Isolated antibody or its antigen-binding fragment specific for human immunoglobulin e (versions), nucleic acid molecule coding it, host cell, method for preparing antibody or its antigen-bindong fragment, pharmaceutical composition, use thereof for treating ige-associated diseases

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to such binding elements specific for IgE as antibody molecules or its antigen-binding fragments.

EFFECT: antibody or its versions specific for IgE are effective for treating IgE-associated disorders, such as allergy and asthma.

17 cl, 17 dwg, 8 tbl, 9 ex

 

The scope of the invention

The invention relates to connecting elements, in particular the molecules of the antibody to IgE. Connecting elements, among other things, are useful for treating disorders mediated by IgE, including allergies and asthma.

IgE is a member of the family of immunoglobulins and mediates allergic responses such as asthma, food allergies, hypersensitivity type 1 and inflammation of the nasal sinuses.

IgE is secreted and expressed on the surface of b-cells. Briefly, IgE is attached to the membrane In cells through transmembrane domain, which is associated with the Mature molecule IgE by short membranosvyazannaya plot. IgE may also be associated with your Fc site with b-cells, monocytes, eosinophils and platelets through the IgE receptor with low affinity (FcεRII, which is also known as CD23). When exposed to an allergen In the cells that produce specific allergen IgE, clonal breeding. Specific allergen IgE then released into the systemic blood stream Into cells, where it, in turn, binds to b-cells using FcεRII, as well as to mast cells and basophils through the receptor with high affinity (FcεRI). Such mast cells and basophils, thus, sencibilisiruet using the Llerena. Subsequent exposure to the allergen cross-links the FcεRI on mast cells and basophils, thus activating the release their histamine and other factors responsible for clinical hypersensitivity and anaphylaxis.

Connecting elements which inhibit the binding and functional activity through FcERI with simultaneous inhibition of FcERII or without inhibition are useful for inhibition of IgE mediated illnesses, such as allergies and asthma.

It is clear that the FcεRI and FcεRII associated with the site(s) of recognition in constant (Fc) domain of IgE. Were undertaken various studies to identify such sites recognition. For example, peptides corresponding to specific parts of the IgE molecule, used either as competitive inhibitors of the binding of IgE to its receptor (Burt and others, Eur. J. Immun, 17: 437-440 [1987]; Helm and others, Nature, 331:180-183 [1988]; Helm and others, Proc. Natl. Acad. Sci., 86: 9465-9469 [1989]; Vercelli and others, Nature, 338: 649-651 [1989]; Nio, etc., Peptide Chemistry, 203-208 [1990]), or to stimulate production of anti-IgE antibodies that can block the interaction with IgE receptor (Burt and others, Molec. Immun. 24: 379-389 [1987]; Robertson and others, Molec. Immun., 25:103-113 [1988]; Baniyash, etc., Molec. Immun. 25: 705-711 [1988]).

Was recently received Xolair® (Omalizumab), which is sold for the treatment of patients with asthma. Xolair® is humaniterian the second IgG1k monoclonal antibody, which selectively binds to IgE, reducing, thus, the binding of IgE, at least with FcεRI on the surface of mast cells and basophils. By reducing the level associated with the surface IgE on cells that are FcεRI, Xolair®, to some extent reduces the degree of release of mediators of the allergic response. Xolair® is disclosed in publications of international patent applications: WO 93/04173 and WO 97/04807.

However, there is a need in the other connecting elements for IgE, those that have a higher affinity and/or efficacy in comparison with Xolair®, for promising therapeutic strategies.

Invention

Through the use of acceptable methods of selection and analysis we have improved binding elements that inhibit the binding and functional activity through FcERI (IgE receptor presented on fat cells) with simultaneous inhibition of FcERII or in the absence of such inhibition.

Connecting element in accordance with the invention inhibits binding and functional activity through FcERI with simultaneous inhibition of FcERII or in the absence of such inhibition. Inhibition of binding can be a direct inhibition of, for example, by neutralization of IgE. Connecting element in accordance with the invention, the typical is about neutralizes human IgE with IC50 value, less than approximately 10 nm, as determined using, for example, RBL-ER51 analysis of the transmission of the calcium signal. In some embodiments of the connecting element in accordance with the invention neutralizes human IgE with , the IC50 value less than about 1 nm, or less than about 0.5 nm, or less than about 0.2 nm, as determined, for example, using RBL-ER51 analysis of the transmission of the calcium signal.

Connecting elements in accordance with the invention can also be contacted with and neutralize inhuman IgE, meaning IgE orthologues, which exist in nature in a species other than human.

Connecting elements in accordance with the invention are typically specific IgE compared with other immunoglobulins and, thus, bind IgE selectively. Such selectivity can be defined or demonstrated, for example, in the standard analysis of competitive binding.

The binding members are useful for the treatment and/or prevention of disorders which are mediated by IgE, in particular, various allergies and asthma.

Connecting elements are useful for reducing circulating free IgE in a mammal, and is also useful for inhibition of allergen-induced degranulation of mast cells in vivo or in vitro.

Connecting elements are also useful for inhibiting the biological responses mediated by the binding of IgE to FcERI with odnovremenno inhibition of biological responses mediated by IgE binding with FcERII or in the absence of such inhibition, either in vivo or in vitro.

Connecting elements in accordance with the invention may also have diagnostic utility, as, for example, to determine the presence or amount of IgE, or presence, or number of specific allergen IgE in the sample that is of interest, such as a sample obtained from a patient with asthma or allergies.

May use any reasonable method to determine the sequence of residues associated connecting element. For example, you might scan of peptide binding, such as enzyme-linked immunosorbent assay (ELISA) based on the PEPSCAN, as described elsewhere in this application. When scanning peptide linkage, such as a form, which is provided by PEPSCAN systems, short overlapping peptides, originating from antigen system are screened for binding the binding element. The peptides can be covalently linked to the surface of the substrate with the formation of the kit for the analysis of peptides. The peptides can be linear or have a compressed con is ormatio. A compressed conformation can be obtained from the use of peptides containing terminal Cys residue at each end of the peptide sequence. Cys residues can be covalently linked directly or indirectly with the surface of the base so that the peptide is held in a twisted conformation. Thus, the peptides used in the method may contain Cys residues attached to each end of the peptide sequence corresponding to a fragment of the antigen. Can also be used peptides with two loops, in which the Cys residue optionally is placed at or near the middle part of the peptide sequence. Cys residues can be covalently linked directly or indirectly with the surface of the base so that the peptides form conformation with two loops, with one loop on each side of the Central Cys residue. The peptides can be obtained synthetically, and Cys residues may thus be embedded in the desired positions, despite the fact that this sequence of IgE does not exist in nature. Not necessarily, both linear and short peptides can be subjected to screening in the analysis of peptide binding. Scanning peptide binding may involve identification (for example, when using a test set of peptides bound to binding the th element, where the peptides have amino acid sequences corresponding to fragments of IgE (for example, peptides containing approximately 5, 10, or 15 contiguous residues IgE), and comparison of peptides in order to determine the fingerprint residues associated connecting element, where the imprint includes balances that are common to overlapping peptides.

Alternative or additional method of peptide scan may involve the identification of peptides bound to the binding element, at least with a preset value signal: background signal. A more acceptable method of peptide scan to determine the binding are known in the art. Other methods, which are well known in the art and such that can be used to determine residues linked by antibody and/or to confirm the results of peptide scanning, include site-directed mutagenesis, the exchange of hydrogen-deuterium, mass spectrometry, NMR and x-ray crystallography.

Connecting element in accordance with the invention may or may not bind and/or neutralize IgE variants. Thus, the connecting element in accordance with the invention may or may not inhibit the binding of IgE variants with FcERIc simultaneous inhibition of FcERII, or if unavailable or is under such inhibition.

The sequence of the linear IgE epitope, for example in the form of isolated peptide fragments or polypeptides that include them can be used to identify, obtain, isolation and/or analysis of binding elements in accordance with the present invention.

As described in more detail below, connecting elements in accordance with the invention have been demonstrated as such that neutralize IgE with high efficiency. Neutralization means the inhibition of the biological activity of IgE. Connecting elements in accordance with the invention can neutralize one or more biological activities of IgE, but typically inhibit the binding of IgE to FcERI with simultaneous inhibition of binding FcERII or in the absence of such inhibition.

Neutralization of binding of IgE to FcERI with simultaneous inhibition of FcERII, or in his absence may not necessarily be measured as a function of the biological activity of the receptor, such as induced by allergen degranulation of mast cells.

Acceptable tests for measuring neutralization of IgE binding elements in accordance with the invention include, for example, biochemical assays of ligand and receptor and surface plasma resonance (SPR) (e.g., BIACORE).

Inhibition of biological activity can be casticin the m or complete. Connecting elements can inhibit the biological activity of IgE, such as receptor binding or degranulation of mast cells, 100%, or alternatively, on: at least 95 %, at least 90 %, at least 85 %, at least 80 %, at least 75 %, at least 70 %, at least 60 % or at least 50 % activity in the absence of a connecting element.

Neutralizing the effectiveness of a connecting element is usually expressed as the value IC50in nm, unless otherwise specified. In functional assays IC50represents the concentration of a connecting element, which reduces the biological response to 50% of its maximum. In studies of ligand binding IC50represents the concentration that reduces the binding of the receptor by 50 % of maximum specific binding. The value of the IC50can be calculated by building % of the maximum biological response as a function of log concentration of a connecting element and using software such as Prism (GraphPad) for building the sigmoid function in accordance with the data to obtain values IC50. Efficiency can be defined or measured by using one or more assays known to the skilled technician, and/or described in the question is the application or those referred to in this application.

Neutralizing the effectiveness of a connecting element can be expressed as geometric mean values. Geometric mean value (which is also known as the geometric mean value), as used in this application means the average of the logarithmic values of the data set, converted back to base 10. This requires at least two dimensions, for example, at least 2, preferably at least 5, more preferably at least 10 replicates. Specialist in the art can understand that the greater the number of replicates, the more reliable will be the geometric mean value. The choice of the number of replications can be attributed to the competence of a specialist skilled in the art.

Neutralization activity of IgE binding element in the analysis described in this application shows that the binding element binds and neutralizes IgE. Other methods that can be used to determine the binding of a connecting element with IgE include ELISA, Western blotting, immunoprecipitation, affinity chromatography and biochemical tests.

In another embodiment in accordance with the invention provides an isolated binding element that is specific is the cue for immunoglobulin E. when this connecting element has an IC50 value for binding of the specified connecting element with immunoglobulin E in serum at least 10 times lower than for Xolair™, or alternatively, at least 20-fold lower, at least 50-fold lower, at least 75 times, at least 100 times lower, at least 125 times lower than at least 150 times lower, at least 200 times lower, at least 300 times lower, at least 400 times lower, or at least 500 times lower.

Neutralizing the effectiveness of a connecting element, as calculated in the analysis by using the IgE of the first species (e.g., human), can be compared with the neutralizing efficiency of the connecting element in such analysis under similar conditions, the analysis by using the IgE from the second species (e.g., monkeys of cynomolgus) in order to assess the degree of cross-reactivity of a connecting element for IgE of two kinds. Alternatively, the cross-reactivity can be assessed in a competitive analysis of binding, as described in more detail elsewhere in this application.

Connecting element in accordance with the invention can have a higher neutralizing effectiveness against the binding of human IgE or biological analysis than when using IgE from species other than man. Thus, neutralizing the effectiveness of a connecting element in the analysis of human IgE may be greater than in a similar analysis with IgE from species other than human. The efficiency in the binding of human IgE in biological analysis may, for example, be approximately 5 times greater than in a similar analysis, using IgE monkeys of cynomolgus, or in another embodiment may be in 15 or 20 times. In particular, the efficiency in the transfer analysis of the calcium signal for human RBL-ER51 can be determined for concentrations of human IgE 25 ng/ml, while it is compared with the efficiency when using 100 ng/ml of IgE cynomolgus under other similar conditions. Examples of data obtained in such tests the transfer of calcium signal RBL-ER51 when using human IgE and IgE of cynomolgus shown in Table 2b.

Connecting element in accordance with the invention can have a higher affinity for human IgE than IgE. The affinity of the binding element for human IgE may be, for example, approximately 5 or 10 times higher than for IgE monkeys of cynomolgus, or, in another embodiment, may be approximately 100 times higher. Examples of data obtained for IgE, both human and monkey of cynomolgus shown in Table 2A and b.

Connecting elements is in accordance with the invention may have a neutralizing effectiveness against IgE or IC 50approximately 10 nm or less, with concentrations of human IgE 25 ng/ml, for example, in RBL-ER51 the analysis of the transmission of the calcium signal. Alternatively, the value of the IC50is less than approximately 3 nm. In other embodiments of the IC50is less than about 1 nm, or less than about 0.5 nm, or less than about 0.2 nm.

In another embodiment in accordance with the invention provides an isolated binding element specific for immunoglobulin E, where the connecting element has a geometric mean IC50 value for inhibition of transmission of the calcium signal induced by 25 ng/ml of IgE in RBL-ER51 cells less than 1 nm, or alternatively less than 0.6 nm, less than 0.5 nm, less than 0.4 nm, less than 0.3 nm, less than 0.2 nm 0.1 nm or less.

Kinetics and binding affinity (expressed as the equilibrium constant of dissociation KD) elements, binding of IgE to r human IgE may be determined, for example, using surface plasmon resonance (BIACORE). Connecting elements in accordance with the invention typically have an affinity for human IgE (KD) less than about 10 nm, and in some embodiments have a KD less than about 5 nm, in other embodiments have a KD of less than 2 nm. The affinity for IgE monkeys of cynomolgus usually less than approx is Ino 20 nm, in some embodiments, the KD is less than about 10 nm.

Is available a number of techniques for measuring binding affinity of an antibody to its antigen, one of these techniques is a KinExA. Kinetic exclusion assay (KinExA) is immunoanalytical platform for General purposes (essentially using the flowing an), which is capable of measuring the equilibrium constants of dissociation, the rate constants of Association and dissociation of the interactions of antigen/antibody. Because KinExA carried out after reaching the equilibrium state, the methodology is a pre-emptive for use when measuring the KDinteraction multivalent antigen/mAb. The binding of an antibody to the IgE molecule is an example of binding of multivalent antigen. Application KinExA is especially acceptable when multivalent antigen means that multimeric antibody and antigen, which are formed, include more than one antibody and more than one antigen. In such models the complex interaction between the determination of the exact values of KDcan be difficult. The KinExA method can be carried out as described by Drake and others (2004) Analytical Biochemistry 328, 35-43, as measured by KinExA method. Antibody 11 has a value of K 6,3 gr, which is substantially lower than for Xolair™, which has a value of KD353 PM.

In another embodiment in accordance with the invention provides an isolated binding element specific for immunoglobulin E, with KD300 PM or less, as measured using the methods of KinExA. Alternatively, KD200 PM or less, 100 PM or less, 50 PM or less, 20 PM or less, or 10 PM or below.

In vivo endogenous IgE may be glycosylated and, thus, glycosylated human IgE is a therapeutic model of human therapy. Although recombinant human IgE, which may be of bacterial origin and is not to be glycosylated, can be used in the analyses described in this application. Connecting elements in accordance with the invention can bind glycosylated human IgE, such as IgE, obtained using myeloma cell line, such as U266.B1. This is a significant advantage of connecting elements in accordance with the invention, as glycosylated human IgE is a target antigen for in vivo applications in humans.

Connecting element in accordance with the invention may include molecules are antibodies, preferably human antibody molecules or humanitarianly the molecule antibodies. In one aspect in accordance with the invention, the molecule antibodies are monoclonal antibody.

The binding site of the antigen is typically formed using a variable heavy (VH) and variable light (VL) domain of an immunoglobulin, with antigennegative area surface formed by six polypeptide loops, which are called sections, complementarity determining (CDR). There are three CDRs in each VH (HCDR1, HCDR2, HCDR3) and each VL LCDR1, LCDR2, LCDR3), together with portions of the frame plot (FR).

Connecting element in accordance with the invention typically includes a VH domain and/or VL of the antibody. VH domain includes a set of HCDR, and the VL domain comprises a set of LCDR. The antibody molecule may include a VH domain of the antibody comprising the VH CDR1, CDR2, and CDR3, and a frame area. It can alternatively or additionally include a VL domain of the antibody containing the VL CDR1, CDR2 and CDR3, and a frame area. Examples of VH domains of antibodies (SEQ ID NOS:2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 288, 300, and 306) and VL domains of antibodies (SEQ ID NOS:318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378 and 380) and CDRs (SEQ ID NOS:3-5, 8-10, 13-15, 18-20, 23-25, 28-30, 33-35, 38-40, 43-45, 48-50, 53-55, 58-60, 63-65, 68-70, 73-75, 78-80, 83-85, 88-90, 93-95, 98-100, 103-105, 108-110, 113-115, 118-120, 123-125, 128-130, 133-135, 138-140, 143-145, 148-150, 153 to 155, 158-160, 163-165, 168-170, 173-175, 178-180, 183-185, 188-190, 193-195, 198-200, 203-205, 208-210, 213-215, 218220, 223-225, 228-230, 233-235, 238-240, 243-245, 248-250, 253-255, 258-260, 263-265, 268-270, 273-275, 278-280, 283-285, 296-298, 289-291, 296-298, 301-303, 307-309, and 314-316) in accordance with the present invention shown in the attached list of sequences, which forms part of the present disclosure (see also Table 3A). Additional CDR disclosed below and in Table 1. All VH and VL sequences, CDR sequences, CDR sets, sets HCDR and sets LCDR disclosed in this application are the aspects and embodiments in accordance with the invention.

As described in this application, the set of CDR comprises CDR1, CDR2 and CDR3. Thus, the set of HCDR refers to the HCDR1, HCDR2 and HCDR3, and set LCDR refers to the LCDR1, LCDR2 and LCDR3. Unless otherwise specified, the "CDR set includes HCDR and LCDR.

Alternatively, the connecting element in accordance with the invention may include the binding site of the antigen within molecules other than antibodies, usually provided by one or more CDR, for example, a set of CDR in the protein cage molecules other than antibodies, as discussed further below.

As described in this application, a molecule of the original antibody was isolated as such, which contains a set of CDR sequences, as shown in Table 1 (see 1 Antibody), Through the implementation of the optimization process, we received a panel of clones of antibodies under non-2-28, CDR sequences, which shall originate from the original CDR sequences and contain modifications in the provisions listed in Table 1. Thus, for example, from Table 1 we can see that Antibody 2 has the original sequence of HCDR1, HCDR2, LCDR1, LCDR2 and LCDR3, and contains the original HCDR3 sequence in which the residue Kabata 96 is replaced by S, the remainder Kabata 97 replaced by L, the remainder Kabata 99 replaced by S, and the remainder Kabata 100 is replaced by A.

Described in this application connecting element is comprises a set of CDRs as shown in Table 1 (Antibody 1), where HCDR1 represents SEQ ID NO:3 (residues Kabata 31-35), HCDR2 represents SEQ ID NO:4 (residues Kabata 50-65), HCDR3 represents SEQ ID NO:5 (residues Kabata 95-102), LCDR1 represents SEQ ID NO:8 (residues Kabata 24-34), LCDR2 represents SEQ ID NO:9 (residues Kabata 50-56) and LCDR3 represents SEQ ID NO:10 (residues Kabata 89-97). Connecting element in accordance with the invention can be a source connecting element, as shown in Table 1, where one or more CDRs contain one or more amino acid additions, substitutions, deletions and/or insertions. In some embodiments of the connecting element includes a set of CDR containing from one to ten of additions, substitutions, deletions and/or insertions relative to the original sequences of the Antibodies 11. In another embodiment, from one to ten substitutions compared with the Antibody 11. In another embodiment is formed from one to eleven is pribavlenie, substitutions, deletions and/or insertions relative to the original sequence of the Antibody 1. In another embodiment, from one to ten substitutions relative to Antibody 1.

In some embodiments of the connecting element in accordance with the invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3; where HCDR3 has amino acid sequence SEQ ID NO:5, optionally containing from 1 to 5 amino acid additions, substitutions, deletions and/or insertions; and LCDR3 has amino acid sequence SEQ ID NO:10, optionally containing from 1 to 6 amino acid additions, substitutions, deletions and/or insertions. In these embodiments HCDR1 may contain the amino acid sequence of SEQ ID NO:3; HCDR2 may contain the amino acid sequence of SEQ ID NO:4; LCDR1 may contain the amino acid sequence of SEQ ID NO:8; and a LCDR2 may contain the amino acid sequence of SEQ ID NO:9. Alternatively, HCDR1, HCDR2, LCDR1 and LCDR2 may also collectively contain one or more amino acid additions, substitutions, deletions and/or insertions relative to the original sequence (Antibody 1), as, for example, from one to ten substitutions.

Connecting element in accordance with the invention may include one or a combination of CDR, as described in this application. For example, a connecting element in accordance with the invention may include HCDR1 having the amino acid after outermost SEQ ID NO:3; HCDR2 having the amino acid sequence of SEQ ID NO:4; HCDR3 has amino acid sequence selected from the group consisting of SEQ ID NOS:5, 15, 25, 65, 75, 85, 95, 145, 155, 175 and 255; LCDR1 having the amino acid sequence of SEQ ID NO:8; LCDR2 having the amino acid sequence of SEQ ID NO:9; and LCDR3 having the amino acid sequence selected from the group consisting of SEQ ID NOS:10, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 and 280.

In some embodiments of the connecting element or VH domain in accordance with the invention includes the original HCDR3 (SEQ ID NO:5) with one or more of the following substitutions:

the remainder Kabata 96 replaced with S, M or T;

the remainder Kabata 97 replaced by L or G;

the remainder Kabata 98 replaced by K;

the remainder Kabata 99 replaced by S, W, a, T or E;

the remainder Kabata 100 is replaced by a or I.

In some embodiments of the connecting element or VL domain may include the source LCDR3 (SEQ ID NO 10) with the remainder Kabata 94, zamenjenim on T, R, D, P, E, N, H, Q or A.

In some embodiments of the connecting element or VL domain in accordance with the invention includes the original LCDR3 (SEQ ID NO 10) with one or more of the following substitutions:

the remainder Kabata 94 is replaced by T, R, D, P, E, N, H, Q or A;

the remainder Kabata 95 replaced by T, K, S, I, G, H, M, F, R, N, K or Q;

the remainder Kabata 95A replaced by L, H, D, G, R, N, Q, K, or E;

the remainder Kabata V replaced by T, H,S, Y, L, or N;

the remainder Kabata 96 replaced by G or A;

the remainder Kabata 97 replaced by R, S, or G.

In one embodiment of the invention is a connecting element, in which the HCDR1 has amino acid sequence SEQ ID NO:103, HCDR2 has amino acid sequence SEQ ID NO:104, HCDR3 has amino acid sequence SEQ ID NO:105, LCDR1 has amino acid sequence SEQ ID NO:108, LCDR2 has amino acid sequence SEQ ID NO:109, and LCDR3 has amino acid sequence SEQ ID NO:110. For example, see Antibody 11 Table 1.

Other embodiments in accordance with the invention are connecting elements, such as antibody molecules, able to compete with antibodies in accordance with the invention, such as an Antibody (11) of Table 1, for binding to human IgE, thus connecting elements neutralize human IgE with the value of the IC50less than about 1 nm in the analysis described in this application, or with the value of the IC50less than approximately 0.5 nm. In some embodiments of the IC50is the lesser of approximately 0.2 nm.

The invention provides binding elements comprising HCDR1, and/or HCDR2 and/or HCDR3 of any of antibodies 1-28 and/or LCDR1, and/or LCDR2 and/or LCDR3 of any of antibodies 1-28, e.g. a set of CDRs of any of antibodies 1-28 shown in Table 1. Connecting the element is provided may include a set of VH CDRs of one of these antibodies. Optionally, it may also include the set VL CDR of one of these antibodies, and VL CDR can be from the same or from different antibodies that VH CDR. VH domain comprising a set of HCDR any of the antibodies 1-28 and/or VL domain comprising a set of LCDR any of the antibodies 1-28, also provided by the invention.

Typically, when VH domain is coupled with the VL domain by providing antigennegative site antibodies, although, as discussed in this application below, to bind antigen can be used only VH or VL domain. VH domain of the Antibody 1 (see Table 1) can mate with the VL domain of the Antibody 1, so as to form antigennegative site antibodies, comprising both VH and VL domains of antibody 1. Similar embodiments are provided for the other VH and VL domain disclosed in this application. In other embodiments of the VH of the Antibody 1 mates with the VL domain that is different from the VL of the Antibody 1. Promiscuity light chains is well established in the field of engineering. In addition, similar to the embodiment provided by the invention for other VH and VL domain disclosed in this application. Thus, the VH of the original or any of the antibodies 2-28 may mate with VL source or any of the antibodies 2-28.

Connecting element may include a set of H and/or L CDRs of the source antibody or any of antibodies 2-28 twenty, sixteen, ten, nine or less, for example, one, two, the, three, four or five amino acid additions, substitutions, divisions, and/or insertions within the disclosed set of H and/or L CDRs. Alternatively, the connecting element may include a set of H and/or L CDRs of the source antibody or any of antibodies 2-28 twenty, sixteen, ten, nine or less, for example one, two, three, four, or five amino acid substitutions within the disclosed set of H and/or L CDRs. Such modifications can potentially be implemented at any residue within the set of CDR. For example, modifications can be made in the provisions modified by any of the other antibodies 2-28, as shown in Table 1. Thus, one or more modifications can include one or more substitutions at the following residues: residues Kabata 96, 97, 98, 99 and 100 in HCDR; residues Kabata 94, 95, 95A, B, 96 and 97 LCDR.

Connecting element can include an antibody molecule containing one or more CDRs, such as a set of CDRs in wireframe plot antibodies. For example, one or more CDR or set of CDRs of the antibodies can be transplanted in the frame area (for example, human skeleton plot) to ensure molecule antibodies. The frame sections can be an embryonic genes sequences person. Thus, the frame area can be germination, in accordance with one Il is more residues in the frame area change to match the sections in the equivalent provision in the most similar embryonic skeleton plot person. Thus, the connecting element in accordance with the invention can be an isolated molecule human antibodies contain the VH domain comprising a set of HCDR in human embryonic skeleton plot, for example, human embryonic skeleton plot IgG VH. Connecting element also contains a VL domain comprising a set of LCDR, for example, in human embryonic skeleton plot IgG VL.

The remains of the VH and/or VL frame section can be modified, as discussed and shown in the examples in this application, for example, by using site-directed mutagenesis. VH or VL domain in accordance with the invention or binding element comprising such a VL domain, preferably has the sequence of the VH and/or VL domain of the antibody, as shown in Table 3.

Permissiona molecule antibody has the same CDRs, but a great frame sections, compared to herminiimonas molecule antibodies. Harmonisierung antibody can be obtained by harmonizatio frame sections of the sequences of the VH and VL domains referred to in this application for these antibodies.

Connecting element in accordance with the invention may be such that competes for binding to IgE with any linking element, which both bind IgE and include a connecting element, such as a VH and/or VL domain, CDR, for example, HCDR3, and/or a set of CDRs disclosed in this application. Competition between binding elements can be easily analyzed in vitro, for example, using ELISA and/or by labeling using specific reporter molecule to one binding element, which can be determined in the presence of one or more other untagged binding elements, which allows the identification of binding elements that bind the same epitope or an overlapping epitope. Such methods are well known to the average expert in the art and are described in more detail in this application. Thus, an additional aspect in accordance with the present invention provides a binding element comprising a binding site antigen human antibody that competes with antibody molecule, such as, in particular, a molecule antibody comprising VH and/or VL domain, CDR e.g. HCDR3 or set of CDRs of the source antibody or any of antibodies 1-28, for binding to human IgE. In one embodiment of the connecting element in accordance with the invention competes with the Antibody 11, as shown in Table 1.

Another embodiment in accordance with the invention provides connecting elements that are associated with specifics what they plot IgE. Binding may be determined, for example, by determining or monitoring of specific interaction between the connecting element and the remnants of IgE, for example, in the structure of the connecting element: IgE complex, which can be determined, for example, using x-ray crystallography. The structure of Antibodies 11 associated with Cε3-Cε4 domains of human IgE is determined using x-ray crystallography, providing the opportunity to study two interactions Fab antibodies 11 with IgE in the crystal. IgE is a bivalent antigen, because there are two light chains and two heavy chains. X-ray crystallographic study showed that the Fab binds to the epitope located across two heavy chains of IgE.

The first interaction indicates that the site of interaction of Antibodies 11 includes balances from Glu390 to Asn394 inclusive and sugar GlcNAc1 and Man6 in one heavy chain of IgE and Leu340, Arg342, from Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 and the remains of the sugar Man 5 in a different heavy chain of IgE.

In one embodiment in accordance with the invention provides an isolated binding element specific for immunoglobulin E, where the specified binding element binds to the epitope in the immunoglobulin E, which includes the remains of Glu390 to Asn394 inclusive in the first the second the heavy chain of IgE and Leu340, Arg342, from Ala428 to Thr434, inclusive, Thr436, Ser437 and Glu472 second heavy chain of IgE; in an additional embodiment of the specified epitope further includes sugar GlcNAc1 and Man6 first heavy chain of IgE and the remains of the sugar Man 5 second heavy chain of IgE.

The second interaction indicates that the site of interaction of Antibodies 11 includes balances Glu390 from Gln392 to Asn394 inclusive, and sugar GlcNAc1 and Man6 first heavy chain of IgE and Leu340, Arg342, from Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 second heavy chain of IgE.

In an additional embodiment in accordance with the invention provides an isolated binding element specific for immunoglobulin E, where the specified binding element binds to the epitope in the immunoglobulin E, which includes:

the remains Glu390 from Gln392 to Asn394, inclusive, in the first heavy chain of IgE and Leu340, Arg342, from Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 second heavy chain of IgE;

in an additional embodiment of the specified epitope further includes sugar GlcNAc1 and Man6 first heavy chain of IgE.

In an additional embodiment in accordance with the invention provides an isolated binding element specific for immunoglobulin E, where the specified binding element binds to the epitope in the immunoglobulin E, which includes:

the remains Glu390 from Gln392 to Asn393 inclusive, first is the first IgE heavy chain and Leu340, Arg342 from Ala428 to Thr434 inclusive, Thr436, Ser437 and Glu472 second heavy chain of IgE;

the specified epitope further includes sugar GlcNAc1 and Man6 first heavy chain of IgE.

In an additional embodiment in accordance with the invention provides an isolated binding element specific for immunoglobulin E, which binds to the epitope that includes elements from a first heavy chain of IgE and the elements of the second heavy chain of IgE.

In additional aspects the present invention provides a binding element comprising a binding site of human antigen antibody which competes with antigennegative site antibodies for binding to human IgE, where antigennegative site of the antibody comprises the VH domain and VL domain, and where the VH and VL domains comprise a set of CDRs of the source (Antibody 1) or any of the antibodies 2-28, as disclosed in this application.

In additional aspects, the invention provides an isolated nucleic acid that includes a sequence encoding a binding element, VH domain and/or VL domain in accordance with the present invention. For example, SEQ ID NOS:1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 287, 299 and 305 encode typical VH domains in accordance with the present invention, a SEQ ID NOS:317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 33, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377 and 379 encode typical VL domains in accordance with the present invention. The invention also includes methods of obtaining a connecting element, VH domain and/or VL domain in accordance with the invention, which include the expression of specified nucleic acid under conditions which provide the specified binding element, VH domain and/or VL domain and restore it using the isolation or purification of the binding element.

Another aspect in accordance with the present invention provides nucleic acid, generally isolated, which encodes a VH CDR and VL CDR

the sequences disclosed in this application.

An additional aspect provides a host cell containing or transformed nucleic acid in accordance with the invention.

Additional aspects in accordance with the present invention provide compositions containing the connecting elements in accordance with the invention, and their use in methods of inhibiting and/or neutralizing IgE, including methods of treatment of the human or animal-assisted therapy.

For example, connecting elements in accordance with the invention can be used in the treatment or prevention of or used in a method for the diagnosis of biological response rates across evania, disorder or condition of the human or animal (e.g., in a patient who is a person) or in vitro.

The method of treatment and/or prevention may include the introduction of the indicated patient a connecting element in accordance with the invention in a quantity sufficient to cause capable of measuring the neutralization of IgE. Conditions that can be treated in accordance with the present invention include any in which IgE plays a role, such as different types of allergies and asthma.

These and other aspects in accordance with the invention are described in more detail below.

Is acceptable to specify here that when this application is used "and/or", this expression is taken as a specific disclosure of each of these two signs or with other components or in the absence of the other. For example, "a and/or b ' is taken as a specific disclosure of each of (i) A, (ii) and (iii) a and b, exactly the same as if each were offered individually in this application.

IgE is an immunoglobulin Amino acid sequence that is a constant area of the human IgE is freely available. In some embodiments IgE may be IgE human or monkey of cynomolgus. As described elsewhere in this application, IgE can be recombi is based, and/or can be either glycosylated or deglycosylated. IgE is expressed naturally in vivo in a glycosylated form, for example in U266.B1 cells. Glycosylated IgE can also be expressed in recombinant systems.

Connecting element generally refers to one member of a pair of molecules that are associated with each other. Members of the binding pair can be existing in nature, or fully or partially obtained synthetically. One member of the pair of molecules has an area on its surface, or a cavity, is associated with and is, thus, complementary to a particular spatial and polar organization of another member of the pair of molecules. Examples of the types of binding pairs are antigen - antibody, Biotin - avidin, hormone - hormone receptor, receptor - ligand and enzyme - substrate. The present invention relates to the type of reactions antigen - antibody.

Connecting element typically includes a molecule having a binding site of the antigen. For example, the binding member can be an antibody molecule or protein that is not derived from antibodies, including binding site of the antigen.

The binding site of the antigen can be provided with adjustment CDR protein structures, other than antibodies, such as fibronectin or cytochrome b and others [1. Haan & Maggos (2004) BiCentury, 12(5): A1-A6; 2. Koide et al. (1998) Journal of Molecular Biology, 284: 1141-1151; 3. Nygren et al. (1997) Current Opinion in Structural Biology, 7: 463-469], or by randomization or mutation of amino acid residues of the loop within the protein frame structure to ensure the binding specificity for the desired target. Frame structure designed for new binding sites in proteins considered Nygren and others [3. Nygren et al. (1997) Current Opinion in Structural Biology, 7: 463-469]. Protein frame structure for imitators antibodies disclosed in the application WO/0034784, to which reference is entered in this application in its entirety. In this paper the authors describe proteins (simulators antibodies), which include domain, fibronectin type III, containing at least one randomised loop. Acceptable frame structure, in which transplanted one or more CDRs, such as a set of HCDR may be provided in any domain member of the immunoglobulin gene superfamily. The frame structure may be a human or nonhuman protein. The advantage of the frame structure of a protein other than an antibody, is that it can provide the binding site of the antigen in the molecule frame structure, which has smaller dimensions and/or is easier to produce, than at least some molecules of the antibody. The small size of the connecting ele is enta can give useful physiological properties, such as the ability to enter into cells, to penetrate deeply into the tissues or to achieve targets that are located in other structures, or to contact in protein cavities of the target antigen. The application of the binding sites of the antigen in the frame structures of the protein, other than antibodies, discussed in Wess, 2004 [4. Wess, L. In: BioCentury, The Bernstein Report on BioBusiness, 12(42), A1-A7, 2004]. Typical proteins are stable skeleton and one or more variable loops, in which the amino acid sequence of the loop or loops is specifically or randomly mutated with the formation of the binding site of the antigen that binds to the target antigen. Such proteins include IgG-binding domains of protein a from S. aureus, transferrin, tetradactyl, fibronectin (for example, the 10th domain of fibronectin type III), lipocalin and gamma crystalline and other frame structure of Affilin™ (Scil proteins). Examples of other approaches include synthetic "MICROTEL based cyclotide - small proteins with intramolecular disulfide bonds, microbulk (Versabodies™, Amunix) and proteins anchirinah repeats (DAR, Molecular Partners). Such proteins also include small, engineered protein domains, such as, for example, immunodominant (see, for example, patent publication US No. 2003082630 and No. 2003157561.

In addition to the sequences of the antibodies is a and/or the binding site of the antigen-binding element in accordance with the present invention may include other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. Connecting elements in accordance with the invention can carry capable of determining a label or can be conjugated with a toxin or target residue or an enzyme (e.g., using peptidyl communication or linker). For example, the connecting element may include a catalytic site (for example, the enzyme domain), and the binding site of the antigen, where the binding site of the antigen binds to the antigen and, thus, directs the catalytic site on the antigen. The catalytic site may inhibit the biological function of antigen, for example, by cleavage.

Although, as noted earlier, the CDR may be stored in frame structures, other than antibodies, structure for moving the CDR or set of CDRs in accordance with the invention will in General be represented as a sequence of the heavy or light chain of the antibody or its significant part, in which the CDR or set of CDRs is located in the area corresponding to the CDR or set of CDRs existing in the nature of the variable domains VH and VL of the antibody encoded rebuilt immunoglobulin genes. Structure and location of immunoglobulin variable to the ENES can be determined by reference to Kabat and others, 1987 [5. Kabat, E.A. et al. Sequences of Proteins of Immunological Interest. 4thEdition. US Department of Health and Human Services. 1987] and their specifications, which can be found as "Kabat (Kabat)" using any search engine Internet.

Using plot CDR or CDR is expected to identify hypervariable parts of the heavy and light chains of immunoglobulin, as defined by Kabat and others 1991 [6. Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington] and later editions. Antibody generally includes the 3 CDRs of the heavy chain and 3 CDR light chain. The term CDR or more CDR is used in this application in order to specify, in accordance with the particular case, one of these sites or multiple sites, or even all of these areas, which contain most of the amino acid residues responsible for binding due to the affinity of the antibody to the antigen or the epitope it recognizes.

Among the six short sequences of CDR third CDR of the heavy chain (HCDR3) has a high degree of variability (greater variety, essentially due to the mechanisms of gene rearrangement, which give it a start). He may be short, for example the size of 2 amino acids, despite the fact that the largest size, which is known, is 26. The length of the CDR may also be varied in accordance with the length, which can be the t to be adapted with specific underlying frame of the plot. Functionally HCDR3 partly plays a role in determining the specificity of antibodies [7. Segal et al., PNAS, 71:4298-4302, 1974; 8. Amit et al., Science. 233:747-753, 1986; 9. Chothia et al., J. Mol. Biol., 196:901-917, 1987; 10. Chothia et al., Nature, 342:877-883, 1989; 11. Caton et al., J. Immunol., 144:1965-1968, 1990; 12. Sharon et al., PNAS, 87:4814-4817, 1990; 13. Sharon et al., J. Immunol., 144:4863-4869, 1990; 14. Kabat et al., J. Immunol., 147:1709-1719, 1991]. In another embodiment in accordance with the invention provides an isolated binding element comprising a HCDR3 sequence selected from Table 3A.

The term "molecule antibody" refers to immunoglobulin or natural, or partly or completely obtained synthetically. The term also covers any polypeptide or protein, including antigennegative site antibodies. In this application it is clear that the invention does not relate to the antibody in its natural form, in other words, they are not in their natural environment, but they can be isolated or obtained by purification from natural sources or obtained by genetic recombination or by chemical synthesis, including the modification by using unnatural amino acids. Fragments of antibodies, which include antigennegative site antibodies include, but without limitation, molecules such as Fab, Fab', Fab'-SH, scFv, Fv, dAb and Fd. Were constructed of various other molecules antibodies comprising one or bol is e antigenspecific sites of the antibodies, including, for example, Fabz, RAES, diately, Triatel, tetrathele and Minitel. The antibody molecules and methods for their preparation and their use are opisanie in [15. Holliger & Hudson, Nature Biotechnology 23(9):1126-1136 2005].

Is also possible to use monoclonal and other antibodies and methods of recombinant DNA technology to produce other antibodies or chimeric molecules that bind the target antigen. Such techniques may also involve the introduction of DNA that encodes a variable segment of immunoglobulin, or CDR of the antibody constant regions or the constant parts plus frame areas other than immunoglobulin. See, for example, EP-A-184187, GB 2188638A or EP-A-239400 and a number of other literature. Hybridoma or other cell producing the antibody may be a genetic mutation or other changes that may or may not alter the binding specificity of the resulting antibodies.

As antibodies can be modified in a number of ways, the term "antibody molecule" is to be treated as such, which covers any binding member or substance having antigennegative site antibodies with the desired specificity and/or binding to the antigen. Thus, this term encompasses fragments of the antibodies and derivatives, including any polypeptide comprising what aka any antigennegative site antibodies, both natural and wholly or partially synthetic. Here, therefore, also include chimeric molecules comprising antigennegative site of the antibody, or equivalent, fused to another polypeptide (e.g., one that is derived from other species, or belongs to another class or subclass of antibody). Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, as well as in a number of other literature.

Additional techniques that are available in the design area antibodies allowed us to isolate human and humanized antibodies. For example, human hybridoma can be obtained as described Kontermann and Dubel [16. Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag New York, LLC; 2001, ISBN: 3540413545]. Phage display, another common technique for receiving connecting elements has been described in many publications, such as Kontermann and Dubel and WO92/01047 (discussed further below), and patents US5969108, US5565332, US5733743, US5858657, US5871907. US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US6291650, US6492160, US6521404.

Transgenic mouse in which the mouse antibody genes are inactivated and functionally replaced by the genes of a human antibody, while the other components remain intact murine immune system, can be used to isolate human antibodies [17. Mendez, M. et al. (1997) Nature Gnet, 15(2): 146-156]. Humanized antibodies can be produced using techniques known in the art such as those disclosed, for example, WO91/09967, US 5,585,089, EP592106, US 565,332 and WO93/17105. In addition, WO2004/006955 describes methods for humanization of antibodies is based on the selection frame sequence of the variable region from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable segment of the antibody species other than human, with canonical CDR structure types for the corresponding CDR library sequences of human antibodies, such as embryonic gene segments antibodies. Variable parts of the human antibodies with similar canonical CDR structure types compared to CDR species other than human, form a subset of the members of the sequences of human antibodies from which to choose the sequence of the human skeleton plot. A subset of the members may be additionally ordered when using amino acid similarity between the CDR sequences of human and CDR species other than human. In the method according to WO2004/006955 choose highly quoted human sequence to provide a sequence of frame section is designed for the project chimeric antibodies, which functionally replaces the CDR sequence of the human CDR equivalents species other than human, when using the selected subset of the members of the human frame sections, providing, thus, humanitariannet antibody with high affinity and low immunogenicity in the absence of the need to compare sequences of frame section between the antibodies of human and species other than human. Chimeric antibodies obtained in accordance with this method, are also disclosed.

Synthetic molecules antibodies may be those that are created by the expression of the genes obtained using oligonucleotides synthesized and integrated within an acceptable expression vectors, for example, as described in Knappik and others [18. Knappik et al. J. Mol. Biol. (2000) 296, 57-86] or Krebs and others [19. Krebs et al. Journal of Immunological Methods 254 2001 67-84].

It has been shown that fragments of whole antibodies may perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CN domains; (ii) the Fd fragment consisting of the VH and CN domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment [20 Ward, E.S. et al., Nature 341, 544-546 (1989); 21. McCafferty et al (1990) Nature, 348, 552-554; 22. Holt et al (2003) Trends in Biotechnology 21, 484-490], which consists of a VH or VL domain; (v) izolirovani the e CDR areas; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single-chain Fv molecules (scFv), where VH domain and a VL domain are connected via a peptide linker which allows the two domains to associate with the formation of the binding site of the antigen [23. Bird et al. Science, 242, 423-426, 1988; 24. Huston et al, PNAS USA, 85, 5879-5883, 1988]; (viii) bespecifically single-chain Fv dimers (PCT/US92/09965) and (ix) "diately", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; [25. Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993]). Molecules, Fv, scFv or ditelo can be stabilized by the introduction of disulfide bridges linking the VH and VL domains [26. Reiter, Y., et al. Nature Biotech, 14, 1239-1245, 1996]. Can also be obtained Minitel, including scFv, coupled with CH3 domain [27. Hu, S. et al. Cancer Res., 56, 3055-3061, 1996]. Other examples of binding fragments are Fab', which differ from Fab fragments by the accession of several residues on carboxyterminal the end SN domain of the heavy chain, including one or more cysteines from the hinge area antibodies, and Fab'-SH, which is a Fab' fragment in which the cysteine(s) residue(remainder) of the constant domains bear a free thiol group.

Fragments of the antibodies in accordance with the invention can be derived from antibody molecules 1-28, Porosozero, such as digestion by enzymes, such as pepsin or papain and/or by cleavage of disulfide bridges by chemical recovery. In accordance with another method, fragments of antibodies, which are included in the present invention, can be obtained using the techniques of genetic recombination, a well-known specialist in the art, or by peptide synthesis using, for example, automated peptide synthesizers, such as those supplied by Applied Biosystems and others, or by the synthesis and expression of nucleic acids.

Functional fragments of the antibodies in accordance with the present invention include any functional fragment whose half-life is increased by chemical modification, in particular by Paglierani, or by embedding in the liposome.

dAb (domain antibody) is a small Monomeric antigen binding fragment of the antibody, in particular variable plot heavy or light chain antibody [22]. VH dAb is formed in the nature of the camel (for example, camel, llama) and can be obtained by immunization camel with the target antigen, isolating specific antigen In cells and direct cloning dAb genes from individual cells. dAb is also in zmeinym to get in the cell culture. Its small size, good solubility and thermal stability make it especially physiologically acceptable and useful for selection and affinity maturation. Camel VH dAb were improved for therapeutic applications entitled "nanotesla™". Connecting element in accordance with the present invention may be a dAb, including VH or VL domain, substantially such as set forth in this application, or VH or VL domain comprising a set of CDRs, substantially such as set forth in this application.

Bespecifically or bifunctional antibodies form a second generation monoclonal antibodies, in which two different variable segment are connected in the same molecule [28. Holliger and Bohlen 1999 Cancer and metastasis rev. 18:411-419]. Their use has been demonstrated both in the field of diagnosis and therapy due to their ability to bring new effector functions or to some target molecules on the surface of tumor cells. If you use bespecifically antibodies, they can be a traditional bespecifically antibodies that can be obtained using different methods [29. Holliger, P. and Winter, G. Current Opinion Biotechnol. 4, 446-449 1993], for example, obtained chemically or from hybrid hybridomas, or can be any of bespecifically fragment is in antibodies, mentioned above. These antibodies can be produced using chemical methods [30. Glennie M J et al., 1987 J. Immunol. 139, 2367-2375; 31. Repp R. et al., 1995 J. Hemat. 377-382] or somatic methods [32. Staerz U.D. and Bevan M. J. 1986 PNAS 83; 33. Suresh M.R. et al., 1986 Method Enzymol. 121: 210-228], but also and preferably using the techniques of genetic engineering, which enable you to induce heterodimerization and, thus, to improve the process of purification of the desired antibodies [34. Merchand et al., 1998, Nature Biotech. 16:677-681]. Examples bespecifically antibodies include, in accordance with the BiTE technology™, which you can use binding domains of the two antibodies with different specificnosti and directly link them with short flexible peptide. This allows you to combine two antibodies one short polypeptide chains. Diately and scFv can be constructed without Fc plot, using only the variable domains, thereby potentially reducing the effects antiidiotypic reaction.

Bespecifically antibodies can be constructed as whole IgG, as bespecifically Fab'2, as Fb'PEG as diately or as bespecifically scFv. In addition, two bespecifically antibodies may be associated with the conventional methods known in the field of engineering, with the formation of tetravalent antibodies.

Bespecifically of diately, unlike bespecifically C is selected antibodies can also be particularly useful because they can be easily constructed and expressed in E. coli. Diately (and many other polypeptides, such as fragments of antibodies) with acceptable binding specificnosti can be easily selected using phage display (WO94/13804) from libraries. If one shoulder diately remains constant, for example, with a specificity directed against IgE, the library can be obtained when the other shoulder is subjected to variations, and select the acceptable antibody specificity. Bespecifically whole antibodies may be those obtained by alternative methods of design, as described by Ridgeway and others, 1996 [35. Ridgeway, J. B. B. et al. Protein Eng., 9, 616-621, 1996].

Various methods are available in the field of technology to generate antibodies against IgE. The antibody can be a monoclonal antibody, particularly a human, murine, chimeric or gumanitarnogo origin, which can be obtained in accordance with standard methods well known to the person skilled in the technical field.

In General, to obtain monoclonal antibodies or their functional fragments, especially of murine origin, it is possible to refer to techniques which are described, in particular in the manual "Antibodies" [36. Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., pp.726, 1988] or the method of obtaining from hybridomas described by Kohler and Milstein [37. Kohler and Milstein, Nature, 256:495-497, 1975].

Monoclonal antibodies can be obtained, for example, from a cell of an animal immunized with IgE or one of its fragments containing the epitope which is recognized by the specified monoclonal antibody. Acceptable fragments and peptides or polypeptides comprising them are described in this application and can be used to immunize animals to generate antibodies against IgE. Specified IgE or one of its fragments can, in particular, to be obtained according to the usual working methods, by genetic recombination, based on the nucleic acid sequence contained in the cDNA sequence that encodes IgE or a fragment, by peptide synthesis starting from the sequence of amino acids contained in the peptide sequence of IgE and/or its fragment.

Monoclonal antibodies can be, for example, purified on affinity column, which was pre-immobilized IgE or one of its fragments containing the epitope which is recognized by the specified monoclonal antibody. In particular, monoclonal antibodies can be purified by chromatography on protein a and/or G, then implement or not implement estlat ion-exchange chromatography, aimed at the elimination of residual protein contamination, as well as DNA and LPS, as such, and then carry out or not carry out pressure chromatography on sepharose the gel in order to eliminate the potential aggregates due to the presence of dimers or other multimers. In one embodiment of these techniques may be used simultaneously or sequentially.

Antigennegative site is part of a molecule which binds to and is complementary to all or part of the target antigen. In the molecule of antibody, this term refers to antigennegative site antibodies and includes part of the antibody that binds to and is complementary to all or part of the target antigen. If the antigen is large, then the antibody can bind only to a specific part of an antigen, this part is called the epitope. Antigennegative site antibodies can be provided by one or more variable domains of antibodies. Antigennegative site antibodies may include variable area light chain antibody (VL) and variable plot heavy chain antibodies (VH).

Isolated refers to a state in which the connecting elements in accordance with the invention, or nucleic acid encoding such binding elements will be in the General case is to predstavljati an arrangement in accordance with the invention. Thus, connecting elements, VH and/or VL domains, and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes that have an origin other than the sequence that encodes a polypeptide with the required function. Isolated elements and isolated nucleic acid will be free or substantially free of material with which they are the naturally associated such as other polypeptides or nucleic acids with which they occur in the natural environment, or the environment in which they were received (e.g. cell culture)when such reception is carried out using recombinant DNA technology, carried out in vitro or in vivo. Elements and nucleic acid may constitute such receptionand with diluents or excipients, and still for practical purposes, isolated, for example, the elements will usually be mixed with gelatin or other media, if used for coating microtiter tablets for use in immune the analyses or will be mixed with pharmaceutically acceptable carriers or diluents, when used for diagnosis or therapy. Connecting elements may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (such as Cho or NS0 (ESAS 85110503) cells), or they can be (for example, when obtained using the expression in a prokaryotic cell) deglycosylation.

Heterogeneous medications including molecules antibodies anti-IgE, also form part of the in accordance with the invention. For example, such preparations may represent a mixture of antibodies with heavy chains of the full length and heavy chains with a missing C-terminal lysine, with varying degrees of glycosylation and/or derivationally amino acids, such as cyclization of N-terminal glutamic acid with the formation of residue pyroglutamic acid.

As used in this application, the phrase "substantially as set out" refers to the characteristic(s) of the relevant CDR of VH or VL domain binding elements described in this application, which will be either identical or highly similar to the above sections, the sequence of which is presented in this application. As described in this application, the phrase "highly similar" to the specified(s) parcel(s) of one or more variable domains implies that 1 d is approximately 6, for example, from 1 to 5, including from 1 to 3, or 1, or 2, or 3, or 4 amino acid substitutions may be those which have been made in the CDRs and/or VH or VL domain.

Detailed description

As mentioned above, the connecting element in accordance with the present invention modulates and can neutralize the biological activity of IgE. As described in this application, IgE binding elements in accordance with the present invention can be optimized by neutralizing efficiency. In the General case, the optimization activity involves matirovanie sequence selected connecting element (usually sequence of the variable domain of the antibody) to obtain library binding elements, which are then subjected to analysis on the activity and take a more active binding elements. Thus, the selected optimized the efficiency of the connecting elements tend to be more high efficiency than connecting element, from which was obtained the library. However, the high efficiency of binding elements may constitute such obtained without optimization, such as the high efficiency of the connecting element can be obtained directly in the implementation of the initial screening, such as biochemical analysis of neutralization. "On timisoreana activity connecting element refers to the linking element with optimized activity neutralize certain activity, or with a reduction function. Analyses and effectiveness are described in more detail elsewhere in this application. The present invention provides optimized for efficiency, and not optimized connecting elements, as well as ways to optimize the effectiveness for the selected connecting element. The present invention thus allows qualified in this field specialist to receive connecting elements having a high efficiency.

Despite the fact that the optimization can be used for connecting the elements with the higher efficiency of this connecting element, it should also be noted that the connecting elements with high efficiency can be obtained even without optimization of efficiency.

In an additional aspect, the present invention provides a method of obtaining one or more connecting elements which are able to bind to the antigen, the method comprises bringing into contact library binding elements in accordance with the invention and the antigen, as well as the selection of the one or more connecting elements library, the ability to communicate with the specified antigen.

The library can be viewed on the particle or molecular complexes, such as replication competent genetic fashion the s, such as yeast, bacteria or bacteriophage particles (e.g., T7), viruses, cells or covalent ribosomal or other in vitro system display, each particle or molecular complex containing nucleic acid encoding a VH variable domain antibodies can be viewed on her, and not necessarily viewed as VL domain, in case of its presence. Phage display is described in WO92/01047, for example, patent US5969108, US5565332, US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US6291650, US6492160 and US6521404, each of which is entered as the reference in this application in its entirety.

After the selection of linking elements which are able to bind to the antigen and viewed on bacteriophage or library other particles or molecular complexes, nucleic acid can be derived from the bacteriophage or other particle or molecular complex, showing the specified selected connecting element. Such nucleic acid can be used in the subsequent receipt of a connecting element or VH or VL variable domain of antibodies by expression of the nucleic acid sequence of nucleic acid extracted from bacteriophage, or other particle or molecular complex, showing the specified selected binding element.

Variable domain VH ant the body from the amino acid sequence of the variable domain VH antibody specified selected connecting element may be provided in an isolated form as a binding element, with such a VH domain.

The ability to bind to IgE can be further analyzed, can also be defined ability to compete, for example, with the original molecule, antibody or antibody molecule 2-28 (e.g. in scFv format and/or IgG format, for example, IgG1) for binding to IgE. The ability to neutralize IgE can be analyzed, as discussed separately in any place in the application.

Connecting element in accordance with the present invention can neutralize the biological activity of IgE with the efficiency of the original molecule, antibody or other molecule antibodies, one of the antibodies 2-28, such as scFv or IgG1, or with higher efficiency.

Binding affinity and neutralization efficiency of different linking elements can be compared with acceptable terms.

Variants of the VH and VL domains and CDRs in accordance with the present invention, including those for which amino acid sequences are set forth in this application and which can be used in connecting elements for IgE, can be obtained by means of changing the sequence or mutation and screening for antigenspecific elements with the desired characteristics. Examples of the desired characteristics include, but are not limited to, the following:

- High St. the binding affinity for antigen as compared with known antibodies which are specific for the antigen.

- Increased neutralization activity of the antigen in comparison with the known antibodies that are specific for the antigen, if it is known.

- Provided competitive with known antibody or ligand for the antigen at a certain molar ratio.

The ability to thus complex.

- The ability to bind to a specified epitope.

Linear epitope, e.g. peptide sequence identified using scanning peptide binding, as described in this application, for example, when using sifted peptides, linear and/or curved conformation.

- Conformational epitope formed remains open.

- Ability to modulate a new biological activity of IGE or molecules with a down-regulation. Such methods are also provided in this application.

Options molecule antibodies disclosed in this application can be obtained and used in the present invention. Following the guidelines for computational chemistry in the application of techniques for the analysis of multivariate data to the relationship structure/features activity [38. Wold, et al. Multivariate data analysis in chemistry. Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski), D. Reidel Publishig Company, Dordrecht, Holland, 1984 (ISBN 90-277-1846-6)] can be derived quantitative relationships characteristic of antibody activity using well known mathematical techniques such as statistical regression, recognition and classification of images [39. Norman et al. Applied Regression Analysis. Wiley-Interscience; 3rdedition (April 1998) ISBN: 0471170828; 40. Kandel, Abraham & Backer, Eric. Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR, (May 11,1995), ISBN: 0133418847; 41. Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089; 42. Witten, Ian H. & Frank, Eibe. Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations. Morgan Kaufmann; (October 11, 1999), ISBN: 1558605525; 43. Denison David G. T. (Editor), Christopher C. Holmes, Bani K. Mallick, Adrian F. M. Smith. Bayesian Methods for Nonlinear Classification and Regression (Wiley Series in Probability and Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369; 44. Ghose, Arup K. & Viswanadhan, Vellarkad N.. Combinatorial Library Design and Evaluation Principles, Software, Tools, and Applications in Drug Discovery. ISBN: 0-8247-0487-8]. Properties of antibodies can be derived from empirical and theoretical models (e.g., analysis of likely contact residues or calculated physico-chemical properties) sequence antibodies, functional and spatial structures and their properties, which may be considered separately or in combination.

Antigennegative website antibody comprising the VH domain and VL domain is, as a rule, consists of six loops polypeptid is a: three of the variable domain of the light chain (VL) and three of the variable domain of the heavy chain (VH). Analysis of antibodies with known atomic structure determines the relationship between the sequence and spatial structure antigenspecific active centers antibodies [45. Chothia C. et al. Journal Molecular Biology (1992) 227, 799-817, 46 Al-Lazikani et al. Journal Molecular Biology (1997) 273(4), 927-948]. These relationships suggest that, with the exception of the third segment (loop) in the VH domain, loop binding site are one of a small number of main chain conformations: the canonical structure. Canonical structure formed in a particular loop, as shown, is defined by its size and by the presence of certain residues at key sites in the loop and frame sections [45. Chothia C. et al. Journal Molecular Biology (1992) 227, 799-817; 46. Al-Lazikani, et al. Journal Molecular Biology (1997) 273(4), 927-948].

This study is the relationship between the sequence - structure can be used to predict those residues in the antibody of known sequence but unknown spatial structure, which are important for maintaining the spatial structure of the CDR loops and, thus, maintain the binding specificity. These predictions can be supported by comparing predictions and results obtained by experiments on optimization. In the structural approach can be used to create the model molecule antibodies [47. Chothia, et l. Science, 223, 755-758 (1986)] when using any freely available or commercial software package, such as WAM [48. Whitelegg, N.R.u. and Rees, A.R (2000). Prot. Eng., 12, 815-824]. Software for visualization and analysis of protein, such as Insight II (Accelrys, Inc.) or Deep View [49. Guex, N. and Peitsch, M.C. Electrophoresis (1997) 18, 2714-2723] can then be used to evaluate possible substitutions at each position of CDR. This information can then be used to perform substitutions, which may have a minimal or beneficial effect on activity.

Methods required for the implementation of substitutions in the amino acid sequence of CDR of VH or VL domains of the antibodies and binding elements in General are available in the field of engineering. Variant sequences may be obtained by using replacement that may or may not be predicted as such which have a minimal or beneficial effect on activity and assessed for the ability to bind and/or neutralize IgE and/or any other desired property.

Variants of the amino acid sequence of the variable domain of any of the VH and VL domains whose sequences are in particular disclosed in this application can be used in accordance with the present invention, as discussed above.

An additional aspect in accordance with the invention, not only is em a molecule antibody comprising a VH domain that is at least, 60, 70, 80, 85, 90, 95, 98 or 99%identity with the amino acid sequence of the VH domain of the antibody 1, shown in the attached list of sequences, and/or comprising a VL domain that is at least, 60, 70, 80, 85, 90, 95, 98 or 99%identity with the amino acid sequence of the VL domain of the antibody 1, shown in the attached list of sequences. Algorithms that can be used to calculate percent identity of two amino acid sequences include, for example, BLAST [50. Altschul et al. (1990) J. Mol. Biol. 215:405-410], FASTA [51. Pearson and Lipman (1988) PNAS USA 85:2444-2448], or the algorithm of Smith-Waterman [52. Smith and Waterman (1981) J. Mol Biol. 147:195-197], for example, using the default settings.

Special options may include one or more changes in amino acid sequence (addition, division, substitution and/or insertion of amino acid residue). In some embodiments, options are less than approximately 20 changes.

Changes may be those that are implemented in one or more frame sections and/or one or more CDR. Change does not usually cause loss of function, so that the connecting element includes the thus modified amino acid sequence, may retain the ability to bind and/or neutralize Ig. They can keep the same quantitative value binding and/or neutralizing ability, as connecting element, in which there was a change, for example, as measured in the analysis described in this application. Connecting element includes a modified thus amino acid sequence may have an improved ability to bind and/or neutralize IgE.

The change may include replacing one or more amino acid (s) residue(s) do not exist in nature or non-standard amino acids, modification of one or more amino acid residues to non-existent in nature or non-standard form or embedding one or more non-existent in nature or non-standard amino acids in the sequence. Examples of rooms and localization of changes in the sequence in accordance with the invention are described elsewhere in this application. Existing in the nature of the amino acids include the 20 standard L-amino acids, identified as G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, S, K, R, N, D, E in accordance with their standard one-letter codes. Nonstandard amino acids include any other residue that may be a built-in polypeptide skeleton or the modification of existing amino acid residues. Custom aminoxy the lots can be represented as existing in nature or non-existent in nature. Some existing in the nature of non-standard amino acids are known in the technical field, such as 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N-acetylserine etc. [53. Voet &Voet, Biochemistry, 2nd Edition, (Wiley) 1995.]. Those amino acid residues that are derivationally in their N-alpha positions will be located only on the N-terminal end amino acid sequence. Typically in the present invention the amino acid is an L-amino acid, but it can represent D-amino acid. The change may, therefore, include the modification of L-amino acids on or replace it with a D-amino acid. Methylated, acetylated and/or phosphorylated forms of amino acids are also known amino acids in the present invention can be a subject of any such modification.

Amino acid sequence of domain antibody and connecting elements in accordance with the invention may include unnatural or unusual amino acids described above. Non-standard amino acids (such as D-amino acids can be embedded in the amino acid sequence during synthesis or obtained by modifying or replacing the "source" of the standard amino acids after the synthesis of the amino acid sequence.

The use of Nestan artnik and/or not existing in the nature of amino acids enhances the structural and functional diversity and may, thus, to enhance the capacity to achieve the desired properties of the IgE binding and neutralization of the binding element in accordance with the invention. Additionally, D-amino acids and analogues have been demonstrated as such, which have different pharmacokinetic profiles compared with standard L-amino acids, due to in vivo degradation of polypeptides containing L-amino acids, after the introduction of the animal, such as man.

New VH or VL parts, bearing derived CDR sequences in accordance with the invention, can be obtained by using random mutagenesis of one or more selected VH and/or VL genes to obtain mutations within the variable domain. This technique is described Gram and others [54. Gram et al., 1992, Proc. Natl. Acad. Sci., USA. 89:3576-3580], who used error-prone PCR. In some embodiments, one or two amino acid substitutions carried out within the whole variable domain CDR or set.

Another method that can be used is a directed CDR areas VH or VL gene mutagenesis. Such techniques are disclosed Barbas and others [55. Barbas et al., 1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813] and Schier and others [56. Schier et al., 1996, J. Mol. Biol. 263:551-567].

All the above methods are known in the art, and a qualified technician will be able to use these techniques to provide connecting elements in accordance with the invention using conventional methodology in the prior art.

An additional aspect in accordance with the invention provides a method for obtaining antigennegative site antibodies to IgE, the method comprising providing by addition, deletions, substitutions or insertions of one or more amino acids in the amino acid sequence of the VH domain listed in the application, VH domain, which is a variant amino acid sequence of the VH domain, an optional connection VH domain, secured thereby, with one or more VL domains, and analysis of the VH domain or VH/VL combination or combinations to identify a binding member or antigennegative site antibodies to IgE and optionally with one or more the desired characteristics, such as the ability to neutralize the activity of IgE. Specified VL domain may have an amino acid sequence that is substantially provided in this application. Can be used the same way in which one or more variants of a sequence of the VL domain disclosed in this application, together with one or more VH domains.

As mentioned above, the amino acid sequence of CDR substantially the same as presented in this application can be in the form of CDRs in the variable domain of a human antibody or its substantial part. HCDR3 sequence, with the public such as presented in this application represent an embodiment in accordance with the present invention and each of them can be in the form of HCDR3 in the variable domain of the heavy chain of a human or its essential part.

The variable domains used in this invention can be obtained or arising from any embryonic lines, or obtained by rebuilding the human variable domain, or can be a synthetic variable domain on the basis of consensus or actually existing known sequences of variable domains of a human. Variable domain may be derived from an antibody of a species other than human. CDR sequence in accordance with the invention (e.g., CDR3) may be a built-in repertoire of variable domains, in which there is no CDR (e.g., CDR3), using recombinant DNA technology. For example, Marks and others [57. Marks et al, Bio/Technology, 1992, 10:779-783] describe how to obtain a repertoire of variable domains of antibodies, in which consensus primers directed to or adjacent to the 5' end of the variable domain are used in conjunction with consensus primers to the third frame section of human VH genes to provide repertoire of VH variable domains, which are not CDR3. Marks, etc. additionally described how this repertoire can be connected to the CDR3 specific antibodies. Using similar techniques derived from the CDR3 sequences in accordance with the present invention may be shuffled with repertoires of VH or VL domains which lack a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide connecting elements in accordance with the invention. The repertoire may then be viewed in a suitable system of the host, such as a phage display system in accordance with the application WO92/01047, which is introduced in this application as a reference in its entirety, or any subsequent literary source, including Kay, Winter and McCafferty [58. Kay, VK, Winter, J., and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press], so that can be selected acceptable linking elements. The repertoire may consist of any number from 104individual members and above, for example, at least 105at least 106at least 107at least 108at least 109or, at least, 1010members or more. Other acceptable system of the host include, but are not limited to, yeast display, bacterial display, T7 display, viral display, cell display, ribosomal display is she and covalent display.

Provided is a method of obtaining a connecting element for IgE antigen, which includes:

(a) providing a source of repertoire of nucleic acids encoding the VH domain that either includes a CDR3 to be replaced or lacks a plot, encoding CDR3;

(b) the connection specified repertoire with donor nucleic acid that encodes the amino acid sequence substantially such as shown in this application for VH CDR3, so that the donor nucleic acid is embedded in the CDR3 of land in the repertoire to ensure repertory product nucleic acids encoding VH domain;

(c) expression of nucleic acids specified repertoire of the product;

(d) the selection of a connecting element for IgE;

(e) recovering the specified binding member or nucleic acid that encodes it.

In addition, can be used the same way in which VL CDR3 in accordance with the invention is combined with a repertoire of nucleic acids encoding a VL domain that either includes a CDR3 to be replaced or lacks a plot, CDR3 encoding.

Like this one, or more, or all three CDRs may be introduced into a repertoire of VH or VL domains, which are then subjected to screening for binding element or binding elements for IgE.

Nab is emer, can be used one or more HCDR1, HCDR2 and HCDR3 of the original antibody or antibody 2-28, or set of HCDR source of the antibody or antibody 2-28, and/or may use one or more LCDR1, LCDR2 and LCDR3 of the original antibody or antibody 2-28, or set LCDR source of the antibody or antibody 2-28.

Like this you may use the VH and VL domains, sets of CDR and sets HCDR and/or sets LCDR disclosed in this application.

A substantial part of the variable domain of the immunoglobulin may include at least three CDRs of the site, together with their intermediate frame sections. The part may also include at least about 50 % of either or both of the first and fourth frame sections, while 50 % are C-terminal 50 % of the first frame section and the N-terminal 50 % are fourth frame section. Additional residues at the N-terminal or C-terminal end of a substantial part of the variable domain may be those that are not normally associated with existing nature areas variable domain. For example, the design of the connecting elements in accordance with the present invention, implemented using the techniques of recombinant DNA, can lead to the embedding of the N - or C-terminal residues encoded by linkers introduced to facilitate a clone of the application and other stages of manipulation. The other stages of manipulation include the introduction of linkers to join variable domains in accordance with the invention with additional protein sequences, including constant plots antibodies, other variable domains (for example, when receiving diatel) or is able to identify functional labels, as discussed in more detail elsewhere in this application.

Despite the fact that in some respects, in accordance with the invention, the connecting elements include a pair of VH and VL domains, single binding domains based on either sequence of VH or VL domain, form further aspects in accordance with the invention. It is known that a single immunoglobulin domains, in particular VH domains capable of binding target antigens in a specific way. For example, see the discussion dAb above.

In the case of any single from binding domains, these domains may be used to screen for complementary domains capable of forming dual-domain binding element capable of binding to IgE. This can be obtained by the methods of screening of the phage display technique using the so-called hierarchising dual combinatorial approach as disclosed in the application WO92/01047, entered into this application by reference in its integral is t, in which an individual colony containing either clones H or L chain, is used to infect a complete library of clones encoding the other chain (L or H), and the resulting double-stranded binding element is subjected to selection in accordance with the techniques of phage display, such as those described in this link. This technique is also disclosed in Marks and others, above.

Connecting elements in accordance with the present invention may additionally include a constant areas of the antibodies or portions thereof, for example the constant parts of the human antibody or portion thereof. For example, a VL domain may be joined at its C-terminal end to the constant domains of the light chain of the antibodies, including human Cκ or Cλ chains. Likewise connecting element based on a VH domain may be joined at its C-terminal end to all or part (for example, CN domain) the heavy chain immunoglobulin having origins from any of the isotype of the antibody, such as IgG, IgA, IgE and IgM and any of the isotype subclasses, particularly IgG1 and IgG4. IgG1 is preferred due to its effector functions and ease of production. Any synthetic or other variant const plot, which possesses such properties and stabilizes the variable sites may also be useful in the present from which britanii.

Connecting elements in accordance with the invention can be a labeled using capable of determining or functional label. Thus, the connecting element or molecule antibodies may be present in the form immunoconjugate so, to get capable of detecting and/or quantifying a signal. Immunoconjugate may include a molecule antibodies in accordance with the invention, a conjugate with capable of determining or functional label. The label may be any molecule that produces or can be induced secretion signal, including, but without limitation, fluorescent substance, radioisotope labels, enzymes, chemiluminescent substances or photosensitizing agents. Thus, the binding can be a detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or the absorption of light.

Acceptable labels include, for example, but without limitation,

- enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucoseoxidase, glucoamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malatdegidrogenaza and peroxidase, such as horseradish peroxidase;

- dyes;

- fluorescent the e labels and fluorescent substances, such as fluorescein and its derivatives, fluorochrome, connections, rhodamine and derivatives, GFP (GFP is an abbreviation for "green fluorescent protein"), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthalic aldehyde and fluorescamine; fluorophores such as entandrophragma and chelates, for example, europium etc (Perkin Elmer and Cis Biointernational),

- chemoluminiscence label or chemiluminescense substances such as isoluminol, luminal and dioxetane;

- bioluminescent labels such as luciferase and luciferin;

- sensitizing substances;

- coenzymes;

- enzyme substrates;

- radioactive labels, including, but without limitation, Brom, plered, cobalt, ftor, Galli, Galli, ogorod (tritium), India, indium, IDM, IOD, IOD, IOD, IOD, rtot, rtot, Fosfor, renin, Reni, Reni, ruteni, ruteni, ruteni, ruteni, Scandi, selen, sera, tehnici, technetium, Tellur, Tellur, Tellur, tuli, tuli, tuli, yttrium 199 and other radioactive label referred to in this application;

particles, such as latex or carbon particles; metal salts; crystalloids; liposomes; cells, etc, which can be further labeled with a dye, catalyst or other capable of defining groups;

- molecules such as Biotin, digoxigenin and the 5-bromodeoxyuridine;

remains of toxins, such as, for example, the residue of the toxin selected from the group consisting of Pseudomonas exotoxin (PE or a cytotoxic fragment or mutant), Diptheria toxin or a cytotoxic fragment or mutant, botulinum toxin a, b, C, D, E or F, ricin or a cytotoxic fragment, such as ricin A, abrin or cytotoxic fragment, saporin or cytotoxic fragment, anti-toxin, Phytolacca American or cytotoxic fragment and brodina 1 or cytotoxic fragment.

Acceptable enzymes and coenzymes are disclosed in Litman, and others, US4275149, and Boguslaski, and others, US4318980, each of which is introduced in this application by reference in its entirety. Acceptable fluorescent substance and chemiluminescense substances are disclosed in Litman, and others, patent US4275149, which was put into this application by reference in its entirety. Labels include chemical residues, such as Biotin, which can be determined by linking to the specific related, capable of determining a remainder, for example, labeled Avidya or streptavidin. Capable of defining labels can join the antibodies in accordance with the invention with conventional chemical reactions known in the sphere of the technology.

Immunoconjugate or their functional fragments can be obtained using methods known to the skilled technician in the art. They can be merged with enzymes or fluorescent labels, directly or through the use of an auxiliary spacer elements group or linking group, such as polyallelic such as glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA), or in the presence of condensing agents such as those mentioned above for therapeutic conjugates. Conjugates containing fluorescent labels can be obtained by reaction with isothiocyanates.

Methods known to a qualified specialist in this field techniques for condensation of therapeutic radioisotopes to antibodies, either directly or using a chelating agent such as EDTA, DTPA, mentioned above, can be used for radioactive elements that can be used in diagnosis. It is also possible to carry out the tagging natrium using the method on the basis of chloramine T [59. Hunter W. M. and Greenwood F. C. (1962) Nature 194:495] or technician using techniques Crockford and others (US4424200 entered into this application by reference in its entirety) or attach DTPA as described natowich (US4479930, entered into this application by reference in its entirety).

There are many ways by which a label can produce a signal detectable by external means, for example by means of visual observation, electromagnetic radiation, heat and chemicals. The label may also contact other binding element that binds to the antibody in accordance with the invention or with the media.

The label can directly generate the signal, and, thus, to generate a signal requires no additional components. Many organic molecules, such as fluorescent substances are able to absorb ultraviolet and visible light, where the absorption of light transfers energy to these molecules and raises them to an excited energy state. This absorbed energy is then dissipated by radiation of light at the second wavelength. This radiation of light with the second wavelength may also transfer energy labeled acceptor molecule, and the received energy is dissipated from the acceptor molecule by light radiation, such as resonance energy transfer fluorescence (FRET). Other labels that directly produce a signal include radioactive isotopes and dyes.

Alternatively, the label may require the work of other components to generate a signal, and the signal produced by the system will include all components required to produce the measured signal, which may include substrates, coenzymes, enhancers, additional enzymes, substances that react with enzymic products, catalysts, activators, cofactors, inhibitors, scavenger, metal ions and specific binding substance required for binding substances, generating a signal. A detailed discussion of acceptable systems that produce a signal can be found in Ullman, and other US5185243, which was put into this application by reference in its entirety.

The present invention provides a process involving the induction or allowing binding of a connecting element, as is provided in this application, and IgE. As noted, such binding may take place in vivo, for example, after the introduction of a binding member or nucleic acid that encodes a binding element, or it may take place in vitro, for example, ELISA assays, Western blotting, immunocytochemistry, thus, affinity chromatography and biochemical assays or assays based on cell.

In General, the complexes formed between the connecting element in accordance with the invention and IgE, can be defined, among others, using enzyme immunoassay, radio is Aliza, thus, a fluorescent immunoassay, a chemiluminescent analysis of Western blot turns, analysis on the basis of spreading in the radial direction, analysis agglutination or analysis based particles.

The present invention also provides for a direct measurement of the levels of antigen by use of a connecting element in accordance with the invention, for example, in the biosensor system. For example, the present invention includes a method for identifying and/or measuring the binding of IgE, including (i) the interaction indicated a connecting element with IgE and (ii) determining the binding of the specified connecting element with IgE, where binding is determined using any method or detectable labels described in this application. This and any other method of determining the binding described in this application can be interpreted directly by the person carrying out this method, for example, using visual observation-defined labels. Alternatively, this method or any other method of determining the binding described in this application can provide the report in the form of autoradiography, photographs, computer printouts, report flow cytometry, graphics, charts, researched tube, or container, or the cell that contains the result, or any other the th visual or physical representation of the method.

May be determined by the amount of binding of the binding element with IgE. Quantitative assessment related to the amount of antigen in the sample, which may be of diagnostic interest. Screening for IgE binding and/or quantitative evaluation can be useful, for example, when selecting patients for diseases and disorders that are referenced in this application and/or any other disease or disorder involving abberant expression and/or activity of IgE, the expression and/or activity.

The diagnostic method in accordance with the invention may include (i) obtaining a sample of tissue or fluid from the subject, (ii) the impact on the specified sample of tissue or fluid by one or more connecting elements in accordance with the present invention; (iii) determining the associated IgE compared with the control sample, where an increase in IgE binding compared to a control may be indicative of Abbasanta the level of expression or activity of IgE. Samples of tissue or fluid that is subjected to analysis, include blood, serum, urine, biopsy material, a tumor, or any tissue that is suspected of contents abberant levels of IgE. Positive test results abrantie levels or activity of IgE actors can also benefit from the JV is to get treatment, disclosed later in this application.

The diagnostic method in accordance with the invention may additionally include a capture complex binding element and IgE using immobilized antigen. For example, the antigen may be immobilized on a side bar of the analytical device to capture the antigen-specific IgE in the sample that is of interest.

Specialist in the art can choose the appropriate method for determining the binding of the binding element with the antigen in accordance with his preference and General knowledge in the light of the methods presented in this application.

Reactivity binding elements in the sample can be determined using any reasonable method. The radioimmunoassay (RIA) represents one possible. Radioactively labeled antigen is mixed with unlabeled antigen (the sample) and to bind to the binding element. Associated antigen is physically separated from unbound antigen and determine the amount of radioactive antigen associated with a connecting element. The more antigen in the sample, the less radioactive antigen will be contacted with the connecting element. Analysis of competitive binding can also be used when using non-radioactive antigen when COI is whether the antigen or analog, associated with reporter molecule. Reporter molecule can be fluorochrome, phosphorus and laser dye with isolated spectral characteristics of absorption or emission. Acceptable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red, and chelates and cryptate lanthanides. Acceptable chromogenic dyes include diaminobenzidin.

Other reporters include macromolecular colloidal particles or granular material, such as latex beads that are painted, magnetic and paramagnetic, and biologically or chemically active agents that can directly or indirectly cause capable of determining the signals that are subjected to visual observation, the electron is determined or recorded by other means. Such molecules may, for example, to represent enzymes that catalyze reactions that develop, or change the color or cause changes in electrical properties. They may represent a molecular excited, so that the electronic transitions between energy States lead to characteristic spectral absorption or emission. These can include chemicals used in conjunction with biosensors. System definition based on the Biotin/avidin or Biotin/strept the Vidin and alkaline phosphatase may also be used.

The signals generated by the conjugates of individual binding element-reporter, can be used to obtain capable of quantifying absolute or relative data linking relevant connecting element in the samples (normal and analyzed).

The set includes a connecting element in accordance with any of the aspects or embodiments in accordance with the present invention, may also be provided as an aspect of the present invention. In this set connecting element can be labeled in order to be able to carry out its reactivity in a sample, for example as described in the application below. In addition, the connecting element may or may not be attached to a solid support. The components of the kit in General are sterile and kept in sealed vials or other containers. Sets can be used in diagnostic analysis or other methods, to which connecting elements are useful. The kit may contain instructions for use of the components in the way, for example the method in accordance with the present invention. Supporting materials that help in the way or allow such a method may also be included in the set in accordance with the invention. Auxiliary materials include Deut is th, excellent binding element that binds to the first binding element and is conjugated with a detectable label (e.g. a fluorescent label, radioactive isotope or enzyme). Sets based on the antibodies can also include beads for implementation thus. Each component sets in the General case is presented in its own container. Thus, these sets generally include a variety of containers that are acceptable for each connecting element. In addition, these kits can include instructions for analysis and how to interpret and analyze the data resulting from the analysis.

The present invention also provides the use of a connecting element, as described above, to measure the levels of antigen in competitive analysis, in other words, the method of measuring the level of antigen in the sample by use of a connecting element, as provided by the present invention in the competitive analysis. This method may be such that does not require physical separation of the bound and unbound antigen. The accession of the reporter molecule to the connecting element so that when this happens the physical or optical change represents one of the possibilities. Reporter molecule may not orestano or indirectly generating a detectable signal, which can be quantified. The relationship of reporter molecules may be directly or indirectly covalently, for example, through a peptide bond or ecovalence. Communication with the peptide bond may be a result of recombinant expression of gene fusion encoding the antibody and reporter molecule.

In various aspects and embodiments of the present invention comprises a connecting element, which competes for binding to IgE, such as human IgE, with any binding element defined in this application, for example, antibody 1, for example, in IgG1 format. Competition between binding elements can be easily analyzed in vitro, for example, by marking a specific reporter molecule to one binding element, which can be determined in the presence of another(their) unmarked(s) bind(s) item(s) to allow determination of the binding elements that bind to the same epitope or an overlapping epitope. Competition may be determined, for example, using ELISA, in which IgE is immobilized on the tablet, and the first marked or labeled binding element, together with one or more other unmarked or its associate is their items are added to the tablet. The presence of unlabelled binding member that competes with the labeled binding element, see if the reduction of the signal emitted by the labeled binding member. In one example, can be used HTRF® epitope competitive analysis.

For example, the present invention includes a method of identifying compounds that bind IgE, including (i) immobilization of IgE-based, (ii) the contact specified immobilized IgE simultaneously or in stages, at least one marked or labeled binding element in accordance with the invention and one or more unmarked or its investigational linking compound, and (iii) identification of a new binding of IgE connection by monitoring the reduction of the associated marker labeled binding element. Such methods can be carried out in high yield when used in the format of advance of the tablet or in the form of arrays. Such analyses can also be carried out in solution. See, for example, patent US5814468, which was put into this application by reference in its entirety. As described above, the binding definition can be interpreted directly by the person carrying out the method, for example, using visual observation-defined labels or reduce its Pris is tste. Alternatively, the linking techniques in accordance with the invention may present the report in the form of autoradiogram, photographs, computer printouts, report the results of flow cytometry, graphics, charts, researched tube, or container, or the cell that contains the result, or any other visual or physical representation of the method.

Competitive analysis can also be used in the mapping of epitopes. In one case, the mapping of epitopes can be used to identify the epitope associated with the element that binds IgE, which may not necessarily have optimized characteristics of neutralization and/or modulation. Such an epitope can be a linear or conformational. Conformational epitope may include at least two different fragment of IgE, where these fragments are placed close to each other, when IgE is folded in its tertiary or Quaternary structure with the formation of a conformational epitope that is recognized by IgE inhibitor, such as IgE binding element. In the analysis on competition can be used peptide fragment of the antigen, in particular a peptide comprising or essentially consisting of an epitope of interest. Can be used peptide containing the epitope sequence plus one and the or more amino acids at either end. Connecting elements in accordance with the present invention may be such that their binding to the antigen is inhibited by a peptide having a sequence comprising the sequence.

The present invention also provides an isolated nucleic acid encoding a connecting element in accordance with the present invention. The nucleic acid may include DNA and/or RNA. In one aspect the present invention provides nucleic acid which encodes a CDR or set of CDRs or VH domain or VL domain, or antigennegative site of the antibody, or antibody molecule, e.g. scFv or IgG1, in accordance with the invention, as defined above.

The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide, as shown above.

The present invention also provides a recombinant host cell that includes one or more structures, as described above. Nucleic acid encoding any CDR or set of CDRs or VH domain or VL domain, or antigennegative site of the antibody, or antibody molecule, e.g. scFv or IgG1, as provided in this application is, in itself forms an aspect in accordance with the present invention, as methods for the receipt of the encoded product, where the method accordingly includes the expression using the coding nucleic acid. Expression can be achieved by culturing under suitable conditions a recombinant host cell containing the nucleic acid. After receipt by the expression of VH or VL domain or binding element can be isolated and/or purified using any acceptable technique, and then can be used as it is acceptable.

Nucleic acid in accordance with the present invention may include DNA or RNA and may be a wholly or partially synthetic. Link to the nucleotide sequence as represented in this application encompasses a DNA molecule with the specified sequence and covers the RNA molecule with the specified sequence in which U is substituted for T, if the context requires another.

One additional aspect provides a method of obtaining VH variable domain of the antibody, where the method includes the induction of the expression of the coding nucleic acid. Such a method can include culturing the host cell under conditions suitable to obtain the VH variable domain of the antibody.

Similar methods of obtaining VL variable domains and linking elements, including the operating VH and/or VL domain, are provided as further aspects in accordance with the present invention.

The method of obtaining may include the step of isolating and/or purifying the product. The method of obtaining may include receptionarea product in the form of a composition comprising at least one additional component, such as a pharmaceutically acceptable filler.

Systems for cloning and expression of the polypeptide in a variety of host cells are well known. Acceptable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of the antibodies and fragments of antibodies in prokaryotic cells is well studied in the field of engineering. For review see, for example, Plilckthun [60 Pluckthun, A. Bio/Technology 9:545-551 (1991)]. Common bacterial host is an E. coli.

Expression in eukaryotic cells in culture is also available to qualified specialists in this field of technology as the opportunity to receive a connecting element [61. Chadd HE and Chamow SM (2001) Current Opinion in Biotechnology 12:188-194; 62. Andersen DC and Krummen L (2002) Current Opinion in Biotechnology 13:117; 63. Larrick JW and Thomas DW (2001) Current Opinion in Biotechnology 12:411-418]. Lines of mammalian cells available in the field of machinery for the expression of heterologous polypeptide, including the t cells of the Chinese hamster ovary (Cho), HeLa cells, kidney cells baby hamster, NS0 cells, mouse melanoma cells, YB2/0 cells of rat melanoma, embryonic cells, human kidney, human germ cells of the retina and many others.

Can be selected or designed acceptable vectors with appropriate regulatory sequences, including promoter sequences, terminator sequence, polyadenylation sequence, enhancer sequences, marker genes and other sequences that are appropriate. Vectors can be plasmids, for example phasmidae, or to be viral, such as phage, if this is acceptable [64. Sambrook and Russell, Molecular Cloning: a Laboratory Manual: 3rd edition, 2001, Cold Spring Harbor Laboratory Press]. Any known techniques and recipes for working with nucleic acid, for example, upon receipt of the structures on the basis of nucleic acid, mutagenesis, sequencing, introduction of DNA into cells and gene expression, as well as the analysis of proteins, are described in detail in Ausubel and others [65. Ausubel et al. eds., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, 4thedition 1999].

An additional aspect of the present invention provides a host cell containing nucleic acid as disclosed in this application. Such a host cell can be in vitro and may be in ku is ture. Such a host cell can be in vivo. In vivo the presence of the host cell may allow for intracellular expression of the connecting elements in accordance with the present invention as intracel" or intracellular antibodies. Intracel can be used for gene therapy.

Another aspect provides a method comprising introducing a nucleic acid in accordance with the invention in a host cell. In the introduction you can use any available method. For eukaryotic cells acceptable methods may include transfection mediated by calcium phosphate, DEAE-dextran, electroporation-mediated liposomes transfection and transduction using retrovirus or other virus, for example, vaccines, or for insect cells - baculovirus. With the introduction of a nucleic acid in a host cell, in particular in the eukaryotic cell, you can use viral system or system-based plasmids. Plasmid system can be maintained episomally or may be injected into host cell or an artificial chromosome. The introduction can be either random or targeted integration of one or more copies at a single locus or multiple loci. For bacterial cells acceptable methods may include mediated chloride, calc the I transformation, electroporation and transfection using bacteriophage.

The introduction may be followed by induction or permit expression of the nucleic acid, for example, by culturing the host cell under conditions suitable for gene expression. Purification of expressed product can be achieved using methods known to the person skilled in the technical field.

Nucleic acid in accordance with the invention can be an integrated into the genome (e.g., chromosome) of the host cell. The integration can be promotarova by including sequence, which promotes recombination in the genome according to standard methods.

The present invention also provides a method that involves using a construction as described above, in the expression system to Express the connecting element or polypeptide, as described above.

Connecting elements in accordance with the present invention can be used in methods of diagnosis or treatment of humans or animals, in particular humans. Connecting elements for IgE can be used to treat disorders characterized biological effects mediated IgE, in particular, various types of allergies and asthma. For example, connecting elements in soo is according to the invention can be used for the treatment of allergic rhinitis, allergic contact dermatitis, atopic dermatitis, anaphylaxis, food Allergy, urticaria, inflammatory bowel disease, eosinophilic gastroenteritis induced drug rash, allergic ophthalmopathy or allergic conjunctivitis.

Connecting elements for IgE can be used for inhibition of allergen-induced degranulation of mast cells in vivo or in vitro, reduction of FcεR1-mediated biological responses in vivo or in vitro, as well as to reduce the level of circulating IgE in a patient who is a person or an animal.

In accordance with this invention provides a method for inhibiting allergen-induced degranulation of mast cells in mammals, including the introduction of a given mammal connecting element, such as an antibody, VH domain, or VL domain in accordance with the invention, in a quantity sufficient to neutralize IgE.

The invention additionally provides a method of reducing FcεRI-biological response while reducing indirect FcεRII biological responses, including contact cells expressing FcεRI and/or FcεRII with a connecting element, for example an antibody, VH domain, or VL domain in accordance with the invention, in the presence of IgE.

The invention additionally provides a method of reducing FcERI mediated biological responses with simultaneous reduction mediated FcERII biological responses, including contact cells expressing FcεRI and/or FcεRII with a connecting element, for example an antibody, VH domain, or VL domain in accordance with the invention, in the presence of IgE.

When the analyzed cells are in contact with a connecting element in accordance with the invention in vitro, control(s) cell (cells) can also be used for positive controls (for example, reactions that do not contain binding element) and/or negative controls (for example, reactions that do not contain IgE and/or antigen).

When cells are subjected to contact with a connecting element in vivo, for example, by introducing a connecting element in accordance with the invention to a mammal, showing FcεRI-mediated and/or FcεRII-biological responses connecting element in accordance with the invention is introduced in a quantity sufficient to neutralize IgE.

In addition, the invention provides a method of reducing the level of circulating IgE in a mammal, such as man, including the introduction of a connecting element, such as an antibody, VH domain or VL domain in accordance with the invention, in a quantity sufficient to neutralize m red eye reduction is of circulating IgE.

Connecting elements in accordance with the invention can be used in the diagnosis and treatment of diseases or disorders, including, but not limited to, one or more of the following: allergic rhinitis, allergic contact dermatitis, atopic dermatitis, anaphylactic reaction, food Allergy, urticaria, inflammatory bowel disease, eosinophilic gastroenteritis induced drug rash, allergic ophthalmopathy rhinoconjunctivitis, allergic conjunctivitis, bronchial asthma, hypersensitivity of the respiratory tract, cosmetic Allergy, Allergy induced by drugs, hypersensitivity syndrome, induced by drugs, allergies to metals, professional hypersensitive pneumonitis, chronic hypersensitive pneumonitis, cold hypersensitivity, hypersensitivity induced by helminths, allergic to latex Allergy and pollen.

The data presented in this application with respect to binding and neutralization of IgE, thus, show that the connecting elements in accordance with the invention can be used to treat or prevent such disorders, including reducing the severity of disorders. Accordingly, the invention provides with the persons of treating or reducing the severity of, at least one symptom of any disorder referred to in this application, including the introduction to a patient in need an effective amount of one or more connecting elements in accordance with the present invention separately or in a combined therapeutic regimen with other acceptable drug, known in the field of engineering or described in this application, so that the weight of at least one symptom of any of the above disorders is reduced.

Connecting elements in accordance with the invention can be used in acceptable animals and in animal models of disease, in particular in monkeys.

Thus, the connecting elements in accordance with the present invention are useful as therapeutic agents in the treatment of diseases or disorders involving IgE, for example, production of IgE, the expression and/or activity, in particular abberant products, the expression or activity. The method of treatment may include the introduction of an effective amount of a connecting element in accordance with the invention to a patient in need where production, expression and/or activity of IgE thus reduced. The treatment method may include (i) identification of the patient showing elevated levels or activity of IgE, e.g. the R, when using the diagnostic methods described above, and (ii) introducing an effective amount of a connecting element in accordance with the invention to a patient, where the increased production, expression and/or activity of IgE is reduced. An alternative method of treatment may include (i) identification of the patient who has no obvious increase levels of IgE, but which, as expected, can benefit from the introduction of a connecting element in accordance with the invention, and (ii) introducing an effective amount of a connecting element in accordance with the invention the patient. The effective amount in accordance with the invention is an amount that reduces the increased production, expression and/or activity of IgE so that it decreases or reduces the severity of at least one symptom of a particular disease or disorder, which is subjected to treatment, but without necessarily cure the disease or disorder.

The invention also provides a method of antagonization at least one effect of IgE, including contact with or the introduction of an effective amount of one or more connecting elements in accordance with the present invention so that at least one effect of IgE exposed antagonization. The effects of IgE, which can be antagonisation with what omashu methods in accordance with the invention, include biological responses, mediated FcεRI and/or FcεRII, and reduce any effects that arise as a result of these reactions the binding.

In accordance with this additional aspect in accordance with the invention provides an isolated binding element, such as an antibody, VH domain or VL domain in accordance with the invention for the manufacture of a medicinal product for the treatment of disorders associated with or mediated by IgE, as discussed in this application. Such use or methods of obtaining drugs or pharmaceutical compositions include receptionarea a connecting element with a pharmaceutically acceptable filler.

Pharmaceutically acceptable carrier may be a compound or combination of compounds included in the pharmaceutical composition, does not cause secondary reactions, and allowing, for example, to improve the introduction of the active(s) compounds), to increase his (their) life period and/or its (their) effectiveness in the body, to increase the solubility in solution or to improve its transformation. Such pharmaceutically acceptable carriers are well known and will be adapted by a skilled person in the art as a function of the nature and methods of implementation of the selected(s) act the main(s) connections).

Connecting elements in accordance with the present invention will usually be administered in the form of pharmaceutical compositions which may comprise at least one component in addition to the connecting element. Thus, pharmaceutical compositions in accordance with the present invention, and for use in accordance with the present invention may include, in addition to the active ingredient, pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to the skilled technician in the art. Such material should not be toxic and should not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material will depend on the method of administration, which may be an oral, by inhalation, intratracheal, local, intravesicularly or by injection, as discussed below.

Pharmaceutical compositions for oral administration, such as, for example, a single domain antibody molecules (e.g., "nanotesla™"), etc. are also provided in the present invention. Such oral compositions may be in the form of tablets, capsules, powder, contained in a liquid or semi-liquid form. The tablet may include a solid carrier, such as Gelati is, or adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, hydrocarbons, animal and vegetable oils, mineral oil or synthetic oil. May also include physiological saline solution, dextrose or a solution of another saccharide, or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol.

For intravenous injection or injection into the injury site of the active ingredient will be in the form of parenteral available aqueous solution which is free from pyrogen and has an acceptable pH value, isotonicity and stability. Skilled professionals in the art will be able to prepare acceptable solutions when using, for example, isotonic medium, such as injectable solution of sodium chloride, injectable solution, ringer's injection ringer's solution with lactate. Preservatives, stabilizers, buffers, antioxidants and/or other additives can be used if it is necessary, including buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecylsilane ammonium chloride; chloride hexadecane; benzalkonium chloride; chloride benzene; phenol, butyl who or benzyl alcohol; alkylarene, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); polypeptides of low molecular weight; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; soleobrazutaya counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Connecting elements in accordance with the present invention can be receptionby in the form of liquid, semi-solid or solid form, depending on the physicochemical properties of the molecule and the method of delivery. The composition may include fillers or combinations of fillers, for example: sugars, amino acids and surfactants. The liquid composition can have a wide range of concentrations of antibodies and pH. Solid compositions can be obtained by lyophilization, spray drying or dried, for example, using the supercritical liquid is barb. Formulations of anti-CXCL13 will depend on the intended method of delivery: for example, formulations for pulmonary delivery may consist of particles with physical properties that provide deep penetration into the lungs during inhalation; formulations for topical administration (for example, for the treatment of scarring, for example, dermal scarring) may include agents, viscosity modifier, which prolong the time during which the drug is in the scene. Connecting element can be prepared with a carrier that will protect the connecting element from the quick release, as the composition of controlled release, including implants, transdermal patches, and microencapsulation delivery system. Can be used biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polychaetes, and polylactic acid. Numerous methods for obtaining such compositions are known to the skilled person skilled in the technical field [66. Robinson, J. R. ed., Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York, 1978].

Anti-IgE treatment can be provided orally (as, for example, for a single domain antibody molecules (e.g., "nanotesla™"), by injection (e.g. subcutaneously, intra ticular, intravenously administered intraperitoneally, intraarterially or intramuscularly), via inhalation, intratrahealno using intravesicular path (installation into the bladder) or topically (for example, intraocular, internasals, rectal, into wounds, on skin). Treatment may be given mode pulse infusion, particularly with decreasing doses of a connecting element. The route of administration can be determined by physical and chemical characteristics treatment, special considerations for the disease or the requirement of optimising the efficiency or to minimize side effects. Private route of administration is an intravenous. Another method of administration of the pharmaceutical compositions in accordance with the present invention is a hypodermic. This is provided that anti-IgE treatment will not be limited to application in the clinic. Thus, subcutaneous injection when used without needle device is also preferred.

Examples of intravenous compositions include:

Composition (1) includes

Insulated connecting element in accordance with the invention (optional 10, 50, 100, or 150 mg/ml indicated a connecting element, for example, antibodies)

50 mm sodium acetate

100 mm NaCl

pH 5.5

Composition (2) includes

Insulated connecting e the ment in accordance with the invention (optional 10, 50, 100, or 150 mg/ml indicated a connecting element, for example, antibodies)

20 mm succinate

105 mm NaCl

10 mm arginine

pH 6,00

The composition can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated.

Connecting element for IgE can be used as part of combination therapy in combination with additional medical component. How combinational treatment can be used to provide significant synergistic effects, in particular the combination of anti-IgE binding element with one or more other drugs. Connecting element for IgE may be injected simultaneously or sequentially or as a combined preparation with another therapeutic agent or agents, to treat one or more conditions listed in this application.

Connecting element in accordance with the present invention may be provided in combination or in addition to one or more of the following agents:

- cytokine or agonist or antagonist of cytokine function, (for example, an agent that acts on the transmission path of cytokine signal, such as a modulator SOCS system), such as alpha, beta and/or gamma-interferon; insulinogenic growth factor type I (IGF-1) receptors and associated binding proteins; interleukins (IL), for example, one or more of IL-1 to -33, and/or interleukines antagonist or inhibitor, such as anakinra; inhibitors of receptors of members of the family of interleukins or inhibitors of specific subunits of these receptors, inhibitors of tumor necrosis factor alpha (TNF-α), such as anti-TNF monoclonal antibodies (for example infliximab, adalimumab and/or CDP-870) and/or antagonist of TNF receptor, such as the immunoglobulin molecule (such as etanercept) and/or agent with a low molecular weight, such as pentoxifylline;

- modulator In cells, for example, a monoclonal antibody directed against b-lymphocytes (such as CD20 (rituximab, MRA-aIL16R or belimumab) or T-lymphocytes (for example, CTLA4-Ig (Abatacept), HuMax I1-15);

the modulator activation, maturation or survival of cells (such as Atacicept);

the modulator of immune function, for example, the antagonist lymphotoxin LIGHT transmission path (such as LTBR-Fc)

a modulator that inhibits osteoclast activity, for example, antibody to RANKL;

- modulator function of chemokine or chemokine receptor, such as an antagonist of CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11 (for the C-C family); CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5 and CXCR6 (C-X-C family) and CX3CR1 for C-X3-C family;

- inhibitor of the matrix metalloprotease (DFID), i.e. one or more of stromelysin,collagenase and gelatinase, and arakanese, in particular collagenase-1 (MMP-1), collagenase-2 (MMP-8), collagenase-3 (MMP-13), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10) and/or stromelysin-3 (MMP-11) and/or MMP-9 and/or MMP-12, for example, an agent such as doxycycline;

- inhibitor of leukotriene biosynthesis, an inhibitor of 5-lipoxygenase (5-LO) or antagonist of a protein that activates 5-lipoxygenase (FLAP), such as zileuton; ABT-761; Finlayson; tepoxalin; Abbott-79175; Abbott-85761; N-(5-substituted)-thiophene-2-alkylsulfonamides; 2,6-di-tert-butylbenzothiazole; methoxyethylamine, such as Zeneca ZD-2138; the compound SB-210661; substituted pyridinyl compound 2-cyanonaphthalene, such as L-739,010; compound 2-cyanohydrin, such as L-746,530; indole and/or quinoline compound such as MK-591, MK-886 and/or BAY x 1005;

- a receptor antagonist for leukotrienes (LT) B4, LTC4, LTD4, and LTE4 selected from the group consisting of phenothiazines-3-1, such as L-651,392; ajdinovic compounds such as CGS-25019c; benzoxazepine, such as ontazolast; benzoquinoneimine, such as BIIL 284/260; and compounds such as zafirlukast, ablukast, montelukast, pranlukast, verlukast (MK-679), RG-12525, Ro-245913, iralukast (CGP 45715A), and BAY x 7195;

- inhibitor of phosphodiesterase (PDE), such as methylxanthines, for example, theophylline and/or aminophylline; and/or a selective inhibitor of PDE isoenzyme, for example, a PDE4 inhibitor and/or an inhibitor of the isoform PDE4D, and/or hibitor PDE5;

antagonist of the histamine receptor type 1, such as cetirizine, loratadine, desloratadine, Fexofenadine, acrivastine, terfenadine, astemizole, azelastine, levocabastine, chlorpheniramine, promethazine, cyclizine, and/or mizolastine (in the General case is applied orally, topically or parenterally);

- proton pump inhibitor (such as omeprazole) or gastroprotective receptor antagonist of the histamine type 2;

antagonist histamine receptor type 4;

- shoudougai sympathomimetic agent agonist alpha-1/alpha-2 adrenergic receptors, such as propylhexedrine, phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine, napolitain hydrochloride, Oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, Xylometazoline hydrochloride, tramazoline hydrochloride and itinerarary hydrochloride;

- anticholinergic agent, for example, an antagonist of muscarinic receptor (M1, M2, and M3), such as atropine, hyoscine, glycopyrrolate, ipratropium bromide, Tiotropium bromide, bromide oxytrope, pirenzepine, telenzepine;

agonist beta adrenergic receptor subtypes 1-4 beta receptor), such as izoprenalin, salbutamol, formoterol, salmeterol, terbutaline, ortsiprenalin, bitolterol mesilate and/or pirbuterol, such as its chiral enantiomer;

- chrome, for example, sodium cromoglycate and/or nedocromil n is sodium;

- glucocorticoid, such as flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide, and/or mometazon furoate;

- an agent that modulates nuclear hormone receptors such as PPAR;

immunoglobulin (Ig) or Ig preparation or an antagonist or antibody modulating the function Ig, such as anti-IgE (e.g. omalizumab);

another applied systemically or topically anti-inflammatory agent, e.g., thalidomide or its derivative, a retinoid, dithranol and/or calcipotriol;

- combination of aminosalicylates and sulfapiridina, such as sulfasalazine, mesalazine, balsalazide, and olsalazine; and immunomodulatory agents, such as thiopurine; and corticosteroids such as budesonide;

is an antibacterial agent, for example, a derivative of penicillin, tetracycline, macrolide, beta-covenants, fluoroquinolones, metronidazole, and/or input via inhalation aminoglycosides; and/or an antiviral agent such as acyclovir, famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine, rimantadine; ribavirin; zanamavir and/or oseltamivir; a protease inhibitor such as indinavir, nelfinavir, ritonavir and/or saquinavir; inhibitor nucleoside reverse transcriptase inhibitors, such as didanosine, lamivudine, stavudine, zalcitabine, zidovudine; the non-nucleoside reverse transcr PTZ, such as nevirapine, efavirenz;

- cardiovascular agent, such as a blocker calcium channel blocker beta-adrenergic receptors, angiotensin-converting enzyme (ACE)receptor antagonist of angiotensin-2; an agent that reduces the level of lipids, such as statin and/or fibrate; modulator morphology of blood cells, such as pentoxifylline; thrombolytic and/or an anticoagulant, for example, a sedimentation inhibitor of platelets;

- an agent that affects the Central nervous system, such as an antidepressant (such as sertraline), a drug against Parkinson's disease (such as deprenyl, L-DOPA, ropinirole, pramipexol; inhibitor IAIA, such as selgin and rasagiline; inhibitor comP, such as tasmar; And-2 inhibitor, an inhibitor of the reuptake of dopamine, NMDA antagonist, agonist of nicotinic acid, dopamine agonist and/or an inhibitor of neural synthase nitrous oxide) and the drug against Alzheimer's disease, such as donepezil, rivastigmine, taken, SOH-2 inhibitor, propentofylline or metrifonate;

agent for the treatment of acute and chronic pain, for example acting on the Central and peripheral nervous system analgesics, such as an opioid analogue or derivative, carbamazepine, phenytoin, valproate sodium, amitriptyline or another antidepressant agent, paraceta the ol, or nonsteroidal anti-inflammatory agent;

- parenterally or topically applied (including by inhalation) local analgesic agents, such as lignocaine or equivalent;

agent against osteoporosis, for example, a hormonal agent such as raloxifene, or biphosphonate, such as alendronate;

(i) tryptase inhibitor; (ii) an antagonist of platelet activating factor (PAF); (iii) an inhibitor of the interleukin converting enzyme (ICE); (iv) IMPDH inhibitor; (v) inhibitors of adhesion molecules, including VLA-4 antagonist; (vi) cathepsin; (vii) kinase inhibitor, for example, the inhibitor tyrosinekinase (such as Btk, Itk, Jak3 MAP examples of inhibitors may include gefitinib, imatinib mesilate), serine/threonine kinases (for example, an inhibitor of MAP kinase, such as R, JNK, protein kinase A, b and C and IKK), or a kinase, which is involved in cell cycle regulation (e.g., cyclin-dependent kinase); (viii) an inhibitor of glucose-6 phosphate dehydrogenase; (ix) receptor antagonist cenina-B.sub1. and/or B.sub2.; (x) antipodagriceski agent, for example colchicine; (xi) an inhibitor of xanthine oxidase, for example allopurinol; (xii) an agent that promotes the excretion of uric acid, for example probenecid, sulfinpirazon and/or benzbromarone; (xiii) a means of enhancing the secretion of growth hormone; (xiv) transforming growth factor (TGFβ); (xv) platelet-derived growth factor (PDGF); (xvi) a growth factor, fibreboard the flappers, for example basic fibroblast growth factor (bFGF); (xvii) colony-stimulating factor granulocyte-macrophage (GM-CSF); (xviii) capsaicinoids cream; (xix) receptor antagonist tachykinin NK.sub1. and/or NK.sub3., such as NKP-S, SB-233412 (talnetant) and/or D-4418; (XX) elastase inhibitor such as UT-77 and/or ZD-0892; (xxi) an inhibitor of the enzyme, converting TNF-alpha (TACE); (xxii) inhibitor induced nitrous oxide synthase (iNOS) or (xxiii) chemoattractant homologous to the receptor molecule, which is expressed on T cells (such as a CRTH2 antagonist); (xxiv) inhibitor R (xxv) agent modulating the function of Toll-like receptors (TLR), (xxvi) agent modulating the activity of purinergic receptors such as RH; (xxvii) inhibitor of factor activation of transcription, such as NFkB, API, and/or STATS.

The inhibitor may be specific or may be a mixed inhibitor such as an inhibitor, is aimed at more than one molecule (e.g. receptor), or a class of molecules referred to above.

Connecting element can also be used in Association with a chemotherapeutic agent or another tyrosine kinase inhibitor with a joint introduction or in the form immunoconjugate. Fragments of the indicated antibodies can also be used in bespecifically antibodies, obtained through recombination mechanisms or biochemical merge and then is Sociali specificity described above antibodies with specificity the other antibodies, able to recognize other molecules involved in the activity for which IgE is associated.

For the treatment of inflammatory diseases such as rheumatoid arthritis, osteoarthritis, asthma, allergic rhinitis, chronic obstructive pulmonary disease (COPD), or psoriasis connecting element in accordance with the invention can represent this, coupled with one or more agents, such as nonsteroidal anti-inflammatory agents (in this application are referred to as NSAIDs)including non-selective cyclo-oxygenase inhibitors(SOH)-1/SOH-2, which are used either topically or systemically (such as piroxicam, diclofenac, propionic acids such as naproxen, flurbiprofen, fenoprofen, Ketoprofen and ibuprofen, fenamate, such as marennikova acid, indomethacin, sulindac, azapropazone, pyrazolones such as phenylbutazone, salicylates such as aspirin); selective inhibitors MOR-2 (such as meloxicam, celecoxib, rofecoksib, valdecoxib, lumiracoxib, parecoxib, etoricoxib); oxide donors nitric acid, inhibiting collagenase (CINOD); glucocorticosteroids (injected or oral, intramuscular, intravenous or intra-articular path); methotrexate, Leflunomide; hydroxychloroquine, d-penicillamine, auranofin or other parenteral or oral drugs is Olot; analgesics; diacerein; intra-articular therapies such as derivatives of hyaluronic acid; and nutritional supplements such as glucosamine.

Connecting element in accordance with the invention and one or more of the above additional medical components can be used in the production of medicines. The drug can represent such individual or combined introduction of the individual, and accordingly may include a connecting element and an additional component in the form of a combined preparation or in the form of individual drugs. Certain drugs can be used to improve individual and sequential or simultaneous administration, and allow you to enter the components in various ways, for example with oral and parenteral administration.

In accordance with the present invention provide compositions may be introduced mammals. The introduction is usually carried out in a "therapeutically effective amount", which is sufficient to be useful to the patient. Such use may constitute at least alleviating at least one symptom. Actually enter the amount and the speed and dynamics of the introduction will depend on the nature and severity of the condition is, which they were treated, the particular mammal, which they were treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the type of binding element, method of administration, mode of administration and other factors known to the doctors. The prescription of treatment, e.g. decisions regarding dose, are within the competence of doctors and other medical doctors and may depend on the severity of symptoms and/or development of diseases that are being treated. Acceptable doses of antibodies are well known in the technical field [67. Ledermann J.A. et al. (1991) Int. J. Cancer 47:659-664; 68. K.D. Bagshawe et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4:915-922]. Can be used in specific dosages specified in this application or in a doctor's office recommendations (2003), as is appropriate for the type of medication that is injected. Therapeutically effective amount or acceptable dose of a connecting element in accordance with the invention can be determined by comparing its in vitro activity and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice and other animals researched on humans are known. The exact dose will depend on a number of factors, including whether the antibody as for diagnostics is, prevention or for treatment, the size and location of the site, which is subjected to processing, the exact nature of the antibody (e.g., a whole antibody, fragment or diatel) and of any nature used for the detection of a label or other molecule attached to the antibody. The typical dose of the antibody will be in the range from 100 μg to 1 g for system applications and from 1 μg to 1 mg for local applications. May be given higher initial loading dose, followed by one or more lower doses. Typically, the antibody will be a whole antibody, such as IgG1 isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other formats antibodies proportional to molecular weight. Treatment may be repeated daily, twice a week, weekly, or monthly intervals, at the discretion of the attending physician. Treatment may be repeated every two to four weeks for subcutaneous injection every four to eight weeks for intravenous injection. Treatment may be periodic, and the period between the introduction of approximately two weeks or more, for example about three weeks or more, approximately four weeks or more, or bring the flax once a month. The treatment may be performed before and/or after surgical treatment, and/or may be injected or applied directly to the anatomical site of surgical treatment.

A brief description of the tables and figures

Table 1 list of amino acid sequences of the CDRs of the heavy chain and light chain CDR of each of the antibodies 1-28.

Table 2A shows an example of the efficiencies identified from libraries of site-directed mutagenesis in the study using HTRF® assays binding of receptor-ligand. Table 2b shows the binding affinity (KD) for a typical connecting elements in accordance with the invention against human IgE and IgE monkeys of cynomolgus using SPR (BIACORE). Table 2b also shows the efficiency, expressed as IC50, for a typical connecting elements in accordance with the invention in RBL-ER51 the analysis of the transmission of the calcium signal (time - 4 hours with 25 ng/ml human IgE or 100 ng/ml IgE monkeys of cynomolgus).

Table 3A shows the typical sequence of connecting elements in accordance with the invention, shown in the list of sequences, where SEQ ID NOS correspond as shown in Table 3A.

Table 3b shows the VL DNA and amino acid sequence of VL of a typical connecting elements in accordance with the invention from the prior bid, medium, small is, shown in the attached list of sequences, where SEQ ID NOS correspond as shown in Table 3b.

Table 4. Example of calculation of binding affinity using BIAcore analysis and measurement efficiency in the use of RBL-ER51 analysis of the transmission of the calcium signal for harmonisierung antibodies.

Table 5 shows the direct interaction between IgE Cε3-Cε4 and Fab Antibodies 11 in the x-ray crystallographic studies for interaction 1.

Table 6 direct interaction between IgE Cε3-Cε4 and Fab Antibodies 11 in the x-ray crystallographic studies of the interaction of 2.

Table 7 shows the crystal parameters and data processing x-ray studies and refinement statistics of x-ray crystallographic studies.

Table 8 shows the final model studies security studies (Example 8).

Figure 1: the Example 2.7 shows the molar concentration of the antibody expressed as log on the X-axis and the peak height in RBL-ER51 the analysis of the transmission of the calcium signal on the y-axis where there's no shading squares refer to the antibody 11, the shaded - irrelevant IgG1 control antibody, and where there's no shading inverted triangles - to anti-IgE cross-linking antibody (Biosource). Should be about the mark, that is shaded and where there's no shading shapes overlap in this figure.

Figure 2 shows the sequence ε3-4 FLAG His10 of cynomolgus.

Figure 3 shows the sequence of the variable heavy chain, which encodes the scFv (D12_VH) human antisdruciolo and one haplotype of a gene IgHE of cynomolgus (cyIgHE TQ).

Figure 4 shows the sequence of the variable heavy chain, which encodes the scFv (D12_VH) human antisdruciolo and one haplotype of a gene IgHE of cynomolgus (cyIgHE ME).

Figure 5 shows the sequence of the variable light chain scFv (D12_VL) human antisdruciolo genes cyIGLC 4 lambda constant area of cynomolgus, a number of sequences: 1 to 708.

Figure 6 shows the sequence of the variable light chain scFv (D12_VL) human antisdruciolo genes D12_VL cyIGLC 7 lambda constant area of cynomolgus.

Figure 7 refers to the Example 3, and shows the percentage of maximum inhibition of IgE expression in cells not treated with anti-IgE, where the X axis represents the concentration of Antibody 11 nm, and the Y axis represents the percentage of inhibition. The top chart refers to the Antibody 11, and the bottom chart is for reference antibody.

Figure 8 is for Example 4 and shows the percentage inhibition of total release of β-hexosaminidase +/- the standard is the first error of the mean, where the X axis represents the concentration of Antibody 11 nm, and the Y - axis the percentage of inhibition. The top chart refers to the Antibody 11, and the bottom chart is for reference antibody.

Figure 9 refers to the Example 5. The figure shows the percentage of bound IgE in human serum with increasing concentrations of anti-IgE antibodies, Antibody 11. The X-axis measures the concentration of Antibodies 11 micrograms/ml, and the Y axis shows the amount of bound IgE as a percentage of free IgE, analyzed and built as a function of total IgE.

Figure 10 refers to the Example 7, and shows

a) representation of IgE ε3-ε4 dimer in the form of strips, where the two monomers are referred to as IgE1 and IgE2 and interacts with two molecules of Fab Antibodies 11. Glycosylation in Asn394 shown as ball and stick model; and

b) rotated 90 degrees to the top view from the top, showing that the majority of interactions with IgE Fab fragment is provided by the heavy chain. The figure obtained when using the program PyMOL (DeLano Scientific LLC, San Carlos, California, U.S.A)

Figure 11 refers to the Example 7 and shows glycosylation at position Asn394 IgE.

Figure 12 relates to Example 8 and shows the average value toxicokinetics profiles 11 Antibodies IgG1Antibodies 11 IgG2and E after administration of dose 1 mg/kg (Day 1), 30 mg/kg (Day 8) and 100 mg/kg (Day 16 and above) of obese the us cynomolgus. Error bar represents standard deviation. The Y-axis represents the serum concentration of the antibody, and the Y - axis is time in days after the first dose. Group 1 (Antibody 11 IgG1) are shown as shaded circles. Group 2 (Antibody 11 IgG2) where there's no shading shown as triangles, and Group 3 (various anti-IgE molecules E) are shown as shaded squares.

Figure 13 refers to Example 8 and shows the profiles of the average value of free IgE in monkeys, cynomolgus who received weekly doses of 11 Antibodies IgG1Antibodies 11 IgG2and E (1 mg/kg / day of 1.30 mg/kg / day 8 and 100 mg/kg / day 16 and above). Error bar represents standard deviation. The Y-axis represents the concentration of IgE in ng/ml and the X axis represents the time in days. Group 1 (Antibody 11 IgG1) are shown as shaded circles. Group 2 (Antibody 11 IgG2) where there's no shading shown as triangles and Group 3 (various anti-IgE molecules E) are shown as shaded squares.

Figure 14 relates to Example 8 and shows a graph of the number of platelets (X109/l), expressed as percentage change compared with the average value volume 2 prior doses against the concentration in the blood plasma of the animals in Group 1 (treated with Antibody 11). This curve is representative for the other 16 animals within 3 groups, which demonstrates that the absence of any significant effect on platelets. The X-axis shows time in hours, the left Y - axis the level of platelets as the percentage change compared with the average level before treatment, and the left Y - axis the concentration of anti-IgE antibodies in nmol/L. the Shaded squares show the concentration of platelets and shaded diamonds - the concentration of anti-IgE antibodies. The shaded triangles indicate the dose of anti-IgE antibodies in mg/kg

Figure 15 relates to Example 8 and shows a graph of the number of platelets (X109/l), expressed as percentage change compared with the average value volume 2 prior doses against the concentration in the blood plasma of the animals in Group 1 (treated with Antibody 11 IgG1), which demonstrates temporarily drop (35% below the baseline) in the number of platelets on day 29. The X-axis shows time in hours, the left Y - axis the level of platelets as the percentage change compared with the average level before treatment, and the left Y - axis the concentration of anti-IgE antibodies. The shaded triangles indicate the dose of anti-IgE antibodies in mg/kg

Figure 16 refers to Example 9 and shows the inhibition of Antibody 11 mediated IgE/FceRI cytotoxicity. The X-axis represents the molar concentration of Antibody 11, and the Y - axis the percentage of cytotoxicity. On the chart where there's no shading triangles and the dashed circles refer to Mov18 IgE experiments is, where where there's no shading triangles represent the isotype control, and the shaded circles - Antibody 11, the diagram also shown in bold where there's no shading triangle (which is hidden under the points on the right side of the chart) and where there's no shading a circle belong to NIP IgE control, where where there's no shading, bold triangle represents the isotype control, and where there's no shading a circle is an Antibody 11.

Figure 17 refers to Example 9 and shows the inhibition of Antibody 11 mediated IgE/CD23 phagocytosis. The X-axis represents the molar concentration of Antibody 11, and the Y - axis the percentage of phagocytosis. On the chart where there's no shading triangles and the dashed circles refer to Mov18 IgE experiment, where there's no shading where the triangles represent the isotype control, and the shaded circles - Antibody 11, the diagram also shown in bold where there's no shading triangle where there's no shading and the circle belong to NIP IgE control, where where there's no shading, bold triangle represents the isotype control, and where there's no shading a circle is an Antibody 11.

Examples

Libraries of phage display of intact human single-chain Fv (scFv) cloned in formigny vector based on the filamentous phage M13 and ispolzovaniya selection [69. Vaughan, T. J., et al. (1996). Nature Biotechnology 14, 309-314; 70. Hutchings, C. Generation of Naive Human Antibody Libraries, in Antibody Engineering, R. Kontermann and S. Dubel, Editors. 2001, Springer Laboratory Manuals, Berlin, p.93]).

Specific anti-IgE scFv antibody isolated from libraries of phage display using a series of cycles of selection for recombinant human IgE.

Selected scFv antibodies optimized in respect of binding to human IgE and/or in relation to efficiency and has been reformatted as IgG antibodies.

SEQUENCE

The typical sequence of connecting elements in accordance with the invention shown in the attached list of sequences, where SEQ ID NOS conform, as shown in Table 3A below, where:

i) if the number of antibodies is accompanied by an indication GL, for example 8GL, it refers to an antibody in which one or more residues have been reverse mutations to the fetal configuration, in the General case, if you use the GL, it means that all degermination residues, which may be reverse mutation to without embryonic capable of determining loss of activity, were germanisraeli; and

ii) if the number of antibodies is accompanied by an indication PGL, for example 11PGL, it refers to an antibody in which one or more residues have been reverse mutations to the fetal configuration is tion, in General if you are using PGL, it means that more residue was subjected to reverse mutations to the fetal than in the case of GL, but it led to some loss of activity compared to degermination antibody.

Table 3A
AntibodySEQ ID No.Description
11VH/DNA
12VH/amino acid
13HCDR1
14HCDR2
15HCDR3
1317VL/DNA
1318VL/amino acid
18LCDR1
19LCDR2
110LCDR3
211VH/DNA
212VH/amino acid
213HCDR1
214HCDR2
215HCDR3
2319VL/DNA
2320VL/amino acid
218LCDR1
219LCDR2
220LCDR3
321VH/DNA
322VH/amino acid
323HCDR1
324HCDR2
325HCDR3
3321VL/DNA
3322VL/amino acid
328LCDR1
329LCDR2
330LCDR3
431VH/DNA
432VH/amino acid
433HCDR1
434HCDR2
435HCDR3
4323VL/DNA
4324 VL/amino acid
438LCDR1
439LCDR2
440LCDR3
541VH/DNA
542VH/amino acid
543HCDR1
544HCDR2
545HCDR3
5325VL/DNA
5326VL/amino acid
548LCDR1
549LCDR2
550LCDR3
6 VH/DNA
652VH/amino acid
653HCDR1
654HCDR2
655HCDR3
6327VL/DNA
6328VL/amino acid
658LCDR1
659LCDR2
660LCDR3
761VH/DNA
762VH/amino acid
763HCDR1
764HCDR2
65HCDR3
7329VL/DNA
7330VL/amino acid
768LCDR1
769LCDR2
770LCDR3
871VH/DNA
872VH/amino acid
873HCDR1

td align="center"> VH/DNA
AntibodySEQ ID No.Description
874HCDR2
875HCDR3
8331VL/DNA
8 332VL/amino acid
878LCDR1
879LCDR2
880LCDR3
981VH/DNA
982VH/amino acid
983HCDR1
984HCDR2
985HCDR3
9333VL/DNA
9334VL/amino acid
988LCDR1
989LCDR2
990LCDR3
1091VH/DNA
1092VH/amino acid
1093HCDR1
1094HCDR2
1095HCDR3
10335VL/DNA
10336VL/amino acid
1098LCDR1
1099LCDR2
10100LCDR3
11101VH/DNA
11102VH/amino acid
1103HCDR1
1104 HCDR2
1105HCDR3
1337VL/DNA
1338VL/amino acid
1108LCDR1
1109LCDR2
1110LCDR3
12111VH/DNA
12112VH/amino acid
12113HCDR1
12114HCDR2
12115HCDR3
12339VL/DNA
12340VL/amino acid
12 118LCDR1
12119LCDR2
12120LCDR3
13121VH/DNA
13122VH/amino acid
13123HCDR1
13124HCDR2
13125HCDR3
13341VL/DNA
13342VL/amino acid
13128LCDR1
13129LCDR2
13130LCDR3
14131VH/DNA
14132VH/amino acid
14133HCDR1
14134HCDR2
14135HCDR3
14343VL/DNA
14344VL/amino acid
14138LCDR1
14139LCDR2
14140LCDR3
15141VH/DNA
15142VH/amino acid
15143HCDR1
15144HCDR2
15145 HCDR3
15345VL/DNA
15346VL/amino acid
15148LCDR1
15149LCDR2
15150LCDR3
16151VH/DNA
16152VH/amino acid
16153HCDR1
16154HCDR2
16155HCDR3
16347VL/DNA
16348VL/amino acid
16158LCDR1
16 159LCDR2
16160LCDR3
17161VH/DNA
17162VH/amino acid
17163HCDR1
17164HCDR2
17165HCDR3
17349VL/DNA
17350VL/amino acid
17168LCDR1
17169LCDR2
17170LCDR3
18171VH/DNA
18172VH/amino acid
18173HCDR1
18174HCDR2
18175HCDR3
18351VL/DNA
18352VL/amino acid
18178LCDR1
18179LCDR2
18180LCDR3
19181VH/DNA
19182VH/amino acid
19183HCDR1
19184HCDR2
19185HCDR3
19353VL/DNA
19354VL/amino acid
19188LCDR1
19189LCDR2
19190LCDR3
20191VH/DNA
20192VH/amino acid
20193HCDR1
20194HCDR2
20195HCDR3
20355VL/DNA
20356VL/amino acid
20198LCDR1
20199LCDR2
20 200LCDR3
21201VH/DNA
21202VH/amino acid
21203HCDR1
21204HCDR2
21205HCDR3
21357VL/DNA
21358VL/amino acid
21208LCDR1
21209LCDR2
21210LCDR3
22211VH/DNA
22212VH/amino acid
22213HCDR1
22214HCDR2
22215HCDR3
22359VL/DNA
22360VL/amino acid
22218LCDR1
22219LCDR2
22220LCDR3
23221VH/DNA
23222VH/amino acid
23223HCDR1
23224HCDR2
23225HCDR3
23361VL/DNA
23362VL/s is nakikita
23228LCDR1
23229LCDR2
23230LCDR3
24231VH/DNA
24232VH/amino acid
24233HCDR1
24234HCDR2
24235HCDR3
24363VL/DNA
24364VL/amino acid
24238LCDR1
24239LCDR2
24240LCDR3
25241
25242VH/amino acid
25243HCDR1
25244HCDR2
25245HCDR3
25365VL/DNA
25366VL/amino acid
25248LCDR1
25249LCDR2
25250LCDR3
26251VH/DNA
26252VH/amino acid
26253HCDR1
26254HCDR2
26 255HCDR3
26367VL/DNA
26368VL/amino acid
26258LCDR1
26259LCDR2
26260LCDR3
27261VH/DNA
27262VH/amino acid
27263HCDR1
27264HCDR2
27265HCDR3
27369VL/DNA
27370VL/amino acid
27268LCDR1
27269LCDR2
27270LCDR3
28271VH/DNA
28272VH/amino acid
28273HCDR1
28274HCDR2
28275HCDR3
28371VL/DNA
28372VL/amino acid
28278LCDR1
28279LCDR2
28280LCDR3
8GL281VH/DNA
8GL282VH/and inability
8GL283HCDR1
8GL284HCDR2
8GL285HCDR3
8GL373VL/DNA
8GL374VL/amino acid
8GL296LCDR1
8GL297LCDR2
8GL298LCDR3
8PGL287VH/DNA
8PGL288VH/amino acid
8PGL289HCDR1
8PGL290HCDR2
8PGL291HCDR3
8PGL 375VL/DNA
8PGL376VL/amino acid
8PGL296LCDR1
8PGL297LCDR2
8PGL298LCDR3
11GL299VH/DNA
11GL300VH/amino acid
11GL301HCDR1
11GL302HCDR2
11GL303HCDR3
11GL377VL/DNA
11GL378VL/amino acid
11GL314LCDR1
11GL315LCDR2
11GL316LCDR3
11PGL305VH/DNA
11PGL306VH/amino acid
11PGL307HCDR1
11PGL308HCDR2
11PGL309HCDR3
11PGL379VL/DNA
11PGL380VL/amino acid
11PGL314LCDR1
11PGL315LCDR2
11PGL316LCDR3
381Se-4 FLAG of cynomolgus His10 nucleotide
382Ce3-4 FLAG of cynomolgus His0 protein
383D12_VHcyIgHE TQ nucleotide
384D12_VH cylgHE TQ protein
385D12_HE cyIgHE ME nucleotomy
386D12_VH cylgHE ME protein
387D12_VL cylgLC 4 nucleotide
388D12_VL cylgLC 4 protein
389D12_VL cylgLC 7 nucleotide
390D12_VL cylgLC 7 protein
391FceRI_Fc (NSO) nucleotide
392FceRI_Fc (NSO) protein

In the list of sequences that have been submitted since the time of application (provisional application US number: 60/901304) sequence 3' ggt codon and the corresponding residue of glycine, shown in the nucleotide and linakis is now the sequences for the VL DNA and corresponding amino acid sequence of VL, included in expressed sequences of scFv and IgG of this antibody. C-terminal glycine residue sequence corresponds to the residue 108 Kabata. This terminal glycine is not part of the VL sequence was deleted from the sequence shown in Table 3A. Sequences for DNA VL and VL amino acid from the temporary application included in the list of sequences and are shown in Table 3b below. The origin of this residue and coded them triplet ggt is explained below.

For expression of the light chain of IgG is provided a nucleotide sequence encoding a light chain of the antibody, including the first exon that encodes a VL domain, and the second exon encoding the CL domain and the intron separating the first exon of the second exon. Under normal conditions intron visiplate through the mechanism of mRNA processing, splicing 3' end of the first exon to the 5' end of the second exon. Thus, when DNA containing the specified nucleotide sequence, is expressed as RNA, the first and second exons spiceroads together. Broadcast splanirovannaya RNA produces a polypeptide comprising a VL domain and a CL domain. After splicing Gly at residue Kabata 108 is encoded by the last base (g) sequence of the VL domain of the frame section 4 and the first two bases (gt) CL d the MENA.

Thus, the glycine residue at residue Kabata 108 was included in the list of sequences VL sequences in the time of the application, but as described above, cannot be considered a C-terminal residue VL domain of the antibody molecules and, thus, was removed from the list of sequences in Table 3A.

107
Table 3b
SEQ ID NOAntibodyDescription
61VL/DNA
71VL/amino acid
162VL/DNA
172VL/amino acid
263VL/DNA
273VL/amino acid
364VL/DNA
374VL/amino acid is
465VL/DNA
475VL/amino acid
566VL/DNA
576VL/amino acid
667VL/DNA
677VL/amino acid
768VL/DNA
778VL/amino acid
869VL/DNA
879VL/amino acid
9610VL/DNA
9710VL/amino acid
10611VL/DNA
11VL/amino acid
11612VL/DNA
11712VL/amino acid
12613VL/DNA
12713VL/amino acid
13614VL/DNA
13714VL/amino acid
14615VL/DNA
14715VL/amino acid
15616VL/DNA
15716VL/amino acid
16617VL/DNA
16717VL/amino acid
17618VL/DNA
17718VL/amino acid
18619VL/DNA
18719VL/amino acid
19620VL/DNA
19720VL/amino acid
20621VL/DNA
20721VL/amino acid
21622VL/DNA
21722VL/amino acid
22623VL/DNA
22723VL/amino acid
23624VL/DNA
23724VL/amino acid
24625VL/DNA
24725VL/amino acid
25626VL/DNA
25726VL/amino acid
26627VL/DNA
26727VL/amino acid
27628VL/DNA
27728VL/amino acid
29429 (8 GL)VL/DNA
29529 (8 GL)VL/amino acid
29430 (8 PGL)VL/DNA
29530 (8 PGL)VL/amino acid
31232 (11 GL)VL/DNA
31332 (11 GL)VL/amino acid
31233 (11 PGL)VL/DNA
31333 (11 PGL)VL/amino acid

In the list of sequences temporary application of the sequence listed as Antibodies 29-34, are shown in Table 3A as Antibodies 8GL, 8PGL, 11GL and 11PGL. Some of these antibodies have a common VL domain and, as a result, some of the identification number of sequences in the list of sequences provided in the provisional application, are empty. The correct composition of antibodies is as follows:

Antibody 29 corresponds 8GL VH domain.

Antibody 30 corresponds 8PGL VH domain.

The antibody 31 corresponds to a VL domain that is common to 8GL and 8PGL.

The antibody 32 corresponds 11GL VH domain.

Antibody 33 corresponds 11PGL VH domain.

Antibody 34 corresponds to a VL domain that is common to 11GL and 11PGL.

Sequence identification number 286, 292, 293, 304, 310 and 311 are empty. This has been corrected in Table 3A.

The invention will be further presented in the following examples the AMI, which do not limit it:

Example 1. Recipe for isolation

1.1 Selection

Libraries of phage display of intact human single-chain Fv (scFv), cloned in formigny vector based on filamentous phage, used for selection (Vaughan and others, Nature Biotechnology 14:309-314 (1996), Hutchings, Antibody Engineering, R-Kontermann and S. Dubel, ed 2001, Springer Laboratory Manuals, Berlin. P93). Anti-IgE specific scFv antibodies were isolated from libraries of phage display using a series of cycles of selection or plasma purified human IgEK (Calbiochem), or plasma purified human IgEλ (Biodesign), essentially as previously described by Vaughan and others (Vaughan and others, Nature Biotechnology 14:309-314 (1996). Briefly, the breeding of penninga human IgE in PBS (PBS Dulbecco, pH 7.4) was adsorbing on microtiter tablet Maxisorp (Nunc) overnight at 4°C. Cells were washed with PBS, and then blocked for one hour using PBS-Marvel (3% wt./vol.). Purified phage in PBS-Marvel (3% wt./about.) was added to the cells and carried out the binding of antigen adsorbed to cells for 1 hour. Unbound phage were removed by using a series of cycles of washing using PBS-tween (0.1 percent.about.) and PBS. Particles bound phage were suirable, was introduced into the bacterium and "saved" for the next cycle of selection (Vaughan and others, Nature Biotechnology 14:309-314 (1996)). Carried out the Pope is the temporal cycles of selection when using Kappa and lambda forms IgE.

1.2 Inhibition of binding of IgE to FcεRI when using unpurified scFv

A representative number of individual scFv from the second cycle of selection were grown in 96 tablets cells. ScFv expressed in periplasmic bacteria were screened for their inhibitory activity in a homogeneous analysis based on FRET (resonance energy transfer fluorescence) binding of human IgE/human FceRI. In this analysis, the samples were competing for binding to human IgE (Calbiochem 401152)labeled with europium chelate (Perkin Elmer 1244-302), with human FcεRI-Fc (NSO cells, obtained at the company). A detailed analysis is provided in section "Materials and methods".

1.3 Inhibition of binding of IgE to FcεRI when using purified scFv

ScFv, which showed a significant inhibitory effect on the interaction of IgE:FcεRI, in the form of crude periplasmatic extracts were subjected to DNA sequencing (Vaughan and others, 1996, Nature Biotechnology 14: 309-314), (Osboum 1996; Immunotechnology. 2, 181-196). Unique scFv again expressed in bacteria and purified using affinity chromatography (as described Bannister and others (2006) Biotechnology and bioengineering, 94. 931-937). The effectiveness of these samples was determined by competition serial dilution of purified drug against FcεRI (NS0 cells received at the company), for binding to human IgE (Calbiochem 401152), labeled hela is ω europium (Perkin Elmer 1244-302). Purified scFv preparations, for example. Antibody 1 is able to inhibit the interaction of IgE-FcεRI. Detailed recipe is provided in the section "Materials and methods".

1.4 Reformatting scFv to IgG1

The clones were transferred from the scFv format in IgG format using sublimirovanny VHand VLdomain vectors expressing the heavy and light chain antibodies, respectively. VHdomain cloned into a vector (pEU15.1 or pEU9.2)containing the constant domains of the heavy chain of human and regulatory elements for the expression of whole heavy chain IgG1 or IgG2 in mammalian cells, respectively. Similarly, VLdomain cloned either in vector pEU3.4 for the expression of the Kappa light chain of human or pEU4.4 for the expression of the constant domain of the lambda light chain of human regulatory elements for the expression of whole light chain of IgG in mammalian cells. Vectors for the expression of heavy chains and light chains were described Persic and others (Persic, L., and others (1997) Gene 187, 9-18). Vectors Cambridge Antibody Technology designed to enable EBV OriP element, which, in combination with EBNA1 protein enables episomal replication of the plasmid. To obtain the IgG vectors expressing the heavy and light chain IgG, was transfusional in EBNA-HEK293 mammalian cells. IgG expressed and secretively on Wednesday. The products obtained obyedinenie filtered before cleaning. IgG was purified using chromatography on the basis of protein A. Supernatant cultures were loaded on a ceramic column with protein A (BioSepra) and washed with 50 mm Tris-HCl pH 8,0,250 mm NaCl. Bound IgG was suirable from the column using 0.1 M sodium citrate (pH 3.0) and neutralized by adding Tris-HCl (pH 9,0). Replace the buffer in buyowner material on PBS when using Nap10 columns (Amersham, #17-0854-02) and determined the IgG concentration spectrophotometrically using the extinction coefficient based on the amino acid sequence of IgG (Mach and other Anal. Biochem. 200(1):20-26, 1992). Purified IgG were analyzed by aggregation or degradation when using SEC-HPLC and by SDS-PAGE.

1.5 the Inhibition of the transfer of calcium signaling in RBL-ER51 cells with purified scFv and IgG

The efficiency of neutralization for purified preparations of scFv and IgG bioactivity against human IgE, FcεRI mediated through, was evaluated using RBL-ER51 analysis of the transmission of the calcium signal. RBL-2H3 cells (line basophils rats) was stable transfusional using human FcεRI (RBL-ER51 cells). Free IgE, nearby cells, binds to FcεRI on the cell surface and the subsequent cross-linking associated with the IgE receptor leads to mobilization of calcium, which can be determined by using the mouth of the STS to read the fluorescence images (FLIPR). Detailed description of the recipe provided in the section "Materials and methods".

Purified preparations of scFv Antibodies 1 are capable of inhibition induced IgE signal RBL-ER51 cells at the maximum of the studied concentrations. In the analysis of purified IgG values IC50for Antibody 1 was counted as such, which is 34 nm.

1.6 Selectivity and species cross reactivity of antibodies in kits are used ® assays competition epitopes

Species cross-reactivity and selectivity of antibodies against IgE and structurally related molecules, IgA, IgM, IgD and IgG were determined using kits are used® analyses competition epitopes. The analysis determines the relative cross-reactivity by measuring the inhibition of biotinylated IgE (purified from blood plasma, BIODESIGN International), linking each immobilizovannoi anti-IgE antibody.

Titration of purified IgA, IgM, IgD, and IgG (all Calbiochem) were analyzed in each assay to establish the specificity profile for each group of structurally related protein, as measured by IC50 values in this assay.

Titration specific IgE, including IgE ε3-ε4 domain of cynomolgus (received by the company from HEK-EBNA cells), human IgE Cε3-Cε4 domain (obtained by the company from HEK-EBNA cells) and human IgE lambda (BIODESIGN International)were subjected to testing the each assay to establish the species cross-reactivity of antibodies. A full-sized human IgEλ, together with IgE ε3-ε4 domains of human and cynomolgus, provided curves of inhibition. Did not observe any inhibition for any of the structurally related proteins. These data demonstrate the fact that Antibody 1 binds to human IgEλ, ε3-ε4 domain of IgE and is cross-reactive with IgE of cynomolgus. However, Antibody 1 is not associated with any other greatest affinity IgE proteins person. Detailed description of the recipe provided in the section "Materials and methods".

1.7 Inhibition of IgE binding to CD23 using purified IgG

IM9 cell line of human cells) have been demonstrated as such, which Express CD23 but not FcεRI, under basic conditions. IgE binds to CD23 on the surface of IM9 cells. Associated with CD23 IgE may then be contacted with the anti-IgE-phycoerythrin (Caltag) and determined using flow cytometry (FACSCalibur, BD Biosciences).

Antibodies can be assessed for inhibition of the interaction of IgE/CD23. Detailed description of the recipe provided in the section "Materials and methods". Briefly, titration investigated IgG was mixed with IgE before incubation with IM9 cells. After one-hour incubation, the cells were washed, and bound IgE was determined with the help of anti-IgE-phycoerythrin (Caltag). The 1 antibody inhibited the interaction of IgE/CD23 with a value of IC50 of 16 nm (n=3)./p>

1.8 Cross-linking associated with FCεRI IgE

Antibodies were subjected to evaluation on the potential of cross-linking associated with FcεRI IgE using RBL-ER51 analysis of calcium signaling. RBL-ER51 cells, as described in "Materials and methods", loaded IgE. Antibodies were incubated with IgE loaded cells and assessed their ability to stimulate calcium response. Antibody 1 was not able to induce capable of determining calcium response.

Example 2. Optimization of antibody

2.1 Optimization of source clone using site-directed mutagenesis

Antibody 1 was subjected to optimization by using the approach of site-directed mutagenesis with selection of phage display-based affinity. For the approach of site-directed mutagenesis large scFv phage libraries, which are the origin from the master clone, created by oligonucleotide-directed mutagenesis sections 3, defining complementarity (CDR3), variable heavy (VH) and light (VL) chain, as described in Clackson and Lowman 2004 (A Practical Approach, 2004. Oxford University Press).

Libraries were subjected to selection of phage display-based affinity in order to select variants with higher affinity for IgE. As a result, they will show improved inhibitory aktivnosti respect to the binding of IgE to FcεRI. Such selection may shall be carried out substantially so, as described previously (Thompson 1996; Journal of Molecular Biology. 256. 77-88). Briefly, scFv phage particles were incubated in solution with biotinylated human IgE λ (obtained from U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167] and modified at the company). ScFv-phage bound to the antigen, then captured using paramagnetic granules, coated with streptavidin (Dynabeads® M280) in accordance with the manufacturer's recommendations. Selected particles of scFv-phage were then recovered as described previously (Vaughan and others, Nature Biotechnology 14:309-314 (1996)), and the selection process was repeated in the presence of increasing concentrations of bio-human IgE (from 250 nm to 25 PM for 5 cycles).

After 5 cycles of selection VH and VL randomized libraries were subjected to recombination with the formation of one library, in which clones contained randomly paired individually randomized VH and VL sequences. The selection is then continued as described previously in the presence of increasing concentrations biologicheskogo IgE (from 100 PM to 1 PM for an additional 3 cycles).

2.2 Identification of superior clones obtained by site-directed mutagenesis, using biochemical analysis of antibody-ligand

ScFv obtained from breeding products site-directed mutagenesis, expressed in bacterial periplasm and subjected to the screening format epitope competitive HTRF® (homogeneous fluorescence with time resolution) analysis of inhibition of binding of human IgE (obtained from U266 [Ikeyama and other 1986. Molecular Immunology 23 (2); pp. 159-167]), labeled with cryptate europium (CIS bio International 62EUSPEA), with anti-human IgE (Antibody 1, isolated in Example 1). A detailed analysis is provided in section "Materials and methods". ScFv, which showed a significant inhibitory effect were subjected to DNA sequencing, and the only scFv was obtained in the form of refined products.

2.3 Inhibition of binding of IgE to FcεRI with purified scFv

Purified scFv was investigated in the format HTRF® analysis of the binding of the receptor-ligand (homogeneous fluorescence with time resolution) for inhibition of binding of human IgE (obtained from U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167]) or IgE of cynomolgus (recombinant, see section "Materials and methods"), labeled with cryptate europium (CIS bio International 62EUSPEA), with the human FcεR1-Fc (obtained by the company from NSO cells). An example of data on the effectiveness of scFv is included in Table 2A

Table 2A
An example of the efficiencies of clones identified from libraries of site-directed mutagenesis, the study analyses HTRF® binding receptor-ligand
Clone (permissively) The geometric mean value (95% CI) IC50for scFv (nm)
Analysis of human IgEAnalysis of IgE cynomolgus
Antibody 1475 (399-565)Weak/incomplete
Antibody 228 (n=1)317 (n=1)
Antibody 35 (n=1)18 (n=1)
Antibody 42 (0.4 to 14)5 (2-17)
Antibody 53 (0,2-34)6 (1-30)
Antibody 63 (n=2)11 (n=2)
Antibody 79(n=1)186 (n=1)
Antibody 85 (2-9)12 (8-20)
Antibody 99 (n=1)132 (n=1)
Antibody 1010 (n=1)116 (n=1)
Antibody 11 2 (0,5-7)7 (3-15)
Antibody 123 (n=2)7 (n=2)
Antibody 132 (n=2)7 (n=2)
Antibody 148 (n=1)15 (n=1)
Antibody 157 (n=1)172 (n=1)
Antibody 165 (2-11)63 (46-87)
Antibody 176 (n=1)109 (n=1)
Antibody 1811 (n=1)110 (n=1)
Antibody 216 (n=1)65 (n=1)
Antibody 226 (n=1)68 (n=1)
Antibody 231 (n=1)6 (n=1)
Antibody 249 (n=1)111 (n=1)
Antibody 258 (n=1) 86 (n=1)
Antibody 2612 (n=1)121 (n=1)
Antibody 279 (n=1)117 (n=1)
Antibody 281 (n=1)7 (n=1)

2.4 Inhibition of the transfer of calcium signaling in RBL-ER51 cells using purified IgG

After reformatting as IgG efficiency optimized clones was determined using a modified RBL-ER51 analysis of the transmission of the calcium signal. This analysis was adapted from the method to be used when directed isolation to improve the sensitivity with the aim of identifying more effective antibodies. Detailed description of the recipe provided in the section "Materials and methods". Data on the IC50effectiveness against human IgE and cynomolgus are shown in Table 2b.

Table 2b
The calculation of binding affinity using BIAcore and measure the effectiveness of using RBL-ER51 analysis of the transmission of the calcium signal for optimized antibodies.
Antibody Biacore KD (nm) (geometric mean value)RBL-ER51 analysis of the transmission of the calcium signal IC50(nm) geometric mean value (95% confidence. int)
Human IgEIgE of cynomolgusHuman IgEIgE of cynomolgus
42,18,00,088 (0,039-0,199)0,151 (0,086-0,265)
52,47,90,091 (0,005-1,79)0,181
62,13,70,096has 0.168
82,66,30,112 (0,02-0,62)0,188 (0,103-0,340)
111,69,30.069 (0,042-0,12)0,153 (0,068-0,34)
122,3230 0,1340,334
132,39,90.0880,244
(0,038-0,02)(0,134-0,43)
164,6620,292 (0,10-0,85)of 4.38
188,00,5322,97
192,57,90,095 (0,043-0,21)0,111 (0,008-1,55)
203,310,50,1910,31
210,306to 3.58
220,2622,66
232,74,30,1090,523
260,3985,1
283,27,20,099 (0,017-0,586)0,253

2.5. Garmonizacija

Amino acid sequence of VHand VLdomains optimized anti-IgE embryonic lines in the VBASE database (Tomlinson 1997; Journal of Molecular biology. 224. 487-499), and the closest germ line identified in relation to the similarity of the sequence. For VHdomain Antibodies 1 this cell line was a Vh1 DP-3 (1-f). For VLdomains such line was a VU DPL8 (1E).

Excluding Vernier residues (Foote & Winter 1992), which remained unchanged, it was made 10 changes from the embryonic line in frame sections VH domain and 2 changes in the VL domain of the Antibody 1. Five changes in the VH domain, and both changes in the VL domain was subjected to reverse transformation to the nearest embryonic sequence in respect of identical aligned human antibodies. Changes in the balances Kabata with the numbers 1, 20 a, 83 and 89 VH domain was left unchanged to maintain the effectiveness (Antibody 8 GL and Antibody 11 GL). Germanization these amino acid residues was carried out using standard techniques of site-directed mutagenesis with an acceptable mutagenic primers. Harmonisierung IgG was then subjected to re-analysis to confirm the absence of a decrease in the affinity or efficacy. An example of affinely and efficiencies for harmonisierung (GL) antibodies is provided in Table 4.

Table 4
Example of calculation of binding affinity using BIAcore and measure the effectiveness of using RBL-ER51 analysis of the transmission of the calcium signal for harmonisierung antibodies.
Antibody (harmonisierung)Biacore KD (nm) (geometric mean value) of the human IgERBL-ER51 transfer calcium signal IC50(nm) human IgE geometric mean value (95% confidence interval)
Antibody 8 GL2,50,085 (0,057-0,13)
Antibody 11 GL1,5 0,084 (0,063-0,11)

2.6 Inhibition of IgE binding c CD23 using purified IgG

Some optimized antibodies were evaluated for inhibition of the interaction of IgE/CD23 using IM9 analysis of the binding, as described previously. Antibodies were investigated in this system, have been identified as those which inhibit the interaction of IgE/CD23. The values of the IC50for 8 Antibodies and Antibody 11 was 16.5 nm and 23 nm, respectively.

2.7 Cross-linking of IgE bound to FcεRI

Some optimized antibodies were examined for cross-ability to communicate with IgE bound to FcεRI, when using RBL-ER51 analysis of the transmission of the calcium channel. RBL-ER51 cells, as described in "Materials and methods"maximally loaded with IgE. Optimized antibodies were incubated with IgE loaded cells and subjected to evaluation on their ability to stimulate calcium response. Not determined no signal (Figure 1).

2.8. Selectivity and species cross reactivity optimized antibody kits are used in® assays competition epitopes

Selectivity and species cross-reactivity directed antibodies was re-estimated using kits are used® competition analysis epitopes as described previously (see section 1.6 "Materials and methods")

Titration of purified IgA, IgM, IgD and IgG (all Calbiochem) analysera the Ali in each assay to establish the specificity profile for each group of structurally related protein, as measured by IC50 values in this assay.

Titration specific IgE, including human IgEλ (obtained from U266), human IgEκ (Calbiochem), IgE ε3-ε4 domain of cynomolgus (received by the company from HEK-EBNA cells) and human IgE ε3-ε4 domain (obtained by the company from HEK-EBNA cells) were tested in each assay to establish the species cross-reactivity of antibodies. A full-sized human IgEλ and κ, together with IgE ε3-ε4 domains provided curves of inhibition. Did not observe any inhibition for any of the structurally related human proteins (IgA, IgM, IgD and IgG). These data demonstrate the fact that the panel analyzed the antibody binds to human IgEλ and κ, ε3-ε4 domain of IgE and is cross-reactive with respect to IgE of cynomolgus. However, antibodies are not associated with greatest affinity IgE proteins.

2.9 Calculation of binding affinity on the basis of affinity for optimized clones using BIAcore

Binding affinity purified IgG samples from a representative number of clones of human IgE and cynomolgus were determined using surface plasma resonance using BIAcore 2000 biosensor (BIAcore AB), essentially as described by Karlsson and others 1991; Journal oflmmunological Methods 145 (1-2) 229-240. Briefly, protein G' (Sigma Aldrich, P4689) covalently linked to the surface SM of sensor chip using standard reagents for amine binding in accordance with the manufacturer's instructions (BIAcore). This surface protein G' was used to capture the purified anti-IgE antibody Fc domain, ensuring the surface density 50RU. Human IgEX or IgE of cynomolgus prepared in HBS-EP buffer (BIAcore AB), in the concentration range from 125 nm and 7.6 nm, was passed over the surface of sensor chip. Restored surface using 10 mm glycine, pH of 1.75 between each introduction antibodies. Received sensogram was estimated using the software BIA evaluation 3.1 and customized to bivalent analytical model to provide relative data binding. Examples of affinely analyzed for IgG are shown in Table 2b, and Table 4.

Materials and methods Example 1 and 2

Inhibition of binding of IgE to FcεRI using unpurified scFv

The results of the selection were subjected to screening in the analysis of FRET (resonance energy transfer fluorescence) homogeneous binding receptor-ligand-based analytical format for inhibition of the binding of human IgE (Calbiochem 401152)labeled with europium chelate (Perkin Elmer 1244-302), with human FcεRI-Fc (obtained from NS0 cells on the company).

The results obtained in guided isolation were subjected to screening in the form of undiluted periplasmatic extracts containing unpurified scFv made in: 50 mm MOPS buffer pH 7,4,0,5 mm EDTA and 0.5 is sorbitol.

15 μl of the crude scFv sample was added to the analytical tablet 384 cells (Perkin Elmer 6006280). After this was performed by adding 15 ál of 11 nm human FceRI-Fc (based on the molecular weight of 260 kDa), 15 μl of 40 nm to human IgG Fc labeled with XL665 (CIS Bio International 61HFCXLA), and then was added 15 μl of 0.75 nm labeled with europium human IgE. Control of nonspecific binding was determined using 300 nm human IgE (Calbiochem). All breeding was prepared in 50 mm Tris-HCl (pH 7,8), containing 250 mm sodium chloride and 0.05% tween-20 (analytical buffer).

Then incubated analytical tablets for 1.5 hours at room temperature before reading permitted in time of fluorescence at wavelengths of 615 nm and 665 nm consistently when using reader tablets VICTOR2 (Perkin Elmer).

Data were normalized using the software VICTOR2 for counting the number of pulses per second (CPS). Values CPS consistently used to calculate percent specific binding, as described in equation 1.

Equation 1:

Inhibition of binding of IgE to FcεRI with purified scFv

Purified scFv from positive clones identified by screening were subjected to study in the analysis of FRET (resonance energy transfer fluorescence) homogeneous with whom Azania receptor-ligand-based analytical format for inhibition of the binding of human IgE (Calbiochem 401152), labeled with europium chelate (Perkin Elmer 1244-302), with human FcεRI-Fc (obtained from NSO cells on the company).

Titration of scFv concentrations used to establish the effectiveness of scFv, as measured by values of the IC50in analysis. 15 ál titration of purified scFv sample was added to the analytical tablet 384 cells (Perkin Elmer 6006280). After this was performed by adding 15 ál of 11 nm human FcεRI-Fc (based on the molecular weight of 260 kDa) and 15 μl of 40 nm to human IgG Fc labeled with XL665 (CIS Bio International 61HFCXLA), and then was added 15 μl of 0.75 nm labeled with europium human IgE. Control of nonspecific binding was determined using 300 nm human IgE (Calbiochem). All breeding was prepared in 50 mm Tris-HCl (pH 7,8), containing 250 mm sodium chloride and 0.05% tween-20 (analytical buffer).

Then incubated analytical tablets for 1.5 hours at room temperature before reading permitted in time of fluorescence at wavelengths of 615 nm and 665 nm consistently when using reader tablets VICTOR2 (Perkin Elmer).

Data were normalized using the software VICTOR2 for counting the number of pulses per second (CPS). Values CPS consistently used to calculate percent specific binding, as described in equation 1.

Equation 1:

Defined IC50the values when using the software GraphPad Prism by curve fitting using the logistic equation with four parameters (Equation 2).

Equation 2:

Y=Bottom+(Bepx-Bottom)/(1+10^((LogEC50-X)*slope))

X represents the logarithm of concentration. Y represents specific binding.

Y starts from the Bottom and rises to the Top in the form of a sigmoidal function.

Inhibition of transmission of the calcium signal by using purified scFv and IgG in RBL-2H3 cells stably transfected with the human FcεR1 (RBL-ER51 cells)

The effectiveness of the neutralization of purified preparations of scFv and IgG regarding the bioactivity of human IgE mediated through FcεR1, was evaluated using RBL-ER51 analysis of the transmission of the calcium signal. Human FcεRI cloned from peripheral blood lymphocytes of human rights in rdnk vector and transfusional in RBL-2H3 cells (cell line rat basophils) using standard electroporation techniques. Transfetsirovannyh cells were cloned by the method of serial dilution and analyzed for expression of surface FcεRI. Received RBL-ER51 cells maintained in medium containing G418 (Invitrogen 10131-027) to maintain stable expression of the receptor. Free IgE, nearby cells, binds to FcεRI on the surface of the glue is OK, and subsequent cross-linking associated with the IgE receptor leads to mobilization of calcium, which can be determined by using the device for reading fluorescent image (FLIPR).

RBL-ER51 cells were sown at a concentration of 5×104/100 μl/cell in culture medium [DMEM (Invitrogen 41966) with 9% vol./about. mainactivity with warm FBS (Invitrogen 10100-147) and 400 μg/ml G418 (Invitrogen 10131-027)] black tablets in 96 wells flat bottom containing tissue culture (Costar) and incubated at 37°C, 5% CO2within 18-24 hours. After it was seized Wednesday, leaving intact monolayer of cells and replaced its 100 μl/cell FLUO-4AM loading buffer [DMEM with 0.1% FBS, 20 mm HEPES and 2.5 mm probenecid and 2 μg/ml FLUO-4AM (TeffLabs)] for 1-2 hours at 37°C, 5% CO2. Remove the loading buffer and the cells washed three times with 200 μl/cell PBS. Remove the final wash solution was replaced with his 70 μl/cell FLIPR buffer [125 mm NaCl25 nm KCl, 1 mm MgCl2, 1.5 mm CaCl2, 30 mm Hepes and 2.5 mm probenecid 5 mm glucose, 0.01% by vol./about. FCS]. The plates were incubated at 37°C, 5% CO2within 5-45 minutes.

Researched solutions of purified scFv or IgG (double frequency) was diluted to the desired concentration in FLIPR buffer in tablets with V-shaped bottom (Greiner). Irrelevant antibodies, aiming at IgE, was used as the negative counter is La. IgE (obtained from Calbiochem or U266 cells [Ikeyama, etc.. 1986. Molecular Immunology 23 (2); pp. 159-167]) received in FLIPR buffer and mixed with an acceptable investigational antibody to obtain IgE concentration of 3.33 mg/ml in a total volume of 40 μl/cell. Used in this analysis the concentration of IgE was chosen as the dose that when the final concentration analysis provides approximately 80% of the maximum calcium response. All samples were incubated for 30 minutes at room temperature before transfer 30 ál of the mixture IgE / antibody to cells loaded with dye prepared as described above. Analytical plates were incubated at 37°C for 10 minutes in order to allow free IgE to contact the RBL-ER51 cells.

To measure the mobilization of calcium after addition of cross-linking anti-IgE FLIPR (Molecular Devices) otkalibrovani for acceptable exposure in accordance with the manufacturer's instructions. Anti-IgE (Biosource AHI0501)diluted in FLIPR buffer, was added to the analytical tablets to obtain a final concentration of 10 μg/ml was Detected fluorescence of FLUO-4AM dye at 1-second intervals for 80 measurements, then used an 8-second intervals for 40 measurements. Drew peak response for each cell, and the data then were analyzed using about the testing the software Graphpad Prism.

Measurement of cross-linking anti-IgE in RBL-ER51 cells

To measure the ability of purified IgG cross-link IgE bound to FcεRI, prepared RBL-ER51 cells and loading dye as described in the analysis of inhibition. Cells were incubated for 10 minutes in 100 μl of 1 μg/ml human IgE (obtained from Calbiochem or U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167])diluted in FLIPR buffer, in order to allow IgE contact FcεRI on the cell surface. Used in this analysis the concentration of IgE was chosen as the dose that when the final concentration analysis provides approximately 80% of the maximum calcium response. To measure the mobilization of calcium after addition of cross-linking anti-IgE, FLIPR (Molecular Devices) otkalibrovani for acceptable exposure in accordance with the manufacturer's instructions. 30 µl sample antibody, diluted to an appropriate concentration in FLIPR buffer, was added to the analytical tablets loaded with IgE. Anti-IgE (Biosource AHI0501) was used as positive control. Recorded fluorescence dye FLUO-4AM (TeffLabs) at 1-second intervals for 80 measurements, then used an 8-second intervals for 40 measurements. Drew peak response for each cell, and the data then were analyzed using programme the welfare Graphpad Prism.

Selectivity and species cross reactivity of antibodies in kits are used® assays competition epitopes

Purified IgG was adsorbing on microtiter tablets on Maxisorp 96 wells (Nunc) in PBS at a concentration that provides a meaningful signal, when added to biotinylated human IgE at a concentration that is approximately equal to the estimated value of KD for the private IgG. Excess IgG was washed with PBS-tween (0.1 percent .about.) and blocked cells with PBS-Marvel (3% wt./about.) within 1 hour. Was prepared by serial dilution for each of the following competing compounds in PBS, starting from a concentration approximately equal to 1000 times the KD value of the interaction between biotinylated human IgE and the corresponding IgG; human IgE lambda (obtained from U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167]), human IgE Kappa (Calbiochem), human IgE Cε3-Cε4 domain (derived from HEK-EBNA cells at the firm), IgE Cε3-Cε4 domain of cynomolgus (obtained from HEK-EBNA cells on the company), human IgA, IgM, IgD and IgD (all from Calbiochem). These serial dilutions were added to an equal volume of biotinylated human IgE at a concentration approximately equal to KD (which led to the obtaining of the series, starting from a ratio of the competing antigen:biotinylated human IgE approximately 1000:1). These compounds then shift the and blocked IgG and left for equilibration for 1 hour. Unbound antigen was removed by washing with PBS-tween (0.1 percent.vol.), while remaining biotinylated human IgE was determined using the conjugate streptavidin-Europe+ (kits are used® define, PerkinElmer). Measured fluorescence with a time resolution of at 620 nm on the reader tablets EnVision (PerkinElmer). The data of fluorescence were analyzed using either the software Graphpad Prism, or Microsoft™ Excel.

Identification of superior clones using biochemical analysis of antibody-ligand

The products obtained as a result of selection were subjected to screening in the format epitope competitive HTRF® (homogeneous fluorescence with time resolution) analysis of inhibition of binding of labeled using cryptate europium human IgE (obtained from U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); p159-167]), (CIS bio International 62EUSPEA) with anti-human IgE (Antibody 1).

In guided optimization products selection were subjected to screening in the form of undiluted and diluted periplasmatic extracts containing unpurified scFv prepared in 50 mm MOPS buffer pH 7,4,0,5 mm EDTA and 0.5 M sorbitol.

4 nm anti-human IgE was pre-mixed with 20 nm IgG against human Fc labelled with XL665 (CIS Bio International 61HFCXLA). Was added 10 μl sample of the crude scFv to the tablet the bottom of the CSO volume 384 cells (Costar 3676). After this was added 5 μl of a mixture of antibodies to human IgE and anti-Fc-XL665, and then added 5 μl 1/245 cultivation labeled Cryptocom human IgE (approximately 2.3 nm labeled Cryptocom human IgE). Control of nonspecific binding was determined using 300 nm human IgE (obtained from U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167]). All cultivation was carried out in phosphate buffer saline (PBS)containing 0.4 M potassium fluoride and 0.1% BSA (analytical buffer).

Analytical tablets then centrifuged at 1000 rpm at room temperature for 1 minute and incubated for 3 hours at room temperature before reading fluorescence with a time resolution at wavelengths of 615 nm and 665 nm consistently when using reader tablets EnVision (Perkin Elmer).

Data were analyzed by counting value % Delta F for each sample. Delta F% was determined in accordance with equation 1.

Equation 1:

Delta F% values consistently used to calculate percent specific binding, as described in equation 2

Equation 2:

Inhibition of binding IgEc FcεRIc through improved scFv (peeled)

Purified scFv was investigated in the format HTRF® analysis of St. the statements of receptor-ligand (homogeneous fluorescence with time resolution) for inhibition of binding of human IgE (obtained from U266 [Ikeyama and other 1986. Molecular Immunology 23 (2); pp. 159-167]), or IgE of cynomolgus (recombinant, see section "Materials and methods"), labeled with cryptate europium (CIS bio International 62EUSPEA), with human FcER1-Fc (obtained by the company from NSO cells).

Titration of scFv concentrations used to establish the effectiveness of scFv, as measured by values of the IC50in analysis. 1,9 nm human FcεR1-Fc (based on the molecular weight of 260 kDa) was pre-mixed with 20 nm IgG against human Fc labeled with XL665 (CIS Bio International 61HFCXLA). 10 ál titration of purified scFv sample was added to the analytical tablet low volume 384 cells (Costar 3676). After this was performed by adding 5 μl of a mixture of FcεR1-Fc anti-Fc-XL665, and then 5 µl of the dilution 1/197 labeled Cryptocom IgE person or cynomolgus). Control of nonspecific binding was determined using 300 nm human IgE or cynomolgus (firm). All breeding was prepared in phosphate buffered saline (PBS)containing 0.4 M potassium fluoride and 0.1% BSA (analytical buffer).

Analytical tablets then centrifuged at 1000 rpm at room temperature for 1 minute and incubated for 3 hours at room temperature before reading fluorescence with a time resolution in the wavelength 615 nm and 665 nm consistently when used in the Sri reader tablets EnVision (Perkin Elmer).

Data were analyzed by counting value % Delta F for each sample. Delta F% was determined in accordance with equation 1.

Equation 1:

Delta F% values consistently used to calculate percent specific binding, as described in equation 2

Equation 2:

Defined IC50the values when using the software GraphPad Pnsm by curve fitting using the logistic equation with four parameters (Equation 3).

Equation 2:

Y=Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*slope))

X represents the logarithm of concentration. Y represents specific binding.

Y starts from the Bottom and rises to the Top in the form of a sigmoidal function.

Identification of superior clones in RBL-ER51 the transfer analysis calcium signaling

The effectiveness of the neutralization of purified IgG preparations from superior antibodies was evaluated in a modified version of transfer analysis of the calcium signal when using RBL-ER51, as described in the recipe for isolation.

RBL-ER51 cells were sown at a concentration of 5×104/100 μl/cell in culture medium [DMEM (Invitrogen 41966) with 9% vol./about. mainactivity with warm FBS (Invitrogen 10100-147) and 400 μg/ml G418 (Invitrogen 10131-027)] black tablets in 96 wells flat bottom is, containing tissue culture (Costar)and incubated at 37°C, 5% CO2within 18-24 hours. After this was removed Wednesday and replaced it with dilutions of the investigated antibodies (6,67 nm to 1.33 PM) for analysis [DMEM (Invitrogen 41966) with 9% vol./about. mainactivity with warm FBS (Invitrogen 10100-147) and 400 μg/ml G418 (Invitrogen 10131-027) and 1.6% of a mixture of penicillin / streptomycin (Invitrogen 15140-122)], after which was added IgE [derived from human (U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167]) or cynomolgus (recombinant, see section "Materials and methods")], dissolved in an analytical environment to obtain the final concentration of IgE 25 ng/ml and 100 ng/ml, respectively. The plates were incubated at 37°C, 5% CO2within 4 hours.

After this was removed the mixture of antibody/IgE, leaving intact monolayer of cells and replaced with 100 μl/cell FLUO-4AM loading buffer [DMEM with 0.1% FBS, 20 mm HEPES and 2.5 mm probenecid and 2 μg/ml FLUO-4AM (Invitrogen)] for 1-2 hours at 37°C, 5% CO2. Removed loading buffer, and the cells washed three times with 200 μl/cell PBS. Remove the final wash solution was replaced with 100 μl/cell FLIPR buffer [125 mm NaCl, 5 nm KCl, 1 mm MgCl2, 1.5 mm CaCl, 30 mm Hepes and 2.5 mm probenecid 5 mm glucose, 0.01% by vol./about. FCS]. The plates were incubated at 37°C, 5% CO2within 5-45 minutes.

To measure the mobilization of calcium after addition of the cross-wired the th anti-IgE FLIPR (Molecular Devices) otkalibrovani for acceptable exposure in accordance with the manufacturer's instructions. Anti-IgE (Biosource AHI0501)diluted in FLIPR buffer, was added to the analytical tablets to obtain a final concentration of 2.3 μg/ml was Detected fluorescence of FLUO-4AM dye at 1-second intervals for 80 measurements, and then used a 3-second intervals for 40 measurements. Drew peak response for each cell, and the data then were analyzed using the software Graphpad Prism.

Measurement of cross-linking of Ig associated with FceRI, using superior antibodies

To measure the ability of purified IgG cross-link IgE bound to FcεRI, prepared RBL-ER51 cells as described in the analysis of inhibition to assess superior antibodies. RBL-ER51 cells were sown at a concentration of 5x104/100 μl/cell in culture medium [DMEM (Invitrogen 41966) with 9% vol./about. mainactivity with warm FBS (Invitrogen 10100-147) and 400 μg/ml G418 (Invitrogen 10131-027)] black tablets in 96 wells flat bottom containing tissue culture (Costar) and incubated at 37°C, 5% CO2within 18-24 hours. After that, remove the medium and replace it 100 μl/cell human IgE diluted (obtained from U266 [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167]), diluted to a concentration of 1 μg/ml in the medium for analysis [DMEM (Invitrogen 41966) with 9% vol./about. mainactivity with warm FBS (Invitrogen 10100-147) and 400 μg/ml G18 (Invitrogen 10131-027) and 1.6% penicillin / streptomycin (Invitrogen 15140-122). The IgE concentration was selected to ensure maximum download RBL-ER51 cells. The plates were incubated for 4 hours at 37°C, 5% CO2.

Then remove the solution IgE, leaving intact monolayer of cells, and replaced it 100 μl/cell FLUO-4AM loading buffer [DMEM with 0.1% FBS, 20 mm HEPES and 2.5 mm probenecid and 2 μg/ml FLUO-4AM (Invitrogen)] for 1-2 hours at 37°C, 5% CO2. Removed loading buffer, and the cells washed three times with 200 μl/cell PBS. Remove the final wash solution was replaced with 100 μl/cell FLIPR buffer [125 mm NaCl25 nm KCl, 1 mm MgCl2, 1.5 mm CaCl2, 30 mm Hepes and 2.5 mm probenecid 5 mm glucose, 0.01% by vol./about. FCS]. The plates were incubated at 37°C, 5% CO2within 5-45 minutes.

To measure the mobilization of calcium after addition of cross-linked anti-IgE, FLIPR (from 1.53 μm to 2.33 nm) FLIPR (Molecular Devices) otkalibrovani for acceptable exposure in accordance with the manufacturer's instructions. 30 µl sample antibody, diluted to appropriate concentrations in FLIPR buffer, was added to the analytical tablets. Anti-IgE (Biosource AHI0501) was used as a positive control. Recorded fluorescence of FLUO-4AM dye at 1-second intervals for 80 measurements, and then used a 3-second intervals for 40 measurements. Drew peak response for each cell, and Dan is haunted then subjected to analysis by using the software Graphpad Prism.

Inhibition of IgE binding to CD23 on IM9 cells using purified IgG

Antibodies were evaluated for inhibition of IgE / CD23 interaction when using the analysis of binding on IM9 cells. IM9 cell line of human cells) were maintained in culture medium [RPMI 1640 glutamax (Invitrogen 61870-010); 9% vol./about. inactivated by heating with FBS (Invitrogen 10100-147)] using standard procedures tissue culture

To study optimized IgG IM9 cells pre-treated with 25 ng/ml human IL-4 (Peprotech, 200-04) for 3 days at 37°C/5%CO2in order to increase the expression of CD23.

Collected IM9 cells and resuspendable in running buffer [PBS with 1% goat]. Blocking Fc receptor was carried out by addition of Fc fragments (TEBU-bio) to obtain a final concentration of 5 µg/ml of This cell suspension were planted in the amount of 100 μl/cell in polypropylene tablet containing cells with a U-shaped bottom (Greiner) and incubated on ice for 30 minutes.

Breeding antibodies (667 nm to 1 nm) were prepared in polypropylene tablets containing cells with a U-shaped bottom (Greiner) and mixed with IgE (obtained from U266 cells [Ikeyama and others, 1986. Molecular Immunology 23 (2); pp. 159-167]) to obtain a final concentration of IgE 10 µg/ml for 30 minutes at room temperature. Tablets with cells was centrifuged is at 2000 rpm for 2 minutes, delete supernatant, leaving cellular precipitate. Cells resuspendable in a mixture containing 100 μl/cell antibodies and IgE, and incubated on ice for 1 hour. The precipitated cells were centrifuged at 2000 rpm for 2 minutes and removed supernatant antibody/IgE. Cells were washed by resuspendable 200 μl/cell running buffer and centrifuged as described above.

IgE is associated with cell surface was determined with the help of anti-IgE-phycoerythrin (Caltag)diluted to 1/30, vol./about., 100 μl/cell. Analytical plates were incubated on ice for 20 minutes in the dark before centrifugation at 2000 rpm for 2 minutes and washed with 2×200 μl of running buffer as described above. Cells resuspendable in 100 ál of Cell Fix (BD biosciences) and were analyzed using FACSCalibur (BD Biosciences) to determine FL2 staining.

Data were analyzed using the software CellQuest Software (BD biosciences). Dismissing the geometric mean of the fluorescence FL2, and the data is then analyzed using Microsoft Excel and Graphpad Prism software.

Obtaining human IgE ε3-4, the terminal marked with His FLAG 10. A fragment of human IgE ε3-4 was the same as described previously in Wurzburg and others (2000). The structure of the human IgE-Fc Cε3-C4 revealed the conformation of girlstv affecting domains of antibodies. The cDNA fragment, which encompasses nucleotides 2135-2868 (GenBank Deposit number J00222), amplified using RT-PCR from total RNA stimulated with human IL13 RVMS (mononuclear cells in peripheral blood). This PCR product was cloned in pCR2.1 (Invitrogen).

To implement the secretion of the expressed protein and receiving sequence, which includes in frame C-terminal FLAG epitope and polyhistidine label (His10), IgE ε3-4 fragment amplified by PCR using primers that include 5' BssHII site, 3' FLAG epitope, polyhistidine label (His10) and XbaI site, and consistently embedded in pEU8.2 vector. Modified pEU8.2 a vector containing the EF-1 promoter, genomic sequence for murine leader peptide IgG1, oriP the source of replication to allow episomal replication of the plasmid in the transfection in cell lines expressing the EBNA-1 gene product (such as HEK293-EBNA cells).

Protein was purified from conditioned medium using IMAC chromatography, and then was carried out by gel-filtration (SEC).

Getting IgE ε3 4 cynomolgus, C-terminal labeled with FLAG His10

Const plot of IgE cynomolgus determined by direct sequencing of PCR products amplified from genomic DNA using the two primers, covering nucleotides 1174-2989 human locus IgHE (heavy chain of IgE) (GenBank access number J00222). The exons identified by homology with the sequence of human and, thus, it is possible to predict the sequence of the cDNA for a constant area of the heavy chain of IgE cynomolgus.

Synthesized cDNA encoding the mouse sequence of the leader peptide of IgG1 cynomolgus, ε3-4 (Figure 2), C-terminal FLAG epitope and polyhistidine label (DNC), and cloned into pDONR221 (Invitrogen). Then when using LR Gateway® reaction (Invitrogen) gene, which is of interest tolerated in the expression pDEST12.2 vector (Invitrogen)modified by the insertion of oriP replication source of rser vector (Invitrogen) to allow episomal plasmid replication during transfection in cell lines expressing the gene product EBNA-1 (such as HEK293-EBNA cells).

Protein was purified from conditioned medium using IMAC chromatography, and then was carried out by gel-filtration (SEC).

Obtaining chimeric D12 variable segment and a constant area of IgE cynomolgus

Synthesized cDNA encoding variable plot heavy chain scFv anti-estradiol person (D12_VH) and one of two different haplotypes of the gene IgHE of cynomolgus (cyIGHE TQ and cyIGHE ME) (DNC) and cloned it into the vector pDONR221 (Invitrogen. Also synthesized cDNA representing the variable area light chain scFv anti-estradiol person (D12_VL) and one of the two genes lambda constant area of cynomolgus (cyIGLC4 and cyIGLC7) (DNC) and cloned it into the vector pDONR221 (Invitrogen).

Then when using LR Gateway® reaction (Invitrogen) gene, which is of interest tolerated in the expression pDEST12.2 vector (Invitrogen)modified by the insertion of oriP replication source of rser vector (Invitrogen) to allow episomal plasmid replication during transfection in cell lines expressing the gene product EBNA-1 (such as HEK293-EBNA cells).

Recombinant chimeric IgE protein representing variable plot heavy chain human scFv anti-estradiol, merged with ε1-4 IgE of cynomolgus (Figures 3 and 4), and variable area light chain scFv human anti-estradiol, merged with lambda constant area of cynomolgus (Figures 5 and 6), downregulation of HEK293 EBNA cells was purified using the method as described Ikeyam and others (1986) Mol Immunol 23 pp. 159-67.

Human FcεRI-Fc (obtained from NS0 cells in the firm) FcεRI, covering nucleotides 67-711 (GenBank access number NM_002001), cloned from above genomic area Fc of human IgGI, as well as from pEU1.2 first described Persic and others (1997) Gene 187; 9-18. The product was cloned in rdnk EcoRI - XbaI (SEQ ID NO:391 and 392). Expr is the Russia recombinant fused protein FcεRI_Fc was carried out by stable transfection NS0 cells using rdnc FcεRI_Fc design. Stable expression was established by selection using G418, isolation of clones using the serial cultivation and identification of clones with high expression level. FcεRI_Fc protein was then purified from the conditioned medium using affinity chromatography with protein A, and then provodili gel-filtering.

Example 3. In human cells - inhibition of intracellular IgE

Mononuclear cells from peripheral blood (RVMS) were isolated from heparinized whole human blood by centrifugation using ficoll-Pak gradient (Phannacia). B cells consistently isolated from the population RPMS when using positive anti-CD19 selection using magnetic particles (Miltenyi). Both positive and negative fraction In cells, collected as cells that passed through a magnetic column. Cells from the negative fraction of cells containing all RVMS cells, except the cells were treated with mitomycin C to prevent proliferation. Cells were incubated with 50 μg/ml of mitomycin C for 30 minutes, then washed with culture medium (RPMI 1640 with glutamax (Gibco) /10% FCS (Gibco) /50 units/ml penicillin /50 μg/ml streptomycin (Gibco)) and further incubated in PBS for 70 minutes before the final rinse to remove all of mitomycin C. For induction d is fferential b-cells, 4×104B-cells and 9.2×105cells from the negative fraction b cells were planted on the tablet of the 96 wells in a medium for cell culture, complemented by a 3.5 μm beta-mercaptoethanol (Sigma) and 20 μg/ml transferrin (Chemicon), and pre-incubated with anti-IgE monoclonal antibody or control antibody at a concentration of 0.001-100 nm for 30 minutes before addition of human interleukin-4 (IL-4) at a concentration of 10 ng/ml Cells sequentially incubated in a humidified chamber with CO2within 12-14 days. 12-14 days the tablets were subjected to a short cycle of centrifugation, collected supernatant, and cells were stained for the detection of intracellular IgE using the following recipe.

First, cells were incubated with PBS and 1% human serum for 10 minutes to block Fc receptor binding. Then the cells were fixed and subjected to disruption of membrane permeability on ice when using a set of Cytofix/Cytoperm from Becton Dickinson. Then the cells were washed before the addition of polyclonal anti-human IgE-FITC antibodies Kroll (DAKO) at a final dilution of 1:6, and mouse monoclonal anti-human CD19 - RPE/Cy5 antibody (DAKO) at a final dilution of 1:10. It is important that the cells were carefully washed in order to avoid exposure to residual anti-IgE MAb (monoclonal EN is ITIL). After 30 minutes incubation, the cells were washed and samples were analyzed on a FACS Calibur using HTS devices for loading tablets for 96 wells. Then recorded the percentage of cells in the CD19+ population, which together expresses IgE, and the expression of intracellular IgE were represented as % inhibition of the maximum IgE expression in cells not treated with a blocking anti-IgE monoclonal antibodies. Antibody 11 inhibited the induction of IgE positive cells with IC50 value of 1.6 nm (Figure 7 - top chart). Irrelevant antibody of the same format was used as negative control (CAT-002), it does not inhibit the induction of IgE positive cells(Figure 7 - lower chart).

Example 4. Line of fat cells (LAD2) - inhibition of release of β-hexosaminidase

LAD2 cells [A.S.Kirshenbaum and other Leukemia research 27 (2003)] were cultured at a density of cells of 0.25 to 0.6×106cells/ml in medium free from serum (StemPro-34, Life Technologies), supplemented nutritional Supplement StemPro-34,2 mm L-glutamine and 100 ng/ml of recombinant factor of human stem cells (rhSCF, R&D). To analyze the determination of β-hexosaminidase cells were sown at a density of 2.5×104cell/cell and pre-incubated in polypropylene plate of 96 cells with blocking anti-IgE b in the concentration range of 0.0001-100 nm. the entrances were incubated at 37°C for 30 minutes before addition of IgE at a concentration of 0.15 nm, then cells were incubated for additional 4 hours. After incubation with IgE cells were washed with buffer to remove the rest of IgE, and after that IgE is associated with FceR on LAD2 cells, cross-linked with αIgE (600 μg/ml goat anti-human IgE, Sigma) for 30 minutes at 37°C. the Incubation was stopped by cold centrifugation, collected cell supernatant and transferred to the plate of 96 wells. The content of β-hexosaminidase were analyzed using a slightly modified version of the method published by Smith and others [J Smith and others Biochem. J. (1997)223, 321-328]. Briefly, 2 mm p-nitrophenyl-N-acetyl-D-glucosaminide in 0.2 M citrate buffer, pH 4.5 was used as substrate for hexosaminidase. The reaction was stopped by adding 1M Trio-buffer pH to 9.0. The optical density was measured spectrophotometrically at a wavelength of 405 nm (minus value at a wavelength of 570 nm) using reader Spectramax from Molecular Devices. Calculated effect of anti-IgE MAb against inhibition of β-hexosaminidase and represented as percent inhibition of total release +/- standard error of the mean. Antibody 11 inhibited β-hexosaminidase with IC50 value of 0.04 nm (Figure 8 - top chart), while irrelevant MAb of the same format (CAT-002) is not inhibited β-hexo is Amiridze (Figure 8 - the bottom chart).

Example 5. Binding of anti-IgE antibodies and IgE in serum using ELISA

Description analysis

Serum samples were prepared from blood samples obtained from human donors. Tablets ELISA on 96 wells (Nunc Maxisorp, No. 442404) were coated with 150 μl/cell 1 µg/ml FcERI-Fc-His, diluted in PBS, and incubated at 4°C over night. After incubation over night tablets thrice washed with PBS containing 0.05% tween-20 (PBST, Medicago 09-9401-100). To reduce background binding tablets sequentially incubated with 200 μl/cell blocking buffer consisting of PBS containing 0.5% BSA, incubated at room temperature for 2 hours and washed three times with PBST as described above. Samples (human serum or plasma with varying concentrations of anti-IgE antibodies. Antibody 11) and standards (ImmunoCAP total IgE (human) calibration standard, Phadia, Uppsala) were diluted in PBS containing 0.05% tween-20, and kept on ice until such time as they are not used for analytical tablets in the amount of 150 μl/cell. The tablets were closed, and the samples were incubated at room temperature for 2 hours. To remove unbound sample tablets thrice washed with PBST as described previously. After this was added 150 μl/cell anti-human IgE rabbit (D€1, 30-1917-00, 420036-02, 841204, 9911302 from Phadia, Uppsaa) at a concentration of 0.25 μg/ml, diluted in PBST, to determine the associated human IgE. Then the tablets were again closed, and incubated during 1 hour at room temperature. To remove unbound anti-human IgE antibodies rabbit, tablets, three times washed with PBST as described above. Conjugated with HRP-secondary antibody (anti-human IgE goats). The conjugate was diluted 1:25000 in PBST, was added 150 μl of the cell, closed the tablets and incubated for 1 hour at room temperature. Then the tablets three times washed with PBST as described above. Then was added a solution of TMB substrate (DAKO substrate-Chromogen. No. S1599) to each cell in the amount of 150 μl/cell and the plates were incubated for 10 minutes at room temperature. The reaction was stopped by adding 150 μl/cell solution to stop reaction (2M H2SO4and read the absorbance at 450 nm on the device Tecan SAFIRE. Since the IC50 measurement is dependent on the concentration of ligand (i.e. IgE) in the analysis, in this analysis IC50will depend on the number of IgE ligand present in the sample of human serum. In a representative experiment, Antibody 11 mattered IC50202 PM (Figure 9). In the same experiment Xolair™ mattered IC5057 nm.

Example 6. The measurement of complex formation between IgE and purified IgG

The character is eristic immune complexes, formed between purified human IgE and purified anti-IgE IgG (Antibody 11), carried out in a high pressure liquid chromatography. In addition, the continuous scattering of light at different angles (MALS) was used to estimate the size of the complex. Complexes were formed by incubation of IgE and IgG together at three different molar ratios (3:1, 1:1 and 1:3, respectively) in PBS Dulbecco at 18°C for 1 hour. For molar ratio 1:1 concentration of each protein was 2.5 μm. A higher ratio was achieved by increasing the concentration of the relevant protein to 7.5 μm. These samples were analyzed by two columns Bio-Sep-SEC-S 4000 (300×7.8 mm)connected together. Balanced columns and the samples were analyzed in PBS Dulbecco at a flow rate of 1 ml/min, the system Agilent HP1100 HPLC. Peaks were identified at 220 and 280 nm with the use of the detector diode matrix, and the eluate was also passed through the detectors Wyatt Technologies DAWN EOS (MALS) and Optilab rEX (index of refraction).

Chromatography of the sample with a molar ratio of 1:1 gave a doublet of peaks (determined by UV absorption)that are not fully separated and corresponded to the retention time 13,88 minutes and 14.9 minutes. These values hold show the formation of non-covalent complexes with IgE IgG. MALS were given molecular weight 1,085 to the a (peak 13,88 min) and 702 kDa (peak of 14.9 min). These masses are consistent with the complexes according to heterotetramer (predicted mass 674 kDa, 2IgE:2IgG) and heterohexameric (predicted mass of 1010 kDa, 3IgE:3IgG). Chromatographic analysis and MALS analysis for samples with molar ratio as 3:1 and 1:3 (IgE:IgG) provided a similar profile with the sample 1:1 with peaks corresponding to heterotetramer and heterohexameric determined using UV absorption. Additional peaks were identified as meeting the excess IgE or IgG in the samples.

Example 7. Determination of binding epitopes using harmonisierung Antibodies 11

The use of x-ray crystallography to determine the precise three-dimensional structures of proteins at atomic resolution is well-known to specialists in this field of technology. This method was used for detailed visualization of proteins that interact with antibodies (Padavattan and others, 2007; Karpusas and others, 2001). Such is the most accurate method of epitope mapping, however, requires considerable effort and is based on the possibility of obtaining crystals of satisfactory quality, which, in turn, depends on the purity and quality of the protein sample and competence in order to find acceptable conditions of crystallization. After obtaining crystals of protein-antibody were irradiated using rentgenovsk the x-rays for obtaining diffraction model, which depends on the exact distribution of the atoms. The diffraction model can be analyzed using crystallography to determine the three-dimensional positional coordinates of the atoms in the structure. This allows you to carefully investigate the sites of interaction between a protein and an antibody.

7.1 Definition of the x-ray crystal structure of the complex of IgE ε3-ε4 antibody

IgE domain ε2-ε3 cloned, expressed and purified, with the goal of structural definition. Likewise derived Fab fragment was obtained by digestion and purification of whole Antibodies 11, advanced to bind to IgE. The complex was prepared by mixing and purified using exclusion chromatography to remove IgE domains and Fab molecules that do not form complexes. The resulting crystals of the complex of IgE ε3-ε4/Fab treated triangular spatial group R. They were subjected to an analysis at the European laboratory for synchrotron radiation (ESRF) in Grenoble, France. Received a complete diffraction data to a resolution to 2.85 Å. The structure can be solved using molecular replacement (Rossman, 1972) when using variable and constant part of the Fab fragment as a separate research models, whereby oriented and positioned Fab fragments in the crystallographic asymmetric the units. In total, there were identified three Fab fragments in the asymmetric unit. After this three IgE ε3-ε4 molecules can be placed in the asymmetric unit. Each IgE dimer binds to two Fab molecules and, thus, in total, the asymmetric unit comprises one half of a full complex of IgE/Fab.

7.1.1 General description of the complex IgE Cε3-Cε4/Fab Antibodies 11

The crystal structure shows that each of IgE Cε3-Cε4 dimer is associated symmetric or asymmetric way with two Fab fragments (Figure 10). Since the asymmetric unit of the crystal contains one and a half IgE/Fab complex partial complex forms a dimer with symmetrically associated partner in the adjacent asymmetric unit with dual axes.

Both molecules IgE dimer, denoted as IgE1 and IgE2, interact with the Fab fragment of Antibody 11. The majority of interactions is provided by Fab heavy chain, which interacts with IgE1 and IgE2, while the light chain, as observed, interacts only with IgE1. The epitope of the antigen is mainly in the domain ε3, with the contribution of one amino acid, which is placed close to the hinge area of the domain ε4.

Three sites of interaction between IgE Cε3-Cε4 and Fab in the asymmetric unit of the crystal are very similar. However, after verification with the Alo clear one of the Fab molecules is much less ordered than the other two Fab molecules. It is usually observed in the crystal structures and due to the fact that a certain area is flexible and acquires a different orientation in the crystal so that the electron density is less certain. Such a Fab molecule and the interactions it has with IgE Cε3-Cε4 molecule, thus, not considered in this analysis.

IgE is known to be glycosylated at site Fc with the remainder Asn394 (Wurzburg and others).

Characterization of the glycosylation of Fc carried out using mass spectrometric analysis after digestion with trypsin showed three different ways glican associated with Asn394 consisting of cow patterns Man3GlcNAc2extension 2,3 or 4 hexoses, presumably all mannose (Figure 11). In fact, of the balance Asn394 in all three IgE ε3 domains enhanced electron density is distributed in the cavity between the two IgE molecules in the dimer. Electron density suggests vysokomernoy structure type with two N-acetylglucosamine (GlcNAc) and three or four Mennonite units, which are observed in each circuit, which is consistent with the data of mass-spectrometric analysis. Only one out of hexoses cow patterns, Man6, which is merged with Ma4, is visible in the electron density, which suggests that the rest of 1-3 hexose are flexible.

7.1.2 description of the epitope and paratope

This crystal structure allows you to explore epitope interaction between IgE ε3-ε4 and Fab with atomic detail. There are two independent interaction of IgE/Fab permitted structure, with the exception of the third Fab molecules because of its poorly defined electron density maps, which will be described later. They are very similar, as evidenced by the standard deviation between the two is equivalent to the Fab fragments of variable chain 031 Å, calculated using ε provisions (overlay, SR 1994) and between equivalent IgE monomers of 0.82 and 0.96 Å, respectively. Despite this high similarity of the two interactions will be described separately and will be referred to as IgE/Fab1 and IgE/Fab2. The details of the interactions are given in Table 5 and Table 6, where the number of the residue contains a pointer chain (HC: Fab heavy chain, LC: Fab light chain, IgE1, IgE2). The numbering of amino acid residues of the Antibody 11 is in accordance with the system Kabat (Kabat and others 1991). Distances were obtained using SSR program CONTACT (SSR, 1994).

Both involve interaction plots, complementarity determining (CDR) from both heavy and light chain fragm the NTA antibodies the remains of the frame area (the area outside of the CDR Fab) and amino acid residues of the two monomers in IgE ε3-ε4 monomer. Light chain of the antibody interacts with IgE1 in IgE ε3-ε4 the dimer, while the heavy chain interacts with both monomers. However, most of the contacts are between the heavy chain and the monomer IgE2 antigen. These two interactions are described in detail below.

7.1.3 Detailed description of the interaction between Fab1 and IgE, interaction 1

The interaction site determining the epitope of IgE ε3-ε4, covers an area of 1100 Å2(calculated using the program Areaimol, see the link SSR, 1994) and by using amino acid residues from Glu390 to Asn394, including for IgE1 and Leu340, Arg342, from AA to Thr434, inclusive Thr436, Ser437 and Glu472 in IgE2 antigen. In addition, the remains of sugar GlcNAc1 and Man6 in IgE1 and Man5 in IgE are in contact with the Fab molecules. Amino acid residues of the heavy chain that interact with the antigen, including sugar residues are those of the CDR1: Tyr32, CDR2: Asp53 and Asn54, CDR3: Val95, Met96, Ile100, Gly100b, Gly100c, Asp101 and Tyr102 and wireframe plot: Glu1, Lys23, Thr30, Ala71 to Arg77, inclusive, and Tyr79. The remains of carrying out the contribution from the light chain of the Fab are Asp50 and Ser56 of CDR2 and Leu 46 and Tyr49 of the frame section. Interaction includes 19 hydrogen bonds in addition to non-polar contacts van-der In which also.

7.1.4 Detailed description of the interaction between Fab1 and IgE, the interaction of 2

The interaction site determining the epitope of IgE ε3-ε4, covers an area of 1165 Å2(calculated using the program Areaimol, see the link SSR, 1994) and is carried out using the amino acid residues Glu390, from Gln392 to Asn394, inclusive IgE1 and Leu340, Arg342, from Ala428 to Thr434, inclusive Thr436, Ser437 and Glu472 in IgE2 antigen. In addition, the remains of sugar GlcNAc1 and Man6 in IgE1 are in contact with the heavy chain Fab. Amino acid residues of the heavy chain that interact with the antigen, including sugar residues are those of the CDR1: Tyr32, CDR2: Pro52a, Asp53 and Asn54, CDR3: Val95, Met96, Ile100, Gly100b, Gly100c, Asp101 and Tyr102, and from wireframe plot: Glu1, Lys19, Lys23, Thr30, Ala71 to Ser75, inclusive, Arg77 and Tyr79, and from wireframe plot: Glu1, Lys23, Thr30, Ala71 to Arg77 inclusive and Tyr79. The remains of carrying out the contribution from the light chain of the Fab are Ser56 of CDR2 and Tyr49 of the frame section. Interaction includes 19 hydrogen bonds in addition to non-polar contacts of the van der Waals forces.

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Table 5
Direct interaction between IgE ε3-Ce4 and Fab Antibodies 11, interaction 1
Chain FabThe remainder Fab The monomer IgEThe remainder of IgEDistance (Å)
Hydrogen bonds
NATyr 32 HEGlcNAc 1 O72,58
NAMet 96 OIgE1Asn 394 ND2is 3.08
NAGly 100b OIgE1Arg 393 NH12,60
NAGly 100C OIgE1Arg 393 NE2,80
NATyr 102 HEIgE1GlcNAc 1O6is 3.08
NAAsp 53 OIgE2Met 430 N2,83
NAAsp 53 OIgE2Arg 431 NH1to 2.67
NAAsp 53 OD1IgE2Arg 431 NH12,77
NAAsp 53 OD1IgE2Arg 431 NH22,60
NAAla 71 OIgE2Ser 432 OG2,64
NAAsp 72 OD2IgE2Arg 342 NH13,06
NAAsp 72 OD2IgE2Thr 434 N3,02
NAAsp 72 OD2IgE2Thr 434 O2,97
NAThr 73 OG1IgE2 Ser 432 N2,79
NAThr 73 OG1IgE2Ser 432 O3,05
NASer 74 NIgE2Ser 432 Ois 3.08
NASer 74 OGIgE2Arg 342 NH13,10
NASer 74 OGIgE2Thr 433 OG12,87
NAArg 77 NH1IgE2Glu 472 OE22,56
Nonpolar contacts <4Å
LCLeu 46IgE1Arg 3933,79
LCTyr 49IgE1Gln 3923,85
LCTyr 49IgE1Arg 3933,66
LCAsp 50IgE1Arg 3933,71
LCSer 56IgE1Glu 3903,47
LCSer 56IgE1Lys 391a 3.87
NAGlu 1IgE1Man 63,40
NAVal 95IgE1Arg 3933,49
NAIle 100IgE1Arg 3933,55
NAAsp 101IgE1 Arg 3933,50
NAMet 96IgE1GlcNAc 1to 3.67
NALys 23IgE2Glu 4723,43
NAThr 30IgE2Arg 4313,80
NAAsp 53IgE2Leu 4293,34
NAAsn 54IgE2Ala 4283,45
NAAsp 72IgE2Ser 4323,22
NAAsp 72IgE2Thr 4333,53
NAThr 73 IgE2Met 4303,98
NAThr 73IgE2Arg 4313.22
NASer 74IgE2Leu 3403,33
NASer 75IgE2Arg 3423,32
NAAsp 76IgE2Man 5with 3.27
NAArg 77IgE2Thr 4363,84
NATyr 79IgE2Thr 4363,43
NATyr 79IgE2Ser 437with 3.27
The distance from what your interaction, used for hydrogen bonds is 3,2Å, for nonpolar interactions - 4,0Å

Table 6
Direct interaction between IgE Cε3-Cε4 and Fab Antibodies 11, the interaction of 2
Chain FabThe remainder FabThe monomer IgEThe remainder of IgEDistance (Å)
Hydrogen bonds
LCSer 56 OGIgE1Glu 390 OE12,44
LCSer 56 OGIgE1Glu 390 OE22,99
NATyr 32 HEIgE1GlcNAc 1072,41
NA Gly 100b OIgE1Arg 393 NH22,58
NAGly 100c OIgE1Arg 393 NE3,01
NATyr 102 HEIgE1GlcNAc 1 O62,73
NAAsp 53 OIgE2Met 430 N2,74
NAAsp 53 OIgE2Arg 431 NH12.62
NAAsp 53 OD1IgE2Arg 431 NH12,88
NAAsp 53 OD1IgE2Arg 431 NH22,65
NA Ala 71 OIgE2Ser 432 OG2,87
NAAsp 72 OD1IgE2Ser 432 O3,13
NAAsp 72 OD2IgE2Arg 342 NH13,17
NAAsp 72 OD2IgE2Thr 434 O3,11
NAThr 73 OG1IgE2Ser 432 N2,94
NASer 74 NIgE2Ser 432 O3,17
NASer 74 OGIgE2Arg 342 NH13,00
NA Ser 74 OGIgE2Thr 433 OG12,77
NATyr 79 HEIgE2Ser 437 N3,09
Nonpolar contacts <4Å
LCTyr 49IgE1Gln 3923,76
LCTyr 49IgE1Arg 3933,63
LCSer 56IgE1Gln 3923,89
NAGlu 1IgE1Man 63,25
NAVal 95IgE1 Arg 3933,48
NAMet 96IgE1GlcNAc 13,68
NAMet 96IgE1Asn 3943,31
NAIle 100IgE1Arg 3933,44
NAAsp 101IgE1Arg 3933,81
NALys 19IgE2Ser 4373,53
NALys 23IgE2Glu 4723,70
NAThr 3 IgE2Arg 4313,88
NAPro 52aIgE2Met 4303,95
NAAsp 53IgE2Leu 4293,51
NAAsn 54IgE2Met 4303,90
NAAsn 54IgE2Ala 4283,47
NAAsp 72IgE2Thr 4333,61
NAThr 73IgE2Arg 4313,26
NASer 74IgE2Leu 3403,30
NASer 75IgE2Arg 3423,55
NAArg 77IgE2Thr 4363,88
NAArg 77IgE2Glu 472with 3.27
NATyr 79IgE2Thr 4363,37
Distance off communication used for hydrogen bonds is 3,2Å, for nonpolar interactions - 4,0Å

Materials and methods experiment 7

Overexpression Of IgE ε3-ε4.

Cell line and culture medium.

In this work we used the original adhesive cell line HEK293-EBNA (nvitrogen, Stockholm, Sweden), stably expressing nuclear antigen-1 of Epstein-Barr. Cells adapted to growth in suspension before transferring to the environment DHI using a phased replacement of the environment (Davies and others 2005). Used DHI environment differed slightly from the original description that was free of CA2+. After adaptation was created by the working cell Bank and both cell lines were grown in the usual way in the environment DHI-free CA2+, with the addition of 4 mm glutamine, 2% vol./about., IgG fetal calf serum ultra low molekulyarnym weight, 250 μg/ml G418 (all from Invitrogen, Stockholm, Sweden) and 0.1% wt./about. Pluronic F68 (Sigma-Aldrich, Stockholm, Sweden) up to a maximum of 20 passages.

The transfection procedure

Cooked in water mother solution 1 mg/ml linear 25 kDa of polyethylenimine (Polysciences Europe, Eppenheim, Germany), pH was brought to 7.0, sterile filtered and stored in the form of a small aliquot at a temperature of -80°C until use. Mixture for transfection were obtained immediately before transfection in the environment DHI without additives, equivalent to 1/10 transfection volume. To obtain a mixture for transfection DHI was divided into two halves. Was added 0.8 µg DNA per ml transfection volume to one half of the DHI environment, while the other half was added 2 μg PEI per ml transfection volume. After a brief shaking of the two solutions and incubating them for 5 minutes slowly when alali the DNA solution to a solution of PEI. Transfection the mixture incubated for 20-30 minutes at room temperature before making the wave bioreactor (Wave Biotech AG, Tagelswangen, Switzerland). 4 hours after transfection the culture was added to the volume for the final products containing DHI with additives and Durrer (Kerry Bio-science, Almere, the Netherlands) to obtain a final concentration of 0.3% (wt./vol.).

Culture for inoculation

For breeding crops for seeding cells were grown in plastic bottles for shaking at 37°C in an atmosphere of 5% CO2placed in a thermostat equipped with orbital shaker (Infers AG, Bottmingen, Switzerland). The cells were transplanted twice a week in the normal way when reaching a concentration of approximately 2×106cells/ml prior to separation. Density and cell viability was evaluated by using an automatic device for counting cells (Innovatis AG, Bielefeld, Germany). For the wave of culture the cells were divided to 1×106cells/ml one day before transfection to ensure that they are in the logarithmic growth phase at the beginning of the experiment. Wave culture was inoculable directly from the rapper. All cultures for inoculation were concentrated by centrifugation and resuspendable in fresh culture medium before addition to the bioreactors.

The wave of culture

The expression wasp is actulaly in wave bioreactors (Wave Biotech AG, Tagelswangen, Switzerland) with a working volume of 10 l Wave bioreactors were seeded to a density of 1×106cells/ml in 4.5 l of enriched DHI environment. 2 hours after implementation phases of cultural adaptation was transfusional 0.5 l mixture for transfection. 4 hours after tranfection culture fueled 10 l total volume enriched DHI environment and Durer to obtain a final concentration of 0.3% (wt./vol.). Daily taking samples to determine the density of cells, viability and protein concentration.

The expression vector of

The vector expressing human IgE Cε3-Cε4, with C-terminal Flag tag and a 10-his-tag tag received from the vector described Persic and others (1997). The system was under the control of the EF1-a promoter.

Purification of IgE Cε3-Cε4

20 l of cell supernatant five times concentrated and subjected diafiltration to 2xPBS (308 mm NaCl, 20 mm phosphate, pH 7.4) using a filter with a cross-flow cut-off molecular weight of 10 kDa (Pellicon 2, Millipore). Wednesday was subjected to binding in a total volume of 30 ml NiSepharose (GE Healthcare) for 2 hours at 4°C, washed with five volumes 2xPBS and loaded in a XK26 column. Then the column was washed with five column volumes with 40 mm imidazole in 2xPBS to flush contaminating proteins. In conclusion, IgE was suirable when using 400 mm imidazole in 2xPB. Combined fractions containing IgE with high degree of purity, concentrated approximately four times (up to ~5 mg/ml) before skipping through a column of Superdex 200 50/60 SEC (1200 ml, GE Healthcare) using 2xPBS as a rolling buffer. Separated some large proteins, and IgE were found in the main peak. Collected only a fraction of the main peak, as in other factions were present contamination. This step increased the purity of the sample to ~99%. The total number of product was 42 mg, and purified IgE has a concentration of 2 mg/ml

Analysis of glycosylation IgE ε3-ε4

Digestion in solution using trypsin

Human minimal domain of IgE, IgE ε3-ε4, 2 mg/ml, in 2xPBS (composition: 308 mm NaCl, 20 mm phosphate, pH 7.4) was mixed with 100 μl of trypsin 0.02 mg/ml in 25 mm NH4HCO3. The digestion was carried out overnight at 37°C and stopped him using 2 μl of formic acid (67%) in N2O.

Nano-LC MS/MS:

The analysis was carried out on a column of 20 cm x 50 μm (inner diameter) quartz capillary column filled with ReproSil-Pur C18-AQ porous particles with a size of 3 μm, associated with the LTQ-Orbitrap mass spectrometer (Thermo). Was carried out by injection of 8 μl of the sample (automatic sampler Agilent), and the peptides were subjected to capture on pre-column 4.5 cm x 100 μm (inner diameter )before separating. Che is ez 5 minutes linear run when using a 0.1% formic acid, the gradient was 10-50% acetonitrile within 5-30 minutes (Agilent), 200 nl/min and the eluent was subjected to electrorefining using the emitting tip. The device is operated in a dependent from data mode to switch between Orbitrap FT-MS) control scanning and ion trap (IT-MS/MS) of the three most excessively protonated ions.

Static elektrorazpredelenie MS/MS:

For checking a charged state glycopeptides fragments were subjected to analysis of selected precursors using ESI needle 1.6 kV, fragmentarily and defined in the device Orbitrap, oppositely directed to the linear ion trap, in nano-LC analysis.

Analysis of data in respect of glycopeptides:

Calculated MN+the mass of potential glycopeptides were analyzed for the presence of glycosylation using device GlycoMod (http:expasy.org/tools/glycomod) (Cooper and others 2001). Introduced protein sequence and the tolerance on weight for 10 frequent. in million All of the glycopeptides were examined for the presence of fragments containing glycan.

Getting Fab Antibodies 11.

Purification of IgG

The 11 antibody was purified from CHO-EBNA transition material when using MabSelect SuRe (GE Healthcare) chromatography medium with protein A. Replace the buffer in the protein a eluate on PBS, pH to 7.2 using PD-10 columns (GE Healthcare), and then filtered through a 0.22 μm filter when used in the research Institute Millex-GP syringe filter type (Millipore).

Digestion and purification of Fab

Preparing a buffer for digestion, containing 30 mm hydrochloride DL-cysteine dissolved in GIBCO PBS (Invitrogen). Papain, derived from papaya latex (Sigma), was restored in the buffer for digestion with obtaining a solution with a concentration of 10 mg/ml, which was kept at room temperature for at least 30 minutes before using. Added cysteine to IgG Antibodies 11 with a 30 mm solution then was added papain in the ratio of 1 mg of papain 100 mg of IgG. The digestion was stopped after 90 minutes by adding 0.5 M iodated (Sigma) with a 50 mm iodated in the final mixture for digestion. Fab was purified from the medium for digestion when using MabSelect SuRe (GE Healthcare) chromatography medium with protein And neisvaziuosiu mode. The Fab fraction obtained from MabSelect SuRe the stage was subjected to the shift buffer of 50 mm sodium acetate/100 mm NaCl, pH 5.5 using PD-10 columns (GE Healthcare) and subjected to concentration to ~10 mg/ml using Amicon Ultra-15 5 kDa MWCO devices for filtering in a centrifuge (Millipore). The final product was additionally purified using Mustang Q acrodisc (Pall) and then filtered through a 0.22 μm filter using Millex-GP syringe filter type (Millipore).

Obtaining a complex of IgE Cε3-Cε4 Fab Antibodies 11

A solution containing 2 mg/ml IgE Cε3-Cε4 the office 308 mm NaCl and 20 mm phosphate, pH 7.4, was mixed with a solution containing 10,6 mg/ml Fab Antibodies 11 in 50 mm sodium acetate, 100 mm NaCl, pH 5.5 at a stoichiometric ratio of 1 to 1.1 IgE Fc3-4-glycosilated and heterodimer Fab Antibodies 11, respectively. The mixture was left on ice overnight, and then was carried out by gel filtration on a HiLoad Superdex200 16/60 column (GE Healthcare), equilibrated with 20 mm Tris HCl pH 7.6 and 0.15 M NaCl. Collected the main peak containing the complex, and concentrated to 10.4 mg/ml prior to use in experiments on crystallization.

Crystallization of the complex of IgE Cε3-Cε4 Fab Antibodies 11.

Crystallization was carried out in accordance with the method drop vapor diffusion. Drops containing equal amount of protein and solution tanks (150+150 ll) were placed in the Crystal tablet Quick on 96 wells (Greiner Bio-one) with the volume of the tank 80 ál. The complex crystals were grown in a drop of solution capacity 100 mm MgCl2, 100 mm sodium citrate pH 5.0 and 15% PEG 4000 in a time period of 2-3 weeks at 4°C. the Crystals were collected in a cryoprotective solution (100 mm MgCl2, 100 mm sodium citrate pH 5.0 and 15% PEG 4000, which is brought up to a content of 20% glycerol by adding 100% glycerol) and was slowly cooled with liquid nitrogen.

Data collection and structure solution of the complex of IgE Cε3-Cε4 Fab Antibodies 11.

Collected diffraction data from single crystals at the European laboratory synchrotron is wow radiation (ESRF) in Grenoble using a flat beam ID-29. The original dataset (dataset 1, table 7) were recorded to a resolution of 3 Å, later collected data at higher resolution to 2.85 Å, both belonged to space group P3221. The data were processed using autoPROC (Global Phasing Limited GPhL, Cambridge, UK). Statistical treatment of the data presented in Table 7. The asymmetric unit contains three Fab molecule and three molecules of IgE Cε3-Cε4, which corresponds to a solvent content of 54 %. The structure was solved with molecular replacement using the program PHASER (Read, 2001, Storoni and others 2004, MSO and others 2005). The original model for the Fab fragment of IgE and Fc domain were obtained from the previously described structures 1AQK (Faber and others 1998) and 1FP5 (Wurzburg and others 2000).

The whole Fab as a model study was unsuccessful due to variations of the angles in the hinge area between the variable domain and a constant domain. Instead, was prepared and identified in the initial run PHASER two separate research model consisting of the variable domains and constant domains, respectively (Read 2001, Storoni and others 2004, MSO and others 2005). This last was added until the completion of Fab fragments. A total of two Fab fragments and one variable domain identified in this way. Subsequent runs of molecular substitution placed three IgE Cε3-Cε4 molecules, one to whom that should again be shortened to enable a single domain 4. At this stage, data were collected best quality (dataset 2, table 7), and this model was optimized with the new data using the program autoBUSTER (Global PHMeering Limited GPhL, Cambridge, UK). Further amino acids Fab molecules were subjected to change in manual mode to adjust the sequence of the Antibody 11 when using the graphics program COOT (Emsley &Cowtan 2004). After the second cycle limited maximum plausible optimization using isotropic factors In optimization in Refmac (CCP4 1994) the remaining domains of the latest Fab and IgE molecules manually built into the electron density. Two additional characteristic of the enhanced electron density was observed protruding from the amino acid residue Asn394 in the cavity between IgE molecules. This was interpreted as glycosylation and, thus, was added N-acetylglucosamine and three to four units of mannose to models of IgE. Additional cycles of optimization included the building in manual mode plots loops in COOT (Emsley &Cowtan 2004), carried out by optimizing either in autoBUSTER (Global Phasing Limited GPhL, Cambridge, UK)or in Refmac5 (Murshudov and others 1997) when using TLS for individual domains and limitations cristallografia symmetry (NCS) for IgE molecules. In total were built 213 water by using the option of taking water in Refmac5 (Murshudov and the R. 1997), after which it was carried out by visual inspection. In the final cycle optimization eliminated NCS constraints that led to obtaining the final model with R=20.0 % and R.=27,0%.

Table 7
Crystal parameters and data processing x-ray and optimization statistics
The array data 1The array data 2
Space groupP3221P3221
Wavelength (Å)0,9760,976
Cell constant a (Å)140,62141,562
b (Å)140,62141,562
c (Å)244,65245,562
Interval resolution (Å)2,93-35,162,85-109,11
The optimal resolution of the frame (Å)2,93-3,01 2,85 of 2.92
Overall completeness (%)99,9100,0
The completeness of the optimal frame (%)100,0100,0
Image, unique6060166338
The ratio of provisions6,610.7
The multiplicity of the provisions of the optimal frame6,811,0
Rtotal(%)1of 0.1330,099
R optimal frame (%)0,9140,75
The average value (I)/Wed off. (I)13,120,2
The average value (I)/Wed off. (I) the optimal frame2,03,4
R.total(%)2do not use20,0
R.free(%) do not use27,0
1 Rthe mergerhkl[(Σi|Ii-<I>|)/ΣiIi]
2 Rvaluehkl||FOBS.|-|Fposcit.||/Σhkl|FOBS.|
Rthe loose.represents the cross-validation R factor computed for the analyzed set of 5 % of unique images

References cited in Example 7

Davies A. Greene, A. Lullau E. Abbott WM. Optimisation and evaluation of a high-throughput mammalian protein expression system. Protein Expression &Purification. 42(1):111-21. (2005).

CCP4 (Collaborative Computational Project, Number 4) (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50:760-763.

Cooper, C.A., Gasteiger E., Packer N.

GlycoMod - A software Tool for Determining Glycosylation Compositions from Mass.

Spectrometric Data.

Proteomics 1:340-349 (2001).

Emsley, P. &Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D60:2126-2132 (2004).

Faber, C., Shan, L., Fan, Z., Guddat, L.W., Furebring, C., Ohlin, M., Borrebaeck, C.A., Edmundson, A.B. Three-dimensional structure of a human Fab with high affinity for tetanus toxoid. Immunotechnology: 253-270 (1998).

Kabat, E.A., Wu, T.T., Perry, H., Gottesman, K. and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. NIH Publication No. 91-3242.

Karpusas, M., Lucci, J., Ferrante, J., Benjamin, C., Taylor, F.R., Strauch, K., Garber, E., Hsu, Y.M. (2001) Structure ofCD40 ligand in complex with the Fab fragment of a humanized neutralizing antibody. Structure 9, 321, (2001).

Leslie A. (1991) Macromolecular data processing. In Moras, D., Podjarny, A.D. and Thierry, J.C. (eds), Crystallographic Computing V. Oxford Universty Press, Oxford, UK, pp.27-38.

McCoy, A.J., Grosse-Kunstleve, R.W., Storoni, L.C. & Read, R.J. Likelihood-enhanced fast translation functions. Acta Cryst D61, 458-464 (2005).

Murshudov, G.N., Vagin, A. A., and Dodson, E.J. (1997) Refinement of Macromolecular structures by the maximum-likelihood method, Acta Crystallogr D53:240-255.

Padavattan, S., Schirmer, T., Schmidt, M., Akdis, C., Valenta, R., Mittermann, I., Soldatova, L., Slater, J., Mueller, U. & Markovic-Housley, Z. Identification of a B-cell an epitope of Hyaluronidase, a Major Bee Venom Allergen, from its Crystal Structure in Complex with a Specific Fab. J Mol Biol 368, 742-752. (2007).

Persic L. Roberts A. Wilton J. Cattaneo A. Bradbury A. Hoogenboom HR. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene. 187(1):9-18. (1997).

Read, R.J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Cryst. D57, 1373-1382 (2001).

Rossmann, M.G. (edt): "The Molecular Replacement Method", Gordon &Breach, New York (1972).

Storoni, L.C., McCoy, A.J. & Read, R.J. Likelihood-enhanced fast rotation functions. Acta Cryst D60, 432-438 (2004).

Wurzburg, B.A., Garman, S.C. and Jerdetzky, H.S. Structure of the human IgE-Fc epsilon With 3-C epsilon 4 reveals conformational flexibility in the antibody effector domains. Immunity, 13, 375-385 (2000).

Example 8. Assessment of the common security and ability harmonisierung anti-IgE mAb to induce a reduction in the number of platelets in immature animals monkeys, cynomolgus

Studying (not appropriate GLP) study was carried out in immature monkeys, cynomolgus to assess the overall safety and the relative ability of the antibodies in accordance with the invention, an anti-IgE mAb, 11 Antibodies IgG1Antibodies 11 IgG2and another anti-IgE antibodies E to cause a decrease in the number thrombocyto the blood.

The objectives of this study were: 1) to determine the overall safety and relative abilities of the three candidate anti-IgE mAb to induce a reduction in the number of platelets/TCP and associated effects in immature monkeys, cynomolgus; 2) to determine the preliminary pharmacokinetic parameters for mAb in monkeys; 3) to evaluate the ability of three candidate mAb to cause a decrease in the level of free IgE and determine (PK/PD) relationship between the concentration of mAb and levels of free IgE.

Materials and methods of Example 8

Eighteen bred for this purpose monkeys, cynomolgus (Masasa fascicularis) received from Bioculture, Mauritius. Animals were age from 63 to 67 weeks at the beginning doses. Monkeys were subjected to pre-selection (from a large pool consisting of 100 animals) in the presence of high levels of IgE (units/ml) and were divided into 3 groups, each of which contained 3 males and 3 female monkeys who received 11 Antibody IgG1(Group 1), 11 Antibody IgG2(Group 2) or E (Group 3). Each of the 3 mAb receptively in 50 mm sodium acetate, 100 mm NaCl, pH 5.5 and introduced animals in the dose volume 2 ml/kg by slow intravenous injection (using mechanized syringe/pump for infusion) at the speed of 1 ml/min

Animals received a dose once a week (for 5 weeks/5 doses) with increasing UB is, gives doses of 1 mg/kg, 30 mg/kg and 100 mg/kg (×3) on Days 1, 8, 16, 22 and 29 (table 8). Additional doses of 11 Antibodies IgG1and 11 Antibodies IgG2was administered to Groups 1 and 2, respectively, at Day 37. 2 the most high doses were theoretically defined as such, which can lead to concentrations in serum, which were previously shown as leading to thrombocytopenia (TCP) with Xolair immature monkeys, cynomolgus. Low dose, as expected, was it possible to determine the ability of the mAb to cause reduced levels of IgE.

Table 8
The resulting model studies
GroupDescriptionLevel dose (mg/kg/day) on Day:The number of animals
1816222937MalesFemale
1Ab 11 IgG1130 10010010010033
2Ab 11 IgG213010010010010033
3E48130100100100-33
(Ab 11 = 11 Antibody)

The animals were followed for 8 weeks after a dose at Day 28 and searched for a cure from any Toxicological effects

All animals were subjected to daily examination for signs of discomfort or obvious toxicity, and recorded the body weight and food consumption. In addition, each animal underwent a thorough physical examination daily during periods of dose and at least for one week during periods without the introduction of the eskers. All animals were also subjected to surveillance prior to each dose, and through 0, 5, 2, 6, 24, 48 and 168 hours after administration of the dose.

Blood samples for analysis of standard haematological parameters (including the number of platelets collected in EDTA) and coagulation parameters (collected in trisodium citrate) was taken from the femoral vein/artery twice during pre-treatment (weeks -2 and -1). Additional samples for counting platelets and standard Hematology were collected after 24 hours and 144 hours after each dose (Days 2, 7, 9, 14, 17, 22, 23, 26, 30 and 35; samples from Groups 1 and 2 only in the Days of 38 and 43) and every 2 weeks during the 8-week recovery period (Days 43, 57, 71 and 82). Samples for the determination of coagulation collected through 144 hours after each dose (Days 7, 14, 22, 26 and 35) and at the end of the recovery period (Day 82). Samples for the determination of coagulation were also collected at Day 57. Blood samples for activation of the compliment (Sa, C3b and BB fragments) were taken once during pre-treatment(week -1) and approximately 24 hours after completion of the treatment period (Day 30).

Serum samples for TK analysis were taken from all three groups on Day 1 before the introduction of the dose through 0, 5, 6, 12, 24, 48, 144 hours after the dose, on Days 8, 16, 22 and 29 after 0, 5, 24 and 144 hours after administration of the dose, Day 29 (only Groups 3 and 4) through 336, 672, 1008, 1272 hours after the dose, in the Yan 37 (only Group 1 and 2) through 0, 5, 24, 144, 480, 816, 1080 hours after a dose. The samples were analyzed for mAb using generic sandwich immunoassay (using biotinylated human IgE for mAb and labeled l-647 mouse anti-human IgG agent for definitions) and Gyrolab Bioaffy equipment (including speakers with beads streptavidin). Additional samples for analysis of IgE were collected on Day 1 before the introduction of the dose through 0, 5, 6, 12, 24, 48, 144 hours after the dose, on Days 8, 16, 22 and 29 after 0, 5, 144 hours post dose and at study end (Day 82) through 1272 hours (Groups 3 and 4) or 1080 hours (Groups 1 and 2) after administration of the final dose. Samples were analyzed on a free IgE using immunoassay using the ImmunoCap system (Phadia AB, Uppsala, Sweden) using human IgG-FcεRIa to capture free IgE and anti-human IgE rabbit (PCS-conjugate) for definitions.

At the end of the experiment at Day 85 exercised full macroscopic examination under the General supervision of pathomorphology and recorded all of the damage. Determined the absolute weight of the bodies and the ratio of the body weight: the body weight. Tissue from a number of bodies collected and stored in frozen form, but has not carried out any microscopic studies (except for macroscopic abnormalities or unexpected death, see above.

The results for Example 8

Observations in relation to General security

All 3 mAb were well-tolerated in the absence of any clinical signs of illness throughout the study, except for one animal treated with the Antibody 11, which was sent for autopsy prematurely due to a General deterioration in clinical status and reduced body weight. Because the status of this animal has deteriorated during the recovery period and there were no data during pathological and hematological studies of these animals, the observed effects are not expected as such, are bound mAb. In all groups were cases of soft or liquid faeces, however, because these observations were not associated with dose and was not observed in all animals or all of the time points in the same animal, as well as observed many times during the period of dose and recovery periods, it is unlikely that they are related to the mAb. The average body weight and average weight gain of the body showed some individual variation among animals within each group during the whole study period. However, all animals gained weight as expected, during the treatment period (with the exception of one animal, which was discussed above) and was not edogo differences between groups. Was noted to have distinct effects associated with treatment, in terms of absolute or relative weights of organs in any of the groups. In General pathological examination and microscopic examination of the pathology found no observations in a limited range of tissues examined, which would give the opportunity to suggest any effect of mAb treatment.

Toxicokinetic (TK) and the levels of IgE

Not observed-dependent sex differences in TK in this study. In the General case, the effect was similar for these 3 mAb and was linear in the range of doses of 1-100 mg/kg Average value profiles TK Antibody 11 IgG1Antibodies 11 IgG2and E shown in Figure 12. Did not observe an obvious impact of the falling levels of IgE on TK, even at very low doses. TK these 3 antibodies were typical of human IgG in monkeys, cynomolgus.

The average value of the maximum observed concentration (Cmax) after administration of the last dose of 100 mg/kg was 18700, 15900 and 24000 nm for 11 Antibodies IgG1Antibodies 11 IgG2and E respectively. The average value of the final half-life for TK after the last dose of 100 mg/kg was approximately 10-13 days. It has not been confirm reduced TK impact due to the potential development of anti-human antibodies of the primacy of these animals.

CPE is the profile values of free IgE after weekly dose of 11 Antibodies IgG 1Antibodies 11 IgG2and E at different dose levels in monkeys, cynomolgus shown in Figure 13. Average baseline IgE before the animals received the first dose was 514, 414 and 690 ng/ml for groups receiving 11 Antibody IgG1, 11 antibody IgG2and E respectively. On Day 1 dose 1 mg/kg induced 75-80% reduction in the level of free IgE in 1 hour after a dose. Because of the low impact after a dose of 1 mg/kg level of free IgE returned to baseline within 1 week. Higher doses lead to permanent suppression of the level of free IgE during the treatment period. The level of free IgE returned to baseline for the two groups 2 Antibodies 11 at the end of the study, while the level of free IgE in A group remained supression.

Effects on platelets

None of the 3 mAb (Antibody 11 IgG1, 11 antibody IgG2and E) not induced a significant decrease in the number of platelets in any time in any animal, except for one animal that received 11 Antibody IgG1, which had a low blood platelet count (34,9%) in a single point in time Day 29 (24 hours after a dose of 100 mg/kg / Day 28). The fourth dose of 11 Antibodies IgG1and 11 Antibodies IgG2Day 37 not induced any complement the form of further reducing the level of platelets in this or other animal in these 2 groups.

Figure 14 shows a chart of the number of platelets (×109/l), expressed as a percentage difference from the mean of the two pre-dose versus concentration differences from animals in Gruppe 1 (treated with Antibody 11 IgG1). This chart is typical of 16 animals from 3 groups that did not show a significant influence on platelets [change for the animal group Antibody 11]. Figure 15 shows a similar diagram for the animals of Group 1 (treated with 11 Antibody IgG1), which showed a temporary significant reduction (35% below the baseline) in the number of platelets on Day 29.

It is interesting to note that monkeys treated with Antibody 11 IgG1that showed a temporary significant reduction in the platelet count after the dose on Day 29 had the highest value Cmax (29400 nmol/l) (but not exposure) at this time. Levels in plasma consistently fell sharply, and the platelet count returned to the levels observed before the introduction of the prior dose. This indicates a possibility that may require a higher threshold concentration in the blood plasma to induce a reduction in the number of platelets. However, one animal treated E, reached similar levels (28500 nm/l) in the absence of the tvii corresponding effect on platelets (Figure 14).

Other hematological effects

Except for platelet count (see below), did not report the impact of treatment with mAb for most hematological parameters (hemoglobin concentration, hematocrit, mean volume of erythrocytes, the average amount of hemoglobin in the erythrocytes distribution width red cell volumes, thrombocytosis, distribution width platelet volumes, number of erythrocytes, the average amount of hemoglobin in the erythrocyte, the width of the distribution of hemoglobin, the average volume of the platelet, reticulocyte count, total and differential WBC) and parameters of blood coagulation (prothrombin time, activated partial thromboplastin time). The increase in the number of reticulocytes was observed in all groups, but the changes were not associated with the relationship dose/impact have not been agreed within the group (animals in the group had a higher, lower or unchanged levels compared with the values of the period before the introduction of doses) or from the same animal (values for the same animal was moving up and down between different points in time regardless of the impact), and in the absence of a concurrent control group relationship with mAb treatment could not be fully settled Tue determined at this time. Any of these changes had a reverse development by the end of the recovery period. Not noted any significant effects of treatment on the activation of complement (Sa, Sa or BB fragments).

Discussion and conclusions

The study showed that anti-IgE mAb, 11 Antibody IgG1, 11 antibody IgG2and I were well-tolerated with the introduction of high levels of repeated doses (up to 100 mg/kg) immature monkeys, cynomolgus in the absence of reliable harmful Toxicological effects. Only one animal of 18 monkeys showed a decrease in the number of platelets in the only moment of time after injection of 100 mg/kg of Antibody 11 IgG1when the mAb concentration in plasma was reached almost 30,000 nmol/L. Values Cmax concentrations in plasma, which was achieved by using all 3 mAb in this study were much greater than those expected to be achieved in clinical use (for example, 200 nmol/l).

Example 9. Functional inhibition effect of IgE to FcεRI and CD23

The ability of optimized Antibodies 11 to functionally inhibit the interaction of IgE to FcεRI and CD23 were evaluated in IgE mediated analysis of cell killing, adapted compared with Bracher and others (Journal Immunol. Methods 2007 323:160-171). U937, pre-treated with IL-4, were shown the AK such which produce both FcεRI and CD23. By co-cultivation with tumor cells of the ovary IGROV1 in the presence of IgE specific for the antigen, which is expressed on IGROV1 cells, U937 cells were able to kill tumor cells. This destruction was mediated by mechanisms of cytotoxicity and phagocytosis, which has been shown to be triggered by interaction of IgE to FcεRI and CD23, respectively, on U937 effector cells.

The 11 antibody and isotype control were evaluated in this analysis for the inhibition mediated destruction with or FcεRI, or CD23. Detailed recipe for this procedure is provided in section "Materials and methods". Briefly, titration investigated IgG were mixed with target-specific (MOv18) or irrelevant (NIP) IgE before incubation with U937 effector cells stimulated with IL-4, and labeled IGROV1 target cells. After 2.5 hours of incubation were washed cells were stained with anti-CD89-phycoerythrin antibody (BD Biosciences) and iodide of propecia (Molecular Probes). After washing, the fluorescence of the cells were analyzed using FACSCalibur flow cytometer (BD Biosciences). Fluorescent dyes mentioned above are used for the separation of living cells, cells, destroyed with the help of cytotoxicity, and the cells are destroyed by phagocytosis. Unlike and atopicheskogo control antibodies, the 11 antibody is able to inhibit mediated IgE/FcεRI cytotoxicity (Figure 16) and mediated by IgE/CD23-phagocytosis (Figure 17).

Materials and methods - Example 9

Antibodies were evaluated for inhibition of IgE mediated destruction of tumor cells IGROV1 using U937 cells. IGROV1 cells (cell line carcinoma of the ovary) and U937 cells (myelomonocytic cell line) was maintained in culture medium [RPMI1640,10% vol./about. FCS, 2 mm glutamine, 5000 units/ml penicillin, 100 μg/ml streptomycin (all from Invitrogen)] using standard procedures tissue culture. MOv18 IgE directed against FBP (linking folate protein), which is expressed on IGROV1 cells, used as a specific for tumor antibodies. NIP (the hapten 4-hydroxy-3-nitrophenacyl)-specific IgE was used as an irrelevant control antibody. MOv18 and NIP antibodies were obtained as described above in Gould and others, (1999) Eur. J. Immunol. 29:3527-3537.

U937 cells pre-treated for 4 days before the experiment on the elimination of cells with 10 ng/ml recombinant human IL-4 (R&D Systems) in order to increase the expression of CD23. The day before the experiment, the destruction of IGROV1 cells target cells were labeled with fluorescent dye CFSE (carboxyfluorescein diacetate Succinimidyl Esther. Moleular Probes). Briefly, cells were subjected to trypsinization (trypsin/EDTA, Gibco), washed in culture medium and resuspendable in PBS at a concentration of 50×106cells/ml Then the cells were incubated at 37°C for 10 minutes with CFSE at a concentration of 0.01 mm. After labeling, the cells were washed once in ice-cold culture medium and then incubated over night at 37°C, 5% CO2.

To assess the inhibitory effect of Antibody 11 received dilution of antibody in the test tubes (12×75 mm (Falcon, BD Biosciences) and 2 µg MOv1 or NIP IgE was added to obtain a final volume of 80 μl. This mixture is incubated without cells within 30 minutes. Stimulated with IL-4 U937 cells were once washed in the environment and resuspendable at a concentration of 1.33×106cells/ml CFSE Labeled IGROV1 cells were trypsinization, once washed in the environment and resuspendable at a concentration of 4×105cells/ml cells were Added to tubes containing antibodies (120 μl for U937 cells and 200 μl for IGROV1 cells), mixed and incubated for 2.5 hours at 37°C, 5% CO2. Then the cells were washed in ice-cold FACS buffer (PBS, free from calcium/magnesium, 5% goat serum) and incubated for 25 minutes with anti-CD89-phycoerythrin antibody (BD Biosciences, 10 μg/ml) for tagging U937 effector cells. Cells were again washed in ice-cold FACS buffer and dead cells were stained put the m addition of 0.25 μg/ml of iodide of propecia (Molecular Probes). After 15 minutes at 4°C cells were washed degalim FACS buffer, resuspendable in 250 μl of ice-cold FACS buffer and were analyzed by fluorescence using FACSCalibur flow device for counting cells (BD Biosciences) according to manufacturer's instructions. Cells with relevant monotonous staining was used to establish the voltage and compensation definition channels (FL1, FL2 and FL3). The combination of the dyes used in this analysis allowed the selection of populations of different cells [live effector cells (positive for phycoerythrin), favoritemovie IGROV1 tumor cells (positive for phycoerythrin and CFSE), live tumor cells (positive for CFSE), dead tumor cells (positive for CFSE and iodide of propecia), dead effector cells (positive for phycoerythrin and iodide of propecia)]. This selection was used to calculate the target cells destroyed by FcεRI-mediated cytotoxicity (equation 1) and mediated by CD23 phagocytosis (equation 2).

Equation 1: %cytotoxicity={[(R1SL-R1)+R3]/R1SL}×100

where R1 = the total number of CFSE positive tumor cells

R3 = number destroyed, but intact tumor cells (without fragmentation or phagocytosis)

R1SL control = Average R1 3 control samples of effector and target cells is to without antibodies (R1 control spontaneous death).

Equation 2: %phagocytosis=(R2/R1SL)×100

where R2 = the number of tumor cells, phagocytosing effector cells

R1SL control = Average R1 3 control samples of effector and target cells without antibody (R1 control spontaneous death).

Links

All references cited anywhere in this description, including those cited elsewhere above, is introduced in this application as references in their entirety and for all purposes.

Table 1b
Kabat numberingHCDR1HCDR2HCDR3
313233343550515252A535455565758596061 626364659596979899100100A100B100C100D101102
DYNIYLIDPDNGETPYAEKFQGVMGKWIK GGYDY
Antibody 25tLSAnd
Antibody 21 tLSAnd
Antibody 7 tLSAnd
Antibody 18LL/td> RSAnd
Antibody 11 PGL
Antibodies the 22 TLSAnd
Antibody 16 SLSAnd
Antibody 26 SLEAnd
Antibody 2SLRSAnd
Antibody 10SLRAAnd
Antibody 1 LHRRL
Antibody 8 GL LR
Antibody 24TL TAnd
Antibody 15TLTAnd
Antibody 8 PGL LR

1. An isolated antibody or antigennegative fragment specific(s) to human immunoglobulin E, where the aforementioned antibody or antigennegative fragment includes
a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where in which:
HCDR1 has amino acid sequence SEQ ID NO: 103;
HCDR2 has aminokislotnoi sequence of SEQ ID NO: 104;
HCDR3 has amino acid sequence SEQ ID NO: 105;
LCDR1 has amino acid sequence SEQ ID NO: 108;
LCDR2 has amino acid sequence SEQ ID NO: 109;
LCDR3 has amino acid sequence SEQ ID NO: 110.

2. An isolated antibody or antigennegative fragment specific(s) to human immunoglobulin E, an antibody molecule includes a set of CDRs: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, in which:
HCDR1 has amino acid sequence SEQ ID NO: 103;
HCDR2 has amino acid sequence SEQ ID NO: 104;
HCDR3 has amino acid sequence SEQ ID NO: 105, where SEQ ID NO: 105 may include one or more of the following substitutions: M in position 2 is replaced by L, S, or T; G at position 3 replaced by L or H; K at position 4 is replaced by R; W in position 5 is replaced by E, S, A, R or T and I at position 6 is replaced And or L;
LCDR1 has amino acid sequence SEQ ID NO: 108;
LCDR2 has amino acid sequence SEQ ID NO: 109;
LCDR3 has amino acid sequence SEQ ID NO: 110 where SEQ ID NO: 110 may include one or more of the following substitutions P in position 6 T, S, N, R, B, Q, D, or E; T at position 7 is replaced by L, K, S, I, G, H, M, F, R, Q or N; L at position 8 is replaced by S, N, D, R, N, or G; T at position 9 is replaced by N, S, Y, L, N, or G; S at position 10 is replaced by a G or a and P at position 11 is replaced by S, L or G.

3. An isolated antibody or antigennegative frag the UNT according to claim 1 or 2, where the specified antibody or antigennegative fragment specific(EN) for immunoglobulin E and binds to the epitope that includes elements from the first heavy chain of IgE and the elements of the second heavy chain of IgE.

4. An isolated antibody or antigennegative fragment according to claim 3, where the aforementioned antibody or antigennegative fragment specific(EN) for immunoglobulin E and binds to the epitope in the immunoglobulin E, including:
the remains of Glu390 to Asn394, inclusive, in the first heavy chain of IgE and Leu340, Arg342, from AA to Thr434, inclusive, Thr436, Ser437 and Glu472 second heavy chain of IgE.

5. An isolated antibody or antigennegative fragment specific(s) for immunoglobulin E in accordance with claim 4, where the epitope further includes the remains of the sugar GlcNAcl and Man6 first heavy chain of IgE and the remainder of the sugar Man 5 second heavy chain of IgE.

6. An isolated antibody or antigennegative fragment according to claim 3, where the aforementioned antibody or antigennegative fragment specific(EN) for immunoglobulin E and binds to the epitope in the immunoglobulin E, including:
the remains Glu390, from Gln392 to Asn394, inclusive, in the first heavy chain of IgE and Leu340, Arg342, from l428 to Thr434, inclusive, Thr436, Ser437 and Glu472 second heavy chain of IgE.

7. An isolated antibody or antigennegative fragment specific(s) for immunoglobu is on E in accordance with claim 5, where the epitope further includes the remains of the sugar GlcNAcl and Man6 first heavy chain of IgE.

8. The antibody according to any one of claims 1, 2 and 3-7, where the connecting element is a monoclonal antibody.

9. An isolated nucleic acid molecule encoding an isolated antibody or antigennegative fragment according to any one of claims 1, 2 and 3-8.

10. Host cell transformed with the nucleic acid molecule in accordance with claim 9 for obtaining antibodies or antigennegative fragment according to claims 1, 2 and 3-8.

11. The method of obtaining an isolated antibody or antigennegative fragment according to any one of claims 1, 2 and 3-8 for the treatment of disorders associated with IgE, comprising culturing the host cell of claim 10 under conditions suitable for production of the indicated antibodies or antigennegative fragment.

12. Pharmaceutical composition for treating disorders associated with IgE, comprising the antibody or antigennegative fragment according to any one of claims 1, 2 and 3-8 and pharmaceutically acceptable filler.

13. The pharmaceutical composition according to item 12, including:
the antibody or antigennegative fragment according to claims 1, 2 and 3-8
20 mm succinate
105 mm NaCl
10 mm arginine
pH 6,00.

14. The composition according to item 12 or 13 for use as a medicine.

15. The use of a composition according to 14 for l the treatment of disorders associated with IgE.

16. The use of a composition according to item 12 or 13, where the disorder is one or more of allergies, asthma and bronchitis.

17. The use of a composition according to item 12 or 13, where the disorder is one or more of allergic rhinitis, allergic contact dermatitis, atopic dermatitis, anaphylaxis, food Allergy, urticaria, inflammatory bowel disease, eosinophilic gastroenteritis induced drug rash, allergic ophthalmopathy, allergic conjunctivitis, bronchial asthma, hypersensitivity of the respiratory tract, cosmetic Allergy, Allergy-induced drugs, hypersensitivity syndrome induced by drugs, allergies to metals, professional hypersensitive pneumonitis, chronic hypersensitive pneumonitis, cold hypersensitivity, hypersensitivity induced by helminths, latex allergies and allergies the pollen.



 

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