Recombinant allergen

This invention relates to a recombinant allergen and in particular to a recombinant Fel d 1, the major cat allergen, which is functionally equivalent to the natural allergen.

Exposure to airborne particles derived from household cats (Felis domesticus) is a common cause of IgE-mediated allergy in, for example, Europe and the US (see Ichikawa, K., et al. (1999) Clin Exp Allergy 29, 754-761; Roost, H. P., et al. (1999) J Allergy Clin Immunol 104, 941-947 and Lau, S., et al. (2000) Lancet 356, 1392-1397). Indeed, cats are found in about 25% of households in Western countries and allergy to cats is found in a large part of the population, e.g. about 10% in the US. The severity of symptoms range from relatively mild rhinitis and conjunctivitis to potentially life-threatening asthmatic exacerbation.

Treatment of cat allergy by allergen injections is often employed but clinical results are variable since for cat allergy, only crude dander extract is available for treatment (see Lilja, G., et al. (1989) J Allergy Clin Immunol 83, 37-44 and Hedlin, G., et al. (1991) J Allergy Clin Immunol 87, 95.5-964) although alternative formulations have been proposed (see Norman, P. S., et al. (1996) Am J Respir Crit Care Med 154, 1623-1628 and Oldfield, W. L., et al. (2002) Lancet 360, 47-53).

Although patients are occasionally sensitised to several different molecules in cat dander and pelts, e.g. albumin and cystatin, the major allergen is Fel d 1 (i.e. Felis domesticus allergen 1, formerly termed Cat 1). The importance of this allergen has been emphasised in numerous studies. In fact more than 80% of cat allergic patients exhibit IgE antibodies to this potent allergen (see Ohman, J. L., Jr., et al. (1977) J Allergy Clin Immunol 60, 317-323 and van Ree, R., et al. (1999) J Allergy Clin Immunol 104, 1223-1230).

Fel d 1, was first described 25 years ago as the dominant cat allergen and several subsequent studies have characterised the biochemical and immunological nature of Fel d 1 (see Ohman, J. L., Jr., et al. (1974) J Immunol 113, 1668-1677; Leitermann, K., et al. (1984) J Allergy Clin Immunol 74, 147-153; Chapman, M. D., et al. (1988) J Immunol 140, 812-818; Duffort, O. A., et al. (1991) Mol Immunol 28, 301-309; Morgenstern, J. P., et al. (1991) Proc Natl Acad Sci USA 88, 9690-9694; van\'t Hof, W., et al. (1993) Allergy 48, 255-263; van Milligen, F. J et al. (1994) J Allergy Clin Immunol 93, 34-43; Vailes, L. D., et al. (1994) J Allergy Clin Immunol 93, 22-33; Counsell, C. M., et al. (1996) J Allergy Clin Immunol 98, 884-894; Kristensen, A. K., et al. (1997) Biol Chem 378, 899-908 and Batard, T., et al. (2000) J Allergy Clin Immunol 106, 669-676).

The allergen is a 35-39 kDa acidic glycoprotein containing 10-20% N-linked carbohydrates and is found in the pelt, saliva and lachrymal glands of cats. It is formed by two non-covalently linked heterodimers, each consisting of one 70 residue peptide (known as “chain 1”) and one 85, 90 or 92 residue peptide (known as “chain 2”) encoded by separate genes (see Duffort, O. A., et al. (1991) Mol Immunol 28, 301-309; Kristensen, A. K., et al. (1997) Biol Chem 378, 899-908; Morgenstern, J. P., et al. (1991) Proc Natl Acad Sci USA 88, 9690-9694 and Griffith, I. J., et al. (1992) Gene 113, 263-268). (Chain 1 shares limited (30%) sequence homology with rabbit uteroglobulin/human Clara cell 10 kDa protein (CC10) and the mature natural (n)Fel d 1 has been associated with gelatin and fibronectin degrading activity.)

Several variants of Fel d 1 have been observed and are reported in the references cited hereinabove. The primary structure of chain 1 is the sequence of SEQ ID NO 1. Reported variants of chain 1 are Lys₂₉→Arg or Asn and Val₃₃→Ser. The primary structure of chain 2 is the sequence of SEQ ID NO 2 or 3. Reported variants of chain 2 are Asn₁₉→Ser, Gly₂₀→Leu, Ile₅₅→Val, Arg₅₇→Lys, Val₅₈→Phe, Glu₆₉→Val, Tyr₇₂→Asp, Gln₇₉→Glu and Asn₈₈→Lys (see Kristensen, A. K, et al. (1997) Biol Chem 378, 899-908).

Furthermore, three inter-chain disulfide bridges linking the two peptides in native Fel d 1 have been identified, i.e. ³Cys(1)-⁷³Cys(2), ⁴⁴Cys(1)-⁴⁸Cys(2) and ⁷⁰Cys(1)-⁷Cys(2), suggesting an anti-parallel orientation of Fel d 1 peptides. See Kristensen, A. K., et al. (1997) Biol Chem 378, 899-908.

Several attempts have been made to associate the separate peptides into a native-like allergen in E coli with only partial success (see Bond, J. F., et al. (1993) Mol Immunol 30, 1529-1541; Keating, K. M., et al. (1995) Mol Immunol 32, 287-293 and Slunt, J. B., et al. (1995) J Allergy Clin Immunol 95, 1221-1228) and recently a soluble and immunoreactive chain 1-linker-chain 2-fusion expressed in baculovirus has been described in which the linker is a peptide having 19 amino acid residues.

A mix of the separate chains has proven to be useful for in vitro allergy diagnosis, but so far no soluble, stable and correctly folded recombinant (r)Fel d 1 homodimer with retained disulfide formation has been available (see van Ree, R., et al. (1999) J Allergy Clin Immunol 104, 1223-1230 and Slunt, J. B., et al. (1995) J Allergy Clin Immunol 95, 1221-1228).

Accordingly, the present invention provides a recombinant Fel d 1 fusion product comprising a Fel d 1 chain 1, a Fel d 1 chain 2 and a linker selected from a carbon-nitrogen bond or a short-peptide linker which links the N-terminal amino, acid of one chain to the C-terminal amino acid of the other chain.

The present invention will now be described with reference to the following drawings, in which

FIG. 1 shows the primary structure and disulfide bridges of the recombinant His-tagged rFel d 1(2+1) fusion molecule;

FIG. 2 shows the results of size exclusion chromatography of rFel d 1(2+1);

FIG. 3 shows the deconvoluted electrospray mass spectrum from analysis of rFel d 1(2+1);

FIG. 4 shows the electrospray mass spectrum from analysis of non-reduced rFel d 1(2+1) digested with trypsin;

FIG. 5 shows the time-dependent dissociation analysis of immobilised rFel d 1(2+1) by surface plasmon resonance;

FIG. 6 shows the result of a far-UV CD analysis of rFel d 1 and nFel d 1;

FIG. 7 shows the IgE responses to Fel d 1 in individuals sensitised to cats in direct ELISA;

FIG. 8 shows the dose-dependent inhibition of serum IgE from a pool of individuals sensitised to cats; and

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