Isolated glycoprotein ib alpha polypeptide of human thrombocytes, fused protein, dna molecule (versions), expression vector (versions), cell (versions), polypeptide expression method, fused protein expression method, pharmaceutical composition (versions), method of inhibiting boding of blood cells to biological tissue in biological system, method of inhibiting bonding of protein to biological tissue in biological system and disorder treatment method
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.
EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.
41 cl, 3 dwg, 8 ex
The LEVEL of TECHNOLOGY
It is believed that the deleterious effects associated with vascular disorders such as stroke, heart attack and arteriosclerosis caused, at least partially, inappropriate running the inflammation and repair reaction vessels. Inflammation and repair reaction vessels include adhesive interactions between different cell types that commonly occur freely circulating in the blood. Examples of such interactions include interactions that can occur between platelets, leukocytes and the inner wall of blood vessels (i.e. vascular endothelium). In conditions of high friction forces in the fluid platelets attach to the endothelium through interaction between the complex of glycoprotein (GP) Ib-IX-V on their surface and the factor a background of Villebranda (vWF), present at the exposed subendothelial vessels. In addition, platelets, is attached to the endothelium of blood vessels, can bind and capture freely circulating platelets mediated by vWF binding that allows for the growth of thrombus through successive layers of platelets. GPIbα chain complex GPIb-IX-V may also facilitate the binding of α-thrombin to the surface of platelets, increasing the level mediated by thrombin cleavage GPV and protease-activated receptors (PAR).
In response to vascular damage, such as atherosclerotic plaques, or mechanical damage, such as damage caused by angioplasty, the establishment of a stent procedures, cardiopulmonary bypass, ischemic lesion or stenosis, leukocytes and platelets accumulate at the site of damage to the vessel and to provide multiple adhesive substrates to each other. This accumulation of leukocytes and platelets leads to local production factors, such as mitogens, cytokines and chemokines, causing additional unwanted progression of vascular disease.
Were described therapeutic polypeptides, including vWF-binding region derived from GPIbα. One such polypeptide basis of the sequence, containing two amino acid substitutions (G233V M239V) in the human amino acid sequence of wild-type GPIbα. Ig-protein containing 290 amino acids of the extracellular vWF-binding domain of this option (called GPIb2V-Ig), inhibits thrombosis of the coronary artery.
This invention provides an improved polypeptide variants glycoprotein-Ibα, which is applicable as therapeutic agents for the treatment of vascular disorders. In different variants of implementation of these options find reduced aggregation, increased stability, reduced binding to thrombin or two or more of these properties. One application of these polypeptide variants of the protein glycoprotein-Ibα is used as a fused protein for the treatment of vascular conditions associated with inflammation of blood vessels, thrombosis, atherosclerosis and related to the angioplasty restenosis.
In one aspect the polypeptide binds to alpha-thrombin with a lower affinity compared to the binding of alpha-thrombin polypeptide, which includes the naturally occurring amino acid sequence of human GPIbα (SEQ ID NO:1), or polypeptide, named GPIb2V (SEQ ID NO:2), which includes amino acid substitutions (G233V and M239V) relative to the amino acid serial is a major SEQ ID NO:1. When the binding of the polypeptide with thrombin with lower affinity of more thrombin becomes available for the formation of a blood clot, and undesirable bleeding of the subject is minimized. Examples of polypeptides which exhibit reduced binding of thrombin compared with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 include polypeptides that include one, two or three amino acid substitutions Y276F, Y278F, Y279V or conservative option.
In some embodiments, implementation of aggregation of the polypeptide is reduced relative to the aggregation of the polypeptide that includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, implementation of the reduced aggregation observed during the synthesis of this polypeptide in the cell. An example of a polypeptide that detects reduced aggregation, is a polypeptide that includes the amino acid replacement C65S or a conservative variant relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
In some embodiments, the implementation of this polypeptide is more resistant to proteolysis than the polypeptide that includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. An example of such a polypeptide is a polypeptide that includes amino acids is th replacement K237V, or a conservative variant relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
Examples of suitable polypeptides are polypeptides that have the following replacement amino acid sequence relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2: Y276F, Y276F K237V, Y276F C65S, Y276F Y278F Y279F, Y276F Y278F Y279F K237V and Y276F Y278F Y279F K237V C65S.
The polypeptides according to the invention can be provided in the form of a fused protein. For example, protein glycoprotein Ibα may include a first polypeptide containing at least a region of a variant polypeptide glycoprotein Ibα associated with the second polypeptide. In some embodiments of the second polypeptide forms of multimer, such as a dimer. In some embodiments of the second polypeptide includes at least a region of the polypeptide immunoglobulin.
The invention also provides nucleic acid encoding the polypeptide variant of glycoprotein Ibα, as well as vectors, cells and methods of expression of the polypeptide variant of glycoprotein Ibα using encoding the polypeptide variant of glycoprotein Ibα nucleic acids. The invention additionally features nucleic acid encoding a polypeptide fused protein variant of glycoprotein Ibα, as well as a vector containing coding polypep the d fused protein glycoprotein Ibα nucleic acid, described herein, and a cell containing the vectors or nucleic acids described herein.
This invention also provides a method of inhibiting leukocyte adhesion in biological tissue by contacting the leukocyte with the merged polypeptide glycoprotein Ibα. This leukocyte contact sufficient for inhibition of leukocyte adhesion and biological tissue.
In another aspect, the invention provides a method of treating disorders associated with platelet activation. The method includes introducing to a subject an effective amount of the fused polypeptide glycoprotein Ibα.
This invention also includes pharmaceutical compositions that include the polypeptide variants of the glycoprotein Ibα or fused polypeptides variants of glycoprotein Ibα.
If there are no other uczany, all technical and scientific terms used herein have the same meaning that is commonly understood by a person of ordinary skill in the field to which this invention. Although in the practice or testing according to the invention can be used in the methods and materials similar or equivalent to the methods and materials described here, the following describes the appropriate methods and materials. All publications, patent applications, patents and other ssy is key, mentioned herein incorporated by reference in their entirety. In the event of a conflict should be guided by this description, including definitions according to the invention. In addition, the materials, methods and examples are illustrative only and are not intended to limit the invention.
Other characteristics and advantages according to the invention will be apparent from the following detailed description and from the claims.
BRIEF description of the GRAPHICAL MATERIAL
Figure 1 is a graph showing the inhibition of chimeras GPIbα on ristocetin-induced platelet aggregation. Shows the transmittance of light (y-axis) for wild-type GPIbα ("Normal") and chimeras GPIb-Ig WT, GPIbα-290/2V-Ig, GPIbα-290/2V/FFF-Ig.
Figure 2 is a graph showing the antithrombotic efficacy of chimeras GPIbα in a canine model of coronary thrombosis Volts (y-axis: blood LCX).
Figure 3 is a graph showing the inhibition of the function of platelet GPIbα-290/2V-Ig and GPIbα-290/2V/FFF-Ig in a rabbit model of coronary thrombosis assessed by PFA-100.
DETAILED description of the INVENTION
Made of glycoprotein Ibα polypeptide with reduced binding to alpha thrombin, reduced aggregation and/or increased resistance to proteolysis in accordance with this invention is applicable as terapeuticas what their agents, for example, in the treatment of different conditions that benefit as a result of inhibition of binding of activated cells platelets with vWF in the cells of blood vessels. In some embodiments, the implementation of these polypeptides is provided in the form of a fused protein.
Protein variants according to the invention with a reduced binding of thrombin best way lead to reduced bleeding. The binding of thrombin to its receptor GPIbα platelet count is necessary for clotting. The binding of thrombin with soluble therapeutically GPIb may exacerbate bleeding in vivo isolation of thrombin from receptor GPIbα platelet, thereby reducing induced by thrombin platelet aggregation. Thus, protein variants, which displays a lower affinity towards thrombin, do thrombin available for interaction with the receptor Ibα of platelets, which, in turn, stimulates coagulation.
Suitable polypeptide includes the amino acid sequence from 1 to 15 amino acid substitutions, deletions or insertions relative to the amino acids in the district 65-279 amino acids, inclusive, of the naturally occurring protein sequence GPIbα person shown in SEQ ID NO:1, below, or variant GPIb2V, the amino acid sequence of which is shown in SEQ ID NO:2, below. In one or more options, which the ants implementation of this polypeptide has one or more of the following activities: (i) a lower affinity binding to alpha thrombin relative to binding to alpha thrombin polypeptide, which includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 person; (ii) a lower aggregation regarding aggregation of the polypeptide that includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2; or (iii) increased resistance to proteolysis relative to the polypeptide that includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
While not wishing to be bound by theory, the authors believe that these properties are based on the results of substitutions in regions of the target polypeptide sequence variants GPIb2V (SEQ ID NO:2). The change in the balance of cysteine at residue serine at position 65 (i.e. C65S) leads to inhibition of the aggregation of GPIbα molecules, in particular during the process of recombinant receipt. Residues of tyrosine, found in the provisions 276, 278 and 279 in the sequence of the wild type, usually modified excision in sulfotyrosine that creates anionic electrostatic interaction with alpha-thrombin. Selective elimination of these tyrosine residues by converting them to phenylalanine (i.e. replacement Y276F, Y278F and Y279F) reduces binding to alpha thrombin (Marchese et al., J. Biol. Chem. 270:9571-78) while maintaining the desired binding to vWF. Replacement of lysine valine at position 237 to prevent proteolysis at this site during the process of recombinant receipt.
A fragment of the sequence of 290 amino acids of the naturally occurring chain of glycoprotein Ibα person is presented below:
Amino acid sequence variants GPIb2V below as SEQ ID NO:2. This option is also discussed in the published patent application U.S. No. 20030091576 and copies of WO 02/063003.
In some embodiments, the implementation of this polypeptide has no more than 12 substitutions, insertions or deletions relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. For example, it may have 10, 8, 7, 6, 5 or less substitutions, insertions or deletions relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the implementation of these substitutions, insertions or deletions are in the region of amino acids 65-279 inclusive.
In some embodiments, the implementation of this polypeptide binds with lower affinity to alpha-thrombin relative to binding to alpha thrombin polypeptide that includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. Examples of polypeptides which exhibit reduced binding, are polypeptides that include one, two or three amino acid substitutions Y276F, Y278F, Y279V, or a conservative variant relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
In some embodiments, implementation of aggregation of the polypeptide is reduced relative to the aggregation of the polypeptide is th includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, implementation of the reduced aggregation observed during the synthesis of this polypeptide in the cell. An example of a polypeptide that detects reduced aggregation, is a polypeptide that includes the amino acid replacement C65S, or a conservative variant relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
In some embodiments, the implementation of this polypeptide is more resistant to proteolysis than the polypeptide that includes the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2. An example of such a polypeptide is a polypeptide that includes the amino acid replacement K237V, or a conservative variant relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.
In some embodiments, the implementation of this polypeptide includes an amino acid sequence with 1-10 amino acid substitutions, insertions or deletions relative to amino acids 1-290 SEQ ID NO:1 or SEQ ID NO:2, provided that at least one of these amino acid substitutions is C65S, K237V, Y276F, Y278F, Y279F, K237V.
Examples of suitable polypeptides are polypeptides that have the following replacement amino acid sequence relative to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2: Y276F, Y276F K237V, Y276F C65S, Y276F Y278F Y279F, Y276F Y278F Y279F K237V and Y276F Y278F Y279F K237 C65S.
The invention also includes polypeptides with one or more, for example 2, 3, 5, 6, 8, 10, 15 or more amino acid substitutions in the polypeptide produced from sequences of glycoprotein Ibα, along with the polypeptides corresponding to SEQ ID NO:1 and SEQ ID NO:2. They include, for example, GPIb302 (SEQ ID NO:3), GPIb302/2A (SEQ ID NO:4), GPIb/4 (SEQ ID NO:5) and GPIb290/1A (SEQ ID NO:6).
Variant protein glycoprotein may be provided as part of a fused protein. For example, fused proteins protein glycoprotein Ibα immunoglobulin applicable for inhibiting the attachment of platelets and leukocytes to the biological tissue, such as, for example, the vascular endothelium. Slit proteins according to the invention or nucleic acids encoding these fused proteins, can be incorporated into pharmaceutical compositions and administered to the subject to inhibit the interaction between the ligand glycoprotein Ibα (such as the factor a background of Villebranda, Wt-1, P-selectin or thrombin) and protein glycoprotein Ibα on the cell surface, such as a platelet. Inhibition of binding suppresses mediated protein glycoprotein Ibα platelet aggregation and associated signal transduction in vivo.
Merged the tree squirrels of glycoprotein Ibα immunoglobulin can be used to modulate the bioavailability of a cognate ligand protein glycoprotein Ibα. Inhibition of the interaction of the ligand protein glycoprotein Ibα/protein glycoprotein Ibα therapeutically applicable, inter alia, for the treatment of vascular inflammation and other vascular disorders associated with platelet activation.
Fused polypeptides variants of glycoprotein Ibα
In various aspects, the invention provides fused proteins, which include the first polypeptide containing at least a portion of the variant polypeptide glycoprotein Ibα, functionally associated with the second polypeptide. In this context, a “protein” or “chimeric protein” glycoprotein Ibα includes at least a portion of the variant polypeptide glycoprotein Ibα, functionally-related polypeptide, which is not a glycoprotein Ibα. The terms “polypeptide glycoprotein Ibα or variant of the polypeptide glycoprotein Ibα” refer to the polypeptide having the amino acid sequence corresponding to at least part of the polypeptide glycoprotein Ibα, whereas the term “polypeptide that is not a glycoprotein Ibα”refers to a polypeptide having the amino acid sequence corresponding to a protein which is essentially homologous to the protein of glycoprotein Ibα, for example, corresponding to a protein that differs from the polypeptide or glycoprotein Ibα and obtained and the same or a different organism. In fused protein, glycoprotein Ibα polypeptide glycoprotein Ibα may correspond to the entire protein or part protein Ibα.
In one embodiment, protein glycoprotein Ibα contains at least one biologically active portion of a protein glycoprotein Ibα. In another embodiment, protein glycoprotein Ibα contains at least two biologically active portion of a protein glycoprotein Ibα. In another embodiment, protein glycoprotein Ibα contains at least three biologically active portion of a protein glycoprotein Ibα. In the fused protein, the term “functionally linked” is intended to indicate that the first and second polypeptides are linked in a manner that enables at least one function associated with the polypeptide glycoprotein Ibα. When used to refer to a nucleic acid encoding the fused polypeptide glycoprotein Ibα, the term "functionally linked" means that a nucleic acid encoding a polypeptide glycoprotein Ibα, and nucleic acid encoding a polypeptide, which is not a glycoprotein Ibα, fused in the same reading frame to each other. Polypeptide that is not a glycoprotein Ibα, may be merged with the N-end or From the end of the polypeptide glycoprotein Ibα.
In the following embodiment, the protein glycopro the ina Ibα may be associated with one or more additional parts of the molecule. For example, protein glycoprotein Ibα may optionally be associated with the fused protein GST, which sequence fused protein glycoprotein Ibα merged with the end of the GST sequences (i.e. glutathione-S-transferase). Such fused proteins can facilitate the purification of fused protein glycoprotein Ibα.
In another embodiment, this protein comprises a heterologous signal sequence (i.e polypeptide sequence that is not present in the polypeptide encoded by the nucleic acid glycoprotein Ibα) at its N end. For example, the signal sequence of the native glycoprotein Ibα may be removed and replaced with a signal sequence from another protein. In some cells of the host (for example, cells of the host mammal) expression and/or secretion of the glycoprotein Ibα may be increased through use of a heterologous signal sequence. Representative signal sequence is MPLLLLLLLLPSPLHP (SEQ ID NO:8). Another representative of the signal sequence is MPLQLLLLLILLGPGNSLQL WDTWADEAEK ALGPLLARDRR (SEQ ID NO:9). If desirable, one or more amino acids can be included between the first polypeptide molecules containing GPIbα-part, and part of the second polypeptide.
Chimeric or fused boe is OK according to the invention can be obtained by standard methods of recombinant DNA. Nucleic acid sequences encoding polypeptides GPIbα and amino acid sequences of these polypeptides, of which design options GPIbα polypeptide described in WO02/063003, the content of which is incorporated herein by reference in its entirety.
For example, DNA fragments encoding the polypeptide variant sequences are ligated together in-frame reading in accordance with a conventional method, for example, with blunt ends or staggered ends for ligation, cleavage with restriction enzymes (restrictable) to ensure appropriate ends, building sticky ends, as necessary, processing of alkaline phosphatase in order to avoid undesirable joining, and enzymatic ligation. In another embodiment, the slit gene can be synthesized by conventional methods, including automated DNA synthesizers. Alternatively, can be carried out PCR amplification of gene fragments using anchor primers that result in the formation of complementary overhangs between two consecutive gene fragments which can then be subjected to annealing and re-amplified to generate a sequence of a chimeric gene (see, for example, Ausubel et al., (eds.), Current Protocols in Molecular Biology, Johm Wiley and Sons, 199). Also available are numerous expression vectors that encode part of the merger (for example, Fc-region heavy chain immunoglobulin). Nucleic acid glycoprotein Ibα may be cloned into such an expression vector such that this part of the merge linked in reading frame with the protein of the immunoglobulin.
Fused polypeptides of the glycoprotein Ibα may exist as oligomers, such as dimers or trimers. In some embodiments, the implementation of the fused polypeptide glycoprotein Ibα is a dimer.
In some embodiments, the implementation of the polypeptide part of the glycoprotein Ibα provided in the form of a variant polypeptide glycoprotein Ibα, having a mutation in the naturally occurring sequence GPIbα (wild type)that results in higher affinity (relative to neutrogenas sequence) binding of GPIbα polypeptide with a molecule on the cell surface leukocyte. For example, this mutant polypeptide may bind with higher affinity to factor von Willebrand's disease (vWF). This increased reactivity, or Hyper-reactivity can be estimated using low concentrations of ristocetin. Alternatively, any other method for determining the reactivity of the polypeptide with vWF can also be used to identify polypep the Idov, which are more reactive with vWF, i.e. more reactive than the naturally occurring wild-type GPIbα. Examples of polypeptide glycoprotein Ibα that are associated with a higher activity with vWF include options GPIbα, which include changes in the sequence of the hinge region of GPIbα polypeptide. The hinge area is defined as the area including the remains of 220-310, and reportedly is the main binding site for vWF to GPIbα polypeptide. Mutations in the hinge region include mutations at residue 233, which in wild-type GPIbα encodes glycine. An example of a suitable replacement is the replacement, in which the glycine at position 233 is replaced with valine (i.e. G233V). Second site mutations in the hinge region is the site at residue 239, which in wild-type GPIbα encodes methionine. Replacement of methionine 239 with valine is representative replacement, but can also be replaced by other amino acids. In addition, variants of the hinge region of GPIbα polypeptide suitable for use in the merged polypeptide according to the invention have mutations in the residue as position 233, and provisions 239 (see, for example, Dong et al., JBC 275:36 27663-27670 (2000)). Thus, the invention includes a fused proteins, which have a substitution at position 239, for example replacement M239V version of the GPIbα polypeptide. The invention from OSISA also fused protein, having a substitution at position 233, for example G233V, and fused protein, which includes a variant polypeptide GPIbα with the provisions of substitutions as 233 and 239, for example, replacing G233V and M239V.
In some embodiments, the first polypeptide includes a full-size GPIbα polypeptide. Alternatively, the first polypeptide contains less of the polypeptide than the full-sized GPIbα polypeptide. For example, the first polypeptide has a length smaller than 600 amino acids, for example less than 500, 250, 150, 100, 50 or 25 amino acids, or equal to these amounts of amino acids.
In some embodiments of the second polypeptide is soluble. In some embodiments of the second polypeptide increases the half-period of existence in the body (for example, the period of existence in serum) associated polypeptide. In some embodiments of the second polypeptide includes a sequence that facilitates the binding of this fused polypeptide with the second polypeptide GPIbα. In some embodiments of the second polypeptide includes at least a region of the immunoglobulin polypeptide. Merged immunoglobulin polypeptides are known in this field and are described, for example, in U.S. patent numbers 5516964, 5225538, 5428130, 5514582, 5714147 and 5455165.
In some embodiments of the second polypeptide soda which provides full-immunoglobulin polypeptide. Alternatively, the second polypeptide contains a smaller polypeptide than full-polypeptide immunoglobulin, for example, heavy chain, light chain, Fab, Fab2, Fv or Fc fragments. For example, in some embodiments of the second polypeptide comprises a heavy chain polypeptide of an antibody. In other embodiments of the second polypeptide comprises the Fc-region of an immunoglobulin polypeptide.
In another aspect according to the invention, the second polypeptide has less effector function than the effector function of the Fc region of the heavy chain immunoglobulin of the wild type. The effector function of the Fc includes, for example, binding to Fc receptors, complement fixation and active elimination of T cells (see, for example, U.S. patent No. 6136310). Methods of activity analysis, elimination of T cells, Fc-effector functions and stability of antibodies known in the field. In one embodiment, the second polypeptide has a low affinity or no affinity against Fc receptor. In another embodiment, the second polypeptide has a low affinity or no affinity in relation to protein complement C1q.
A representative sequence of the second polypeptide comprises amino acid sequence SEQ ID NO:10. This sequence includes the Fc-region. Nepoddelnuyu am is necessaty are amino acids, which differ from the amino acid found at the corresponding position of the sequence of wild-type immunoglobulin:
Options GPIbα polypeptide may include, in addition to this substituted amino, an alternative amino acid, which performs the same function as the specified amino acid. In some embodiments, this alternative amino acid is related to the specified amino acid, as a conservative amino acid replacement of this amino acid. “Conservative amino acid substitution” is a substitution in which a specific amino acid residue substituted amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been identified in this area. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromaticheski and side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Thus, in some embodiments, implementation, instead of replacing tyrosine with phenylalanine at position 276, 278 or 279 (Y276F, Y278F or Y279F), the tyrosine residue may be replaced by tryptophan or histidine. Similarly for option C65S cysteine can be replaced by alternative glycine, asparagine, glutamine, threonine, or tyrosine. To replace L237V lysine can be an alternative replaced by alanine, leucine, isoleucine, Proline, phenylalanine, methionine or tryptophan.
The invention also includes a polypeptide sequence which is at 80%, 85%, 90%, 95%, 98% or 99% homologous to the sequence presented in SEQ ID NO:1 or SEQ ID NO:2, provided that it includes one or more of the amino acid substitutions C65S, K237V, Y276F, Y278F, Y279F, K237V. Derivatives or analogs of the nucleic acids or proteins according to the invention include, but are not limited to, molecules that contain areas that are essentially homologous nucleic acids or proteins according to the invention, in various embodiments, at least about 70%, 80%, 85%, 90%, 95%, 98% or even 99% for nucleic acid sequence or amino acid sequence of identical size when compared with the matched sequence, when the eat this mapping is performed by using a computer program homology, known in this field, or molecule encoding a nucleic acid which is capable of gibridizatsiya with the complement of the sequence encoding the aforementioned proteins under stringent, moderately stringent conditions or low severity. An example of a program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, WI), using default settings, which uses the algorithm of Smith and Waterman.
According to another aspect of the invention relates to vectors (including expressing vectors, containing a nucleic acid encoding a fused polypeptide glycoprotein Ibα or its derivatives, fragments, analogs or homologs. Recombinant expressing the vectors according to the invention can be designed for expression of the fused polypeptide glycoprotein Ibα in prokaryotic or eukaryotic cells.
In another embodiment expressing vector fused polypeptide glycoprotein Ibα is yeast expressing vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae is known in this field.
Alternatively, the fused polypeptide glycoprotein Ibα can be expressed in insect cells using methods known in this field, e.g., using baculovirus expresser the participating vectors.
In another embodiment, the nucleic acid according to the invention is expressed in mammalian cells using the expressing vector mammals.
In another embodiment, recombinant expressing vector mammals capable of driving expression of the nucleic acid is predominantly in a particular cell type (e.g., used tissue-specific elements for the expression of nucleic acids). Tissue-specific regulatory elements are known in this field.
Further, this invention provides a recombinant expressing a vector containing a DNA molecule according to the invention, cloned in expressing this vector in the antisense orientation. That is, the DNA molecule is functionally linked to a regulatory sequence in such a way that it makes possible the expression (by transcription of this DNA molecule) of an RNA molecule which is antisense relative mRNA fused polypeptide glycoprotein Ibα. Can be selected regulatory sequence functionally linked to a nucleic acid cloned in the antisense orientation, which control the continuous expression of the antisense RNA molecule in a variety of types of cells, such as promoters and/or enhancers viruses,or can be selected regulatory sequences, that control constitutive, tissue-specific or cleocinonline by expression of antisense RNA. This antisense expressing the vector may be in the form of recombinant plasmids, family or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the type of cell in which you entered this vector.
The host-cell can be any prokaryotic or eukaryotic cell. For example, fused proteins, glycoprotein Ibα can be expressed in bacterial cells such as E. coli, insect cells, yeast cells, or mammalian cells (such as human cells, cells of the Chinese hamster ovary (Cho) or COS cells). Other suitable cells are the owners of well-known specialists with expertise in this field.
The DNA vector can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection.
The cells of the host mammal, such as cells of the Chinese hamster ovary (Cho) or COS cells, can be transliterowany expressing vectors to create opportunities through post-translational modification, generation of epitope sialyl LewisXN-linked and O-linked Glick is nah fused polypeptide glycoprotein Ibα. In the case of cells SNO this requires the co-expression of the enzymes α-1,3-/1,4-fucosyltransferase (Kukowska-Latallo et al., Genes Dev. 4:1288-303, 1990) and Core2 β-1,6-N-acetylglucosaminyltransferase (Kumar et al., Blood 88:3872-79, 1996). The presence of the epitope sialyl LewisXN-linked and O-linked picano fused polypeptide glycoprotein Ibα will enhance the binding of selectins.
In stable transfection of mammalian cells it is known that depending on expressing vector and transfection methods, only a small fraction of cells may integrate the foreign DNA into their genome. For identification and selection of these integrants gene, which encodes a breeding marker (for example, the gene of resistance to antibiotics), is usually introduced into the cells of host-together with the gene of interest. Various breeding markers include markers that give resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a breeding marker can be introduced into the cell host on the same vector as the vector that encodes a fused polypeptides of glycoprotein Ibα, or can be introduced on a separate vector. Cells stably transfetsirovannyh introduced nucleic acid can be identified by drug selection means (for example, cells that are on the Chile itself breeding gene marker will be survive at the time as the other cells die).
A host cell according to the invention, such as a prokaryotic or eukaryotic cell is the master in culture, can be used for producing (i.e. expression) fused polypeptide glycoprotein Ibα. Thus, this invention provides additional methods of obtaining the fused polypeptide glycoprotein Ibα using host cells according to the invention. In one embodiment, the method comprises culturing the host cell according to the invention (into which was introduced expressing recombinant vector that encodes a fused polypeptides of the glycoprotein Ibα) in a suitable medium, so that the produced fused polypeptides of glycoprotein Ibα. In another embodiment, this method further provides for the allocation of the fused polypeptide glycoprotein Ibα from the medium or the host cell.
Fused polypeptides can be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis or the like
Chemical synthesis of polypeptides facilitates the incorporation of modified or unnatural amino acids, including D-amino acids and other small organic molecules. Replacement of one or more L-amino acids in the peptide is relevant isoform of D-amino acids can be used to increase the stability of peptides to enzymatic hydrolysis, and to enhance one or more properties of biologically active peptides, i.e. binding receptor functional activity or duration of action.
The introduction of covalent cross-links in the peptide sequence is conformationally and topographically strain polypeptide molecular skeleton. This strategy can be used for the development of peptide analogs of these fused polypeptides with enhanced activity, selectivity and stability. Because the conformational entropy of the cyclic peptide is lower than its line-up, the acquisition of the specific conformation can occur with a smaller decrease in entropy for the cyclic analogue than for acyclic analogue, by making it more preferable for binding free energy. Macrocyclization often perform the formation of amide bond between the N - and C-ends of the peptide, between the side chain and the N - or C-end [for example, using the K3Fc(CN)6at pH 8.5] or between the side chains of two amino acids. Disulfide bridges are also injected into a linear sequence to reduce their flexibility. In addition, the replacement of cysteine residues penitsillaminom (Pen, 3-mercapto-(D)valine) was used to increase the selectivity of some interactions opioid-receptor.
Pharmaceutical compositions comprising fused polypeptides Ibα or coding their nucleic acids
Slit proteins glycoprotein Ibα or molecules of nucleic acids encoding these fused proteins (also referred to as "therapeutic agents" or "active compounds"), according to the invention and their derivatives, fragments, analogs and homologs that can be entered into pharmaceutical compositions suitable for administration. Such compositions typically contain a molecule of nucleic acid, protein, or antibody and a pharmaceutically acceptable carrier. In this context, it is assumed that the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and delaying absorption (suction) agents and the like, compatible with pharmaceutical administration. Suitable carrier materials are described in the most recent edition Remington''s Pharmaceutical Sciences, a standard reference in this field, which is incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline solution, solutions, ringer's solution, dextrose and 5% human serum albumin. Can also be used liposomes and non-aqueous media, such as non-volatile oil. The use of such media and agents for pharmaceutically active substances is well known in this field. Except the hen any conventional medium or agent is nesovmestimymi with an active connection, discusses the use of any agent and any environment in these compositions.
Described here, the active agents can also be prepared in the form of liposomes. Liposomes are prepared by methods known in this field. Liposomes with enhanced circulation time is described in U.S. patent No. 5013556.
Especially applicable liposomes can be obtained by the process of evaporation with reversed phase with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivationally the phosphatidylethanolamine (PEG-PE). Liposomes ekstragiruyut through filters with defined pore size to obtain liposomes of the desired diameter.
The pharmaceutical composition according to the invention is prepared so that it was compatible with the intended method of administration. Examples of routes of administration include parenteral, for example intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e. local), transmucosal and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterialagents, such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; hepatoblastoma agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of toychest, such as sodium chloride or dextrose. the pH can be adjusted with acids or alkalis, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or mnogorazovye vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if the components are water soluble) or dispersions and sterile powders for unintended preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsipanni, N.J.) or phosphate buffered saline (SFR). In all cases, the composition must be sterile and must be fluid to the extent that it was easy to enter the syringe. It must be stable under the conditions of preparation and storage and must be preserved against the contaminating action the Oia microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol and the like), and suitable mixtures. The proper fluidity can, for example, be maintained by applying a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved with the use of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like In many cases in this song can be enabled isotonicity agents, such as sugar, polyalcohol, such as mannitol and sorbitol; and sodium chloride. Prolonged absorption of injectable compositions can be caused by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating active compound (e.g., fused protein, glycoprotein Ibα) in the required amount in an appropriate solvent with one ingredient or combination of ingredients enumerated above, as required, with subsequent filter of STERI what izala. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are drying in vacuum and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from its pre-sterile filtered through microporous membrane) solution.
Oral compositions generally include an inert diluent or suitable for food media. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic injection of the active compound may be incorporated with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared in liquid media for use in a liquid for rinsing the mouth, where this compound in a liquid carrier is applied orally and rinsed his mouth and connection otherquot or swallow. Pharmaceutically compatible binding agents, and/or substance-adjuvants can be included as part of this composition. Tablets, pills, capsules, lozenges and the like can contain any of the following and is gredients or compounds of a similar nature: a binder, such as microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch or lactose, disintegrity agent, such as alginic acid, Primogel or corn starch; lubricating substance, such as magnesium stearate or Sterotes; a sliding agent, such as colloidally silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.
For administration by inhalation of these compounds are delivered in the form of an aerosol spray from a pressurized container or dispensing device, which contains a suitable propellant, for example a gas, such as carbon dioxide, or a nebulizer.
Systemic administration can also be performed using transmucosal or transdermal means. For transmucosal or transdermal administration of penetrants, suitable for barrier through which the composition must penetrate, used in the composition. Such penetrants are usually known in this field and include, for example, for transmucosal introduction, surfactants, bile salts and derivatives of fuseboy acid. Transmucosal introduction can be performed with the use of sprays for the nose, or suppositories. For transdermal introduction the of active compound is prepared in the form of ointments, healing ointments, gels, or creams as generally known in this field.
These compounds can also be prepared in the form of suppositories (e.g., with conventional bases for suppositories, such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compound are prepared with carriers that will protect the connection against the rapid removal from the body, for example the composition of controlled release, including implants and microencapsulation delivery system. Can be used biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyarteritis and polylactic acid. Methods of preparing such compositions will be evident to specialists with expertise in this area. These materials can be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. As pharmaceutically acceptable carriers can also be used liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens). They can be prepared according to methods known to specialists with expertise in this field.
In some embodiments, the implementation of the pen is sexual or parenteral composition is prepared in dosage unified form for ease of administration and uniformity of dosage. Dosed standardized form called here physically discrete units suitable as single doses to be treatment of the subject; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect, together with the required pharmaceutical carrier. Detail in relation to dosage of unified forms according to the invention is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the field of compounding such an active compound for the treatment of individuals, and depends on the above.
Molecules of nucleic acids according to the invention can be incorporated into vectors and used as gene therapy vectors. The gene therapy vectors can be delivered to a subject, for example, intravenous injection, local introduction or stereotactic injection. The pharmaceutical preparation of the vector gene therapy may include vector gene therapy in an acceptable diluent, or can contain a slow release matrix in which the immersed carrier delivery. Alternatively, when the vector of the full delivery of the gene can be obtained in intact from recombinant glue is OK, for example, retroviral vectors, this pharmaceutical preparation can include one or more cells which produce the gene delivery system.
If desirable, can be cooked preparations continuous release. Suitable examples of drugs continuous release include a semi-permeable matrices of solid hydrophobic polymers containing the antibody, and these matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of matrices of continuous release include polyesters, hydrogels (for example, poly(2-hydroxyethylmethacrylate) or poly(vinyl alcohol)), polylactide, copolymers of L-glutamic acid and γ-ethyl-L-glutamate, degradiruem the ethylene vinyl acetate, degradiruete copolymers of lactic acid and glycolic acid, such as the LUPRON DEPOT™ (injectable microspheres composed of a copolymer of lactic acid - glycolic acid and acetate leuprolide) and poly-D-(-)-3-hydroxipropionic acid. While polymers such as ethylene vinyl acetate and lactic acid - glycolic acid capable of release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
The pharmaceutical composition can be enclosed in a container, package or transfer device is istwo together with instructions for administration.
Methods of inhibiting the attachment in a biological system
This invention relates to a method of inhibiting the attachment of blood cells to biological tissue within a biological system. This method involves adding to a biological system fused polypeptide according to the invention in a quantity sufficient to inhibit the attachment of blood cells to the biological tissue.
Blood cell can be, for example, leukocyte, platelet or erythrocyte. The leukocyte can be any leukocyte, which can be attached to biological tissue. In various aspects of this leukocyte is a granulocyte (i.e., the neutrophil, basophil or eosinophil), monocyte (i.e. macrophage or lymphocyte (e.g., T-lymphocyte, b-lymphocyte, infiltrating the tumor lymphocytes or natural cell-killer). In some embodiments, the implementation of these leukocytes Express the β2 integrin, such as Wt-1. Alternatively, the leukocyte expresses the selectin ligand.
This invention also includes methods of inhibiting the attachment of proteins to biological tissue within a biological system. This method involves adding to a biological system fused polypeptide according to the invention in a quantity sufficient to inhibit the attachment of this protein to the biological tissue.
This protein may be embryopathy (for example, covalently, ecovalence or ion). Alternatively, the protein can be in a soluble form (i.e. in solution). This protein is a factor a background of Villebranda, thrombin, P-selectin glycoprotein Ibα.
In this context, the biological system mean any system that contains biological components, e.g. cells, proteins, carbohydrates, lipids or nucleic acids. This biological system may be a system in vivo, ex vivo or in vitro.
"Assignment" includes any biological interaction of leukocyte, such as rolling, solid attachment or specific interaction.
Inhibition of attachment of blood cells or protein to the biological tissue can be measured using methods known in this field. For example, assays for detecting binding of glycoprotein Ibα with biological tissue is described in Simon et al., J. Exp. Med. 192:193-204, 2000, cited in this article the links. In various embodiments, the implementation of binding merged GPIbα protein inhibits the binding of blood cells or protein in the biological tissue of at least 30%, 50%, 75%, 90%, 95%, 99% or 99.9%.
The attachment may also be evaluated in terms of larger or smaller flow than the physiological conditions of flow, including static conditions and consistently applying staticheskii shear (cutting) conditions. Attachment can be defined, for example, colorimetrically, fluorometrically flow cytometry or using analysis in a flow chamber with parallel plate.
This invention relates also to methods of treatment associated with activation of platelet disorders in a subject by introducing the subject of biologically active therapeutic compounds (“therapeutics”). Alternatively, the subject is administered one or more of the following ingredients: statin; acetylsalicylic acid (aspirin); heparin (including nefrackzionirovannam or low molecular weight heparins); antagonists of glycoprotein IIb/IIIa; clopidogrel; antagonists of P-selectin; thrombin inhibitors, and thrombolytic enzymes.
The subject can be, e.g., any mammal, such as human, Primate, mouse, rat, dog, cat, cow, horse, pig.
Terapeutica include, for example: (i) any one or more of the polypeptide glycoprotein Ibα and its derivatives, fragments, analogs and homologues; (ii) antibodies directed against a polypeptide glycoprotein Ibα described in (i)and (iii) nucleic acids encoding a polypeptide glycoprotein Ibα and its derivatives, fragments, analogs and homologues described in (i) above.
Essentially, any disorder which can etiological associated with activation of thrombosis is itow, considered preventable or treatable. This disorder can be, for example, inflammation of blood vessels; atherosclerosis; restenosis (e.g., associated with angioplasty restenosis); and/or status associated with thrombotic disease, such as angina (including stable angina or unstable angina), acute myocardial infarction, stroke, venous thrombosis or arterial thrombosis.
Hereinafter the invention will be illustrated in the following non-restrictive examples.
Example 1. The sulfation of tyrosine GPIb-Ig plays an important role in the binding of thrombin, but is less important for binding vWF
The role of acid carboxyl-terminal region of GPIbα binding to vWF and thrombin experienced research tyrosine residues at positions 276, 278 and/or 279 of the three types of slit proteins GPIbα-IgG1Fc: proteins with GPIbα sequence of human wild type; fused protein with replacement M239V (1V) and a fused protein with a double replacement G233V M239V (2V). These proteins were produced in the cells of the Chinese hamster ovary (Cho), then directs the Protein A-affinity chromatography and anion exchange chromatography. Allocated diarizonae form fused protein in which the state of sulfation was varied from 0 to 6.
Binding of these isoforms with the A1 domain of vWF (A1) with intact vWF and α-thrombin was tested with the use of the cation of surface plasmon resonance (BiaCore).
1V - 2V-polypeptides were associated with a significantly higher affinity to the A1 domain in comparison with WT polypeptides. 1V - 2V-polypeptides were found significantly enhanced affinity in relation to the intact vWF relatively fused protein with the sequence of wild-type GPIbα. Kdfor 2V-variant was in the range of 4 nm to 12 nm, and Kdfor 1V-variant was in the range of 119 nm and 284 nm for the fully sulfated relatively desulfation isoforms. The GPIbα binding of wild-type vWF is not detected under the same conditions, indicating that Kd>1 mm. The variation of the state of sulfation in WT, 1V and 2V are not correlated with a significant change in the binding domain A1 or intact vWF. Finally, the interaction of GPIbα-Ig-vWF showed the kinetics of the slow-on/slow-off (on/off) slow, typical for hydrophobic interaction (Kaand Kd~105M-1with-1and ~10-3with-1respectively.
For molecules GPIb-Ig239V and GPIb-Ig2V the binding affinity of fully sulfated isoforms in relation to thrombin was ~80 times higher than the binding affinity of desulfuromonas isoforms (Kd0.5 µm and 40 µm, respectively). To bind vWF fully sulfated molecules were only 3-4 times more active than desulfation form of both mutants (Kd= 3,4 - 15 nm for 2V 6-0-sulfated, 113-521 nm for 1V 6-0-sulfated). Investigated the relationship between the state of sulfation and the binding factor a background of Villebranda and binding to thrombin.
How bleeding from the tail of the rat: used adult male rats Sprague-Dawley (Talconic) weighing 250-300 g Supplier supplied the animals catheter jugular vein before delivery. Animals were kept in individual cages at room temperature with a light period of 12 hours (6 am - 6 PM) in the Institute's equipment and provided standard food for laboratory animals and water ad libitum. Animals acclimatized to their new environment for at least three days before using.
Procedure sample introduction GPIb-Ig and bleeding from the tail vein of rats on the day of the experiment the receiver extension tube (20 cm tube D 50) with a three-way shutoff valve and syringe in 1 cubic centimeter filled with sterile isotonic 0.9% saline, was added to the permanent jugular vein catheter. The test sample of the original solution GPIb-Ig was diluted in isotonic 0.9% saline and was administered at the indicated doses in a volume of 0.1 ml per 100 g of body weight over the other shoulder three-way shut-off valve. The catheter was rinsed with 0.2 ml of saline solution. Ten minutes after injection, rats were removed from their cells and gently placed in a restraining device is O. The distal segment 1-2 mm of the tail was cut off with a razor blade and the tail was quickly immersed in chemical beaker of 300 ml, pre-filled with 0.9% isotonic saline solution (temperature 37aboutC). Bleeding time was defined as the period of time from the start bleeding the tail to a full stop bleeding (no re-bleeding within 30 seconds after you stop bleeding). At the end of this experiment, rats were killed by inhalation of CO2.
In the analysis of bleeding from the tail vein of rats after 10 minutes after i.v. doses of 200 µg/kg were detected bleeding time 4 minutes in variants GPIbα with the state of sulfation 0, 1 or 2. Bleeding time was increased by increasing the status of sulfation to 6, where the bleeding time was 10 minutes.
These data demonstrate that the sulfation plays a role in the binding of thrombin, but a much smaller role in the binding of vWF. However, the condition of sulfation significantly affect the binding of α-thrombin. The stoichiometry of binding of the GPIbα-Ig with thrombin was higher than 2 α-thrombin on GPIbα-Ig. The interaction of α-thrombin-GPIbα is much more sensitive to the state of sulfation GPIbα-Ig compared to the hydrophobicity of the loop Cys208-Cys249 GPIbα, which binds to vWF. These results indicate that GPIbα contains two functionally featuring the, for example by transferring them subdomain: one to bind vWF and one to bind thrombin.
Example 2. Cys65 in GPIbα is involved in aggregation
The crystal structure reveals that Cys65 GPIbα is hidden in the LRR region. Very low coefficient of tagging (approximately 5%) with thiol-reactive probes under natural conditions showed the unavailability of this Cys residue. Analysis of Allman and pre peptide mapping showed that Cys65 no modification. However, even a very small amount of exposed thiol could form intermolecular disulfide bonds and cause aggregation. Investigated the role of this residue in the aggregation.
When the exposure of the sample GPIbα stress at 40aboutWith over 80 hours approximately 25% of the total protein formed aggregates. These units were reversible, but most were LTOs-stable. Analytical ultracentrifugation showed that these units were in the range from dimer to octamer. Map unrestored, alkyl Achro-K-peptides of these units showed that this map has gone free Cys-containing peptide (peptide K4), which can form a large peptide with another C4 and other Cys-containing peptides. To confirm the role of free Cys in the aggregation sample GPIbα were subjected to thermal stress in 1 M GnHCl, which led to the total covalent aggregation. In the presence of 1 M GnHCl structure of the protein is apparently shattered, ek is Peyrouse through this hidden Cys65 and accelerating the aggregation. These data suggest that under these conditions the free Cys residue may participate in the aggregation.
Example 3. Slit proteins GPIbα interact with vWF polypeptide
Investigated the direct interaction of variants fused protein GPIbα and vWF polypeptide.
Tested variants fused protein GPIbα included 1V, 2V, 3V, 3V/C65S, 2V/FFF and option called "cut 2V". Control reactions included GPIbα protein of 290 amino acids, one of the vWF polypeptide and outside the control polypeptide (47.mFc, protein PSGL-1).
Fused polypeptide or a control polypeptide was mixed with Protein granules And-Sepharose that were associated with parts of the molecule immunoglobulin fused protein. Protein associated with Protein granules And-Sepharose, suirable and subjected to electrophoresis in a 3-8% Tris-acetate gel. Then the gel was immunoblotting with polyclonal antibody to vWF and this antibody were detected. The gels were subjected to electrophoresis as in regenerating and non conditions. This procedure was performed twice.
The results of binding to A1-vWF fragment or intact vWF fragment shown below. vWF showed the strongest binding options GPIbα 3V and 3V/C65S in the analysis as in regenerating and non conditions. Strong binding was observed as variant 2V at that time, the AK is relatively less binding was detected with option 1V and option 2V/FFF. The GPIbα polypeptide wild type showed weak binding to vWF in these experiments.
|The binding constant of the vWF A1 (KAndM-1)||3,6E+07||1,9F+08||1,5E+09||9,1E+07||1,5E+09|
|The binding constant of the vWF (KAndM-1)||7,7E+07||5,6E+08||1,7E+09||2,4E+08||1,7E+09|
Example 5: Inhibition of in vivo repeated thrombosis coronary artery
The ability to merge Ibα protein/protein of immunoglobulin inhibition of thrombosis of the coronary artery in vivo determined using the procedure described Folts et al., Circulation 54:365-70, 1976. This protein includes a sequence of GPIbα with substitutions G233V K237V and M239V relative to the amino acid sequence of SEQ ID NO:1.
Half-dogs (mongrels) weighing 20-25 kg anaesthetize pentobarbital sodium (30 mg/kg i.v.), then incubated and ventilated anatrim air with the use of a respirator. Put venous and arterial catheters. Heart reach a left thoracotomy through the fifth intercostal space. The pericardium open and sew to the edges of the wound to ensure a "frame" without displacement of the heart. Allocate approximately 2 cm of the left circumflex coronary artery (LCX). The average and dynamic for LCX subjected to continuous monitoring using a perivascular ultrasonic probe for blood flow, placed proximally on the artery. After a period of stabilization of the endothelial LCX hurt compression hemostatic clip. Plastic constrictor is placed distal to the overlapping areas of damaged endothelium to provide approximately 70-80% stenosis of the vessel. When reducing blood flow to zero flow restored by shaking the constrictor to offset the aggregated platelets. This reduction and the restoration of blood flow is called the CFR. At least five consecutive CFR register before the introduction of the tested drugs.
The increase in the number 3V leads to higher blood flow. These results show that merged glycoproteins inhibit thrombosis in this animal model.
Example 6. The treatment in vivo of the subject with unstable angina (UA) or myocardial infarction with navysenim ST (NSTEMI)
Option GPIbα-Ig in this invention the input is t in the form of a fused protein of a single intravenous bolus injection (10 mg) patients with unstable angina or myocardial infarction c navysenim ST (NSTEMI). Treatment with this option weakens UA or NSTEMI. For monitoring activity GPIb-Ig using analysis of induced ristocetin platelet aggregation (RIPA).
Example 7. The comparison of time periods bleeding with the use of clopidogrel and options GPIbα 2V or GPIbα 2V/FFF
Effect on bleeding time were tested by comparison of the bleeding time in rats receiving clopidogrel and GPIbα 2V or two different doses of GPIbα 2V/FFF, which has no binding sites of thrombin.
Option GPIbα was injected intravenously two hours after administration of clopidogrel (4.3 mg/kg, po). Test options GPIbα included GPIbα 2V/290/FFF GPIbα 2V/290 GPIbα 2V/290 GPIbα 2V/290 GPIbα 2V/290, GPIbα 2V/290/FFF (100 µg/kg) and GPIbα 2V/290 (50 µg/kg). RBT (bleeding time in rats) was measured 15 minutes after the introduction of option GPIbα. For each treatment used six or seven animals.
Bleeding time was increased from 8.2 minutes (N=7) in rats treated with a single clopidogrel, up to 24 minutes in rats treated with clopidogrel and 50 mg/kg GPIbα 2V/290 (N=6). Bleeding time was increased to 13.9 minutes in rats treated with 50 mg/kg GPIbα 2V/290/FFF (N=6), and to 19 minutes in rats treated with 100 μg GPIbα 2V/290/FFF (N=7). Thus, a shorter bleeding time was observed in animals treated with a double dose of GPIbα 2V/290/FFF in comparison with animals treated with option GPIbα 2V/290.
Example 8. Against the antithrombotic efficacy sulfated and desulfuromonas forms soluble recombinant chimeras GPIbα in a canine model of coronary thrombosis artery
The ability of binding ligand and antithrombotic efficacy desulfuromonas recombinant chimeras of human GPIbα (GPIb-290/2V/FFF-Ig) was compared to the ability of binding ligand and antithrombotic activity of sulfated recombinant chimeras of human GPIbα (GPIb-290/2V-Ig).
Used soluble recombinant chimeras GPIbα included GPIb-290/2V-Ig, which consists of 290 N-terminal amino acids with GPIbα increasing function substitutions of valine at positions 233 and 239, fused with the Fc domain of human IgG1. GPIbα-290/2V/FFF-Ig received additional substitutions of the three tyrosine residues (Tyr-276, 278, 279) phenylalanine residues for the removal of sulfation.
Reduction of cyclic flow (CFR) induced constrictive injury of the left circumflex coronary artery in shot the male half of the dogs (mongrels). Coronary blood flow was subjected to continuous monitoring using a perivascular ultrasonic blood flow probe. Template bleeding time was measured when the surface of the inner lower lip using an automated cutting device. Prolongation in bleeding time caused by sulfated and desulfuromonas chimeras GPIbα, was evaluated using a rat model of bleeding from the tail. The function of platelets in whole blood before and after FS is cnyh time points after treatment in the model of Volts was measured using the analyzer-100 platelet function (PFA-100).
The ability of the GPIbα binding-290/2V-Ig and GPIbα-290/2V/FFF-Ig with vWF and α-thrombin was evaluated in vitro using Biacore analysis. The results are presented below:
|Mutant||Linking vWFA1||The binding of thrombin|
|GPIb wild-type||266 nm||0,76 µm|
|GPIb-290/2V/FFF-Ig||to 15.4 nm||35,8 mcm|
The effect of wild-type or chimeras on GPIb-induced ristocetin aggregation of human platelets is shown in figa-1D. Shown GPIbα wild-type ("normal"), GPIb-Ig WT, GPIbα-290/2V-Ig and GPIbα-290/2V/FFF-Ig.
Response to dose GPIb-290/2V-Ig (“2V”) and GPIb-290/2V/FFF-Ig (“FFF”) in a canine model of coronary thrombosis Volts shown below. A score of 4 is given if the CFR were completely eliminated, and a score of 3 is given if the CFR have not been fully resolved, but the coronary artery was able to spontaneously open.
|2V (μg/kg, iv)||Score reaction||Bleeding time (minutes)||FFF (μg/kg, iv)||Score reaction||Bleeding time (minutes)|
|50||4 (6/6)||2,0||50||3 (3/3)||2,4|
|100||4 (6/6)||3,7||100||4 (3/3)||2,3|
|500||4 (2/2)||of 10.25||500||4 (3/3)||of 5.4|
Felt also the prolongation of bleeding time from the tail of rats caused GPIb-290/2V-Ig and GPIb-290/2V/FFF-Ig:
|2V (μg/kg, iv)||Bleeding time|
|N||FFF (μg/kg, iv)||Bleeding time|
Figure 2 is a graph showing the antithrombotic efficacy of chimeras GPIbα in a canine model of coronary thrombosis Volts (y-axis: LCX).
Figure 3 is a graph showing the inhibition of platelet function GPIb-290/2V-Ig and GPIb-290/2V/FFF-Ig in a rabbit model of coronary thrombosis assessed by PFA-100.
These results demonstrate that the transformation of the six sulfated tyrosines in phenylalanine reduces the binding of domain A1 of vWF in ~3 times in these studies, and almost completely eliminates binding of thrombin. GPIb-290/2V/FFF-Ig showed a 50% reduction in efficiency in comparison with the GPIb-290/2V-Ig in the reduction of cyclic flow, prolongation of bleeding time and shutdown time ADP estimated using the PFA-100. To prevent the formation of blood clot by means of chimeras GPIbα at these doses is x occurs through the inhibition of the A1-domain of vWF, and this activity is independent of the binding of thrombin.
Although this invention has been described together with its detailed description, the preceding description is intended to illustrate and not to limit the scope of the invention defined by the scope of the attached claims. Other aspects, advantages, and modifications are within the scope of the following claims.
1. The selected polypeptide containing one or more substitutions in the amino acid sequence of SEQ ID NO: 2, where the specified one or more substitutions are
Y276F K237V C65S;
Y276F C65S or
Y276F Y278F Y279F K237V C65S
and where the specified polypeptide binds to factor a background of Villebranda affinity that is at least 10 times higher than that of the GPIbα polypeptide containing SEQ ID NO: 1, and has at least one activity selected from the group consisting of
(a) a lower affinity binding to alpha thrombin relative to binding to alpha-thrombin protein sequence with amino acid sequence SEQ ID NO: 2, and
(b) a lower aggregation relative to aggregation of the protein sequence with the amino acid sequence SEQ ID NO: 2; and
(c) increased resistance to proteolysis relative stability of a protein sequence from amino acid after what outlineto SEQ ID NO: 2.
2. The polypeptide according to claim 1, where the specified one or more substitutions is a Y276F K237V C65S.
3. The polypeptide according to claim 1, where the specified one or more substitutions is a Y276F C65S.
4. The polypeptide according to claim 1, where the specified one or more substitutions is a Y276F Y278F Y279F K237V C65S.
5. Protein containing a polypeptide according to any one of claims 1 to 4 and the region of the polypeptide heavy chains of immunoglobulin, for inhibiting the attachment of leukocytes to the biological tissue or to treat disorders associated with platelet activation.
6. Fused protein according to claim 5, where the region polypeptide heavy chain immunoglobulin is an Fc-region.
7. Fused protein according to claim 6, where the Fc-region has less effector function than the effector function of the Fc-region of a heavy chain immunoglobulin of the wild type.
8. Fused polypeptide according to claim 1, where Fc region binds with low affinity Fc receptor or no affinity against Fc receptor.
9. Fused polypeptide according to claim 7, where Fc region binds with low affinity to protein complement C1q or no affinity in relation to protein complement C1q.
10. Fused polypeptide according to claim 7, where the Fc-region contains the sequence of SEQ ID NO: 10.
11. Fused protein according to claim 6, further containing a signal sequence.
12. Fused protein according to claim 11, where this signal sequence is lnost contains the sequence of SEQ ID NO: 8 or SEQ ID NO: 9.
13. A DNA molecule encoding a variant of glycoprotein Ibα of human platelets (GPIbα) and characterized by nucleotide sequence determines the amino acid sequence of the polypeptide according to claim 1.
14. Expressing the vector nucleic acid containing a DNA molecule according to item 13, for expression of the polypeptide encoded by the specified DNA.
15. The cell containing the vector nucleic acid by 14, for expression of the polypeptide encoded by the DNA molecule according to item 13.
16. The way of expression of the polypeptide according to claim 1, providing for the cultivation of cells indicated in paragraph 15 under conditions suitable for expression of the polypeptide.
17. A DNA molecule encoding a protein containing variant of glycoprotein Ibα of human platelets (GPIbα), and characterized by nucleotide sequence determines the amino acid sequence of the polypeptide of claim 6.
18. The vector nucleic acid containing a DNA molecule according to 17, suitable for the expression of fused protein encoded by the specified DNA molecule.
19. The cell containing the vector nucleic acid by p, for expression of the polypeptide encoded by the DNA molecule by 17.
20. The way of the expression of the fused protein according to claim 6, providing for culturing cells according to claim 19 under conditions suitable for expression of the indicated fused protein.
21. Pharmaceutical composition, with whom containing a series of an effective amount of the polypeptide according to claim 1 and a pharmaceutically acceptable carrier, for inhibiting the attachment of leukocytes to the biological tissue or to treat disorders associated with platelet activation.
22. Pharmaceutical composition comprising an effective amount of the fused protein according to claim 6 and a pharmaceutically acceptable carrier, for inhibiting the attachment of leukocytes to the biological tissue or to treat disorders associated with platelet activation.
23. Method of inhibiting the attachment of blood cells to the biological tissue in a biological system, providing added to this system fused protein according to claim 6 in an amount sufficient to inhibit the attachment of blood cells to the tissues, where biological tissue forms a complex with factor a background of Villebranda, thrombin, glycoprotein Ibα or P-selectin.
24. The method according to item 23, where the blood cell is a platelet.
25. The method according to item 23, where the platelet expresses the glycoprotein Ibα, P-selectin or thrombin.
26. The method according to item 23, where the blood cell is a leukocyte.
27. The method according to p, where leukocyte expresses Wt-1 or a selectin ligand.
28. The method according to any of PP-27, where the biological system is an in vitro system, the system in vivo or ex vivo.
29. Method of inhibiting the attachment of proteins to biological tissue within a biological system, providing an Addendum to the biological the th system fused protein according to claim 6 in number, sufficient for inhibition of attachment of the protein to the tissue, where biological tissue forms a complex with factor a background of Villebranda, thrombin, glycoprotein Ibα or P-selectin.
30. The method according to clause 29, where the protein is a membrane-bound protein.
31. The method according to clause 29, where the protein is in solution.
32. The method according to item 30, where the protein is a factor a background of Villebranda, thrombin, glycoprotein Ibα or P-selectin.
33. The method according to p where the protein is a factor a background of Villebranda or thrombin.
34. The method according to any of PP-33, where the biological system is an in vitro system, the system in vivo or ex vivo.
35. A method of treating disorders associated with platelet activation in need of this subject, introducing the subject of the fused protein according to claim 6.
36. The method according to p, where the breach is associated with thrombotic disease.
37. The method according to p, where the specified violation is coronary heart disease, angina, acute myocardial infarction, stroke, venous thrombosis, atherosclerosis or arterial thrombosis.
38. The method according to p, where the specified violation is angina.
39. The method according to p where specified angina is unstable angina.
40. The method according to any of p-39, where the specified subject is people.
41. The method according to any of p-39, to omnitele introducing the subject compounds selected from the group consisting of acetylsalicylic acid, heparin antagonist of glycoprotein IIb/IIIa inhibitors, clopidogrel, antagonist P-selectin, thrombin inhibitor, and thrombolytic enzyme.
SUBSTANCE: invention discloses a pharmaceutical composition which contains TAT-HOXB4 protein as an effective component. Said composition has stimulating effect on production of hematopoietic stem cells. More specifically, the recombinant protein TAT-HOXB4 enhances acceptance of intramedullary transplants, hematopoietic reconstruction, repopulation and number of circulating stem cells, specifically after chemotherapy or exposure.
EFFECT: higher protein output and stability.
24 cl, 11 dwg, 2 tbl, 9 ex
SUBSTANCE: invention can be used in medical and biologic industry for preparing antineoplastic drugs. Plasmid DNA pFK2 providing synthesis of recombinant analogue of human kappa casein fragment, in Escherichia coli cells is designed; and a method for preparing a recombinant product with using it is described. The recombinant analogue of human kappa-casein fragment recovered from Escherichia coli cells transformed by recombinant plasmid DNA pFK2 has molecular weight of approximately 16 kDa; consists of residual methionine, human kappa-casein fragment with 24 on 134 amino acid residue and C-terminal histidine path and exhibits apoptotic activity in relation to malignant cells.
EFFECT: higher anticancer activity of the compounds.
3 cl, 6 dwg, 4 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.
EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.
8 cl, 10 dwg, 14 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention pertains to bioengineering. In particular, the invention relates to method of obtaining recombinant mutant horse cytochrome c. This method is realised by introduction of K27E/E69K/K72E/K86E/K87E/E90K or K8E/E62K/E69K/K72E/K86E/K87E or K8E/K27E/E62K/E69K/K72E/K86E/K87E/E90K mutations through site-directed mutagenesis into the horse cytochrome c gene which is contained in pBPCYCS/3 plasmid DNA. Further, the Escherichia coli JM-109 strain of the obtained recombinant plasmid DNA is transformed and the target protein is expressed and introduced through cation-exchange and adsorption chromatography.
EFFECT: invention enables use of recombinant mutant horse cytochrome c as a test system for measuring the rate of generation of superoxide in membrane preparations.
3 dwg, 5 ex
SUBSTANCE: in modified molecule IL-4RA, which inhibits mediated IL-4 and IL-13 activity, amino-acid remains 37, 38 or 104 represent cysteine. Polynucleotide, which codes specified antagonist, in composition of expression vector, is used to transform host cell and produce IL-4RA. Produced molecule IL-4RA is PEGylated and used to eliminate abnormalities that are related to high activity of IL-4 and IL-13.
EFFECT: invention makes it possible to produce antagonist with longer period of half-decay compared to non-modified IL-4RA.
17 cl, 1 dwg, 7 tbl, 7 ex
SUBSTANCE: in dissolvent, which contains from 55% to 70% of water (wt/wt), precursor of insulin or precursor of insulin derivative is exposed to fermentative splitting at alkaline values of pH. In process of fermentative splitting, they use tripsin or lysil-specific protease, preferably Achromobacter lyticus protease I. Then without separation of intermediate product from reaction mixture, mentioned intermediate product is fermentatively complemented with nucleophilic compound, which represents aminoacid ether, aminoacid amide, peptide, peptide ether or peptide amide in reaction mixture, having water content in the range from 10% to 50% of water (wt/wt), at acidic values of pH, close to neutral pH value. If required, protective group (s) is/are removed.
EFFECT: preparation of insulin compound from its precursor by efficient improved method.
24 cl, 5 ex
SUBSTANCE: invention is related to nucleic acids and multidomain proteins, which are able to bind vessel endotheliocyte growth factor (VEGF), and may be used in medicine. Recombinant method is used to produce polypeptide, which consists of component (R1R2)X and, unnecessarily, multidomain component (MC), which represents aminoacid sequence with length from 1 to 200 of amino acids, having at least one remainder of cysteine, where X≥1, R1 means antibody-like (Ig) domain 2 of VEGF receptor Llt-1, and R2 means Ig-domain 3 of VEGF receptor Flk-1. Produced fused polypeptide does not contain multidomain component in case, when X=2, and in case when X=1, multidomain component represents aminoacid sequence with length from 1 to 15 amino acids. Produced polypeptide is used in composition of pharmaceutical compound for VEGF-mediated disease or condition.
EFFECT: invention makes it possible to produce highly efficient trap of VEGF, special structure of which is suitable for local introduction into specific organs, tissues or cells.
16 cl, 3 tbl, 7 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology, specifically to a method of producing recombinant protein human albumin-interleukin-2 or recombinant protein human albumin-alpha 16-interferon, modified by attachment of human albumin. The method involves technology of culturing yeast strain Pichia pastoris PS106/pPIC9HAbIL-2 or yeast strain Pichia pastoris PS106/pPIC9HAbIFNa-16 in modified culture medium BMGY, after which induction synthesis of target proteins is carried out at low temperature. Further, cells are removed and the medium is concentrated. Target proteins are then precipitated using ammonium sulphate or polyethyleneglycol 3350. Target proteins are then separated by gel filtration on Sephacryl HR 200 or BioRad P-300 sorbents. Finally, affinity chromatography is then done on Cibacron F3GA sorbent.
EFFECT: invention simplifies and increases efficiency of the technology of purifying target proteins, and also allows for obtaining biologically active hybrid proteins, suitable for making medicinal agents.
3 cl, 1 tbl, 5 ex
SUBSTANCE: vitamin K dependent protein is made by separating a cultivated eukaryotic cell that contains an expressing vector that contains a nucleic acid molecule coding vitamin K dependent protein and associated sequences regulating expression. The associated sequences contain the first promoter and the nucleic acid molecule coding gamma-glutamylcarboxylase, and the second promoter. The first promoter represents a pre-early promoter of human cytomegalovirus (hCMV), and the second promoter is a pre-early promoter SV40. Herewith the expressing relation of vitamin K dependent protein and gamma-glutamylcarboxylase is 10:1 to 250:1.
EFFECT: invention allows for making gamma-carboxylated vitamin K dependent protein in production quantities.
29 cl, 5 dwg, 6 tbl, 7 ex
FIELD: medicine; microbiology.
SUBSTANCE: way is intended for reception of functionally active LF form, the basic toxic protein defining cellular disturbances, leading to death of an organism at infection with a malignant anthrax bacterium. For realisation of the way a recombinant plasmid pETHIS-LF (7816 items) is designed, containing a full-size gene of the lethal factor (LF) of malignant anthrax under the control of the promotor of bacteriophage T7 and to a determinant of ampicillin tolerance. The plasmide provides effective synthesis of LF protein of malignant anthrax merged with sequence of six Histidinums for clearing with the metal-chelate chromatography. The strain Escherichia coli BL-HISLF is designed using transformation of the specified plasmid DNA in the strain E.coli BL21 (DE3), synthesizing active LF protein. The target product is separated with the way including clearing on a metal-chelate sorbent with the subsequent additional clearing of the LF protein by gel-filtration.
EFFECT: reception of active recombinant protein LF on the simplified technology and with a high output of synthesised protein of the lethal factor.
3 cl, 3 dwg, 3 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: present invention relates to molecular biology and can be used in designing agent and methods of modulating body functions associated with HGF/c-met signalling pathway. The invention discloses HGF/c-met polypeptide-antagonists which are mutant forms of HGF which contain a mutation in the N-terminal part of the β-chain and/or in its dimerisation part. The disclosed polypeptides have lower biological activity compared to wild type HGG and can be used in modulating activity of c-met, cell proliferation, cell migration and angiogenic cell activity.
EFFECT: invention describes a method of obtaining HGF muteins using DNA recombinant technology and agents which are necessary for its existence.
22 cl, 8 dwg, 1 ex
SUBSTANCE: described is strain of hybrid cultivated cells of animals Mus. Musculus RIM9 - producer of monoclonal antibodies, specific to peptide of human milk - lactaptin and its recombinant analogues, which possess apoptotic activity with respect to human cancer cells. Invention allows to obtain monoclonal antibodies, recognising general linear antigen determinant of recombinant and natural lactaptin.
EFFECT: possibility to elaborate sensitive and specific test-systems for detecting lactaptin, antibodies to it and for isolation of lactaptin from human milk by method of affinity chromatography.
2 tbl, 4 ex
SUBSTANCE: cell line of humans melanoma mel H has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of vertebrates of Russian collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 715 Д".
EFFECT: invention allows to extend arsenal of human melanoma lines, which are used for creation of antitumour vaccine, applied for treatment of melanoma and other malignant neoplasms.
1 tbl, 2 ex
SUBSTANCE: cell line of human melanoma mel Me has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of vertebrates of Russian collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 712 Д".
EFFECT: invention allows to extend arsenal of cell lines of human melanoma, applied for creation of antitumour vaccine, which are applied for treatment of melanoma and other malignant neoplasms.
1 tbl, 2 ex
SUBSTANCE: cell line of human melanoma mel Rac has stable cultural and morphological characteristics, is stored in Specialised collection of cell cultures of Institute of Cytology RAS by number "РККК (П) 710 Д".
EFFECT: invention allows to extend arsenal of cell lines of human melanoma, used for creation of antitumour vaccines, which are applied for treatment of melanoma and other malignant neoplasms.
1 tbl, 2 ex
SUBSTANCE: invention refers to medicine and biology, more specifically to technology and methods for recovery of leukaemia cell suspension from spleens of high-leukaemia mice, and can be used in experimental oncological haematology for storage of low-frequent subtypes of murine leukaemia. There is offered a method for preparing a leukaemia cell suspension from spleens of inbred mice AKR/JY involving skin preparation with an antiseptic, splenectomy, decapsulation and homogenisation of spleen, filtration of the prepared suspension to be diluted in a sterile cooled artificial extracellular medium to the required concentration of leukaemia cells, prepackaging of the prepared suspension in ampoules for storage and transportation with decapsulation, grinding and homogenisation of fragmented spleen pulp performed in polyglucin solution containing ozone 25.72 mg/l and cooled to 24.0±1.0°C, leukaemia cells are filtered and diluted in polyglucin solution containing ozone 25.72 mg/l and cooled to 15.0±2.0°C.
EFFECT: invention provides maximum safety of specific functional adequacy of the transplanted leukaemia cells.
1 ex, 1 tbl
SUBSTANCE: invention relates to biotechnology and can be used in treatment of diseases, namely oncologic ones. Method is based on sampling from patient immature dendrite cells, their cultivation ex vivo for maturing and formation of allostimulating activity. Cells are additionally collected with antigen and are introduced to the same patient for formation of adaptive immunity. During cultivation ex vivo of immature dendrite cells fragmented allogenic double-stranded genomic DNA with fragments with size 200-6000 bp is introduced into culture medium in amount 5 mcg/ml of medium.
EFFECT: invention allows to extend field of obtained product application with increase of general immune state of patients.
SUBSTANCE: there is produced a new CT-F5/H3 hybrid cell clone producing monoclonal cholera toxin antibody (MCAB) in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises MCAB specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:20000 1:40000 in the cultural fluid, 1:4000000 in ascitic.
EFFECT: antibodies can be used for designing immunobiological systems of cholera toxin detection exceeding available analogues in sensitivity.
2 dwg, 4 ex
SUBSTANCE: there is produced a new CT-E6/E10 hybrid cell clone producing monoclonal cholera toxin antibody in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises monoclonal antibodies specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:10000 in the cultural fluid, 1:1000000 in ascitic.
EFFECT: produced antibodies exceeds the analogues available in sensitivity.
2 dwg, 4 ex
SUBSTANCE: there is produced a new CT-B1/F8 hybrid cell clone producing monoclonal cholera toxin antibody (MCAB) in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises MCAB specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:20000 in the cultural fluid, 1:2000000 in ascitic.
EFFECT: antibodies can be used for designing immunobiological systems of cholera toxin detection exceeding available analogues in sensitivity.
2 dwg, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: to obtain microbe-resistant plants plant cells are transformed with vector, which contains polynucleotide structure, which codes defensin polypeptide, which has amino-acid sequence C-x (3)-C-x (7, 9)-C-C, C-C-(8)-C-x-C and C-x-C-x (8, 11)- C.
EFFECT: obtaining polypeptide, which can be also applied as drug or veterinary medication, as well as in forage for animals.
23 cl, 4 tbl, 6 ex