Human anti-cd40 antagonist monoclonal antibody

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a low-immunogenicity anti-CD40 monoclonal antibody prepared of a chimeric 5D12 (ch5D12) antibody. There are also described: a nucleic acid, a cell and a cell culture for preparing the antibody according to the invention, a method for preparing it, a pharmaceutical composition, using the antibody for preparing a drug and a method for administering the antibody according to the invention to an individual for the purpose of relieving the symptoms of an autoimmune disease, an inflammatory disease, for the purpose of suppressing a graft rejection reaction and/or treating CD40-positive cancer.

EFFECT: what is described is a method for selecting the high-expression human anti-CD40 antibodies containing insertion, deletion, inversion and/or replacement of 1 to 5 amino acids as compared to the antibody according to the invention, and the antibody prepared by the above method for selecting.

31 cl, 21 dwg, 14 tbl, 10 ex

 

The present invention relates to human antibodies, and humanized monoclonal antibodies with reduced immunogenicity, their use and methods of obtaining. The invention in particular relates to a monoclonal antibody antagonists against human CD40.

Molecule CD40 is a membrane glycoprotein type I with a molecular mass of 50 kDa and is expressed on b cells, monocytes/macrophages, dendritic cells (DC) and activated endothelial cells.1-6Under certain conditions the CD40 can also be found on fibroblasts, epithelial cells and keratinocytes.7The CD40 ligand (CD40L, CD154), integral membrane glycoprotein of type II with a molecular mass of 32 kDa, transtorno is expressed on activated T-cells CD4+and a small population of activated T-cells CD8+.8,9In addition, CD40L was detected in other cell types upon activation, including mast cells, basophils, b cells, eosinophils, DC, and platelets.10,11

Studies in mouse models have clearly demonstrated the involvement of the interaction between CD40L-CD40 in the pathophysiology of various autoimmune diseases (for review see reference12). The data obtained in transgenic CD40L on mice, which become lethal inflammatory bowel disease, provided the first d is the proof, the interaction of CD40-CD40L may also play a role in the pathogenesis of inflammatory bowel disease.13Monoclonal antibody (MAB) against CD40 mouse is effective against inflammation of the mucous membranes and production of interferon-γ by T-cells CD4+own layer (lamina propria) in TNBS-induced colitis.14In the mouse model of inflammatory bowel disease severe combined immunodeficiency (SCID) have shown that treatment with anti-CD40L from the date of recovery of T cells completely prevented clinical and histological manifestation of experimental colitis.15Moreover, with the introduction of anti-CD40L 5 weeks after recovery of T cells it was possible to prevent progression of the disease, and treated animals showed improvement of symptoms and histology compared to control animals.15In addition, recovery of T cells of SCID mice with T cells from mice with off CD40L further demonstrated the significant role of expressing CD40L T cells in disease development and production of interleukin-12.16

The interaction of CD40-CD40L can be prevented by using monoclonal antibodies (MAB) against or CD40L or CD40. The expression of CD40L on activated platelets led to thromboembolic events during treatment people who CD40L MAB against human rights in the form of IgG 1at the highest dose levels and stop the development of these Mat.17,19Therefore, the antagonism against CD40 is a much more attractive approach. Restimulating antagonistic activity Mat 5D12 (against human CD40) has been demonstrated in various in vitro studies using various types of bearing CD40 cells20,22and antagonistic activity of chimeric 5D12 (ch5D12) was confirmed in vivo using a model of the disease on various non-humans primates.23,27ch5D12 is a molecular engineered antibody as IgG4man, containing the variable regions of the heavy and light chains 5D12 muscles, and was designed to reduce the ability to immunogenicity and increased half-life existence in vivo mouse Mat 5D12 when used for people.

Patients with Crohn's disease suffer from calling the weakness of inflammatory disorders of the gastrointestinal tract, the exact etiology and pathogenesis of which remains unknown.28,29The disease is characterized by the receipt of the diseased mucous membrane of activated T-cells, b-cells and macrophages,30,31local production of soluble mediators of inflammation and damage to the tissues involved.28,29It is shown that T-cells CD4+and macrophages in the mucosa and Fokine, such as tumor necrosis factor (TNF)-α and IL-12 play a Central role in initiating the inflammatory cycle in Crohn's disease.32,38T-cells of inflamed mucous membranes exhibit higher proliferative capacity28,29and secrete increased amounts of IFN-γ and IL-2. Increased levels of mRNA transcripts for cytokines associated with T-cells found in the biopsy specimens of the mucous membranes of patients with Crohn's disease.33In the study of the expression of CD40/CD40L in the lesions in Crohn's disease, conducted by the authors of the present invention, it has been suggested that CD40L plays a major role on activated T-cells CD4+.39CD40L can mediate strong activation of bearing CD40 cells, mainly b cells and macrophages, thereby leading to increased production of TNF-α and IL-12 in the lesion. Using immunohistochemistry increased staining 5D12 was detected in all samples of diseased regions of patients with Crohn's disease compared with unaffected areas. Double staining for CD40 and CD20 (b-cells) or CD68 (macrophages) showed that in the sliced samples of patients with Crohn's disease cells CD40+were mainly b-cells in lymphoid follicles and macrophages in its own layer. T cells own layer inflamed mucous membranes of ballygrant Krona induced production by monocytes significant amounts of IL-12 and TNF-α after 48 h co-culture. Adding 5D12 has led to reduced production of IL-12 and TNF-α; levels of production decreased to levels observed at the control T cells own layer as in the absence, in the presence of IFN-γ.39

The aim of the present invention to provide an alternative molecules having at least the same safety and/or effectiveness that 5D12 in vivo, but not necessarily the same degree specified safety and/or effectiveness. Antibody 5D12, or at least its variable regions, has mouse origin. The present invention relates to variants of the variable regions of the heavy and light chains 5D12. As a result, the present invention relates to a polypeptide comprising the amino acid sequence of the formula (I)

In which

X1represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;

X2represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;

X3represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;

X4represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R, or H; and

X5represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H.

The specified polypeptide contains a sequence that is identical to the sequence of the variable regions of the heavy price and antibody 5D12, however, the polypeptide is less immunogenic for a person whom it is administered by itself or as part of an antibody containing the specified polypeptide. In these positions X1-X5some amino acids may be present as indicated. Linking molecule contains the polypeptide of the present invention has satisfactory properties bind CD40. It is noted that the production of the antibody in the cell applies to some extent depending on the type of amino acids in these positions. This will be described in detail later.

The present invention also relates to the polypeptide of the present invention containing the amino acid sequence

The above polypeptide essentially overlaps the variable region of the heavy chain of the antibody 5D12 mouse. Amino acid sequence modified in several positions relative to the sequence of the mouse. The modified polypeptide has good binding properties and good tolerability people who receive the polypeptide. In these provisions can be integrated with various amino acids without substantially reducing and/or modifying at least immunological properties of the polypeptide compared to the original polyp is predom mouse. Preferably, the polypeptide of the present invention contains G, A, V, L, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H at position X1. Thus, it is not reduced and/or does not significantly change at least the ability to produce the polypeptide in a cell of the mammal compared with the original polypeptide mouse or a chimeric polypeptide. In a particularly preferred embodiment of the present invention

X1represents G, A, V, L, P, F, or M;

X2represents G, A, V, L, I, P, F, or M;

X3represents G, A, V, L, I, P, F, M;

X4represents G, A, V, L, I, P, F, M; and

X5represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T or Y.

Such polypeptides are preferred for products at a high level in the cell of a mammal, in particular, in the structure of antibodies.

In addition, the present invention relates to a polypeptide according to the present invention, in which

X1represents G, A, V, L or M;

X2represents G, A, V, L, I or M;

X3represents G, A, V, L, I, P, F, M;

X4represents G, A, V, L, I or M; and

X5represents P, F, W, N, Q, S, T or Y.

Such polypeptides are more preferred for the production of antibodies at a high level and well tolerated by humans. In addition, the present invention relates to polypep the DN of the formula (I), in which X1represents L; X2represents I; X3is a P; X4is a M; and/or X5represents S. Such a polypeptide is especially preferred due to their ability to effectively produced in the structure of antibodies, which reproduces the binding and pharmacological properties of antibodies ch5D12, while the immunological properties of the people improved compared with the antibody or mouse antibody ch5D12, and at least not significantly reduced production of the polypeptide compared to murine or chimeric analogue.

In addition, the present invention relates to a polypeptide of the formula (I)in which X1represents I; X2is a V; X3is a P; X4is a M; and/or X5represents S. This polypeptide is especially preferred because of its ability to effectively produced in the structure of antibodies, which reproduces the binding and pharmacological properties of antibodies ch5D12, while improved immunological properties in humans compared with the antibody or mouse antibody ch5D12, and at least not significantly reduced production of the polypeptide compared to murine or chimeric analogue.

The antibody that differs edge is least one position X 1-X5from chimeric 5D12 in the relevant provisions, has the best immunological properties compared to the chimeric antibody 5D12 people. In a preferred embodiment, such antibodies of the present invention, preferably, refers to a polypeptide of the present invention, in which X1is a I and X2is a V; X1is a I and X2represents I; X1is a L, and X2represents I; or X1is a L, and X2represents V. Particularly preferred such polypeptides in combination with X3that is a P; X4that is a M; and X5that represents F or S. In one embodiment implementing the present invention relates to a polypeptide according to the present invention, in which X1represents L, X2is a V; X3represents L; X4is a L; and X5represents F. Production of antibodies containing a specified polypeptide, is extremely effective in the simultaneous presence of improved immunological properties in humans compared with ch5D12. In addition, the present invention relates to a polypeptide containing the amino acid sequence GFS 1SRYSVYWX2R, where X1is a L, and X2represents I; or X1is a I and X2is a V. This polypeptide includes a modified variable regions CDR1 of the heavy chain 5D12. This CDR1 contains at least one excellent amino acid compared with the variable regions CDR1 of the heavy chain of the antibody 5D12. This amino acid substitution leads to the improvement of the immunological properties of the modified antibody 5D12 or ch5D12, the modification includes at least replace the corresponding sequence of the specified polypeptide in 5D12 or ch5D12 the polypeptide of the present invention, but provides a high production of antibodies in the cell of a mammal.

In addition, the present invention relates to variable regions of the heavy chain containing a polypeptide of the formula (I). The specified variable region contains preferably 90-130, more preferably, 100 to 120, more preferably, 105-115, most preferably, 113 amino acids. The polypeptide may be obtained synthetically or produced by the cell. Preferably, the specified variable region of the heavy chain is produced by the cell. In nature there are at least five types of heavy chains: γ, δ, α, µ and ε, where each type defines a class of immunoglobulins. Polypeptide really the invention can be used as a binder directly or it can enter into the composition of the antibodies. With the introduction of the antibody polypeptide, preferably, combined with the constant region of the heavy chain of the antibody. Thus, the present invention also relates to a heavy chain antibody containing a polypeptide of the formula (I). In the art there are many derivatives and analogs of antibodies with variable regions. However, currently, many different parts, derivatives and/or analogs of antibodies are used. Non-limiting examples of such parts, derivatives and/or analogues are single-chain Fv fragments, Manuela, VHH, Fab fragments, artificial binding proteins, such as, for example, Avineri, etc. are a Common feature of such specific binding substances is the presence of variable regions of the heavy chain. Thus, the present invention also relates to a connecting structure that contains the polypeptide of the formula (I).

The preferred connecting structure of the present invention is the antibody as the antibody contains natural structure. Therefore, the present invention in a preferred embodiment, refers to an antibody containing the polypeptide of the present invention.

Connecting structure of the present invention, preferably, represents a binding is th design, which is well tolerated by animals. The tolerance of the animal in respect of the polypeptide is regulated by many different aspects. Immunity mediated by T-cells, mediated by b-cells or other, is one of the parameters that are included in the tolerance of the animal in respect of the polypeptide. As noted above, the antibody 5D12 is of murine origin. The polypeptide of formula (I) has reduced immunogenicity to humans. Therefore, he is sometimes listed as demonizirovannyj option variable regions of heavy 5D12. Thus, in one aspect the present invention relates to the antibody with epitope specificity of the antibody 5D12, and a heavy chain of the indicated antibody is a polypeptide of formula (I). As used in the present description, the term "demonizirovannyj" is defined as less immunogenic to the animal than the original antibody. The polypeptide of formula (I) is neimmunizirovannah compared to the heavy chain in 5D12 due to the removal of known T-cell epitopes of human rights. T-cell epitopes are amino acid sequences within proteins, capable of binding molecules MHC (major histocompatibility complex) class II. After removal of T-cell epitopes antibody becomes less immunogenic. Preferably, the flexible about the art of the present invention is, in addition, humanized, for example, generovanou. When using methods of generowania the remains of the outer part, which are present in abundance for the immune system to selectively replace the remnants of the person to provide a hybrid molecule that has either weakly immunogenic or essentially non-immunogenic generovanou surface. Used in the present invention an animal, preferably is a mammal, more preferably a Primate, most preferably, human.

The antibody of the present invention, preferably, contains a constant region of human antibodies. In accordance with the differences in the constant regions of the heavy chains of the antibodies are grouped in five classes or isotypes: IgG, IgA, IgM, IgD and IgE. These classes or isotypes include at least one of these heavy chains, which represent the corresponding Greek letter. In a preferred embodiment, the present invention relates to the antibody of the present invention, in which the specified constant region selected from the group constant regions of IgG, IgA, IgM, IgD and IgE, more preferably, the specified constant region comprises a constant region of IgG, more preferably, the constant region of IgG1preferably, the mutated constant region of IgG1most predpochtitelno, the specified constant region is a constant region of IgG4. Moreover, the specified constant region of IgG4is, preferably, the constant region of IgG4man. Preferably, the antibody IgG4according to the present invention contains a constant region amino acid sequences of the heavy and light chain depicted in Fig. Preferably, the antibody IgG4according to the present invention contains the amino acid sequence of the heavy and light chain depicted in Fig. Some variation of the constant region of IgG4occurs in nature and/or Pets without changing the immunological properties of the resulting antibodies. As a rule, in the constant region are allowed approximately 1-5 amino acid substitutions. Antibody with a constant region of IgG4or mutated constant region of IgG1has at least the greater part of the pharmacological properties of the antibodies, but does not bind complement and will not, therefore, cause a depletion of the cells with which it is associated in vivo. Preferably, the specified constant region is a constant region of human antibodies.

In one of the embodiments the present invention relates to a nucleic acid that encodes a polypeptide of the present invention is/or connecting structure according to the present invention, and/or antibody of the present invention. Used in the present invention the nucleic acid is typically, but not exclusively, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). For the skilled in the art specialist available alternative nucleic acid, such as, for example, peptide-nucleic acid (NCP). Nucleic acid of the present invention, for example, is injected into the cell. When the specified nucleic acid is expressed in a given cell, the cell produces the polypeptide and/or linking design, and/or antibody of the present invention. Therefore, the present invention in one of the embodiments refers to the cell containing the polypeptide of the present invention, a connecting structure according to the present invention, the antibody of the present invention and/or nucleic acid of the present invention. The specified cell, preferably, is a cell of an animal, more preferably, the cell is mammalian, more preferably, the cell is a Primate, most preferably a human cell. For the purposes of the present invention a suitable cell is any cell that can include, preferably, to produce the polypeptide of the present invention, connecting the con is tructio of the present invention, the antibody of the present invention and/or nucleic acid of the present invention.

In addition, the present invention relates to a cell containing the antibody of the present invention. Preferably, the cell produces the indicated antibody. In a preferred embodiment, the specified cell is a hybridoma cell, a cell of Chinese hamster ovary (Cho, NSO cell or cell PER-C6TM. In a particularly preferred embodiment, the specified cell is a cell SNO. In addition, the invention relates to a cell culture containing the cell of the present invention. Various institutions and companies have developed cell lines for large-scale production of antibodies, for example, for clinical application. Non-limiting examples of such cell lines are cells of Cho, NSO cells, or PER.C6 cellsTM. These cells can also be used for other purposes, for example, for the production of proteins. Cell line, developed for the production on an industrial scale proteins and antibodies, specified in the present description as an industrial cell line. Thus, in a preferred embodiment, the present invention relates to the use of the cell line, developed for large-scale production of the protein and/or antibodies for the production of intitulado the present invention.

In addition, the present invention relates to a method of producing an antibody comprising culturing the cell according to the present invention and collecting the specified antibodies from the specified culture. Preferably, the specified cell cultured in serum-free medium. Preferably, the cell adapted for growth in suspension. In addition, the invention relates to an antibody obtained by the method of producing the antibody of the present invention. The antibody is preferably purified from the medium of culture. Preferably, this antibody is subjected to affinity purification.

The cell of the present invention is, for example, a line of hybridoma cells, cell, NSS, NSO cell or another cell type, which are suitable for the production of antibodies for clinical purposes. In a particularly preferred embodiment, the specified cell is a human cell, preferably a cell transformed area E1 adenovirus or its functional equivalent. A preferred example of such a cell line is the cell line PER.C6TMor its equivalent. In a particularly preferred embodiment, the specified cell is a cell of SSC or its variant, preferably, a variant that uses glutamylcysteine (GS) vector system for the expression of antibodies.

Marked some amino acids in the referenced provisions X1-X5less suitable for the production of high antibody comprising the polypeptide of formula (I), in producing the antibody the cell. In a preferred embodiment, the polypeptide of formula (I) in the specified antibody contains X1-X5and

X1represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H. In a particularly preferred embodiment, X1represents G, A, V, L, I, P, F, or M; X2represents G, A, V, L, I, P, F, or M; X3represents G, A, V, L, I, P, F, M; X4represents G, A, V, L, I, P, F, M; and X5represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T or Y. More preferably, the said antibody comprises a polypeptide of the formula (I), where X1represents G, A, V, L, I or M; X2represents G, A, V, L, I or M; X3represents G, A, V, L, I, P, F, M; X4represents G, A, V, L, I or M; and X5represents P, F, W, N, Q, S, T or Y. More preferably, X1represents L; X2represents I; X3is a P; X4is a M; and/or X5represents S. Especially preferably, when X1is a I and X2is a V; X1is a I and X2represents I; the 1is a L, and X2represents I; X1is a L, and X2represents L; X1is a V, and X2represents I; X1is a V, and X2is a V; X1is a L, and X2represents L; X1is a V, and X2is a L; or X1is a L, and X2is a V. These last polypeptides are particularly preferred in combination with X3that is a P; X4that is a M; and X5that is a or F, or S, preferably s In one of the embodiments the present invention relates to a polypeptide according to the present invention, in which X1represents L; X2is a V; X3represents L; X4is a L; and X5represents F. Production of antibodies, including specified polypeptide, is good with the parallel provision of improved compared to ch5D12 immunological properties of the people.

In another preferred embodiment, the present invention relates to a polypeptide according to the present invention, in which at least one of X1, X2, X3, X4or X5the two which is the same amino acid, as the amino acid in the corresponding position of the sequence, which, as demonstrated in the present invention, gives a truly significant levels of expression, and where, in addition, at least one of X1, X2, X3, X4or X5is the same amino acid as the amino acid in the corresponding position of the amino acid sequence 5D12. The advantage of the polypeptide of the present invention, in which at least one of X1, X2, X3, X4or X5is the same as the amino acid in the corresponding position of the amino acid sequence 5D12, is that the connecting structure of the present invention comprising such a polypeptide has the best level of expression than the polypeptide of the present invention, where none of X1, X2, X3, X4or X5is not the same amino acid as the amino acid in the corresponding position of the amino acid sequence 5D12. It was expressed not associated with any theory, the assumption that such a high level of expression due to the fact that the amino acid at position X1, X2, X3, X4and/or X5promotes proper Assembly connecting structure of the present invention, if the specified is minociclina is the same amino acid, as the amino acid in the corresponding position of the amino acid sequence 5D12.

Antibody containing a polypeptide of the formula (I), has restimulating antagonistic activity. Because the interaction of CD40L-CD40 involved in the pathophysiology of various inflammatory diseases, such as autoimmune diseases and transplant rejection, the polypeptide of the present invention, therefore, is suitable, in particular, to reduce the intensity of the symptom of inflammatory diseases. In one of the embodiments, the antibody contains a connecting structure according to the present invention. In a preferred embodiment, the specified antibody is a monoclonal antibody. The technology of monoclonal antibodies allows us to produce large quantities of essentially pure antibodies, and thus, the predicted products. Therefore, the present invention in one of the embodiments relates to a monoclonal antibody-antagonist against human CD40 containing the polypeptide of the present invention. Connecting structure of the present invention is more suitable for this purpose, as it is in one of the embodiments is neimmunizirovannah compared to mouse 5D12 and/or chimeric 5D12. Therefore, the connecting structure on n the present invention has reduced immunogenicity and increased half-life existence in humans compared with mice 5D12 and/or chimeric 5D12. In this connecting structure according to the present invention has a stable pharmaceutical efficacy against various inflammatory diseases. Thus, in a preferred embodiment, the present invention relates to neimmunizirovannah monoclonal antibody-antagonist against CD40 according to the present invention.

As previously indicated, the present invention relates to such 5D12 molecules that contain amino acid modifications compared with the amino acid sequence 5D12, and modifications are present at least in the variable regions of the heavy chain and, preferably, also in the variable region of the light chain. In this context, the present invention also relates to a connecting structure of the present invention, comprising the amino acid sequence of the formula (II)

In which

X6represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;

X7represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;

X8represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;

X9represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R, or H; and

X10represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H, and

X11PR is dstanley a G A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H.

The specified binding composition preferably is a monoclonal antibody antagonist against human CD40 according to the present invention. In a preferred embodiment, X is chosen from X6, X7, X8, X9, X10or X11selected from the group similar to the amino acid in the corresponding position in the amino acid sequence 5D12 and/or equivalent amino acid in the corresponding position in the sequence, which, as shown in the present application, provides high levels of expression, as shown in example 2. Therefore, the connecting structure of the present invention preferably contains the amino acid sequence of the formula (II), in which

X6represents N, Q, S, T, Y, W, or C;

X7represents the D, E, N, Q, S, T, Y, W, or C;

X8represents N, Q, S, T, Y, G, A, V, L, I, P, F, M, W, or C;

X9represents G, A, V, L, I, P, F, M;

X10represents G, A, V, L, I, P, F, M; and

X11represents N, Q, S, T, Y, G, A, V, L, I, P, F, M, W or C.

In another embodiment, the present invention relates to a monoclonal antibody-antagonist against human CD40 according to the present invention containing a polypeptide of the formula (II) according to the present invention, containing the following amino acid sequence:

In a preferred embodiment, the present invention relates to a polypeptide of the formula (II) according to the present invention, in which X6is a T or S, X7represents the D or Q, X8represents Q or P, X9is a V or A, X10is a V or L and X11represents F or Y. More preferably, X6represents T, X7represents Q, X8represents P, X9represents A, X10is a V, and X11is a y Such 5D12 antibodies containing the polypeptide of the formula (I) and the preferred polypeptide of the formula (II), combine high levels of expression in the producing cell and tolerability, as well as a good pharmacodynamic properties in humans.

Antibody antagonist against human CD40 according to the present invention preferably contains the amino acid sequence of the variable region of the heavy chain of the formula (I) and the amino acid sequence of the variable region of the light chain of the formula (II). This antibody has good features. Of course, you can create variations of this source of antibodies by modifying one or more amino acids in it. The activity of many of these options the options will be more or less similar to the specified source antibody. Such variants are also included in the scope of the present invention. There are many ways to modify the antibody of the present invention. A non-limiting example of such modification is the antibody containing pyroglutamic instead of glutamate. Other non-limiting examples of such modifications include insertion, deletion, inversion and/or replacing one or more amino acids compared with the specified source antibody. The present invention relates to means and methods of creating such an option. The invention relates to the tests for determining the characteristics of the specified option. In a preferred embodiment, the present invention relates to a variant of the original antibody of the present invention, containing the insertion, deletion, inversion and/or replacement of approximately 1 to 10 amino acids compared to the amino acid sequence specified source of antibodies. Preferably, the above insertion, deletion, inversion and/or replacement does not include the amino acid at position X1and the position X2variable regions of the heavy chain of the formula (I) of the original antibody.

In a preferred embodiment, the present invention relates to a method of selecting antibodies antagonist against human CD40, comprising creating a first cell line which produces and is output to the antibody antagonist against human CD40, and determining the amount of the original antibody, which is produced by the specified first line of cells, with the specified initial antibody includes the amino acid sequence of the variable region of the heavy chain

in which X1and X2pairs are selected from the group consisting of X1= I and X2= V; X1= L and X2= I; X1= V and X2= V; X1= L and X2= L; or X1= L and X2= V,

while this method also includes the

creating at least one additional cell line, which produces a variant of the specified source of the antibodies, and the specified variant antibody is a modified original antibody containing the insertion, deletion, inversion and/or replacement of approximately 1-5 amino acids compared with the specified source antibody, and this modification does not consist of the amino acid modifications at position(s)defined by X1and X2and the determination of the number of variant antibodies, which produces the specified at least one additional cell lines, with this method, in addition, includes the selection of a variant antibody, which was produced in amounts that were at least 50% of the original antic the La. Preferably, the specified initial antibody contains amino acid sequence of the light chain

These insertion, deletion, inversion and/or replacement of approximately 1-5 amino acids can be in any part of the antibody that is not or does not include the amino acids at positions X1and X2. Preferably, the above insertion, deletion, inversion and/or replacement of approximately 1-5 amino acids are specified amino acid sequence of the heavy chain or the specified amino acid sequence of the light chain compared with the amino acid sequence of the corresponding chain in the specified source antibody. Preferably, the above insertion, deletion, inversion and/or replacement of approximately 1-5 amino acids are specified amino acid sequence of the heavy chain compared with the specified sequence of the heavy chain of the specified source of antibodies. Preferably, the method also includes the establishment of a line of cells producing antibodies, which produces the specified selected antibody. This cell line producing antibodies can be specified additional cell line, or even a different cell line, which produces the specified selected antibody. Predpochtitel is about, this method also involves collecting the specified selected antibodies. In addition, the present invention relates to isolated and/or recombinant antibody-antagonist against human CD40, obtained by the method according to the present invention. In a preferred embodiment, the specified antibody antagonist against human CD40 contains a modified amino acid sequence of the heavy chain

In which X1and X2pairs are selected from the group consisting of X1= I and X2= V; X1= L and X2= I; X1= V and X2= V; X1= L and X2= L; or X1= L and X2= V,

while this modification includes insertion, deletion, inversion and/or replacement of approximately 1-5 amino acids compared with the specified amino acid sequence of the heavy chain, and this modification does not consist of the amino acid modifications at position(s)defined by X1and X2.

In one of the embodiments the present invention relates to pharmaceutical compositions containing the polypeptide of the present invention, the binder of the present invention, the antibody of the present invention, the nucleic acid of the present invention and/or a cell according to the present invention. Also present from retina refers to the polypeptide of the present invention, the binding substance of the present invention, the antibody of the present invention, the nucleic acid of the present invention and/or the cell of the present invention for use as a drug, preferably, medicines to reduce the intensity of symptoms of autoimmune disease and/or inflammatory diseases and/or reduce transplant rejection, and/or for the treatment of CD40-positive cancers. In a preferred embodiment, the autoimmune and/or inflammatory disease is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis, bullous of pemphigoid and atopic dermatitis.

Since the polypeptide of the present invention, particularly suitable for reducing the intensity of the symptom of inflammatory diseases due to its netymology antagonistic against CD40 properties, the polypeptide of the present invention is suitable for reducing the intensity of the symptom of several disorders. Used in the present invention, the term "inflammatory disease" is defined as any disease that includes an inflammatory component. In the interest of the present invention inflammatory disease in castnet is, includes autoimmune disease and/or graft rejection. A Central role in the interaction of CD40-CD40L to initiate, enhance and prolong the immune response plays a polypeptide of the present invention, in particular, it is suitable for immunomodulation of autoimmune disease.

See below for information on CD40 and CD40L to illustrate the role of CD40 and its ligand in inflammatory diseases. Molecule CD40 is a membrane glycoprotein type I with a molecular mass of 50 kDa and is expressed on b cells, monocytes/macrophages and dendritic cells (DC).45-50In addition, under pathological conditions CD40 can be found on endothelial cells (EC), fibroblasts, epithelial cells and keratinocytes.51The CD40 ligand (gp39, TBAM, TRAP, CD40L, CD154), integral membrane glycoprotein of type II with a molecular mass of 32 kDa, transtorno is expressed on activated T-cells CD4+and a small population of activated T-cells CD8+.52,53In addition, CD40L was detected in other cell types upon activation, including mast cells, basophils, b cells, eosinophils, DC, and platelets.54,55

Contacts CD40 and CD40L activates a series of biological events in b cells, including proliferation, expression of activation markers, production of immunoglobulins (Ig)switch isotypes, d is a logical adhesion and rescue from apoptosis. 56,57However, as described above, the distribution of molecules CD40 is not limited to b-cells, as originally claimed. Freshly isolated human monocytes Express low levels of molecules CD40, which can be improved through the cultivation in the presence of IFN-γ.47-49,58The CD40 binding of monocytes/macrophages induces the secretion of large amounts of proinflammatory mediators, such as IL-1, TNF-α and IL-12, which induce inflammatory reactions and possess destroying tumor cells activity47-49,58and save them from apoptosis.48The binding of CD40 also causes increased differentiation and activation of DC, the increased expression of co-stimulating molecules, such as CD86, CD80 and CD58, increased cytokine production and inhibition of apoptosis.50,59In addition, when the expression under conditions of inflammation signal transmission from CD40 can induce the expression of molecules 1 intercellular adhesion (ICAM-1), molecules 1 adhesion to cells of the vascular (VCAM-1) and E-selectin on EC.55These results suggest that signaling through CD40 during interactions with T-cell-EU can be an important step in the regulation of the activation of the EU and attract leukocytes in non-lymphoid tissues. In vivo studies have shown the importance of the interactions of CD40-CD40L in the development of humoral immune responses,60,61in premirovanii and activation of antig the n-specific T-cells 62temporary activation of macrophages,63and in a protective cell-mediated immune responses mediated through the T cell activation of macrophages against intracellular infection parasitic organisms, such as Pneumocystis, Cryptosporidium, Leishmania.64-66

The specific role of CD40 and its ligand shown in murine models of autoimmune disease. Studies in animal models have clearly demonstrated the involvement of the interaction between CD40L-CD40 in the pathophysiology of various autoimmune diseases. In these studies, using mice suffering from natural or experimental autoimmune diseases, preventing interactions CD40L-CD40 had a clearly beneficial effects. It is shown that CD40L MAB against mouse prevents or reduces the symptoms of the disease when induced by collagen arthritis, experimental allergic encephalomyelitis (EAE, a model of MS on the animal), mice (SWR x NZB)F1 ordinary erythematosus and in mice with diabetes in non-obese (NOD), which spontaneously developed-dependent T-cell autoimmune diabetes. The data indicate that the interaction of CD40-CD40L also play a role in the pathogenesis of inflammatory bowel disease, which includes Crohn's disease and ulcerative colitis. It is shown that transgenic against CD40L mouse with a large number of copies of the transgene, which become lethal inflammatory bowel disease, marked infiltration of cells CD40+and T-cell CD40L+in the diseased tissue.67Mat against CD40L effectively prevent inflammation of the mucous membranes and the production of IFN-γ by T-cells CD4+own layer in animals with colitis, which was induced by 2,4,6-trinitrobenzenesulfonic acid.44Recently conducted a direct comparison between treatment with anti-TNF-α and hinder the path of CD40-CD40L using MAB against CD40L, on experimental models of inflammatory bowel disease in mice with SCID. In this model of syngeneic cells CD45RBhighCD4+injected mice with SCID, which subsequently develops diarrhea or loose stools and have progressive weight loss, starting with 3-5 weeks after recovery of T cells, as symptoms of experimental inflammatory bowel disease. Treatment or anti-TNF-α, or anti-CD40L from the date of recovery of T cells completely prevented clinical and histological manifestation of experimental inflammatory bowel disease. In addition, the introduction of anti-CD40L 5 weeks after recovery of T cells could prevent progression of the disease, and the treated animals showed improvement of symptoms and histology compared with the control animals (unpublished observations).

Recently the work has been demonstrated, what is impeding the path of CD40-CD40L leads to severe immunosuppression in transplantation models. Combined treatment of allogeneic small lymphocytes or depleted on T-cells, small lymphocytes plus antibody against CD40L mouse made possible the survival of an allograft in a null islets of Langerhans in 37 of the 40 recipients, characterized by major and minor histocompatibility loci.68On the basis of these experiments it was concluded that the effective prevention of interaction of CD40L-CD40, most likely, led to the prevention of the induction of co-stimulating molecules on small resting lymphocytes alloreactive T-cells of the host. In another recent study demonstrated that the introduction of CD40L MAB against mouse during transplantation significantly prolonged survival of fully various murine cardiac allografts as desensibilisation and sensitized hosts. However, if anti-CD40L therapy was delayed until 5 days after surgery, anti-CD40L not prolonged graft survival. From this study it was concluded that anti-CD40L therapy inhibited rejection of allografts, mainly by preventing T-cell effector functions. In addition, it was shown the, that obstruction of the ways CD80/CD86-CD28 and CD40-CD40L at the same time effectively ceases T-cell clonal increase in vitro and in vivo, increases long-term survival of fully allogeneic skin grafts and inhibits the development of chronic vascular rejection source vascularized cardiac allografts. In addition, obstructing the path of CD40-CD40L, optionally in combination with obstructing the path of CD80/CD86-CD28, prevents rejection of renal allograft model of renal allograft on macaque RH.69,70Additional information regarding the effects 5D12 inflammatory conditions are given, for example, in references71-81.

Multiple sclerosis is an autoimmune disease of the Central (spinal) nerve system. This violation of the white substance that surrounds nerve fibers and hardens. The term "multiple sclerosis" (MS) literally means "many scars". The hardened areas of nervous tissue called plaques. Symptoms, the severity and course of multiple sclerosis are highly variable and depend in part on locations of the plaques and the degree of destruction of the white matter. The defeat of the white matter of the nervous system slows down nerve impulses, which leads to inconsistency of the nervous system.

The experimental the initial autoimmune encephalomyelitis (EAE) in the common marmoset (Callithriz jacchus) is a useful preclinical model of multiple sclerosis (review of which is given in references 86,87,102,104). Lesions of the white matter of the Central nervous system (CNS), which develop at different variants of this model, EAE, have in common with MS pathological, radiological and immunological characteristics.95,98,101Therefore, the model of EAE on grunke is a bridge in broad immunological gap between humans and rodents, which complicates the selection of promising drugs in the pipeline of their products in the preclinical stage.96,99,103

Marmosets immunized rhMOG, recombinant protein representing the extracellular fragment MOG person (amino acids 1-125), developing EAE in 100% of cases, due to the presence of monomorphic element sensitivity MHC class II Caja-DRB∗W1201 in the repertoire of each marmoset.82,84,85,107A particularly effective aspect of this model for the development of therapy is that lesions developing in the white matter of the brain, it is possible to visualize and experiment to characterize using clinically relevant imaging modalities magnetic resonance.92,105Longitudinal analysis of white matter lesions of the brain with the help display of magnetic resonance imaging (MRI) demonstrated a progressive increase in the volume and chronic inflammatory activity in most lesions. More tor the, using the characteristics of the pathology of the Central nervous system using MRI and previously described histological criteria101found that the majority of the lesions are in the early active stage.106

Model rhMOG-induced EAE was used to test whether antibodies aimed at co-stimulating molecules of antigen presenting cells (APCS) and T cells, a possible treatment for MS. The interaction of CD40 with its ligand CD154 plays an important role in various immunopathogenic processes that operate during EAE, including the activation of b cells, activation of antigen presenting cells (APC), the emergence of the answers in the form of antigen-specific T cells and the induction of effector functions of macrophages.90,93,97,7A study conducted in 1996, confirmed that mice treated with antibody against CD154, protected from EAE.88. However, a clinical trial on patients with MS using antibody against CD154 was stopped due to unexpected side effects, which were not observed in experiments on animals.17

Monoclonal antibody mouse (Mat) 5D12 (mu5D12) was raised against human CD40. Antibody 5D12 was a strong inhibitor mediated CD40-CD40L activation in several cell types and, unlike most other MAB against CD40, does not show CD40-stimulating asset is barb. 21,22,39As a murine antibody against human CD40 mu5D12 and chimeric version of the antibody ch5D12 showed strong suppressive effects on the development of lesions in the white matter of the CNS and neurological disorders in models of EAE on grunke and did not show mark of side effects.23,24The same study showed that intravenous administration of MAB against CD40 affected EAE ordinary igranka can pass to the damage of the white matter of the brain, where molecules CD40 clearly expressed on infiltrating macrophages and activated microglia,95what was discovered earlier in MS.88This raised the question on how does ch5D12 therapeutic effect on existing lesions.

In the article 't Hart and others described the development of brain lesions in 7 immunized rhMOG monkeys using sequential display of magnetic resonance imaging (MRI) with an interval in 2 weeks.76The results of this study demonstrated suppression of inflammation at the site of the lesion in all 3 treated ch5D12 monkeys, whereas the increase in lesion was reduced in 2 of 3 treated ch5D12 monkeys.

Preventing contact of CD40 with its ligand CD154 on activated T-cells early in the disease process has a significant in Janie on clinical and neuropathological expression of EAE in rodents. 88,89,91,94,100in models with non-humans primates.23,24CD40 is clearly expressed in lesions of the white matter in the CNS of patients with MS and EAE affected rodents88and non-humans primates.95What bearing CD40 APC within the Central nervous system, such as infiltrating macrophages and perivascular and parenchymal helicity, make a significant contribution to the pathogenesis of EAE, was elegantly demonstrated by chimeric within the bone marrow of mice.83

The results shown 't Hart and others, Laman, etc. and Boon and other76,79,81indicates that blocking CD40 antibody is a potentially effective treatment for MS. It is important that the Mat ch5D12 has no visible side effects in a model of EAE on igranka, no other species of primates.80Favorable clinical effect of antibodies against CD40 has been demonstrated in placebo-controlled experiments on two models of EAE on igranka, namely, induced by myelin human79or rhMOG81. In addition, in the article 't Hart and others indicated the inhibitory effect of treatment with antibody against CD40 on existing lesions.76

Psoriasis is an inflammatory skin disorder affecting 1-2% of the population. In this disease, T cells and keratinocytes in the lesions activated and Express markers of activation and costimulatory the e molecule. I believe that some co-stimulating molecules expressed on keratinocytes and T cells interact with each other, and that these interactions contribute to the disease activity.108-110One such set of molecules can be CD40, which is expressed on activated keratinocytes, and CD154 (CD40 ligand), which transtorno is expressed on activated T-cells CD4+. The binding of CD40-CD154 between T-cells and keratinocytes can lead to the release of these cells, inflammatory mediators, which are observed in abundance in places lesions of psoriasis. Recently presented an overview of CD40, CD154 and interactions of CD40-CD154.111

Cultured keratinocytes also Express CD40; expression increases during IFN treatment. The binding of CD40, expressed in a high degree on the treated IFN-gamma keratinocytes (referred to throughout this work as keratinocytes CD40++), CD154 leads to an increase in ICAM and increased production of cytokines.112-114

Still, there was only one message, which indicates that this binding is involved in the pathogenesis of psoriasis.113The types of cells that Express CD40 in the lesions of psoriasis were not identified in the latter study, and their frequency, and the defeat also not described. Also not investigated Pris DTIE T cell CD154+ in the defeat of psoriasis. Thus, it remains unknown whether CD154 as one of the signals for production by keratinocytes chemokines and complement, which in excess are found in places lesions of psoriasis.115

Recently Pasch et al. demonstrated the presence and localization of cells CD40+ and CD154+ by immunohistochemistry in the affected and unaffected skin taken from ten patients with psoriasis.116Increase positivity in respect of CD40 was observed in clusters of keratinocytes, and high expression of CD40 was shown on almost all cells of Langerhans CD1a+ in normal, unaffected and affected epidermis. In addition, high expression of CD40 was presented on almost all cells CD83a+ in the defeat of psoriasis; they have rarely been observed in unaffected and normal skin.116Also a small part of T-cells demonstrated the expression of CD154 in the majority of patients in connection with cells CD40+. These results increase the possibility that T cells CD154+ may be binding to keratinocytes CD40+, Langerhans cells and dendritic cells CD83+ and release of these mediators at the site of lesion. In addition, they demonstrated that binding of CD40 induces the release of chemokines (IL-8, RANTES and MCP-1).116In the same publication, Pasch et al. demonstrated that is associated with CD40, visualaid is the chemokines IL-8, MCP-1 and to a lesser extent RANTES inhibited by the antibody is an antagonist against CD40 5D12.116These data suggest that antagonistic MAB against CD40 5D12 may at least partially have an effect on inflammation observed in the defeat of psoriasis.

In the application US2003/0165499 reported moderate antipsoriatics effect 5D12 and other antibody antagonists against CD40 in the model system xenogenic transplant in mice with SCID, which is used as a model for the treatment of psoriasis, demonstrating that the antibody antagonists against CD40 can be used for the treatment of psoriasis. In this in vivo system was demonstrated therapeutic effect 5D12.

In a particularly preferred embodiment, the specified autoimmune and/or inflammatory diseases include inflammatory bowel disease. Another preferred variant implementation refers to the application of the present invention, in which the specified inflammatory bowel disease includes ulcerative colitis (UC) or Crohn's disease (CD).

As illustrated above, the polypeptide of the present invention is suitable for treatment of various inflammatory diseases, including autoimmune diseases and transplant rejection. Therefore, the present invention in one of the embodiments include the method of reducing the intensity of the symptom of the autoimmune disease and/or inflammatory diseases and/or reduce transplant rejection. In an additional embodiment, the present invention relates to the use of the polypeptide of the present invention, a connecting structure of the present invention, antibodies of the present invention, nucleic acids of the present invention and/or cells of the present invention to obtain medicines that reduce the intensity of the symptom of the autoimmune disease and/or inflammatory diseases and/or reduce graft rejection. As used in the present description, the reduction in intensity of the symptom is defined as the decrease of intensity of at least one symptom of the disease, at least partially. The present invention is of particular interest for the treatment of autoimmune and/or inflammatory diseases, for which there is currently no effective treatment. Examples of such diseases are rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis, inflammatory bowel disease, bullous pemphigoid and atopic dermatitis. The polypeptide of the present invention, optional part of the connecting structure or the cell according to the present invention and/or encoded by the nucleic acid of the present invention, is suitable, in particular, to reduce symptom auto monogo and/or inflammatory diseases, because certain properties of the polypeptide of the present invention help to prevent the path of CD40-CD40L in a specific way. In addition, since the connecting structure of the present invention, preferably, is neimmunizirovannah connecting structure of the present invention is present for a long period of time and, thus, exerts its antagonistic activity in a patient for a considerable period of time. Therefore, the present invention in one of the embodiments relates to the use of the polypeptide of the present invention, a connecting structure of the present invention, antibodies of the present invention, nucleic acids of the present invention and/or cells of the present invention to obtain medicines that reduce the intensity of the symptom of the autoimmune disease and/or inflammatory diseases and/or reduce graft rejection, and specified autoimmune and/or inflammatory disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis, inflammatory bowel disease, bullous of pemphigoid and allergic dermatitis.

Brief description of figures

Figure 1. Concentration ch5D12 in sivaram the e after a single injection on day 0 of the four dose levels, these characters. Values are given as µg ch5D12/ml of serum was determined by enzyme linked immunosorbent assay, the details of which are given in the link27.

Figure 2. Changes of activity indicators of Crohn's disease (CDAI) during the 28-day period after infusion ch5D12. The shaded area indicates the period in which the levels ch5D12 exceed a concentration of 10 μg/ml (see also figure 1), which, as found, is a functional antagonistic serum levels in primates.24-270.3 mg/kg group levels in the serum never exceeded 10 ág/ml

Figure 3. The histological indicators of disease activity at day 0 and day 28. The maximum rate of activity is 16, and the determination of the rate of activity conducted in accordance with reference40. At day 0 and day 28 were received samples of ileum (diamonds) of nine subjects and samples of the colon (squares) 11 subjects, and the results were presented for each dose level ch5D12 [(a) 0.3 mg/kg; (b) 1.0 mg/kg; (c) 3.0 mg/kg and (d) 10.0 mg/kg

Figure 4. Biopsy samples of the colon of the patient 012 (3.0 mg/kg) and biopsy of the ileum of a patient 011 (3.0 mg/kg) are presented as examples of the reduction of the inflammatory response. The samples were stained with antibodies recognizing all T-lymphocytes (CD3), b cells (CD19), macrophages (CD68) and cells CD40+, $ (a, C) and the day 28 after injection ch5D12. NO, hematoxylin-eosin.

Figure 5. A consensus DNA sequence and deduced amino acid sequence regions VH and VL 5D12 mouse.

6. Comparison of the amino acid murine V-regions with generowanie and daimonizomai V-regions 5D12.

7. Alignment 12 scanned options amino acids (Q5E, K13A, E16Q, T17S, I29L, I37V, P45L, M48L, STS60NSA, T68S, S79F and T108S) neimmunizirovannah VH 5D12 with the original murine sequence (ch5D12) and fully neimmunizirovannah sequence (DI5D12).

Fig. FACS analysis using JY cells. The supernatant of the cells, PER.C6, transtorno expressing variants (Q5E, K13A, E16Q, T17S, I29L, I37V, P45L, M48L, STS60NSA, T68S, S79F and T108S) 5D12, collected after 48 h In the quality control also collected supernatant of cells transfected or ch5D12, or DI5D12, together with the supernatant from false (without plasmid) transfected cells. The binding of the expressed antibodies was investigated using FACS using JY cells with anti-human labeled FITS the second antibody (diluted 1/100). As a control FACS JY cells were incubated only with the secondary antibody labeled FITS.

Fig.9. The additional alignment options V-L-I in positions 29 and 37 (29I-37V, 29V-37i indigenous, 29I-37L, 29V-37V, 29L-37L, 29V-37L, 29L-37V) neimmunizirovannah VH 5D12 with the original chimeric sequence (ch5D12), fully neimmunizirovannah sequence (DI5D12; 29I-37i indigenous) and G102 (29L-37i indigenous).

Figure 10. Distribution or antibody-based test productivity of cell lines GS-CHO, producing antibody PG102, and "over-age" cultures in 24-hole plates. The number of cell lines deferred on axis X, the ranges of concentrations of antibodies pending on the Y-axis (the ranges of concentrations of antibodies are allocated in ascending groups, ranging from 0-25 µg/ml).

11. Profiles of growth and accumulation of antibodies to cell lines: (A) L107 (DC1; top panel), B) L25 (DC2; middle panel) and C) M (DC3; top panel) fueled culture CDACF in shake flask.

Fig. Comparison of binding PG102 (red) and ch5D12 (blue) at 10 μg/ml CD40 immobilized on 3 different levels.

Fig. Inhibition antiidiotypic Mat 173-36-1 binding of MAB against CD40 with JY cells expressing human CD40. MAB against CD40 investigated at a concentration of 1 μg/ml Data show the arithmetic mean ± standard error value 4 separate definitions for each antibody.

Fig. ELISA CD40-Fc. Depending on the concentration of binding ch5D12 (PG100) and PG102 with CD40-Fc man. Tablets overnight covered 250 ng/ml CD40-Fc man. Mat isotype control, chFUN-1, directed against human CD86. Data show the arithmetic mean ± standard error (n=3).

Fig. Quantitative determination using a FACS binding of MAB against CD40 with JY cells. ch5D12 (PG100) and PG102 comparable concentrations for premaxillae binding. Mat against CD86, chFUN-1, also demonstrated the binding of JY cells in these experiments because of the expression on the surface of this cell line human CD86. Data show the arithmetic mean of double determinations for each antibody.

Fig. Inhibition of binding ch5D12 (PG100)-RE and PG102-RE with JY cells unlabeled antibodies ch5D12 (PG100) and PG102. Labeled ch5D12 (PG100) (a) and PG102 (C) were incubated with 1 μg/ml in the presence of increasing concentrations of the competing unlabeled antibodies. The binding of the labeled antibodies was determined as the average fluorescence intensity (MFI) using flow cytometry. Maximum MFI to link ch5D12 (PG100)-RE and PG102-RE amounted to 369 units of fluorescence (a) and 305 units of fluorescence (B), respectively. Data show the arithmetic mean ± standard error value of 4 separate experiments.

Fig. Inhibition of release of IL-8 cells TNR-1 after cocultivation with Jurkat cells. Cells TNR-1-stimulated IFN-γ, cocultured with Jurkat cells in the presence of increasing concentrations ch5D12 (PG100) or PG102. The release of IL-8 from cells TNR-1-induced binding of Jurkat cells with cells TNR-1 through CD40-CD40L, were determined using ELISA. Data show the result of one experiment.

Fig. The sequence of the nucleic acid and amino acid placentas is of heavy and light chain IgG 4PG102.

EXAMPLES

Example 1

Materials and methods

For inclusion in the study were selected eighteen adult subjects (ages 18-60 years) with clinical diagnosis of Crohn's disease confirmed by radiologic, endoscopic, or histological data, and with the index of activity of Crohn's disease (CDAI), comprising at least 220, but not more than 450 (celebrated for 7 days prior to the introduction of the investigational medicinal product). Subjects were allowed to undergo the following treatments before and during the study: treatment mesalamine for 8 or more weeks in the dose remains constant during the 4 weeks before screening; a maximum of 30 mg of corticosteroid per day (or 9 mg of budesonide per day) for 8 weeks or more in the dose remains constant during the 2 weeks before screening; mercaptopurine or azathioprine for 4 or more months in the dose remains constant throughout the 8 weeks before screening. Subjects could not receive treatment with cyclosporine or methotrexate within 3 months before screening, and were not allowed to they are pre-treated Mat. The average age of all registered entities amounted to 35.8 years, including seven of the subjects were men and 11 were women. All subjects were of Caucasian ethnicity. Have been arisen visible differences between patients in different groups at inclusion (table 1) except three groups of low doses of most of the subjects were women, while the group of the highest dose (10.0 mg/kg) consisted only of which male subjects. No outstanding differences between the four groups with respect to the electrocardiogram (ECG), signs of life, physical examination, history, signs and symptoms of Crohn's disease and baseline laboratory values baseline. No significant differences in the parameters of the basic line among the four groups doses. However, the largest dose also showed the highest index CDAI at baseline. This study was authorized by the Committee on medical ethics, University Hospitals Leuven, Belgium, Leiden University Medical Center, the Netherlands, Medizinisch clinic, Kiel, Germany, and Hadassah Medical Center, Jerusalem, Israel. All patients gave informed consent to the study.

Study design and treatment Protocol

ch5D12 is a molecular engineered antibody IgG4man, containing the variable regions of the heavy and light chains of the initial version of a Mat 5D12. It is shown that this Mat is associated with bearing CD40 cells and prevents mediated CD40 activation of different cells.20-27ch5D12 was administered in an open, conducted in many centres the test once the input dose, which examined four levels of doses. The distance between the th treatment group, there were five subjects except for the final dose, which were registered only three of the subject. A single injection at dose levels that comprise of 0.3, and 1.0, 3.0 and 10.0 mg/kg ch5D12, carried out intravenously. After completion of a set of one-dose group, set the following group started only after receipt of proof of safety at the current dose level. Clinical disease activity was assessed at every visit weekly during the first 28 days later last visit to day 56. Two subjects (group a dose of 3.0 mg/kg) were excluded from the study after assessments in 28 day, but their data are also included in the analysis. The subjects were required to remain on the same dose of their current medications throughout the study. While research has not been used for unauthorized concomitant drug. Response to treatment ch5D12 was defined as CDAI reduction, amounting to ≥100 units, and clinical remission was defined as CDAI ≤ 150 (total score) and the decrease in CDAI, amounting to at least 100. All subjects who underwent endoscopy for screening and at day 28 (n=11), were analyzed to reduce endoscopic index of severity of Crohn's disease (CDEIS). For histopathology and immunohistochemistry of biopsies taken from these 11 patients before and at day 28 after injection ch5D12. Safety assessments included physical surveyed the I, signs of life, ECG and laboratory data (biochemistry, Hematology and urinalysis), including evaluation of anti-dsDNA, pANCA and human antibodies against chimeric antibodies (NASA). NASA were determined using enzyme linked immunosorbent assay (ELISA), as explained in reference (27).

Pharmacokinetics

Concentration ch5D12 in serum were determined using (ELISA), as previously described.27To determine coverage CD40 injected ch5D12 blood was collected in heparinized tubes and diluted two times in PBS. Mononuclear cells from peripheral blood (PBMC) were isolated by centrifugation in a gradient (Lymphoprep, Nycomed, Roskilde, Denmark), and 500,000 cells were stained with FITC-labeled ch5D12 and PerCP-labeled antibody against CD20 (Becton-Dickinson, Mountain View, CA, USA) and incubated on ice for 30 minutes as a background control for binding 5D12-FITC separate tube PBMC were stained only PerCP-labeled antibody against CD20. Not bound peroxidase cells were washed, and cells were analyzed using flow cytometry (FACSort; Becton-Dickinson). The event collection was carried out by setting discriminatory open and get expressing CD20 cells for a total of 5000 events. If the number expressing CD20 cells was low for a set of samples, took care of getting the same number of events as in the unpainted and painted products.

Histology and immunohistochemistry

Bi is PSII mucosa of the ileum and colon, received during ileocolonoscopy before treatment on day 0 and day 28 using standard forceps were fixed in 6% formalin for routine analysis. Additional samples instantly froze in connection with optimal cutting temperature Tissue-Tek (Miles Laboratories Inc., Naperville, IL, USA) cooled in liquid nitrogen the isopentane. The samples were stored at -80°C until further use. Fixed in formalin samples were usually processed by paraffin. Slices with a thickness of five microns were prepared and stained with hematoxylin and eosin. Sections were analyzed using a microscope Leitz Wetzlar (Wetzler, Germany). Generally analyzed four semi-sequential controlling cutoff for each sample. Frozen samples were used for immunohistochemical analysis, which was performed using a set Mat to assess the presence of different subpopulations of lymphocytes and monocytes/macrophages. The set is completed Mat directed against CD40 and CD40L. Immunohistochemical staining was performed on cryostat sections were dried overnight at room temperature and fixed in absolute acetone for 10 minutes Rehydration preparations were incubated for 30 min with the following Mat: CD3 (clone UCHT1, 1/10 dilution) (Dako, Glostrup, Denmark), CD4 (clone MT, 1/10 dilution) (Dako), CD8 (clone 144B, 1/20 dilution) (Dako), CD19 (clone HD37, 1/30 dilution) (Dako), CD40 (clone 5D12, asvadana 1/100) (PanGenetics, Amsterdam, the Netherlands), CD68 (clone KR1, 1/50 dilution) (Dako), CD154 (clone M90, dilution 1/10) (Serotec, Oxford, United Kingdom). The second mate was labeled with Biotin antibody against mouse IgG (1/400 dilution; Dako)used within 30 minutes For effective blocking of endogenous peroxidase, the sections were also incubated in a solution of methanol containing 0.3% (vol./about.) H2About2within 20 minutes After three washes with PBS was added labeled with peroxidase complex avidin/Biotin (Dako). Between incubations, the sections were washed in phosphate buffered saline at pH 7 for 15 minutes the reaction Product was visualized by incubation of the slice for 10 min in 0.05 M acetate buffer at pH of 4.9, containing 0.05% 3-amino-9-ethylcarbazole (Janssen, Beerse, Belgium) and 0.01% H2About2that led to the emergence of bright red immunoreactive site. Subsequently, the sections were subjected to weak contrast staining with Harris hematoxylin, rinsed with distilled water and covered using glycerol cover glass. The controls, which were negative, consisted of the absence of the first or second antibody, the use of separately Chromogen and use irrelevant matched isotype mouse IgG (vimentin; Dako).

All processed paraffin-embedded sections stained with hematoxylin and eosin, analisia is Alice the same pathologist (K.G.), not knowing about the origin of the samples. The disease activity was evaluated using histological figures40from which maximum activity corresponded to 16. Also analyzed blindly immunohistochemically painted environment. For 15 sets of biopsies cells with positive staining were counted in fields high magnification (magnification X40). Fields were chosen in accordance with the highest density of stromal cells. The number was expressed as percentage of the total number of mononuclear cells in the stroma. These estimates are used to create a system of quantitative assessment, whereby the intensity of the cells with positive staining were subdivided into four categories: -, +/-, + and ++. Normal values for CD3+, CD4+, CD8+, CD19+ and CD68+ for ileum and colon is a +. Normal values for cells CD40+ and CD154+ is negative (-).

Immunological security settings

To assess any nonspecific immunosuppression caused ch5D12, evaluated the proliferation of T-cells in peripheral blood in response to mitogen of phytohemagglutinin (RNA). Stimulation of RNA (1 μg/ml) was carried out in selected PBMC as previously reported.41In addition, to avoid depletion of circulating cells was determined by the percent of circulating cells CD3+, CD4+, CD8+ and CD20+. For proton the th cytometry were collected at specified time points in tubes with heparin blood, from which 100 μl of whole blood in each test tube were stained as follows (all Mat were purchased from Becton-Dickinson): anti-CD3-FITC, anti-CD19-PE, anti-CD45-PerCP, anti-CD45-FITC, anti-CD4-PE, anti-CD8-PE and anti-CD14-PE. Stained cells were then analyzed in a flow cytometer FACScan (Becton-Dickinson). This Protocol was performed to calculate the relationship of CD4+ to CD8+ and determine the percentage of b-cells in a population of lymphocytes. Reported for CD3 and CD19 interest were the percentage of T-cells and b-cells in a population of lymphocytes, while on CD4 and CD8 were reported as percentages of cells CD3. Only CD14 (population of monocytes) are presented as percentage of the total population of CD45.

Results

pharmacokinetics

The average maximum levels ch5D12 after a single intravenous injection were dose-dependent and proportional to the dose (figure 1). After 24 h, approximately 50% of the introduced ch5D12 can be detected in serum in the groups at highest three doses. 0.3 mg/kg group, only 15% were present in the serum after 24 hours Of research on non-humans primates was calculated indicator t1/2β 8-10 days27which is confirmed by current data on the people. Full coverage CD40 on b cells in peripheral blood was not achieved by infusion of 0.3 mg/kg, as determined using ex vivo competitive analysis using FITC-labeled ch5D12 (not shown). In the 1.0 mg/kg group is the discovery of CD40 on circulating b cells could be observed for approximately 1 week, at that time in the two highest dose groups this period was increased to 2-3 weeks. This indicates that the group of lowest dose of antagonism against CD40 was incomplete, while full of antagonism was achieved at 1.0 mg/kg group during the 1 week or even longer in the groups of higher doses.

Clinical responses

The analysis of the evolution CDAI shows that 13 of the 18 subjects (72%) was observed favorable response after infusions ch5D12. Similarly, four of the 18 subjects (22%) was observed remission during this period. At day 21 favorable response was observed in 10 out of 18 subjects. Groups 2 and 4 showed the greatest average decrease in CDAI, and clear relationship dose-effect was observed (figure 2; table 2). After this analysis of variance of repeated measurements showed a statistically significant decrease in CDAI during the 56-day observation period (P<0,001). The difference between groups was not statistically significant. Only in groups 3 and 4 could be assessed CDEIS (as endoscopy was performed in only three subjects in group 1 and 2). In group 3, two of the five subjects had a decrease in CDEIS, while in group 4 two of the three subjects had a decrease. The rest of the subjects in these two groups showed no changes (not shown).

Histological changes

For patients who had taken bio is these (n=11), one researcher conducted the evaluation of changes in histopathology and immunohistochemistry. Evaluation on the biopsy material showed a clear effect of treatment on microscopic disease activity, as well as on the intensity of the infiltration of mononuclear cells in its own layer.

Change from baseline to measure histopathological activity at day 28 are shown in figure 3. There were samples of ileum nine patients and samples of the colon 11 patients at day 0. In four cases, the terminal ileum was not included in the screening, despite the fact that five patients had active disease of the ileum. The average histological index at day 0 for patients with active disease was 4.6 (range 3-7). The average histological score for the five patients declined to 1.0 (range 0-3) at day 28. Samples of the colon from four of the 11 patients not included in day 0, and samples of the colon from these patients remained normal at day 28. In seven patients the colon was clearly affected, while the average histological index at day 0 was 4.5 (range 2-12). He fell at day 28 to an average average of 1.7 (range 0-7). Five of the seven patients rate at day 28 was equal to or 0, which is an indicator of a normal biopsy, or 1 : the indicator only architectural abnormality. In the group of lowest dose (panel a) indicator for colon one subject dropped, but figures for other colon in this group remained high, and the indicator for the ileum has even increased. In groups of higher doses (panels b-d) the rate of activity for or ileum, or colon, or for both of these guts fell at day 28 after treatment ch5D12. In conclusion, a positive answer [as defined in reference (41)] was observed in 81% (9/11) of the subjects. In addition, seven of the subjects with active disease at day 0 demonstrated reduced activity of neutrophils essentially to the fact that neutrophils are not present in their biopsies at day 28.

To determine the cells that are targeted by the treatment ch5D12, each Protocol was performed immunohistochemical evaluation using existing biopsies, using markers of T cells (CD3, CD4 and CD8) and b-cells (CD19) and a marker of macrophages (CD68). The results are listed in table 3. In the group of lowest dose reduction was not observed at day 28, whereas all other groups showed decreases in infiltration. In samples of ileum at baseline increased the number of T-cells CD3+ in six of the nine cases. In the colon increased the number of T-cells CD3+ was present in four out of 11 patients, all of whom were also active for Alemania according to the results of conventional histology. At day 28 the number of T-cells CD3+ were normal in all except one patient. This patient (patient 001; group the lowest dose of 0.3 mg/kg) was observed growing cells CD3+ in the ileum and colon in parallel with chronic inflammatory proliferative activity and inflammation according to the results of conventional histology. CD4+ and CD8+ subordinated similar to CD3+ movie.

Picture for b-cells CD19+ was comparable to the pattern observed for T cells. At day 0 was increased In cells from six of the nine patients, which had a biopsy of the ileum, and in four of the 11 samples of the colon. At day 28, the number of b cells was normalized in all cases, except one patient in the low dose group.

A similar decrease at day 28 was observed for cells as monocytes/macrophages CD68+. At day 0 was marked by an increased number of cells as monocytes/macrophages CD68+ in five of the nine patients, which had a biopsy of the ileum, and in four of the 11 samples of the colon. Again, all of the increase is normalized at day 28, except for one patient in the low dose group, in a sample of the colon which is the number of cells as monocytes/macrophages CD68+ remained high, and in biopsies of the ileum was observed even increase that decrees the AET, the patient still had active disease at day 28.

Figure 4 presents typical examples of the reduction of all three major cell types (T cells, b cells and cells as monocytes/macrophages) in the biopsies of the ileum of a patient 011 (activity index: 7) and biopsies of the colon of the patient 012 (activity index: 12) prior to the introduction of ch5D12 and day 28 after injection ch5D12.

Security

Subjects were not excluded from the study because of adverse effects (AE), although the severity of most of these AE was mild to moderate. By evaluating AE occurring in ≥5% of the total number of AE, found that the most frequent AE were pyrexia, arthralgia, myalgia, and headache. The frequency of arthralgia, often taking place both at baseline and during the study period, decreased during the observation period. Flu-like symptoms and itchy rash that occurs in four subjects, of which only one subject was observed moderate rash that began soon after the introduction of the investigational medicinal product (3.0 mg/kg). In General there is no explicit response to the dose to any individual AE, with the possible exception of headache/migraine.

Six of the subjects were noted six serious adverse effects (SAE); 0.3 mg/kg group, such effects did not occur, three such effect was observed at 1.0 mg/chgroup, two such effect was observed at 3.0 mg/kg group, and one such effect was observed at 10.0 mg/kg group. Five of the six SAE was considered possibly related to the investigational medicinal product, one SAE not considered. All except one such effect was related to the gastro-intestinal tract (two to the ileum two 1.0 mg/kg-subjects; two to an exacerbation of Crohn's disease at 1.0 mg/kg of the subject and 3.0 mg/kg of the subject; and one - to strengthen abdominal pain at 3.0 mg/kg of the subject). However, two of these effects occurred at such time that a causal relationship with the investigational medicinal product is very incredible (one of the subject on the day of infusion, and another subject after exposure to endoscopy after 29 days after administration of the investigational medicinal product). None of the observed variance does not seem related to the investigational drug or dose.

The immunological parameters security

Because ch5D12 inhibits the activation of b-cells in vitro, we determined the levels of immunoglobulins in General, and, as discovered, they were constant during the observation period. To exclude the possibility that ch5D12 can Deplete b cells from the circulation, the authors of the present invention conducted an analysis of a subpopulation of PBMS in the course of the study. Introduction ch5D12 had no effect on the interest and AB is alumnae number of CD3-, CD4, CD8 and CD14-positive cells. The absolute number of circulating b-cells CD19+ transtorno decreased soon after the introduction of ch5D12 and restored to their original values after ch5D12 were withdrawn from circulation (not shown).

To investigate whether caused if the treatment ch5D12 General immunosuppressive effect, PBMS, selected at different points in time before and after treatment, stimulated polyclonal T cell activator of RNA. Unfortunately, cells from group a dose of 10.0 mg/kg, including cells obtained before the introduction of ch5D12, were not frozen properly and therefore were not able to answer RNA. The stimulation index induced by incubation with RNA, was highly variable, but the tendency to decrease after treatment ch5D12 were not found (data not shown).

Throughout the study, antibodies against ch5D12 were not found in two groups of low doses. At 3.0 mg/kg group found that one entity was established response against ch5D12 response, although the signal was very low, and it could only be detected in undiluted sample of this subject at day 28. In the largest dose other entity had antibodies against ch5D12 at day 28 and day 56 of the study. They can be detected at a dilution of 1:64. Therefore, only two subjects had a positive immune response n the introduction ch5D12.

Discussion

In the open, conducted in multiple centers test four levels of doses ch5D12 were administered to subjects with Crohn's disease from mild to severe. Was registered eighteen subjects in each treatment group had five of the subjects, except for the final dose, which were recorded only three of the subject. Levels ch5D12 in serum was increased depending on the doses and prolonged half-life existence in serum observed in cynomolgus macaques,27has been confirmed in humans. This study shows the ability to inhibit CD40-mediated activation of cells using ch5D12 in the absence of major side effects. Analysis CDAI shows that 72% of the subjects showed a clinical response, and 22% were in remission during the study. These results are promising; however, the open design of the study and the lack of a control group makes it impossible to definitely attribute the observed changes to the introduction of the investigational medicinal product. However, the authors of the present invention actually has some evidence in favor of the anti-inflammatory effect ch5D12. First, the effectiveness of treatment confirms the observed decrease in CDEIS among the subjects in the two highest dose groups. Secondly, the results from the ATA histological and immunohistochemical analyses performed on biopsies show that ch5D12 reduces inflammatory activity of the colon or ileum. In the group of low-dose obvious effect was not observed. Subjects in the 10.0 mg/kg group had the lowest rates of histological activity at day 0, and therefore the decrease in activity is limited in this group. Most indicators at baseline were found in the 1.0 mg/kg and 3.0 mg/kg group, and these patients was apparent reduction in histological indices at day 28.

Although two patients were identified antibodies against ch5D12 in the present study, the levels of antibodies NASA remained very low and were found only at very low dilutions of serum. It remains to establish whether responses against ch5D12 be detected in subsequent studies.

This test represents the first introduction people MAB against CD40, in General, and ch5D12, in particular. However, evidence of biological activity Mat 5D12 was previously obtained in vivo in experimental autoimmune encephalomyelitis (EAE) in igranka. Introduction 5D12, starting from 2-4 weeks from induction of the disease, had a clearly beneficial effects.23This in vivo proof of concept stimulated with recombinant humanization and further development 5D12. Biological activity ch5D12 also defined on m is Delhi EAE on igranka. At day 50 after MOG-immunization, the frequency of the disease in the placebo group was 100% (50% of the animals were killed because of the severity of EAE), whereas in the group treated ch5D12 signs of disease were observed.24Moreover, ch5D12 showed functional activity in a model of renal transplantation on macaque RH25and when used as immunosuppressive strategies to prevent immune responses against adenoviral particles and their products.26The study of tissue cross-reactivity in the tissue of humans and cynomolgus macaques showed that ch5D12 associated with the cell surface of b-cells and DC in lymphoid organs. Unexpected cross-reactivity was not observed neither in human tissue, or the tissue of cynomolgus macaques. Assessment of safety and tolerability for ch5D12 conducted in cynomolgus monkeys, for which, as has been shown, weekly introduction ch5D12 for 4 weeks is safe and without any side effects for all macaques. In this study, was obtained functional evidence that ch5D12 can prevent the activation and proliferation of b-cells.27Taken together these studies show that antagonistic MAB against CD40 ch5D12 not have unexpected cross-reactivity, safe limits immunoreactivity in Evensk the x macaques and inhibits prototype chronic inflammatory disease of the Central nervous system marmosets. Therefore, preclinical evaluation supported the further development for use for patients.

Using animal models was evaluated by co-stimulatory path CD40-CD154 (CD40L) as a promising clinical target for the treatment of autoimmune diseases and rejection of the graft.15Potentially the interaction of CD40-CD154 can inhibit with targeting or CD40 or CD154. All previous studies have focused on the implementation of antagonism against CD154, which is selectively expressed on activated T-cells and platelets.8-11Clinical studies in humans using MAB against human CD154 were stopped due to thromboembolic events.12-14Because this Mat against CD154 developed in the form of gumanitarnogo IgG1 that binds extremely well with Fc-receptors, Mat against CD154 can cross-link CD154 with Fc-receptors, leading to the formation of blood clots. Recently it was reported that constitutive CD40 is expressed on platelets and, as found, is functionally important when using soluble CD154 as a stimulus for platelet activation. These stimulating effects, leading to activation of platelets were cancelled when adding the Mat against CD154 or CD40.42The authors present what about the inventions confirmed nelinumeroisia properties ch5D12 also in experiments with human platelets, because adding ch5D12 to pre-stimulated with the use of suboptimal concentrations of ADP or collagen platelets was not accompanied by any effect on the activation state of platelets (M.F.Hoylaerts, unpublished observations, Leuven, Belgium). In addition, the Fc-part of IgG4fundamentals Mat ch5D12 has reduced ability to bind to Fc receptors expressed on platelets, compared with the base IgG1. A further difference between the two approaches comes from the fact that ch5D12 focused on b cells, monocytes, macrophages and DC, while the Mat against CD154 focused mainly on activated T-cells, which leads to markedly different clinical strategies. Signs of thromboembolism was not observed in any of the patients treated in this study.

The effect ch5D12 largely based on preventing mediated CD40 cell activation. Stimulation of CD40 induces the secretion of cytokines and chemokines by macrophages and DC and increases antigen presenting ability of the latter. ch5D12 DC shows antagonistic activity against receptor CD40, whereby induced production of TNF-α,39he could also inhibit the secretion of TNF-α, which, without doubt, an important cytokine in Crohn's disease.43However, upon stimulation with CD40 also the Indus is ciruit a number of other inflammatory cytokines, such as IL-8 and IL-12,39and, therefore, ch5D12 could have anti-inflammatory properties in addition to preventing the production of TNF-α. Figuring out depend on whether the observed effects are primarily of a decrease in the activation of T cells, or reduce the release of cytokines and chemokines, awaits further research.

This is the first study in humans using ch5D12 proves promising clinical benefit in the absence of serious side effects. Will require additional clinical studies to establish the role ch5D12 in maintaining remission and in monitoring the security of multiple infusions and of optimal doses of ch5D12. It is obvious that therapeutic benefit of antagonism against CD40 will not be limited to Crohn's disease, and it can potentially be extended to many other inflammatory diseases.

Example 2

Materials and methods

The creation of chimeric variant 5D12

Starting from the hybridoma cell line 5D12, cloned murine variable regions 5D12. In short, from the lysed cells using Qiagen was isolated total RNA. Using RT-PCR by using the primers 5' CAG GTS MAR CTS SAG SAG TC W GG 3' and 5'GCA TGT ACT AGT AAT TTT TVT TGT CCA CYT TGG TGC T 3' amplified region VH and using primers 5' CGA TAC GAS MTY CAG CTG ACC CAG TCT CCA-3' and 5' GAC TCA TCT AGA TAC ACT CAT TCC TGT TGA AGC TCT TG-3' amplif is provoked region VL mu5D12. After sequence analysis identified a consensus sequence for the V-regions. Subsequently, the V-region was reklamirovali in pcDNA3002 (Marissen et al., J. of Virol., 2005: 79: 4672-4678) with genomic sequences, encoding the IgG4and Kappa person to construct expressing the chimeric immunoglobulin IgG4plasmids.

Creating neimmunizirovannah option 5D12

In cooperation with Biovation, using their patented technology reimmunization, within V-regions were identified potential T-cell epitopes. Epitopes of T cells-helper cells include a short amino acid sequences in proteins with the ability to bind to MHC molecules of class II. After removal of T-cell epitopes antibody can no longer initiate the help of T-cells and subsequent immunogenicity against it. Identification was performed using the threading peptides. The approach of threading peptides is a computer-based method for the prediction of binding peptides (short amino acid sequences) with 18 different molecules MHC class II person, based on known structures resolved by x-ray crystallography. Moreover, carried out the analysis to search for the V-scope presence known is the shaft connecting the T-cells of human peptides in the database http://www.wehil.wehi.edu.au).

Based on the original murine amino acid sequences of the V-regions created surface humanized (generowanie) V-region. Identified 8 potential T-cell epitopes within this generovanou sequence VH, and 4 T-cell epitopes were identified within this generovanou region VL. Identified T-cell epitopes removed by using amino acid replacement. Based on this analysis, the DNA sequence encoding demonizirovannyj region VH and VL, synthesized and reklamirovali in expressing plasmid pcDNA3002-hIgG4-Kappa.

Site-directed mutagenesis

To restore mouse sequences within the VH region subjected reimmunization expressing plasmids pcDNA3002 used in accordance with the Protocol of the manufacturer set for Stratagene site-directed mutagenesis Quick-Change. Table 4 presents the used oligonucleotides. Briefly, the plasmid was denaturiruet followed by annealing sense and antisense primers containing the desired mutation. Using Cycling (30 sec 95°C, 30 sec 55°C; 600 sec 68°C) 16 times using the device for PCR Biometra T gradient included mutagenic sequence. Nemotional original plasmids were digested DpnI, which only cleaves methylated DNA. Bafter the following PCR mixture, containing the mutated plasmid was transformed into competent cells XL1-Blue. Colonies were selected and analyzed for DNA sequences to the correct plasmid. The second set of experiments focused on the creation of additional variants of the provisions of 29 and 37, in particular, by replacing I by structurally related V or L. Similar to the manner described above was created plasmid using oligonucleotides presented in table 6. To create double mutants took two rounds of mutagenesis (table 7). The plasmids used for transformation of competent cells XL1-Blue.

Expression in PER.C6TM

The expression of different variants 5D12 carried out transtorno in PER.C6TM(Crucell). Briefly, the day before transfection of PER.C6TMsubjected trypsinization and counted. The cells were placed in DMEM, replaced with 10% FCS at a density of 4×105cells per well in hole tablet T24. The next day the medium was replaced with 0.5 ml fresh medium. For each well, 1 μg of plasmid DNA was dissolved in 50 ál of OPTI-MEM I and combined with an equal volume of OPTI-MEM I containing 3 ál LipoFectAMINE 2000 (LF2000) (Invitrogen). The mixture was incubated for 20 min at room temperature to allow the formation of complexes between DNA-LF2000 reagent. Subsequently, the complexes of DNA-LF2000 reagent (100 ál) was directly added to each LUN is in. Transfetsirovannyh cells were incubated at 37οWith the incubator with CO2. After 48 h supernatant analyzed for the expression of antibodies.

ELISA

The number of expressed antibodies were determined using a sandwich ELISA. Briefly, 96-well tablets EIA/RIA (Corning 3590) were coated overnight at 4°C With polyclonal antibody against human IgG (Jackson 109-005-088)diluted 1/100 PBS (100 µl/well). After blocking PBS containing 0.05% Tween and 1% BSA, tablets, three times washed with PBS-0.05% of Tween. Subsequently, as the standard used purified chimeric 5D12 during the titration starting at 400 ng/ml to zero in three repetitions. Samples (transient supernatant) also used during the titration. All cultivation was carried out in PBS-0.05% of Tween. The plates were incubated for 1 hour at 37°C. After three washes in each well was brought diluted 1/5000 antibody for detection (labeled with alkaline phosphatase against the human Kappa (Southern Biotech Associated 2060-04)). The plates were incubated for 1 hour at 37°C. After removal of the incubation mixture tablets were washed 5 times. Staining was performed using substrate PNP (Sigma N-2765) (1 mg PNP/ml substrate buffer : 100 mm Tris/HCl, 100 mm NaCl, 5 mm MgCl2·6H2Oh, pH of 9.5). OP nm was measured on a BioRad 550 Titertek using 655 nm as the reference point.

FACS

Using FACS analysis were analyzed by anti the war specificity of the expressed antibody. The JY cells are EBV transformed b-cells that Express CD-40. Briefly, 100,000 cells JY incubated with 100 μl of the supernatant from the experiment of transient expression at 4°C for 20 minutes. To remove unbound antibody, the cells were washed in PBS containing 0.05% Na-azide and 1% BSA. Used second antibody, diluted 1/100, labeled with FITC, anti human IgG (Jackson 109-095-127). After incubation for 20 minutes at 4°C. cells were washed. Finally, the samples were measured on a FACScan (BD).

Results

After analysis of several clones identified a consensus sequence for the murine V-regions 5D12 (figure 5).

Using human surface modeling V-region were reconstructed, while maintaining critical murine amino acids. Using the method of threading peptides and database searches identified potential T-cell epitopes within these reconstructed sequences. Identified 8 potential epitopes (the first basis of the potential epitope: position 27, 30, 43, 46, 57, 61, 77 and 83) within the scope of the VH and 4 potential epitope (position 7, 13, 28 and 86) within the region VL. Based on the mapping T-cell epitopes and process generowania determined optimal demonizirovannyj amino acid VH and VL (6). To remove T-cell epitope at position 61 in the VH region, special attention was paid not to the introduction of a potential site of N-glycosylation (N-X-T/S): NSA replaced STS. At the end of this design were changed 12 positions in the VH region (including provisions 60-62 as 1 replacement). For the region of VL have been changed 8 provisions.

In preliminary experiments, comparing the levels of expression of fully neimmunizirovannah 5D12 (DI5D12) ch5D12 (renamed PG100)found that because of the introduced mutations DI5D12 expressionalism at lower levels than the original sequence (data not shown).

In principle, each replacement can affect the properties of the expressed antibody. Therefore, to study the influence of each replaced by the provisions of the VH region on the properties of the antibodies were constructed 12 additional options (Fig.7). In each case 1 the situation was changed back to the original murine amino acid sequence.

For validation of expression levels and functionality of different variants were constructed 14 expressing plasmids pcDNA3002. Each plasmid contains the basis IgG4person involved in the VH region, and the region Kappa man, which linked the region VL. One plasmid encodes h5D12 that used the murine V-region. Other 13 loft the ID were daimonizomai options in which neimmunizirovannah region VL is used in combination with neimmunizirovannah the VH region or one of the 12 variants of the VH presented on Fig.7. Subsequently, PER.C6 cellsTMwas transfusional 14 plasmids using LipoFectamine 2000. Also included false negative transfection (without plasmid). After 48 hours transient supernatant from each transfection were collected and analyzed for the expression of antibodies. On Fig presents FACS data. It is clear that all of the tested structures associated with JY cells, which indicates that the specificity of binding CD40 5D12 saved.

In addition, the FACS data indicate that the levels of produced antibodies differ. Obviously the difference in expression level between DI5D12 and ch5D12 (PG100). Options Q5E, K13A, E16Q, T17S, STS60NSA T68S and demonstrate the same DI5D12 characters expression. Other options seems to increase levels of expression. For exact definitions of the different levels of expression analyzed all supernatant by quantitative ELISA, which are defined by the levels of antibodies. As shown in table 5, the tested variants can be divided into 3 groups: no or weak improvement in the level of expression (Q5E, K13A, E16Q, T17S and STS60NSA), options P45L, M48L, S79F and T108S restore the levels of expression compared to ch5D12 (PG100) up to 40%, while options I29L and I37V Vosstania liveout level above 50%. Especially the substitution at position 29 (I to V), appears to have the greatest impact on levels of expression. Later variant I29L was renamed antibody PG102.

The observation that the substitution at position 29 (I to L) or 37 (I to V) has the greatest effect on the levels of expression were among other unexpected observations, because the amino acids I, L and V are structurally closely related, and all 3 form the group of branched-chain amino acids. In a subsequent study, it was further established, what is the effect of the combined substitutions of amino acid I in positions 29 and 37 of structurally related amino acids V, or L.

Figure 9 demonstrates the ways V-L-I, which were constructed coaxially with ch5D12 (PG100), DI5D12 and PG102 (option I29L).

For validation of expression levels and functionality of these variants V-L-I were designed for more expressing plasmids pcDNA3002 that contain the basis IgG4man, which is connected to the VH region of one of the variants, and the region Kappa person involved in the field of VL, as described above. Subsequently, PER.C6 cellsTMwas transfusional plasmids using LipoFectamine 2000. After 48 hours transient supernatant from each transfection were collected and analyzed for the expression of antibodies. For precise definitions of the various levels of expressi who reviewed all supernatant by quantitative ELISA, which are defined by the levels of antibodies. As shown in table 8, various combinations of V, L or I at positions 29 and 37 are accompanied by different levels of expression. This further shows that not all replacement V, L or I ultimately lead to higher levels of production.

Discussion

Properties can be modified by reducing the immunogenicity of therapeutic antibodies. In this study it is shown that the scheme reimmunization leads to an antibody that still has the ability to bind to the antigen, however, the antibody can only be produced at low levels. Most likely that when the expression there is the problem of Assembly neimmunizirovannah regions VH and VL. By changing each substituted position back to the original murine sequence within the region VH discovered that some provisions are essential for the proper expression of the antibody. Especially the site, based on the approach of threading peptides had a significant influence on the expression level. N-terminal replacement, all entered on the basis of generowania, had little influence. It is remarkable that the replacement I L in position 29 has had the biggest impact, what was unexpected, since both amino acids are structurally closely related. Subsequently, the var is ant I29L was renamed antibody PG102.

Subsequent studies have demonstrated that different combinations of V, L or I at positions 29 and 37 lead to variations in levels of production, demonstrating that although the 3 branched chain amino acids V, L or I are structurally closely related, replacing each of them in positions 29 and 37 has an impact on the levels of full production of antibodies.

Example 3

Materials and methods

Design, selection and development of cell line GS-CHO for the production of PG102 (option I29L)

PanGenetics BV asked Lonza Biologics (Lonza) to perform the design, selection and evaluation cell line GS-CHO expressing human recombinant IgG4/Kappa antibody PG102 (option I29L).

Cell line designed by transfection of host cells CHOK1SV vectors created using Expressway gene glutamine synthase (GS) system Lonza (WO2003054172). Sequences of the genes were delivered PanGenetics. The CHOK1SV cell line is a suspension variant cell line of Chinese hamster ovary SNACK adapted to chemically defined, does not contain animal ingredients (CDACF) environment (Bebbington et al. (1992) Biotechnology 10: 169-75; de la Cruz Edmonds MC et al. (2006) Mol. Biotechnol. 34: 179-90).

All reagents, components, environments and materials received from accredited suppliers. Drug nutrient SF40 and SF41, Wednesday CM42 and addition to environment SPE were obtained from Lonza. Environment CD CO (Invitrogen) was also used with the addition of a selective agent L-methanesulfonamide (MSX) for routine subculture adapted to CDACF cell lines and phenol red for transfection. Selective agent MSX was delivered Sigma. These environment and nutrients do not contain antibiotics. Designing GS-expressing vectors encoding genes for PG102, also performed Lonza. Cells are the owners of CHOK1SV received from a test tube cryopreserved working cell Bank 269-W Lonza.

Cell culture, concentration and vitality

Cells were recovered from the vials of cryopreserved stocks with fast heat up to 37°C and cultivation accordingly in 50 ml of medium for growth. Createsite substances were removed by centrifugation of the cells, discard supernatant and resuspending cells in fresh medium for growth. For static culture, cells were grown in flasks KZT25, with the volume of the culture that constitutes 5-15 ml. These static culture was placed in an incubator at 35,5 is 37.0°C, containing an atmosphere of 10% (vol./about.) CO2in the air.

For suspension culture culture increased in an appropriate volume of medium for growth and subcultural every 4 days for growth in the medium CDACF. Used vessels were 125 ml shake flasks (volume of culture, comprising 10-30 ml), 250 ml shake flask (volume of culture, constituting 30-50 ml), 500 ml shake flask (volume of culture average of 50-100 ml), 1 l of the rotating bottle (volume of culture average of 100-200 ml) or 2 l rotate is iesa bottles (volume of culture, average of 200-400 ml). The free space of each culture was aziraphale 5% of the volume of/the amount of CO2in the air, and the flask was sealed. Then the culture was incubated at 35,5 is 37.0°C on the platform for shaking, rotating at 140±5 rpm

The concentration of cells in General, and viable cells were obtained using aseptic sampling sample culture, staining viable cells Trifanova blue and microscopic inspection using the modified hemocytometer Fuchs-Rosenthal. When appropriate, the samples were diluted to count.

In the alternative case, the concentration of cells in General and of viable cells was determined using a CASY particle counter is 1 or automatic meter cells Vi-CELL XR. To counter CASY-1 sample of the culture was aseptically collected from each flask, diluted with buffer Casyton, and determined the number of viable cells and cells in General. Viable and non-viable cells were distinguished by size. The percentage viability was calculated as the ratio of viable cells to cells in General, multiplied by 100. To counter the Vi-CELL XR 0.7 ml of cell culture was collected aseptically, and perform automatic calculation using Trypanosoma blue. For the identification and enumeration of viable and non-viable cells using a number of photos and a computer algorithm Ana the AIA. For all methods of calculating the percentage of viability was calculated as the ratio of viable cells to cells in General, multiplied by 100.

Transfection of host cells CHOK1SV

On the day of transfection cells growing in non-selective medium, collected by centrifugation and resuspendable at a concentration of 1.43×107viable cells/ml For each transfection into a cuvette for electroporation was added to approximately 0.7 ml of cell suspension and 40 μg of plasmid DNA. A cuvette for electroporation was then placed in the apparatus for electroporation, and sent a single pulse of 300 volts, 900 μf. After electroporation the cells from the cell distribution in 96-well plate at 5000 cells-owners/well, using a medium CD-CHO/phenol red. The plates were incubated at 35,5 is 37.0°C in an atmosphere of 10% vol./about. CO2in the air. One day after transfection in each well was added 150 μl of selective medium CD-CHO/phenol red/66,6 μm MSX. The final concentration of MSX in each well was 50 μm. Tablets controlled to determine when nitrostilbene cells died, leaving tricks transfected cells. Tricks transfected cells were visible after approximately 3-4 weeks after transfection. All transfectants tested and further improve, came from the UNOC, containing only one colony that was determined by visual assessment.

Assessment of the productivity of cell lines in static culture

96-well plates were incubated for approximately 3-4 weeks after transfection in order to make possible the formation of colonies. Colonies were examined microscopically to confirm that they are of a suitable size (covering more than 60% of the bottom of the wells) and that only one colony is present in each well. The supernatant of the culture is then examined for the production of antibodies using the ELISA Lonza for the collected antibodies described under the name "ELISA"Assembly. The percentage of confluently wells were evaluated during the sample. The value obtained by dividing the result of the analysis on the percentage of confluently used for distribution in a number of cell lines.

Culture of cell lines, occupying the top places in the series, was increased in 24-hole tablets in the environment CD-CHO/phenol red/25 μm MSX and left to achieve confluently. Upon reaching confluently this culture was used for inoculation of the flasks KZT25 environment CD-CHO/phenol red/25 μm MSX, while in the rest of the culture was again fresh environment CD-CHO/phenol red/25 μm MSX, and incubation was carried out for an additional 14 days. At this point in time the culture supernatant Sobir the Lee of 24-well plates, and determined the concentration of antibodies using ELISA Lonza for the assembled antibody. Made the selection of the most productive cell lines. Upon reaching confluently in flasks KZT25, selected cell lines were provided the opportunity to form many layers and then re-filed the environment CD-CHO/25 μm MSX. After the fluid cultures were returned to the incubator for an additional four to seven days until the environment became again orange/yellow, and cells were removed from flasks.

The capacity of cell lines in suspension culture CDACF

Suspension cultures were initiated from confluent cultures in flasks KZT25. Suspension cultures were inoculable at a concentration of 0.05-0.2×106viable cells/ml in 125 ml shake flasks containing medium CD-CHO/25 μm MSX. If the concentration of viable cells did not reach the 0.05×106viable cells/ml after a maximum of 14 days in flasks KZT25, 15 ml of each culture automatically transferred in 15 ml of medium CD-CHO/25 μm MSX 125 ml shake flasks. Cell line then has subculturally in fresh medium CD-CHO/25 μm MSX, at a concentration at inoculation, component of 0.2×106viable cells/ml in the 4-day subculture, to obtain an acceptable and reproducible characteristics of growth.

Assessment of growth and productivity of cell lines in susp nionoy culture

Fueled culture in shake flask

Singlet culture of each selected cell line was prepared in 500 ml shake flasks with 100 ml of cell suspension using environment CM42/4×SPE. Culture was inoculable at 0.2×106viable cells/ml, and free space of each culture was balanced 5% vol./about. CO2in the air. Cultures were incubated at 35,5 is 37.0°C on the platform for shaking, rotating at 140±5 rpm, to achieve the concentration of viable cells after the peak equal to or less than 0,6×106viable cells/ml, or reach day 15 ("pererasti"). At this time the culture was collected. The cell concentration was determined daily using an automatic counter cells Vi-CELL. At a cell concentration, constituent 1,4-2,2×106viable cells/ml, was carried out by bolus addition of nutrients SF40. Further bolus additions were made after approximately 24, 48 and 72 hours after the initial addition. The second nutrient (SF41) used only upon receipt of the concentration of cells in excess of 4.0×106viable cells/ml samples of the cultures were taken at appropriate intervals, centrifuged, and supernatant kept at -20±5°C until analysis on the concentration of antibodies using HPLC with the use of the m protein A. Supernatant collected from each of overgrown culture was stored at -20±5°C. Ten overgrown crops with the highest concentrations of the antibodies was further assessed by the quality of the produced antibodies. The samples were examined for levels of aggregates using the method of gel liquid chromatography high resolution (GP-HPLC) prior to purification using protein a and characteristics of antibodies by gel-electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE) in a reducing or non conditions and analysis using isoelectric focusing (IEF) and analysis of oligosaccharides by time of flight mass spectrometry with ionization by laser desorption, with the assistance of the matrix (MALDI TOF-MS).

Cryopreservation

Cells were subjected to cryoconservation at key points in time during the working of the program. Cells in growing cultures were isolated by centrifugation and resuspendable in the mixture for cryopreservation. It consisted of a suitable environment for the growth of 92.5%.about.) and dimethyl sulfoxide (7,5% vol./vol.).

Aliquots of cells were then divided into labeled test tubes, with each aliquot contained approximately 1.5 ml of medium for cryopreservation and approximately 0.5 to 1.5×107viable cells. The tubes were then frozen in the machine is programmable logical device for freezing cells and subsequently transferred to the cryostat with liquid nitrogen.

Number generation

For each selected cell line generation number of cells was defined as zero at the point in time when the culture was transferred into a suspension. Subsequently, the generation number expected in each subculture, using a procedure derived from the following equation (rounded down to the nearest 0,5 generation).

where Nf= the number of generations in time f

Ni= generation number at time i

Xf= concentration of viable cells (cells/ml) at time f

Xi= concentration of viable cells (cells/ml) at time i

and f>i.

Data analysis

The integral over time of the area under the growth curve (the integral over time of the concentration of viable cells (IVC); 109kletok/l) was calculated by summing the square (approximated to a rectangular line) between adjacent values of cell concentration. Area was calculated by multiplying the arithmetic mean of the two concentrations of viable cells (cells/ml) on the time elapsed between the two definitions (h). This method is based on the method described Renard and others (1988) Biotechnology Letters 10(2) pages 91-96.

When appropriate, the expected total rate of specific products (qp General: mg/109cells/h) using a linear analysis of regressi is the antibody concentration (mg/l) against the integral over time of the concentration of viable cells. The slope of this line is equal to qp total. Speed specific products during collection (qp collection) was calculated by dividing the concentration of the antibodies at the time of collecting the value of the IVC during the collection.

ELISA Assembly

The concentration of antibodies in the sample supernatants was determined using ELISA, which is assembled IgG person. It included the capture of the harvested antibodies in the samples and the standard 96-well pad, covered with goat anti-Ig Fc man. Bound antibody was detected using goat antibodies against the light chain of the human, linked to horseradish peroxidase and a chromogenic substrate tetramethylbenzidine. The development of color was proportional to the mass of the antibodies present in the sample. The concentration was determined using a standard curve, constructed using the standard IgG (lot number L07387/5/2).

HPLC with protein And

Product concentration was determined using liquid chromatography high-resolution protein A (HPLC) on an Agilent 1100 HPLC. The antibody selectively contacted with the column for immunodetection with protein AND POROS. Unbound material was suirable with columns, and the remaining bound antibody was released, lowering the pH of the solvent. The elution was monitored by optical density at 280 nm, using a detector of multiple wavelengths. The amount of antibody was determined (using p is ogromnie Chemstation software) against standard antibodies (batch number L07385/4/10) and introduced the amendment on the extinction coefficient E 280 nm0,1%=1,52.

Affinity purification using protein a

Prepared column (5 ml) of Sepharose-protein a rmp feed by gravity. Before use, the column was cleaned 6M guanidine HCl and balanced buffer 50 mm glycine/glycinate - 250 mm NaCl, pH 8.0. pH applied to the column material was brought to pH 8.0±0,1 prior to application to the column. Affinity column with protein And rmp were washed with buffer to balance and suirable in 0.1m glycine buffer, pH 3.5. Eluate with speakers neutralized to approximately pH to 7.0 with 2M Tris-base and dialyzed against phosphate buffered saline, Dulbecco in preparation for IEF analysis and electrophoresis in SDS-page.

Analysis by electrophoresis in SDS-PAG antibody, subjected to affinity purification using protein a

Samples of cell cultures were prepared for analysis using purification using protein A. Electrophoresis was performed using 4-20% polyacrylamide gels manufacturing Novex. For electrophoresis in SDS-page in reducing conditions, samples were recovered using 2-mercaptoethanol, and denaturiruet using SDS at pH 8.0. The samples were heated over a 2.0±0.5 minutes before loading into the gel (10 μg per line). Electrophoresis in SDS-page in non conditions was performed using the same gels and non of Novex sample buffer (2-what mercaptoethanol not added). The samples were heated during standard time of 1.0±0.5 minutes before loading into the gel (4 µg per line). After electrophoresis the proteins were visualized by staining for 75 minutes Kumasi brilliant blue R250 and discoloration methanol/acetic acid.

Analysis of IEF antibodies, and subjected to affinity purification using protein a

Samples of cell cultures were prepared for analysis using purification using protein A. the IFA Analysis was performed using agarose gels for IFA production and manufacturing, pH 3-10. Approximately 10 μg of protein in the sample line was loaded into the gel and then subjected to pre-focus at 70 volt-hours (VC). After removing the plate for loading samples, electrophoresis was continued at 1500 Concerns (1500 V, 20 mA, 25 W per gel). IFA-gels were fixed in a solution of trichloroacetic acid: sulfosalicylic acid: methanol and then stained Kumasi brilliant blue R250 (batch number, Kumasi blue 003/L11905/003 118 and/L11905/131). The gels were then decolorized and dried (room party tools for discoloration 003/L11905/117 003 and/L11905/121).

GP-HPLC

By using method GP-HPLC protein components are separated according to their size. Large components, such as protein aggregates are too large to penetrate into the particle matrix in any big step is no, and therefore, suiryudan with speakers in front of smaller components such as a monomer protein. Smaller components, such as fragments that penetrate the matrix more easily and suiryudan after monomer. Therefore, this method was used to separate the antibody monomer as aggregates and fragments. Monomeric component is identified by its characteristic retention time and position, relative to the calibration markers.

Undiluted samples were injected with getting 50-250 µg boot, and the product concentration was 1-5 mg/ml (estimated range load analysis Lonza). The range of molecular masses for speakers GF-250 was 4-400 kDa.

Components of the sample was detected at 280 nm, and the peak chromatogram were analyzed using software Agilent Chemstation. The integration was performed using the method of vertical drop for units and touching film fragments.

The proportion of components in the sample was determined by calculating the peak areas of each component relative to the total integrated peak area.

Analysis of MALDI TOF-MS

Analysis of oligosaccharide antibodies PG102 produced by each cell line, was performed using MALDI-TOF-MS using a MicromassTMMALDI-LR with delayed extraction. MALDI TOF-MS was performed as part of the equipment is of electrona way of positive ions. The instrument was also calibrated using a standard mixture of N-glycans.

The antibody was purified from the collected supernatants, using a column with protein AND POROS. Purified fractions were collected, and disulfide bonds were reduced by dithiothreitol, followed by alkylation of thiols using iodoacetate. Oligosaccharides were released using the enzyme N-glycanase (PNGaseF) and placed between two layers of the matrix in the form of a super-dihydroxybenzoic acid (super-DHB) on mounting plate for analysis of MALDI-TOF-MS.

Results

Transfection of host cells CHOK1ST

The sequence of the gene PG102 was used Lonza to construct a vector with two genes pPG102/DGV (not shown), which was used for transfection of the cell line host CHOK1ST using electroporation. Using vector pPG102/DGV, have completed six rows of transpency, and supernatant 255 transfectants were skanirovali on the production of antibodies in 96-well tablets using ELISA Assembly. Detectable levels of antibodies produced 99.6% of prokreditovannym of transfectants. Forty-three transfectant across the top seats in the row were selected for further evaluation in 24-hole tablets.

The build-up of static cultures and adaptation to suspension

Culture of selected cell lines GS-CHO first increased in 24-hole tablets, and then abrasively in static flasks KZT25. The rest of the cells in 24-hole tablets fresh medium, and subjected there is overgrowth" or antibody-based test to assess productivity. Selected cell lines showed a range of concentrations of the antibodies of 8.3 to 120 mg/l (figure 10).

Culture 23 cell lines with the highest concentrations of the products as determined by ELISA Assembly, were selected for further evaluation. Selected culture was transferred on Wednesday CDACF in suspension culture. All cell lines were successfully adapted to suspension culture and were selected for further evaluation in a recharging cultures.

Assessment of growth and productivity characteristics of selected cell lines in a recharging culture in shake flask

Growth and characteristics of productivity 23 selected cell lines was evaluated in a recharging culture in shake flask. For each cell line were obtained singlet culture. Two nutrients, SF40 and SF41, was added to the cultures, when the concentration of viable cells was consistent with the criterion recharge. The Protocol used for the recharge mode, replays, as far as technically feasible generalized fermentation GS-CHO Lonza. Data concerning the growth and productivity of 23 selected cell lines are presented in table 9. Analysis by HPLC using a protein a of sobran the x supernatants fed cultures in shake flasks showed four cell lines were produced antibodies at concentrations exceeding 1000 mg/l cell Line L107 have produced antibodies in the greatest concentration, 1674 mg/L.

Characterization of antibodies purified from recharging culture in shake flask selected cell lines (Cho

Samples of ten cell lines with the highest concentration of antibody was purified using chromatography on Sepharose-protein a rmp, prior to analysis. Purified using protein a antibodies and then analyzed using electrophoresis in SDS-page and IEF. Visual analysis of purified using protein a samples analyzed by electrophoresis in SDS-page in non conditions showed that all samples were comparable with each other, showing a strip of intact antibody with a molecular weight of approximately 200 kDa (data not shown). Visible additional minor bands, although not all of them can be seen on the gel image. Was also observed three bands with molecular masses between 116 and 200 kDa, a single band with a molecular mass between 66 and 97 kDa, a single band with a molecular mass of between 37 and 55 kDa and two bands with molecular masses between 22 and 31 kDa. Additional band observed between 66 and 97 kDa, is half of antibodies for antibody IgG4in non conditions. Same with the may half antibodies was also observed in control assays IgG 4(IAC).

Purified using protein a samples analyzed by electrophoresis in SDS-page in reducing conditions, also comparable to each other, showing a strip of heavy chain with a molecular mass of approximately 50 kDa and a band of light chain with a molecular mass of approximately 25 kDa (data not shown). The IEF analysis showed that the integrity of the samples purified using protein a antibodies from selected cell lines demonstrates comparable safety profiles, with six main bands (3 major and 3 minor bands) in the range of pI 8,15-of 9.30 for all cell lines except L97, M92 and M59. These three cell lines showed three major and two minor bands. In samples J3 and J4 were observed extra band, although not all of them can be seen on the gel image.

Oligosaccharide analysis of antibodies

Oligosaccharide analysis of the antibodies produced by each of the ten cell lines selected for further evaluation in a recharging cultures in shake flasks, was performed using MALDI-TOF-MS. The predominant oligosaccharide structures in antibodies derived from the ten cell lines were G0F and G1F, which are typical N-linked oligosaccharide structures observed in antibodies. Were found relatively low levels of structures in the form of oligomannose, this is very high level, at 6.7% of the glycans in General, was determined for antibodies derived from cell line L52.

All samples were observed structure oligomannose-5 man-5) in the range of 1.1 to 4.6% of the glycans in General. All analyzed samples contained comparable levels of oligosaccharides and relatively low concentrations of oligomannose-5 (data not shown).

GP-HPLC

When the antibody produced by each of the ten cell lines selected for further evaluation were analysed using GP-HPLC, components with low molecular masses (LMWC) were at least critical, but preferred was the level below 25%. All samples showed levels below 25%, except L52, which is demonstrated by 27.1% (table 10).

Two different calculation of share units is not normally used, but, as I thought necessary, since the levels of LMWC varied too much between samples.

Further analysis of the data, excluding LMWC and includes only the monomer and larger peaks showed that the peak areas of the aggregates are less than 2.2% for all samples (table 11). Levels below 5% are considered acceptable.

Selection of cell lines

Three cell lines were selected for preparation of preliminary sources-inventory (PSS). Used selection criteria were a combination of high concentrations of sobiraemosti, acceptable growth characteristics and acceptable quality characteristics of the product.

Cell line L107 was selected as a host cell line, because it showed the greatest concentration of the collected antibodies from 23 estimated cell lines, together with acceptable characteristics of growth and product quality (figa).

Cell line L25 showed the second greatest concentration of the collected antibodies of the estimated twenty-three of the cell lines, together with acceptable characteristics of growth and product quality (pigv). Therefore, the cell line L25 was selected as the first compensating cell line.

Cell line M showed the third largest concentration of the collected antibodies of twenty-three evaluated cell lines and had acceptable characteristics of growth and product quality (figs). On this basis, the cell line M was selected as the second compensating cell line.

Cryopreservation prior sources-stocks

A preliminary source inventory (20 tubes per line) were subjected to cryoconservation for each of the three selected cell lines. These stocks were stored in the cryostat with liquid nitrogen vapor phase. Cell line L107, L25 and M were renamed in DC1, DC2 and DC3, respectively.

Discussion

Lonza Biologics asked to perform designed the e, selection and evaluation cell line GS-CHO expressing human recombinant IgG4/Kappa antibody PG102.

The sequence of the gene was delivered PanGenetics, and the vectors were created using expressing the gene of glutamine synthase (GS) system Lonza. The CHOK1SV cell line, suspension variant cell line of Chinese hamster ovary (Cho) SNACK adapted to chemically defined, does not contain animal components of the environment, was subjected to transfection by electroporation.

From transfectants CHOK1SV were obtained 254 cell line, which secretively antibody. Forty-three cell lines were assessed for growth and productivity in 24-hole plates. Were defined as the antibody concentration up to 120 mcg/ml Twenty-three cell lines were evaluated in a singlet fed cultures in shake flasks. Were obtained concentration of the collected antibodies in the range that makes 121-1674 µg/ml For the four cell lines were obtained concentration of the collected antibody excess of 1000 mg/L. Ten cell lines with the highest concentrations of antibodies were selected for analysis the quality of the product. Product quality - purified antibodies from each of the selected cell lines was comparable with each other when analyzed by polyacrylamide gel electrophoresis with sodium dodecyl sulfate in reducing or newos tanavlivaetsya conditions and analysis using isoelectric focusing. Oligosaccharide analysis of purified antibodies from each of the ten selected cell lines showed relatively low (<4,6%) levels oligomannose-5.

Analysis of GP-HPLC showed that the levels of aggregates were below 1% for each of the top three cell lines.

Three cell lines (L107, L25 and M) were selected for further evaluation based on the data relating to the growth, productivity and product quality. A preliminary source inventory of 20 tubes for each cell line was subjected to cryoconservation. Three cell lines were renamed in DC1, DC2 and DC3, respectively.

Example 4

Determination of the kinetics of binding ch5D12 (PG100) and PG102 (option I29L) with CD40-Fc man using surface plasmon resonance (Biacore)

Materials and methods

The kinetics of binding ch5D12 (PG100) and PG102 (option I29L) with human CD40 were compared using the resonance of surface plasmons at a constant temperature, component 25,0°C using a Biacore instrument 300. Recombinant human extracellular domain of CD40, fused with the Fc domain of Ig man (huCD40-Fc; catalog number R&D Systems - 149-CD-50) was used as the target antigen in these experiments. HuCD40-Fc (1 μg/ml in 10 mm acetate buffer, pH 5.0) were linked through the amino groups of Lys residues c activated by carbodiimide chips SM Biacore, receiving the level of immobilization of the antigen, comprising 127,4 EN (conventional units). Telekinetically analyses were prepared serial dilution ch5D12 (PG100) and PG102 (in the range of 10.0-0,313 µg/ml) in operating Biacore buffer (HEPES buffered saline solution, containing EDTA and surfactant; HBS-EP). Kinetic analyses were performed at a flow rate of 30 µl/min the Samples were given the opportunity to contact for 4 min and dissociativity for 5 min at the same flow rate. The surface of the chips then regenerates within 30 seconds, using 30 mm NaOH, and the baseline was restored in HBS-EP for 2.5 min before injection of the next sample. Were injected with alternating samples PG102 and ch5D12 (PG100) with increasing concentration for the first set of duplicates for each dilution and then with a decrease for the second set of duplicates.

Data collection software has been used to control BIAcore (version 3.2), and for the analysis of kinetic data software has been used to assess BIAevaluation version 4.1). The binding parameters were determined using two different models of molecular interactions: model bivalent substances as determined by analysis, and model binding in a ratio of 1:1 Langmuir.

Results

The maximum levels observed binding to antibodies ch5D12 (PG100) and PG102 were 152,5 RU and 140,2 RU, respectively.

Calculated kinetic rate constants in accordance with the models of Langmuir and bivalent binding (ka and kd or ka1 and kd1, respectively) were slightly different for the two antibodies. Thus the m PG102 showed a higher rate of Association and dissociation than ch5D12 (PG100) (Fig), although the affinity in General (KD) of the two antibodies did not differ significantly in these models. Kinetic parameters of these experiments are summarized below.

The model of Langmuir (1:1)

ch5D12 (PG100)PG102
kaof 1.88×106M-1with-1of 2.51×106M-1with-1

kd5,07×10-4with-15,96×10-4with-1
KDa 2.71×10-10Mis 2.37×10-10M

Model bivalent binding (1:2)

ch5D12 (PG100)PG102
ka16,41×105M-1with-1of 7.82×105M-1with-1
kd11,60×10-3with-12,47×10-3with-1
ka2to 1.21×10-3RU-1is 1.31×10-3RU-1
Kd2 are usedto 2.55×10-3with-1of 2.24×10-3with-1
KDof 5.29×10-9M5,41×10-9M

Analysis of the degree of agreement for the two methods showed that the data are better described by a model of bivalent binding.

The calculated affinity of two variants of antibodies differ between models, but estimates of the dissociation constants (KD)obtained for each antibody using the same model were comparable; a 2.71×10-10M and 2.37×10-10M for ch5D12 (PG100) and PG102, respectively, using the Langmuir model, and the 5.25×10-9M5,41×10-9M, using the model of bivalent binding.

Example 5

Inhibition of binding ch5D12 (PG100) and PG102 with JY cells using antiidiotypic antibody

To demonstrate that PG102 (option I29L) binds to the same epitope CD40, the original molecule ch5D12 (PG100), used antiidiotypic antibody to inhibit the binding of PG102 and ch5D12 with expressing CD40 JY cells as measured by FACS.

Materials and methods

The JY cells expressing CD40, blocked with 5% savoro the key man for 30 min at room temperature. Antiidiotypic antibody against mouse Mat 5D12 (clone 173-36-1) pre-incubated with various concentrations (0-10 μg/ml) or with ch5D12 (PG100) (1 μg/ml), PG102 (1 μg/ml), or chimeric antibody against human CD86 (chFUN-1, 1 μg/ml) as a negative control in a total volume of 50 μl/tube and incubated for 15 min JY Cells were washed in FACS buffer (1×PBS, 1% BSA and 0.05% sodium azide), and the supernatant was discarded. Cells JY resuspendable in FACS buffer to a concentration of 2×106cells/ml 50 ál of cell suspension JY was added to the pre-preincubating mixtures of antibodies with obtaining a final volume of 100 μl/tube. Cells were incubated for 30 min at 4°C before washing with 4 ml of buffer for FACS for each tube, and the supernatant was discarded. Cellular precipitate resuspendable 100 μl of diluted goat antibodies against human IgG conjugated with FITC (Jackson Immunoresearch Labs, catalog number 109-095-127) in FACS buffer (1:100) and incubated for 30 min at 4°C. Cells were washed in 4 ml of buffer for FACS, and the supernatant was discarded. Sediment resuspendable in 200 µl of buffer for FACS, and bound antibody was determined using flow cytometer FACScan (Becton Dickinson).

Results

Antiidiotypic antibody Mat 173-36-1 inhibited binding of PG102 and ch5D12 (PG100) huCD40 expressed in JY cells, dependent on the concentration clicks the zoom (Fig). Mat 173-36-1 had no effect on the binding chFUN-1 and CD86, which is also expressed on cells JY. No significant differences in blocking the effect antiidiotypic Mat 173-36-1 on the specific binding against CD40 antibodies to CD40 on the cells JY. For example, the calculated values of log IC50 for mediated Mat 173-36-1 inhibition ch5D12 (PG100) (party 1) and PG102 (batch 2) were -5,74±0.14 and -5,76±0,11, respectively (p>0,05; both n=4). These values correspond to concentrations antiidiotypic antibodies, comprising approximately 1.8 μg/ml (~12 nm). Table 12 presents a summary of the calculated values of-log IC50 for mediated antiidiotypic antibody inhibition of binding ch5D12 (PG100) and PG102 with JY cells.

Example 6

Linking ch5D12 (PG100) and PG102 with CD 40F determined using ELISA

Materials and methods

Tablets for ELISA (tablet Costar EIA/RIA, catalog number Corning - 3590) were coated with 100 µl/well huCD40-muIg (Ancell, catalogue number 504-020)diluted to 250 ng/ml in PBS, and incubated overnight at 4°C in an atmosphere of humidity. The tablets were washed 3 times with buffer washes (0,05% Tween-20 in PBS), 200 μl/well. Subsequently, the plates were blocked with a buffer for blocking (5% fraction V BSA [Roche, catalogue number 735094] in the buffer for leaching), 200 μl/well, and incubated for 1 h at 37°C in an atmosphere of humidity). The tablets were washed 3 times with buffer is La leaching. Check antibodies (ch5D12 (PG100), PG102 and chimeric antibody against human CD86 chFUN-1) was diluted in the range of 0-1200 ng/ml in the buffer to block and transferred into tablets for analysis in the final volume of 100 μl/well followed by incubation for 1 h at 37°C in an atmosphere of humidity). The tablets were washed 3 times with buffer for washing, followed by addition of 100 μl/well goat antibodies against human Kappa conjugated with AR for detection (Southern Biotech Associates, catalogue number 2060-0)diluted 1:1000 in buffer for blocking. The plates were incubated for 1 h at 37°C in an atmosphere of moisture before washing 3 times with buffer for washing and one additional washing PBS. The substrate PNP was prepared by adding 1 tablet para-nitrophenylphosphate (Sigma, catalog number N-2765) in 15 ml of buffer for substrate PNP (12.1 g Tris, of 5.84 g NaCl, 1,02 g MgCl2.6H2O in 1 liter of H2O (brought to pH to 9.6 with HCl). In tablets for analysis were added 100 μl/well of substrate PNP. The plates were incubated for several minutes (maximum 30 minutes) at 37°C to allow the development of painting before stopping the reaction by addition of 3M NaOH. The intensity of staining was determined at 405 nm using a card reader for microplates (Biorad, model 550).

Results

In several independent experiments found that serially diluted ch5D12 PG100) and PG102 (option I29L) comparable way contacted with immobilized CD40-Fc (Fig), while the control antibody against CD86 did not show appreciable binding to CD40-Fc. Calculated concentrations for premaxillae binding for all experiments are summarized in table 13. Significant differences were observed in concentrations for premaxillae binding for any of the estimated quantities of antibodies against CD40.

Example 7

Linking ch5D12 (PG100) and PG102 with huCD40 expressed in JY cells determined by FACS

Materials and methods

Expressing CD40 JY cells were washed in FACS buffer (1×PBS, 1% BSA and 0.05% sodium azide). The supernatant was discarded, and cells JY resuspendable in FACS buffer to a concentration of 2×106cells/ml Subsequently, 50 μl of the suspension of JY cells were added to 50 μl each of the test antibodies (ch5D12 (PG100), PG102 and chimeric antibodies against CD86 person chFUN-1 as a negative control), prepared in the concentration range 0-900 ng/ml, and incubated for 30 min at 4°C. Cells were washed in 4 ml of buffer for FACS for each tube, and the supernatant was discarded. Cellular precipitate resuspendable in 100 μl of goat antibodies against human IgG conjugated with FITC (Jackson Immunoresearch Labs, catalog number 109-095-127), diluted 1:100 in FACS buffer, and incubated for 30 min at 4°C. Cells were washed in 4 ml of buffer for FACS, and the supernatant was discarded. Precipitate resuspended is ovale in 200 µl of buffer for FACS, and bound antibody was determined using flow cytometer FACScan (Becton Dickinson).

Results

All of the tested party ch5D12 (PG100) and PG102 showed similar properties to communicate with CD40 expressed on cells JY, (Fig) with comparable concentrations for premaxillae antibody binding sites (table 14). chFUN-1 was also associated with JY cells, although with lower average fluorescence intensity (MFI)than ch5D12 (PG100) and PG102. This happened because of JY cells also Express CD86 person.

Discussion

Properties contact CD40 various parties PG102 (option I29L) and ch5D12 (PG100) was determined using in vitro assays cell-based and ELISA. Linking PG102 and ch5D12 (PG100) with JY cells expressing CD40, inhibited with equal efficiency antiidiotypic Mat 173-36-1. Thus, the range of calculated average values-logIC50 for parties PG102 and ch5D12 (PG100) was 5.51-6,11 (n=8) and 5,43-6,04 (n=10), respectively (p>0,05). Antibody 173-36-1 directed against the variable regions predecessor ch5D12 (PG100), mouse Mat 5D12, and did not inhibit the binding of an antibody isotype control against CD86. In further experiments it was determined the concentration of PG102 and ch5D12 (PG100) for premaxillae binding using ELISA analysis of the binding of an antibody to CD40-Fc man and with the help of FACS analysis of binding of JY cells. In ELISA assays was not C is uchitelnoj difference between the concentrations of PG102 and ch5D12 (PG100) for premaxillae link for any reliable party. For example, we calculated the average values of log concentrations for premaxillae binding on the parties PG102 and ch5D12 (PG100) was 7,47±0.04 and 7,54±0,03, respectively (P>0,05, both n=4), corresponding to approximately 30 ng/ml Similar concentrations for premaxillae binding was observed in FACS experiments linking PG102 and ch5D12 (PG100) with CD40 expressed on cells JY. These results indicate that PG102 and ch5D12 (PG100) have the same binding the CD40 Pratap that demonstrates comparable performance activity antiidiotypic antibodies against inhibition of binding of PG102 and ch5D12 (PG100) with CD40. Moreover, both antibodies show similar in vitro activity against binding to CD40 in assays based on ELISA and cells. Together, the presented data suggest that the ability of PG102 and ch5D12 (PG100) to bind CD40 person are the same.

Example 8

Competitive inhibition of binding ch5D12 (PG100)-RE and PG102-RE with JY cells expressing CD40, using unlabeled antibodies against CD40

Materials and methods

The JY cells transformed by Epstein-Barr line human lymphoblastoid b-cells) were grown in modified according to the method of Claims environment Dulbecco (IMDM)containing 10% V / V heat inactivated fetal calf serum (FCS), 2 mm L-glutamine and 50 μg/ml gentamicin, at which osphere humidified air, containing 5% CO2at 37°C. Cells were collected at day measurements using flow cytometry. In these experiments we used unlabeled antibodies ch5D12 (PG100) (part 4) and PG102 (part 2). Both antibodies also used in labelled reform for the direct determination of binding with JY cells using flow cytometry. Tagging antibodies PE order was carried out AbD Serotec (Oxford, United Kingdom). For each FACS analysis used the following Protocol. The JY cells were collected from cell cultures and counted. Subsequently, 1×105CD40-expressing cells JY/200 ál buffer for incubation (PBS, 1% BSA, 0.05% of sodium azide) were incubated with 1 µg/ml of labeled antibodies ch5D12 (PG100) or PG102 in the presence of varying concentrations (0-10 μg/ml) of unlabeled competing ch5D12 (PG100) or PG102 for 30 min at 4-8°C. Cells were washed, and then subjected to the analysis of flow cytometry, in which the MFI was determined at each concentration using flow cytometer FACScan (Becton &Dickinson). Five thousand events per sample were measured and analyzed using the software CellQuest® software (Becton &Dickinson).

Results

Unlabeled antibodies ch5D12 (PG100) and PG102 showed similar inhibitory properties in these studies (Fig). Average values-logIC50for inhibition of binding of 1 μg/ml ch5D12 (PG100) unlabeled ch5D12 (PG100) IS PG102 was 7,50±0.03 and 7,63±0,03, respectively (both n=4), while average values-logIC50for inhibition of binding of 1 μg/ml PG102 unlabeled ch5D12 (PG100) and PG102 was 7,54±0.01 and 7,65±0,02, respectively (both n=4). These values-logIC50correspond to the concentrations of PG102 and ch5D12 (PG100), constituting approximately 20-30 ng/ml (i.e. ~130-200 PM).

Example 9

Inhibition of release of IL-8 from cells TNR-1

Materials and methods

The functionality of PG102 was determined using the functional bioanalysis based cells. Briefly, on day 1 of human cells TNR-1 and Jurkat 39,8/50 were cultured in modified according to the method of Claims environment Dulbecco (IMDM, BioWhittaker, catalogue number - BE12-722F), supplemented with 10% fetal calf serum (Gibco, catalog number - 10270-106) and 50 μg/ml gentamicin (Invitrogen, catalogue number - 15750-045). Cells TNR-1 stimulated rhuIFN-γ (1000 units/ml cell culture.

Day 3 bioanalysis cells TNR-1 and Jurkat 39,8/50 required at a concentration of 4×105cells/ml Cells TNR-1 and J39,8/50 calculated, and determined their viability. The cells were then diluted to the concentration of that component of approximately 1×106cells/ml of cell Suspension were entertained at a ratio of 1:1 and incubated at 37°C in humidified atmosphere with 5% CO2within 48 hours

On day 3 cells TNR-1 and J39,8/50 washed as follows (in the case of cells TNR-1 stage flushing Ave is naznachen to remove IFN-γ): 5 ml of cell suspension TNR-1, stimulated IFN-γ, and J39,8/50 was transferred into a 50 ml test tube Falcon's Twister. Added 40 ml of a balanced salt solution Hank (HBSS), and cells were centrifuged at 1500 rpm (using a centrifuge IEC) within 6 minutes the Supernatant was discarded. Sediment from cells resuspendable in a preheated IMDM, supplemented with 10% FCS. Cells TNR-1 and J39,8/50 brought to a concentration of 4×105cells/ml

For analysis of inhibition of the tested sample ch5D12 (PG100) or PG102 (option I29L) were serially diluted in a hot environment IMDM, supplemented with 10% fetal bovine serum, to obtain the final of the studied concentrations in the range 0-160 ng/ml TNR-1, J39,8/50 and check the sample (the samples) was added in three repetitions in tablets for cell culture with a round bottom (NunclonTMin the following order: 50 μl of cells TNR-1 (equivalent to 2×104cells per well), 50 µl of tested sample and 50 μl of cell J39,8/50. The total volume was 150 μl per well. Tablets for cell culture were Packed in a porous adhesive film and incubated at 37°C in humidified atmosphere with 5% CO2within 48 hours

On day 5, after a period of cultivation, amounting to 48 h, 70 μl of supernatant cell cultures aspirated and transferred to microtiter plate with a round bottom. The collected supernatant cell cultures were analyzed to provide the e IL-8, using a commercial ELISA (Biosourse, ritonavir for IL-8 person, catalogue number - SNS) according to the manufacturer's instructions.

Results

PG102 (option I29L) and ch5D12 (PG100) showed similar inhibitory effects on stimulated release of IL-8 cells TNR-1 (Fig). The calculated values of-log IC50for the two antibodies was 8,42 and 8,28, respectively. These values correspond to the concentrations of the IC50constituting approximately 30 PM.

Example 10

Prior study of tissue cross-reactivity in the tissue of humans and cynomolgus macaques showed that ch5D12 (PG100) associated with the cell surface of b-cells and DC in lymphoid organs. Unexpected cross-reactivity was not observed neither in human tissue, or the tissue of cynomolgus macaques. This study was repeated for PG102, and similar results were achieved, which indicates that ch5D12 (PG100) and PG102 associated with different tissue sections in a similar way.

Previous assessment of safety and tolerability for ch5D12 conducted in cynomolgus monkeys, for which, as has been shown, weekly introduction ch5D12 for 4 weeks is safe and without any side effects for all macaques. In this study, was obtained functional evidence that ch5D12 can prevent the activation and proliferation of the cells. 27Safety studies were repeated for PG102, using the long Protocol. Briefly, we developed a toxicity study with intravenously for 13 weeks in cynomolgus monkeys with a recovery period for 14 weeks using level 3 doses (0, 25 and 100 mg/kg, intravenously) and 26 macaques (6 monkeys (3 males, 3 females) in each group processing, plus 4 monkeys (2 males, 2 females) as recovery groups in each branch of the active processing). Conducted the following measurements: TC, anti-PG102 response, flow cytometry (including floor PBMC CD40), biopsy of lymph nodes and standard Toxicological assessment, such as Hematology and immunohistochemistry.

The results of these studies showed that for any of the tested dose levels toxicity is not observed. PG102 was safe and well tolerated.

Taken together these studies show that antagonistic MAB against CD40 PG102 man, as his original antibody ch5D12 (PG100), will not have unexpected cross-reactivity, safe and well tolerated in vivo.

Table 6

Mutagenic oligonucleotides. The oligonucleotides used to introduce mutations in the positions of amino acids 29 and 37. Lists of sense and antisense (rev) oligonucleotides.

Table 7

The creation of the dual is utanov, requires 2 rounds of mutagenesis. Listed below are what sets of primers were used in stage 1 and stage 2, respectively, to equal the additional options DI5D12.

Table 8

Data concerning expression, measured by quantitative ELISA. For each additional option 5D12 (29I-37V, 29V-37i indigenous, 29I-37L, 29V-37V, 29L-37L, 29V-37L and 29L-37V) together with ch5D12 (PG100), DI5D12 and PG102 (option I29L) shows the concentration of antibodies in the collected supernatant.

Table 9

Summary data concerning the growth and productivity of cell lines Cho in fed cultures CDACF in shake flasks

Table 10

Analysis of GP-HPCL (including components with low molecular masses (LMWC))

ND = not detected.

Table 11

Analysis of GP-HPCL (only IgG products)

ND = not detected.

Table 12

Summary of calculated values-logIC50 for mediated antiidiotypic antibody inhibition of binding ch5D12 (PG100) and PG102 with JY cells. Data show the arithmetic mean ± standard error value n definitions. No significant difference in inhibitory activity antiidiotypic antibodies with any of the tested batches ant the phone (p> of 0.05, analysis of variance (ANOVA) with subsequent analysis of the Same for multiple comparisons).

Table 13

A summary of the calculated values of-log concentration for premaxillae binding to bind ch5D12 (PG100) and PG102 with CD40-Fc human, as determined by ELISA. Data show the arithmetic mean ± standard error value n definitions. No significant difference in the binding of any of the tested batches of antibodies (R>of 0.05, analysis of variance (ANOVA) with subsequent analysis of the Same for multiple comparisons). For reference, the value of-log concentration for premaxillae binding, comprising 7,45, concentration, component approximately 35 ng/ml (~200 pmol).

Table 14

A summary of the calculated values of-log concentration for premaxillae binding to bind ch5D12 (PG100) and PG102 with JY cells expressing CD40 person, as determined using ELISA. Data show the arithmetic mean of double determinations. For reference, the value of-log concentration for premaxillae binding, comprising 7,45, concentration, component approximately 35 ng/ml (~200 pmol).

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92. E.K. Jordan, H.I. McFarland, B.K. Lewis, N. Tresser, M.A. Gates, M. Johnson, M. Lenardo, L.A. Matis, H.F. McFarland and J.A. Frank, Serial MR imaging of experimental autoimmune encephalomyelitis induced by human white matter or by chimeric myelin-basic and proteolipid protein in the common known as marmoset, AJNR Am. J. Neuroradiol. (1999); 20: 965-976.

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94. J.D. Laman, C.B. Maassen, M.M. Schellekens, L. Visser, M. Kap, E. de Jong, M. van Puijenbroek, M.J. van Stipdonk, M. van Meurs, C. Schwarzler and U. Gunthert, Therapy with antibodies against CD40L (CD154) and CD44-variant isoforms reduces experimental autoimmune encephalomyelitis induced by proteolipid protein peptide, Mult. Scler. (1998); 4: 147-153.

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107. P. Villoslada, K. Abel, N. Heald, R. Goertsches, S.L. Hauser and C.P. Genain, Frequeny, heterogeneity and encephalitogenicity of T cells specific for myelin oligodendrocyte glycoprotein in naive outbred primates, Eur. J. Immunol. (2001); 31: 2942-2950.

108. Bata-Csorgo Z, Hammerberg C, Voorhees JJ, Cooper KD. Intralesional T-lymphocyte activation as a mediator of psoriatic epidermal hyperplasia. J Invest Dermatol (1995); 105(Suppl 1): 89S-94S.

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1. Antibody against CD40, containing the variable region of the light chain with the amino acid sequence

where X6represents N, Q, S, T, Y, W, or C;
X7represents the D, E, N, Q, S, T, Y, W, or C;
X8represents N, Q, S, T, Y, G, A, V, L, I, P, F, M, W, or C;
X9represents A, V;
X10represents G, A, V, L, I, P, F, M; and
X11is the N, Q, S, T, Y, G, A, V, L, I, P, F, M, W or C,
and the variable region of the heavy chain with the amino acid sequence

where X1is a I and X2is a V;
X1is a L, and X2represents I;
X1is a V, and X2is a V;
X1is a L, and X2is a L; or
X1is a L, and X2is a V; and
X3represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H;
X4represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R, or H; and
X5represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T, Y, D, E, K, R or H.

2. The antibody according to claim 1,
where X3represents G, A, V, L, I, P, F, M;
X4represents G, A, V, L, I, P, F, M; and
X5represents G, A, V, L, I, P, F, M, W, C, N, Q, S, T or Y.

3. The antibody according to any one of claims 1 or 2,
where X3represents G, A, V, L, I, P, F, M;
X4represents G, A, V, L, I or M; and
X5represents P, F, W, N, Q, S, T or Y.

4. The antibody according to claim 3,
in which X1represents L;
X2represents I;
X3is a P; and
X4is a M; or X5represents S.

5. The antibody according to claim 1,
in which X3is a P;
X4is the battle M; and
X5represents F or X5represents S, preferably S.

6. The antibody according to claim 1,
in which X1represents L; X2is a V; X3represents L; X4is a L; and X5is a F.

7. The antibody according to any one of claims 1 to 6, containing the constant region of human antibodies, preferably, the constant region of IgG.

8. The antibody according to claim 7, in which the specified constant region is an area with a lack of activation of complement, preferably constant region of IgG4person or mutated constant region of IgG1person.

9. Nucleic acid encoding the antibody according to any one of claims 1 to 8.

10. The cell containing the nucleic acid according to claim 9, which expresses the antibody according to any one of claims 1 to 8.

11. The antibody according to any one of claims 1 to 8, wherein the antibody is a monoclonal antibody against human CD40.

12. Monoclonal antibody against CD40 man in claim 11, where the antibody is neimmunizirovannah.

13. The antibody according to claim 1, in which X6is a T or S, X7represents the D or Q, X8represents Q or P, X9is a V or A, X10is a V or L and X11represents F or Y.

14. The antibody according to item 13, in which X6pre is is a T, X7represents Q, X8represents P, X9represents A, X10represents V and X11represents Y.

15. Cell of claim 10, which is hybridoma cell, a cell of the ovary of the Chinese hamster, a NSO cell, or cells, PER-C6TM.

16. Cell culture for 15 to obtain the antibody according to any one of claims 1 to 8, and 11-14.

17. A method of producing an antibody comprising culturing the cell according to any one of p or 15 and the collection of the specified antibodies from the specified culture.

18. Pharmaceutical composition for reducing the intensity of symptoms of autoimmune disease and/or inflammatory disease, and/or reduce transplant rejection, and/or for the treatment of CD40-positive cancer, containing a therapeutically effective amount of antibodies against CD40 according to any one of claims 1 to 8.

19. The antibody according to any one of claims 1 to 8 and 11 to 14 for use as pharmaceuticals.

20. The use of antibodies according to any one of claims 1 to 8 and 11 to 14 for receiving drugs that reduce the intensity of the symptom of the autoimmune disease and/or inflammatory disease, and/or reduce transplant rejection, and/or for the treatment of CD40-positive cancers.

21. The application of claim 20, wherein the specified autoimmune and/or inflammatory disease is selected from the group consisting of rheumatoid the CSOs arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis, bullous of pemphigoid and allergic dermatitis.

22. The application of item 21, wherein the specified autoimmune and/or inflammatory disease is inflammatory bowel disease.

23. The application of article 22, which specified inflammatory bowel disease includes ulcerative colitis (UC) or Crohn's disease (CD).

24. The method of introducing the individual antibodies against CD40, including the introduction of a specified individual a therapeutically effective amount of the antibody according to any one of § § 11 to 14 or 19 to relieve symptoms of autoimmune disease and/or inflammatory disease, and/or to decrease the reaction transplant rejection, and/or for the treatment of cancer, positive for CD40.

25. The method of selecting antibodies against human CD40 with enhanced expression that includes a first cell line which produces the original antibody against human CD40, and determining the amount of the original antibody, which is produced by the specified first line of cells, with the specified initial antibody contains a variable region light chain containing a sequence

and variable regions of the heavy chain, which contains a sequence of

in which X1and X2pop the RNO is chosen from the group consisting of X1=I and X2=V; X1=L and X2=I; X1=V and X2=V; X1=L and X2=L; or X1=L and X2=V, with the specified method further includes creating at least one additional cell line, which produces a variant of the specified source of the antibodies, and the specified variant antibody is a modified original antibody, including insertion, deletion, inversion and/or replacement of approximately 1-5 amino acids compared with the specified source antibody, and this modification does not consist of the amino acid modifications at position(s)defined by X1and X2and the determination of the number of variant antibody, which is produced by the specified at least one additional cell line,
while this method includes the selection of variant antibodies against human CD40 with enhanced expression.

26. The method according A.25 which includes the insertion, deletion, inversion and/or replacement of approximately 1-5 amino acids are specified amino acid sequence of the heavy chain or the specified amino acid sequence of the light chain compared with the amino acid sequence of the corresponding chain in the specified source antibody.

27. The method according to p which includes the insertion, deletion, inversion and/or replacement of approximately the nutrient 1-5 amino acids are specified amino acid sequence of the heavy chain compared with the specified sequence of the heavy chain of the specified source of antibodies.

28. The method according to any of PP-27, where the specified insertion, deletion, inversion and/or replacement represents a variable amino acid in the sequence of the heavy chain or variable sequence of the light chain of the original antibody with the corresponding amino acid sequence variable sequence of the heavy or light chain of chimeric 5D12, respectively.

29. The method according to any of PP-28, further comprising obtaining a cell line producing the antibody, which produces the specified selected antibody.

30. The method according to clause 29, further comprising collecting the specified selected antibodies.

31. Antibody against human CD40, which are collected by the method according to item 30, where the antibody contains a variable domain of the light chain containing the sequence

and the variable domain of the heavy chain, which contains a sequence of

where X1and X2are a pair selected from the group consisting of X1=I and X2=V; X1=L and X2=I; X1=V and X2=V; or X1=L and X2=L,
where the antibody contains an insertion, a deletion, inversion and/or replacement of between about 1-5 amino acids, where this modification does not consist of modification of amino acid position(s)defined as X1and X2

 

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85 cl, 5 ex, 29 dwg, 38 tbl

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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

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