Antiidiotypical antibody for antibodies inhibiting immunoglobulin bonding to its high affinity receptor
FIELD: immunology, biotechnology, medicine.
SUBSTANCE: invention relates to antiidiotypical monoclonal antibody or fragment thereof for BSW17 antibody effecting on LgE Cε3-region bonding to high affinity LgE receptor. Amino acid sequence is as described in specification. antiidiotypical antibody is useful as pharmaceutical composition ingredient for LgE-mediated disease treatment. Invention make in possible to prevent allergic disorders and inflammations due to inhibiting interaction between LgE Cε3-region with high affinity receptor by claimed antibody.
EFFECT: new agent for allergic and inflammation disorder treatment.
7 cl, 32 dwg, 5 tbl, 10 ex
The present invention relates to antiidiotypic antibodies. It refers to inhibition of the interactions that cause the stimulation of mast cells and basophils induced by binding to cell allergen, stitched with IgE, which leads to release of histamine and other mediators, as well as de novo synthesis of cytokines involved in the regulation of inflammatory and allergic reactions. The invention relates to antiidiotypic the antibodies or fragments of antibodies that have an impact on the bindingε 3-region of IgE to its receptor with high affinity for IgE (high-affinity IgE receptor.). Identification of specific binding sites for IgE, which interact with its high-affinity receptor (Fcε RI), provides a basis for obtaining antibodies that interfere with this interaction by detecting binding epitopes. Induction of such antibodies by vaccination allows you to create new and with full implementation method of Allergy treatment. In particular, the present invention relates to the identification and obtaining of recombinant fragments of antibodies, which can be included in a vaccine composition for the production of antibodies to IgE, which may provide protection against the induction of IgE mediated allergic Ricci.
Allergic symptoms are caused by separation from cells of the surrounding tissue and vascular structures vasoactive amines (neurotransmitters), first of all, histamine. Histamine in normal is stored in specialized cells called mast cells and basophilic granulocytes. Fat cells are distributed in the tissues of the animal, and basophils circulate in the vascular system. These cells synthesize and store histamine inside the cell until not is a certain sequence of events, stimulating its secretion.
The role of antibodies in the form of IgE in mediating allergic reactions is well known. IgE is a complex consisting of a polypeptide chain structure, which like other immunoglobulins, consists of two light and two heavy chains, stitched together by disulfide bonds in a "Y-shaped" configuration. Each light chain has two regions, one variable region (VL), made with a region having a relatively constant amino acid sequence, which is called the constant region (CL). In contrast, the heavy chains have one variable region (VH), and in the case of IgE, four constant region (CH1, CH2, CH3, CH4, also known asε 1,ε 2,ε 3ε 4). Two "shoulder" of the antibodies responsible for binding to the antigen, have areas with variable polypeptide structure, and they are called Fab'-fragments or F(AB')2d≤10-10M). In addition, it is believed that this curved shape also responsible for the formation of an equimolar complex between IgE and associated with the cell or soluble Fcε RIα though on the IgE molecule can form identical epitopes on twoε 3-domains responsible for binding to the receptor. This monovalently is functionally necessary, if you want to avoid stimulation of the receptor in the absence of allergen.
Interactive sites, depending on their functions, may already be available and, therefore, possess the ability to bind to cellular receptors. An alternative to this, they can be hidden until the binding of the antibody to the antigen, after which the antibody can change the structure and thus will become available to other active sites, which can further stimulate specific immune activity. It has been suggested that conformational transformation, affecting the binding ofε 3 receptor, is the cause of the stoichiometric ratio 1:1 which of the complex Fcε /Fcε RI on the cell surface.
Allergic (immunologic) the selection of mediators in the body of fat cells and basophils can occur only under the following circumstances: the IgE molecule must join or be attached via its Fc fragment to the site of cellular Fc receptor, resulting in the attachment of the molecules of IgE to mast cell or basophil; and Fab'-fragments associated with cell IgE molecules should be cross-stitched with certain compatible antigen (allergen). When this interaction occurs in the fat cell or basophile automatically stimulated histamine release in the local environment, causing a known Allergy symptoms (figure 1). In the last phase reactions occur other biochemical processes that lead to de novo synthesis and release of cytokines and other mediators.
Conventional approaches to the treatment of allergies include systemic therapy with the use of antihistamines or desensitization of patients; these approaches are not aimed at intervening in the main interaction of IgE - mast cell/basophil. Another approach is based on obtaining the polypeptide chains that can block the binding of antibodies in the form of IgE to Fc-receptors on the surfaces of cells, and the displacement of IgE binding sites, which IgE in the e-connected, in addition, studies have been conducted in order to clarify the nature of the alleged "effector" of the site within the Fc region of IgE, which probably creates immunological signal, stimulating the release of histamine mast cells/basophils.
Also, attempts were made use of recombinant fragments of IgE as immunogens to obtain a protective anti-IgE vaccine, and they were effective. The main argument against this vaccine due to the fear that used for immunization of large fragments of IgE initiates not only the production of inhibitory antibodies, but may also lead to cross-linking and thereby to produce the education of patients anaphylactogenic antibodies.
The purpose of strategies to overcome this problem is to identify the smallest possible fragment of IgE, ideally consisting only of the binding site of the receptor, which upon binding is hidden inside the complex IgE/IgERI and thus no longer available for cross-linking when generated by the vaccine immune response. It is unlikely that attempts to reconstruct such a complex molecular structure has been successful, given the removal of different spatial regions Withε 3, involved in the interaction of IgE/IgERI.
Problems inherent in "CL is a classical" approach, based on the use of vaccines can be overcome by using for the active immunization of short mimotope peptides or chemically synthesized peptides crosslinked with appropriate media, either in the form of a recombinant fused structures, for example, ovalbumin or IgG. Such peptides are structural mimic the epitope recognized by monoclonal antibody BSW17, which recognizes a conformational epitope on the Fcε including those parts of it that are inε 3-field and who are Withε 4-region. The cell line of hybridoma producing BSW17, deposited on December 19, 1996, in accordance with the Budapest Treaty (Deposit of micro-organisms in the European collection of cultured animal cells (ESAS) under registration number 96121916. This antibody has an interest profile of biological activities, which are summarized in figure 2. It is nanofilament and protects mast cells and basophils man from IgE-dependent excretion of histamine induced trigger signals. BSW17 or BSW17-like antibodies circulating in the vascular system, protect against allergic reactions by (a) inhibiting the stimulation of mast cells and basophils through competitive inhibition of the interaction of IgE/IgERI, and b) reduce browneye in serum using a down-regulation of IgE synthesis In the cells. As structural mimikou epitope of IgE antibodies to chemically synthesized BSW17-mimotope peptides, induce an immune response leading to the production of BSW17-like antibodies in the host. Since it is established that BSW17 are nerafinirovannye, inhibit the binding of IgE/IgERI and synthesis of IgE on the surface of b-cells, these antibodies secreted in response to the introduction of vaccines on the basis of BSW17-mimotope peptide have similar protective properties. A possible disadvantage of these vaccines on the basis of SW17-mimotope peptide may be a need to link chemically synthesized peptides with native proteins to enhance immunogenicity of the peptides. In addition, the structural flexibility of short peptides allows them to take different steric conformation. Thus, only a fraction of polyclonal antimonotone immune response may be therapeutically active in cross-reacts with human IgE.
The present invention is devoid of any defects inherent in the approach based on the use of mimotope peptides. It is based on the use of antibodies or fragments of antibodies, which are antiadiotipiceskih with respect to antibodies, which affect the binding of IgE to its high-affinity receptor, in particular on the application of recom is anantnag antiidiotypic antibodies to BSW17. According to the schematic of theory of Jerne (Jerne, N., Ann. Immunol. 125C  373), hypervariable sites of antibodies (At) themselves can be antigens. Produced by such antibodies known as antiidiotypic (anti-id) antibodies (At), because they are associated with idiotypical region of the first antibody (figure 3). Such anti-id antibodies directed to antigennegative center (paratope) a first antibody (At) and, thus, represent the "internal image" of the original antigen. Therefore, anti-id antibodies (At), called antibodies "internal image" or Itβ possess ability to stimulate the formation of antibodies using their hypervariable sites. These antibodies anti-id antibodies (At) resemble the structure of Pratap At and therefore have biological properties similar to antibody At. In the case of system hIgE/BSW17, IgE is the source of antigen, and BSW17 - antibody At. Pratap antiidiotypic antibodies to BSW17, i.e. At, therefore, is a structural MINICOM hIgE-region (epitope)recognized BSW17. Structural Pratap Et is equivalent to the above-described chemically synthesized BSW17-mimotope peptides. If this (recombinant) antiidiotypic antibody to BSW17 used as a vaccine, the vaccinated patient should induction SW17 under the tion of the immune response (At). Like BSW17, these polyclonal immunoglobulins in the form of At can affect the binding of IgE to its high-affinity receptor, thus acting as anti-allergic agents. In contrast to the flexible synthetic mimotope peptides Pratap At must be present in the environment in structurally rigid conformation. Thus, the immune response to a specific epitope hIgE should be more specific. In addition, this approach eliminates the need for heterologous immunogenic carrier. Thus it is possible to avoid possible side effects that are associated with protein carrier type tetanus toxin or toxoid diphtheria.
The present invention relates to antibodies or fragments of antibodies, which are antiadiotipiceskih in relation to such antibodies, as E25 (alsuma) or CGP56901 or preferably BSW17 affecting bindingε 3-region of IgE with high affinity IgE receptor; later in the context of the present description they briefly designated as "mimeole according to the invention". When they are antiadiotipiceskih towards BSW17, they are briefly referred to as BSW17-mimeole".
Thus, mimeole according to the invention are antiidiotypic antibodies or antibody fragments that are specific Saint is called epitope, which is Pratap antibodies to IgE that recognizes the siteε 3-sphere molecules of IgE binding to high-affinity IgE receptor (Fcε RI).
If mimeole according to the invention are intended for administration to humans, they are mainly human. Preferably they are recombinant. Preferably they are monoclonal. Preferably they represent fragments of antibodies, for example, contain or include:
or both heavy and light chains (e.g., Fab fragments)or only the heavy or light chain (e.g., dimers light chain), preferably in combination with the component parts of their constant region, which, for example, presented in figure 4 (Seq.id. no. 35, 36, 37 and 38), where the term "constant region" you should also understand the constant region with little steric modifications such as those present in alltimecase variants, for example in the 1-5 positions, as a rule, only in 1 position of amino acids in the constant region;
or parts thereof, in particular, at least they define the specificity of the part, for example, presented on figa-5g (Seq.id. no. 2, 4, 6, 8);
- or Subpart, for example, at least their hypervariable sites, such as peptides, consisting of sections amino acid sequence comprising at m is d one CDR, for example, including at least one CDR, or preferably two or more, preferably three CDRs presented on figa, 5B, 5B or 5C (Seq.id. no. 2, 4, 6, 8), optional in conjunction with the adjacent frame sequences, e.g., up to about 10 amino acids at one or both ends of the CDR.
Mimeole according to the invention are isolated and almost pure antibodies or antibody fragments derived from naturally occurring anti-id antibodies to IgE. In particular, they contain virtually no other antibodies. Under "practical purity" should be understood purity constituting at least about 60 wt.%, preferably about 90 wt.%, more preferably about 99 wt.% or more.
The invention also relates to pharmaceutical compositions, especially vaccines, including mimeole according to the invention either as single molecules or in the form of a conjugate protein, a chemically-related molecule immunogenic carrier, optionally in combination with an adjuvant or other conventional excipients.
It also refers to mimetism according to the invention, intended for use as pharmaceutical agents, particularly as vaccines, primarily for the treatment of IgE mediated diseases.
It also refers to the skin is of antibodies which affect the binding ofε 3-region of IgE with high affinity IgE receptor, such as BSW17, to identify mimeole according to the invention using conventional methods, such as the technology of phage reproduction.
In addition, it relates to a method for treatment of IgE mediated diseases, in particular, by vaccination, introducing a therapeutically effective amount mimeole according to the invention to a patient in need of such treatment or vaccination.
It also relates to the use of mimeole according to the invention for the preparation of drugs for the treatment of IgE mediated diseases, in particular vaccines.
It also relates to the use of mimeole according to the invention to generate polyclonal or monoclonal antibodies intended for passive immunization; to obtain polyclonal or monoclonal antibodies to mimetism according to the invention for passive immunization or by introducing mimeole according to the invention acceptable animal (not human) and isolation and purification of the thus obtained antibody, or by using conventional methods based on hybrid; and to the use of polyclonal or monoclonal antibodies obtained using mimeole according to the invention, for the treatment of IgE mediated diseases by passive immunization.It also relates to a method of identifying mimeole according to the invention, providing
- identification of naturally occurring antiidiotypic antibodies to IgE;
- allocation of their fragments; and
- selection of their recombinant fragments by linking with acceptable monoclonal antibody to IgE, such as BSW17, which affects the binding ofε 3-region of IgE with high affinity IgE receptor.
After their identification and study of properties mimeole according to the invention can be obtained in the usual way, for example, using recombinant DNA or by chemical synthesis.
To identify antiidiotypic antibodies having the same specificity as mentioned above mimotope peptides (i.e. selected epitopes in the IgE molecule), can be used available library of bacteriophage that expresses Fab-area population of human antibodies. This library design, for example, from the group of b-cells isolated from tonsils of people, and the immobilized antibody BSW17 used as targets for biological penninga. Phage particles expressing human Fab fragments specific recognition BSW17, allocate and increase their number. Thus, these recombinant Fab-fragments are pilotelli according to the present invention and particularly antiidiotype to hypervariable areas BSW17. When used as a vaccine they induce an immune response, which leads to the production of BSW17-like antibodies in suffering allergies of the patient. Because BSW17 is nanofilament and inhibits the binding of IgE/IgERI and synthesis of IgE on the surface of b-cells, polyclonal antibodies, secreted in the body of the patient in response to the introduction of vaccines based on antiidiotypic Fab fragments of BSW17, have similar properties. The immune response is highly specific and safe, as opposed to the "classical approach based on the use of vaccines" are not originating from any IgE fragments of the protein, which could provide cross-linking of antibodies in immunized patients, in addition, can be applied compositions, in which the missing media.
These BSW17-mimeole are recombinant antibodies or antibody fragments consisting of the variable regions (V regions) and constant regions (P-regions)derived from human immunoglobulin G. Two different clone (clone 52 and 43), which are different fragments mimeole on their surface, were identified by biological penninga libraries of phage antibodies on immobilized antibody BSW17. Structure mimeole simulates BSW17-epitope fragments of the human gE within hypervariable sites (CDR) and the adjacent frame sections (FR) V-regions. cDNA and amino acid sequences of the heavy and light chain V-regions of clone 52 and clone 43 presents on figa-5g (Seq. id. no. 1-8). The design of the light chain of clone 52 (L.C.)2includes dimeric "Fab-like fragment of the light chain. The full structure of these BSW17-mimeole schematically represented in figa-4B, it should be understood that the portions of constant region may also have a slight steric modifications such as those present in the above-described alltimecase options. Amino acid sequence of each complete heavy and light chains of these clones represented on figa-12g (Seq.id. no. 35-38).
Mimeole according to the invention possess pharmacological activity. As a result, they can be used as pharmaceutical agents such as antigens for vaccines. Being virtually unable to call naritaiwatashi histamine release, they are able to stimulate the appearance of antibodies with strong serological cross-reactivity towards amino acid sequences of the target Fc fragment of IgE.
Initial dose mimeole according to the invention is, for example, from about 0.05 mg to about 5 mg, preferably about 1 mg; it may be, for example, nasal or subcutaneously or intramuscularly, and then after 14-28 days imposed on the Torno (booster) dose of the same drug. Dose, of course, must depend to some extent on the age, weight and General health of the patient and can be adjusted accordingly.
Immediate vaccination, which is called active immunization pilotelli according to the invention is preferably carried out using recombinant peptides (Fab fragment light chain or heavy chain), which can be obtained using conventional methods in various expression systems hosts, such as bacteria, fungi or eukaryotic cells.
Preferred is the introduction of free recombinant mimeutil. However, it is also possible to further improve the immunogenicity of immunogen by chemical bonding with the immunogenic carrier. The term "immunogenic carrier" in the context of the present description mean materials that have the ability to independently cause an immunogenic response in an animal host and which can be covalently cross-linked to the polypeptide either directly through the formation of peptide or ester links between free carboxyl, amino or hydroxyl groups in the polypeptide and the corresponding groups on the material immunogenic carrier, or alternatively, by communication through a conventional bifunctional cross-linking group./p>
Examples of such carriers include serum albumin animals, globulins serum of animals, thyroglobulin animals, hemoglobin animals, hemocyanine animals (in particular, hemocyanin lymph snails [KLH]); proteins extracted from Ascaris, for example extracts of Ascaris described in J. Immun. 111  260-268, J. Immun. 122  302-308, J. Immun. 98  893-900 and Am. J. Physiol. 199  575-578, or refined products; polylysin, polyglutamine acid, copolymers of lysine and glutamic acid, copolymers containing lysine or ornithine, etc. currently, vaccines are manufactured using quality material immunogenic carrier of diphtheria toxoid, such as CRM197 or tetanus toxoid (Lepow M.L., and others, J. Infectious Diseases 150  402-406; Coen Beuvery, E., and others, Infection and Immunity 40  39-45), and these toxaemia materials can also be used according to the present invention. In contrast to chemically detektirovaniem diphtheria toxin, preferably used recombinant mutant diphtheria toxin CRM197. In CRM197 the glycine residue at position 52 is replaced by glutamic acid with the formation of non-toxic product. CRM197 is a well known non-toxic carrier protein, and it is used in the registered human vaccine. Purified protein derivative of tuberculin (PPD) is particularly preferred for the COI is whether in the scheme of "active" immunization, because (1) it is not itself induces T-cell response (i.e., it is in fact, "T-cell hapten"), but still behaves like a fully processionary antigen and therefore is recognized by T-cells; (2) it is known that it is one of the most powerful Gapanovich "media" from the point of view of the detection mechanism; and (3) it can be used for immunization of people without additional testing.
As agents, linking the hapten to a carrier, can be used by agents commonly used in the preparations of antigens. Covalent crosslinking mimeole according to the invention with immunogenic material of the carrier may be made the conventional method. For example, for direct covalent crosslinking is preferably used as a crosslinking agent derivatives of bis-N-Succinimidyl, more preferably bis(sulfosuccinimidyl)suberate (BSS), or glutaric aldehyde or carbodiimide, most preferably dicyclohexylcarbodiimide (DC) or 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
The ratio of the hapten, the agent linking the hapten to a carrier, and the carrier can be easily evaluated by the conventional method, but preferably, applied to the mass ratio of the carrier and the hapten ranged from about 1 to about 6, preferably from priblizitel is but 1 to about 5, calculated on the weight of the hapten, and the ratio of molar equivalents of the agent linking the hapten to the carrier and the hapten ranged from approximately 5 to approximately 10. Using the reaction described above combination of media associated with the hapten with an agent that binds the hapten to a carrier, receiving the desired composition of the antigen in the form of a complex peptide-carrier consisting of mimotope according to the invention and a carrier.
After completion of the reaction the resulting immunogen can be easily isolated and purified conventional method, such as dialysis, gel filtration or by fractionated precipitation.
The preferred embodiment of the present invention is the active immunization by direct vaccination; however, it is also possible and passive immunization. In the latter case mimeole according to the invention introduce a suitable animal (not human) and the resulting antibodies are isolated and purified and then administered to a person in order to cause the relief of allergic symptoms.
Mimeole according to the invention can be used as pharmaceutical agents, especially vaccines, in particular, for the treatment of IgE mediated diseases, such as allergies, for example, asthma, atopic dermatitis, allergic forms of eosinophilia, rhinitis, chronic urticaria and food allergies.
The term "treatment" should p is take preventive, and therapeutic immunization. The host is preferably a human, however, when amending the invention is also applicable to almost any mammal, such as a cat or dog. When considering this application in the international phase as references addressed the following prototype:
Dl Progr. Allergy Clin. Immunol. (Proc. Int. Congress Allergol. Clin. Immunol., 16) 4  339-342;
D2 Int. Arch. Allergy Immunol. 118  119-121;
D3 Tumor Biology 18  Suppl. 2, 59;
D4 Tanox WO 89/06138;
D5 Panent Abstracts of Japan 4  [S.A. 130  138296k] [JP 11/000174].
The explanation of the drawings:
Figure 1: Interaction between IgE and its high affinity receptor.
Figure 2: properties of monoclonal antibodies BSW17 to hIgE.
Figure 3: Scheme antiidiotypic interaction:
Epitope hIgE recognized BSW17, and antiidiotypic Pratap schematically shown as black dots. Black circles marked hypervariable region of the antibody BSW17 (At) and polyclonal antibodies 3 (At)induced by immunization using antiidiotypic antibodies At.
Figure 4: Structure of the three recombinant BSW17-mimeole:
A: Anti-id-BSW17 (archidiocese antibody to BSW17),
clone 52 (SDS426); light chain: (L.C.)2(Seq.id. no. 36)
B: Anti-id-BSW17, clone 52 (SDS427); Fab: FAB(Seq.id. no. 35 and 36)
In: Anti-id-BSW17, clone 43 (SDS463); Fab: FAB(Seq.id. no. 37 and 38)
Figure 5.: Clones Fab-fragm new antibodies to BSW17: DNA sequence of a human immunoglobulin, identified using bacteriophage and deduced amino acid sequence:
Hypervariable sites (CDR) in italics:
Figa: clone 52; variable region heavy chain (Seq.id. no. 1 and 2) (CDR1: Seq.id. no. 39 and 40; CDR2: Seq.id. no. 41 and 42; CDR3: Seq.id. no. 43 and 44);
Figb: clone 52; variable region light chain (Seq.id. no. 3 and 4) (CDR1: Seq.id. no. 45 and 46; CDR2: Seq.id. no. 47 and 48; CDR3: Seq.id. no. 49 and 50);
Figw: clone 43; variable region heavy chain (Seq.id. no. 5 and 6) (CDR1: Seq.id. no. 51 and 52; CDR2: Seq.id. no. 53 and 54; CDR3: Seq.id.no. 55 and 56);
Figg: clone 43; variable region light chain (Seq.id. no. 7 and 8) (CDR1: Seq.id. no. 57 and 58; CDR2: Seq.id. no. 59 and 60; CDR3: Seq.id. no. 61 and 62).
6: Homology of amino acid sequences rFab-fragments of antibodies to BSW17 andε 3 domain of human IgE:
A: anti-id Fab, clone 52, a heavy chain (hIgE,ε 3: Seq.id. no. 25; clone 52: Seq.id. no.26):
anti-id Fab, clone 52, light chain, a comparative analysis of the sequences 1 (hIgE, Cε 3: Seq.id. no. 27; clone 52: Seq.id. no.28);
anti-id Fab, clone 52, light chain, a comparative analysis of the sequences 2 (hIgE, Cε 3: Seq.id. no. 29; clone 52: Seq.id. no.30), respectively;
B: anti-id Fab, clone 43, a heavy chain (hIgE,ε 3: Seq.id. no. 31; clone 43: Seq.id. no.32);
anti-id Fab, clone 43, light chain (hIgE,ε 3: Seq.id. no. 33; clone 43: Seq.id. no.34), respectively.
A comparative analysis of the amino acid sequence: identical residues denoted by shaded rectangles similar to the amino acids marked with gray rectangles (Lipman and Pearson). Provisions Fcε residues indicated above each pair of compared sequences. The position of the hypervariable (CDR) and frame sections (FR) recombinant Fab-fragments indicated under each pair of sequences.
Fig.7: Competitive binding of rFab-fragment anti-id-BSW1 on the surface premirovany IgE SNOα -cells using labeled with FITZ BSW17.
Fig: Binding of human IgE purified using affinity chromatography rabbit immunoglobulines antibodies to SW17-mimeole:
The detection of complexes hIgE/antibody to mimeole using sandwich ELISA: hIgE and purified using immunoaffinity chromatography preparations of antibodies to SW17-mimeole were mixed in equimolar concentrations and incubated at 4° C. After incubation the mixture was added to the wells tiralongo microplate sensitized with a monoclonal antibody to hIgE LE27 (1 μg/ml) as immobilizing antibodies. Bound IgG mimeole were revealed using complex: goat anti-rabbit IgG-HRP:
□ denotes complexes hIgE/SDS410;
Ο denotes complexes hIgE/SDS411.
Fig.9: the Immune response to the antibody to SW17-mimeole in mice of Balb/c line:
▪ refers to the mouse 1; • refers to the mouse 2; ▿ refers to the mouse 3; Δ refers to the mouse 4;indicates the mouse 5
A: anti-id-BSW17.52; Legazpi (SDS426);
B: anti-id-BSW17.52; Fab (SDS427);
In: anti-id-BSW17.43; Fab (SDS463).
Value OP represent the values of the optical density corrected for background binding in desensibilisation holes. The average values of two replicates. Variations are usually <0,05 OP.
Figure 10: Inhibition of binding hIgE/Fcε RIα using purified using immunoaffinity chromatography of antibodies to mimeole:
A: ▪ means BSW17;
• refers to the clone 52 antibody; light chain (SDS410);
Δ refers to the clone 52 antibodies; Fab (SDS411);
▿ refers to the clone 52 antibody; light chain (passed through the column);
B: ▪ means BSW17;
• refers to the clone 43 antibodies; Fab (SDS476).
11: Profiles PKA-points groups of rhesus monkeys (n=2), immunized with various preparations mimeole:
A: Immunogen: anti-id-BSW17.52; light chain (SDS426);
B: Immunogen: anti-id-BSW17.52; Fab (SDS427);
In: Immunogen: anti-id-BSW17.43; Fab (SDS463).
Presents data on passive cutaneous anaphylactic (PKA) skin reactions rhesus monkeys through various time intervals after immunization. The values of PKA-points indicate the intensity of PKA reactions, calculated on the basis of the values of the areas under the curves (AUC)obtained by plotting the dependence of the diameters of the blue spots on the skin from the concentrations injected IgE (JW8). The ball is s denote the average values for each group, consisting of 2 monkeys that were immunized with the same drug mimeutil, and is calculated based on the data for each monkey are shown in table 4. Variations are shown on the graph in the form of line segments describing the value of the error of the result. Statistical values of R is shown on straight lines describing the value of the error of the result. Moments in time that were produced booster injections are indicated under the x-axis.
Fig: Complete amino acid sequence of the heavy and light chains of the three recombinant BSW17-mimeole:
Fig.l2a: Anti-id-BSW17, clone 52: variable and first constant region of the heavy chain (Seq.id. no. 35);
Figb: Anti-id-BSW17, clone 52: variable and constant region of the light chains of Kappa-type (Seq.id. no. 36);
Figw: Anti-id-BSW17, clone 43: mariaelena and first constant region of the heavy chain (Seq.id. no. 37);
Figg: Anti-id-BSW17, clone 43: variable and constant region of the light chains of lambda-type (Seq.id. no. 38).
Clone 52 mimeole (L.C.)2includes light chain, presented on figb (Seq.id. no. 36)connected by a disulfide bridges, as shown in figa.
Fab-fragment of clone 52 mimeole includes light chain, presented on figb (Seq.id. no. 36), and a heavy chain, presented on figa (Seq.id. no. 35)connected by a disulfide bridges, as shown in Fig.
Fab-fragments the UNT clone 43 mimeole includes a light chain, presented at Figg (Seq.id. no. 38), and a heavy chain, presented on FIGU (Seq.id. no. 37)connected by a disulfide bridges, as shown in figv.
Below the invention is illustrated in the examples, not ohranyaemih its volume. Temperatures are given in degrees Celsius. In the description uses the following abbreviations:
anti-id = antiidiotypic
ABTS = [2,2'-asindi(3-ethylbenzothiazoline)sulfonate]
BSA = bovine serum albumin
BSW17 = mouse monoclonal antibody to human IgE;ε 3 - specific
CDR = hypervariable sites
Withε 3 = third domain of the constant region of the heavy chain of IgE;
Withε 4 = fourth domain of the constant region of the heavy chain of IgE
Withε F = mimotopes peptide that mimics the sε 3-epitope region recognized BSW17
Withε M = mimotopes peptide that mimics the sε 4-epitope region recognized BSW17
CFU = colony forming unit
ELISA = enzyme-linked immunosorbent assay
Fab = fragment antibodies devoid of constant regions 2 and 3 of the heavy chain
Fcε RI; IgERI = high-affinity IgE receptor
Fcε RIα = high-affinity receptor for IgE, α chain
TPS = fetal calf serum
FR = frame sections
FITZ = conjugated with fluoresceinisothiocyanate
HRP = peroxidizability of horseradish
CSA = human Suvereto the hydrated albumin
(h)IgE = (human) immunoglobulin E
Mat = monoclonal antibody
MNC = mononuclear cells
NIP = 3-nitro-4-hydroxydiphenylamine acid
p.c. = polyclonal
Phab = phage providing Fab-fragments (Fab-expressing bacteriophage)
SFR = phosphate buffered saline
PKA = passive cutaneous anaphylaxis
PWM =mitogen of lacunosa
r = recombinantly
KT = room temperature
SPR = surface plasma resonance
Example 1: Construction of libraries of phage
a) a Source of lymphocytes
As a donor to obtain mononuclear cells (MNC) from peripheral blood using the two adult men. The first adult male, who was atopic donor, with clinical allergic symptoms, was administered via intramuscular booster injections of 0.5 ml adsorbed on alum tetanus toxoid (Te Anatoxal Berna, Swiss Serum and Vaccine Institute, Berne, Switzerland). After 7 days MNC were isolated by centrifugation in a gradient of Ficoll (Lymphoprep, the firm Pharmacia, Milwaukee, Wisconsin, USA) and then were cultured for 3 days in medium RPMI-1640 (firm Seromed, Basel, Switzerland)containing 103units/ml IL-2 (firm Sigma, St. Louis, Missouri, USA), 50 μg/ml of cell line Pansorbin (Staphylococcus aureus Cowan 1, the firm Calbiochem, La JOLLA, California, USA) and stolbnyake, diluted in the ratio 1:1000 medium RPM1-1640. Then from these cells received total RNA using phenol-chloroform and guanidinosuccinic (Chomczynsi R. and Sacci, N., Anal. Biochem. 162  156). The second donor, who was an adult hyperimmune man with RH D, was administered via an intravenous booster injection of 2 ml of compacted red blood cells, obtained from the known blood donor-men who have blood group 0 RhD+. MNC were isolated by centrifugation in a gradient of Ficoll on the 18th day after revaccination. Cells before RNA extraction were cultured for 3 days in medium RPM1-1640 containing 103units/ml IL-2 and 10 μg/ml of the mitogen of lacunosa (PWM; firm Sigma, L9379, Buchs, Switzerland).
Samples of human tonsils were obtained after removal of the tonsils have three children. Tonsils were softened in the medium RPM1-1640 in a sterile Petri dish and cut into small pieces. Then, tissue, cells and medium was transferred into a sterile test tubes, allowed to settle tissue debris and supernatant MNC were isolated by centrifugation in a gradient of Ficoll. Selected b cells by incubation MNC covered (sensitized) CD19 paramagnetic granules and then received RNA using the above method using phenol-chloroform and guanidinosuccinic.
For cloning circuits all mimeole use the Wali vector omb3, which was obtained from the Scripps Research Institute La Jolla, California, USA (Barbas III, C.F., and Lerner, R.A., Companion Methods Enzymol. 2  119). The Escherichia coli strain XL1-Blue, which was used for transformation vector mb3, and the phage-VCSM13 helper, received from the company Stratacyte (La JOLLA, California, USA).
b) creating a library of bacteriophages
Designed three different libraries: the first, denoted as BS from MNC isolated from the blood of the first atopic donor-men, the second, designated as LD2, MNC second donor-men obtained on day +18 after intravenous booster injection, and the third, denoted as ST, enriched In the cell population MNC isolated from the tonsils of children. Of these cells received total RNA using the method based on phenol-chloroform and guanidinosuccinic. 10 μg of this RNA was used to generate cDNA using oligo(dt)-primer (400 ng) was subjected to reverse transcription using reverse transcriptase M-MuLV and conditions described by the supplier (firm Boehringer Mannheim, Germany). PCR amplificatio carried out according to the method described by M. Vogel and others, E.J. of Immunol. 24 (1994) 1200. In General, the method consists in the following: used 100 μl of medium for PCR buffer containing Perkin-Elmer with 10 mm MgCl25 µl cDNA, 150 ng each of the appropriate 5'- and 3'-primer, all 4 dNTP, each at a concentration of 200 μm, and 2 units/ml Taq polymerase (firm Perkin Elmer, who translated Jersey USA). PCR amplification of the heavy and light chains of the Fab molecules were carried out separately using a set of primers company Stratacyte (details below). For amplification of the heavy chain used 6 primers for synthesis of progress against transcription, which hybridize with each of the 6 families VHgenes and amplification of light chains used one primer for chains of Kappa-type and one primer for chain lambda type. Primers for synthesis during transcription, designed in such a way that they are compatible with the hinge constant region domains γ 1 and γ 3 heavy chain. For amplification of the light chain primers for synthesis during transcription, should correspond to the 3'-end of the constant regions of Kappa chain and lambda chain. PCR products of the heavy and light chains were combined separately, was subjected to purification on a gel and digested respectively with restricted XhoI/SpeI and SacI/XbaI (firm Boehringer Mannheim, Germany). After cleavage of the PCR products were extracted once with a mixture of phenol:chloroform:isoamyl alcohol and purified using the gel excision. Embedding cleaved with XhoI/SpeI Fd-fragment and subsequent embedding by ligating the cleaved with SacI/XbaI light chain vector mb3, transformation of cells of strain XLl-Blu and receiving phages was carried out with the public method described by Barbas III, C.F., and R.A. Lerner, Companion Methods Enzymol. 2  119. After transformation of strain XLl-Blue E.coli cells were isolated and subjected to titration on tablets to determine the size of the libraries. These results indicate that the level of expression libraries is 1× 107, 7,7× 106and 3× 106CFU (colony forming units) for BS, LD2 and ARTICLE, respectively.
C) PCR primers
VHI 5'-CAC TCC CAG GTG CAG CTG CTC GAG TCT GG-3' (Seq.id. no. 9):
VHII 5'-GTC CTG TCC CAG GTC AAC TTA CTC GAG TCT GG-3' (Seq.id. no. 10);
VHIII 5'-GTC CAG GTG GAG GTG CAG CTG CTC GAG TCT GG-3' (Seq.id. no. 11);
VHIV 5'-GTC CTG TCC CAG GTG CAG CTG CTC GAG TCG GG-3' (Seq.id. no. 12):
VHV 5'-GTC TGT GCC GAG GTG CAG CTG CTC GAG TCT GG-3' (Seq.id. no. 13):
VHVI 5'-GTC CTG TCA CAG GTA CAG CTG CTC GAG TCA GG-3' (Seq.id. no. 14):
CHI(γ I) 5'-AGC ATC ACT AGT ACA AGA TTT GGG CTC-3' (Seq.id. no. 15);
VL(κ ) 5'- GT GCC AGA TGT GAG CTC GTG ATG ACC CAG TCT CCA-3' (Seq.id. no. 16);
CL(κ ) 5'- T CCT TCT AGA TTA HUNDRED ACA CTC TCC CCT GTT GAA GCT CTT TGT GAC GGG CGA ACT C-3' (Seq.id. no. 17);
VL(λ ) 5'- TGC ACA GGG TCC TGG GCC GAG CTC GTG GTG ACT CA-3' (Seq.id. no. 18);
CL(λ ) 5'- G CAT TCT AGA HUNDRED TTA TGA ACA TTC TGT AGG GGC-3' (Seq.id. no. 19).
Example 2: Selection of recombinant fragments SW17-specific antibodies (BSW-17-mimeole) from phage libraries
Selection SW17-specific phages was performed using 4 cycles of panning. Each cycle consisted of two pre-absorption on the Mat to IgE Le27 before absorption on the Mat to IgE BSW17. Preliminary absorption was carried out as follows: 2 tubes on the I immunoassay (Maxisorp, Nunc) were senzibilizirani 4 ml Le27 (20 μg/ml) overnight at 4° then blocked for 2 h at 37° 4 ml of a mixture of SFR/2% skim milk. The first test tube was incubated on a rotating downward upward horizontal platform at RT for 30 min with 4 ml of blocking solution containing 2× 1012SOME of each library phage (BS, LD2 and ARTICLE). Then the phage was transferred to the second tube and the process repeated again. After the second pre-nonspecific absorption against Le27 phages were added in a test tube, sensitised 4 ml BSW17 (20 μg/ml)and blocked with a mixture SFR/2% skim milk, according to the method described above. After incubation for 2 h at RT on a rotating downward upward horizontal platform, the test tube is then washed 10 times SFR/0.1% tween and 10 times SFR. Adherent phage consistently suirable first with 500 μl of 0.1 M triethylamine, and then after three times washing SFR with 500 μl of 0.1 M HCl solution, the pH value is increased to 2.2 with glycine, which was added 1 mg/ml BSA. Each stage of the elution was carried out for 10 min at RT and erwerbende phage was neutralized with 250 μl of 1 M Tris-HCl, pH 7.4, and 30 μl of 2 M Tris-base, respectively. Selected phages amplified using cells of E. coli strain XLl-Blue according to the method described by Barbas and Lerner is ISE (1991), then they were used in three cycles of panning. After each cycle of panning monitored titer eleirovania phages, determining CFU (table 2):
The increase in titer SW17-specific Phab by successive cycles of panning
|The cycle of panninga)||Title buervenich phages (CFU)|
|a)For each cycle of panning 6× 1012phage particles pre-absorbed twice in test tubes sensitized with 20 µg/ml Le27, and then incubated in the same test tube sensitized with 20 µg/ml BSW17.|
Example 3: the Nucleotide sequence recombinantly BSW17-mimeutil.
Plasmid DNA of selected phage clones were obtained using the Nucleotrap kit (firm Machery-Nagel, düren, Germany) and sequencing of the nucleotide sequences was performed using the system for sequencing type ABI 373A using set for termination circuit PRISM Ready Reactin DyeDeoxy Terminator Cycle Sequencing (company Applied Biosystems, Germany).
For sequencing the consequences of the successive heavy chain used the following primers:
CHγ l (5'-CGCTGTGCCCCCAGAGGT-3') (Seq.id. no. 20) and
DCL (5'-GGCCGCAAATTCTATTTCAAGG-3') (Seq.id. no. 21).
To obtain sequences of the light chain used the following primers:
Withλ (5'-GAGACACACCAGTGTGGC-3') (Seq.id. no. 22).
Withκ (5'-CACAACAGAGGCAGTTCC-3') (Seq.id. no. 23) and
pCL (5'-CTAAACTAGCTAGTCGCC-3') (Seq.id. no. 24).
The primers were synthesized using Microsynth (Balgach, Switzerland), based on DNA sequences of different selected phage clones were withdrawn 2 different amino acid sequences of the heavy and light chains of recombinant SW17-specific antibody (clone 52, clone 43). Sequences and their localization in hypervariable sites (CDR) and the sequence of frame sections shown in figa-5g (Seq.id. no. 1-8).
Comparative analysis of amino acid sequences of fragments of recombinant BSW17-specific antibodies, represented by clones of 52 and 43, and a human IgE allowed to reveal homology with human sequences Withε 3-domain, which is involved in binding with high-affinity receptor (6) (Seq.id. no. 25-34). Thus, paratope characteristic fragments, recombinant antibodies that mimic certain structures present in the human IgE ("mimeutil"). Thus, antibodies that are produced in allergic patients by vaccination with these recombinant pilotelli, the debtor is advised to recognize human IgE and prevent allergic reactions by inhibiting the binding of IgE/IgERI.
Example 4: production and purification of recombinant SW17-mimeole
Received instant mimeole of phage clones 43 and 52 (figure 5) (Seq.id. no. 1-8). To obtain soluble Fab fragments of the sequence gIII encoding tail protein pIII ragovoy particles were removed and replaced consisting of 6 residues histidine tag to facilitate purification of Fab-fragment using Ni2+-chelate affinity chromatography.
Fastenau DNA was obtained using Nucleotrap kit (company Macherey-Nagel, düren, Germany) and were digested with SpeI and NheI. The DNA fragment with a length of 4.7 TPP devoid gIII-region, was treated with alkaline phosphatase and purified by electrophoresis on agarose gel. The linearized DNA is ligated with a DNA fragment coding for six histidine residues at the 5'- and 3'-ends of the restriction sites SpeI and NheI, respectively. After ligating the DNA was used to transform cells of E. coli XL1-Blue and selected individual clones producing soluble mimeutil. One of these clones was selected for large-scale purification and were grown in 1 l SB-environment (Super broth)containing 50 μg/ml carbenicillin at 37° to achieve the OD of 1.0 at 600 nm. Then the culture was induced 1 mm isopropyl-β -D-thiogalactopyranoside (IPTG) (firm Biofinex, Preroman, Switzerland) and grown for 4 h at 37° . Bacteria were palletizable at 6000 rpm for 20 min at 4° resuspendable in 30 ml of buffer, which produce ultrasound (0.1 M Na3PO4, 8 M urea, pH 8.0), and then treated with ultrasound on ice. Thereafter, insoluble components were removed by centrifugation at 15,000 rpm for 30 min at 4° and containing Fab-fragment of the supernatant was purified on a 1 ml Nickel-nitroacetate column. The column was washed with buffer, which produce ultrasonic treatment, to remove impurities, and then carried out a two-step elution buffer, which produce ultrasound at pH 5.1 and 4, respectively. Monitored fractions at OD 280 nm and aliquots were analyzed using LTO-page (12%, not restoring) to confirm purity and identity mimeutil. Containing mimeole fractions were then pooled, concentrated and dialyzed in a counter SFR.
The purity of the final preparations mimeole (shown in figure 4 and 12) (Seq.id. no. 35, 36, 37, 38) were evaluated by exposing a sample of the LTO-PAG. Band of protein was assessed by staining Kumasi brilliant blue. The concentration was determined by comparing the colored Kumasi blue diamond bands mimeole with known amounts of a standard protein (BSA)and spectrophotometrically. These drugs mimeole further used for analysis of competitive binding and immunizat and rabbits.
Example 5: Inhibition mediated BSW17 substitution associated with the IgE receptor using recombinant BSW17-mimeole
BSW17 recognizes and replaces IgE bound to its high-affinity receptor. To determine whether rFab-fragments of anti-id-BSW17 to inhibit this reaction substitution was carried out by analysis of competitive binding using IgE attached to the cell surface, which is IgERI. Used for the analysis of recombinant cell line of Chinese hamster ovary (Cho), stable transfectional DNA coding α -chain of human IgERI [cell line Choα ; Blank U. and others, Eur.J.Biol.Chem. 266 (1991) 2639]. A series of test samples containing 5× 104CHOα cells were incubated in FACS-buffer (buffer for cell sorting device with excitation fluorescence) (SFR, 0.3% BSA, 0.02% of NaN3with 48 ng of hybridoma IgE B11 (Zrcher A.W., and others, Immunol.Lett. 46 (1995) 49-57] for 15 min at RT. After a single washing FACS-buffer, each sample was incubated for 15 min at RT with preexisting complexes BSW17 conjugated to fluoresceinisothiocyanate (SW17-FITZ), and with increasing concentrations of rFab-fragments of anti-id BSW17. The complexes were obtained as follows: 50 μl SW17-FITZ at a concentration of 1.3 nm was incubated with different amounts of rFab-fragments of anti-id-BSW17 (40 nm; 200 nm; 1 μm; 4 μm; and 40 μm) in those who tell 30 min at RT. Control sample containing only the SNOα cells were incubated for 15 min at RT with BSW17-FITZ to determine nonspecific binding. SNOα cells once were washed in FACS buffer and after adding 100 μl of FACS buffer, cells were analyzed in a flow cytometer type FACSCalibur, Becton Dickinson)equipped with argon laser tuned at a wavelength of 488 nm. Discriminatory open for scattering in the forward direction/lateral direction for the dot-blot was established around the Monomeric cells and fluorescence was evaluated quantitatively and were expressed as the mean values of the fluorescence channel (mcf). The percentage of positive cells was calculated as the percentage of binding with BSW17 SNOα -cells. As can be seen from Fig.7, linking with BSW17 SNOα -cells decreased with increasing concentrations of Fab fragments of anti-id-BSW17, which suggests that 2 clone Fab fragments of anti-id BSW17 have the ability to inhibit the binding of BSW17 with IgE.
Example 6: Immunization of rabbits with recombinant BSW17-pilotelli
This example demonstrated that immunization or rFab-fragments of attela to BSW17 (which contain heavy plus light chain of clone 52, as shown in figure 4, and figa and 12B) (Seq.id. no. 35 and 36), or moreover, addition of mimetism that includes only the light chain (figa and figb) (Seq.id. no. 36), induced in rabbits se the oral immune response, which gives cross-react with human IgE. Two female new Zealand white rabbits were senzibilizirani (primarily were immunized), injecting subcutaneously 300 mcg/kg rFab-fragment antibodies to BSW17 or fragment of the light chain of clone 52, emulsified in a ratio of 1:1 in complete Freund's adjuvant, and then three times every 2 weeks were subjected to a booster injection of the same number of mimeutil, emulsified in a ratio of 1:1 in incomplete Freund's adjuvant. Serum samples were obtained on day 0 (before treatment) and in animals took blood samples after 7 days after the last injection.
Rabbit immune serum was purified using immunoaffinity chromatography using human IgE (SUS-11 IgE), chemically cross-linked with columns of Sepharose 4B. Using this procedure, the fraction of hIgE antibody can be selected from the total fraction of immunoglobulins, which gives the ability to accurately characterize therapeutically important immune response from the point of view of antibody titers and affinity.
Immunoaffinity purification of antibodies to mimeutil, which give cross-react with human IgE, includes two stages. In the first stage of IgG-fraction was isolated from rabbit antisera by precipitation with ammonium sulphate, in the second stage hIgE-specific antibodies to SW17-mimeole associated with human IgE (SUS-11 IgE), covalently cross-linked to CH-Sepharose 4B, the placenta is brilliant elution, dialysis and concentration.
Depending on the concentration of the formation in the solution of complex purified using immunoaffinity chromatography immunoglobulins with human IgE was confirmed using ELISA: SUS-11 IgE were incubated at 4° during the night with equimolar quantities of immunoglobulins to mimeutil. Formed in solution, the complexes were made in the wells tiralongo microplate sensitized with a monoclonal antibody LE27 to hIgE as immobilizing antibodies. Bound IgG to mimeole were revealed using complex: polyclonal anti-rabbit IgG-HRP. The results are shown in Fig.
Example 7: Immunization of mice with recombinant SW17-pilotelli
Recombinant mimeole originating from both clones 43 and 52, can induce the appearance of antibodies to mimetism. Mice were subjected to immunization. Groups of 5 mice lb/s was injected subcutaneously with 5 μg in 5 mouse recombinante SW17-mimeutil, which was received in the bacteria E. coli and purified using Nickel-affinity chromatography. Adjuvant used aluminum hydroxide:
group 1 was immunized with a sample SDS426 corresponding anti-id-BSW17.52; light chain;
group 2 were immunized with a sample SDS427 corresponding anti-id-BSW17.52; Fab-fragment.
group 3 were immunized with a sample SDS463 corresponding anti-id-BS17.43; Fab-fragment.
On days 21 and 41 after the primary immunization was carried out by 2 booster injections (5 μg per mouse). Blood samples were taken in the days 0, 20, 28, 35, 42, 49 and 56 after the primary immunization. Got the serum and probed her on the presence of antibodies to mimeole using ELISA.
Wells tiralongo microplate was senzibilizirani with 1 μg/ml polyclonal human IgG, and incubated with diluted at a ratio of 1:50 mouse serum obtained from blood samples taken at the indicated time points after primary immunization. Bound antibodies to mimeole (ahIgG to the human frame sections and constant regions of a human antibody) was determined using a second incubation with horseradish peroxidase conjugated rabbit artemisinin IgG (gamIgG-HRP). The color change reaction mixture was obtained using a chromogenic substrate ABTS.
Found that all mice developed antibodies to mimetism after the second booster injection with all of recombinant drugs mimeole (Fig.9).
Example 8: Antibodies to recombinant SW17-mimetism received in the body of rabbits, bind with high affinity human IgE
It is established that vaccination with recombinant BSW17-mimeole induces a humoral immune response with high affinity in respect to the research Institute of human IgE. Kinetic parameters characteristic of the binding of purified using immunoaffinity chromatography immunoglobulin to mimetism with human IgE was analyzed using surface plasma resonance (SPR).
SPR analysis was performed using devices such as BIAcore, Biacore, Uppsala, Sweden). Specific binding surface was obtained by stitching together using amine combination of human IgE or mouse IgG3touch-type element SM according to the manufacturer's instructions. Using this procedure, the biological molecules are connected via primary amino groups with carboxymethylamino dextranase surface of the sensor element. 10 pmole human myeloma, including IgE or SUS-11 IgE was associated with various mobile cell element. Mouse Mat in the form of IgG3, ABL 364 (ATSS HB 9324), immobilizerpower on different tracks of the same sensor element. ABL 364 used as a reference for determining the binding of antibodies to mimetism with structures not related to the immunoglobulin hIgE, and for the correction of possible changes in reflectance caused by changes of the buffer. Density combination was ~ EN 13000.
All of biomolecular interactions, which were evaluated using the BIAcore device, was performed at 25° using HBS (10 mM Hepes, pH=7,4, 150 mm aCl, 3.4 mm etc and 0.005% vol. surfactant P-20) as a constantly current buffer. The concentration of each of the analyzed sample, noise-order kinetic analysis on the surface of the sensor element, ranged from 33 mm to 499 mm. The flow rate was 5 μl/min test samples were injected with over 1200, and then entered HBS for about 1800 to estimate the dissociation of the bound of the analyzed sample. The item regenerates, passing within 120 with 10 mm Hcl. Nonspecific binding was assessed by skipping the analyzed samples through a reference path containing ABL 364, and before carrying out the kinetic analysis subtract the resulting value from the data obtained for specific binding.
Curves of binding obtained with SPR, were analyzed using the software package for analysis BIAevaluation 3.0. For the comparative analysis of the interactions of antibodies to mimeutil/IgE used single-phase model. For each curve was calculated rate constant of Association ka, the rate constants of dissociation of kdand equilibrium constants for the dissociation of KD=1/KA(KA=constant affinity), and these data are summarized in table 3:
Obviously, antibodies with high affinity for human IgE, can induzione the change in rabbits, immunized recombinant SW17-mimeole (values of KDare in the nanomolar range). Fab clone 43 (SDS463; figv; anti-id-BSW17.43) possessed the ability to induce an immune response with very high affinity to human IgE (KD<1 nm).
Thus, using monoclonal antiidiotypic antibodies can induce a strong polyclonal response against IgE, which is the main component of the strategy of vaccination of a person.
Example 9: Antibodies to recombinant BSW17-mimetism received in the body of rabbits, inhibit the binding of human IgE to its high-affinity receptor
In order for the vaccine was active anti-allergic vaccine should be formed complex between antibodies to mimetism and hIgE, which should prevent the binding of IgE to its high-affinity receptor. In binding with high-affinity receptor is involved Withε 3-epitope region Val(370) - Gly(379) in the sequence Val(370)-Asn(383) (6) (Seq.id. no. 25). for the identification of homology of amino acid sequences from the CDR SW17-mimeole. Thus, it can be expected that IgE-specific antibodies, which are produced by immunization with recombinant SW17-pilotelli, Express their therapeutic effect by inhibiting the binding of IgE to its high is finny receptor by blocking the binding domain. To confirm this purified antibody mimeole obtained from immunized rabbits were tested in respect to inhibition of binding hIgE/Fcε RIα using competitive ELISA. As typical for the disease trait was assessed by the ability of free IgE to contact recombinant Fcε RIα (RIα -CSA-RIα double fused protein; DFP) (figure 10).
hIgE (SUS-11; 1 µg/ml figa and 0.1 µg/ml figb) were subjected to pre-incubation for 16 h at +4° with increasing concentrations of immunoglobulins to mimetism or Mat BSW17 as a reference. The group of antibodies present in the fractions passing through the column of the drug SDS410, was included as a negative control. The formed complexes were made in the wells tiralongo microplate sensitized with 1 μg/ml antibody Le27 to IgE as binding antibodies, and incubated with dual fused protein Fcε RIα - CSA - Fcε RIα labelled with horseradish peroxidase for 1 h at 37° . Associated DFP was determined using the chromogenic substrate. The OD values were expressed as % binding. Linking nesteriak competitor SUS-11 IgE was taken as 100%. Presents the average values of measurements made with duplication. Variations typically accounted for less than 2%.
The results of the certificate shall listout about that immunization of rodents using SW17-mimeole leads to the formation of specific high-affinity antibodies to hIgE. These antibodies mimetism inhibit the binding of IgE to its high-affinity receptor in vitro, indicating the importance of vaccination strategy using SW7-mimeole for the development of anti-allergic vaccine.
Example 10: Inhibition of passive cutaneous anaphylaxis by vaccination of rhesus monkeys with recombinant SW17-pilotelli
Inhibition of passive cutaneous anaphylaxis (PKA) in monkeys can be used to test anti-allergic activity of the compounds in vivo.
Vaccination pilotelli leads to inhibition of the cutaneous anaphylactic reactions in rhesus monkeys. Groups consisting of 2 monkeys were injected subcutaneously with 500 μg per animal recombinant anti-id-SW17-mimeutil. After primary immunization was carried out by 2 booster injections. About 10 days after each booster injection was carried out by testing PKA. Monkeys 91 VI, VI 92, 93 VI and VI 95 tested against PKA reaction again 3 months after the last booster injection. The immunization scheme are summarized below in table 1:
|Monkey||Immunogen||Booster injection (day|
|VI 91||anti-id-BSW17.52; light chain (SDS426) (figa)||0, 21, 54, 92||0, 30, 61, 105, 203|
|Monkey||Immunogen||Booster injection (day|
|VI 92||anti-id-BSW17.52; light chain (SDS426) (figa)||0, 21, 54, 92||0, 30, 61, 105, 203|
|VI 93||anti-id-BSW17.52; Fab (SDS427) (figb)||0, 21, 54, 92||0, 30, 61, 105, 203|
|VI 95||anti-id-BSW17.52; Fab (SDS427) (figb)||0, 21, 54, 92||0, 30, 61, 105, 203|
|VI 2||anti-id-BSW17.43; Fab (SDS463) (pigv)||0, 21, 54, 112||0, 33, 64, 123|
|VI 75||anti-id-BSW17.43; Fab (SDS463) (pigv)||0, 21, 54, 112||0, 33, 64, 123|
Makaka RH, pre-treated with low doses of ketamine hydrochloride, 10-15 mg/kg, intramuscularly (Parke-Davis, UK) in order to maintain them in a fixed position, was administered by intradermal injection of various doses of IgE (JW8) (firm Serotec, Oxford, UK) in the skin of the abdominal. IgE (JW8) is a chimeric antibody comprising murine antigennegative site, specific for hapten NIP, and h is human Feε fragment of the heavy chain. Using needle 30-th size cephalocaudal in a volume of 100 μl was injected increasing number of(0, 2, 10, 50, 250 ng/ml saline) IgE (JW8). After 2 h the animal was intravenously injected 25 mg NIP conjugated to BSA. After control of infection conjugate NIP-BSL animals were again treated with sedative agent. For visualization of skin reactions immediately after the control of infection by antigen, the animals were injected subcutaneously dye Evans blue (1%, 0.5 ml/kg). Skin reactions were evaluated after 20 min after injection of antigen by measuring duplication diameters blue spots and calculate their average values in mm.
PKA was estimated at defined time points after primary immunization and booster injections. The intensity of PKA was calculated from the values of the areas under the curves (AUC)obtained by plotting the dependence of the diameters of the blue spots on the skin from the concentrations injected IgE (JW8). PKA-points are given in table 4 for each monkey, represent the calculated AUC values:
On the basis of PKA-points for each animal to its immunization (values at day 0), used as a benchmark for comparison, we can conclude that all the monkeys responded to vaccination SW17-mimetism reduction of intensity of PKA. Neilus the e results obtained with the use of designs based on the light chain of clone 52 (SDS426) (figa), the level of inhibition was 60-83% (PKA-40 points and 17). In monkeys immunized Fab-fragment of clone 52 (sample SDS427) (figb), the RCA reaction was suppressed 55-65% (PKA-points 45 and 35). Slightly lower inhibition of PKA, components 18-38% (PKA-points 82 and 62), detected using Fab-fragment of clone 43 (sample SDS463) (pigv), despite its higher compared to clone mimeole 52 characteristics in respect of BSW17-binding and induction of high-affinity antibodies to hIgE in rabbits.
According to the Protocol of vaccination vaccination using clone mimeole 52 becomes effective after the third booster injection (except monkeys VI92) and partial suppression of PKA is maintained even after 3 months. In contrast, the Fab-fragment of clone 43 was effective after the second booster injection, while the third control infection mimetism had no action.
Profile of average PKA-scores for each group of 2 monkeys immunized with the same drug mimeole calculated for obtained for each monkey data (table 4), see figure 11.
Positive PKA results obtained by immunization of monkeys (compared with scores obtained before processing each animal or compared with untreated control animals), it is clear on the show the effectiveness of vaccination pilotelli and confirm the concept of their mechanism of action. Vaccination of rhesus monkeys with recombinant BSW17-pilotelli leads to the formation of high-affinity antibodies to human IgE, which inhibit PKA in vivo.
1. Recombinant monoclonal antibody (mimeole) or a fragment of an antibody, which antiidiotypic against the antibody BSW17, which affects the binding ofε3-region of IgE with high affinity IgE receptor, characterized in that it constitutes or includes the light chain dimer or one of the two Fab fragments presented on figure 4 (SEQ ID NO : 35, 36, 37 and 38), with their constant region may additionally include steric modifications to 5 amino acids, such as those present in alltimecase options, and where SEQ ID NO: 36 corresponds to the light chain dimer (L..)2Minitel clone 52 SDS426;
SEQ ID NO : 35 and 36 correspond to the Fab fragment Minitel clone 52 SDS427;
SEQ ID NO : 37 and 38 correspond to the Fab fragment Minitel clone 43SDS463.
2. Mi is Sotelo according to claim 1, characterized in that represents or comprises the amino acid sequence represented by figa, 5B, 5C, or 5 g (SEQ ID NO : 2, 4, 6 and 8), the constant region may additionally include steric modifications to 5 amino acids, such as those present in alltimecase options.
3. Mimeole according to claim 1, characterized in that represents or includes at least one CDR or preferably 2 or more, preferably 3, CDR, presented at Figo (SEQ ID NO : 2, 40, 42 and 44), 5B (SEQ ID NO : 4, 46, 48 and 50), 5B (SEQ ID NO : 6, 52, 54 and 56) or 5 g (SEQ ID NO : 8, 58, 60 and 62), optionally in combination with neighboring sequences frame section, comprising up to 10 amino acids at one or both ends of the CDR.
4. Mimeole according to claim 1, characterized in that represents or includes at least one of the sequences shown in Fig.6 for clones 52 and 43, which are homologues Withε3 domain of human IgE (SEQ ID NO: 26, 28, 30, 32 and 34).
5. Militello according to any one of claims 1 to 4, characterized in that it is intended for use as an active ingredient in pharmaceutical compositions for the treatment of IgE mediated diseases.
6. Pharmaceutical composition for the treatment of IgE mediated diseases, characterized in that it includes mimeole according to claim 1 or as a separate, full molecule or protein is the first conjugate, chemically associated with the molecule immunogenic carrier, optionally with adjuvant and additional conventional excipients.
7. The pharmaceutical composition according to claim 6, characterized in that it is a vaccine.
FIELD: pharmaceutical industry.
SUBSTANCE: composition contains steroid as active principle, in particular 11-β,16-α,17-α,21-tetrahydroxy-9-α-fluoro-1,4-pregnadiene-3,20-dione or pharmaceutically acceptable salt thereof and special-destination additives including microcrystalline cellulose, crospovidone, and magnesium stearate.
EFFECT: optimized bioavailability, increased storage stability, and improved organoleptic properties.
3 cl, 1 tbl
FIELD: pharmaceutical industry.
SUBSTANCE: drug composition includes antihistamine agent as active principle, in particular 1-(p-chlorophenyl)-1-(pyrid-2-yl)-3-N,N-dimethylpropylamine or pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carrier including microcrystalline cellulose, lactose, crospovidone, and magnesium stearate.
EFFECT: optimized bioavailability, increased storage stability, and improved organoleptic properties.
6 cl, 5 ex
FIELD: drugs, medicine.
SUBSTANCE: invention relates to application of 1-methylindolyl-3-thioacetic acid tris-(2-hydroxyethyl)ammonia salt, which is known hypolipidemic agent, as immunosuppressor. Present invention ales it possible to produce pharmaceuticals for transplanted organ and tissue rejection prophylaxis or treatment of various immune-associated diseases.
EFFECT: new drug for transplant rejection prophylaxis or treatment of immune-associated diseases.
3 tbl, 3 ex
FIELD: drugs, medicine.
SUBSTANCE: invention relates to application of 4-chlorophenoxyacetic acid tris-(2-hydroxyethyl)ammonia salt, which represents known antioxidant, as immunomodulator. Present invention makes it possible to produce drugs for treatment of infections, inflammation, and autoimmunity disorders, wherein stimulating action to leukocyte amount and primary humoral response is combined with hypersensitivity inhibiting.
EFFECT: new drug for treatment of infections, inflammation, and autoimmune disorders.
3 tbl, 3 ex
FIELD: drugs, medicine.
SUBSTANCE: invention relates to application of 2-hydroxyphenoxyacetic acid tris-(2-hydroxyethyl)ammonia salt, which represents known antioxidant, as immunomodulator. Present invention makes it possible to produce drugs for treatment of infections, inflammation, autoimmune and lymphoproliferative disorders due to selective action on cell and humoral immunity.
EFFECT: new drug for treatment of infections, inflammation, and other diseases.
3 tbl, 3 ex
FIELD: drugs, medicine.
SUBSTANCE: invention relates to application of 2-chlorophenoxyacetic acid tris-(2-hydroxyethyl)ammonia salt, which represents known herbicide, as immunomodulator. Present invention makes it possible to produce drugs for mieloactivity stimulation and hypersensitivity inhibiting when treatment of infections, inflammation, autoimmune and lymphoproliferative disorders.
EFFECT: new drug for mieloactivity stimulation and hypersensitivity inhibiting.
3 tbl, 3 ex
FIELD: medicine, pediatrics, pulmonology.
SUBSTANCE: in blood serum under testing one should detect the level of specific IgE-AB to S. pneumoniae antigen and at its value being above 0 kU/l it is necessary to perform single vaccination with "Pneumo 23" preparation at the dosage of 0.5 ml intramuscularly regardless of the period of exacerbation at the background of basic therapy. The present innovation enables to increase immunity that in its turn increases efficiency of pharmacotherapy at bronchial asthma and decreases sickness rate at ARVI.
EFFECT: higher efficiency of prophylaxis.
FIELD: medicine, pediatrics.
SUBSTANCE: one should perform single vaccination with "Pneumo 23" preparation at the dosage of 0.5 ml intramuscularly 2 d after the end of basic therapy. The present innovation increases the duration of remission period in case of chronic inflammatory pulmonary disease in children, decreases the frequency of acute respiratory viral diseases due to adequate immunocorrection.
EFFECT: higher efficiency of prophylaxis.
SUBSTANCE: it is suggested to apply tris-(2-hydroxyethyl)ammonium salt of 1-benzylindolyl-3-thioacetic acid earlier known as a stabilizer of cell membrane as preparation to treat autoimmune diseases. The property of the above-mentioned salt to inhibit T-dependent activation of B-lymphocytes, under conditions of decreased medullary function and body leukopenia should enable to develop new pharmacological preparation for treating autoimmune diseases, such as, for example, systemic lupus, rheumatoid polyarthritis, transplant's detachment at transplanting either organs or bony marrow.
EFFECT: higher efficiency of application.
4 ex, 3 tbl
SUBSTANCE: invention relates to application of 2-Br-phenoxyacetic acid tris-(2-oxiethyl)ammonium salt with antioxidant activity as immunomodulator. Claimed compound gas stimulates action on mieloactivity and original humoral response, which was estimated on the base of amount of lgM-antibody forming cells, as well as depressive action on cell immune response and cell proliferation of spleen. Compound of present invention with immunological properties is useful in production of pharmaceuticals for treatment of various immune pathological conditions.
EFFECT: new application of known antioxidant as immunomodulator.
4 ex, 4 tbl
FIELD: veterinary science.
SUBSTANCE: animals should be introduced with antihistamine serum (AHS) subcutaneously at the dosage of 4.0-5.0 ml in combination with myxoferon at the quantity of 60-75 dosages and vitamin C at the dosage of 1.0-1.5 ml/animal, once daily, thrice at interval of 5-7 d. Application of AHS in combination with myxoferon and ascorbic acid provides active stimulation of immunological reactivity, increases total body resistance I animals and causes no toxic effects and allergic reactions.
EFFECT: higher efficiency of correction.
FIELD: medicine, pharmaceutics, pharmacology.
SUBSTANCE: one should apply mammalian anti-HBP-antibodies. The ways are being suggested to identify monoclonal antibody bound, at least, with one epitope upon native HBP (heparin-binding protein) and methods to detect whether a mammal produces HBR being bound with a monoclonal antibody and, also, the kits for the above-mentioned purpose. The present innovation provides the opportunity to apply the mentioned antibodies in preventing and treating disorders associated with bradykinin releasing.
EFFECT: higher efficiency of application.
25 cl, 11 dwg, 3 ex, 1 tbl
SUBSTANCE: the present innovation deals with cryoprotective ointment containing recombinant interferon-α2. The suggested cryoprotective ointment contains recombinant interferon-α2, glycerol, polyethylene glycol 300-6000, polyglucin, buffered 0.02%-Trilon B solution at pH of 5.5-7.0 and ointment foundation at a certain content of components per 1.0 g ointment. Additionally, cryoprotective ointment could contain glycine 3,7-bis(dimethylamino)phenothiazonium chloride, dry immunoglobulin preparation or dry immunoglobulin preparation for enteral application. Ointment foundation of cryoprotective ointment could contain water-free lanolin, Vaseline and Vaseline oil, at the following ratio of components: 2.5;3.5:1 - 6.5:0.5:1. The innovation provides maximal safety of recombinant interferon-α2 activity in cryoprotective ointment at multiple alteration of positive and negative environmental temperature and at keeping cryoprotective ointment under these conditions.
EFFECT: higher efficiency of application.
8 cl, 8 ex
FIELD: medicine and immunology, in particular treatment and prevention immunodeficiency conditions and diseases associated with bacterial or viral aggression.
SUBSTANCE: claimed method includes administration to a patient immunoglobulin drug (e.g., pharmaceutical composition containing 6-12 % of specific heterologous secreted immunoglobulin A, isolated from milk or foremilk of immunized ungulates). Administration is performed parenterally wherein single dose is at least 10 IU/kg of patient weight for treatment or at least 5 IU/kg for prophylaxis; or perorally in dose of 0.2-0.5 g and/or topically one-two times per day for 1-5 days. Method of present invention makes it possible to decrease dose of administrating immunoglobulin due to prolonged retention of its high titers in body fluids.
EFFECT: enlarged range of application and assortment of immunoglobulin drugs.
4 cl, 5 ex
FIELD: microbiology and immunology, in particular immunodiagnosis.
SUBSTANCE: atypical strain of melioidose Burkholderia pseudomallei-111-6-1 with altered phenotype defected with respect to synthesis of 8 antigen and acting as immunosuppressor is used as antigen for animal immunization. Immune serum is obtained after 2 immunization cycles of animal-producer with titer in gel immunodiffusion reaction not less than 1:128.
EFFECT: immune serum with increased specific activity.
2 tbl, 2 ex
SUBSTANCE: the innovation deals with new immunogenic conjugates of beta-propionamide-bound polysaccharide and N-propionamide-bound oligosaccharide with protein, and the method to obtain these conjugates has been suggested, as well. Conjugates should be applied to obtain vaccines against infectious diseases and cancer that enables to broaden the number of preparations applied in treating the above-mentioned diseases.
EFFECT: higher efficiency.
1 dwg, 2 ex, 8 tbl