Recombinant il4 antibodies used for treating disorders associated with the activity il4

 

(57) Abstract:

The invention relates to biotechnology, in particular to recombinant IL4-antibodies used for treating disorders associated with the effect of IL4. The invention includes chimeric and humanized IL4 Mab-antibodies that are derived from highly b antibodies, as well as containing pharmaceutical compositions and methods of diagnosis and treatment. The advantage of the invention is to create a high-affinity IL4-antagonist, is able to reduce the level of eosinophilic inflammation. 22 C. and 3 h.p. f-crystals, 10 ill., 3 table.

This request is partially continued by the application regarding the application of U.S. serial number 08/136,783 dated October 14, 1993, which is a continuation of application U.S. serial number 08/117.366 dated September 7, 1993, and on both of these applications are referred to in the present invention.

The present invention mainly relates to the field of fused proteins, and proteins used for the treatment and diagnosis of pathological States associated with the action of IL 4 and excess production of IgE, more specifically the present invention relates to chimeric and humanized IL 4 antibodies.

Altacee as seasonal rhinitis and conjunctivitis, and more serious diseases such as atopic dermatitis and atopic asthma, as well as such diseases, life-threatening, as anaphylactic shock. The common factor of these painful conditions is the body's immune response to allergens, and this reaction is the production of immunoglobulin E (IgE) antibodies in genetically predisposed patients (atopy). Inhibition of IgE production in a long time was the purpose of specific immunotherapy of allergic diseases using desensitizing vaccines. However, in recent years the safety and effectiveness of vaccine therapy began to be questioned, however, the need of lowering IgE levels did not fall.

Interleukin 4 (IL 4) is a protein mediator in the lymphoid system. Studies of lymphocytes from atopic patients revealed an increased number of T-lymphocytes, which are able to secrete IL 4 in response to stimulation and increased amounts of IL 4, secreted after stimulation.

It was found that anti-IL 4 antibody inhibits IgE, but not IgG inhibits1or IgG2a/Finkelman with al., Ann. Rev. Immunol. 8:303(1990)/, as well as products in order to influence the accumulation of eosinophil in the tissues. See, for example, Tepper with TCS. Cell, 62:457(1990); Tepper with TCS. Cell, 57; 503(1989).

In the art at the present time, there remains a need in creating a high-affinity IL 4 antagonist, is able to reduce the level casinopalace inflammation by decreasing proliferation IL 5 secreting cells and inhibition mechanism of adhesion, with the result that Sosnoviy could accumulate in the tissues and used for the treatment, prevention and diagnosis of allergic reactions.

In accordance with the first aspect of the present invention provides a protein having affinity for binding to the interleukin-4, which contains plots complementarity determining (CDR) inhuman neutralizing monoclonal antibodies (Mab) characterized by a dissociation constant equal to or less 210-10M for human IL 4, and the first partner of the merger, in which at least one and preferably all areas, complementarity determining (CDR) are replaced with CDR of the monoclonal antibody nonhuman origin (Mab). Such neutralizing monoclonal antibody of nonhuman origin can be selected from the group consisting of V and 6A1, sootvetstvenno associated with the second fused protein, including all or part of the immunoglobulin constant chain.

In accordance with a related aspect the present invention provides CDR neutralizing monoclonal antibodies of human origin (Mab) characterized by a dissociation constant equal to or less 210-10M for human IL 4 and nucleic acid molecules encoding such CDR.

According to another aspect of the present invention provides humanized (humanized) antibodies containing at least one and preferably six plots, complementarity determining (CDR) derived neutralizing monoclonal antibodies nonhuman origin (Mab) characterized by a dissociation constant equal to or less 210-10M for human IL 4.

According to another aspect provides a chimeric antibody containing human constant region of the heavy and light chains variable regions of the heavy and light chains produced from inhuman neutralizing monoclonal antibodies (Mab) characterized by a dissociation constant equal to or less 210-10M for human IL 4.

Another aspect of this is the cast of protein or Mab (for example, humanized, chimeric, etc.,) and pharmaceutically applicable carrier.

Another aspect of the present invention provides a method of treatment and/or prophylaxis of allergic conditions in the application of an effective amount of the pharmaceutical composition of the invention.

Another aspect of the present invention provides methods and components used for the recombinant production of fused protein, Mab (e.g., humanized, chimeric, etc.,), CDR, Fab or F (ab')2or their analogues derived inhuman neutralizing monoclonal antibodies (Mab) characterized by a dissociation constant equal to or less 210-10M IL 4. Such components include isolated encoding nucleic acid sequence, a recombinant plasmid containing a nucleic acid sequence selected controlled by regulatory sequences capable of directing their expression in the cells of the host, as well as cell hosts (preferably mammalian), transfetsirovannyh recombinant plasmids. The method of obtaining includes the cultivation transtitional line host cell this is: in these cells, and selection of the expression product.

Another aspect of the present invention encompasses a method for diagnosing allergies and other medical conditions associated with excess formation of immunoglobulin E in human body, which consists in contacting of the sample of biological fluid from the slit proteins, Mab's (for example, humanitarianism, chimeric, etc.,) and Fab's of the present invention and the analysis of the implementation of binding between the said fused protein, Mab or Fab and human interleukin 4.

Another aspect provides a method of screening a monoclonal antibody with high titer against human interleukin 4, which includes:

a) obtaining a hybridoma cell line, characterized by secretion of monoclonal antibodies against human interleukin 4; and (b) the specified screening hybridoma cell line with the aldehyde-conjugated interleukin-4 or biotinylated human interleukin-4. Preferably conduct screening of hybridoma cell lines with biotinylated human interleukin-4.

The present invention also provides a neutralizing Mab with high affinity is the absence of aldehyde-conjugated human interleukin-4 or biotinylated human IL 4.

According to another aspect of the present invention provides a neutralizing monoclonal antibodies rodents that are specific for human interleukin-4 and having affinity for binding characterized by a dissociation constant equal to or less 210-10M. Examples of such monoclonal antibodies can serve murine Mab, W, rat Mab, 6A1 and other Mab with the same characteristics (i.e., the ability to bind to epitopes V or 6A1 specificity for human IL 4 and the dissociation constant equal to or less 210-10M). Another aspect of the present invention is hybridoma 3426A11C1B9.

Other aspects of the present invention are presented below in the detailed description of its preferred embodiments.

In Fig. 1 (sequence SEQ ID N: 1,2) illustrates the variable area light chain (amino acids 21-132) mouse 114 antibodies V, human/mouse W chimeric antibody, as well as the native signal sequence (amino acids 1-20). The underlined portions are CDR (Sequence SEQ ID N: 15, 16; SEQ ID N: 17 and 18; and SEQ ID N: 19 and 20).

In Fig. 2 (SEQ ID N: 3 and 4) illustrates the variable plot heavy chain (amino acids 20-140) mouse V, as well as 23 and 24; and SEQ ID N: 25,26).

In Fig. 3 (SEQ ID N: 9, 10) is illustrated variable plot heavy chain (amino acids 21-141) human/mouse W chimeric antibodies and its signal sequence (amino acids 1-19; SEQ ID N: 5 and 6). The underlined parts indicate the CDR derived V (SEQ ID N: 21, 22; SEQ ID N: 23, 24, and SEQ ID N: 25, 26).

In Fig. 4 illustrates (SEQ ID N: 11, 12) variable plot heavy chain (amino acids 20-141) gumanitarnogo V antibodies with signal sequence (amino acids 1-19; SEQ ID N: 5, 6). The underlined portions are CDR derived V (SEQ ID N: 54, 22; SEQ ID N: 55, 24; and SEQ ID N: 56, 26).

In Fig. 5 (SEQ ID N: 13, 14) illustrates the variable area light chain (amino acids 21-131) gumanitarnogo V antibodies and signal sequence (amino acids 1-20; SEQ ID N: 7, 8). The underlined portions are CDR derived V (SEQ ID N: 53, 16; SEQ ID N: 17, 18, and SEQ ID N: 27, 28).

In Fig. 6A (SEQ ID N: 5, 6) shows the signal sequence of the heavy chain used in the example below, 4.

In Fig. 6B (SEQ ID N: 7, 8) shows the signal sequence of the light chain used in the example below, 4.

In Fig. 7 given schematic illustration of the plasma is holding beta lactamase gene (BETA LAC), the replication origin SV40 (SV40), cytomegalovirus promotor sequence (CMV), signal sequence, chimeric variable heavy chain from SEQ ID No. 9, 10, a constant area of the human heavy chain, a poly A signal of the bovine growth hormone (BGH), betaglobin promoter (beto glopro), digidrofolyatreduktazy gene (DHFR) and another BGH sequence, a poly A signal in the environment pVC19.

In Fig. 8 presents a diagram of the plasmid pIL 4 chlc-pcdn used to ekspressirovali chimeric IL 4 leggiadro variable segment SEQ ID No. 1 and 2 in mammalian cells. This plasmid differs from that shown in Fig. 7 fact that it contains a chimeric Legazpi variable plot, non-chimeric heavy chain constant area of the human light chain and neomicina gene (Neo) in addition to the DHFP.

In Fig. 9 presents a diagram of the plasmid pIL 4 hzhc-1-pcd used for expression of the synthetic IL 4 variable segment of the heavy chain SEQ ID No. 11 and 12 in mammalian cells. This plasmid differs from that shown in Fig. 7 in that it includes a variable area humanized heavy chain other than the one which is present in the chimeric heavy chain.

In Fig. 10 presents a diagram of the plasmid pIL mammal. This plasmid differs from that shown in Fig. 8 in that it includes a variable area humanized light chain that is different from that present in the chimeric light chain and does not encode DHFP gene.

The present invention provides a number of antibodies, their fragments and fused proteins, mainly humanized antibodies which have binding specificity against human IL 4, neutralizing activity and high affinity to human IL 4, as shown in the examples related to murine Mab or mouse Mab 6A1. Such products find use in therapeutic and pharmaceutical compositions for the treatment of reactions caused by the action of IL 4 and IgE. Such products are also used for diagnosis caused by the action of IL 4 painful condition in the measurement result (for example, by the method of enzyme-linked immunosorbent assay (EL1SA) circulating endogenous level of IL 4 people.

1. The definition adopted.

The term "fused protein" refers to a protein encoded by a fused molecule, which can be obtained by expression in the selected cell host. Such fused proteins are genetically engineered antibodies, such as t const plot immunoglobulin, for example, Fy, Fab or F(ab)2and so on

The term "fusion molecule" refers to a nucleic acid sequence that encodes plots, complementarity determining (CDR) nonhuman immunoglobulin, which is inserted into the first partner of the merger, including the human variable framework sequence. Sometimes, the first merge partner operatively associated with the second partner of the merger.

The term "first merge partner" refers to a nucleic acid sequence that encodes a human skeleton or variable area of the human immunoglobulin, where the native (or naturally occurring) CDRS are replaced with CDR donor antibody. Human variable region can be an immunoglobulin heavy chain, light chain or both such circuits), as well as similar to their functional fragments. Such CDR or CDR region inside the variable regions of antibodies (immunoglobulins) can determenirovana (to be defined) by known methods. So, for example, Kabat with TCS. /Sequences of Protein of Immunological Interest, 4th ed. The U.S. Department of health, national Institute of health (1987)/ describes the rules of the location of the CDR. Besides the well-known computer what the eye affinato binding, characterized by a Kd equal to or less 210-10M for human IL 4.

The term "binding specificity relative to human IL 4" refers to high titer (or affinity) in the case of human, but not bovine or murine IL 4.

The term "second partner merge" refers to other nucleotide sequences coding for the protein or peptide, conjugated with whom the first merge partner in a frame or by using the optional traditional linker sequence (for example, by rapid binding). Preferably, this substance is an immunoglobulin. The second merge partner can include a nucleic acid sequence encoding a full constant region of the same (for example, homology, when the first and second fusion proteins have the same origin) or additional (e.g., heterologous) of the desired antibodies. This substance may be an immunoglobulin heavy or light chain (or both such circuits as part of a single polypeptide). The second partner of the merger is not limited to a particular class of immunoglobulin or isotype. In addition, the second partner p, the discrete part of the corresponding human constant section or portion of the frame). This second partner of merger may also include a sequence encoding an integral membrane protein exposed on the outer surface of the host cell, for example, as part of ragovoy library, or a sequence encoding a protein for diagnostic purposes, such as horseradish peroxidase, galactosidase, etc.

Terms Fy, Fc, Fab or F(ab)2are used in the standard values (see , for example, with Harlow al., Antibodies Alaboratore Manual, laboratory cold spring Harbor (1988).

Used in the text, the term "antibody engineering" refers to the type of fused protein, i.e., synthetic antibodies (e.g., chimeric or gumanitarnogo antibodies), in which part of the variable domains of the light and/or heavy chains of a selected acceptor antibody are replaced by analogous parts of one or more donor antibody having specificity against the selected epitope. For example, these molecules may include antibodies, characterized by the presence of humanized heavy chain associated with unmodified light chain or chimeric light chain), or Vice versa. Engineering Antalya domain frame areas of light and/or heavy chain acceptor antibody with the in order to preserve the specificity of binding of the donor antibody. Such antibodies may include replacing one or more CDR (preferably all) of the acceptor antibody CDRs from the donor antibody, described in this document.

The term "chimeric antibody", as used herein, refers to the type of engineering antibodies that contain natural variable region (light chain and heavy chain) produced from donor antibodies associated with constant regions of light and heavy chains produced from the acceptor antibody.

The term "humanitariannet antibody" refers to antibody engineering type with its CDR of nonhuman donor immunoglobulin, and the other remaining part of the immunoglobulin molecule are derived from one or more human immunoglobulin. It should be noted that the frame of reference remains subject to change in order to preserve binding affinity (see , for example, with Queen al., Proc. Natl. Acad. Sci. UCA 86, 10029-10032 (1989), with Hodgson al., Bio /Technology, 9:421 (1991).

Used in the text, the term "donor antibody" refers to an antibody (polyclonal, monoclonal or recombinant), which sposobleny fragments or analogues to the first partner of the merger, in the resulting merged molecule and expressed fused protein with antigenic specificity and neutralizing ability, characteristic of the donor antibody. An example of donor antibodies suitable for use in the present invention, can serve as inhuman neutralizing monoclonal antibody (e.g., mouse), denoted as W. Antibody V is defined as having a high titer, human-IL 4 specific (i.e. not recognize bovine or murine IL 4), neutralizing antibody isotype IgG1with DNA variable light chain and the amino acid sequence of SEQ ID No. 1 and 2, as well as DNA variable heavy chain and the amino acid sequence of SEQ ID No. 3 and 4 in the constant region of a suitable mouse IgG.

The term "acceptor antibody" refers to an antibody (polyclonal, monoclonal or recombinant), heterologous donor antibody, which facilitates the transfer of all (or any part thereof, but preferably all) of nucleic acid sequences encoding the frame regions of the heavy and/or light chain and/or constant regions of the heavy and/or light chain of the second merge partner. Predpochtiteljno complementarity segment amino acid sequences of the antibodies, which are hypervariable area of immunoglobulin light and heavy chains. See, for example, Kabat with TCS. Sequences of proteins having immunological importance, 4th ed., The U.S. Department of health, national Institute of health (1987). There are three CDR (or CDR regions in the heavy and light chains in the variable portion of an antibody. In this regard, as used in the text, the term "CDR" refers to all three CDRs of the heavy chain, or all three CDRs of a light chain (or all CDRs both light and heavy chains).

CDR provides a set of constant values for binding of the antibody to the antigen or epitope. CDR of the present invention are derived from sequences of the variable heavy and light chains of donor antibodies and contain analogues of naturally occurring CDR, and such counterparts have part or retain the same antigencoated specificity and/or neutralizing ability, and that of the donor antibody from which they originated.

The term "distribution antigenemia specificity or neutralizing ability is assumed, for example, that although Mab V can be characterized by a certain level of antigenic affinity, and the CDR is to give lower or higher affinity, it is assumed that the CDR V in such circumstances, the same will recognize the same agricap that W. Examples tagliani CDR V can serve as sequence ID No. 22; sequence ID # 24; sequence ID No. 26; and examples Legazpi CDR V can serve as sequence ID No. 16; sequence ID No. 18 and sequence ID No. 20.

The term "functional fragment" refers to an incomplete variable sequence of the heavy or light chain (e.g., small deletions at the amino - or carboxylic immunoglobulin variable segment), preserving the same antigencoated specificity and/or neutralizing ability, as the antibody from which was obtained the fragment.

The term "analog" refers to an amino acid sequence modified by at least one amino acid, and this modification can be carried out chemically, to be a replacement or rearrangement of several amino acids (usually not more than 10) and this modification allows amino acid sequence to preserve the biological characteristics such as specificity to the antigen, high titer or affinity inherent to unmodified p is riccioni endonuclease sites inside or within the surrounding CDR region.

Analogs can also arise in the course of allelic variations. The term "allelic variation or modification" means a change in the nucleic acid sequence that encodes an amino acid or peptide sequence of the invention. Such variations or modifications may be associated with degeneration of the genetic code or result from a deliberate design in order to give the desired characteristics. Such variations or modifications can result in lead or not to lead to a change in any amino acid coding sequence. For example, the amino acid sequence of CDR of the light chain, sequence ID No. 16 is identical to the native murine sequence and gumanitarnogo V antibodies. However, such CDR sequence encoded in SEQ ID N: 15, and SEQ ID N: 53. Similarly, CDR SEQ ID N: 22 encoded as SEQ ID N: 21, and SEQ ID N:54, CDR SEQ ID N: 24 encoded as SEQ ID N: 23, and SEQ ID N: 55; and CDR SEQ ID N: 26 encoded as SEQ ID N: 25, and SEQ ID N:56.

The term "effector agents" refers to deproteinised molecules vehicles that conventional methods can be linked fused proteins and/or natural or synthetic, light or Taulat traditional media used in diagnostics, for example polystyrene or other plastic beads, polysaccharides, such as those used in the system BlAcope /Pharmacia / or other non-protein substances used in medicine and are safe for use on humans and animals. Other agents-effectors can include the macrocycle for chelation atom of heavy metal, or a radioisotope. These effectors can also be used to increase the time of half-fused proteins; such agents include polyethylene glycol.

II. High-affinity IL 4-monoclonal antibodies.

To implement the design of the antibodies, fragments and fused proteins of the invention can be applied to non-human species (for example, materials obtained from cows, sheep, primates, rodents (e.g. mice and rats), and so on) and they are used to generate the desired immunoglobulin by submission with native human IL-4 or its peptide epitope. Conventional hybridoma techniques are used to provide a hybridoma cell line secreting inhuman Mab to IL 4. Then such hybridoma subjected to screening using IL 4, covalently linked with a 96-hole of plastie with methods described in detail in the following example 2. Thus, one of the distinguishing features of the present invention is a method for the detection Mab to human IL 4, which used analytical tools allow you to avoid denaturation of IL 4. According to this method, it was found that can be detected Mab with a high titer (or high affinity to human IL 4.

As one of the examples will first be described getting a neutralizing Mab with high titers from murine donor. Mab V representing the desired mouse (donor) antibody for use in the development of chimeric or gumanitarnogo antibodies, are described in detail in following example 1. V Mab is characterized antigenemia specificity for human IL 4 with Kd< 2 10-1M (about 1.8 10-10M) in relation to IL 4. Kdfor 114 of the Fab fragment of such V has a value of less than 310-10Refer to the Epitope of this antibody cannot be mapped using IL 4 linear peptides and, therefore, such an epitope is considered to bind to the non-contiguous epitope. Picture link suggests the presence of a binding site in region B-C loop (residues 60-69) ---> C-helix (residues 70-93). In the from the with al., J. Biol. Chem. 267: 20371-20376 (1992), Wlodaver with al., FEBS Lett. 309: 59-64(1992), Redfield with TCS. Biochem 30: 11029-11035 (1991), Smith al., J. Mol. Biol. 224: 899-904(1992), with Garrett al., (1992) and Rowers c al. Biochem. 31: 4334-4346 (1992) and Science 256:1673-1677 (1992).

Other desirable donor antibody is a rat Mab, 6A1. Obtaining this Mab is described below in example 7. This Mab is characterized in that it is the isotype IgG2aand has constant dissocia against IL 4 less 2 10-10M (approximately 1,610-10M). As in the case of W epitope target such 6A1 not mapped to IL 4 linear peptides and therefore, this epitope is considered as non-contiguous and three-dimensional. Picture of binding to IL 4 mottainai and its biological activity indicate the binding of the D-helix of human IL 4 (amino acid residues 109-127), apparently in the region of the tyrosine at amino acid residue # 124.

The present invention is not limited to the use of V Mab, 6A1 Mab or hypervariable (i.e. CDR) sequences. Any other suitable IL-4 antibodies with high titer, characterized by a dissociation constant equal to or less 2 10-10M against human IL 4, and the corresponding anti-IL 4 CDR can serve as a substitute for these materials. The following description of the simplification of the description.

Ill. Antibody fragments

The present invention also encompasses the use of Fab fragments or F(ab)2fragments derived Mab directed against human IL 4. Such fragments are used as agents that protect in vivo against IL 4 - IgE-mediator States or in vitro as part IL 4 diagnostic tools. The Fab fragment contains the complete light chain and aminoterminal part of the heavy chain; and the fragment F(ab')2is a fragment formed by two Fab fragments linked by a disulfide bond. Mab W, 6A1, and other similar high-affinity, IL 4 binding antibodies are sources of Fab fragments and F(ab'), which can be obtained by traditional methods, for example by cleavage of Mab with the appropriate proteolytic enzymes, papain and/or pepsin, or recombinant methods. Such fragments Fab and F(ab')2themselves are used as a therapeutic, prophylactic or diagnostic agents, as well as donor sequences containing variable plots and CDR sequences used for the formation of recombinant or humanized antibodies, as described in Nasli other above-described antibodies may provide sequence, for example, variable peptide sequence of the heavy and light chain framework sequences, CDR sequences, functional fragments and analogues, as well as iadarola their nucleic acid sequences used to design and produce different fused proteins (including genetically engineered antibodies), which are characterized by antigenemia specificity of the donor antibody.

Thus, as one example of the present invention provides the sequence of the variable light chain and variable heavy chain of the IL 4 mouse antibodies V and sequence derived. Variable plot heavy chain V characterized by amino acid residues 20-140 SEQ ID N: 4. CDR region indicated by underlining in Fig. 2 and represented in SEQ ID N: 22; SEQ ID N: 24 and SEQ ID N: 26. Variable region of the light chain clone is characterized by amino acid residues 21-132 of Fig. 1 /SEQ ID N: 2/. CDR region are amino acid residues 44-58 /SEQ ID N: 16/; 74-80/ SEQ ID N: 18/ and 113-121 / SEQ ID N: 20/.

Also provided by the variable region of the chimeric heavy chain and signal nucleotide and amino acid sequences. These posledovatelnostei the heavy chain shown in SEQ ID N: 5 and 6. Region CDRs are underlined in Fig. 3 and they are identical in amino acid sequence of native murine CDR /SEQ ID N: 21-26/. Nucleotide and amino acid sequences of variable region of the chimeric light chain is identical unmodified V sequences (amino acid residues 21-132 SEQ ID N: 2), which makes possible the use of natural murine signal sequence (amino acid residues 1-20 SEQ ID N: 2).

The variable region of the humanized heavy chain and the signal sequence illustrated in Fig. 4 /SEQ ID N: 11 and 12/. The signal sequence is also shown in SEQ ID N: 5 and 6. Other suitable signal sequences known to specialists in this field, can serve as a substitute for the signal sequences disclosed in the examples. CDR amino acid sequences of this design is identical to CDR native murine and chimeric heavy chains and they are shown in SEQ ID N: 22 (encoded by SEQ ID N: 54), SEQ ID N: 24 (encoded by SEQ ID N: 55) and SEQ ID N: 56 (encoded by SEQ ID N: 26).

Given as example (synthetic) variable sequence of the humanized light chain is illustrated in Fig. 5 /SEQ ID N: 13 and 14/. This signal Portege indicated by underlining and they differ from CDR native murine CDR signal amino acid sequence of SEQ ID N: 20. Thus, the CDR of the humanized light chain represented by SEQ ID N: 53 and 16, SEQ ID N: 17 and 18 and SEQ ID No. 27 and 28. The existing difference is described in detail in example 3.

Nucleic acid sequences of the present invention or fragments thereof, encoding the peptide sequence of the variable light chain and heavy chain are used in unmodified form or can be synthesized with the aim of introducing the desired modifications, such as restriction sites. The selected nucleic acid sequence of natural or synthetic origin, derived Mab V or other desirable IL 4 antibodies with high titer, may not necessarily contain restriction sites to facilitate insertion or ligation into a suitable nucleic acid sequence, such as one that encodes a frame region of the desired antibody ligation with mutated CDR or merging with a nucleic acid sequence that encodes a selected second partner of the merger.

Taking into account the degeneracy of the genetic code, can be constructed of different coding sequences that can encode variable amino acid consistently that share the antigenic specificity of the donor antibody. The selected nucleic acid sequence of the invention or fragments thereof, encoding the peptide sequence of the variable chains or CDRs can be used to obtain the fused proteins, chimeric or humanized antibodies or other engineering antibodies of the present invention in operative connection with the second merger partner.

These sequences are also used for mutagenic introducing specific changes in nucleic acid sequence encoding a CDR or framework region, and introducing the resulting modified or fused nucleic acid sequence into a plasmid for expression. For example, silent substitutions in a nucleotide sequence of the frame and CDR-coding regions used to create restriction sites that facilitate the insertion of mutagenically CDR (and/or frame) areas. Such CDR region used to construct gumanitarnogo antibodies of the invention.

It should be noted that in addition to the selected nucleic acid sequences encoding part of the fused protein and antibodies described herein can be used Used DNA sequences include those sequences, which hybridize under strict conditions (see T. Maniatis with TCS. Molecular cloning (a Laboratory manual), laboratory cold spring Harbor (1982), pp. 387-389/ with DNA sequences. One example of such hybridization in stringent conditions is hybridization at 4XSSC at 65oC followed by washing in 0.1 XSSC at 65oC for one hour. Another example of hybridization in simple terms can serve as a treatment in 50% formamide, 4 XSSC at the 42oC. Preferably, such hybrid DNA sequences contained at least 18 nucleotides, i.e., have a size close to the size of the CDR.

V. Merged molecules and fused proteins

Merged molecules can encode fused proteins, which include engineering antibodies as chimeric antibodies and humanized antibodies. Desirable merged molecule contains the CDR sequences encoding peptides with antigenic specificity of IL-4 antibodies, preferably high-affinity antibodies provided by the present invention, inserted in the first merge partner (human frame region or human immunoglobulin variable region).

Preferably, the first part is no enable sequence, the coding region of the second antibody, for example the Fc region. The second partners of merger may also include sequences encoding other immunoglobulins, which, preserving the reading frame fused constant region of the light or heavy chain, or the merger is carried out by means of the linker sequence. Engineering antibodies directed against functional fragments or analogs of IL 4, can be designed to provide increased binding of the same antibody.

The second partner mergers may also be connected to the above described agent-affector, including non-protein molecules carrier, with which the second partner of the merger may be operatively connected by traditional means.

Fusion or adhesion between the second partners of a merger, for example sequences of the antibody and agent-affector may be any suitable methods, for example by creating the traditional covalent or ionic bonds, fusion protein or with the use of such hetero-bifunctional staplers, as carbodiimide, glutaric aldehyde, etc., Such techniques are known in this area and is easily accessible from traditional hee is provide the desired space between the second merge partner and agent-affector, can also be designed in the fused molecule. The design of such linkers are well known to specialists in this field.

It should be noted that the signal sequence of the molecules of the invention can be modified to enhance expression. As one of the examples can be mentioned protein having the amino acid sequence the sequence of the murine heavy chain, which is identical chimeric variable heavy chain (Yn) Fig. 2 / SEQ ID N: 4/ contains the original signal peptide replaced with another signal sequence (amino acid residues 1-20) / SEQ ID N: 6/.

Example of a fused protein contains a peptide or protein sequence of the variable heavy and/or light chain having antigenic specificity of Mab W, for example, Yn/amino acid residues 21-141 sequence SEQ ID N: 9 and 10/ and YLchain /amino acid residues 21-132 of sequences SEQ ID N: 1 and 2/. Another desirable protein of the invention is characterized by the amino acid sequence containing at least one, and preferably all CDR variable regions of the heavy and/or light chains of murine antibodies V, and the remaining pair, humanized YHand YLthe field of SEQ ID N: 11 and 12 and SEQ ID N: 13 and 14 (Fig. 4 and 5).

According to another embodiment of the invention engineering antibody may contain attached additional agent. For example, recombinant DNA technology can be used for engineering antibodies of the invention, in which the Fc fragment CH3 domain molecules of all antibodies is replaced by an enzyme or other detektiruya molecule (e.g., polypeptide effector or molecule-reporter).

The second partner mergers may also be operatively associated with nimmanahaeminda peptide, protein or fragment heterologous CDR-containing sequence having the antigen specificity of the murine B. The resulting protein may, in the course of expression to show how anti-IL 4 antigenic specificity and characteristics of nimmanapalli. Characteristics of such partner mergers may include such functional characteristics as other binding or receptor domain, or therapeutic characteristics, if the merge partner itself is a therapeutic protein, or more antigenic characteristics.

Another desirable th length, or any of its discrete fragment, such as Fab or F(ab')2the heavy chain dimer, or any minimal recombinant fragments, such as Fyor single-chain antibody (SCA) or any other molecule with the same specificity that the selected donor Mab, e.g. Mab V or 6A1. This protein can be used in the form of a fused protein or can be used in nakitai form.

In each case, when the second merge partner comes from a different antibody, for example, of any isotype or class of immunoglobulin frame or constant region, there is a genetically engineered antibody. Engineering antibodies may contain immunoglobulin (Ig) constant region and a variable framework region from a single source, such as the acceptor antibody, and one or more (preferably all) of the CDR donor antibody, for example, described in the text anti-IL 4 antibody. In addition, there may be changes, such as deletions, substitutions or insertions of variable domain framework region of the heavy and/or light and/or tagliani variable domain spanning region of the acceptor chain acceptor Mab on the nucleic acid or amino acid levels or areas of donor CDR so, eating constructed in such a way, to use one (or both) of the variable heavy and/or light chain IL 4 Mab (optionally modified as described) or one or more neraidodhmiourgies CDR of the heavy or light chain (see example 3). Engineering antibodies of the present invention are neutralizing antibodies, i.e., they can block the binding of IL 4 receptor protein. For example, the antibody derived from Mab W, directed against specific tertiary protein epitope of human IL 4, which is believed to be in region B-C loop----> C-helix, as noted above.

Such engineering antibodies may include humanitariannet antibody containing wireframe plots of selected human immunoglobulin or subtype, or chimeric antibody containing human constant region of the heavy or light chain fused with functional fragments of IL-4 antibodies. Suitable human (or other animal) acceptanoe antibody may be selected from conventional database, such as database KAVATLos-Lamoshi database and the Swiss protein database, based on homology with the nucleotide and amino acid sequences donarska (amino acid basis), may be suitable for providing a constant region of the heavy chain and/or variable framework region of the heavy chain to insert the donor CDR. Suitable acceptor antibody, is able to be a donor is constant or variable frame regions of the light chain may be selected similarly. It should be noted that the heavy and light chain acceptor antibody does not have to have origin from one and the same acceptor antibody.

Preferably, heterologous frame and a constant region selected from a human immunoglobulin classes and isotypes, such as IgG (subtypes 1-4), IgM, IgA, and IgE. However, the acceptor antibody does not need to contain only human immunoglobulin protein sequences. So, for example, can be constructed gene in which a DNA sequence encoding a part of the chain of human immunoglobulin fused to a DNA sequence that encodes nimmanahaeminda amino acid sequence, for example the polypeptide effector or reporter molecule.

One example of a particularly desirable gumanitarnogo antibody contains CDR, W inserted in the frame region, the VA or preferably three CDRs of the variable regions of the heavy and/or light chain IL-4 antibodies are inserted in the frame region sequence selected human antibody, replacing the native CDR latest antibodies.

Preferably humanitariannet the antibody variable domains of both heavy and light chains of the person subjected to genetic engineering operations by replacing one or more CDR. You can use all six CDR or various combinations of less than six CDR. Preferably replace all six CDR. You can replace the CDR only in the human heavy chain, using as unmodified light chain light chain of the human acceptor antibody. On the other hand, compatible light chain may be chosen from other human antibodies, using traditional database antibodies. The remaining part of engineering antibodies may be derived from any suitable human acceptor immunoglobulin.

Thus, genetically engineered humanitariannet antibody preferably has the structure of a natural human antibody or its fragment and possesses the combination of properties required for effective therapeutic the ski application.

As another example, engineering the antibody that contains the three CDRs of the variable region of the light chain W /SEQ ID 1: 16, 18, 20, and 28/, and three CDRs, variable regions of the heavy chain W /SEQ ID No. 22, 24 and 26/.

The resulting humanitariannet antibody is characterized antigenemia specificity and high affinity Mab W.

Professionals in this field should be clear that engineering the antibody may be subject to additional modifications result in changes of the amino acids of the variable domains, and it does not have to influence the specificity and high affinity of the donor antibody (i.e., analog). So, for example, were designed humanized monoclonal antibody in which the amino acid residue at position 120 light chain represented arginine / SEQ ID N: 13 and 14 and/ or the threonine /SEQ ID N: 57 and 58/. It should be noted that the amino acids of the heavy and light chains can be substituted by other amino acids or variable domain frameworks, either CDR or in both areas.

In addition, the constant region may be modified, aimed at raising or lowering of the selective properties of molecules present is whether the change in the ability to bind and activate complement (see for example, Angal with TCS. Mol. Immunol. 30: 105-108(1993), with Xu al., J. Biol. Chem. 299: 3469-3474(1994), Winter with al., EP 307,434-B).

Fused protein, representing a chimeric antibody differs from the above humanized antibody that has variable regions of the heavy and light chains completely inhuman donor antibody includes a frame region associated with human immunoglobulin constant regions of both chains. It should be noted that the chimeric antibody retains more inhuman sequence relative to the humanized antibodies of the invention may cause a significant immune response in humans.

As discussed below, such antibodies are useful in the prevention and treatment-related effects of IL 4 allergic disorders.

VI. Getting fused protein engineering of antibodies.

Preferably variable sequences of the light and/or heavy chain and the CDR of Mab W /SEQ ID N: 16, 18, 20, 22, 24 and 26 and/ or other suitable donor Mab (for example, 6A1) and their encoding nucleic acid sequences are used to construct a fused protein engineering of antibodies, preferably humanized antibodies izobreteniya technical solutions of the present invention.

Hybridoma producing the selected donor Mab, e.g murine antibody W, is subjected to conventional cloning and DNA its variable regions of the heavy and light chains to be made by methods known to experts in this field, for example by the methods described by Sambrook with TCS. Molecular cloning (a Laboratory manual), 2nd ed., Laboratory Cold Spring. Harbor (1989). Variable regions of the heavy and light chains V containing at least the CDR and those portions of the variable domain of the frame region of the light and/or heavy chain acceptor Mab required to maintain a binding specificity in relation to donor Mab, and the remaining immunoglobulin side chain antibody derived from a human immunoglobulin, is obtained using polynucleotide primers and reverse transcriptase. CDR identify using well-known database and compared with other antibodies.

Can then be obtained mouse/human chimeric antibody and its analysis is conducted on the ability to bind. Such chimeric antibody contains YHand Y1the area is completely inhuman donor antibodies associated with constant parts of the human is wow antibodies identified using a computerized database, for example KAVATand a human antibody having homology to V, was chosen as the acceptor antibody. Sequence synthetic variable regions of the heavy chain, containing V CDR within the frameworks of human antibody, designed with optional substitutions of nucleotides in frame areas with the aim of introducing restriction sites. Such a constructed sequence is then synthesized using overlapping oligonucleotides, amplified using polymerase chain reaction (PCR) and bug fixes.

Similarly was designed suitable variable frame region light chain.

Humanitariannet antibody may be derived from a chimeric antibody, or preferably may be obtained synthetically, the insert donor CDR of Mab heavy and light chains in the selected frame of the heavy and light chain. On the other hand humanitariannet antibody of the invention may be obtained using standard mutagenesis techniques. Thus, the resulting humanitariannet antibody contains human framework region and CDR donor Mab. Can be carried out subsequent manipul cells masters, such as COS cells or CHO. Other details of this method are presented in example 4. Other humanized antibodies can be obtained using this method on other suitable IL 4-specific, neutralizing, non-human antibodies with high titer.

The traditional expression vector or recombinant plasmid receive, conducting operative Association such coding sequences fused protein with traditional regulatory control sequences capable of controlling the replication and expression and/or secretion from the host cell. Regulatory sequences include promoter sequences, for example the CMV promoter and signal sequences that originate from other known antibodies. Similarly receive a second expression vector containing a DNA sequence encoding a light or heavy chain complementary antibodies. Preferably this second expression vector identical to the first, except in those cases where the coding sequences and capable of selection markers are designed to the highest possible functional expression of each polypeptide chain.

Suitable vectors for cloning and sublimirovanny design as well as the compositions of the present invention may be selected by the specialists in this field of technology. So, for example, can be applied generally accepted pYC series of cloning vectors. One of the vectors is a pYC19 and he produced such suppliers as Amersham (Buckinghamshire, UK) or Pharmacia (Uppsala, Sweden). In addition, any vector that can easily be replicated, contains multiple cloning sites and marker genes and cloning can easily be done any manipulation. Thus, the selection cloning vector is not the limiting factor present soobramoney, can be selected by the person skilled in the art from any of the traditional vector. Such vectors also contain the selected regulatory sequences in operative Association with the DNA coding sequences of immunoglobulin regions and capable of directing the replication and expression of heterologous DNA sequences in the selected cells of the host, such as CMV-promoter. Such vectors contain the above-described DNA sequences that encode engineering antibody fused molecules. On the other hand, such vectors may contain a selected immunoglobulin sequence, modified by the insertion of the desired restriction sites to facilitate manipulation.

The expression vectors can also be characterized marker genes suitable for enhancing the expression of heterologous DNA sequences, for example digidrofolyatreduktazy gene of a mammal (DHFP) or gene neomechanisticism (neo). Other preferred vector sequences include poly-A signal sequence, such as bovine growth hormone (BGH), and beta-globalparameters sequence (betaglopro). Used vectors ek the components of such vectors, for example, the replicons, genes selection, enhancers, promoters, signal sequences, etc., can be obtained from natural sources or synthesized by known methods with a view to their use for the expression and/or secretion of the product of recombinant DNA in the selected host. Other suitable expression vectors, numerous types are known in this area for expression in mammalian, bacterial, insect, yeast and fungi, can also be selected for use for the purposes described above.

The present invention also encompasses a cell line, transtitional recombinant plasmid containing the coding sequence for engineering of antibodies or their fused molecules. Cell owners used for cloning or other manipulation of such cloning vectors, are also traditional. However, the most desirable to use cells of various strains of E. coli for replication cloning vectors and other stages of the design fused protein of the present invention.

Suitable cell host or cell lines for the expression of engineered antibodies or fused proteins of the invention preferably are telpochcalli is such a cell of the mammal, as CHO-cell or myeloid cell. Can be used in human cells, that allows to modify the molecule profiles of human glycosylation. Can be used and other eukaryotic cell lines. Selection of suitable host cells from the mammal, methods for transformation, culture, amplification, screening, and receive the product and its purification are known from the literature. See, for example, the above-cited work Sambrook with TCS.

Bacterial cells may be useful as host cells suitable for expression of recombinante Mab of the present invention. However, due to the tendency of proteins for expression in bacterial cells in neurogenous or misfolded form or in deglycosylation form, any of the recombinant Mab obtained in the bacterial cell, shall be subjected to screening to preserve the ability to bind antigen. If the molecule expressed by the bacterial cell, obtained in a properly stacked form, such a bacterial cell may be considered desirable by the owner. So, for example, various strains of E. coli used for expression, as is well known, can be used as cell-Hostellerie etc.

As we all know the strains of yeast cells can also be used as host cells, as the cells of insects, for example Drasophila and Lepidoptera, and also expressing viral system. See, for example, Miller with al. Genetic engineering, 8:277-298, Plenum Press (1986) and cited in this work literature.

The main methods of constructing vectors of the present invention, transfection methods required for obtaining host cells of the invention, and methods of cultivation, necessary to obtain the fused protein or engineering antibodies of the present invention from a host cell are well-known techniques. Similarly, after receiving the fused proteins or engineering antibodies of the invention can be purified from cell culture using standard methods, including precipitation with ammonium sulfate, purification on affinity columns, chromatographic columns, gel electrophoresis, etc., These methods are known in the art and do not limit the scope of the invention.

Another way of expression of humanized antibody may comprise expression in transgenic animal as described in U.S. patent N 4873316. This method is to Express the General allows the female to produce the desired recombinant protein in its milk.

After the expression of the desired engineered antibody is examined for in vitro activity using the appropriate analysis. Currently, the common scheme of analysis of the ELISA used to assess qualitative and quantitative binding of engineering antibodies with IL 4-epitope. In addition, other in vitro tests, for example Blacore/Pharmacia/, can also be used to test the neutralizing efficiency before a subsequent clinical study in humans, carried out to assess persisted engineering antibodies in the human body, regardless of the normal clearance mechanisms.

Following the methods described for obtaining humanized antibodies of V, the person skilled in the art can also construct humanized antibodies from other donor IL 4-antibody sequences of the variable regions and CDRs of the peptides described in this application. Engineering antibodies can be obtained with variable frame sections that are potentially recognized as "their" recipients of engineering antibodies. In variable frame region can be made small modifications to implement a strong increase antigenic svyazyvanie what to use to treat people with painful conditions associated with IL 4. These antibodies can also be used to diagnose these conditions.

VII. Therapeutic and prophylactic use.

The present invention also relates to a method of treatment of people experiencing allergic disorders, which consists in applying an effective dose of an antibody comprising one or more engineered antibodies or fused proteins described in this application, or their fragments.

Therapeutic reaction caused by the use of the molecules of the present invention arises from binding to human IL 4 and subsequent block allocation IgE. Thus, molecules of the present invention in the form of drugs and formulations for therapeutic use, are highly desirable for use in patients with allergic disorders such as allergic rhinitis, conjunctivitis, atopic dermatitis, atopic asthma, and anaphylactic shock.

Slit proteins, antibodies, engineering antibodies or fragments thereof of the present invention can also be used in conjunction with other antibodies, particularly human Mab reactive with other markers (epitopes) that are responsible for painful innoperable against epitopes, responsible for the disease state is selected animal, against whom the action of the antibodies of the invention can also be used in veterinary compositions.

It is assumed that therapeutic agents of the present invention will be useful for the treatment of allergic conditions during the period of time from 2 days to 3 weeks or as needed. For example, a longer treatment times may be desirable in the treatment of seasonal rhinitis, etc., This method represents a significant step forward compared to the currently used method investments in accordance with known methods of treating disorders caused by the action of IL 4. Dosage and duration of treatment depend on the relative duration of the molecules of the invention in the body and they may be regulated by a specialist, depending on the type of disease and General health of the patient.

The type of application therapeutic agent of the invention can be any that ensures the delivery of such agent in the host organism. Slit proteins, antibody engineering antibody fragments, and pharmaceutical compositions of the invention are particularly postherpetische agents of the present invention can be prepared as pharmaceutical compositions, containing an effective amount of engineering (for example, gumanitarnogo) antibodies of the present invention as an active ingredient in a pharmaceutically applicable carrier. Prophylactic use of an agent of the invention I prefer to use aqueous suspension or solution containing antibody engineering, preferably in the exchange buffer at physiological pH, in a form ready for injection. Compositions for parenteral administration will typically contain the solution of engineering antibodies of the invention or a mixture dissolved in a pharmaceutically applicable carrier, preferably in an aqueous medium. Can be used a large number of water carriers, for example of 0.4% saline, 0,31% glycine solution, etc., Such solutions must be sterile and, as a rule, they do not contain fine particles. These solutions can be sterilized well-known methods (e.g. by filtering). Such compositions may contain pharmaceutically applicable auxiliary connections required to approximate physiological conditions, and they can be pH regulators, buffering agents, etc., the concentration of the antibodies of the invention in such pharmaceutical formulation mabuiag concentration choose based on the required volume of fluid, viscosity, etc. in accordance with the specific type of application.

For example, the pharmaceutical composition of the present invention for intramuscular injection is prepared so that it contained 1 ml of sterile buffered water and 1 ng to 100 mg, typically, 50 ng of 30 mg or more preferably 5-25 mg of engineering antibodies of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion should contain 250 ml of sterile ringer's solution and 1 to 30 mg, preferably 5-25 mg of engineering antibodies of the present invention. Preparation methods used parenteral compositions is well known in the art and they are described in more detail, for example, in Remington''s Pharmaceutical Science, 15th ed. Mack Publishing Company, Easton, PA.

Preferably, therapeutic agent of the invention was present in the pharmaceutical preparation in the form of single dose. The appropriate therapeutically effective dose can be easily determined by the person skilled in the art. For the effective treatment of inflammatory disorders in humans and animals should be used parenterally, preferably nutramerica dose, if necessary, can be repeated after an appropriate period of time during the inflammatory response in accordance with the suggestions of therapist.

The present invention also encompasses the administration of IL 4 fused protein of the present invention simultaneously or sequentially with other antibodies or fused proteins, characterized by anti-IL 4 activity, such as factor activity of antitumor necrosis or other pharmaceutical activities, compatible with IL 4 receptor binding ability of the fused protein of the invention. Other such antibodies are produced by the industry or can be constructed in accordance with the method similar to that described in this application.

Slit proteins and engineering antibodies of the present invention can also be used for diagnostic purposes, for example for identifying disorders related to the effects of IL 4, or track the progress of treatment of such disorders. As diagnostic reagents such fused proteins can be the traditional way is marked for use in ELISA and other conventional tests, designed to measure the levels of IL 4 in serum, plasma, or other appropriate dia.

Antibody engineering antibody or fragments thereof, described in this application can be lyophilized for storage and re-compiled in a suitable carrier prior to use. It was shown that this technique is effective for normal immunoglobulins and known methods for lyophilization and reconstitution can be used.

The following examples illustrate various aspects of the present invention, including the construction of the representatives of the engineering of antibodies and their expression in suitable vectors and cell-hosts, and these examples do not limit the scope of the invention. All amino acids are marked traditional three-letter or one - letter codes. All the necessary restriction enzymes, plasmids, and other reagents and materials were obtained from commercial sources, if it is not specifically mentioned. All common methods cloning ligation and other methods of recombinant DNA technology was carried out in accordance with described in T. Maniatis with al., cited above, or the second edition of this book (1989), as amended Sombrook with al., the same publishers (Sambrook with labour).

Example 1.-Receiving Mab V

A. Methods of immunization.

Four mice (F1 hybrids BaIb/c and C57B1/6) podkin the Dili re inside the peritoneal immunization, 50 μg IL 4 in incomplete adjuvant's adjuvant. Based on the good value of the titer of serum antibodies against IL 4, one of the mice additionally were immunized 8 weeks 200 µg IL 4 (intraperitoneally, saline), two days later, 100 μg IL 4 (intraperitoneally in saline solution) and two days later, 50 μg IL 4 (intraperitoneally in saline solution). Two days after the final immunization was performed splenectomy.

B. Methodology mergers and screening system.

Cells of the mouse spleen was used to obtain hybrid (by a standard method, for example, described with Kohler al., Nature 256; 495(1975), of which > 250 cell clones were screened for secretion of antibodies to IL 4, using a commercially available system B1Acore and ELISA, as described below, in order to bind IL 4. Five holes were received positive response. Only 1 clone mice, V, was strongly positive. All of the secondary clones derived V were positive.

Example 2. The ELISA assays and affinity constants.

A. ELISA

Screening analysis carried out as described below, was designed to measure the affinity of the native human IL 4. In experiment 1 aktivirovani who was inkubirovali over night at room temperature. To the plate was covalently attached hIL 4. The solution IL 4 was removed and nonspecific related (NSB) sites were blocked by 1% bovine serum albumin (BSA) in TBS buffer (50 mm Tris, 150 mm NaCl, 1 mm MgCl2, of 0.02% NaN3, a pH of 7.4) for 60 minutes at 37oC. After this and each of the subsequent stages, the plate was washed 4 times proryvnym buffer (10 mm Tris, 150 mm NaCl, 0.05% tween-20, 0.02% of NaN3, pH 7,4). After this was added 50 μl of hybridoma medium (purified V or Fab fragments) and 50 ál analytical buffer (0,5% bovine gamma globulin in TBS buffer) and plates were incubated for 60 minutes at 37oC. 100 μl of biotinylated antimelanoma antibody was added to each well in an environment analytical buffer and incubation was performed as described above. 100 μl of streptavidin conjugated with alkaline phosphatase, was added to the wells and were incubated (30 min at 37oC). Added 100 μl/well of PNP and incubation was performed for 30 min at 37oC. Readings were shot on the optical density at a wavelength of 405 nm. In experiment 2 streptavidin coated plates (100 μl/well, 1 μg/ml in phosphate buffer solution (PBS)) were incubated overnight at 4oC and analyzed as described below. Streptavidine the solution was removed, the plates four times washed proryvnym buffer 50 ál of biotinylated IL 4, added together with 50 μl of analytical buffer and the system was incubated for 30 minutes at 37oC. was then added 50 μl of purified V IgG or Fab fragment (or hybridoma medium) plus 50 μl analytical buffer and incubation was performed for 60 minutes at 37oC. was Added 100 μl antimisting IgG-alkaline-phosphatase conjugate and incubation was performed for 60 minutes at 37oC. was Added 100 μl of PNP substrate and incubation was performed for 30 minutes at 37oC. Count readings were performed as described above.

B. Calculation V affinity to IL 4.

Using the results from the above experiments and summing them, as described below, the expected Kdfor V in accordance with the method Beadty with TCS. J. Immunol. Methods 100 : 173-179 (1987): Kaff=1/2(2[Abx]-[Ab],

Abx= concentration of bound Ab at 150 ng/ml biotinylated hIL 4;

Ab = concentration of bound Ab at 300 ng/ml biotinylated hIL 4.

Dissociation constants were calculated from the following equation: Kd= 1/Kaff.

Experiment 1: analysis of ELISA on the plate with 96 wells, coated with streptavidin (100 ng/well). Kd= 2,2 10-10M (V Fab).

Experiment 2: analysis of ELISA on the plate with 96SS="ptx2">

Mab V recognizes human IL 4, but does not recognize bovine or murine IL 4. One of the ways to determine this phenomenon consists in the following. The ELISA can be performed using the plate with 96 wells, coated with artemisinin IgG and then blocked by bovine serum albumin, to which 50 μl W (100 ng/ml), 25 μl of inhuman IL 4 and 25 ál of Biotin-IL 4 were incubated for 60 min at 37oC, and then were rinsed with streptavidin conjugated with alkaline phosphatase and PNP.

Similarly, it was found that Mab 6A1 does not recognize bovine or murine IL 4.

Example 3. Humanitariannet antibody

One humanitariannet antibody designed in such a way that it contained the murine CDR within the framework of a human antibody. Such humanitarian version of IL 4-specific mouse antibodies V was prepared by carrying out the following procedures.

A. cDNA cloning.

The cDNA clones were obtained from mRNA V heavy and light chains extracted from V hybridoma cell line /example 1/, using a set of Boehringer Mannheim. Primers specific as mouse articulated section, and Kappa constant region, ispolzovalas-3'

Primer gamma heavy-chain represents /SEQ ID N: 30/:

5' - CTACATATCCAACCCTTACAACCACAATC3'.

Double-strand cDNA was cloned directly into the plasmid pGEM7f+/Promega/, which then transformed E. coli DN-5A /Bethesda Research Labs/.

B. DNA Sequencing.

cDNA clones eight murine heavy and one light chain mouse from part A was subjected to sequencing. The results of the sequencing of the variable regions of these clones are shown in SEQ ID N: 1, 2, 3 and 4. Each clone contains amino acids, known as conservative among the variable regions of murine heavy chains or light chains, and the mouse signal sequence. Amino acid sequence of CDR are listed below.

CDR region of the heavy chain are SEQ ID N: 22, 24 and 26 (amino acids 50-56, 71-86 and 119-129 sequence SEQ ID N: 4) Cm. Fig. 2. Such sequences encoded by the sequences SEQ ID N: 21, SEQ ID No. 23 and SEQ ID N: 25, respectively. CDR region of the light chain are SEQ ID N: 16, 18 and 20 (amino acids 45-58, 74-80 and 113-121 sequence SEQ ID N: 2. Cm. Fig. 1. Such sequences encoded by the sequences SEQ ID N: 15, 17 and 19, respectively.

C. Selection of human skeletons.

After lots of 20-140 SEQ ID N: 4) were compared with database sequences of the human immunoglobulin using databases KAVATand SWiSS for the purpose of identification of the human skeleton for both heavy and light chain, which most closely compatible with mouse source on the homology of the sequence. In addition to these studies on homology sequence of the light and heavy chains were also evaluated relative positional database developed from structural models of the Fab domain with the aim of identifying potential conflicts associated with substitutions of amino acids, which can influence CDR presentation. In this case, structural studies found no obvious conflicts; therefore used DNA encoding inferred on the basis of studies of amino acid sequence homology.

Used frame region heavy chain antibodies derived from human myeloma immunoglobulin (COR) /E. M. Press, N. M. Hogg Biochem. J. 117: 641-660 (1970)). This sequence was approximately 77% homologous (69,4% identity) V variable region chains on the amino acid level.

For a suitable variable frame region of the light chain used a variable frame sequence light chain of a human antibody, identifitsirovana 80.2% homology (identity 72,0%) murine variable region light chain at the amino acid level.

Using mouse W CDR /SEQ ID N: 15-26/ and the sequence of the human antibody, got synthetic heavy chain and carried out PCR for DNA amplification. Such sequences synthesized using the following overlapping oligonucleotides and amplified using PCR. SEQ ID N: 31-37 provides five overlapping oligo and 2 PCR primer. Oligo 1 /SEQ ID N: 31/, as it contains the base 5-121. Oligo 2 /SEQ ID N: 32/ contains the base range 122-241 and oligo 3 /SEQ ID N: 33/ contains the base 242-361. The two lower filament primer SEQ ID N: 34 and SEQ ID N: 35 contain the base 134-110, and base 134-110, and base 253-230. All errors in the mapped sequences that were included PCR, have been fixed. PCR was carried out again using as 5' primer nucleotides 1-25 SEQ ID N: 36, and as the 3' primer - nucleotide 361-341 sequence SEQ ID N: 37.

Synthetic variable plot ligated in expressing vector pCD together with the synthetic signal sequence SEQ ID N; 5 and 6 of the chimeric constructs of the heavy chain together with IgG1human constant region. Synthetic YHand nucleotide and amino acid sequences of the signal posledovatel V BDR. However, the coding sequences for CDR /SEQ ID N: 54, 55 and 56/ differ from mouse V coding sequences /SEQ ID N: 21, 23 and 25/. The resulting expression vector, 114hzhc 1-1-Pcd shown in Fig. 9.

CDR gene region pressestelle frame light chain was removed restriction digestion and replaced with the following synthetic IL 4 CDR genes, which were obtained synthetically.

For CDR, 1:

SEO IDNO: 38: 5'CTAGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGG 3'

SEQ IDNO: 39: CCTTGCAGTTGATGGTGGCCCTCTGGCCCAGAGACACAG

SEQ IDNO: 40: TCGAGAGGCCTCCCAAAGTGTTGATTATGATGGTGATAG

TTATATGAACTGGTATCAGCAGAAACCC

SEQ IDNO: 41: GGGTTTCTGCTGATACCAGTTCATATAACTATCACCATCATA

ATCAACACTTTGGGAGGCCTC

for CDR2:

SEQ IDNO: 44:

GGGCAGCCTCCTAAGTTGCTCATTTACGCTCGATGCAATCTA

GAATCTGGGGTAC

SEQ 1DNO: 45;

CCCAGATTCTAGATTGGATGCAGCGTAAATGAGCAACTTAGG

AGGCTGCCC

For CDR3:

SEQ 1DN: 42:

ATACTACTGTCAGCAAAGTAATGAGGATGGTCCGAGGTTCGG

CGGAGGGAC

SEQ 1DNO: 43:

CTTGGTCCCTCCGCCGAACCTCGGAGGATCCTCATTACTTTG

CTGACAGTAGT

Synthetic YLand nucleotide and amino acid sequence of the signal sequence shown in Fig. 5 /SEQ ID N: 13 and 14/. Amino acid sequence of the first two CDR /SEQ ID N: 16 and 18/ identical to the corresponding murine V CDR. However, the coding sequence for the first CDR /SEQ ID N: 53/ differs from mouse V coding posilutely sequence. One of them /SEQ ID N: 28/ differs in amino acid signal /SEQ ID N: 20/ from native murine V sequence. SEQ ID N: 28 encoded by the sequence SEQ ID N: 27. Synthetic variable light region ligated into the expression vector together with its signal sequence /SEQ ID N: 7 and 8/. One of the obtained expression vectors, 114hzic1-O-Pcn shown in Fig. 10.

Such synthetic variable sequences of the light and/or heavy chains used in the construction of gumanitarnogo antibodies.

Example 4. Expression gumanitarnogo Mab in COS cells and CHO.

pUC18-subclones for YHreceived thereby, to add a signal sequence, originally derived from a human antibody SEQ ID N: 5. For YLreceived pUC18 subclones to add a signal sequence SEQ ID N: 7.

Humanitariannet heavy chain derived IgG1isotype, shows 89: 3% homology (identity 84,3%) at the amino acid level to mouse heavy chains from V. Synthetic YHprovided by amino acids 20-141 sequence SEQ ID N: 11 and 12. Humanitariannet light chain, human Kappa-chain, demonstrates 92,0% homology ( SEQ ID N: 13 and 14/, containing V CDR, was designed and synthesized as described for synthetic heavy chains.

DNA fragments corresponding signals which are associated with variable areas as humanized heavy and light chain, inserted pUC19 - based expressing plasmids in mammalian cells, with CMY promoters and a constant region of a human heavy or light chain Chimera, obtained in example 5, using traditional methods /Maniatis, cited above/ to obtain plasmid 114 hzhc1 - 1Pcd (heavy chain) /Fig. 9/ and 114 hz1c1 - oPcn) (light chain) /Fig. 10/. Plasmids HZHC and HZLC was cotranslationally in COS cells and supernatant analyzed by the method of ELISA for the presence of gumanitarnogo antibodies after 3 and 5 days. Another humanitariannet antibody designed using IgG4 isotype.

In the present example retrieves engineering antibodies. Other techniques can be used to create other engineering antibodies using other anti-IL 4 antibodies (for example, 6A1 - example 7) obtained by traditional methods.

Example 5. Construction of chimeric antibodies

A. Chimeric heavy chain designed by separation of the Oh fragment. Designed and synthesized a small oligomer DNA for the connection of mouse variable segment with a human IgG1 constant region (Bst EII - Apa1):

5' primer: SEQ ID N: 50: GTCACCGTCTCCTCAGCTAGCACCAAGGGGG

3' primer: SEQ ID N: 51: CTTGGTGCTAGCTGAGGAGACG.

Two such fragment is ligated into a plasmid pCD (see Fig. 7) (cleaved with EcoR1 and Apa1), which already encodes the human IgG1 constant plot. This clone not expressively, so 5' UTP wild-type and signal sequence was deleterule and replaced with SEQ ID N: 5 and 6.

Since the traditional restriction endonucleases the site is not available on the 3' end of the signal sequence, was introduced Bst EII site (i.e., a silent mutation) using PCR. Used the following PCR primers;

SEQ IDNO: 48: 5' primer: 5'CACCTTACCCTCAAACACTC 3'

SEQ IDNO: 49: 3' primer: 5'GAAGTAGTCCTTGACCAG 3'

Then from this plasmid was isolated Bst EII- > PST restriction fragment. Then designed and synthesized a new signal sequence and 5 ITR containing EcoRI and Bst E ends.

SEQ IDNO: 46: 5 primer: AATTCGAGGACGCCAGCAACATGGTGTTGCA GACCCAGGTCTTCATTTCTCTGTTGCTCTGGAATCTCTGGTGCCTACGGGC

AG

SEQ IDNO: 47: 3' primer: GTAACCTGCCCGTAGGCACGAGAGATCCAGA

GCAACAGAGAAATGAAGACCTGGGTCTGCAACACCATGTTGCTCGCGTC

CTCG

Chimeric light chain is designed with an intermittent consisted of a commercially available p UC18 universal reverse primer at the 5' end (EcoR1) and 3' primer, introducing the website Nar1:

[5'CATCTAGATGGCGCCGCCACAGTACGTTTGATCTCCAGCTTGGTCCC3'

used to merge murine variable segment with a human constant plot. Then this variable region is ligated in the expression vector PcDN (EcoR1 Nar1) (Fig. 8), which already contains the human Kappa-area.

After 3 and 5 days were collected supernatant and perform the analysis using ELISA method described below: the ELISA plate was coated with 0.1 μg of goat antibodies specific for the Fc region of a human antibody. Supernatant medium was added for one hour. Add horseradish peroxidase conjugated goat antibody specific for full human IgG antibody. Then within an hour was added ABTS peroxidase substrate (Kirkegaard and Perry laboratories Inc. Gaithersburg, MD). Has detected the expression of chimeric antibodies. In the second ELISA analysis of COS cell supernate containing chimeric antibody specifically associated with recombinant human IL 4 protein. This result confirms the fact that the genes encoding the antibody specific to IL 4 was subjected to cloning.

B. Humanitariannet heavy chain may be derived from such a chimeric heavy chain. Humanitarian heavy chain is an above the most homologous protein sequence in the Swiss database of protein on murine V YH(amino acids 20-140 of the sequence SEQ ID N: 4). Such humanitarian sequence of the heavy chain (EcoRI ApaI) was obtained synthetically and was carried out by PCR for DNA amplification as described above. Synthetic variable region is ligated in expressing vector pCD (EcoR1 Apa1) together with the synthetic signal sequence SEQ ID N: 5 and 6 of the chimeric constructs of the heavy chain and human IgG constant plot.

Similarly humanitariannet light chain can be obtained from the chimeric light chain as described above for the heavy chain. This gene (RY Nar1) was also obtained synthetically. Humanitarianly YLligated in expressing vector pCN, digested in the presence of EcoR1 Nar1, together with its signal sequence (EcoR1 EcoRY). The expression vector contains the human Kappa constant region.

Example 6. Cleaning and thermodynamics gumanitarnogo Mab.

Purification of CHO expressed chimeric and gumanitarnogo V can be achieved by traditional methods of protein A (or G) affibody been successfully used to clean up purity > 95% of other Mab (e.g., respiratory syncytial virus and malaria circumsporozoite antigens).

The affinity and detailed thermodynamics of IL-4 binding humanized Mab W and mouse V (example 1) was determined by the method of titration microcalorimeter. In this method, the measured binding assays by their internal heats of reaction (see, for example, Wiseman with TCS. Anal. Biochem. 179: 131-137 (1989). The affinity of both Mab were so strong that it was not possible to carry out measurements at ambient temperature. Then was applied thermodynamic approach: (I) the affinity was measured at 60oC when it is low enough to be measured directly; and (II) filmed temperature dependence of the enthalpy of binding for 30-60oC. Together the results obtained made it possible to calculate the affinity in a wide temperature range using equation Gibbs-Helmholtz.

The results of thermodynamics IL-4 binding to gumanitarnogo and mouse V antibodies are presented in table 1. On

the basis of the differences in free energy, enthalpy, entropy and heat capacity of two Mab can conclude that the fuzzy their thermodynamic behavior.

Example 7. Paymentsavannah rats using the Protocol of immunization, described for mice in example 1. 6A1 chose from a hybrid (specifically, hybridoma SV) obtained from rats immunized with human IL 4.

Kdfor 6A1 was calculated as described Beaty with TCS. J. Immunol. Methods. 100: 173-179(1987) and the obtained value was equal to 2 10-10M

Hybridoma 3426A11C1B9 deposited October 6, 1993, at the European collection of cell cultures, animals (ECACC), health Laboratory service centre for applied Microbiology and research, Norton Down, Salisbury, Wiltshire, SP4 O1G, UK, catalogue number 93100620 and accepted as patent Deposit in accordance with the Budapest Treaty of 1977 governing the Deposit of microorganisms for the purposes of patenting.

Example 8. The biological activity of Mabs: V (gumanitarnogo) V (mouse) and 6A1.

The following analyses were conducted using the following methods.

A. Binding to glycosylated rhIL 4.

The above antibodies induced to deglycosylation recombinant human IL 4 (rh IL 4), which was produced in E. coli. Because native human IL 4 is glycosylated, it is important to confirm the binding of mA is to glycosylated, and deglycosylation human recombinant IL 4 and so on is directed to the epitope, which can be masked by natural human IL 4.

B. Inhibition of IL-4 binding to the receptor.

The ability V to inhibit the binding of IL 4 with its receptor was studied using125I rhIL 4 binding to cell line Gibbon, M1a /ATSC T1B201/, which carries approximately 6,000 receptors per cell. MA cells were incubated with125I-IL 4 for 30 minutes at 37oC. Absorption of radioactivity was determined in a gamma counter after separation of the cell-associated 125I-IL 4 by centrifugation through an oil gradient. Nonspecific binding was determined by incubation in the presence of 100-fold molar excess of its IL 4/Park al., J. Exp. Med. 166: 476-488 (1987)/. The value of the IC50for its IL 4 this analysis was PM when the number of added IL 4 PM. For the intact murine (IgG) V value IC50was PM and PM for Fab fragment. When different concentrations of IL 4 (PM) obtained in the analysis the number for murine (IgG) V amounted to 109 gr.

C. Inhibition of lymphocyte proliferation.

Using the method described Spits with sutstvie of phytohemaglutinin, T-cell mitogen, to the activation of IL 4 receptor. The resulting blastoidea cells then stimulated for three days with IL 4. Proliferation was measured by the introduction of3H thymidine. The proliferation of B-cells was measured by the method of analysis in accordance with Collard al., Lymphokines and interferons. A practical approach, mainly 19, page 345, with the following modifications. Purified B cells from human tonsils were stimulated for 3 days with IL 4 and immobilized anti-IgM. Proliferation was measured by the introduction of3H thymidine.

Mouse W inhibited the introduction of 3H-thymidine in human peripheral T-lymphocytes stimulated PM IL 4, and B-lymphocytes in human tonsils, stimulated PM IL 4. IL 2-stimulated T-lymphocytes are not exposed to. The value of the IC50for inhibition of proliferation of T-cells was 30pm, and proliferation of B-cells PM. The corresponding values for the Fab fragment of mouse V was 108 and 393 PM.

D. Inhibition of CD23 induction.

CD23 is a low affinity receptor for IgE (FcER11) and is induced on the membrane of B-lymphocytes to low concentrations of IL 4, which is a necessary prerequisite for IgE Produzione is in the cells expressing CD23 receptor was determined by flow cytometry /Defrance with al., J. Exp. Med. 165:1459-1467 (1987)/. Mouse W inhibit CD23 expression on B lymphocytes of human tonsils, stimulated 8.3 gr IL 4 when the value of the IC50PM.

E. Inhibition of IgE secretion.

Unlike other analyses, in which IL 4 was added when EC50the concentrations of /with Pere al., Proc. Natl. Acad, Sci. 85: 6880-6884(1988), IgE secretion was investigated in the presence of concentrations of IL 4, provides the maximum secretion in order to reduce the variability inherent in such a system. The proliferation of T-cells was measured as follows. Human peripheral blood lymphocytes were incubated with IL 4 within 10 to 18, preferably 12 days. The IgE concentration in the culture supernatant was determined by ELISA method.

IgE secretion inhibited murine V and Fab fragment V in the presence of 1.7 nm IL 4, giving the value of the IC50of 1.9 and 5.0 nm, respectively. The experiment was repeated using a lower concentration of IL 4, 667 PM, eroding the value of the IC50to 0.65 nm in the case of mouse W. In the studied concentration within the molar ratio of antibody (IgC) to IL 4 remained unchanged (1:1).

F. Summary and interpretation of the data.

In all analyses, except IgE secretion, IL 4 was added with about ED50the concentration. The molar ratio between the antibody and IL 4 required for 50% inhibition, close to gumanitarnogo V, mouse V and 6A1 in two analyses of lymphocyte proliferation, but higher for gumanitarnogo V in CD23 induction analysis. The last analysis is particularly sensitive, requiring very low (~5%) levels of employment receptor (Kruse with al., EMBO J, 12:5121, 1993) and, as can be seen from the results obtained when using the mouse V and therefore susceptible to analytical variation.

Comparison of activities of rat 6A1 and mouse V demonstrates a similar pattern of functional effects, and unexpected inability 6A1 to completely inhibit the binding radioiodinated IL 4 with its receptor. It is assumed that radioterminal IL 4 used in receptore-binding analysis, jodiramon affordable tyrosine residue 124. When comparing the ability 6A1 to inhibit CD23 expression induced or its iodirovannoi IL 4, it was found that the inhibition is less effective in the case iodirovannoi ligand. These results show that 6A1 associated with IL 4 in the area of 6A1 is a neutralizing antibody to the high affinity, the ability to communicate with a completely different plot IL 4 than U.

Example 9. Pharmacokinetics

Pharmacokinetic properties gumanitarnogo V studied on male rats varieties Sprague Dawley, humanitariannet V applied to the four beasts in the form of iv spherical mass with a dosage of 1 mg/kg, and sampling of blood and was continued for 5 weeks after application. The concentration gumanitarnogo V in plasma was determined using IL 4/anti-human IgG sandwich EILSA intended to confirm not only the presence of circulating human IgG, but also its ability to bind to recombinant human IL 4.

The results obtained are summarized in table 3.

The obtained data show that the variability between animals is relatively small and the disappearance gumanitarnogo V from plasma, apparently, is two-phase. The apparent purification of plasma was low (0.5 ml/h/kg). The half-life was 11 days. Thus, the pharmacokinetic characteristics gumanitarnogo V derived CHO cells, consistent with other humanitarianism monoclonal antibodies in rats. A long period of circulation provioe V, apparently, will be effective for a long period of time.

Numerous modifications and variations of the present invention are covered by the description above and they should be obvious to specialists in this field. For example, a human framework region, or their modifications other than the examples above antibodies can be used to design humanized antibodies. Such modifications and changes in the compositions and methods of the present invention are covered by the scope of the attached claims.

1. Protein that has binding specificity against human interleukin-4 (IL4), containing region complementarity determining (CDR), where amino acid sequence of fields that define the complementary heavy chain, are

(a) ThrSerGlyMetGlyValSer: SEQ ID NO:22;

(b) HisIle TyrTrpAspAspLysArgTyrAsnProSerLeulysser: SEQ ID NO:24 or

(c) ArgGluThrVal PheTyrTrpPheAspVal: SEQ ID NO:26,

where the amino acid sequence of fields that define a complementary light chain, are

(a) LeuAlaSerGlnSerValAspTyrAspGlyAspsertyrmetasn: SEQ ID NO:16;

(b) AlaAlaSerAsnLeuGluSer: SEQ ID NO:18, or

(c) GlnGlnSerAsnGluAspProProArg: SEQ ID NO:28,

where the amino acid is ysAlaSerGlnSerValAspTyrAspGlyAspsertyrmetasn: SEQ ID NO:16;

(b) AlaAlaSerAsnLeuGluSer: SEQ ID NO:18, or

(c) GlnGlnSerAsnGluAspProProThr: SEQ ID NO:20,

originating from nonhuman monoclonal antibodies selected from the group consisting of 3B9 and A characterized by a dissociation constant equal to or less 2 x 10-10M with respect to human IL4, and the first partner of a merger, where the sequence of the first partner to merge contains the sequence of the heavy chain: amino acids 21-50, 56-71, 88-119 and 131-141 sequence SEQ ID NO: 12, where the sequence of the first partner to merge contains a sequence of light chain: amino acids 20-42, 58-72, 80-111 and 121-131 and the sequence SEQ ID NO:14.

2. Region complementarity determining (CDR) of the heavy chain of the immunoglobulin amino acid sequence which is selected from the group consisting of

(a) ThrSerGlyMetGlyValSer: SEQ ID NO:22;

(b) HisIle TyrTrpAspAspAspLysArgTyrAsnProSerleulysser: SEQ ID NO: 24, and

(c) ArgGluThrValPheTyrTrpPheAspVal SEQ ID NO:26.

3. Region complementarity determining (CDR) the light chain of the immunoglobulin amino acid sequence which is selected from the group consisting of

(a) LeuAlaSerGlnSerValAspTyrAspGlyAspsertyrmetasn: SEQ ID NO:16;

(b) AlaAlaSerAsnLeuGluSer: SEQ ID NO: 18;

(c) GlnGlnSerAsnGluAspProProArg: SEQ ID NO:28 and

(c) GlnGlnSerAsnGluAspProProThr: SEQ ID NO:20.

4 is Abolina, a sequence which is selected from the group consisting of:

(a) ACT TCT GGT ATG GGT GTG AGC: SEQ ID NO:21;

(b) CAC ATT TAC TGG GAT GAT GAC AAG CGC TAT; AACCCATCCCTGAAGAGC: SEQ ID NO:23;

(c)AGA GAG ACT GTG TTC TAC TGG TAC TTC GAT GTC: SEQ ID NO:25;

(d) ACC TCC GGT ATG GGT GTT TCC: SEQ ID NO:54;

(e) CAC ATC TAC TGG GAC GAC GAC AAA CGT TAC AAC CCG AGC CTG AAA TCC: SEQ ID NO: 55 and

(f) CGC GAA ACC GTT TTC TAC TGG TAC TTC GAC GTT: SEQ ID NO:56.

5. The nucleic acid molecule encoding a region complementarity determining (CDR) the light chain immunoglobulin sequence which is selected from the group consisting of

(a) AAG GCC AGC CAA AGT GTT GAT TAT GAT GCT GAT AGT TAT ATG AAC: SEQ ID NO:15;

(b) AAG GCC TCC CAA AGT GTT GAT TAT GAT GGT GAT AGT TAT ATG AAC: SEQ ID NO:53;

(c) GCT GCA TCC AAT CTA GAA TCT: SEQ ID NO:17;

(d) CAG CAA AGT AAT GAG GAT CCT CCG ACG: SEQ ID NO:19, and

(e) CAG CAA AGT AAT GAG GAT CCT CCG AGG: SEQ ID NO:27.

6. Humanitariannet antibody containing heavy and light chain, characterized by a dissociation constant equal to or less 2 x 10-10M against IL4, in which the frame of the said heavy and light chains originate from at least one of the selected human antibody and the amino acid sequence of fields, determining the complementarity of each of the specified chain, come from inhuman neutralizing monoclonal antibodies specific to Cellino IL4, and with identifying characteristics of monoclonal antibodies W.

7. The antibody under item 6, characterized in that it is not necessarily associated with agent-effector selected from the group consisting of a protein molecule carrier, polystyrene and plastic granules.

8. Chimeric antibody containing heavy and light chain, characterized by a dissociation constant equal to or less approximately 2 x 10-10M relative to human IL4, in which the amino acid sequence of fields, determining the complementarity of these heavy and light chains are from inhuman neutralizing monoclonal antibodies specific to human IL4, characterized by a dissociation constant equal to or less approximately 2 x 10-10M relative to human IL4, and with identifying characteristics of monoclonal antibodies W.

9. Pharmaceutical composition comprising an effective amount of the fused protein under item 1 and a pharmaceutically acceptable carrier.

10. Method for the treatment of allergies and other medical conditions associated with excessive production of IgE in the human body, including the stage of introduction nucleinate acid sequence, which is chosen from the group consisting of a) a nucleotide sequence that encodes a protein under item 1, having the sequence shown in Fig.5 SEQ ID NO: 13, and Fig.4 SEQ ID NO:11; C) a nucleotide sequence complementary to (a); (C) a nucleic acid sequence of at least 18 nucleotides capable of hybridisierung with (a) or (b) under strict conditions; (d) a fragment or analog (a), (b) or (C), which encodes a protein characterized by the specificity of human interleukin-4; and the specified sequence does not necessarily contain the restriction site.

12. The selected nucleic acid sequence which is selected from the group consisting of:

a) SEQ ID NO:21; SEQ ID NO:23;

SEQ ID NO:25; SEQ ID NO:54;

SEQ ID NO:55; SEQ ID NO:56;

SEQ ID NO:15; SEQ ID NO:53;

SEQ ID NO:17; SEQ ID NO:19, and

SEQ ID NO:27;

(b) nuclein acid sequence complementary to a), C) nucleic acid sequence of at least 18 nucleotides capable of hybridisierung in stringent conditions with (a) or (b), and

(d) a fragment or analog (a), (b) or (C) encoding a protein characterized by the specificity of human interleukin-4.

13. Recombinant plasma the pIl4chlc-Cdn, comprising the nucleic acid sequence under item 12.

15. Recombinant plasmid pIl4hzcll-Pcd, including nucleic acid sequence under item 12.

16. Recombinant plasmid pIl4hzlc1-0-Pcn, including nucleic acid sequence under item 12.

17. The method of obtaining gumanitarnogo antibodies specific for human interleukin-4, which lies in the cultivation of cell lines COS and CHO transtitional recombinant plasmid by PP.13 - 16, under control of the selected regulatory sequences capable of directing its expression in these cells.

18. The way to diagnose allergies and other medical conditions associated with excessive formation of immunoglobulin E in humans, which consists in contacting of the sample of biological fluid with a high titer of monoclonal antibodies to human IL-4, having means for binding, characterized by a dissociation constant equal to or less 2 x 10-10M, and analyzed for the presence of binding between the specified monoclonal antibody and human interleukin-4.

19. The method of screening of monoclonal antibodies to human IL-4 with an affinity for binding britaney cell lines, characterized by the secretion of monoclonal antibodies to human interleukin 4 and (b) the specified screening hybridoma cell line using illegitimating human interleukin-4 or biotinylated human Il-4.

20. A neutralizing monoclonal antibody to human IL-4, having means for binding, characterized by a dissociation constant equal to or less 2 x 10-10M, its Fab fragment or (Fab')2a fragment obtained by screening the hybridoma library products using illegitimating human interleukin-4 or biotinylated human Il-4.

21. Neutralizing monoclonal antibody mouse specific for human IL-4 and having affinity for binding characterized by a dissociation constant equal to or less 2 x 10-10M

22. The antibody according to p. 21, characterized in that it comprises the amino acid sequence of the light chain of SEQ ID NO:2.

23. Neutralizing monoclonal antibody rat specific to human IL-4 and having affinity for binding characterized by a dissociation constant equal to or less 2 x 10-10M

full line hybridoma, having the identifying characteristics AS, with access number 93100620, deposited on 6 October 1993 in the European Collection of Cultures of animal Cells.

 

Same patents:
The invention relates to the field of medicine and is designed to forecast the development of endometriosis in girls with oligomenorrhea and chronic pelvic pain

The invention relates to medicine, namely to urology, and can be used to predict the development of septic complications in the early postoperative period in children with abnormal development of the kidneys and upper urinary tract (VMP)

The invention relates to the field of medical genetics, in particular to somatic mutations in the gene multifunctional tumor suppressor (MTS) in the case of neoplasms of human rights and the use of these mutations for diagnosis and prognosis of these diseases
The invention relates to medical Microbiology

The invention relates to medicine, in particular to clinical diagnosis

The invention relates to diagnostic methods for detecting natural antibodies, binding proteins, extracted from the liver of goats having a molecular weight of approximately 14 kDa, with antitumor activity with the introduction of xenogeneic species, as well as to compositions containing an effective amount of the antibodies or hyperimmune serum, induced by immunization of animals or human proteins with M

The invention relates to the field of medical ecology and is intended to establish individual and population susceptibility to cancer
The invention relates to medicine, in particular to methods for study of the immune system, and can be used to determine the functional state of cellular immune system in patients with thermal injury

The invention relates to ophthalmology, namely oftalmologii, and is intended to predict the clinical course of uveal melanoma to increase the length and quality of life category of patients

The invention relates to a monoclonal antibody having the ability to inhibit homing hematopoietic stem cells and to identify surface antigen stromal cells, having the ability to maintain homing hematopoietic stem cells, as well as to hybridoma producing monoclonal antibody

The invention relates to the complementarity determining regions (CDR, hypervariable regions) and variable regions (V regions) of murine monoclonal antibodies to human interleukin-8 (IL-8), human/mouse chimeric antibody to human IL-8, and reconstructed human antibodies, and region, defining a complementary variable region, a human light chain (L-chain) and variable regions of the heavy chain (H-chain) of the person replaced the CDR of mouse monoclonal antibodies to human IL-8

The invention relates to the field of medicine and biotechnology, namely to new proteins, which factors in the growth and development of megakaryocytes (MGDFs; mostly labeled Mp1-ligands), the biological activity of which is to stimulate the growth of megakaryocytes and their differentiation or maturation, which ultimately leads to the formation of platelets

The invention relates to biotechnology and concerns gumanitarnogo immunoglobulin specific for the protein L-selectin person

The invention relates to propertytaxsession systems that require cleavage product a predecessor to the new polypeptide capable of restoring dichloroindophenol and oxidized glutathione to DNA that encodes this polypetide, farmkompanijam comprising the polypeptide, monoclonal antibodies against the indicated polypeptide

The invention relates to medicine, in particular to Oncology and immunology, and for the treatment of b-cell lymphoma

The invention relates to a monoclonal antibody having the ability to inhibit homing hematopoietic stem cells and to identify surface antigen stromal cells, having the ability to maintain homing hematopoietic stem cells, as well as to hybridoma producing monoclonal antibody
Up!