Il2 antibodies

FIELD: medicine, pharmaceutics.

SUBSTANCE: humanised monoclonal antibody and its active fragment under the invention neutralises human IL2 activity by binding with said human IL2 prior to, during and/or after binding of said human IL2 with human IL2 receptor. A variable light chain region of said antibody contains an adherent amino acid KAPKA sequence in its second frame region, and in addition, in the CDR1-CDR3 regions, contains the amino acid sequences presented in SEQ ID NO 1-3 disclosed in the description, while a variable heavy chain region contains in the CDR1 - CDR3 regions, amino acid sequences presented in SEQ ID NO 4-6 disclosed in the description. The invention describes a polynecleotide molecule coding the antibody under the invention or its active fragment, a pharmaceutical composition based on said antibody or its active fragment exhibiting human IL2 neutralising action, and also an application of said antibody or its active fragment or the polynecleotide molecule coding it for preparing a drug which optionally contains an auxiliary anti-inflammatory or anticancer agent for treating inflammatory diseases or tumours, respectively.

EFFECT: production of the alternative specific IL2 activity inhibitors which directly bind with human IL2.

24 cl, 18 dwg, 4 tbl, 9 ex

 

This invention relates to antibodies and their fragments, which are specifically associated with human cytokine IL2 (interleukin 2). The invention additionally relates to polynucleotides, encoding such antibodies and fragments thereof, to pharmaceutical compositions containing them, and to medical applications, comprising such antibodies and their fragments.

Human IL2 is a protein of 133 amino acids (of 15.4 kDa), which has no significant sequence homology with any other factors. IL2 is synthesized as the precursor protein of 153 amino acids with the first 20 aminobenzamide amino acids that functions as a hydrophobic secretory signal sequence. This protein contains a single disulfide bond (connecting position Cys58/105), essential for biological activity.

Biological activity of IL2-mediated membrane receptor, which is expressed almost exclusively on activated but not in resting stage (Go), The t-cells. Full IL2-receptor consists of three transmembrane protein subunit of the type I: alpha, beta and gamma; functional receptor with lower affinity can be formed only beta - and gamma-receptor proteins. B-cells in the stage of Goand mononuclear leukocytes in the stage of GoR is the RCD Express this receptor. The expression of IL2-receptor, in particular alpha-subunit, is modulated by many factors, such as IL5, IL6 and inducer L2R/p55.

Both murine and human IL2 cause proliferation of T cells homologous species with high efficiency. Human IL2 is also functional in mouse cells, but not Vice versa. IL2 is a growth factor for all subpopulations of T-lymphocytes. It is a factor antigen-specific proliferation of T cells, which induces the progression of the cell cycle in cells in the stage of Goand, thus, provides clonal reproduction activated T-lymphocytes. IL2 also stimulates the proliferation and differentiation of activated b-cells. As in the case of proliferation of T-cells, this activity also requires the presence of additional factors, such as IL4.

Due to these effects on T-cells and b-cells IL2 is a Central regulator of immune responses. The Central value of IL2 in the initiation and strengthening of adaptive immune responses is well illustrated clinical efficacy of medicines, which are often used to suppress unwanted immune responses such as, for example, when rejection of transplants. Immunosuppressive drugs cyclosporin a and FK506 (tacrolimus) ingibiruet the production of IL2 by disrupting signaling through the T-cell receptor, whereas rapamycin (sirolimus) inhibits signaling through the IL2-receptor. Cyclosporine a and rapamycin act synergistically to limit immune responses by preventing propagation of clones of T cells that run IL2. However, all these compounds targeted to intracellular signaling pathways that inhibit not exclusively, IL, but also other factors. This implies that the clinical application of these drugs involves a significant risk of unwanted side effects because of their limited specificity to the target.

In this area also known for the numerous examples of antibody inhibitors of the activity of IL2, for example, a commercially available antibody daclizumab (Zenapax®, Protein Design Lab, Inc.). However, the known antibody inhibitors of the activity of the IL2 exert their biological effects by binding with IL2-receptor, but not with the antigen. Given the important clinical application of inhibitors of the activity of IL2, the present invention is to offer an alternative specific inhibitors of the activity of IL2.

Accordingly, according to one aspect of the present invention proposed humanitariannet monoclonal antibody or fragment that specifically bind to the human interleukin-2 (IL2), where the specified humanitariannet monoclonal antibody which neutralizes the activity of human IL2 by binding with the specified human IL2 before, during and/or after bonding of the specified human with human IL2 IL2-receptor, and where the variable region of the light chain of the specified gumanitarnogo monoclonal antibodies contains in its second frame region sequence of contiguous amino acids KARKA, preferably at amino acid positions 42-46.

The terms "humanitariannet monoclonal antibody"or "humanitariannet antibody"or "humanitarianly immunoglobulin"or grammatically related options, as they are used here, are used interchangeably in relation to a molecule containing the antigen-binding site derived from one or more than one immunoglobulin, non-human, and this molecule further comprises at least part of, for example, at least one of the frame regions of the variable domain of the light or heavy chains derived from one or more than one human immunoglobulin or germline sequences. The term "humanitariannet antibody", as used here, includes humanitarianly variable domain light chain immunoglobulin and humanitarianly variable domain of the heavy chain of immunoglobulin. Humanitariannet antibody can include a constant region, partially or on the whole of the action (including synthetic analogs) from one or more than one sequence of the human gene. It is expected that humanitariannet antibody will bind to the same target antigen, and that the donor antibody, which provides CDR. Typically, all of the segments or sections of gumanitarnogo antibody or immunoglobulin, except CDR, are essentially identical or substantially homologous to the corresponding segments or parts of naturally occurring or consensus sequences of human immunoglobulins.

Variable region light chain (VL) of the specified gumanitarnogo monoclonal antibodies contains in its second frame region sequence of contiguous amino acids KARKA, preferably at amino acid positions 42-46. This preferred sequence numbering, i.e. provisions 42-46, refers to the numbering as set forth in database "VBase" (© MRC Centre for Protein Engineering), available at the Internet address http://www.mrc-cpe.cam.ac.uk. For clarity, sequence alignment frame VL-regions (such as the sequence V-Kappa and V-lambda) and a VH-region of the germ line is incorporated into the present application in the form as they are in Vbase (see respectively 7, 8 and 9; in particular, Figa and 9a in respect of the numbering of the second frame regions of light chains in human sequences V-Kappa and V-lambda germline).

While here privatradiostationen numbering (i.e. the provisions of amino acids 42-46) amino acid sequence KARKA (i.e. Lys-Ala-Pro-Lys-Ala), for ease of correlation with the above-quoted reference should be understood that the nature of the amino acids in this partial sequence, not the sequence numbering in it and is itself the sequence is crucial for activity gumanitarnogo monoclonal antibodies according to the invention. As is known to the person skilled in the art, there are many ways numbering sequence of the human antibody germline, and the above-quoted reference (VBase) is only one of them. Therefore, a partial amino acid sequence KARKA contained in the second frame region of some variable regions of the light chain of the human germ line can be assigned to a different source under a different numbering system than the one defined in the above link. In this case, the partial amino acid sequence KARKA would carry numbering, other than the provisions of the preferred amino acids 42-46, whereas the sequence corresponding to the preferred amino acid positions 42-46 thus another way of numbering probably would have been amino acid sequence that differs from KARKA. In this case, as nudeporno specialist in this field, partial amino acid sequence with the "correct" sequence (KARKA), but with deviating numbering (any other than the preferred amino acid positions 42-46) should be considered as significant distinctive feature of the present invention, and not the other partial amino acid sequence, the numbering of which is "correct" (the preferred amino acid positions 42-46), but the nature of which is different from KARKA.

Suddenly discovered that antibodies or fragments thereof, having the consensus sequence KARKA in the second frame region of the light chain, in particular do not have in this segment terminal alanine residue, can specifically bind with IL2, but not able to neutralize its activity. This takes place especially when the CDR region contained in the variable regions of light and heavy chains gumanitarnogo monoclonal antibodies, are such as represented in SEQ ID nos 1-3 (respectively for CDR 1-3 variable region of the light chain) and SEQ ID nos 4-6 (respectively for CDR 1-3 variable regions of the heavy chain). Without being bound by theory, the authors present invention attributed this loss of neutralizing activity with the exception of the consensus sequence KARKA, in particular, with the substitution terminal residue Ala is in this segment by another amino acid, other than alanine, destabilization and/or conformational alterations adverse effect on neutralizing, but not binding activity.

The term "specifically binds" or grammatically related expressions such as "specific binding", "specific binding", "specific binder" etc. as they are used here, refers to the ability gumanitarnogo monoclonal antibody or its fragment to distinguish human IL2 and any number of other potential antigens other than human IL2, to such an extent that from a pool of many different antigens as potential binding partners contacts or substantially contacts only human IL2. Within the value on a given invention of the human IL2 "significantly" associated when in the pool of many equally available to different antigens as potential binding partners of human IL2 binds at least 10 times, preferably 50-fold, most preferably 100 times or more (in the kinetic sense)than other antigens other than human IL2. As it is clear to the ordinary person skilled in the art, such kinetic measurements can be carried out, for example, on the installation Biacore.

Humanitariannet antibody, Riego fragment according to this invention is monoclonal. The term "monoclonal" as it is used here, should be understood as having a value, usually attributed to him in this area, namely, an antibody that recognizes a single epitope on the linked antigen. It differs in this way from a polyclonal antibody, which is a set of different antibodies bind to the same antigen, albeit with different epitopes on the antigen. For this reason, one molecule of the antigen can contact many molecules polyclonal antibodies specific to that antigen, but only one molecule of specific monoclonal antibodies specific to that antigen; after binding one molecule monoclonal antibodies linked epitope is blocked and therefore no longer available to bind another molecule identical monoclonal antibodies. Monoclonal nature of antibodies makes it particularly suitable for use as a therapeutic agent, such as antibody will be in the form of a single homogeneous molecular particles, which can be well characterized, reproducible and get clear. These factors result in a product, the biological activity of which can be predicted with a high level of accuracy, which is a very important facto the Ohm, if such molecules are going to get the approval of the regulatory body for therapeutic introduction of mammals, in particular humans.

The term "neutralization", "Converter", "neutralised" and grammatically related options in the form as it is used here, refers to the partial or complete attenuation of biological(their) effect(s) IL2. Such partial or complete attenuation of biological(their) effect(s) IL2 is the result of the modification, interruption and/or cancellation of IL2-mediated signal transduction, which is manifested, for example, intracellular signaling, cell proliferation, release of soluble substances, increasing or decreasing regulation of intracellular activation of genes, for example, which leads to the expression of surface receptors for ligands other than IL2. As is clear to a person skilled in the art, there are many ways of determining whether the agent, for example under consideration, the antibody or its fragment, be classified as a neutralizer. Usually this can be done standard test in vitro, which is usually carried out as follows. In the first experiment proliferation of the cell line for which it is known that the level of proliferation depends on the activity of IL2, incubated in a series of samples with varying what koncentracije IL2, then measure the level of proliferation of this cell line. From this measurement to determine the concentration of IL2, which provided premaxillary cell proliferation. Then spend the second experiment proliferation using in each series of samples of the same number of cells, which is used in the first experiment proliferation, concentrations of IL2 defined above, and, this time, varying concentrations of the antibody or its fragment, which, as I suspect, is a neutralizer for IL2. Again measure cell proliferation to determine the concentration of the antibody or its fragment sufficient for implementation premaxillae inhibition of growth. If the resulting graph of inhibition of growth relative to the concentration of the antibody (or fragment) has a sigmoidal shape, was then carried out some degree of antibody-dependent growth inhibition, i.e. the activity of the IL2 was reduced somewhat. In this case, the antibody or its fragment should be considered as a "Converter" in the context of the present invention. One example of a cell line, for which it is known that the level of proliferation depends on the activity of IL2 is the cell line CTLL-2, commercially available from LGC Promochem. Another example of a suitable cell line is NK92 (DSMZ).

Neo is idunno, humanitariannet monoclonal antibody according to the invention neutralizes the activity of human IL2 by binding with the specified human IL2 before, during and/or after bonding of the specified human with human IL2 IL2-receptor. This method of neutralization is quite unexpected. Traditionally, antibody-mediated neutralization of biological activity of the ligand, where the specified biological activity depends on the binding of the indicated ligand to the receptor, by preventing the formation of such ligand-receptor complex. According to this classical scenario neutralizing neutralizing antibody binds to the ligand or receptor in the section on the contact boundary between ligand and receptor. Thus, the presence of antibodies to sterically and/or electrostatically prevents the formation of ligand-receptor complexes: ligand-receptor complex is not formed, and biological activity, usually attributed to binding of ligand to its receptor, is not implemented.

Methods of neutralization observed for gumanitarnogo monoclonal antibodies according to this invention, differs sharply from that classic scenario that the abolition of the biological activity, usually attributed to IL2, does not depend on preventing the formation of a complex between IL2 and its prescriptions the op. This means that the biological activity of IL2 is cancelled regardless of whether IL2 is already associated with IL2-receptor or not, implying that the epitope recognized humanized monoclonal antibody according to the invention, is not located on a plot of IL2, which interacts with IL2-receptor. Neutralization of IL2, as such, can be achieved with humanized monoclonal antibody according to the invention in several ways.

According to the first method, the antibody binds with IL2 in the solution prior to formation of the complex between IL2 and its receptor, so that IL2-mediated signal transduction does not occur when IL2 binds to its receptor.

According to the second method, the antibody binds with IL2 simultaneously with the formation of the complex IL2 with its receptor. Here the simultaneous formation of a ternary complex receptor-IL2-antibody again does not lead to any, or at least to any significant signal transduction.

According to the third method of IL2 already formed a complex with its receptor, and antibody binds to IL2, while IL2 is in complex with its receptor on the cell surface, the carrier IL2-receptor. In this third scenario, any IL2-mediated signal transduction that already have a place to bind IL2 antibody is cancelled as soon as suasive the camping antibody.

Such non-classical neutralization, i.e. neutralization, as it is with humanized monoclonal antibody according to the invention is quite unexpected and has several therapeutic benefits.

First, because humanitariannet monoclonal antibody of the invention recognizes an epitope IL2, which is not directly involved in contact with IL2-receptor, between therapeutic antibody, on the one hand, and IL2-receptor, on the other hand, there is competition. The effect is that the patient can enter a lower concentration of therapeutic antibodies, than would be possible otherwise, if between the antibody and IL2-receptor competed for binding to the same epitope. This effectively increases therapeutic efficacy gumanitarnogo monoclonal antibodies according to the invention, since it is possible to reduce the input concentration (relative to that required when the condition of the classical method of neutralization) without loss of biological effect. The introduction of a smaller amount of therapeutic agent is highly desirable not only from the point of view of the tolerability of the patient, but also from an economic point of view, as the cost burden of this therapy is reduced or, on the other hand, a greater num which patients may benefit from a given amount of therapeutic antibodies.

Secondly, as mentioned above, the ability gumanitarnogo monoclonal antibodies according to the invention to contact with IL2 and neutralize its biological activity in complex with its receptor has a huge advantage in what is already happening IL2-mediated signal transduction can be turned off without having initial dissociation IL2 from its receptor-binding partner. The final effect of this is that the desired neutralizing the biological activity gumanitarnogo monoclonal antibodies according to the invention is implemented in vivo more rapidly than is possible for other "classic" antibody-converters, which must first compete with IL2-receptor for the bindable epitope on IL2 before you cause any therapeutic effect. This speed can be especially useful in acute scenarios, such as immune rejection of transplants of organs, which is known for anti-IL2 therapy.

The third advantage of this unusual method of neutralization, as described above, refers to the fact that IL2 receptors are localized on the surface of T cells. T cells themselves produce IL2 and respond to IL2 proliferation, potentsiruya thus their own proliferation. When some of the acute inflammatory reaction is s, such as tissue rejection after transplantation, it is desirable not only to reduce the magnitude of the inflammatory response, attributed to the existing T-cells, but also to reduce the number of T cells, generating an immune response. Humanitariannet monoclonal antibody according to the invention is particularly effective in achieving this goal. As explained above, the biological activity of IL2, already associated with its receptor on the surface of T cells, will be canceled. However, upon such termination, humanitariannet monoclonal antibody according to the invention will usually be associated with IL2 (which itself is associated with IL2-receptor) for some period of time, programming, thus, T-cell destruction by antibody-dependent cellular cytotoxicity ("ADCC", antibody-dependent cellular cytotoxicity). When ADCC cell-target, which is covered with immunoglobulin, killed effector cell Fc receptors that recognize the Fc-part of an immunoglobulin, covering the target cell. In most cases, the effector cells involved in ADCC, are natural killer ("NK", natural killer)that bear on its surface, among others, Fc receptor Fc gamma RIII. Thus killing cells coated with immunoglobulin, so that the specificity of killing cells directly correlates with specificness is using antibody binding sites. Then in the context of the present invention T-cells, which are decorated with humanized monoclonal antibody according to the invention through IL2 in complex with its receptor, become target cells in the above sense, then lyse, among others, NK-cells. This effect is rapid and effective immune response, attributed to cells bearing IL2 receptors, such as T-cells.

According to one embodiment of the present invention at least one of the first, third and/or fourth region(s) of the frame region of the light chain contained in a human monoclonal antibody or its fragment complies(comply) with the human germline sequence for this area/these areas.

According to another embodiment of the present invention variable region light chain gumanitarnogo monoclonal antibody or its fragment according to the invention contains in its CDR1 region amino acid sequence represented in SEQ ID NO:1. According to another embodiment of the present invention variable region light chain gumanitarnogo monoclonal antibody or its fragment according to the invention contains in its CDR2 region, the amino acid sequence represented in SEQ ID NO:2. According to another embodiment of the present invention, variable about the art light chain gumanitarnogo monoclonal antibody or its fragment according to the invention contains in its CDR3 region amino acid sequence, presented in SEQ ID NO:3.

According to another embodiment of the present invention variable region light chain gumanitarnogo monoclonal antibody or its fragment according to the invention further comprises in its CDR1 region amino acid sequence represented in SEQ ID NO:1, in its CDR2-region amino acid sequence represented in SEQ ID NO:2, and in its CDR3-region amino acid sequence represented in SEQ ID NO:3.

According to another embodiment of the present invention the variable region of the heavy chain contains in its CDR1 region amino acid sequence represented in SEQ ID NO:4. According to another embodiment of the present invention the variable region of the heavy chain contains in its CDR2 region, the amino acid sequence represented in SEQ ID NO:5. According to another embodiment of the present invention the variable region of the heavy chain contains in its CDR3 region amino acid sequence represented in SEQ ID NO:6.

According to another embodiment of the present invention the variable region of the heavy chain contains in its CDR1 region amino acid sequence represented in SEQ ID NO:4, in its CDR2-region amino acid sequence represented in SEQ ID NO:5, and in its CDR3-region amino acid sequence represented in SEQ ID NO:6.

According to D. the natives embodiment of the present invention variable region light chain gumanitarnogo monoclonal antibody or its fragment according to the invention further comprises in its CDR1 region amino acid sequence, presented in SEQ ID NO:1, in its CDR2-region amino acid sequence represented in SEQ ID NO:2, and in its CDR3-region amino acid sequence represented in SEQ ID NO:3, and the variable region of the heavy chain contains in its CDR1 region amino acid sequence represented in SEQ ID NO:4, in its CDR2-region amino acid sequence represented in SEQ ID NO:5, and in its CDR3-region amino acid sequence represented in SEQ ID NO:6. It was found that these CDR region are especially useful when linking with IL2 and neutralize the biological effect of IL2 in the manner described above.

According to another embodiment of the present invention, the amino acid sequence of the first frame region of the light chain, the remaining amino acid sequence of the second frame region of the light chain and the amino acid sequence of the third frame region of the light chain match any of the sequences of subgroups VKI human germline loci in ø12, O2, O18, O8, A30, L1, L15, L4, L18, L5, L19, L8, L23, L9, L11 or L12; or subgroup VL1 human germ line 1A locus; or any of the sequences of subgroups VL2 human germline loci in 2C, 2E, a or 2b2. In this embodiment the expression "the rest of the amino acid sequence of the second frame region Le is coy chain" refers to those amino acids in the second frame region of the light chain, which differ from the sequence KARKA. Thus, when using numbering database Vbase expression "the rest of the amino acid sequence of the second frame region of the light chain" refers to amino acids in the provisions of the second frame region of the light chain 35-41 and 47-49, inclusive, regardless of whether the frame region of this light chain V-Kappa or V-lambda frame area (see Figa and 8A in respect of the numbering sequences of the human germline, referring respectively to the frame region V-Kappa and V-lambda). Preferred in this embodiment is an optional inclusion in the fourth frame region light chain sequence corresponding to the sequence found in the sequence JK4 human germ line, in particular FGGGTKVEIK.

Other amino acid sequences that are suitable for use as the fourth frame region of the light chain include, but are not limited to FGQGTKVEIK, FGQGTKLEIK, FGPGTKVDIK, FGQGTRLEIK, FGTGTKVTVL, FGGGTKLTVL and FGGGTQLTVL.

According to another embodiment of at least one of the first, second and/or third frame region(s) of the heavy chain contained in a human monoclonal antibody or its fragment corresponds to the sequence of this area/these areas in the human embryo is the second line.

According to another embodiment of the amino acid sequence of the first frame region of the heavy chain, the amino acid sequence of the second frame region of the heavy chain and the amino acid sequence of the third frame region of the heavy chain matches any sequence of subgroups human VH3 germline, in particular in the locus 3-07, where amino acid sequence of the first frame region of the heavy chain is a EVQLVESGGGLVQPGGSLRLSCAASGFTFS, the amino acid sequence of the second frame region of the heavy chain is a WVRQAPGKGLEWVA, and amino acid sequence of the third frame region of the heavy chain is a RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR. Preferably, you can choose the amino acid sequence of the fourth framework region of the heavy chain, for example, in combination with three frame sequences cited above, the locus 3-07 the germ line of one of the following sequences: WGQGTLVTVSS, WGRGTLVTVSS, WGQGTMVTVSS, WGQGTLVTVSS, WGQGTLVTVSS and WGQGTTVTVSS.

In the preferred embodiment humanitariannet monoclonal antibody or its fragment contains a variable region light chain containing the amino acid sequence represented in SEQ ID NO:7 and the variable region of the heavy chain containing the amino acid sequence is lnost, presented in SEQ ID NO:8. Especially preferred is humanitariannet monoclonal antibody, which contains a light chain containing the amino acid sequence represented in SEQ ID NO:9 and a heavy chain containing the amino acid sequence represented in SEQ ID NO:10. Next humanitariannet IgG1 antibody against IL1, containing SEQ ID NO 9 and 10 and/or SEQ ID NO 7 and 8, will be referred to as "Ant-IL2".

This humanitariannet monoclonal antibody can be in the form of IgG antibodies, in particular antibodies IgG1 or IgG4. As is well known in this field, IgG contains not only the variable regions of the antibodies responsible for the highly selective recognition and binding to the antigen, but also the constant region of the heavy and light polypeptide chains of antibodies are usually present in endogenously produced antibodies, and, in some cases, even the decoration in one or more than one site carbohydrates, and this glycosylation is usually present on the Fc-plot of IgG. It is known that these Fc-sections cause different effector functions in vivo, such as ADCC and complement-dependent cytotoxicity ("CDC"). The ADCC mechanism described here above. When CDC two identical immunoglobulin associated with two identical antigens (for example, in the case of IL2 in T-cell) on the surface of target cells, so that the corresponding Fc-plots are very close to each other. This scenario involves proteins of the complement, among them complement proteins such as C1q, C3, C4 and C9, the latter of which creates a pore in the target cell. Cell-target dies as a result of this perforation. At the same time, the cell(s)target(s) also R(I)has been furnished in other sections on its/their surface(s). This decorating involves effector cells, which then kill the cell(s)target(s) in a manner analogous to the method described above in the context of the mechanism of ADCC (see, for example, Gelderman et al. (2004), Trends in Immunology 25, 158-64).

Preferably, the IgG antibody is an IgG1 antibody or antibody IgG4, formats that are preferred, since their mechanism of action in vivo is particularly well understood and characterized. This is especially true for IgG1 antibodies.

According to another embodiment of the present invention the fragment gumanitarnogo monoclonal antibodies can be a scFv, a single domain antibody, Fv, ditelo, tandem diatel, Fab, Fab' or F(ab)2. These formats can usually be divided into two subclasses, namely the subclass formats that consist of a single polypeptide chain, and a subclass of format, that contain at least two polypeptide chains. The members of the first subclass includes scFv (including one VH region and VL region, United in a single polypeptide chain pose the STV polypeptide linker); and a single domain antibody (comprising a single variable domain antibody that specifically binds human IL2). Members of the latter subclass include Fv (including one VH region and the VL-region as a separate polypeptide chains, which ecovalence related to each other); ditelo (including two ecovalence associated polypeptide chains, each of which contains two variable regions of the antibodies typically one VH and one VL on the polypeptide chain and arranged so that when non-covalent connection VH on a single polypeptide chain with VL corresponding to the other polypeptide chain and Vice versa is formed molecule bivalent antibodies); tandem ditelo (bespecifically single-chain Fv-antibodies containing four covalently linked variable immunoglobulin VH - and VL-region of two different specificdate forming glycosilated, which is twice more than ditelo described above); Fab (containing a single polypeptide chain, the entire light chain of the antibody, which itself contains a VL-region and a constant region light chain and as the other polypeptide portion of the heavy chain of the antibody containing the entire VH region, and part of the constant region of the heavy chain, and these two polypeptide chains linked through messagewall disulfide bond; Fab' (same as Fab, described above, except that the heavy chain of the antibody contains additional restored disulfide bond); and F(ab)2 (containing two molecules of Fab', and each molecule of Fab' is associated with the other molecule Fab' by miaocheng disulfide bonds). In General, fragments of the antibodies described herein above types provide more flexibility in the formation of, for example, the pharmacokinetic properties of the antibodies are desirable for therapeutic administration, in accordance with the particular existing queries. For example, it may be desirable to reduce the size entered antibodies in order to increase the degree of penetration into the tissue in the treatment of tissue for which it is known that they are poorly vascularsurgery (for example, joints). In some circumstances it may also be desirable uvelichenie the speed at which therapeutic antibody is eliminated from the body, and the specified speed is usually faster by reducing the size of the injected antibodies.

According to another embodiment of the present invention humanitariannet monoclonal antibody may be present in monovalent monospecific or multivalent mono - or multispecificity, in particular, divalent mono - or bespecifically forms. In General, nohoval ntoe monospecific, in particular bivalent monospecific antibody may provide a therapeutic advantage in that the neutralization done this antibody increases the avidity effects, i.e. the linking of many molecules of the same antigen, here human IL2, the same antibody. Several monovalent forms monospecific antibodies according to the invention described above (for example, scFv, Fv, or a single domain antibody). Multivalent multispecificity, in particular the divalent bespecifically form gumanitarnogo monoclonal antibodies against human IL2 according to the invention can include a whole IgG, in which one binding domain binds to human IL2, whereas the second binding domain binds to a different antigen than the human IL2. Other multivalent multispecific, in particular the divalent bespecifically form can mainly be humanitariannet single-stranded bespecifically antibody, i.e. the construction of recombinant gumanitarnogo antibodies containing two structures scFv as described above United into one continuous polypeptide chain short polypeptide spacer between the two entities scFv, as is well known in this field. Here is one of scFv-plot especifismo single-chain antibodies containing the different in this bespecifically single-chain antibody, will specifically bind human IL2, as described above, while the other corresponding scFv-plot of this especifismo single-chain antibodies will bind to a different antigen for which it is determined that he will have a therapeutic benefit.

According to another embodiment of gumanitarnogo monoclonal antibody or its fragment may be derived, for example, organic polymer, for example with one or more than one molecule of polyethylene glycol ("PEG"). As is well known in this field, such receiving derivatives can be useful for modulation of the pharmacodynamic properties of antibodies or their fragments.

scFv is especially preferred (monovalent monospecific) fragment of an antibody, particularly scFv containing the amino acid sequence represented in SEQ ID NO:11 or SEQ ID NO:12.

According to another aspect of the present invention proposed a human monoclonal antibody or its fragment containing amino acid sequence having at least 70%homology, preferably at least 80, 90 or even more than at least 95%homology with the amino acid sequence represented in any of SEQ ID nos:1-12. Homology can be determined by standard programs for sequence alignment, the mi as Vector NTI (InforMax™, Maryland, USA). Such programs are comparing the aligned sequences according to the principle of the amino acid at amino acid, and can set different sensitivity levels for comparison (for example, identical to the amino acid that is a conservative amino acid substitution, etc.). In fact, as here used this term, consider two amino acids are considered "conservative substitution" of each other if they belong to the same major group. As a non-limiting example, two different amino acids belonging to the group of nonpolar amino acids, would be considered "conservative substitutions" each other, even if these two amino acids would not be identical, whereas non-polar amino acid, on the one hand, and the essential amino acid, on the other hand, was not considered to be "conservative substitution" of each other. Panel 3.1 "Molecular Biology of the Cell", 4thEdition (2002), by Alberts, Johnson, Lewis, Raff, Roberts and Walter divides amino acids into four main groups: acidic, non-polar, uncharged polar and basic. This grouping can be used to determine, for the purposes of the present invention, whether a particular amino acid "conservative substitution" is another question amino acids.

According to another aspect of the present invention proposed polynucleotide molecule. This polynucleotide mo is Akula contains the nucleotide sequence, encoding the amino acid sequence represented in any of SEQ ID nos:1-12, or the nucleotide sequence showing at least 60%, preferably 65, 70, 75, 80, 85, 90 or 95%homology with the specified nucleotide sequence. Here, the homology can be determined by comparing the polynucleotide molecule containing the nucleotide sequence encoding the amino acid sequence of any of SEQ ID nos:1-12, with a polynucleotide molecule having considered the nucleotide sequence ("test sequence"), by sequence alignment, and where the nucleotide in the test sequence is considered to be homologous if it is either identical to the corresponding nucleotide in the nucleotide sequence that encodes the appropriate amino acid sequence of any of SEQ ID nos:1-12, or if one or more than one nucleotide variance in the test sequence from the corresponding(s), nucleotide(s) in nucleotide sequence, the coding amino acid sequence of any of SEQ ID nos:1-12, leads to the formation of a triplet of nucleotides that, when broadcast network amino acid that is either identical (due to the degenerate triplet) corresponding amino acid in the corresponding amino acid is the sequence of any of SEQ ID nos:1-12, or a conservative substitution. Here, the term "conservative substitution" should be understood as described above.

According to another aspect of the present invention proposed a pharmaceutical composition comprising humanitariannet monoclonal antibody or its fragment, or a polynucleotide molecule having a nucleotide sequence encoding the amino acid sequence represented in any of SEQ ID nos:1-12, or encoding the amino acid sequence containing amino acid sequence having at least 70%homology with any of SEQ ID nos:1-12, where "homology" is to be understood, as explained here above.

According to this invention, the term "pharmaceutical composition" refers to compositions for administration to the patient, which are mammals, primarily the individual who is the man. In the preferred embodiment, this pharmaceutical composition comprises a composition for parenteral injection or infusion. Such parenteral injection or infusion can take advantage of the process of resorbtive in the form of, for example, intradermal, subcutaneous, intramuscular and/or intraperitoneal injection or infusion. Alternatively, such a parenteral injection or infusion can bypass the processes of resorbtive and be in the form of, for example, intracardiac,intra-arterial, intravenous, vnutribolnychnoy and/or intracranial injection or infusion. In another preferred embodiment, this pharmaceutical composition comprises a composition for injection through the skin. One example of insertion through the skin skin is the introduction, in which the pharmaceutical composition is applied, for example, in the form of a solution, suspension, emulsion, foam, rubbing, ointments, pastes and/or patch on the skin. Alternatively, the introduction of the pharmaceutical composition can be accomplished through one or more than one mucous membrane. For example, the introduction may be transbukkalno, lingual or sublingual, i.e. through the mucous(s) cover(s) of the mouth and/or tongue, and may be, for example, in the form of tablets, lozenges, coated with sugar pills (i.e. pills) and/or mouthwash. Alternatively, the introduction may be enteral, i.e. through the mucous(s) cover(s) of the stomach and/or intestinal tract, and may be, for example, in the form of tablets, coated sugar pill (i.e. tablets), capsules, solution, suspension and/or emulsion. Alternatively, the introduction may be rectal and may be, for example, in the form of suppositories, rectal capsules, and/or ointments, or rubbing. Alternatively, the introduction may be intranasal and may be, for example, in the form of a cap is l, ointment or rubbing and/or spray. Alternatively, the introduction may be pulmonary, i.e. through the mucous(s) cover(s) of the bronchi and/or alveoli, and can be, for example, in the form of an aerosol and/or inhalation. Alternatively, the introduction may be conjunctival and may be, for example, in the form of eye drops, ointments for the eyes and/or rinsing eye. Alternatively, the introduction can be carried out through the mucous(s) cover(s) of the urogenital tract, for example, can be intravaginal or intraurethral, and may be, for example, in the form of a suppository, ointment and/or pencil. It should be understood that the above alternatives introduction are not mutually exclusive, and that the combination of any of them may be effective treatment regimen.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the field and include physiological solutions with phosphate buffer, water, emulsions such as emulsions of oil/water, different types of humidifiers, sterile solutions, etc. Compositions containing such media can be prepared in the form of drugs is well known in traditional ways. This is farmacevticheskie song you can enter the subject in a suitable dose. Dosage schedule will be determined by the attending physician and clinical factors. As is well known in the field of medicine, dosage for any patient depends on many factors, including the size of the patient, the surface area, age, the particular compound to be introduction, sex, time and route of administration, General health and other drugs that are administered simultaneously. For example, preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, emulsions and liposomes. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters, such as etiloleat. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered environment. Fillers suitable for conventional parenteral administration include sodium chloride, dextrose and ringer, dextrose and sodium chloride, lactate ringer's or fixed oils. Fillers suitable for intravenous or intraarterial administration include liquid and nutritious fillers, electrolyte fillers (such as fillers on the basis of dextrose ringer's) and the like. Can also contain preservatives and other doba is key, such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases and the like. In addition, the pharmaceutical composition of the present invention may contain protein carriers, such, for example, serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the pharmaceutical composition according to the invention, in addition to gumanitarnogo monoclonal antibody or its fragment (as described in this invention may contain additional biologically active agents, depending on the intended use of this pharmaceutical composition. Such agents can represent medicines acting on the gastrointestinal system, drugs that act as drugs, medicines, preventing hyperuricemia, drugs inhibiting immunoreactive (e.g., corticosteroids), medicines, modulating inflammatory response, medicines acting on the circulatory system, and/or such agents as cytokines, are known in this field.

According to another aspect of the present invention proposed the use of gumanitarnogo monoclonal antibody or its fragment, as described above, or polynucleotide the molecule, as described above, in the manufacture of a medicinal product may contain one or more than one additional anti-inflammatory agent for the treatment of inflammatory diseases in mammals, preferably humans. Mostly such inflammatory diseases selected from the group consisting of rheumatoid arthritis (RA), asthma, multiple sclerosis (MS), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (IBD), uveitis, macular degeneration, colitis, psoriasis, Malerovskoj degeneration, antiphospholipid syndrome (APS), acute coronary syndrome, restenosis, atherosclerosis, recurrent polychondritis (RP), acute or chronic hepatitis, unsuccessful implantation of orthopedic implants, glomerulonephritis, erythematosus, autoimmune disorders, acute pancreatitis, or ankylosing spondylitis (AS).

According to another aspect of the present invention proposed the use of gumanitarnogo monoclonal antibody or its fragment, as described above, or a polynucleotide molecule as described above, in the manufacture of a medicinal product may contain one or more than one additional anticancer agent for treatment of tumor is th disease or other condition with delayed apoptosis of cells, increased survival or cell proliferation in mammals, preferably humans. Preferably the tumor disease is a cancer, and specified preferably cancer is a leukemia, multiple myeloma, gastric carcinoma or carcinoma of the skin.

According to another aspect of the present invention, a method for treatment of inflammatory diseases, in which humanitariannet monoclonal antibody or its fragment, as described above, or a polynucleotide molecule, as described above, introducing (possibly together with one or more additional anti-inflammatory agent) to a mammal, preferably to a subject, which is man, in sufficient quantity and for a sufficient period of time to prevent and/or facilitate specified inflammatory diseases. Mostly such inflammatory diseases selected from the group consisting of rheumatoid arthritis (RA), asthma, multiple sclerosis (MS), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (IBD), uveitis, macular degeneration, colitis, psoriasis, Malerovskoj degeneration, antiphospholipid syndrome (APS), acute coronary syndrome,restenosis, atherosclerosis, recurrent polychondritis (RP), acute or chronic hepatitis, unsuccessful implantation of orthopedic implants, glomerulonephritis, lupus, autoimmune disorders, acute pancreatitis, or ankylosing spondylitis (AS).

According to another aspect of the present invention, a method for treatment of neoplastic disease where humanitariannet monoclonal antibody or its fragment, as described above, or a polynucleotide molecule, as described above, introducing (possibly together with one or more than one additional anticancer agent) to a mammal, preferably to a subject, which is man, in sufficient quantity and for a sufficient period of time to prevent and/or facilitate specified neoplastic disease or other condition with delayed apoptosis, increased survival or proliferation of cells. Preferably the tumor disease is a cancer, and specified preferably cancer is a leukemia, multiple myeloma, gastric carcinoma or carcinoma of the skin.

The invention will now be illustrated by the following non-limiting graphical materials and examples. A brief description of the figures is given below:

Figure 1. Persistent binding to the antigen after the gum is establishing a VL-region.

Figure 2. The loss of neutralizing activity after humanization of the VL-region.

Figure 3. The binding of IL2 is not affected by the substitutions in the human/mouse frame regions in the VL-region.

Figure 4. The loss of neutralizing activity after the introduction of the second frame region of the light chain of a human.

Figure 5. Amino acid substitutions at positions 42-46 VL-region (in the second frame region of the light chain) do not affect antigen binding.

6. Mutation of leucine to alanine at position 46 of the second frame region of the light chain leads to the restoration of neutralizing activity.

Figa. Amino acid sequences of human germline for the first and second frame regions of the light chain (V-Kappa). CDR regions were excluded; the rest of the numbering of the remaining frame is the same as published in the database of the online "Vbase" (see above Web link).

Figb. Amino acid sequences of human germline for the third frame region of the light chain (V-Kappa). CDR regions were excluded; the rest of the numbering of the remaining frame is the same as published in the database of the online "Vbase" (see above Web link).

Figa. Amino acid sequences of human germline for the first and second frame regions of the light chain V-lambda). CDR regions were excluded;the rest of the numbering of the remaining frame is such, as published in the database of the online "Vbase" (see above Web link).

Figb. Amino acid sequences of human germline for the third frame region of the light chain (V-Kappa). CDR regions were excluded; the rest of the numbering of the remaining frame is the same as published in the database of the online "Vbase" (see above Web link).

Figa. Amino acid sequences of human germline for the first and second frame regions of the heavy chain. CDR regions were excluded; the rest of the numbering of the remaining frame is the same as published in the database of the online "Vbase" (see above Web link).

Figb. Amino acid sequences of human germline for the third framework region of the heavy chain. CDR regions were excluded; the rest of the numbering of the remaining frame is the same as published in the database of the online "Vbase" (see above Web link).

Figure 10. The binding specificity gumanitarnogo antibodies against IL2 "Anti-IL2 with limfomoj cell line NKL natural killer cells.

11. Anti-IL2 cancels IL2-dependent increase regulation of the expression of CD124 on the surface of cells CTLL-2.

Fig. Anti-IL2 specifically blocks the signal transduction IL2 below IL2-receptor in cell signaling pathways.

Fig. On the effectiveness of anti-IL2 and DACL Zumba differentially affects the level of expression of CD25.

Fig. Results showing the effect of anti-IL2 and daclizumab on IL2-dependent proliferation of primary human NK cells.

Fig. Results showing the effect of anti-IL2 and daclizumab on IL2-dependent release of IFN-gamma NK-cells.

Example 1: Obtaining antigen - human IL2 ("hIL2")

The aim of the experimental approaches described below in Examples 1A, 1B and 1C, were receiving recombinant IL2 antigenic material originating from different sources: antigen, expressed in prokaryotic cells, eukaryotic cells, and recombinant protein antigen is commercially available as a certified therapeutic agent.

Example 1A: Recombinant expression of the E. coli

Mature hIL2 (i.e. encoding amino acid residues APTSSS...IISTLT) cloned in a single open reading frame ("ORF") of prokaryotic expressing vector pBAD (Invitrogen), using standard PCR and molecular biology. At the 5'end were added three nucleotides coding for the methionine at the 3'end before the stop codon was incorporated nucleotide sequence that merges hexaglycine label with the end of the protein.

This construction was used to transform competent E. coli strain (strain BL21(DE3), Stratagene)using the instructions provided by the manufacturer. Bacteria you asifali in standard LB medium to a density OD(600 nm), equal to 0.5, then for 5 h was added L-arabinose to a concentration of 0.2% wt./about. in order to run the expression. Collection of E. coli was carried out by centrifugation at 10,000 g for 15 minutes Then received insoluble fraction (bullock inclusions), using reagent and Protocol BugBuster (Novagen) according to manufacturer's instructions.

Taurus inclusions were solubilizers in 6 M guanidine-hydrochloride ("GuHCl) and then diluted to 0.1 mg/ml with buffer containing 2 M GuHCl (pH of 8.0)/1 mm glutathione ocil./10 mm glutathione reset., and incubated for 16 h at 20°C. After incubation, the pH is brought to 6.0 by slow addition of glacial acetic acid with vigorous stirring. Finally, used three sequential chromatographic approach for obtaining highly purified and homogeneous protein preparation hIL2his: affinity chromatography with immobilized metal (IMAC), HPLC with reversed-phase, ion-exchange chromatography. The functionality of the purified protein was confirmed in the experiment on cell proliferation (see below).

Example 1B: Recombinant expression of mammalian cell

Mature hIL2 (i.e. encoding amino acid residues APTSSS...IISTLT) cloned in eukaryotic expressing vector pEFdhfr (Mack M, et al. (1995) PNAS 92, 7021-5), using standard PCR and molecular biology. At the 5'-end was added to the nucleus is IGNOU sequence, encoding a leader peptide of human IgG to ensure efficient processing and secretion, on the 3'-end before the stop codon was incorporated nucleotide sequence that merges hexaglycine label with the end of this protein.

The 293 cells (DSMZ, order code ESS) planted with such density to cover 25-35% of the surface of the Cup, and were cultured for 24 h and Then these cells were transfusional expressing vector pEFdhfr-hIL2, using reagent "Transfast" (Promega) according to manufacturer's instructions. After an additional 60-hour cultivation period were collected cell supernatant and protein hIL2-his was purified using IMAC with subsequent ion exchange chromatography. The functionality of the purified protein was confirmed in the experiment on cell proliferation.

Example 1B: Purchase Proleukin

Proleukin (prepared as recombinant hIL2, expressed in E. coli) were purchased from Chiron.

When using the above approaches have received three different sources fully functional recombinant antigen hIL2.

Example 2: Production gumanitarnogo monoclonal antibodies against hIL2

It was desirable to produce humanitariannet monoclonal antibody ("mAb") with a particularly appropriate way of action, which has as a specific target person is a mini-hIL2 and neutralizes its biological activity. Usually neutralizing mAb, having as target the secretory soluble protein such as a cytokine hIL2, recognize the epitope, which at least partially overlaps with the epitope recognized component of the corresponding cytokine receptor. Thus, mAb directly competes with the receptor for binding of the cytokine. This mechanism of action implies that it is possible to achieve effective neutralization. This mAb should be used in a high enough dose to compete for the cytokine receptor.

Example 2A: the starting point → commercially available monoclonal antibody against hIL2 in the form of protein

To achieve understanding of the extent to which different mAb against hIL2 could carry out neutralization, as mentioned above, received mAb against hIL2 by immunization of mice with subsequent selection of spleen cells and hybridoma merge, and everything was done according to standard protocols. In addition, acquired a commercially available mAb against hIL2. Used the pool of available mAb to compare the properties of different antibodies in three assays: binding to soluble antigen tested in ELISA (enzyme linked immunosorbent assay), binding to the antigen associated with the cell surface, tested in FACS, and the ability to neutralize the bioactivity hI2, tested using analysis of cell proliferation.

The ELISA was carried out as follows.

All incubation was carried out at 20°C. Used streptavidin coated 96-well plates to ELISA (Nunc) for attaching PEG-biotinylated Proleukin, 0.1 ág in 100 ál of PBS-TB (phosphate buffered saline, pH=7,4, 0,05% vol./about. Tween-20, 1% wt./about. BSA) per well for 30 minutes. The tablet then washed 3 times with 200 μl per well PBS-T (phosphate buffered saline, pH=7,4, 0,05% vol./about. Tween-20). Added samples of different mAb, 100 μl per well, and incubated the samples for 1 hour. The tablet then washed 3 times with 200 μl per well of PBS-T. Used in determining antibody was mAb goats against human IgG conjugated with HRP (Jackson Immunoresearch)diluted 1:1000 in PBS-TB, 100 μl per well, and incubation was carried out for 1 hour. The tablet then washed 3 times with 200 μl per well of PBS-T. Finally, to quantify the binding of an antibody to an antigen by incubation with the substrate for HRP: 100 µl of substrate buffer 2,2'-Azino-di[3-ethyl-benzothiazoline-6-sulfonic acid] ("ABTS") (Roche Diagnostics, tablets ABTS), and incubated tablet for 5-10 minutes until until developed green staining. This staining was measured at 405 nm onto the reader for 96-well plates.

Analysis of ACS was carried out as follows.

For optimal growth in culture conditions cells lymphoma cell line of human natural killer NKL depends on the presence of approximately 5 ng/ml hIL2 in the medium (minimal medium in the modification of Iscove (Biochrom AG); 10% vol./about. fetal bovine serum (Biochrom AG); 100 μg/ml penicillin/streptomycin (Biochrom AG). When preparing the experiment NKL cells, 1×106on ml, deprived of hlL2 for 24 hours by culturing in medium containing no hIL2. Immediately before the experiment, these cells were washed with medium not containing hIL2. All following incubation was performed at 4°C for 30 minutes; washing the used buffer PBS-F (phosphate buffered saline, 3% vol./about. fetal bovine serum) at 4°C. First 2×105NKL cells in 200 μl of medium were incubated with 1 μg of recombinant human hIL2 or left without hIL2 in the same conditions. Then washed the cells 3 times, each wash was carried out with 2 ml PBS-F. Then 2×105cells were incubated with different mouse mAb against hIL2, 1 μg in 200 μl of medium at 4°C for 30 minutes. These cells were again washed three times as above, and finally incubated with FITC-conjugated detecting mAb goat against mouse IgG (Jackson Immunoresearch), diluted 1:1000 in 200 µl of PBS-F. After three additional washes at the facility FACS analyzed tile is know fluorescence of the cells, holding hIL2 on its surface, compared to normal cells.

Analysis of cell proliferation was performed as follows.

For optimal growth in culture conditions mouse CTL cell line CTLL-2 (LGCPromochem) depends on the presence of approximately 5 ng/ml hIL2 in the medium (minimal medium in the modification of Iscove (Biochrom AG); 10% vol./about. fetal bovine serum (Biochrom AG); 100 μg/ml penicillin/streptomycin (Biochrom AG); 0.5 mm 2-mercaptoethanol (Gibco)). Both murine and human hIL2 work equally well, supporting the survival and proliferation of cells CTLL-2. In the preparation of the experiment, cells CTLL-2, 1×106on ml, deprived of hIL2 12 hours by culturing in medium containing no hIL2.

Immediately before the experiment, these cells were washed with medium not containing hIL2. For the experiment on proliferation and evaluation of inhibition of bioactivity of hIL2 different mAb used 96-well plate for tissue culture. Used final analytical volume of 200 ál per well, with this amount consisted of: 5×104cells CTLL-2, 2 ng/ml hIL2 (for approximately premaxillae proliferation) and various mAb against hIL2 at a concentration of 5000 ng/ml, 1000 ng/ml, 200 ng/ml and 40 ng/ml All samples were received in the triple repetition. The respective mixtures were incubated 48 hours at 37°C in a humidified chamber in risotti 5% carbon dioxide. Then was determined by viable cells, using the data of the fluorescent staining of AlamarBlue (Biosource International) and fluorescence reader for 96-well plates in accordance with manufacturer's recommendations.

Found that mAb202 (commercially available from the firm R&D Systems) (1) binds to soluble antigen, (2) binds to the antigen associated with the cell surface, and (3) effectively neutralizes the bioactivity hIL2. Among the tested antibodies only mAb 202 gave positive results in all three tests and, therefore, was considered as a promising candidate according to the properties defined above and, therefore, was chosen as a starting point for further experiments.

Example 2B: determination of the primary sequence of the antibody against hIL2 by sequencing: identification of sequences of the variable regions of the heavy chain ("VH") and a variable region light chain ("VL")

Due to the lack of available hybridoma clone mAb202, mAb sequenced to identify the VH and VL amino acid sequences. This has been Fab-fragments mAb202. These fragments were subjected to proteolytic cleavage and subsequent-line HPLC for the separation of peptides. Then separate the peptides were analyzed in relation to the amino acid composition and serial is a major using MS/MS mass spectrometry. This approach led to the identification of the VH - and VL-protein sequences.

Example 2B: Controlling for maintaining functionality: merge sequenced regions of VH/VL of known murine constant regions

It was desirable functional confirmation of the sequencing results obtained from sequencing of the protein mAb202 described above. Thus, the gene encoding the sequenced VH, synthesized and cloned in expressing vector, providing constant region IgG1 mouse. Similarly, the gene encoding the sequenced VL, synthesized and cloned in expressing vector, providing C-Kappa domain of the mouse. These two expressing vector ideally would allow to reconstruct the original mAb202, functionality which then could be retested as described above. After the co-expression of both vectors in 293 cells in cell supernatant was determined by mAb against IL2 with properties comparable to the properties that watched the original mAb202. Compliance activity (determined using ELISA, as well as in the analysis of cell proliferation using cell line CTLL-2)observed in the reconstructed mAb after sequencing of a protein with the activity of the parent mAb202 can be taken as confirmation that the sequence defined for the VH - and VL-regions e is th antibodies were correct.

Example 2G: the Humanization of the heavy chain

The goal of humanization is the complete preservation of the binding specificity and biological activity of antibodies while minimizing the content of sequences of non-human present in the mAb. The last goal leads to the production of antibodies, which are less likely to cause an immune response when administered to a subject, which is man, than its parent antibody of nonhuman origin. Initially received expressing vector for the chimeric heavy chain that contains the original VH mouse together with domains C1, C2 and C3 of the human IgG1 isotype. After expression of a chimeric heavy chain in Association with the chimeric light chain (see below) the properties of the original murine mAb could be reproduced (see below). The next logical step was the humanization of the VH-region. In order to avoid changes in the specificity of the CDR sequences are left unchanged. Therefore, based on the original murine VH was looking for the most closely related human VH frame sequence. Found that of all human VH frame regions of the human frame area 1-3/3-07/J6 has the highest degree of homology with the original murine frame area. Found that the frame area 1-3/3-07/J6 16 different amino acids OS is Adam from the corresponding murine VH frame areas. The alignment below shows a direct comparison between the original murine and human 1-3/3-07/J6 VH frame areas; the original CDR sequences are underlined, and the identity of amino acids between the two sequences are indicated by an asterisk.

Constructs containing the original murine VH or humanitarian VH, in the subsequent text will respectively be referred to as SNA (chimeric heavy chain containing a VH mouse and C1, C2, C3) and hHC (humanitariannet a heavy chain comprising the VH-containing CDR region of the mouse in the frame region of human VH and C1, C2, C3 person). For expression of recombinant protein humanized heavy chain was cloned open reading frames encoding humanitarian VH in combination with C1, C2 and C3 domains of human IgG1 isotype in a suitable vector (Raum T et al. (2001) Cancer Immunol Immunother. 50, 141-50).

Example 2D: Humanized light chain

Humanization was performed similarly to the approach described above for the heavy chain. Briefly, received expressing vector for chimeric light chain containing the source VL mouse together with the IC domain of man, and tested after co-expression with chimeric heavy chain (see above). Again, as in the second stage, based on the original VL mouse found most closely related to the frame sequence VL. All three CDR remained. Frame region ø12/Jk4 VL person turned out to be the most related sequences. When comparing VL mouse and ø12/Jk4 person in frame VL regions differed only 22 amino acid residue. The alignment below shows a direct comparison between the original murine and human frame areas ø12/Jk4; original CDR sequences are underlined, the identity of amino acids between the two sequences are indicated by an asterisk.

Constructs containing the original murine VL or humanitarian VL, in the subsequent text will respectively be referred to as cLC (chimeric light chain containing a VL mouse and Scappa person) and hLC (humanitariannet light chain comprising the VL containing murine CDR region in the frame region VL person and Scappa person). For expression of recombinant protein humanized light chain was cloned open reading frames encoding humanitarian VL in combination with the IC domain of man, into a suitable vector (Raum T et al. (2001) Cancer Immunol Immunother. 50, 141-50).

Example d: Permutation human and mouse sequences in the form of whole frame fields: assessment of binding and neutralization by analysis of cell proliferation

After a successful humanization and heavy and light chain testireba and properties of the resulting humanized mAb compared with chimeric mAb, i.e. antibody molecule containing the entire murine variable domains. As a chimeric mAb showed neutralization of bioactivity IL2 comparable to the original mAb, was used in these experiments as a control. For the time cotransfected 293 cells (the Protocol used was identical to the transfection method described in Example 1B except that the cells were transfusional simultaneously the two plasmids used pair expressing vectors encoding the heavy and light chains.

After expression in 293 cells comparable number of different variantov mAb in cell supernatant was confirmed using anti-hIgG ELISA, which was performed as follows.

In 96-hole tablet for ELISA (Nunc) were incubated with mAb against hIgG (Abcam LTD.) at a dilution of 1:2000 in PBS, 100 μl per well for 12 hours at 4°C. Each well three times washed with 200 ál of buffer PBS-T, then collected 100 ml of pure supernatant from 293 cells, filled each well serial dilution of supernatants in the medium and incubated for 1 hour at 20°C., Again washed each well 3 times with 200 μl of buffer PBS-t was Added to the wells mAb goat IgG against human conjugated with HRP (Jackson Immunoresearch)diluted 1:1000 in PBS-TB, 100 μl per well, and incubated for 1 hour at 20°C. the tablet Then washed 3 times with 200 μl per well of PBS-T. Finally, quantitatively evaluated St is the statements antibodies with hIgG by incubation with HRP substrate: 100 µl buffer with ABTS substrate (Roche Diagnostics, tablets ABTS), and incubated tablet for 5-10 minutes, until it developed a green color. This staining was measured at 405 nm onto the reader for 96-well plates. For all subsequent experiments used only supernatant with comparable amounts of mAb. Linking different antigen mAb were tested using ELISA (see above).

Properties of the resulting mAb against binding of soluble antigen and neutralize the bioactivity of IL2 were analyzed respectively using ELISA and analysis of proliferation of CTLL-2 (see Example 2A above for detailed experimental protocols). In ELISA experiments with increasing absorption unit will indicate increased amounts mAb that recognize the antigen, hIL2. In the analysis of the proliferation of CTLL-2 increasing units of fluorescence will indicate an increased amount of metabolically active (=living) cells. All cell supernatant ("SN"), containing different variants of mAb, controlled by the same concentration of mAb with anti-hIgG ELISA before exposed them to the following experiments. The results of a typical experiment is shown in figure 1. Here both combinations hLC+hHC (hLC=humanitariannet light chain, hHC=humanitariannet heavy chain) and cLC+hHC (cLC=chimeric light chain) show comparable binding to the antigen hIL2. The results of the s analysis CTLL-2 (Figure 2) show that hLC+hHC does not lead to any detectable neutralizing the bioactivity hIL2, as detected by fluorescence does not differ from the control SN. In contrast, cLC+hHC reduces the number of living cells, which is evident from the dependent on the concentration of mAb reduce fluorescence. The use of two separate circuits cLC or hHC in this analysis had no effect on hIL2-dependent survival of cells (data not shown). Each experimental point in a typical experiment are presented in figure 2, represents the average result for samples in duplicate. The results of this analysis are summarized in Table 1:

Table 1
light chainheavy chainthe binding of antigenneutralization
hLCHHC+-
cLCHHC++

These results show that, although the binding of soluble antigen, apparently, is no different, neutralization is lost as soon as used with hLC VA what Ianto humanized heavy chain. Conclusion: the humanization VL entails some functional impairment mAb.

In order to determine where, i.e. in which a frame region was introduced this deterioration, humanization of the VL frame region cLC carried out in different segments, changing at one time only one frame region (i.e. frame 1, frame 2 or frame 3), and not all three frame region at the same time from mouse to man. Was developed short range, and it will be used here when referring to these options humanization. According to this nomenclature three uppercase letters are used to create triplets, indicating each of the first three frame regions 1, 2 and 3, where the first position in the triplet indicates the nature of the frame 1, the second position in the triplet indicates the nature of the frame area 2, and the third position in the triplet indicates the nature of the frame region 3. For example, "MMO" would indicate human frame area 1 in VL, which otherwise is of murine origin, while the "CMA" indicates that only the frame area 2 is human, while the frame region 1 and 3 are of mouse origin.

Properties of different human/murine hybrid VL domains were analyzed with regard to their effects. Again the binding of antigen hIL2 and neutrons is the realisation of bioactivity IL2 were analyzed using ELISA and analysis of proliferation of CTLL-2, respectively (see Example 2A above for detailed experimental protocols). In the experiment with ELISA increasing unit absorption indicate increased amounts mAb that recognize the antigen, hIL2. In the analysis of the proliferation of CTLL-2 increasing units of fluorescence indicate an increased amount of metabolically active (=living) cells. All cell supernatant ("SN"), containing different variants of mAb, controlled by the same concentration of mAb using anti-hIgG ELISA before subjecting them to the following experiments. Figure 3 shows the typical results of the comparative experiment. Here you can see that all human/murine hybrid options VL exhibit comparable binding to the antigen hIL2 when combined with hHC. The results of the analysis of CTLL-2 (Figure 4) show that neutralization of bioactivity hIL2 is observed only under the condition that the frame area 2 VL is a mouse; MHM+hHC does not change the number of living cells compared to control SN. Each experimental point in a typical experiment are shown in Figure 4 represents the average value for samples in duplicate. The results of this analysis are summarized in Table 2:

Table 2
light chain heavy chainthe binding of antigenneutralization
MMOHHC++
CMAHHC+-
NMMHHC++
LmnsHHC++

These experiments are quite clearly show that the frame area 2 VL determines whether a mAb to neutralize the bioactivity IL2 or not. A more detailed comparison of mouse and human sequences of the frame area 2 reveals that these sequences differ by three amino acids. In particular, murine frame area 2 contains a partial amino acid sequenceQSPKAwhereas the corresponding human sequence is aKAPKL(amino acids, which differ in humans and mice, were highlighted for clarity).

Example d: Permutation human and any mouse sequences in the frame area 2; evaluation of binding and neutral is Itachi analysis of proliferation and analysis of the induction of the target genes

To determine whether all three amino acid substitutions or only some of them crucial in the context of obtaining a neutralizing mAb, performed an additional series of experiments. For this amino acid residues QS And or murine origin was re-introduced in the hLC.

Properties of the resulting mAb against binding of soluble antigen and neutralize the bioactivity of IL2 were analyzed respectively using ELISA and analysis of proliferation of CTLL-2 (see Example 2A above with respect to detailed experimental protocols). In the experiment with ELISA increasing unit absorption indicate increased amounts mAb that recognize the antigen, hIL2. In the analysis of the proliferation of CTLL-2 increasing units of fluorescence indicate an increased amount of metabolically active (=living) cells. All cell SN containing different variants of mAb, controlled by the same concentration of mAb with anti-hIgG ELISA before subjecting them to the following experiments. Figure 5 shows the results of a typical experiment. As you can clearly see, both options: QSPKL+hHC and KARKA+hHC show comparable binding to the antigen hIL2. Analysis of CTLL-2 (Fig.6) shows that QSPKL+hHC does not lead to any detectable neutralizing the bioactivity hIL2, as some fluorescence is not different from the control SN. On the contrary, KAPKAhHC reduces the number of living cells, what is evident from the dependent on the concentration of mAb reduce fluorescence. Each experimental point in a typical experiment are shown in Fig.6 represents the average value for samples in duplicate. The results of this analysis are summarized in Table 3:

Table 3
light chainheavy chainthe binding of antigenneutralization
hLCHHC+-
hLC_QSPKLHHC+-
hLC_KAPKAHHC++

This shows that one amino acid residue localized in the frame area 2 VL, determines will neutralize whether mAb bioactivity IL2: alanine residue at this position, originating from murine frame region 2, provides the neutralization, and the residue is leucine derived from human framework region 2, no.

Example 3: Determine how to eliminate

The term"anti-IL2", as used below, means humanitariannet antibody against IL2 containing the light chain containing the amino acid sequence represented in SEQ ID NO:9 (which itself contains a VL region amino acid sequence represented in SEQ ID NO:7), and a heavy chain containing the amino acid sequence represented in SEQ ID NO:10 (which itself contains a VL region amino acid sequence represented in SEQ ID NO:8). VL antibodies against IL2 contains the amino acid sequence "KARKA", as explained above in Example HD.

It was desirable to better understand the neutralization method of hIL2 with anti-IL2. With this in mind, we conducted experiments to study the nature of the binding hIL2 components hIL2-receptor, on the one hand, and anti-IL2, on the other hand.

As for the survival of NKL cells required IL2, we can conclude that these cells indeed Express a functional receptor for IL2. Conducted an experiment with FACS according to the procedures described above. Briefly, these cells were incubated with a mixture of anti-hIL2 mAb and species-specific secondary detection antibody (pre-cooked mixture"). The secondary antibody was conjugated with a fluorescent label. Cellular fluorescence was monitored in the presence and in the absence of NL2 using FACS. You shall anjali following experimental scenarios to answer the question, does it require a certain order of incubation for the observed effects.

In the first scenario, a pre-prepared mixture was incubated in the presence or in the absence of hIL2 for 30 min, then was added NKL cells. Cellular fluorescence was observed hIL2-dependent manner. In the second scenario, NKL cells were incubated in the presence or in the absence of hIL2 for 30 min, then was added the previously prepared mixture. Again watched cellular fluorescence hIL-2-dependent manner. These experiments demonstrate that hIL2 when linking with anti-IL2 can still contact their receptor, and, moreover, that hIL2 in Association with its receptor can still interact with anti-IL2.

These results demonstrate that the epitope hIL2 associated antibody against IL2, obtained as described above, is different, at least in part, on the epitope hIL2 associated hIL2-receptor. This method of neutralization is notable in that it implies that the neutralization of hIL2 can be performed by binding of this molecule in its soluble form, and in the receptor-bound form. Then with a chronological point of view, this means that the event binding between hIL2 and anti-IL2 can occur either before or after the formation of the complex between hIL2 and hIL2-receptor; in any case, the neutralization is ioactivity hIL2. Therefore, by extrapolation, we can assume that neutralization is also carried out when two relevant events linking the formation of the complex between hIL2 and hIL2-receptor and the complex between hIL2 and anti-IL2 - occur simultaneously.

It should be noted that this method of neutralization, which is observed for anti-IL2, sharply differs from other known methods of neutralization, in which the epitope bound by a neutralizing antibody against the ligand and the receptor to the ligand, is one and the same. In this traditional scenario is impossible the existence of a ternary complex between the ligand, the ligand-receptor and a neutralizing antibody against the ligand. In other words, in this traditional way of neutralization of the ligand should contact a neutralizing antibody against the ligand at a time when the ligand is still in soluble form, so that the formation of the complex between the ligand and the ligand-receptor prevented.

Example 4: IL2-dependent binding of anti-IL2 with human limfomoj cell line NKL natural killer cells

In this example investigated the binding specificity of anti-IL2 with hIL2 associated with the cell surface. Parent Ab against IL2 (mAb202) showed strictly IL2-dependent binding with cell surface NKL cells. Therefore, it is particularly the property must be confirmed for anti-IL2.

NKL cells were deprived of hIL2 for 24 h before the experiment. Anti-IL2 or human control antibody isotype IgG1 were incubated in the absence or in the presence of 2-fold molar excess of hIL2 at 20°C for 60 minutes Appropriate mixture was then added to NKL cells (105cells per sample) and then incubated for 30 min on ice. Then these cells were thoroughly washed and added fluorescently labeled detection antibody goat against human IgG, followed by incubation for 30 min on ice. Again washed cells and then subjected to FACS analysis for the study of cell-associated fluorescence.

As expected, in the absence of hIL2 was not determined significant cell-associated fluorescence with anti-IL2, and with a control antibody (Figure 10, left). In the presence of hIL2 cell-associated fluorescence with the control antibody did not (Figure 10, right graph, the shaded peak). In contrast, incubation with hIL2 and anti-IL2 resulted in a significant increase in fluorescence (Figure 10, right panel, outlined with black peak), indicating specific IL2-dependent binding of anti-IL2 with the cell surface. Thus, anti-IL2 was preserved ability to recognize hIL2 associated with the cell surface. This experiment provided evidence that anti-IL2 hIL2 not only in solution, but also recognizes hIL2, which is associated with one or more components of its receptor. Therefore, hIL2 can contact the anti-IL2 and component(s) IL2-receptor newsamerican way.

Example 5: Anti-IL2 cancels IL2-dependent increase regulation of the expression of CD124 on the cell surface of cells CTLL-2

After stimulation with hIL2 cells CTLL-2 proliferate and they are under increasing regulation of expression of CD124 on cell surfaces (IL-4R alpha) (Puri, R.K., et al. (1990). Immunology 70, 492). Therefore, cells CTLL-2 receive sensitivity concomitant stimulation via IL-4 by IL2-stimulated. Therefore, anti-IL2 may not only limit IL2-mediated proliferation, but also to influence the expression of CD124.

To test this hypothesis, cells CTLL-2 were cultured in the absence of hIL2 for 12 h before the experiment and then stimulated with 0.5 ng/ml hIL2 for 5 h in the presence or in the absence of titrated concentrations of anti-IL2. The levels of expression of CD124 was assessed using FACS analysis using fluorescently labeled CD124-specific antibody. The average values of specific fluorescence intensities were plotted on a graph against different concentrations of anti-IL2 (11, empty squares with black line); the average values of fluorescence, registered is consistent in the absence of anti-IL2 (11, the filled diamond) or in the absence of IL2 (11, filled triangle), were included as controls. As follows from 11, anti-IL2 reduced expression of CD124 dose-dependent manner; IC50calculated from this analysis, was approximately 3.3×10-10M. These data imply that anti-IL2 not only affects cell proliferation CTLL-2, but also other IL2-dependent cellular responses, such as expression of CD124.

Example 6: Anti-IL2 specifically blocks IL2 signal transduction below IL2R in cellular signaling pathways

This experiment was performed to further exclude the possibility that anti-IL2 mediates its effects on hIL2-dependent cellular responses in part through a cytotoxic mechanism, and to confirm that the mechanism of action of anti-IL2 is highly specific for signaling stimulated hIL2, but has no effect on the related path. Among the most rapid cellular events IL2-mediated cellular signals is the tyrosine phosphorylation of the transcription factor STAT3 (Leonard, W.J. 2000. IL2 Family Cytokines and their Receptors). Other cytokines, such as IL-6, trigger partially overlapping cellular signaling pathways, which also include STAT3 (Hemmann, U., et al. (1996). J Biol Chem 271, 12999; Stahl, N., et al. (1995). Science 267, 1349).

Therefore, anti-IL2 tested relative to it, the effects on the phosphorylation of STAT3 tyrosine, run IL2 and IL6. Peripheral blood lymphocytes were isolated from fresh blood, incubated at a concentration of 2×106cells/ml, pre-stimulated 48 h lecithin, and then was left alone in the medium for 24 h before stimulation. The cells are then stimulated with saturating concentrations of IL2 or IL6/sIL6Rα without mAb, or in the presence of anti-IL2, or izotopicheskogo control monoclonal antibodies for 15 minutes. After separation of the cytoplasmic extracts using PAG-ordinator (polyacrylamide gel electrophoresis with sodium dodecyl sulfate), investigated the status of STAT3 phosphorylation using Western blot turns using antibodies specific to STAT, phosphorylated at tyrosine (Fig, top panel). To control the comparable boot also provided blotting total protein STAT3 (Fig, bottom panel). Electrophoretic mobility of standard proteins are indicated on Fig to the left of each panel.

The stimulation of IL2 and IL6 was significantly increased cellular phosphorylation of STAT3 at tyrosine in the absence of anti-IL2 (Fig, lanes 2 and 3 compared to 1 or strips 6 and 7 compared to 1). Therefore, anti-IL2 specifically affects the phosphorylation of STAT3 on tyrosine after IL2 stimulation, but not after IL6 stimulation (Fig, lane 4 compared with lane 5). These data demonstrate that anti-IL2 are the two which is highly specific for intervention in biology hIL2 and has no effect on the path, regulated by other factors, and anti-IL2 has no obvious cytotoxic effects.

Example 7: On the effectiveness of anti-IL2 and daclizumab differentially affect levels of expression of CD25

The inhibitory activity of daclizumab, gumanitarnogo mAb against CD25, in parallel compared with the inhibitory activity of anti-IL2 and izotopicheskogo control antibodies in the analysis of cell proliferation using IL2-dependent cell line NKL (Fig).

To study the effect of expression levels of CD25 on the cell surface to the inhibition of IL2-induced cell proliferation in any of the anti-IL2 or daclizumab NKL cells were sorted using FACS with low or high levels of expression of CD25, and both cell populations was studied simultaneously in this experiment. mAb against CD25 used for FACS-sorting, did not prevent binding of IL2 or daclizumab with CD25 (data not shown). Immediately after sorting was possible a clear distinction CD25low.and CD25high.populations using FACS; during petisteho experiment two data populations were closer together, resulting in levels of expression of CD25, comparable with populations before sorting. This implies that the results obtained in this analysis reflect only clearly separated populations of CD25low.compared to CD25high.for the original FA is s, but not for the later phases of this experiment. Therefore, differences that will be observed in relation to inhibition of proliferation antibody against IL2 and daclizumab when comparing populations of CD25low.and CD25high.is limited due to the unstable levels of CD25 expression; all of these data you can display a clear trend indicating a differential dependence of the efficiency of daclizumab and anti-IL2 from CD25. In the preparation of this experiment NKL cells lacked in hIL2 for 16 hours under cultivation in an environment that does not contain hIL2. Used finite volume environment for the analysis of 200 µl per well, which included: 1×104NKL cells, 2 ng/ml hIL2 (to ensure premaxillae proliferation) and different monoclonal antibodies titrated concentrations. All samples were prepared in duplicate. Incubation of the respective mixtures occurred within 120 hours, and then viable cells were visualized using a fluorescent dye.

In General, the analysis of anti-IL2 was more effective in neutralizing IL2-mediated proliferation compared with daclizumab. As expected, the effectiveness of anti-IL2 did not affect the levels of expression of CD25 in NKL cells CD25low.and CD25high.curves obtained with anti-IL2, essentially superimposed on each other in Front, curves obtained with daclizumab, show a clear difference in NKL cells with CD25low.compared to CD25high.. Izotopicheskie control Ab had no effect (Fig). Thus, this experiment provided evidence in vitro that the effectiveness of daclizumab, but not anti-IL2, depends on the levels of CD25.

Example 8: Effect of anti-IL2 or daclizumab on IL2-dependent proliferation of primary NK cells

Not only primary T cells, but also primary NK cells can proliferate in response to IL2-stimulation. Thus, in an additional experiment investigated the inhibition of IL2-induced proliferation of freshly isolated NK cells.

These cells were obtained by negative selection from blood and incubated with hIL2 (5.5 ng/ml) in the presence or in the absence of titrated anti-IL2 or daclizumab. The control antibody was used only in very high concentrations; the other control was carried out with cells in the absence of IL2 and antibodies. Viable cells was quantitatively assessed using a fluorescent dye at the end of a one-week incubation period. Anti-IL2 in this experiment was significantly reduced IL2-induced proliferation of primary NK cells. At high concentrations of anti-IL2 proliferation essentially limited to the levels observed is in the absence of IL2, that indicates that anti-IL2 affects all IL2-responsible NK-cells that are present in this analysis. On the contrary, daclizumab showed the effect only at a significantly reduced amplitude, which suggests that the presence of this antibody was affected only on the fraction of NK-cells (Fig). For further investigation of this finding tracked the levels of expression of CD25 on during the week-long incubation with IL2 and antibodies: only about 11% of all NK cells shows a donor was found expression of CD25 with a maximum on the day 3 and with a reduction to 2% on the 7th day. Accordingly freshly isolated NK cells from all donors did not show detectable expression of CD25, and NK-cells in all analyzed donors were found similar to the levels and kinetics of expression of CD25 (data not shown). That would explain why daclizumab could inhibit the proliferation of only a fraction of NK-cells (Fig). Anti-IL2 again demonstrated its independence from the levels of expression of CD25 and blocked the proliferation of all NK cells with the value of the IC50approximately 3×10-10M. These results strictly indicate that anti-IL2, but not daclizumab able to inhibit IL2-mediated signals via the IL2-receptor CD122/CD132 with intermediate affinity, independent of CD25.

Example 9: Effect of anti-IL2 or daclizumab on IL2-chief of the independent release of IFN-gamma NK-cells

In addition to the proliferation of typical and quick response of primary NK cells to stimulation by the cytokine is the release of IFN-gamma. The release of the latter, as dependent anti-IL2 and daclizumab, were measured in an additional experiment.

In this analysis freshly isolated MK-human cells stimulated with a mixture containing hIL2 (5.5 ng/ml), hIL12 (5 ng/ml) and hIL18 (5 ng/ml), running efficient production and release of IFN-gamma by these cells. Compared the effects of titrated anti-IL2, daclizumab and izotopicheskogo control antibodies on the release of IFN-gamma during the first 48 h of incubation. And anti-IL2, and daclizumab reduced expression of IFN-gamma dose-dependent manner, whereas the control antibody had no effect (Fig). Anti-IL2 was a more potent inhibitor of the release of IFN-gamma, with IC50approximately 1.3×10-10M compared to about 1.1×10-9M for daclizumab (Fig). In contrast to the experiment described in the previous example, all NK cells in this experiment demonstrated the expression of CD25 (data not shown), explaining the deeper effect daclizumab on IFN-gamma compared with proliferation of NK-cells.

Table 4 summarizes the equilibrium binding constant (KDfor anti-IL2 and daclizumab, and values of the IC50received in parallel comparative exp is ramento with both Ab, as described above in Examples 8 and 9.

Table 4
DescriptionUnitAnti-IL2Daclizumab
The binding affinity ofThe equilibrium dissociation constant (KD)6,8±6,1×10-10M (BiaCore)
2,5±1,6×10-9M (Surface cells)
3,0×10-9M#
Proliferation of primary NK cellsIC501,0±0,6×10-10M1,4±0,4×10-9M*
Production of IFN-gamma NK-cellsIC501,3±1,0×10-10M1,1±0,8×10-9M
#according to Junghans, R.P., et al. (1990). Cancer Res 50, 1495.
*based on ~10% of the total NK cell population that Express CD25.

1. Humanitariannet monoclonal antibody or its active fragment, which specifically binds to human interle the Keene-2 (IL2),
where the specified humanitariannet monoclonal antibody neutralizes the activity of human IL2 by binding with the specified human IL2 before, during and/or after bonding of the specified human IL2 with human IL2 receptor,
where the variable region of the light chain of the specified gumanitarnogo monoclonal antibodies contains in its second frame region sequence of contiguous amino acids KARKA, and
where the variable region of the light chain further comprises in its CDR1 region amino acid sequence represented in SEQ ID NO:1, its DR2-region amino acid sequence represented in SEQ ID NO:2, and in its CDR3 region amino acid sequence represented in SEQ ID NO:3; and where the variable region of the heavy chain contains in its CDR1 region amino acid sequence represented in SEQ ID NO:4, in its DR2-region amino acid sequence represented in SEQ ID NO:5, and in its CDR3-region amino acid sequence represented in SEQ ID NO:6.

2. Humanitariannet monoclonal antibody or its active fragment according to claim 1, where the sequence of contiguous amino acids CHARKA is located at amino acid positions 42-46 second frame region.

3. Humanitariannet monoclonal antibody or its active fragment according to claim 1, where at least one is erway, the third and/or fourth frame region of the light chain complies(comply) with the sequence this area/these areas in the human germ line.

4. Humanitariannet monoclonal antibody or its active fragment according to claim 1, where at least one of the first, third and/or fourth frame region of the light chain complies(comply) with the sequence this area/these areas in the human germ line.

5. Humanitariannet monoclonal antibody or its active fragment according to claim 1, where the amino acid sequence of the first frame region of the light chain, the remaining amino acid sequence of the second frame region of the light chain (i.e. amino acid positions 35 to 41 and 47 to 49 inclusive) and amino acid sequence of the third frame region of the light chain match any of the sequences of subgroups VKI human germline loci in ø12, O2, O18, O8, A30, L1, L15, L4, L18, L5, L19, L8, L23, L9, L11 or L12; or subgroup VL1 human germ line 1A locus; or any sequence of subgroups VL2 human germline loci in 2C, 2E, a or 2b2.

6. Humanitariannet monoclonal antibody or its active fragment according to claim 1, where the amino acid sequence of the first frame region of the heavy chain, the amino acid sequence coronaries region of the heavy chain and the amino acid sequence of the third frame region of the heavy chain independently correspond to any of the sequences of subgroups human VH3 germline.

7. Humanitariannet monoclonal antibody or its active fragment of claim 6, where the amino acid sequence of the first frame region of the heavy chain, the amino acid sequence of the second frame region of the heavy chain and the amino acid sequence of the third frame region of the heavy chain such as in the locus 3-07 subgroups human VH3 germline.

8. Humanitariannet monoclonal antibody or its active fragment according to any one of pp.5-7, where the amino acid sequence of the fourth framework region of the light chain corresponds to the amino acid sequence of human JK4 (FGGGTKVEIK).

9. Humanitariannet monoclonal antibody or its active fragment according to claim 1, where the specified humanitariannet monoclonal antibody contains a variable region light chain containing the amino acid sequence represented in SEQ ID NO:7, and the variable region of the heavy chain containing the amino acid sequence represented in SEQ ID NO:8.

10. Humanitariannet monoclonal antibody according to claim 1, where the specified humanitariannet monoclonal antibody includes a light chain containing the amino acid sequence represented in SEQ ID NO:9 and a heavy chain containing the amino acid sequence represented in SEQ ID NO:10.

11. Humanitariannet mo is olonline antibody according to claim 1, representing IgG.

12. Humanitariannet monoclonal antibody according to claim 11, where the specified IgG is an IgGl or IgG4.

13. Humanitariannet monoclonal antibody according to any one of claims 1 to 9, where the specified active fragment is an scFv, a single domain antibody, Fv, ditelo, tandem diatel, Fab, Fab' or F(ab)2.

14. Humanitariannet monoclonal antibody according to item 13, where the specified active fragment is an scFv, in particular, where the indicated scFv contains variable regions of its light chain amino acid sequence represented in SEQ ID NO:7, and the variable region heavy chain amino acid sequence represented in SEQ ID NO:8.

15. Humanitariannet monoclonal antibody according to 14, where the indicated scFv contains the amino acid sequence represented in SEQ ID NO:11 or SEQ ID NO:12.

16. Humanitariannet monoclonal antibody or its active fragment according to claim 1, containing an amino acid sequence having at least 70%homology, preferably at least 80, 90 or even more than at least 95%homology with the corresponding amino acid sequence represented in any of SEQ ID nos:7-12.

17. Polynucleotide molecule encoding humanitariannet monoclonal antibody or its active fragment, which is specificity the ski binds to human interleukin-2 (IL2), according to any one of claims 1 to 16, where this molecule contains a nucleotide sequence encoding the amino acid sequence represented in any of SEQ ID nos:7-12, or the nucleotide sequence showing at least 95%homology with the specified nucleotide sequence, where the homology can be determined by comparing the polynucleotide molecule containing the nucleotide sequence encoding the amino acid sequence of any of SEQ ID N0:7-12, with a polynucleotide molecule having considered the nucleotide sequence by sequence alignment, where the nucleotide in the sequence is considered to be homologous if it is either identical to the corresponding nucleotide in the nucleotide sequence that encodes the appropriate amino acid sequence of any of SEQ ID nos:7-12, or if one or more than one nucleotide variance in this sequence from the corresponding one or more than one nucleotide in the nucleotide sequence that encodes the amino acid sequence of any of SEQ ID nos:7-12, leads to the formation of a triplet of nucleotides that, when broadcast network amino acid that is either identical (due to the degenerate triplet of corresponding AMI is ocelote in the corresponding amino acid sequence of any of SEQ ID nos:7-12, or a conservative substitution.

18. Pharmaceutical composition having a neutralizing activity against human interleukin-2 (IL2) in the human body and contains humanitariannet monoclonal antibody or its active fragment according to any one of claims 1 to 16 or polynucleotide molecule by 17.

19. The pharmaceutical composition according p, optionally containing one or more anti-inflammatory or anti-cancer drug.

20. Application gumanitarnogo monoclonal antibody or its active fragment according to any one of claims 1 to 16 or polynucleotide molecules 17 in the manufacture of a medicinal product may contain one or more than one additional anti-inflammatory agent for the treatment of inflammatory diseases in mammals, preferably humans.

21. The application of claim 20, where specified inflammatory diseases selected from the group consisting of rheumatoid arthritis (RA), asthma, multiple sclerosis (MS), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (IBD), uveitis, macular degeneration, colitis, psoriasis, Malerovskoj degeneration, antiphospholipid syndrome (APS), the acute coronary syndrome is a, restenosis, atherosclerosis, recurrent polychondritis (RP), acute or chronic hepatitis, unsuccessful implantation of orthopedic implants, glomerulonephritis, lupus, autoimmune disorders, acute pancreatitis, or ankylosing spondylitis (AS).

22. Application gumanitarnogo monoclonal antibody or its active fragment according to any one of claims 1 to 16 or polynucleotide molecules 17 in the manufacture of a medicinal product may contain one or more than one additional anticancer agent, for treatment of neoplastic disease or other condition with delayed apoptosis, increased survival or cell proliferation in mammals, preferably humans.

23. The application of article 22, where the specified tumor disease is a cancer.

24. The application of item 23, where the specified cancer is a leukemia, multiple myeloma, gastric carcinoma or carcinoma of the skin.



 

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11 cl, 6 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology and represents stable and soluble scFv-antibody and Fab-fragment, specific with respect to TNFα, which contain specific sequences of light chain and heavy chain, optimised as to stability, solubility, in vitro and in vivo TNFα binding and low immunogenity. Also claimed are antibody-coding DNA-sequence, vectors, host-cell, as well as method of obtaining antibody with application of said cells. In addition claimed are therapeutic and diagnostic compositions and stable water pharmaceutically ready form based on antibodies, as well as method of treating TNFα-associated disease.

EFFECT: invention can be efficiently used for diagnostics and treatment of TNFα-associated disorders.

61 cl, 18 dwg, 5 tbl, 5 ex

Immunoglobulins // 2404192

FIELD: medicine.

SUBSTANCE: invention represents immunoglobulins, in particular antibodies, which specifically bind to human interleukin 13 (hIL-13). Claimed are recombinant or transfected mammalian host cell, for cloning, as well as for expression of vectors, coding antibodies. Claimed are antibody-containing pharmaceutical composition, set, containing said pharmaceutical composition and pharmaceutical composition, containing monoclonal antibody against IL-4, such as pascolizumab. Claimed is method ob obtaining antibody, and applications of said antibodies.

EFFECT: antibodies can be used for treatment of various diseases to disorders, responsible for modulation of hIL-13 interaction with human IL-13 receptor.

51 cl, 29 dwg, 22 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention relates to molecular pharmacology and specifically to a peptide which is part of an interleukine-15 (IL-15) sequence which can inhibit biological activity of the said molecule.

EFFECT: obtaining a peptide which inhibits T cell proliferation induced by IL-15, and apoptosis caused by tumour necrosis factor when bonding with the alpha subunit of the (IL-15R) receptor.

8 cl, 4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.

EFFECT: applicable in medicine for preparing a vaccine.

52 cl, 32 dwg, 7 tbl, 29 ex

FIELD: medicine.

SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.

EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.

14 cl, 1 tbl, 6 ex

FIELD: pharmacology.

SUBSTANCE: invention concerns immunology and biotechnology. There is offered human monoclonal antibody specific to TNF-alpha containing light and heavy chain with appropriate CDR3 sites. There are described versions thereof including those based on heavy and light chains and coded by human genes VH3-33 and A30VK1 or VH3-53 and L2VK3 respectively. There are disclosed: the method for estimating the TNF-alpha content in the patient's sample with using specified antibodies, and application of antibodies for preparing a medical product. There are described: compositions for diagnostics and treatment of the conditions associated with TNF-alpha activity on the basis of antibodies. There is disclosed coding nucleic acid, a cell for making said antibodies and the method for making said antibodies.

EFFECT: application of the invention ensured high-affinity neutralizing monoclonal antibodies with improved Kd and IC50 in comparison with Infliximab, Adalimumab or Etanercept that can find application in medicine for treatment and diagnostics of the diseases associated with TNF-alpha hyperactivity.

35 cl, 13 dwg, 36 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: there are disclosed polypeptide variants containing Fc-areas IgG, having amino acid modifications providing changed effector functions Fc in specified polypeptides. There is disclosed composition for antibody targeting on antigen, containing the specified polypeptide. There is described method for preparing the specified polypeptide. Also, there are disclosed the methods for treating V-cell tumour or a malignant disease characterised by V-cell expression of CD20, treating chronic lymphocytic leukosis, relieving the symptoms of the V-cell controlled autoimmune disease, treating a angiogenesis-associated disorder, treating HER2-expressing cancer, treating LFA-1-mediated involvement, treating IgE-mediated involvement wherein specified methods imply introduction to the patient of the therapeutically effective amount of said polypeptide.

EFFECT: higher clinical effectiveness.

63 cl, 6 ex, 13 dwg, 10 tbl

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology. Proposed is an antibody against interferon-α/β-binding protein I or against its mutein, obtained through conservative substitution. Proteins, against which the antibody is directed, are extracted from urine and link interferon-α/β-binding protein I with Kd constant from 3.6x10-9 to 1.6x10-10 M.

EFFECT: use of the invention allows for detecting presence of interferon-α/β-binding protein I in different samples.

5 cl, 10 dwg, 6 tbl, 17 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: claimed invention relates to field of biotechnology. Claimed is antibody against interferon-α/β-connecting protein II or against its mutein, obtained by conservative substitutions. Here protein, against which antibody is directed, is separated from urine and connects interferon-α/β-connecting protein II with constant Kd equal 2.12x10-9 M.

EFFECT: obtaining possibility to detect interferon-α/β-connecting protein II presence in different samples.

5 cl, 10 dwg, 6 tbl, 17 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and medicine. What is offered is ProThrThrLysThrTyrPheProHisPhe peptide, a based pharmaceutical composition which is used for antitumour immune response stimulation, and also to methods of treating a mammal and immune response modulation.

EFFECT: range of products for cancer treatment is extended.

19 cl, 49 tbl, 3 ex

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