Methods and means for modifying complement activation

Details Methods and means for modifying complement activation Methods and means for modifying complement activation

1999-06-11

2001-12-11

Saunders, David (Department: 1644)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

C514S012200

Reexamination Certificate

active

06328968

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to the field of immunology, in particular molecular immunology and uses thereof in modern medicine. In particular the invention provides methods and means that find their use in the field of modifying the complement activation cascade and provides methods and means for interference with immune reactions by manipulation of CD97-CD55 interaction”.
SUMMARY OF THE INVENTION
The invention provides means and methods to (down) regulate the complement activation pathway and other interactions of the immune system. This is of particular interest in the area of transplantations of organs and/or tissues and/or cells from donors to recipients. To avoid immunologic responses from the host against the transplanted material (the graft) careful matching of the immunological profile of both host and recipient is required. This requirement makes the availability of transplants or grafts a limiting factor in transplantation medicine. It would therefore be a major step forward if this requirement could be avoided so that many more donors become available for transplantation purposes. In working towards a suitable source for transplantable organs and tissues porcine material has been considered. Transplanting porcine material into humans would normally result in an immunogenic reaction, usually a hyperactive rejection of the transplant. For this reason transgenic pigs have been developed which express CD55 decay accelerating factor on the surface of their cells. CD55 plays an important role in complement inactivation as explained below. In short CD55 inhibits two important convertases in the complement activation pathway.
Thus it was thought that by providing the porcine transplant with CD55 the complement activation could be inhibited. It has now been found that the solution for avoiding the hyperimmune rejection may not be that simple, because of the role of the CD97 protein. The present invention identifies that problem and solves it.
The invention also provides means and methods to regulate early and late interactions that play a role in (auto)immune disease. Autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, SLE, diabetes and others, are diseases of which the pathogenetic mechanisms are generally not well understood, but whereby a complex of immune reactions as diverse as cytokines activation, activated cells of the immune system, antibody formation and complement activation are alone and in combination directed against “self” components of the patient's body. Such a cascade of immune reactions can lead to a disturbance of the patient's immunological homeostasis which, when not sufficiently mitigated by the body's own system of immune regulation, lead to serious and life threatening disease. Also, acquired immune deficiencies or normal immunologic reactions to infectious agents and the like can lead to a similar immune reaction that is in itself harmful to a patient. CD97 is an antigen which becomes immediately upregulated on most leucocytes during activation (1). We recently identified CD97 as a 7-TM molecule whose membrane-spanning region is homologous to the secretin receptor superfamily (2). CD97 is different from this group of mammalian and insect peptide hormone receptors (3), in that it has an extended extracellular region with three to five EGF domains at the N-terminus. The finding of a highly similar architecture in EMR
1
(4), which possesses six EGF domains, and its probable murine homologue F4/80 (5) indicates the existence of a new group of 7-TM receptors characterized by several N-terminal EGF domains. We have demonstrated that this new type of 7-TM molecule has recently evolved by exon shuffling to the upstream region of an ancestral gene from the secretin receptor superfamily (6). All EGF domains in CD97 and EMR1, except the most N-terminal ones, possess a calcium binding site. The Ca
2+
in this subgroup of EGF domains stabilizes the conformation of the domain and can mediate contact to other proteins (7). The rather recent acquisition of EGF domains raised the possibility that CD97, in parallel with its molecular evolution, has acquired the ability to bind cellular ligands. To explore this, COS cells transfected with CD97 cDNA were incubated with different cells of haematopoietic origin. As shown in
FIG. 1
, adherence of peripheral blood lymphocytes (PBL) and erythrocytes to COS cells expressing CD97 was observed which was completely abolished by CD97 mAbs CLB-CD97/1 (see the experimental part) or BL-Ac/F2 (1). Once the binding studies indicated that a ligand for CD97 is expressed on erythrocytes, immunization of mice with human erythrocytes was used to generate a ligand-specific mAb. One mAb (CLB-CD97L/1) was identified that blocked the adherence of both, erythrocytes and PBL to CD97-transfected COS cells. This mAb recognizes a 70-kD protein (FIG.
2
A). Among antigens of this size studied within the Fifth International Leucocyte Typing Workshop, a small number was found to be expressed by both erythrocytes and lymphocytes (8). When we tested the capacity of mAbs directed against these antigens to block the binding of erythrocytes to CD97 transfectants, IA10, a mAb recognizing CD55 (9) turned out to be inhibitory. A direct comparison of CLB-CD97L/1 with IA10 revealed that both mAbs immunoprecipitate not only the same major protein of 70 kD which characterizes the CD55 antigen (10), but also a minor band at 140 kD which represents dimeric CD55 (11) (FIG.
2
A). Furthermore, IA10 completely blocks the binding of biotinylated CLB-CD97L/1 to PBL (FIG.
2
B), indicating that the mAb generated to the ligand of CD97 recognizes the same epitope as IA10 that has been previously mapped to the first (of four) SCR of CD55 (12). CD55 or decay accelerating factor (DAF) is a GPI-anchored molecule expressed by all blood cells and cells in contact with blood and tissue fluid that protects from complement-mediated damage by inhibiting C3/C5 convertases (10). To investigate the specificity of the interaction between CD97 and CD55, a larger panel of CD55 mAbs, directed against distinct SCR domains within the molecule, was tested in the above described adherence assay. As shown in
FIG. 3
, inhibition of erythrocyte adhesion to CD97-transfected COS cells between 23 and 92% was observed. The finding that also mAbs mapping to the second (BRIC110) and third (BRIC216) SCR of CD55 are able to block suggests that these domains in addition to the first SCR are involved in ligation of CD97. The ability to dissociate and prevent assembly of C3/C5 convertases in both the classical and alternative pathway of the complement cascade has recently been mapped to the SCR domains two, three and four (12,13). It was shown that SCR1 is not required for DAF function, but SCR2, SCR3 and SCR4 are each necessary for DAF to exert its protective effect with regard to complement activation. Removal of SCR1 was associated with a small increase in inhibitory activity. The finding that the first SCR of CD55 is involved in adhesion to CD97 is the first demonstration of a molecular function for this domain.
Further evidence that CD97 specifically interacts with CD55 came from the observations that erythrocytes lacking CD55 expression fail to adhere to CD97 transfectants. First, paroxysmal nocturnal hemoglobinuria (PNH) is an acquired somatic defect in GPI-anchor synthesis that leads to the absence of GPI-anchored molecules (14). Due to the clonal character of this hematopoietic stem cell disorder, CD55-positive erythrocytes remain that can adhere to CD97 transfectants. However, after depletion of the unaffected, CD55-expressing erythrocytes, a complete abrogation of adherence was seen (FIG.
4
A). Second, the Inab phenotype represents an inherited deficiency in CD55 expression due to truncative mutations in the CD55 gene (15). Erythrocytes with this phenotype were not able to bind to COS cells expressing CD97 (FIG.
4
B). Further investigation towards the specificity of the interaction between CD97 and CD55 demonstrated the specific

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