WO2012059112A1 - Development of medical products based on a new discovered function of red blood cells - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6006—Cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- mice • A number of white mice were slaughtered to collect their blood on sodium citrate and their organs (liver, kidney and spleen) were preserved on 10 % formalin.
- RBCs hemolysate was prepared from TB patients showing positive TB smear and from cured TB patients.
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention is based on a newly discovered function of RBCs. It is discovered that RBCs contain antigens which are different from their proteome. Those antigens can be precipitated/separated using plasma of blood from which RBCs were taken. It is found that those antigens consist of HLA antigens, tissue specific antigens, and foreign antigens. The foreign antigens can be fetus antigens in pregnant females, tumor antigens, microorganisms antigens, food, insects or other antigens from environment. The collection of those transported antigens represent a dynamic store. Consequently, RBCs may play role in tolerance through transporting those antigens to central organs of the immune system. The idea, in the presented invention, is to exploit this store in producing a number of products. Those products include vaccines, clinical laboratory kits and treatment components. The vaccine products cover: vaccines for bacteria, viruses and parasites in humans and animals; and vaccines for the prevention of malignant tumors in patients showing malignant transformation or as a supportive treatment of patients already having a tumor. The clinical laboratory kits covers: diagnostic kits for efficient diagnosis of diseases caused by microorganisms in humans and animals; diagnostic kits for malignant tumors; tissue typing kits for organ transplantation; and monitoring kits for organ rejection and hypersensitivity. The treatment components will help in treatment of difficult disorders including, among others, hypersensitivity disorders, chronic graft rejection, and autoimmune disorders. In effect, the products that can be produced based on this discovery are innumerable. The invention methods are based on selecting specific disease transported antigens, identify those antigens, manufacture those antigens, and then produce an appropriate product.
Description
Description
DEVELOPMENT OF MEDICAL PRODUCTS BASED ON A NEW DISCOVERED FUNCTION OF RED BLOOD CELLS
Technical Field
[ 1 ] Human and animal vaccines
[2] Human and animal laboratory kits
[3] Human components
Background Art
[4] Normally, the immune system distinguishes between what is self and what is non- self (foreign body): tolerates self antigens and responds to non-self antigens. The phenomenon of tolerating antigens is called immune tolerance or simply tolerance. It can be defined as "a specific unresponsive state induced by prior exposure to an antigen which is due to either a programmed failure of the immune system or active suppression." Tolerance is a basic function of the immune system that is important to maintain body integrity and health.
[5] Exposure to antigens during intrauterine life induces tolerance. During postnatal life, tolerance to a particular antigen depends on dose, antigen-nature, and root of administration. Generally artificial induced tolerance is of finite duration. Basically, there is no absolute tolerance. Low-affinity auto-antibodies exist normally against self antigens.
[6] Consequently, tolerance to self antigens is a process that continues throughout life but begins during fetal development. There are a number of mechanisms that describe how tolerance occurs, which can be classified into: central, peripheral, and anergy. In central tolerance immature self-reactive T lymphocytes recognize antigens in the thymus and undergo negative selection (deletion). In peripheral tolerance mature self- reactive T lymphocytes that escape central tolerance and recognize self antigens in peripheral tissues can be inactivated (anergy), killed (deletion) or regulated (suppressed). In anergy mature self-reactive lymphocytes do not respond to antigens in non-inflamed environment. Those mechanisms are explained through the following theories: clonal deletion, clonal anergy, clonal ignorance, receptor editing, and suppressor cells.
[7] RBCs are specialized for transport. Besides, RBGs have pinocytic activity which is well documented. Their cell membrane antigens can function as receptors, e.g., Duffy antigen has been proved to act as a receptor for plasmodium vivax malaria to invade RBCs. Consequently, RBCs easily absorb soluble antigens through pinocytosis while particulate antigen need receptor sites on RBCs in order to be absorbed.
[8] It should be remarked that there is a lot of research on RBCs or erythrocytes
proteome. This research is directed towards the identification of proteins from both erythrocyte membrane and cytoplasm. Besides studying the identified proteins, the clinical goal is to make a relation between proteins and diseases to discover a
biomarker which is used in diagnosis.
Disclosure of Invention
[9] We found that the critical issue in the process of tolerance is the transport
mechanism of antigens. This mechanism should be capable of hiding those antigens from various immune system cells and/or antibodies during their journey in the blood circulation until they reach their target immune system organs. We have proved that Red Blood Cells (RBCs) do this job.
[ 10] RBCs' absorbed antigens will reach the central organ of the immune system (e.g., thymus and bone marrow) and induce central tolerance. The degree of induced tolerance is dependant on the size of the RBCs antigens store. In effect, the larger the size of the store, the higher is the probability these antigens meet their corresponding premature lymphocytes in thymus and bone marrow with consequent induction of their apoptosis. Antigens are tolerated as far as there are enough stores of those antigens in RBCs. In this way, the degree of immune response to a particular antigen may vary.
[1 1 ] We proved that during pregnancy, male spouse antigens are transported to placenta where they neutralize any harmful antibodies. In prenatal life, we expect that if antigens are introduced to a fetus while the immune system is still incapable to respond, there are good chances for those antigens to be processed by phagocytic cells and absorbed by RBCs. When mature lymphocytes production starts, later in life, antigens stores of RBCs are used to induce tolerance.
[12] In addition to proving that RBCs transport function is beneficial for body integrity and health, we also found that it is a security hole that gets exploited in a harmful way, for instance, by microorganisms, and malignant tumors.
[13] An important criteria which differentiate transported antigens from RBCs proteome content is the existence of antibodies against transported antigens in plasma of the blood from which RBCs are taken. This criteria is used to separate and concentrate those transported antigens through using affinity column chromatography loaded with protein G Sepharose or protein A Sepharose beads. Usually, those transported antigens are missed while studying RBCs proteome because their concentration is very low. Another reason is related to the method used for the interpretation of the mass spectrometry data and the search engine used for the identification of the proteins in the different types of sequence data banks available. The search is targeted towards finding proteins which are related a particular taxonomic category. It is not expected to find foreign protein. Consequently, those proteins are reported to be unknown.
[14] It is important that the discovered function of RBCs fill a gap in understanding of tolerance. Notice that there is a general agreement about this gap. Part of this gap can be expressed in the following questions:
Why soluble antigens administered intravenously favor tolerance while particulate antigens injected into the skin favor immunity.
Why ingested large doses of soluble proteins induce systemic T lymphocyte
tolerance, whereas the components of vaccines such as the Sabin polio vaccine induce an effective local immune response.
• Why tolerance is easier to induce in prenatal than postnatal life.
Further, the discovered function of RBCs answers some other questions or research puzzles including:
• How circulating IgG antibodies against blood groups 'A' and 'B' in a pregnant woman with blood group 'O' do not harm the fetus whose blood group is not 'Ό'.
• How microorganisms and malignant tumors overcome the immune system's arsenal.
• Why some hosts are susceptible to particular microorganisms while others are resistant to them.
Consequently, the discovered function of RBCs will help in:
• Diagnosis of diseases caused by microorganisms
• Diagnosis of malignant transformation and tumors
• Diagnosis and monitoring of chronic rejection syndrome for different organs transplantations
• Diagnosis of autoimmune disorders
• Monitoring desensitization in patients with hypersensitivity
• Typing of donor and recipient for organ transplantation
• Vaccination of humans and animals against different microorganisms • Vaccination of humans against malignant tumors
• Treatment of autoimmune disorders
• Treatment of chronic rejection syndrome
• Treatment of hypersensitivity to particular antigens
• Treatment of some fertility problems
• Diagnosis and treatment of disorders caused by abnormal proteins, e.g.,
Alzheimer syndrome
• Describing some idiopathic diseases and help in their diagnosis and treatment.
Experiments
We have proved that RBCs transport self and foreign antigens through the following experiments.
Experiment I : We have demonstrated that pregnant women store the 'A' and/or 'B' blood group antigens in their RBCs, if and only if their male spouse is not blood group 'O'. The technique is based on competitive inhibition of RBCs agglutination. If the hemolysate contains ABO specific antigens, then those antigens will compete with RBCs and prevent their agglutination. The experiment is done as follows:
• Blood samples were taken on heparin. RBCs and plasma were separated in two tubes.
• Females spouse, RBCs were washed in normal saline several times, frozen until they are ruptured, and then centrifuged.
• Males spouse RBCs were washed several times in saline then some of the RBCs were used to prepare a 5% suspension.
• Some of the washed male spouse RBCs were frozen until they are ruptured, and then centrifuged.
• As a positive control, serial dilutions of female spouse plasma were made using normal saline. A drop of the male spouse's hemolysate is added before adding his RBCs suspension.
• In test tubes, serial dilutions of the female spouse's plasma were made using normal saline. A drop of the female spouse's hemolysate was added before adding a drop of the male spouse's RBCs suspension.
• It was observed that agglutination was inhibited by female spouse hemolysate and was not inhibited by male spouse hemolysate. In most cases agglutination was inhibited in the first tube. However, agglutination was never observed in subsequent tubes.
Experiment II: RBCs store self HLA antigens and the male spouse HLA antigens. In effect, a pregnant woman tolerates her fetus and placenta using the same mechanism by which her body tolerates herself antigens, i.e., a fetus is part of self. The experiment was done using commercial HLA Typing Trays for the identification and definition of HLA Class I Antigens using the microlymphocytotoxicity assay. It is, also, based on competitive inhibition. Consequently, if typing wells that show positive reaction were inhibited in corresponding testing wells by adding hemolysate, this proves the existence of specific competing antigens. The experiment is done as follows:
• Blood samples were taken on heparin. The lymphocytes were separated using the Ficoll hypaque technique. The RBCs were then separated to prepare the hemolysate. The hemolysate is prepared by washing RBCs in phosphate buffer saline several times, and then frozen until RBCs are ruptured.
• For each couple, four typing trays were used:
• The first is used for ordinary typing of the female spouse
• The second is used for ordinary typing of male spouse
• The third is used to detect self HLA antigens in male hemolysate • The fourth is used to detect male spouse HLA antigens in female spouse hemolysate • Positive controls: In the first and second trays, positive controls are as usual.
In the third and fourth trays a hemolysate from a third person is added to control wells. Notice that the whole experiment is better to be done in two steps; first do the typing then test for the antigen existence in a next step. This gives a chance to select a hemolysate of a person that has a different HLA
typing. Notice that hemolysate can be kept for a long time in deep freeze. • The first and second trays are used for typing of HLA Class I for the couple. • In the third tray, we added male hemolysate (diluted 1/16) in typing wells before adding male lymphocytes, and then examine wells that gave positive reaction in the second tray. It was observed that male spouse hemolysate inhibited the typing reaction indicating the existence of self HLA antigens. • In the fourth tray, we added female hemolysate (diluted 1/16) before adding male lymphocytes, and then examine wells that gave positive reaction in the second tray, too. It was observed that female spouse hemolysate inhibited the typing reaction indicating the existence of male spouse HLA antigens.
Experiment III: Because RBCs transport antigens to central organs of the immune system, then RBCs will transport Tissue Specific Antigens (TSAs). The objective of this experiment is to prepare antibodies against TSAs through injecting white mice RBCs into rabbits. The experiment is done as follows:
• A number of white mice were slaughtered to collect their blood on sodium citrate and their organs (liver, kidney and spleen) were preserved on 10 % formalin.
• The separated RBCs which were washed many times with sodium citrate and then washed with 3% formol-saline to kill any bacterial contamination.
• The washed RBCs were hemolysed with 3% formolin in sterile distilled water and centrifuged for 10 min. at 13000 rpm.
• RBCs hemolysate were injected subcutaneously into a number of rabbits for four times on weekly intervals.
• Blood was collected from those rabbits ear-vein after 28 days from the first injection.
• Then, sera were examined for antibodies against TSAs of liver, kidney and spleen using sandwich technique in histopathology sections.
• All sections show florescence which means that sera have antibodies against TSAs
Experiment IV: If a host is susceptible to a particular microorganism, then host RBCs will transport its antigens. The experiment is done as follows:
• A number of rabbits were infected by Escherichia coli 0157 through subcutaneous injection to prepare hyper immune serum.
• A sheep was infected in the same time through oral route.
• Blood was collected from the rabbit ear-vein after 21 days from the injection. • Blood was collected from the infected sheep on weekly basis for 4 weeks. • Rabbits sera were separated and examined for antibodies against E. coli 01 57 using direct bacteria slide agglutination test.
• Ouchterlony agarose gel antigen-antibody precipitation test was done using
rabbit serum in a central well and each week sheep washed RBCs hemolysate in peripheral wells. Precipitation lines were observed in the second and third weeks which means that the transported antigens store is dynamic as it changes with the health state of the animal.
Experiment V: This experiment is similar to Experiment V. The purpose of the experiment is to investigate a different type of bacteria which produces cellular immune response. Mycobacterium tuberculosis strain H37Rv was selected. The experiment was done as follows:
• A bacterial extract was prepared by sonication in ice of a water bacterial suspension.
• The suspension was filtered using, first 0.45 urn millipore filter followed by 0.22 urn millipore filter.
• Rabbit were injected subcutaneously with 0.1 mg protein from the filtered extract mixed with oil and flagellin adjuvant.
Two more similar injections were done. The interval was two weeks.
• Blood was collected two weeks after the last injection and serum was
separated immediately after clot retraction.
• Affinity column chromatography was prepared from Protein A Sepaharose beads and the prepared rabbit hyper immune serum
• RBCs hemolysate was prepared from TB patients showing positive TB smear and from cured TB patients.
• The hemolysate was applied to the affinity column. After washing the
column, hemolysate antigens were eluted.
• The eluted antigens were separated using two-dimemensional gel electrophoresis.
• Spots were identified by mass spectrometry which reveals eleven Mycobacterium tuberculosis H37Rv proteins in patients and not revealed in cured patients.
Description of Invention
The present invention exploits RBCs transported antigens store which can be defined as: All the antigens that can precipitated by plasma or serum of blood from which RBCs are taken. The following technologies and resources will be used:
1. Techniques from genomics and proteomics: these are used for separation, identification and synthesis of transported antigens.
2. Public databases of already identified proteins: those databases are provided by organizations like: European Bioinformatics Institute (EBI), which provides the universal protein database (UniProt), the American National Institute of Health (NIH), and Human Proteome Organization (HUPO)
3. Data Mining (DM) in locally built database: DM can be targeted towards the identification of RBCs' transported antigens related to a particular disease or
towards the identification of RBCs' antigens that are missing in a particular disease.
In this invention, the first step is to prepare / separate transported antigens store from diseased patients or animals. The invention of new generation of microorganisms' vaccines depends on identifying RBCs transported antigen(s), which belong(s) to a particular microorganism. Antibodies against those antigens will be used in the diagnosis of their corresponding diseases' conditions. Those antibodies can be prepared to be used as passive vaccines, too. In this regard, new technologies, such as Affibody, can be efficiently used to replace natural antibodies. Similarly, separation of transported antigens of malignant rumors from patients' RBCs will enable the preparation of antibodies against those antigens. Those antibodies can be used as passive vaccines and/or in the detection of tumors' antigens in the RBCs of patients. This detection test will be very sensitive as it can detect malignant transformation even before the tumor exists. Also, the separated antigens can be used to vaccinate those patients to prevent the development of the tumor or as a supportive treatment.
Generally, the separation of self and non-self RBCs transported antigens from normal individuals and patients will help in many directions that cannot be enumerated.
Among those directions:
1. Preparation of antibodies against tissues specific antigens; this will enable better typing of donor and recipient
2. Preparation of tissues specific antigens for desensitization treatment of autoimmune diseases or graft rejection syndrome
3. Building of databases that enable data mining for discovering general health important information which is different from bioinformatics databases in their content and purpose.
4. Preparation of antigens related to environment and food; this will help to desensitize patients that are sensitive to those antigens while preparing antibodies against those antigens will help to monitor the level of RBCs store for those antigens.
Important Remarks
In general, the technology of separation, identification, and synthesis of proteins are not the issue in the described methods. Also, the use of monoclonal, polyclonal antibodies, Affibody, or any other similar products are not the issue. The issue is to exploit the RBCs transported antigens store, as defined in [24], in diagnosis, monitoring, treatment, or prevention of a particular disease condition.
Description Of Drawings
Fig. 1 , depicts the methods of preparing laboratory kits and vaccines. In animals, it o can be applied on microorganisms products. These methods can be described in the following steps:
1. The resources are:
• [A] RBCs samples of patients or animals
• [B] microorganisms / tumor cell line.
2. For each sample:
• Prepare hemolysate (step 1 )
• Purify transported antigens using sample plasma (step 2)
3. Pool all the purified antigens
4. Prepare hyper immune serum for microorganism or tumor cell line (step 3)
5. Purify antibodies using extracted antigens from microorganisms / tumor cell line (step 4)
6. The purified antibodies are then used to separate disease related antigens from purified hemolysate antigens (step 5)
7. The disease related antigens are identified and manufactures (step 6)
8. Prepare products (step 7)
Fig. 2, depicts the methods of preparing laboratory kits and treatment components for autoimmune disorders and chronic rejection syndrome . These methods can be described using the following steps:
1. The resources are:
• [A] patients' RBCs, and patients' plasma
• [B] normals' RBCs.
2. Hemolysate of both patients (step 1 ) and normals (step 3) are prepared
3. Transported antigens are separated using the sample plasma and pooled ([C] and [D]).
4. Antibodies of patient plasma are separated (step 2) and pooled ([E])
5. The plasma of patients contains auto-antibodies while the transported antigens contains their corresponding antigens but in small quantities. Consequently, using affinity column loaded with patients' hemolysate will bind most of the antibodies and the wash out will contain the auto-antibodies and definitely with other antibodies (step 4). Meanwhile, this minimizes the search for disease related antigens.
6. The column wash out is used to prepare another affinity column to separate the possible disease related antigens (step 5).
7. The possible disease related antigens are identified and the possible related antigens are manufactured (step 6).
8. The manufactured antigens are further tested against patients plasma to select antigens that have high antibody titer (step 7)
9. Use those selected antigens in preparing products (step 8)
Claims
Claims
RBCs of humans and animals transport antigens, which can be precipitated by plasma or serum of the blood of human or animal from which RBCs are taken. Those transported antigens represent a dynamic store in which antigens concentration change over time and some antigens may disappear without leading to a disease, e.g., food antigens and bacterial antigens. The sources of antigens are ingested food, tissues breakdown, and environment. The environment is the source of microorganisms, insects, animals, and other antigens. In effect, this store reflects the health state of individual or animal. Consequently, the transported antigens of this store can be used in preparation of innumerable products including but not limited to: laboratory kits, vaccines, and components which can be used in many purposes including but not limited to treatment. Transported antigens store of claim [ 1 ] does not include and is not related to RBCs membrane antigens as those antigens cannot be precipitated by plasma of the blood from which RBCs are collected and are not dynamic in nature, i.e., they do not change with time. Also, they cannot be used in treatment or vaccination as they are not related to the cause of the disease.
Transported antigens store of claim [1 ] does not include and is not related to proteins of RBCs proteome which cannot be precipitated in the same way like transported antigens and are not dynamic in the same sense as the transported antigens store. In all normal individual the RBCs proteome is the same while transported antigens store is not the same. Also, they cannot be used in treatment or vaccination as they are not related to the cause of the disease.
Transported antigens store of claim [ 1 ] does not include antigens of parasites of RBCs, e.g., Plasmodium malaria and Babesia. In babesiosis, sporozoites enter RBCs which are the target place to continue their life cycle. In malaria, during the liver stage, antigens that belong to schizont are part of the transported antigens store while antigens of merozoites which invade RBCs are not part of the transported antigens store. This is because antigens of sporozoites or merozoites which are revealed in infected RBCs have not been precipitated by plasma or serum of blood from which RBCs are taken. The separated antigens can be due to either disintegration of or excretion by the parasite, inside RBCs. Consequently, their antigens are not considered part of the transported antigens store.
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| PCT/EG2010/000041 WO2012059112A1 (en) | 2010-11-01 | 2010-11-01 | Development of medical products based on a new discovered function of red blood cells |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018210395A1 (en) | 2017-05-16 | 2018-11-22 | Mahmoud Abdel Wahed Rafea | Lateral flow chromatographic assay for tuberculosis (tb) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991016073A1 (en) * | 1990-04-24 | 1991-10-31 | The University Of Newcastle Research Associates Limited | Oral vaccine comprising antigen surface-associated with red blood cells |
| US20030045001A1 (en) * | 2001-08-29 | 2003-03-06 | Deborah Burgess | Immunochromatographic test strip with arcuate sample application zone for ease-of-use in the field |
| WO2009019317A1 (en) * | 2007-08-08 | 2009-02-12 | Erytech Pharma | Composition and therapeutic anti-tumour vaccine |
| WO2009066131A1 (en) * | 2007-11-19 | 2009-05-28 | Mahmoud Rafea | Methods for preparation of vaccines, laboratory kits, and treatment components |
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2010
- 2010-11-01 WO PCT/EG2010/000041 patent/WO2012059112A1/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991016073A1 (en) * | 1990-04-24 | 1991-10-31 | The University Of Newcastle Research Associates Limited | Oral vaccine comprising antigen surface-associated with red blood cells |
| US20030045001A1 (en) * | 2001-08-29 | 2003-03-06 | Deborah Burgess | Immunochromatographic test strip with arcuate sample application zone for ease-of-use in the field |
| WO2009019317A1 (en) * | 2007-08-08 | 2009-02-12 | Erytech Pharma | Composition and therapeutic anti-tumour vaccine |
| WO2009066131A1 (en) * | 2007-11-19 | 2009-05-28 | Mahmoud Rafea | Methods for preparation of vaccines, laboratory kits, and treatment components |
Non-Patent Citations (1)
| Title |
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| LUTZ, H. U.: "An age-specific cell antigen is present on senescent human red blood cell membranes.", MECHANISMS OF AGEING AND DEVELOPMENT, vol. 15, no. 1, 1981, pages 65 - 75 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018210395A1 (en) | 2017-05-16 | 2018-11-22 | Mahmoud Abdel Wahed Rafea | Lateral flow chromatographic assay for tuberculosis (tb) |
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