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Very special antibodies

Substances produced by a Brazilian company will facilitate the production of new medicines

proteimaxVisual effect caused by the action of the antibodies on the receptors of the cellsproteimax

The good results attained in cooperative work between Brazilian researchers from Proteimax, a biotechnology company from the city of Cotia, in Greater São Paulo, and the Mount Sinai School of Medicine, in New York, should facilitate the development of new medicines by the pharmaceutical industry such as analgesics, antihypertensives and antidepressants, amongst others. The group succeeded, in the laboratory, to produce a series of antibodies that are substances that bind themselves to the receptors existing in the membranes of the cells and known as GPCRs (G-Protein-Coupled Receptors). It is by activating or blocking these receptors that many drugs act. These proteins do the communication between the extracellular medium and the intracellular medium, allowing , or not, a drug to have its effect on a given organ of the body.

Besides being a tool for the development of new drugs and for studying the action of these products in laboratory tests with animals, the antibodies serve to discover precisely which G-protein coupled receptors are related to certain diseases. Knowing which sensors are affected, it will be possible to create and to test new drugs that have their action directed towards these receptors.
The new antibodies are intended for researchers from industries or research institutions that are going to use these tests on animals, in vitro or in vivo, during the development of new drugs, to determine the action and the duration of the new medicines in the organism, or to identify in advance possible side effects, besides understanding better the biochemical workings of diseases.

Proteimax has now developed 11 types of antibodies that recognize different GPCRs in the activated form and have as their commercial target institutions from all over the world. The market for this type of antibodies is very large and covers the whole of the pharmaceutical industry worldwide, because the use of this input is going to decrease the time for developing and approving new medicines, besides reducing production costs and make the end product cheaper for the consumer market. “We have already been sought out by researchers and companies from other countries, who want more information about the product”, says Andrea Sterman Heimann, who graduated in molecular sciences at USP and is a director of Proteimax. In mid-December, she celebrated with her team the publication of an article that details the use of the antibodies in the electronic version of the Journal of Biological Chemistry, a scientific periodical of high impact in the biological area.

Among the 11 antibodies is the angiotensin II receptor, a protein compound involved in hypertension, which, amongst other functions, makes the arteries contract, leading to an increase in blood pressure. The angiotensin II anti-receptor antibody can be used to determine whether a given drug – an antihypertensive or other medicine that one wants to test – inhibits or activates the angiotensin II receptor in the cells of the heart and of the blood vessels, for example. So, in the case of an antihypertensive, the test serves to verify whether the medicine really works. If the experiment is being done during the development of a new antihistamine, a remedy against allergies, and its acts by activating the angiotensin II receptor, the chemical action of this medicine may not be welcome, because the patient may have an unnecessary increase in pressure.

Another antibody developed at Proteimax recognizes the activated dopamine receptor, a neurotransmitting substance, the lack of which in the organism is related to Parkinson’s disease, a disease characterized by tremors, or to bipolar disorder, in which the patients alternates symptoms of depression and euphoria. One of the applications of the antibodies that recognize these activated antibodies is their use as a tool in the work on the development of, for example, antidepressant medicines, using in this case the antibody for the GPCR receptor of serotonin, a natural substance with the neurotransmitting function connected with feelings of pleasure. With the antibody that recognizes the activated serotonin receptor, produced at Proteimax, the researchers may be able to know whether the cells that have serotonin receptors are seriously affected by an anti-inflammatory, identifying an unwanted side effect.

PROTEIMAXAnother antibody produced by the company recognizes the action of Delta-9-THC, the active principle of marijuana, in the cannabinoid receptor, responsible for the effects of the drug on the organism. Actually, this receptor was given its name during studies on the effects of marijuana on the organism, but other drugs can activate or inhibit this receptor, particularly in medicines that act in the control of pain, a symptom also related to it. The antibody, in this case, will serve to test whether new compounds with a medicinal purpose that act in the control of pain can act on this receptor, activating it or inhibiting it. The objective is to identify a medicine that activates this receptor, in the case of reducing pain, but that does not bring about the psychotropic effects of marijuana. The same line of thinking is valid with the antibody of the opioid receptor, activated by endorphin, a hormone released into the bloodstream when we exercise ourselves. It is also connected with fighting pain, like the drug morphine (an opioid receptor activator). “With the antiopioid antibody, it will be possible, in an easier and quicker way, to choose better other types of morphine, without the side effects, like addition and the tolerance to the medicine that it shows”, Andrea says.

Nowadays, about 40% of the medicines most used in the world act directly or indirectly activating or blocking the receptors of the GPCR kind. “For some time, researchers from all over the world have been looking for better methods of testing to verify the activity of the G-protein-coupled receptors. And the antibodies that we have managed to develop serve precisely for this”, Andrea Sterman Heimann says. The GPCRs are considered the most important class of receptors. A possible analogy for understanding their workings and the importance they have in the organism is the use of an interphone installed in the gatehouse of residential or office buildings. Someone inside the building communicates, by the interphone, with another person who is outside. Depending on what the person outside says, the one inside will do something, such as open the door, call someone, receive something, get rid of the stranger, etc. Receptors in general correspond to the interphone, while the G-protein-coupled receptors would be equivalent to the make of a given interphone. And each type of the latter, the GPCR, would be a different model of that make of interphone. In all, there are now about 400 of these receptors known.

What does it mean to activate or to block a GPCR? Activation occurs when some substance, for example, a drug, binds itself to it, and, from then on, sends a signal inside the cell. In the comparison with the interphone, it would be when someone picks up the apparatus and talks, setting off some response. This someone is called the agonist, which makes the chemical information pass through the wall of the cell. Blocking the receptor means inhibiting the possibility of it receiving the stimulus of the agonist. The blockage would occur, for example, if the wire were cut, which would prevent any possibility of communication between whoever is inside and whoever is outside the building, or the cell.

The production of antibodies that recognize a specific conformation of the GPCRs (active or inactive) represents a powerful new technique that can be used to examine the duration and extension of physiopathological stimuli, such as, for example, the action of morphine on the central nervous system. ?This is important for basic and clinical research, besides creating a new tool for screening – a scanning method normally used by the pharmaceutical laboratories to test new drugs and checking their effects and their effectiveness. This new technique is quicker and cheaper for the work of identifying new drugs that act on a certain type of GPCR”, says Andrea.

PROTEIMAXA green color, in the membrane of a rat’s cell, shows that the antibody has recognized the receptor of angiotensin II, a substance connected with high blood pressure. The nucleus of the cell is identified in redPROTEIMAX

The production of antibodies in itself is now a technique known and established in various countries. The existing antibodies normally recognize protein independently of its state, whether active or inactive. The great innovation of the group was to succeed in making the antibodies recognize the receptor in a specific state, in this case, activated. “That is difficult to do, and involves studies in the sphere of bioinformatics and the production of the antibody in itself, which has a certain degree of complexity”, Andrea explains. The secret, the researcher says, was to manage to identify the region of the GPCR receptor that undergoes a change in conformation (or structure) when it is activated. “The antibody that we created binds itself precisely to this specific region of the receptor that is altered. All the commercial antibodies stick to regions of the receptor that do not change, whether in the active or inactive form. With this, they merely have the function of detecting the presence of the receptor without revealing its state of activation in relation to a drug, for example.”

New antibodies
Fundamental to the success of the research was the partnership established with researcher Lakshmi Arehole Devi, from the Mount Sinai School of Medicine, one of the world?s greatest authority in GPCRs. Everything began in 2003, when she came to Brazil to take part in the congress of the Brazilian Pharmacology Society, at the invitation of Professor Emer Suavinho Ferro, from the Biomedical Sciences Institute of the University of São Paulo (ICB/USP), who is also a partner in Proteimax. “In a conversation with her, I said that I was beginning to produce antibodies to give an impulse to Proteimax, and she was interested in collaborating. We made an antibody for her to test, and such was the success that the partnership did not stop any more. Last year, I spent three months in New York testing the new antibodies produced by us in Brazil. These tests are necessary for checking whether they do in fact recognize the activated form of the receptor, both in vitro and in vivo”, says Proteimax’s director.

Besides tests of new drugs, the company’s products will also be useful in basic research. With them, researchers from research institutions, in institutes or universities, will be able to evaluate which receptors are involved in the appearance of a disease. “It is possible, for example, to find out which receptors of the brain are affected by Parkinson’s disease and to test possible drugs to revert the damaged receptors”, Andrea says.

In practice, the experiments with the antibodies begin when the researcher puts the cells or just the membranes with the receptors onto a polyethylene plate (a transparent test plate) and sensitizes them with the drug that he wants to test. Afterwards, he adds the antibody. If it binds itself to the receptor, a yellow color is revealed in the sample. The researcher knows that the receptor was activated and that the drug has worked. In in vivo tests, the process is a bit longer. To begin with, the drug is administered to an animal. Time for it to act is awaited, the animal is killed, and a tissue where, supposedly, the drug acted, is removed from the body. This tissue is put into contact with the antibodies. If the drug has worked, colored pigments, normally green or red, arise in the sample. “The standard technique used by laboratories for testing the effectiveness of new drugs, called binding, is troublesome and offers a certain hazard. A drug marked with radioactive molecules has to be used, which exposes the researchers to the risk of contamination. Not to mention that it is about ten times more expensive than the process that we have created”, Andrea says.

The method for manufacturing the antibodies follows the standard protocol for producing these substances in rabbits. The first step is to choose the part of the protein (peptide) that the antibody must recognize. Next, this part of the protein has to be synthesized and coupled to a carrier protein, known by the acronym KLH (which stands for Keyhole Limpet Hemocyanin). This combination (the peptide plus the carrier protein) is given the name of antigen. The next step is to inject the antigen into the rabbit and wait for the animal to produce the antibody against it. The last stage is taking blood from the animal and submitting it to a process of purification. “The secret of the process is to choose the right part of the protein”, Andrea says. Each rabbit produces, in its useful life, of about 4 months, 50 milliliters of antibodies, which have the appearance of a reddish viscous liquid substance. This quantity is sufficient for the pharmaceutical industry to carry out 500 thousand assays.

The project for developing antibodies was financed by FAPESP’s Small Business Innovation Research Program (PIPE), which provides for the production of 50 different types of these substances. The financial support of the Foundation also makes it possible for Proteimax to carry out the tests to verify the effectiveness of the antibodies done in New York. Since they are molecules in the public domain, the antibodies are not patentable, but Proteimax can patent the idea of using antibodies that recognize the conformation of the receptors to identify the action of substances and stimuli, as well as in the treatment of diseases. By the end of January, the patent request should be deposited with the National Institute of Industrial Property (INPI).

The Project
Specific antibody conformation: proposal for generating directed antibodies and G-protein-coupled receptors (GPCRS) (nº 04/14258-0); Modality Small Business Innovation Research Program (PIPE); Coordinator Andrea Sterman Heimann – Proteimax; Investment
R$ 111,922.2 and US$ 102,102.83 (FAPESP)