Antibodies against E-selectin

Details Antibodies against E-selectin Antibodies against E-selectin

1996-11-25

2001-03-20

Saunders, David (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C530S387300, C530S388220, C530S416000

Reexamination Certificate

active

06204007

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to antibodies having specificity for E-selectin characterised in that said antibodies are whole antibodies of neutral isotype, to processes for preparing said antibodies, to pharmaceutical compositions containing said antibodies, and to medical uses of said antibodies.
BACKGROUND OF THE INVENTION
Selectins are a family of structurally related transmembrane glycoproteins implicated in the adhesion of leukocytes to vascular endothelial cells. The three known members, designated E-, P- and L- selectin are composed of three types of domain, an amino terminal C-type lectin domain, one EGF-like domain and between two and nine complementary regulatory repeats. Stimulation of endothelium by inflammatory cytokines e.g. II-1, or TNF results in the upregulation of E-selectin expression on the cell surface.
Experiments in vitro have shown that E-selectin can support the adhesion of polymorphonuclear cells, monocytes and a subpopulation of T-lymphocytes (see for example, Bevilacqua et al (1989) Science 243 1160-1165; Picker et al (1991) Nature 349 796-799 and Leeuwenberg et al (1992) Scant. J. Immunol 35 335-341). Mouse antibodies to E-selectin that block PMN binding in vitro have been shown to reduce extravasation of PMNs (neutrophils) in animal models (Mulligan, M. et al J. Clinical Investigation 88, 1396-1406 (1991) and Gundel, R. et al J. Clinical Investigation 88, 1407-1411 (1991)).
E-selectin thus appears to play a key role in the movement of leukocytes to sites of inflammation due to injury or infection. A corollary of this is that the expression of E-selectin is increased in certain inflammatory diseases. Hence E-selectin contributes to the disease process by supporting the adhesion of leukocytes which in turn cause tissue damage. It follows that an antibody to E-selectin that blocks this process would attenuate the extent or severity of the inflammation and hence be of therapeutic benefit.
Since most available monoclonal antibodies are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response. Therefore, the use of rodent monoclonal antibodies as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological response to the antibody and will either remove it entirely or at least reduce its effectiveness.
Proposals have been made for making non-human MAbs less antigenic in humans using engineering techniques. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule. A simple form of engineering antibodies involves the replacement of the constant regions of the murine antibody with those from a human antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81 6851-55; Whittle et al (1987) Prot. Eng. 1 499-505). The lowering of the level of the HAMA response to the chimeric antibodies leads to the expectation that further engineering of the variable region outside of the antigen binding site may abolish the response to these regions and further reduce any adverse response.
A more complex form of engineering of an antibody involves the redesign of the variable region domain so that the amino acids constituting the murine antibody binding site are integrated into the framework of a human antibody variable region. This has led to the reconstitution of full antigen binding activity in a number of cases (Co et al (1990) J. Immunol. 148 1149-1154; Co et al (1992) Proc. Natl. Acad. Sci. USA 88 2869-2873; Carter et al (1992) Proc. Natl. Acad. Sci. 89 4285-4289; Routledge et al (1991) Eur. J. Immunol. 21 2717-2725 and International Patent Specifications Nos. WO 91/09967; WO 91/09968 and WO 92/11383).
Naturally occurring and engineered human antibodies may be regarded as bifunctional agents, with the N-terminal variable region responsible for antigen binding and sequences within the C-terminal part responsible for determining interactions with the various cell types which participate in immune responses. Recognition of these effector sites on antibodies by specific cell surface receptors on cytotoxic cells can result in antibody-dependent cellular cytotoxicity and complement mediated lysis. This can result in killing of the cell presenting the antigen.
E-selectin is expressed on the surface of endothelial cells. The loss of endothelial cells as a result of antibody bound to target antigen is highly undesirable. Endothelial cells make up the endothelium which forms a barrier between the tissues of the body and the vascular system. The loss of or damage to the structural integrity of the endothelium is extremely disadvantageous and can lead to oedema and vasculitis. It is highly advantageous therefore to avoid depletion of the endothelial cell population while blocking the target antigen. Recent papers by Podolsky et al (J. Clin. Invest. 92 (1993) 372-380), Westphal et al Clin. Exp. Immunol. 96 444-449 (1994), and the Editorial Lancet 337 (1991) confirm that the use of whole antibody is undesirable due to undesirable effector functions mediated via the Fc region of the antibody. Another group has attempted to overcome the problem of undesirable effector functions by the use of antibody fragments lacking the effector signals which result in antibody-dependent cellular cytotoxicity (Mulligan et al J. Clinical Investigation 88, 1396-1406 (1991)). Antibody fragments are known, however, to have a short half-life (Pimm et al Nuclear Medicine Communication 10, 585-593 (1989); Molthoff et al Br. J. Cancer 65, 677-683 (1992) and Buist et al Cancer Res. 53 5413-5418 (1993)) making their therapeutic usefulness in the treatment of many diseases extremely limited.
BRIEF SUMMARY
The present invention provides a novel solution to this problem of preventing depletion of the target endothelial cell population. We have found that by making a whole anti-E-selectin antibody of neutral isotype it is possible to produce a therapeutically useful antibody which does not result in endothelial cell depletion.
In a first aspect the invention therefore provides an antibody having specificity for E-selectin characterised in that said antibody is a whole antibody of neutral isotype.
In a preferred embodiment of the first aspect of the invention the antibody has specificity for human E-selectin.
The antibodies according to the invention preferably recognise the E-selectin lectin or EGF domain.
As used herein the term ‘whole’ antibody is used to denote an antibody comprising substantially full length heavy and light chains, and antibodies to which amino acids have been substituted, altered, added and/or deleted.
The approach of using a whole antibody of neutral isotype has not been tried before in this area. The term ‘neutral isotype’ means that the interactions with antibody Fc receptors i.e. FcRI, FcRII and FcRIII and complement are absent or so weak as to cause minimal detrimental physiological effects such as antibody dependent cellular cytotoxicity (ADCC) and/or complement mediated lysis and also the antibody produces a minimal immune response in the host. As used herein the term ‘minimal immune response’ is used to denote a typical primate immune response to an iv injection of a human or engineered human antibody.
Anti-E-selectin antibody may be prepared using well-known immunological techniques employing E-selectin as antigen. Any suitable host may, for example, be immunised with E-selectin or activated HUVEC (human umbilical vein endothelial cells) and splenocytes or lymphocytes recovered and immortalised using for example the method of Kohler et al, Eur. J. Immunol. 6, 511 (1976). The resulting cells are diluted and cloned to obtain a single genetic line producing anti-E-selectin antibodies in accordance with conventional practice. Where it is desired to produce recombinant anti-E-selectin antibodies these may be produced using methods well known in the art.
Several regions of the Fc region of antibodies have been

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