CA2148007A1

CA2148007A1 - Allergenic extracts

Info

Publication number: CA2148007A1
Application number: CA002148007A
Authority: CA
Inventors: Vinay Lucas Koshte
Original assignee: Individual
Current assignee: Individual
Priority date: 1992-11-03
Filing date: 1993-11-03
Publication date: 1994-05-11
Also published as: JPH08505126A; EP0667788A1; AU682669B2; WO1994009824A1; AU5372694A

Abstract

This invention relates to a preparation of allergenic (antigenic) extracts useful in a systematic approach to diagnosis, immunotherapy and the monitoring thereof of allergic patients, and also to be used in diagnosis of other immunological disorders. The method comprises of an initial quick extraction step, wherein most of the immunologically irrelevant material is removed in the solute and discarded. The allergenic residue is recovered and is repeatedly extracted while retaining the solute, resulting in a sequence of allergenic extracts that are immunologically potent.

Description

2 ~ (3 r~

Aller enic extracts The present invention relates to allergenic (antigenic) extracts to be used in diagnostics of immunological disorders preferably allergic diseases, the immunotherapy and the monitoring of the immunotherapy in allergic patients and to a method for the preparation of the same.
People who suffer from allergy have allergic (reaginic) antibodies, called IgE antibodies. These antibodies combine with the offending substances, called allergens (antigens) to manifest an allergic reaction. It has been a practice to ext~act these allergens from their source to be used for diagnostic and therapeutic purposes.
The prior art methods used in preparation of an allergenic extract especially for inhalation allergens such as pollens, mites, insects, molds, aller~ens from foods and other sources are to extract the source material in water - or various solutions from two hours to overnight or more.
This extract is then dialyzed to remove low molecular compounds and then lyophilized. In vivo and in vitro diagnostic test systems based on this so called crude 2S extract show manifestation of allergic reaction and presence of IqE antibodies but fails to give more information which the clinician requires for the proper management of the patient. The extract so obtained contains about 90~ irrelevant waste material consisting of carbohydrates, lipids, enzymes, toxic materials, irritants and number of other molecules which have no significance in immune response. This makes the test systems less sensitive and inaccurate. When used for the purpose of immunotherapy, the crude extract has not delivered any significant results and the presence of the waste materials causes mild to serious side effects during immunotherapy. Moreover this crude extract is not suitable in immunoassays to monitor the levels of IgG antibodies, the so called "blocking ~ ~ ~ 2 antibodies" during immunotherapy, because of the interference and high background level caused by irrelevant nonimmunological materials in the crude extract.
Among the irrelevant waste materials, the crude extract contains only about 10% of number of different allergenic molecules. Attempts are also made to obtain purified allergens from these crude preparations through techniques such as gel filtration, ion exchange chromatography, monoclonal antibodies, DNA technology, synthetic peptides etc. Until now the use of these purified allergens have not led to any significant improvement in diagnosis, immunotherapy or monitoring the effects of immunotherapy. Because the purified allergens become denatured as a result of the purification processes and the immune system fails to recognize these denatured purified allergens.
Extensive literature is available on ~he subject of methods for the preparation of allergenic extracts. The U.S. patents 3.591.677; 3.798.319; 4.031.199; 4.774.226 and 4.963.356 describe various methods and processes to prepare allergenic extract. In all of these methods the aller~enic source material is initially extracted durinq 16 hours to as long as 5 days. Contrary to the conventional methods, the U.S. patent 4.364.938 employs a method wherein the source material is repeatedly extracted. The extracted solutes containing most of the allergenic activity is discarded and the residue is recovered. This residue either directly or after further treatment is suspended in 0.9%
saline and administered to the patient by way of injections. The allergen release from the source material has also been studied by a number of investigators among them Marsh et al. J. Allergy Clin. Immunol. 1981:67.206-16 and Baraniuk et al. J. Allergy Clin.Immunol 1988: 81.1126-34.
To summarize problems in allergy, the crude extract contains too much irrelevant material and the purified allergens add to the complexity of the problem because most patients have IgE antibodies to more than one wo g4,09824 ~ ~ 4 8 ~ O I PCT/EP93/03041 . .

allergen in that particular source material. The method of the present invention overcomes these problems and it has neither been disclosed nor suggested in the prior art.
It is the object of this invention to provide a novel process for the preparation of allergenic (antigenic) extracts which contain almost none of the immuno1Ogically irrelevant waste materials.
It is the object of this invention to provide a systematic approach to diagnosis of patients with immunological disorders preferably al~ergies, a systematic approach to immunotherapy of an allergic patient and a systematic approach to monitoring the allergic patients who are undergoing immunotherapy.
I have unexpectedly found that after the extraction of ~he source material according to the conventional methods, the residue which is left behind and discarded contains more immunological activity than the extract itself. After further`experimentation it became quite clear that most of this immunogenic activity was in fact of allergenic nature and it depended upon the rate of release of allerqenic molecules from the source material.
The present invention provides a method to prepare an allergenic residue obtainable by extracting the allergenic source in an aqueous solvent whereby this extraction does not last longer than about five minutes.
The solute containing most of the immunologically irrelevant material is discarded and the allergenic residue is recovered. Optionally this procedure may be repeated depending upon!the allergenic source material. As soon as the extraction medium comes in contact with the source material, most of the irrelevant material immediately enters into the solution whereas there is a slight delay of the allergenic molecules to enter into the solution. This time of delay of the entry of the allergenic molecules into the solution is variable in each particular allergenic source material. Therefore outermost care should be taken to minimize the duration of this extraction step. The purpose of this step is the removal of most of the W094/09824 ~ PCr/EP93/0304l 2 ~

irrelevant waste material into the solute with minimum or no loss of allergenic molecules. The allergenic residue so obtained is then subjected to repeated extractions while retaining the solute in the following order to obtain a series of sequential extracts:
A. At least one extraction in a suitable aqueous solvent, the total extraction time lasting not longer than four hours. These extracts contain the rapidly released allergens.
B. At least one extraction in said solvent, each lasting four hours or more till almost no allergenic activity is released in extracted solute from the preceding allergenic residue. This can last longer than one day.
These extracts contain the slowly releasing allergens.
C. At least one extraction in acidic solution, each extraction lasting not longer than thirty minutes; or by the stepwise gradual increase of the molarity of the acidic solution resulting in a series of sequential extracts, till almost no immunogenic activity is released in the extracted solute from the preceding residue. These extracts contain the basic allergens.
D. At least one extraction in alkaline solution, each extraction lasting not longer than thirty minutes; or by the stepwise gradual increase of the molarity of the alkaline solution resulting in a series of sequential extracts, till almost no immunogenic activity is released in the extracted solute from the preceding residue. These extracts contain the acidic allergens.
Each allergenic source material has a different composition of aliergenic molecules with varying degrees of rate of release into a given solution. In some allergenic source materials all the allergenic molecules are released into the solution within 36 hours, while in others it continues to be released even after 4 days. Therefore the parameters of time, volume and pH for all the steps during extraction need to be adapted to the characteristics of each particular allergenic source material so as to obtain the extracts with optimum allergen content and functional 2 1 ~ 7 e~ficiency. However, It should be noted that during each extraction step the allergen is constantly being released in to the extraction medium and therefore even a shortest extraction step will contain some allergenic activity.
In a preferred embodiment the material from which the allergens are to be extracted is gently shaken 1:10 w~v in a solvent such as water or any physiological buffer system at about neutral pH, preferably physiological phosphate saline (pH 7.4) for not longer than about five minutes. This solution is immediately filtered. The solute is discarded and the allergenic residue is recovered. If necessary this step can be repeated one more time depending upon the source material. This procedure is very important and critical, and extreme care should be taken to minimize the time duration. All these procedures can be performed at room temperature but preferably at 4C. The recovered allergenic residue is then extracted with the same solvent of about the same volume. The extraction carried out for at least one hour. The solution immediately filtered~ The filtrate is retained and is called Extract A-I and the allergenic residue is recovered. This step is repeated one more time to obtain Extract A-II. The two filtrates mixed together and named Extract A.
The recovered allergenic residue is then extracted in the same manner except for longer than 4 hours and this procedure is repeated over the period of 24 hours or more till the last filtrate contains almost no allergenic activity. The number of times this procedure is to be repeated, d!epends upon the allergen release from the repeatedly extracted allergenic residue which is the function of that particular allergenic source material. The - filtrates so obtained are mixed together in one pool to prepare Extract B or more pools in their sequential order to prepar~ Extract B-I or Extract B-II, etc.
The recovered allergenic residue is quickly washed with distilled water. Then It is extracted with an acidic solution such as acetic acid of about 0.1 M for not 2 ~ ~ PCTJEP93/03041 longer than thirty minutes, preferably about 10 minutes.
The solution is filtered. The filtrate is retained and the pH brought to neutral. The allergenic residue is recovered.
This is repeated for 2 times or more. These flltrates pooled togethPr to obtain Extract C Acidic or more pools in their sequential order.
The allergenic residue is then quickly washed twice with water and then extracted with alkaline solution such as sodium hydroxide solution of about 0.1 M for not longer than thirty minutes, preferably about 10 minutes.
The solution is filtered. The filtrate is retained and the pH thereof brought to neutral. The allergenic residue is recovered. This is repeated for 2 times or more. These filtrates pooled together to obtain Extract D Basic or more pools in their sequential order.
It is preferred that Extract C Acidic and Extract - D Basic should be equilibrated against physiological solution before being used.
Extract A contains rapidly released allergens 2G which are mostly so called minor allergens and part of slowly released allergens from the allergenic residue.
Extract B contains slowly released allergens which are mostly so called major allergens. About 80% of allergic patients have reaginic i.e. IgE antibodies to major allergens. Therefore Extract A optionally combined with the first solute of Extract B is to be used for skin prick test and in immunoassays as a screening test for detection of IgE antibodies in allergic patients. Extract B is the follow up test to the Extract A. Since Extract B contains major allergens, It detects approximately 80% patients from Extract A positive patients. On basis of the result of Extract B test, the clinician will be able to decide and select the extract to be used for immunotherapy. The presence of allergenic activity in the Extract C Acidic depends upon the source material, whereas the Extract D
Basic besides the allergenic molecules also contains a large quantity of antigenic proteins. These extracts play a significant role in the assessment, planning and management 2 1 ~ ~ ~ O 1 of the allergic disease~ Moreover Extract C Acidic, Extract 3 Basic and their fractions are of great importance in the diagnosis of patients with diseases of other immunological disorders.
These extracts are prepared for immunotherapy according to standard methods that are known for the purpose of their administration to allergic patients. These extracts may be ~urther subjected to mild chemical treatment by various prior art methods wherein the extracts lose their allergenic reactivity while retaining their full potency for immunogenicity. The patients who have IgE
- antibodies only to the screening test and not to Extract should be treated with Extract A or Extract A-I and the immunotherapy will be monitored on the same extract. The patients who have IgE antibodies to Extract B will be initially treated with Extract B and monitored on the same extract. If necessary this immunotherapy should be followed by Extract A or Extract A-I. The patients with low titer of IgE antibodies to Extract B could be considered for the treatment with ~xtract A. Extract C Acidic and Extract D
Basic can be used alone or in mixture with other extracts of the same source material. This depends upon the presence of IgE antibodies in the allergic subject. But since Extract D Basic contains large quantity of antigens other than allergens, the use of this extract must be approached with extreme caution. These extracts are also to be used to monitor the patient during immunotherapy to their respective extract.
The present invention is not restricted to any particular source material. However, plant pollen from all kinds of grasses, weeds, trees, flowers, etc. can be extracted by this method. Animal epithelia from whole skin, hair, feather and dander can be extracted by this ~!~NlENDE~ SHEET

7 ~
~ 8 method. All kinds of fungi like moulds, yeasts, rusts, smuts, etc. can be extracted by this method. All bacteria and viruses may also be extracted by this method. All kinds of insect bodies and all kinds of mites can be extracted according to this method. This method can be used to extract allergens of the house dust. Food material of plant and animal origin can also be extracted by this method. For each particular source material the parameters of time, volume and pH for all the steps during extraction should be determined skillfully and should be carefully selected.
These extracts are better than any conventional extracts until now, because the initially discarded solute as a result of a very quick extraction step removes most of the irrelevant material which has no immunological role to play in immune response. As a result the major cause related to the the problems of false background in the diagnostic and the monitoring test systems is removed as well as the cause of serious side effects during immunotherapy is removed. These extracts provide a unique advantage that it makes it possible to test, treat and monitor the patient on the same material. The extracts obtained from the recovered allergenic residue in subsequent steps are almost purified, immunologically potent and far superior as compared to any conventional extract. The allergens in these extracts are in a native state as opposed to all other methods of allergenic preparation and their purification, where at times the allergen extract has undergone a harsh treatment resulting in denaturation of the allergenic molecules. The extracts obtained with this method have a much longer shelf life, because of the absence of carbohydrates and proteolytic enzyme activity which is often the cause of deterioration ' of the quality of the conventional extracts.
The allergenic extracts so obtained are very comparable to the situation in vivo. During immunotherapy these allergenic molecules are presented to the antigen presenting cells in their native form. The processes within the antigen presenting cells will be able to produce a W094/09824 2 1 4~ PCT/EP93/03041 right kind of antigen peptides ~hich are in a recognizable form to the T-cell, so that the T-cell can modulate a correct immune response and now the immune system can perform it's task.
Furthermore the extracts obtained by this method are economically and immunologically much more relevant for it's practical application and clinical consequences than the purified allergens obtained by other techniques such as monoclonal antibodies, DNA technology or making synthetic peptides, etc. This is because of the simple reason that most allergic individuals have IgE antibodies to more than one allergen.
After the encounter between an allergic subject and allergenic source material the allergens start releasing into the body fluids. By virtu of it's slow releasing character, the allergens of Extract B get a longer period to sensitize the patient. Therefore the Extract B obtained by this method identifies approximately 80% of the patients with aller~y clearly establishing itself as a source of major allergens. The extracts prepared by this method may also reveal the presence of allergens that have not been found until now. These - extracts and particularly Extract D Basic extend the field of diagnosis of patients with other immunological disorders. The systematic approach provided by this invention gives a much clearer understanding of allergic diseases and helps the clinician to a better treatment and management of allergic patient.

The foLlowing examples are only given by illustration.

EXAMPLE t 1. 1 gram of defatted dry pollen of Dactylus glomerata (orchard grass) was put in a test tube. 10 ml of phosphate buffered saline (PBS~ was added. The test tube was shaken gently for one minute and the content was filtered through a glass filter funnel of 10-11 um retention type. To hasten the filtration a slight vacuum was applied on the side where the filtrate came out. The filtration was complete within 15 seconds. Immediately another 10 ml of PBS was added to the funnel. The funnel was shaken for 30 seconds and the solution filtered within 15 seconds. The filtrate discarded and the pollen residue recovered.
2. The pollen residue so obtained was transferred to a another test tube and extracted with 10 ml PBS for 30 minutes. It was then filtered. The filtrate was retained and passed through 0.22 um filter. This is called Extract A-I. The residue recovered and extracted again in the same 2C manner for another 30 minutes to obtain Extract A-II.
Extract A-I and Extract A-II mixed to obtain Extract A.
3. The recovered pollen residue from the last step was extracted again in the same manner as above. But this time for 4 hours. The filtrate retained and passed through 0.22 um filter. This procedure was repeated for 2 times. All the last 3 filtrates were mixed together to obtain Extract B-I. The recovered pollen residue was extracted in the same manner as above to obtain Extract B-II. Extract B-I and Extract B-II mixed to obtain Extract B.

4. The polleniresidue recovered from the last extraction step of Extract B-II was quickly washed 2 times with 5 ml distilled water. It was then extracted with 0.1 M
acetic acid for 10 minutes. The filtrate retained and the pH was neutralized gently with sodium hydroxide. The pollen residue was recovered. This procedure was repeated for 2 times. The filtrates mixed to obtain Extract C Acidic.

5. The recovered pollen residue from the last step was quickly washed 2 times with 5 ml distilled water.

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W094/0982~ 21~ 3 7 PCT/EP93/0~041 It was then extracted with 3 times 0.1 M NaOH as in the last step. The filtrates were gently neutralized with acetic acid and mixed to obtain Extract D Basic.~
All the extracts so obtained and concentrations or dilutions thereof are to be used in vivo and vitro diagnostic methods. These extracts are also to be used for immunotherapy and for the monitoring thereof.

Repeating the procedure of example 1, but replacing the pollen Dactylus glomerata with english rye grasspollen (Lolium perrene). In this case step 3 was repeated only one time t different from two times in 15 example 1). -Repeating the procedure of example 1, but replacing the pollen Dactylus glomerata with timothy pollen (Phelum pratense).

Repeating the procedure of example 1, but replacing the pollen Dactylus glomerata with birch tree pollen (Betula verrucosa). In this case step 3 was repeated only one time ( different from two times in example 1).
"
EX~MPLE 5 Repeating the procedure of example 1, but replacing the pollen Dactylus glomerata with house dust mite (Dermatophagoides pteronyssinus). In this case step 3 was repeated four times ( different from only two times in example 1). `

Claims

1. A process for preparing an allergenic residue from an allergenic source material comprising: subjecting the said allergenic source material to at least one extraction step in an aqueous solvent, said extraction step lasts not longer than five minutes, said extraction step resulting in removal of most of the immunologically irrelevant material in the extracted solute and minimum or no loss of allergenic activity from the recovered allergenic residue.

2. An allergenic residue obtainable according to the process of claim 1.

3. A process for preparing at least one allergenic extract comprising: subjecting the residue of claim 2 to at least one subsequent extraction step (a) in an aqueous solvent, the total extraction time of said subsequent extraction (a) being shorter than four hours and said extraction step (a) resulting in the release of rapidly releasing allergenic activity in the extracted solute.

4. A process according to claim 3 wherein the total extraction time of extraction step (a) is shorter than two hours.

5. A process according to claim 3 or 4 wherein said extraction (a) comprises at least two sequential extractions.

6. A process according to claim 5 wherein the subsequent extractions of extraction (a) each lasts less than an hour.

7. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claims 3 to 6 are combined in a pool.

8. A process for preparing at least one allergenic extract comprising: subjecting the residue obtainable from any of the subsequent extraction steps (a) according to any of claims 3 to 6 to at least one further extraction step (b) in an aqueous solvent, said further extraction step (b) lasting longer than four hours and said extraction step (b) resulting in release of most of the slowly releasing allergenic activity in the extracted solute.

9. A process according to claim 8 wherein said further extraction (b) comprises at least two sequential extractions.

10. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claim 8 or 9 are combined in a pool.

11. A process for preparing an acidic allergenic extract wherein the allergenic residue obtainable from any of the extraction steps (b) of claims 8 or 9 is subjected to at least one additional extraction step (c) in an acidic solution and said extraction step (c) lasting not longer than thirty minutes, resulting in release of most of the immunogenic activity in the extracted solute.

12. A process for preparing a series of acidic allergenic extracts according to claim 11 comprising carrying out repeated sequential extractions each lasting not longer than thirty minutes.

13. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claims 11 and 12 are combined in a pool.

14. A process for preparing an alkaline allergenic extract wherein allergenic residue obtainable from any of the extraction steps (b) of claims 8 or 9, or any of the extraction steps (c) of any of claims 11 or 12 is subjected to at least one additional extraction step (d) in an alkaline solution and said extraction step (d) lasting not longer than thirty minutes, resulting in release of most of the immunogenic activity in the extracted solute.

15. A process for preparing a series of alkaline allergenic extracts according to claim 14 comprising carrying out repeated sequential extractions each lasting not longer than thirty minutes.

16. A process to prepare at least one allergenic extract wherein the extracts obtainable according to any of claims 14 and 15 are combined in a pool.

17. The allergenic extract obtainable according to the process of any one of claims 3 to 16.

18. The use of at least one allergenic extract according to claim 17 in diagnostics for immunological disorders, preferably to detect allergic disease.

19. The use of at least one allergenic extract according to claim 17 in therapeutic treatment of patients, preferably in immunotherapy.

20. The use of at least one allergenic extract according to claim 17 in monitoring the patients during immunotherapy.