WO2017142493A1

WO2017142493A1 - Fc part removed incomplete antibody (fab) for medical use

Info

Publication number: WO2017142493A1
Application number: PCT/TR2016/050352
Authority: WO
Inventors: Mehmet ÖZEN
Original assignee: Özen Mehmet
Priority date: 2016-02-15
Filing date: 2016-09-26
Publication date: 2017-08-24
Also published as: TR201601910A1

Abstract

The present invention is related to the use of antibodies having only the Fab part, formed following the removal of the Fc part after fractioning the antibody into Fab and Fc sections, in order to be used in treating antibody related diseases. More specifically, the Fab section of antibodies obtained from serum samples in order to prevent the incompatibility between blood groups.

Description

DESCRIPTION

FC PART REMOVED INCOMPLETE ANTIBODY (FAB) FOR MEDICAL USE

Technical Field

The present invention is related to the use of incomplete antibodies having only the Fab part, which formed fractioning the antibody into Fab and Fc section and removal of the Fc part , in order to use in treating antibody related diseases. More specifically, the Fab sections of antibodies obtained from serum samples have been used for preventing the incompatibility between blood groups.

Known State of the Art

In accordance with the American Autoimmune Related Diseases Association (AARDA), there are more than 80 diseases related to the immune system, and approximately 50 million persons suffer from such diseases in the USA. Due to these diseases USA is obliged to cover an annual health cost of 100 billion dollars.

A large section of the immune system diseases have been classified as "Antibody Related Diseases". Excessive immune response is shown against to a stimulant named "antigen" in such antibody related diseases. These responses are provided by means of various proteins named "antibodies". This system is extremely activated in said diseases which normallyprotects our body and leads to the damaging of various organs in our body by the antibodies.

Various medicines which are immunosuppressants are used in treating these antibody related diseases. However the effects of these medicines are usually transpire late and as they need to be used for a long period of time, the side effects of these medicines can be frequently observed in patients. Another method used in such diseases is the plasmapheresis method and antibodies are removed from the body by means of this method. However patients show a temporary recovery with said plasmapheresis method and this method does not provide a permanent treatment. The reason for this is that, the antigen is not eliminated by means of this method and therefore antibodies are continuously and repeatedly formed. l The antibody molecule shaped like the letter "Y" has a Fab fragment (Fragment Antigen Binding section) and a body section which is responsible of several biological activities. The body section is defined as Fc (Freagment Crystallizable section) due to its crystallization feature.

Antibodies which lead to immune system diseases are formed from Fc section which binds to the complementary article which is a protein which stimulates the immune response and the Fabs which bind the antigen like other antibodies. While the antibody is bound to the antigen with the Fab section it is bound to the complement by means of the Fc section. Binding to the complement leads to the activation of the immune response. In order to activate the complement both the Fab and the Fc section of the antibody is needed. The immune response that is formed as a result of these is called the humoral immune response.

As it has been mentioned above humoral immune response does not only lead to several diseases but it is also observed in our body. The most typical situation where the strongest antibody response is observed is the blood groups. These antibodies and therefore the immune response is the reason which blood transfusion cannot be performed between people having different blood groups. When erythrocyte is transfused to a person having the blood group A from a person with the blood group B, the antigens located on the erythrocytes of the B blood group are recognized by the antibodies which are present in the circulation system of the person with the A blood group and therefore they form a humoral immune response. Created humoral immune response forms a fatal state known as transfusion reaction in the person who has received the blood, in order to prevent the formation of such a transfusion reaction before it is formed, a test that is called the "crossmatch test" is performed in a laboratory and the compatibility of the persons giving and receiving the blood can be determined beforehand.

Monoclonal antibody fragments that have been mentioned below can be commercially obtained in USA. Generic Name Initial

Indication (Initial

(Commercial Definition approval Sponsor approval)

Name) date

abciximab Anti-GPIIb/llla Preventing clotting in

12/22/94 Centocor

(Reopro) chimeric Fab Angioplasty

ranibizumab Anti-VEGF-A

Macula degeneration 06/30/06 Genentech

(Lucentis) humanized Fab

Anti-TNFalpha

certolizumab Medium to severe

pegylated 04/22/08 UCB pegol (Cimzia)

humanized Fab Crohn's disease

However a treatment study for antibody related diseases regarding the usage of the incomplete antibody having only the Fab section which is obtained by fragmenting the antibody into Fab and Fc sections and then removing the Fc section is needed.

Pepsin and papain enzymes are used in order to divide the antibodies in to Fab and Fc parts. Another enzyme used for antibody fragmentation is IdeS enzyme (3).

Any study regarding the usage of incomplete antibodies whose Fc section has been removed in antibody related diseases is not present. However, the usage of the Fab section separated from the antibodies obtained via using other methods presently, is known in toxicology (4). Initially an antibody is obtained in Digoxin toxicity and following this the antibody obtained against digoxin is fragmented and the Fab section is isolated and this incomplete antibody that is formed is used as a medicine to cure digoxin toxicity. This medicine called D!G!BIND has not been shown any kind of serious side effects until today (5). Rabbits are injected with digoxin in order to obtain Fab to cure digoxin toxicity. The antibodies formed against digoxin in the rabbit are isolated. The Fc section is removed from the antibody obtained and Fab is given to the patient. The Fab that is given binds the digoxin and is discharged from the kidneys. The Fab's used in treatment nowadays are obtained by using this method.

1 - Fragmentation of immunoglobulin G. Andrew SM, Titus JA. Curr Protoc Cell Biol.

2003 Feb;Chapter 16:Unif 16.4.

2- Fragmentation of immunoglobulin M. Andrew SM, Titus JA. Curr Protoc Immunol.

2001 May;Chapter 2:Unit 2.1 OA. 3- A monoclonal antibody against hinge-cleaved IgG restores effector function to proteolytically-inactivated IgGs in vitro and in vivo. Brezski RJ, Kinder M, Grugan KD, Soring KL, Carton J, Greenplate AR, Petley T, Capaidi D, Brosnan K, Emmeli E, Watson S, Jordan RE. MAbs. 2014;6(5):1265-73.

4- Fab antibody fragments: some applications in clinical toxicology. Flanagan RJ1 , Jones AL. Drug Saf. 2004;27(14):1 1 15-33.

5- Digoxin-specific antibody fragments in the treatment of digoxin toxicity. Chan BS, Buckley NA. Clin Toxicol (Phila). 2014 Sep-Oct;52(8):824-36.

Description of the Invention

The A and B genes which define the blood group, determine which one of the A and B type proteins (agglutinogen) will be presented in the cell membranes of the red blood cells found in our blood. In the blood type A, only A type protein is found, in the blood type B, only B type protein is found and in the AB blood type both A and B proteins are found surface of the red blood cell membrane. In zero (0) blood type neither of the proteins are present at the red blood cell membrane. Antibodies named agglutinins which protect the organism are found in the blood; agglutitin prevents the entrance of red blood cells from a different blood type into the body. Agglutinins are formed as protein are divided into A and B types, similar to agglutinogens.

The present invention comprises the usage of Fab sections of antibodies in order to prevent blood incompatibility. The antibodies in the blood serum are fragmented by enzymes in order to obtain Fab sections of antibodies and the Fc sections are removed. Pepsin or papain is used as an enzyme during this process. As a result the Fab sections of the antibodies are isolated. The Fab sections that are isolated, are treated with normally incompatible the blood type erythrocytes . According to this process either the Fab sections isolated from antibodies obtained from those people having the blood group A are treated with B group erythrocytes, or the Fab sections isolated from antibodies obtained from those people having the blood group B are treated with A group erythrocytes. The method of the present invention basically comprises the following steps:

a) The antibodies inside the blood serum are fragmented by enzymes and the Fc sections are removed from the medium,

b) The Fab sections are isolated,

c) The Fab sections that are isolated are treated with erythrocytes that are from normally incompatible blood group.

In more general term, the method of the invention comprises the following steps:

1 . Obtaining an antibody

a. Several antibodies which are known in the art are can be obtained commercially. Processes can be carried out using these antibodies. If commercially available antibodies are going to be used, antibodies shall be directly obtained.

b. When the antibody cannot be commercially available, if the antibody is a natural antibody, in other words if it is formed on its own (blood group antibodies are good examples of such antibodies), plasma can be isolated via using the plasmapheresis method from healthy individuals or serum is obtained from the blood.

c. When the antibody is not commercially available and is disease related, plasma is obtained by means of plasmapheresis from individuals carrying antibodies that are related to the disease or serum is obtained from their blood.

2. Fragmenting the antibody and isolating the Fab section:

The plasma and serum or antibodies that are obtained according to the methods mentioned above, are separated into Fab and Fc sections by being treated in vitro with the mentioned enzymes that fragment antibodies. The Fab section is isolated. Therefore a drug specific to the disease is obtained from which the Fab is isolated.

Example:

The antibodies from the serum of a person having the blood group A are fragmented with pepsin and the Fc sections have been removed; the remaining Fab sections are processed with erythrocytes obtained from B blood group. When these processed B blood group erythrocytes and the total blood sample of the person from the A blood group have been subjected to crossmatch test a severe reaction was not observed. In the crossmatch test performed on a card, it was clearly observed that the reaction that has occurred against the monoclonal antibodies was prevented with Fab.

The efficiency of those drugs that are to be obtained by this method are clearly proved as ever incompatibility between the blood groups are able to eliminated by using of the present invention.

industrial Application of the Invention

It has been discovered that the Fab sections to be used in treatment are efficient in accordance with the results of the study that has been carried out. For this reason, it is possible that the isolated Fab sections may be used on the person having antibody related diseases. Moreover the present invention is a novel treatment method for the above mentioned antibody related diseases and the advantages of this treatment is that it is efficient, and does not have any serious side effects.

It is possible to fragment the antibodies obtained from healthy people and patients suffering from said diseases and to isolate the Fab sections, therefore obtaining medicines that comprise the Fab sections specific to each antibody and to present said medicines to the use of patients.

More specifically the Fab sections to be obtained from blood samples from A and B blood groups can be used in order to prevent blood incompatibility and the Fab sections can be produced as medicines and/or reactants.

Claims

1 . Fab sections of antibodies to be used in preventing blood incompatibility.

2. The Fab sections of antibodies according to claim 1 are obtained according to the below mentioned process:

a) The antibodies in the blood serum are fragmented by means of an enzyme and the Fc section is removed from the medium,

b) The Fab sections are isolated,

c) The Fab sections that have been isolated are treated with erythrocytes which are from incompatible blood groups.

3. An enzyme according to claim 2, wherein said enzyme is pepsin or papain.

4. Antibodies according to claim 2, which have been obtained from a person with the blood group A and the isolated Fab sections are treated with B blood group eryhthrocytes.

5. Antibodies according to claim 2, which have been obtained from a person with the blood group B and the isolated Fab sections are treated with A blood group erythrocytes.