Recombinant allergens provide new opportunities

The diagnostic tools of tomorrow are already here

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Today allergenic proteins can be identified and produced in large quantities by recombinant DNA technology. Recombinant allergens can be produced with consistent quality and without genetic and biological variation. Using recombinantly produced allergenic proteins in in vitro testing provides new opportunities for designing improved tests and offers new tools to address clinical and immuno-logical questions. Recombinant allergens provide new opportunities to refine the diagnostic procedures of IgE mediated

allergy. In addition to pointing out the offending allergen source, e.g. birch pollen, it is now possible to also identify the actual protein components eliciting the allergic symptoms. Thus, in vitro tests based on recombinant allergens are useful tools to collect information on symptom triggers at the molecular level. Recombinant in vitro tests make it possible to study more complicated phenomena, such as geographic differences in clinical reactivity and cross-reactions towards seemingly distant allergens.

Recombinant allergens provide new opportunitiesThe diagnostic tools of tomorrow are already here

Birch allergen componentsProtein Common name Biological function Mw (kDa)

Bet v 1 PR-10 protein Pathogen response protein 17

Bet v 2 Profilin Actin-binding protein 15

Bet v 3 4-EF-hand calcium- Calcium-binding protein 23 binding protein

Bet v 4 2-EF-hand Calcium-binding protein 8 calcium-binding protein, polcalcin

Bet v 6 Isoflavone Isoflavone reductases 34 reductase

Bet v 7 Cyclophilin Petidylprolyl isomerase 18

Traditional allergy tests merely point out the offending allergen source, e.g. birch pollen. But such an allergen source in fact contains a number of allergenic components. With tests based on recombinant allergens the specific proteins responsible for the patient’s sensitization can be identified.

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Improved allergy testsRecombinant allergens can be used to design improved allergy in vitro tests:

Improving the clinical sensitivity of natural extractsIn cases when the natural extract has a scarcity of a specific allergenic component, addition of this recombinant protein to the extract improves the clinical sensitivity and the quantitative performance of the test. Phadia has already used this strategy with great success to design an improved version of ImmunoCAP™ Allergen k82, Latex, ensuring that all sensitized patients are really captured.

Designing optimized recombinant “extracts”Recombinant allergens may be combined to form a well-characterized composition containing an optimal amount of relevant allergenic components of a natural extract, but excluding components of little or no diagnostic value. This offers interesting opportunities for future test development.

The use of recombinant allergens in IgE antibody tests

As an additive to extracts As extract replacement As single allergen components

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Characteristic symptoms

Local(OAS)

Systemic PR-10proteins

Ns-LTPs

Sensitization profiles

Profilins

By using tests for single allergenic components as a complement to more traditional IgE antibody tests, further clinically relevant information can be gained. The possibility to investigate the sensitization to single allergenic components can shed light on phenomena that have hitherto been difficult to explain. Some examples:

Explaining geographic differences in clinical reactionsIn the northern part of Europe the characteristic symptoms of allergy to fruit and vegetables are local reactions in the mouth or throat (oral allergy syndrome, OAS), while patients in southern Europe more frequently have systemic symptoms. The explanation appears to be different sensitization profiles. Testing with recombinant single component tests shows that Bet v 1-sensitization, in all proba-bility caused by birch pollen, dominates in the north. In the southern parts, on the other hand, antibodies to lipid transfer proteins (LTPs)* dominate, indicating the predominance of true food allergy.

Explaining clinical reactivity to enable better advice to patientsTesting with single components is a useful tool to investigate and explain allergic reactions more in detail and to determine if they are caused by cross-reacting IgE antibodies to different allergens. For a patient showing symptoms when eating apples or other fruits, traditional extract-based tests will determine the source of the allergen triggering the reaction, such as apple, pear or cherry. However, the original source of sensitization could also be tree or grass pollen and the symptoms due to a

cross-reaction between allergenic components with similar structures present in both plant pollen and food proteins. Tests for single allergenic components can be used to give additional information on the source of sensitization on the molecular level – e.g. pollen molecules, LTPs or profilins – and make it possible for the physician to draw conclusions as to the clinical implications. Whereas pollen sensitized patients with symptoms during specific seasons may benefit from symptomatic treatment during the pollen season, avoidance of the offending foods may be essential for patients with LTP sensitization. These patients are also likely to develop more severe symptoms.

New tools to answer clinical questions– single component tests

Geographic differences in allergy to fruits & vegetables and sensitization profiles.

* LTPs are major allergen components in many fruits, such as peach, apple, apricots, and widely distributed throughout the plant kingdom. LTPs from botanically unrelated vegetables show a moderate to high degree of sequence homology.

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Optimized patient selection for immunotherapy

Recombinant allergens enable a more specific diagnosis which greatly improves the diagnostic base for prescribing specific immunotherapy (SIT):

Determining the sensitization profile before treatmentSingle recombinant allergen tests offer new oppor-tunities to determine whether a patient is a good candidate for SIT or not, and for suggesting the optimal therapy. If the patient’s allergic reactions are caused by sensitization to the major allergen components of a common allergen source (e.g. Bet v 1 in birch pollen), the patient is likely to respond well to immunotherapy with common extracts, as these contain a high amount of this component. On the other hand, if the patient is sensitized to another component than the major one, e.g. Bet v 2 or Bet v 4 in birch pollen, immunotherapy with extracts heavy on Bet v 1 will probably not be effective enough. There are even concerns that immunotherapy with allergenic components to which the patient is not sensitized may in fact induce new sensitization that may worsen the symptoms rather than reducing them.

Monitoring treatmentWhen SIT is used, the immunological effect of the treatment can be followed periodically by deter-mining IgE and IgG antibodies to major recombinant allergens. Furthermore, periodic determinations also make it possible to spot potential development of sensitization to minor cross-reacting components.

A possible decision tree for the treatment of birch pollen allergy

Confirm birch pollen sensitizationImmunoCAP™ Allergen t3, Birch

Component resolved diagnostics:

IgE-mediated birch pollen allergy confirmed

Clinically relevant birch pollen allergy unlikely

Allergen: Result:

rBet v 1 +rBet v 2, 4 –

High

Allergen: Result:

rBet v 1 +rBet v 2, 4 +

Medium

Allergen: Result:

rBet v 1 –rBet v 2, 4 +/–

Low

• Major birch pollen component: ImmunoCAP™ Allergen t215, rBet v 1• Cross-reactive minor birch pollen components: ImmunoCAP™ Allergen t216, rBet v 2 and t220, rBet v 4

Suitability for a birch pollen specific immunotherapy?

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CRD – the new approach in allergy diagnostics

Recombinant allergens make component resolved diagnostics, CRD, possible. With CRD the total antibody reactivity profile of an allergic patient may be identified, along with the disease-eliciting allergens and potential cross-reactivity interactions. CRD has the potential to revolutionize allergy

diagnostics. In the future, diagnoses based on detailed individual reactivity profiles at molecular level, may enable specific immunotherapy with the exact proteins that the individual has become sensitized to, and much better and more precise advice on allergen avoidance.

Phadia leads the way

Phadia has long been at the frontline in the field of recombinant allergens, offering IgE antibody tests with recombinant and purified natural allergen components. Over 10 years ago, the first recombinant allergen on ImmunoCAP™ assay platform was intro-duced. Since then, the number of single component diagnostic in vitro tests is continuously growing. Our scientists are busy developing new recombinant allergen tests, investigating their clinical importance and exploring ways to utilize them. We are convinced that recombinant allergens will become important tools not only for research purposes and specialized applications, but also in clinical routine IgE antibody testing, for the benefit of wider circles of physicians and patients.

Phadia AB. P O Box 6460, SE-751 37 Uppsala, SwedenTel +46 18 16 50 00. www.phadia.com

Head office Sweden +46 18 16 50 00 Austria +43 1 270 20 20 Belgium +32 2 749 55 15 Brazil +55 11 3345 5050 Denmark +45 7023 3306 Finland +358 9 8520 2560 France +33 1 61 37 34 30 Germany +49 761 47 8050 Great Britain +44 1 908 84 70 34 Italy +39 02 641 634 11 Japan +81 3 5365 8332 Netherlands +31 30 602 3700 Norway +47 21 67 32 80 Portugal +351 21 423 5350 Spain +34 935 765 800 Sweden +46 18 16 50 00 Switzerland +41 43 343 4050 Taiwan +886 2 2516 0925 United States +1 800 346 4364 Other countries +46 18 16 56 16

A recombinant allergen is a biotechnologically

produced allergen molecule originally identified

from an allergen extract. Most of the existing

recombinant allergens have been expressed in

Escherichia coli and are usually comparable with

their natural templates (proteins) in structural

features and immunological properties. Other

high-technological expression systems have

been developed to produce recombinant

allergens through bacteria, yeast and insect cells.

Recombinant allergens mostly have immuno-

globulin E (IgE) antibody binding capacity

comparable to that of the natural allergen and

generally show good reactivity in in vitro and

in vivo diagnostic tests.

To date, many different allergen components

from various allergen sources have been cloned,

sequenced and expressed as recombinant proteins.

What is a recombinant allergen?

1. Asero R et al. Plant Food Allergies: A Suggested Approach to Allergen-resolved Diagnosis in the Clinical Practice by Identifying Easily Available Sensitization Markers. Int Arch Allergy Immunol 2005;138:1-11.

2. Asero R et al. Immunological cross-reactivity between lipid transfer proteins from botanically unrelated plant-derived foods: a clinical study. Allergy 2002;57:900-6.

3. Mari A et al. The oral allergy syndrome: improved diagnostic and treatment methods. Curr Opin Allergy Clin Immunol 2005;5:267-273

4. Valenta R et al. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy 1999;29:896-904.

5. Kazemi-Shirazi L et al. Recombinant Marker Allergens: Diagnostic Gatekeepers for the Treatment of Allergy. Int Arch Allergy Immunol 2002;127:259-68.

6. Moverare R et al. Development of new IgE specificities to allergenic components in birch pollen extract during specific immunotherapy studied with immunoblotting and Pharmacia CAP System™. Allergy 2002;57:423-30.

7. Jutel M et al. Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol 2005;116:608-13.

8. Reisinger J et al. Allergen-specific nasal IgG antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity. Clin Immunol 2005;116:347-54.

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