Bivalent bispecific antibodies

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to method of obtaining bivalent bispecific antibody, which includes transformation of host cell by vectors, containing molecules of nucleic acids, coding first light chain and first heavy chain of bivalent bispecific antibody, and vectors, containing molecules of nucleic acids, coding second light chain and second heavy chain of bivalent bispecific antibody, cultivation of host cell under conditions, providing synthesis of molecule of bivalent bispecific antibody from said culture. Said antibody contains first light chain and first heavy chain of antibody, specifically binding with first antigen, and second light chain and second heavy chain of antibody, specifically binding with second antigen, in which variable domains VL and VH of second light chain and second heavy chain are replaced by each other and constant domains CL and CH1 of second light chain and second heavy chain are replaced by each other.

EFFECT: invention makes it possible to increase output of correct bispecific antibody by increasing the level of correct heterodimerisation of heavy chains of wild type and modification of heavy chains resulting from crossing over.

2 cl, 31 dwg, 3 tbl, 4 ex

 

The present invention relates to new bivalent bespecifically antibodies, their obtaining and application.

Background of the invention

In this area known engineered proteins, such as bi - and multispecific antibodies, which can bind two or more antigens. These multispecificity binding proteins can be created on the basis of methods of cell fusion, chemical conjugation or recombinant DNA.

In recent years a wide variety of formats recombinant bespecifically antibodies, for example tetravalent bespecifically antibody obtained by fusion, for example IgG antibody format and single chain domains (see e.g. Coloma, M. J., etc., Nature Biotech 15, 1997, cc.159-163; WO 001/077342 and Morrison S. L., Nature Biotech 25, 2007, cc.1233-1234).

Also developed several other new formats that are no longer preserved the basic structure of antibodies (IgA, IgD, IgE, IgG or IgM), such as dia-, Tria - or tetrathele, mini body, several single chain formats (scFv, bis-scFv), which can bind two or more antigens (Holliger R. et al., Nature Biotech 23, 2005, cc.1126-1136; Fischer, N., Leger, O., Pathobiology 74, 2007, cc.3-14; Shen J, etc., Journal of Immunological Methods 318, 2007, cc.65-74; Wu s et al., Nature Biotech 25, 2007, cc.1290-1297).

In all of these formats use linkers either to merge the basic structure of the ant�body (IgA, IgD, IgE, IgG or IgM) with a further binding protein (e.g. scFv) or to merge, e.g. two Fab fragments or scFv (Fischer N., Leger, O., Pathobiology 74, 2007, cc.3-14). While it is clear that the linkers have the advantage when creating bespecifically antibodies, they can be related also to the problem of therapeutic plan. In fact, these foreign peptides can induce an immune response against the linker or the area of the junction between the protein and the linker. In addition, the flexible nature of these peptides makes them more sensitive to proteolytic cleavage, which can lead to poor stability, aggregation and increased immunogenicity of the antibody. In addition, there may be a need to maintain effector functions such as complement-dependent cytotoxicity (CDC) or antibody-induced cell dependent immune cytotoxicity (ADCC) mediated by Fc-region, by maintaining a high degree of similarity to naturally occurring antibodies.

Thus, ideally, you must create bespecifically antibodies, which have a very similar overall structure with naturally occurring antibodies (type IgA, IgD, IgE, IgG or IgM), with minimal deviation from human sequences.

In accordance with one approach bespecifically antibodies with high�coy degree of similarity to naturally occurring antibodies created using technology quadrom (quadrigemina) (see Milstein C. and A. C. Cuello, Nature, 305, 1983, cc.537-540) based on the somatic fusion of two different hybrid cell lines expressing murine monoclonal antibodies with the desired specificnosti especifismo antibodies. As a result of random pairing of heavy and light chains from two different antibodies produced in cell lines of hybrid hybridomas (or quadroma) get up to 10 different kinds of antibodies, of which only one represents the desired functional bespecifically antibody. Due to the presence of incorrectly paired of by-products and significantly reduced product yield, require more complex cleaning procedures (see, e.g., Morrison S. L., Nature Biotech 25, 2007, cc.1233-1234). In General, this problem of incorrectly paired byproducts, persists in applying the methods of recombinant expression.

Approach with which you can work around the problem of having incorrect mating byproducts, known as the "knobs-into-holes" (interaction type protrusion-depression"), aims to strengthen the coupling of two different heavy chains of the antibody by introduction of mutations in the CH3 domains for the modification of the interface in the contact area. On one chain with the large size of the amino acids were replaced by amino acids with short side chain�s to create "hollow". Conversely, amino acids with larger side chains were introducible in the other CH3 domain, creating a "ledge". By co-expression of two heavy chains (and two identical light chains, which must comply with both heavy chains), received a high output heterodimer formation ("protrusion-depression") with respect to homodimer formation ("depression is a depression or ledge-ledge") (Ridgway J. B., Presta L. G., R. Carter and WO 1996/027011). The percentage of heterodimer can be further improved by remodeling of the boundary surfaces of the two CH3 domains using a phage display technology and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant, A. M. et al., Nature Biotech 16, 1998, cc.677-681; Atwell, S., Ridgway J. B., Wells J. A., Carter P., J Mol Biol 270, 1997, cc.26-35). New approaches to technology "knobs-into-holes" described for example in EP 1870459 A1. Although this format is probably very attractive, there are currently no data on its development towards clinical application. One important limitation of this strategy is that the light chains of the two parental antibodies must be identical to prevent incorrect mating and formation of inactive molecules. Thus, this technology is not suitable for easy creation of recombinant bivalent bespecifically antibodies to two antigens� using as the source of the two antibodies to the first and second antigen, as should be optimized or the heavy chain of these antibodies, and/or identical light chains.

In WO 2006/093794 compositions described heterodimeric binding proteins. In WO 99/37791 multipurpose described derivatives of antibodies. In Morrison, etc., J. Immunolog, 160, 1998, cc.2802-2808 describes the impact of the replacement of variable regions on the functional properties of IgG.

A brief summary of the invention

The present invention relates to bivalent especificacao the antibody containing:

a) light chain and heavy chain of an antibody specifically binding to a first antigen; and

b) the light chain and heavy chain of an antibody specifically binding to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which CL and CH1 domains of the constant regions (constant domains CL and CH1) are replaced by each other.

The following variant embodiment of the invention is a method of producing bivalent especifismo antibodies, proposed in the invention consists in the fact that

(a) transforming a host cell

vectors that contain nucleic acid molecules encoding the light chain and heavy chain of an antibody that specifically binds to the first antigen,

vectors that contain nucleic acid molecules encoding the light chain and heavy chain of the antibodies�and, that specifically binds to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which the constant domains CL and CH1 are replaced at each other;

b) cultivate a host cell under conditions that allow to synthesize the specified antibody molecule; and

b) secrete the antibody molecule from the culture.

The following variant embodiment of the invention is a host that contains

- vectors that contain nucleic acid molecules encoding the light chain and heavy chain of an antibody that specifically binds to the first antigen,

- vectors that contain nucleic acid molecules encoding the light chain and heavy chain of an antibody that specifically binds to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which the constant domains CL and CH1 are replaced at each other.

The following variant embodiment of the invention is a composition, preferably a pharmaceutical or diagnostic composition of the antibody, proposed in the invention.

The following variant embodiment of the invention is a pharmaceutical composition, which contains the antibody, proposed in the invention, and at least one pharmaceutically acceptable excipient.

The alternative of implementing�t of the invention is a method of treating a patient, in need of therapy, characterized in that is administered to the patient in a therapeutically effective amount of the antibody, proposed in the invention.

Detailed description of the invention

The invention relates to bivalent especificacao the antibody containing:

a) light chain and heavy chain of an antibody specifically binding to a first antigen; and

b) the light chain and heavy chain of an antibody specifically binding to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which the constant domains CL and CH1 are replaced at each other.

Thus, divalent bespecifically antibody contains:

a) the first light chain and the first heavy chain of an antibody specifically binding to a first antigen; and

b) a second light chain and the second heavy chain of an antibody specifically binding to a second antigen,

in which the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other

and

in which the constant domains CL and CH1 of the second light chain and the second heavy chain is replaced at each other.

Thus, to generate antibodies specifically binding to a second antigen, apply the following processes:

in light chain

variable domain VL light chain is replaced by variable domain VH of the heavy chain �indicated antibody and the constant domain CL light chain is replaced by the constant domain CH1 of the heavy chain of the indicated antibody;

and in the heavy chain

variable domain VH of the heavy chain is replaced by variable domain VL light chain of the indicated antibody and the constant domain CH1 of the heavy chain is replaced by a constant domain CL light chain of the indicated antibody.

The term "antibody" in the context of the present description refers to the full monoclonal antibodies. Such antibodies are composed of two pairs, each of which includes a light chain (LC) and heavy chain (HC) (these pairs of light chain (LC)/heavy chain abbreviated denote in the context of the present description as LC/HC). Light chains and heavy chains of these antibodies are polypeptides consisting of multiple domains (areas). In a complete antibody, each heavy chain contains a variable region heavy chain (abbreviated as designated in the context of the present description as HCVR or VH) and a constant region of the heavy chain. The constant region of the heavy chain contains a constant CH1-, CH2 - and CH3 domains of the heavy chain (antibody, which belongs to the classes IgA, IgD and IgG) and do not necessarily constant CH4-domain of the heavy chain (antibody, which refers to classes of IgE and IgM). Each light chain contains a variable domain light chain VL and a constant domain of the light chain CL. The structure of one class of naturally occurring antibodies, namely antibodies of the IgG class, are shown, e.g.�, Fig.1. The variable domains VH and VL can be further subdivided into the field of hypervariability designated as hypervariable sites (CDR), interspersed more conservative areas, which are called frame sections (FR). Each VH - and VL-region is composed of three CDR and four FR located from aminobenzo to carboxilic in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Janeway C. A., Jr. etc., Immunobiology., 5-e Izd., publishing Garland Publishing, 2001; and J. Woof, Burton D., Nat Rev Immunol 4, 2004, cc.89-99). Two pairs, each comprising a heavy chain and light chain (HC/LC), possess the ability to specifically contact with the same antigen. Thus, a complete antibody is a bivalent monospecifičeskoj antibody. These "antibodies" are, for example, mouse antibodies, human antibodies, chimeric antibodies, humanized antibodies and developed by using genetic engineering of the antibody (antibody or the mutant antibody), if they retain their characteristic properties. Most preferred are human or humanized antibodies, primarily in the form of a recombinant human or humanized antibodies.

Know the five types of heavy chains of antibodies of mammals, which represent the Greek letters: α, δ, ε, γ and µ (Janeway C. A., Jr. etc., Immunobiology., 5-e Izd., �ZD-Garland Publishing, 2001). Present the type of heavy chain determines the class of antibody; these chains are detected in the antibody type IgA, IgD, IgE, IgG and IgM, respectively (Rhoades R. A., R. G. Pflanzer, Human Physiology, 4th ed., publishing house Thomson Learning, 2002). Different heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε is composed of approximately 550 amino acids.

Each heavy chain contains two domains, i.e., a constant region and a variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotype. Heavy chains γ, α and δ contain a constant region that consists of three constant domains CH1, CH2 and CH3 (ranked), and hinge region, which gives flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); heavy chains μ and ε contain a constant region that consists of four constant domains CH1, CH2, CH3 and CH4 (Janeway C. A., Jr. etc., Immunobiology., 5-e Izd., publishing Garland Publishing, 2001). The variable region of the heavy chain differs in antibodies that are produced by different b-cells, but is the same for all antibodies produced by individual b-cell or B-cell clone. Variable region of each heavy chain of the antibody contains approximately 110 amino acids and consists of a single domain.

In mammals there are only two types of light chains, which is about�administered as lambda (λ) and Kappa (κ). A light chain has two successive domains: one constant domain CL and one variable domain VL. Approximate length light chain is 211-217 amino acids. Preferably, the light chain is a Kappa light (κ) chain, and the constant domain CL preferably represents C-Kappa (κ).

The concept of "monoclonal antibody" or "monoclonal antibody composition" in the context of the present description relate to a preparation of antibody molecules of identical amino acid composition.

"Antibodies" according to the invention can be an antibody of any class (e.g., IgA, IgD, IgE, IgG and IgM, preferably IgG or IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1), and both antibodies, which is output from divalent bespecifically antibody, proposed in the invention, have the Fc-region of the same subclass (e.g., IgG1, IgG4, etc., preferably IgG1), preferably of the same allotype (e.g., Caucasian).

The term "Fc region (fragment) antibody" is well known to specialists in this field and it is determined based on the cleavage of antibodies with papain. Antibodies, proposed in the invention contain as the Fc-region is preferably the Fc-region derived from human antibody, and preferably all other parts of the human to�nstantly areas. Fc-region of an antibody is directly involved in complement activation, C1q-binding, C3 activation and binding to Fc-receptor. While the effect of the antibodies of the complement system is subject to certain conditions, binding to C1q is due to specific binding sites in the Fc-region. These binding sites are known in this field and are described, for example, T. J. Lukas, etc., J. Immunol. 127, 1981, cc.2555-2560; Brunhouse, R., and Cebra, J. J., Mol. Immunol. 16, 1979, cc.907-917; Burton D. R., etc., Nature 288, 1980, cc.338-344; Thommesen, J. E., and others, Mol. Immunol. 37, 2000, cc.995-1004; Idusogie, E. E., etc., J. Immunol. 164, 2000, cc.4178-4184; Hezareh M et al., J. Virol. 75, 2001, cc.12161-12168; Morgan A. et al., Immunology 86, 1995, cc.319-324; and EP 0307434. These binding sites represent, for example, L234, L235, D270, N297, E, K320, K322, R331 and R329 (numbering is given according to the EU numbering Cabot, see below). Antibodies of subclasses IgG1, IgG2 and IgG3 usually characterized by their ability to complement activation, C1q-binding and C3 activation, whereas IgG4 do not activate the complement system, do not bind to C1q and activate C3. Preferably, the Fc region is a human Fc-region.

The term "chimeric antibody" refers to an antibody containing the variable region, i.e. binding region derived from a single source or from the same species, and at least a portion of a constant region derived from a different source, or other species, and how great�ILO, get using the methods of recombinantly DNA. Preferred are chimeric antibodies that contain murine variable region and human constant region. Other preferred forms of "chimeric antibodies", falling under the scope of the present invention are antibody constant region which is modified or changed compared to the original antibody for the purpose of obtaining the properties proposed in the invention, particularly about the C1q binding and/or binding of Fc-receptor (FcR). Such chimeric antibodies is also referred to as "switched antibodies of class. Chimeric antibodies are the product of expression of immunoglobulin genes containing the DNA segments that encode the variable regions of immunoglobulins, and the segments of DNA that encode the constant region of immunoglobulins. Methods of producing chimeric antibodies involve conventional methods of recombinant DNA and gene transfection, which are well known in the art (see, e.g., Morrison S. L., etc., Proc. Natl. Acad. Sci. USA 81, 1984, cc.6851-6855; US 5202238 and US 5204244).

The term "humanized antibody" refers to antibodies in which a frame or a hypervariable sites (CDR) has been modified so that they contain a CDR of an immunoglobulin of different specificity as compared with the specificity of the parent immunoglobulin�. In a preferred embodiment of the invention for obtaining a "humanized antibody" of mice transplanted CDR frame in the area of human antibodies (see, e.g., L. Riechmann et, Nature 332, 1988, cc.323 to 327; and M. S. Neuberger et, Nature 314, 1985, cc.268-270). Especially preferred CDRs correspond to CDR, which are sequences recognizing the antigens listed above for chimeric antibodies. Other forms of "humanized antibodies", falling under the scope of the present invention are antibody constant region which is further modified or changed compared to the original antibody for the purpose of obtaining the properties proposed in the invention, particularly about the C1q binding and/or binding of Fc-receptor (FcR).

The term "human antibody" in the context of the present description refers to the antibody variable and constant region which is derived from immunoglobulin sequences of the human germ line. Human antibodies are well known in the art (M. A. van Dijk and van de Winkel J. G., Curr. Opin. Chem. Biol. 5, 2001, cc.368-374). Human antibodies can also be obtained in transgenic animals (e.g. mice) that are a result of immunization can produce a full spectrum or a certain part of the human antibodies in the absence of production endogeno� immunoglobulin. Transfer of genes of the human immunoglobulin germ line in this mutant germ line of mice should lead to the production of human antibodies after antigenic stimulation (see, e.g., Jakobovits, A., and others, Proc. Natl. Acad. Sci. USA 90, 1993, cc.2551-2555; Jakobovits A. et al., Nature 362, 1993, cc.255-258; Bruggemann M., etc., Year Immunol. 7, 1993, cc.33-40). Human antibodies can also be obtained using phage display libraries (Hoogenboom, H. R. and Winter, G., J. Mol. Biol. 227, 1992, cc.381-388; J. D. Marks et, J. Mol. Biol. 222, 1991, cc.581-597). To obtain human monoclonal antibodies can also be used methods Cole with co-workers and Boerner with co-authors (Cole et, Monoclonal Antibodies and Cancer Therapy, ed. by Alan R. Liss, 1985, p. 77; and Boerner, R., and others, J. Immunol. 147, 1991, cc.86-95). As already mentioned for chimeric and humanized antibodies, proposed in the invention, the term "human antibody" also includes such antibody constant region which is modified for the purpose of obtaining the properties proposed in the invention, particularly about the C1q binding and/or FcR binding, e.g. by "class switching" i.e. change or mutation of Fc-regions (e.g., IgG1 to IgG4 and/or IgG1/IgG4 mutation).

The term "recombinant human antibody" in the context of the present description refers to all human antibodies that receive, Express, or create isolated using the methods of recomb�nation, such as antibodies isolated from a host cell, such as NS0 or Cho cell or from an animal (e.g. a mouse) that is transgenic due to the presence of human immunoglobulin genes or antibodies expressed using a recombinant expression vector, which transfection the host. Such recombinant human antibodies have variable and constant region, which are located in a converted form. Recombinant human antibodies, proposed in the invention, is subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of VH and VL regions of the recombinant antibodies are sequences that, while derived from sequences of VH and VL of the human germ line and related lines, may not exist in natural conditions in the spectrum of the human germ line antibody in vivo.

The term "variable region (domain)" (variable domain light chain (VL) variable region heavy chain (VH)) in the context of the present description relates to the field of each pair of light and heavy chains, which are involved directly in binding the antibody to the antigen. The variable domains of human light and heavy chains have the same General structure and each domain contains h�tire frame plot (FR), sequences which are very conservative, connected by three "hypervariable sites (or complementarity determining areas, CDR). Frame sections adapted to β-folded conformation and the CDRs may form loops connecting the β-folded structure. The CDR in each chain retain their three dimensional structure using skeleton plots and form together with the CDRs from the other chain the antigen-binding center. CDR3-areas of heavy and light chains of the antibodies play a particularly important role in the binding specificity/affinity of antibodies, proposed in the invention, and are therefore an additional object of the invention.

The concept of "hypervariable site" or "antigen-binding center of the antibody" in the context of the present description relate to amino acid residues of an antibody which are responsible for binding antigen. Hypervariable portion contains amino acid residues from a "complementarity determining areas" or "CDR". "Framework" or "FR"-areas represent areas of variable regions, other than those specified in the present description of the residues of the hypervariable area. Thus, the light and heavy chain antibodies contain in the direction from N - to C-Terminus sites FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The CDR in each chain amino acids separated by a specified frame of the site In particular CDR3 of the heavy chain represents the area that makes the greatest contribution to the binding to the antigen. CDR and FR-plots determined using standard nomenclature of Cabot (Kabat et, Sequences of Proteins of Immunological Interest, 5th ed., publishing house of the Public Health Service, National Institutes of Health, Bethesda, MD (1991).

"Constant region (domains) of the heavy chain and light chain are not directly involved in the binding of the antibody to an antigen, but exhibit different effector functions. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins are divided into the classes mentioned above.

The term "divalent bespecifically antibody" in the context of the present description refers to an antibody, as described above, in which each of the two pairs comprising a heavy chain and light chain (HC/LC), specifically binds to a different antigen, i.e., the first heavy and the first light chain (derived from antibody to the first antigen) specifically associated with both the first antigen and the second heavy and the second light chain (derived from antibody to a second antigen) are specifically associated both with a second antigen (as shown in Fig.2); these divalent bespecifically antibodies have the ability to specifically communicate simultaneously with two different antigens and not more than �two antigens unlike, first, from monospecifičeskoj antibody that has the ability to bind to only one antigen, and, secondly, from tetravalent terspecific antibody that has the ability to communicate simultaneously with four molecules of antigens.

According to the invention the ratio between the desired divalent bespecifically antibody and unwanted side products can be improved by replacing some domains only in one pair comprising a heavy chain and light chain (HC/LC). Although the first of the two HC/LC pairs, obtained from an antibody that specifically binds to the first antigen, remains virtually unchanged, the second of the two HC/LC pairs, obtained from an antibody that specifically binds to a second antigen, modified using the following replacements:

light chain: replacement of the variable domain VL light chain variable domain VH of the heavy chain of the indicated antibody that specifically binds to a second antigen, and a constant domain CL light chain constant CH1 domain of the heavy chain of the indicated antibody that specifically binds to a second antigen,

heavy chain: replacement of the variable domain VH of the heavy chain variable domain VL light chain of the indicated antibody that specifically binds to a second antigen, and cons�antago CH1 domain of the heavy chain constant domain CL light chain of the indicated antibody which specifically binds to the second antigen.

Thus, the resulting bivalent bespecifically antibodies are artificial antibodies, which contains

a) light chain and heavy chain of an antibody specifically binding to a first antigen; and

b) the light chain and heavy chain of an antibody specifically binding to a second antigen,

where the specified light chain (of an antibody specifically binding to a second antigen) contains a variable domain VH is VL and a constant domain CH1 is CL,

where the specified heavy chain (of an antibody specifically binding to a second antigen) contains a variable domain VL instead of VH and a constant domain CL instead of CH1.

According to another object of the invention specified improved the ratio between the desired divalent bespecifically antibody and unwanted side products can be improved even further by using one of the following two alternative approaches.

A) the First alternative approach (see Fig.3)

CH3 domains of the indicated divalent especifismo antibodies, proposed in the invention, it is possible to change via technology "knob-into-holes", which is described in detail with several examples, for example in WO 96/027011, Ridgway J. B., etc., Protein Eng 9, 1996, cc.617-621; and Merchant, A. M. et, Nat Biotechnol 16, 1998, cc.677-681. P�and using this method of interacting surfaces of the two CH3 domains change with the aim of increasing heterodimerization both heavy chains, containing these two CH3 domain. Each of the two CH3 domains (two heavy chains) can be a "ledge" and the other to be a "depression". The introduction of a disulfide bridge stabilizes heterodimer (Merchant A. M, et, Nature Biotech 16, 1998, cc.677-681; Atwell, S., Ridgway J. B., Wells J. A., Carter P., J Mol Biol 270, 1997, cc.26-35), and increases product yield.

Thus, according to a preferred embodiment, the CH3 domains of bivalent especifismo antibodies, in which the first CH3 domain and second CH3 domain each in contact with each other at the interface, which represents the original boundary surface between the CH3-domain of an antibody, change through technology "knob-into-holes", including additional stabilization by introduction of a disulfide bridge in the CH3 domains (as described in WO 96/027011, Ridgway J. B., etc., Protein Eng 9, 1996, cc.617-621; Merchant A. M, etc., Nature Biotech 16, 1998, cc.677-681; Atwell, S., Ridgway J. B., Wells J. A., Carter P., J Mol Biol 270, 1997, cc.26-35) to activate the formation of divalent especifismo antibodies.

Thus, according to one of the objects of the invention divalent bespecifically antibody is characterized in that the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each in contact with each other at the interface, which represents the original boundary surface between the CH3-domain of an antibody; e�Ohm interface change to enable the formation of divalent especifismo antibodies, where change is characterized in that:

a) change the CH3 domain of one heavy chain so that the original surface of the partition CH3 domain of one heavy chain, which is in contact with the original surface section of CH3 domain of the second heavy chain in divalent bespecifically the antibody, amino acid residue substituted with amino acid residue which has a larger side chain volume, thereby creating a bulge on the surface of the partition CH3 domain of one heavy chain that can fit into the cavity at the interface of CH3 domain of the other heavy chain, and

b) change the CH3 domain of the other heavy chain so that the original interface of the second CH3-domain, which is in contact with the original surface section of the first CH3-domain dyadic bespecifically the antibody, amino acid residue substituted with amino acid residue having a smaller side chain volume, thereby creating a cavity at the interface of the second CH3 domain that can fit a bulge at the interface of the first CH3-domain.

Preferably, the specified amino acid residue, which has a larger volume side chain, selected from the group comprising arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

Preferably, the specified amino acid residue having a smaller volume�Moo side chain, selected from the group including alanine (A), series (S), threonine (T) valine (V).

According to one of the objects of the invention both CH3 domains, optionally modified by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain, so that could form a disulfide bridge between both CH3-domains.

In another preferred embodiment of the invention both CH3 domains are altered, using the remains of R409D; K370E (K409D) as constituting a "ledge" residues and D399K; E357K as constituting a "depression" residues, as described for example in EP A.

Or

B) the Second alternative approach (see Fig.4)

This approach involves the replacement of the CH3 domain of the constant region of one heavy chain CH1 domain of the constant region of the heavy chain; and replacement of the CH3 domain of the constant region heavy chain CL domain of a constant region light chain.

CH1 domain of the constant region of the heavy chain, which replace the CH3 domain of the heavy chain, may be a domain of any Ig class (e.g., IgA, IgD, IgE, IgG and IgM) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

CL-domain of a constant region of light chain, which replace the CH3 domain of the heavy chain, may be the lambda (λ) or Kappa (κ) type, preferably Kappa (κ) type.

Thus, a preferred option implementation�ia of the invention is a bivalent bespecifically antibody, which contains:

a) light chain and heavy chain of an antibody specifically binding to a first antigen; and

b) the light chain and heavy chain of an antibody specifically binding to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which the constant domains CL and CH1 are replaced at each other, and is optional

in) the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain, each of which is in contact with each other at the interface, which represents the original boundary surface between the CH3-domain of an antibody; wherein the surface of the partition change to enable the formation of divalent especifismo antibodies, where change is characterized in that:

VA modified CH3 domain of one heavy chain so that the original surface of the partition CH3 domain of one heavy chain, which is in contact with the original surface section of CH3 domain of the second heavy chain in divalent bespecifically the antibody, amino acid residue substituted with amino acid residue which has a larger side chain volume, thereby creating a bulge on the surface of the partition CH3 domain of one heavy chain that can fit into the cavity at the interface of CH3 domain of the other heavy chain,

and

WB, are modified CH3 domain of the other heavy chain so that the IP�one surface section of the second CH3-domain, which is in contact with the original surface section of the first CH3-domain dyadic bespecifically the antibody, amino acid residue substituted with amino acid residue having a smaller side chain volume, thereby creating a cavity at the interface of the second CH3 domain that can fit a bulge at the interface of the first CH3 domain;

or

d) the CH3 domain of the constant region of one heavy chain is replaced by CH1 domain of the constant region of the heavy chain; and CH3 domain of the constant region of the other heavy chain is replaced by the CL domain of a constant region light chain.

The term "antigen" or "antigen molecule" in the context of the present description are used interchangeably, and they refer to all molecules that can specifically contact the antibody. Divalent bespecifically antibody specifically binds to the first antigen and the second different from the first antigen. The term "antigen" in the context of the present description include, for example, proteins, various epitopes of proteins (as different antigens according to the invention) and polysaccharides. They mainly includes parts (coats, capsules, cell walls, flagella, fimbrae and toxins) of bacteria, viruses and other microorganisms. Lipids and nucleic acids are the antigens only when they join�nenii with proteins and polysaccharides. Non-microbial exogenous (foreign) antigens can include pollen, egg protein, and proteins from transplanted tissues or organs, or antigens, on the surface transfusiona blood cells. Preferably, the antigen is selected from the group including cytokines, cell surface proteins, enzymes and receptors of cytokines, cell surface proteins, enzymes and other receptors.

Tumor antigens are antigens that are presented by molecules I or GCG GCG II on the surface of tumor cells. Sometimes these antigens are presented by tumor cells and never the healthy cells. In this case, they are called specific for tumor antigens (TSA), and they usually are the result of specific tumor mutations. More common are antigens that are presented by tumor cells and healthy cells, and they are called associated with tumor antigens (TAA). Cytotoxic T-lymphocytes that recognize these antigens, may have the ability to destroy tumor cells before their proliferation or metastasis. Tumor antigens can also be provided on the surface of the tumor in the form modified by mutation of the receptor, in this case, they should be recognized by b-cells.

In one preferred �of aryanto the invention, at least one of the two different antigens (first and second antigen), which divalent bespecifically antibody specifically binds, is a tumor antigen.

In another preferred embodiment, both different antigens (first and second antigen), to which divalent bespecifically antibody specifically binds represent tumor antigens; in this case, the first and second antigen may represent two different epitope of the same specific for a tumor protein.

In another preferred embodiment, at least one of the two different antigens (first and second antigen), to which divalent bespecifically antibody specifically binds, is a tumor antigen and the other is an antigen effector cells, such as T-cell receptor, CD3, CD16, etc.

In another preferred embodiment, at least one of the two different antigens (first and second antigen), to which divalent bespecifically antibody specifically binds, is a tumor antigen and the other is an anti-cancer substance such as a toxin or inhibitor of the kinase.

In the context of the present description, the term "specifically binds" or "svyazyvaete� specifically to" refers to the antibody, specifically binding to the antigen. Preferably, the binding affinity to antibodies specifically binding with said antigen, characterized by a KD value of 10-9mol/l or lower (e.g., 10-10mol/l), preferably a KD value of 10-10mol/l or lower (e.g., 10-12mol/l). The affinity of the binding is determined using standard analysis of binding, such as surface plasma resonance (Biacore®).

The term "epitope" includes any polypeptide determinant having the ability to specifically contact the antibody. In some embodiments, epitope of determinants include chemically active surface groups of molecules such as amino acids, side chains of sugars, phosphoryl or sulfonyl, and in some embodiments, may have specific three dimensional structural characteristics and/or specific charge characteristics. The epitope is a region of the antigen that binds the antibody. In some embodiments, it is believed that the antibody specifically binds to an antigen when it recognizes its antigen target in a complex mixture of proteins and/or macromolecules.

Another option of the invention is a method of producing �muhametovo especifismo antibodies, proposed in the invention consists in the fact that

(a) transforming a host cell

vectors that contain nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen,

vectors that contain nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which the constant domains CL and CH1 are replaced at each other;

b) cultivate a host cell under conditions that allow to synthesize the specified antibody molecule; and

b) secrete the antibody molecule from the culture.

In General, apply two vectors encoding the light chain and heavy chain of an antibody that specifically binds to the first antigen, and two vector encoding the light chain and heavy chain of an antibody that specifically binds to the second antigen. One of the two vectors encodes the corresponding light chain and the other of the two vectors encodes the corresponding heavy chain. However, in an alternative embodiment of a method for producing divalent especifismo antibodies, proposed in the invention, can be used for transformation of a host cell only one first vector encoding a light chain� and heavy chain antibodies, specifically binding to a first antigen, and only one second vector encoding a light chain and heavy chain of an antibody specifically binding to a second antigen.

The invention relates also to a method for producing antibodies, namely that cultivate appropriate to the host cell under conditions that allow to synthesize antibody molecules, and secrete the antibodies from the culture, for example, providing for the expression

the first nucleotide sequence which encodes a light chain of an antibody specifically binding to a first antigen,

second nucleotide sequence that encodes a heavy chain of an antibody specifically binding to a first antigen,

- a third nucleotide sequence that encodes a light chain of an antibody specifically binding to a second antigen, in which the variable domain light chain VL are replaced by variable domain VH of the heavy chain and the constant domain CL light chain is replaced by the constant domain CH1 of the heavy chain, and

- fourth nucleotide sequence that encodes a heavy chain of an antibody specifically binding to a second antigen, in which the variable domain VH of the heavy chain is replaced by a variable domain light chain VL and a constant domain CH1 of the heavy chain samananda constant domain CL light chain.

Another option of the invention is a host that contains

- vectors that contain nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a first antigen,

- vectors that contain nucleic acid molecules encoding the light chain and heavy chain of an antibody specifically binding to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which the constant domains CL and CH1 are replaced at each other.

Another option of the invention is a host that contains

a) a vector containing a nucleic acid molecule which encodes a light chain and a vector containing a nucleic acid molecule that encodes a heavy chain of an antibody specifically binding to a first antigen,

b) a vector containing a nucleic acid molecule which encodes a light chain and a vector containing a nucleic acid molecule which encodes a heavy chain of an antibody specifically binding to a second antigen, in which the variable domains VL and VH are replaced on each other

and

in which the constant domains CL and CH1 are replaced at each other.

Another option of the invention is a composition, preferably the pharmaceutical�technical or diagnostic composition divalent especifismo antibodies, proposed in the invention.

Another option of the invention is a pharmaceutical composition comprising divalent bespecifically antibody, proposed in the invention, and at least one pharmaceutically acceptable excipient.

Another option of the invention is a method of treating a patient who is in need of therapy, characterized in that is administered to the patient in a therapeutically effective amount of divalent bespecifically antibody, proposed in the invention.

The term "nucleic acid or nucleic acid molecule" in the context of the present description relates to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is a double-stranded DNA.

In the context of the present description the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such definitions include the offspring. Thus, the terms "transformants" and "transformed cells" refers to the primary cell and obtained from cultures without regard to the number of transfers. It should be understood that all progeny may not be identical in composition DNA due to deliberate or inadvertent mutations. Under the volume of�of bretania falls variant offspring, having the same function or biological activity, which is found in the original transformed cells. If other definitions are used, it will be obvious from the context.

The concept of "transformation" in the context of the present description refers to the process of transfer of the vector/nucleic acid in a host cell. If the host cells used cells without formidable barriers of cell membranes, the transfection is carried out, for example, by the method of deposition of calcium phosphate, which is described by Graham and Van der Eh, Virology 52, 1978, cc.546 FF. However, you can also apply other methods of introduction of DNA into cells, such as injection of nuclei or the fusion of protoplasts. If used prokaryotic cells or cells which contain the expressed structure of cell membranes, can be applied, for example, transfection method, based on treatment with calcium using calcium chloride, which is described by Cohen, F. N. et, PNAS. 69, 1972, cc.2110-2114.

Recombinant antibodies with the help of transformation is well known in this field and are described, for example, in the review articles of Makrides, S. C., Protein Expr. Purif. 17, 1999, cc.183-202; S. Geisse, etc., Protein Expr. Purif. 8, 1996, cc.271-282; Kaufman R. J., Mol. Biotechnol. 16, 2000, cc.151-161; R. G. Werner, etc., Arzneimittelforschung 48, 1998, cc.870-880, as well as in US 6331415 and US 4816567.

In the context of the present description, the term "expression" relative�designed to the process wherein the nucleic acid is transcribed into mRNA, and/or to the process by which the transcribed mRNA (also referred to as a transcript) can then be translated into peptides, polypeptides or proteins. And transcripts, and the encoded polypeptides refer in General as gene product. If the polynucleotide is removed from genomic DNA, expression in eukaryotic cells may include splicing of the mRNA.

"Vector" is a nucleic acid molecule, in particular self-replicating, which takes the integrated nucleic acid molecule in the host cell and/or between cells-hosts. The term includes vectors that function primarily consists of incorporating DNA or RNA into the cell (for example, chromosomal integration), replication vectors, the function of which consists primarily of DNA replication or RNA, and expression vectors, the function of which consists primarily of transcription and/or translation of DNA or RNA. Under the concept are also vectors that possess more of the said functions.

"Expression vector" is a polynucleotide that is introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. The term "expression system" usually refers to the acceptable cell-master, soda�containing expression vector, function which can be the yield of the desired product expression.

Divalent bespecifically antibodies, proposed in the invention, preferably get recombination methods. These methods are well known in this field and provide protein expression in prokaryotic and eukaryotic cells followed by isolation of polypeptide antibody and, as a rule, cleaning to pharmaceutically acceptable purity. For protein expression of nucleic acids encoding light and heavy chains, or fragments thereof, are inserted into expression vectors using standard methods. The expression is carried out in a suitable prokaryotic or eukaryotic cells-the owners type Cho cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). Divalent bespecifically antibodies may be present in whole cells, in cell lysates or in partially purified or substantially purified form. Cleaning is carried out to remove other cellular components or other contaminants, e.g. other cellular nucleic acids or proteins, by standard methods which include treatment with alkali/LTOs, chromatography on columns and other methods well known in the art (see urrent Protocols in Molecular Biology, edited by Ausubel F., and others, publ Greene Publishing and Wiley Interscience, New York, 1987).

Expression in NS0 cells is described, for example, Barnes L. M., etc., Cytotechnology 32, 2000, cc.109-123; and L. M. Barnes, etc., Biotech. Bioeng. 73, 2001, cc.261-270. Transient expression is described, for example, Durocher Y., etc., Nucl. Acids. Res. 30, 2002, p. E9. Cloning of variable regions is described in R. Orlandi, etc., Proc. Natl. Acad. Sci. USA 86, 1989, cc.3833-3837; P. Carter et, Proc. Natl. Acad. Sci. USA 89, 1992, cc.4285-4289; and L. Norderhaug, etc., J. Immunol. Methods 204, 1997, cc.77-87. The preferred short-time expression system (HEK 293) is described by Schlaeger, E.-J., and Christensen, K., in Cytotechnology 30, 1999, cc.71-83 and Schlaeger, E.-J., in J. Immunol. Methods 194, 1996, cc.191-199.

Regulatory sequences that can be used for prokaryotes represent, for example, a promoter, optionally an operator sequence and the binding site of ribosomes. It is known that eukaryotic cells may use promoters, enhancers and polyadenylation signals.

Nucleic acid is functionally linked" when it is placed under the functional control of another nucleotide sequence. For example, the DNA of predpolagavshegosja or secretion leader is functionally linked to DNA that encodes a polypeptide if it is expressed in the form of predella, which is involved in secretion of the polypeptide; a promoter or enhancer functionally linked to the coding sequence if it has vozdeystviyna the transcription of the sequence; or the binding site of the ribosome is functionally linked to a coding sequence if it is positioned so that it was easier streaming. As a rule, "functionally linked" means that the DNA sequences being linked are contiguous and, in the case of a secretion leader, contiguous reading frame. However, you do not want the enhancers were related. The binding is effected by ligation at suitable restriction sites. If such sites do not exist, then according to common practice, the use of synthetic oligonucleotide adapters or linkers.

Divalent bespecifically antibodies can be separated from the culture medium using standard methods purify immunoglobulins, such as, for example, chromatography on protein a-sepharose, chromatography on hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography. DNA or RNA that encode a monoclonal antibody, easily identified and sequenced using standard methods. The source of such DNA and RNA can serve as cells of the hybrid. After DNA extraction can be integrated into expression vectors, which are then transfecting the host cell, such as cells HEK 293 and Cho cells, or myeloma cells that otherwise are not able to produce a protein of the immunoglobulin, for the synthesis of recombinant monoclonal antibodies in the cell�Ah-hosts.

Amino acid sequence variants (or mutants) divalent especificacao antibody is produced by introduction of the appropriate nucleotide substitutions in DNA antibodies or by the synthesis of nucleotides. However, such modifications are only possible in a very limited range, for example, as described above. For example, the modification should not alter the above-mentioned characteristics of the antibody, such as IgG isotype and binding to the antigen, but can increase the yield of recombination, protein stability or facilitate cleaning.

The following examples, sequence listing and drawings are given with the aim of better understanding the present invention, the full scope of which is presented below in the claims. It is obvious that can be done the modifications set out in the procedures without deviation from the essence of the invention.

Sequence listing

SEQ ID NO:1 amino acid sequence of heavy chain antibodies < IGF-1R> wild-type.

SEQ ID NO:2 amino acid sequence of light chain antibodies < IGF-1R> wild-type.

SEQ ID NO:3 amino acid sequence of the heavy chain * (NS*) antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, in which the VH domain of the heavy chain is replaced by the VL domain of the light chain and CH1 domain of the heavy chain is replaced by the CL domain of the light chain.

SEQ ID NO:4 amino acid n�the sequence of the light chain * (LC*) of antibody < IGF-1R> bearing replacement VL-VH/CL-CH1, in which the VL domain of the light chain is replaced by the VH domain of the heavy chain and the CL domain of the light chain is replaced by a CH1 domain of the heavy chain.

SEQ ID NO:5 amino acid sequence of ectodomain (ECD) IGF-1R bearing a label that represents His-streptavidin-binding peptide (ECD IGF-1R-His-SBP).

SEO ID NO:6 amino acid sequence of the heavy chain of antibody to angiopoietin-2 <ANGPT2> wild-type.

SEO ID NO:7 amino acid sequence of the light chain of antibody to angiopoietin-2 <ANGPT2> wild-type.

SEQ ID NO:8 amino acid sequence of the heavy chain * (HC*) of antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, in which the VH domain of the heavy chain is replaced by the VL domain of the light chain and CH1 domain of the heavy chain is replaced by the CL domain of the light chain.

SEQ ID NO:9 amino acid sequence of the light chain * (LC*) of antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, in which the VL domain of the light chain is replaced by the VH domain of the heavy chain and the CL domain of the light chain is replaced by a CH1 domain of the heavy chain.

SEQ ID NO:10 amino acid sequence of the CH3 domain ("tabs") with the substitution T366W for use in technologies "knobs-into-holes".

SEQ ID NO:11 amino acid sequence of the CH3 domain ("depression") with replacement T366S, L368A, Y407V for use in technologies "knobs-into-holes".

SEQ ID NO:12 amino acid sequence of the heavy chain of the antibody < VEGF> wild-type.

SEQID NO:13 and 14 amino acid sequence of the light chain of the antibody < VEGF> wild-type leader sequence.

SEQ ID NO:15 amino acid sequence of the heavy chain * (HC*) of antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, in which the VH domain of the heavy chain is replaced by the VL domain of the light chain and CH1 domain of the heavy chain is replaced by the CL domain of the light chain and the CH3 domain carries an amino acid sequence with the substitution of T366S, L368A, Y407V ("depression") for use in technologies "knobs-into-holes".

SEQ ID NO:16 amino acid sequence of the heavy chain of the antibody < VEGF> wild type, in which CH3-domain carries an amino acid sequence with the substitution T366W (the"projections") for use in technologies "knobs-into-holes".

SEQ ID NO:17 amino acid sequence of the heavy chain(C)* (HC*) of antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, in which the VH domain of the heavy chain is replaced by the VL domain of the light chain and CH1 domain of the heavy chain is replaced by the CL domain of the light chain, with built-in advanced glycine.

SEO ID NO:18 amino acid sequence of the light chain(G)* (LC*) of antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, in which the VL domain of the light chain is replaced by the VH domain of the heavy chain and the CL domain of the light chain is replaced by a CH1 domain of the heavy chain, with built-in advanced glycine.

Description of the drawings

The drawings show:

Fig.1 - schematic representation of the naturally occurring full antibody type IgG, especiall�quarter against a single antigen, with two pairs of heavy and light chains, each of which contains located in the generally accepted order of variable and constant domains.

Fig.2 - schematic representation of divalent especificacao antibody, comprising: (a) light chain and heavy chain of an antibody that specifically binds to a first antigen; and b) the light chain and heavy chain of an antibody that specifically binds to a second antigen, in which the variable domains VL and VH are replaced at each other and in which the constant domains CL and CH1 are replaced at each other;

Fig.3 - schematic representation of the divalent especificacao antibody, comprising: (a) light chain and heavy chain of an antibody that specifically binds to a first antigen; and b) the light chain and heavy chain of an antibody that specifically binds to a second antigen, in which the variable domains VL and VH are replaced at each other and in which the constant domains CL and CH1 are replaced at each other, and in which the CH3 domains of both heavy chains modified with the help of technology "knobs-into-holes";

Fig.4 - schematic representation of the divalent especificacao antibody, comprising: (a) light chain and heavy chain of an antibody that specifically binds to a first antigen; and b) the light chain and heavy chain of an antibody that specifically binds with a second and�Tigana, in which the variable domains VL and VH are replaced at each other and in which the constant domains CL and CH1 are replaced at each other, and in which one of the CH3 domains of the constant region of both heavy chains is replaced by the CH1 domain of the constant region of the heavy chain; and the other CH3 domain of the constant region of the heavy chain is replaced by the CL domain of a constant region light chain;

Fig.5 - schematic representation of the protein sequence of the heavy chain *<IGF-1R > HC* antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1;

Fig.6 - schematic representation of the protein sequences of light chain *<IGF-1R > LC* antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1;

Fig.7 - plasmid map of the expression vector pUC-HC*-IGF-1R heavy chain *<IGF-1R > HC*;

Fig.8 - plasmid map of the expression vector pUC-LC*-IGF-1R light chain *<IGF-1R > LC*;

Fig.9 - plasmid map of the expression vector 4700-Hyg-OriP;

Fig.10 - the results obtained using LTOs-page for purified monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1 (IgG1*) where HC* and LC* allocated using the immunoprecipitation using protein A-agarose from cell culture supernatants after transient transfection of the cell line HEK293-F;

Fig.11 - data binding monospecifičeskoj antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, and antibodies wildly�of type < IGF-1R> with ECD IGF-1R, obtained based on the evaluation of binding using ELISA;

Fig.12 - plasmid map of the expression vector pUC-HC*-ANGPT2> the heavy chain* <ANGPT2 > HC*;

Fig.13 - plasmid map of the expression vector pUC-LC*-ANGPT2> light chain* <ANGPT2 > LC*;

Fig.14 data obtained by LTOs-page in reducing and Sevostyanova conditions for purified mixture which contains (A) monospecifičeskoj antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, B) bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, and b) antibodies <ANGPT2> wild-type ("a mixture containing bespecifically antibody, bearing replacement VL-VH/CL-CH1"), from cell culture supernatants using affinity chromatography on protein A, followed by gel filtration and concentration;

Fig.15 - implementation of the principle of cellular FACS analysis to detect the formation of the bridging connection type IGF-1R-ANGPT2, using the cell line I24 expressing IGF-1R, to identify the presence of functional especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1;

Fig.16 - the results obtained for samples A-G, using a cellular FACS analysis to detect the formation of the bridging connection type IGF-1R-ANGPT2, using the cell line I24 expressing IGF-1R, to identify the presence of functional bispace�quarter antibody < ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, in the supernatants of cell cultures;

SampleLine cells expressing IGF-1RAntibodyhANGPT2Identifying antibodyDetection
AndI24untreated-<Ang-2>migG1-BiotinSA-PE
BI24untreatedhANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
InI24a mixture containing bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1hANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
GI24a mixture of "wild-type"hANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
DI24antibody <ANGPT2 > wild typehANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
EI24antibody < IGF-1R > wild typehANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
WI24monospecifičeskoj antibody < IGF-1R > bearing replacement VL-VH/CL-CH1hANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE

Fig.17 - the results obtained for samples A-G, using a cellular FACS analysis to detect the formation of the bridging connection type IGF-1R-ANGPT2 using the cell line I24 expressing IGF-1R, to identify the presence of functional especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1;

SampleLine cells expressing IGF-1RAntibodyhANGPT2Identifying an�Italo Detection
AndI24untreated-<Ang-2>migG1-BiotinSA-PE
BI24untreatedhANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
InI24a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1hANGPT2, 2 mg/mlmigG1-Biotin-isotype controlSA-PE
GI24a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1hANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
DI24the mixture is an antibody of wild-typehANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
EI24 antibody <ANGPT2 > wild typehANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE
WI24antibody < IGF-1R > wild typehANGPT2, 2 mg/ml<Ang-2>migG1-BiotinSA-PE

Fig.18 - schematic representation of the principle of the analysis of ECD binding IGF-1R on the basis of ELISA;

Fig.19 - schematic representation of the principle of analysis of binding of ANGPT2-based ELISA;

Fig.20 - schematic representation of the principle of analysis, to detect the formation of the bridging connection type VEGF-ANGPT2, based on ELISA;

Fig.21 - schematic representation of the principle of analysis, to detect the formation of the bridging connection type ANGPT2-VEGF-based Biacore;

Fig.22 - A) obtained by LTOs-page data cleaning antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, where the purified antibody < IGF-1R > bearing replacement VL-VH/CL-CH1 corresponds concentrated, obtained by gel filtration (SEC) pool; B) the data obtained by gel filtration of the purified antibodies < IGF-1R>, bearing replacement VL-VH/CL-CH1;

Fig.23 - A) obtained by LTOs-page data cleaning antibody <ANGPT2> bearing replacement VL-VH/CL-CH1,where the purified antibody < ANGPT2> bearing replacement VL-VH/CL-CH1 corresponds concentrated, obtained using SEC pool; B) the data obtained by gel filtration of the purified antibodies<ANGPT2> bearing replacement VL-VH/CL-CH1;

Fig.24 - data binding with ANGPT2 monospecifičeskoj antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, and antibodies <ANGPT2> wild type, obtained by analysis based binding ELISA;

Fig.25 - data binding with ANGPT2 monospecifičeskoj antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, and antibodies <ANGPT2> wild type, obtained using Biacore analysis;

Fig.26 data obtained using LTOs-page in reducing and Sevostyanova terms of fractions, - eluted after gel filtration especificacao antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1;

Fig.27 - the transcript of the bands obtained by carrying LTOs-page, using mass spectrometry of the native factions. Shows the position of the proteins identified by mass spectrometry in samples of relevant LTOs-page in Sevostyanova conditions;

Fig.28 - ELISA data analysis-eluted fractions 5 and 9, obtained by carrying out gel filtration, especificacao antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1, which allows to detect the formation of the bridging connection type VEGF-ANGPT2. Bespecifically and the tetravalent antibodies�about TvG6-Ang23, recognizing at the same time ANGPT2 and VEGF, included as a positive control;

Fig.29 - data obtained using surface plasma resonance, fractions 5 and 9, obtained by carrying out gel filtration, especificacao antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1, based on the Biacore analysis, to detect the formation of the bridging connection type VEGF-ANGPT2. Bespecifically and tetravalent antibody TvG6-Ang23, recognizing at the same time ANGPT2 and VEGF, included as a positive control;

Fig.30 is a schematic representation especificacao antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1, for heterodimerization which used the technology of "knobs-in-holes";

Fig.31 - data obtained using Biacore analysis of the binding ANGPT2 monospecifičeskoj antibodies <ANGPT2> bearing replacement VL-VH(G)/CL-CH1(G), and antibodies <ANGPT2> wild-type.

Examples

Materials and General methods

General information regarding the nucleotide sequences of light and heavy chains of human immunoglobulin, represented in: Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., publishing house of the Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acids of antibody chains are numbered according to EU numbering (Edelman, G. M., etc., Proc. Natl. Acad. Sci. USA 63, 1969, cc.78-85; Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., publishing house of the Public Health Service, National Institutes of Health, Bthesda, MD, 1991).

Methods of recombinant DNA

For manipulation of DNA were used standard methods described in J. Sambrook et, Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Reagents for molecular biology were used according to the manufacturers instructions.

The gene synthesis

Desired gene segments were obtained from oligonucleotides that are created by chemical synthesis. Gene segments 600-1800 T. D., which are flanked by unique restriction sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequent cloning, using these restriction sites, e.g. KpnI/SacI or AscI/PacI into a cloning vector pGA4, which is based on plasmid pPCRScript (firm Stratagene). The DNA sequence sublimirovanny gene fragments was confirmed by DNA sequencing. Synthesis of gene fragments was performed in the order presented in the specifications of the company Geneart (Regensburg, Germany).

Determination of DNA sequences

The DNA sequence was determined by sequencing two chains at the firm MediGenomix GmbH (Martinsried, Germany) or firm Sequiserve GmbH (Vaterstetten, Germany).

Analysis of DNA sequences and proteins and processing sequence data

Applied the software package firm GCG (Genetics Computer Group, Madison, NJ) Wi), version 10.2 � Infomax's Vector NT1 Advance suite version 8.0 for creating, mapping, analysis, annotation and illustration of sequences.

Expression vectors

For expression of the described antibodies we used variants of expression plasmids designed for transient expression (e.g. in cells HEK293 EBNA or HEK293-F) or on the basis of cDNA constructs using the CMV promoter-intron A, or on the basis of the genomic structure using the CMV promoter.

In addition to the cassette of the antibody expression vectors contain:

- the site of initiation of replication provides replication of this plasmid in E. coli, and

- the gene for β-lactamase which confers resistance in E. coli to ampicillin.

The transcriptional unit of the gene of the antibody consisted of the following elements:

- unique(s) site(s) restriction enzymes at the 5'-end,

- immediate-early enhancer and promoter from the human cytomegalovirus,

- located behind the sequence of the intron And in the case of expression based on cDNA-design,

- 5'-noncoding region of the gene of the human antibody

- signal sequence of the heavy chain of immunoglobulin,

- chain of human antibody (wild type or with the replacement of the domains) or in the form of cDNA constructs, either in the form of genomic constructs with exon-intron structure of immunoglobulin,

- 3'-noncoding region with �ignalinos sequence and polyadenylation

- unique(s) site(s) of restriction at the 3'-end.

The fusion gene containing the described chain antibodies, as described below, was performed using PCR and/or synthesis and Assembly of genes using known methods and procedures of recombination by connecting corresponding segments of nucleic acids, for example, using unique restriction sites in the respective vectors. Sublimirovanny nucleotide sequences were confirmed by DNA sequencing. For a short transpency created a large number of plasmids by obtaining plasmids from transformed E. coli cultures (kit Nucleobond AX, the company Macherey-Nagel).

Methods cell culture

Applied standard methods of culturing cells as described in Current Protocols in Cell Biology, ed. by J. S. Bonifacino, M. Dasso, J. B. Harford, Lippincott-Schwartz, J. and Yamada K. M., publishing house of John Wiley & Sons, Inc., 2000. Bespecifically antibodies expressively by short-term cotransfection respective expression plasmids grown in attached cell cultures HEK293-EBNA or grown in suspension cell cultures HEK29-F, as will be described below.

Short transpency in the system of HEK293-EBNA

Bespecifically antibodies expressively by short-term cotransfection respective expression plasmids (e.g. encoding�sponding heavy chain and modified heavy chain, and the corresponding light chain and modified light chain) grown in attached cell cultures HEK293-EBNA (cell line of human embryonic kidney 293 expressing the nuclear antigen of Epstein-Barr; American type culture collection, deposited under the number of ATSS No. CRL-10852, lot's 959 218), which were cultured in DMEM (modified by the method of Dulbecco medium eagle, the company Gibco), supplemented with 10% PCS (fetal calf serum, firm Gibco) with ultra-low IgG content (Ultra Low IgG PCS), 2 mm L-glutamine (firm Gibco) and 250 µg/ml geneticin (firm Gibco). For transpency used reagent for transpency type FuGENE™ 6 (Roche Molecular Biochemicals) using the reagent FuGENE™ (ál) and DNA (μg) of 4:1 (range from 3:1 to 6:1). Proteins expressively with appropriate plasmids using a molar ratio of plasmids encoding (modified and wild-type) light chain and heavy chain, 1:1 (equimolar ratio) in the range from 1:2 to 2:1, respectively. Cells were fed on day 3 of L-glutamine to a concentration of 4 mm glucose (Sigma) and NAA (nicotinic acid amide) (firm Gibco). Collected cell culture supernatants containing bespecifically antibody, on days 5 to 11 after transfection by centrifugation and stored at -20°C. General information regarding the recombinant stock�these human immunoglobulins, for example, in HEK293 cells, see Meissner P., etc., Biotechnol. Bioeng. 75, 2001, cc.197-203.

Transient transfection in the HEK293 system-F

Bespecifically antibody was produced by transient transfection of the corresponding plasmids (e.g. encoding the heavy chain and modified heavy chain and corresponding light chain and modified light chain) using the HEK293-F (firm Invitrogen) according to the manufacturer's instructions. In General, the method consisted of the following: HEK293-F cells (firm Invitrogen) grown in suspension cultures in either shake flask or in a fermenter with a stirrer in serum-free medium for expression type FreeStyle 293 (Invitrogen company), transfectional a mixture of four expression plasmids and 293-pectina or pectina (firm Invitrogen). In 2-liter shake flask (Corning company) were seeded cells HEK293-F with a density of 1.0×106cells/ml in 600 ml and incubated at 120 rpm, 8% CO2. After 1 day the cells were transfectional at cell density of approximately 1.5×106cells/ml with about 42 ml of a mixture (A) containing 20 ml of Opti-MEM (firm Invitrogen) with 600 μg total plasmid DNA (1 μg/ml), which encodes a heavy chain or a modified heavy chain, respectively and the corresponding light chain in an equimolar ratio, and the mixture B) containing 20 ml Opti-MEM + 1.2 ml 293-pectin or pectin (2 μl/ml). In dependence�completely from the absorption of glucose in the fermentation process added a solution of glucose. After 5-10 days of collecting the supernatant containing the secreted antibody, and either of the supernatant was directly purified antibody or supernatant were frozen and stored.

Determination of protein

The protein concentration of the purified antibodies and their derivatives were determined based on optical density (OD) at 280 nm using the molar extinction coefficient calculated on the basis of amino acid sequence, according to the method described by Race, etc., Protein Science, 1995, 4, cc.2411-1423.

Determination of the concentration of antibodies in the supernatants

The concentration of antibodies and their derivatives in supernatants of cell cultures was determined by immunoprecipitation with protein A-agarose granules (Roche). 60 µl of protein A-agarose pellets were washed three times in TBS-NP40 (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet-P40). Then 1-15 ml of cell culture supernatant were applied to protein A-agarose pellets, pre-balanced in TBS-NP40. After incubation for 1 h at room temperature, the pellets were washed on a filter column type Ultrafree-MC (firm Amicon) using a single 0.5 ml TBS-NP40, twice with 0.5 ml of double (2×) with phosphate-buffered saline (2×'s SGA, Roche), and briefly four times with 0.5 ml 100 mm Na-citrate buffer, pH 5.0. Bound antibody was suirable by adding 35 ál of sample buffer NuPAGE® LDS (firm Invitrogen). Half clicks�SCA was combined with the reducing agent for samples NuPAGE® or left in unrestored form, respectively, and kept for 10 min at 70°C. Then 5-30 µl was used for the implementation of LTOs-page using 4-12% bis-Tris gels NuPAGE® (Invitrogen company) (applying the MOPS buffer for the implementation of LTOs-page in Sevostyanova conditions and the MES buffer in the form of a movable buffer with antioxidant additive NuPAGE® (Invitrogen company) for the implementation of LTOs-page in reducing conditions and stained Kumasi blue diamond.

The concentration of antibodies and their derivatives in supernatants of cell cultures was assessed quantitatively using affine GHWR chromatography. In General, the method consisted of the following: the supernatants containing antibodies and their derivatives, which were associated protein a, was introduced column (Applied Biosystems Poros A/20 200 mm KH2PO4, 100 mm sodium citrate buffer, pH 7.4 and suirable from matrix with 200 mm NaCl, 100 mm citric acid, pH of 2.5 using the Agilent 1100 HPLC. - Eluted protein was assessed quantitatively using UV absorption and integration areas of peaks. Purified standard antibody in the form of IgG1 was used as a standard.

In an alternative embodiment, the concentration of antibodies and their derivatives in supernatants of cell cultures was assessed using the "sandwich"IgG-ELISA. In General, the method consisted of the following: 96-well titration microplates with high ability to bind streptavidin And (tablets type Strepta Well High Bind Strepataidin A (Roche)), were sensibilized rate of 100 μl/well biotinylated anti-human IgG F(ab')2 < h-Fcγ>BI is used as the immobilized molecules (company Dianova), in a concentration of 0.1 μg/ml for 1 h at room temperature or alternatively over night at 4°C and then washed three times using 200 μl/well's SGA, 0,05% tween (SRT, Sigma). Was added to the wells at 100 μl/well of the serial dilution in's SGA (Sigma), containing the appropriate antibody supernatants of cell cultures, and incubated for 1-2 h on a shaker for titration microplates at room temperature. The wells were washed three times using 200 μl/well SFRT, and bound antibody was detected with 100 μl of F(ab')2<hFcγ>POD (firm Dianova) at a concentration of 0.1 μg/ml as an identifying antibody for 1-2 h on a shaker for titration microplates at room temperature. Unrelated identifying antibody was washed three times using 200 μl/well SFRT, and identifying the associated antibody was detected by adding 100 μl of ABTS/well. Determination of absorbance was performed on the spectrometer type Tecan Fluor at a wavelength of 405 nm (the length of the reference wavelength 492 nm).

Purification of proteins

Proteins were purified from filtered supernatant cell cultures according to standard protocols. In General, the method consisted of the following: proteins were applied to a column Packed with protein a-separate (firm GE Healthcare), and laundered's SGA. The elution of antibodies was carried out at pH of 2.8, followed immediately by neutralization of the sample. Aggregated protein was separated from Monomeric antibodies by gel filtration (Superdex 200, GE Healthcare) in a's SGA or 20 mm histidine, 150 mm NaCl, pH 6.0. The fraction of Monomeric antibodies were pooled, if necessary, concentrated using, for example, centrifuge concentrator type MILLIPORE Amicon Ultra (30 MWCO (molecularbiology limit), were frozen and stored at -20°C or -80°C. Part of the samples was left for subsequent analytical study of the protein and its analytical characterization, for example, using LTOs-page, gel filtration, or mass spectrometry.

LTOs-page

Was applying gel NuPAGE® Pre-Cast (firm Invitrogen) according to the manufacturer's instructions. In particular, used 10% or 4-12% bis-Tris Pre-Cast gels NuPAGE® Novex® (pH 6.4) and mobile buffers such as NuPAGE® MES (gels applied in reducing conditions, the movable buffer with antioxidant additive NuPAGE®) or MOPS (gels applied in Sevostyanova conditions).

Analytical gel filtration

As gel filtration, designed to determine the aggregate and oligomeric state of antibodies, used GHUR-chromatography. In General, the method consisted of the following: purified on protein A antibody was introduced into the column type Tosoh TSKgel G3000SW 300 mm NaCl, 50 mm KH2PO4/K 2HPO4, pH 7.5 in the system of Agilent 1100 HPLC or column Superdex 200 (GE Healthcare) in 2×'s SGA in GHUR system Dionex. The number-eluted protein was determined based on UV absorption and integration areas of peaks. As the standard used standard for gel filtration company BioRad Gel Filtration Standard 151-1901).

Mass spectrometry

General deglycosylated a lot of antibodies, obtained as a result of crossing-over was determined and confirmed using mass spectrometry with ionization by electrospray (ESI-MC). In General, the method consisted of the following: 100 µg of purified antibodies were deglycosylated using 50 med. N-glycosidase F (PNGF, firm ProZyme) in 100 mm KH2PO4/K2HPO4, pH 7 at 37°C for 12-24 h at a protein concentration up to 2 mg/ml and then were desalted using GHUR on a column of Sephadex G25 (GE Healthcare). The mass of the respective heavy and light chains were determined using ESI-MC after deglycosylation and recovery. Overall, the method was as follows: 50 µg of antibody in 115 μl were incubated with 60 μl of 1M TSAR and 50 ál of 8M hydrochloride of guanidine after desalting. The total mass and the mass of the recovered heavy and light chains were determined using ESI-MC using MC-Q-Star Elite, equipped with the nanomate source.

ELISA for the evaluation of ECD binding IGF-1R

Particular binding created antibodies to the extracellular domain (ECD) IG-1R was analyzed by ELISA. For this purpose, extracellular domain of IGF-1R (residues 1-462) containing the naturally occurring leader sequence and 12-rich LI-cysteine domains of ectodomain alpha-chain of human IGF-1R (according to McKern and others, 1997; Ward and others, 2001), fused with N-terminal tag representing His-streptavidin-binding peptide (His-SBP), cloned into a derivative with pcDNA3 vector and short-term expressively in NCR-cells. The protein sequence of the ECD of IGF-1R-His-SBP is presented in SEQ ID NO:10. 96-well titration microplates with high ability to bind streptavidin And (tablets type Strepta Well High Bind Strepatavidin A (Roche)), were sensibilized by using 100 μl/well of cell culture supernatant containing soluble protein IGF-1R-ECD-SBP, over night at 4°C and washed three times using 200 μl/well's SGA, 0,05% tween (SRT, Sigma). Was then added to the wells at the rate of 100 μl/well of serial dilutions of the respective antibody and the reference antibody antibody wild type < IGF-1R> 's SGA (Sigma), which included 1% BSA (fraction V, Roche), and incubated for 1-2 h on a shaker for titration microplates at room temperature. In the implementation of serial dilutions in the wells were added the same amount of purified antibodies. The wells were washed three times using 200 μl/well SFRT, and with�asanee antibody detected, using 100 µl/well of F(ab')2<hFcγ>POD (firm Dianova) at a concentration of 0.1 µg/ml (1:8000) as an identifying antibody for 1-2 h on a shaker for titration microplates at room temperature. Unrelated identifying antibody was washed three times using 200 μl/well SFRT, and identifying the associated antibody was detected by adding 100 μl of ABTS/well. Determination of absorbance was performed on the spectrometer Tecan Fluor at a wavelength of 405 nm (the length of the reference wavelength 492 nm).

Biacore analysis using BCD IGF-1R (IGF-1R-ECD)

Link the newly created antibodies to human IGF-1R-ECD was investigated also by means of surface plasma resonance, using a device type BIACORE T100 (GE Healthcare Biosciences AB, Uppsala, Sweden). In General, the method consisted of the following: to assess the affinity goat anti-human antibody type IgG JIR 109-005-098 immobilizovana on CM 5 chip through amine combinations for the presentation of antibodies to human IGF-1R-ECD, carrier Fc. Binding was assessed in HBS buffer (HBS-P (10 mm HEPES, 150 mm NaCl, 0.005% tween 20, pH 7.4) at 25°C. IGF-1R-ECD (R&D Systems, or obtained the inventors) was added to the solution in various concentrations. The Association was assessed by injection of IGF-1R-ECD during the time interval from 80 C to 3 minutes; dissociation was assessed by washing the chip surface HBS buffer for 3-10 minutes and a KD value Oprah�elali, using the model of Langmuir binding at a 1:1 ratio. Due to the low density of the load and the level of capture antibodies < IGF-1R > data monovalent binding IGF-1R-ECD. The data obtained for negative control (e.g., curves, obtained only for the buffer) were subtracted from the curves obtained for sample, for correction inherent in the shift of the main level and to reduce noise signals. Applied software Biacore T100 Evaluation, version 1.1.1 for analysis sensogram and to calculate data concerning the affinity (Fig.18).

ELISA to assess the binding of ANGPT2

Features of the binding of the generated antibodies with full-size ANGPT2 protein-His (R&D Systems) was assessed using ELISA. For this purpose, transparent polystyrene advanced titration microplates company Falcon were sensibilized 100 μl (1 μg/ml) of recombinant human ANGPT2 (R&D Systems, without media) 's SGA for 2 h at room temperature or over night at 4°C. the Wells were washed three times using 300 ál SRT (0,2% tween 20) and blocked with 200 μl 2% BSA, 0.1% tween 20 for 30 min at room temperature and then washed three times using 300 ál SRT. Was added to the wells at 100 μl/well of serial dilutions of purified antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, and as a reference antibody antibody <ANGPT2> in wild-type firm's SGA Sigma) and incubated for 1 h on a shaker for titration microplates at room temperature. The wells were washed three times using 300 ál/well SPRT (0,2% tween 20) and bound antibody was detected using 100 μl/well (0.1 ug/ml) F(ab') < hk > POD (firm Biozol, catalogue No. 206005) or 100 μl/well (0.1 ug/ml) F(ab')<hFcγ>POD (firm Immuno research) in 2% BSA 0,1% tween 20 as an identifying antibodies, within 1 h on a shaker for titration microplates at room temperature. Unrelated identifying antibody was washed three times using 300 ál/well SFRT, and identifying the associated antibody was detected by adding 100 μl of ABTS/well. Determination of absorbance was performed on the spectrometer Tecan Fluor at a wavelength of 405 nm (the length of the reference wavelength 492 nm).

Biacore analysis to assess the binding of ANGPT2

Link the newly created antibodies to human ANGPT2 also investigated using surface plasma resonance, using a device type BIACORE T100 (GE Healthcare Biosciences AB, Uppsala, Sweden). In General, the method consisted of the following: to assess the affinity goat polyclonal antibody <hIgG-Fcg> immobilizovana on CM 5-chip or 4-chip via amine combinations for the presentation of antibodies to human ANGPT2. Binding was assessed in HBS buffer (HBS-P (10 mm HEPES, 150 mm NaCl, 0.005% tween 20, pH 7.4) supplemented with 5 mm CA2+or without, at 25°C. Purified ANGPT2-His (R&D Systems or obtained and purified in the claimed invention) was added to the solution in various�different concentrations. The Association was assessed by injection ANGPT2 for 3 minutes; dissociation was assessed by washing the chip surface HBS buffer for 3-5 min and the KD value was determined using the model of Langmuir binding at a 1:1 ratio. Due to the heterogeneity of the drug ANGPT2 binding in the application of ingredients in the ratios of 1:1 was not detected; as a result, spent only a relative estimate KD values. The data obtained for negative control (e.g., curves, obtained only for the buffer), subtracted from the curves obtained for sample, for correction inherent in the shift of the main level and to reduce noise signals. Used the program Biacore T 100 Evaluation, version 1.1.1 for analysis sensogram and to calculate data concerning the affinity (Fig.19).

The inhibition of binding of hANGPT2 with Tie-2-ECD (ELISA)

To evaluate the capacity of ANGPT2 antibodies to provide interfering effect on the binding to Tie-2 was carried out by ELISA described below. The test was conducted in 384-well direzioni the microplates (firm MicroCoat, Germany, catalogue No. 464718) at room temperature (CT). After each stage of incubation the plates were washed three times SPRT. In the beginning of the experiment, the plates were sensitized with 0.5 μg/ml protein Tie-2 (R&D Systems, UK, catalogue No. .313-T1) for at least 2 hours (h). Then the wells were blocked's SGA, supplemented� 0,2% tween 20 and 2% BSA (Roche Diagnostics GmbH, Germany) for 1 h. Dilutions of purified antibodies in's SGA were incubated together with 0.2 μg/ml huANGPT2 (firm R&D Systems, UK, catalogue No. 623-AN) for 1 h at CT. After washing was added over 1 h, the mixture containing 0.5 μg/ml biotinylating ANGPT2 antibody, clone VM (firm R&D Systems, UK) and diluted in the ratio 1:3000 conjugate streptavidin-HRP (Roche Diagnostics GmbH, Germany, catalogue No. 11089153001). Plates were then washed 6 times SPRT. Tablets showed, using freshly prepared ABTS reagent (Roche Diagnostics GmbH, Germany, buffer catalog no 204530001, tablet catalogue No. 11112422001), for 30 min at CT. The absorbance was evaluated at 405 nm.

ELISA, to detect the formation of the bridging connection type ANGPT2-VEGF

Particular binding created bespecifically antibodies was assessed using ELISA using immobilized full-sized protein VEGF165-His (R&D Systems) and protein of human ANGPT2, conjugated with His (human ANGPT2-His) (firm R&D Systems) to detect simultaneous binding especifismo antibodies. Only bespecifically antibody possessed the ability to bind simultaneously to VEGF and ANGPT2 and tie "bridge" two of the antigen, while monospecifičeskoj "standard" antibody type IgG1 did not possess the ability to bind simultaneously to VEGF and ANGPT2. For wire�Denia mentioned analysis transparent polystyrene advanced titration microplates company Falcon were sensibilized 100 μl (2 μg/ml) of recombinant human VEGF 165 (R& D Systems) 's SGA for 2 h at room temperature or over night at 4°C. the Wells were washed three times using 300 ál SRT (0,2% tween 20) and blocked with 200 μl 2% BSA, 0.1% tween 20 for 30 min at room temperature and then washed three times using 300 ál SRT. 100 µl/well of a series of dilutions especificacao antibody and control antibodies in's SGA (Sigma) was added to the wells and incubated for 1 h on a shaker for titration microplates at room temperature. The wells were washed three times using 300 ál SRT (0,2% tween 20) and bound antibody was detected by adding 100 μl (0.5 μg/ml) of human ANGPT2-His (R&D Systems) 's SGA. The wells were washed three times using 300 ál SRT (0,2% tween 20), and associated ANGPT2 were detected using 100 μl (0.5 μg/ml) antibodies <ANGPT2> mIgG1-Biotin (BAM0981, firm R&D Systems) for 1 h at room temperature. Unrelated identifying antibody was washed three times using 300 ál SRT (0,2% tween 20) and bound antibody was detected by adding 100 μl diluted in the ratio 1:2000 conjugate streptavidin-POD (Roche Diagnostics GmbH, catalog no. No. 11089153) that is diluted in the ratio 1:4 with blocking buffer, for 1 h at room temperature. Unbound conjugate streptavidin-POD were washed 3-6 times using 300 ál SRT (0,2% tween 20) and bound conjugate streptavidin-POD was revealed by adding 100 μl of ABTS/well. �distribution of absorption was performed with the spectrometer Tecan Fluor at a wavelength of 405 nm (the length of the reference wavelength 492 nm) (Fig.20).

Biacore analysis intended to detect simultaneous binding bespecifically antibodies to VEGF and ANGPT2

To further confirm the data obtained by ELISA, to detect the formation of bridging ties, conducted another analysis using the technology of surface plasma resonance and the device type Biacore T100 evidence for simultaneous binding to VEGF and ANGPT2 according to the following Protocol. Data were analyzed using the software package T 100: ANGPT2 immobilizovana level "capture" 2000-1700 RU (relative units) using an antibody bearing a label of five atoms of histidine (His-antibody) (His-At not containing BSA, firm Qiagen No. 34660) that immobilizovana on CM 5 chip by aminooctane (containing BSA). Buffer HBS-N served as a rolling buffer for activation used a mixture of EDC/NHS. Immobilized antibody His-At not containing BSA, diluted in the buffer for the combination representing NaAc, pH 4.5, concentration = 30 mg/ml, a carboxyl group, which was still activated at the end of the process, blocked by injection of 1M ethanolamine, the experiment was carried out at a density of ligands 5000 and 17000 RU. ANGPT2 at a concentration of 500 nm, diluted in the movable buffer + 1 mg/ml BSA, have been "seized" His-AB at a flow rate of 5 µl/min Then to �demonstratie binding especificacao antibodies with ANGPT2 and VEGF was carried out by incubation with rhVEGF. For this bespecifically antibody was associated with ANGPT2 at a flow rate of 50 µl/min and at a concentration of 100-500 nm, diluted in the movable buffer + 1 mg/ml BSA, and the presence of simultaneous binding was detected by incubation with VEGF (rhVEGF, R&D Systems, catalog No. 293-VE) in a's SGA + movable buffer, consisting of 0.005 vol.% Tween-20, + 1 mg/ml BSA, a flow rate of 50 μl/min and the use of VEGF at a concentration of 100-150 nm. In the analysis the Association was 120 s, dissociation time of 1200 s. the Regeneration was carried out at a flow rate of 50 µl/min using a 2×10 mm glycine buffer, pH 2.0 and at contact time of 60 s Sensogram corrected using the common use of two controls (the reference control: linking especificacao antibodies and rhVEGF with immobilized molecule His-At). As reference sample"blanks" for each of the At served as variants in which the concentration of rhVEGF was "0". Schematic representation of the Biacore analysis are shown in Fig.21. Sensogram corrected using the common use of two controls (the reference control: linking especificacao antibodies and rhVEGF with immobilized molecule His-At). As reference sample"blanks" for each of the At served as variants in which the concentration of rhVEGF was "0".

Examples 1

Receiving, expression, clear�and characterization monospecifičeskoj bivalent antibody < IGF-1R> in which the variable domains VL and VH are replaced at each other and in which the constant domains CL and CH1 are replaced with each other (abbreviated as designated in the context of the present description as antibody < IGF-1R > bearing replacement VL-VH/CL-CH1)

Example 1A

The creation of gene-expression plasmids for monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1

The sequence of the variable domains of the heavy and light chain monospecifičeskoj bivalent antibodies to the receptor insulin-like factor-1 <IGF-1R> bearing replacement VL-VH/CL-CH1, including the respective leader sequences described in this example, taken from the heavy chain of a human antibody < IGF-1R> (SEQ ID NO:1, plasmid 4843-pUC-HC-IGF-1R) and its light chain (SEQ ID NO:2, plasmid 4842-pUC-LC-IGF-1R), which are described in WO 2005/005635, and the constant domains of the heavy and light chain were taken from a human antibody (C-Kappa and IgG1).

Gene segments encoding the leader sequence, the variable domain light chain (VL) and a constant domain of human Kappa light chain (CL) antibodies < IGF-1R > was linked and fused with the 5'-end of the Fc domains of the constant regions of the human γ1 heavy chain (hinge-CH2-CH3). DNA encoding the relevant protein obtained by replacing the VH and CH1 domains on VL and CL domains, created by gene synthesis and is denoted Dale� as < IGF-1R > HC* (heavy chain*) (SEQ ID NO:3).

Gene segments encoding leader sequence, variable domain of the heavy chain (VH) and a constant domain (CH1) of the human γ1 heavy-chain antibodies < IGF-1R > was linked to an independent circuit. DNA encoding the relevant protein obtained by replacing the VL and CL domains in the VH and CH1 domains, created by gene synthesis and is denoted hereafter as < IGF-1R > LC* (light chain*) (SEQ ID NO:4).

Fig.5 and Fig.6 is a schematic view of the protein sequence of the modified heavy chains <IGF-1R > HC* and the modified light chain <IGF-1R > LC*.

The following summarizes the relevant expression vectors:

Vector DW047-pUC-HC*-IGF-1R

Vector DW047-pUC-HC*-IGF-1R is a gene-expression plasmid, for example, intended for short-time expression of the heavy chain* <IGF-1R > HC* (expression cassette based on the cDNA-design; with CMV-intron A), in cell line HEK293 (EBNA) or for stable expression in Cho-cells.

In addition to gene expression cassette <IGF-1R > HC* this vector contains

- the site of initiation of replication of pUC18 that provides replication of this plasmid in E. coli, and

- the gene for β-lactamase which confers resistance in E. coli to ampicillin.

The transcriptional unit of a gene <IGF-1R > HC* contained the following elements:

- unique SAI� AscI restriction at the 5'-end,

- immediate-early enhancer and promoter from the human cytomegalovirus,

- next to him the sequence of intron A,

- 5'-noncoding region of the gene of the human antibody

- signal sequence of the light chain of the immunoglobulin,

- gene human Mature NS*-chain <IGF-1R>, which encodes the variable domain of the fusion of the human light chain (VL) and a constant domain of human Kappa light chain (CL), fused with the 5'-end of the Fc domains of the constant domains of the human γ1 heavy chain (hinge-CH2-CH3),

- 3'-noncoding region with signal sequence and polyadenylation

- unique restriction site SgrAI at the 3'-end.

Plasmid map of the expression vector DW047-pUC-HC*-IGF-1R intended for <IGF-1R > HC* shown in Fig.7. Amino acid sequence <IGF-1R > HC* (including signal sequence) is presented in SEQ ID NO:3.

Vector DW048-pUC-LC*-IGF-1R

Vector DW048-pUC-LC*-IGF-1R is a gene-expression plasmid, for example, designed for short-expression carrier replacement VL-VH/CL-CH1 light chain* <IGF-1R > LC* expression cassette based on the cDNA-design; with CMV-intron A), in cell line HEK293 (EBNA) or for stable expression in Cho-cells.

In addition to gene expression cassette <IGF-1R > LC* this vector contains:

website initiatives�AI replication of pUC18, provides replication of this plasmid in E. coli, and

- the gene for β-lactamase which confers resistance in E. coli to ampicillin.

The transcriptional unit of a gene <IGF-1R > LC** consisted of the following elements:

- unique site restriction enzymes Sse8387I at the 5'-end,

- immediate-early enhancer and promoter from the human cytomegalovirus,

- located behind the sequence of intron A,

- 5'-noncoding region of the gene of the human antibody

- signal sequence of the heavy chain of immunoglobulin,

- gene human Mature LC*-chain <IGF-1R>, which encodes the variable domain of the fusion of the human heavy chain (VH) and constant domains of the human γ1 heavy chain (CH1),

- 3'-noncoding region with signal sequence and polyadenylation

unique restriction sites SalI and FseI at the 3'-end.

Plasmid map of the expression vector DW048-pUC-LC*-IGF-1R is designed for light chain* <IGF-1R > LC*, shown in Fig.8. Amino acid sequence <IGF-1R > LC* (including signal sequence) is presented in SEQ ID NO:4.

Plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R can be used for transient or stable cotransfected, for example, HEK293 cells, HEK293 EBNA or Cho (2-vector system). The purpose of the comparison was carried out by transient expression of antibodies� < IGF-1R> wild type using plasmids 4842-pUC-LC-IGF-1R (SEQ ID NO:2) and 4843-pUC-HC-IGF-1R (SEQ ID NO:1) is similar to the method described in this example.

To achieve higher levels of expression during short-term expressions in the cells HEK293 EBNA gene expression cassette <IGF-1R > HC* can subclinical using AscI sites, SgrAI, and gene expression cassette <IGF-1R > LC* can subclinical with Sse8387I sites and Fsel at the gene-expression vector 4700-pUC-Hyg_OriP that contains

- OriP element and

- a gene causing resistance to hygromycin, as a selectable marker.

Transcription units of heavy and light chains can either be subclinical in two independent vectors 4700-pUC-Hyg-OriP to cotransfected (2-vector system), or they can be cloned in the same vector 4700-pUC-Hyg-OriP (1-vector system) for subsequent transient or stable transpency using the resulting vectors. Fig.9 shows a plasmid map of the main vector 4700-pUC-OriP.

Example 1B

The creation of gene-expression plasmids for monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1

Fused genes <IGF-1R> (fused genes HC* and LC*) containing the modified Fab-formed antibodies < IGF-1R> wild type, were collected using known methods and processes of recombination p�the connection of the respective segments of nucleic acids.

Each of the nucleotide sequences encoding HC* and LC* IGF-1R, synthesized by chemical synthesis and then cloned into a cloning vector pGA4, which is based on plasmid pPCRScript (firm Stratagene), at the company Geneart (Regensburg, Germany). The expression cassette encoding the IGF-1R NS* built by ligating suitable for E. coli the plasmid, using the restriction sites PvuII and BmgBI, with the final vector DW047-pUC-HC*-IGF-1R; the expression cassette encoding the corresponding IGF-1R LC* built by ligating suitable for E. coli the plasmid, using the restriction sites PvuII and SalI, to obtain the final vector DW048-pUC-LC*-IGF-1R. Sublimirovanny nucleotide sequences were confirmed by DNA sequencing. For transient and stable transpency used a large number of plasmids, which were obtained from transformed E. coli cultures (kit Nucleobond AX, the company Macherey-Nagel).

Example 1B

Transient expression monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, in cell line HEK293 EBNA

Recombinant antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, expressively by short-term cotransfection plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R is grown in an attached cell culture the HEK293-EBNA (cell line of human embryonic kidney 293 expressing nuclear�th antigen of Epstein-Barr; American type culture collection, deposited under the number of ATSS No. CRL-10852, lot's 959218), which were cultured in DMEM (modified by the method of Dulbecco medium eagle, the company Gibco), supplemented with 10% FCS (fetal calf serum, firm Gibco) with ultra-low IgG content (Ultra Low IgG FCS), 2 mm L-glutamine (firm Gibco) and 250 µg/ml geneticin (firm Gibco). For transpency used reagent for transpency type FuGENE™ 6 (Roche Molecular Biochemicals) using the reagent FuGENE™ (ál) and DNA (μg) of 4:1 (range from 3:1 to 6:1). Plasmids encoding the light and heavy chain <IGF-1R > HC* and LC* (plasmids DW047-pUC-HC*-IGF-1R d DW048-pUC-LC*-IGF-1R), expressively using two different plasmids using a molar ratio of plasmids encoding the light chain and heavy chain, 1:1 (equimolar ratio) in the range from 1:2 to 2:1, respectively. Cells were fed on day 3 of L-glutamine to a concentration of 4 mm glucose (Sigma) and NAA (nicotinic acid amide) (firm Gibco). Collected cell culture supernatants containing antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, in the days from 5 to 11 after transfection by centrifugation and stored at -20°C. General information regarding the recombinant expression of human immunoglobulins, for example, in HEK293 cells, see Meissner R. et al., Biotechnol. Bioeng. 75, 2001, cc.197-203.

Example 1G

Immunoprecipitate monospecifičeskoj d�uchulenskogo antibodies < IGF-1R> bearing replacement VL-VH/CL-CH1

Monospecifičeskoj bivalent antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, was isolated from the supernatants of cell cultures (example 1B) by immunoprecipitation using protein A-agarose granules (Roche). 60 µl of protein A-agarose pellets were washed three times in TBS-NP40 (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet-P40). Then 1-15 ml of cell culture supernatant were applied to protein A-agarose pellets, pre-balanced in TBS-NP40. After incubation for 1 h at room temperature, the pellets were washed on a filter column type Ultrafree-MC (firm Amicon) using a single 0.5 ml TBS-NP40, twice with 0.5 ml of double (2×) with phosphate-buffered saline (2×'s SGA, Roche), and briefly four times with 0.5 ml 100 mm Na-citrate buffer, pH 5.0. Bound antibody was suirable by adding 35 ál of sample buffer NuPAGE® LDS (firm Invitrogen). Half of the sample was combined with the reducing agent for samples NuPAGE® or left in unrestored form, respectively, and kept for 10 min at 70°C. Then 20 μl was used for the implementation of LTOs-page using 4-12% bis-Tris gels NuPAGE® (Invitrogen company) (applying the MOPS buffer for the implementation of LTOs-page in Sevostyanova conditions and the MES buffer in the form of a movable buffer with antioxidant additive NuPAGE® (Invitrogen company) for the implementation of LTOs-page in postanal�living conditions) and stained Kumasi blue diamond. Fig.10 shows obtained using LTOs-page results of the standard experiment of immunoprecipitation. Monospecifičeskoj bivalent antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, resembled the characteristics of a typical antibody type IgG1, wherein the light chain LC* was present in the strip, which corresponds to a molecular mass of approximately 25 kDa and a heavy chain NS* was present in the strip, which corresponds to a molecular mass of 50 kDa. When using Sevostyanova conditions can be detected band, which corresponds to a molecular mass of approximately 150 kDa, characteristic of the full antibody.

Example 1D

Cleaning monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, and confirm his identity using mass spectrometry

Expressed and secreted monospecifičeskoj bivalent antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, was purified from the filtered cell culture supernatants using affinity chromatography on protein A according to known standard methods. In General, the method consisted of the following: containing antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, the supernatants of cell cultures after short-term transpency clarified by centrifugation (10000×g for 10 min) and filtration through a filter with a pore size of 0.45 μm and depositing�whether containing protein A column (HiTrap MabSelect Xtra (GE Healthcare), balanced's SGA buffer (10 mm Na2HPO4, 1 mm KH2PO4, 137 mm NaCl and 2.7 mm KCl, pH 7.4). Unbound proteins were washed countervailing's SGA buffer, and then 0.1 M sodium citrate buffer, pH 5.5 and laundered's SGA. For elution of the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, used 100 mm sodium citratis buffer, pH of 2.8, after which the samples were immediately neutralized by adding 300 μl of 2M Tris, pH of 9.0 for 2-ml fraction. Aggregated protein was separated from Monomeric antibodies by gel-filtration on a column (HiLoad 26/60 Superdex 200 prep grade (GE Healthcare) in 20 mm histidine, 150 mm NaCl, pH 6.0, and the fraction of Monomeric antibodies were then concentrated using a centrifugal concentrator MILLIPORE Amicon Ultra-15. Antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, were frozen and stored at -20°C or -80°C. the integrity of the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, was analyzed by LTOs-page in the presence of a reducing agent and then stained Kumasi blue diamond according to the method described above in example 1G. The purified antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, resembled the characteristics of a typical antibody type IgG1, wherein the light chain LC* was present in the strip, which corresponds to a molecular mass of approximately 25 kDa and a heavy chain NS* was present in the strip, which corresponds to a molecular mass of 50 kDa. When using�AI Sevostyanova conditions can be detected band which corresponds to a molecular mass of approximately 150 kDa, characteristic of the full antibody. Aggregation and oligomeric state of the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, was investigated using analytical gel filtration and found that the purified antibody with Fab obtained as a result of crossing-over, was in a Monomeric state. Characterized samples were used for further analytical studies of the protein and the study of functional characteristics. Using ESI-mass spectrometry confirmed theoretical molecular weight (which was calculated excluding the C-terminal lysine residue of the heavy chain and excluding the C-terminal residue of Pyroglutamate light chain) fully deglycosylated antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1 detected in major peaks (Fig 22).

Example 1E

Analysis of the ability monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, bind IGF-1R with used to assess binding of IGF-1R-ECD ELISA and Biacore analysis

Binding capacity monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, was assessed using ELISA with the use of the extracellular domain (ECD) of IGF-1R according to the method described above. Fig.11 presents the standardized results obtained by using the "sandwich"IgG-ELISA on �Snov, antibody titration < IGF-1R> bearing replacement VL-VH/CL-CH1, and antibodies < IGF-1R> wild type, from supernatants after transient expression in HEK293E cells. Clearly demonstrated that the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, possessed functional activity and revealed to him the characteristics of binding and kinetic characteristics comparable with the characteristics of the antibody < IGF-1R> wild type, and therefore it was fully functional. Small differences were within the error of the method and could, for example, be the result of a slight variability in the concentrations of protein.

These data were confirmed using Biacore analysis, which was used for the respective purified antibodies, it was found that monospecifičeskoj bivalent antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, had affinity and kinetic binding characteristics of IGF-1R-ECD, comparable with the original wild-type antibody, within error of the method. Kinetic data are presented in the table below:

ka [1/Mc]kd [1/s]K(D) [M]
antibody < IGF-1R > wild type3,18 E+065,521 E-3 of 1.74 E-09
antibody < IGF-1R > bearing replacement VL-VH/CL-CH12,65 E+066,258 E-32,36 E-09

Example 1G

Analysis of the ability monospecifičeskoj bivalent antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, link 1GF-1R by FACS using sverkhekspressiya IGF-1R I24-cells

To confirm the binding activity of the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, IGF-1R, sverkhekspressiya on the surface I24-cells (NIH3T3 cells expressing recombinant human IGF-1R, Roche) using FACS-analysis. In General, the method consisted of the following: 5×105I24-cells in each tube intended for the implementation of FACS, were incubated with a dilution of purified antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, and antibodies < IGF-1R> wild type as the reference antibody and incubated on ice for 1 h. Unbound antibody was washed with 4 ml of chilled on ice's SGA (firm Gibco) 4-2% FCS (firm Gibco). Then the cells were centrifuged (5 min at 400 g) and bound antibody was revealed using conjugate F(ab')2<hFcγ>PE (company Dianova) on ice for 1 h, protected from light. Unrelated identifying the antibody was washed with 4 ml of chilled on ice's SGA + 2% FCS. Then the cells were centrifuged (5 min at 400 g), �was suspendirovanie in 300-500 µl's SGA and associated identifying antibody was evaluated quantitatively using the device FACSCalibur or FACS Canto (BD company (FL2-a channel 10000 cells per process). The experiment was included as control antibodies of the appropriate isotype for the exclusion of any cases of nonspecific binding. The binding of an antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, and antibodies < IGF-1R> wild-type IGF-1R on the surface I24-cells was compared by comparing the concentration-dependent shift of the mean intensity of fluorescence.

Additional experiments demonstrated that the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, retained the ability to induce internalization of IGF-1R in MCF7 cells and had only minor ADCC activity in DU145 cells upon incubation with human RVMS, comparable with the activity of antibodies < IGF-1R> wild-type.

Combined data obtained in the experiments described in example 1, indicate that on the basis of replacement domains, you can create a fully functional antibodies with properties comparable with the properties of wild-type antibodies. The preservation of all functional properties are also confirmed by using biochemical and cellular assays. These antibodies with substitutions of domains was the basis for the creation described below bespecifically antibodies.

Examples 2

Receiving, expression, purification and characterization monospecifičeskoj bivalent antibody <ANGPT2> where variabel�nye domains VL and VH are replaced at each other and in which the constant domains CL and CH1 are replaced with each other (abbreviated as designated in the context of the present description as the antibody < ANGPT2> bearing replacement VL-VH/CL-CH1)

Example 2A

The creation of gene-expression plasmids for option monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1

Sequences of variable regions of heavy and light chain monospecifičeskoj bivalent antibody to angiopoietin-2 (<ANGPT2> bearing replacement VL-VH/CL-CH1), including the respective leader sequences described in this example, taken from the heavy chain of human antibodies <ANGPT2> (SEQ ID NO:6) and light chain (SEQ ID NO:7) described in WO 2006/045049 and constant domains of the heavy and light chain were taken from a human antibody (C-Kappa and IgG1).

Gene segments encoding the leader sequence, the variable domain light chain (VL) and a constant domain of human Kappa light chain (CL) antibodies <ANGPT2> were linked and fused with the 5'-end of the Fc domains of the constant regions of the human γ1 heavy chain (hinge-CH2-CH3). DNA encoding the relevant protein obtained by replacing the VH and CH1 domains on VL and CL domains, created by gene synthesis and is denoted hereafter as < ANGPT2 > HC* (heavy chain*) (SEQ ID NO:8).

Gene segments encoding leader sequence, variable domain of the heavy chain (VH) and a constant domain (CH1) of the human γ1 heavy-chain antibodies <ANGPT2> was linked to an independent circuit. DNA code�tion of the corresponding protein, the resulting replacement of the VL and CL domains in the VH and CH1 domains, created by gene synthesis and is denoted hereafter as < ANGPT2 > LC* (light chain*) (SEQ ID NO:9).

Appropriate expression vectors described in example 1A. Plasmid map of the expression vector pUC-HC*-ANGPT2> the heavy chain* <ANGPT2 > HC* shown in Fig.12. Amino acid sequence <ANGPT2 > HC* (including signal sequence) is presented in SEQ ID NO:8.

Plasmid map of the expression vector pUC-LC*-ANGPT2> light chain* <ANGPT2 > LC* is shown in Fig.13. Amino acid sequence <ANGPT2 > LC* (including signal sequence) is presented in SEQ ID NO:9. Plasmids pUC-HC*-ANGPT2> and pUC-LC*-ANGPT2> can be used for transient or stable cotransfected, for example, cells HEK293-F, HEK293 EBNA or Cho (2-vector system).

To achieve higher levels of expression during short-term expressions in the cells HEK293 EBNA gene expression cassette <ANGPT2 > HC* can subclinical using AscI sites, SgrAI, and gene expression cassette <ANGPT2 > LC* can subclinical with Sse8387I sites and FseI at the gene-expression vector 4700 pUC-Hyg_OriP that described in example 1A. Transcription units of heavy and light chains can either be subclinical in two independent vectors 4700-pUC-Hyg-OriP to cotransfected (2-vector systems�), or they can be cloned in the same vector 4700-pUC-Hyg-OriP (1-vector system) for subsequent transient or stable transpency using vectors formed through the sites FseI, SgrAI, Sse8387I and AscI.

Antibody <ANGPT2> wild-type cloned in plasmids SB04-pUC-HC-ANGPT2 (SEQ ID NO:6) and SB06-pUC-LC-ANGPT2 (SEQ ID NO:7), which are analogues of the vectors described in this example above in example 1A. Transcription units of heavy and light chain antibody <ANGPT2> wild type was subclinically generated from the basis vectors plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2 through the sites FseI, SgrAI, Sse8387I and AscI in plasmids SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2 to achieve higher levels of transient expression in HEK293E cells. For comparison and in the implementation of joint experiments expression (see example 3) antibody <ANGPT2> wild-type or short (Ko)expressively using either plasmid SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2, or plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2.

Example 2B

The creation of gene-expression plasmids for monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1

Fused genes antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1 (fused genes HC* and LC*) containing the modified Fab-formed antibodies <ANGPT2> wild type, were collected using known methods and processorcount by connecting corresponding segments of nucleic acids.

Each of the nucleotide sequences encoding HC* and LC* antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, synthesized by chemical synthesis and then cloned into a cloning vector pGA4, which is based on plasmid pPCRScript (firm Stratagene), at the company Geneart (Regensburg, Germany). The expression cassette encoding the NA* antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, was built by ligating suitable for E. coli the plasmid, using the restriction sites PstI and EcoNI, with the final vector pUC-HC*-<ANGPT2>; the expression cassette encoding the corresponding <ANGPT2 > LC* built by ligating suitable for E. coli the plasmid, using the restriction sites PvuII and FseI, the final vector pUC-LC*-<ANGPT2>. Sublimirovanny nucleotide sequences were confirmed by DNA sequencing. For transient and stable transpency used a large number of plasmids, which were obtained from transformed E. coli cultures (kit Nucleobond AX, the company Macherey-Nagel).

Example 2B

Transient expression monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, in cell line HEK293 EBNA

Recombinant antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, expressively by short-term cotransfection plasmids pUC-HC*-ANGPT2> and pUC-LC*-ANGPT2> grown as attached cultures glue�OK HEK293-EBNA (cell line of human embryonic kidney 293, expressing the nuclear antigen of Epstein-Barr; American type culture collection, deposited under the number of ATSS No. CRL-10852, lot's 959218), which were cultured in DMEM (modified by the method of Dulbecco medium eagle, the company Gibco), supplemented with 10% FCS (fetal calf serum, firm Gibco) with ultra-low IgG content (Ultra Low IgG FCS), 2 mm L-glutamine (firm Gibco) and 250 µg/ml geneticin (firm Gibco). For transpency used reagent for transpency type FuGENE™ 6 (Roche Molecular Biochemicals) using the reagent FuGENE™ (ál) and DNA (μg) of 4:1 (range from 3:1 to 6:1). Plasmids encoding the light and heavy chain <ANGPT2 > HC* and LC* (plasmids pUC-HC*-ANGPT2> and pUC-LC*-ANGPT2>), expressively using two different plasmids using a molar ratio of plasmids encoding the light chain and heavy chain, 1:1 (equimolar ratio) in the range from 1:2 to 2:1, respectively. Cells were fed on day 3 of L-glutamine to a concentration of 4 mm glucose (Sigma) and NAA (firm Gibco). Collected cell culture supernatants containing antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, in the days from 5 to 11 after transfection by centrifugation and stored at -20°C. General information regarding the recombinant expression of human immunoglobulins, for example, in HEK293 cells, see Meissner R. et al., Biotechnol. Bioeng. 75, 2001, cc.197-203.

Example 2G

Cleaning monop�citiescape bivalent antibody < ANGPT2> bearing replacement VL-VH/CL-CH1, and confirm his identity using mass spectrometry

Expressed and secreted monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, was purified from the filtered cell culture supernatants using affinity chromatography on protein A according to known standard methods. In General, the method consisted of the following: containing antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, cell culture supernatants after transient transfection, clarified by centrifugation (10000 g for 10 min) and filtered through a filter with a pore size of 0.45 μm and was introduced containing protein A column (HiTrap MabSelect Xtra (GE Healthcare), 's SGA balanced-buffer (10 mm Na2HPO4, 1 mm KH2PO4, 137 mm NaCl and 2.7 mm KCl, pH 7.4). Unbound proteins were washed countervailing's SGA buffer, and then 0.1 M sodium citrate buffer, pH 5.5 and laundered's SGA. For elution of the antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, used 100 mm sodium-citrate buffer, pH of 2.8, after which the samples were immediately neutralized by adding 300 μl of 2M Tris, pH of 9.0 for 2-ml fraction. Aggregated protein was separated from Monomeric antibodies by gel-filtration on a column (HiLoad 26/60 Superdex 200 prep grade (GE Healthcare) in 20 mm histidine, 150 mm NaCl, pH 6.0, and the fraction of Monomeric antibodies then concentrated to panostandalone hub MILLIPORE Amicon Ultra-15. Antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, were frozen and stored at -20°C or -80°C. the integrity of the antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, was analyzed by LTOs-page in the presence of a reducing agent and then stained Kumasi brilliant blue (Fig.23A). Aggregation and oligomeric state of the antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, was investigated using analytical gel filtration (Fig.23B). Characterized samples were used for further analytical studies of the protein and the study of functional characteristics. Using ESI-mass spectrometry confirmed theoretical molecular weight of fully deglycosylated antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, found in the major peaks. No signs of pollution reminiscent of antibody proteins.

Example 2E

Analysis of the ability monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, link ANGPT2 using used to assess binding of ANGPT2 ELISA and Biacore analysis

Binding capacity monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, was assessed using ELISA using full size ANGPT2 protein-His (R&D Systems) according to the method described above. Fig.24 presents the results obtained by using intended for the assessment�key binding "sandwich"-ELISA based on the titration of purified antibody < ANGPT2> bearing replacement VL-VH/CL-CH1, and antibodies <ANGPT2> wild-type. Clearly demonstrated that the antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, possessed functional activity and revealed to him the characteristics of binding and kinetic characteristics comparable with the characteristics of the antibody <ANGPT2> wild type, and therefore it was fully functional. Small differences were within the error of the method and could, for example, be the result of a slight variability in the concentrations of protein.

These data are confirmed by Biacore analysis, which was used for the respective purified antibodies, it was found that monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, characterized by the value of KD equal to 13 PM, and thus, is comparable within the errors of the method, the parameters of the affinity and kinetic binding characteristics with ANGPT2 with the original antibody <ANGPT2> wild type, for which the value of KD is 12 PM (Fig.25)

Example 2G

Analysis of functional properties monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1

To demonstrate that the functional properties monospecifičeskoj bivalent antibody <ANGPT2> bearing replacement VL-VH/CL-CH1 comparable to the properties of the original antibody <ANGPT2> d�who type, compared the ability of two antibodies to provide interfering effect on the binding of ANGPT2 with the extracellular domain of its Tie2 receptor by using designed to assess binding ELISA described above. According to the respective intended to assess binding ELISA / EC50 value characterizing the impact of interfering antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, for binding to human ANGPT2 was 135 ng/ml, which is comparable with the value EU50 145 nm obtained for the original antibody <ANGPT2> wild-type. These data were confirmed using competitive analysis of cell binding ligand, which was performed using HEK293 cells, sverkhekspressiya on the surface of Tie2, which establishes that the value EU50 characterizing the impact of interfering antibodies <ANGPT2> bearing replacement VL-VH/CL-CH1, for binding to human ANGPT2 was 225 ng/ml, which is comparable with the value / EC50 of 205 nm obtained for the original antibody <ANGPT2> wild type (data not shown).

Combined data obtained in the experiments described in example 2, indicate that on the basis of replacement domains, you can create a fully functional antibodies with properties comparable with the properties of wild-type antibodies. The preservation of all functional properties confirmed�eno also using biochemical and cellular assays. These antibodies with substitutions of domains was the basis for the creation described below bespecifically antibodies, which are presented below in examples 3 and 4.

Example 2B

Molecular modelling of the antibodies with replacement of domains allowed to establish that the steric replaced by the space between domains can limit the styling. For this reason, when creating the present invention was created designs that were introducible additional glycine residue at the C-end is replaced with domains of Fab-fragments, for example, between the VL-CL and binding hinge region or at the end of the free VH-CH1-domain, respectively. Corresponding monospecifičeskoj bivalent antibody designated hereinafter as the antibody <ANGPT2> bearing replacement VL-VH(G)/CL-CH1(G).

Sequences of variable regions of heavy and light chain monospecifičeskoj bivalent antibody to angiopoietin-2 (<ANGPT2> bearing replacement VL-VH/CL-CH1), including the respective leader sequences described in this example, taken from the heavy chain of human antibodies <ANGPT2> (SEQ ID NO:6) and light chain (SEQ ID NO:7) described in WO 2006/045049 and constant domains of the heavy and light chain were taken from a human antibody (C-Kappa and IgG1).

Gene segments encoding the leader sequence, the variable domain light chain (VL) and constant house� human Kappa light chain (CL) antibodies < ANGPT2> were linked and fused with the 5'-end of the Fc domains of the constant regions of the human γ1 heavy chain (hinge-CH2-CH3) and included an additional glycine residue. DNA encoding the relevant protein obtained by replacing the VH and CH1 domains on VL and CL domains, created by gene synthesis and is denoted hereafter as < ANGPT2 > HC(G)* (heavy chain*) (SEQ ID NO:17).

Gene segments encoding leader sequence, variable domain of the heavy chain (VH) and a constant domain (CH1) of the human γ1 heavy-chain antibodies <ANGPT2> was linked to an independent circuit that includes an additional glycine residue. DNA encoding the relevant protein obtained by replacing the VL and CL domains in the VH and CH1 domains, created by gene synthesis and is denoted hereafter as < ANGPT2 > LC(G)* (light chain*) (SEQ ID NO:9). <ANGPT2 > HC(G)* (heavy chain*) (SEQ ID NO:18).

Appropriate expression vectors similar to those described above in example 2. Then these expression vectors were used for transient joint expression of the corresponding antibodies <ANGPT2> bearing replacement domains VL-VH(G)/CL-CH1(G), in HEK293-F cells. The corresponding antibody <ANGPT2> bearing replacement VL-VH(G)/CL-CH1(G), was purified after short-expression according to the method described above and analyzed using LTOs-page, gel filtration and mass spectrometry rela�estoodeeva antibodies. Levels of protein expression were quite high and similar to the levels obtained for conventional antibodies bearing replacement VL-VH/CL-CH1 described above. For all studied properties not identified any unexpected results, and they were comparable with the properties of the corresponding antibodies <ANGPT2> wild-type. Fig.31 shows data demonstrating comparable properties antibodies <ANGPT2> wild-type and antibodies <ANGPT2> bearing replacement VL-VH(G)/CL-CH1(G), which are obtained using the dedicated to assess the binding of ANGPT2 Biacore analysis. Within the limits of error of the method, the affinity of the binding of both antibodies to human ANGPT2 was comparable and was characterized by KD values (average of two replications), constituting approximately 38 PM for antibodies <ANGPT2> wild-type and PM for about 45 antibodies <ANGPT2> bearing replacement VL-VH(G)/CL-CH1(G).

ka [1/Mc]kd [1/s]K(D) [M]
antibody <ANGPT2 > wild type5,26 E+052,0 E-43,79 E-10
antibody <ANGPT2> bearing replacement VL-VH(G)/CL-CH1(G)of 5.29 E+06 2,358 E-34,45 E-09

Examples 3

Expression especificacao bivalent antibody <ANGPT2-IGF-1R>, in which the heavy and light chains bind specifically to IGF-1R, the variable domains VL and VH are replaced at each other and the constant domains CL and CH1 are replaced with each other (abbreviated as designated in the context of the present description as the antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1)

Example 3A

Short-term joint expression of antibody <ANGPT2> wild-type and antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1 cell line HEK293 EBNA obtaining especifismo antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1

To create a functional especificacao antibodies that recognize ANGPT2, on the basis of Fab-fragment antibodies <ANGPT2> wild-type, on the one hand, and recognizing IGF-1R, on the basis of Fab-fragment antibodies < IGF-1R > bearing replacement VL-VH/CL-CH1, on the other hand, two expression plasmids encoding the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1 (example 1A), together expressively with two expression plasmids, encoding the antibody <ANGPT2> wild-type (example 2A). The statistical Association of the heavy chains of wild-type (NA) and heavy chains, obtained as a result of crossing-over Fab (HC*), this resulted in the receipt of especificacao and bivalent antibody <ANGPT2-IGF-1R> bear�replacement VL-VH/CL-CH1. Assuming that both antibodies equally well expressibility and thus did not form by-products in quantities that can affect the result, this process should result in the following three main products in the ratio 1:2:1: monospecifičeskoj antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, and antibody <ANGPT2> wild-type. In addition, you can expect to receive several by-products such as dimers LC-LC* (Fab2-fragment), NS-NS* (monovalent antibody) and HC*-LC.

In contrast, co-expression of two gene-expression plasmids encoding the antibody < IGF-1R> wild-type (example 1A), two expression plasmids encoding the antibody <ANGPT2> wild type, as reference antibodies should be generated only a small amount of functional especificacao antibodies < IGF-1R-ANGPT2> because of the statistical Association of the heavy chains, but uncontrolled Association of light chains, both heavy chains of the antibody wild type < IGF-1R > and <ANGPT2>.

To create a mixture of basic products, such as (A) antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, B) bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, and b) the antibody <ANGPT2> wild-type, four plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R � or plasmids SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2 (or plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2) was short cotransfection grown as attached cultures cells HEK293-EBNA (cell line kidney of a human embryo 293 expressing the nuclear antigen of Epstein-Barr; American type culture collection, deposited under the number of ATSS No. CRL-10852, lot's 959218), which were cultured in DMEM (modified by the method of Dulbecco medium eagle, the company Gibco), supplemented with 10% FCS (fetal calf serum, firm Gibco) with ultra-low IgG content (Ultra Low IgG FCS), 2 mm L-glutamine (firm Gibco) and 250 µg/ml geneticin (firm Gibco). For transfection reagent used for transfection type FuGENE™ 6 (Roche Molecular Biochemicals) using the reagent FuGENE™ (ál) and DNA (μg) of 4:1 (range from 3:1 to 6:1). Plasmids encoding the light and heavy chain HC* and LC* antibodies < IGF-1R > (plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R), and HC and LC of antibody <ANGPT2> (plasmids SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2 or plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2, respectively), expressively using four different plasmids in a molar ratio of plasmids encoding the light and heavy chain, comprising 1:1 (equimolar ratio). Cells were fed on day 3 of L-glutamine to a concentration of 4 mm glucose (Sigma) and NAA (firm Gibco). The collected supernatant containing a mixture of basic products, such as (A) antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, B) bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, and b) the antibody <ANGPT2> wild type were identified as a mixture containing especificamente, bearing replacement VL-VH/CL-CH1". The supernatants of cell cultures, which included a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1, collected on days 5 to 11 after transfection by centrifugation and stored at -20°C.

For comparison, antibody < IGF-1R> wild type short-term together expressively using encoding HC and LC plasmids (4842-pUC-LC-IGF-1R and 4843-pUC-HC-IGF-1R, example 1A) in combination with the antibody <ANGPT2> wild type using the encoding HC and LC plasmids (SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2 or SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT2, respectively). Cells were fed on day 3 of L-glutamine to a concentration of 4 mm glucose (Sigma) and NAA (firm Gibco). The collected supernatant containing a mixture of different variants of the antibodies of the wild type < IGF-1R > and < ANGPT2> that were either monospecifičeskoj or bespecifically or incapable of binding options due to the uncontrolled Association of light chains, both heavy chains of the antibody wild type < IGF-1R > and < ANGPT2>, leading to the incorrect Association of light chains, designated as "a mixture of wild-type. The supernatants of cell cultures, which included a mixture of wild-type, were collected on days 5 to 11 after transfection by centrifugation and stored at -20°C.

Since antibody <ANGPT2> wild type detected higher output briefly�belt expression compared with the antibody < IGF-1R> wild type and antibody < IGF-1R> carrying Fab obtained as a result of crossing-over, the ratio of plasmids encoding the antibody <ANGPT2> wild type, and plasmids encoding the antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, was shifted to the expression of antibodies <ANGPT2> wild-type. The ratio of plasmids should be adapted in subsequent experiments so as to obtain the same level of expression of both specificdate and a more even distribution of different antibodies.

Example 3B

Immunoprecipitate especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1

A mixture containing major products, namely (A) monospecifičeskoj antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, B) bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, and b) the antibody <ANGPT2> wild-type ("a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1"), were isolated from the supernatants of cell cultures (example 3A) by immunoprecipitation using protein A-agarose granules (Roche). 60 µl of protein A-agarose pellets were washed three times in TBS-NP40 (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet-P40). Then 1-15 ml of cell culture supernatant were applied to protein A-agarose pellets, pre-balanced in TBS-NP40. After incubation for 1 h at room temperature, the pellets were washed on a filter column type ltrafree-MC (firm Amicon), using single 0.5 ml TBS-NP40, twice with 0.5 ml of double (2×) with phosphate-buffered saline (2×'s SGA, Roche), and briefly four times with 0.5 ml 100 mm Na-citrate buffer, pH 5.0. Bound antibody was suirable by adding 35 ál of sample buffer NuPAGE® LDS (firm Invitrogen). Half of the sample was combined with the reducing agent for samples NuPAGE® or left in unrestored form, respectively, and kept for 10 min at 70°C. Then 20 μl was used for the implementation of LTOs-page using 4-12% bis-Tris gels NuPAGE® (Invitrogen company) (applying the MOPS buffer for the implementation of LTOs-page in Sevostyanova conditions and the MES buffer in the form of a movable buffer with antioxidant additive NuPAGE® (Invitrogen company) for the implementation of LTOs-page in reducing conditions and stained Kumasi blue diamond. According to LTOs-page for three studied species of antibodies no differences detected. All antibodies resembled the characteristics of a typical antibody in the form of IgG1, while the light chain was present in the strip, which corresponds to a molecular mass of approximately 25 kDa and a heavy chain was present in the strip, which corresponds to a molecular mass of 50 kDa. When using Sevostyanova conditions can be detected band, which corresponds to a molecular mass of approximately 150 kDa, characteristic of Paul�wow antibodies. Therefore, for evidence of the presence of functional especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1 obtained as a result of crossing-over, a mixture of three types of antibodies were purified (example 3B) and subjected to analysis using a FACS cell, to detect the formation of bridging ties (example 3D).

Example 3B

Cleaning especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1

A mixture containing major products, namely (A) monospecifičeskoj antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, B) bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, and b) the antibody <ANGPT2> wild-type ("a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1"), described in example 3A, was purified from the filtered cell culture supernatants using affinity chromatography on protein A according to known standard methods. In General, the method consisted of the following: contains a mixture of antibodies, the supernatants of cell cultures resulting from short-term transpency plasmids DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R and SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2, clarified by centrifugation (10000 g for 10 min) and filtered through a filter with a pore size of 0.45 μm and was introduced containing protein A column (HiTrap MabSelect Xtra (GE Healthcare), 's SGA balanced-buffer (10 mm Na2HPO4, 1 mm KH2/sub> PO4, 137 mm NaCl and 2.7 mm KCl, pH 7.4). Unbound proteins were washed countervailing's SGA buffer, and then 0.1 M sodium citrate buffer, pH 5.5 and laundered's SGA. For elution especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, used 100 mm sodium citratis buffer, pH of 2.8, after which the samples were immediately neutralized by adding 300 μl of 2M Tris, pH of 9.0 for 2-ml fraction. Aggregated protein was separated from Monomeric antibodies by gel-filtration on a column (HiLoad 26/60 Superdex 200 prep grade (GE Healthcare) in 20 mm histidine, 150 mm NaCl, pH 6.0, and the fraction of Monomeric antibodies were then concentrated using a centrifugal concentrator MILLIPORE Amicon Ultra-15. A mixture containing the antibody bearing replacement VL-VH/CL-CH1, were frozen and stored at -20°C or -80°C. the integrity of the species of antibodies was analyzed by LTOs-page (see Fig.14) in the presence of a reducing agent and then stained Kumasi blue diamond according to the method described in example 1G. Part of the mixture of antibodies resembled the characteristics of a typical antibody in the form of IgG1, while the light chain was present in the strip, which corresponds to a molecular mass of approximately 25 kDa and a heavy chain was present in the strip, which corresponds to a molecular mass of 50 kDa. When using Sevostyanova conditions can be detected band, which conforms to the� molecular mass of approximately 150 kDa, characteristic of the full antibody. Using LTOs-page after purification on protein And found no noticeable side products, such as monovalent antibodies, etc. Aggregation and oligomeric state especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, was investigated using analytical gel filtration and found that the purified types of antibodies were in a Monomeric state. Characterized samples were used for further analytical studies of the protein and the study of functional characteristics. For evidence of the presence of functional especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, a mixture of three types of antibodies were analyzed using a FACS cell, to detect the formation of bridging ties (example 3D).

For the purpose of comparison, a mixture of wild-type, resulting from co-expression plasmids for HC - and LC-chain antibodies < IGF-1R> wild-type (4842-pUC-LC-IGF-1R and 4843-pUC-HC-IGF-1R, example 1A) in combination with plasmids for HC - and LC-chain antibodies <ANGPT2> wild-type (SB07-pUC-Hyg-OriP-HC-ANGPT2 and SB09-pUC-Hyg-OriP-LC-ANGPT2), was purified from the filtered cell culture supernatants using affinity chromatography on protein A, followed by gel filtration according to the method described above.

Example 3A

Identification of functional especificacao �ntitle < ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1 using a cellular FACS analysis to detect the formation of bridging ties, using expressing IGF-1R cell line I24

To confirm the presence of functional especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, in a mixture that contained bespecifically antibody bearing replacement VL-VH/CL-CH1, comprising the following main products: A) monospecifičeskoj antibody < IGF-1R > bearing replacement VL-VH/CL-CH1, B) bespecifically antibody <ANGPT2-IGF-1R>, bearing replacement VL-VH/CL-CH1, and b) the antibody <ANGPT2> wild type, obtained after short-joint expression according to the method described in example 3A was carried out cell FACS-analysis, to detect the formation of bridging ties such as IGF-1R-ANGPT2, using the cell line I24 (NIH3T3 cells expressing recombinant human IGF-1R, Roche). Principle of the test is shown in Fig.15. Bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, which is present in the supernatant (example 3A) or in a mixture of purified antibodies (example 3B), respectively, possessed the ability to bind to IGF-1R on the surface I24-cells and to ANGPT2 simultaneously with; and thus had the ability to connect a bridged connection two antigen target with two opposing Fab-fragments.

Overall, meth�d consisted of the following: 5×10 5cell line I24 to the tube intended for the implementation of FACS, were incubated with either 50 μl of undiluted cell culture supernatant, or 160 μg/ml dilution of the total mixture of purified antibody on ice for 1 h. In the first case, cells were incubated with (A) a cell culture supernatants, obtained after co-expression resulting from crossing-over plasmid <IGF-1R> (DW047-pUC-HC*-IGF-1R and DW048-pUC-LC*-IGF-1R) and plasmid <ANGPT2> wild-type (SB04-pUC-HC-ANGPT2> and SB06-pUC-LC-ANGPT2), which was identified as a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1", or B) the supernatants of the cell cultures obtained after co-expression plasmid <IGF-1R> wild-type (4842-pUC-LC-IGF-1R and 4843-pUC-HC-IGF-1R) and plasmid <ANGPT2> wild-type (SB04-pUC-HC-ANGPT2> and SB06-pUC-LC-ANGPT2), which was identified as "a mixture of wild-type (Fig.16). In the second case, corresponding to the purified mixture of antibodies from a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1, or a mixture of "a mixture of wild-type inflicted on I24 cells (example 3B, Fig.17). Unbound antibody was washed with 4 ml of chilled on ice's SGA (firm Gibco) + 2% FCS (firm Gibco), cells were centrifuged (5 min at 400 g) and bound bespecifically antibody was detected using 50 μl (2 μg/ml) of human angiopoietin-2 (ANGPT2) (firm R&D Systems) for 1 h on ice. Then unbound angiopoietin-2 (ANGP2) were washed once (Fig.16) or twice (Fig.17), using 4 ml of chilled on ice's SGA (firm Gibco) + 2% FCS (firm Gibco), cells were centrifuged (5 min at 400 g) and bound angiopoietin-2 was detected using 50 μl (5 μg/ml) antibodies < Ang-2> mIgG1-Biotin (BAM0981, firm R&D Systems) for 45 min on ice; alternatively, cells were incubated with 50 µl (5 µg/ml) conjugate mIgG1-Biotin as an isotype control (R&D Systems). Unrelated identifying the antibody was washed with 4 ml of chilled on ice's SGA (firm Gibco) + 2% FCS (firm Gibco), cells were centrifuged (5 min at 400 g) and identifying the associated antibody was detected using 50 μl of conjugate 1:400 streptavidin-PE (SA-PE) (firm Invitrogen/Zymed) for 45 min on ice, protect from light. Unbound conjugate streptavidin-PE were washed with 4 ml of chilled on ice's SGA + 2% FCS. Then the cells were centrifuged (5 min at 400 g), were resuspended in 300-500 µl's SGA and bound conjugate streptavidin-PE was evaluated quantitatively using a FACSCalibur device (BD company (FL2 channel, 10000 cells per process). The experiment was included as control antibodies of the appropriate isotype for the exclusion of any cases of nonspecific binding. In addition, as controls included purified monospecifičeskoj bivalent antibodies in the IgG1 form, such as <IGF-1R > and < ANGPT2>.

Fig.16 presents the results obtained by FACS cell-anal�for, detecting the formation of bridging ties, using I24-cells, for which was used the supernatants of cell cultures; Fig.17 presents the results obtained using the purified mixture of antibodies. In both cases, applied technology VL-VH/CL-CH1-replacement, incubation with the supernatant or the purified antibody from a mixture containing bespecifically antibody bearing replacement VL-VH/CL-CH1", which was obtained after co-expression of antibody bearing replacement VL-VH/CL-CH1, and the wild-type antibody, led to a significant shift in fluorescence, indicating the presence of functional especificacao antibodies <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, which had the ability to communicate simultaneously with IGF-1R on the surface I24 cells and to ANGPT2; and thus had the ability to connect a bridged connection two antigen target with two opposing Fab-fragments. In contrast and as predicted, in the case of joint expression of two antibodies wild-type, formed only a very small relative number of functional bespecifically antibodies, which only led to a slight shift of fluorescence in FACS analysis, to detect the formation of bridging ties, when you made the cell culture supernatant or cleared�TES the antibody from a mixture of wild-type after co-expression plasmid < IGF-1R> wild-type and plasmid <ANGPT2> wild type, indicating the presence of only a small relative amount of functional especificacao antibodies < IGF-1R-ANGPT2> wild-type.

These results show that by co-expression of plasmids carrying the replacement VL-VH/CL-CH1 that encode the antibody < IGF-1R > and the wild-type plasmid that encode the antibody <ANGPT2>, you can easily create functional bespecifically antibody <ANGPT2-IGF-1R> bearing replacement VL-VH/CL-CH1, which simultaneously detects two different targets. In contrast, the joint expression of two plasmids of wild-type encoding antibodies < IGF-1R > and < ANGPT2>, leads to high variability of the resulting antibodies, and only to a very small content of functional especificacao antibodies < IGF-1R-ANGPT2> wild-type.

Examples 4

The creation of a divalent especificacao antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1, with modified CH3-domains (knobs-into-holes") (Fig.30)

As can be seen from the above example, co-expression of plasmids encoding the wild-type antibody and plasmids encoding antibodies carrying the replacement VL-VH/CL-CH1, led to the creation of a mixture that contained bespecifically antibody bearing replacement VL-VH/CL-CH1, comprising the following main products: A) monop�nificence antibody bearing replacement VL-VH/CL-CH1, B) bespecifically antibody bearing replacement VL-VH/CL-CH1, and b) the wild-type antibody. To further increase output especificacao antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1, applied technology "knobs-into-holes" for co-expression of antibody <ANGPT2> bearing replacement VL-VH/CL-CH1, and antibodies <VEGF> wild-type with the aim of increasing heterodimerization corresponding unmodified and modified heavy chain (using HC and HC*) and more homogeneous and functional drug especifismo antibodies. This example describes the creation of especifismo antibody that recognizes simultaneously VEGF and ANGPT2, on the basis of antibody <VEGF> wild type G6-31 and described above Mat representing bearing replacement VL-VH/CL-CH1 antibody <ANGPT2>.

As described above, the gene segments encoding the leader sequence, the variable domain light chain (VL) and a constant domain of the Kappa light chain (CL) antibodies <ANGPT2>, interlocked and fused with the 5'-end of the Fc domains of the constant regions of the human γ1 heavy chain (hinge-CH2-CH3). DNA encoding the relevant protein obtained by replacing the VH and CH1 domains on VL and CL domains, created by gene synthesis and is denoted hereafter as < ANGPT2 > HC* (heavy chain*) (SEQ ID NO:8).

Gene segments encoding leader sequence, variable d�Myung heavy chain (VH) and a constant domain (CH1) of the human γ1 heavy-chain antibodies < ANGPT2>, was linked to an independent circuit. DNA encoding the relevant protein obtained by replacing the VL and CL domains in the VH and CH1 domains, created by gene synthesis and is denoted hereafter as < ANGPT2 > LC* (light chain*) (SEQ ID NO:9).

The sequence of the variable domains of the heavy and light chain monospecifičeskoj bivalent antibody < VEGF> wild type, denoted as G6-31, including the respective leader sequences described in this example, taken out of the variable domains of the heavy chain (SEQ ID NO:12), and the variable domain light chain (SEQ ID NO:13) of human antibody < VEGF> such as G6-31, they both are derived from the obtained by using a phage display human antibody to VEGF, such as G6-31, which is described in Liang et, J Biol Chem. 13, 281(2), January 2006, cc.951-961 and in US 2007/0141065 and constant domains of the heavy and light chain were taken from a human antibody (C-Kappa and IgG1).

For this purpose, the plasmids encoding heavy chain antibodies < VEGF> wild type G6-31, modified by introduction of gene segment CH3 created through the synthesis of genes encoding and forming the "flush" with the rest T366W (SEQ ID NO:10), CH3-domain of the corresponding heavy chain antibodies < VEGF> wild type G6-31 receiving the sequence presented in SEQ ID NO:16; in contrast, the plasmid, encoding�th heavy chain* HC* bearing replacement VL-VH/CL-CH1 antibody < ANGPT2> Mat, modified by introduction of gene segment CH3 created through the synthesis of genes encoding and which form of "depression" remains T366S, L368A, Y407V (SEQ ID NO:11), CH3-domain of the corresponding heavy chain* HC* bearing replacement VL-VH/CL-CH1 antibody <ANGPT2> Mat, receiving the sequence of which is presented in SEQ ID NO:15. In this case, the plasmid encoding support "tabs" antibody < VEGF> wild type G6-31, created in the form of genomic vectors, and plasmids that encode bearing replacement VL-VH/CL-CH1 antibody <ANGPT2> Mat, created in the form of carrier intron-cDNA vectors.

Then the four plasmids that encode a heavy chain HC with "projections" (SEQ ID NO:16) antibody < VEGF > G6-31 and the light chain of the wild-type LC (SEQ ID NO:14), and a modified heavy chain* HC* "depressions" bearing replacement VL-VH/CL-CH1 antibody <ANGPT2> (SEQ ID NO:15) and a modified light chain* LC* (SEQ ID NO:8), together expressively in equimolar ratio by short-term transpency system HEK293-F according to the method described above, the 2.6-liter scale in a fermenter type Quad. Antibody was purified using column HiTrap MabSelect (GE), and then subjected to gel filtration on a column (HiLoad Superdex 200 26/60 (GE) according to the method described above. Output after the SEC was approximately 38 mg of purified fractions of antibodies after transient expression in 2-lit�new scale. Fig.26 presents data obtained using LTOs-page in reducing and Sevostyanova conditions, the fractions obtained after purification using gel filtration. Using LTOs-page in reducing conditions can be demonstrated that were synthesized corresponding wild-type and modified heavy and light chains, and they are purified to a homogeneous state. However, when applying LTOs-page in Sevostyanova conditions shows that there are several types of antibodies.

Further fractions obtained by gel filtration, were analyzed by mass spectrometric method (or deglycosylation, but not recovered, or deglycosylation and restored samples). With the help of mass spectrometry in fractions it is possible to identify several types of antibodies and to identify the corresponding bands in applying appropriate LTOs-page (Fig.27). The following table summarises the types of antibodies that can be identified:

Types1. Shoulder straps 5 fraction, deglycosylation1. Shoulder straps fraction 9, deglycosylation
LC G6-31/23255 Yes/(SS): 23251 Yes--
G6-31/49149 Yes /(SS):49136 Yes --
LC* bearing replacement VL-VH/CL-CH1 antibody <ANGPT2>/23919 Yes/ (SS): 23914 Yes--
NS* bearing replacement VL-VH/CL-CH1 antibody <ANGPT2> Mat/ 49245 Yes/(SS): 49231 Yes-
LC G6-31+LC bearing replacement VL-VH/CL-CH1 antibody <ANGPT2> Mat=>(SS): 47165 YesXX

Types1. Shoulder straps 5 fraction, deglycosylation1. Shoulder straps fraction 9, deglycosylation
Full ATA G6-31 (SS), 144774 YesX
Full bespecifically At <VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1 (SS), 145532 Yes (LC+HC G6-31)+(LC*+HC*)X
½ At G6-31 (SS), 72387 YesXX
(NS G6-31) + (NS bearing replacement VL-VH/CL-CH1 antibody <ANGPT2> Mit)=>(SS): 98367 YesXX
(SH) 2 × (�With bearing replacement VL-VH/CL-CH1 antibody < ANGPT2> Mac) + LC G6-31 => 121745 YesX
2 × (NS G6-31) + LC G6-31+NS bearing replacement VL-VH/CL-CH1 antibody <ANGPT2> Mat => (SS): 170754 YesX+20 Yes

Thus, the required bespecifically antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1, which recognizes, on the one hand, VEGF, and on the other hand, ANGPT2, can be accurately identified using mass spectrometry and identified using appropriate LTOs-page. Schematic representation of the requested especificacao antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1 shown in Fig.30. The highest concentration of the specified desired antibodies can be detected in fractions 5 and 6, coming later fractions its concentration is decreased.

Moreover, the presence of especifismo antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1, which had the ability to simultaneously communicate with ANGPT2 and VEGF was confirmed using the above ELISA test, which can detect the formation of bridging ties (Fig.28) described above and Biacore analysis, which reveals the formation of bridging ties (Fig.29), which demonstrated that the required bespecifically antibody possessed the ability to communicate simultaneously�military with ANGPT2 and VEGF. In these analyses tetravalent bespecifically antibody TvG6-Ang23 served as control.

It should be noted that due to the relatively high level of sverkhekspressiya antibody < VEGF> wild type, such as G6-31, for example, result in a higher yield of products expression using genomic vectors encoding derivatives G6-31, the formation of inactive by-products increased, leading to an excess of inactive dimers G6-31 and consisting of two halves antibody (poloitical). To achieve maximum yield of the desired especifismo antibody < VEGF-ANGPT2> bearing replacement VL-VH/CL-CH1 initiated additional studies designed to achieve a uniform expression of all four chains of the antibody and reduce the formation of byproducts. These studies include (I) optimization of an equimolar stoichiometry of 4 plasmids, which are used for co-expression, for example, by combining transcriptional units in one or two plasmids with the introduction of appropriate regulatory elements; and (II) optimization of heterodimerization, for example, using a variety of technologies "knobs-in-holes", such as the introduction of a disulfide bridge in the CH3 domain, for example, Y349C in carrying a "ledge" chain and D356C in a carrier "depression" chain and/or their Association with the use of ostatka� R409D; K370E (K409D) as constituting "the ledges" residues and D399K; E357K as constituting a "depression" residues, which are described in EP A.

1. A method of producing bivalent especificacao antibody, where the antibody contains:
a) the first light chain and the first heavy chain of an antibody specifically binding to a first antigen; and
b) a second light chain and the second heavy chain of an antibody specifically binding to a second antigen,
in which the variable domains VL and VH of the second light chain and the second heavy chain is replaced at each other and the constant domains CL and CH1 of the second light chain and the second heavy chain is replaced at each other,
where the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain each in contact with each other at the interface, which represents the original boundary surface between the CH3-domain of an antibody; wherein the surface section is modified to enable the formation of divalent especifismo antibodies, where the change is different because:
a) modified CH3 domain of one heavy chain
so that the original surface of the partition CH3 domain of one heavy chain, which is in contact with the original surface section of CH3 domain of the second heavy chain in divalent bespecifically the antibody, amino acid residue substituted at amino acid residue that has a large p� the volume of the side chain, which leads to the creation of convexity on the surface of the partition CH3 domain of one heavy chain that can fit into the cavity at the interface of CH3 domain of the other heavy chain, and
b) modified CH3 domain of the other heavy chain
so that the original interface of the second CH3-domain, which is in contact with the original surface section of the first CH3-domain dyadic bespecifically the antibody, amino acid residue substituted at amino acid residue having a smaller side chain volume, which leads to the creation of a cavity at the interface of the second CH3 domain that can fit a bulge at the interface of the first CH3-domain, with the specified amino acid residue, which has a larger volume side chain selected from the group comprising arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W) and the specified amino acid residue having a smaller side chain volume, selected from the group including alanine (A), serine (S), threonine (T) valine (V),
the method includes the following steps:
a) transformation of host cell
vectors containing the nucleic acid molecule encoding a first light chain and the first heavy chain divalent especificacao antibodies;
vectors containing the nucleic acid molecule encoding a second light chain and the second �agelou chain divalent especifismo antibodies,
b) culturing the host cell under conditions that provide for synthesis of the bivalent molecules especificacao antibodies;
b) the allocation of the bivalent molecules especificacao antibodies from the specified culture.

2. A method according to claim 1, distinguish fact that both CH3-domain of an antibody modified by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain, so there may be formed a disulfide bridge between both CH3 domains.



 

Same patents:

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to antibodies including human antibodies and their antigen-binding portions, which specifically bind to CCR2, in particular to human CCR2, and can act as CCR2 inhibitors. Anti-CCR2 antibodies are those binding to first and/or second extra-cellular CCR2 loops. The present invention also refers to human anti-CCR2 antibodies and to their antigen-binding portions. The present invention refers to the recovered heavy and light chains of immunoglobulin initiated from human anti-CCR2 antibodies, and to nucleic acid molecules coding such immunoglobulins. The present invention also refers to methods for preparing human anti-CCR2 antibodies and their antigen-binding portions, to compositions containing such antibodies or their antigen-binding portions, and to methods for using antibodies and their antigen-binding portions, and compositions for diagnosing and treating.

EFFECT: invention refers to methods for gene therapy with the use of nucleic acid molecules coding molecules of heavy and light chains of immunoglobulin, wherein the above molecules contain anti-CCR2 antibodies and their antigen-binding portions.

25 cl, 24 dwg, 8 tbl, 17 ex

Csf-1r antibody // 2547586

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. There are presented an antibody and its antigen-binding fragment specifically binding human colony-stimulating factor-1 receptor (CSF-1R) characterised by sequences of complementary-determining regions (CDR). There are also disclosed a nucleic acid coding the antibody according to the invention or its antigen-binding fragment, a vector providing the expression of the antibody and its antigen-binding fragment, and a pharmaceutical composition applicable in treating the diseases associated with an inflammation or an autoimmunity, or cancer.

EFFECT: invention can find further application in diagnosing and therapy of the CSF-1 associated diseases.

23 cl, 18 dwg, 4 tbl

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, in particular to peptydoglycane hydrolase biosynthesis, and represents a protein with the peptydoglycane hydrolase activity, a plasmid, containing a peptydoglycane hydrolase-coding fragment, a bacterium-producer, a method of microbiological peptydoglycane hydrolase synthesis, as well as a pharmaceutical composition, containing the obtained peptydoglycane hydrolase, for the therapy of diseases, caused by Gram-negative microflora.

EFFECT: elaborated method of microbiological synthesis makes it possible to obtain bacteriophage S-394 peptydoglycane hydrolase in an effective way.

22 cl, 2 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.

EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.

28 cl, 4 dwg, 7 tbl, 10 ex

FIELD: chemistry.

SUBSTANCE: inventions relate to chimeric proteins, nucleic acid, coding such a protein, an expression cassette, providing the expression of nucleic acid, a vector, including the expression cassette, a method of diagnostics and a set for diagnostics. The characterised chimeric Borrelia protein includes at least one sequence of an extracellular domain of the VlsE Borrelia protein of the first type, corresponding to a certain strain, and at least one sequence of IR6 area of the VlsE Borrelia protein of the second type or Borrelia of the first type, but corresponding to a strain, different from the strain of the first type, with Borrelia being selected from Borrelia stricto-sensu, Borrelia afzelii and Borrelia garinii.

EFFECT: claimed inventions make it possible to carry out diagnostics of Lyme-borreliosis with an increased specificity and sensitivity.

15 cl, 8 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. Presented are antibodies targeting integrin α2β1 containing humanised anti-integrin alpha-2 (α2) antibodies, as well as a method of treating by the integrin α2 antibodies. The humanised integrin α2 antibodies comprise a variable region of a light chain domain, a constant human light chain domain and a variable constant heavy chain domain of human IgG1, which exhibit the altered effector function. The variable constant heavy chain domain of human IgG1 comprises an S324N substitution. The invention can be used in medicine.

EFFECT: antibodies exhibit complement-dependent cytotoxicity, improved antibody-dependent cell-mediated cytotoxicity and improved CDC and ADCC.

33 cl, 3 dwg, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is a host cell of Bordetella pertussis, Bordetella bronchiseptica or Bordetella parapertussis, which is used as an adjuvant or for preventing or treating whooping cough, having the low activity of endogenous glycosyltransferase at least 98% identical to the amino acid sequence SEQ ID NO: 2 as compared to the activity of glycosyltransferase of a relative parent strain, wherein the low activity is ensured by using an inactivating vector, which causes the inactivation of expression of a sequence of endogenous nucleic acid coding glycosyltransferase, or reduces to a low level of expression of the sequence of endogenous nucleic acid coding glycosyltransferase by the fusion of nucleic acid coding glycosyltransferase with a low-level or inducible promotor. What is disclosed is a preparation consisting of LPS of the above host cell with an increased replacement of hexosamine 1' or 4' phosphate groups of LPS referred to a lipid A, as compared to a LPS preparation from the related parent strain; thereby LPS is characterised by producing at least 8 ions in the ESI-MS spectrum, wherein the preparation is used as an adjuvant or for preventing or treating whooping cough. What is presented is using the above host cells or the LPS preparation for producing the preparation for preventing and/or treating whooping cough, or producing the drug preparation for immunising a mammal, wherein the host cells or LPS is used as an adjuvant. What is described is a pharmaceutical composition used as the adjuvant or for preventing or treating Bordetella infection containing the above host cell or above LPS preparation in an effective amount and a pharmaceutically acceptable carrier.

EFFECT: invention enables producing the pharmaceutical preparation of Bordetella cells or LPS, possessing the high immunogenicity as compared to the preparation of the related parent strain Bordetella.

12 cl, 7 dwg, 3 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. Presented are versions of an antigen-binding polypeptide specific to LIGHT polypeptide characterised by a variable domain of heavy and light chains, as well as versions of based conjugates for treating a disease or a condition. What is described is a pharmaceutical composition for inhibiting apoptosis induced by human LIGHT based on a therapeutically effective amount of the polypeptide. There are disclosed: a method of treating or a diagnosing the disease or condition on the basis of the composition. There are described versions of the recovered polynucleotide or a cell transformed by the polynucleotide for preparing the antigen-binding polypeptide, as well as a method for producing the antigen-binding polypeptide on the basis of the cell.

EFFECT: using the invention provides the antibodies, which block the human or macaque LIGHT interaction to LIGHT receptors that can find application in treating various diseases related to high T-cell activity.

23 cl, 11 dwg, 8 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology.

EFFECT: bispecific anti-human vascular endothelial growth factor VEGF and human angiopoietin-2 ANG-2 antibodies, methods for producing them, pharmaceutical compositions containing the above antibodies, and using them are described.

13 cl, 26 dwg, 15 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a polypeptide containing two binding fragments presented by antibodies; the first of them binds to CD3e(epsilon) chain epitope of a human or a primate, other than a chimpanzee, particularly Callithrix jacchus, Saguinus oedipus and Saimiri sciureus; the second one - to EGFR, Her2/neu or IgE of a human or a primate, other than a chimpanzee, with the above CD3e epitope containing the amino acid sequence Gln-Asp-Gly-Asn-Glu. There are also disclosed a coding sequence of the nucleic acid, a vector, a host cell and a method for preparing the above peptide, as well as a pharmaceutical composition and using the polypeptide in preventing, treating or relieving a proliferative disease, a malignant disease or an immunological disorder.

EFFECT: invention provides the clinical improvement of T-cell redistribution and the enhanced safety profile.

17 cl, 8 tbl, 26 dwg, 26 ex

FIELD: biotechnology.

SUBSTANCE: bispecific antibody is proposed, that binds to both the blood coagulation factor IX/activated blood coagulation factor IX and with the blood coagulation factor X, and functionally replaces the function of blood coagulation factor VIII. The nucleic acid is considered, encoding the antibody of the invention, a vector, a cell and a method of producing the antibody, and also a pharmaceutical composition and a kit for use in the method of preventing and/or treating bleeding or diseases associated with or caused by bleeding.

EFFECT: invention may find further application in the treatment of diseases associated with impaired blood clotting.

16 cl, 2 ex, 6 dwg

FIELD: chemistry.

SUBSTANCE: claimed is bispecific antibody, which is bound with both blood coagulation factor IX/activated blood coagulation factor IX and with blood coagulation factor X and functionally replaced function of blood coagulation factor VIII. Described are nucleic acid, coding antibody by invention, vector, cell and method of obtaining antibody, as well as pharmaceutical composition and set for application in method of prevention and/or treatment of bleeding or diseases, associated with or induced by bleeding.

EFFECT: invention can be applied in therapy of diseases, associated with blood coagulation disorders.

16 cl, 2 ex, 6 dwg

FIELD: medicine.

SUBSTANCE: what is presented is a fused protein that is a Notch1 antagonist, which consists of a human Fc region fused with the EGF-like repeat 1-13 of Notch1 or the EGF-like repeat 1-24 of Notch1. Fc-portion is localised on a carboxy-terminal portion of the EGF-repeat. There are described a pharmaceutical composition for the protein-based Notch signal transmission inhibition and using it for preparing the pharmaceutical composition for treating an individual suffering from: tumour; ovarian cancer; metabolic disorder; vascular proliferative retinopathy. What is presented is using the fused protein for producing the pharmaceutical composition for inhibition: angiogenesis in the individual; physiological lymphangiogenesis or pathological lymphangiogenesis in the individual; tumour deposits in the individual.

EFFECT: using the invention provides the proteins expressed in a supernatant at a level by several times more than the fused protein containing the EGF-like repeats 1-36 of Notch1; they penetrate into the tumour better, maintain a ligand-binding ability with the fused protein containing the repeats 1-24, binds to DLL4 and JAG1, whereas the fused protein containing the repeats 1-13 only binds to DLL4, but not to JAG1 that can find application in therapy of various diseases related to the Notch1 activity.

18 cl, 124 dwg, 10 ex

FIELD: medicine.

SUBSTANCE: inventions relate to the field of immunology. Claimed are a single-chain antibody, specific to a carcinoembryonic antigen, a chimeric mononuclear T-cell receptor, a vector, a host cell and a method of diagnostics or treatment of diseases, characterised by the presence of antigens, capable of binding with the chimeric receptor. Described is a genetic construction, coding chimeric monomolecular T-cell receptors, in which an effector fragment of the T-cell receptor is combined with an antigen-recognising part, which represents variable fragments of two different antibodies to the carcinoembryonic antigen (CEA).

EFFECT: claimed inventions can be used in T-cell cancer therapy.

7 cl, 4 dwg, 3 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.

EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.

13 cl, 10 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: claimed invention relates to immunology and biotechnology. Claimed is binding protein for binding one or more targets, which contains four polypeptide chains forming four functional antigen-binding sites. Four polypeptide chains contain VD1-(X1)n-VD2-C-(X2)n. VD1 stands for first variable domain of heavy chain, VD2 stands for second variable domain of heavy chain, C stands for CH1 domain, X1 stands for polypeptide linker, on condition that it is not constant domain, and X2 stands for Fc-region, and n equals 0 or 1. Two polypeptide chains contain VD1-(X1)n-VD2-C. VD1 stands for first variable domain of light chain, VD2 stands for second variable domain of light chain, C stands for CL domain, X1 stands for linker, on condition that it is not constant domain; and n equals 0 or 1. Conjugate of binding protein with visualising detecting cytotoxic or therapeutic agent is described. Disclosed are: nucleic acids (NA), coding polypeptide chains, as well as expressing vectors, vectors for replication, host cells which contain them, and method of obtaining antibody applying cells. Described is pharmaceutical composition for treatment or preventing target-associated disease or disorder based on binding protein. Method of treatment by introduction of binding protein is described.

EFFECT: application of invention provides new format (DVD-Ig) of antigen-binding molecules, which in the same dosage possess higher activity with respect to target than respective full-size antibodies, which can be applied in medicine for prevention and treatment of various diseases.

45 cl, 27 tbl, 5 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a method for obtaining an antibody, the pharmacokinetic properties of which have been changed at maintaining antigen-binding activity of a variable area, which provides for the following stages: (a) obtaining antibodies in which there has been modified a charge of amino-acid residues chosen from amino-acid residues in positions 31, 61, 62, 64 and 65 of the variable area of a heavy chain and in positions 24, 27, 53, 54 and 55 of the variable area of a light chain in compliance with numbering as per Kabat system, where modification of the charge of amino-acid residues leads to the change of 1.0 or more at a theoretical isoelectric point of the variable area of the antibody, and (b) extracting an antibody with stored antigen-binding activity from antibodies obtained at stage (a).

EFFECT: invention allows effective change in pharmacokinetic properties of an antibody, thus maintaining its antigen-binding activity.

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology and immunology. Claimed is antibody, specifically binding with form A FcγRIII (CD16) (FcγRIIIA, CD16A) and not-binding specifically with form B (FcγRIIIB, CD16B), its antigen-binding fragment and multi-specific antibody, which includes antigen-binding fragment of antibody by invention. Compositions, which contain antibody by invention or its antigen-binding fragment, and their application in treatment of autoimmune, inflammatory, inflectious diseases, allergy and cancer, as well as set for detection of FcγRIIIA are described. Polynucleotides, vectors and host cells and method of obtaining antibody by invention or its antigen-binding fragment are described.

EFFECT: claimed invention provides novel antibodies to FcγRIIIA and, in that way, can find further application in therapy of FcγRIIIA-mediated diseases.

51 cl, 24 ex, 8 dwg, 5 tbl

FIELD: medicine.

SUBSTANCE: present invention refers to immunology. Presented is a molecule of bispecific single-chain antibody containing a first binding domain able to bind to epitope of CD3-epsilon-chain of human and Callithrix jacchus (tamarin), Saguinus oedipus (cotton-top tamarin) and Saimiri sciureus (squirrel monkey), and a second binding domain able to bind to an antigen specified in a group consisting of: PSCA, CD19, C-MET, endosialin, EGF-like domain 1 EpCAM coded by exon 2, FAP-alpha or IGF-IR (or IGF-1R) or a human and/or a primate. The epitope CD3e contains an amino acid sequence disclosed in the description. Disclosed are a nucleic acid coding the above molecule of the bispecific single-chain antibody, an expression vector, a host cell and a method for producing the antibody, as well as the antibody produced by the method. Described is a based pharmaceutical composition containing the molecule of the bispecific single-chain antibody and a method for preventing, treating or relieving cancer or an autoimmune antibody. Presented is using the above molecule of the bispecific single-chain antibody for making the pharmaceutical composition for preventing, treating or relieving cancer or the autoimmune disease.

EFFECT: using the invention provides the clinical improvement in relation to T-cell redistribution, reducing it, and the improved safety profile.

23 cl, 74 dwg, 17 tbl, 33 ex

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