Complexes of antibodies with some cytokines

FIELD: medicine, oncology, biochemistry.

SUBSTANCE: invention relates to fused proteins, namely to the multifunctional fused protein cytokine-antibody. This fused protein involves immunoglobulin region and cytokine fused protein of the formula IL-12-X or X-IL-12 wherein interleukin-12 (IL-12) represents the first cytokine and X represents the second cytokine taken among the group comprising IL-2, IL-4 and GM-CSF bound covalently either by amino-end or carboxyl-end to subunit p35 or p40 of interleukin-12 (IL-12) in its heterodimeric or a single-chain form. Indicated fused cytokine protein is fused by either its amino-end or carboxyl-end with indicated region of immunoglobulin. Multifunctional fused protein cytokine-antibody shows an anticancer activity.

EFFECT: valuable medicinal properties of protein complexes.

13 cl, 40 dwg, 18 ex

 

This application is based on priority of U.S. patent No. 60/147924, filed August 9, 1999, the contents of which are incorporated here by reference.

The technical FIELD

The present invention relates to methods construction and expression of protein complexes with multiple cytokines and songs with their participation. More specifically, the invention relates to a fused protein composed of several cytokines and addressing component, and methods of use thereof in treating diseases, such as cancer and viral infection.

PRIOR art

The regulatory circuit that controls the immune system, based on secreted molecules of signaling proteins called cytokines, designed for on and off functions of immune cells and the regulation of their proliferation. In these reactions in the General case involved several cytokines that are required to achieve a biological effect are consistent. Some cytokines, such as interleukin-2 (IL-2), can induce the proliferation of immune cells and can activate other functions, including the secretion of secondary cytokines. Another cytokine, interleukin-12 (IL-12) (see Review: Trinchieri // Stood. 1994. So 84. S-4027) can induce proliferation of certain immune cells and to induce the other is th immunomodulator - interferon-γ (IFN-γ). This induction of IFN-γ is the main function of IL-12, although IL-12 has other important functions that are not associated with IFN-γ. Because IL-12 in the case of infectious disease is induced at an early stage, I believe that it links the innate and acquired immune system.

Numerous studies in vitro and in murine and human immune cells demonstrated the importance of combinations of cytokines in the development of optimal immune responses. For example, most T cells do not Express the receptor of IL-12 (IL-12R) as long as they do not induce mitogen or not cultured in the presence of high concentrations of IL-2 (Desai and others // J. Immunol. 1992. So 148. S-3132). Once the receptors are expressed, cells become more sensitive to IL-12. In addition, IL-12 induces the transcription of IFN-γbut the mRNA of IFN-γ then quickly collapses. In the presence of IL-2 mRNA is stabilized, which leads to a sharp increase in production of IFN-γ (Slept, etc. // J. Immunol. 1992. So 148. Pp.92-98). In other studies it was found that combinations of cytokines: IL-3 plus IL-11 or IL-3 plus factor Steele have synergene with IL-12 effect on the proliferation of early progenitor cells of the blood (Trinchieri, 1994; cited above). The combination of interleukin-4 and granulocytes factor stimulation image is of the colonies by macrophages (granulocyte macrophage colony stimulating factor GM-CSF) is particularly useful for the stimulation of dendritic cells (Palucka, etc. // J. Immunology. 1998. So 160. S-4595). For stimulation mediated by cells of the immune reaction is also useful to combine IL-12 and IL-18 is a recently open running path TP lymphokines with some functions, complementary to the functions of IL-12 (Hashimoto and others // J. Immunol. 1999. I. 163. S-589; Barbulescu, etc. // J. Immunol. 1998. So 160. S-3647). In addition, IL-2 and IFN-γ in some situations have synergene action (M.A. Palladino // U.S. Patent No. 5082658).

In many such studies of synergism was found that it is very important relative concentration of each cytokine. Despite the fact that the addition of IL-12 in the presence of suboptimal amounts of IL-2 synergize acted on the induction of proliferation, cytolytic activity and the induction of IFN-γ, it was shown that the combination of IL-2 with IL-12 with a high dose of one of the cytokines gave antagonistic effect (Perussia, etc. // J. Immunol. 1992. So 149. S-3502; Mehrotra, etc. // J. Immunol. 1993. So 151. S-2452). A similar situation was found with combinations of IL-12 and IL-7.

Studies of the synergistic action of IL-12 and other cytokines in the generation of antitumor responses in mice also yielded mixed results. In some models, when suboptimal doses of each cytokine was detected synergies and higher doses were given increased toxicity. On other models, comb the Nations of IL-12 and IL-2 had very weak or no had synergene action (see, for example, Nastala, etc. // J. Immunol. 1994. So 153. S-1706). These results may be due to the fact that in vivo, it is difficult to combine two potentially synergenix agent, especially if you want to maintain a fixed ratio of the activities of the two agents with different pharmacokinetic properties such as half-life in blood flow and distribution in the body.

In experiments in cell cultures in vitro enough to control the levels of cytokines, but in vivo the relative distribution in the body and localization of cytokines can act many factors that affect their immunostimulirutuyu ability. The most important of these factors is the half-life. The half-life of IL-2 in blood flow after injection of the bolus is about 10 minutes In contrast, it was noted that the half-life of IL-12 in the blood stream is more than 3 h in mice (Wysocka, etc. // Eur. J. Immunol. 1995. V.25. S) and from 5 to 10 h in humans (Lotze and others // Ann NY Acad Sci. 1996. T. S-454).

I believe that this difference is due to the relatively small size of IL-2 and GM-CSF (15-25 kDa to 75 kDa in IL-12), resulting in IL-2 and GM-CSF are derived by filtering through the kidneys. Normally filtered by the kidneys and excreted proteins with a molecular weight of less than 50 kDa. Almost all cytokines have a molecular weight below 50 kDa and are similar to the rapid removal of the filter h is through the kidneys. If you want treatment with the two small, fast out by cytokines, simply enter cytokines together. However, for cytokines with significantly different at times half-life joint introduction is not optimal.

Systemic injection of cytokines is difficult because of their harmful side effects. For example, significant side effects observed with the introduction of large quantities of interferon-alpha, including toxicity to the skin, the nervous, immune and endocrine systems. It is expected that the merger of several cytokines may provide a particularly serious side effects.

One strategy to reduce the side effects of systemic administration of cytokines is the confluence of the cytokine with the second molecule with the ability to purposely addressing. Fused protein in which the Fc region of immunoglobulin placed at the N end of another protein (called "immunizing" or merger "Fc-X", where X denotes a ligand, such as interferon-alpha), have many distinct useful biological properties (Lo etc. // U.S. Patent No. 5726044 and 5541087; Lo, etc. // Protein Engineering. T. 11. S). In particular, such fused proteins can be contacted with the appropriate receptors for the Fc to the cell surface. However, the binding of the ligand with its receptor on the cell surface orientation of the Fc region is inalsa, and it turns out that it prevents the action sequences, which define the dependent antibody-mediated cell cytotoxicity (ADCC) and binding of complement. As a result, the Fc region of the molecule in the Fc-X stops effectively direct ADCC and binding of complement. The cytotoxic effect due to the merger of the N-terminal cytokine and C-terminal Fc region is well known. For example, the fusion of IL-2 N-end Fc region gives the molecule, is able to contact the cells with the receptor for IL-2, bind complement and lyse cells (Landolfi N.F. // U.S. Patent No. 5349053, 1993). On the contrary, fused proteins Fc-IL-2 does not have this property. Thus, it is expected that the merger of Fc-X will have a useful quality - increased half-life in serum and preferential concentration in the liver without unwanted effects in the form of ADCC and binding of complement.

It was demonstrated that with the Fc region in the configuration of the Fc-X can be merged many diverse proteins with a short half-life in serum, and the resulting mergers have significantly longer half-life in serum. However, the half-life in serum of two different fusions with the Fc in General will not be the same. Therefore, if necessary, the delivery of two different components of X, the joint introduction of two different fused Fc-X in most cases will not be optimal.

In some situations the best way adresovano direct effect of cytokines on the cell surface antigen is to merge it with the antibody (or its derived fragment)having specificity and affinity (affinity) to this antigen (Gillies // U.S. Patent No. 5650150; Gillies, etc. // OEWG. Natl. Acad. Sci. USA. T. 89. S), or in connection peptide bond of the protein antigen and stimulating cytokine in the form of a fused protein (Hazama, etc. // Vaccine. T. S). Although antibodies may themselves increase the half-life merged with them cytokine, between mergers of various cytokines with the same antibody can be differences (see, for example, Gillies, etc. // Bioconjugate Chem. 1993. V.4. S-235; Gillies, etc. // J. Immunol. T.160. S-6203)which complicate their joint localization at the site of target. As discussed above, this can lead to an imbalance in the activities of cytokines and decrease the desired energichnogo actions. In addition, by using two different fused proteins it is necessary to test the safety and efficiency parameters of each merging separately, and then to test their mixtures.

The INVENTION

The present invention provides complexes or fusion of two or more different cytokines, useful both for General and addressed to immune therapy. These complexes or merger optional include the other protein components. One of the features of such complexes or mergers is that they provide a given ratio of activities of cytokine components.

In General, the present invention relates to protein complexes containing at least two different cytokines. Cytokines can be in the same polypeptide chain or to be bound by the covalent bond such as a disulfide bond, or a bond formed by chemical crosslinking. Alternatively, cytokines may be in a state of stable non-covalent Association. In some preferred implementations protein complex contains addressing component, such as an antibody or antibody fragment, which adresovano directs the complex to a specific location (locus) of a mammal.

In the preferred embodiment, the present invention provides a protein complex that combines the biological activity of double-stranded cytokine, such as IL-12 biological activity of a second cytokine. Cytokines can be covalently linked (e.g., fused) to each other. Cytokines can also be connected through other components. For example, polypeptide chain containing a second cytokine may have a binding component that specifically binds to IL-12, such as tetelo to IL-12 or the receptor for IL-12. Alternatively, the communicating component can communicate with the second component, which is associated with IL-12. For example, if the polypeptide chain, containing subunit of IL-12, also contains avidin, a polypeptide that contains a second cytokine, may also contain Biotin as addressing component. In one of the preferred embodiments the second cytokine is IL-2.

The present invention provides methods of obtaining fused proteins containing IL-12, which have the activity of IL-12, and the second activity of the cytokine, and provide more long-term, single pharmacokinetic behavior similar to the behavior of the IL-12, in result prolonging the duration of activity of the second cytokine and keeping the balance of the activities of the two cytokines after the injection the animal.

In another example embodiment of the invention fused proteins contain heterodimeric form of IL-12, in which subunit of IL-12 R35 and R40 are connected by a disulfide bond and covalently attached to the second cytokine or amino end or at the carboxyl end of the b subunit R35 or R40 IL-12, with the following General formula fused protein: IL-12-X or X-IL-12, where X is a second cytokine.

In another example embodiment of the invention fused proteins contain a second cytokine, covalently attached to either the amino-end, or to carbox is linoma the end of single-stranded (single-chain - sc) form of IL-12 containing two polypeptide subunits, United flexible peptide bridge, with the following General formula fused protein: sclL-12-X or X-sclL-12.

In another embodiment, two cytokine optionally fused with a protein that can form dimeric or multimeric structure, either at the amino-end, or at the carboxyl end of the specified protein chain. In a preferred variant of this embodiment, one of the forms fused protein of IL-12 with a second cytokine later merged with part of a chain of immunoglobulin (Ig), such as the Fc region, which is capable of dimerization. Other embodiments encompass a fusion of at least one polypeptide chain of the IL-12 on either end component Ig and second cytokine, is attached at the other end.

In the following example, the implementation of two or more cytokine fused protein with addressing capability in the strength of binding with a specific receptor. For example, the Fc region can bind with receptors for the Fc, strewn in the liver. Merge Fc region with multiple cytokines illustrates the advantages of combining the dimerization and targeting, but in some cases it is useful to construct a merge multiple cytokines, which can only multimerization or only to be senders, but not combine both abilities.

<> In yet another embodiment, a protein containing several cytokines, optionally fused with the amino - or carboxyl end of the class representative molecules with a variety of addressing capacity, such as an antibody or peptide aptamers with or without frame (Colas etc. // Proc NatI Acad Sci USA. 1998. T. S-14277). A specific example is the merging of multiple cytokines at least part of the antibodies, which are capable of binding antigen, such as the intact antibody, single-chain antibody or single-chain Fv region. Additional embodiments include a merger of at least one polypeptide chain of the IL-12 with any end at least part of the chain antibodies, capable of binding the antigen, and with a second cytokine, is attached at the other end.

According to the above descriptions, in General it is preferable to construct the fused proteins of several cytokines and fused proteins of several cytokines with the antibody by genetic engineering so that the protein components are connected by covalent bonds such as amide bond or a disulfide bond. However, it is possible to construct such protein complexes to the use of chemical crosslinking agents. Such methods are well known in the chemistry of proteins. Alternatively, sometimes just POPs the AMB protein complexes fusion of different cytokines with protein partners, forming a stable non-covalent complexes. For example, use ecovalence linked heterodimeric protein-carrier: the first cytokine is fused with one of the subunits of heterodimer, the second cytokine is fused with the second subunit of heterodimer, and both fused protein mixed in suitable conditions. For example, in the same cell Express a nucleic acid encoding the fused protein with docsubject cytokine. In this way can be constructed protein complex with multiple cytokines, cytokine which components can be connected ecovalence, either directly or indirectly. To meet the purposes of the present invention, this complex should be stable enough to persist with the introduction of the animal and to provide a biological effect.

The present invention also provides for nucleic acids encoding the fused protein containing two or more cytokines, where one of the cytokines, preferably IL-12, and the encoded nucleic acid protein contains optional other protein components. Preferred embodiments include nucleic acids that encode the fusion of two or more cytokines with dimerizes protein, such as the Fc region chain antibodies. Another set of preferred embodiments are nuclein what s acid, encoding the merger of two or more cytokines protein with addressing capability, such as an antibody.

The present invention also provides for methods of constructing mergers with two or more cytokines, and how the expression of such fused proteins.

The invention also provides for methods of treating diseases and other pathological States in which the treatment includes a suitable combination of activities of two or more proteins. In one example implementation, at least one protein has a short (e.g. less than 20 minutes) or only moderately long (for example, less than 40 minutes) half-life in serum. Proteins fused by methods of genetic engineering or other methods and are human or animal. Thus, the activity of the two proteins are present in a predetermined ratio, and are not required to enter two proteins separately with different dosing regimens. In addition, the half-life in serum fused protein is generally closer to the half-life of the protein component with a longer half-life in serum, resulting in a longer effective time of the half-life of the protein or proteins with shorter half-life in serum.

More specifically, the invention provides methods of immunotherapy treatment of diseases, such as cancer or infects the district of disease, or other diseases that are treated double-stranded cytokine, such as IL-12, in combination with a second cytokine. In the preferred embodiment, IL-12 merge with IL-2 or GM-CSF and enter the animal or human. In other preferred embodiments GM-CSF is drained with IL-4 and enter the animal or human. In another embodiment, IL-12 merge with IL-18 and enter the animal or human. Such treatment can be used in conjunction with other methods of treatment of diseases. In addition, the present invention provides methods of vaccination against a variety of antigens, which can be used for the prevention or treatment of various diseases.

In other embodiments of these methods, two different cytokine merge with dimeric protein component, such as the Fc region of antibodies, and enter the animal or human. In the preferred form of these ways cytokine IL-12 merge with the Fc region with a second cytokine, which preferably is an IL-2 or GM-CSF.

In some other embodiments of these methods, two different cytokine fused to the intact antibody and found the animal or person. In the preferred form of these ways cytokine IL-12 is drained to the antibody with a second cytokine, which preferably is an IL-2 and GM-CSF. The present invention also provides a mixture of fused proteins antibody-cytokine suitable for the treatment of diseases. In one example implementation for the treatment of diseases of a mixture of fused protein antibody-IL-2 fused protein antibody-IL-12. For example, treating cancer, viral infection or bacterial infection.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

All of the above and other objectives of the present invention and its various distinctive features can be better understood from the following description accompanied by the attached drawings. In all the drawings the same numbers indicate the same structure.

Figo gives a schematic illustration of a merger of two cytokines in its simplest form: one cytokine is fused with a second cytokine, optional through the bridge. Figures from 1B 1I show the different ways in which the second cytokine (marked "cyt") can be attached to the heterodimeric cytokine IL-12. Specifically, the second cytokine can be merged with the end of R40 (pigv), N-end R40 (figs), With the end of the R35 (fig.1D) or N-end R35 (fige). In addition, figure 1 shows how the second cytokine may be merged with the single-chain variant of IL-12. Specifically, single-stranded molecules IL-12 may be a subunit of the R35 attached to the N-end of the subunit R40, with a second cytokine-end (fig.1F) or N-end (fig.1G). As a researcher who as alternatives, single-stranded molecules IL-12 may have R40 N-end R35, with a second cytokine-end (fign) or N-end (Fig).

In figures 2A-2C schematically shows how to merge multiple cytokines (enclosed in a border)are presented in figure 1, may be further fused with the Fc region of antibodies, shown here as a hinge (H), CH2 domain and CH3 domain (ovals). Specifically, each of the eight molecules of figure 1 may be merged or With the end (figa), or N-end (pigv) Fc region. In addition, there is no need first cytokine and a second cytokine (each enclosed in a box) to connect directly with each other. They can be connected through the Fc region (figs).

In figures 3A - 3G schematically shows the set of ways in which protein with multiple cytokines may be optionally fused with intact immunoglobulin, such as IgG. Region of the heavy chain V presents an oval shape, marked Vn, the region of the light chain V presents an oval shape, marked VLand the constant region is represented by the empty ovals. Merge multiple cytokines, shown in figure 1, can be placed on the C-end of the heavy chain (figa), on the N end of the heavy chain (pigv), on the N end of the light chain (figs), or With the end of the light chain (fig.2D). In addition, there are many ways in which the first and second cytokines can be since the June attached to the N - and C-ends of the heavy and light chains, three of them are shown in figures 3E - 3G.

In figures 4A - 4C schematically shows how the first and second cytokines can be merged with the "single-stranded" antibody, which merged variable light and variable heavy chains and the protein is expressed as a single polypeptide, which is then homodimerization. Specifically, the merging of multiple cytokines can be placed on the C-end (tiga) or N-end (pigv). In addition, there is no need first cytokine and a second cytokine to connect directly with each other. They can be linked via a single-stranded antibody (figs).

In figures 5A - 5C schematically shows how the first and second cytokines can be merged with the single-stranded region of the Fv, consisting of a merged variable regions of the heavy chain and light chain. Specifically, the merger of the first cytokine with a second cytokine can be placed on the C-end (tiga) or N-end (pigv). In addition, there is no need first cytokine and a second cytokine to connect directly with each other. They can be linked via a single-chain Fv region (figs).

Figures 6A and 6B show the synergistic action of IL-12 and IL-2 in the induction of IFN-γ in mononuclear cells of peripheral blood (peripheral blood mononuclear cells - PBMC) in response to the addition of individual cytokines or fused proteins. On figa, cells were treated human is Kim IL-12 before (squares) or after (crosses) activation phytohemagglutinin, or fused protein of IL-12-IL-2 (diamonds) or after (triangles) activation phytohemagglutinin. On FIGU presents the experience in which cells were treated with a mixture of IL-12 and IL-2 added to their molar ratio of 1:1 (black diamonds), fused protein (Fc-IL-12-IL-2 (shaded squares) and fused protein (antibody-IL-12-IL-2 (lightly shaded triangles). On the X-axis indicates the concentration of IL-12 PG/ml, if it is present as an intact protein or composition of the fused protein. Y-axis indicates the concentration of IFN-γ (ng/ml), determined by ELISA method.

Figure 7 presents a typical biological analysis of IL-12, which allows to separately measure the activity of the fused protein and compare it with the activity nakitai molecules IL-12. Pictured stimulation includeCH-thymidine by human PBMC in response to the introduction of murine IL-12 (empty circles), a mixture of murine IL-12 and IL-2, added in a molar ratio of 1:1 (black squares), murine IL-2 (empty triangles) and fused protein containing the antibody, murine IL-12 and murine IL-2 (black diamonds). On the X-axis indicates the concentration (PM) Monomeric cytokines (cytokines) in the form of intact protein or composition of the fused protein on the Y-axis indicated the inclusion labeled with tritium thymidine in pulses per minute (pulse/min).

On Fig presents the standard analysis of biological engineering the th activity of IL-2. The graph shows the stimulation of the proliferation of murine CTLL cells in response to murine IL-2 (circles), on protein antibody-mu.IL-12-mu.IL-2 (diamonds) and murine IL-12 (squares). On the X-axis indicates the concentration (PM) Monomeric cytokines (cytokines) in the form of intact protein or composition of the fused protein. Cells were incubated in medium containing within 48 h different amounts of the cytokine or fused protein, and then determined the number of viable cells using the MTT test/MTS. On the Y-axis indicated the absorption at 490 nm in units of optical density (OD).

Fig.9 shows the stimulation include3H-thymidine human RVMS in response to murine IL-12 (empty circles), a mixture of murine IL-12 and IL-2, added in a molar ratio of 1:1 (black circles), protein (mouse Fc)-(single-chain IL-12)IL-2 (black triangles) and murine single-chain IL-12 fused with murine IL-2 (black diamonds). On the X-axis indicates the concentration (PM) Monomeric cytokines (cytokines) in the form of intact protein or composition of the fused protein on the Y-axis indicated the inclusion labeled with tritium thymidine in pulses per minute (pulse/min).

Figure 10 shows the stimulation include3H-thymidine human RVMS in response to murine IL-12 (empty circles), a mixture of murine IL-12 GM-CSF added when a molar ratio of 1:1 (black circles), murine GM-CSF (black triangles) and merged the protein mu.Fc-mu.IL-12-GM-CSF (crosses). On the X-axis indicates the concentration (PM) Monomeric cytokines (cytokines) in the form of intact protein or composition of the fused protein on the Y-axis indicated the inclusion labeled with tritium thymidine (imp/min).

11 shows the effect of introducing fused protein antibody-cytokine-cytokine mice Balb/C mice with subcutaneous tumors derived from cells ST carcinoma of the rectum, which due to gennoingenernyi manipulation Express human Arcam - antigen for KS-1/4. Black diamond shows the average volume of tumors in the control mice, which were injected in PBS days: 0th, 1st, 2nd, 3rd and 4th. Triangles shows the average volume of tumors in mice that were injected 6 µg KS-IL-12-IL-2. Squares indicated the average volume of tumors in mice that were injected 3,4 µg KS-IL-2 and 5.3 micrograms of KS-IL-12. Injections were performed inside tumors. On the X-axis indicates the time (days)elapsed after the first injection; Y-axis indicated the average volume of tumors in cubic mm

Fig demonstrates the effect of the introduction of the fused protein antibody-cytokine-cytokine SCID mice with subcutaneous tumors derived from cells ST carcinoma of the rectum, which due to gennoingenernyi manipulation Express human Arcam. Diamond shows the average volume of tumors in the control mice, which were injected in PBS days: 0th, 1st, 2nd, 3rd and 4th. Triangles shows the average volume of tumors in mice, which is the Odile 6 µg KS-IL-12-IL-2. Squares indicated the average volume of tumors in mice that were injected 3,4 µg KS-IL-2 and 5.3 micrograms of KS-IL-12. Injections were performed inside tumors. On the X-axis indicates the time (days)elapsed after the first injection; Y-axis indicated the average volume of tumors in mm3.

Fig shows the results of the comparison steps of introducing a fused protein antibody-cytokine and antibody-cytokine-cytokine mice with subcutaneous tumors from cell carcinoma, Lewis lung (LLC), which due to gennoingenernyi manipulation Express human Arcam. Diamond shows the average volume of tumors in the control mice, which inside tumors were injected PBS on days: 0, 1-St, 2-nd, 3-th and 4-th. The squares show the average volume of tumors in mice, which inside tumors in the day: 0th, 1st, 2nd, 3rd and 4th were administered 20 μg KS-IL-2. Triangles shows the average volume of tumors in mice, which inside tumors in the day: 0th, 1st, 2nd, 3rd and 4th were administered 20 μg KS-IL-12. The crosses indicated the average volume of tumors in mice, which inside tumors in the day: 0th, 1st, 2nd, 3rd and 4th were administered 20 μg KS-IL-12-IL-2. X-axis indicates the time (days)elapsed after the first injection; Y-axis indicated the average volume of tumors in mm3.

On Fig shows the effect of the introduction of the fused protein antibody-cytokine-cytokine mice with subcutaneous tumors derived from cell carcinoma of the lung Lewis, who, due to gennoingenernyi manip is the visible Express human Arcam. Diamond shows the average volume of tumors in the control mice, which inside tumors were injected PBS on days: 0, 1-St, 2-nd, 3-th and 4-th. Triangles shows the average volume of tumors in mice which were administered 20 μg KS-IL-12-IL-2. Squares show the average volume of tumors in mice that were injected with 11.5 µg KS-IL-2 and 18 µg KS-IL-12. Injections were performed inside tumors. On the X-axis indicates the time (days)elapsed after the first injection; Y-axis indicated the average volume of tumors in mm3.

On Fig shows the effect of the introduction of the fused protein antibody-cytokine-cytokine mice with subcutaneous tumors derived from cell carcinoma, Lewis lung that Express or do not Express the human Arcam. Black squares show the average volume of tumors in mice whose tumors were obtained from cells LLC/KSA. Black diamond shows the average volume of tumors in mice whose tumors were obtained from LLC cells. Mice were administered 20 μg KS-IL-12-IL-2 per day: 0th, 1st, 2nd, 3rd and 4th. Injections were performed inside tumors. On the X-axis indicates the time (days)elapsed after the first injection; Y-axis indicated the average volume of tumors in mm3.

On Fig shows the effect of the introduction of the fused protein antibody-cytokine-cytokine mice with subcutaneous tumors derived from cell carcinoma of the lung Lewis. 0-day was administered by subcutaneous injection approximately 106cells. Diamond and shows the average volume of tumors in native mice. Squares show the average volume of tumors in mice that had subcutaneous tumor cell carcinoma, Lewis lung (LLC), expressing the result gennoingenernyi manipulation of human Ersam, and who have been spared these tumors introduction KS-IL-12-IL-2. On the X-axis indicates the time (days)elapsed after injection; Y-axis indicated the average volume of tumors in mm3.

In figures 17A and 17B shows the effect of the secretion of tumor cell protein with a single cytokine or more cytokines on the ability of cells to form tumors in animals with normal immune system. On figa provides a comparison of the 4 groups of mice: mice C57BL/6, which was subcutaneously injected 1×106the LLC tumor cells (black diamonds); mouse C57BL/6, which was subcutaneously injected 5×106tumor cells LLC (empty diamonds); mouse C57BL/6, which was subcutaneously injected 1×106tumor cells LLC, expressing sclL-12 (black triangles); and mouse C57BL/6, which was subcutaneously injected 5x106tumor cells LLC, expressing sclL-12 (empty triangles). On figv a comparison of C57BL/6 mice, which were injected subcutaneously 1×106the LLC tumor cells (black diamonds); C57BL/6 mice, which were injected subcutaneously 5x106tumor cells LLC (empty diamonds); C57BL/6 mice, which were injected subcutaneously 1×106the LLC tumor cells expressing sclL-12-IL-2 (crosses), and m is Shea C57BL/6, which was subcutaneously injected 5×106the LLC tumor cells expressing sclL-12 (empty circles). On the X-axis indicates the time (days)elapsed after the injection of tumor cells. On the Y-axis indicated the average volume of tumors in mm3.

On Fig shows the effect of the secretion of tumor cell protein with a single cytokine or more cytokines on the ability of cells to form tumors in immunodeficient animal. This figure shows a comparison of SCID mice, which were injected subcutaneously 1×106the LLC tumor cells (black diamonds); SCID mice, which were injected subcutaneously 1×106the LLC tumor cells expressing sclL-12 (black triangles), and SCID mice, which were injected subcutaneously 1×106the LLC tumor cells expressing sclL-12-IL-2 (empty circles). On the X-axis indicates the time (days)elapsed after the injection of tumor cells. On the Y-axis indicated the average volume of tumors in mm3.

INFORMATION CONFIRMING the POSSIBILITY of carrying out the INVENTION

The present invention provides protein molecules, in which two or more different cytokine fused or form a complex. Protein complexes or fused proteins can optionally include additional protein components, including components capable of multimerization and addressing, such as the Fc region and parts of the antibody containing sites join the antigen. The invention also provides nucleic acids encoding the fused protein with multiple cytokines. The invention also provides for methods of constructing the nucleic acid encoding the fused protein with multiple cytokines, methods of producing fused proteins with multiple cytokines and methods of using fused proteins with multiple cytokines for the treatment of diseases and illnesses.

In the same sense as it is used here, the term "cytokine" refers to Sekretareva protein or active fragment or mutant form, which modulates the activity of cells of the immune system. Examples of cytokines include interleukins, interferons, chemokines, tumor necrosis, stimulating the formation of colonies factors for precursors of immune cells, etc.

In the same sense as it is used here, the term "heterodimeric cytokine" refers to a cytokine composed of two different protein subunits. IL-12 is the only known up to the present time natural heterodimeric cytokine. However, there may be constructed artificial heterodimeric cytokines. For example, you can combine IL-6 and soluble fragment of the IL-6R, getting a heterodimeric cytokine. The same can be done with CNTF and CNTF-Rα (Trinchieri // Blood. 1994. T. S).

In the same sense as it is used here, the term is h "interleukin-12" (IL-12) denotes docsubject cytokine, consisting of subunits R35 and R40, or active single-chain fusion R35 and R40, or a variant, fragment or derivative.

In the same sense as it is used here, the term "interleukin-2" (IL-2) denotes IL-2 any mammal, such as human IL-2 mouse IL-2, or its active species or allelic variant, fragment or derivative.

In the same sense as it is used here, the term "GM-CSF" refers to the cytokine protein mammalian factor stimulation of the formation of colonies of granulocytes/monocytes (Granulocyte/Monocyte-Colony Stimulating Factor), such as human GM-CSF, murine GM-CSF, or its active species or allelic variant, fragment or derivative.

In the same sense as it is used here, the term "Fc region of immunoglobulin" means carboxyl-terminal site of the constant region of the heavy chain of the immunoglobulin or its analogue or share. For example, the Fc region of immunoglobulin IgG may contain at least part of the hinge region, CH2 domain and CH3 domain. In the preferred embodiment, the Fc region comprises at least part of the hinge region and CH3 domain. In another preferred embodiment, the Fc region comprises at least the CH2 domain, and more preferably includes at least part of the hinge region.

In the same sense as it is used here, the term "pepti the hydrated bridge" means one or more peptides, used to join together two proteins (e.g. protein and the Fc region). Peptide bridge is often a sequence of amino acids, such as, for example, with a predominance of glycine and/or serine. Preferably the peptide bridge is a mixed sequence with a predominance of residues glycine and serine length of approximately 10-15 amino acids.

In the same sense as it is used here, the term "multimeric" refers to a stable connection between two or more protein subunits by covalent or non-covalent interactions, for example through linking disulfide bonds.

In the same sense as it is used here, the term "dimer" refers to a specific multimeric molecule, in which two protein subunits stably connected with the participation of covalent or non-covalent interactions. A stable complex is a complex with a dissociation rate, or rate of decay in that it fails for at least several minutes (i.e., the complex must be stable long enough when used in vivo, in order to reach the tissues where it is addressed, and to provide a biological effect). The Fc fragment is typically creates dimer fragments of the heavy chain, containing part of the hinge region, CH2 domain and/or CH3 domain. However, it is known that many Belkova the ligands bind to their receptors in the form of dimers. If the cytokine X dimerized naturally, component X in the molecule Fc-X will timeresults to a much greater extent, because the process of dimerization depends on the concentration. Physical sblizhenie two elements X, United Fc, turns dimerization in an intramolecular process, greatly shifting the equilibrium towards the formation of dimer and enhancing its binding to the receptor.

In the same sense as it is used here, the term "vector" refers to any nucleic acid that represents the nucleotide sequence, which can be introduced into the cell host, recombine with the genome of the host cell and integrated into the genome of the host cell or autonomously replicated in the form of an episome. Such vectors include linear nucleic acids, plasmids, phagemid, Comedy, RNA vectors, viral vectors and other non-limiting examples of viral vectors include retrovirus, adenovirus and adeno-associated virus.

In the same sense as it is used here, the term "gene expression" or "expression of a protein" is understood as meaning the transcription of the DNA sequence, broadcast transcript mRNA and or secretion of the protein product, or obtaining protein product in the form available for selection.

In the same sense as it is used here, the term "immunocyto the h" means a protein, containing the antibody and the cytokine, as disclosed in U.S. patent No. 5650150.

In the same sense as it is used here, the term "leader sequence" means a protein sequence that is attached, usually at the N-end, the second protein sequence, and thus makes the second protein sequence be excreted from the cell. Usually the leader sequence is cleaved and removed from the second protein sequence, which is becoming Mature protein. The term "leader sequence" is usually synonymous with the term "signal sequence".

In the same sense as it is used here, the term "Arcam" means a molecule having the property of adhesion to epithelial cells (Cirulli and others 1998. T. S-1534) and is synonymous with the designation "KSA", denoting the antigen, bound monoclonal antibody KS 1/4. Arcam is a protein on the cell surface that are redundantly expressed on cancer cells derived from epithelial cells.

In the same sense as it is used here, the term "KS-1/4" refers to the specific monoclonal antibody binding to Arcam.

In the sense as used here, the terms "KS-IL-2", "KS-IL-12 and KS-IL-12-IL-2" (and similar) are fused proteins antibody-cytokine consisting respectively of the KS-1/4 with interleukin-2, KS-1/4 with interle the Keene-12 and KS-1/4 together with interleukin-12, and with interleukin-2. Here are also other design fused proteins with similar names. Because you can merge cytokines with a molecule antibodies in different positions, such a designation as "KS-IL-12-IL-2"refers (unless stated otherwise) to the class of proteins containing KS-1/4 together with interleukin-12 and interleukin-2, merged in any possible position.

In the same sense as it is used here, the term "14.18" refers to a specific monoclonal antibody, which binds to ofwholesale antigen GD2.

Some typical examples of the implementation of protein structures embodying the present invention, illustrated in figures 1-5. Part of the molecule is depicted in figures 2-5, identified as 1A-1I; it carries them to the fused proteins shown in figures 1A-1I, and illustrates that any of the fused protein of figure 1 may additionally be fused, as indicated, with other proteins. Cytokines are indicated by rectangles, the constant region of the antibody are indicated by ovals, and the variable region of the heavy chain and the variable region of the light chain marked marked by ovals.

The present invention describes protein complexes containing two different cytokine and optionally including other protein components. Homodimeric cytokine (for example, interferon-alpha, interferon-beta, interferon-gamma, IL-5, IL-8 and so on), although it contains several subunits, however, is a single cytokine. Similarly heterodimeric cytokine, such as IL-12, while providing different subunits, is a single cytokine. Moreover, heterodimeric form homodimeric normal cytokines, such as heterodimer MCP-1/MCP-2, or form of the two alleles homodimeric normal cytokine (e.g., Zhang // J. Biol. Chem.1994. T. S-15924) represent a single cytokine. The complexes of the present invention contain two different cytokine, each of which (e.g., IL-2 and IL-12; IL-4 and GM-CSF; MCP-1 and eotaxin; and so on) capable of modulating the activity of cells of the immune system.

On figa shown a preferred embodiment of the invention: fused protein (10)-the end of the first cytokine (12) fused to the N-end of the second cytokine (14), optional across the area of the bridge (not shown). In some implementations of the present invention protein complex according to the invention contains at least two cytokine with significantly different half-life in serum. For example, the use of small and large proteins often results in a protein with a half-life in blood flow characteristic of a larger protein. Thus, in cases where it is necessary to combine the effects of IL-12 and a second cytokine, conveniently EC is to preservati both cytokine as protein General formula IL-12-X or X-IL-12, where X is a second cytokine. Here you can see two deserve special attention benefits. First, it increases the half-life in serum more rapidly displayed protein. Secondly, the time of half-life in serum of both cytokines become very close.

Double-stranded cytokine, such as IL-12, can be merged with a second cytokine in the N - or C-end of each of the chains of double-stranded cytokine. In one example implementation of the second cytokine is fused to either the N-end or From the end of the subunit of IL-12 - or R35, or R40 (FIGU-1E). In the fused protein on 16 FIGU N-end of the first cytokine (12) merged with the end of the subunit of IL-12 R40 (18). Subunit R40 (18) connected to the subunit of IL-12 R35 (20) covalent bond (22). In the fused protein on 24 pigs N-terminal subunit R40 (18) merged with the end of the first cytokine (12) and is connected to the subunit of the R35 (20) covalent bond (22). On fig.1D shows the protein (26)where N is the end of the first cytokine (12) merged with the end of subunit R35 (20), which are connected by covalent bond (22) subunit R40 (18). On five protein 28 includes a subunit of the R35 (20), merged its N-end C-end of the first cytokine (12) and connected by covalent linkage 22 with subunit R40 (18).

In the second embodiment subunit of IL-12 can be merged with the formation of single-stranded protein sclL-12 with N-end or subunit R35 or suryadinata, and the second cytokine can be attached to the N - or C-end received sclL-12 (figures 1F-1I). Thus, in the preferred embodiment shown in fig.1F, protein contains 30 single-chain IL-12, in which the N-terminal subunit R40 (18) merged with the end of subunit R35 (20), optionally via a peptide bridge. In this example, the implementation of the N-end of cytokine (12) merged with the end of subunit R40 (18). In the shown fig.1G example implementation of the N-terminal subunit R35 (20) merged with the end of cytokine (12), optionally via a peptide bridge. On the figures 1H and 1I shows the fused proteins 34 and 36 containing a different single-stranded version of IL-12, in which the N-terminal subunit R35 merged with the end of subunit R40, optionally via a peptide bridge. In the shown fign fused protein 34 N-end-of-12 cytokine is fused with the end of subunit R35 (20). In the shown fig.1l fused protein 36 N-terminal subunit R40 (18) merged with the end of cytokine 12. In the preferred embodiment, IL-12 fused with IL-2.

The production of such molecules is illustrated further by the examples.

It is often convenient to Express heteropolymer molecules, such as IL-12 or antibody, as single-stranded molecules, in which non-identical subunits are connected by a short amino acid bridges (Huston, etc. // OEWG. Nat. Acad. Sci. 1988. So 85. C. 5879; Lieschke and other // Nat. Biotechnol. 1997. So 15. S. 35; G.J. Lieschke and Muligan R.C. // U.S. patent No. 5891680). Design of gene fusion, and then the desired protein can be expressed in cells containing a single design of recombinant DNA. Such single-stranded version heteropolymeric cytokine may be optionally fused with a second cytokine, which allows the expression of a fused protein with the desired set of activities for a single design of recombinant DNA. The expression of these molecules is illustrated further by the examples.

The present invention also describes a protein containing IL-4 and GM-CSF. This combination is particularly useful for functional stimulation presentation ("presentation") of the antigen by dendritic cells. Other useful mergers contain IL-12 and IL-18. Both of these include cytokine Th1 immune response, but are slightly different, complementary activity.

The present invention also describes a fused protein in which several different merged cytokines optionally fused with a protein that can form multimer, such as homodimer or heterodimer. The advantage of such molecules is that the activity of one or more cytokines can be enhanced by dimerization. In some cases, the increased activity of the dimerization can occur due to the fact that the cytokine binds to its receptor in the form of dime is and. In one example implementation of several cytokines merged with part of the molecule antibodies, such as the Fc region (figure 2). In another embodiment, IL-12, and a second cytokine is fused with a protein component, capable of formation of homodimers. In the preferred embodiment, the second cytokine is IL-2 or GM-CSF. Fused proteins can be created in a variety of ways, using all possible interleavings of several different protein components from N-Terminus to the C-end in the slit protein. For example, if the interleukin-12 and the second cytokine is fused with the Fc region, both cytokine can be merged in any order from the N - or C-end Fc region, or a single cytokine may be merged with the N-end, and the second - To-end.

Some of these cyclic permutations (permutations) is shown in figure 2. For example, shown in figa example implementation protein 44 of the present invention merged with the end of the Fc region containing the hinge region (38), plot CH2 (40) and the plot CH3 (42). Protein 44 can have many different structures, including, for example, the structure of the fused proteins 10, 16, 24, 26, 28, 30, 32, 34 or 36 shown in figures 1A-1I. If protein 44 has more than one N-end and C-end, as in the fused proteins 16, 24, 26 and 28, the Fc region can be merged with any N-end fused protein 44. As shown in figv, protein 44 may be merged with the N-end areas and Fc. In the shown figs example, the first cytokine 12 may be merged with the N-end Fc region, and a second cytokine 14 can be merged with the end of the Fc region.

Structural considerations

It is important to note that cytokines, as a class of proteins according to their size and core collapse options are similar to each other. So here specific examples show how you can construct fused proteins with multiple cytokine family cytokine proteins. For example, many cytokines are a class of collapsible proteins called "chetyrehstvolnym beam". Protein type chetyrekhmernogo beam include: factor stimulation of colonies of granulocytes (G-CSF), interleukin-6 (IL-6), the factor inhibiting leukemia (LIF), growth hormone, factor neurotrophin cilia (CNTF), leptin, erythropoietin, factor stimulation of granulocyte colony-macrophage (GM-CSF), interleukin-5 (IL-5), factor stimulation colonies of macrophages (M-CSF), IL-2, IL-4, interleukin-3 (IL-3), IL-10, interferon-beta, interferon-alpha and closely related interferon Tau, and interferon-gamma (IFN-γ).

In addition to IL-5 and IFN-γall of these proteins are collapsed as monomers with four approximately parallel alpha-helices and two intersections. In all cases, except for IL-5 and IFN-γ, N-end and C-end are on the same surface protein. Since the proteins of type h is directparallel beam, in addition to IL-5 and IFN-γ, are collapsed in the same way described here, the methods for IL-2, IL-4 and GM-CSF is also applicable for other protein type chetyrekhmernogo beam and for other small cytokine proteins that fold as monomers.

Chemokines are a specific class of cytokines that are believed to have extracellular gradient distribution and mediate the chemotaxis of specific classes of immune cells. For example, MCP-1 is a chemoattractant for monocytes, macrophages and activated T-cells; eotaxin is a chemoattractant for eosinophils and interleukin-8 is a chemoattractant for neutrophils. In addition to their function as chemoattractants, chemokines and other cytokines capable of inducing the expression of certain genes in certain target cells. For example, suppose that MCP-1 induces the expression of tissue factor in cells of vascular smooth muscle (Schecter, etc. // J Biol Chem.1997. T. S-28573).

The present invention discloses merge cytokine-cytokine and fusion of antibody-cytokine-cytokine, in which one or more cytokine is a chemokine(s). The invention discloses also protein structures with three or more cytokines, one or more cytokine is a chemokine(s). For example, chemokines IP-10, RANTES, MIP-1α, MIP-1β, chemoattractant proteins of macrophages, eotaxin, lymphotactin, BC can be merged with a second cytokine, connected or not connected with other components, such as an antibody.

The human genome, for example, encodes at least 50 cytokines. Known cytokines have similar three-dimensional structure of monomers and similar type protein folding. Therefore disclosed in the present invention the main types of protein structures and strategies for design can be applied to various known or not open until this time cytokines.

Chemokines are a special type of collapse with three beta-strands and one alpha helix. Chemokines are collapsed as monomers and in some (but not all) cases, then timeresults after folding. All chemokines way folding of the Monomeric subunits are identical and the overall structure is extremely similar. For example, by x-ray crystallography and/or NMR were determined three-dimensional structure of interleukin-8, platelet factor 4, factor stimulating the growth of melanoma (MGSA), macrophage inflammatory protein (MIP), RANTES (regulated upon activation, expressed and secreted by T-cells in normal), monolitnogo chemoattractant protein-1 (MCP-1, MCAF), eotaxin, monolitnogo chemoattractant protein-3 (MCP-3), chemokine domain fractalkine, activating neutrophil peptide-2 (NAP-2), factor-1 from stroma cells (SDF-1), macrophage inflammatory b the LCA-2, the chemokine hcc-2 (macrophage inflammatory protein-5), Gro-beta-induced cytokine chemoattractant neutrophils and CINC/Gro. In all these structures the same styling, and they are basically similar. Due to the fact that the method of folding chemokines same described herein are methods lymphotactin applicable also to other chemokine proteins.

Often for the function of chemokine important its free N-end. Therefore, in some embodiments it is advantageous to construct such a merger, in which the second cytokine, antibody or other protein component can be merged with the end of the chemokine. To construct a protein complex that contains two active chemokine, useful, for example, to merge two different chemokine N-ends of the heavy and light chains of the antibody. Some chemokines, such as IL-8, under physiological conditions are dimers. For some applications it is useful to co-Express a fusion of several cytokines with the antibody, such as a merger IL-8-antibody-cytokine, together with nakitai component of the IL-8 or component of the IL-8 with another partner in the merger, which does not interact with the protein component is an antibody. In this case, the various components of the IL-8 fused protein can homodimerization without spatial obstacles or polymerization, which can occur if the aircraft is components of IL-8 were merged with chain antibodies. The desired protein with multiple cytokines can then allocate fractionation by size or by binding with binding with the antibody protein, such as protein a of Staphylococcus.

In a preferred example of execution of the fused proteins with multiple cytokines containing a chemokine, a protein also includes a component having a function of targeting, such as an antibody, to bind to the antigen. Without going into theory, one would expect the prevalence of chemokine throughout the body will not work or will not lead to a General reduction of sensitivity of the cells to this chemokine. In addition, I believe that chemoattractant function of the chemokine can be manifested only when there is a concentration gradient of the chemokine.

The preferred embodiment is a protein lymphokine-antibody-interleukin-2. Another preferred embodiment is one in which a and chemokine, and a second cytokine include Th1 immune response. For example, the most preferred example is a protein that contains IP-10 and IL-12.

Extension of time half-life of several cytokines with short times sex life

The present invention also describes a fused protein containing two cytokines, both of which have low half-life in serum, merged with a third component having a bol is choe half-life in serum. For example, if you want the stimulation of dendritic cells, it is useful to combine the activity of IL-4 and GM-CSF (Thurner // J Immunol. Methods. 1999. T. P.1-15; Palucka, etc. // J. Immunol. 1998. T. S-4595). Because IL-4 and GM-CSF are small molecules with short times of half-life in serum, it is useful to construct a fused protein containing the Fc region, IL-4 and GM-CSF. The resulting molecule is a potent stimulator of proliferation and activity of dendritic cells. Similarly, for the delivery of cytokines joint activities to cells expressing a predetermined antigen, IL-4 and GM-CSF can be drained from addressing component, such as an antibody.

The Fc region, one or as part of an intact antibody, can give mergers with several cytokines, some of the properties that can be beneficial or adverse depending on the particular application. These properties include dimerization, extension of time half-life in serum, the ability to bind complement, the ability to participate in activity-dependent antibody-mediated cell cytotoxicity (ADCC) and binding to receptors for the Fc. If the most desirable characteristic is the extension of time half-life in serum, and immunological properties of the Fc region is not important or desirable, it is preferable to use the Fc region, which is a natural cook who ntom or mutant lost one or more immunological properties. For example, if you want to equalize and extend the half-life in serum of two or more cytokines short at times half-life in serum, it is preferable to construct a protein with multiple cytokines containing the Fc region of lgG2 or lgG4, which has a relatively reduced affinity or no affinity receptors for the Fc, or Fc region containing a mutation in the binding site with receptors for the Fc. In fact, it has already been shown that the merging of some cytokines with antibodies increases the affinity of the fused protein to receptors for Fc and that this increases their rate of excretion in animals. It was found that the use of Fc regions with low affinity receptors for the Fc significantly increases the half-life in serum of these molecules (Gillies, etc. // Cancer Res. 1999. So 59. C. 2159-2166). In some cases, and depending on the use of cytokines Fc region binding to the receptor for Fc, leads to the internalization of the fused protein with multiple cytokines and to the destruction of one or more cytokine components.

Addressing

The present invention also describes a fused protein in which two or more cytokines attached to a protein, is able to localize the cytokines on the specific molecule is ishani, the cell or bodily division. The preferred molecule with localizing ability is an antibody or part of a molecule containing recognize the antigen variable regions of antibodies. However, there may be used other localizing molecules or domains, such as specific ligands or receptors that exist in the nature of binding proteins that bind to specific substrates, enzymes, artificial peptides that were selected on the basis of binding capacity or localization peptides with specific physico-chemical properties of the resulting addressing the ability of proteins exhibiting the capacity for addressing, by associating with addressable molecule, or other types of proteins. In the event of a merger with addressable molecule two cytokines preferred the first cytokine is IL-12. If you are using IL-12, the second preferred cytokine is IL-2 or GM-CSF.

In the event of a merger with the antibody, there are many ways that can be flushed two or more cytokines, as for joining, there are several possible places. For example, the antibody IgG consists of two heavy and two light chains. Two of the cytokine can be merged with each other and then attached to the N - or C-end of either the heavy or light chain. Alternatively, each cytokine can be slapo separately with one of the N - or C-ends of the molecule antibodies.

Figure 3 illustrates the set of ways in which two of the cytokine can be fused with the antibody molecule. For example, according pigs, protein 44 of the present invention may be merged with the C-end of the heavy chain of immunoglobulin (46)which is connected to a light chain immunoglobulin (48). As in figure 2, protein 44 may have a different structure, including, for example, the structure of the fused proteins 10, 16, 24, 26, 28, 30, 32, 34 or 36, shown in figures 1A-1I. As shown in figv, protein 44 may be merged with the N-end of the heavy chain of immunoglobulin (46)connected to a light chain immunoglobulin (48). Shown in figures 3C and 3D examples of implementation protein 44 is fused to the N-end (figs) or C-end (fig.3D) the light chain immunoglobulin (48)connected to the heavy chain of immunoglobulin (46). As shown in figures 3E and 3F, the first cytokine (12) may be merged with the light chain of immunoglobulin (48)connected to the heavy chain of immunoglobulin (46), merged with a second cytokine (14). Cytokines 12 and 14 can be merged with N-ends (figs) or with C-tips (fig.3F) chains of immunoglobulin. Alternatively, as shown in fig.3G, the first cytokine (12) may be merged with the N-end of the light chain of immunoglobulin (48), while the second cytokine (14) merged with the end of the heavy chain of immunoglobulin (46).

Merge with single-chain antibodies

Sometimes it is convenient to Express the activated antibodies in the form of single-stranded molecules. The present invention also provides fused proteins in which two or more cytokines fused to single-chain antibody. This has the advantage that it reduces the number of DNA structures used ekspressirovannoj desired fused protein, which can be particularly useful for gene therapy. In particular, if cytokines are single-stranded molecules, then the merge of cytokines with single-chain antibody will allow you to Express the protein as a single protein chain.

As shown in figures 4A-4C, in some embodiments, the cytokines can be merged with the single-chain antibody on its N end, it is the end or at both ends. For example, as shown in figa, protein 44 may be merged with the C-end of single-chain antibodies (50)containing the variable region of light chain (52) and the variable region of the heavy chain (54). As shown in figv, protein 44 may also be fused to the N-end single-chain antibodies (50). In the example implementation shown in figs, the first cytokine (12) fused to the N-end single-chain antibodies (50), and a second cytokine (14) merged with the end of single-chain antibodies (50).

The preferred embodiment is a fusion of IL-12 and a second cytokine with single-chain antibody. The preferred embodiment includes as the second cyto is in IL-2 or GM-CSF.

The constant region of the antibodies have the ability to participate in many effector functions. For example, lgG1 involved in the binding of complement, ADCC and binding with the receptor for Fc. The position at which the cytokine is fused, can alter the effector function of the constant region of the antibody, which is useful if the modulation of these effector functions are required.

In some cases it may be necessary to construct a merger of two or more cytokines with the component having the addressing region of the antibody, but devoid of constant regions. This protein is smaller than the full merge antibodies to two or more cytokines, which for some purposes is an advantage. In addition, this protein will be deprived of one or more effector functions of intact antibodies.

Therefore, the present invention is featured in fused proteins in which two or more cytokine merged with the single-stranded region of the Fv. As shown in the embodiments shown in figures 5A-5C, two cytokine can be merged with the N-end or the end of the region, Fv, or cytokines can be merged on one with each of the ends. For example, as shown in figa, protein 44 of the present invention may be merged with the C-end of single-chain Fv region containing the variable region of light chain of immunol bolina (52) and the variable region of the heavy chain of immunoglobulin (54). Protein 44 may also be fused to the N-end region Fv, as shown in figv. As shown in figs, the first cytokine (12) may be merged with the N-end of the Fv region and the second cytokine (14) can be merged with the end of the Fv region.

Antibodies as heterodimeric media for several cytokines

In some circumstances you may need to construct a merger of two or more cytokines, which both cytokines activity needed for the same end. For example, it may be that for each activity of the cytokine required naturally existing N-end. It is impossible to construct a fused protein in a single polypeptide chain, in which both cytokine components will be active.

The antibodies are heterodimeric proteins consisting of heavy and light chains covalently linked by disulfide bonds. If you want to construct a protein with multiple cytokines, which for both cytokine components required intact, nality N-end, then it is preferable to merge two cytokine with N-ends of the heavy and light chains of antibodies (fige). Similarly, if you want to construct a protein with multiple cytokines, which for both cytokine components required intact, nality With the end, then it is preferable to merge two of the cytokine with the ends of Aheloy and light chains of antibodies (fig.3F). If the antibody is used only as a carrier for the connection in this way the two cytokines, then it may be useful mutational change or to delegate those parts of the antibody, which adds additional properties related to immune function. For example, it may be preferable to use as the carrier of the Fab region, because the region Fab preserves the ability of the antibodies to homodimerization, but loses the features of the Fc region. May also be useful to use the antibody or fragment antibodies in which failed the binding site to the antigen.

Merge multiple cytokines with antibodies combine many of the new distinctive features of the present invention. In mergers of several cytokines with the antibody is equalized and prolonged the half-life of cytokines in the serum; the activity of both cytokines is localized in the area of the target and it is particularly important that excluded toxic effects due to systemic injections several synergize acting cytokines, there is no need to directly drain the cytokines with each other, but they can be merged with different places of the heavy and light chains of antibody molecules.

When designing a fused protein containing multiple cytokines and antibody, there are many choices, structures and config the Nations, which can be differentiated using conventional experiments. Also useful consideration of the positions of structural biology. For example, many cytokines fall into a class called 4-helix bundles. These structures consist of four alpha-helices, and their N-end and C-end close neighbors to each other. In General, the surface of the cytokine around the N - and C-end is not used in the binding of cytokine receptor, so both ends can be used to merge with the antibody or with a second cytokine. However, due to the spatial (physical) limitations it is sometimes difficult to directly drain and N-and C-end of the cytokine type 4-helical bundle with different parts of the molecules. So if you want to merge two different cytokine type 4-helical bundle with the antibody, it is useful to merge cytokines with different sites of antibodies. Alternatively, if you want to construct a polypeptide chain in the form of a chain-lg)-cytokine-cytokine, to overcome the steric problems, you can use one or more flexible joints.

To attach several cytokines instead of antibodies to use other secreted heterodimeric molecule. For example, you can use complex containing prostate-specific antigen and an inhibitor of proteases, with which it forms a complex, heavy chain lgA and the chain J, the members of the families of the TGF-beta and their partners linking type astatine, or IL-12.

Nucleic acids

The present invention also takes an important place of nucleic acids that can Express each of the above types of proteins. They include a nucleic acid encoding the fused protein containing two or more cytokine; mergers involving two or more cytokines and dimerization domain, such as an Fc region; merging containing two or more cytokine, merged with antibody and two or more cytokine, merged with the Fv region. The preferred forms of nucleic acids are DNA vectors, which can be bacterial cells or mammalian cells expressed fused protein. For fused proteins containing multiple polypeptide chains, you can use multiple nucleic acids. Alternatively, it may be useful to align two or more sequences encoding a protein, one molecule of nucleic acid. The examples illustrated specific forms of attention-grabbing nucleic acids encoding combining multiple cytokines.

Nucleic acids of the present invention is particularly useful for the expression of the fused proteins with multiple cytokines or for producing these proteins, or for the purposes of gene therapy.

Methods of synthesis of useful embodiments of the present izobreteny is, and analysis methods suitable for testing their pharmacological activity described in the examples.

The present invention also provides pharmaceutical compositions and methods of their use for the treatment and prevention of a wide range of diseases, including (but not limited to) for the treatment of various infectious diseases and cancer, and vaccination against various diseases.

Fused proteins with multiple cytokines can be applied for the treatment of diseases caused by bacteria, parasites, fungi or viruses, or cancer. For example, it is known that IL-12 has a protective effect against many types of infections, including (but not limited to) infection by Listeria monocytogenes bacteria; parasites Toxoplasma gondii, Leishmania major and Schistosoma mansoni; the fungus Candida albicans, and viruses: virus choriomeningitis and cytomegalovirus. Some cytokines generally operate in combination, it is often useful to apply the fused proteins containing two or more cytokines, about which we know that they are synergize. For example, since it is known that IL-2 enhances the action of IL-12, it is useful to combine these cytokines in the treatment of diseases caused by bacteria, parasites, fungi and viruses.

The preferred method of treatment of infectious diseases is to use merged Bel is impressive with several cytokines, additionally merged with addressing agent, which places several cytokines at the site of infection. Different strategies of addressing are described below.

The pharmaceutical compositions of the present invention can be used in the form of solids, liquefied solids or in liquid dosage forms. Such forms include, for example, pills, capsules, powders, liquids, suspensions and the like, preferably in the form of a single dosage, suitable for administration of precise dosages. Songs include suitable pharmaceutical carrier or excipient and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc. Such fillers may include other proteins such as human serum albumin or plasma proteins. Actual methods of preparing such dosage forms are known or will be apparent to experts in this field. Compositions or formulations for administration in any case will contain such an amount of the active component (components), which is effective to achieve the desired effect in the subject to treatment of a subject.

The introduction of these compositions may be made by any of the accepted for agents capable of such activity, ways of administration. These methods include oral, parenter is the emotional or local injection and other routes of administration in the body. The preferred method of administration is injection.

The amount of the active component will, of course, depend on the subject to the treatment of the subject, the severity of the disease, the route of administration and a doctor prescribing the medication.

As described above, cytokines, such as IL-2, IL-12, GM-CSF, IL-4, and others, have been studied in the treatment of cancer. In some circumstances, in cancer treatment has the advantage of using a fused protein with multiple cytokines, due to the greater ease of administration, increased time half-life in serum is one of cytokine components and/or enhance the modulation of the relative activities of the two cytokines.

The preferred method of cancer treatment is to adresovano to send cytokines in specific organ or tissue, so that the action of cytokines can be concentrated, and side effects of their distribution in the body can be eliminated. For example, it is expected that the merger of several cytokines with the Fc region are concentrated in the liver, which can be useful in the treatment of cancer, localized in the liver. The preferred method is to use a fused protein with multiple cytokines, which is optionally fused with addressing agent such as an antibody. In particular, antibody KS 1/4 and 14.18 directed against ofwholesale antigens (N.M. Varki andetc. // Cancer Res 1984. T. S-687; Gillies, etc. // Journal of Immunological Methods. 1989. T. S; U.S. patent No. 4975369 and 5650150). When using merge antibodies with multiple cytokines is often useful to investigate the tumor type and to choose the antibody to the antigen, which is likely present in this tumor type. For example, it may be useful to characterize the tumor by the method of FACS analysis, to conduct Western blokirovanie, to explore the DNA of tumor cells, or simply to identify the type of tumor cells. Such research methods characteristics of the tumors are well known to specialists in the field of the study of tumors, such as oncologists and specialists in tumor biology. You can also adresovano to direct fused proteins with multiple cytokines in various other ways, such as a merger with components representing specific ligands or receptors, fusion with a peptide aptamers with a pre-selected by the ability to bind, chemical conjugation with small molecules localizing ability, etc. These ways of addressing can also be used in treating other diseases, such as infectious diseases.

Treatment with gene therapy of cancer and other cell disorders

Nucleic acids of the present invention can be used as a means of gene therapy for cancer treatment what other diseases where you need to target the immune system to a specific cell type. For example, cancer cells extracted from human or animal cancer cells enter one or more nucleic acids encoding a protein with multiple cytokines, and then the cancer cells are injected back to the human or animal. Alternatively, you can enter the DNA in the cancer cells in situ. After that the person or animal is formed immune response to cancer cells that can lead to cure or reduce the severity of the cancer. Gene merge multiple cytokines, United with the appropriate regulatory elements to ensure expression in mammalian cells can be transliterowany in cancer cells by any of many methods, including using coprecipitation with calcium phosphate, "gene gun", adenoviral vectors, cationic liposomes, retroviral vectors, or using any other effective method of transfection. The nucleic acid can encode a protein of several cytokines, optionally fused with other components.

Anticancer hemotherapy with a nucleic acid expressing a fusion of several cytokines, can be combined with other cancer therapies, such as ways of enhancing immunostimulatory properties kitoh the cytokine protein. For example, a nucleic acid of the present invention may also Express other protein components, which may contribute to the development of the immune response to antigens expressed by cancer cells, or may be transliterowany together with other nucleic acids expressing such protein components. Specifically, in cancer cells it is possible to transliterate nucleic acid expressing together a stimulating surface protein V7 (Robinson and others // U.S. Patent No. 5738852). Transfection of cancer cells nucleic acid expressing a fusion of several cytokines, may also be accompanied by the introduction of antibodies or immunocytokine, aimed at cancer cells (Lode, etc. // Proc. Nat. Acad. Sci.1998. T. S). Transfection of cancer cells nucleic acid expressing a fusion of several cytokines, may also be accompanied by the introduction of inhibitors of angiogenesis (Lode, etc. // Proc. Nat. Acad. Sci.1999. V.9. S).

Methods of treatment using additional Immunostimulants and/or antagonists of angiogenesis can also be combined with systemic introduction of fused proteins of several cytokines. The advantage of the joint introduction of additional Immunostimulants or angiogenesis blockers is that such impacts, in contrast to the destructive DNA Agay is tov and blockers of the cell cycle, don't kill immune cells, which can be divided due to stimulation of fused protein with multiple cytokines.

The preferred embodiment of this method of gene therapy is the introduction to the cancer cells of one or more nucleic acids coding (coding) of IL-12 and a second cytokine, and then in the reverse introduction cancer cells are human or animal. Preferably the second cytokine is an IL-2 or GM-CSF.

The present invention provides a new vaccine compositions and methods to enhance the effect of vaccines designed to provide the vaccinated mammal hosts protective mediated by cells of the immune response to certain pathogens, using as adjuvant of two or more cytokines that have been merged. For example, if you want Th1 immune response, you can merge several contributing reactions Th1 cytokines and enter animal derived protein in combination with the antigen.

In particular, it is possible to merge and enter together with the antigen IL-12 and IL-2. Alternatively, IL-12 and IL-2 can optionally be merged with itself antigenic protein and use it to stimulate the immune response. In this case, the invention is directed to vaccines, which is implemented through mediated by cells of the immune system of the host, i.e. the appearance of qi is otoxicity T-lymphocytes and activated phagocytes to provide protection against infection by a specific pathogen. Especially useful to conduct vaccination fused proteins containing IL-12, IL-2 and antigen, because this combination controls the Th1 response to the antigen. Used in humans conventional adjuvants, such as alum, tend to induce the reaction Th2 dysbalance.

If you want immune response Th2 dysbalance, you can use the merged combination of cytokines that control the Th2 response. For example, it may be useful to merge IL-4 and IL-10 to get one molecule, where the resulting protein is used as adjuvant. In particular, if it is necessary to mobilize the animal dendritic cells can be merged combination of IL-4 and GM-CSF or to merge with Fc region to ensure binding to antigen-presenting cells, or to merge with an antibody capable of directing the slit cytokines in the target tissue such as a tumor.

The present invention provides new therapeutic compositions and methods of adjuvant strengthen actions aimed at ensuring synergistic action with certain therapeutic compositions, including the so-called "cancer vaccines", which may contain a selected antigen, present on a cancer cell. For example, a protein containing two or more fused cytokines can directly enter the appropriate way along with the desired image processing is different cancer cells.

EXAMPLES

Example 1. Construction of gene fusions are able to Express the fused protein cytokine-cytokine

To create a multifunctional protein that contains several cytokines, were synthesized gene fusion subunit R40 interleukin IL-12 interleukin IL-2 and subunit R40 interleukin IL-12 GM-CSF. Additionally, the coding sequence for the Mature murine R35 (SEQ ID NO:1) was merged with the promoter and leader sequence that provides high level expression and efficient secretion. Coding sequences for m mouse components p40 IL-2 and p40-GM-CSF are shown respectively as SEQ ID NO:2 and SEQ ID NO:3. It was also engineered the merger (the human p40)IL-2 (SEQ ID NO: 4). Were designed to merge the Fc region of murine lgG2a mouse subunit R35 (SEQ ID NO:5) and human subunit R35 with the Fc region of human lgG1 (SEQ ID NO:6), using a previously disclosed plasmids (Lo etc. // Protein Engineering. 1998. T. S-500; Lo, etc. // U.S. Patent No. 5726087).

The fusion of the Mature subunit R35 mouse and human C-end of the heavy chain of the antibody KS 1/4 described Gillies and others (J. Immunology. 1998. T. S-6203). Merge Mature subunit R35 mouse and human C-end of the heavy chain of the antibody 14.18 were constructed in a similar way (publication of the international application WO99/29732).

Discussed here type of strategy cleaner with IL-2 and GM-CSF are generally applicable to the merger of two or more cytokines. Characteristically, the coding sequence for most N-terminal part contains a signal sequence for secretion, whereas the C-terminal parts of the signal sequence is not required. In some circumstances it may be useful to place the coding sequence for a short petidomo bridge, preferably a length of 10-15 amino acids and rich in glycine and alanine, between the coding sequences for the two cytokines. Manipulation of DNA used to create all of these types of mergers, consistent with the level of training of specialists in this field.

For example, the details of constructing a merge subunit R40 murine IL-12 murine IL-2 was as follows. Fully subunit cDNA R40 murine IL-12 was cloned using the polymerase chain reaction (PCR) from spleen cells of a mouse activated by concanavalin And (concentration in culture medium with 5 μg/ml for 3 days). Direct primer had the sequence AA GCT AGC ACC ATG TGT CCT CAG AAG HUNDRED ACC (SEQ ID NO:7), in which the Nhel site GCL4GC (nucleotides 3-8 sequence SEQ ID NO:7) was placed in the ascending side of the initiation codon ATG broadcast, and the reverse primer had the sequence CTC GAG HUNDRED GGA TCG GAC CCT GCA GGG (SEQ ID NO: 8), in which the Xhol site CTCGAG (1-6 nucleotides of the sequence SEQ ID NO:8) was placed in the descending side of the once behind the stop codon broadcast TAG (the anticodon of a HUNDRED). After checking the nucleotide sequence of the fragment Nhel-Xhol containing cDNA mu-p40 their natural leader, sewn by ligase in split restrictase Xbal-Xhol vector pdCs (Lo etc. // Protein Engineering. 1998. So 11. C. 495-500). Restriction enzymes cut sites Nhel and Xbal had compatible sticky ends, and the Nhel site was used to clone mu.p40 as mu.p40 had an inner Xbal site.

To construct a DNA that encodes a fusion mu.p40-mu.lL-2, used oligonucleotide bridge to connect DNA mu-p40 its website > PST (TGC AG) with Smal fragment-Xhol containing the cDNA of the Mature murine IL-2. The DNA sequence at the junction of the fused protein has been With TGC AGG GTC CGA TCC CCG GGT AAA GCA CCC (SEQ ID NO: 9), where TGC AG (1-6 nucleotides of the sequence SEQ ID NO: 9) is a website > PST, CCG GG (15-20 nucleotides of the sequence SEQ ID NO: 9) is the Smal site, TSS - residue C-terminal amino acid sequence of murine subunit R40 and GCA encodes the N-terminal residue of the Mature murine IL-2.

DNA encoding a single-chain fusion mu.lL-12-mu.GM-CSF was obtained from DNA constructs, the coding above single-chain fusion mu.lL-12-mu.lL-2 replacement cDNA mu.lL-2 cDNA mu.GM-CSF in the Smal site. The DNA sequence at the junction of single-stranded mu.lL-12 mu.GM-CSF has been With TGC AGG GTC CGA TCC CCG GGA AAA GCA (SEQ ID NO:10), where TGC AG (nucleotides 1 sequence SEQ ID NO:10) is a website > PST, CCG GG (17-22 nucleotides of the sequence is SEQ ID NO:10) - the Smal site, SHH encodes the C-terminal amino acid residue of murine subunit R40 and GCA encodes the N-terminal residue of the Mature murine GM-CSF.

Example 2. The expression of the fused proteins with IL-12

Slit proteins IL-12-IL-2 expressed in the following way. Different combinations of individual vectors encoding fusion with the participation of R40, and vectors containing the coding R35 proteins, introduced a joint transfection in 293 cells carcinoma of the epidermis of a person for a temporary expression of the fused proteins. DNA was purified using preparative sets (company Wizard, Promega Inc.), precipitated with ethanol for sterilization and resuspendable in sterile water.

For the expression of biologically active heterodimeric fused protein with IL-12 different combinations of individual vectors encoding merged and naslite form subunits, temporarily expressed co-transfection of 293 cells carcinoma of the human epidermis. DNA was purified using preparative sets (company Wizard, Promega Inc.), precipitated with ethanol for sterilization and resuspendable in sterile water. Precipitation with calcium phosphate was obtained by standard methods, using 10 µg DNA per ml (5 µg each, if conducted joint transfection of the two plasmids), and was added in the amount of 0.5 ml per Cup to the cultures of 293 cells grown on plates with a diameter of 60 mm to about 70% confluence (Molecular Cloning: A Lboratory Manual, 2ndEd., Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, 1989). After 16 h, the medium containing the precipitate was removed and replaced with fresh medium. After 3 days the culture fluid was collected and analysed the presence of the product of expression of transfected genes by the method of ELISA, the biological determination of the activity of IL-12 or immunoprecipitates and analysis of labeled radioactivity of proteins by electrophoresis in gels in the presence of SDS. For the introduction of a label when replacing the culture medium on the second day of cultivation used the medium without methionine and added35S-methionine (100 mccoury/ml). After incubation for 16 h, the medium was collected, cleared by centrifugation (5 min at 13,000 rpm in a tabletop microcentrifuge) and incubated with beads of sorbent (protein a)-sepharose (10 ál pellet in 1 ml of the supernatant culture fluid). After 1 h incubation at room temperature, the pellets were washed by repeated centrifugation and resuspending in PBS buffer containing 1% nonidet-R40 (NP-40). The final precipitate resuspendable in gel buffer with SDS and boiled 2 minutes After removal of the pellet by centrifugation, the supernatant was divided into two aliquots. To one sample was added reducing agent (5% 2-mercaptoethanol), and both samples were boiled 5 min, and then was applied to a polyacrylamide gel with SDS. After electrophoresis the spruce exhibited the x-ray film (autoradiography).

The transfection was carried out by expressing the following plasmids: mu.p35 plus mu.p40-IL-2, KS-1/4-mu.p35 plus mu.p40, KS-1/4-mu.p35 plus mu.p40-IL-2, 14.18-mu.p35 plus mu.p40-IL-2, hu.Fc-hu.p35 plus hu.p40-IL-2, KS-1/4-hu.p35 plus hu.p40-IL-2, and 14.18-hu.p35 plus hu.p40-IL-2, where "mu" refers to murine proteins, a "hu" to the human.

When cells were labeled in the process of metabolism35S-methionine and explored secreted proteins by gel-electrophoresis in reducing conditions in the presence of SDS and autoradiography, in each case, the observed high levels of expression. The values of molecular weight restored fused proteins were predicted based on the values of molecular weight protein components, i.e. 35 kDa for subunit R35 of IL-12; 40 kDa for subunit R40 IL-12; 16 kDa for IL-2; 32 kDa for Fc; 55 kDa heavy chain immunoglobulin; and 28 kDa for the light chain of the immunoglobulin. In the analysis of observed proteins migrating respectively the values of molecular weight close to predicted.

There were also highlighted consistently transfetsirovannyh cell line expressing the fused protein with multiple cytokines. For heterodimeric structures of IL-12 p40 IL-2 or IL-12 p40-GM-CSF expressing vectors encoding the fused protein was constructed as described previously for a single subunit R40 IL-12 (Gillies, etc. // J. Immunol. 1998. So 160: C. 6195-6203). Line transfected cells, expressyou the e fused proteins with R40, the second time was transfusional expressing vectors, encoding the subunit of the R35 IL-12, or protein Fc-p35, or protein antibody-R35 described in Gillies, etc. // J. Immunol. 1998. T: S-6203).

Collected supernatant from stably transfected cells expressing hu.Fc-IL-12-IL-2 (i.e. expressing KS-p35 and p40 IL-2). Purification of products was performed on the sorbent (protein a)-sepharose binding and elution in accordance with the instructions of the supplier (Repligen, Needham, MA, USA). Levels of IL-12 and IL-2 in purified proteins was determined by ELISA method. The results showed that the content of individual cytokines differed in mass by approximately 4 times, which corresponds to 4-fold difference in molecular weights of IL-12 and IL-2. Similarly, the analysis by the method of ELISA for levels of IL-12 and IL-2 in the products of the transfected cells expressing hu.KS-IL-12-IL-2 was given for IL-12 and IL-2 are the same value. Thus, within the accuracy of the definitions using ELISA method, the measured values show that IL-12 and IL-2 are produced in a molar ratio of near 1:1. The same results were obtained for fused proteins IL-12-GM-CSF fused with either Fc or with the whole antibody.

Example 3. Synergene action fused proteins in the analysis of the induction of IFN-γ

Measured biological activity of fused proteins IL-12-IL-2 in the test for the induction of IFN-γ using the-W or resting, or mitogen-activated mononuclear cells of peripheral blood (RVMS)obtained from volunteers (6). The production of IFN-γ measured using ELISA method.

Mononuclear cells of peripheral blood was obtained from healthy volunteers and were purified by centrifugation at 1700 rpm for 20 min in a gradient of Ficoll-Hypaque (Pharmacia). Containing RVMS "leathery layer was diluted in culture medium without serum (SF-RPMI) to 50 ml, the cells were collected by centrifugation at 1500 rpm for 5 minutes After centrifugation in a gradient cells resuspendable environment for cell cultures containing 10% serum fetal cow (RPMI-10) with addition or without addition of phytohemagglutinin (PHA, 10 μg/ml) to the density of cells 5 x 106cells/ml and cultured for 3 days at 37°in CO2-thermostat with moisture. The cells were collected by centrifugation, washed three times with equal volumes of SF-RPMI and resuspendable in fresh medium RPM-10 (1 x 106cells/ml). Aliquots (100 µl) were distributed in the cells of tablets with 96 cells, receiving a finite number of cells 105cells on the cell. Samples from the culture medium serially diluted and added into the cells of the tablet with 96 cells. In the control cells contributed IL-12 (figa) or equimolar mixture of commercial preparations of IL-2 and IL-12 (pigv). Cytokines were obtained from firm R & D Systems. Tablets Inka is Aravali for 48 h at 37° With CO2thermostat. In selected for analysis aliquot (20 µl) using ELISA method was determined by the concentration of IFN-γ according to the supplier's instructions (Endogen, Inc., Woburn, MA, USA).

On figa presents a comparison of the activities of the fused protein of IL-12-IL-2 and one IL-12. The results show that one IL-12 induces moderate levels of IFN-γwhereas protein IL-12-IL-2 in a high degree induces the synthesis of IFN-γ. As it is also known that IL-2 is ineffective in the synthesis of IFN-γ, these results show that in the fused protein of both IL-12 and IL-2 functional and act synergize.

Further, the activity of the fused protein is Fc-IL-12-IL-2, fused protein KS-IL-12-IL-2 and a mixture of equimolar amounts of IL-12 and IL-2 were compared for their ability to induce IFN-γ. Presented at figv the results show that the fused protein is Fc-IL-12-IL-2 and protein KS-IL-12-IL-2 have approximately the same activity as equimolar mixture of IL-12 and IL-2. The same results were obtained when constructing fused proteins were used murine forms of IL-2 and IL-12, and construction was produced in the same manner as described in example 1 for the human form.

Example 4. The biological activity of IL-2 and IL-12 in the slit proteins IL-12-IL-2

Activity of IL-2 and IL-12 fused proteins was compared with the activity of free cytokines in tests on the proliferation. Actively the TB molecules (mouse antibody 14.18)-IL-12-IL-2 was tested in a typical analysis of IL-12 in the test cell proliferation. Human PBMCs were obtained from volunteers and were cultured for 3 days in the presence of 5 μg/ml of phytohemagglutinin-P, washed with medium HBSS Hank and were sown in tablets for micrometrology at a density of 105cells in the cell, according to the standard procedure (Gately, M.K., R. Chizzonite, and D. H. Presky // Current Protocols in Immunology. 1995. S-6.16.15). Cells were incubated in the presence of different subjects proteins for 48 h, 10 h to determine the level of incorporation of radioactivity was added 0.3 mccoury3H-thymidine. IL-12 and equimolar mixture of IL-12 and IL-2 stimulated the incorporation in cells3H-thymidine with dose dependence and protein 4.18-IL-12-IL-2 was approximately equally effective in stimulation include3H-thymidine. IL-2 stimulated the inclusion of3H-thymidine only at higher molar concentration, which indicates that the observed inclusion of3H-thymidine stimulated fused protein 14.18-IL-12-IL-2, caused mainly by the activity of IL-12. The results are shown in Fig.7.

Additionally tested the biological activity of a component of IL-2 in another test on the proliferation of cells according to the standard procedure (Davis L.S., Lipsky R.E. and Bottomly, K. // Current Protocols in Molecular Immunology. 1995. S-6.3.7). For proliferation of the cell line of mouse CTLL-2 IL-2. Cell line CTLL-2 will also proliferated in response to IL-4, but it is not the feeling is sustained fashion to the action of IL-12. Cells CTLL-2 active in logarithmic growth phase were washed twice in medium without IL-2 and were sown in tablets for micrometrology with density 1×104cells the cell in the presence of varying amounts of commercial murine IL-2, fused protein mu.14.18-IL-12-IL-2 or commercial murine IL-12 and continued the cultivation period of 48 hours At the end of the period growth method MTT/MTS determined the number of viable cells. On Fig presents experience, which varied levels of IL-2, IL-12 or fused protein 14.18-IL-12-IL-2. The results show that murine IL-2 and protein mu.14.18-IL-12-IL-2 are approximately equally active in stimulating the proliferation, whereas the increase in the number of murine IL-12 caused a marked stimulation of cell proliferation. This result shows that stimulation of cell proliferation CTLL-2 fused protein 14.18-IL-12-IL-2 induced component of the IL-2 and not a component of IL-12.

Example 5. Construction and expression of single-stranded and mnogotirazhnogo fused proteins IL-12-IL-2 antibody and without antibodies

Single-chain protein with murine cytokines IL-12-IL-2 was designed as follows. The fusion of coding sequences for p40 IL-2 were designed using methods similar to the one used in the construction of fusion of human proteins p40 IL-2 in example 1. To connect with DNA encoding the subunits of the R35 and R40 IL-12 and to create a single coding sequence, synthesized DNA encoding the bridge, with Xhol site at the 5'end and a BamHI site on the 3'-end. the 5'-End sequence that encodes the fusion of mouse subunit R40 with IL-2, was modified by the introduction of a restriction site and then Legerova with 3'-end of the bridge. 3'-End sequence that encodes a murine subunit R35, was modified by creating a restriction site and Legerova into the Xhol site of the bridge. cDNA encoding the single-stranded mu.lL-12 and mu.p40-mu.lL-2 described in example 1, was combined with a convenient restriction site in the sequence for R40 to produce a third DNA construct encoding a single-chain fusion mu.lL-12-mu.lL-2. These stages was performed using various necessary vectors and the selected DNA fragments. Sequence of SEQ ID NO: 11 represents a region of nucleotide sequence that encodes the fusion of mouse components R35-bridge-R40-IL-2.

At the same time, using appropriate methods was designed sequence encoding the corresponding single-chain murine IL-12. The coding sequence represented by the sequence SEQ ID NO: 12.

Moreover, it was additionally constructed DNA sequence encoding the Fc region of murine lgG2a, fused with the N-end merger R35-linker-p40 IL-2. The coding sequence is presented as SEQ ID NO: 13.

Coltivirus the nnye 293 cells were transfusional expressing plasmids, encoding the single-chain protein with murine partners: Fc-IL-12-IL-2 and Fc-IL-12. The expression of the fused proteins were analyzed as described in example 2. Cleaning merged with Fc proteins was performed by linking with the sorbent (protein a)-sepharose. Observed high levels of expression of Fc-IL-12-IL-2 and Fc-IL-12. Based on the values of the apparent molecular weight obtained for migration in SDS-gels, it was concluded that the protein was synthesized intact. These values were 123 kDa for Fc-IL-12-IL-2 and 107 kDa for Fc-IL-12.

Protein KS-sclL-12-IL-2 described in example 1, is a tetramer with two different polypeptide chains: a light chain of KS-1/4 and heavy chain of KS-1/4 connected With the end part of sclL-12-IL-2. To determine which sites on the antibody molecule required for attaching cytokine components, was designed second protein, in which the components: antibody KS 1/4, IL-12 and IL-2 was in a configuration other than the configuration merge KS-IL-12-IL-2 in example 1. This second protein was tetramer and consisted of two different polypeptides. One polypeptide consisted of the light chain of the antibody KS 1/4. Another polypeptide consisted of single-stranded mu. IL-12, merged with Mature M-end of the heavy chain of the antibody KS1/4, for which the carboxyl end of the heavy chain was located murine IL-2.

cDNA encoding a subunit of the R35 mouse is on IL-12, cloned by PCR from spleen cells of a mouse activated by concanavalin And (5 μg/ml of culture medium for 3 days). Direct primer had the sequence AGCTT GCTAGCAGC ATG TGT CAA TCA CGC TAC (SEQ ID NO: 14), where the Hindlll site AAGCTT (1-6 nucleotides of the sequence SEQ ID NO: 14) placed in the ascending side of the initiation codon ATG broadcast, a reverse primer had the sequence CTCGAG CTT TCA GGC GGA GCT CAG ATA GCC (SEQ ID NO: 15), where the Xhol site CTCGAG (1-6 nucleotides of the sequence SEQ ID NO: 15) is placed in a downward position relative to the stop-codon broadcast TGA (anticodon TCA).

DNA encoding a single-chain IL-12, is a DNA mu.p35 connected with the oligonucleotide coding for a rich residues glycine and serine bridge, followed by DNA mu.p40. The resulting design has the place of attachment of the oligonucleotide with the following sequence:

where G AGC TC (1-6 nucleotides of the sequence SEQ ID NO: 16) is a Sacl restriction site located directly in the ascending direction from the stop codon broadcast mouse subunit R35, GCG encodes the C-terminal amino acid residue of murine subunit R35, GGA TCC (50-55 nucleotides of the sequence SEQ ID NO: 16) - the restriction site BamHI entered for ligating and ATG encodes N-terminal amino acid residue of the Mature mu.p40.

DNA encoding one who ipotechnoe merge mu.IL-12-(heavy chain KS)-mu.GM-CSF, had at the junction of mu.p40 and Mature N-Terminus of the heavy chain KS the following sequence:

where TGC AG (1-6 nucleotides of the sequence SEQ ID NO: 18) is the restriction site > PST, located directly in the ascending direction from the stop codon broadcast R40, TCC encodes the C-terminal amino acid residue of murine subunit R40, and CAG encodes N-terminal amino acid residue of the heavy chain Mature KS. The obtained DNA encoding a single-chain fusion mu.IL-12-(heavy chain KS)-mu.lL-2, then jointly expressed with a light chain KS.

For further research which the ends of the molecule antibodies suitable for forming a connection with the merger, and how many polypeptides can be joined in a fused protein with multiple cytokines, was expressed and tested for activity third protein containing KS-1/4, IL-12 and IL-2, namely IL-12-KS(light chain) + KS(heavy chain)-IL2. This protein - hexamer and contains three different polypeptide. One polypeptide comprises a mouse subunit R35, merged with the light chain of the antibody KS1/4. The second polypeptide comprises a heavy chain antibody KS1/4, fused with human IL-2 (Gillies, etc. // Proc. Natl. Acad. Sci. USA. 1992. T. S), and the third polypeptide is a mouse subunit R40. When the expression of two light chains and two heavy chains are connected with the formation of tetrameristaceae antibody-cytokine. In addition, the R35 on the N end of the light chain is also linked by disulfide bonds with R40.

DNA encoding the fusion Mir-C8(light chain) is at the junction of the following sequence:

where G AGC TC (1-6 nucleotides of the sequence SEQ ID NO: 20) - Sacl restriction site located directly in the ascending direction from the stop codon broadcast R35, GCG encodes the C-terminal amino acid residue R35, GGA TCC (50-55 nucleotides of the sequence SEQ ID NO: 20) is the restriction site BamHI, introduced to provide ligation, a GAG encodes the N-terminal amino acid residue of the light chain.

For ekspressirovali this review of the fused protein was generated by transfection expressing vector containing the gene of resistance to neomycin, and selection in G418 cell line expressing mu.p40. Expressing mu.p40 cell line was then transfusional expressing vector containing the transcription unit and the light chain and heavy chain and a selective marker dihydrofolate-reductase, allowing selection on a background of methotrexate (Gillies, etc. // J. Immunol. 1998. So 160. C. 6195).

Example 6. The activity of the fused protein (murine single-chain IL-12)IL-2

The same methods that were used in example 4 were used to test the activity of murine single-chain fused protein of IL-2-IL-2, produced by transient expression. The amount of each cytokine in the supernatant of the cell culture was first determined using ELISA method, and then used it to establish dose-response relationships. Activity values were very close to the established and described above for the fused protein of IL-12-IL-2 with Fc and an antibody.

Specifically, the activity of IL-12 molecules fused protein with murine partners: mu.sclL-12-IL-2 and mu.Fc-sclL-12-IL-2 was tested in a test cell proliferation RVMS person described in example 4. IL-12 and equimolar mixture of IL-12 and IL-2 stimulated the inclusion of3H-thymidine into cells with dose dependence. Per mol of both fused protein and sclL-12-IL-2, and Fc-sclL-12-IL-2 stimulation include3H-thymidine was approximately as effective as IL-12 (figure 9). As described in example 4, IL-2 stimulates the inclusion of3H-thymidine only at a much higher molar concentrations, suggesting that the observed inclusion of3H-thymidine stimulated fused proteins sclL-12-IL-2, is mainly determined by the activity contained IL-12.

In addition, in tests based on cells was evaluated biological activity component of the IL-2 fused proteins sclL-12-IL-2, and found that it is within the accuracy of per mole approximately the same as the activity to Marchenkova IL-2. Biological activity component of the IL-2 was determined by cell proliferation CTLL-2, as described in example 4. The results show that murine IL-2, a fusion of murine proteins sclL-12-IL-2 fusion of mouse protein is Fc-IL-12-IL-2 were approximately active in the stimulation of cell proliferation. Murine IL-12 did not stimulate cell proliferation in CTLL-2. These results indicate that stimulation of cell proliferation CTLL-2 fused proteins sclL-12-IL-2 is a component of IL-2, but not part of IL-12.

The activity of IL-12 and IL-2 protein is Fc-IL-12-IL-2, IL-12-KS-IL-2 and IL-12-KS(light chain) + KS(heavy chain)-IL-2, described in example 5, were also determined by a test on a cellular basis. Analysis of cell proliferation RVMS and to set labeled with tritium thymidine was shown that the protein is Fc-IL-12-IL-2, IL-12-KS-IL-2 and IL-12-KS(light chain) + KS(heavy chain)-IL-2 all have high activity of IL-12. Similarly with a test for cell proliferation CTLL-2 has been shown that the protein is Fc-IL-12-IL-2, IL-12-KS-IL-2 and IL-12-KS(heavy chain) + KS(light chain)-IL-2 all have high activity of IL-2. Furthermore, ELISA analysis of both protein and IL-12-KS-IL-2 and IL-12-KS(light chain) + KS(heavy chain)-IL-2 was closely associated with antigen Ersam, even though the V-region, respectively, the heavy and light chains fused their N-ends with other proteins.

Example 7. The activity of the fused protein with murine components of the IL-12-M-CSF

The activity of IL-12 murine molecules Fc-IL-12-GM-CSF was determined in the test for cell proliferation (figure 10). Human RUMS received from three volunteers were cultured with 5 μg/ml of phytohemagglutinin-R for 3 days, washed with medium HBSS Hank and were sown in tablets for micrometrology at a density of 105cells in the cell, according to the standard procedure (M.K. Gately, R. Chizzonite, and D. H. Presky // Current Protocols in Immunology. 1995 S-6.16.15). Cells were incubated in the presence of different subjects proteins for 48 h and 10 h to determine the levels of incorporation of radioactivity was added 0.3 mccoury3H-thymidine. IL-12 and equimolar mixture of IL-12 and GM-CSF stimulated the incorporation in cells3H thymidine with dose-dependent and protein 14.18-IL-12-GM-CSF was approximately equally active in promoting the inclusion of3H-t amidine. GM-CSF did not stimulate the inclusion of3H-thymidine at the tested concentrations, which indicates that the observed inclusion of3H-thymidine stimulated fused protein 14.18-IL-12-GM-CSF, mainly due to the contained IL-12.

In addition, in cell tests was tested biological activity component of the GM-CSF various fused proteins IL-12-GM-CSF. It was found that the component of the fused protein of GM-CSF is active, and the activity per mole is in the same range as the commercial activity of M-CSF. For example, the biological activity component of the GM-CSF was tested in another test on cell proliferation, following well-known specialists in the field of molecular immunology procedure (S.C. Cooper and Broxmeyer N.E. // Current Protocols in Molecular Immunology. 1996. S-6.4.20). Cell line mouse 32D(GM) for proliferation needs GM-CSF. This line is derived from the original cell line 32D, described Cooper and Broxmeyer, and is partially sensitive to GM-CSF (Faas, etc. // Eur. J. Immunol. 1993. V.23. S-1214). Cell line 32D(GM) is not sensitive to IL-12. The 32D cells(GM) active in the logarithmic growth phase was twice washed with medium without GM-CSF, were sown in cell tablet for micrometrology at a density of approximately 5×103cells the cell in the presence of varying amounts of commercial murine GM-CSF or fused protein with murine partners of IL-12-GM-CSF and were grown for 48 h For 16 h to determine the level of incorporation of radioactivity was added 0.3 mccoury3H-thymidine. When the number of fused protein of IL-12-GM-CSF was obtained dependence include3H-thymidine dose of this protein. This indicates that the activity has a component of GM-CSF fused protein of IL-12-GM-CSF. Moreover, the biological activity of GM-CSF in fused protein, calculated on mol, comparable to the commercial activity of murine GM-CSF.

Example 8. Effects on carcinoma of the rectum, immunotropic the different mice fused protein with multiple cytokines

To test whether protein of several cytokines with the antibody to be used for the treatment of carcinoma of the rectum in mammals with intact immune systems, were carried out the following experiments. ST is obtained from mice Balb/C cell line carcinoma of the rectum. Using standard techniques of genetic engineering this line was given the ability to Express the adhesion molecule of epithelial cells of human (Eram), which is the antigen recognized by the antibody KS 1/4; these cells was designated CT26/KSA.

Mice of Balb/C were inoculable subcutaneously 2×106cells CT26/KSA. When tumors reached a volume of approximately 100-200 cubic millimeters mice for further research were divided randomly into three groups. Starting from day 0, groups of mice with tumors were administered PBS, about 3.4 MGK KS-IL-2, mixed approximately 5.3 µg KS-IL-12, or about 6 μg KS-IL-2-IL-12. These doses taken the introduction to each group of mice is equal to the number of molecules of IL-12 and IL-2. Injections were performed into the tumour, once a day for 5 days. The size of the tumors were measured by caliper.

The results of one such experiment is shown figure 11. In this experiment, the protein KS-IL-2-IL-12 completely inhibited the growth of tumors. Mix KS-IL-12 and KS-IL-2 also gave a significant suppression of tumor growth, but not so full is e, as KS-IL-2-IL-12. In the group of mice treated with KS-IL-2-IL-12, 6 mice of 9 were almost cured of tumors: these 6 mice survived up to a 93-day, when the experiment was terminated, and the tumors in these mice decreased and disappeared, so between days 39 and 93 subcutaneous tumors could be detected. The remaining 3 mice, tumor growth was delayed, so the volume of the tumors exceeded 4000 mm3only after 87 days.

Of mice exposed to a mixture of KS-IL-12 and KS-IL-2 two mice were clearly cured of subcutaneous tumors and survived until the end of the experience. Tumors in the remaining 7 mice has not disappeared and has increased over time, reaching a volume of 1000 mm3one mouse and more than 4000 mm3at 6 mice.

The fact that KS-IL-2-IL-12 is more effective than equimolar mixture of KS-IL-12 and KS-IL-2 was unexpected. Applied in this experiment, the doses were introduced about 15 picomoles fused protein per dose, which corresponds to about 9×1012the molecules. At the beginning of treatment, the volume of each tumor was approximately 160 mm3that corresponds to about 160 million cells. Each cell expresses about 106molecules Ersam, so there is about 1.6×1014molecules of antigen Ersam, which may be contacted antibodies KS. Therefore, it is unlikely that mixing KS-IL-12 and KS-IL-2 and injected them into mice with tumors of these two immunocytokines fused protein compete with each other the om for binding to the antigen. Thus, the effective dose of IL-12 and IL-2 should be at least equally high for both mixtures KS-IL-12 and KS-IL-2 and KS-IL-12-IL-2.

Example 9. The impact of the fused protein with multiple cytokines in carcinoma of the rectum in immunodeficient mammals

Many forms of cancer treatment to kill dividing cells, including cells of the immune system. As a result, cancer patients often suppressed immunity. To check whether you can use fused proteins with multiple cytokines for the treatment of mammals with a suppressed immune system, SCID mice with tumor CT26/KSA, introduced KS-IL-12-IL-2, mix KS-IL-12 and KS-IL-2 or PBS. Mouse SCID have deficiency In T-cells, and to fight infections, they need a branch of the innate immune system such as natural killer cells (NK).

Mice with subcutaneous tumors CT26/KSA received, as described in example 8. Three groups of 8 mice each with tumors of approximately 100-200 mm3introduced by injection into the tumor drugs in the same dosage and the same mode as in example 8. The results are presented on Fig. In this case, protein KS-IL-12-IL-2 mix KS-IL-12 and KS-IL-2 were approximately equally effective in each group to the 25-th day 5 of 8 mice were cured. However, in 5 of 6 untreated animals, the tumor began to grow with the speed characteristic on what I tumors in control animals, with an effective delay of about 14 to 21 days. It's not like the behavior of tumors in immunoprofiling mice in example 8: even if the tumor has not been completely destroyed by the processing of the fused protein KS-IL12-IL2, the tumor had not started vigorously growing up to 60 days after the start of the experiment.

These experiments show that the protein of several cytokines with the antibody can be used for the treatment of cancer in immunosuppressive animal.

Example 10. Treatment of carcinoma of the lung by intratumoral injection of a fused protein with multiple cytokines: a comparison with the treatment of individual immunocytokine

To assess efficacy in lung cancer cell of origin fused proteins with multiple cytokines and immunocytokines, bearing a single cytokine component, was conducted the following experiment.

Carcinoma, Lewis lung (LLC) is an aggressive tumor derived from the C57BL/6 mice. Line of LLC cells expressing the human protein Ersam, designed by standard methods of genetic engineering, these cells are called LLC/KSA.

Of C57BL/6 mice with subcutaneous tumors LLC/KSA received, as described in example 8 (the number of cells was checked by the method KML). Five groups of 5 mice each with tumors of approximately from 100 to 200 mm3introduced drugs by injection into the tumor within 5 days. Mice enter and PBS, about 20 μg KS-IL-12, about 20 μg KS-IL-2 or about 20 μg KS-IL-12-IL-2.

The results are shown in Fig. In this case, protein KS-IL-12-IL-2 was much more effective than either KS-IL-12, or KS-IL-2. All mice receiving protein KS-IL-12-IL-2, tumor to 27 th day disappeared. In the 74th day in these mice was tested for pulmonary metastases as described in example 14; source subcutaneous tumors did not appear neither in the period of exposure, nor in the second experiment. In contrast, the introduction or KS-IL-2 or KS-IL-12 led to some noticeable Sheibani tumors and a significant delay in tumor growth, but the tumor over time, continued to grow. Comparison of the results of this example with the previous examples shows that in some types of sickness and the ways of introducing the treatment with a mixture of immunocytokines carrying different cytokine components, is more effective than individual treatment type immunocytokine.

Example 12. Treatment of carcinoma of the lung by intratumoral injection of a fused protein with multiple cytokines: comparison with treatment with a mixture of immunocytokines

To assess the effectiveness of fused proteins with multiple cytokines and mixtures of immunocytokines carrying different cytokine components, obtained from cell lung cancer was conducted the following experiment.

Of C57BL/6 mice with subcutaneous tumors LLCKSA received, as described in example 11. Three groups of 7 mice each with tumors of approximately 100-200 mm3injected drugs by injection into the tumor within 5 days. Mice were administered PBS, a mixture of approximately 18 μg KS-IL-12 and approximately 11,5 µg KS-IL-2 or 20 µg KS-IL-12-IL-2.

The results are shown in Fig. In this case, protein KS-IL-12-IL-2 was much more effective than the mixture KS-IL-12 and KS-IL-2. All mice receiving protein KS-IL-12-IL-2, tumor disappeared to 27 th day. In contrast, the introduction of a mixture of KS-IL-12 and KS-IL-2 resulted in a slight shrinkage of tumors and significant delays in their growth, but the growth of all tumors eventually resumed.

Example 13. The dependence of the antitumor activity of the fused protein of several cytokines with the antibody from the antigen

To assess whether the effectiveness of the fused protein of several cytokines with the antibody in the treatment of tumors from tumor-specific expression of the antigen recognized by the antibody was carried out the following experiment.

Were created as described in example 11, a group of 7 mice with subcutaneous tumors LLC/KSA and the second group of 9 mice with tumors developing from the original cell line LLC. These two groups of mice with tumors of approximately 100-200 mm3injected drugs by injection into the tumor within 5 days. Mice received approximately 20 micrograms of KS-IL-12-IL-2

The results are shown in Fig. In this case, the mouse had a tumor LLC/KSA, were all fully healed from cancer. In contrast, only two mice with LLC tumors were cured, and all other mice with LLC tumors were observed temporary reduction of tumors, but over time their tumors were grown to a large amount.

These results show that the detection of the surface antigen Arcam promotes attachment KS-IL-12-IL-2 to the surface of tumor cells LLC/KSA, resulting immune response is enhanced. Some antitumor effect was also observed and compared to tumors derived from cells LLC. Without binding themselves to theoretical considerations, we can assume that in this case, the antitumor effect of KS-IL-12-IL-2 due to the fact that injection of fused protein was produced directly in the tumor, and he therefore temporarily localized in the tumor.

Example 14. The formation of immune memory against a certain type of tumor cells

In the treatment of cancer, the main problem is the development of metastases. To check out whether the treatment of the fused protein of several cytokines with antibody to lead to the formation of long-term immune memory about the type of tumor cells and prevent the development of metastases was conducted the following experiment.

Five C57BL/6 mice from example 11 enter the and KS-IL-12-IL-2, and they were clearly cured of subcutaneous tumors. In the 74th day of the start of treatment described in example 14, these five mice were administered an intravenous injection of 106cells LLC/KSA. As control 8 C57BL/6 mice also was injected intravenously 106cells LLC/KSA.

On the 28th day, the mice were killed and their lungs examined for the presence of metastases. Light eight control mice were 70-100% covered with metastases, the average coating the surface of the lung was 85%. The average weight of the lungs in these mice was equal 0,86, on the Contrary, on the surface of the lungs of five pre-treated mice, there was no metastasis, and the average lung weight was 0.28 g, which corresponds to a weight of normal mouse lung. These results indicate that a therapeutic effect on the original tumor cells leads to the development of long-term immune memory on tumor cells; this memory prevents the formation of metastases by this type of tumor cells.

Table 1

Protection of mice with repressirovannymi tumors from lung metastases LLC/KSA
Preliminary treatmentThe degree of metastasisLung weight (g)
Without treatment4, 4, 4, 4, 4, 4, 3, 30,88±0,27
0, 0, 0, 0, 00,27±0,03

The average lung weight in the control group without tumors was of 0.2, the Degree of metastasis was evaluated by the percentage coverage of the surface of light-fused metastatic nodules, where 0 indicates the absence of metastases, 1 - floor 1-25%; 2 - cover 25-50%; 3 - floor 50-75%; 4 - cover 75-100%.

In the second experiment to study the formation of immune memory were used 6 out of 7 mice from example 12, which were introduced tumor cells LLC/KSA who developed subcutaneous tumors, and these tumors disappeared. After 62 days after the start of treatment in example 12 six-treated mice and 10 fresh not receiving treatment, subcutaneous injection was introduced 106cells LLC. These cells do not Express Arcam - antigen KS man.

Fresh mice injected LLC cells formed tumors that grew in all mice at high speed. In contrast, tumors in pre-treated mice grew much slower, and one of the mice subcutaneous tumor was not detected. The results are presented on Fig.

Because the human antigen KS, Ersam, is not expressed in LLC cells, the immune response to LLC based on other antigens expressed by these cells.

Example 15. Fused proteins with multiple cytokines as vaccine

Fused proteins with several is likemy cytokines can be used as vaccines, if they are fused with a protein antigen. Specific order components from N-Terminus to the C-end, there is a fused protein is a single polypeptide chain or oligomer may vary, depending on the facilities to construct expressing plasmids. Protein can be introduced in a variety of ways, such as intravenous, subcutaneous, intraperitoneal, etc. similarly, the dose and frequency of injection generally require empirical selection, which is standard practice in vaccination of people and is well known to experts in the field of vaccine development.

For example, mice injected protein in the form of antigen-IL-12 is a cytokine, and a cytokine in the slit protein is the second cytokine other than IL-12. Control mice injected with antigen-cytokine, antigen-IL-2 or one antigen. At different points in time during and/or after administration of the antigen fused protein selected blood samples from a vein and receive and analyze the plasma contains antibodies to this antigen. It is shown that produces antibodies to the antigen. Moreover, the nature of the immune response to the antigen corresponds to a Th1 response. Antibody stronger and type of produced antibodies other than when certain control immunizations.

More specifically, the mice of Balb/c administered intravenously (5 mg/day, 5 days) in PBS buffer protein composition (the antibody is of ne)-(murine IL-12)-a(murine IL-2). Control mice injected with the same antibody, but not attached to the merger of IL-12-IL-2. None of the entered injection solutions do not contain any other type of adjuvant. On the 10th day of the veins take blood samples in plastic microcentrifuge tubes with sodium citrate and get the plasma by centrifugation at maximum speed in a tabletop microcentrifuge Eppendorf. Cell tablet method ELISA (96 wells) coated with human antibodies containing the constant region of human immunoglobulin. These tablets are used to catch any mouse antibodies produced in response to immunization. After washing unbound material detection related mouse antibodies produced using goat antibodies to mouse Fc region (Jackson ImmunoResearch)conjugated with horseradish peroxidase. All associated antibodies may be specific or constant regions, or to the variable regions of a human immunoglobulin, both of these areas are presented in a human antibody and fused proteins.

Human antibody without merging with IL-12-IL-2 response almost no or no. In contrast, protein induces a strong immune response with antibodies in the absence of exogenous adjuvants and despite the fact that the intravenous route of administration is extremely unfavorable in comparison with subcutaneous or inside the peritoneal introduction to induction of such reactions. In the group of mice that were administered a protein a antibody - IL-12-IL-2, observed the appearance of antibodies lgG2a isotype, which is typical for reactions with the stimulation of IL-12. In the group of mice immunized with human antibody, such antibodies are not observed.

Immunogenicity fused proteins with multiple cytokines containing the fusion antigen-IL-2 and introduced in different ways, checked by injection of a solution of fused protein (such as described above) in PBS or other biocompatible buffer, or known Freund, such as part-time or full beta-blockers. For example, every two weeks, you can produce one or more subcutaneous, intradermal or intraperitoneal injection. Alternatively, the protein can enter the first subcutaneous injection, and then intraperitoneal injection. Beta-blockers cannot be used in humans due to irritation at the injection site. Approved for use in humans and can be used in the present invention, alternative adjuvants, such as the precipitation of aluminium hydroxide (alum, "Aluminum"). For intradermal injections can also be used new organic chemical adjuvants on the basis of squalane and lipids.

Example 16. Gene therapy fused proteins with multiple cytokines

It was also shown provided methods of gene therapy PR is Tiviakov activity of the fused protein with multiple cytokines in the treatment of lung cancer. Cell carcinoma of the lung Lewis were consistently transliterowany using the above system viral vectors (DNA pLNCX-sclL-12-IL-2 or pLNCX-sclL-12, transfusiona in the packaging cell line RA). These constructs encode a single-chain version of IL-12, in which the subunit R35 and R40 are connected by a bridge. The selection of clones was performed in vitro in a medium with G418, clones stably expressing about 50-60 ng/ml IL-12, were identified using ELISA method (R & D Systems).

Approximately 1x106and about 5×106the LLC cells expressing sclL-12 or sclL-12-IL-2 was administered by subcutaneous injection to mice C57BL/6 and SCID mice. As control mice C57BL/6 and SCID mice were injected 2×106cells LLC. The LLC cells expressing IL-12, they form tumors that grow about the same rate as tumors derived from cells LLC, which has not been given the ability to Express cytokines. However, the C57BL/6 mice, and in mice SCID LLC cells expressing sclL-12-IL-2, or do not form tumors or form tumors, which soon diminish and disappear (figures 17 and 18).

Example 17. Design fused proteins with multiple cytokines containing IL-4 and GM-CSF

The cytokines IL-4 and GM-CSF when their combined use are potent activators of dendritic cells. Protein with multiple cytokines with AK is ewnetu IL-4 and GM-CSF, can be constructed as follows. The coding sequence for GM-CSF was merged with preservation of the reading frame with the 3'-end of the coding sequence for the heavy chain of the antibody KS 1/4, in front of which was placed a leader sequence. In addition, the coding sequence for IL-4, including a leader sequence, was merged with the preservation of the reading frame to the 5'-end of the coding sequence for the light chain of the Mature antibody KS 1/4.

Specifically, to construct DNA encoding a fused protein of murine IL-4 and light chain antibody KS 1/4, cDNA of murine IL-4 was adapted by PCR using direct primer TCTAGACC ATG GGT CTC AAC CCC CAG (SEQ ID NO: 22), in which the Xbal site TCTAGA (1-6 nucleotides of the sequence SEQ ID NO: 22) was placed with the rising side of the initiation codon ATG broadcast, and the reverse primer With GGA TCC CGA GTA ATC CAT TTG CAT GAT GCT CTT TAG GCT TTC CAG G (SEQ ID NO: 23), which contains a BamHI site GGA TCC (nucleotides 2-7 of the sequence SEQ ID NO: 23) with the 3'-side of the codon TCG (anticodon CGA)encoding the C-terminal amino acid residue of murine IL-4. After cloning of the PCR fragment and verification of the nucleotide sequence of the fragment Xbal-BamHI containing cDNA of murine IL-4, ligated with oligonucleotide duplex BamHI-Aflll coding rich residues glycine and serine flexible peptide bridge. The end Aflll who was b in turn is attached to artificially embedded website Aflll, previous N-end of the light chain of the Mature antibody KS 1/4. The DNA sequence and protein in place of the compound obtained by the two legirovanii below.

In this sequence DNA sites GGATCC (nucleotides 4-9 sequence SEQ ID NO: 24) and CTTAAG (nucleotides 48-53 sequence SEQ ID NO: 24) are the two restriction sites BamHI, respectively, and Aflll used for reconstruction; TCG encodes the C-terminal serine residue in a murine IL-4; GAG encodes the Mature N-terminal light chain of KS-1/4, and the amino acid sequence rich GlySer peptide bridge is shown below the DNA sequence. Around the DNA segments coding for both the fused polypeptide in a suitable manner were placed auxiliary sequence for highly efficient expression, including strong promoters; this was done with the use described in the previous examples, receptions and other standard techniques of molecular biology.

The DNA sequence encoding the fused protein of IL-4-KS(light chain) and KS(heavy chain)-GM-CSF, were transliterowany in cells, NS/0, and there has been a high level of expression of the corresponding polypeptide. SDS-PAGE in reducing conditions showed a diffuse (diffuse) zone with a molecular weight of about 80 kDa, corresponding to the polypeptide (heavy chain)-GM-CSF, and not the several zones near 50 kDa, relevant merger of IL-4-(light chain). The presence of diffuse zone and mentioned several areas reflects the varying degrees of glycosylation, respectively, IL-4 and GM-CSF.

Subunits are combined in associated by disulfide bonds tetramer protein with a structure corresponding to the General structure on fig.3G. This protein is the antigen-binding activity of KS-1/4, the ability to contact their Fc regions with protein And staphylococci and cytokine activity of IL-4 and GM-CSF. The activity of IL-4 was measured dependent on IL-4 stimulation include labeled with tritium thymidine into cells of CTLL-2. The activity of GM-CSF was measured dependent on GM-CSF stimulation include labeled with tritium thymidine into the cells 32(D)GM. Per mole activity of IL-4 and activity of GM-CSF in fused protein KS-IL-4-GM-CSF was close to the activity of the purified IL-4 and GM-CSF.

The merger of the cytokines IL-4 and GM-CSF without the attached sequences of the antibody was designed as follows. Murine IL-4 was cloned by PCR from RNA of cells in the spleen of a mouse. Direct primer had the sequence TCTAGACC ATG GGT CTC AAC CCC CAG (SEQ ID NO: 26), in which the Xbal site TCTAGA (1-6 nucleotides of the sequence SEQ ID NO: 26) was placed with the rising side of the initiation codon ATG broadcast, and the reverse primer had the sequence CGA TAT CCC GGA CGA GTA ATC CAT TTG CAT GAT GCT CTT TAG GCT TTC CAG G (SEQ ID NO: 27), in which the EcoRV site GAT ATC (nucleotide is 2-7 sequence SEQ ID NO: 27) is placed directly in the 3'-away from the codon TCG (anticodon CGA), encoding the C-terminal amino acid residue of murine IL-4. After checking the nucleotide sequence of the fragment Xbal-EcoRV containing cDNA mu.lL-4 with its native leader, was Legerova in the Smal fragment-Xhol containing cDNA mu. GM-CSF to obtain the following sequence at the junction of the merger between mu.lL-4 and mu.GM-CSF: ATG GAT TAC TCG TCC GGG ATG GGA AAA GCA CCC GCC CGC (SEQ ID NO:28), where the C-terminal sequence of mu.lL-4 and N-terminal sequence mu.GM-CSF bold, a G ATG GG (17-22 nucleotides SEQ ID NO: 28) is a sequence resulting prishivki the ligase end with smooth trim EcoRV to end with smooth trim Smal. The resulting DNA encoding the fusion mu.lL-4-mu.GM-CSF, and then cloned in expressing vector. Expressed protein was analyzed using SDS-PAGE. It was found that it migrates as a diffuse zone with apparent molecular weight of 45 to 50 kDa. Per mole activity of IL-4 and GM-CSF in the fusion of IL-4-GM-CSF was close to the activity of the purified IL-4 and GM-CSF.

Example 18. The design DNA that encodes a protein lymphotactin-KS-IL-2 and expression of the protein lymphotactin-KS-IL-2

Chemokines is a special class of cytokines that are believed to form gradients and are intermediaries in the chemotaxis of immune cells. In addition, like other cytokines, chemokines can induce the expression of specific the fir genes in target cells. One of the characteristic features of chemokines is that their activity need a free N-end, which may limit the choice of ways in which you can construct fused proteins.

Was designed protein cytokine-antibody-cytokine consisting of a cytokine lymphotactin, which is a chemokine, an antibody KS 1/4 and the cytokine IL-2. This protein was the tetramer and contained two different polypeptide. One polypeptide consisted of mouse lymphotactin, fused with the N-end of the heavy chain of the antibody KS 1/4, followed by on-end was a IL-2. Fusion heavy chain KS-1/4 to-end with IL-2, i.e. KS(heavy chain)-IL2 was described previously (Gillies, etc. // Proc. Natl. Acad. Sci. USA. 1992. So 89. S. 1428). Another polypeptide consisted of the light chain of the antibody KS1/4.

Complete coding sequence of mouse lymphotactin was published in Kelner and ZIotnik // Science. 1998. I. 266. S. 1395. To construct DNA encoding a fused protein of murine lymphotactin and merge (heavy chain KS)-IL-2 cDNA mouse lymphotactin adapted by PCR using direct primer TCTAGAGCCACC ATG AGA CTT CTC CTC CTG AC (SEQ ID NO: 29), in which the Xbal site TCTAGA (1-6 nucleotides of the sequence SEQ ID NO: 29) was placed with the rising side of the initiation codon ATG broadcast, and the reverse primer GGA TCC CCC AGT CAG GGT TAC TGC TG (SEQ ID NO: 30), which introduced a BamHI site GGA TCC (1-6 nucleotides of the sequence SE ID NO: 30) directly from the 3'-side of the codon GGG (anticodon CCC), encoding the C-terminal amino acid residue of murine lymphotactin. After cloning the PCR fragment and verification of the nucleotide sequence of the fragment Xbal-BamHI containing cDNA of mouse lymphotactin was Legerova with oligonucleotide duplex BamHI-Aflll coding rich residues glycine and serine flexible peptide bridge. The end Aflll was in turn connected with artificial site Aflll, in front of the Mature N-end merger KS(heavy chain)-IL-2. The DNA sequence in the connecting area, the resulting two legirovanii below:

where GGATCC (nucleotides 4-9 sequence SEQ ID NO: 31) and CTTAAG (nucleotides 48-53 sequence SEQ ID NO: 31) are two restriction site, respectively BamHI and Aflll used for reconstruction; SSS encodes the C-terminal amino acid residue of murine lymphotactin; CAG encodes the Mature N-terminal heavy chain KS-IL-2; amino acid sequence rich GlySer peptide bridge shown above the DNA sequence. Then DNA encoding the fusion (mouse lymphotactin)-(heavy chain KS)-IL-2, cloned in expressing vector, then expressed together with a light chain KS1/4.

Lymphotactin activity expressed fused protein lymphotactin-KS-IL-2 was tested in a migration test in the Boyden chamber on the cells (Leonard and others // Current Protocols in Immunology. 1999. C. 6.12.3). Alternatively, using the NK cells (natural killer cells). Alternatively, the activity lymphotactin was determined in a standard test cell on calcium flux in response to activation coupled with G-protein receptor (Maghazachi, etc. // FASEB J. 1997. So 11. C. 765-774). In addition, experienced the protein lymphotactin-KS-IL-2 and found that he is active in the analysis on the ability to contact Arcam, and he was also active in the tests on the activity of IL-2, such as a test for cell proliferation CTLL-2.

Incorporation by reference

All the above publications are fully incorporated in this application by reference.

Equivalents

The invention can be implemented in other specific forms without going beyond the limits of his ideas and essential characteristics. Therefore, the previous implementation should be considered in all respects as illustrating but not limiting the invention described here. Thus, the scope of the invention is indicated not so much the previous description as supplied by the formula, and therefore assumes that it contains all changes within the meaning and range of equivalence of formulas.

1. Multifunctional protein cytokine-antibody, including the region of immunoglobulin and protein cytokine formula IL-12-X or X-IL-12, where interlay the in-12 (IL-12) is a first cytokine, and X is a second cytokine selected from the group comprising IL-2, IL-4 and GM-CSF, covalently attached to either the amino-end, or at the carboxyl end of the subunit p35 or p40 interleukin-12 (IL-12) in its heterodimeric or single-stranded form, and the specified protein cytokine fused or its amino end or carboxyl end of the specified region of the immunoglobulin.

2. Multifunctional protein cytokine-antibody according to claim 1, characterized in that the IL-12 and a second cytokine specified fused protein cytokine fused via a polypeptide of the bridge.

3. Multifunctional protein cytokine-antibody according to claim 1 or 2, characterized in that the specified region of the immunoglobulin and the specified protein cytokine fused via a polypeptide of the bridge.

4. Multifunctional protein cytokine-antibody according to claim 1, characterized in that the second cytokine in the specified fused protein cytokine is an IL-2.

5. Multifunctional protein cytokine-antibody according to claim 1, characterized in that region of an antibody contains a constant region of a heavy chain immunoglobulin comprising the hinge region, CH2 domain and CH3 domain.

6. Multifunctional protein cytokine-antibody according to claim 5, characterized in that region of the immunoglobulin is an Fc region.

7. Megafunction the local protein cytokine-antibody according to claim 5, characterized in that region of the immunoglobulin further comprises a CH1 domain.

8. Multifunctional protein cytokine-antibody according to claim 7, wherein the specified region of the immunoglobulin further comprises a domain of the variable region of the heavy chain of immunoglobulin (VH), which is immunologically capable of reaction with specific for a cancer antigen or a viral antigen.

9. Multifunctional protein cytokine-antibody of claim 8, wherein the specified region of the immunoglobulin further comprises a light chain immunoglobulin associated with the heavy chain of immunoglobulin, forming thus the intact antibody.

10. Multifunctional protein cytokine-antibody according to claim 9, characterized in that said antibody is a single-chain antibody.

11. Nucleic acid encoding a multifunctional protein cytokine-antibody as defined in any one of claims 1, 6, 9, or 10.

12. Pharmaceutical composition having anti-cancer activity, including multifunctional protein cytokine-antibody as defined in any one of claims 1,6,9 or 10, optionally with a pharmaceutical carrier or excipient.

13. A method of treating cancer in a mammal, comprising administration to the mammal of an effective amount of a pharmaceutical com is osili, as defined in item 12.



 

Same patents:

FIELD: genetic engineering, proteins, medicine, pharmacy.

SUBSTANCE: invention relates to a method for preparing a fused protein representing immunoglobulin Fc-fragment and interferon-alpha and can be used in treatment of hepatitis. Method involves construction of a fused protein comprising immunoglobulin Fc-fragment prepared from Ig G1 or Ig G3 in direction from N-end to C-end and the end protein comprising at least one interferon-alpha. Fc-fragment and the end protein are joined directly or by a polypeptide bridge. The fused protein is used for preparing a pharmaceutical composition used in treatment of liver diseases and in a method for targeting interferon-alpha into liver tissues. Invention provides preparing the fused protein eliciting with biological activity of interferon-alpha providing its concentrating in liver and showing enhanced solubility, prolonged half-time life in serum blood and enhanced binding with specific receptors.

EFFECT: improved targeting method, valuable biological properties of fused protein.

10 cl, 5 dwg, 9 ex

FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: method involves selection of signal sequence suitable for the effective expression of Leu-hirudine in E. coli cells by the polymerase chain reaction-screening method. Method involves construction of a protein as a precursor of hirudine based on the selected signal sequence of surface membrane protein from Serratia marcescens, oprF protein from Pseudomonas fluorescens or fumarate reductase from Shewanella putrifaciens by joining the Leu-hirudine amino acid sequence with C-end of selected signal sequence. Prepared precursor of Leu-hirudine is used in a method for preparing Leu-hirudine. Invention provides preparing Leu-hirudine by the direct secretion in E. coli cells with the high yield. Invention can be used in preparing the hirudine precursor.

EFFECT: improved preparing method.

4 cl, 1 dwg, 2 tbl, 12 ex

FIELD: biotechnology, medicine.

SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.

EFFECT: valuable properties of new cytokine.

18 cl, 5 tbl, 1 dwg, 55 ex

FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.

SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.

EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.

7 cl, 12 dwg, 4 tbl, 16 ex

FIELD: genetic engineering, molecular biology.

SUBSTANCE: invention proposes a method for detecting genes encoding membrane-bound transmembrane proteins. Method involves expression of the nucleic acid chimeric sequence in the cell-host consisting of DNA fragment encoding secreting protein that is able to bind antigen and DNA fragment to be tested; interaction of cells expressing the fused protein with antigen; selection of cells on surface of that indicated antigen is bound; isolation of recombinant vector containing in selected cells of DNA fragment to be tested and, if necessary, determination of its sequence. Also, invention proposes the developed vector constructions and comprising their sets designated for realization of the proposed method. Invention provides significant simplifying the screening process of libraries and cloning genes encoding transmembrane proteins. Invention can be used for detecting and preparing genes encoding any membrane-bound proteins used in different branches of science and practice.

EFFECT: improved isolating method, valuable biological properties of protein.

27 cl, 7 dwg, 1 tbl, 8 ex

FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

SUBSTANCE: the hemopoietic protein comprises the amino acid sequence of the formula: R1-L1-R1, R2-L1-R1, R1-R2 or R2-R1 wherein R1 represents the modified ligand flt-3; R2 represents the modified human IL-3, the modified or unmodified colony-stimulating factor. Modification of R1 is carried out by addition of N-end with C-end directly or through linker (L2) that is able to join N-end with C-end to form new C- and N-ends. The modified human IL-3 is prepared by replacing amino acids at positions 17-123. The human G-CSF is modified by exchange of amino acids. The hemopoietic protein is prepared by culturing cells transformed with vector comprising DNA that encodes the hemopoietic protein. The hemopoietic protein stimulates producing hemopoietic cells and this protein is used as a component of pharmaceutical composition used in treatment of humans suffering with tumor, infectious or autoimmune disease. Invention provides preparing multifunctional hemopoietic proteins eliciting the enhanced activity with respect to stimulation of hemopoietic cells and eliciting the improved physical indices. Invention can be used for preparing chimeric multifunctional hemopoietic proteins.

EFFECT: improved preparing and producing method, valuable medicinal properties of protein.

22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

The invention relates to genetic engineering and can be used for therapeutic purposes, in particular in the treatment of neoplastic processes

FIELD: biotechnology, medicine.

SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.

EFFECT: valuable properties of new cytokine.

18 cl, 5 tbl, 1 dwg, 55 ex

FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.

SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.

EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.

7 cl, 12 dwg, 4 tbl, 16 ex

The invention relates to genetic engineering and can be used for therapeutic purposes, in particular in the treatment of neoplastic processes

The invention relates to biotechnology and can be used to obtain a properly curled, containing the precursor of insulin chimeric protein

The invention relates to the field of biotechnology and can be used in medicine

The invention relates to the field of immunobiotechnology and may find application in medicine
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