Single-stranded cyclic tri-specific antibody

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology and immunology. Proposed here is a polynucleotide, encoding a cyclic single-stranded tri-specific antibody. The antibody is directed against human ovarian carcinoma in vitro, has mass of approximately 84 kD and consists of three components: an antibody against human ovarian carcinoma cells, anti-CD3 antibody and anti-CD28 antibody, which are joined together by peptide interlinks such that, they form a cyclic antibody. Invented is an expression vector, containing a coding polynucleotide and versions of E.coli host cell based on the polynucleotide and expression vector.

EFFECT: use of the invention provides for a stable antibody molecule, optimum for activation of T-cells, which can be used in curing human ovarian carcinoma.

8 cl, 12 dwg

 

Background of the present invention

1. scope of the present invention

The present invention relates obtained by genetic engineering circular single-stranded trapezitinae antibodies coded its DNA sequences, expression vectors containing these sequences, as well as cells of the host containing the indicated expression vectors.

2. Description related technologies

The strategy of constructing a circular single-stranded trapezitinae antibodies based on the introduction into the same molecule three different antigenspecific sites. Since the genes of antibodies selected to design antibodies, different circular single-stranded trapezitinae antibody has different biological functions. Known trapezitinae antibodies designed mainly by means of chemical bonding, the method interspecific hybridoma or genetic expression at the confluence, and antibodies contained three Fab fragment or a single chain antibody (scFv) of the three antibodies (Fay T N et al., 1988; Tutt A, et al., 1991; Jung G, et al., 1991; Schott M E et al., 1993; French R R, 1998; Somasundaram With et al., 1999; Schoonjans R, et al., 2000a; Schoonjans R, et al., 2000b; Wong W M et al., 2000).

Referred to in the present invention a circular single-stranded trapezitinae antibody represents a new type of antibody obtained is about genetic engineering, it is designed based on the model of two-signal activation of T-cells in immunotherapy of cancer a key factor. In immunotherapy of tumors cellular immunity mediated by T-cells, plays an important role. For the full activation of T cells requires two signals: the primary signal is generated by a complex of TCR/CD3-related antigen specificity; the second signal, also called co-stimulatory signal, creates a co-stimulatory molecules on the surface of APC. CD3 person contains 5 different polypeptide chains. CD3 and TCR constitute the CD3/TCR complex with non-covalent bonds. If T cells receive only the primary signal (TCR-binding), co-stimulatory signal is antigen-non-specific and has no restrictions from the point of view of the major histocompatibility complex, however, he is involved in the indexing of cytokine secretion, proliferation and influence the work of T cells. CD28 is the most important receptor co-stimulatory signal on the surface of T cells. From various receptors, co-stimulatory signal to T-cells (a type of CD2, CD4, CD8 etc) only CD28 able to prevent the induction of immunological tolerance T cells (Slavik et al., 1999). On the basis of these principles it is possible to activate T-cells using anti-CD3 antibody and anti-CD8 antibodies as l is gandow these molecules, respectively.

The development of gene technology, in particular the use of gene technology to modify antibodies, promotes the rebuilding of the antibodies in accordance with the requirements of a particular application. In recent years we have received some bispecific antibodies and bispecific single-chain antibodies having various properties of the target cells that can recognize cancer cells and stimulate immune cells-effectors.

Most reports on the use of antibodies obtained by genetic engineering, in immunotherapy of tumors concerned bispecific antibodies (BsAb) or monoclonal antibodies specific for tumor-specific antigen (TAA) and CD3 or CD28. In early clinical trials for immunotherapy of tumors BsAb was produced by the concatenation of two antibodies against the trigger molecule TCR-CD3 and TAA. However, the effect of therapy has been disappointing, because it could lead to immunological tolerance clone of activated T-cells, and apoptosis. This problem can be circumvented by using ex vivo activation of T cells through the use of co-stimulatory molecules IL-2 or lectin. With more in-depth study of molecules CD28 and the development of theory of dual stimulating signal was found that monoclonal anti-SF antibody, as V7-family able for avati co-stimulatory signal and to unite with anti-CD3/TAA, launching into action optimal activation of T cells.

In the work Demanet et al (1996) have shown that in the model in mice (Balb/C) lymphoma, loaded with 10.sup.5 cells BCL1 disappeared due to repeated injections BsAb anti-CD3/anti-Id with monoclonal anti-CD28 antibody. Compared with a single application of BsAb, therapeutic effect was higher in 20 times, while the dose of BsAb was only 10% of a single dose of BsAb. Using the model in mice (SCID) In chronic limfocitna leukemia person Bohlen et al (1997) showed that the combined injection of anti-CD3/anti-CD19 BsAb and anti-CD28 McAb can mediate autologous T cells to suppress the growth of tumor cells and prevent recurrence of the tumor process. In further studies of anti-CD3/anti-TAA BsAb and anti-CD28/anti-TAA BsAb used together in order to clarify the specificity to tumor cells. For example, in Renner et al (1994) reported that the combined injection of anti-CD3/anti-CD30 BsAb and anti-CD28/anti-CD30 BsAb made SCID mice suffering from disease Hodgkin had a lethal effect. Mazzoni et al (1996) was coadministered with anti-CD3/anti-FBP (carcinoma of the ovary TAA) and anti-CD28/anti-FBP BsAb in the analysis of cytolysis in vitro, the results showed that the double signals can effectively be activated CD8.sup.+T-cells, destroying cell carcinoma of the ovary with antigen FBP. Comparing the results, obtained using only anti-CD3/anti-CD19 BsAb, Manzke et al (1999) received a promising effect in the simultaneous application of anti-CD3/anti-CD19 BsAb and bivalent anti-SU antibodies for the treatment mediated by b-cell lymphocytes of cancer. Received also a significant effect of the treatment of solid tumors using BsAb. In mice with transplanted with B16 melanoma and lung cancer in the work of Grosse-Hovest et al (1999) have shown that anti-CD3/anti BsAb has significant curative effect, but the impact on the speed of destruction of cancer cells can be significantly enhanced by simultaneous injection of anti-S BsAb and anti-CD3/anti BsAb. In addition, when you attack the cancer cells, the number of surviving for a long time individuals was significantly higher in mice treated with intravenous injection BsAb, which proves the fact that the BsAb is able to induce long-term protective immunity.

Thus, the antibody, the resulting joint coupling of genes of anti-TAA antibodies, anti-CD3 antibody and anti-CD28 antibodies, and their expression on the technology of genetic engineering, should activate effector cells and more effectively treat the tumor. At the same time all methods of production must become more simple and effective, and less expensive. However, if these three EN is the body will be concatenated only one after the other, forming a linear molecule, the antibody will be unstable and is difficult to transport in vivo. In order to solve this problem in the present invention, a method of constructing a circular single-stranded trapezitinae antibodies (TsAb) by cyclization of the linear molecule antibody fragments antibody hinge region.

In addition, there are many problems concerning the use of mAb mice in clinical therapy, and these problems require solutions. One of the main tasks is the HAMA (human anti-mouse antibody) response of human antibodies to the antigen of the mouse caused to the patient due to the immunogenicity of mAb mouse. This response creates a rapid clearance of mouse antibodies from the blood, neutralizes and inhibits the activity of antibodies, it is cause an allergic reaction patient. Since creating a monoclonal antibody human technology hybridoma very difficult, for the full application rodent monoclonal antibodies using methods of easing antibodies by gene technology and protein engineering required selective method.

The mitigation strategies of antibody based on the information recognizability structure and a clear domain antibodies. Form antibodies resembles a capital letter "Y" and contains two identical heavy chains and two identical light chains. Each circuit has one of the variations is entrusted domain, and one or more conservative domains. The variable domains are primarily responsible for binding to the antigen, while conservative domains responsible for the binding of effector molecules. The variable domains contain three areas of flexible loops, sequence and crystal structure hypervariability, these areas are directly linked with antigens and are called hypervariable parts of the molecule antibodies (CDRs). The rest of the field variable domains have less variability, they consist of antiparallel β sheets and are called frame sections (FRs). The sequence of the CDRs and FRs are arranged one behind the other, forming a sandwich structure. This structure is linked by a disulfide bond between the heavy and light chains or between two heavy chains. Conservatism antibody structures makes possible changes in methods of gene and protein engineering while maintaining antigennegative specificity and effector functions; while at the same time is reduced as much as possible, its immunogenicity for use in clinical therapy.

The first generation of weak antibody is a hybrid antibody human mouse containing the variable areas of the rodent and constant parts of the person. It was shown that the hybrid antibody method is about to significantly increase the pharmacodynamic factor and to reduce the immunogenicity; some hybrid antibodies have been used in clinical trials. However, the results of these experiments showed that more than half of the patients treated with hybrid antibody response NAMA was developed only after repeated injections. The second generation of weak antibodies called antibody grafted on the hypervariable segment ("CDR-grafted" antibody), in this case hypervariable areas of rodent antibodies were transplanted in the skeletal parts of the human antibodies. Compared with the hybrid antibody this antibody is further weakened, this is done to ensure that the specified antibody was largely identical to a human, but it would antigennegative specificity of the antibodies of the rodent. In principle, the same frame parts of the human antibodies can be transplanted different hypervariable areas of rodent antibodies with varying transformed antibodies that contain different sequence. However, this super simple transplant usually produces antibodies with weak activity (or non-activity), because the additional transitions of single amino acid residues within the same framework can influence the conformation antigennegative site. Therefore, there is postanaesthetic to take into account possible interactions with amino acid residues of FRs and CDRs. It is necessary to preserve the inherent rodents amino acid residues, which serve to hold the CDRs in the appropriate spatial antigenspecific designs. For example, if a human antibody directed to the frame antigenic molecules, transplantation of the patient's only the CDRs of rodent antibodies against the surface antigen of human lymphocytes, the resulting transformed antibody has unacceptable affinity. In the application of computer models to VH-region CDRs and FRs, it was found that in the rodent antibody Phe27 on FR1 is in direct contact with CDR1, but with Ser27 human antibodies. When the specified residue serine at position 27 was substituted with phenylalanine, transformed thus the antibody retained the original binding activity of the antibodies of the rodent. In fact, after several mutations separate FR residues affinity of some transformed antibody can be increased more than 3 times. The first transformed antibody SAMRAT-1H been successfully used in clinical therapy of lymphoma (not representing lymphoma Hodgkin) and rheumatoid arthritis. Similar results were also obtained from HuRSV-19 and D1.3VHFNS/VK.

The overall strategy for the substitution of the amino acid residues includes: choice of acceptor most homologous follower of the spine of the human FRs, based on the known crystal structure of the variable regions and the amino acid sequence of their respective families; build molecular models using the computer; and then determining which amino acid residues needs replacement. However, it should be noted that the selection of the main FR residues may increase the heterogeneity of the antibodies, as well as his affinity. Therefore, when designing suitable therapeutic antibodies against you repeatedly to optimize the balance between affinity and immunogenicity. In the present invention we have designed a cyclic trapezitinae antibody transformed with anti-CD3 scFv and transformed single domain anti-CD28 antibody; they were designed in our own library. For further use in clinical therapy, it would be useful to reduce the immunogenicity of the whole molecule as a whole.

When designing cyclic trapezitinae antibodies is very important interlinker, because he must determine whether a particular design is so successful as was hoped. In our invention as interlinker we chose a part of the Fc fragment of human IgG, part of the serum albumin of human rights and the hinge region of the immunoglobulin IgG3' CL. Between these and what terrenkurami and fragments of antibodies was used flexible short leptin (Gly 4Ser), which is designed to facilitate accurate retention of various fragments of antibodies.

Interlinker Fc: Failure to activate a cascade of effector due to the lack of Fc is the main disadvantage of small molecules antibodies. It was proved that the four subclasses of human IgG is most effective to mediate ADCC CDC capable of IgGl. Some C-terminal residues IgGl CH2 can connect with Clq, starting classical chromosomal way exchange. Of these residues remains Glu318, Lys320 and Lys322 are spatially confined and they are located on the surface of the Fc to be directly connected with Clq. Some studies have also shown that the Fc glycosylation at Asn297 very important for ADCC and CDC, this was not observed any effect on antigennegative activity of the antibodies. Therefore, when designing trapezitinae antibodies of the present invention as interlinker was selected IgGl CH2 person, containing 26 amino acid fragments starting with Asn 297 to Lys322 (including glycosylation site and the site Clq binding).

Interlinker HSA: Another problem of using small molecules antibodies in clinical practice is their short half-life and rapid elimination from the blood, which is a significant defect for immunotherapy, but is with the combat advantage for immunodiagnostics and neutralize toxins. Serum albumin human is the most important protein and it is widely distributed in the human body. Not having enzymatic activity, immune activity and side effects, HSA is excreted from the liver slowly and is in a state in vivo for several weeks. It was shown that the stability of the protein, coupled with HSA, can be increased by 20-40 times, and protein, in order to cleanse, mainly absorbed by the liver, which significantly reduced the toxicity of the kidneys. The HSA molecule contains 3 domain, which includes a 585 amino acid residues and 17 disulfide bonds. Was. confirmed that the domain III can execute the function of the intact HSA protein. So as interlinker of the present invention was selected fragment of the 25 amino acid residues (with 403 at 425) from domain III.

The hinge region of human immunoglobulin IgG3' CL: Cysteine of the hinge region capable of forming a disulfide bond, which facilitates the connection of the two heavy chains in the formation of antibodies with natural spatial structure. The hinge region of human immunoglobulin IgG3' consists of 17 amino acid residues, including two cysteine, and this makes it suitable functions interlinker. In the present invention a fragment of 17 amino acid residues of the hinge region of the immunoglobulin man IgG3' CL is used for cyclization trapezitinae antibodies.

Literature: [0018] [Reterences: I. Huang H L. Gene engineering antibody. Monoclonal Antibody Communication, 1991, 7(3): 1-4; 2. Liu X F, Huang HL. Progress in gene engineering antibody. Biotechnol Progress, 1994, 14(1): 54; 3) Huang H L. Humanized antibody small molecule antibody and tumor therapy. Monoclonal Antibody Communication, 1993, 9(3): 19; 4. Slavik, M. J, Hutchcroft, J E. & Bierer, B. E.(1999): CD28/CTLA-4 and CD80/CD86 families, signaling and function. Immunologic Research. 19/1:1-24; 5. Demanet C, Brissinck J, Leo O, et al.: Bispecific antiboddy-mediated immunotherapy of the BCL1 lymphoma: increasd efficacy with multiple injections and CD28-induced costimulation. Blood 1996; 87: 4390-4398; 6. Bohlen H, Manzke O, Titzer S et al.: Prevention of Epstein-Barr virus-induced human B-cell lymphoma in severe combined immunodeficient mice treated with CD3.times.CD19 bispecific antibodies, CD28 monospecific antibodies, and autologous T cells. Cancer Res. 1997; 57: 1704-1709; 7. Reimer S, Jang W, Sahin U, et al. Science 1994; 264: 833-835; 8. Mazzoni A, Mezzanzanica D, Jung G, et al.: CD3-CD28 costimulation as a means to avoiding T cell preactivation in bispecific monoclonal antibody-based treatment of ovarian carcinoma. Cancer Res. 1996; 56: 5443-5449; 9. Manzke, O., Berthold F, Huebe, et al. (1999): CD3.times.Cd19 bispecific antibodies and CD28 left-hand drive vehicles antibodies enhance T-cell reactivity against autologous leukemic cells in pediatric B-A11 bone marrow. Int. J. Cancer, 80: 715-722; 10. Grosse-Hovest L, Brandl M, Dohlsten M, et al.: Int. J. Cancer 1999; 80: 138-144; 11. Bouliannc, G L., Hozumi, N. & Shulman, M J (1984): Production of functional chimeric mouse/human antibody. Nature. 312, 643-646; 12. Neubcrger, M S., Williams, G T & Fox, R.. (1984): Recombinant antibodies possessing novel effector functions. Nature 312,604-608; 13. Jones, P T, Dear, P H., Foote, J et al. (1986): Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature, 321,522-525; 14. Riechmann, L., dark, M, Waldmann, H. et al. (1988): Reshaping human antibodies for therapy. Nature, 332,323-327; 15. Fay T N, Jacobs I, Teisner B. ct al.(1988): Two fetal antigens (FA-1 and FA-2) and endometrial proteins (12 PP and PP 14) isolated from amniotic fluid; preliminary observations in fetal and maternal tissues. Eur JObstet Gynecol Reprod Biol, 29(1):73-85; 16. Tutt A, Stevenson G T, Glennie M J(1991): Trispecific F(ab') 3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells. J Immunol 147(1):60-9; 17. Jung G, Frcimann U, Von Marschall z, et al.(1991): Target cell-induced T cell activation with bi - and trispecific antibody fragments. Eur J Immunol 21(10):2431-5: 18. French R R, (1998): Production of bispecific and trispecific F(ab)2 and F(ab)3 antibody derivatives. Methods Mol Biol, 80: 121-134; 19. Somasundaram S, Sundarapandiyan, Keler T. et al., (1999): Development of a trispecific antibody conjugate that directs two distinct tumor-associated antigens to CD64 on myeloid effector cells. Hum Antibodies, 9(1):47-54; 20. Schoonjans R, Willems A, Schoonooghe S, et al.(2000a): Fab chains As an efficient heterodimerization scaffold for the production of recombinant bispecific and trispeciiic antibody derivatives. J Immunol, 165(12):7050-7; 21. Schoonjans R, Willems A, Grooten J, ct al., (2000b): Efficient heterodimerization of recombinant bi - and trispecific antibodies. Bioseparation, 9(3): 179-83; 22. Wong W M, Vakis S A, Ayre To R, et al., (2000): Rheumatoid arthritis T cells produce Thi cytokines in response to stimulation with a novel trispecific antibody directed against CD2, CD3, and CD28. Scand J Rheumatol, 29(5):282-7; 23. Schott M E, Frazier A, Pollock D K, et al., (1993): Preparation, characterization, and in vivo biodistribution properties of synthetically cross-linked multivalent antitumor antibody fragments. Bioconjug Chem, 4(2): 153-65].

Carcinoma of the ovary occupies the second place among malignant tumors in gynecology. The most serious difficulty of this disease is the lack of symptoms at an early stage, the propensity for recurrence and lower survival rate (30%). To improve the situation after treatment of this disease, it would be important to develop a sensitive method for early diagnosis and effective approach to addressing the pockets, the remaining after surgery. In this case, when performing immunotherapy of carcinoma of the ovary should be useful circular single-stranded trapezitinae antibody.

Summary of the present invention

The aim of the present invention is the provision of a specially designed genetic engineering circular single-stranded trapezitinae antibodies, which has low toxicity and high efficiency; and simplification of the method of its production.

In a preferred embodiment, the present invention provides an expression vector that can be used in the design of universal circular single-stranded trapezitinae antibodies.

In another aspect the present invention provides a vector host cell containing the expression vector used in the construction of circular single-stranded trapezitinae antibodies.

In another aspect the present invention provides a nucleotide sequence encoding a specified circular single-stranded trapezitinae antibody.

The antibody molecule has two identical pairs of heavy chains and light chains. Each chain comprises one variable region (V) and one or more constant regions (C). Variable regions V responsible for binding to the antigen and a constant region is t - for binding to the effector molecule. Within each variable region has three short flexible loop-like segments, which are called hypervariable sites (CDRs), they abberant serial arrangement and crystal structure. Other intermediate stations, called frame (FRs), a relatively stable and are composed of β-sheets. These areas CDRs and FRs are located at intervals, forming a "sandwich"structure. The following terms used in the present invention.

"Fab antibody" is an antibody fragment that contains Fd fragment

(Vh heavy chain+ CH1), and the entire light chain. Through disulfide bond they form heterodimer. Its size is approximately 1/3 the size of the whole molecule antibodies and has only one antigennegative site.

"Odnotsepochechnoi antibody (scFv)" is a recombinant protein produced by genetic engineering. It consists of a VH and VL connected by a peptide linker. Its size is approximately 1/6 of the size of the whole molecule antibodies.

"Single domain antibody" is variable plot heavy or light chain. This type of fragment antibodies obtained by genetic engineering, has a single domain, and its size is approximately 1/12 the size of the entire mole is uly antibodies.

"Minimally recognized by the unit (MRU) is any single hypervariable area CDRs of the variable regions of the heavy or light chain. Its size ranges from approximately to approximately 1/70 1/80 size of the whole molecule antibodies.

In the "modified antibody" (also known as CDR-grafted antibody) replacement rodent CDRs on the CDRs of human conduct artificial synthesis or site-directed mutagenesis, so antigennegative activity of the original monoclonal antibodies rodent remains. Some amino acid residues of FRs person can prevent conformation antigennegative site, so you must change them in order to get weakened the antibody with the highest affinity.

The present invention provides a circular single-stranded trapezitinae antitumor antibody. It contains three parts: antitumor Fab, single domain antibody or scFv, transformed Fab, single domain antibody or scFv against human CD3, and transformed Fab transformed single-domain antibody or scFv against human CD28; these parts are stitched peptide interlinkable, forming a circular single-stranded molecule.

Antitumor antibody having tsiklicheskuyu single-stranded traspecific structure, by mentioning the case in the present invention, can be Fab-fragment, a single domain antibody or single-chain antibody against ovarian cancer man.

Best of all, if the specified circular single-stranded trapezitinae antibody consists of single-chain antibodies against carcinoma transformed single-chain antibodies against human CD3 and transformed single-chain antibodies against CD28 person who stitched peptide interlinkable with the formation of circular single-stranded molecule.

Better if the specified circular single-stranded trapezitinae antibody, referred to in the present invention, is a VNantibodies against CD28, having one of the following amino acid sequence:

Better if in a circular single-stranded respecifies the antibody referred to in the present invention, there are several peptide interlinker located between the specified antitumor antibody (Fab, single domain antibody or scFv), transformed CD3 antibody (Fab, single domain antibody or scFv) and transformed CD28 antibody (Fab, single domain antibody or scFv). These peptide interlinker can have one of the following amino acid sequences:

It would be better chtobybiznes trapezitinae antibody was cross-linked to the formation of cyclic molecules using the following peptide interlinker:

3 (1) HINGE (reverse): HUMAN-IgG3'CL PCRPCTHTTDGLPTKLE (2) HINGE (forward): HUMAN-IgG3'CL ELKTPLGDTTHTCPRCP

The present invention provides a nucleotide sequence encoding a circular single-stranded trapezitinae antibody referred to in the invention.

In other aspects the present invention provides an expression vector containing the above-mentioned nucleotide sequences. The expression vector can be pTRI.

In other aspects the present invention provides a cell host containing the above-mentioned expression vector. Specified the host-cell may be Escherichia coli.

Designing and building referred to in the present invention a circular single-stranded trapezitinae antibodies is based on the following theory. To activate T-lymphocytes necessary co-stimulatory signal. The gene encoding the antibody against carcinoma of the person, merge with sequences of two transformed antibodies against the two main molecules that stimulate signal. The present trapezitinae antibody differs from other traspecific antibodies the following characteristics:

1. Trapezitinae cyclic antibody is a protein molecule. The hinge area of human antibodies is injected into a flanking region of the molecule linear trapezitinae antibodies, and using posledovatel the surface of the hinged section of this antibody closes the circle by disulfide bonds. The formation of cyclic molecules reduces interference from various antigenspecific sites in the same molecule, makes it more stable and easier transport in vivo.

2. All three antibodies trapezitinae antibodies (particularly single-domain antibody against human CD28) are antibody molecules which are small. Molecular weight trapezitinae antibodies is quite low (84 kDa), which makes it suitable for immunotherapy of cancer.

3. Antibodies against CD28 and CD3 responsible for the activation of T-cells are weakened transformed antibodies with significantly lower immunogenicity.

4. Between each of the two antibodies is specially designed interlinker, which provides retention of the antibody in the desired conformation, and also introduces many other biological functions.

5. All three molecules are antibodies linked with the whole molecule and endow it with three different functions.

6. Antitumor antibody trapezitinae antibodies can easily be replaced by another tumor-specific or zanocin-specific antibody; this should extend the boundaries of its application.

7. This antibody is designed so that it can produce E. coli, and the resulting products do not require further transformation in vitro. So the WMD specified antibody easy to obtain at a low cost.

Brief description of drawings

Figure 1 represents a scheme for the construction and expression of a circular single-stranded trapezitinae antibodies;

Figure 2 illustrates the binding of different antibodies (anti-tumor scFv x anti-CD3 x anti-CD28) and interlinker;

Figure 3 (a, b) represents two DNA sequences and putative amino acid sequence of the transformed single-chain antibodies (VH) in respect of CD28;

Figure 4 (a, b) represents the DNA and amino acid sequence interlinker;

Figure 5 represents the products of the polymerase chain reaction (PCR);

6 represents a genetic map of universal expression vector pTRI used for circular single-stranded trapezitinae antibodies;

Fig.7 presents the results denaturing electrophoresis in polyacrylamide gel cyclic trapezitinae antibodies against carcinoma of the ovary, subjected expression in pTRI;

Fig represents the results of the analysis of cyclic trapezitinae antibodies against carcinoma of the ovary, subjected to expression in E. coli, made by the method of Western blotting; on the left the projection presents Lane 1, supernatant of the vector pTMF, and Lane 2, supernatant TsAb; on the right the projection presents Lane 1, supernatant TsAb (400 µg/ml); Lane 2, supernatant TsAb (40 μg/ml); Lane 3, superna the ant TsAb (4 μg/ml);

Fig.9 presents the results of the interaction of a circular single-stranded trapezitinae antibodies against carcinoma of the ovary with CD28 antigen; the results obtained by the method of enzyme-linked immunosorbent assay (ELISA), served as control supernatant vector pTMF; TRI was supernatant TsAb;

Figure 10 represents the results of the interaction of a circular single-stranded trapezitinae antibodies against carcinoma of the ovary with antigen cell membrane of carcinoma of the ovary or with CD3 antigen; the results obtained by the method of enzyme-linked immunosorbent assay (ELISA); and PTMFSKOV - interaction supernatant vector pTMF with antigen cell membrane of carcinoma of the ovary; TRISKOV - interaction TsAb with antigen cell membrane of carcinoma of the ovary; PTMFJUR - interaction supernatant vector pTMF with antigen cell membrane Juekat; TRIJUR - interaction TsAb with antigen cell membrane Jurkat;

11 illustrates the cytotoxicity in vitro of cyclic single-chain trapezitinae antibodies against carcinoma of the ovary to the carcinoma cells of the ovary (OCCD3CD28: anti-ovary carcinoma scFv+anti-CD3 antibody+anti-CD28 antibody; OCCD3: anti-ovary carcinoma scFv+anti-CD3 antibody; TRI: circular single-stranded trapezitinae antibody; Control: antibodies are absent; Vector CK: supernatant vector);

Fig presents the analysis of circular single-stranded trapezitinae ant the body's reaction rosethorne.

Detailed description of the present invention

The sequence of interlinker was artificially synthesized using the polymerase chain reaction. By implementing this sequence in pUC19 was granted a new plasmid, named pUHM1. The DNA fragment bispecific antibodies against carcinoma of the ovary CD3 received by cleavage of the plasmid pALM-Fc with XhoI and BamHI and subsequent implementation in pUHMl. The plasmid containing this sequence, called pUHM2. Another expression plasmid pTCH1 received, introducing the transformed single-domain antibody against CD28 and interlinked in pTMF. Fragment bispecific antibodies (anti-ovarian carcinoma x anti-CD3) and interlinker uncoupled from pUHM2, and then implemented in pTCH1. The resulting vector expression, called pTRI, used to modify competent cells BL21. After it was confirmed that the obtained clones are pTRI-positive, conducted inoculation in LB medium containing 50 μg/ml kanamycin. Cultivation was carried out at 37°C With vigorous shaking until the optical density component from about 0.4 to about 0.5. The culture was treated with IPTG for 4 h to a final concentration of 0, 8 mmol/l, and then collected by centrifugation. The resulting cells were subjected to lysis using ultrasound, and the lysate was centrifuged in accordance with the s 10 min at a speed of 12000 rpm Supernatant and dense clumps were separated by denaturing electrophoresis in 8% and 12% polyacrylamide gel. The samples were subjected to standard analysis, including Western blot turns, the analysis of immunological activity and cytological analysis (see figure 1-6).

Below is a detailed Protocol cloning vector.

1. Construction of cloning vector pUMH1

The specified cloning vector was derived from pUC19. The sequence of the linker (5'-HmdIII-pelB-human IgG3'CL hinge(complementary)-Gly4Ser-HSA-Gly4Ser-NdeI-EcoRI-3') was introduced into pUC19, Linearisation HindIII and EcoRI. 6 oligonucleotide fragments, named P1-P3 and RE1-RE3, used as template/primer for a complete 285 base pairs of the fragment linker:

1.1 Using overlapping PCR products when designing linker sequence

The process was carried out in two stages, as shown in figure 5. First stage: two-product of M1 and M2, with double helix, combined with P1, P2, RE2 and RE3, P3, and RE1, respectively. Second stage: the entire linker was obtained using overlapping PCR products with equimolar amounts of M1 and M2 (as a matrix).

Obtaining M1: in 30 µl reaction by adding 4 μl (approximately 10 pmol/l) P1, P2, RE2 and RE3, respectively, 3 μl of 10 × pfu buffer, DNA polymerase, 4 μl of dNTPs (2 mmol/l each), 1 μl of pfu is NC polymerase, the introduction of deionized H2O to volume of 30 μl, and the imposition of top 100 μl of paraffin oil. 30 PCR thermal sensor cycles. thermal cycle was as follows: 94°C for 1 min, 55°C for 30 s and 72°C for 40 C. the amplified DNA Fragments were analyzed by 2.5% agarose gel. The target band was excised and recovered using the kit for DNA purification (Gel DNA purification kit; Watson Inc. Shanghai, China).

Obtaining M2: in 30 µl reaction by adding 10 ál of P3, RE1 (approximately 10 pmol each), 3 μl 10 × PCR buffer, 4 µl dNTPs (2 mmol/l each), 1 μl of 10 pfu buffer, DNA polymerase, the introduction of deionized H2O to volume of 30 μl, and the imposition of top 100 μl of paraffin oil. 30 PCR thermal sensor cycles. thermal cycle was as follows: 94°C for 1 min, 60°C for 30 s and 72°C for 40 C. the amplified DNA Fragments were analyzed by 2.5% agarose gel. The target band was excised and recovered using the kit for DNA purification (Gel DNA purification kit; Watson Inc. Shanghai, China).

Obtaining the full length of the linker: in 30 μl reactions by adding 5 ál of recovered M1 and M2 as matrices, 2 μl of P1 and RE1 as primers, 3 μl of 10 × pfu buffer, 4 ál dNTPs (2 mm each), 1 μl of DNA polymerase, the introduction of deionized H2About to full on the of Yama, of 30 μl, and the imposition of top 100 μl of paraffin oil. 30 PCR thermal sensor cycles. thermal cycle was as follows: 94°C for 1 min, 55°C for 30 s and 72°C for 40 C. the amplified DNA Fragments were analyzed by 2.5% agarose gel. The target band was excised and recovered using the kit for DNA purification (Gel DNA purification kit; Watson Inc. Shanghai, China).

1.2 Splitting restriction endonuclease and purification of PCR products

The PCR product along its entire length were digested for 4 h with HindIII and EcoRI at 37°C. the Cleaved DNA fragments were separated on 1% agarose gel. The target band was restored with the help of the kit for DNA purification (Gel DNA purification Kit; Watson Inc. Shanghai, China).

1.3 Mini-preparation of pUC19

Odnoklikovoy DH5α containing plasmid pUC19 was inoculable in 5 ml of LB medium containing 100 μg/ml ampicillin. The culture was incubated overnight with vigorous shaking at 37°C. 1.5 ml of the culture was poured into a test tube and within 1 min centrifugation was performed at a speed of 12000 rpm, the Medium was removed and repeated the bacterial suspension of the precipitate in 100 μl of solution 1 (50 mmol/l glucose, 10 mmol/l ATDC, 25 mmol/l Tris-C18.0) with vigorous vortex shaking. To the resulting suspension of bacteria was added to 200 ál of freshly prepared solution II (0.2 mol/l NaOH, 1% sodium dodecyl sulphate), tube the pilot closed, the contents were mixed several times inverting the tube. The tube was kept for 3 min on ice and then it was added 150 μl of ice solution III (3 mol/l CAS, pH 4.8). The tube was closed and gently several times turned. The tube was kept on ice 5 minutes Lysate of bacteria was centrifuged at 12000 rpm for 10 min, the supernatant was transferred into a test tube. Added double volume of chilled on ice ethanol and the obtained supernatant was besieged DNA. The solution was mixed with the vortex shaking and put aside for 10 min at room temperature. Spent centrifugation at 12000 rpm for 20 min, for a single washing of the precipitate DNA was added 75% ethanol and an open test tube was left for drying at room temperature. The precipitate was dissolved in 100 μl of N2O. containing 50 μg/ml of RNA-free DNA. The tube was kept for 30 min, added equal volumes of phenol and chloroform (1:1). When shaken, mixed organic and aqueous phase and spent centrifugation at 12000 rpm for 2 min at room temperature. The upper aqueous layer was transferred into a clean test tube and added equal volume of chloroform: isopentanol (24:1; vol/vol). Added 1/10 volume of NaAc (3 mol/l, pH 5.2) and 2 volumes of chilled on ice ethanol. The resulting solution was mixed with the vortex Stradivari and put aside for 10 min at room temperature. Spent centrifugation at 12000 rpm for 60 minutes For a single washing of the precipitate DNA was added 75% ethanol and an open test tube was left for drying at room temperature. The precipitate was dissolved in 25 µl of H2O.

1.4 the Splitting of the restriction endonuclease and purification of plasmid pUC19

1 ág of pUC19 DNA was digested with HindIII and EcoRI, the process was performed in 40 μl of system (4 μl of × 10 buffer, 30 units of HindIII and 30 units of EcoRI) at 37°C for 4 h the Reaction mixture was analyzed on a 1% agarose gel. The target band was restored with the help of the kit for DNA purification (Gel DNA purification Kit;Watson Inc. Shanghai, China).

1.5 Stitching linearizing pUC19 fragment linker

20 μl of the reactor for stitching used approximately 40 ng of the recovered fragment double-cleaved pUC19 and approximately 20 ng of double-cleaved PCR products (2 μl of × 10 buffer T4 DNA ligase buffer T4 DNA ligase). Incubation was performed over night at 1,6°C.

1.6 Obtaining competent E. coli cells using the calcium chloride

With plates assembled one colony top 10. This colony was transferred into 3 ml of LB medium containing no antibiotic. The culture was incubated with shaking overnight at 37°C. 300 μl of the resulting culture was transferred into 30 ml of LB medium. Incubation was carried out at 37°C under conditions of vigorous shaking until the optical is some density OD 550approximately component of from 0.3 to about 0.4; process took approximately 3 hours the Culture was kept on ice for 10 minutes to cool, and then spent the centrifugation speed 4000 rpm at 4°C. the Supernatant was removed. For re-suspension was added 20 ml of chilled on ice with 0.1 mol/l CaCl2while creating a slight twist and a soft shake. The culture was cooled, leaving the tube for 30 min on ice. Spent centrifugation for 10 min with a speed of 4000 rpm at 4°C. the Supernatant was removed and re-suspension of sediment was added 2 ml of chilled on ice with 0.1 mol/l CaCl2containing 12% glycerol. In a test tube was added 200 μl of competent cells and kept at -70°C.

1.7 Transformation of E. coli, the screening of positive colonies of bacteria and identification by sequencing

Gently mixed 10 μl of the product of stitching with 200 ál of DH5 α chemically competent cells, 30 min, kept on ice, and then incubated at 42°C in a water bath for exactly 90 C. and Then for 5 min, cooled on ice and 200 ál of transformed competent cells were transferred to the agar LB medium containing 100 μg/ml ampicillin. After absorption of the liquid, the plate is turned over and incubation was performed over night at 37°C. Collected one colony to the method SEL knogo lysis to make mini-preparation of plasmid DNA. Performed identification of plasmids in her cleavage with Hind and EcoR, and then carried out amplificatoare using P1 and RE1 as primers. Then the positive plasmid was identified by sequencing and built as pUMH1.

2. Construction of cloning vector pUMH2

Cloning vector pUMH2 received from plasmids pUMH1 when implementing its fragment bispecific antibodies (5'-XhoI-anti-ovarian carcinoma scFv-Fc linker-anti-CD3 scFv-BamHI-3') between the XhoI and BamHI. The resulting fragment (anti-ovarian x anti-CD3) bispecific single-chain antibodies, rasalila of pALM-Fc, constructed in our laboratory.

2.1 production and purification of the fragment bispecific antibodies

Plasmid DNA was extracted from pALM-Fc by the method of alkaline lysis. Spent the splitting of 1 µg pALM-Fc with XhoI and BamHI, the process was carried out in 40 μl reaction containing 30 units of XhoI and BamHI (TaKaRa, Dalian, China), 4 μl × buffer, incubation was performed at 37°C for 4 h the resulting product was separated on 1% agarose gel. The target band was restored with the help of the kit for DNA purification (Gel DNA purification Kit; Watson Inc. Shanghai, China).

2.2 Cleavage and purification of plasmids pUMH1

Spent splitting 1 ál pUMH1 DNA with XhoI and BamHI, the process was carried out in 40 μl volume as described above; the incubation was carried out at 37°C for 4 h Cleaved fragment was extracted using a kit ocistring (Gel DNA purification Kit; Watson Inc. Shanghai, China).

2.3 Stitching, transformation and screening of positive colonies

The crosslinking reaction was carried out under the following conditions:

Double-split pUMH140 ng
Double-split fragment bispecific antibodies20 ng
Cross-linking buffer 10×T42 ál
T4 DNA ligase2 ál
water that does not contain nucleases,
to the final amount20 ál

Incubation was performed overnight at 16°C. 10 μl of stitched mixture gently mixed with 200 ál of DH5 α competent cells. Placed for 30 min on ice, then incubated at 42°C in a water bath for exactly 90 C. then cooled on ice for 5 min and 200 ál of transformed competent cells were transferred to LB agar medium containing 100 μg/ml ampicillin. After absorption of the liquid, the plate is turned over and incubation was performed over night at 37°C. Collected one colony to carry out mini-preparation of plasmid DNA. Inspection and identification of embedded fragment with XhoI, BamHI, HindIII and EcoR. Identified positive plasmid was a pUMH2.

3. Construction and expression of a circular single-stranded trapezitinae antibodies

Building a circular single-stranded trapezitinae antibody-based expression vector RDN and bispecific fragment pUMH2. rtsn was obtained from vector pTMF, which contained - NdeI-(VH of anti-CD28 scFv)-(c-myc)-Gly4Ser-Human IgG3'CL(17aa, Forward)-BamHI.

3.1 Construction of expression plasmids pTCH1

Spent the extraction of the recombinant plasmid pUC19 containing the fragment - NdeI-(anti-CD28 VH)-(c-myc)-Gly4Ser-Human IgG3'CL (17AA, Forward)-BamHI-using the method of alkaline lysis. Then realized the splitting of 1 µg plasmid DNA with NdeI and BamHI, carried out in 40 μl of system (4 μl 10 × buffer, 30 units of NdeI, BamHI). Incubated in a water bath at 37°C for 4 h Simultaneously conducted splitting vector pTMF. The resulting product was separated on 1% agarose gel. The target band was restored with the help of the kit for DNA purification (Gel DNA purification Kit; Watson Inc. Shanghai, China).

The crosslinking reaction was carried out under the following conditions:

Double-split pTMF40 ng
Double-cleaved fragment of anti-CD28 scFv20 ng
2 ál
T4 DNA ligase2 ál
water that does not contain a nuclease,
to the final amount20 ál

Incubated over night at 16°C.

10 μl stitched mixture gently mixed with 200 μl of BL21 competent cells. Put on ice for 30 min, then incubated at 42°C in a water bath for exactly 90 C. then cooled on ice for 5 min and 200 ál of transformed competent cells BL21 moved on LB agar medium containing 50 μg/ml kanamycin. After absorption of the liquid, the plate is turned over and incubation was performed over night at 37°C. Collected one colony to carry out mini-preparation of plasmid DNA. Performed identification of the embedded fragment using NdeI and HindIII. Received positive plasmid was named pTCH1.

3.2 Splitting restriction endonuclease and purification of the vector pTCH1

1 μg DNA pTCH1 were digested with NdeI and HindIII, the process was performed in 40 μl of system (4 μl of × 10 buffer, 30 units of NdeI and HindIII) at 37°C for 4 h, the Product was separated on 1% agarose gel. The target band was restored with the help of the kit for DNA purification (Gel DNA purification Kit; Waton Inc. Shanghai, China).

3.3 production and purification of interlinker containing bispecific antibody

Using the method of alkaline lysis had a mini-dissection pUMH2. 1 μg DNA pUMH2 was digested with NdeI and HindIII, the process was performed in 40 μl of system (4 μl 10 × buffer, 30 units of NdeI and HindIII). Incubated at 37°C for 4 h, the Product was separated on 1% agarose gel. The target band was restored with the help of the kit for DNA purification (Gel DNA purification Kit; Watson Inc. Shanghai, China).

3.3 Stitching, transformation and screening of positive colonies

The crosslinking reaction was carried out under the following conditions:

Double-split pTCH140 ng
Double-split fragment bispecific antibodies20 ng
Cross-linking buffer 10×T42 ál
T4 DNA ligase2 ál
The addition of water to the final volume average20 ál

Incubation was performed overnight at 16°C. 10 μl of stitched mixture gently mixed with 200 μl of BL21 competent cells. Put on ice for 30 min, then incubated at 42°C in a water bath for exactly 90 spoke this is about cooled on ice for 5 min and 200 ál of transformed competent cells were transferred to LB agar medium, containing 50 μg/ml kanamycin. After absorption of the liquid, the plate is turned over and incubation was performed over night at 37°C. Collected one colony to carry out mini-preparation of plasmid DNA. Performed identification of the embedded fragment using NdeI and HindIII. The positive plasmid was named pTR1.

3.5 Expression of circular single-stranded trapezitinae antibodies

Collected one freshly made positive colony pTR1, were transferred to LB medium containing 50 μg/ml kanamycin, and incubated overnight at 37°C. the Obtained overnight culture was diluted in the ratio 1:100 in 50 ml of fresh LB medium, the process was carried out in 250-ml flask. The culture was incubated at 37°C for its growth until it reaches the middle of its logarithmic growth (OD600of 0.4-0.5). Then in the culture additionally made IPTG to a final concentration component of 0.8 mm, and continued the incubation for an additional 4 h the resulting microorganisms collected and processed by the ultrasound on ice, then spent centrifugation for 10 min at a speed of 12000 rpm Obtained supernatant and the precipitate were analyzed by the method of denaturing electrophoresis in 8%, 12% polyacrylamide gel.

The molded gel sodium dodecyl sulphate-polyacrylamide (VAT-PAA) (table 1).

Table 1

30% acrylamide raw material: (29:1) (ml)Khel**RHEL***RHELUgel****
5%12%8%
0,5
2,1
6,0
a 4.9
4,0
6,9
0,53
0,93
H2(Ml):
1M Tris-HCl: pH (8.8)(ml)0,38--0,05
1.5M Tris-HCl: pH (6.8) (ml)-the 3.8the 3.8-
10% VAT (ml):0,030,150,150,02
10% Peroxydisulfate (ml):0,030,150,150,02
TEMED (ml):0,0030,0060,0012
* 29:1 weight:weight of acrylamide to N,N'-methylene bis-acrylamide.
** Because the gel - concentrating gel; *** & gel separating gel; ****U. gel - sealing gel)

Sample preparation: protein sample is mixed with an equal volume of buffer (100 mm Tris-HCl pH 6.8, 200 mm DTT, 4% sodium dodecyl sulfate, 20% glycerol, 0.2% Bromphenol blue), heated for 5 min at 100°C. Then, each sample is loaded into polyacrylamide denaturing gel electrophoresis. It is carried out at 60V in concentrating gel, then at 120V into the separating gel in a special buffer (25 mm Tris, 0.1% sodium dodecyl sulfate, 250 mm glycine (rn)). Proteins were stained for 1-2 h in Crusader solution of Kumasi brilliant blue R-250 (0.25% (weight/volume) of the dye R 250, 50% methanol, 10% acetic acid). This is followed by bleaching with 10% acetic acid (50% methanol, 10% acetic acid), the solution was changed every 30 min until then, while the substrate is translucent (3-5 times). Take pictures and analyze gels (shown in Fig.7).

Western blotting: using electrophoresis, the protein samples are transferred from the polyacrylamide gel to PVDF membrane. Electrophoresis is carried out in accordance with the Protocol supplied by the m by the manufacturer. Within 2 h in blocking solution (5% skim milk) carry out fast incubation of the membrane, then three times (5 min each wash) wash using TBST. The membrane was transferred in TBST containing mouse immunoglobulin (anti-c-myc IgG) at a dilution of 1:1000, and incubation was performed for 1 h at room temperature. Then the membrane is washed three times in TBST (5 min each wash) and transfer it in TBST containing conjugate antibodies goat and mouse (goat anti-mouse IgG-HRP). In conclusion, spend incubation of the membrane in the substrate solution (6 mg/ml DAB, 1% H2About2), which carry out up until the intensity of the target bands will not be sufficient. The reaction is stopped by repeated washing of the specified membrane in deionized water. Molecular weight scTsAb was 84 kDa (shown in Fig).

4. Chemical characterization of cyclic odnotsepochechnoi trapezitinae antibodies in vitro

4.1 Antigennegative activity circular single-stranded trapezitinae antibodies

Antigennegative activity sTRI against the antigen rhCD28/Fc and the antigen of the cell membrane of cells Jurkat cells and SKOV-3 were examined by the method of enzyme-linked immunosorbent assay (ELISA). The antigen of the cell membrane was obtained by ultrasonic destruction of tumor cells. ELISA was performed on the antigen, immobilis the bath 96-cellular plate. As primary antibodies used antibody mouse anti-c-myc antibody, (9E10), and as a secondary antibody - HRP-conjugate antibody goat and mouse IgG (HRP-conjugated goat anti-mouse IgG). In conclusion, the results visualized using as substrate the optical path difference (OPD); the absorption was measured at 490 nm. As shown in figure 9 and figure 10, a circular single-stranded trapezitinae antibody capable of forming specific chemical bonds with three types of antigens.

4.2 Assessment of cytotoxicity in vitro

Lymphocytes in human peripheral blood (PBL) were obtained from healthy donors by gradient Ficoll separation; the fraction of monocytes/macrophages iactiveaware method adhesion to glass (37°C, 2 h). For preparation of a monolayer of cells on 96-cellular flat-bottomed plate was placed cells SKOV-3. Simultaneously to this monolayer of tumor cells was added to freshly isolated effector cells (PBL)with different dilutions of the supernatant containing sTRI, used the appropriate relationship. Incubation was carried out for 3 days at 37°C incubator with 5% CO2. To remove cell effector plate washed twice with RPMI medium 1640. Added 200 μl of medium RPMI 1640 and 20 μl of MTT solution (0.5 mg/ml, sigma) and incubation was performed for 4 h at 37°C. the Supernatant MTT was discarded, EXT the wheelie 100 μl of DMSO and the readings were read at OD 570. Adding only one environment, prepared idle experience, as control was added to target cells and effector cells. For each sample did three repeats. The percentage of cytotoxicity was calculated according to the following formula: % cytotoxicity = (absorption control cells - absorption of the investigated cells/acquisition of control cells - absorption of idle cells) × 100. As shown in figure 11, the addition of a circular single-stranded trapezitinae antibodies led to the killing effect of tumor cells. The percentage of cytotoxicity for a group of circular single-stranded trapezitinae antibodies is significantly higher than in the other two experimental groups (oc-scFv+CD3scFv and oc-scFv+CD3 scFv+CD28 scFv).

4.3 Analysis of rosethorne

Treated with trypsin, the cells SKOV-3 was centrifuged for 5 min at a speed of 1000 rpm, the supernatant was discarded and spent re-suspension of the cells in complete medium (10% bovine serum, RPMI 1640). In each cell added 2×102cells SKOV-3 and held incubation over night at 37°C in an incubator with 5% CO2. Peripheral blood lymphocytes were isolated as described above, simultaneously held their activation by adding 100 IU of IL-2, the process was carried out at 37°C over night. To remove residual IL-2, PBLs thrice washed with RPMI 1640, and then entered them on the plate SKOV-3, is compared with E/T which was 20:1 (E: effector cells, PBLs; T: target cells, cells SKOV-3). Meanwhile, added to the supernatant circular single-stranded trapezitinae antibodies, spent incubating the mixture at 37°C in an incubator with 5% CO2. Intervals comprising 2 h, were pictures taken under the inverted microscope. As shown in Fig and in table 2, after 2 h cells effector began to stick with trusses target. After 4 h began the destruction of the majority of target cells, and after 10 h the majority of target cells disintegrated into fragments.

Table 2
The percentage of formation of rosettes, indirect circular single-stranded traspecific antibody
Concentration sample400 μg/ml antibodies40 µg/ml antibody4 μg/ml antibodies40 mg/ml of vector supernatantIdle
The percentage of formation outlets40%30%20%10%0

1. Polynucleotide encoding circular single-stranded thespecification antibody against carcinoma of the ovary human in vitro, which has a molecular weight of about 84 kDa, includes arranged in the following order from aminobenzo to carboxylic following components:
interlinker selected from reverse hinge interlinker of IgG3 human, comprising the amino acid sequence PCRPCTHTTDGLPTKLE or hinged interlinker of IgG3 human, comprising the amino acid sequence ELKTPLGDTTHTCPRCP;
peptide with amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 5;
(i) the antibody immunospecificity linking cell carcinoma of the ovary, where the specified antibody is a Fab fragment, a single domain or humanitariannet single-chain (scFv) antibody;
interlinker consisting of amino acid sequences with Asn297 on Lys 322 of the IgG1 CH2;
(ii) an anti-CD3 antibody, where the aforementioned antibody is a Fab fragment, a single domain or humanitariannet single-chain (scFv) antibody;
peptide with amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 5;
interlinker consisting of amino acid sequences with 403 at 425 amino acid residue of the domain III of serum albumin person;
peptide with amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 5;
(iii) anti-CD28 EN is Italo, where the specified antibody is a Fab fragment, a single domain or humanitariannet single-stranded (scFy) antibody;
peptide with amino acid sequence SEQ ID NO: 4 or SEQ ID NO: 5;
interlinker selected from the hinge interlinker of human IgG3 with the amino acid sequence ELKTPLGDTTHTCPRCP of reverse hinge interlinker of human IgG3 with the amino acid sequence PCRPCTHTTDGLPTKLE,
the first interlinker and last interlinker thespecification antibodies chosen so that they form disulfide bonds.

2. Polynucleotide according to claim 1, where the specified anti-CD28 segment specified thespecification antibody includes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

3. Polynucleotide according to claim 1 or 2, characterized in that the antibody that is encoded by the specified polynucleotide, reverse hinge interlinker has the amino acid sequence of SEQ ID NO: 9; interlinker consisting of amino acid sequences with Asn297 on Lys 322 of CH2 LgG1, has the amino acid sequence of SEQ ID NO: 6; interlinker consisting of amino acid sequences with 403 at 425 amino acid residue of the domain III of serum albumin human, has the amino acid sequence of SEQ ID NO: 7; and a hinged interlinker has the amino acid sequence of SEQ ID NO: 10./p>

4. Polynucleotide according to claim 3, characterized in that the antibody that is encoded by the specified polynucleotide, articulated interlinker further includes a segment of the C-myc comprising amino acid sequence SEQ ID NO: 8; and the reverse pivot interlinker further includes the amino acid sequence of SEQ ID NO: 3.

5. Expressing a vector comprising polynucleotide according to claim 1 or 2.

6. Expressing the vector according to claim 5, characterized in that it is pTRI.

7. A host cell E. coli, including polynucleotide according to claim 1 or 2.

8. A host cell E. coli containing expressing vector according to claim 5.



 

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11 cl, 14 dwg, 1 tbl, 2 ex

FIELD: medicine; microbiology.

SUBSTANCE: efficiency of the transducing phage crops multiplied on the strain-donor, and strains of the future recipients. Quality of the recipient is checked at statement of an intraspecific and interspecific transduction, by addition of 1 ml transducing phage of various concentration to 1 ml recipient cultures B.anthracis, B.thuringiensis and B.cereus with the various characteristic of efficiency of the transducing phage crops, and the control - 1 ml recipient culture admix about 1 ml of a phage buffer. Incubate during 20 minutes at 33°C in a shaker at 100 sh. rot./minute, then in all assays it is brought on 1 ml of a preparation of cellular receptors of corresponding recipients. Again incubate during 30 minutes at 33°C. Then in each assay bring on 1 ml of corresponding receptors and inoculate on 0.2 ml from skilled and control assay on the selective Hottinger mediums, grown TetR colonies - transductant are calculated in 48-72 hours after incubation of crops at 35°C. The recipient is considered optimum for transduction if efficiency of the transducing phage crops is peer 0.5-1.0 (relation of an indicator of concentration transducing phage received on the strain-donor to an indicator of concentration received on the selected strain-recipient).

EFFECT: selection of optimum recipients for realisation of a transduction from an available collection of strains Banthracis and closely related bacilli.

1 dwg, 2 tbl, 2 ex

FIELD: medicine; molecular biology.

SUBSTANCE: invention concerns molecular biology, genetic engineering and can be used in medicine. Quantitative express identification of the HIV-1 genome in assay is detected with the help of oligonucleotide primers, complementary to a conservative site 5'-LtR-part of the HIV-1 genome, fluorescently marked probes and the RNA-modified fragment of a conservative site of 5'LTR area of the HIV-1 genome as the internal quantitative standard. The analysis is performed in real time in "the closed test tube" format by means of the calibration curves received with the help plasmides pVarl5-HIV-LTR and pBluSK-HIV-LTR mod, containing the native and modified kdnk-fragment virus genome from conservative 5'-LTR-pocledovatelnocti accordingly. Plasmide pVar15-HTV-LTR it is received on the basis of the pVar15 plasmides. The pBluSK-HIV-LTR mod plasmides is received on the basis of pBluKSM plasmide.

EFFECT: possibility of determination of HIV-1 presence in assay irrespective of type of a virus, a combination of types and presence of related kinds.

4 cl, 10 dwg, 9 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: current invention relates to the field of biotechnology and immunology. Proposed is an antibody, specific to the human ED-B. Antibody specified is a molecule in the form of either dimerizated mini-immunoglobulin or IgG1, whose variable region comes from the antibody L19. In case the mini-immunoglobulin variable region L19 is merged with εS2-CH4, then as in the case IgG1, the variable region L19 is merged with the constant domain of IgG1. Conjugates of antibodies with radioisotopes have been discovered. Described is the coding nucleic acid, carrying its host cell, capable of producing antibodies, and method of obtaining antibodies from cells. Discovered is a method of determining the degree of bonding of antibodies, also compositions based on antibodies. Described is the use of antibodies for preparing medicine for treating either damage related to angiogenesis, or for treating tumours. Utilisation of the invention provides antibodies, which possess high accumulating capacity to tumours, improved capability to bonding with radioactive labels and unexpectedly retains immunoreactivity in the plasma, in comparison to scFv L19. Antibody specified can be used in diagnostics and treatment of tumours.

EFFECT: obtaining antibodies which can be used in diagnostics and treatment of tumours.

22 cl, 13 dwg, 8 tbl

FIELD: chemistry.

SUBSTANCE: invention concerns biotechnology, particularly obtaining cytokine proteins, and can be applied in cell technology. Polypeptide binding Zalpha11 ligand receptor is obtained. Nucleotide sequence coding new polypeptide is introduced into host cell as part of expression vector, and polypeptide is produced.

EFFECT: possibility of efficient adjustment of proliferation and/or development of hemopoietic cells in vitro and in vivo.

14 cl, 1 dwg, 55 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to biotechnology and to methods of producing vaccine preparations with the help of gene engineering and immunology. The invention proposes the expressive plasmid DNA p-BMC-gag (A)-hum, containing an artificial gene gag (A)-hum, for expression of p55 protein in eukaryote cell. The invention can be used in medicine and allied industries ensure a 10-12-fold increase in expression of p55 protein of the human immunodeficiency virus of the 1st type in eukaryote cells.

EFFECT: high-efficiency method of producing vaccine preparations gene engineering and immunology.

1 cl, 2 dwg, 2 tbl, 4 ex

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to area of gene engineering and biotechnology and can be used for labeling biological objects. The nucleic acid molecule was extracted that encoded the fluorescing protein selected from fluorescing proteins represented by the Copepoda Crustacea biological kinds and fluorescing mutants of the aforesaid proteins. The aforesaid nucleic acid was functionally bonded to appropriate elements of expression regulation to be used in the method of producing aforementioned fluorescing protein. On the basis of the aforesaid extracted nucleic acid cloning and expressing vectors are produced as well as the expressing cassette. The cell and a stable cellular lineage, containing the aforesaid expressing cassette produce the fluorescing protein. Note that the said fluorescing protein, the nucleic acid encoding the protein above and expressive genetic structures containing the said nucleic acid, are used incorporated with the set designed to label a biological molecule. The fluorescing protein is also used in the methods of labeling a biological molecule, a cell or a cellular organella.

EFFECT: expanded biological objects labeling means.

12 cl, 9 dwg, 1 tbl, 7 ex

FIELD: biology; biotechnology.

SUBSTANCE: invention concerns biotechnology. It claims linking molecule represented by monoclonal antibody or its fragment capable of binding human NogoA polypeptide, human NogoA 623-640. Polynucleotide encoding the claimed molecule is presented. Expression vector including indicated polynucleotide is described. Host cell including indicated polynucleotide or vector is described. Pharmaceutical composition for treatment of diseases related to nerve reconstruction and containing indicated molecule is described.

EFFECT: obtainment of antibodies capable of binding NogoA 623-640.

9 cl, 1 dwg, 3 tbl, 7 ex

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