Antibody igg with affinity binding with respect to antigenic complex cd3, recombinant nucleic acids encoding antibody light and heavy chain, method for preparing system, method for preparing antibody, method for treatment of patient

FIELD: genetic engineering, immunology, medicine.

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

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

33 cl, 5 dwg, 1 ex

 

The present invention relates to novel antibodies directed against antigenic CD3 complex to DNA and RNA, coding for the production of these antibodies, cell lines containing such DNA and/or RNA capable of their production, and to methods of producing antibodies using the above DNA, RNA and/or cells.

Antigen human CD3 consists of a minimum of four invariant polypeptide chains that ecovalence associated with T-cell receptors on the surface of T cells, and it is now called, as a rule, antigen CD3 complex. This complex is very deeply involved in T-cell activation in response to antigen recognition of T-cell receptors. All monoclonal antibodies against CD3 can be used to increase the sensitivity of T cells to secondary proliferative stimuli, such as IL1 (interleukin-1) and IL2 (interleukin-2). In addition, some monoclonal antibodies against CD3 are mitogenic in relation to T cells. This property is izotopicheskii dependent and is the result of interaction between Fc-domain antibodies against CD3 with receptors Fcon the surface of cells.

Antibodies against CD3 use for effects on the immune status by suppressing, enhancing or re-orientation of T-cell responses to antigens. The investigator is about, they have considerable therapeutic potential for use as immunosuppressive means a person, for example for the treatment of rejection after transplantation, renal, hepatic or cardiac allografts.

WO 92/06193 and equivalent application (GB 2249310A, application No. ER, JP 516117/91 and application for U.S. patent, registration number №07/862543, the contents of which are incorporated in the present description as links) belong to the antiglobulin reaction of antibodies against CD3 using reverse engineering or "humanization" gene variable regions of antibodies and ekspressirovali in combination with the corresponding genes of constant domains of human antibodies. The content of monoclonal antibodies, non-human, is reduced so that the antiglobulin reaction is unlikely.

WO 93/19196 and its equivalents (e.g., European patent 0586617, U.S. patent 5585097 and application for U.S. patent, registration No. 08/478684, all included in the present description as links) belong to the primary immunization. They describe the use of glycosylamine humanized antibodies against CD3 IgG subclass, which unexpectedly retain their antigen-binding specificity and immunosuppressive properties and still do not induce T-cell mitogens in vitro and induce a lower level of release of titoki the s in vivo, at the same time keeping some affinity in relation to the Fc.

Although such antibodies against CD3 possess high therapeutic value, to get them into the cellular structure is not easy. In practice, it is revealed that bad exit antibodies associated with poor growth transtitional cell lines. After continuous operation for several years, the best level for obtaining antibodies is approximately 10 μg/ml, and cells expressing antibodies against CD3, grow very slowly. In addition, these cells in systems with hollow cartridges used in large-scale production, will eventually become unproductive.

Vector system REE Celltech Glutamine Synthesis, used in the above expression of antibodies against CD3, usually provides the expression of other humanized antibodies at about 200 μg/ml of the Original hybridoma cell line rat (YTH 12.5) expressives in cell culture at a relatively normal level of 100 µg/ml, which indicates poor production of antibodies associated with humanized form. It turns out that there are one or more of the expressed humanized proteins, which are toxic to cells because after transfection cells become productive faster than grow.

AB the ora of the present invention unexpectedly discovered, that by obtaining chimeric forms antibodies against CD3, binding variable region of the light chain D rats with a constant region of a lambda man and cloning the resulting form in REE containing glycosylamine heavy chain gumanitarnogo CD3, you can get a myeloid cell line, ensuring the expression of functional glycosylamine antibodies against CD3 in the amount of 60-100 µg per ml of culture. Through the use of cloning by the method of serial dilution is possible to select some clones and to provide an even higher level of expression, for example about 120 μg/ml, and when receiving on a large scale to maintain sustainable long-lived culture without adverse effects on cell growth. Thus, the chimeric antibodies of the present invention show a good capacity for production in the absence of antiglobulin reaction, usually associated with antibodies obtained from rats.

Accordingly, the present invention relates to antibody IgG with affinato binding against antigenic CD3 complex, in which the heavy chain contains the frame variable regions together with at least one CDR selected from the amino acid sequence SEQ ID NO: 2, 4 and 6 and their respective conservatively modified variants, and a light chain zodariidae variable regions together at least one CDR, selected from the amino acid sequence SEQ ID NO: 8, 10 and 12 and their respective conservatively modified variants, characterized in that the frame variable regions of the heavy chain corresponds to the sequence typical of the man, and the variable region of the light chain includes one or more specific amino acids characteristic of the sequence, typical of rodent.

Preferably, the variable region of the light chain comprises enough of the amino acids that are specific for the sequence, typical of rodent, so that the light and heavy chains are associated stronger than in the case when variable region light chain is fully consistent with the field, the typical human. Convenient to the variable region of the light chain is fully consistent with the area, typical of rodent, for example a sequence of a rat. On the other hand, may be included only a few or even one of the amino acids characteristic of rats.

Specific amino acids in the sequence of the variable region of the light chain are rather typical rodent, and not for a person to choose among the amino acids represented in SEQ ID NO 14 in the list of sequences attached to this description, and the preferred sequence of frame variable regions easily the chain, which includes all possible amino acids characteristic of a skeleton of the rat, together with the corresponding CDR sequences. So amino acids characteristic of the frame of the variable segment light chain of the rat, SEQ ID NO 14 are Gln-1, Ala-2, Val-3, Val-4, Ala-7, Asn-8, Thr-12, Leu-14, Ser-16, Lys-19, Leu-20, Leu-39, Tyr-40, Glu-41, Ser-44, Met-48, Tyr-50, Phe-75, His-79, Asn-80, Val-81, Ala-82, Ile-83, Ile-88 and Phe-90. The corresponding amino acids of human rights in each case are Asp-1, Phe-2, Met-3, Leu-4, Pro-7, His-8, Glu-12, Pro-14, Lys-15, Ile-19, LEU-20, Gln-39, Arg-40, Pro-41, Ala-44, Val-48, Phe-50, Ser-75, Ser-79, Gly-80, Leu-81, Gln-82, Thr-83, Asp-88 and Tyr-90. The last sequence of a person is illustrated in EP 0586617 IN page 6 and in the corresponding patent application in the USA.

Conveniently, when the frame is variable regions of the heavy chain is typical of the man, and the variable region of the light chain is an area typical of rodent, all of the above amino acids are SEQ ID NO 14, typical for rats. However, one or more of these provisions SEQ ID NO 14, but not all, can correspond to the positions of the typical person, subject to the adequacy of compliance sequences such rodents, such as rats, to ensure the stable interaction of light and heavy chains, compared with that provided by a fully humanized form known from the prior art. This interaction is preferably Taco is about, when the antibody is expressed in cells REE using the instructions of the manufacturers (Celltech), achieved a higher level of 50 μg/ml, preferably exceeding the level of 100 µg/ml Preferably, such cells should not become unproductive in large numbers after using for several weeks.

Specialists in the art will understand that methods such as site-directed mutagenesis using PCR allows to obtain such different variable light chain, so that all their variants according to the invention can be obtained without excessive effort and skanirovat the level of expression in cells REE.

Amino acid sequence of CDR of SEQ ID NO 2, 4, 6, 8, 10 and 12 correspond to the CDR (a), (b), (C), (d), (e) and (f) in WO 93/19196, and CDR can also be attributed to the corresponding CDR (a)to(f)shown below.

Preferably, each of the heavy chain and/or light chain contains all three of their respective CDR of SEQ ID NO 2, 4 and 6 and SEQ ID NO 8, 10 and 12.

Preferably, antibodies are glycosylamine. The term "glycosylamines" is used in its ordinary meaning to indicate that antibodies corresponding to the invention, are not glycosylated.

The term "typical person" in relation to the frame of the plot indicate the frame, which is quite similar to the frame antibodies che is oweka, which is essentially non-immunogenic to humans when present in the original antibody. Preferably, the antibody of the invention with a heavy chain to the frame, the typical human has a value of affinity towards the antigen CD3 60-140%, often 80 to 100%, from that of rodent antibodies. Characteristics of humanized monoclonal antibodies and methods for their production of rodent monoclonal antibodies described in U.S. patent 5585089 included in the present description by reference. Comparison of the variable regions of the heavy chain, the typical human, mouse analogue can be done by comparing SEQ ID NO 16 (rat) with the corresponding region detected in the N-end of SEQ ID NO 20. SEQ ID NO 15 is a DNA sequence that encodes SEQ ID NO 16. Thus, frame the site, the typical human can have, for example, seven or more of the thirteen changes that distinguish the sequence N-terminal 119 amino acids of SEQ ID NO 20 from the N-terminal 119 amino acids of SEQ ID NO 16. Preferable to include all amino acids, the typical human. These changes can take place, for example, in any of the provisions 5, 18, 19, 42, 49, 75, 77, 78, 88, 93, 97, 98 and 114 of these sequences.

The term "typical rodent" in relation to the frame of the plot indicates the frame corresponding in amino acid sequentially the ti frame antibodies rodent, for example, rat or mouse. In the case of antibodies against CD3 comfortable amino acid skeleton of the site are amino acids antibodies rat.

Other information about D3-antigens can be found in the message of the First international workshop and conference on differentiation of human leukocytes. Information about the antigens and description of various glycosylated antibodies against CD3-antigen also can be found in the messages of the workshops and conferences of this series, in particular the Third and Fourth, published by Oxford University press. Specific examples of such antibodies are antibodies described Van Ller et al., Euro.J.Imminol., 1987, 17, 1599-1604; Alegre et al., J. Immunol., 1991, 140, 1184 and Smith et al., ibid, 1986, 16, 478, and the last publication refers to antibody-IgG UCHT1 and its options.

However, of particular interest as the basis for the antibodies of the present invention are CDR contained in the antibody ACTS and YTH 12.5.14.2. Antibodies ACT are described in publications such as Chatenaud et al., Transplantation, 1991, 51, 334, and New England Journal of Medicine paper, 1985, 313, 339, as well as in patents EP 0018795 and USA 4361539. Antibody YTH 12.5.14.2 (hereinafter called YTH 12.5) are described in such publications as Clark et al., European J. Immunol., 1989, 19, 381-388, and reconstructed YTH 12.5 are the subject of EP 0504350 and equal application for U.S. patent, registration No. 08362780 and U.S. patent 5585097, where a detailed description is given CDR, available in the specified antibody. Application for U.S. patent, registration No. 08362780 and U.S. patent 5585097 and 4361539 included in the present description as a reference.

The term "conservatively modified variants" is a term well known in the art, showing variants containing modifications, in essence, does not affect the affinity of antibodies and antigens. This term essentially has the meaning set forth in U.S. patent 5380712 included for this purpose in the present description by reference.

As for the CDR, of the greatest importance CDR of the heavy chain (a), (b) and (C). For specialists in this field of technology is also understood that the antibodies of the invention also contain a constant domains.

CDR (a), (b) and (C) are located in the heavy chain in the following order: frame plot of human antibodies 1/(a)/wireframe plot of human antibodies 2/(b)/wireframe plot antibodies man 3/(C)/wireframe plot of human antibodies 4 in the top position to the direction of the constant domain (n-end - To-end), and CDR (d), (e) and (f) are located in the light chain sequence of frame section antibodies rodent 1/(d)/wireframe plot of rodent antibodies 2/(e)/wireframe plot of rodent antibodies 3/(f)/wireframe plot of rodent antibodies 4 in the top position to the direction of the constant domain. Therefore, it is preferable that when all three CDR is available is t in the heavy chain CDR, they are arranged in a sequence (a), (b) and (C) in the leader position to the direction of the constant domain and light chain CDR are arranged in a sequence (d), (e) and (f) in the leader position to the direction of the constant domain. Wireframe plot antibodies rodent is a preferred frame section antibodies rat.

However, it should be borne in mind that the antibodies of the present invention can contain the CDR, completely different from the CDR described above, and that, even when this does not happen, there may be heavy chains and, particularly, light chains containing only one or two CDRs from among CDR (a), (b) and (C) and (d), (e) and (f), respectively. However, although the presence of all six of the above chains in the antibody of the present invention is not required, often in the most preferred antibodies will be present all six CDR.

Therefore, particularly preferred antibody contains a heavy chain, typical for a person with three CDRs (a), (b) and (C)containing the amino acid sequence of SEQ ID NO 2, 4 and 6 or their respective conservatively modified variants, and light chain, typical for rats, with three CDRs (d), (e) and (f)containing the amino acid sequence of SEQ ID NO 8, 10 and 12 or their respective conservatively modified variants, where the CDR of the heavy chain are alright (a), (b) and (C) leader in the direction of the constant region and light chain CDR are in the order (d), (e) and (f) leader in the direction of the constant field.

The preferred form of the first aspect of the present invention is an antibody, in particular glycosylamine having binding affinity of respect D3-antigen human constant region of the antibody is or is derived from a constant region, characteristic of man, and in particular represents a constant region of the lambda attached to the variable region of the light chain of the rat.

One of the suitable options is the presence of the antibody wireframe plot variable domain of the light chain of the rat, the corresponding amino acid sequence of the sequence in hybridoma YTH 12.5, i.e. SEQ ID NO 14, although constant region, still preferably is formed from one of the constant regions of a human, for example, will be a constant region of a lambda man. Preferred amino acid sequence of the chimeric light chain of the rat/human and the constant region of the lambda is SEQ ID NO 18. Recombinant nucleic acid, for example DNA encoding YTH 12.5, contains the amino acid sequence of SEQ ID NO 13, while the DNA encoding a variable region light chain is Rysy and a constant region of a lambda man, contains SEQ ID NO. 17.

Some sequences of the framework variable domain of the heavy chain of man will be preferred in the case of transplantation the preferred CDR sequences, as the 3-dimensional conformation of CDR will be better preserved in these sequences and the antibody will maintain a high level of affinity towards the antigen. The frames of variable (V) region of the heavy chain are preferably frames, which are encoded by gene III VY26/D/J VH, typical for a person of the hybridoma cell lines of b-cells 18/2 (Huminghat, Dersimonian et al., Journal of Immunology, 139, 2496-2501; WO 93/19196 and application for U.S. patent, registration No. 08/478684).

Therefore, in a preferred embodiment of the first aspect of the present invention, one or more preferred CDR heavy chain antibodies rats against CD3 present in the framework of the variable domain of a person having the following amino acid sequence that is read in the leader position to the direction of the constant field, and the CDR is a CDR (a), (b) and (C)as defined above, it is conservatively modified variant or another CDR:

SEQ ID NO 21/CDR/SEQ ID NO 22/CDR/SEQ ID NO 23/CDR/SEQ ID NO 24.

Similarly, one or more preferred CDR light chain antibodies against CD3 rats are present in the framework of the variable domain of a rodent, having given d is more amino acid sequence, read in the leader position to the direction of the constant field, and the CDR is a CDR (d), (e) and (f), as defined above, it is conservatively modified variant or another CDR:

SEQ ID NO 25/CDR/SEQ ID NO 26/CDR/SEQ ID NO 27/CDR/SEQ ID NO 28.

In glycosylamines the antibody containing all three preferred CDR light chain, variable region light chain contains SEQ ID NO 14.

Constant region heavy and light chain can be based on antibodies of various types desired for antibody representing the antibody IgG, but, although they can occur from rodent antibodies, such as rat or mouse, they are preferably derived from human antibodies. As described above, in the case of a light chain constant region is preferably a region of the lambda, and in the case of heavy chains, it preferably represents isotype IgG, particularly IgG1, suitably modified to implement glycosylamine.

In glycosylamines the antibody containing all three preferred CDR variable region of the heavy chain and CH1-hinge-glycosyl-CH2CH3IgG1 human contains SEQ ID NO 20 and is encoded by the DNA of SEQ ID NO 19.

Know that all of the constant region of human IgG isotype are glycosylated by aspartic residue at position 297, which is part of the N-glycosylate the Oia motive asparagine 297-X298-serine299or threonine299where X represents any amino acid residue except Proline. Thus, the antibody of the invention can be applicationaware by replacing the asparagine in such constant region to another amino acid that cannot be glycosylamine. You can potentially use any other amino acid residue, but the most preferred alanine. On the other hand, glycosylation at the asparagine297can be prevented by changing one of the other residues of the motif, for example by replacing the residue 298 on Proline or residue 299 to an amino acid other than serine or threonine. Methods of implementation of such site-directed mutagenesis is well known to specialists in this field of technology and may be implemented, for example, by using a kit for site-directed mutagenesis, such as, for example, the set of commercially available from Amersham. An example of such a procedure is also described further here.

In engineering it is well known that the substitution of one amino acids in the CDR to another with similar properties, for example the replacement of a glutamic acid residue at the residue is aspartic acid, can significantly change the properties or structure of the peptide or protein, in which the replacement or replacement.

Thus, aplikasyon the bathrooms antibodies of the present invention include antibodies, containing the preferred CDR, but with a specific amino acid sequence in which produced such replacement or substitution without changing essentially the affinity of binding and specificity CDR. On the other hand, in the sequence of amino acid residues of the CDR, you can perform deletions or sequence it is possible to increase the N - or C-ends, or both, and at the same time, the activity still persisted.

Preferred glycosylamine antibodies in accordance with the present invention are those in which the constant affinity towards the antigen is 105mol-1or more, for example up to 1012mol-1. Ligands with different affinity may be suitable for different applications, so that, for example, in some cases, it may be appropriate affinity in 106, 107or 108mol-1or more. However, as a rule, are suitable antibodies with affinity in the range of 106-108mol-1. Typically, such antibodies also do not detect any significant affinity binding against other antigens. The binding affinity of antibodies and antibody specificity can be checked using the methods of analysis, such as described in the "Examples" section in EP 0586617 and in the application for U.S. patent, registration No. 08/478684, and in the patent of the USA 5585097, included in this description as references (see, for example, example 5 Analysis of changes in orientation of the cells effector), or by using methods such as ELISA and other immunoassays.

Antibodies of the present invention are glycosylamine IgG antibodies against CD3 configuration in the form of "Y"which can have two identical light and two identical heavy chains, and, thus, are divalent in respect of each antigen-binding site with affinity against D3-antigen. On the other hand, the invention is also applicable to antibodies in which only one of the fragments shoulders antibody has a binding affinity of respect D3-antigen. Such antibodies can take various forms. Thus, another fragment antibodies can be affinity binding against antigen other than CD3, so that the antibody is bespecifically antibody, for example as described in U.S. patent No. 4474893 (incorporated in the present description by reference) and applications to the European patent No. 87907123.1 and 87907124.9. On the other hand, the antibody can have only one fragment, showing the affinity of binding, and this antibody is referred to as "monovalent".

Monovalent antibodies (or antibody fragments) can be obtained in many ways. Glenine and Stevenson (Nature, 295, 712-713 (1982)describe the method according to the teachings of the monovalent antibodies by enzymatic cleavage. Stevenson et al. describe another approach to obtaining monoclonal antibodies, which received the enzymatic fragments, Fab' and Fc bound chemically (Anticancer Drug Design, 3, 219-230 (1989)). In this way received monovalent antibodies lose one of their Fab'-fragments. The third method of obtaining a monovalent antibody is described in European patent No. 131424. In the approach described in this patent, "Y" antibody is retained, but only one of the two Fab'-domains will bind to the antigen. This is achieved through the introduction of hybridoma gene encoding alien light chain, which will be connected to the heavy chain of the antibody with the formation of a mixture of products in which the monovalent antibodies are one of the products of interest.

However, it is preferable monoclonal glycosylamine antibodies against CD3 invention receive the following way. This method includes the introduction of expressing in a suitable system, such as a cellular system, such as is described hereafter together with the genes encoding the heavy and light chain gene encoding a "shortened" heavy chain that lacks a domain variable region and first constant domain of the heavy chain, and a gene loses exon for each of these domains. This leads to the production cell system of the mixture is (a) antibodies which are full of bivalent antibodies, (b) fragments of antibodies, consisting only of two truncated heavy chains (i.e., Fc fragment), and (C) fragments of antibodies, monovalent for D3-antigen consisting of a truncated heavy chain and light chain in combination with a normal heavy chain. This fragment of the antibody (s) is a monovalent, because it only has one Fab'-fragment. Getting monovalent antibodies in the form of such fragments by means of the above method is preferable for several reasons. The so-obtained antibody fragments are easily cleared from the mixture of antibodies produced by a cellular system, as, for example, they can be separated, based simply on their molecular weight. This option is not possible according to the method of European patent No. 131424, where the obtained monoclonal antibodies have characteristics similar to the characteristics of bivalent antibodies in relation to the size and appearance of signs.

In addition, upon receipt of the fragments of the monoclonal antibodies according to the new method uses terms that can be more easily adjusted, and thus the process is not as arbitrary as a method of enzymatic cleavage and chemical crosslinking, in which you want to divide a complex reaction product, and an additional advantage is that the used cleoc the second line will produce fragments of monoclonal antibodies in the absence of the necessary procedures for the continuous synthesis, required in the method of enzymatic cleavage and chemical crosslinking.

It is believed that glycosylamine antibodies according to the invention do not occur in nature and such glycosylamine antibodies can be obtained synthetically in many ways. However, the most convenient appropriate gene construct for constant and variable regions of the heavy and light chains present in the antibody, to obtain separately and then embed it in a suitable expressing system.

Genes encoding the variable domains of the ligand of the desired structure can be obtained and accordingly attach to genes coding for the constant domains of the antibody, subjected to site-directed mutagenesis. Such constant genes can be obtained from hybridoma cDNA or chromosomal DNA to be subjected to site-directed mutagenesis to obtain glycosylamine constant regions. The genes encoding the variable regions can also provide methods of synthesis of genes used in the identification contained in the CDR. Suitable cloning vectors for DNA can be vectors of different types.

Specialists in this field of technology will present that such genes can be obtained in different ways. For example, (i) in a known manner to create a number of hybridomas against D3 antigen, (ii) to obtain DNA from these hybrid with what omashu procedures described in WO 92/06193 and WO 93/19196 and corresponding U.S. patents by mRNA extraction and transformation into cDNA using PCR, (iii) to skanirovat this cDNA oligonucleotide probes that correspond in sequence to the DNA sequence complementary CDR, (iv) to sequence any positively identified hybridoma and (v) to reconstruct the sequence of the rat methods of humanization, as described in the above patents. In order to be able to get some, and preferably all six CDR, you can use site-directed mutagenesis to embed the desired DNA into the appropriate places in the DNA encoding the frame.

The expression of these genes through the cell cultivation system for the production of functional CD3 ligand is most conveniently done by transformation of a suitable prokaryotic system or, in particular, eukaryotic cells, in particular lines, immortalized mammalian cells, such as line of myeloma cells, for example cells of rat myeloma YB2/3.01/Ad (hereinafter called YO), myeloma cells NS20 or ovary cells Chinese hamster (although also of interest is the use of plant cells) expressing vectors containing DNA encoding the different areas of the antibodies with subsequent cultive is the Finance system of transformed cells, producing the desired antibody. Such common methods of application for obtaining ligands of the present invention, are well known in the art and are described in publications such as "Molecular Cloning", Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989 (2nd edition). These methods are also illustrated by the examples in WO 93/19196 and the application for U.S. patent, registration No. 08/478684 included in this description as a reference.

Thus, another aspect of the present invention relates to a method for glycosylating IgG antibodies, corresponding to the first aspect, having a binding affinity of respect D3-antigen comprising culturing cells that can Express the antibody in order to make their expression. The third aspect of the present invention also relates to cell lines, directly expressing glycosylamine antibodies according to the invention.

Among these cell lines are preferred line containing the DNA sequence encoding the preferred CDR described herein previously. The group of nucleotide sequences encoding CDR (a)to(f)described herein previously shown below, respectively, as (a)-(f), but it should be borne in mind that the degeneracy of the genetic code allows changes to be made in these sequences, still deruosi amino acid sequence of CDR.

(a) SEQ ID NO 1; (b) SEQ ID NO 1; (C) SEQ ID NO 5;

(d) SEQ ID NO 7; (e) SEQ ID NO 9; (f) SEQ ID NO 11.

Such cell lines will, in particular, contain a larger DNA sequence comprising (1) DNA expressing variable frame sections of the heavy chain of a person that contains one or more CDRs from among (a), (b) and (C), and (2) DNA expressing variable frame sections of the light chain of rodents, such as rats, containing one or more CDRs from among (d), (e) and (f).

A specific example of such DNA is SEQ ID NO 19, encoding a CDR (a), (b) and (C), localized in the framework of the heavy chain, encoded gene VH26.D.J human VH type III associated with SN-hinge-glycosyl-CH2CH3IgG-man, as described above, and SEQ ID NO 17 encoding a CDR (d), (e) and (f), localized in the framework of the light chain, the encoded chimeric protein constant region of human lambda YTH 12.5.

Chimeric, partially humanized, glycosylamine antibodies, in accordance with the present invention have a therapeutic value, particularly when immunosuppression, in particular to suppress transplant rejection, especially when it is desirable that the immunosuppression was temporary, and not full, and so that T cells are not completely destroyed, but instead turned out to be non-functional due to the blockade by antibody complex antigen-CD3-TCR. In addition,the use glycosylamine antibodies against CD3 may have potential in other areas, such as the treatment of malignant tumors, specifically when designing bespecifically antibodies (to redirect effector cells) or conjugates of the antibody-toxin, where the efficacy of the medicinal product could be complicated Fc-mediated killing of effector cells or non-specific killing of cells containing the receptor-Fc, respectively.

Thus, in its fourth aspect, the present invention relates to a method of treating patients suffering from malignant tumors, in particular lymphoma, or use, for immunosuppression, for example in the case of a threat transplant rejection, including the introduction of a therapeutically effective amount glycosylamine antibodies in accordance with the first aspect of the invention.

Glycosylamine antibodies in accordance with the first aspect of the invention can be included in compositions for administration to patients by injection of these antibodies together with a physiologically acceptable diluent or carrier. Antibodies injected, preferably in the form of compositions for injection together with such diluent or carrier, which is sterile or pyrogen-free. The instructions may be stated that a suitable dose of the antibody is about 1-10 mg daily injections, for example within 10 days, although SLE is a result of the elimination of the primary effect of dose can be if necessary, enter a larger number of antibodies, for example, even at 100 mg per day, depending on the individual needs of the patient. Use in veterinary medicine is based on the same dosage.

Now the invention will be described with reference to the following non-limiting examples, figures and list of sequences. Other implementation of the present invention falling under the scope of the present claims, will be apparent to the person skilled in the art in light of the following sections.

Figures

The Figure 1 shows a diagram FACS analysis of binding of a fully humanized glycosyl-D3 (EP 0586617, application for U.S. patent, registration No. 08/478684 and U.S. patent 558097) and chimeric antibodies of the present invention, which uses a lightweight flexible frame section antibodies rats. One light chain YTH 12.5 LAG1 not show a normal link, so as not associated with a heavy chain.

The Figure 2 presents the diagram FACS analysis of binding of two chimeric transfectants obtained using vector pOXD52neo, which thus Express D52-antigen on their surface. The charts illustrate the use of vector pOXD52neo as a way to check whether the clonal transfectants population. TF 12.5L/CD3A..27 has one peak at colouring CD52, showing that all cells cont ciruit antibodies against CD3, while TF 12.5L/CD3A.34 has two peaks, indicating unproductive population of cells producing antibodies against CD3.

The Figure 3 presents graphs of the OD 492 from the dilution ELISA to compare the production of human IgG as measures of output antibodies in the case of chimeric CD3 present invention and gumanitarnogo HELICOIL-CD3 prior art. Supernatant CD3 tested three weeks after transfection, show that cells containing DNA encoding chimeric antibody of the present invention produce about 120 μg/ml of antibody, a fully humanized CD3 produce less than 10 μg/ml.

In Figures 4 and 5 show diagrams of binding of chimeric antibodies of the present invention and fully humanized CD3 with Jurkat cells to assess the affinity of antibodies. Start with a known concentration of 100 µg/ml, and then diluted to 1/20, and seven of titrate to 1/2560. Painting staining show that the affinity is the same.

The list of sequences

SEQ ID NO 1 is a DNA sequence that encodes a CDR (a).

SEQ ID NO 2 represents the amino acid sequence of CDR (a).

SEQ ID NO 3 is the DNA sequence that encodes a CDR (b).

SEQ ID NO 4 is the amino acid sequence of CDR (b).

SEQ ID NO 5 is the FDS is the first DNA sequence, coding CDR (s).

SEQ ID NO 6 is the amino acid sequence of the CDR (s).

SEQ ID NO 7 is the DNA sequence that encodes a CDR (d).

SEQ ID NO 8 represents the amino acid sequence of CDR (d).

SEQ ID NO 9 is a DNA sequence that encodes a CDR (e).

SEQ ID NO 10 is an amino acid sequence of CDR (e).

SEQ ID NO 11 is a DNA sequence that encodes a CDR (f).

SEQ ID NO 12 is an amino acid sequence of CDR (f).

SEQ ID NO 13 is a DNA sequence that encodes a variable region light chain of the rat.

SEQ ID NO 14 is an amino acid sequence of the variable region of the light chain of the rat.

SEQ ID NO 15 is a DNA sequence that encodes a variable region of the heavy chain of the rat, containing the corresponding CDR.

SEQ ID NO 16 represents the amino acid sequence of the variable region of the heavy chain of the rat, containing the corresponding CDR.

SEQ ID NO 17 is a DNA sequence that encodes a variable region light chain of the rat with the corresponding CDR and a constant region of a lambda man.

SEQ ID NO 18 is an amino acid sequence of the variable region of the light chain of the rat with the corresponding the CDR and a constant region of a lambda man.

SEQ ID NO 19 is a DNA sequence that encodes a variable region of the heavy chain CDR and SN-hinge-glycosyl-CH2CH3person.

SEQ ID NO 20 is an amino acid sequence of the variable region of the heavy chain CDR and SN-hinge-glycosyl-CH2CH3person.

SEQ ID NO 21-24 represent the amino acid sequence of the framework variable domain of the heavy chain of a person without CDR.

SEQ ID NO 25-28 represent the amino acid sequence of the framework variable domain of the light chain of the rat without CDR.

SEQ ID NO 29 and 30 represent the primers used for cloning the variable region of the light chain of the CD3 rats in YARD 12.

General methodology

General methods for obtaining monoclonal antibodies against CD3 with humanized heavy chain

Cloning and reverse engineering gene V-region antibody rat YTH 12.5, specific CD3 antigen of human conduct as described Routledge et al., 1991, Eur.J.Immunol., 21, 2717 and in the application for patent in the United Kingdom No. 9121126.8 and its equivalents. YTH 12.5 is a hybridoma cell line secreting monoclonal IgG2b antibodies specific against the antigen CD3 complex, but the methodology is applicable to other cells secreting antibodies against CD3 with the same CDR (see the end the relevant description).

Briefly, the methodology is based on the methodology Oriandi et al., 1989, P.N.A.S. USA, 86, 3833, using polymerase chain reaction (PCR). Gene VH(gene variable regions of the heavy chain) clone using oligonucleotide primers VH1FOR and VH1BACK (see above patents included in this description as references). The PCR products alloyed into the vector M13-VHPCR1, where they perform site-directed mutagenesis using 6 oligonucleotide primers. Gene VL(gene variable light chain) clone using primers designed on the basis of published sequences VLλ. Gene clone into the vector M13-VKPCR together with the constant region of the light chain lambda man. In this vector mutagenesis frame vlcarried out using the oligonucleotides 5. Then humanitarianly gene vlembed in expressroute vector pHβ Arg-1.

Get the vector R, in which the reconstructed gene VH CD3 can be expressed in combination with various genes constant region of H chain of the immunoglobulin, and this vector is based vector pHβ Apr-qpt (Gunning et al., 1987, P.N.A.S. USA, 85, 7719-7723). The DNA fragment 1.65 TPN containing gene digidrofolatreduktazy (dhft) and signals the expression of SV 40 (Page & Sydenham, 1991, Biotechnology, 9, 64)is inserted into the unique EcoRI site pHa Apr-gpt. Then the DNA fragment HindIII-Amnv 700 BP, encoding the reconstructed gene CD3-VH, clone in the multiple cloning site of the vector, after and under control β -actin promoter. Then the desired gene constant region of H chain (genomic configuration) can be embedded in a unique BamH1 site of restrictase after gene CD3-VH.

Glycosylamine constant region of human IgG1 derived from gene G1m (1, 17) wild type described by Takahashi et al. (1982, Cell 29, 671-679), as follows. Gene clone in the vector M13 tgl31, where they perform site-directed mutagenesis (set Amersham International PLC), and mutate amino acid residue at position 297 with aspartic residue at the residue is alanine.

Oligosaccharide at Asn-297 is a characteristic of all normal human IgG antibodies (Kabat et al., 1987, Sequence of Proteins of Immunological Interest, publication of the U.S. Department of health), and each of the two heavy chains of the IgG molecules contains one carbohydrate group branched chain, which connects with the amide group of the aspartic residue (Rademacher and Dwek, 1984, Prog. Immunol., 5, 95-112). Replacement of asparagine to alanine prevents the glycosylation of antibodies.

Alcovision constant region of IgG 2.3 TPN cut out from M13 by double decomposition with the use of BamHI and BgIII and are ligated into the BamHI site of the vector R and get the clone R.

Subconfluent monolayers ovary cells Chinese x is Macca dhfr -nontransferred vector R, containing the gene for the heavy chain and the second vector R containing the reconstructed light chain λ person (Routlege et al., 1991, Eur. J. Iminunol., 21, 2717-2725). Before transfection of both plasmids linearized using Pvul restriction endonuclease. Transfection is performed with the use of reagent DOTMA (Boehringer, Germany), following the manufacturer's recommendations.

The transfectants heavy and light chain are selected in free from xanthine and gipoksantina environment IDMD, containing 5% (V/o) cialisbuynow fetal calf serum.

Also described getting a similar vector of the heavy chain IgG1-D3 human wild-type R (Routlege et al., 1991, Eur.J.Immunol., 21, 2717-2725, and GB9121126.8 included in this description as references). Vectors for the expression of H-chain, containing the genes for the constant region neutropenia human IgG2 (Flanagan & Rabbitts, 1982, Nature, 300, 709-713), IgG3 (Huck et al., Nuc.Acid.Re5., 14, 1779-1789), IgG4 (Flanagan & Rabbitts, 1982, Nature, 300, 709-713), Epsilon (Flanagan & Rabbitts, 1982, EMBO Journal 1, 655-660) and alpha 2 (Flanagan & Rabbitts, 1982, Nature, 300, 709-713) (vectors R, R, R, R and R, respectively), obtained from vector R. The introduction of these vectors in combination with the vector light chain R in Cho-dhfr cells-as previously described , provides cell lines secreting antibodies against CD3 isotype γ 1, γ 2, γ 3, γ 4, ε and α 2, respectively. Cells expressing EN is the body against CD3, subjected to two cycles of cloning in soft agar and then multiply as roller culture. The immunoglobulin of approximately 4 l of tissue culture supernatant from each cell line are concentrated by ammonium sulfate precipitation, extensive cialiswhat against PBS and then determine quantitatively as follows.

Because antibodies are not in pure form, use competitive analysis, developed specifically to determine the concentration of the antibodies with binding ability against D3-antigen. T-lymphoblasts person incubated with UCHT-1, labeled with fluorescein (FITZ)-antibodies that bind the same epitope D3-antigen, and chimeric samples. The concentration used FITZ set in advance to polonesian. Unlabeled YTH 12.5 (purified by HPLC) titrated from a known initial concentration and added to wells containing T cells and UCHT-1-FITZ. Unlabeled antibodies serve as a competitor for the antigen-binding centre. It is defined as the decrease in the average intensity of the visible fluorescence when cells are examined using FACS analysis. Accordingly, the titration of chimeric antibodies with unknown initial concentration gives the number of sigmoidal curves when plotting the dependence of the average fluorescence intensity divorced from what I antibodies. These curves can be compared directly with a standard curve for YTH 12.5 and you can use equivalent antibodies.

EXAMPLE.

Getting glycosylamine antibodies specific against D3-antigen person containing the CDRs of the corresponding CDR sequence of the antibody rat YTH 12.5, variable frame with the heavy chain of the person associated with the constant region of IgG1 and the variable framework the light chain of the rat associated with the constant region of the human lambda.

In case of loss of the light chain of the CD3 chose insufficient chain YTH 12.5LAG1 expressing only the heavy chain of the CD3 rats, and used for transfection in a fully humanized glycosylamines heavy chain of CD3. The DNA fragment BamH1-Hind111 1.4 TNP encoding the design of the humanized heavy chain applicationvantage CD3 IgG1, cloned in the multiple cloning site of two different expressing vectors pHβ Apr-1 gpt (Gunning et al., (1987), P.N.A.S USA, 84, 4831, and 85, 7719-7723) and pOXCD52neo (Frewin, unpublished data)containing various breeding markers.

Expressing the vector pOXCD52neo obtained using a strong promoter "factor elongation of polypeptide chain 1" (EF1)producing antibodies in large quantities (see Shigekazu Nagata NAR, Vol. 18, No. 17, page 5322). Paste it into the design together with neomitsinovoy selectivity marcero is. The vector also includes cDNA for expressed on the surface of antigen CD52 Campath-driven TC-promoter (all of these promoters and markers described and available). Expression of CD52 on the surface of cells allows the identification of transformants using CD52 antibodies.

Then separately have transfusional YTH 12.5LAG1 two plasmids by electroporation and heavy transfectants selected with IMDM containing 5% fetal calf serum, MPA and xanthine in the case of pHβ Apr-Igpt, and IMDM containing 5% fetal calf serum and 1 mg/ml G418 in the case pOXD52neo, were cultured for two weeks until I grew up living colonies for testing. Both transfectant gave positive clones by screening for the production of human IgG1 using ELISA. Functional antibodies against CD3 was tested for binding to the line Jurkat T-cells (ATSC TIB 152 (J.Immunol., 133, 123-128 (1984)) and analyzed by the method of FACS (Becton Dickinson), both analyses showed the output of the antibody in an amount of from 30 to 50 mcg/ml

Vector pOXD52neo allows control over transfitsirovannykh cells producing antibodies using marker of cell surface CD52. Only cells containing the marker, secrete antibodies, so using antibodies to CD52 Campath-related FITZ-transfitsirovannykh cells, FACS method, you can implement an is Liz on the percentage of cells producing antibodies, and can confirm the clonal status. Negative producing cells was not detected, and the output of the antibodies remained at the level of 50 µg/ml in normal cell growth.

Chimeric form glycosylamine antibodies against CD3 were obtained using PCR with the accession of the variable region of the light chain of the CD3 rats to constant region of human lambda using primers introducing sites restricts Hind III and EcoR1 cloning in expressing vector REE, Celltech (see Bebbington et al., (1992), Biotechnology, 10, 169). Sequences of primers SEQ ID NO 29 and 30 are also listed in the attached list of sequences.

The final design sequenced and cloned in REE already containing humanitarian glycosylamine CD3 heavy chain, and transfusional in the myeloid cell line N30 (ECACC No 85110503-Galfre and Milstwin (1981), Enzymology, 73(B), 3-46) by electroporation. The resulting clones were skanirovali on the production of antibodies using ELISA on human IgGI and light chain lambda person and by FACS for binding with cell line Jurkat clone of T-cells. When ELISA using goat antibody against human IgFc (Sigma 12136) as the immobilized antibody and biotinylated goat antibody against human IgG (Amersham RPN 1003) or biotinylated goat antibody against human the light chain lambda (Amersham RPN 1188) as identifying antibodies (see Routledge et al., Eur.J.Immunol., (1991), 21:2717-2725).

After a single transfection of 16 clones expressed from 60 μg/ml to 100 μg/ml, significantly more than any other transfection reconstructed glycosyl-D3. These transfectants were then cloned by serial dilution method and some of them have improved the expression of up to 120 µg/ml of This remained stable during long-term cultivation and large scale production of antibodies did not cause problems with cell growth.

Figures 1-4 illustrate the ability of these antibodies to bind CD3 with the same capacity as described previously fully humanized glycosylamine antibodies against CD3 prior art.

1. The IgG antibody with a binding affinity of against antigenic CD3 complex, where the heavy chain contains the frame variable regions together at least one CDR chosen among the amino acid sequences of SEQ ID NO: 2, 4 and 6 and their respective conservatively modified variants, and light chain contains the frame variable regions together at least one CDR chosen among the amino acid sequences of SEQ ID NO: 8, 10 and 12 and their respective conservatively modified variants, wherein the sequence of frame variable regions of the heavy chain corresponds to the sequence, typical of the Sabbath.Oh for a man, and the carcass of the variable region of the light chain contains one or more amino acids, characteristic sequence typical of rodent.

2. The IgG antibody according to claim 1, characterized in that the variable region of the light chain contains a sufficient number of amino acids characteristic of the sequence, typical of rodent, for light and heavy chains were associated more strongly than in the case when variable region light chain is fully consistent with the area, typical of the man.

3. The IgG antibody according to claim 1 or 2, characterized in that the variable region of the light chain contains a sufficient number of amino acids characteristic of the sequence, typical of the rodent order, when DNA encoding the antibody is expressed in cells of RAY 12 receive the output of antibody excess of 50 mg/ml

4. The antibody according to any one of the preceding paragraphs, characterized in that the variable region light chain fully corresponds to the sequence of the rat.

5. The antibody according to any one of the preceding paragraphs, characterized in that the variable region light chain corresponds to SEQ ID NO: 14 or a sequence modified so that one or more, but not all, amino acids from a number Gln-1, Ala-2, Val-3, Val-4, Ala-7, Asn-8, Thr-12, Leu-14, Ser-16, Lys-19, Leu-20, Leu-39, Tyr-40, Glu-41, Ser-44, Met-48, Tyr-50, Phe-75, His-79, Asn-80, Val-81, Aa-82, Ile-83, Ile-88 and Phe-90 replaced by the corresponding amino acids of a human, selected from a number of Asp-1, Phe-2, Met-3, Leu-4, Pro-7, His-8, Glu-12, Pro-14, Lys-15, Ile-19, LEU-20, Gln-39, Arg-40, Pro-41, Ala-44, Val-48, Phe-50, Ser-75, Ser-79, Gly-80, Leu-81, Gln-82, Thr-83, Asp-88 and Tyr-90.

6. The antibody according to any one of the preceding paragraphs, characterized in that each of the heavy chain and/or light chain contains all three of their respective CDR of SEQ ID NO: 2, 4, and 6 and SEQ ID NO: 8, 10 and 12.

7. The antibody according to any one of the preceding paragraphs, wherein the CDRs in the heavy chain are arranged in sequence in the following order: frame plot of human antibodies 1 /SEQ ID NO: 2/ wireframe plot of human antibodies 2 /SEQ ID NO: 4/ wireframe plot antibodies man 3 /SEQ ID NO: 6/ wireframe plot of human antibodies 4 in the top position to the direction of the constant domain from N-Terminus to the C-end, and the light chain sequence of frame section antibodies rodent 1 /SEQ ID NO: 8/ wireframe plot antibodies rodent 2 /SEQ ID NO: 10/ wireframe plot of rodent antibodies 3 /SEQ ID NO: 12/ wireframe plot of rodent antibodies 4 in the top position to the direction of the constant domain.

8. The antibody according to any one of the preceding paragraphs, characterized in that the frame parts of the human antibodies contain the amino acid sequence of SEQ ID NO: 21, 22, 23 and/or 24.

9. The antibody according to any one of the preceding paragraphs, characterized in that one or a few who preferred to CDR heavy chain antibodies against CD3 rats are present in the framework variable domain of the human having an amino acid sequence that is read in the direction from the top to the constant region containing SEQ ID NO: 21/ CDR / SEQ ID NO: 22 / CDR / SEQ ID NO: 23 / CDR / SEQ ID NO: 24.

10. The antibody according to any one of the preceding paragraphs, characterized in that the constant region of the antibody is typical for a person.

11. The antibody of claim 10, wherein the constant region of the antibody corresponds to a constant region derived from human.

12. The antibody according to any one of the preceding paragraphs, characterized in that the constant region of the antibody contains a constant region of the lambda, the typical human.

13. The antibody according to any one of the preceding paragraphs, characterized in that the variable region of the light chain of the rodent is linked to a constant region of lambda, the typical human.

14. The antibody according to any one of the preceding paragraphs, characterized in that the frame contains plot variable domain of the light chain of the rat with SEQ ID NO: 14.

15. The antibody according to any one of the preceding paragraphs, characterized in that it contains a chimeric light chain of human/rat and amino acid sequence of SEQ ID NO: 18 constant region of the lambda.

16. The antibody according to any one of the preceding paragraphs, characterized in that the constant region of the heavy chain belongs to the IgG1 isotype.

17. The antibody according to any one of the preceding points is, wherein the aspartic residue at position 297 of the heavy chain constant region is replaced by another amino acid residue.

18. The antibody according to 17, wherein the aspartic residue is replaced by an alanine residue.

19. The antibody according to any one of the preceding paragraphs, characterized in that only one of the shoulders has an affinity against CD3 antigen.

20. The antibody according to claim 19, characterized in that is monovalent.

21. The antibody according to claim 20, characterized in that one half of the antibody consists of the complete heavy chain and light chain, and the other half consists of a similar, but shortened heavy chain, no longer binding site for the light chain.

22. The antibody according to any one of the preceding paragraphs, characterized in that it is in the form of a pharmaceutical composition comprising a physiologically acceptable diluent or carrier.

23. The antibody according to any one of the preceding paragraphs, characterized in that applicable in therapy.

24. The antibody according to any one of the preceding paragraphs, characterized in that applicable for the manufacture of a medicinal product for use in immunosuppression or treatment of malignant tumors.

25. The antibody according to any one of the preceding paragraphs, characterized in that the antibody glycosylamine.

26. Recombinant nucleic acid coding for the common light chain antibodies according to claim 1, containing the nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO: 18.

27. Recombinant nucleic acid encoding a heavy chain of the antibody according to claim 1, containing a nucleotide sequence corresponding to the amino acid sequence of SEQ ID NO: 20.

28. The method of obtaining system, expressing the light and/or heavy chains of the antibody according to claim 1, involving the transformation of a host cell a recombinant nucleic acid according p and/or 27.

29. The method according to p, wherein the transforming is performed by the vector containing the recombinant nucleic acid according p or 27.

30. The method according to p or 29, characterized in that the transform is performed with the vector containing a recombinant nucleic acid encoding a heavy chain and a vector containing a recombinant nucleic acid encoding a light chain.

31. The method according to item 30, wherein the specified design code chain constant regions.

32. A method of producing an antibody according to any one of claims 1 to 25, providing for the cultivation of prokaryotic or eukaryotic cells, cotransformation a vector containing a recombinant nucleic acid according p, and a vector containing a recombinant nucleic acid according to item 27.

33. The method of treatment of a patient, with raduege malignant tumor or in need of immunosuppression, introducing the indicated patient a therapeutically effective amount of the antibody according to any one of claims 1 to 25.



 

Same patents:

FIELD: biotechnology, microbiology, agriculture.

SUBSTANCE: the strain Lactobacillus plantarum 578/25 is obtained by method of step-by-step selection and selected by its ability to produce significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.09.-DEP. Invention provides eliminating the pollution of environment in producing the protein fodder, to elevate the protein specific yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process of its preparing, to simplify apparatus equipment, to utilize waste in manufacturing using the natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: biotechnology, agriculture, microbiology.

SUBSTANCE: invention relates to a new isolated strain of Lactobacillus acidophilus 1660/08 as a producer of the protein fodder. The strain Lactobacillus acidophilus 1660/08 is obtained by the selection method and selected by its ability to form significant amount of crude protein and to accumulate the biomass. The strain is deposited in the VGNKI collection at number VGNKI-03.04.10.-DEP. Invention provides eliminating the environment pollution in producing the protein fodder, to enhance the specific protein yield, to reduce energy consumptions in preparing protein fodder, to simplify and to accelerate the process in its preparing, to simplify equipment fitting out and to utilize waste in manufactures using natural raw.

EFFECT: valuable properties of strain.

2 tbl, 10 ex

FIELD: fodder industry.

SUBSTANCE: invention relates to a method for preparing protein-vitamin fodder that involves solid or liquid waste in production and processing the natural raw (grains, milling waste, post-alcoholic distillery grains, beer pellets, fruit pulps or whey). Enzyme lysates are prepared from solid waste and starch waste. Cobalt salt is added to liquid waste or enzyme lysates. Prepared nutrient medium is used in incubation of lactobacillus and propionibacillus microorganisms taken by the following pairs: Lactobacillus acidophilus 1660/02 with Propionibacterium freudenreichii subsp. shermanii 103/12; or Lactobacillus acidophilus 1660/02 with Propionibacterium acnes 1450/28; or Lactobacillus plantarum 578/25 with Propionibacterium freudenreichii subsp. shermanii 103/12; or Lactobacillus plantarum 578/25 with Propionibacterium acnes 1450/28. This method provides preparing fodder enriched with vitamins and proteins and containing live cells of lactobacillus and propionibacillus microorganisms. Method enriches animal intestine microflora after feeding the prepared fodder to animals. Fodder comprises protective substances (organic acids, enzyme systems) and can be stored as crude form for the prolonged time.

EFFECT: improved preparing method, valuable properties of fodder.

13 cl, 1 tbl, 12 ex

The invention relates to the field of biotechnology, specifically to obtain protein/factor inhibiting osteoclastogenesis (OCIF), and can be used for treatment and immunological diagnosis of diseases involving bone resorption

The invention relates to biotechnology and can be used for separation of proteins with desired properties from large pools and nucleic acids (NC)

FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

The invention relates to biotechnology and can be used to obtain Malinov human IL-4 with an activating T-cell activity and reduced activating endothelial cells activity

The invention relates to biotechnology, in particular to the creation of transgenic plants with insecticidal properties

The invention relates to the field of genetic engineering and can be used in the biomedical industry

The invention relates to the field of genetic engineering and can be used in the biomedical industry for preparation of medicinal products for gene therapy

The invention relates to the medical industry and refers to polypeptides that are specific against CD19 and CD3, and their application

The invention relates to biotechnology and can be used to create a functioning tyrosine-specific chimeras proteinkinase
Up!