clone is a clone originating from an animal or human cell line transfected with a vector containing a gene encoding a human IgG immunoglobulin.
4. The method of claim 1 , wherein the polyvalent IgGs inhibit a mechanism of lysis of the effector cells via a FcγRIII receptor.
5. The method of claim 1 , wherein the target cells are red blood cells treated with papain.
6. The method of claim 1 , wherein the adding comprises adding
100 μl of the purified monoclonal antibody at a concentration of 20 to 200 ng/ml, to the reaction mixture comprising
25 μl of papain-treated red blood cells,
25 μl of the effector cells,
and 50 μl of the polyvalent IgGs at a concentration of between 1 and 20 mg/ml.
7. The method of claim 1 , wherein the determining and selecting comprises comparing the lysis percentage of the target cells for the monoclonal antibody to that for two positive controls, which are a chemical compound and a reference antibody and a negative control, which is an antibody.
8. The method of claim 1 , wherein the selected monoclonal antibody is an anti-Rh(D) antibody and the target cells are rhesus D red blood cells.
9. The method of claim 7 , wherein the reference antibody is a polyclonal antibody and the negative control is a monoclonal antibody incapable of inducing clearance in vivo.