Humanised antibody (h14,18) based on mouse antibody 14,18, contacting gd2, and its fusion with il-2

FIELD: medicine.

SUBSTANCE: there is offered application of humanised fused protein for making a medicine used for stimulation of immune response and stabilisation of disease progressing in patients with GD2-positive tumours. The antibody contains antibody H14.18 caught with surface glycosphingolipid GD2 of human cells, and cytokine IL2. There is disclosed method of increase in ADCC and lysis activity of natural killers in cancer patients by introduction of the fused protein. The invention can be applied in GD2-overexpression cancer therapy.

EFFECT: application of the invention provides low-immunogenicity antibody.

2 cl, 8 dwg, 1 tbl, 2 ex

 

The technical FIELD

The present invention generally relates to modified antibodies. More specifically, the invention relates to modified antibodies with reduced immunogenicity that are specifically associated with the surface glycosphingolipids GD2 human cells, and to their use as therapeutic agents.

PRIOR art

In recent years, significant progress has been made in the development of drugs based on antibodies. For example, researchers have identified not only a variety of markers that are specific to cancer, but also a variety of antibodies that specifically bind to these markers. Antibodies can be used for delivery of certain molecules, such as a toxin or immune stimulator, such as a cytokine, to cancer cells expressing a marker to selectively kill the cancer cell.

Antibody 14.18 - is derived from a mouse monoclonal antibody to glycosphingolipid GD2 cell surface. GD2 is disialogangliosides, which normally is expressed in large numbers only on the outer surface membranes of neuronal cells, and its presentation to the immune system is limited to the blood-brain barrier.

In contrast, for many of tumor cells characterized by abnormal uraniastrasse of cell surface glycosphingolipids. GD2, for example, is expressed on the surface of many tumor cells, including neuroblastoma, Protocol, astrocytomas, melanoma, small cell lung cancer, osteosarcoma and other soft tissue sarcoma. Accordingly, GD2 is convenient ofwholesale marker for targeting domains of immunostimulatory proteins on tumor cells in order to enhance effective immune response against tumor cells for destruction. Although antibody mouse 14.18 (antibody m14.18) can facilitate the targeting of these domains of proteins on tumor cells, its amino acid sequence derived from a mouse can have a harmful effect on the desired therapeutic effect.

With the introduction of patient antibodies may have concomitant immunogenicity for mammalian recipient. This is more likely to occur if antibodies are not autologous. Accordingly, the effectiveness of drugs based on antibodies is often limited immunogenic reaction (response)directed against therapeutic antibodies. This immunogenic response in a typical case is strengthened if the antibody is fully or partially obtained from a mammal that is different from the mammalian recipient, for example, if an antibody derived from a mouse, and the recipient is the person./p>

For clinical use in humans, it may be useful to modify antibodies derived from a mouse, so that they resembled human antibodies, to reduce or minimize the immunogenicity of antibodies derived from mouse. Immunogenicity antibodies derived from a mouse, can be reduced through the creation of chimeric antibodies in which the constant region of human antibodies merged with variable domains of the mouse. However, surviving the variable domains of the mouse usually remain immunogenic to humans, and therefore they can reduce the effectiveness of drugs based on antibodies.

Some approaches to reduce immunogenicity, such as "veneering" and "humanization", include the replacement of many amino acids and can disrupt the binding of antibody to the antigen. Antibody m14.18 binds to GD2 with moderate affinity. Therefore, it is expected that mutations that significantly reduce the affinity m14.18 to GD2, will reduce its effectiveness in therapeutic applications in humans. Accordingly, in the art there is a need for therapeutic antibodies that can effectively target GD2 and have reduced immunogenicity when administered to humans.

The INVENTION

In General, the present invention provides a modified form of the antibody m14.18, the cat heaven is less immunogenic to humans, but retains affinity binding m14.18 with GD2 person.

More specifically, the invention provides humanitarian form antibodies m14.18 (hu14.18 antibody)in which several specific for mouse amino acids in one or more frame sections replaced with other amino acids to reduce their immunogenicity in humans. The invention also provides products of the merging of hu14.18 antibody with one or more nimmanahaeminda parts to enhance the effect of targeted immune therapy.

In one aspect the present invention provides a variable region of the antibody comprising the amino acid sequence presented as SEQ ID NO: 1, which defines a variable region light chain of immunoglobulin (VLthe region). In another aspect the invention relates to variable regions of the antibodies comprising the amino acid sequence presented as SEQ ID NO: 2, which defines the variable region of the heavy chain of immunoglobulin (VHthe region). In one example implementation of the invention relates to variable regions of the antibody in which the amino acid sequence of SEQ ID NO: 1 is connected to the amino acid sequence presented as SEQ ID NO: 2. Amino acid sequence can be connected, for example, by a disulfide bond or peptide is a link.

The following aspect of the invention relates to variable regions of the antibody that specifically binds to GD2 and contains at least amino acids 1-23 of SEQ ID NO: 1, amino acids 1-25 of SEQ ID NO: 2 or amino acids 67-98 of SEQ ID NO: 2. These sequences define frame sections in the variable regions of immunoglobulin antibodies hu14.18. Frame sections are described in more detail below.

One aspect of the invention relates to a method for targeting a cell surface, the carrier GD2, and includes an introduction to the patient variable regions of the antibodies according to the present invention. In one example of the invention, the cell-target is a tumor cell. Other aspects of the invention include a nucleic acid encoding a variable region of an antibody, or a cell containing the nucleic acid that can be administered to the patient or used for the production of a protein in vitro.

The invention also provides a polypeptide that contains the variable region of the antibody according to the present invention and Fc-fragment, containing at least the CH2 domain, nucleic acid encoding the polypeptide, the cells containing the nucleic acids, and methods for targeting a cell carrier GD2 on their surface, through the introduction of a polypeptide, nucleic acid or cells patsie the Tu. In some examples of the invention, the Fc-fragment derived from IgG1.

Variable region of the antibody can be connected (with the introduction of the Fc-fragment or without implementation) with nimmanahaeminda part. More specifically, nimmanahaeminda part can be a cytokine, such as interleukin, hematopoietic factor, lymphokine, interferon or a chemokine. Interleukin may, for example, be interleukin-2 or interleukin-12. Hematopoietic factor and lymphokine can be, for example, granulocyte-macrophage colony-stimulating factor (GM-CSF) and lymphotoxin, respectively. Interferon may be, for example, interferon-α, interferon-β or interferon-γ. In some examples of the invention, the protein comprises a second nimmanahaeminda part, for example a second cytokine. In a specific example of the invention, the protein comprises the variable region of the antibody, IL-2 and IL-12.

It should be understood that the features of various embodiments of the invention described in this work are not mutually exclusive and can exist in various combinations and permutations.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

Figure 1 illustrates the amino acid sequence of variable region of the light chain of the immunoglobulin according to the present from which bretania.

Figure 1 illustrates the amino acid sequence of variable region of the heavy chain of the immunoglobulin according to the present invention.

Figures 2A-D illustrate the nucleotide sequence of the expression vector comprising a hybrid nucleic acid encoding the protein consisting of the light chain of the immunoglobulin heavy chain of an antibody and IL-2, according to the present invention.

Figure 3A illustrates the amino acid sequence of the light chain of the immunoglobulin according to the present invention.

Figure 3 illustrates the amino acid sequence of the heavy chain of the immunoglobulin according to the present invention.

INFORMATION CONFIRMING the POSSIBILITY of carrying out the INVENTION

The present invention provides a modified form of the antibody m14.18, which is less immunogenic for people, but still can specifically bind to GD2 person. Reduction of immunogenicity is achieved through one or more modified amino acid sequences of the variable domains of immunoglobulin. The antibody can be used for the treatment of GD2-positive tumors, particularly when merging it with the cytokine or other immunomodulator.

When used in this report, the terms "antibody" and "immunoglobulin" should be understood as meaning: (a) the intact antibody (for example - monoclonal antibody or polyclonal antibody), (2) it antigennegative areas, including, for example, Fab fragment, Fab'fragment, (Fab')2-fragment, an Fv fragment, a single-stranded binding site antibodies, sFv, (3) bispecific antibodies and their antigennegative areas, and (4) multispecific antibodies and their antigennegative areas.

When used in this report, the terms "specifically binds", "specifically binds" and "specific binding" should be understood as meaning that the antibody has an affinity of binding to a specific antigen at least about 106M-1more preferably at least about 107M-1even more preferably at least about 108M-1and most preferably at least about 1010M-1.

When used in this report, the terms "frame parts" and "FR" should be understood as meaning parts of the variable region of the immunoglobulin, adjacent to areas complementarity determining (CDR). CDR represent areas of variable regions of immunoglobulin, primarily interacting with the antigen. As shown in figure 1, VHand VLthe plots contain four FR and are located in areas amino acid sequence taken in the scope.

Bol is e specifically, with reference to the amino acid sequence depicted in Figa (SEQ ID NO: 1), FR-sections of the light chain of the determined amino acid sequences from Asp1 to Cys23 (huVLFR1), His39 to His54 (huVLFR2), Gly62 to Cys93 (huVLFR3) and Phe104 to Lys113 (huVLFR4). With reference to the amino acid sequence depicted In figure 1 (SEQ ID NO: 2), FR-sites of the heavy chain determined amino acid sequences from Glu1 to Ser25 (huVHFR1), Trp36 to Gly49 (huVHFR2), Arg67 to Ser98 (huVHFR3) and from Trp103 to Ser113 (huVHFR4).

The protein sequence according to the present invention

The hallmark of the present invention are antibodies that bind, preferably specifically, to surface glycosphingolipids GD2 human cells and have modified region derived from an antibody of a mouse m14.18. Amino acid sequence of VHor VL(or both) are modified or humanitarian to reduce their immunogenicity when administered to man. According to the present invention, the antibody m14.18 can be humanitarian, for example, using methods such as reimmunization, in which the potential epitopes for T-cells are removed or attenuated by introducing mutations that reduce binding epitope peptide molecules and MHC class II (see, for example, WO98/52976 and WO00/34317). Alternatively, mutate epitopes T cell animals so that they match your own epitopes person present in human antibodies (see, for example, U.S. Patent No. 5,712,120). The present invention provides antibodies to GD2 containing VLand VHareas that contain at least one humanitarian FR-sequence, thereby reducing their immunogenicity when administered to a human.

I. Variable regions of the heavy and light chains

As stated above, hu14.18 contains humanized variable regions derived from m14.18 that retain the ability to specific binding to the antigen GD2 person. In some embodiments of the invention VLthe area of hu14.18 antibody contains the following polypeptide:

D-V-V-M-T-Q-T-P-L-S-L-P-V-T-P-G-E-P-A-S-I-S-C-R-S-S-Q-S-L-V-H-R-N-G-N-T-Y-L-H-W-Y-L-Q-K-P-G-Q-S-P-K-L-L-I-H-K-V-S-N-R-F-S-G-V-P-D-R-F-S-G-S-G-S-G-T-D-F-T-L-K-I-S-R-V-E-A-E-D-L-G-V-Y-F-C-S-Q-S-T-H-V-P-P-L-T-F-G-A-G-T-K-L-E-L-K (SEQ ID NO: 1).

In specific embodiments of the invention hu14.18 antibody contains a light chain FR1, which is defined residues 1-23 of SEQ ID NO: 1, namely, D-V-V-M-T-Q-T-P-L-S-L-P-V-T-P-G-E-P-A-S-I-S-C (huVLFR1).

In another example embodiment of the invention VH-the area of the hu14.18 antibody contains the following polypeptide:

E-V-Q-L-V-Q-S-G-A-E-V-E-K-P-G-A-S-V-K-I-S-C-K-A-S-G-S-S-F-T-G-Y-N-M-N-W-V-R-Q-N-I-G-K-S-L-E-W-I-G-A-I-D-P-Y-Y-G-G-T-S-Y-N-Q-K-F-K-G-R-A-T-L-T-V-D-K-S-T-S-T-A-Y-M-H-L-K-S-L-R-S-E-D-T-A-V-Y-Y-C-V-S-G-M-E-Y-W-G-Q-G-TS-V-T-V-S-S (SEQ ID NO: 2).

In specific embodiments of the invention hu14.18 antibody contains FR1 of the heavy chain, which is defined residues 1-25 of SEQ ID NO: 2, namely: E-V-Q-L-V-Q-S-G-A-E-V-E-K-P-G-A-S-V-K-I-S-C-K-A-S (huVNFR1).

In other embodiments of the invention hu14.18 antibody contains FR3 of the heavy chain which is represented by residues 67-98 SEQ ID NO: 2, namely: R-A-T-L-T-V-D-K-S-T-S-T-A-Y-M-H-L-K-S-L-R-S-E-D-T-A-V-Y-Y-C-V-S (huVNFR3).

In the scope of the present invention also includes various combinations of the above embodiments of the invention. For example, the hu14.18 antibody can include VLthe sequence presented as SEQ ID NO: 1, and VHthe sequence presented as SEQ ID NO: 2. VLand VH-region can be connected by a disulfide bond or a peptide bond, depending on how the sequence coding their nucleic kislotno V-region are linked by a disulfide bond, if their sequences are encoded by different recombinant DNA. In contrast, the V-region typically associated peptide bond, if their sequences are encoded single-stranded structure of DNA.

The present invention also includes an antibody that specifically binds to GD2 and contains at least one area of humanized V regions. For example, the hu14.18 antibody may contain VLthe region is, defined as SEQ ID NO: 1, and VH-the area containing at least one humanitarianly FR, for example - huVNFR1 or huVNFR2. Alternatively, an antibody according to the present invention may contain VN-the area identified as SEQ ID NO: 2, and VL-the area containing at least one humanitarianly FR, for example - huVLFR1. Hu14.18 antibody may also contain VH-the area containing at least one humanitarianly FR, and/or VL-the area containing at least one humanitarianly FR.

In some examples of the invention, the variable region light chain and the variable region of the heavy chain can be made, respectively, with a constant region light chain and the constant region of the heavy chain of immunoglobulin. Light chain of an antibody has a constant region that are designated as Kappa or lambda chain. In a particular example embodiment of the invention the constant region of the light chain is a Kappa chain. The constant region of the heavy chain and their various modifications and combinations are discussed in more detail below.

II. Fc-fragment

The variable domains of the antibodies according to the present invention by selecting merge with Fc-fragment. Used in this paper Fc-fragment covers the domains derived from the constant region of the heavy the ETUI immunoglobulin, preferably immunoglobulin, including fragments, analogs, variants, mutants or derivatives of the constant region. The constant region of the heavy chain of immunoglobulin is defined as a naturally existing or obtained through the synthesis of a polypeptide that is homologous to at least one section of the C-terminal region of the heavy chain, including SN, hinge, CH2, CH3, and, for some classes of heavy chains, CH4 domains. The area of the hinge connects SN-domain CH2-CH3-land Fc-fragment. The constant region of the heavy chains of all mammalian immunoglobulins shows great similarity sequence aminonitrotoluenes DNA sequences of these regions of immunoglobulins are well known in the art. (See, for example, Gillies et al., (1989) J. Immunol. Meth. 125:191).

In the present invention Fc-fragment typically comprises at least the CH2 domain. For example, Fc-fragment may include the entire constant region of the heavy chain of immunoglobulin (SN-hinge-CH2-CH3). Alternatively, the Fc-fragment can include all of the hinge region or portion thereof, CH2 domain and CH3 domain.

The constant region of immunoglobulin responsible for many important effector functions of antibodies, including binding to Fc receptor (FcR) and complement fixation. There are 5 main classes of constant region heavy the chain, classified as IgA, IgG, IgD, IgE and IgM, each of which has distinct effector functions, referred to as isotypes.

For example, IgG is divided into four γ-isotype: γ1, γ2, γ3 and γ4, also known as IgG1, IgG2, IgG3 and IgG4, respectively. The IgG molecule can interact with many classes of cellular receptors, including three classes of Fcγ receptors (FcγR), specific for the IgG class of antibodies, namely FcγRI, FcγRII and FcγRIII. It was reported that sequences important for IgG binding to FcγR receptors are located in the CH2 and CH3 domains.

On the half-life of antibodies in the serum affects the ability of this antibody to connect with Fc-receptor (FcR). Similarly, the half-life of fused proteins containing immunoglobulins, also influenced by their inability to communicate with these receptors (Gillies et al., Cancer Research (1999) 59:2159-66). CH2 and CH3 domains of IgG2 and IgG4 have undetectable or reduced affinity binding to Fc-receptors compared with IgG1 domains. Accordingly, the half-life in serum is similar antibodies can be increased through the use of CH2 and/or CH3 domains from IgG2 or IgG4 isotypes. Alternatively, the antibody may contain a CH2 and/or CH3 domain from IgG1 or IgG3 with a modification of one or several amino acids in these domains to reduce the affinity of binding to Fc-receptors (see, for example, Application n the U.S. patent 09/256,156, published as patent Application U.S. 2003-0105294-A1).

The hinge region of the Fc fragment in the norm is close to the end SN-domain constant region of the heavy chain. When proteins according to the present invention, the hinge homologous naturally existing region of immunoglobulin and typically includes cysteine residues linking the two heavy chain disulfide bonds, as in the natural immunoglobulins. Representative sequences of the hinge regions of human immunoglobulins and mouse can be found in ANTIBODY ENGINEERING, A PRACTICAL GUIDE (Borrebaeck, ed., W.H. Freeman and Co., 1992).

Suitable for the present invention the hinge region can be obtained from IgG1, IgG2, IgG3, IgG4 and other isotypes of immunoglobulins. The isotype IgG1 has two hinge disulfide bonds, providing education effective and sustainable disulfide bonds. Therefore, the preferred hinge region according to the present invention derived from IgG1. Optionally, the first, closest to the N-end cysteine IgG1 hinge mutate to increase the expression and Assembly of antibodies or containing antibodies fused protein according to the present invention (see, for example, the patent Application U.S. 10/093,958, published as patent Application U.S. 2003-0044423-A1).

It is known that, in contrast to IgG1, the hinge region of IgG4 inefficient forms a disulfide St. the bond between the chains (Angal et al., (1993), Mol. Immunol. 30:105-8). Similarly, the hinge region of IgG2 contains four disulfide bonds that contribute to the oligomerization and probably incorrect disulfide bonding during secretion in recombinant systems. Suitable for the present invention the hinge region can be derived from the hinge region of IgG4, preferably containing a mutation that enhances the correct formation of disulfide bonds between parts of the molecule derived from the heavy chain (Angal et al., (1993), Mol. Immunol. 30(1):105-8). Another preferred hinge region derived from the hinge region of IgG2, in which the first two cysteine motivovany in other amino acids, for example, in order of preference, serine, alanine, threonine, Proline, glutamic acid, glutamine, lysine, histidine, arginine, asparagine, aspartic acid, glycine, methionine, valine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan or selenocysteine (see, for example, the publication of the patent Application U.S. 2003-0044423-A1).

Fc-fragment is merged with the variable region of the antibodies according to the present invention may contain a CH2 and/or CH3 domains and the hinge region, which are obtained from the different isotypes of antibodies. For example, Fc-fragment may contain a CH2 and/or CH3 domains of IgG2 or IgG4 and the hinge region of IgG1. The Assembly of such hybrid Fc-fragments described in the publication For the Cai on U.S. patent 2003-0044423-A1.

At the confluence with the variable region of the antibodies according to the present invention Fc-fragment preferably contains one or more modifications of amino acids, which tend to increase the half-life of fused protein containing the Fc-fragment in serum. Such amino acid modifications include mutations that significantly reduce or eliminate the activity of the protein in relation to binding to Fc receptor or complement fixation. For example, one of the types of mutations removes a glycosylation site of the Fc-fragment of the heavy chain of immunoglobulin. In IgG1 glycosylation site is Asn297 (see, for example, the patent Application U.S. 10/310,719, published as patent Application U.S. 2003-0166163-A1).

III. The connecting region fused protein

Variable regions according to the present invention may, optionally, be connected or merged with nimmanahaeminda part, directly or indirectly, such as through a linker peptide (for example -(Gly4-Ser)3(SEQ ID NO: 3)). Immunogenicity fused protein according to the invention can be reduced by disrupting the ability of the connecting region fused protein or connective epitope to interact with receptor T cells, as described in published Patent Application U.S. 2003-0166877-A1. Even with the merger of two proteins person, for example - Fc fragment of human IL-2 human, the area surrounding the flanged connecting region or connecting epitope contains a peptide sequence that is absent in the norm in the human body, and therefore it can be immunogenic. Immunogenicity connecting epitope can be reduced, for example, by introducing one or more glycosylation sites near the connecting region or through the identification of possible epitope T-cell overlapping the connecting region, as described in published Patent Application U.S. 2003-0166877-A1, and the replacement of amino acids near the connection to reduce the ability of probable epitope T cells to interact with receptor T cells.

The half-life of the protein in the serum can also be increased by introducing mutations in the connection region fused protein. For example, a protein containing the CH3 domain, merged with nimmanahaeminda part, you can replace the C-terminal lysine CH3 domain to another amino acid such as alanine, which can provide a significant increase in the half-life of the resulting fused protein in serum.

In some embodiments of the invention proteolytic cleavage of the connecting region fused protein is desirable. Accordingly, the intergenic region may include a nucleotide sequence encoding the site of proteolytic cleavage. This website, located between immunol what Abolina and cytokine, can be designed to provide proteolytic selection of the cytokine in the target. For example, it is well known that plasmin and trypsin digested peptide chain after residues lysine and arginine in areas accessible to proteases. Also known by other site-specific endoprotease and amino acid sequences that they recognize.

IV. The treatment of human diseases fused proteins containing antibody hu14.18

Variable regions of the antibodies according to the present invention can be connected to diagnostic and/or therapeutic agent. The agent can be fused with the antibody with obtaining the fused protein. Alternatively, the agent can be chemically cross-linked with the antibody obtaining immunoconjugate. The agent may be, for example, a toxin, a radioactive label, radiopharmaceutical preparation, immune, etc.

Variable region of the antibody according to the present invention can be attached to the cytokine. Preferred cytokines include interleukins such as interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16 and IL-18, hematopoietic factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and erythropoietin, tumor necrosis (TNF)such as TNF-α, lymphokines, such kalenterin, regulators of metabolic processes, such as leptin, interferons such as interferon-α, interferon-β, interferon-γ, and chemokines. Preferably, a fused protein consisting of antibodies and cytokines, or immunoconjugate showed biological activity of the cytokine. In one example of the invention, the variable domain antibodies fused with IL-2. Preferably, several amino acids in IL-2-part mutate to reduce the toxicity, as described in published Patent Application U.S. 2003-0166163-A1.

For example, on Figa and 3 depicts the amino acid sequence of a specific example of implementation of the fused protein on the basis of the antibodies according to the present invention. More specifically, Figa shows the peptide sequence of the humanized light chain immunoglobulin which comprises the variable and constant region. Figure 3 is depicted In the peptide sequence of the humanized heavy chain immunoglobulin combined with IL-2. Polypeptides provide a protein-based gumanitarnogo antibodies that can specifically bind to GD2 and stimulate the immune system.

Optionally, protein complexes can optionally contain a second substance, for example a second cytokine. In one example of the invention, the protein-based antibodies hu14.18 sod is RIT IL-12 and IL-2. Getting protein complexes containing immunoglobulin domain and two different cytokine, is described in detail in U.S. Patent No. 6,617,135.

Slit proteins according to the present invention can be used for the treatment of human diseases, such as cancer. In the treatment of human cancers especially advisable to introduce protein-based antibodies and IL-2 containing V-regions according to the present invention, by infusion or subcutaneous injection using a dose of from 0.1 to 100 mg/m2/patient. In the preferred embodiment of the invention, in particular, it is expedient to introduce protein-based antibodies and IL-2 containing V-regions according to the present invention, by infusion or subcutaneous injection using a dose of from 0.1 to 10 mg/m2/patient, and more preferably from about 3 to 6 mg/m2/patient.

Clinical trials have shown that after the introduction of protein hu14.18-IL-2 retains its ability to activate IL-2-reactive cells via IL-2 receptor and the ability to bind to GD2-positive tumor cells and deliver IL-2 to the surface. In addition, the introduction of a fused protein hu14.18-IL-2 cancer patients led to the cessation of progression of the disease in an unexpectedly large number of patients (see Example 1).

The pharmacy is practical composition according to the present invention can be used in the form of a solid, semi-solid or liquid dosage forms, such as tablets, capsules, powders, liquids, suspensions and the like, preferably in the form of a single dosage forms suitable for injection with precise dosages. Songs include standard pharmaceutical carrier or excipient, and, in addition, they may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc. Such fillers can contain other proteins such as human serum albumin or plasma proteins. Existing methods of preparing such dosage forms are known or obvious to experts in the given field of technology. In any event, the composition or formulation for the introduction will contain the active component (or components) in a quantity sufficient to produce a desired effect in the subject to be treated.

To enter the composition according to the present invention by using any recognized method of introducing substances possessing such activity. These methods include oral, parenteral or local injection and other systemic forms. The preferred method of administration is intravenous injection in a pharmaceutically acceptable carrier (see Example 1).

Put the number of active connections will, of course, depend on the subject is, to be treated, the severity of the disease, the route of administration and the opinion of the prescribing doctor.

Nucleic acids according to the present invention

I. Recombinant DNA encoding the antibody hu14.18

The invention also includes nucleic acids that can Express all the above types of proteins. These include, for example, nucleic acid encoding the amino acid sequence presented as SEQ ID NO: 1; amino acid sequence presented as SEQ ID NO: 2; VL-the area of the hu14.18 antibody that comprises the amino acid sequence huVLFR1; VH-the area of the hu14.18 antibody that comprises the amino acid sequence huVHFR1; VH-the area of the hu14.18 antibody that comprises the amino acid sequence huVHFR3; and fused proteins containing the hu14.18 antibody, comprising at least one of the above humanized FR sequences and one or more therapeutic agents.

The hu14.18 antibody according to the present invention can be obtained through the techniques of genetic engineering, i.e. by obtaining a recombinant nucleic acid that encodes the amino acid sequence of GD2-specific antibodies containing the desired FR according to the present invention. One note is ditch the invention, the gene construct, encoding the antibody according to the present invention contains (5'-3' orientation) segment of DNA encoding the variable region of the heavy chain, containing at least one humanitarianly FR, and the segment of DNA encoding the constant region of the heavy chain. In another example embodiment of the invention another DNA segment encoding a cytokine, fused with the 3'-end of the DNA segment encoding the constant region of the heavy chain. In the following example of the invention, the gene construct comprises in 5'-3' orientation) segment of DNA encoding the variable region of the heavy chain, containing at least one humanitarianly FR, and a DNA segment encoding a cytokine. Alternatively, the nucleic acid according to the present invention may include (in 5'-3' orientation) segment of DNA encoding the variable region of the light chain containing at least one humanitarianly FR, and a DNA segment encoding a cytokine. In some embodiments of the invention nucleic acid encoding a cytokine, join in reading frame to the 3'-end of the gene encoding the constant region (e.g., CH3 exon), either directly or through interesnuyu area (for example, using appropriate linkers, such as DNA encoding a (Gly4-Ser)3(SEQ ID NO: 3)).

II. Expression of the recombinant DNA encoding the antibody is hu14.18

Nucleic acid encoding a protein according to the present invention, can be introduced or inserted into one or more expression vectors for introduction into an appropriate recipient cell in which it is expressed. Introduction of nucleic acids into expression vectors may be carried out using standard methods of molecular biology. Preferred expression vectors are vectors with which the encoded protein can be expressed in a bacterial cell or a mammalian cell.

According to the present invention, the variable region of the heavy chain of the antibody preferably is expressed together with the corresponding light chain in the same cell. For fused proteins, which contain several polypeptide chains, you can use more than one expression vector. Ways transfection, in which it is used, for example, two vector expression, often deliver both vectors in the target cell. Alternatively, sometimes it may be appropriate to use a single vector encoding multiple polypeptides, for their joint expression in one cell.

For example, on Figa-D shows the sequence of a nucleic acid vector encoding heavy and light chain immunoglobulin according to this is the overarching invention. The vector also includes a nucleic acid encoding IL-2, fused with the 3'-end of the heavy chain of immunoglobulin. Accordingly, when introduced into the cell, and this vector can provide protein consisting of gumanitarnogo antibodies and IL-2, which specifically binds to GD2 and stimulates immune function.

In addition, it may be convenient to Express the proteins according to the present invention as single-stranded molecules. For example, the variable region of the antibody may be expressed as single-chain antibody or sFv, optionally fused with nimmanahaeminda protein. In another example of the invention, the heavy chain (since merged with her or without cytokine cytokine) combine with complementary light (or heavy) chain (since merged with her or without cytokine cytokine) with the formation of monovalent or divalent immunoconjugates.

Recipient cell lines preferably are lymphoid cells, such as myeloma cells (or hybridoma). Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded transfitsirovannykh genes, and can glycosylate proteins. Particularly preferred recipient cells are cells Sp2/0 myeloma, which normally do not produce endogenous immunoglobulin. After transfection, the cell will produce only what about the immunoglobulins, encoded transfitsirovannykh gene structures. Transfetsirovannyh myeloma cells can be grown in culture or in the peritoneum of mice, in the latter case, secreted immunoconjugate can be isolated from ascitic fluid. As recipient cells, you can use other lymphoid cells, such as b-lymphocytes.

There are several ways transfection of lymphoid cells with vectors containing the recombinant nucleic acid encoding the chimeric Ig chain. The preferred method of introducing the vector into lymphoid cells is merging with spheroplasts (see, e.g., Gillies et al. (1989) Biotechnol. 7:798-804). Alternative methods include electroporation or precipitation (deposition) calcium phosphate. Other suitable methods of getting immunoconjugates are obtaining RNA sequence that encodes a recombinant construct, and its translation in the corresponding system in vivo or in vitro. After the expression of the proteins according to the present invention can be isolated using standard procedures purification of proteins (see, for example, U.S. Patent No. 5,650,150).

III. Treatment of cancer through gene therapy

Nucleic acids according to the present invention can be used as gene therapeutic agents for the treatment of cancer and other diseases, in which a is s, it is desirable to Orient the immune system to fight specific cell type. For example, from a person or animal can be derived cells, and these cells can be transliterowany one or more nucleic acids encoding an antibody according to the present invention. The cells are then injected back to the human or animal. Transfitsirovannykh cells can be normal or cancer cells. Alternatively, the nucleic acid can be introduced into cells in situ. After this a man or an animal gives the immune response to cancer cells that can cure cancer or to reduce the severity of the disease. Variable region of the antibody according to the present invention, stitched (connected) with the appropriate regulatory elements to stimulate expression in mammalian cells can be transliterowany in cells using a variety of methods, including using calcium phosphate, "gene guns", adenoviral vectors, cationic liposomes, retroviral vectors or any other effective methods of transfection.

In the specific example embodiment of the invention hu14.18 antibody is used for the selective delivery of the cytokine to the target cell in vivo, so that the cytokine may have a local biological effect, for example to cause a local inflammatory response, stimulating the growth and activation of T cells or activate ADCC (antibody-dependent cellular cytotoxic is here). In the circulatory system of the subject, bearing target cells, is administered a therapeutically effective amount of the antibody.

The invention is further illustrated is not limiting examples.

DESCRIPTION of embodiments of the INVENTION

Example 1

Purification of hu14.18-IL-2 formulation

In one of the tests hu14.18-IL-2 expressed in NS/0 cells, supernatant tissue culture was collected and identified protein hu14.18-IL-2 using (series) chromatography on a column with Abx Mixed Resin, chromatography with recombinant Protein a and chromatography on a column of Q Separate followed by diafiltration in tangential flow through a Pellicon 2 to replace the buffer in the composition. The details of these stages of purification are described below. Stage inactivation and virus removal alternated with these stages, as described below. Stage inactivation and virus removal is not needed for the cleanup, but were used in order to ensure compliance with regulatory requirements.

pH two liters of supernatant tissue culture NS/0 containing hu14.18-IL-2, brought to 5.9 using a 1M solution of acetic acid was passed through Abx column (J.T.Baker), washed with a solution containing 10 mm MES and 100 mm sodium acetate, pH 6.2, and was suirable solution of sodium acetate (500 mm) with a pH of 7. This material was loaded into the column with the recombinant Protein is (Pharmacia), washed with a solution of 100 mm of sodium phosphate and 150 mm NaCl with a pH of 7, was washed with a solution of 100 mm of sodium phosphate and 150 mm NaCl with pH 6, was washed with a solution of 10 mm sodium phosphate with a pH of 7 and was suirable solution of 100 mm of sodium phosphate and 150 mm NaCl with a pH of 3.5. pH lirovannomu material was equal to 4.2. To contribute to the inactivation of the virus, this pH was lowered to 3.8, and the preparation was incubated for 30 minutes, after which the pH was neutralized to 7 with 1M NaOH solution. To remove nucleic acids, this material was loaded on a column of Q Separate (Pharmacia) and washed with a solution of 100 mm of sodium phosphate and 150 mm NaCl with a pH of 7. Nucleic acid was associated with the column, whereas the protein was detected in the stream, passed through the column, and in the wash water, and washing was repeated until until A not returned to the original line. Diafiltration through a Pellicon 2 (Millipore) was performed according to the manufacturer's instructions, and the end product of hu14.18-IL-2 were placed in the following composition:

pH buffer composition was brought to 7 with 1 M NaOH.

As the final stage of the preparation was filtered through a membrane Viresolve 180 (Millipore), which was cut off substances with a molecular mass of more than 180,000 daltons. This had the effect of "polishing" of the material, resulting in deleted aggregated dimers and oligomers of higher order.

Example 2

The antitumor activity of the fused protein hu14.18-IL-2

Was observed in the clinical trials of Phase I.

To assess the safety and efficacy of hu14.18-IL-2 was completed clinical trial Phase I. Suitable for participation in the trial patients had histologically confirmed diagnosis of melanoma, which was considered incurable surgically and medically. These patients could be suitable for measurement or evaluation of metastatic disease, or they could be signs of disease after surgical resection of distant metastases or regional relapse. Patients with multiple (two or more) of local or regional recurrence were included in the trial only if they had previously obtained evidence of lymph nodes, and if the recurrence were separated by a period of not less than 2 months. All patients were required adequate bone marrow (determined by the total is the number of leukocytes (WBC)> 3.500/ml or the total number of granulocytes>2000/ml, platelet count>100.000/ml and hemoglobin content>10.0 g/DL), adequate liver function (defined by the level of aspartate aminotransferase (AST)<3 x normal and total bilirubin < 2.0 mg/DL) and adequate renal function (defined by the level of creatinine in serum<2.0 mg/DL or creatinine clearance > 60 ml / min). All patients according to electrocorticography (ECOG) had a status of 0 or 1, and the expected life of at least 12 weeks. Patients who received chemotherapy, radiation therapy or other immunosuppressive therapy in the period up to 4 weeks prior to the tests, not included in the test. Patients could have metastases in the Central nervous system (CNS), if they were subjected to treatment and were stable for at least 4 weeks before the test date. From all patients consent was obtained, based on the received information.

This testing phase I was planned as an open, non-randomized trial with dose increase, in which the group consisting of 3-6 patients received hu14.18-IL-2 in one of the following doses: 0,8, 1,6, 3,2, 4,8, 6,0 or 7.5 mg/m2/day. hu14.18-IL-2 was administered during the patient's stay in hospital with a 4-hour intravenous (IV) infusion for 3 consecutive days in the first week of each course of treatment. Merged the trees hu14.18-IL-2 was administered to patients in the composition, containing 4% mannitol, 100 mm arginine hydrochloride, 5 mm citrate and 0.01% tween 80 at pH 7. Patients were discharged from the hospital, provided the stability of their condition, approximately 24 hours after the last infusion. Side effects and toxicity was assessed by common toxicity criteria NCl (version 2.0) and the scale of Wisconsin cancer center for IL-2 (General condition, weight gain, and temperature). Doselimiting toxicity (DLT) was defined as the appearance of toxicity level 3 or 4, except lymphopenia 3 degrees, hyperbilirubinemia, hypophosphatemia or hyperglycemia. Maximum tolerated dose (MTD) was defined as the dose at which two out of six patients during 1 year of treatment were found DLT. Patients with symptoms of toxic level 3 related to treatment, were to be restored, at least to level 1 before they could continue the treatment at 50%dose reduction during the course 2. Patients with progression of the disease ≥ 25% were excluded from the trials. Patients with stable disease were course 2.

Pharmacokinetic properties of hu14.18-IL-2 were evaluated in patients. When the levels of hu14.18-IL-2 were evaluated in serial samples taken from all 33 patients immediately after the first 4-hour infusion (day 1, course 1), it was found that the half-life equal to 3.7 cha is a (+/- SD (standard deviation), 0.9 h). This value is intermediate between the periods of half-life 2 its components (approximately 45 minutes for IL-2 and 3 days for chimeric antibodies m14.18) and comparable with the value that was obtained for the half-life of the chimeric protein m14.18-IL-2 in mice. After clearance of hu14.18-IL-2 from the serum of these patients it was not possible to detect all components of IL-2 and hu14.18 antibody. Peak serum and area under the curve (AUC) during course 1 was found reliable dose-dependent increase (p<0,001).

In this test, the treatment received thirty-three patients. Table 1 lists the clinical outcomes of the disease. Two patients (6%) underwent only the first 2 days of the course 1. One of these patients (dose level 3) on the 2nd day of treatment was detected hyperbilirubinemia of degree 3, and the second patient (dose level 6) there is a hypoxia of degree 3 and hypotension that required discontinuation of treatment. Both patients the disease progressed, and they have not received a second course of therapy. At nineteen patients (58%) after the first course of therapy, the disease was stable, and they received a second course of therapy. Five patients (15% of all patients) for course 2 required a 50%dose reduction due to adverse effects occurred during course 1. Seventeen patients (52% of all patients) completed the course 2. One patient (in over dose 4) refused to receive the latest infusion during course 2, and in one patient (level dose of 6) had to stop the last infusion rate of 2 due to hypotension. Eight patients (24% of all patients) after the second treatment the disease was stable. The results show that hu14.18-IL-2 induces the stabilization of the progress of the disease in an unexpected large number of patients.

Eight of 33 patients, the disease remained stable after 2 courses of therapy, and 4 of these 8 patients were alive without evidence of disease progression (1 with stable disease and 3 with no evidence of disease) for 20-52 months after completion of the treatment Protocol.

Five of 33 patients were taken in a test with no measurable signs of disease after surgical resection of recurrence or metastases. Two of these five patients the disease progressed, whereas the remaining 3 patients were alive without evidence of disease (20-52 months). These data are consistent with the hypothesis that clinical benefit from immunotherapy intervention is most likely in patients with a low tumor burden. In addition, one patient after two courses of treatment were observed objective reducing the size of the node in the lung, but the overall response of the disease was assessed as the progress of the disease due to the growth of distant site. The node was resected after therapy hu14.18-IL- and the disease in this patient has not progressed for more than 3 years.

1. Mannitol4%
2. Arginine hydrochloride, USP/NF100 mm
3. Citric acid USP-FCC5 mm
4. Polysorbate 80of 0.01% (weight/volume)
Table 1
Clinical outcomes
The number of patients
Patients who have completed course 131
Stable disease after course 119
50%dose reduction for course 25
Patients who have completed course 217
Stable disease after course 28

Immunostimulation in vivo with hu14.18-IL-2 in a clinical trial Phase I

Patients receiving treatment hu14.18-IL-2, were also studied for signs of stimulation of the immune system. Lymphopenia peripheral blood was observed on the 2nd-4th day, and it was followed by "return" in the form of limfotsitoz on 5th-24th day. Both of these changes were dose-dependent (p<0.01 and p<0,05, respectively). The number of lymphocytes in the 5th, 8th, 15th and 22nd day was significantly larger than the original values for a course 1. The initial number of lymphocytes for course 2 (29-day course 1) exceeded the initial number l is Mazitov for course 1, that shows that the effect of the first treatment course was still to 29 th day. In addition, during the course 2 the number of lymphocytes in the 5th, the 8th and the 15th day of these 12 patients was larger than the corresponding values for the 5th, 8th and 15th days of the course 1.

The cell surface phenotype of lymphocytes was found expansion of CD16+and CD56+lymphocytes (markers of natural killer cells (NK)) after the first week of therapy hu14.18-IL-2. This effect was still the 29th day of the course 1 (day 1 of course 2). Patients 19-33 (treated 4,8-7,5 mg/m2/day) phenotype on the cell surface of lymphocytes, in addition to the 1-th and 8-th day, was determined on the 15th and 22nd day. The analysis showed that the number of CD56-and CD56/CD16 coexpressing cells remained significantly elevated (p<0,01) on the 8th, 15th and 22nd day.

As a measure of activation of the immune system were measured levels of C-reactive protein (CRP) in patients 13 to 33 and soluble receptor of IL-2 (sIL-2R) in 31 patients who have completed course 1. Significant increase in the mean level of CRP was present at the 3rd-5th day of treatment in course 1 and course 2, compared with the initial level for each course. This increase of CRP levels had returned to baseline levels by 8-th day of each course of treatment. The level of sIL-2R was significantly increased above baseline after 24 hours after infusion hu14.18-IL-2 during course 1 and course 2 and remained elevated until the 8th day. B is lo detected, higher levels of sIL-2R was dose-dependent (p=0.014). Values of sIL-2R for course 2 was increased compared with the corresponding values for rate 1 on days 1-5 in patients receiving the same dose during both courses (p<0,05).

Cell line neuroblastoma LA-N-5 expressing GD2 and linking hu14.18-IL-2, were used to assess activated IL-2 function NC and is caused by antibodies cellular cytotoxicity (ADCC) in mononuclear cells of peripheral blood (PBMC), obtained from 31 patients who have completed course 1. Starting from the 8th day, in these two tests there was a significant increase in cell death mediated by lymphocytes, compared with day 1. 12 patients received a course 2 at the same dose as the rate of 1 showed the results for ADCC, very similar to the results obtained during the course 1. The only parameter that, as it was found, differed in course 2 course 1, there was increased cell death in the presence of IL-2 on day 1, which shows that stimulated cell death in this analysis remained elevated to the 29 th day (day 1 of course 2).

Because LA-N-5-the target is relatively resistant to fresh NK cells, it can be used to measure cell death stimulated IL-2, and ADCC. However, weak destruction of LA-N-5-mediated fresh PBMC in medium (without additional IL-2 in vitro), is not significantly greater the m on the 8th day, compared with day 1.

For patients 19-33 standard analyses with NC were performed in the 1st, 8th, 15th and 22nd day using sensitive to NK cell line-K562 targets. Reliable amplification of lysis by natural killer (NK) cells-targets C, compared to day 1, when tested in the environment or in the presence of IL-2 was observed on the 8th and 22nd day. Serum samples from selected patients were also evaluated to determine the functional activity of IL-2 and the functional activity of antibodies to GD2.

Responsive to IL-2 cell line Tf-1b demonstrated induced IL-2 proliferation in response to serum of patients obtained after infusion of hu14.18-IL-2. Progressive enhancement of proliferation was detected during the first 4 hours after 4-hour infusion. Values returned to baseline after 16 hours after the infusion, which coincides with the half-life of hu14.18-IL-2, equal to approximately 4 hours. Serum samples taken at these time points, were also by flow cytometry investigated for the presence of intact hu14.18-IL-2 immunocytokine (IC), which saves the IL-2 component and activity of antibodies to GD2. hu14.18-IL-2, are capable of binding cell line M21 (GD2-positive), can be detected in serum samples of patients after infusion IC. The IC number, the ability to communicate with M21, progressive vozrasta for the first 4 hours after 4-hour infusion, and then decreased, which also corresponds to the period of half-life, is approximately equal to 4 hours. Finally, analyses were performed in vitro samples taken from patients in order to determine whether the introduction of hu14.18-IL-2 in vivo, the relevant conditions required to achieve the ADCC. PBMC taken at day 8, find an increased cytotoxicity against GD2+cells-targets adding hu14.18-IL-2 to the analysis of cytotoxicity. The same ADCC-analysis was performed with PMBC taken at day 8, but instead of hu14.18-IL-2 to the sample was added patient serum obtained before or after the introduction of hu14.18-IL-2. PBMC obtained from patients on the 8th day of the course 2, were able to mediate the increased destruction of the cell line LA-N-5 in the presence of serum, obtained after the introduction of hu14.18-IL-2, compared with the results obtained with serum taken before infusion. Thus, hu14.18-IL-2 circulating in patients after intravenous injection, can stimulate ADCC with PMBC activated in vivo hu14.18-IL-2 from the same patient.

In short, these results show that with therapy hu14.18-IL-2 associated immunological changes, including the increase in the number of lymphocytes, increase the percentage of CD16+and CD56+PMBC, increased NK lysis and increased ADCC. Additional evidence of the activation of the immune system include increased levels of CRP and sIL-R serum. Laboratory analyses serum and PMBC showed that the molecule hu14.18-IL-2 circulating in the patient's serum after intravenous, retains its ability to activate IL-2-reactive cells through IL-2-receptor, the ability to bind to GD2-positive tumor cells and deliver IL-2 to the surface, which was detected using flow cytometry. NK cells are activated in vivo, as judged by their ability to mediate NK and ADCC function in vitro. In addition, NK cells activated in vivo hu14.18-IL-2, introduced these patients were able to mediate ADCC, increased hu14.18-IL-2 circulating in the serum of these patients. Thus, the conditions for obtaining the activation of the immune system were achieved in all patients in this trial.

1. The use of fused protein consisting of antibodies and cytokines, referred to as the hu14.18-IL2, comprising a light chain having the sequence of SEQ ID NO:5 and a heavy chain imoudu the sequence of SEQ ID NO:6, for the manufacture of a medicine to stimulate the immune response and stabilization of disease progression in patients with GD2-positive tumors, with the indicated antibody exhibits reduced immunogenicity compared to an antibody 14.18 mouse.

2. Method of enhancing antibody-dependent cellular cytotoxicity (ADCC) and gain lizinoj activity of natural killer cells (NK) in a patient having a tumor, by introducing a fused protein consisting of antibodies and cytokines, referred to as the hu14.18-IL2, comprising a light chain having the sequence of SEQ ID NO:5 and a heavy chain having the sequence of SEQ ID NO:6, where the specified antibody exhibits reduced immunogenicity compared to an antibody 14.18 mouse.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.

EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.

4 cl, 6 dwg, 22 tbl, 19 ex

FIELD: chemistry, medicine.

SUBSTANCE: claimed is novel hybrid protein CFP10-ESAT6 from M. tuberculosis, inducing reaction of hypersensitivity of delayed type with respect to M. tuberculosis. Chimeric NA, coding claimed protein is described. Described is method of obtaining claimed protein by cultivating cells of strain BL21(DE3) E.Coli, transformed with constructed recombinant expression vector on the basis of plasmid pET22b(+). Claimed is dosed medicinal form, containing claimed protein, for intracutaneous injection for diagnostics of tuberculosis infection.

EFFECT: high output of protein CFP10-ESAT6, which possesses specific immunogenicity.

8 cl, 2 dwg, 7 tbl, 3 ex

FIELD: genetic engineering.

SUBSTANCE: invention refers to genetic engineering and can be used in medical and biologic industry for making recombinant heterocarpine that is an antagonist of human release factor of growth hormone (GHRH). There is disclosed complete nucleotide sequence coding polypeptide heterocarpine; there are disclosed the related primer sequences to be used in heterocarpine gene cloning, as well as genetic make-ups including specified sequence, particularly hybrid gene coding fused protein containing polypeptide heterocarpine, as well as expression vectors for said hybrid gene. There is described method for making recombinant heterocarpine as His-tag fused protein, providing application of the host cells transformed or transfected with the disclosed genetic make-ups.

EFFECT: recombinant heterocarpine according to the invention can be used in making a medicinal agent for cancer treatment.

9 cl, 6 ex

FIELD: medicine.

SUBSTANCE: polypeptides include single-domain antibody against vWF, A1 domain of vWF, A1 domain of activated vWF, A3 domain of vWF, gp1b and/or collagen. Invention claims methods of obtaining indicated polypeptides, methods of coating devices applied in medical practice (e.g. in X-ray structural analysis, endoprosthetics) with indicated polypeptides.

EFFECT: obtainment of polypeptides for treatment of diseases requiring modulation of thrombocyte-mediated aggregation.

40 cl, 69 ex, 30 dwg, 32 tbl

FIELD: pharmacology.

SUBSTANCE: claimed invention relates to field of biotechnology and immunology. Described is physiologically active protein conjugate. Protein conjugate includes physiologically active polypeptide, which is covalently connected with Fc fragment of immunoglobulin by means of polyethylene glycol. Described is method of obtaining protein conjugate. It can find application in production of various polypeptide medications of prolonged action.

EFFECT: increased physiological activity in vivo construction in comparison with native physiologically active polypeptide and increase of half-life in serum of physically active polypeptide with minimal risk of inducing undesirable immune response.

17 cl, 18 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: method of augmentation of duration of action of physiologically active polypeptide in vivo is described. Physiologically active polypeptide is conjugated with Fc fragment of immunoglobulin by means of PEG. Invention use, in comparison with native physiologically active polypeptide, provides the raised physiological activity in vivo designs and-or augmentation of time of a semilife in Serum of physiologically active polypeptide with the minimum risk of induction of undesirable immune responses.

EFFECT: possibility of application of bond at manufacturing of various polypeptides medicinal preparations of the prolonged action.

11 cl, 18 dwg, 8 ex

FIELD: medicine; pharmacology.

SUBSTANCE: immunogenic hybrid polypeptide includes mimetic peptide of V-cellular epitope of apolypoprotein B-100 in which C-end of mimetic peptide is merged with N-end of T-helper epitope. Amino acid sequences of polypeptide variants are presented in description. Described is method of specified polypeptide production providing application of host cell transformed with recombinant express vector including gene coding specified polypeptide. Besides, invention concerns vaccine composition including specified immunogenic hybrid polypeptide for obesity prevention or treatment, recombinant express vector and host cell.

EFFECT: excellent anti-obesity activity without induction of immune response or severe by-effects.

15 cl, 25 dwg, 4 tbl, 15 ex

FIELD: medicine; pharmacology.

SUBSTANCE: it is obtained a chimerous photo protein (photin), presented by amino-acid sequence of obeline protein, which part (from the rest in position 50 to the rest in position 94) is replaced by a homologous site of amino-acid sequence of clitine protein (the rests 53-97). The method of obtaining new chimerous photo protein by the method of recombinant DNA and vectors applied to it and cells is described. It is offered to use photo protein under the invention as the calcium indicator in various test systems in vitro and in vivo.

EFFECT: increased level of bioluminescence in comparison with natural protein.

11 cl, 6 dwg, 2 tbl, 3 ex

FIELD: medicine; pharmacology.

SUBSTANCE: variants of the combined protein which contain the extracellular domain of a human receptor of a hormone of growth and the domain which includes alarm sequence for joining glycosylphosphatidylynozyte (GPI) anchors are offered.

EFFECT: effective medical product for acromegalia and gigantism treatment.

8 cl, 16 dwg

FIELD: medicine, biotechnologies.

SUBSTANCE: invention can be used for obtaining of the factor VII of blood coagulation. Derivatives of a polypeptide of the factor VII with amino-acid replacements Q250C, R396C and P406C are obtained or with Cysteinum attached to the S-end of native sequence of the factor VII. Obtain derivatives with use of transgene technologies in eucariotic cells-owners of mammals.

EFFECT: invention allows obtaining derivatives of the factor VII with the kept activity of the coagulative factor VII and with increased ability conjugate with PEG, in comparison with the natural form of a polypeptide.

20 cl, 2 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention concerns immunology area. Versions of the artificial fused protein consisting of an antibody (or its fragment) and cytokine, fused through a link peptide are offered. The antibody or its fragment is chosen from an antibody 225, 425, KS 1/4, 14.18, anti-CDx-antibody where x has the whole value 1-25. Each of versions of the fused protein has lowered quantity T-epitopes, at least, in the component of the fused protein presented by an antibody, and as consequence, possesses the lowered adjuvanticity, in comparison with an initial molecule. Identification of T-lymphocyte epitopes is performed by the automated calculation of sizes for the binding centres of class II MHC molecules with the subsequent experimental test of the obtained versions of protein for presence of the lowered adjuvanticity. The automated way of T-epitopes calculation is based on use of the Bjom's function modified in such manner that contribution of Van-der-vaals repulsion and lipophilic interaction in pairs between all lipophilic atoms of the chosen segments of the fused protein and a binding groove of a MHC P molecule is taken into account. Also a way of protein construction on the basis of the modified function Bjom's function with the subsequent experimental test of the received versions for presence of the lowered adjuvanticity is revealed, and also application of the fused protein for preparation of a pharmaceutical composition for tumour treatment is in addition considered.

EFFECT: invention use allows obtaining the fused proteins with the lowered adjuvanticity and, basically, keeping identical biological activity in comparison with a parent molecule; it can be used in treatment of tumours.

4 cl, 6 dwg, 22 tbl, 19 ex

FIELD: chemistry.

SUBSTANCE: proposed is a recombinant single-strand trispecific antibody for treating tumours which express CEA. The said antibody consists of a series of three antibody fragments: anti-CEA-scFv, anti-CD3-scFv and VH CD28-antibody, linked by two intermediate linkers (intermediate linker Fc and intermediate linker HSA). If necessary, a c-myc-mark or (His)6-mark can be added at the C-end. Described is DNA, which codes the antibody, expression vector based on it and E.coli cell, containing the vector.

EFFECT: use of the invention is more beneficial in clinical use compared to bispecific antibodies and known trispecific antibodies, makes easier clearing and expression of an antibody, which can further be used in treating CEA-mediated tumours.

10 cl, 21 dwg, 11 ex

FIELD: medicine.

SUBSTANCE: versions of the bond intended for linkage with the external domain B (ED-B) of a fibronectin are offered. The bond includes an antigen-binding fragment of one-chained antibody L19 and a cysteinum-containing linker for hanging of a radioactive label. Versions of a pharmaceutical composition for diagnostics and treatment of angiogenic diseases on the basis of the specified bond are opened. Application of bond for linkage with radioactive bond is described. The method of reception of bond in eucariotic cells is opened, including in Pichia pastoris and a set for reception is radioactive labelled agents on the basis of bond.

EFFECT: high-avid bond accumulation in solid tumours.

23 cl, 4 dwg, 5 tbl, 15 ex

FIELD: immunology, antibodies.

SUBSTANCE: invention elates to human monoclonal antibodies to MN and antibody fragments to MN that are targeted to repeat sequence GEEDLP within proteoglycan domain. Binding with a desired epitope is confirmed by competitive immunoenzyme analysis method ELISA wherein ELISA signal is attenuated in combined incubation with peptide comprising this repeat sequence (PGEEDLPGEEDLP). Binding inhibition can be confirmed by the Biacore study also wherein binding required antibodies with immobilized MN or proteoglycan peptides can be inhibited by peptide repeat sequence. In addition to binding with human peptide repeat sequence anti-MN can inhibit adhesion of CGL-1 cells to plastic plates covered by MN. Human antibodies anti-MN can be used in treatment of cancer diseases or for diagnosis of cancer diseases wherein the level of MN is increased.

EFFECT: valuable medicinal properties of antibodies.

11 cl, 8 dwg, 2 tbl, 13 ex

FIELD: oncology and biotechnology.

SUBSTANCE: invention concerns conjugates used for treatment of malignant tumor. Conjugate includes staphylococcal or streptococcal wild-type superantigen or modified superantigen and antibody constituent. Bacterial superantigen is modified to reduce serum reactivity with preserved its antigenic activity. Amino acid sequence of superantigen incorporates A-E regions determining binding to TCR and MHC molecules class II. Invention is directed to preparing antitumor drug and also to preparing pharmaceutical composition.

EFFECT: use of the conjugate according to invention activate immune system and, therefore, resistance of mammalian against malignant tumor.

67 cl, 11 dwg, 1 tbl, 11 ex

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing antibodies raised to human leukocyte differentiation factor (HLDF) or to HLDF fragment (31-38) representing peptide of the following structure: Arg-Arg-Trp-His-Arg-Leu-Glu-Lys possessing with antigenic and nucleic acids-hydrolyzing properties, and for diagnostic aims also. Antibodies are prepared from rabbit plasma blood immunized with three injections of antigens wherein synthetic HLDF factor or conjugate is used as antigens. Diagnosis of anaplastic state of human cells is carried out by using solutions of antibodies to HLDF factor or HLDF fragment (31-38) in the concentration 0.0013 mg/ml as biological markers. Invention provides carrying out the differential diagnosis of tumors and normal organs and effective detecting initial stages in cell differentiation disturbances.

EFFECT: improved preparing method of antibody, improved method for diagnosis.

6 cl, 21 dwg, 1 tbl

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

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

FIELD: medicine.

SUBSTANCE: invention refers to new pyridine derivatives or to their pharmaceutically acceptable salts of general formula 1: wherein R1, R2, R3, R4, R5, R6 and R7 are independently chosen from the group including hydrogen atom, halogen, amino, C1-C6lower alkyl, C2-C6lower alkenyl, C1-C6lower alkoxy, C1-C10alkylamino, C4-C9cycloalkylamino, C4-C9heterocycloalkylamino, C1-C10aralkylamino, arylamino, acylamino, saturated heterocyclyl, acyloxy, aryl, heteroaryl, C1-C10aralkyl, aryloxy; X represents oxygen or sulphur atom; Y represents oxygen atom or N-R8, wherein R8 is chosen from the group including hydrogen atom; aforesaid aryl group is chosen from phenyl, naphthyl and condensed phenyl group; aforesaid heteroaryl and saturated heterocyclic groups represent pentagonal or hexagonal heterocyclic ring containing 1 to 2 heteroatoms chosen from oxygen, nitrogen and sulphur atom; or condensed heterocyclic ring; and aforesaid aryl and heteroaryl groups are those that 1 to 4 assistants chosen from group including halogen, C1-C6lower alkyl, C1-C6lower alkoxy are substituted. And specified compounds or their pharmaceutically acceptable salt of formula 1 are not compounds as follows 6-methyl-3,4-dihydro-pyrano[3,4-c]pyridin-1-one, 5-vinyl-3,4-dihydro-pyrano[3,4-c]pyridin-1-one, 6-methyl-8-furan-2-yl-3,4- dihydropyrano[3,4-c]pyridin-1-one, 3-tert-butyl-5,6,7,8-tetrahydro-[2,7]naphthyridine-8-one and dimethyl ether (3S)-6,8-dimethyl-1-oxo-1,2,3,4-tetrahydro-[2,7]naphthyridine-3,5-dicarboxylic acids.

EFFECT: compounds possess inhibitory action with respect to formation of cytokines involved in inflammatory reactions, can be used as a therapeutic agent for treatment of inflammatory diseases, immune diseases, chronic inflammations; it provides antiinflammatory and analgesic action.

21 cl, 7 tbl, 144 ex

FIELD: chemistry.

SUBSTANCE: present invention refers to compounds of general formula (I) in the state of base salt or acid-addition salt, to method of their preparation and to the pharmaceutical composition thereof In the said formula R1 is (C1-C6)alkyl; (C3-C7)cycloalkyl unsubstituted or substituted once or more than once; (C3-C7)cycloalkylmethyl unsubstituted or substituted once or more than once; phenyl unsubstituted or substituted ; benzyl unsubstituted or substituted once or twice ; thienyl unsubstituted or substituted ; R2 is atom hydrogen or (C1-C3)alkyl; R3 is (C1-C5)alkyl; R4, R5, R6, R7, each R8 and R9 independently represents the atom of hydrogen, atom of halogen, (C1-C7)alkyl, (C1-C5)alkoxy or trifluoromethyl radical; n is 0, 1 or 2; Alk is (C1-C4)alkyl.

EFFECT: new compounds possess useful biological activity.

5 cl, 5 tbl, 4 ex

Up!