Method of producing antigen-binding vh domain, application thereof

FIELD: medicine.

SUBSTANCE: method of producing an antigen-binding VH domain wherein an elongated CDR3 loop similar to a camel loop is absent, involves transformation of a mammal cell by a heavy-chain VH heterolocus. The locus contains a gene coding a variable area, at least containing one segment of VH gene, one segment of D gene not being camel's, one segment of J gene not being camel's, one invariable heavy chain region provided that any gene coding the invariable regions does not code a Sn1 functional domain. The segments of V, D and J genes are capable to recombination and formation of the VDJ coding sequence. The transformed cell is capable to express a heavy chain antibody only containing an antigen-binding VH domain and the invariable constant effector region without the functional domain Sn1. The cell is used for producing a transgene animal to be immunised with an antigen of concern. Further the cells or tissues expressing specific antibodies to the heavy chain antigen of concern are recovered; nucleic acid coding the VH domain of the specific heavy chain antibody only is recovered; and the specified antigen-binding VH domain is expressed. The specified VH domains can be used for production of fused proteins or binding complexes of monovalent, bivalent or polyvalent polypeptide.

EFFECT: invention allows producing antigen-specific human antibodies of any class, exhibiting high affinity.

11 cl, 26 dwg, 8 ex

 

The technical field to which the invention relates

The present invention relates to the production of a wide set of functional antibody having a heavy chain that passed the process of affinity maturation, and to their use. The present invention also relates to the production and application of a broad set of class-specific antibodies, with only a heavy chain, and to the production and use of multivalent polypeptide complexes with the functional properties of the heavy chain of the antibodies, preferably, the binding and functional properties of heavy chain antibodies, effector activity constant region and, optionally, additional effector functions.

The present invention also relates to a method for obtaining transgenic mice antibodies having a heavy chain and having a full functional properties in response to antigenic stimulation. In particular, the present invention relates to a method for producing antigen-specific high-affinity human antibodies having a heavy chain of any class or mixture of classes and the selection and expression of fully functional VHantigenspecific domains.

The present invention also relates to the production of multivalent polypeptide complexes having functional properties of the cords is smaller circuits, preferably, the effector activity of the heavy chains and with other binding and effector functions.

Also described antibodies having a heavy chain, and other multivalent binding complexes produced by the methods of the present invention, and their use.

The level of technology

Monoclonal antibodies or their variants will be a high percentage of new drugs that will be releasing in the XXI century. Therapy with monoclinally antibodies has already been adopted as the preferred method of treatment of rheumatoid arthritis and Crohn's disease, and there is considerable progress in the treatment of cancer. Also on the basis of antibodies developed products for the treatment of cardiovascular and infectious diseases. The most sold products based on monoclonal antibodies recognize and bind one well-defined epitope on misheneva ligand (e.g., TNFα). The production of human antibodies for therapy still depends on mammalian cell cultures. The Assembly is complex, consisting of two heavy chains and two light chains (complex H2L2), and the subsequent process of post-translational glycosylation prevent the use of bacterial systems. The cost of production and high costs for the production of antibodies using glue is full of culture mammals are high and may limit the potential therapies based on antibodies in the absence of acceptable alternatives. Many transgenic organisms are able to Express fully functional antibodies. Such organisms include plants, insects, chickens, goats and cattle, but none of them so far has not been used for the production of commercially available therapeutic products.

Functional fragments of antibodies can be generated in E. coli, but the product usually has a low stability in serum, in addition to pegylated in the production process.

Complexes bespecifically antibodies are molecules on the basis of Ig capable of binding to two different epitopes either on the same or on different antigens. Bespecifically antibody containing the binding proteins, alone or in combination with other binding agents that have potential use in the treatment methods, which registered immune function and cause a therapeutic effect, such as the elimination of pathogens (Van Spriel et al., (1999) J. Infect. Diseases, 179, 661-669; Tacken et al., (2004) J. Immunol, 172, 4934-4940; U.S. patent No. 5487890), cancer treatment (Glennie and van der Winkel (2003) Drug Discovery Today, 8, 503-5100); and immunotherapy (Van Spriel et al., (2000) Immunol. Today, 21, 391-397; Segal et al., (2001) J. Immunol. Methods, 248, 1-6; Lyden et al., (2001) Nat. Med., 7, 1194-1201).

Problems in the production occur when the product bespecifically antibodies based on two or more complexes of H2L2. For example, to the expression of two or more sets of genes of heavy and light chains can lead to the formation of up to 10 different combinations, of which only one is a desirable heterodimers (Suresh et al., (1986) Methods Enzymol, 121, 210-228).

To address these problems, several strategies have been designed to obtain bespecifically formats IgG (BsIgG) full length in mammalian cells, which retain the effector function of the heavy chains. For BsIgG requires developed "knob-and-hole" heavy chain to prevent the formation of heterodimers, and are identical to the L-chain, in order to avoid erroneous pairing of L-chains (Carter, (2001) J. Immunol. Methods, 248, 7-15). Also described alternative chemical methods of cross-linking to obtain complexes of the fragments of antibodies, each of which recognizes a different antigen (Ferguson et al., (1995) Arthritis and Rheumatism, 38, 190-200), or cross-binding fragments of antibodies other binding proteins, such as collectino (Tacken et al., (2004) J. Immunol., 172, 4934-4940).

Development of a recombinant dimeric antibodies or combinatory (BsAb), usually lacking effector functions heavy chains, also overcomes heterodimeric excess. Such Minitel contain the minimum single-chain antibodies, with VHand VLbinding sites (scFv), which subsequently collapses and timeresults, forming bivalent bespecifically antibodies, monovalent to each of its nesenevich antigens (Holliger et al., (1993) PNAS, 90, 6444-6448; Muller et al., (998) FEBS Lett, 422, 259-264). At the same time, for education bespecifically combinatory as domains heterodimerization used CH1 and L is a constant domains (Muller et al., (1998) FEBS Lett., 259-264). To obtain BsAb developed a variety of recombinant methods based on systems of expression from E. coli (Hudson, (1999) Curr. Opin. Immunol., 11, 548-557), although it is obvious that the value and volume of production of multivalent antibody purity for clinical use remains a major obstacle for clinical application (Segal et al., (2001) J. Immunol. Methods, 248, 1-6).

In recent years, the concept BsAb was expanded to consider the double recombinant bespecifically antibody, a tetravalent bespecifically antibodies, in which the domains of the VHand VLin each of the H and L chains developed replaced by a pair of binding domains of the scFv. Such structures are difficult to design, can be assembled in mammalian cells in culture in the absence of a heterodimeric redundancy (Lu et al., (2003) J. Immunol. Methods, 279, 219-232).

The structure of immunoglobulins are well known. Most natural immunoglobulins contain two heavy chains and two light chains. Heavy chains are connected to each other via disulfide bonds between the hinge domains that are located approximately in the middle of each heavy chain. The light chain is associated with each heavy chain C-terminal side of the hinge domain. Each light chain is typically associated with the heavy chain by a disulfide bond near the hinge domain.

If the Ig molecule is placed correctly, then each circuit is placed in several distinct globular domains connected by a more linear polypeptide sequence. For example, the light chain is placed in the variable (VL) and constant (CL) domains. Heavy chains have one variable domain VHadjacent to the variable domain light chain first constant domain, a hinge domain, and two or three additional constant domain. The interaction variable domains of the heavy (VH) and light (VL) chains leads to the formation of antigennegative region (Fv). Usually as VHand VLrequired to bind antigen, although it is shown that the heavy chain dimers and aminoterminal fragments remain active in the absence of light chain (Jaton et al., (1968) Biochemistry, 7, 4185-4195).

With the advent of new molecular technologies identified by the presence of antibodies having a heavy chain (deprived of their light chain), in human B-cell proliferative disorders (disease of the heavy chains) and in the mouse models. Analysis of the heavy chain disease at the molecular level have shown that mutations and deletions in the genome level can lead nesootvetstvuyushih expression domain C H1 heavy chain, increasing the expression of antibodies having a heavy chain, which is devoid of binding properties of the light chain (see Hendershot et al., (1987) J. Cell Biol, 104, 761-767; Brandt et al., (1984) Mol. Cell. Biol, 4, 1110-1211).

Independent study of selected domains of the VHman, derived from phage libraries, showed antigen-specific binding domain VHbut it turned out that these domains VHhave low solubility. In addition, it was confirmed that the selection of domains VHthe person with the characteristics of specific binding shown in phage matrices, it is possible to form the connecting blocks for the developed antibodies (Ward et al., (1989) Nature, 341, 544-546).

Studies using other vertebrate species, showed that camels as a result of natural mutations of the gene are synthesized functional dimers IgG2 and IgG3, with only the heavy chain, which are not capable of contact with a light chain due to the lack of binding region CH1 light chains (Hamers-Casterman et al., (1993) Nature, 363, 446-448), and that species such as shark, produce a family of binding proteins, such only the heavy chain, which probably belong to the T-cell receptor mammals or light chain immunoglobulins (Stanfield et al., (2004) Science, 305, 1770-1773).

A distinctive feature of the antibody of camels, with only the heavy is sexless chain, is the domain VHthe camel, which provides improved solubility compared with domain VHman. To improve the solubility characteristics can be developed VHperson (see Davies and Riechmann, (1996) Protein Eng., 9 (6), 531-537; Lutz and Muyldermans, (1999) J. Measurement. Methods, 231, 25-38), or the solubility may be achieved by means of natural selectionin vivo(see Tanha et al., (2001) J. Biol. Chem., 216, 24114-24180). However, if binding domains VHderived from phage libraries, the inherent affinity to the antigen remains in the range of low micromol to high nanomolar despite the use of strategies to improve the affinity, including, for example, the randomization affinity in “hot spot” (Yau et al., (2005) J. Immunol Methods, 291, 213-224).

VHantibodies camel also differ modified CDR3 loop. Specified CDR3 loop on average, longer loops found in antibodies from other animals, and is a feature that, as expected, mainly affects the overall affinity to the antigen and specificity, which is offset by the lack of domain VLthe antibodies of the camel, with only a heavy chain (Desmyter et al., (1996) Nat. Struct. Biol, 3, 803-811, Riechmann and Muyldermans, (1999) J. Immunol. Methods, 23, 25-28).

Structural studies carried out in recent years, camel antibodies confirmed that such mnogoe the Asia antibodies largely run in vivothe processes of maturation-dependent events V(D)J recombination and somatic mutation (De Genst et al., (2005) J. Biol. Chem., 280 (14), 14114-14121).

In recent years we have developed methods for producing antibodies having a heavy chain transgenic mammals (see WO02/085945 and WO02/085944). Functional antibody having a heavy chain, probably, of any class (IgM, IgG, IgD, IgA or IgE) and received from any mammal (including humans) can be produced transgenic mammal (preferably mice) as a result of antigenic stimulation.

The ordinary locus of the heavy chain immunoglobulin contains many gene segments V, several gene segments D and multiple gene segments J. Each gene segment V encodes the domain V from N-end almost to the C-Terminus. C-end of each domain V is encoded by the gene segment D gene segment J. VDJ-rearrangement in B-cells, followed by affinity maturation, gives binding domains VHthat with binding domains VLthen form the plot of antigen recognition or binding site. In the interaction of heavy and light chains involved area CH1 heavy chain and the region κ or λ light chain, which facilitates the interaction.

To obtain antibodies having a heavy chain, the heavy chain locus in the germ line gene contains egment, encoding some or all possible constant region. During maturation rebuilt binding domain VHundergoes splicing education segment encoding a constant region (CH2, providing a reconstructed gene that encodes a heavy chain, which is devoid of domain CH1 and, therefore, not able to contact a light chain immunoglobulin.

Monoclonal antibodies having the heavy chain may be derived from B-cells of the spleen using standard cloning technology or derived from B-cell mRNA using phage display technology resulting in (Ward et al., (1989) Nature, 341, 544-546). Antibodies having a heavy chain derived from camel or transgenic animals, are high-affinity. Sequence analysis of the normal tetramers H2L2showed that diversity, mainly is the result of a combination of VDJ-rearrangement and somatic Hyper-mutations (Xu and Davies, (2000) Immunity, 13, 37-45). Sequence analysis of the expressed mRNA of only heavy chains produced only by camels or transgenic animals, confirms this observation (De Genst et al., (2005) J. Biol. Chem., 280, 14114-14121).

Important and common feature of natural areas VHcamel and man is that each area is bound in the form of a monomer, regardless of the tons of dimerization, with the region VLfor optimal solubility and affinity of binding. This feature is considered to be particularly effective for producing blocking agents and agents of tissue permeability.

Homo - or heterodimer can also be constructed by enzymatic cleavage of antibodies with only a heavy chain, or synthetic methods (Jaton et ah, (1968) Biochemistry, 7, 4185-4195 and U.S. patent No. 2003/0058074 A1). However, the beneficial properties of the binding domain of Monomeric antibodies so far not been used to gain advantages in the construction of multimeric proteins as therapeutic and diagnostic reagents.

VHperson or VHHcamel obtained by the technology of phage display, the advantage of improved performance in the somatic mutations and additional diversity provided by recombination regions D and J region CDR3 binding site of normal antibodies (Xu and Davies, (2000) Immunity, 13, 37-45). Although VHHcamel and has advantages in solubility compared to VHman, for the man he is antigenic and must be synthesized by immunization of a camel or by using phage display technology resulting in.

Introduction binding domain of VHhas a clear advantage over the use of scFv, which is s necessary to design domain V Hand VLwith the associated potential loss of specificity and avidity. Binding domains of the VHobtained from families of related genes, such as T-cell receptors or the family of immunoglobulins sharks, also provide alternatives scFv to generate bi - or multispecific binding molecules. Can also be used for other natural binding proteins and their domains, including, for example, soluble fragments of the receptors.

Classes of antibodies differ in their physiological functions. For example, IgG plays a crucial role in the Mature immune response. IgM is involved in the fixation and agglutination complement. IgA is a vast class of Ig in secretions, i.e. the tear fluid, saliva, colostrum, mucus, and, thus, plays a role in local immunity. The inclusion of class-specific constant regions of the heavy chain when designing multivalent binding complexes provides therapeutic benefits effector functionsin vivodepending on the desired functional properties. Development of individual effector regions can also result in the addition or loss of functional properties (Van Dijk and van der Winkel, Curr. Opin. Chem. Biol., (2001) Aug 5 (4), 368-374). Apparently, for optimum production and selection of antibodies having a heavy chain to the e contain high-affinity binding domains V H(or person, or camel, or have a different origin), will be used alternative approaches for obtaining the antibodies depending on the choice of randomized phage libraries, which do not facilitatein vivorecombination and affinity maturation.

Thus, the inclusion of the functional properties of the constant region IgA could lead to improved functioning of the mucous against pathogens (Leher et al., (1999) Exp. Eye. Res., 69, 75-84), although the functional properties of the constant regions of IgG1 provides increased stabilityin vivoin the serum. The presence of constant domains (CH2 and CH3 heavy chain provides the basis for stable dimerization, which can be seen in natural antibodies, and provides recognition sites for post-translational glycosylation. The presence of CH2 and CH3 also provides the possibility of secondary recognition of antibodies, as reagents and diagnostic tools are used bespecifically and multivalent complexes.

Selected pre-reconstructed sequence of the variable regions of the heavy chain camel pre-clone in front of the hinge region and the effector domain of human IgG1, built-in vector and expressed in COS cells for the production of an is and the body. Antibodies expressed in suchin vitroenvironment, have already been subjected to processes of a switching class (isotype) and maturation (hyperadrenia) the affinity ofin vivothe camel, and they may bind to the antigen (Riechmann and Muyldermans, (1999) J. Immunol. Methods, 231, 25-38).

In the art there remains a need to maximize the diversity of antibodies having a heavy chain and B-cell responsein vivoand, in particular, in obtaining functional set of class-specific antibodies person with only a heavy chain and a functional binding domain of VHonly heavy chains that preserve maximum antigennegative potential for use in a variety of clinical, industrial and academic applications.

In the art, there remains the need for soluble bivalent or multivalent binding polypeptide complexes containing at least part of the heavy chain of the antibody alone or in combination with effector (light) chain, which is physiologically stable and has effector function.

A brief description of the present invention

The present invention relates to a method for producing antibodies having a heavy chain VHor only the heavy chain VH(VHH) camel, transgenic mammalian shall provide, which provides a stage for expression of heterologous heavy chain locus VHor the heavy chain VH(VHH) camel this mammal, and the specified locus of the heavy chain VHor the heavy chain VH(VHH) camel contains a constant region of the heavy chain, which does not encode domain CH1, and the locus, which when expression is able to generate antibodies having a heavy chain of a particular class or classes.

The locus of the heavy chain VHor the locus of the heavy chain VH(VHH) camel may contain one or more gene segments V camel or other animal. Preferably, the gene segment V selected or designed to have improved solubility characteristics. Preferably, the V gene segment extract in humans.

Konstantina region of the heavy chain locus may contain a gene constant region Cα1and/or Cα2, By cε, Cδ, Cγ and/or Cµ heavy chain. In addition, the constant region of the heavy chain locus may contain more than one of the following constant regions: Cα1, Cα2, By Cε, Cδ, Cγ, Cµ.

Preferably the heavy chain locus VHcontains variable region containing at least one gene segment V of a person or a camel, at least one segment D and at least one segm the HT J, moreover, gene segment V man or camel, gene segment D gene segment J is capable of recombination, forming VDJ-coding sequence. The heavy chain locus, preferably, contains twenty or more D gene segments and/or five or more gene segments Preferably J., D and J segments are gene segments of the vertebrate, preferably human. Loop CDR3 can be obtained by use of gene segments D and J, obtained from vertebral or, preferably, human.

The locus of the heavy chain VHmay also contain recombinant sequence (rss), capable of recombination gene segment J directly with gene constant region of the heavy chain.

The constant region of the heavy chain of heterologous heavy chain locus is a constant region of a human or vertebrate, such as camel. Alternatively, the constant region may not be constant region of the heavy chain of immunoglobulin.

Preferably, the methods of the present invention result in normal B-cell maturation. The present invention also relates to an antibody having a heavy chain, or fragment, or a mixture of classes of antibodies having only heavy chains, obtained or obtainable by a method of the present invention. Such an is Italo, having only heavy chains, can be a monoclonal antibody or fragment, such as a binding domain VHman or camel. Binding domain of the VHaccording to the present invention may not contain verbludogorka extended CDR3 loop or, alternatively, may contain verbludogorka extended CDR3 loop.

The present invention also relates to a vector containing a heterologous heavy chain locus of the present invention, and to the cell host transformed by such a vector.

The present invention also relates to a transgenic mammal expressing a heterologous heavy chain locus as described in the present description. Preferably, the transgenic mammal of the present invention has a reduced ability to produce an antibody containing light chain.

The invention relates to the use of antibodies having a heavy chain, or fragment of the present invention for the manufacture of drugs for immunotherapy. Antibodies of the present invention, with only the heavy chain, can also be used as diagnostic tools, reagents, Asimov or inhibitors. The invention relates to pharmaceutical compositions containing antibody having only heavy the s-chain, or its fragment according to the present invention, and a pharmaceutically acceptable carrier.

The present invention also relates to a method for production and selection of antibodies having a heavy chain, which provides the steps:

(a) injection of antigen transgenic mammal as described in the present description;

(b) selection of cells or tissue expressing interest antigen-specific antibody having a heavy chain; and

(c) create hybridoma from a cell or tissue of step (b), and

(d) optionally, cloning, mRNA antibody having a heavy chain, from the specified hybridoma for further production in a heterologous expression system, such as a system of mammals, plants, insects, bacteria, fungi, or in the alternative system.

Then binding domains VHcan be produced by identification and isolation of antigen specific domain VHof cloned mRNA from step (c).

Binding domains of the VHaccording to the present invention can also be obtained by:

(a) injection of antibodies transgenic mammal described in the present description;

(b) selection of cells or tissue expressing interest antigen-specific antibody having a heavy chain;

(c) cloning of the locus VHfrom MRR is, obtained from selected cells or tissues;

(d) display the encoded protein using phage or similar library.

(e) identification of antigen specific domain (domains) VH; and

(f) expression domain (domains) VHseparately or in the form of a fused protein in a bacterial, yeast or alternative systems of expression.

Detailed description of the invention

The present invention allows to overcome the limitations of the prior art and it shows that transgenic animals, in particular mice, can be created using "microlocal for product class-specific antibodies, with only a heavy chain, or a mixture of different classes of antibodies having a heavy chain, which are secreted by plasma or B-cells. Then you can use them either to obtain reliable delivery of class-specific antibodies, with only the heavy chain, using established technology hybrid, or as a source of functional binding domain of VH(VHH) camel or binding domain of VHonly the heavy chain, preferably a soluble binding domain of VHonly the heavy chain of the person deprived of effector functions, but which retain binding function.

Antibodies having only heavy chains (including antibodies camel), the cat is which can be obtained by the methods of the present invention, exhibit high affinity binding, which is the result of a rearrangement of gene segments, V, D and J and somatic mutations, usually without the extended CDR3 loop. Essentially normal B-cell maturation is observed at high levels of antibodies having a heavy chain, a dedicated plasma (it is assumed that the domain is CH1 is absent in all classes of antibodies, present in the recombinant locus). B-cell maturation and secretion collected dimers (e.g., IgG) or multimeric (e.g., IgM) does not depend on the presence or gene expression of light chain.

Analysis of the nucleotide sequence of the antigen-specific mRNA that encodes antigen-specific heavy chain selected from hybridomas obtained from transgenic mice showed that the diversity of antibodies having a heavy chain, is largely a function of VDJ recombination. In addition, the authors present invention has been shown that the diversity of antibodies is formed in a region CDR3 functional antigennegative domain antibody having a heavy chain, with more limited contribution of somatic mutations in domain VH. Using the methods disclosed in the present description, the functional domains of the VNcan be cloned and can be expressed in bacterial systems to obtain binding domain of VH while fully retaining antigennegative ability, specificity, and affinity. In addition, the class-specific dimers or multimer heavy chain can secretariats in the culture of hybridoma cell lines.

On the basis of the present invention is also shown that transgenic mice can be programmed for products preferred classes of antibodies having a heavy chain, in response to antigenic stimulation, such as IgG or anti IgM, or a mixture of, for example, IgA, IgG and IgM.

The authors of the present invention previously described (see patent application WO02/085945 and WO02/085944) creation of transgenic mice expressing minimal devoid of exon CN1 the locus of the constant region of the heavy chain of IgG person connected segments of the human D and J with two genes VHHlamas. Upon antigenic stimulation, they have produced a functional high-affinity antigen-specific IgG antibody having a heavy chain. A mixture of classes of antibodies (IgM and IgG), with only the heavy chain can be obtained by switching classesin vivothrough the use of gene constructs, including the constant region of the heavy chain in tandem (assuming that all genes const areas devoid, domain CH1 and, if present, CH4 domain).

The enhancements described in this the Scripture, show that the mouse is designed with the same locus of the constant region of IgG, connected by segments of the human D and J with two genes VHHLama, and the locus of the constant region of IgM of the person deprived of exon locus CH1, which is connected with the same gene segments of the human D and J genes with VHHLama, also produces high molecular weight (multimeric) antibody IgM, having heavy chains, antibody IgG (dimer), with only the heavy chain. Interestingly, essentially normal B-cell maturation and the production of antibodies depends on the total absence of sequences CH1 in each constant region of the heavy chain, which is available in the transgenic locus. Moreover, if there exon CN4, its removal is not required.

Thus, for example, the transgenic animal carrying the locus of the heavy chain of IgM person with a functional exon CH1, connected with the same gene segments of the human D and J with two gene segments V Lama, and the locus of the constant region of the heavy chain of IgG, devoid of exon CH1, which is connected with the same gene segments of the human D and J with two gene segments V Lama, produces a small amount of an antibody having a heavy chain, and does not show the presence of B-cell maturation.

Other effectory the domains including domain CH4, can be included or not, as desired, with the purpose of introducing or removing from the resulting antibody having a heavy chain effector properties.

The authors of the present invention found that the productive expression of antibodies (i.e. B-cell maturation) can be obtained as a result of the use of any gene segment V located in the structure. The selection and sequencing of mRNA antibodies derived from the B-cells, showed that recombination of gene segments D and J, generera diversity of CDR3. Comparison of the sequences obtained domain VHrevealed somatic mutations, indicating that the recombinant gene segments D and J, as well as in the domain VHreceived downregulation of mRNA antibodies events occurred affinity maturation.

Preferred designs include gene segments V, selected or developed for improved solubility and associated with the cluster chains D and J for recombination and generation of CDR3. Preferably, VDJ sequences associated with constant effector domain (domains) of choice in tandem, each of which lacks exon CN1.

The present invention is not limited to obtaining and production of class-specific human antibodies or camel, with only the heavy chain, or binding the common domain V Hperson (preferably soluble binding domain of VH) (single or associated with effector domain by choice), but also covers the production of chimeric combinations of any gene segment V, obtained from a vertebrate animal (optional, designed with improved solubility characteristics)associated with gene segments D and J. Preferably, the gene segments V are gene segments are human and are not gene segments V camel. The resulting domains VHmay not contain elongated verbludogorka CDR3 loop, except that the segments D and J obtained from the camel. As a result, this gives the domain of VHshowing the diversity of the CDR3 and affinity maturation, not necessarily associated with effector constant region. The latter guarantees a functional secretion and, optionally, the Assembly in the parent transgenic spinal animal of their choice, and also provides further choice effector functions if necessary.

These data have important results for the improved and simplified design class-specificeskich antibody having a heavy chain, and obtain high-affinity soluble domains of the VHthat includes the affinity maturation through somatic mutation. Introduction the selected effector function of the Nations of the constant region of the heavy chain (devoid of C N1) or mixtures thereof makes possible the production of antibodies having a heavy chain of any class or any mixture of antibodies having a heavy chain, without the need for additional development of antibodies. Domains VHcan be expressed separately in bacterial systems or the systems of other organisms or functional antibody having a heavy chain and containing effector domains, which is secreted by hybridomas or transfected cells in culture. Antibodies and binding domains of the VHhumans are widely used in the field of health in the form of medicines, diagnostics and reagents, as well as find application in agriculture, in areas related to environmental protection, and industrial areas.

Thus, in the first aspect of the present invention relates to a method for producing antibodies having a heavy chain VH, transgenic mammal, which provides a stage for the expression of heterologous heavy chain locus VHin the specified mammal. Preferably, the heavy chain locus VHcontains a constant region of the heavy chain, which does not encode domain CH1, and the locus, which can form when the expression of a diverse set of full antibodies having only heavy the chain.

The first aspect of the present invention also relates to a method of production of antibodies, with only the heavy chain VHcamel transgenic mammal in which a stage of the expression of the heavy chain locus VHcamel in that mammal, and the locus of the heavy chain VHcontains a constant region of the heavy chain, which does not encode domain CH1, and the locus of which is when the expression is able to generate a diverse set of full antibody with the heavy chain, including VDJ-restructuring and affinity maturation in response to antigenic stimulation.

Effector molecules of the heavy chains can be designed to be a free functional domains, for example carboxykinase domains CH4, provided that their creation had no effect on secretory mechanisms that prevent the Assembly from the cell surface and, therefore, B-cell maturation. From heterologous locus or remove this locus is missing only the exons of CH1. The locus can be developed with additional features, for example, to improve glycosylation or additional functions.

Preferably, heterologous locus when the expression is able to form functional molecules of IgA, IgE, IgG, IgD or IgM, or their isotopes. Can also be obtained from the separate classes of antibodies or a mixture of classes of antibodies or their isotopes.

Therefore, the heterologous heavy chain locus design for the production of the preferred classes or mixtures of antibodies having a heavy chain, depending on the required class(classes) antibodies, with essentially normal B-cell maturation. The use of gene segments, V, D and J camel and effector regions camel will produce antibodies camel with features specific to camel, for example, antibodies with long CDR3 loops. The use of gene segments of the human V, D and J containing gene segments V, randomly selected, or selected or designed to enhance solubility, will produce human antibodies having a heavy chain.

Antibodies obtained by the present invention have advantages over antibodies of the prior art consists in the fact that the antibodies of the present invention are any one or a known class and, preferably, are human. Antibodies are high affinity due to the combination of VDJ recombination and affinity maturationin vivo. Antibodies and their fragments can be isolated, characterized and obtained using well-known methods known to experts in this field of technology.

The heterologous heavy chain locus

In the context of the present and obrane the term “heterologous” refers to nucleotides sequence or locus, as described in the present description, which is not endogenous to the mammal, in which it is located. “The locus of the heavy chain VH” in the context of the present invention refers to the minimum micrologus, codereuse domain VHcontaining one or more gene segments V, one or more D gene segments and one or more J gene segments, optionally associated with one or more effector regions of the heavy chains (each of which is devoid of domain CH1). Preferably, the primary source of variability of a set of antibodies is the CDR3 region, formed by the selection of gene segments D and J using compounds V-D and D-J.

An advantage of the present invention is the fact that the range and diversity of antibodies obtained in the reconstructed gene sequences VHthat can be maximized through the use of multiple gene segments D and J. Subsequent somatic mutation is achieved, while using the minimum locus (micrologus), without the need of a large number of gene segments loci V or VLand Lc (light chain) immunoglobulin.

Preferably, the heavy chain locus VHcontains from two to five (2, 3, 4 or 5) gene segments V, obtained from any species of vertebrate animal.

Preferably, g is installed segments V are human, not necessarily, selected or designed to improve the solubility.

Preferably, the heavy chain locus VHcontains from two to forty(2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30 or 40) or more gene segments D. D Gene segments may be derived from any species of vertebrate animal, most preferably, D gene segments represent the gene segments of the human D (usually 25 functional gene segment D).

Preferably, the heavy chain locus VHcontains from two to twenty(2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20) or more gene segments J. J Gene segments may be derived from any species of vertebrate animal, but most preferably, the gene segments J represent the J gene segments person (usually 6 gene segments (J).

Preferably, the heavy chain locus VHcontains two or more gene segments V, twenty-five functional gene segments D man and 6 gene segments of human J.

The term “gene segment V” encompasses naturally occurring gene segment V, obtained from a vertebrate animal, including camel and man, which is not necessarily selected, mutated, or designed to improve characteristics, such as solubility. Gene segments V also found in other species, such as shark (see Kokubu et al., (1988) EMBO. J., 7, 3413-3422), or they evolved to provide a variety of VH-like collections of binding proteins, is an illustration of what, for example, is the evolution of a set of immunoglobulin light chain VLor set of VHT-cell receptor.

Preferred ways of improving the solubility of the domain VHinclude logical, as opposed to just random, tools, and described Davies and Reichmann, (1996) Protein Eng., 9 (6), 531-537 and Riechmann and Muyldermans, (1999) J Immunol. Methods, 231, 25-38. Can also occur natural selectionin vivoin the result of affinity maturation and adding useful mutations in the gene VHafter VDJ-adjustment.

Gene segment V must be capable of recombination with a gene segment D gene segment J and constant (effector) region of the heavy chain (which can contain multiple exons, but lacks exon CH1) according to the present invention for generating antibodies having a heavy chain VHin the case of the expression of the nucleic acid.

Gene segment V according to the present invention also includes within its scope any gene sequence encoding a homologous, modified or protein fragment which is capable of recombination with a gene segment D gene segment J, and constant region of the heavy chain (containing the same is or more exons, but lacking exon CH1) according to the present invention for generating antibodies having a heavy chain, as defined in the present description.

Thus, VHcoding sequences can be obtained from a naturally occurring source or they can be synthesized using methods known to experts in this field of technology.

Domain VH” in the context of the present invention relates to the expression product of the gene segment V after recombination with a gene segment D gene segment J, as defined above. Preferably, the domain of VHas used in the present description, remains in solution and is active in physiological environment without the need for any other factor to maintain solubility. Preferably, the ability of the soluble domain of the VHto bind the antigen is improved by VDJ recombination and somatic mutation. There is no dependence on the presence or absence of the extended CDR3 loop, characteristic species of camels. Domain VHalso able to bind the antigen in the form of monomer in the case of Association with effector constant regions can be produced in the form monospecific, bespecifically, multispecificity, bivalent or multivalent forms, depending on the choice and RA is processing used effector molecules (e.g., IgG, IgA, IgM, and so on) or alternative mechanisms of dimerization and multimerization. Any chance of binding to domain VLif expressive as part of a soluble antibody having a heavy chain, the complex is eliminated by deletion of exon CH1 (see Sitia et al., (1990) Cell, 60, 781-790). One domain VHalso can be constructed with different protein domains for producing fused target proteins for therapeutic and diagnostic tasks, for example, toxins, enzymes and agents rendering.

In the context of the present invention, the terms “gene segment D” and “gene segment J” includes naturally occurring sequence of gene segments D and J. Preferably, the gene segments D and J receive the same vertebrate animal from which the genetic segment of V. for Example, if the gene segment V receive from a person and then increase the solubility or develop it, gene D and J segments are also preferably receive from man. Alternatively, gene segments V can be obtained, for example, camel, and gene segments D and J - from the person or Vice versa.

The terms "gene segment D" and "gene segment J" also encompass derivatives, homologues and fragments thereof, provided that the received segment can recombine with the remaining components the customers locus heavy chain antibodies, as disclosed in the present description, to generate antibodies having a heavy chain, as disclosed in the present description. Gene segments D and J can be obtained from naturally occurring sources or they can be synthesized using methods familiar to specialists in the art and disclosed in the present description. Gene segments, V, D and J are capable of recombination and preferably exposed to somatic mutations.

Gene segments, V, D and J, preferably, receive from one type of vertebrate animal. This can be any vertebrate animal, but preferably a human.

In addition, the heterologous heavy chain locus according to the present invention contains the DNA encoding the constant region of the heavy chain, which provides effector functionsin vivo(for example, IgG, IgM, IgA, IgE, IgD or isotypes).

The present invention also provides antigen-specific antibody having a heavy chain, received or obtained by the methods of the present invention.

The constant region of the heavy chain

Functionally, the constant region of the heavy chain is encoded natural or engineered gene segment that is capable of recombination with a gene segment V gene segment D gene segment J in the B-cell. Preferably, the constant on the region of the heavy chain derived from immunoglobulin locus.

According to this aspect of the present invention, each constant region of the heavy chain essentially contains at least one gene constant region of the heavy chain, which is expressed without any functional domain of CH1 so that it was possible to obtain antibodies having a heavy chain. Each constant region of the heavy chain may also contain one or more additional exons constant region of the heavy chain, which is selected from the group consisting of δ, γ1-4, S, ε and α1-2with the condition that additional genes for the constant region of the heavy chain also does not Express a functional domain of CH1. Gene segments of the constant region of the heavy chain is selected depending on the desired preferred class or a mixture of classes of antibodies. Optionally, the heterologous heavy chain locus deprived Cµ and Cδ.

For example, it is known that the molecules of the Ig class M play an important role in the activation of macrophages and complement. Because their binding sites are in close proximity, IgM has a high avidity for pathogens, including viruses. However, it is also known that IgM is difficult to use rapid immunoassay methods, while Ig G class can be used in these ways without difficulty. For such applications it is often useful to choose before actually class antibodies that is, IgG or IgM.

Expresia all or part of the heterologous heavy chain locus γ devoid of CN1, it will not be necessary to produce some or all of the isotypes IgG, depending on the presence of heterologous locus IgG isotypes IgG1, IgG2, IgG3 and IgG4. Alternatively, the heavy chain may contain genes by cε. In therapy can also be used IgE molecule.

Alternatively can be received by the selected mixture of antibodies. For example, IgA and IgM can be obtained if the constant region of the heavy chain contains the genes Cα and Cµ.

Preferably, the constant region of the heavy chain according to the present invention is of human origin, in particular, if the antibody having a heavy chain, should be used for therapeutic applications in respect of the person. Where antibodies having a heavy chain, should be used for diagnostic or veterinary purpose, the constant region of the heavy chain preferably receive from the body of the target, vertebrate or a mammal, in respect of which or over which the conduct of a diagnostic study or veterinary therapy.

During the expression of the constant region of the heavy chain is deprived of the functional domain of CH1. Exon CH1 and need not be constant region Cµ and Cδ can be all goty mutations, deletion or replacement. Preferably, the exon CH1 is subjected to deletions. The presence of, for example, IgM functional domain CH1 inhibits B-cell maturation and, therefore, limits the productive expression of IgG (devoid of CH1), with only the heavy chain within the same locus, as B-cell maturation ingibirovalo.

“Exon constant region of the heavy chain (exon CH”), as disclosed in the present description includes the sequence of naturally occurring exons of CHvertebrate animal, especially a mammal. Exon CHchanged class-specific way. For example, IgG and IgA in a natural way devoid, domain CH4. The term “exon CHalso includes within its scope derivatives, homologues and fragments as the exon CHable to form a functional antibody having a heavy chain, as defined in the present description, if it is a component of the constant region of the heavy chain.

Optional, if present, CH4 or other functional domains can be developed or deleterow inside of the transgene, provided that such a process did not inhibit intracellular secretory process, B-cell maturation or binding activity of the resulting polypeptide antibodies

Mammals

Transgenic mammal used in the methods of the present invention, is not a person. Transgenic mammal is preferably a rodent, such as rabbit, Guinea pig, rat or mouse. Especially preferred mouse. Can also be used in alternative mammals, such as goats, sheep, cats, dogs or other animals.

Preferred transgenic animals created using conventional technology injection of oocytes and, if developed, ES-cell technology or cloning.

Mainly, according to the methods of the present invention, the heavy chain loci and optional light chain immunoglobulins, endogenous regarding animal is subjected to deletions or slesingr, when expressed antibody having a heavy chain.

This approach of generating antibodies having a heavy chain, as described above, can have a particular use for the generation of antibodies for therapeutic applications in relation to a person in the form of frequent introduction of antibodies individuals spine, which is derived from the source of antibodies, which leads to the manifestation of the immune response against these entered antibodies.

Therefore, in an additional aspect, the present invention provides ranchisee mammal, expressing a heterologous heavy chain locus according to the present invention.

Transgenic mammal can be created so that it had a reduced ability to produce antibodies that comprise a light chain.

Antitelomerase cells can be obtained from transgenic animals according to the present invention and used, for example, when creating hybrids for producing antibodies having a heavy chain, as defined in the present invention. Additionally or alternatively, the nucleic acid sequences can be isolated from transgenic mammal according to the present invention and used for the production of antibodies, with only domains VHheavy chains or bespecifically/bifunctional complexes, using recombinant DNA technology, which are well-known specialists in this field of technology.

Alternatively or additionally, according to the present invention, antigen-specific antibodies, with only heavy chains, can be generated by immunization of a transgenic animal.

Thus, in an additional aspect, the present invention provides a method of producing antibodies having a heavy chain, immunization with antigen transgenic mammal according to the ACLs of the present invention.

In a preferred embodiment, the specified aspect of the present invention the mammal is a mouse.

Antibodies having only heavy chains, and fragments

In an additional aspect of the present invention is an antibody having a heavy chain produced according to the method of the present invention, and its functional fragments and derivatives. Fragments covering the binding domain of the VHcan be obtained by enzymatic cleavage or splitting cyanogenmod antibodies of the present invention, with only the heavy chain, that is, devoid of light chains (Jaton et al., (1968) Biochemistry, 7, 4185-4195).

Preferred functional fragment is an antigen-specific binding domain only heavy chains, i.e. binding domain VHas expressed locus VHas a result of recombination between individual gene segments, V, D and J, which later should somatic mutation. According to this aspect of the present invention loci VHcan be cloned from, for example, mRNA is isolated from Enciclopedia cells immunized transgenic animal as described above. Cloned sequences can then be displayed using phage libraries (Ward et al., (1989) Nature, 341, 544-546) or similar is on display, for example, through the use of systems based on yeast (Boder and Wittrup, (1997) Nat. Biotechnol., 15, 553-7), and antigen-specific binding domains VHidentified. Antigen-specific binding domains of the heavy chain can then be made either separately or in the form of a fused protein in dimensional variable bacterial, yeast or alternative systems of expression. The sequence encoding the binding domains of the VHcan also be characterized cloned from hybridomas obtained by classical procedures from immunized transgenic mice. Then they can be used for producing binding domain of VHand their derivatives, including the development of certain classes of antibodies (e.g., IgE, or IgA), and their variants with different effector functions.

Therefore, the present invention also provides a method of producing a binding domain VHcontaining phases in which:

a) allocate a cell or tissue expressing interest antigen-specific antibody having a heavy chain (preferably, interest soluble antigen-specific antibody having a heavy chain);

b) clone sequence encoding a binding domain VHfrom the mRNA obtained from the selected cells Il the tissue;

c) display the encoded protein using phage or similar library.

d) identify antigen-specific binding domains VHand

e) Express binding domains VHseparately or in the form of a fused protein expression systems in bacteria, yeast, mammalian or alternative systems.

Alternatively, the fragments containing the domain VHcan be generated from an antibody of the present invention, with only the heavy chain, by using enzymatic or chemical methods of splitting and subsequent separation of the fragment containing the domain VHfrom other fission products.

Where the binding domain of the VHselected from characterized hybridoma, the cloned sequence of the binding domain of the VHderived from the mRNA can be cloned directly into the expression vector without an additional source of stages of selection using phage system or other display.

System producing antibodies having a heavy chain containing the effector region include mammalian cells in culture (e.g., CHO cells), plants (e.g. maize), transgenic goats, rabbits, cattle, sheep, chickens and insect larvae that are suitable for the technology of mass rezidentura system of production, including infection with virus (e.g. baculovirus in insect larvae and cell lines)represent alternative approaches using cell culture and the germ line. Other methods of production are well known to specialists in this field of technology. Where the Assembly IgA or IgM, having heavy chains, useful coexpressed chain “J”. Suitable methods of producing antibodies of a camel, with only the heavy chain, or some binding domain of VHknown in the art. For example, the binding domains of the VHcamel were produced in bacterial systems, and homodimer camel, with only the heavy chain were produced in hybridomas and transfected mammalian cells (see Reichmann and Muyldermans, (1999) J. Immunol. Methods, 231, 25-38).

Also, it is generally accepted ways of expression developed binding domain of VHman, obtained using phage display technology resulting in (Tanha et al., (2001) J. Biol. Chem., 276, 24774-24780 and references there).

It is shown that insect larvae from lines of transgenic flies produce functional fragments of the antibodies having a heavy chain, in hemolymph with symptoms indistinguishable from the same antibodies produced by mammalian cells (PCT/GB2003/0003319). The present invention also p is dostavljaet antigen-specific Monomeric or dimeric binding domain V Nobtained according to the method of this aspect of the present invention.

The present invention also provides polynucleotide the sequence of the heterologous heavy chain locus, selected polynucleotide encoding the antibody of the present invention, with only the heavy chain, and a vector containing a heterologous heavy chain locus, or a fragment, or a dedicated polynucleotide encoding an antibody having a heavy chain according to the present invention.

The present invention also provides a cell host transformed with a heterologous heavy chain locus or its fragment, or a dedicated polynucleotide coding for the antibody having a heavy chain, or fragment of the antibody according to the present invention.

In a second aspect the present invention provides a polypeptide complex that contains the antigen-specific binding domain VHaccording to the present invention with attached effector site which provides effector activity. This effector activity can be complementary to the effector activity, provide a constant region of the heavy chain and can be located at the amino - or carboxylic molecules. Such polypeptide complexes remain physiologically is such a function, which confers antigen-specific binding domain VHin combination with additional guiding function and effector function of effector sites. Such polypeptide complexes can be in the form of functional monomers or depending on the design and interaction of the effector sites of the dimer, tetramine, pentamerone, multimarine or other complexes comprising different binding domains VNso providing multivalently and multispecificity. Binding domains of the VHcan be at the amino - or carboxylic binding molecules (see figure 1 for an example of the dimer).

If the effector site contains binding domain, it may have specificity, non-antigen-specific binding domain VH. The advantage of this structure is that the polypeptide complex may facilitate the crosslinking of various targets. For example, bespecifically polypeptide complex can be used to enhance interactions cell-cell interactions cell/pathogen. In this embodiment, the polypeptide complex of the present invention can be used, for example, for the formation of a bridge between the two types of cells, such as pathogen and macrophage (see Biburger et al., (2005) J. Mol. Biol, 34, 1299-1311). In such bespecifically developments using binding domain of VHpreferable to use binding domain of scFV. Binding domains of the VHhave a high affinity binding and may be entered in such polypeptide complexes using minimal vector designs and excluding development needed to maintain the specificity and affinity of scFV on their tetramer parent molecule. Where provided by the dimers or multimeric polypeptide complexes, enter the dimerization domains, such as include the domain CH2 and CH3, derived from constant regions of the heavy chains of immunoglobulins (see figure 2).

The term “effector site”, as used in this description includes any site which mediates the cell desired biological effect. Effector site preferably is soluble and can be a peptide, polypeptide or protein, or may be ones structure. For example, the effector site can be an enzyme, a hormone, a cytokine, a drug, a prodrug, a toxin, in particular protein toxin, a radionuclide in the chelate structure, binding domain, dimerization domain or interaction, the agent visualization, albumin or inhibitor.

Albumin can be used is as an effector site for uvelicheniya stability or pharmacokinetic, and/or pharmacodynamic properties of the antigen-specific binding domain VH(Sung et al., (2003) J. Interferon Cytokine Res., 23 (1): 25-36). Alternatively, the effector may be paglinawan structure or naturally glycosylated framework for improving the pharmacodynamic properties.

The effector may be a peptide that is associated with antigen-specific binding domain VHor it can be chemically linked to interspecifically domain VHthe heavy chain, for example, by chemical bonding patterns, such as maleimides the linker. Alternatively, the polypeptide complexes of the present invention can be expressed in the form of a fused protein. Essentially the present invention also encompasses a polynucleotide sequence comprising the heterologous heavy chain locus or dedicated polynucleotide encoding the antibody of the present invention, having only a heavy chain, and polynucleotide further comprises a frame read one or more of the exons encoding the effector phase. This exon may be located at the 5'- or 3'-end of polynucleotide. For example, polynucleotide may contain, in the order of and in reading frame, VHand gene segment binding domain/e is vectorlogo plot. In the case of genetic m attaching different domains can be achieved by using the construction of recombinant DNA that encodes the amino acid sequence of the fused protein, the DNA encoding various domains located in the same reading frame. These designs are valuable as diagnostic and therapeutic tools. As a diagnostic tool, the effector domain can be a fluorescent protein (e.g., GFP) or an enzyme (e.g. β-gal). Alternatively, the effector domain can be a tag to enhance bonding with the substrate (for example, polyhistidine or Biotin) and the antigen to ensure site of attachment for the secondary antibody or lacinova lightning, or similar binding motif, which can serve as the site of attachment of fluorescent markers.

Polypeptide complexes

The authors present invention also suggested that there is a possibility of producing bivalent or multivalent polypeptide complex that contains at least a portion of the heavy chain antibody by one or in combination with a separate effector (light) chain containing complementary domain Assembly and having additional effector activity. Polypeptide complexes according to the present is th invention maintain physiological function, provide constant region of the heavy chain in combination with additional effector functions of the site associated with the effector circuit (figure 3).

Essentially, in the third aspect, the polypeptide complex contains the heavy chain in combination with one or more effector chains (light chain). The second aspect of the present invention provides a polypeptide complex containing a pair of heavy chains and a pair of effector chains and:

the heavy chain pair are connected to each other;

one of the effector circuits associated with one of the heavy chains, and other effector circuit associated with another heavy chain;

each heavy chain contains a binding domain, a dimerization domain, preferably containing at least the domains CH2, CH3 and optional CN4 constant region, and the effector site, the ability to communicate with a complementary domain of the effector Assembly circuit; and

effector circuit includes complementary domain Assembly with attached effector site,

the domain Assembly and the complementary domain of the Assembly are connected to each other through non-covalent interactions.

Preferably, the effector site in the heavy chain differs from the effector site in the effector chain.

Optionally, the polypeptide complex comprises died the s sanirovanii domain at the carboxyl end of the domain C H3 (or domain CH4, if it is present), linking it to the domain of the Assembly. Preferably, the polypeptide complex includes natural hinge domain or flexible designed sanirovanii domain between a binding domain and a domain CH2. The presence of the hinge regions facilitates the independent functioning of the binding domain and the effector sites in the resulting polypeptide complexes.

The effector site in the first polypeptide heavy chain optionally has a specificity that is different from the specificity of the effector site of the second polypeptide heavy chains. According to the present invention effector plot polypeptide complex can be replaced by a binding domain. Preferably, the binding domain contains a domain VH(as defined in the first aspect of the present invention), or a domain that binds a cellular receptor. Received dimeric tetravalent binding protein (polypeptide complex) can contain up to four different effector sites. Preferably the effector sites on aminoterminal the end of the heavy chain are identical, and effector sites on the carboxyl terminal end identical (but recognize the antigen or epitope that is different from the antigen or epitope on aminoterminal the end), facilitating the Assembly of single-glycosilated. Taka is the molecule may be more effective in the capture of pathogens, this effector functionality is provided by the inclusion of suitable functional domains of the heavy chain (e.g., IgA or IgM).

Illustrative polypeptide complex according to the third aspect of the present invention is useful for cytochemical labeling, methods of targeting or therapy, for example, if the effector molecule contains antigen-specific binding domain VHthat targets the surface marker of the cancer cells, and effector site contains a binding domain specific for an enzyme that converts a prodrug (effector circuit). Antigen-specific binding domain VHassociated with the target and delivers effector site in close proximity to the target so that when the binding of the effector chain he can have a biological effect on the target in the presence of prodrugs (e.g., nitroreductase with CB1954). Activate effector functions of the heavy chain of the immunoglobulin domain dimerization can also be useful for eliminating mistaway cells.

Effector chain

Effector circuit includes complementary binding domain and the effector site, which is linked to a heavy chain by using the effector site of the heavy chain, forming the assembled binding polypeptide complex. Complementary the first domain of the effector Assembly of the circuit can be an integral component of the effector phase or protein, or an alternative ligand, fused or chemically bound to the effector site. The heavy chain of the collected binding polypeptide complex is contacted with the target and deliver the effector site (light) chain in close proximity to the target, so you can have an impact on the target.

Effector site

The term “effector site”, as used in this description includes any site which mediates the desired biological effect on the cell. The effector domain can be a cell, such as T-cell, peptide, polypeptide or protein, or may represent ones structure. For example, the effector domain can be an enzyme, a drug, a prodrug, a toxin, in particular protein toxin, a radionuclide chelate in the structure or binding domain. Effector site associated with complementary domain of the Assembly may be cellular, relcovaptan, be organic or inorganic origin, depending on the desired effect.

The term “binding domain”as used in this description in all of the above aspects of the present invention includes a polypeptide domain that is active in the physiological environment. Such a binding domain must also have the ability to connect aniu with the target under physiological conditions.

Such binding domains include domains, which may mediate the binding or adhesion on the cell surface. Suitable domains, which can be used in the polypeptide complexes of the present invention are molecules adhesion of mammalian cells, prokaryotic cells or viruses, cytokines, growth factors, receptor antagonists or agonists, ligands, cell surface receptors, regulatory factors, structural proteins and peptides, whey proteins, secreted proteins, proteins associated with the plasmalemma, viral antigens, bacterial antigens, protozoal antigens, antigens of parasites, lipoproteins, glycoproteins, hormones, neurotransmitters, blood coagulation factors, developed a single chain Fvs, etc. Preferably binding domain is a domain VHvertebrate animal, more preferably a domain VHa mammal, such as a domain VHperson.

Binding domain can contain domain VH(VHH) camel or may contain domain VHreceived from another animal. Preferably the binding domain is a domain VHman. Binding domains of the VHpreferably originate from B-cells derived from transgenic animals or camel (as described above), against which apologist domains V Hderived from synthetic phage libraries, because the first will have a higher affinity in the generation in response to antigenic stimulationin vivothanks VDJ-rearrangement and somatic mutation.

If the effector site contains binding domain, it preferably has a specificity that is distinct from the binding domain of the heavy chain. The advantage of this structure is that the polypeptide complex can facilitate cross-linking of different targets or bind different antigens on the cell-micene (e.g., pathogen).

Binding domain in the first heavy chain can have a specificity that is different from the specificity of the binding domain of the second heavy chain. Thus, the polypeptide complex can be at least bivalent and can cross sew various targets, and the effector domain can have an impact on both targets. Multivalent polypeptide complex can be created by linking such tetravalent heavy chains with effector circuits containing effector domains of different specificity (specificnosti) and functionality. Also the effector part of the first heavy chain can have a specificity that is different from the specificity of the effector part of the second heavy chain, enabling you to capture more odnoyoimennoj circuit, each of which has excellent functionality.

Complementary domain Assembly is associated with effector site

If the heavy chain is associated with effector chain, the term “effector site” and “complementary domain Assembly”, as used in the present description, include any areas that can generate at least attach to each other by non-covalent means. For example, the effector site and the complementary domain of the Assembly can be a protein, peptide fragment, or have a consensus sequence capable of forming interaction protein-protein, for example, which is present between domain CH1 heavy chain immunoglobulin and a constant region light chain immunoglobulin; lacinova lightning; VCAM and VLA-4; integrins and extracellular matrix proteins; integrins and cell surface molecules such as CD54 or CD102; ALCAM and SRCR domains, scFv and the antigen or binding domain VHand the antigen.

The heavy chain

Where the dimerization domains of the heavy chains contain a constant region heavy chain immunoglobulin constant region (exons CH) can provide additional physiological functionality binding polypeptide complex. In particular, the constant domains of the heavy chains immunodeficiency is globulines can provide, among other things, the fixation of complement activation and macrophage binding to Fc-receptors depending on the class or subclass of the constant domains of the antibody.

As discussed above, it is well known that the class of expressed heavy chain plays an important role in effector functionsin vivo. Established cell line can produce polypeptide complex having a useful targeting and biological effects, but the constant region of the heavy chain can refer to the class, which is undesirable from the point of view of diagnosis or therapy, or it may not secretariats in effective amounts. Accordingly, the constant domains of the heavy chain polypeptide complexes of the present invention can specifically be changed or partly or completely excluded for the introduction or removal of components of the heavy chains of immunoglobulins.

For example, it is known that the molecules of the Ig class M play an important role in the activation of macrophages and complement. Due to its location in the immediate vicinity of the binding sites IgM has a high avidity for pathogens, including viruses. However, it is also known that IgM is difficult to use rapid immunoassay methods, whereas Ig G class can easily be used in these ways. For such applications it would be useful to switch to the ACCA heavy chain with µ - γ-domain.

Expression of only the heavy chain locus Cγ will produce IgG, including isotypes IgG1, IgG2, IgG3 and IgG4, some of which will also activate the complement. IgG antibodies bind and activate macrophages and granulocytes and can penetrate through the placenta.

Additional use of different classes of antibodies discussed above.

Constant region heavy chain polypeptide complexes of the present invention can originate from a person, can occur from rabbits, rats or mice, as defined above. Preferably, they come from a man.

Polypeptide complexes of the present invention can also be used to block the binding of ligands to their receptors by dimerization domains, which do not provide effector functions. Using multispecific polypeptide complex can be blocked by a variety of receptors.

In the fourth aspect of the present invention, the effector molecule may contain a dimerization domain, such that the effector molecule may be associated with a single effector molecule. Such dimerization domain may contain one or more domains of CH2, CH3 or CN4 constant region of the antibody and/or j chain In this embodiment of the present invention two or more effectors the e molecules can be associated with the production of dimer or multimer effector molecules. The effector molecule may be the same (allowing the production of glycosilated or homomultimers effector molecules) or different (allowing the production of heterodimer or heteropolymer effector molecules). Preferably, the dimer or multimer effector molecule is bivalent or multivalent. Preferably, the constant region of two or more effector molecules (i.e., the dimerization domains) are identical, thus reducing the likelihood of heterogeneity of the product.

According to a fourth aspect of the present invention provides polypeptide complex containing a dimer of the first heavy chain polypeptide and the second heavy chain polypeptide, and:

each heavy chain polypeptide contains a binding domain and a dimerization domain, which optionally contains at least the domains CH2, CH3 and optional CH4 constant plot antibodies; and, optionally, the effector site, while preferably:

binding domain of the first heavy chain polypeptide has the same specificity as the binding domain and the second heavy chain polypeptide; and

the constant region domains (dimerization) for the two heavy chains of the polypeptide is identical.

Preferably, the first and the second circuit have the same effector sites.

Preferably, the dimerization domain contains at least the domains CH2, CN3 and optional CN4 constant region of the antibody.

A fourth aspect of the present invention also provides polypeptide complex, containing many polypeptide dimers heavy chain and the chain J, and:

many of the dimer polypeptide heavy chains are assembled with the chain J;

each polypeptide heavy chain contains the binding domain and is identical to the domain CH2, CH3 and optional CH4 circuits µ, ε, α or γ; and

in polypeptide complex there are at least two binding domain having a different specificity (see figure 4 and 5).

As defined above for the first aspect of the present invention, each constant region of the heavy chain preferably contains at least one gene constant region of the heavy chain, which is expressed without any functional domain of CN1 so that it can be obtaining antibodies with only the heavy chain. Each constant region of the heavy chain may also contain one or more additional genes for the constant region of the heavy chain, which is selected from the group consisting of Cδ, Cγ1-4; Cµ, by cε and Cα1-2provided that additional genes constant regions of the heavy chain is also not Express the comfort of the functional domain of C H1. The genes for the constant region of the heavy chain is selected depending on the desired preferred class or a mixture of classes of antibodies.

Preferably, there are only two binding domain of various spcifically in downregulation of IgA and IgM.

In one embodiment, the implementation of each of the heavy chains includes domain CH4, and the constant domains are the domains of α and polypeptide complex comprises a j chain

In another embodiment, each of the heavy chains includes domain CH4, and the constant domains are domains µ, and the antibody includes a j chain

Assembly polypeptide complex

The modular domain structure of polypeptide complexes of the present invention gives the possibility to construct them with a large number of possible permutations. Such changes in domain architecture and amino acid sequence of the polypeptide complex can be achieved by appropriate mutation or incomplete synthesis and replacement of candidate areas corresponding to the coding DNA sequences. Replacement or additional domains can be obtained from a compatible recombinant DNA sequences. For example, the heavy chain can include natural hinge or flexible polypeptide domain, both of which nahodatsa is between the binding domain and aminocom.com domain C H2 and between the effector domain and the C-terminal end of the heavy chain (CH3 or CN4).

Heavy chain polypeptide complex of the present invention are expressed in the form of a fused protein. Effector chain polypeptide complex of the specified aspect of the present invention can expressionate in the form of a fused protein or can be built using chemical means or, in the case of their cell of origin, can be isolated from blood or tissue, or captured byin vivo(e.g., albumin).

In the case of genetic m attaching different domains can be achieved by using recombinant constructs of DNA which encodes the amino acid sequence of the fused protein, the DNA encoding various domains that are in the same reading frame.

The effector site, if present as part of a fused protein may be located at either the amino-or carboxylic complementary domain of the Assembly.

Alternatively, the effector domains of the circuit can be built using conventional methods of peptide chemistry, as known in the art, and are not synthesized in the form of a fused protein.

The link can be formed using a peptide bond or by a chemical bond. For example, the effector may be a peptide, wired the th with complementary domain Assembly, or it can be chemically linked to a complementary domain of the Assembly, for example, by chemical bonding patterns, such as maleimides the linker.

Effector site can be located anywhere in the heavy chain. For example, the effector site can be located on the C-terminal end of the heavy chain or between the binding domain and any domain CH2 or hinge domain polypeptide complex. It is preferable that the domain of the Assembly was not between domains CH2 and CH3, because it can interfere with effector function and dimerization domains. Preferably, the effector site was attached to aminobenzo or carboxylic heavy chain using a flexible peptide linker or servirovochnoj region in order to facilitate independent binding/effector function parcel.

Polynucleotide sequences, vectors and cells-owners

The present invention also provides a polynucleotide sequence encoding a heavy chain of any one of the polypeptide complexes of the present invention, a vector containing one or more polynucleotide sequences related to the above sequence, and the cell host transformed by a vector encoding a heavy t the universi polypeptide complex of the present invention. Polynucleotide preferably include sequences that allow the secretion of expressed heavy chain in the form of homodimers in the environment in which growing a host cell. A host cell may be of any origin, including bacterial and yeast cells, but preferred is a host cell of vertebrate animal, more preferred a host cell of a mammal.

Transfection of the same host cell with a second vector encoding a heavy chain containing the binding domain with specificity for a different target, leads to co-expression of the two constructions and the Assembly mixture of homodimers and heterodimers. Homodimer will show specificity for cognate antigen, and heterodimer will bind both antigen.

The present invention also provides a cell host transformed by a vector encoding at least one effector chain polypeptide complex of the present invention. A host cell may be of any origin, including bacterial and yeast cells, but preferred is a host cell of vertebrate animal, more preferred a host cell of the mammal. Alternatively, the effector chain can be synthesized using methods known in the art.

The present invented the e also provides a cell-master, transformed by a vector encoding at least one heavy chain polypeptide complex of the present invention. A host cell may be of any origin, including bacterial and yeast cells, but preferred is a host cell of vertebrate animal, more preferred a host cell of the mammal. Alternatively, the heavy chain can be synthesized using methods known in the art.

The present invention also provides a cell host transformed by a vector encoding at least one heavy chain and at least one effector chain polypeptide complex of the present invention. A host cell may be of any origin, including bacterial and yeast cells, but preferred is a host cell of vertebrate animal, more preferred a host cell of the mammal. Alternatively chain can be synthesized independently and assembled using methods known in the art.

In addition, the present invention provides a transgenic organism expressing at least one Homo - or heterodimeric polypeptide complex of the heavy chains of the present invention. Transgenic organism can be a vertebrate animal is whether the mammal, not a person, plant or insect.

The present invention also provides a method of producing a class-specific antibodies, with only the heavy chain, and their domains VHaccording to the first aspect of the present invention by immunization with antigen transgenic organism of the present invention. In the preferred embodiment of this aspect of the present invention the organism is a mouse.

The production of antibodies and polypeptide complexes for applications in health requires large-scale production systems, examples of which are discussed in more detail above. Such systems include plants (e.g. maize), transgenic cattle and sheep, chickens and insect larvae, suitable for mass-rearing technology. Specialists in the art are also familiar with other systems of production, including infection with virus (e.g. baculovirus insect larvae and cell lines) alternatively, cell cultures and embryonic lines.

These methods and other suitable methods known in the art, can be used for producing binding polypeptide complexes of the present invention. By using these methods can be achieved by getting homodimers and/or gathered the Mer.

The use of antibodies, with only the heavy chain and the polypeptide complexes of the present invention

Antibodies having a heavy chain and binding polypeptide complexes of the present invention have many uses.

Antibodies having the heavy chain and the polypeptide complexes of the present invention include bi - and multispecific polypeptide complexes. These complexes are particularly useful, for example, as therapeutic agents for the treatment and prevention of infectious diseases.

Antibodies having the heavy chain and the polypeptide binding complexes of the present invention is useful for cytochemical labeling, methods of targeting therapy and as diagnostic tools.

When monoclonal antibody therapy evasion of a pathogen, for example, due to mutations leading to the loss of one binding site, is to terminate therapeutic effect of the antibody. The production of heterodimeric polynucleotide complexes that recognize different antigens on the same pathogen, can solve this problem. Use at least two binding domains having different specificity, polypeptide complexes of the present invention can also be used to reinforce the interactions of the cell the years, and interactions cell/pathogen.

In this embodiment, the polypeptide complexes of the present invention can be used, for example, for the formation of bridging polypeptide complexes between the two cell types, such as pathogen and a macrophage or a cancer cell and T-cell. Alternatively, the polypeptide complex can learn two or more epitopes on the same pathogen, while the effector function is provided only by the constant region of the heavy chain.

Alternatively bespecifically binding polypeptide complexes can be used to target cells and tissuesin vivothen to capture circulating effector molecules or agent rendering. For example, bespecifically agents aimed at the tumor, can be used to capture complexes, converting prodrugs, for subsequent localized conversion of prodrugs to the agent visualization. Bi - and multispecific binding complexes in combination with effector agents can also be used to link or destruction of one or more pathogens, depending on the choice of the binding domain. Alternatively, the presence of two or more binding domains that recognize different antigens on the same pathogen, providing the t clinical advantages, reduces the likelihood of escape of the pathogen and the excess of drugs as a result of mutation of the pathogen.

The present invention provides antibodies having only heavy chains, or fragments thereof according to the first aspect of the present invention, polypeptide chains and complexes according to the second aspect of the present invention; effectiye chains and polypeptide complexes according to the third aspect of the present invention. All of them are suitable for pharmaceutical use in relation to man, and thus the present invention provides a pharmaceutical composition comprising an antibody having a heavy chain polypeptide chain, the effector chain or polypeptide complex of the present invention. The present invention also provides the use of antibodies having a heavy chain polypeptide chain, the effector chain or polypeptide complex of the present invention in the manufacture of medicines for the prevention and/or treatment of the disease. Heavy and effector circuit can be used in a medicinal form, together or separately, depending on the method of administration or drug action.

Pharmaceutical compositions and medicaments are usually in the dosage form prior to the introduction of the patient.

N the example, antibodies having a heavy chain, or polypeptide complexes can be mixed with stabilizers, in particular if they are to be dried. Usually during lyophilization stability provides added sugars (e.g. mannitol, sucrose or trehalose), and the preferred stabilizer is mannitol. As a stabilizer may also be added serum albumin human (preferably recombinant). Can also be used a mixture of sugars, such as sucrose and mannitol, trehalose and mannitol, etc.

The composition may be added to the buffer, such as Tris-buffer, the his-tag buffer, glycine buffer, or preferably a phosphate buffer (e.g., containing sodium dihydrophosphate and phosphate sodium). It is preferred to add a buffer to obtain a pH between 7.2 and 7.8, and more specifically a pH of about 7.5.

For recovery after lyophilization can be used sterile water for injection. Also lyophilized cake can be recovered aqueous composition comprising serum albumin human (preferably recombinant).

Usually antibodies, with only the heavy chain and the polypeptide complexes are used in pure form together with pharmacologically acceptable carriers.

Thus, the present image is eenie provides a method of treatment of a patient, contains the introduction to the patient the pharmaceutical composition of the present invention. The patient is preferably a human, and may be the child (for example, a baby nursery or child, teenager or adult, but is usually an adult.

The present invention also provides antibodies having a heavy chain polypeptide effector chain or polypeptide complex of the present invention for use as a medicine.

The present invention also provides the use of antibodies having a heavy chain polypeptide chains, the effector circuits or complexes of the polypeptide chains of the present invention in the production of pharmaceuticals for the treatment of the patient.

These applications, methods and medicines are preferred for treatment of one of the following diseases or disorders: wound healing, cell proliferative diseases, including neoplasm, melanoma, lung tumor, colorectal tumor, osteosarcoma, rectal cancer, ovarian tumor, a sarcoma, cervical cancer, tumor of the esophagus, tumor, breast cancer, pancreatic cancer, bladder cancer, tumors of the head and neck and other solid tumours; myeloproliferative disorders, such as leukemia, lehocki the massive lymphoma, leukopenia, thrombocytopenia, impaired angiogenesis, Kaposi's sarcoma; autoimmune/inflammatory disorders, including Allergy, inflammatory bowel disease, arthritis, psoriasis, and inflammation of the respiratory tract, asthma, immune disorders and rejection of transplanted organs; cardiovascular and vascular disorders, including hypertension, oedema, angina, atherosclerosis, thrombosis, sepsis, shock, impaired reperfusion and ischemia; neuroglial disorders, including diseases of the Central nervous system, Alzheimer's disease, disorders of the brain, amyotrophy lateral sclerosis, and pain; developmental disorders; metabolic disorders, including diabetes mellitus, osteoporosis and obesity, AIDS and renal disease; infections including viral infection, bacterial infection, fungal infection and parasitic infection; pathological condition associated with the placenta and other pathological conditions and for use in immunotherapy.

In an additional aspect, the present invention provides the use of antibodies having a heavy chain, or binding polypeptide complex of the present invention as a diagnostic, prognostic or therapeutic agent imaging. Moreover, the present invention provides the use of the Goma is or heterodimer heavy chains of the present invention alone or in combination with one or more effector (light) chains of the present invention as a therapeutic agent imaging cytochemical reagent, or diagnostic agent.

The present invention provides the use of antibodies having a heavy chain, or fragment, as described above, as the intercellular binding reagent or abzyme. Preferred fragments of the antibody having a heavy chain, are soluble antigenspecific binding domains VH.

The present invention also provides the use of antibodies that have the same antigen-specific chain, or binding domain VHaccording to the present invention as an enzyme inhibitor or receptor blocker. Preferred antibody fragments having only heavy chains are soluble antigen-specific binding domain VH.

The present invention also provides the use of domain VHmerged with the effector molecule for use as a therapeutic agent, agent imaging, diagnostic tools, abzyme or reagent.

Brief description of drawings

Figa and 1B: shows the polypeptide complex containing domain dimerization (optional CH2, CH3 and CH4) binding domain (VH), and effector site (EM). Binding domains and effector sites can be located on the AMI is about - or carboxyterminal ends of the dimerization domains.

Listed flexible linkers (<-) and hinge (is) field.

Figa and 2B: shows the different configuration binding domain and replacing the effector site for more binding domains. A. Preferred option, as are produced by homodimer. Does not require separation of the products. B. is Produced by the mixture of homodimers and heterodimers. Required separation of the products.

Figure 3: shows the polypeptide complex of the heavy chains associated with the effector chain. Effector circuit includes complementary binding domain (CBD) and the effector site (EM). CBD is known EM heavy chain. CBD is fused to an effector or represents the portion of the effector, such as an enzyme, toxin, chelator, agent imaging. Effector chain can be synthesized separately from the heavy chain.

Figure 4 shows a bivalent secretory IgA, associated with the j chain

Figure 5 shows the multivalent IgM-like polypeptide complex having only heavy chains assembled with the chain J.

6: shows the strategy for the creation of transgenic mice expressing the IgG locus, and functional generate antibodies having a heavy chain, and domain VHthe result of antigenic stimulation.

Fig.7: shows the strategy for the creation of transgenic mice expressing IgM locus, and functional generation of antibodies, with only the heavy chain, and domains VHthe result of antigenic stimulation.

Fig: shows the strategy for the creation of transgenic mice expressing locus IgA, and functional generate antibodies having a heavy chain, and domain VHthe result of antigenic stimulation.

Figure 9: sequential alignment of PCR products obtained from bone marrow cDNA using primers VHH1 and VHH2 in combination with primer human Cγ2 from mice containing the locus of constant regions, which have a mutation of splicing camel to remove CH1. The results show that CH1 not deleted.

Figure 10-13: the structure of the VH/construction VH(VHH) camel. 1-n can support any number of genes VHor segments D or J. the Normal complement of the locus person has 51 gene V, 25 functional segments D (plus 2 non-functional segment) and 6 segments J. In the case of the field Cµ (IgM) or by cε (IgE) is absent area H, and has an additional exon CH4 between CH3 and M1. Gene (genes) VHmutated to ensure solubility, as described in publicly available sources.

Genes VHsegments D and J exons C are preferably from a human, but can be from any species, including camel. In the latter case, genes VH(VHH) camel can be it is mounted, because they are naturally soluble.

Fig: schedule of immunization of mice and analysis of antibodies to obtain IgG against E. coli HSP70, with only the heavy chain.

Fig: thread-cytometrics analysis and results of immunochemistry for spleen cells obtained from transgenic mice.

Fig: results of ELISA analysis of transgenic mice immunized with DKTP, and sequence analysis of the obtained library of antibodies.

Fig: examples of somatic mutations and VDJ-realignment observed in immunized transgenic mice.

Fig: the results of the analysis of immunostaining cell lines Tet-on, transfected increases the sensitivity of the plasmid containing the antibody A5.

Fig: the results of Western blot analysis of sera lines of transgenic mice.

Fig: fractionation by size IgM human mixed with IgM person with one chain produced by mice containing the locus IgM plus IgG.

Fig: results of ELISA of IgM and IgG antibodies with one chain generated against human TNFα.

Fig: shows the strategy of creating homodimeric plasmids with affinity binding to HSP70 and αGAG.

Fig: functional expression homodimeric polypeptide complex in CHO cells.

Fig: shows the functional binding and at the same time homodimeric polypeptide complex with alpha αGAG and HSP70 Schematic representation of a bivalent, especifismo antibodies. The second variable region (VHH2 directed against gag) cloned in carboxyterminal the end of antibodies having a heavy chain with a different specificity (VHH1, directed against HSP70). The hinge region between CH3 and VHH2 substituted by a linker region, in which all cysteine replaced by Proline (arrows). Tablet ELISA coated with Gag, blocked with 1% milk/1% BSA in PBS, first incubated in medium with recombinant bespecifically antibodies (dilution 1:2) and then in the cell lysate BI21 (containing HSP70) (dilution 1:2). Was suirable associated proteins with sample buffer =2-mercaptoethanol and dispersed in an 8% gel. Dyed poly/monoclonal antibodies against Gag, recombinant especifismo antibodies and HSP70. αGag: rabbit polyclonal/swine anti-rabbit-AP (blue). αHSP70: monoclonal/goat anti-human IgG-HRP (brown), recombinant bespecifically α antibodies: goat anti-human IgG-HRP (brown). Line 1: Gag/recombinant bespecifically antibody/BI21 cell lysate. Line 2: Gag/cultural environment represents a negative control recombinant bespecifically antibody)/BI21. Line 3: -milk-BSA/recombinant bespecifically antibody/BI21. Line 4: -milk-BSA/cultural environment/BI21. Line 5: Gag/recombinant bispecific the prioritization of antibody/milk-BSA. Line 6: Gag/cultural environment/milk-BSA.

Fig: shows the strategy of creating homodimeric polypeptide complexes, not necessarily associated with effector chains, IgA bearing effector function.

Fig: shows the strategy of creating homodimeric polypeptide complexes, not necessarily associated with effector chains, IgA bearing effector function.

Core technologies

If not stated otherwise, all technical and scientific terms used in this description have the same meaning as generally understood by experts in the art (for example, in the field of cell culture, molecular genetics, chemistry of nucleic acids, hybridization technologies and biochemistry). In molecular, genetic, biochemical methods (see review, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed., John Wiley & Sons, Inc.) and chemical methods use standard technology. Additionally see links Harlow &Lane, A Laboratory Manual, Cold Spring Harbor, N. Y, in the case of a standard immunological techniques.

Can be used with any suitable recombinant DNA technology for the production of bi - and multivalent polypeptide complexes, antibodies having at least one heavy chain, and their fragments according to the present izaberete the Oia. Regular expression vectors, such as plasmids, constructed containing DNA sequences encoding each of the polypeptide chains of the complex or antibody. Can be used with any suitable technology was developed for enzymatic or chemical fragmentation of immunoglobulins and separation of the resulting fragments.

The present invention also provides vectors, including constructs for expression of an antibody having a heavy chain in transgenic mice, construction, and expression of the polypeptide complexes of the present invention.

Obviously, that can be constructed unit vector which contains a DNA sequence encoding more than one polypeptide chain. For example, DNA sequences encoding two different heavy chains, can be put into different positions on the same plasmid.

Alternatively, the DNA sequence encoding each polypeptide chain, may be separately inserted into the plasmid, thus producing several constructed plasmids, each of which encodes a particular polypeptide chain. Preferably, the plasmid, in which the inserted sequences are compatible.

Then each plasmid is used to transform the host cell so that each the years-the host contained a DNA sequence, encoding each of the polypeptide chains in a protein complex.

Suitable expression vectors that can be used for cloning in bacterial systems include plasmids such as Col E1, pcR1, pBR322, pACYC 184 and RP4, phage DNA, or any derivative thereof.

For use when cloning in yeast systems suitable expression vectors include plasmids of size 2 micron.

Any plasmid containing a suitable promoter gene sequence of a mammal, can be used for cloning in the systems of mammals. Promotor sequence insects or baculovirus can be used for gene expression in insect cells. Such vectors include plasmids derived from, for example, pBR322, bovine papilloma virus, retroviruses, DNA viruses and vaccine viruses.

Suitable cell host, which can be used for expression of the polypeptide or antibody, and include bacteria, yeast and eukaryotic cells, such as cell lines insects or mammals, transgenic plants, insects, mammals and other expression systems of vertebrates and posvonotchnik animals.

Polypeptide complexes and antibodies having at least one heavy chain of the present invention

It should be noted that the terms which the polypeptide complex”, “antibody having at least one heavy chain and a heterologous heavy chain locus” of the present invention also include homologous polypeptides and nucleic sequences obtained from any source, for example an appropriate cellular homologues, homologues from other species, and their variants or derivatives.

Thus, the present invention encompasses variants, homologues or derivatives of the polypeptide complexes and antibodies, as disclosed in the present description.

In the context of the present invention homologous sequences include amino acid sequence that is at least 80, 85, 90, 95, 96, 97, 98, 99, 99,5, 99,6, 99,7, 99,8, 99,9% identical, preferably at least 98 or 99% identical at the amino acid level, more than at least 30, preferably 50, 70, 90 or 100 amino acids. Although gomologichnosti can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention, it is preferable to Express gomologichnosti in terms of the identity of the sequence.

The present invention also includes a constructed expression vectors and transformed cell hosts for use in the production of polypeptide complexes and antibodies present the th of the invention.

After expression of the individual chains in the same cell as the host, they can be restored to ensure the full polypeptide or antibody, having only heavy chains in the active form.

Have in mind that in preferred embodiments of the present invention separate the heavy chain can be subjected to processing in the cell host to form the complete polypeptide complex or antibodies, which mostly it is secreted. Preferably the effector circuit is produced separately or the host-cell, or synthetic means.

The methods of obtaining the polypeptide complexes of recombinant antibodies described in the above references, as well as, for example, in EP-A-0623679; EP-A-0368684 and EP-A-0436597.

Immunization of transgenic organism

In an additional aspect, the present invention provides a method of producing antibodies of the present invention, containing the introduction of the antigen to a transgenic organism of the present invention.

Antibodies and polypeptide complexes produced transgenic animals of the present invention include polyclonal and monoclonal antibodies and their fragments. If you need polyclonal antibodies, transgenic animal (e.g., mouse, rabbit, goat, horse, etc) can be immunized with the antigen, and sivaram is the immunized animal is collected and treated by known procedures. If serum containing polyclonal antibodies, contains antibodies to other antigens of interest polyclonal antibodies can be purified using immunoaffinity chromatography and the like by methods known to experts in this field of technology. Methods of producing and processing polyclonal antisera also known in the art.

Application binding polypeptide complexes and antibodies of the present invention

Polypeptide complexes and antibodies, including fragments thereof, according to the present invention can be used in:in vivotherapeutic and prophylactic applications,in vitroandin vivodiagnostic applications, analysesin vitroand the applications of reagents, etc.

Therapeutic and prophylactic use of polypeptide complexes and antibodies of the present invention include the introduction of the above to the recipient mammal, such as man.

Essentially pure polypeptide complexes and antibodies, including fragments thereof at least at 90 to 95% homogeneity are preferred for administration to a mammal, and 98-99% or more homogeneity is most preferred for pharmaceutical use, especially if the mammal is man. After cleaning, incomplete whom or to homogeneity, optionally, the polypeptide complexes and antibodies, with only the heavy chain, as disclosed in the present description, can be used for diagnosis or therapy (including extrakorporale use) or in the development or execution of procedures analysis using methods known to experts in this field of technology.

Usually polypeptide complexes and antibodies of the present invention are used in purified form together with pharmacologically acceptable carriers. Typically, such carriers include water or alcohol/water solvents, emulsions or suspensions, which may include saline solution and/or buffered environment. Injecting fillers include sodium chloride, dextrose and ringer, dextrose and sodium chloride and ringer's lactate.

Suitable physiologically acceptable adjuvant, if you want to maintain polypeptide complex in suspension, can be selected from thickeners, such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous fillers include fluid and nutrient supplements and additives-electrolytes, such as on the basis of dextrose ringer. Can also contain preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents and inert gas is (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

Polypeptide complexes and antibodies, including fragments thereof, according to the present invention can be used in the form of separately entered compositions or in combination with other agents. They may include a variety of immunotherapy drugs, such as cyclosporine, methotrexate, adriamycin, this drug called cisplatin or immunotoxin. Alternatively, polypeptide complexes can be used in combination with enzymes for the conversion of prodrugs in their areas of activity.

Pharmaceutical compositions can include “cocktails” of various cytotoxic or other agents in conjunction with the selected antibodies of the present invention, or even combinations of the selected antibodies of the present invention.

Route of administration of the pharmaceutical compositions of the present invention can be any of the methods known to experts in this field of technology. For therapy, including, without limitation, immunotherapy, polypeptide complexes or antibodies of the present invention can be administered to any patient according to standard techniques. The introduction can be effected by any suitable means, including parenteral, intravenous, venturemusic, intraperitoneal, transdermal, via the pulmonary route, or also appropriately by direct infusion at the same time of the catheter. The dosage and frequency of injection will depend on the age, sex and condition of the patient are also taken into account competitive introduction of other drugs, contraindications, and other parameters.

Polypeptide complexes and antibodies of the present invention can be lyophilized for storage and restored in a suitable carrier prior to use. Can be used known methods of freeze-drying and recovery. Specialists in the art it is obvious that the lyophilization and recovery can result in some loss of functional activity and used these levels should be increased slightly to compensate for such losses.

In addition, polypeptide complexes and antibodies of the present invention can be used for diagnostic purposes. For example, antibodies, as disclosed in the present description, can be formed or obtained against antigens that are specifically expressed during pathological States or whose levels change during these painful conditions.

For some purposes, for example for the purpose of diagnosis or registration, may be added to the label. Suitable labels include, without limitation, any of the following: a radioactive label, NMR spin labels and fluorescent labels. Means detektirovanie the label is well known to specialists in this field of technology.

Compositions containing polypeptide complexes and antibodies of the present invention or their cocktails, can be entered for prophylactic and/or therapeutic treatment.

Compositions containing one or more polypeptide complexes or antibodies of the present invention may be used in prophylactic or therapeutic kits to modify, inactivate, destroy, or remove the selected mistaway cell population in a mammal. In addition, the selected sets of polypeptide complexes and antibodies disclosed in the present invention can be used in vitro orin vitroselectively with the purpose of destruction, purification, or any other way to effectively remove mistaway cell population from a heterogeneous collection of cells.

Example 1

In preparatory experiments, transgenic mice were prepared for expression of the heavy chain locus in which two exons VHHLama connected with the segments of the human variable (D) and joining (J) the heavy chain, and then human genes Cµ, Cδ, Cγ2, Cγ3the constant regions and the 3'LCR human heavy chain immunoglobulin. Human genes Cγ2and Cγ3contained the G-A mutation of splicing. The presence of the Frt site was given the ability to create transgenic mouse c single copy of transg is on from multicopying transgenic arrays by recombination, mediated by Flp. However, the sequence of the transgenic locus with G-A mutation of splicing showed incorrect splicing, but had completely remote CH1 (Fig.9).

Design

To solve this problem was skanirovali kosmidou genomic library for clones containing the genes for VHusing standard methods. One (or more) different VHthe germ line were selected randomly based on their sequence (grades five families in the case of VHperson). Hydrophilic amino acid codons were introduced at position 42, 49, 50 and 52 according to the IMGT numbering (Lefranc et al., (1999)). Genes VHUnited in the BAC vector using standard procedures, such as direct cloning using custom linkers or homologous recombination.

Selected two clones of genomic Pac library RPCI-11 human (BACPAC Recource Center, USA): clone 1065 N8 containing the D and J segments of the heavy chain, Cµ (IgM) and Cδ (IgD), and clone 1115 N15 containing genes Cγ3(IgG3). Bac clone 11771 from another genomic library of a man (Incyte Genomics, CA, USA) was used as the source of the gene Cγ2(IgG2) and LCR heavy chain of immunoglobulin (Mills et al., (1997) J. Exp Med., 15;186(6):845-58).

Using standard technology, Cγ genes3and Cγ2separately was subcloned into the vector pFastBac (Invitrogen). Similarly, any of the other constant regions I could clone of these BAC (IgA, IgE). A complete deletion of exon CH1 was achieved by homologous recombination (Imam et al., (2001)) using sequences, which were located at both ends of the exon CH1 each constant region. Optional you can enter the frt site before field Cµ switch to enable generation odnoletnih loci of mnogokupolnyh loci processing flp-recombinasein vivostandard methods, for example by breeding mice rosa-flp (figure 10).

Individual genes VHsegments D and J exons C and LCR cloned in one BAC or using conventional restriction cleavage and legirovanii or by homologous recombination (or a combination of both), or by using any other technology of cloning.

Then there could be an additional structure.

The locus containing only IgM

To obtain design IgM (11) one or more genes VH(preferably developed genes VHperson to ensure the solubility or genes VHHthe camel), followed by segments of the human D and J heavy chain and Cµ, cloned in BAC. The methodology see above. In this case, in the final BAC cloned only Cµ region.

Locus IgM plus IgG (Cδ is optional)

To obtain design IgM plus IgG (Fig) one or several who are genes V H(preferably developed segments of the VHperson to ensure the solubility or genes VHHthe camel), followed by segments of the human D and J heavy chain, Cµ (without exon CH1, but with exon CH4), (optional Cδ) and modified human genes Cγ2and Cγ3and 3'LCR, cloned in BAC. To generate a locus containing only IgG, loxP sites were introduced during the standard stages of cloning (described above) and BAC were propagated in line E. coli 294 Cre (Buscholz et al.), and posredstvom cre-mediated recombination was received by bacteria, producing locus containing only IgG. Details on for more designs, please see above.

Locus IgM plus IgG (Cδ is optional)

To obtain design IgM plus IgG (Fig) one or more genes VH(preferably developed genes VHperson to ensure the solubility or genes VHHthe camel), followed by segments of the human D and J heavy chain, Cµ (CH1 and CH4), (optional Cδ) and modified human genes Cγ2and Cγ3and 3'LCR, cloned in BAC. To generate a locus containing only IgG, loxP sites were introduced during the standard stages of cloning (described above) and BAC were propagated in line E. coli 294 Cre (Buscholz et al.), and through cre-mediated recombination got the tank is ' series, producing locus containing only IgG.

Transgenic mouse breeding and genotyping

The final BAC was injected transgenic mice using standard injection into fertilized eggs or using technology transferowania embryonic stem cells.

Transgenic loci were checked for integrity and the number of copies using the southern blot analysis of DNA from the tails of mice (Southern 1975), using probes for 5'- and 3'-ends of the locus. Founders were bred in the form of lines on the background MC-/-. Genotyping was performed by standard PCR analysis using primers for each of the different areas of the specified locus. Sequence analysis of RT-PCR products derived from BM cDNA transgenic mice, which was deleterows the entire exon CH1 from both Cγ2and Cγ3(the one with the genes Cµ and Cδ (line HLL) and one without these genes showed that the transgenic loci were not only capable of VDJ recombination, but that IgG transcripts similar to IgG transcripts detected in HCAb llama and camel.

Immunohistochemistry

Put spleen on Wednesday, OCT. Frozen 5-μm cryostate sections were fixed in acetone and was administered by single or double tag, as described previously (Leenen et al., 1998). Anti-B220/RA3-6B2), anti-CD11c/N418 monoclonal antibodies (Steinman et al., 1997) was used in the form of supernatant cultures hybridoma the cells. Used the peroxidase linked goat anti-human IgG and anti-human IgM production Sigma. The reagents of the second phase consisted of peroxidase labeled goat anticrimine Ig (DAKO5 Glostrup, Denmark) or antimachia Ig (Jackson ImmunoResearch Laboratories, West Grove, PA) and alkaline phosphatase goat anticrisisnye Ig (Southern Biotechnology, Birmingam, AL, USA).

On Fig shows immunohistochemical analysis of 5-μm frozen sections of spleen from µMT-/-, WT and HLL and HLL-MD transgenic mice on the background of µMT-/-. Sections were stained with anti-B220 (blue) for B-cells and anti-CD11c/N418 (brown) for dendritic cells. Arrows indicate the position of small clusters of B-cells.

Flow cytometrics analysis

Suspension of single cells were obtained from lymphoid organs in PBS, as described previously (Slieker et al., 1993). Approximately 1×106cells were incubated with antibodies in PBS/0.5% of bovine serum albumin (BSA) in 96-well tablets for 30 minutes at 4°C. the Cells were twice washed in PBS/0.5% of BSA. For each sample counted 3×104events using a FACScan analyzer (Becton Dickinson, Sunnyvale, CA). FACS data were analyzed using CellQuest software, version 1.0. Four-color analysis was performed on a Becton Dickinson FACS Calibur. The following mAb were obtained from BD Pharmingen (San Diego, CA): FITC-conjugated anti-B220-RA3-6B2, PE-conjugated anti-CD19. FACS data skanera the project of spleen cells, painted with anti-CD19 and anti-B220, shown in the lower part Fig.

In the left part of the figure presents the Flp-recombinationin vivoobtained by removing HLL lines in the transgenic line FlpeR, and supporting data FACS scan of recombinant cells of the spleen, showing the survival of B-cells observed directly in the generated source lines HLL-MD. In the right part presents Cre-recombinationin vivoresulting from excretion in transgenic line Cag Cre, and FACS data adenocarinoma recombinant cells of the spleen.

Immunization and production of hybrid (Fig)

Created transgenic mice containing the locus antibody having a heavy chain consisting of two domains VHHLama, areas of human D and J and constant regions IgG2 and 3 (without CH1 domain).

Eight mice were immunized by any heat shock protein 70 (hsp70) from E. coli. 20 μg or 5 μg of antigen with adjuvant Specol (IDDLO, Lelystadt, NL), respectively were injected with s.c. at 0, 14, 28, 42 days and I.P. Pavlova. on the 50th day. Blood was taken at 0, 14 and 45 days. After three booster injections of low titer of antigen specific antibodies were detected in 1 out of 3 mice HLL-MD1 immunized with Hsp70 (Fig).

Conducted standard fusion of spleen cells with cell myeloma line, receiving monoclonal hybridoma cell line, generating monoclonal anti the ate against hsp70 protein. Anti-HSP 70 HCAb consisted of segment VHHLama, located in close proximity to area D (VHH2), recombineering with segment IgHD3-10 person (acc.num. X13972) and segment IgHJ4-02 person (acc.num.X86355). Though not with high frequency, VHHhad a few mutations that resulted in amino acid substitutions, as shown in figa, compared with the configuration of the germ line. Analysis of RT-PCR also showed only one productive IgH transcript in hybridoma, confirming that other transcripts were not formed. IgG2 antibody αHSP70 secretarials in the form of a dimer, with only the heavy chain (Western blot under denaturing gel (dimer) and sedentarism gel (monomer), Fig). Cells of the spleen was merged with myeloma cells Sp2-O-Ag14 (courtesy of R. Haperen) on day 56, using a set of ClonalCellTM-HY (StemCell Technologies, UK) according to manufacturer's instructions.

Transgenic mice containing the locus antibody having a heavy chain consisting of two domains VHHLama, areas of human D and J, the constant regions of human IgM and IgG2 and 3 (all without CH1 domain Fig), were immunized TNFα to generate antibodies HC-IgM. One of the three mice revealed a serum positive in a standard ELISA. Standard fusion of myeloma gave a positive IgM hybridoma (Fig). After gel filtration on sepharose 6B in Sevostyanova the x conditions of each faction, present in the column was loaded into the gel in reducing conditions and detected α-IgM-HRP person (Fig). Fractionation in non conditions showed that HC-IgM is secreted as multimeric antibodies of the same size as a control human IgM (after subtracting the molecular weight of the light chain domain and CH1, which was absent in HC-IgM). Gel fractionation of each fraction in the column in reducing conditions revealed the expected monomer (Fig).

ELISA Ig in serum

Blood from 15-25-week-old mice were collected in tubes coated with EDTA, centrifuged for 15 minutes at room temperature, and the supernatant was diluted 1:5 in PBS. 96-well plates were coated for 2 hours with 5 mg/ml goat anti-human IgG (YES Biotechnology) or goat anti-human IgM (Sigma), washed with PBS, blocked for 1 hour at room temperature in blocking solution (1.5% of BSA/1,5% milk powder/0.1% of Tween-20/PBS) and washed three times with PBS. Downloaded a series of dilutions of serum samples and standards (IgG2 human or human IgM (Sigma, Zwijndrecht, NL)), and incubated for 2-4 hours, and the tablets were washed 6 times with PBS before adding the secondary antibody (1:2000 diluted goat anti-human IgG or goat anti-human IgM linked to HRP (Sigma, Zwijndrecht, NL)). All breeding were made in the blocking RAS is a thief. After 1-2 hours of incubation at room temperature, and washing in PBS was added POD substrate (Roche).

ELISA for detection of antigen-specific soluble sdAb from IgG2 ragovoy library shown in Fig. Soluble sdAb were used as primary antibodies on tablets coated with the antigen, followed by mouse αmyc antibody and HRP-conjugated goat antimurine antibodies. POD was used as substrate. In the lower part of the figure shows the peptide maps of clones with restriction enzyme Hinf I, showing the 5 different inserts encoding sdAb against B. Pertusis.

Construction and screening of libraries of antibodies

Total RNA was isolated from spleens of immunized DKTP mice having odnokorennye IgG (Fig after processing SGE), using an allocation system Ultraspec RNA (Biotecx Laboratories Inc., Houston, Texas, USA). Received cDNA using oligo-dT. DNA fragments encoding fragments of VHHDJ PCR amplified using specific primers: reverse Sfi I primer vh1 (Dekker et al., 2003) in combination with primer hIgG2hingrev (5'-AATCTGGGCAGCGGCCGCCTCGACACAACATTTGCGCTC-3'). Amplificatoare VHHDJ (~400 BP) were digested Sfi I/Not I, purified using the gel and cloned in the vector phagemid pHEN-1, split Sfi I/Not I.

Transformation in electrocompetent TG1 cells gave a library of human antibodies with a single domain. Two were stage select and, using panning on the antigens of the vaccine, adsorbed on plastic (immunoresearch covered with undiluted vaccine). Restriction analysis and sequencing was performed by standard methods.

RT-PCR locus only heavy chains

Then researched, does it work locus HLL MD as normal locus in the production of diverse set of antibodies by sequencing RT-PCR products obtained using IgG2 and IgG3-specific primers for cDNA from Meyerovich plaques. On Fig shows several examples of somatic mutations clones unimmunized mice (left part of the figure) and immunized mice (right part of the figure). Mouse had the loci containing only IgG, and were immunized with hsp70 E. coli lysate Pertussis, tetanus toxin. Grey marked the hinge region of IgG2, since ERKCCV.

Although the analysis of RT-PCR on Meyerovich plaques showed that use both VHall sequenced antibodies had rebuilt VH2. Source of variation is set in the CDR3 region formed by using the selection of segments D and J and using connections V-D and D-J. Using segments person J is similar to the one observed in the reconstructions in humans, and JH4 segments and JH6 are used most often.

This analysis showed that both VHvarious segments of the human D and all the segments of person J contribute to the creation of a diverse set of antibodies. It also shows the presence of IgG3-switched B-cells and the presence of somatic mutations when compared rebuilt each gene with its counterpart in the germ line, i.e. original VHin transgenic constructs (see Fig). Therefore, antigenic receptor IgG person, with only the heavy chain, can provide the necessary signals for B-cell maturation.

Immunoablative

On Fig shows the results of immunostaining of one of the cell lines Tet-on advanced transfected increases the sensitivity of the plasmid containing the A5 antibody (Dekker et al., 2003). In the upper part of the figure shows the doxycycline-induced production of the A5 antibody (red) in the cytoplasm and staining of the cell nuclei DAPI (blue). In the lower part of the figure shows that cells expressing rtTA in nuclei represent cells by induction of producing A5 (upper part of the figure). Staining was performed by one of the HCAb person against rtTA (green)with the following sequence. FITC-conjugated goat anti-human IgG used in the second stage. A5 were detected as previously described in Dekker et al., 2003. rTTA antibody was an IgG3, having the following sequence:

The IgG3 hinge begins with linakis is the notes 198 ELKTPL. For comparison, see the hinge region of IgG2 in Fig.

Western blot analysis

On pig data shown is Western blot analysis of sera of different lines of transgenic mice containing the locus IgM plus IgG (figure 10) after processing cre (i.e. IgM deleterows left only IgG). Serum was purified using protein G was fractionally gel in reducing conditions (Fig, right) and in non conditions (Fig, left). Controls consisted of mice with KO background and a sample of normal human serum. Note the difference in size between the two gels, demonstrating that IgG person, with only the heavy chain, is a dimer.

The signal shown in Fig, were detected using anti-human IgG-antibody using standard procedures.

Fractionation by size human IgM produced by mouse, with the locus of IgM plus IgG

Mouse serum with IgM plus IgG (Fig), was fractionally gel-filtration in non conditions after mixing with the sample of human serum as a control. The results are shown in Fig. Molecular weight complexes in the column decreases with each band representing each faction) from left to right. Fractions (each lane) were analyzed by gel-electrophoresis in reducing conditions.

The ELISA analysis of the implementation of the Yali on several hybridomas, created from immunized human TNFα mice containing the locus IgM plus IgG (Fig). The results are shown in Fig. Two of the top row on Fig analyzed using anti-human IgG, the following two lines using anti-human IgM. Serum samples (arrows) showed that the mouse was generated as IgG and IgM antibodies against TNFα. A single arrow indicates a positive IgM hybridoma. The wells were covered with commercially available human TNFα. All procedures were standard.

Example 2

Bespecifically bivalent antibody generated by the combination of two monospecific antibodies having a heavy chain. The first antibody was formed by the frame, bringing the first specificity and effector functions (variable plot and constant plot, respectively). It was combined with the second antibody with the second specificity using newly designed hinge. This hinge was similar to the existing hinge sequence IgG2, but has been modified by replacing cysteine Proline to prevent cross-linking cysteines in dimer antibodies and to provide additional flexibility due to Proline, which excluded the spatial restriction of the second antibody that otherwise could inhibit its function.

The initial carcenogenic represented antibody produced against HSP70 protein of E. coli. The antigen were injected with HSP70 transgenic mice that contained the locus antibody having a heavy chain, as described above (see Fig). Monoclonal antibody was established from these animals using the standard hybridoma technology of fusion (see above). Then cloned cDNA encoding αHSP-antibody, standard methods RT-PCR of recombinant DNA, obtaining the plasmid containing the full length cDNA, which includes the 5'-end 3'-end of the protein from the N-end to the COOH-end) start codon ATG, the signal peptide sequence of the variable domain VHH1 (see Janssens et al.), nekombinirovannyh region D and J, and constant region Cγ2(without a CH1), but including the stop codon and poly-A-plot (Fig top left). cDNA encoding αHSP70-antibody, PCR amplified for cloning, using direct primer and reverse primer.

Direct primer represented:providing an EcoRI site with the aim of cloning (underlined) sequence (bold), sufficient for the beginning of the broadcast, and the normal start codon (indicated in gray).

The reverse primer was:providing a cloning site (underlined) HindIII and maintaining the normal start codon.

Therefore, the amplification on the no fragment EcoRI/HindIII, containing an EcoRI site (underlined), the sequence (bold), sufficient for the beginning of the broadcast, and the normal start codon of the gene of the antibody αHSP (marked in grey, see Fig).

Reverse the 3'end of the primer represented:providing site cloning HindIII (underlined) and removing the normal stop codon. This gives the fragment (Fig top, second from the left) with an EcoRI site for cloning the promoter sequence and a HindIII site for cloning 5'-end into a plasmid for expression and 3'-end in the new hinge sequence (see below). Finally, the fragment was cut by EcoRI and HindIII to ensure appropriate single-stranded ends for cloning.

Second cloned antibody, adding a second specificity contained domain VHHantibodies Lama to gag antigen retrovirus (PERV) pigs (Dekker et ah, (2003) J Virol, 11 (22): 12132-9, Fig above right). αgag amplified using standard PCR amplification using the following primers:

direct:and reverse primer:. This gives amplificatory fragment (Fig right, second from the top) with an XhoI site (marked in gray) for cloning 5'-end frame with a new hinge (see below) and an EcoRI site (underlined) for cloning the 3'end of the plasmid for expression (Fig, right in the center). Finally, the fragment was cut by EcoRI and XhoI to obtain single-stranded ends for cloning.

Two sequences of the antibodies were combined into a single sequence of recombinant especifismo antibodies using the new hinge. New hinge was created from two oligonucleotides, which together formed a double-strand oligonucleotide with 5'- and 3'-sticky ends (respectively HindIII and XhoI-compatible) with the purpose of cloning. It was designed so that was in the frame, which ended with the sequence αHSP70 and began the sequence αgag. Education sulfide bridges, usually present in the IgG2 hinge person, prevented the replacement cysteine (marked grey) Proline (underlined). Proline added extragalactic hinge, allowing the proper functioning of the domain of a secondary antibody that binds to the COOH-end of the first antibody by means of a hinge.

Normal articulated sequence IgG (cysteine codons indicated by gray prolinnova codons underlined)and its complement deputizedand its complement). It also provided the fragment (hinge in the white rectangle, Fig, center) with two single-stranded ends that are compatible with HindIII sites (bold), and XhI (italics) for cloning.

Three fragments (αHSP70 IgG2, hinge and αgag) sequentially ligated into a plasmid expression bluescript (Pbluescript11 sk+), which contain actin view of the chicken and the sequence of the CMV enhancer (Fig, plasmid expression), using standard recombinant DNA technology. If this expression plasmids (see below) is obtained recombinant bespecifically antibody, shown in the lower part Fig.

Plasmid expression of recombinant especifismo antibodies were propagated and cotranslational the plasmid pGK-hygro (to ensure selection transfetsirovannyh cells) by standard methods (Superfect) in CHO cells (Fig). Positive clones were selected in medium containing hygromycin, and positively identified as expressing recombinant bespecifically antibody by performing standard αgag ELISA (Dekker et al., J.Virol. 2003) environment, growth, containing recombinant bespecifically antibody Sekretareva CHO cells, using the detection of α IgG-HRP person. Positive testing α-gag activity indicates the highest likelihood that this clone expresses recombinant full bespecifically antibody, as gag-specificity is at the back end (COOH-end) recombinant especifismo antibodies. Subsequent ELISA for HSP70 was also positive. Western blot of these clones, using the data, using ELISA, in non and in reducing conditions was performed to show that a protein expressed from a plasmid, is a dimer of 110 kDa (as shown in the lower part Fig) compared to the monomer 55 kDa (and non reducing conditions and Western blot, Fig right). Thus, ELISA and Western blot together showed that recombinant bespecifically antibody is expressed and secreted into the environment in the form of a dimer using transfected CHO cells (>70 ng/ml), and the antibody can bind antigens HSP70 and gag. However, it is shown that the same dimeric molecule of recombinant especifismo antibodies can bind both antigens at the same time.

Respectively, was carried out the following experiment. The first gag antigen fixed on the bottom of the plastic hole (first hole Fig, center). Then took recombinant bespecifically antibody (Fig, top) the first antigen (gag) after applying the CHO cell of supernatant from 1 clone (second hole, Fig, center). Then intensively washed and inflicted a second antigen (HSP 70, Fig, the third hole in the center), then intensively washed. If the molecule recombinant especifismo antibodies could bind both antigens, she had to be captured at the bottom of the wells by linking the first is nigena (gag) and then capture a second antigen (HSP70). When then suirable full range of wells (Fig, in the center of the right hole) on the Western band were seen as recombinant bespecifically antibody and antigens (Fig, below).

To collect secreted recombinant bespecifically antibody, CHO clones were grown in the same standard conditions and in the environment (SIGMA environment for a hybrid, without serum)used for collecting antibodies from hybridomas.

Methods: Wells Nunc-immuno (Maxisorp) were coated with purified recombinant protein gag (12.5 ág/ál in PBS) O/N 4C. Blocked for two hours in 1% milk/1% BSA in PBS. Wednesday CHO-DB clone-1, half-diluted with PBS-milk-BSA (or control), incubated for 3 hours at room temperature. Bacterial cell lysate Bl21 (containing protein HSP70), half razbavlennyi PBS-milk-BSA, incubated for 3 hours at room temperature and washed. Associated proteins were suirable Laemmli buffer for samples containing 2-mercaptoethanol. Samples were analyzed by Western blot analysis and then chased in 10% SDS-PAGE and transferred to nitrocellulose membrane. The blot was blocked for two hours in PBS-milk-BSA, and incubated with primary antibodies. Products were visualized by standard methods using secondary antibodies coupled with enzymes, which allowed visual staining. Use the ALIS the following reagents:

αGag: rabbit polyclonal (1:2000), 2 hours, room temperature

recombinant bespecifically α antibody: goat anti-human IgG-HRP (1:2500), 2 hours, room temperature

αHSP70: monoclonal G20-380 secondary (1:2), 2 hours, room temperature.

Secondary antibodies: goat anti-rabbit-AP (1:2000), 2 hours, room temperature, and goat anti-human IgG-HRP (1:2500), 2 hours, room temperature, against monoclonal HSP70.

For visualization of protein bands used the first substrate NBT/BCIP (red), reacting with alkaline phosphatase (AP), and the second substrate DAB (brown), reacts with horseradish peroxidase (HRP).

All washing steps were performed PBS-0.05% of Tween-20.

The control performed by removing one of the components or adding medium from CHO cells not producing recombinant bespecifically antibodies (Fig), i.e. without the application of recombinant bespecifically antibodies (Wednesday retransferring cells CO) and, therefore, had only gag (lane 2); had no gag at the bottom of the wells and replaced with the protein of milk) and, therefore, should not have been products (lane 3); lack of gag and recombinant especifismo antibodies and the absence of product (lane 4); the absence of HSP70 antigen (replaced by milk protein) and, therefore, had to have only recombinant bespecifically antibody and gag (to ogca 5); the absence of HSP70 and recombinant bespecifically antibody was supposed to have only gag (lane 6).

The fact that all three components (recombinant bespecifically antibody plus both antigen) was only in the hole track 1, which received all three components (see also marked the bottom Fig), shows that a single recombinant bespecifically antibody simultaneously binds both antigen.

Generation bespecifically IgA or multispecificity IgM

Generation especifismo IgA is essentially the same as described for IgG (above), but using in addition Vhsol, D and J, and constant region Cα, leading to the generation of IgA (Fig).

Generation of IgM is largely similar, but shows an additional possibility, because IgM molecules can form large multimeric (with or without J chain). Thus, in addition to molecules, similar to the molecules described above (Fig, bottom right, after elimination sequences multimerization), you can also generate multimeric, just coexpression IgM with different specificnosti (Fig, bottom left).

Example 3

The expression vector encoding the polypeptide complex that contains a heavy chain, including binding domain that binds to PSCA (antigen stem cells, the intercessor is Oh gland), the domain of the Assembly containing the motif latinboy zippers Jun and hinge antibody domains CH2 and CH3; and a light chain comprising the complementary domain of the Assembly consisting of motive latinboy lightning Fos, designed using the techniques of molecular biology as described by Sambrook et al., ((1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press).

The expression vector was then transferrable in a suitable cell host using technology transformations for producing transfected host cell for optimized expression vector. Transfected or transformed cell of the host is then cultured using any suitable technology known to experts in the art, for producing the polypeptide complex of the present invention.

After producing the polypeptide complexes were purified by standard procedures in the art, including cross-filtering, precipitation of ammonium sulfate and affinity column chromatography (e.g., protein A).

Soluble effector domain, consisting of 3,3'-diindolylmethane (DIM), then poured with complementary domain Assembly using techniques known to experts in this field of technology.

Example 4

The expression vector encoding a heavy chain polypeptide complex of the present izaberete the Oia, containing soluble binding domain VHHthat is associated with AFP (Alpha-Fetoprotein), and the domain of the Assembly containing the motif latinboy lightning Jun, and the hinge antibody domains CH2 and CH3, constructed using techniques of molecular biology as described by Sambrook et al.

A second expression vector encoding a light chain polypeptide complex of the present invention is also constructed. It contained a complementary domain of an Assembly consisting of a motive latinboy lightning Fos.

The expression vectors were then transferrable in a suitable cell host the conventional technology for producing cotranslational host cell for optimized expression vector. Transfected or transformed cell of the host is then cultured using any suitable technology known to experts in the art for producing the polypeptide complex of the present invention.

After producing the polypeptide complexes were purified by standard procedures in the art, including cross-filtering, precipitation of ammonium sulfate and affinity column chromatography (e.g., protein A).

Soluble effector domain, consisting of 3,3'-diindolylmethane (DIM), then poured with complementary domain Assembly using techniques known specialized there in this field of technology.

Example 5. VCAM and VLA-4

The expression vector encoding the polypeptide complex that contains a heavy chain comprising a binding domain that binds to PSCA (stem cell antigen of the prostate), the domain of the Assembly containing VCAM and hinge antibody domains CH2 and CH3; and a light chain comprising the complementary domain of the Assembly consisting of VLA-4, merged with the toxin licina, constructed using techniques of molecular biology as described by Sambrook et al.

Then the expression vector was transferrable in a suitable cell host the conventional technology for producing transfected host cell, for optimized expression vector. Transfected or transformed cell of the host is then cultured using any suitable technology known to experts in the art, for producing the polypeptide complex of the present invention.

After producing the polypeptide complexes were purified by standard procedures in the art, including cross-filtration, precipitation with ammonium sulfate and affinity column chromatography (e.g., protein A).

Example 6

The expression vector encoding the polypeptide complex that contains a heavy chain comprising a binding domain that binds to PSCA (stem cell antigen simple is you), the domain of the Assembly containing the motif latinboy zippers Jun and hinge antibody domains CH2 and CH3; and a light chain comprising the complementary domain of the Assembly consisting of motive latinboy lightning Fos and soluble effector domain, encoding the purine nucleoside-phosphorylase (PNP), constructed using techniques of molecular biology as described by Sambrook et al.

The expression vector was then transferrable in a suitable cell host-conventional technologies for producing transfected cell host for optimized expression vector. Transfected or transformed cell of the host is then cultured using any suitable technology known to experts in the art, for producing the polypeptide complex of the present invention. After producing the polypeptide complexes were purified by standard procedures of the present invention, including a cross-filtration, precipitation with ammonium sulfate and affinity column chromatography (e.g., protein A).

PNP turns fludarabine in toxic metabolite 2-ferdinan, which kills cells containing PNP enzyme, and optionally diffuses, killing surrounding uninfected cells, the local observer effect.

Example 7

The expression vector encoding a first heavy chain polyp is pignolo complex of the present invention, containing soluble binding domain VHHthat is associated with the region V3-PND glycoprotein gp120 antigen, and the domain of the Assembly containing the motif latinboy zippers Jun and hinge antigen, domains, CH2 and CH3, constructed using techniques of molecular biology as described by Sambrook et al.

A second expression vector encoding a second heavy chain polypeptide complex of the present invention, which is also constructed, contains soluble binding domain VHHthat is associated with GP-41, domain Assembly consisting of a motive latinboy zippers Jun and hinge antibodies, domain CH2 and CH3.

The third expression vector encoding a light chain polypeptide complex of the present invention is also constructed. It contains complementary domain Assembly consisting of a motive latinboy lightning Fos. Then the expression vectors were transferrable in a suitable cell host the conventional technology for producing cotranslational host cell for optimized expression vector. Transfected or transformed cell of the host is then cultured using any suitable technology known to experts in the art, for producing the polypeptide complex of the present invention.

After producing the polypeptide is the complex was purified by standard procedures of the art, including cross-filtration, precipitation with ammonium sulfate and affinity column chromatography (e.g., protein A).

Soluble effector domain containing HIV-1 MN V3 (PND), a peptide immunogen was then merged with complementary domain Assembly using techniques known to experts in this field of technology.

Example 8

The expression vector encoding a first heavy chain polypeptide complex of the present invention containing a soluble binding domain VHHthat is associated with the region V3-PND glycoprotein antigen, constructed by using the techniques of molecular biology described by Sambrook et al., ((1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press).

A second expression vector encoding a second heavy chain polypeptide complex of the present invention, which is also constructed, contains soluble binding domain VHHthat is associated with GP-41.

Two heavy chains differed in that the constant region of the two heavy chains contain identical domains µ, CH2, CH3 and CH4.

Then the expression vectors were transferrable in the cell host, which constitutively expressed the chain J, using conventional technology for producing cotranslational host cell for optimized expresii vector. Transfected with the th or transformed cell of the host is then cultured, using any suitable technology known to experts in the art for producing the polypeptide complex of the present invention.

After producing the polypeptide complex was purified by standard procedures in the art, including cross-filtration, precipitation with ammonium sulfate and affinity column chromatography (e.g., protein A).

Soluble effector domain consisting of HIV-1 MN V3 (PND), a peptide immunogen, then poured with complementary domain Assembly using techniques known to experts in this field of technology.

All of the above publications included in the present description in its entirety by reference.

Various modifications and changes of the described methods and systems of the present invention are obvious to experts in the art without departure from the scope and essence of the present invention. Although the present invention is described in connection with preferred variant implementation, it should be noted that the present invention as claimed in the claims, is not limited to improper concrete variants of implementation. On the contrary, various modifications of the described embodiments of the present invention which are obvious to experts in biochemistry, molecular Biol the GII and biotechnology or related fields should rassmatrivati as included in the scope of the following claims.

1. The method of obtaining the antigen-binding domain of the VHwhere there is no extended CDR3 loop, similar to the CDR3 loop of a camel, including:
transforming mammalian cells, non-human, a heterologous heavy chain locus VHand:
the locus of the heavy chain VHcontains the gene encoding the variable region containing at least one gene segment VHat least one segment of the gene D, which is not a camel, at least one gene segment J, which is not a camel, and at least one constant region of the heavy chain;
none of the genes encoding the constant region does not encode a functional domain From an1;
the gene segment V segment gene D and gene segment J is capable of recombination with the formation of the coding sequence of the VDJ;
when the expression of the locus capable of forming antibody only with heavy chains containing the antigen-binding domain of the VHand constant effector region, devoid of functional domainn1;
the cultivation of mammalian cells, non-human, to produce a transgenic mammal;
immunization indicated transgenic mammal that is not a person, pre the element of interest antigen;
the selection of cells or tissue expressing interest specific antigen antibodies only with heavy chains,
where the antibody has domain VHwhere there is no extended CDR3 loop, similar to the CDR3 loop of a camel;
identification and selection of the selected cells or tissues nucleic acid that encodes a domain of VHinterest specific antigen antibodies only with heavy chains; and
the expression of the indicated antigen-binding domain of the VHspecified the selected nucleic acid.

2. The method according to claim 1, in which, after immunization with the indicated transgenic mammal that is not a man, grow the affinity of antibodies through somatic mutations in the antigen-binding domain of the VH.

3. The method according to claim 1 or 2, in which the expression of the specified heterologous locus heavy chain VHthe specified transgenic mammal that is not a person, does not depend on the presence or gene expression of light chain.

4. The method according to claim 1, in which the specified transgenic mammal that is not a person, has a reduced ability to produce endogenous antibodies that contain a light chain.

5. The method according to claim 1, in which:
specified transgenic mammal that is not a person, subjected to immunization antigen with the aid of the completion of injection;
nucleic acid encoding the antigen-binding domain of the VHrepresents mRNA, cloned from hybridoma produced by the cell or tissue, producing specific antigen antibodies only with heavy chains; and antigen-specific antigen-binding domain of the VHis expressed in the heterologous expression system.

6. The method according to claim 1, in which:
specified transgenic mammal that is not a person, subjected to immunization with antigen by injection;
nucleic acid encoding the antigen-binding domain of the VHproduced by cloning the antigen-binding domain of the VHof mRNA antibodies only with heavy chains, obtained from selected cells or tissues;
the encoded antigen-binding domain of the VHselected via phage display or similar libraries; and
Express the antigen-binding domain of the VHseparately or in the form of a fused protein in a bacterial, yeast or alternative expression system.

7. The method according to claim 1, in which the locus of the heavy chain VHcontains natural gene segments, V, D and J man.

8. The method according to claim 1, in which the locus of the heavy chain VHcontains more than one gene segment V, more than one gene segment D and more than one segment of a gene J.

9. The method according to claim 1, where at least one segment of the gene , selected or obtained by means of genetic engineering, has improved properties solubility.

10. The method according to claim 1, where at least one of the gene segments V, present in the heavy chain locus, derived from domain VH, which introduced the preferred mutations in the affinity maturation, after shuffling VDJ.

11. Applying one or more binding domain of VHthe same or different specific-binding antigen obtained by the methods according to any one of claims 1 to 10, with or without additional effector domains, to create a fused protein or binding complexes of monovalent, bivalent or multivalent polypeptide.



 

Same patents:

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L.4 A2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Mus musculus L. 1C1 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The invention is also aimed at obtaining monoclonal antibodies 4A2 which are produced by the 4A2 hybridome, (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain) and are used as binding antigens in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain), and monoclonal antibodies 1C1 produced by the 1C1 hybridome (subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain), used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain). The disclosed antibodies are used together in a "sandwich" format immunoenzymometric system for exposing the matrix protein VP40 of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: invention enables to obtain monoclonal antibodies which are specific and do not compete with each other for antigen epitopes and which, when used together in a "sandwich" format immunoenzymometric system, ensure high reliability of results for exposing the matrix protein VP40 of the Ebola virus.

5 cl, 3 dwg, 1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 1B2, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the neucleoprotein of the Ebola virus, Zaire subtype (Mainga strain), and a strain of hybrid animal cells Rattus Norvegicus 7B11 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the nucleoprotein of Ebola virus, Zaire subtype (Mainga strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain). The invention describes monoclonal antibodies 1B2 which are produced by the strain of hybrid animal cells Mus musculus L. 1B2, which relate to the subclass of immunoglobulins IgGl which have a heavy 55 kDa and a light 25 kDa chain, and monoclonal antibodies 7B11 which are produced by the strain of hybrid animal cells Rattus Norvegicus 7B 11 related to the subclass of immunoglobulins IgG. The antibodies are used together in the "sandwich" format immunoenzymometric system for exposing nucleoprotein of the Ebola virus, Zaire subtype (Mainga strain).

EFFECT: use of the invention enables to obtain results during "ВЭ" laboratory reseach and when designing a test system for highly reliable exposure of an antigen.

5 cl, 3 dwg, 2 tbl, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention discloses a strain of hybrid animal cells Mus musculus L. 7D8, which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR, which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as binding antigens in a "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain) and a strain of hybrid animal cells Mus musculus L. 7H10 which is deposited in the Collection of cell cultures of the State Research Center of Virology and Biotechnology VECTOR and which is a producer of monoclonal antibodies which are specific to the VP40 matrix protein of the Marburg virus (Popp strain) and are used as biotin labelled indicators in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain). Monoclonal antibodies 7D8 which are produced by the strain of hybrid animal cells Mus musculus L. 7D8 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7H10 which are produced by the strain of hybrid animal cells Mus musculus L. 7H10 relate to a subclass of immunoglobulins IgGl and have a heavy 55 kDa and a light 25 kDa chain. Monoclonal antibodies 7D8 and antibodies 7D8 and 7H10 are used together in the "sandwich" format immunoenzymometric system for exposing the VP40 matrix protein of the Marburg virus (Popp strain).

EFFECT: use of the invention increases reliability of enzyme immunoassay.

5 cl, 3 dwg,1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of Marburg haemorrhagic fever. A strain of hybrid animal cells Mus musculus L. 3F9 is formed, which is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR. The hybridoma strain produces monoclonal antibodies which are specific to the VP35 protein of the Marburg virus (Popp strain) (hereinafter MCA). MCA 3F9 produced by hybrid animal cells Mus musculus L. 3F9 relate to a subclass of immunoglobulins IgGI, having a heavy 55 kDa and a light 25 kDa chain and having a unique feature of detecting the VP35 protein of the Marburg virus (Popp strain) in a "sandwich" immunoenzymometric system format owing to antigen "capture" properties and simultaneously be an indicator, labeled biotin. The antigen epitope for MCA 3F9 produced by the 3F9 hybridoma is localised between 252 and 278 aminoacid residues.

EFFECT: invention enables to obtain MCA with specificity to VP35 protein of the Marburg virus (Popp strain), suitable for immunodiagnosis of Marburg haemorrhagic fever.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and can be used in immunodiagnosis of human cytomegalovirus. The strain of hybrid animal cells Mus musculus L.5F10 is obtained by merging mouse myeloma cells p3-X63/Ag8.653 (NS/1) with mouse spleen cells BALBc, immunised by an affinity purified recombinant protein pp65. The hybridoma strain is deposited in the collection of cell cultures of The State Research Center of Virology and Biotechnology VECTOR and is used as a producer of monoclonal antibodies for detecting the pp65 protein of human cytomegalovirus.

EFFECT: invention enables to widening of range of strains of hybrid cells Mus musculus L - producers of MCA for detecting the pp65 protein of human cytomegalovirus and production of domestic diagnostic test-systems for detecting cytomegaly.

2 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to humanised anti-TGF-beta-antibody which is linked to TGF-beta. The humanised antibody has a variable domain VH which contains residues of the hypervariable region (non-human), which are contained in the human domain VH which includes a modified framework region (FR) (amino acid and nucleotide sequences are given in the list of sequences). The humanised antibody can contain residues of the complementarity determining region (CDR) of the variable domain of the light strand VL. The invention also relates to a composition for treating TGF-beta mediated disorders, e.g. malignant tumours, nucleic acid, coding monoclonal antibody, and a method of obtaining the latter using host cells. The invention provides a method of treating and detecting TGF-beta in a sample from the body using the disclosed antibody, as well as to a product which contains the humanised antibody and directions for use for treating TGF-beta mediated disorders.

EFFECT: invention enables control of TGF-beta molecules, which can prevent possible changes in antibodies, enables preparation of high-affinity humanised antibodies which act as TGF-beta antagonists.

57 cl, 45 dwg, 4 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention can be used for production of monoclonal antibodies (MCAs) to heat shock protein 70 (HSP 70). A hybridoma strain is made by immunisation of BALB/c mice with bovine HSP 70 within 78 days. For the third days, splenocytes of immune mice (108 cells) are hybridised with murine myeloma cells P3-X63 Ag/8-653 (107 cells). A fusion agent is polyethylene glycol of molecular weight 4000 (Merk, Germany). The hybridisation is followed with selection, screening, cloning and cryopreservation of hybridoma. Hybridoma 6G2 is deposited in the microorganism collections of "ГНТТ ПМБ" under No. H-2. MCA.

EFFECT: produced hybridoma under the invention is more evident to be detected as HSP 70 on the cell surfaces, and change of endocellular HSP 70 level when exposed to the stress factors.

4 dwg, 1 tbl, 6 ex

FIELD: pharmacology.

SUBSTANCE: invention can be used to identify a pseudotuberculosis agent in bacterial cultures, a biological material and environmental objects by applying the indirect hemagglutination test. Substance of the invention consists in development of a new diagnosticum that represents formalinised sheep's erythrocytes sensitised with monoclonal antibodies to lipopolysaccharide antigen of cold version Yersinia pseudotuberculosis serotype I (strain 164/84 serovariant I) and frozen-dried in a protective medium. Shelf life of the preparation is 2 years.

EFFECT: diagnosticum provides high sensitivity, specificity to the UHAT in detecting Yersinia pseudotuberculosis serotype I.

FIELD: veterinary.

SUBSTANCE: strain 5A10 of hybridomal line of cells of mouse Mus. museums, producing monoclonal antibodies to immunoglobulin IgG of cattle (C) is permanent line of cells and is suitable for biotechnology in elaboration of preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 71. Antibody titers in native culture liquid constitute 1:32-1:64, in ascitic liquid 1:640-1:5120 in immuno-enzymatic analysys. Monoclonal antibodiesproduced by strain are specific to immunoglobulin IgG of cattle and do not react with immunoglobulins of sheep. Peroxydase-marked monoclonal antibodies ensure high sensitivity and specificity of IEA for detection of antibodies to C leucosis virus in biological material. Strain 5A10 - producent of monoclonal antibodies to immunoglobulin IgG of cattle can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis.

EFFECT: application of said test-system will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.

5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining genetically engineered vaccines and can be used in medicine. A recombinant gene structure containing a series of human SLC gene, antigen gene and gene Fc-fragment of IgG 1 is obtained.

EFFECT: invention considerably increases efficiency of the immune response of the body to the introduced antigen compared to existing antigen structures.

10 cl, 15 dwg, 1 tbl, 12 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly genetic engineering and can be used in biomedical industry. The invention discloses a method of obtaining a target peptide by splitting a polypeptide or a fused protein using a version of protease OmpT with substitution of the amino acid in position 97, where the amino acid is selected from alanine, leucine, phenylalanine, methionine, serine, threonine, cysteine, asparagine, glutamine, glutaminic acid and histidine, on a site whose structure is characterised by general formula Pn…P2-P1-P1'-P2'…Pn', where P1 is asparagine or lysine, P2 and P2' are an amino acid which is not glutamic or asparagic acid, and P1' is alanine, valine, isoleucine, phenylalanine, methionine, serine, threonine, cysteine, tyrosine or asparagine.

EFFECT: use of the invention can enable efficient and specific isolation of therapeutic peptides with any type of N-terminal sequence.

22 cl, 3 tbl, 20 dwg, 18 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to cell biology, molecular biology, cancer biology and medicine and represents a pharmaceutical composition for targeting to the cells bearing BLyS receptor, containing an effective amount of fused protein consisting of BLyS polypeptide fused with cytotoxic polypeptide where said cytotoxic polypeptide is located on the N-end of fused protein, and BLyS polypeptide is located on the S-end of fused protein, and a pharmaceutically acceptable carrier.

EFFECT: invention provides treatment and prevention of B-cell proliferative disorder.

11 cl, 13 ex, 4 tbl, 20 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to obtaining protein complexes and can be used in medicine. A complex of biologically active recombinant protein is obtained, which is non-covalently bonded to polysialic acid which is inactive in aqueous solution at pH 4.4-4.7 which has prolonged therapeutic effect and has general formula:

where m=20-110; n=1-4; P is biologically active recombinant protein.

EFFECT: invention enables to obtain a complex of biologically active recombinant protein with polysialic acid, having stability and prolonged effect.

6 dwg, 2 tbl, 15 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic and protein engineering and can be used in biomedical industry. A genetic make up is proposed, which codes a peptide in which two domains bonding the growth hormone (GH) receptor are bonded into a tandem by a "semirigid" or "rigid" linker, which consists of at least 1-4 copies of the A(EAAAK)A amino acid sequence.

EFFECT: as a result of expression of the nucleotide sequence coding the said tandem, GH-linker-GH polypeptides are obtained, which exhibit growth hormone receptor agonist properties, which determine the possibility of their use in medicinal agents for treating diseases related to the need to administer the growth hormone.

10 cl, 31 dwg, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to production of new hybrid polypeptide GST-CFP10 by microbiological synthesis with properties of species-specific protein-antigen CFP10 Mycobacterium tuberculosis, which may be used for early species-specific diagnostics of tuberculosis infection. Recombinant plasmid DNA pTB232 has been constructed, which codes hybrid polypeptide GST-CFP10 with properties of mycobacterial antigen CFP10, with average molecular weight (m.w.) 3.4 MDa and having size of 5257 p.n. Recombinant strain of bacteria E. coli BL21/pTB232 contains recombinant plasmid DNA pTB232, is producer of hybrid polypeptide GST-CFP 10 with properties of mycobacterial antigen CFP10 and is deposited in KM GNC VB "Vector" under number B-1027. Recombinant polypeptide GST-CFP10, produced with strain of bacteria E. coli BL21/pTB232, contains as protein-carrier N-end polypeptide fragment glutathione S-transferase S.j. (226 a.o. with m.w. of 26.3 kDa), joined via end site of thrombin hydrolysis (LVPRGS) with C-end polypeptide fragment of antigen CFP10 (100 a.o. with m.w. of 10.8 kDa) and has complete aminoacid sequence with length of 326 a.o. and m.w. of 37.1 kDa, given in text of description.

EFFECT: use of invention provides for the possibility to produce target highly pure hybrid polypeptide GST-CFP10 in preparative amounts with preservation of immunogenic properties of the latter.

3 cl, 4 dwg, 4 tbl, 6 ex

FIELD: pharmacology.

SUBSTANCE: present invention refers to immunology and biotechnology. There is offered recovered human antibody to RG1 polypeptide. There are described versions of antibodies, including one-chain antibody, and immunoconjugate based on said antibodies. There are disclosed methods of selective cell destruction, cell inhibition, treatment of disease state, detection of disease state, detection of RG1, monitoring of clinical course of prostate cancer, prediction in a person with using antibodies and immunoconjugate.

EFFECT: application of the invention provides new antibodies to RG1 polypeptide that can find application in treating tumours with RG1 overexpression.

16 cl, 4 dwg, 1 tbl, 13 ex

FIELD: medicine.

SUBSTANCE: recombinant strain Escherichia coli is produced, which contains plasmid pHINS21 (Escherichia coli JM109/ pHINS21), defining synthesis of hybrid protein, made of N-terminal fragment of human gamma-interferon and human proinsulin, joined by peptide linker, which contains site of splitting with enterokinase (Asp4Lys). Yield of hybrid product that includes proinsulin, provided with new strain-producer, makes at least 30% of total amount of cell protein. Method is suggested for preparation of human proinsulin, including cultivation of strain-producer Escherichia coli JM109/pHINS21, separation of inclusion bodies and their dissolution, renaturation of hybrid protein and its cleaning with ion-exchange chromatography, splitting of hybrid protein with enterokinase or its catalytic subunit and cleaning of proinsulin by ion-exchange chromatography on sorbates with sulfprofile groups.

EFFECT: invention simplifies technological process for production of recombinant human proinsulin and improves conditions of its execution from the point of view of safety engineering.

4 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: protein is constructed, which includes DNA-binding domain SSBTne from thermophile microorganism Termatoga neapolitana, connected to C-end of domain VirD2 from Agrobacterium tumefaciens, which is a signal of nuclear localisation.

EFFECT: efficient transport of transgene into cell nucleus.

6 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method is suggested for production of antibody for binding to NK-cells, which crossly interacts with products of gene KIR2DL1 and KIR2DL2/3 and neutralises inhibitor activity of such KIR. Mentioned method includes selection of such antibodies that crossly interact at least with products of gene KIR2DL1 and KIR2DL2/3, are able to restore lysis with NK cells Cw3+ or Cw4+ target cells and are bound with NK cells or polypeptide of KIR primate. Antibodies produced by this method are described, as well as their derivatives, where antibody is linked with toxin, radionuclide, recognisable aggregation, solid carrier or polyethylene glycol.

EFFECT: invention provides for preparation of single type of antibodies, which controls activity of NK cells of various type, provides for amplification of their cytotoxicity, which may find application in therapy, for increase of activity or cytotoxicity of NK cells in individuals without preliminary detection of HLA type in individual.

7 cl, 13 dwg, 4 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention is a humanised form of mouse anti c-met antibody. The disclosed antibody enables different degrees of impairment of the HGF/c-met signalling pathway.

EFFECT: improved method.

29 cl, 17 dwg, 2 tbl, 1 ex

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