Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
1999-03-25
2001-09-18
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S005000, C435S007100, C435S334000, C530S388100, C530S388350, C424S141100, C424S143100, C424S159100
Reexamination Certificate
active
06291239
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a monoclonal antibody (mAb) against human complement receptor type 2 (CR2, CD21) and a hybridoma as well as process to obtain the same. Furthermore, the invention is directed to therapeutic applications thereof.
2. Description of the Related Arts
Human CR2 (CD21) is a membrane glycoprotein of 145 kDa predominantly expressed on mature B lymphocytes (Tedder, T. F., L. T. Clement, and M. D. Cooper. 1984. Expression of C3 d receptors during human B cell differentiation: immunofluorescence analysis with the HB5 monoclonal antibody.
J. Immunol
. 133:678) and follicular dendritic cells (Reynes, M., J. P. Aubert, J. H. Cohen, J. Audouin, V. Tricottet, J. Diebold, and M. D. Kazatchkine. 1985. Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens.
J. Immunol
. 135:2687), and to a much lesser extent on peripheral blood T lymphocytes, thymocytes or astrocytes. The function of CR2 has been most extensively studied for B-lymphocytes (for a review, see Fearon, D. T. and R. H. Carter. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: Linking natural to acquired immunity.
Annu. Rev. Immunol
. 13:127). CR2 is the receptor for special CR2-binding fragments, in particular for C3dg and (with lower affinity) for iC3b, fragments of C3 that are generated during complement activation and covalently deposited on surfaces e.g. of pathogens. On B-cells, CR2 forms a non-covalent receptor complex with the B-cell specific CD19 molecule and CD81 which is broadly expressed among hematopoietic cells. Coligation of this complex and the BCR-complex lowers the threshold for B-cell activation substantially. The extent of this effect is exponentially correlated to the number of C3dg-residues on the specific antigen.
Of pathophysiological relevance, Epstein-Barr virus infection of human cells is known to require attachment to CR2 as a first step.
CR2 is a member of the family of C3b-binding and/or C4b-binding proteins and its extracellular part is formed by 15 or, as a result of alternative splicing, 16 short consensus repeats (SCR). These structural units of about 60 amino acids share a frame of several strictly conserved amino acids, most importantly four cysteine residues which are linked by disulfide bonds in a way that the first and third Cys and the second and fourth Cys form a bond, respectively.
Characterization of the two N-terminal SCRs involved in ligand binding has extensively made use of the mouse mAb OKB7 which was shown early to inhibit CR2-dependent EAC3d-rosetting of Raji cells (Rao, P. E., S. D. Wright, E. F. Westberg, and G. Goldstein. 1985. OKB7, a monoclonal antibody that reacts at or near the C3d binding site of human CR2
. Cell. Immunol
. 93:549) and to reduce infection of B-cells with EBV (Nemerow, G. R., R. Wolfert, M. E. McNaughton, and N. R. Cooper. 1985. Identification and characterization of the Epstein-Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor (CR2).
J. Virol
. 55:347). OKB7 has also been employed to demonstrate an epitope dependence regarding effects of CR2-mAbs on B-cell proliferation. However, this feature of OKB7 versus other non-blocking mAbs as HB5 (Tedder, T. F., L. T. Clement, and M. D. Cooper. 1984. Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB5 monoclonal antibody.
J. Immunol
. 133:678) is not consistently defined.
Although mAb OKB7 inhibited EAC3d-rosetting with CR2-expressing cells to >95% in our hands, it was rather inefficient in blocking CR2-dependent complement activation on Raji cells. It is, therefore, an object of the invention to generate more effective mAbs. Further objects are the generation of corresponding hybridomas and therapeutic applications. To this end, employing a baculovirus-derived soluble CR2-protein truncated after SCR 4 (Prodinger, W. M., J. Schoch, M. G. Schwendinger, J. Hellwage, W. Parson, P. F. Zipfel, and M. P. Dierich. 1997. Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector.
Immunopharmacology
38:141) as an immunogen and inhibition and removal of the binding of FITC-labelled, C3d-coated agarose microbeads to CR2 as a screening method proved to be a successful strategy.
BRIEF SUMMARY OF THE INVENTION
This and other objects are solved by a monoclonal antibody (FE8) against human complement receptor type 2 (CR2, CD21) which is able to substantially remove C3-derived fragments already attached to CR2, in particular C3dg from CR2 at temperatures of 25° C. and above. The term “substantially removes” means that C3-derived fragments (such as polymeric C3dg) are removed from fully loaded CR2 to an extent of at least 70%, preferably at least 90%. Such a mAb has beneficial properties discussed below. It is essential that the mAb (called FE8) according to the invention is not only able to block C3dg from binding to CR2 but is able to substantially remove C3dg from CR2. This has not been the case for prior art mAb OKB7. Such potential is also not disclosed in EP 358 130 (Götze).
A process to obtain such antibodies (or hybridoma cells producing such antibodies) is characterised by the steps:
a) Preparing a CR2 molecule comprising at least short consensus repeats SCRs 1 to 2 of CR2, and preferably SCRs 1 to 4 of CR2.
b) Immunising rodents, in particular mice, with a solution thereof, which is preferably made in PBS at pH 8.5,
c) Fusing spleen cells from these rodents with a myeloma cell line and culturing these fused cells, preferably with HAT medium,
d) Preparing carrier particles, coating them with a C3-derived fragment binding to CR2, in particular with C3d, and labeling them with tracers, preferably fluoresceinisothiocyanate (FITC),
e) Selecting hybridoma clones which produce monoclonal antibodies by testing, preferably by fluorescence activated cell sorting, their cell culture supernatants for the potential to remove from CR2 said coated carrier particles after they had bound to CR2-expressing cells.
Steps a) to c) are essentially prior art. However, so far it is new to use SCRs 1 up to 4. The technique of step d) is essentially known per se from an earlier publication, i. e. Prodinger, W. M., C. Larcher, M. Schwendinger, and M. P. Dierich. 1996. Ligation of the functional domain of complement receptor type 2 ( CR2, CD21) is relevant for complex formation in T cell lines.
J. Immunol
. 156:2580, but has never been used in the present context. Step e) leads to hybridoma clones (producing antibodies) with the desired properties.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1
graphically demonstrates that FE8 bound to rsCR2, but not to structurally similar FH.
FIG. 2
graphically demonstrates the monoclonal antibody FE8 completely blocks binding of particles carrying polymeric C3dg to CR2.
FIG. 3A
graphically demonstrates the amount of cell-bound monoclonal antibody and cell-bound C3dg present.
FIG. 3B
demonstrates graphically the ability of FE8 to remove bound particles.
FIG. 4A
demonstrates the view as seen by a Axiplan fluorescence microscope when viewing IIB9 under excitation wavelength (fluorescence).
FIG. 4B
demonstrates the view as seen by a Axiplan fluorescence microscope when viewing FE8 under excitation wavelength (fluorescence).
FIG. 4C
demonstrates the view as seen by a Axiplan fluorescence microscope when viewing IIB9 under light transmission (no fluorescence).
FIG. 4D
demonstrates the view as seen by a Axiplan fluorescence microscope when viewing FE8 under light transmission (no fluorescence).
FIG. 5
demonstrates graphically that FE8 reduced
3
H-thymidine incorporation dose dependently and thus the proliferation of B lymphocytes.
FIG. 6
graphically depicts that the monoclonal antibody FE8 inhibits binding of C3dg-coated human immunodeficiency virus (HIV) to CR2-expressing cells and removes C3dg-coated HIV from these cells.
DETAILED DESCRIPTION OF THE INVENTION
According to a preferred embodiment of the invent
Prodinger Wolfgang
Schwendinger Michael
Lorusso & Loud
Park Hankyel T.
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