Preparation of human, humanized or chimaeric antibodies or polypeptides having different binding profiles to Fcgamma receptors
The present invention relates to a method for the production and the selection of human or chimæric or humanized antibodies or molecules that comprise the Fc region of human IgG, capable of modulating the activity of one or several particular Fc receptors, such as the triggering of inhibitory functions through the human type IIB receptors of IgG (FcgammaRIIB/CD32).
1. A method for the preparation of human, humanized or chimæric antibodies or polypeptides having different binding profiles, comprising an Fc region of human IgG, wherein said method comprises:
a. providing candidate human, humanized or chimæric antibodies or polypeptides comprising the Fc region of human IgG produced naturally by or following transfection with a vector comprising the coding sequence for said antibody or polypeptide into animal cell lines comprising, EBV-transformed human B cell lines or from eukaryotic microorganisms,
b. testing the binding of said antibodies or polypeptides on Fcgamma receptors FcgammaRIIIA, FcgammaRIIA and FcgammaRIIB, and
c. selecting antibodies or polypeptides which:
ii. bind to FcgammaRIIA and FcgammaRIIB but do not bind to FcgammaRIIIA or bind to FcgammaRIIIA with an ID50 superior or equal to 0.2 microM.
2. The method according to claim 1 wherein said antibodies or polypeptides selected in step:
c. ii) are produced by cells from a non-lymphoid cell line.
3. The method according to claim 2 , wherein
iii. said non-lymphoid cell line is CHO (ATCC number CCL-61) or a cell line derived thereof.
4. The method according to claim 1 , wherein said binding is performed using:
i. indicator cells from a cell line that express different Fc receptors on their cell surface, or
ii. recombinant Fc receptors comprising FcgammaR ectodomains, Fc receptors derived-peptides.
5. The method according to claim 1 , wherein said antibodies or polypeptides selected for their ability to bind to FcgammaRIIA and FcgammaRIIB are further selected by a functional assays for their ability
ii. to trigger FcgammaRIIB leading to the inhibition of calcium mobilization and to the inhibition of cytokine production such as IL-2 by cells expressing FcgammaRIIB such as B cells and monocytes.
6. The method of claim 5 , wherein said functional assays consist of a calcium mobilization inhibition assay, and/or a cytokine secretion inhibition assay.
7. The method of claim 5 , wherein said functional assay further comprises a specific FcgammaRIIIA ADCC assay.
8. A method of producing antibodies in a non-lymphoid cell line wherein said antibodies bind FcgammaRIIA and FcgammaIIB but do not bind to FcgammaRIIIA or bind to FcgammaRIIIA with an ID50 superior or equal to 0.2 microM.
9. The method of claim 8 , wherein said antibodies are immunomodulatory antibodies.
10. The method of claim 8 , wherein said antibodies induce ADCC and phagocytosis by monocytes and macrophages expressing FcgammaRIIA.
11. The method of claim 8 , wherein said cells from a non-lymphoid cell line is CHO (ATCC number CCL-61).
12. The method of claim 6 , wherein said functional assay further comprises a specific FcgammaRIIIA ADCC assay.
13. The method of claim 9 , wherein said antibodies induce ADCC and phagocytosis by monocytes and macrophages expressing FcgammaRIIA.