How Bacteria and Viruses Help Create Antibodies to Treat Humans

Ekaterina Pavlova, BiomoleculeOne of the most significant dangers to human health are bacteria.

But bacteria also have opponents: viruses are bacteriophages that use the microbial cell as a hotel where everything is included, and when leaving the shelter, they often kill the host. The invention of the phage display method made it possible to use the properties of bacteriophages in the search for new antibodies, which are extremely in demand for improving the diagnosis and therapy of many dangerous diseases.

The target is the structure of the body associated with a certain function, the violation of which leads to the disease. In case of illness, a certain effect can be exerted on the target, which should lead to a therapeutic effect. A drug is a substance that specifically interacts with a target and affects the state of a cell, tissue, or organism [1]. The target can be a receptor on the surface of the cell membrane, an enzyme or a channel that conducts various compounds into the cell. However, the path to the consumer for any drug is long: after confirmation of its functional activity, the stages of preclinical and clinical trials follow, in which small molecules are in danger of never becoming a drug. Under the influence of the patient's enzyme systems, they can become poisonous, or their isomers will turn out to be toxic. A low molecular weight substance can be excreted too quickly or, on the contrary, accumulate in the body, poisoning it. Therefore, in recent years, macromolecules have taken an increasing share in the market of medicines, and among them antibodies – protective proteins of the body - play an important role (Fig. 1).

B cells, after stimulation by T cells or in direct contact with a foreign agent, synthesize antibodies to it. Some of the activated B-lymphocytes – plasma cells – specialize in the production of antibodies, and the rest become memory cells in order to give it a quick and effective rebuff when encountering the same antigen in the future. The antibody synthesized by the plasma cell binds to the "stranger", thereby neutralizing it. This happens in several ways: antibodies specifically bind to toxic sites of the antigen, agglutinate (stick together) with large particles that carry antigens on their surface, or even directly cause the destruction of a bacterial cell. In addition, the antigen "plastered" with antibodies becomes vulnerable to other components of immunity – for example, to macrophages or the complement system [2].

Such important properties as the binding of the antigen, the strength of this binding and the stability of the molecule depend on the structure of the antibody. However, the nature of the creation of antibodies in the body is very complex, and no one can guarantee that in response to even identical antigens, the same structure of antibodies are formed. If antibodies to the same antigen are used to create a drug or diagnostic kit, but with a different structure, then due to the difference in stability and specificity, standardization and reproducibility of the results of work can be forgotten. This means that such antibodies cannot become diagnostic or medicinal in any way. Hence the conclusion: we need antibodies with an identical structure.

Antibodies-"clones" are obtained using cell biology methods from a single progenitor cell. Such antibodies are called monoclonal. Their use as therapeutic agents has become a strategic stage for medicine in changing the concept of treatment – from non-specific therapy to targeted therapy. To date, monoclonal antibodies are most actively used in oncohematology, the treatment of tumors, autoimmune diseases, and especially widely in diagnostics [3].

Obtaining antibodies for human needs, as a rule, begins with the immunization of animals. Several antigen injections are performed, and specific antibodies accumulate in the blood serum. These antibodies, obtained directly from the serum of an immunized animal, are produced by different plasma cells, that is, they are polyclonal. To obtain completely identical – monoclonal – antibodies in the seventies of the last century, scientists Georg Koehler and Cesar Milstein developed a hybrid method [3]. It is based on the fusion of plasma lymphocytes (they produce antibodies, but do not live in culture) and myeloma cells (these are tumor cells that do not produce anything, but are remarkably cultured), as a result of which such a hybrid cell inherits from a B-lymphocyte the ability to secrete the antibodies needed by researchers, and from a tumor cell – immortality (almost infinite division).

Hybridoma has become an outstanding achievement that has opened up huge opportunities for researchers [4]. However, the antibodies that can be obtained using the hybridomic method are still developed by animals and are not suitable for human therapy. Therefore, the researchers faced the task of obtaining completely human antibodies. To solve it, a group of methods called display was developed. What all these methods have in common is that they involve working with the "coupling" of the nucleotide and amino acid sequences of each specific antibody variant. The name "display" comes from the English display – to flaunt, to demonstrate. An integral stage of these methods is the "exposure" of antibody fragments on the surface of the phage particle for further selection of the desired variants by antigens.

The bacteriophage is DNA surrounded by a protein shell – a capsid – and is able to reproduce only inside the host cell. Penetrating there, he shamelessly uses the enzyme systems of the unfortunate bacterium, providing it with his DNA for the synthesis of proteins necessary for its reproduction [6]. A bacterial cell infected with a phage obediently reproduces everything that is encoded in the genome of the virus, so that its offspring assemble their shell from ready-made building blocks. If a nucleotide sequence encoding the desired peptide is introduced into the genome of the progenitor phage by the researcher, several copies of a hybrid capsid protein consisting of its own polypeptide chain and an antibody fragment appear on the surface of its offspring on the surface of the viral particle. A set of bacteriophages, on the surface of which random fragments of antibodies are presented, is called a phage library (Fig. 2).

How to choose from this variety of just a few molecules specific to a single antigen? In the case of a display library, viral particles work as "librarians", and bacterial cells become "readers". If the search for books in an ordinary library was carried out in the same way as antibodies in a display, it would look very unusual. Let's say we are faced with the task of selecting all the books about the subject we are interested in from a library containing 10 billion books: historical, artistic, fairy tales, romance novels in bright covers... To search in the display library, you do not need to get confused in the cards and fill out an application, but you just need to bring this item with you! And then to him (the antigen) librarians (phages) will immediately begin to approach with books in their hands. Specific books (antibodies) that are written only about what we brought with us will "stick" to the antigen tightly, and those in which the subject is mentioned in passing can be easily taken back to the shelf. After the most specific molecules (books) have been found with the help of an antigen (object), they are transmitted to bacteria-"readers". "Readers" turn out to be so conscientious that they not only perceive the information, but also copy it many times. The selection of phages with fragments of antibodies specific to the antigen is called selection (Fig. 3).

According to the source of the material, display libraries can be divided into three groups.

For example, synthetic libraries are based on a small number of structures of variable antibody domains, so it is much easier to work with them than with natural ones, which contain sequences that are diverse in thermodynamic and expression characteristics. But when using variants from natural libraries, the probability of developing an immune response is lower [9].

The molecules obtained in this way can be modified, improving their properties. In addition, a number of therapeutic agents can be created from the same antibody fragment. Depending on the purpose of therapy, it can be associated with a toxin (for example, to fight a tumor), with a cytokine (for targeted delivery to a sore spot) or with another helper fragment, even with a radionuclide.

The success of modern pharmacology largely depends on the development of such fields of science as molecular biology, bioinformatics and genetic engineering. Thanks to these disciplines, it became possible to synthesize the necessary DNA sequences, combine and modify them, as well as to obtain animal proteins in bacterial systems. The undoubted advantage of modern technologies is that they can be used not only to obtain analogues of existing antibodies, but also to create completely new ones [7].

In 2004, it was found that in several cases, taking infliximab (Remicade, Remicade) – anti–inflammatory monoclonal antibodies - was accompanied by the development of lymphomas in patients. In May 2006, the Journal of the American Medical Association (JAMA) published data that remicade increases the risk of cancer by three times [10]. In June 2008, the FDA reported a possible link between the development of lymphomas and other types of tumors in children and adolescents with the use of remicade.

There was an increase in the risk of death in cancer patients when taking avastin (2.5%) – an endothelial growth factor blocker (VEGF) – compared with using chemotherapy alone (1.7%). The fact is that Avastin (bevacizumab) itself does not interact with cancer cells. It blocks endothelial growth factor (vascular lining cells), which secretes the tumor to create more blood vessels around it for intensive nutrition. The tumor secretes the same VEGF as other healthy parts of the body, so blocking the growth of a certain proportion of the vessels needed by the body (for example, vessels for feeding the heart) turns out to be inevitable. Thus, in the case of the use of avastin, the increase in patient mortality is not associated with the underlying disease, but with heart failure [10].

The development of such side effects is predictable. A living organism is a very complex system, and interference aimed at one part of it entails changes in others. Therefore, even with the advent of such a delicate tool as therapeutic antibodies, it is impossible to talk about the invention of the "ideal medicine".

Current protocols are already based on a combined treatment approach, including vaccines, chemotherapy and monoclonal antibodies. Researchers have yet to develop such drugs and therapy regimens that will ensure effective and safe treatment of patients.

08.09.2015