Generics

• Generics

  Interferons

  
  

  Cytokines.

  The family of cytokines includes interleukins, interferons, chemokine, growth factors and colony-stimulating signal polypeptide molecules of the immune system. With a wide spectrum of biological activity, they determine not only adequate level of immune response, but also to regulate the interaction of the main integrative systems of the body - the nervous, immune and endocrine

  The structure and mechanism of action of most cytokines described in detail. Using the techniques of genetic engineering and biotechnology, many modern cytokines are currently produced as recombinant products identical to endogenous molecules in an amount sufficient for their clinical use.

  Many microorganisms - bacteria, yeast, viruses – are used as recipients of foreign genetic material for getting recombinant strains – producers of biotechnological products. Thus obtained recombinant strains of E. coli, producing interferons, insulin, growth hormones, various antigens; B. subtilis strains that produce interferon; yeast, produce interleukins etc.

  Using recombinant cytokines, providing adequate and targeted drug correction of immune dysfunction, increases the effectiveness of immunotherapy and treatment generally. Introduced into the body, make up for the shortage of endogenous cytokines regulatory molecules and reproduce their effects. This is especially important in severe or chronic diseases, where the use of conventional immunomodulators or inducers of cytokine synthesis is useless because of the depletion of compensatory possibilities of the immune system. At present, therapy with recombinant cytokines is one of the most promising and ever-expanding areas of immunopharmacology.

  A special place according to modern ideas about the molecular mechanisms of immune responses belongs to interferon gamma (hereinafter - interferon-y, IFN-y) - regulatory cytokine of the immune response.

  RECOFERON® GAMMA was created on the basis of recombinant IFN-y. The recombinant IFN-y in the body of animals and humans in the treatment and prevention of diseases of different etiologies provides adequate and targeted drug correction of immune dysfunction, filling the deficit of endogenous regulatory molecules and fully reproducing their effects. High immunocorrective efficiency, predictability, and the selectivity of its action caused the availability of specific receptors in the cells, and the existence of natural mechanisms for its elimination. Medicinal products based on recombinant IFN-y are powerful facilities of pathogenetic immune-oriented therapy, have a direct substitute action and have different inductive effects. Currently, they are widely used in the treatment of infectious diseases, cancer and other diseases in animals.

  
  

  The classification and the role of interferons

  In mammals, birds, reptiles and fish are found a broad spectrum antiviral action - interferons. They were first discovered in the study of viral interference, when the animal, infected with a virus, resists to infection with another unrelated virus.

  Interferons (IFN, IFN) -common name, combined number of biologically active proteins or glycoproteins having similar properties which are synthesized in the cells of the organism in process of a protective reaction in response to the invasion of foreign agents: viral infection or antigenic exposure. Due interferons cells become immune to the virus. A factor, defined as an interferon, must be a protein nature, have antiviral activity against various viruses due to cellular metabolic processes involving synthesis of RNA and protein.

  Interferons - multigene family of inducible cytokine, having a variety of functions, including anti-viral, anti-proliferative, anti-tumor and immunomodulatory function.

  Currently, studied more than 20 interferons differing in structure and biological properties of the predominant mechanism of action. IFN divided into three types:

  • Type I - known as virus interferon, including IFN-a (leukocyte synthesized by activated monocytes and B-lymphocytes), IFN-b (fibroblast, synthesized by fibroblasts and epithelial cells, macrophages), IFN-w, IFN-k;
  • Type II -known as immune, includes IFN-y (synthesized by activated T lymphocytes and NK-cells);
  • Type III - has been detected after the type I and type II, information about it demonstrates the importance of the type III IFN in certain types of viral infections.

  First type (IFN-a, IFN-b), mainly characterizes by antiviral and antiproliferative effects, less common - immunomodulator. They are produced directly after the meeting with the pathogen, their action is directed to the localization of the pathogen and prevent its spread in the body. The main effect of IFN-b is local, aimed at preventing the spread of infectious agents from the place of its implementation. If there is no inactivation of the infectious agent in place of the introduction, and it circulates in the body, its contact with lymphocytes and macrophages induces the production of IFN-a. Last quickly spreads with the blood stream and gets into the surrounding tissue, because its main function is to protect the distant organs. These interferons are performed early and non-specific protection of the body against the infectious agent.

  The main action of the second type of interferons (IFN-y) - participation in the reactions of the immune system. It begins to be generated in the subsequent stages of the infectious process by sensitized T-lymphocytes and it is actively involved in the cascade of a specific immune response.

  Viral interferons are induced during viral infections, and the synthesis of type II interferons (IFN-y) induced mitogenic or antigenic stimuli. Most cell types, infected by virus, capable to synthesize IFN-a / b in cell culture. In contrast, IFN-y is synthesized only certain immune system cells, including natural killer (NK) cells, CD4 T cells, CDS cytotoxic suppressor cells.

  
  

  Characteristic of different types of interferon

  Characteristic	IFN-a	IFN-b	IFN-y
  Chemical constitution	Protein	Glycoprotein	Glycoprotein
  Molecular weight, kDa	17,5-23,0	23,0	20,0-23,0
  The number of coding genes	more than 20	1	1
  The number of subtypes	at least 22 in human, several identified in animals	1	1
  The functional form	Monomer	Dimer	Dimer
  Acid resistance	Yes	Yes	No
  Inductors	Viruses (RNA> DNA), B-mitogens	Viruses (RNA> DNA), B-mitogens	Antigens, T-mitogens
  Main sources	Monocytes,B-lymphocytes	Epitheliocytes, Monocytes	T-lymphocytes NK-cells
  Mechanism of action	Inhibits protein synthesis	Inhibits protein synthesis	Enhances MHC antigens; activates cytotoxic T-cells, macrophages and NK-cells

  
  

  The mechanism of action of interferons

  
  

  Antiviral action

  Interferon does not act directly on the virus. Under their influence, the cell becomes resistant to infection. Interferons are the first line of defense against viral infections, because they begin generated immediately after exposure to the virus. This response is directly proportional to the infecting dose.

  At rest, normal cells contain significant amounts of IFN, because the genes encoding them don’t transcribe. Transcription begins after contact with an appropriate inducer cells. Inducers of IFN-a and -b are viruses, RNA (especially double-stranded), lipopolysaccharide (LPS), some components of the bacteria. Among viruses, the most potent inducers of interferons are RNA genome. DNA viruses - weak inducers (except poxviruses). Inducers of IFN-y favor antigens and T-mitogens.

  The blockade of the transcription of genes encoding IFN, is carried out by generating cell suppressor protein, binding to a nucleic acid chain region that controls transcription of these genes. Furthermore, for starting transcription is necessary activator protein, unblocking the activating zone. IFN inducers may affect both the inhibition of production of suppressor protein, and the activation of the activator protein synthesis. Activation of genes actuates protein synthesizing system cells, as a result synthesis and secretion of IFN are implemented.

  As a result of binding of IFN interferon molecules with specific receptors are activated gene group (localized in humans in chromosome 21) on the cell surface. This process is accompanied by the formation of more than 20 new intracellular proteins that contribute to the emergence of resistance to viruses. The main ones are two - 2 ', 5' - oligoadenylate synthetase and protein kinase. 2 ', 5' - oligoadenylate synthetase is an enzyme that converts adenosine triphosphate (ATP), 2 ', 5' - oligoadenylate. Last activates RNAase L, which causes damage to both cellular and viral RNA. By inactivation elF-factor 2 (prolonging factor) protein kinase inactivates elongation of peptide chains of viral proteins.

  Thus, under the action of IFN in cells are synthesized two enzymes, one of which cleaves viral RNA and another inhibits the synthesis of viral proteins. As a result, the new virus particles are either not formed or their number is reduced in tens or hundreds of times.

  Under the influence of IFN becomes damaged protein synthesizing cells system, which may lead to its destruction. However, this applies only to cells infected with the virus. Uninfected cells are indifferent to the effects of IFN, since both of the above protein are activated only in the presence of the viral RNA. Some viruses can block the antiviral activity of IFN. For example, adenoviruses produce specific RNA, which prevents activation of protein kinase.

  IFN with the receptor induces in cell three simultaneous processes that end:

  • activation of the latent endoribonuclease, leading to the destruction of viral RNA;
  • suppression of the synthesis of viral matrix RNA;
  • suppression of the synthesis of the viral envelope proteins.

  These mechanisms are implemented integrally antiviral effect, leading to the suppression of virus replication.

  Иммуномодулирующее действие

  IFN have immunomodulatory effects by influencing the expression of major histocompatibility complex receptor (MHC). IFN molecules increase the expression of class 1 MHC on all cell types, thereby improving the recognition of infected cells by cytotoxic T lymphocytes (CTL). Furthermore, IFN-y enhances expression of molecular class 2 MHC on antigen-presenting cells, thereby improving the presentation of viral antigens CD4 + lymphocytes and natural killer cells (NK-cells) are activated. IFN also stimulate phagocytosis.

  Regulation of immune responses by cytokines (pic.), including interferons, occurs on-ahead principle, cytokine effects on the cell causes the formation of other cytokines - cytokine cascade.

  
  
  

  Anti-proliferative and anti-tumor effect

  Antineoplastic and antiproliferative IFN effects due to the following mechanisms:

  • activation of cytotoxic cells;
  • increased expression of tumor-associated antigens;
  • modulation of antibody production;
  • inhibition of the action of tumor growth factors;
  • inhibition of RNA and protein synthesis of tumor cells;
  • slowing of the cell cycle transition from a phase of "rest";
  • stimulation of tumor cells to mature;
  • restoring deterrent control of proliferation;
  • braking neovascularization in tumor;
  • inhibition of metastasis;
  • biostimulation cytostatic activity: a change in metabolism and decreased clearance;
  • overcoming drug resistance due to the inhibition of multidrug resistance gene.

  
  

  Antibacterial effect

  In recent years, it is shown that IFN also have an antibacterial effect, which is based on the ability of IFN to induce the activity of certain enzymes in the affected cells:

  • Induction of indolamine-2,3- dioxygenase reduces intracellular L-tryptophan, which in turn the death of the bacterial cells due to a metabolic disorder;
  • Induction of NO synthetase leads to production of NO - a powerful bactericidal factor contributing to the destruction of the bacterial cell.

  Additionally, antibacterial role IFN-y is the activation of macrophages, which produce proinflammatory cytokines and reactive oxygen and nitrogen, prostaglandins. These factors contribute to the development of the inflammatory process, leading to the death of the bacteria.

  
  

  Immunobiological role endogenous interferon gamma

  
  

  Endogenous IFN-y(pic., table) was discovered in 1965 (E.F. Wheelock), it’s sensitive to acidic media glycoprotein with a molecular weight of 20 000 - 23 000. The genes encoding IFN-y, are human 12 th pair of chromosomes.

  
  
  

  Major producers of endogenous IFN-y - natural killer (NK) cells and T-lymphocytes. Among the T-lymphocytes IFN-y producers are both cytotoxic CD8 + -, and CD4 + helper T-lymphocytes, but when differentiation Tx1 and Tx2 ability to produce IFN-? They have only Tx1.

  Induce the production of IFN-y can interferonogen substances, antigens, T-mitogens, and some cytokines. IFN-y production is controlled by cytokines. IL-12 and IL-18 enhance its expression, and IL-2 contributes to the implementation of function of CD4 + lymphocytes by activating production of IFN-y.

  Synthesis of IFN-y is inhibitedby IL-4, IL-10, dexamethasone, cyclosporin A viral suppressor proteins, cancer cells.

  Background amount of IFN-yalways present in the body, even if there is no infection, such as interferon status analysis shows that healthy humans and animals always have detectable amount of IFN in the blood, it repeatedly increases due to stimulation or infection. However herpes virus infection and in the last stages of tumor process, the amount of IFN-y approach zero as herpes virus and cancer cells produce proteins that block the synthesis of IFN-y. Therefore, for herpes virus infection and cancer interferon inducers are useless, they must be entered externally.

  IFN-y has similarly to other IFN biological activity (inhibition of viral replication, antiproliferative activity, immunomodulatory effect), but IFN-y closely related with the cytokine system and makes a significant contribution to immunoregulation.

  The biological activity of IFN-y is realized by specific cellular receptors and intracellular signaling protein kinase cascade that leads to activation of the relevant transcription factors and the transcription of the whole gene family encoding resistance factors against infectious agents and complementary cytokines.

  Cells - targets for the action of IFN-y are macrophages, neutrophils, natural killer cells, cytotoxic T-lymphocytes having on their surface receptors for IFN-y

  
  
  

  T-lymphocytes and macrophages.The most important function of IFN-y is its involvement in mediating interactions between lymphocytes and macrophages in the regulation of cellular and humoral correlation components of the adaptive immune response (pic. 1). IFN-y is a stimulator of macrophages, facilitating expression of various functions of cells, including antigen processing and presentation, cytokine production, generation of reactive oxygen and nitrogen. Cytokines whose production is enhanced under the influence of IFN-y, include IL-1 and IL-12 (this cytokine promotes the synthesis of IFN-y and differentiation of T helper in Tx1 side).

  IFN-y increases the expression of MHC class I antigens, which play an important role in the recognition of foreign cells (virus-infected, tumor) CD8 + by cytotoxic T lymphocytes and increases the expression of MHC antigens of class II on antigen presenting cells.

  IFN-y reduces the secretory activity of Th2, inhibits the synthesis of IgE, IgG (2,4) and IgA. At the same time IFN-y enhances the development of an adaptive Th1-dependent immune response. IFN-y with its IL-4 antagonist maintains Th1 / Th2 balance.

  Cytotoxic T-lymphocytes and NK-cells by IFN-y involved in the implementation of a cytotoxic effect (antitumor and antiviral activity). When IFN-y is entered to the body NK-cell activity is increased in a few hours.

  Monocytes.IFN-y stimulates expression of high affinity IL-2 receptor (IL-2R) on the membrane of monocytes, increasing their sensibility to IL-2. In turn, the IL-2 under the action to monocytes stimulates their ability to kill tumor cells and bacteria. As a result of stimulation IFN-y and IL-2 monocytes produce large amounts of biologically active substances and mediators of inflammation: free oxygen species, H2O2, prostaglandin E2, thromboxane B2, TNF-a (tumor necrosis factor a).

  Neutrophils.IFN-y enhances the activity of cytochrome b558 in neutrophils (e.g., at phagocytes failure - for chronic granulomatous disease) that is accompanied by the activation of intracellular destruction of bacteria and reduces the risk of infections.

  IFN-y activates production of acute phase inflammatory proteins, increases the expression of genes C4 and C2 components of complement system.

  B-lymphocytes. IFN-y inhibits B-cell response to IL-4, suppresses the IgE production and expression of CD23-antigen. Thus, at overproduction of IgE syndrome with diffuse neurodermatitis in humans and used IFN-y, it inhibits the synthesis of IL-4 and IL-5 by T-helper cells. IFN-y is an inducer of apoptosis of differentiated B cells, giving rise to autoreactive clones. Cancels the suppressive effect of IL-4 on IL-2-dependent proliferation and the generation of lymphokine-activated killer cells.

  Thus, playing an important role in immunoregulation, IFN-y is a key cytokine of cell immune response and inhibitor of humoral adaptive immune response.

  IFN-y is critical for innate and adaptive immunity against viral, bacterial and several protozoal infections.

  Antiviral activity IFN-y blocks the replication of viral DNA and RNA, viral protein synthesis and assembly of mature virus particles.

  IFN-y influences on the cellular immune response by activating Th1-cells, NK-cells, macrophages, cytotoxic T-lymphocytes. It increases as a non-specific resistance and antigen-specific immune response. Thus IFN-y causes a cytotoxic effect on virus-infected cells.

  Antibacterial action of IFN-y is its ability to induce the activity of several enzymes in the affected cell, which leads to disruption the metabolism and destruction of the bacterial cell. Furthermore, IFN-y-activated cytotoxic T-lymphocytes and NK-cells implement cytotoxic effect and activated macrophages produce inflammatory cytokines, reactive oxygen and nitrogen, prostaglandins. These factors contribute to the development of the inflammatory process, leading to the death of the bacteria.

  The antiproliferative effect of IFN-y is it suppress the growth of tumor cells by inhibiting the synthesis of RNA and of proteins, inhibiting tumor growth factors, that stimulate cell proliferation, slowing down cell cycle transition in the phase "rest", restoring restraining control proliferation, as well as by activation cytotoxic T-lymphocytes and NK-cells that participate in the implement of cytotoxic effect.

  
  
  

  Thus, all the interferons are a group of polyfunctional protein factors with pronounced antiviral and antitumor effect of varying degrees. IFN-a has the strong antiviral activity among all interferons, IFN-y has a more pronounced antiproliferative activity. All interferons have immunoregulatory action of different severity (maximum belongs to IFN-y) - increases the activity of macrophages, T-lymphocytes and NK-cells.

  
  

  The manufacture on recombinant interferons

  nterferons are produced in two ways: leukocyte - derived from human and animal blood after exposure by the virus; Recombinant - is produced by genetic recombination - Molecular Biotechnology. Leukocyte interferon necessarily checked for the presence of antibodies to the virus to avoid possible contamination in their application. The recombinant interferons are absolutely safe, when they are used the probability of patient infection is completely eliminated.

  Genetic engineering is at the heart of biotechnology. It substantially reduces to genetic recombination, i.e. the exchange of genes between two chromosomes. Method of in vitro recombination or genetic engineering involves the separation or synthesis of DNA of different from each other organisms or cells, receiving DNA hybrid molecules, entered recombinant (hybrid) molecules into living cells, creating conditions for the expression and secretion of products encoded by the genes.

  Genes encoding these or other structures, or isolated (cloned) as such (chromosome, plasmid), precisely cleave from these genetic structures by using restriction enzymes. These enzymes and their thousands more already known are able to cut DNA at certain many connections, which is an important tool for genetic engineering. Recently discovered enzymes that cleave RNA at certain constraints, such as DNA endonucleases. These enzymes are called ribozymes.

  Relatively small genes can be obtained by chemical synthesis. At first, occurs a decryption the number and the sequence of amino acids in a protein molecule of the substance, and then by these data will learn the sequence of nucleotides in the gene, because each amino acid correspond to the three nucleotides (codon). With the help of a synthesizer by chemical reactions create a gene, similar to the natural gene.

  The resulting one of ways target gene using ligase enzymes is ligated to other gene which is used as a vector for the insertion of the hybrid gene into a cell. The vector may be plasmids, bacteriophages, human, animal and plants viruses.

  Expressed gene (e.g., IFN-y gene) as a recombinant DNA is incorporated into a bacterial cell of E. coli, which acquires a new property - uncharacteristic cell substance producing (IFN-y), expressed gene encoded.

  
  
  

  As recipients of expressed gene most commonly used E. coli, B. subtilis, Pseudomonas, Salmonella, non-typhoid serovars, yeasts, viruses.

  The method of genetic engineering created hundreds of products for medical and veterinary use, produced recombinant strains superproducer, many of which have found practical application. Already used in medicine genetically engineered hepatitis B vaccines, interleukins-1, 2, 3, 6, insulin, growth hormones, interferons a, b, y, tumor necrosis factor, thymic peptides, mieloleptides, tissue plasminogen activator, erythropoietin, HIV antigens, clotting factor, monoclonal antibodies, and many antigens for diagnostic purposes.

  One of the key components of the immune response is interleukin-2 (IL-2), interferon-gamma (IFN-y) and interferon alpha (IFN-a). On this basis, various recombinant cytokine preparations were established, which are widely used in human and veterinary medicine, including:

  • RECOFERON® GAMMA - IFN-y (LLC "Pharma Gene", Saint-Petersburg),
  • RECOFERON® ALPHA - IFN-a (LLC "Pharma Gene, Saint-Petersburg).

  
  

  Interferon Drugs

  Interferon drugs constitute a separate group of antiviral agents. They were created on the basis of deciphering the biochemical structure of the natural IFN, which are produced by many cells of the blood. IFN is a group of low molecular weight peptides which have antiviral, antiproliferative and immunomodulating activity.

  On the basis of recombinant interferons company LLC "Pharma Gene" (St. Petersburg) developed drugs RECOFERON® GAMMA and RECOFERON® ALPHA for veterinary use. Recoferons® - a recombinant interferons. Currently, the drug REKOFERON® GAMMA successfully completed pre-clinical and clinical trials as well as trials in Federal State Institution "Russian National Center For Quality And Standardization Drugs For Animals And Fodder" for state registration, passed the state registration procedure in the The Federal Service for Veterinary and Phytosanitary Surveillance. Registration certificate number: 78-3-6.15-2710№PVR-3-6.15 / 03 158.

  REKOFERON® GAMMA - recombinant interferon gamma (IFN-y), has a pronounced anti-viral efficiency, relates to immunomodulators.

  IFN-y is a regulatory cytokine, producer of which are natural killer cells, CD4, Th1 cells and cytotoxic CD8 suppressor cells. Interferon gamma receptors belonged to macrophages, neutrophils, natural killer cells, cytotoxic T-lymphocytes. IFN-y activates the effector functions of these cells, their microbicidal, cytotoxicity, cytokine production, nitroxide and superoxide radicals. IFN-y blockes viral DNA and RNA replication, synthesis of viral proteins and assembly of mature virus particles. In this causes cytotoxic effect on virus-infected cells.

  It inhibits B-cell response to IL-4, suppresses the IgE production and expression of CD23-antigen. It is inducer of apoptosis of differentiated B cells, giving rise to autoreactive clones. Cancels the suppressive effect of IL-4 on IL-2-dependent proliferation and the generation of lymphokine activated killer cells. Activates production of acute phase inflammatory proteins, increases the expression of genes C4 and C2 components of complement.

  The antiproliferative effect of IFN-y is the suppression of cell growth by inhibiting the synthesis of RNA and proteins, as well as the inhibition of growth factors stimulating cell proliferation.

  Monoclonal antibodies

  • A chimeric (murine / human) monoclonal antibody to EGFR extracellular domain
  • A chimeric (murine / human) monoclonal antibody against extracellular domain of CD20
  • The humanized monoclonal antibody against VEGF 121-165
  • The humanized monoclonal antibody against extracellular domain of HER2 / neu