Transition of recombinant allergens from bench to clinical application
Introduction
The specific diagnosis and causal treatment of IgE-mediated allergic diseases have relied traditionally on the use of aqueous extracts of various allergenic source materials. The majority of such extracts are complex mixtures of proteins, only some of which exhibit allergenic characteristics. In the cases of two of the most commonly encountered causes of allergic responses, namely grass pollen and the house dust mite Dermatophagoides pteronyssinus, at least 11 and 16 allergens, respectively, have been identified and well characterised [1], [2]. Different allergic patients exhibit different patterns of allergen recognition, and whilst in some cases only one or two allergens may be implicated with an allergic sensitisation, in others a whole spectrum of proteins may be involved. Attempts are often made to define the relative importance of allergens in terms of the frequency with which sensitisation can be identified in populations of allergic patients. So-called major allergens have traditionally been defined as those which can be associated with sensitisation in more than 50% of subjects showing sensitisation to a particular allergenic source material and conversely minor allergens are reactive in relatively few subjects. The major allergens are the focus of attention in the development of recombinant molecules, since these are the ligands for a large proportion of the allergen-specific IgE antibodies that trigger allergic reactions.
The production of allergen extracts of consistent quality from natural source materials places considerable demands on manufacturers. The demonstration of consistent protein patterns and IgE-reactive allergens, together with quantification of individual allergens and measurement of total allergen-specific IgE-reactivity of the extract, represent important aspects in the standardisation, characterisation, and batch consistency of the extracts [3]. Recombinant DNA technology not only facilitates the characterisation and analysis of the allergenic proteins, but also provides the basis for producing allergens and their derivatives that may be able to be used to formulate optimal preparations for both diagnostic applications and specific causal immunotherapy [4], [5].
Diagnostic techniques depend on the ability to demonstrate the existence of specific IgE antibodies directed against an allergen preparation and therefore both in vitro and in vivo diagnostic methods require the use of allergens in their native form that will react with such antibodies [6], [7]. Strategies for allergen-specific immunotherapy rely on either the native forms of the allergens or modified forms with attenuated IgE-reactivity. The latter have traditionally been derived by chemical modification of the allergen extracts, but the advent of DNA technology has provided the opportunity to develop and produce hypoallergenic allergen variants using various strategies for gene mutation [8], [9], [10]. One advantage of such preparations for specific immunotherapy is that the risk of inducing unpleasant side-reactions can be minimised whilst the therapeutic potential of the preparations is retained. Further advantages of recombinant allergens and their derivatives include the production of preparations of consistent pharmaceutical quality; the avoidance of problems of natural extract standardisation; the inclusion of optimal concentrations of the important allergens; the exclusion of non-allergenic proteins; the avoidance of the possible risk of contamination by allergens from other sources; and exclusion of the risk of introducing infectious agents.
Grass pollen is one of the most important causes of allergic rhinitis and is therefore a logical choice to assess the potential of recombinant proteins for allergen-specific immunotherapy. However, there are several major grass pollen allergens and it is probable that a cocktail of various proteins will be required to effect successful treatment. On the other hand, birch pollen has one predominant allergen, Bet v 1, and it is quite conceivable that patients could be desensitised with this allergen alone. Some of the methods used to develop and produce various wild-type allergens of grass pollen, in particular Phl p 1, Phl p 2, Phl p 5b, Phl p 6, and Phl p 13 from Phleum pratense (Timothy grass), are described here, to exemplify various factors that have to be taken into consideration when producing preparations for use in the clinic.
Section snippets
Choice of expression system and design of DNA constructs
The heterologous production of allergen proteins for therapeutic use has the advantage that in most cases one deals with major allergens where the natural counterparts of the recombinant proteins have already been purified and studied in detail. Knowledge about the glycosylation state, the occurrence of internal disulphide bonds, the N-terminal amino acids of the mature allergen, and the overall stability of the protein represents useful information that has to be taken into consideration for
Preparation of first strand complementary DNA
RNA was isolated from 20 mg timothy grass pollen (Phleum pratense) using a GlassMax RNA microisolation system (Life Technologies, Karlsruhe, Germany), while reverse transcription of poly(A+)RNA was performed using a 3′RACE system (rapid amplification of cDNA ends) and an oligo(dT)-containing adaptor primer (Life Technologies) according to manufacturer’s instructions.
PCR-based cloning and sequencing of timothy allergen cDNA from Phl p 13
A degenerate oligodeoxynucleotide primer (Biometra, Göttingen, Germany) was designed according to the reverse translated N-terminal
Practical considerations/potential problems
The transition of recombinant allergens from the bench to clinical application can be divided into three phases. First, the development of a recombinant allergen as a diagnostic tool or agent for specific immunotherapy is carried out at the bench in research laboratory scale, beginning with the identification and cloning of the gene of the allergen in question. Different expression systems have to be tested and different genetic variants of the allergen produced prior to characterisation and
References (42)
Clin. Exp. Allergy
(2000)- et al.
Int. Arch. Allergy Immunol.
(2002) - Allergen Products. Monograph, European Pharmacopoeia (1997)...
Nat. Rev. Immunol.
(2002)- et al.
Int. Arch. Allergy Immunol.
(2001) - et al.
FASEB J.
(2002) - et al.
Allergy
(2002) - et al.
J. Immunol.
(1999) - et al.
Int. Arch. Allergy Immunol.
(1999) - et al.
Int. Arch. Allergy Immunol.
(2002)
Eur. J. Biochem.
Clin. Exp. Allergy
Int. Arch. Allergy Immunol.
Int. Arch. Allergy Immunol.
PNAS
Protein Expr. Purif.
Mol. Biotechnol.
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