Monoclonal antibodies and one-chain fragments of cell-surface prostate-specific membrane antigen antibodies
SUBSTANCE: there are offered versions of: a SSMA monoclonal antibody and its antigen-binding fragment which are bound with a mark or a cytotoxic agent. There are described versions of a pharmaceutical composition and a diagnostic kit based on such antibodies. There are disclosed methods for identification in vitro of tumour cells, as well as for diagnostic identification of tumour cells based on such antibodies. What is described is an isolated polynucleotide for producing monoclonal antibodies.
EFFECT: using the invention provides the antibodies which can bind SSMA in its native form on the surface of tumour cells, are bound with LNCAP, but are not bound with cells with lost SSMA expression that can find further application in prostate cancer therapy.
15 cl, 21 dwg, 3 tbl, 18 ex
Prostate cancer is the most common malignant disease in men and is the second leading cause of death in the Western world. At the present time there is no radical treatment of unresectable prostate. Because of the high mortality and morbidity associated with disease progression, it is necessary the development of new methods for targeted therapies. In contrast to cancer of other organs, prostate cancer, for several reasons, is a good target for therapy with the use of antibodies, because (i) the prostate expresses tissue-specific antigens, (ii) the prostate is a secondary organ and its destruction does not cause harm to the host organism, (iii) areas of metastasis are lymph nodes and bones, which accumulate high levels of circulating antibodies, iv) serum levels of PSA are a means of monitoring therapeutic response.
Among some potential markers that have been identified for prostate cancer, the most famous, apparently, is specific to prostate membrane antigen (PSMA). This transmembrane glycoprotein of type II, a molecular mass of approximately 100 kDa, consists of a short intracellular site (amino acids 1-18), transmembrane domain (amino acids 19-43) and extended extracellular D. the MENA (amino acids 44-750). PSMA can serve as a target for immunotherapy because it meets the following criteria: i) the expression is mainly restricted to the prostate, (ii) as PSMA protein is widely expressed on all stages of the disease, (iii) it is presented on the cell surface, but does not enter into the circulatory system, iv) expression is associated with enzymatic or signaling activity. PSMA is also expressed in the newly formed vascular network of most other solid tumors and therefore may be appropriate as a target for specific delivery of antiangiogenic drugs.
Due to the specificity of monoclonal antibodies (monoclonal antibodies - mAb) against targets, special emphasis was placed on developing them for diagnostic and therapeutic applications in Oncology. However, due to their size and immunogenicity application of mAb in vivo is associated with serious problems. Therefore, the study was focused on developing smaller therapeutic antibodies with fewer side effects, greater availability to the tumor and higher clearance. Thanks to genetic engineering introduces the ability to create single-stranded fragments of antibodies (single chain antibody fragments - scFv), which are potentially powerful tool for cancer therapy. These small antibodies consist of variab the selected domains of the light chain (light chain - VL) and heavy chain (heavy chain - VHassociated linker peptide. They are weak immunogenicity, almost no toxic effects, show a higher level of clearance, increased accumulation in tumors and better penetration into tumor cells. Recombinant murine scFv can be obtained in accordance with standard methods, using either expression library hybrid, or spleen cells specifically immunized mice (Chowdhury and others, Mol. Immunol., 4, 1997, c.9-20).
First published mAb (7E11-C5) against PSMA targeted to the intracellular domain of the protein, and found that it is highly prostatespecific (Horoszewicz and others, Anticancer Res., 7, 1987, c.927-935). In addition, were obtained monoclonal antibodies against the extracellular domain of PSMA after immunization with this antigen. However, there is a difference between binding to the antigen on fixed cells and tissue sections, on the one hand, and linking with viable tumor cells, on the other hand.
Prostatespecific membrane antigen (PSMA) is a marker of prostate cancer, which is highly expressed in normal prostate and prostate cancer. Its expression is prostate cancer increases and mainly found in the prostate.
Prostatespecific membrane antigen (PSMA) I what is unique associated with cell membrane protein, which sverkhekspressiya on cancer cells of the prostate, as well as in the newly formed vascular network of many other solid tumors, in contrast to the vasculature of normal tissues. Thanks to this unique expression of PSMA is an important marker, and a large extracellular target prospective agents. PSMA can serve as a target for delivery of therapeutic agents, such as cytotoxins or radionucleotides. PSMA carries two unique enzyme functions, polytherapy and NAALAD, and found that it is recycled through the lined clatrina fossa, like other membrane-bound receptors.
Anti-PSMA monoclonal antibody (mAb) 7E11 in the form radioimmunoconjugates commercially available as product ProstaScint®"which currently is used for detection of metastases and recurrence of prostate cancer. The PSMA epitope recognized by the monoclonal antibody 7E11-S localized in the cytoplasmic domain prostatespecific membrane antigen.
However, there are reports describing the expression of PSMA in tissues that are not related to the prostate, including the kidneys, liver and brain. A possible explanation for this fact is given O'keefe and others in the Prostate, 58, 2004, s-210, namely, that there is a PSMA-like gene, which has 98% identity with the genome of PMS at the level of nucleotide and which is expressed in the kidney and liver under the control of another promoter, than the PSMA gene.
In WO 01/009192 describes the development of monoclonal antibodies person to prostatespecific membrane antigen. Human monoclonal antibodies were obtained by immunization of mice with purified PSMA or enriched preparations of antigen PSMA. This purified antigen is denatured PSMA, because its purification was carried out by immunoadsorption after cell lysis, ionic detergents.
In WO 97/35616 describes monoclonal antibodies specific for the extracellular domain prostatespecific membrane antigen. Immunization was carried out using C-terminal peptide or a membrane preparation tumors expressing PSMA. These mAb does not bind specifically to PSMA-expressroute cells and therefore cannot be used for diagnostic or therapeutic purposes.
Bander, etc., Seminars in Oncology, 2003, s-677 and US 2004/0213791 respectively, describe monoclonal antibodies directed against prostatespecific membrane antigen. Since immunization was performed using the purified antigen, monoclonal antibodies did not possess in high degree the ability to bind to cells, and of these mAb was impossible to obtain scFv.
In WO 98/03873 describes antibodies, such as those described in US 2004/0213791, or their binding fragments, to whom that recognize the extracellular domain prostatespecific membrane antigen. It cannot be argued that binding fragments of these antibodies actually bind to the antigen. Fracasso and others in The Prostate, 2002, c.9-23 describe monoclonal antibodies against PSMA, which is chemically connected to the a-chain of ricin. The constructs described in this article do not have sufficient binding specificity to the target and are described conventional disadvantages generation immunotoxins.
One of the objectives of the present invention is the provision of the above tools and constructs that will help with higher accuracy to distinguish between tumor cells and healthy cells that Express PSMA or similar protein, and PSMA-negative cells. Such constructs can be used for more specific targeting prostate cancer.
Another objective is to provide constructs that destroy specific cancer prostate cells expressing PSMA.
Prostatespecific membrane antigen (PSMA) is an attractive target for immunotherapy of prostate cancer. However, the cells of the prostate PSMA is expressed with a specific tertiary and Quaternary structure, and antibodies specific for the isolated denatured PSMA, not well recognize tumor cells expressing PSMA. Antibodies and scFv, with asiaasia denatured PSMA, can be obtained after immunization dedicated purified antigen. The present invention, however, allows you to get vysokoaffinnye antibodies and scFv against native cellular PSMA by another method of immunization, which gives rise to only a small number of positive clones. Only antibodies derived from native PSMA can interact with cell-surface PSMA and can be used as diagnostic and therapeutic tools.
Monoclonal antibodies (mAb), single-chain antibody fragments (scFv) and double antibody of the present invention were obtained according to conventional methods from spleen cells of mice. However, mice were immunized with LNCaP cells and lysate of LNCaP cells containing full-sized native PSMA. In a preferred embodiment of the present invention the antigen, namely a full-sized native PSMA, was obtained after treatment of the cells, preferably cells LNCaP, special Lisinym buffer M-PER reagent for extraction of proteins mammals which are commercially manufactured by a company Pierce, Rockford, Illinois. Buffer M-PER uses the proprietary detergent in 25 molarnom bitenova buffer (pH 7,6). Screening and selection of hybridomas and scFv was performed by flow cytometry on PSMA-positive LNCaP cells after the absolute is bcii PSMA-negative cells of the prostate DU 145. Additionally, they were tested for reactivity with purified PSMA. The resulting monoclonal antibodies and scFv were characterized using flow cytometry on LNCaP and PSMA-transfected DU 145 cells and Western blotting with purified glycosylated and deglycosylated PSMA. In addition, conducted immunocytological research with LNCaP cells and immunohistochemical studies on paraffin sections prostate cancers.
In the course of the present invention was able to get three mAb (3/F11 3/A12 and 3/E7), which interacted with a viable LNCaP cells and PSMA-transfected cells, DU 145, but did not possess such ability in relation to other cell lines, which are not expressed PSMA. Binding to LNCaP cells was very strong. When the saturation concentrations (100 nmol) average value of fluorescence intensity (CIF) D ranged from 1000 to 1600. Reactivity against purified PSMA was stronger in the case of the native form (established enzyme-linked immunosorbent assay - ELISA), than in the case of denatured and deglycosylated protein (Western blotting). Immunohistochemistry on paraffin sections was specifically positive for epithelial cells with mAb E7.
By selection of recombinant phages in LNCaP cells and purified PSMA from mAb 3/A12 were obtained is s two scFv, denoted by E8 and A5. The sequence of scFv E8 was identical scFv A4, which was obtained from the library Into cells of the same mouse. ScFv E8 was highly reactive towards LNCaP cells, showing CIF value of approximately 100 at concentrations of saturation, while the value of the CIF for scFv A5 was only about 40 under the same conditions. Was not detected or was detected minimal binding to other cell lines not expressing PSMA. Linking both scFv with purified denatured glycosylated and deglycosylated PSMA was weak. In addition, mAb 3/F11 was obtained scFv, designated D7, and mAb 3/E7 was obtained scFv, designated H12.
In this application describe three mAb, which differ from the monoclonal antibodies described by other authors, high affinity binding and high staining expressing PSMA prostate cancer cells. By immunofluorescence Cytology and detection using confocal laser scanning microscopy it was shown that antibodies 3/F11 3/A12 and 3/E7 not only show a strong binding activity, but also internalized in LNCaP cells. These mAb were obtained after immunization with full-sized native PSMA, in contrast to other published methods of immunization.
After immunization with purified denatured PSMA mAb were obtained, which is yli highly specific against the antigen, but had only limited binding expressing PSMA cells LNCaP and also not included in these cells. These control data are not shown in this application. The literature describes several mAb against PSMA. However, the average values of fluorescence intensity were much lower than with antibodies of the present invention.
Like mAb, after immunization denatured and native PSMA scFv were obtained against PSMA. Using denatured PSMA authors of the invention have been scFv, highly specific to the antigen, but not communicating with LNCaP cells (data not shown in this application). In contrast, using native PSMA authors of the invention have been scFv with high activity binding to cells, but low binding to isolated denatured antigen.
However, the problems identified in this and other tests with chemically related immunotoxins, include the development of antibodies against immunotoxins, hepatotoxicity and leaky vessels, as well as difficulties in obtaining large quantities of a particular material. These problems, at least partially, overcome with the help of genetic engineering, which allows the creation of a less immunogenic smaller immunotoxins, and allows more easily what about getting immunotoxins in large quantities. It is also expected that smaller proteins need to better penetrate into tumors larger than conjugates. So were created recombinant immunotoxins by merging the coding sequence of scFv E8, H12, D7 and A5 and toxin RE. The main finding was that all recombinant immunotoxins effectively killed cultured prostate cancer cells dose-dependent manner. Strong cytolysis was found not only with high binding E8, with IC50 approximately 0.05 nm, but also with the more weakly binding A5-hybrid protein with an IC50 of about 0,09 nm. The cytolysis of cells of prostate cancer not expressing PSMA, was lower by more than 2000 times. This can occur from residual bacterial proteins in preparations of immunotoxins, because the same background could be observed in equally high concentrations of one scFv. The concept of IC50 in the context of the present invention means the concentration of a toxin expressed in nolah, which reduces cell proliferation by 50% compared with cell proliferation without the addition of toxin.
Antibodies and scFv described in this application specifically associated with native PSMA on the cell surface and are therefore valuable for diagnostic and therapeutic applications, focusing on PSMA as a target antigen for prostate cancer.
Because PSMA is expressed on the river of the same cells of the prostate-specific tertiary and Quaternary structure, only antibodies against the cellular conformation can recognize and firmly contact with viable cancer cells of the prostate and PSMA-expressing tissue. Therefore, the purpose of this study was to obtain such mAb and scFv, which can be used for targeted therapy and diagnosis of prostate cancer.
Thus, the present invention provides for an isolated monoclonal antibody, or antigennegative fragment that binds to a specific to prostate membrane antigen in its native form, found on the surface of tumor cells, which is connected with a label or a cytotoxic agent.
The concept of "isolated " monoclonal antibody" refers to a glycoprotein comprising at least two heavy (heavy - H) chains and two light (light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a variable segment of the heavy chain (variable region - VHand constant plot heavy chain. Constant plot heavy chain contains three domains, namely CN, CH2 and CH3. Each light chain contains a variable area light chain (variable region - VL) and the constant-area light chain constant region - CL). Plots of VHand VLadditionally can be divided into hypervariable sites, to the which also called stations, determining complementarity (complementary determining region (CDR), scattered between areas that are more conservative. These areas are also referred to as frame sections (framework region (FR). Each plot VHand VLcomposed of three CDRs and four FR located from amino end to the carboxy-end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Variable parts of the heavy and light chains contain a binding domain that interacts with the antigen.
On Fig, 14 and CDR 20 and 21 are shaded in gray. These areas are important for binding of the monoclonal antibody or its antigennegative fragment. Other areas are of frame sections which can be substituted by other sequences, provided that not violated the three-dimensional structure necessary for binding. If you change the structure of the construct, will not be sufficient binding to the antigen. Monoclonal antibodies derived from mouse, can cause unwanted immunological side effects, because they contain protein from another species that can cause the production of antibodies. To overcome this problem, monoclonal antibodies or their antigennegative fragments can humanize. A method of obtaining a humanized monoclonal antibodies known to specialists in this field. Frame Castlemaine mAb replace the corresponding frame parts person. To save the preferred binding properties, sequence plots CDR need to save. However, it is possible that to improve binding properties will require some amino acid substitutions. This can be performed by the person skilled in the art using conventional methods. In addition, with the introduction of structures to improve properties of the construct may have the amino acid substitutions and/or deletions.
The concept of "antigennegative a fragment of a monoclonal antibody refers to one or more fragments of such antibody which retains the ability to specifically bind to specific membrane antigen prostate in its native form. Examples antigenspecific fragments of an antibody include a Fab fragment, a monovalent fragment consisting of domains VLVHCLand CH1fragment F(ab')2, a bivalent fragment comprising two Fab fragment linked by a disulfide bridge at the hinge region, the Fd fragment consisting of domains VHand CH1fragment FVconsisting of domains VLand VHthe only branch of the antibody, a dAb fragment, which consists of domain VHand the isolated section, complementarity determining (CDR).
An isolated monoclonal antibody or its antigennegative the store fragment of the present invention preferably can be absorbed by a tumor cell if they are used for therapeutic purposes. For diagnostic purposes, the acquisition may not be required.
An isolated monoclonal antibody or its antigennegative fragment of the present invention are strongly linked with LNCAP cells, but not in contact with cells that do not Express specific to prostate membrane antigen.
The binding of the isolated monoclonal antibody or its antigennegative fragment is estimated by the intensity of fluorescence picaridin (PV) (CIF), which is preferably equal to or higher than 40 for scFv and preferably above 1000 for mAb at concentrations of saturation.
Binding properties of monoclonal antibodies or their antigenspecific fragments with native PSMA was compared by treatment of LNCAP cells to increasing concentrations of the primary anti-PSMA antibodies followed by incubation with secondary FE-labeled antibody. On the basis of the obtained curves of saturation can be calculated concentration of antibodies, which achieved 50% saturation plots PSMA. Three mAb 3/F11 3/A12 and 3/E7 showed high binding activity, reaching 50% saturation plots PSMA at a concentration of approximately 16 nm (3/F11), 2 nm (3/A12) and 30 nm (3/E7). In the case of scFv 50% saturation plots PSMA was detected at 10 nm (E8) and 60 nm (A5).
To set the strength of binding wire is elk measurement of fluorescence intensity Feh (picaridin) (CIF). Values Seth put on a graph against the concentration of antibodies (or binding fragments), the value of the plateau Seth corresponds to 100% saturation of the antigen. After you define the top value (plateau corresponding to 100% saturation of the antigen) can be easily set value corresponding to 50% saturation. Using the graph you can find the appropriate concentration in nolah antibodies or their binding fragments.
An isolated monoclonal antibody or its antigennegative fragment includes a label, which may be a particle that emits radioactive radiation. This particle may be a radioactive element in a form that can be attached to the construct, preferably in the form of a complex. For example, for detection of distant metastatic tumors in patients with prostate cancer as immunoscintigraphic agent can be used mAb labeled with111indium. You can also use other suitable radioactive elements, such as35S or131I.
In another embodiment, the isolated monoclonal antibody or its antigennegative fragment can include a cytotoxic agent, which is a cellular toxicant selected from the group consisting of toxins, such as Taxol, cytochalasin B, gramicidin D, ethidium bromide, ameti is a, of mitomycin, etoposide, anapsida, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and/or puromycin.
In a preferred embodiment of the present invention isolated monoclonal antibody or its antigennegative fragment include partial amino acid sequence of at least 10 consecutive amino acids of SEQ ID NO:1 (scFv E8), SEQ ID NO:10 (scFv A5), SEQ ID NO:20 (scFv H12) and/or SEQ ID NO:22 (scFv D7). In a preferred embodiment of the present invention isolated monoclonal antibody or its antigennegative fragment includes at least 25, or more preferably at least 35, and particularly preferably at least 50 consecutive amino acids of SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:20 and/or SEQ ID NO:22, respectively.
In a preferred embodiment of the present invention isolated monoclonal antibody or its antigennegative fragment includes at least one of a CDR having the sequence SEQ ID NO:2 - SEQ ID NO:7 and/or SEQ ID NO:11-16. More preferably, this construct includes at least 3 and more preferably 5, of these CDR sequences. Similar clicks the zoom CDR can be derived from Fig and 21, which marked the CDR sequence.
The present invention also provides DNA sequences that can be used to obtain monoclonal antibodies or their binding fragments. SEQ ID NO:8 and 9 relate to the scFv E8, a SEQ ID NO:17 and 18 refer to the scFv A5. SEQ ID NO:19 and 23 relate to scFv H12, a SEQ ID NO:21 and 24 belong to the scFv D7. The sequence also describe the coding strand and a complementary chain. SEQ ID NO:9 and 18 shown in the 5'→3' orientation. Polynucleotide of the present invention include a continuous sequence of at least 20, preferably 50, more preferably 75, and particularly preferably at least 100 nucleotides from the group consisting of SEQ ID NO: 8, 9, 17, 18, 19, 21, 23 and 24. Particularly preferred sequences encoding CDR.
Another feature of the present invention is the fact that it provides a pharmaceutical composition comprising an isolated monoclonal antibody or its antigennegative fragment described in this application. The pharmaceutical composition of the present invention includes a monoclonal antibody or antigennegative fragment together with pharmaceutically acceptable excipients. Preferably such a composition was prepared for intramuscular or intravenous administration. In another embodiment, the Academy of Sciences of the United Italo may be provided in the composition for prolonged action, which allows slow release of biologically active agent within a certain time, which may range preferably from one to six months. Such a composition sustained release may include a polymer that can be biodegradable, for example, polylactic or copolymer polylactide/polyglycolide, which in the human body decomposes in a long time, thus there is a controlled release over a certain period of time, antibodies, or antigennegative fragment, preferably carrying the toxin.
An isolated monoclonal antibody or its antigennegative fragment can be used for getting medicines for the treatment of cancer, in particular prostate cancer.
In another embodiment, the present invention provides a diagnostic kit for the detection of tumor cells, comprising an isolated monoclonal antibody or its antigennegative fragment. In such scenarios, the implementation of the present invention the label allows you to identify the design using the appropriate devices.
The present invention also provides a method for identifying in vitro tumor cells, wherein the tumor cells to be identified, is subjected to contact with isolated is monoklonalnm antibody or antigennegative fragment bearing a label which can be detected by appropriate analytical equipment. The label allows for diagnostic identification of tumor cells, for example, in the slice of the tumor tissue obtained after surgery or biopsy.
Brief description of figures
Figure 1. FACS analysis of binding of mAb 3/F11 3/A12 and 3/E7-surface PSMA-expressing LNCaP cells at concentrations of saturation.
Figa-century Curves saturation of antigen antibodies 3/F11 (a), 3/A12 (b), 3/E7 ().
Figure 2. Immunofluorescence Cytology: Binding a) mAb 3/F11 b) mAb 3A/12V) E with LNCaP cells. The drawings on the left show the control staining with 4',6-diamidino-2-phenylindole (DAPI).
Figure 3. Immunofluorescence Cytology: Internalization (a) mAb 3/F11 b) mAb 3A/12V) E in LNCaP cells. The drawings on the left show the control staining with 4',6-diamidino-2-phenylindole (DAPI).
Figure 4. Western blotting with purified PSMA and mAb 3/E7, 3/A12 and 3/F11.
Figure 5. Western blotting with glycosylated and deglycosylated PSMA and mAb 3/A12.
6. Immunohistochemistry mAb 3/E7 on paraffin slice of cancer of the prostate.
Figa/b. FACS-analysis of scFv E8 (a) and A5 (b) PSMA-expressing LNCaP cells at concentrations of saturation.
Figv/Curves, saturation antigen scFv E8 (C) and A5 (g).
Fig. Western blotting with purified PSMA and scFv A5 and E8.
Fig.9. Immunocytology scFv E8 on LNCaP cells.
Figure 10. The design and the construction of immunotoxin E8-RE.
11. The cytotoxic effect of recombinant immunotoxin E8-RE on LNCaP cells.
Fig. The cytotoxic effect of recombinant hybrid protein A5-RE on LNCaP cells.
Fig. The sequence of scFv E8. Shows the DNA sequence and amino acid sequence, with marked areas indicate areas CDR.
Fig. The sequence of scFv E5. Shows the DNA sequence and amino acid sequence, with marked areas indicate areas CDR.
Fig. Binding of scFv A5, H12 and D7 with PSMA-negative cells DU145 (a) and PSMA-positive LNCaP cells (A5=A, H12=B, D7=In). Cells were stained with mAb and PE-conjugated mAb to mouse IgG. Histograms represent the logarithm of the fluorescence Feh on a flow cytometer. Negative control was performed only with secondary antibody.
Fig. Binding of scFv A5, H12 and D7 with PSMA-negative cells BOSC (a) and PSMA-transfected BOSC cells (A5=B, H12=D7=D). Cells were stained with scFv anti-C-myc mAb and PV-conjugated artemisinin Ig. Histograms represent the logarithm of the fluorescence Feh on a flow cytometer. Negative control was performed only with secondary antibody.
Fig. The cytotoxic effect of recombinant immunotoxin NE-RE on LNCaP cells (black) and cells DU (white).
Fig. Diagram of the structure of a double anitelea-CD3.
Fig. The cytotoxic effect of the double antibody constructed of scFv A5 (A5/CD3), at various concentrations, and peripheral blood lymphocytes (target ratio of 10:1) on LNCaP cells after incubation for 48 hours
Fig. The sequence of scFv HI 2. Amino acid sequence is shown in single-letter code on the first line (corresponds to the sequence SEQ ID NO:20). The coding circuit is shown on the second line of (SEQ ID NO:19) and the complementary circuit is shown on the third line. This sequence corresponds to SEQ ID NO:23. CDR specifically designated as CDR H1, H2, H3, L1, L2 and L3. Nucleic acid sequences encoding the CDR field shown on a gray background.
Fig. The sequence of scFv D7. Amino acid sequence shown on the first line in the form of a single-letter code. This sequence corresponds to SEQ ID NO:22. The coding chain nucleic acid shown in the first line. This sequence corresponds to SEQ ID NO:21 and a complementary circuit is shown on the third line. This sequence corresponds to SEQ ID NO:24. In the sequence shown region CDR H1, H2, H3, L1 and L2. Nucleic acid sequences encoding these areas are shown on a gray background.
Further, the present invention is illustrated by the following examples.
a) Obtaining PSMA
Cell lines the carcinoma of human prostate LNCaP, DU 145, PC-3 and HeLa, and hybridoma E-S (IgG1-k, PSMA) get in the American type culture collection (ATSS), Rockville, Maryland, USA. Cells LNCaP, DU 145 and HeLa were cultured in medium RPMI 1640, PC-3 were cultured in the medium Nutrimix F12, both environments with the addition of penicillin (100,000 Units/l), streptomycin (100 mg/l) and 10% FTS at 37°C in a humid atmosphere with 5% CO2. To generate LNCaP cells expressing non-PSMA on the surface, in the environment add 2 μg/ml tunicamycin (firm ICN Biomedicals) within 48 hours
Fixed LNCaP cells treated with 4% paraformaldehyde for 10 min at room temperature and then washed thoroughly with the FSB.
To obtain purified PSMA 108cells LNCaP washed FSB and then are lysed in the FSB, containing 1% IGEPAL for 20 min at room temperature. After centrifugation at 10,000 g supernatant applied to a column for affinity chromatography E-C5 (firm Amersham Biosciences, Uppsala, Sweden) and elute PSMA 100 modernim glycine buffer, pH 2.5 and containing 1% Triton X-100. After neutralization extensive protein deleteroute using the FSB.
To prepare deglycosylation PSMA, 1/10 volume of glycoprotein-denaturing buffer (firm BioLabs) are added to a solution containing purified PSMA, and heated for 10 min at 100°C. Then add 1/10 volume of 10% NP-40 (10%) and 50 U f is rment PNGase on ug, PMA and incubated at 37°C for 1 h
To obtain a lysate of LNCaP cells containing full-sized native PSMA, the cells are lysed reagent M-PER, Pierce) for 10 min and then centrifuged at 15,000 rpm for 30 min at 4°C. the Supernatant containing the native full PSMA, collect (M-PER-lysate). High-molecular fraction (100 to 600 kDa) of the lysate separated by HPLC ion-exchange column Biosil 250.
b) Transfection of full-PSMA cells, DU 145 and RS
Full-PSMA clone in two fragments (fragment 1 from 262 BP to specific restriction site EcoRI when 1573 BP and fragment 2 from the position 1574 to 2512) into the vector pCR3.1 (firm Invitrogen). Temporary transfection is obtained by adding a mixture of 4 μg of DNA and 10 ál of lipofectamine (firm Invitrogen) in 500 μl of RPMI medium to 106the cells according to the Protocol of the manufacturer. After incubation for 48 h temporarily transfected cells used for research.
Immunization of mice
Four female mice of Balb/c mice subjected to immunization intraperitoneally 300 µg M-PER-lysate of LNCaP cells, or high molecular weight fraction HPLC lysate, or 106the LNCaP cells, fixed with 2% paraformaldehyde. These drugs are mixed in the ratio of 1:1 with complete adjuvant's adjuvant. Each mouse subjected to immunization 4 or 5 times with 2-week intervals. Later, four days after the last immunization cells Selezen is collected and used for receiving or hybrid or library cells.
Obtaining a library of b-cells
The spleen of the mouse is washed in phosphate-saline buffer solution (FSB), crushed to small pieces, washed again in the FSB and then gently homogenized in a "free" manual homogenizer. The resulting homogeneous cell suspension is layered on Ficoll (firm Pharmacia, Freiburg, Germany) and centrifuged at 400 g for 20 min at room temperature. Interphase b-cells are isolated using beads CD 19 on the instructions of the manufacturer (company Miltenyi, Gerbes Gladbach, Germany). In cells of 106are lysed in 350 μl of a solution consisting of 4 moles guanidinoacetate, 25 mmol of sodium citrate, 0.5% of N-lauroylsarcosinate sodium and 100 mmol 2-mercaptoethanol.
a) Obtaining a hybrid
Spleen aseptic remove and preparing a suspension of individual cells in medium RPMI-1640 containing no serum. Splenocytes add to the myeloma cells SP2/0 in the ratio 10:1 and according to generally accepted methods, conducting mergers and selection (Galfre and others, Nature, 1979, p.131-133).
Supernatant hybrid investigate FACS analysis on cells LNCaP and DU145 and ELISA with purified PSMA as a solid phase. Purification of monoclonal antibodies is performed using a column of protein G (firm Pharmacia).
b) determining the isotype mAb
Ig-isotypes mAb to PSMA define ELISA, IP is by using either unlabeled (solid phase), or peroxidase labeled (traces) of specific antibody isotypes against (company Southern Biotechnology Associates, Birmingham, Alabama).
C) Isolation and characterization of conformational monoclonal antibodies against PSMA
Spend the fusion of spleen cells of Balb/c mice, which were immunized 5 times M-PER-lysate of LNCaP cells with cells of the SP2/0 by conventional methods. Positive hybridoma selected by flow cytometry with LNCaP cells and ELISA on purified PSMA. In this way we obtain three positive clone. The corresponding mAb with their isotypes are 3/F11 (IgG2a), 3/A12 (IgG1) and 3/E7 (IgG2b).
d) Characterization of mAb
By flow cytometry, you notice that three mAb and stained cells LNCaP very good contact with the positive cells, the number of which is in the range from 95% to 98%. The shape of the curves of fluorescence relative to the number of events shows that PSMA is uniformly distributed in the population of cells LNCaP (figure 1). To assess the binding specificity of anti-PSMA mAb, PSMA-negative cells DU145, RS, HeLa cells and Jurkat also stained and analyzed by flow cytometry. None of the three mAb does not stain PSMA-negative cells (percentage of positive cells ranged from 0.04% to 2%).
Binding properties of three antibodies are compared by treating LNCaP cells growing conc what traceme primary anti-PSMA mAb followed by incubation with a saturating amount of secondary PV(phycoerythrin)-labeled goat antibodies and subsequent cytofluorometric analysis. At concentrations of antigenic saturation [100 nm] adjusted mean intensity fluorescence picaridin is approximately 1000 for mAb 3A 12, approximately 1,400 for mAb 3F11 and about 1600 for mAb 3E7. It is shown that for mAb 3A12 Seth 5-times lower than in LNCaP cells expressing non-PSMA (grown with tunicamycin).
By immunofluorescence Cytology and detection using laser scanning confocal microscope can show strong binding of the three mAb with LNCaP cells (figure 2), and their internalization in these cells (figure 3). All mAb positive in ELISA with purified PSMA as a solid phase. Denatured PSMA mAb shows the signal at about 100 kDa Western-blotting (figure 4), and blot with deglycosylation PSMA weak, giving a signal at about 84 kDa, which corresponds to literature data (figure 5).
Immunohistochemistry on paraffin sections cancerous prostate tissues are positive in the case of mAb 3/E7, but negative in the case of mAb 3/F11 and 3/A12 (6). Data are combined in table 1.
Based on these data we can conclude that the 3 mAbs show a very strong and highly specific binding to native and denatured PSMA. Although the binding deglycosylation PSMA is weak, the specificity of sugar can be excluded on the basis of thefact, what is not observed binding to cells that do not Express PSMA.
Obtaining a library of expression of scFv in fahmida pSEX
From the library of b-cells or hybridoma cells, allocate the total RNA and mRNA using membranes on silica gel (reagent Rneasy the company Qiagen, Hilden, Germany) according to the manufacturer's Protocol. Synthesis of cDNA is carried out at 42°C for 60 min in a final volume of 50 μl, containing 25 µl of denatured RNA, 10 μl of 5 × buffer (firm Promega, Heidelberg, Germany), 5 μl of 10 mm dNTP (dATP, dCTP, DSTF, dTTP, the company Promega), and 1.5 µl of RNA-Zina (40 U/ál, the company Promega), and 2.5 ál 150 gr arbitrary review of the primers, and 2.5 μl of AMV reverse transcriptase (10 U/ál, the company Promega). Then the coded parts of the heavy chains and gamma and Kappa-chains amplified by PCR according to the earlier description Orum and others in Nucleic Accept Res., 1993, s-4498. For each chain spend 25 separate reactions, a combination of 25 different primers for direct constant sites with one corresponding reverse primer. Purification of the amplified products for the light chains and heavy chains is performed by agarose gel electrophoresis.
The PCR products for light chains were cleaved by enzymes MluI and NotI, and are ligated to fahmida pSEX81 (Dübel and other Gene, 1993, s-101) at a molar ratio of 1:3 (2 μg of vector, 400 ng of insert). Products one ligating the use of the comfort for electroporation 50 ál electrocompetent the cells of E. coli XL1-blue, Stratagene) according to the Protocol of the supplier. Bacteria plated on nine of cups with a diameter of 80 mm with agarose medium containing 100 μg/ml ampicillin and 0.1 M glucose (SOB-AG), and incubated overnight at 30°C. Bacteria isolated by adding 3 ml of 2×YT medium on each Cup and scraping them sterile glass spatula, and precipitated at 3000 g for 15 minutes Of these bacteria produce plasmid DNA, which represents abilitec Vl. Then the PCR products for the heavy chain and abilitec Vl split by enzymes NcoI and HindIII. Ligation is carried out at a ratio of 3:1 (2 µg of abilitec and 400 ng of insert). Transformation by electroporation, seeding and selection of transformed bacteria is performed according to the description for abilitec Vl. With nine plates SOB-AG with a diameter of 80 mm are generally 18 ml library VHVL.
Obtaining and selection of phage displayed antibody
In the library VHVLin fahmida pSEX genes of antibodies bridesyou in frame with the gene III, which encodes the minor surface protein gIIIp filamentous phage. Therefore, the preparation of recombinant fahmideh particles, exposing on the surface of the antibody requires the Contracting carrier fahmida bacterial cell replication defective phage M13KO7 . M13KO7 added to 10 ml of library culture at a multiplicity of 10. After the Incubus is tion at 37°C for 90 min, the cells precipitated by centrifugation and resuspended in 15 ml of 2×YT-environment, containing 100 μg/ml ampicillin, 10 μg/ml tetracycline and 50 μg/ml kanamycin. The culture is incubated overnight at 37°C at 250 rpm/min, then cooled on ice and centrifuged to remove cells. The supernatant containing the phages are mixed with 1/5 volume of an aqueous solution containing 20% PEG 8000 and 14% NaCl, and incubated for 1 h at 4°C. Then centrifuged for 30 min at 4°C and 6200 g. The precipitate containing the phage, resuspended in 2 ml of 10 mm Tris/HCl, pH 7.5, 20 mm NaCl, 2 mm EDTA, pH 7.5 and used for panning.
b) Panning to select clones that recognize the antigen and the cell
Panning on purified PSMA is carried out in 96-well tablets for micrometrology Maxi-Sorb (firm Nunc)coated with a solution of purified PSMA (100 µl/well, 12 μg/ml of PSMA in the FSB), and blocked with 4% skim milk/FSB. One ml of the purified recombinant phage (approximately 1011) incubated in 1 ml of 4% skim milk/FSB with the addition of 15 μl of 10% Triton X100 for 15 min, then allow linking with 8 wells, coated with PSMA, for 2 h at 37°C. After 20 cycles of washing using a solution FSB/twin room (0,1%) related phage elute with 0.1 M glycine buffer, pH of 2.2. For penninga viable LNCaP cells the pre-phages adsorb to the cells, DU 145. For this procedure, 1 ml (approximately 1011) recombinant phage incubated in 2% fat-free milk/FSB for 15 min and is eaten with cells, DU 145 (10 7) for 1 h at room temperature on a shaker. After that, the cells centrifuged and the supernatant with unabsorbed phage incubated with LNCaP cells (106) for 1 h at room temperature on a shaker. After 10 washes with 2% skim milk/FSB and five washes FSB related phage elute 50 M HCl, followed by neutralization of 1 M Tris-HCl (pH 7.5).
Cells of E. coli TG1 infect lirovannye phages are plated in a Cup with SOB-AG and incubated over night at 30°C. For titration using an aliquot of the eluate. The selection process is repeated from three Yes six times.
in) "Salvation" small phage
Isolate individual colonies from the 96-well plate to micrometrology and each of them is transferred into a single well of 96-well plate with deep wells containing 500 μl of 2×YT-medium containing 100 μg/ml ampicillin and 0.1 mol of glucose (YT-AG), and incubated overnight at 37°C (main plate). Then 40 μl of a saturated culture of each hole of the main tablet is transferred into the corresponding wells of the new tablet containing 400 μl of 2×YT-AG-environment.
To each well add about 1×1010habernig phage MCO and incubated on a shaker for 2 h at 37°C. the tablet Then centrifuged, the precipitate is suspended in the environment 2×YT with the addition of 100 μg/ml ampicillin, 10 μg/ml tetracycline and 50 μg/ml Kana is of CIN and incubated at 29°C and 240 rpm during the night. After centrifugation the supernatant containing the saved family selected and used for the study of phages methods ELISA and flow cytometry.
d) the Study of the phage by ELISA
Tablets for micrometrology cover purified PSMA (1.5 mcg PSMA/ml FSB), incubated overnight and then blocked with 2% skim milk/FSB. To each well was added 200 μl saved fahmid, pre-incubated 1:1 with 2% skim milk/FSB, and incubated for 2 h at room temperature. After five washing steps using the FSB-Twin related phages detected using 200 μl/well anti-M13 antibody conjugated to horseradish peroxidase (firm Pharmacia)for 2 h at room temperature. As the substrate used with 3,3',5',5'-tetramethylbenzidine.
d) isolation and characterization of conformational scFv against PSMA
To generate conformational scFv against PSMA create library VHVLin fahmida pSEX from the library Into cells of a mouse immunized with M-PER-lysate of LNCaP cells. The size of this library is 107. Similarly prepare the library VHVLof monoclonal antibodies 3/A12, which is obtained from the same mouse immunized with lysate of LNCaP cells. The size of this library is 105. To select phages, the former is onerousa on its surface scFv, linking cell PSMA, in another embodiment, spend six cycles of panning in LNCaP cells after absorption cells DU-145 in polystyrene test tubes and microtiter tablets coated with 20 μg/ml purified PSMA. After three, four and six cycles of panning grow separate famiglie colony and phage particles rescued by infection MCO. Analysis 800 phage clones from a library of b-cells by flow cytometry with LNCaP cells and ELISA on purified PSMA revealed one positive clone, called E8. After the fourth cycle of panning library VHVLfrom mAb 3/A12 received two positive clone, called A4 and A5. By sequencing found that A4 is identical to E8.
Coding region of scFv E8 and A5 are transferred from family pSEX in the expression vector pHOG containing C-myc and His-tag at the C-end. The sequence with the corresponding CDR is shown in Fig 13 and Fig. The region encoding the CDR, antigenspecific parts marked on Fig and 14. These sequences should not be changed, while other parts of the sequence that are not marked, you can change. However, should remain the appropriate three-dimensional structure.
Using flow cytometry found that scFv E8 strongly interacts with a viable LNCaP cells, and the CIF value is approximately equal to 100 at concentrations is asimenia, while the A5 binding was much weaker and the CIF value is approximately equal to 40 at concentrations of saturation (Fig 7). In contrast, the binding of purified PSMA as a solid phase in ELISA was weak for E8 and slightly stronger for A5. This character was seen in Western-blots with glycosylated and deglycosylated PSMA (Fig). By immunofluorescence Cytology with LNCaP cells and detection by confocal laser microscopy can show a very good binding and internalization of scFv E8 (Fig.9). Data scFv are summarized in table 2.
|Feature 2 scFv against cell-poverhnostnogo PSMA|
|ScFv||Origin||FACS cells LNCaP [Seth]*||FACS PSMA-transformed cells DU [Seth]*||ELISA PSMA||Blot PSMA||Blot deglycosylation PSMA|
|E8=A4||B-cell library and mAb A12||100||70||floor||(floor)||(floor)|
|Seth = average intensity of fluorescence at a concentration of scFv reaching antigenic saturation (subtract background staining using only the secondary antibody)|
|gender = positive reaction|
|(floor) = weakly positive reaction|
Expression and purification of scFv
The scFv fragments Express in E. coli XL1-Blue, Stratagene), using the vector secretion pHOG 21, which contains sequences for His-6 and c-myc-tag at C-terminal position of scFv (Kipriganov and others, J.Immunol. Methods, 1997, pp.69-77). Bacteria E. coli, transformed with the plasmid pHOG, grown overnight in medium 2×YT-AG, then diluted in the ratio of 1:20 and grown in cultures, 600 ml, at 37°C. When the cultures optical density of 0.8, bacteria precipitated by centrifugation at 1500 g for 10 min and resuspended in the same volume of fresh medium YT containing 50 μg/ml ampicillin, and 0.4 mole of sucrose and 1 mmol isopropylthioxanthone (IPTG). After that, the growth continued at room temperature is f for 18-20 hours Cells are harvested by centrifugation at 5000 g for 10 min and 4°C. For extraction of soluble periplasmatic proteins precipitated bacteria resuspending 5% of the initial volume of ice-cold 50 mm Tris-HCl, 20% sucrose, 1 mm EDTA, pH 8.0. After incubation for 1 h on ice spheroplast centrifuged at 20000 g at 4°C for 30 min, collecting periplasmatic extract in the supernatant. Periplasmatic the extract was concentrated using membrane Amicon YM10 with the top edge cutoff molecular weight of 10 kDa, Amicon, Witten, Germany), followed by dialysis against 50 mm Tris-HCl, 1 M NaCl, pH 7.0.
Cleaning is performed by affinity chromatography with immobilized metal cations. Chromatographic carried out using a column volume of 1 ml with chelat forming separate (firm Pharmacia)loaded with Cu2+and balanced buffer containing 50 mmol Tris-HCl and 1 mol NaCl, pH 7.0. Download periplasmatic the extract was washed with equilibrating buffer, in an amount equal to twenty column volumes containing 30 mmol of imidazole and then elute with the same buffer containing 250 mmol of imidazole. Suirvey material deleteroute against the FSB.
Determination of protein content, make use of a set of Micro BCA Protein Reagent Kit (Pierce firm) according to the manufacturer's instructions.
The induction of protein synthesis in ucaut using IPTG and output scFv is approximately 20 µg 600 ml culture of E. coli XL1.
Vials for tissue cultures are harvested fresh cells LNCaP, DU 145 and RS and prepare a homogeneous suspension of cells in FSB with 3% FCS and 0.1% NaNa. Approximately 105cells incubated with 50 μl saved fahmid, preincubating in the ratio of 1:1 with 2% skim milk/FSB, for 1 h on ice. After 3 washing cycles using the FSB add 25 ál/well of anti-C-myc monoclonal antibodies A (10 μg/ml, the firm Becton Dickinson) or, in the case of testing phages, 25 μg/well anti-M13 antibody (10 μg/ml, the firm Pharmacia) and incubated for 40 min on ice. After triple washing FSB cells incubated with 100 μl of PV-labeled goat antimisting IgG (firm Becton Dickinson) for 40 min on ice. The cells are then washed again and resuspended in 100 μl of a solution containing 1 mcg/ml iodide propidium (firm Sigma, Deisenhofen) in FSB with 3% of FTS and 0.1% NaN3to exclude dead cells. The relative fluorescence of stained cells was measured using a FACScan flow cytometer and the application program CellQuest (firm Becton Dickinson, montain view, California).
The LNCaP cells grown on cover glasses within 24 hours For fixation of cells treated with 2% paraformaldehyde in FSB for 30 min at room temperature, which does not change the permeability to emochnoy membrane, washed with 1% BSA-FSB, quenched for 10 min in 50 mm NH4Cl in the FSB, and rinsed with 1% BSA-FSB. Add primary monoclonal antibody at a concentration of 4 μg/ml in 1% BSA-FSB and incubated for 60 min at 4°C. FITZ-labeled goat antimurine secondary antibody (2 μg/ml, firm Southern Biotechnology Associates Inc., Birmingham, USA) incubated for 30 min and washed thoroughly with 1% BSA-FSB. Microscopic preparations stored in Vectashield (firm Vector Laboratories, Inc. Burlingame, California).
For studies of internalization of the primary antibody incubated for 30 min at 37°C before fixation of the cells with 2% paraformaldehyde and disturbance of permeability of 0.5% Triton X100 in the FSB.
Enclosed in paraffin tissue sections first deparaffinized and then treated with 0.3% Triton X100 in FSB to restore antigen. Frozen sections fixed in cold acetone. Slices treated 30 min with 3% H2About2and 10% methanol to inhibit endogenous peroxidase. After blocking with 1% BSA-FSB add primary antibody at a concentration of 2 μg/ml and incubated for 1 h at room temperature. For scFv add a secondary mouse anti-C-myc antibody and incubated for 1 h at room temperature. Then biotinylated goat antimurine antibody incubated for 30 min at room the Oh temperature and finally show ABC reagent (product Vectastain).
b) Western blot analysis
Western blot analysis is performed using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (LTOs-PAG), purified PSMA and lysate of LNCaP cells and transfer to polyvinylidenedifluoride membrane. The blots block during the night the FSB containing 5% skim milk and incubated with purified mAb or scFv at a concentration of 10 μg/ml for 1 h Then the blots washed 5 times the FSB-Tween (0.5%) and incubated with goat artemisinin IgG, conjugated with horseradish peroxidase for 1 h at room temperature. After 5 washes in the FSB-Twin (0,5%), the blots are using 3,3',5',5'-tetramethylbenzidine as a substrate.
Construction, expression and purification of scFv-RE-proteins
Used according to the present invention, the toxin is a truncated version of the bacteria pseudonomas exotoxin (PE40), which has no domain Ia and which contains only domains Ib, II, and III (Pastan and others, J. Biol. Chem., 1989, her. 15157-15160). DNA coding region in the vector pSW200 received from Prof. W.Wels, Frankfurt (Wels, etc., Biotechnology, 1992, s-1132). The DNA fragment from BP in position from 253 to 613 encoding PE40, amplified by PCR from the plasmid pSW200. Then amplified DNA leading to a vector of pHOG-His-scFv in C-terminal position to the scFv using the restriction site XbaI. All stages of cloning perform according to the accordance with standard methods in E. coli XL1-blue and the products controlled by sequencing.
Protein induction immunotoxin and purification using metal-chelate chromatography (IMAC) similar to those for scFv. The products are examined and characterized by LTOs-SDS page, Western blot and flow cytometry.
Cytotoxicity of immunotoxins scFv-PE40
Metabolism of salt tetrazolium red WST to water-soluble formisano dye is determined in accordance with the manufacturers instructions (firm Boehringer). Target cells (LNCaP and DU 145 as control) inoculated in the amount of 2.5×104/well 96-hole tablet and grown for 24 h before formation of a confluent layer of cells. Add different concentrations of recombinant immunotoxins in the aliquot 50 μl/well and incubated the plates for 48 h at 37°C in an atmosphere of 5% CO2. After this time the culture is mixed with 15 µl/well reagent WST and incubated for 90 min at 37°C in an atmosphere of 5% CO2. Then on the spectrophotometer assess the optical density of the samples at a wavelength of 450 nm (control 690 nm). They calculate the concentration of immunotoxin required for 50% reduction in cell viability relative to that of untreated control cultures (50% inhibitory concentration = IC50).
Cytotoxic assays (WST) immunotoxins E8-R40 and A5-R40 spend with LNCaP cells expressing PS is A, and control cells, DU 145. Figure 11 shows that a high cytotoxic effect can be observed with the immunotoxin E8-RE in LNCaP cells, with an IC50 value of 0.05 nm. On Fig shows the cytotoxic effect of immunotoxin A5-RE, with IC50 is approximately 0,09 nm. Cytotoxic background on the cells, DU 145, not expressing PSMA, is 5% for E8-design and only 0,01% for A5-design, indicating a very good therapeutic window.
Getting scFv H12 and D7 of mAb 3/F11 and 3/E7
Each mAb as described in example 5, receive a library of expression of scFv in fahmida pSEX.
Obtaining and selection of phage displayed antibody is carried out according to example 6.
After six cycles of panning in different types of PSMA and LNCaP cells from mAB 3/E7 received one specific positive clone, called H12, and mAB 3/F11 received one positive clone, named D7. The coding region of each scFv transferred into the expression vector pHOG-21.
Expression and purification of scFv perform according to the description in example 7.
Characterization of scFv H12 and D7
(a) Flow cytometry on PSMA-positive and PSMA-negative cell lines
According to the assessment by flow picometre scFvs H12 and D7 interact with viable LNCaP cells.
On the basis of the saturation curves established that the antibody concentration, d is Schauma 50% saturation plots PSMA, approximately 120 nm (H12) and 20 nm (D7) respectively. At concentrations of saturation is achieved values Seth, representing 70 (H12) and 40 (D7) (Fig).
In order to evaluate the PSMA-binding specificity of the scFv H12 and D7 additionally stained and analyzed by flow cytometry PSMA-negative cancer cells DU145 prostate and RS and other PSMA-negative cell lines (HeLa, MCF7, NST and Jurkat). Neither of the two scFv does not stain PSMA-negative cells.
b) Flow cytometry on the transfectants PSMA
To confirm PSMA-specific binding of scFv H12 and D7 also examined the cells BOSC-23, transfected PSMA. Both scFv show dependent on the concentration of binding to the BOSC cells, transfected PSMA full length, but not associated with atransferrinemia cells (Fig). Conditions of saturation is achieved at 100 nm (D7) and 200 nm (H12). Like mAb, values Seth on transfecting lower than in LNCaP cells, and show a wide scatter, which may correspond to a change of PSMA molecules on the surface of the transfectants.
C) Immunofluorescence Cytology
Immunofluorescence Cytology carried out as described in example 4. When detection using laser scanning confocal microscope observed strong binding of scFv with LNCaP cells and internalization in these cells.
g)the ELISA and Western-blotting
Binding of scFv H12 and D7 with purified PSMA is expressed weakly in the ELISA and Western-blotting.
Sequence (amino acids and nucleic acids) H12 and D7 shown in Fig and Fig.
Design and cytotoxicity of immunotoxins H12-RE and immunotoxin D7-PE40
Designing immunotoxins H12-RE and D7-PE40 carried out by analogy with immunotoxins A5 and E8 as described in example 11. D-40 is a fragment of exotoxin bacteria Pseudomonas.
The cytotoxicity was examined as described in example 12.
Immunotoxin stimulated LNCaP cell death time-dependent manner, with the highest cytotoxicity can be observed after 48 h of incubation.
At this time, the IC50 values of about components 200 PM detected for H12-RE and D7-PE40 (Fig).
Additionally examine the cytotoxicity H12-RE and D7-PE40 on PSMA-negative cell lines DU 145, PC-3, MCF7 and HCT 15. These cell lines cytotoxicity was not detected at concentrations up to 25000 PM.
Design of anti-PSMA/CD3 double antibody
Produce bespecifically double antibody having specificity for PSMA and the chains of the CD3 T-cell receptor complex. Bespecifically double antibody Express in E. coli using the vector containing dicistronic operon for consecratio scFv VhCD3-lA5 and VhA5-V1CD3 (Fig). To construct anti-A5/CD3 double antibody used plasmids pKID19×3 and pKID 3×19 (Kipriyanov, Int. J.Cancer, 1998, s). Bacterial periplasmatic expression and purification similar to these indicators scFv.
The induction of specific toxicity of the double antibody A5-CD3
The ability especifismo double antibody to induce lysis of tumor cells by redirecting mediated T-cell cytotoxicity explore using mononuclear cells of peripheral blood (MCPC) from healthy donors as cell effectors. After incubation with IL-2 or without it for 4 days, the cells are added to the target cells LNCaP, which are seeded at a density of 1.5×104cells/well in 96-hole plate. The ratio of the effector-target is 10:1. Double antibody is added in various concentrations. After incubation for 48 h culture mixed with 15 μg/reagent WST and incubated for 90 min at 37°C in an atmosphere of 5% CO2. Then by spectrophotometry evaluate the optical density of the samples at a wavelength of 450 nm (control 690 nm).
In this test, in vitro dual antibody, apparently, has the clear ability to divert activated and unactivated MCPC to cause lysis of the target cells LNCaP-dependent concentration follows (Fig).
Off the th antibody A5-A5
This bivalent monospecific double antibody receive is similar to getting a double antibody A5-CD3 (example 16). Bacterial periplasmatic expression and purification similar expression and purification of scFv.
By flow cytometry it is possible to show strong and specific binding of the double antibody A5-A5 with LNCaP cells.
1. Isolated monoclonal antibody (mAb) or antigennegative fragment, which
a) bind with specific to prostate membrane antigen (PSMA) in its native form, present on the surface of tumor cells,
b) have the ability to be internalized by the cancer cell,
in) are strongly associated with LNCAP cells, but not in contact with cells that have lost expression specific to prostate membrane antigen
g) associated with a label or a cytotoxic agent, characterized in that
d) they include amino acid sequence selected from the group comprising SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:20 and/or SEQ ID NO:22.
2. An isolated monoclonal antibody or its antigennegative fragment according to claim 1, wherein the monoclonal antibody or its antigennegative fragment have Feh degree of intensity of fluorescence (SIF), which exceeds 1000, and Feh degree of fluorescence intensity (CIF) scFv that p is Evesham 40 when the saturation of the antigen.
3. An isolated monoclonal antibody or its antigennegative fragment according to claim 1 or 2, which show high activity binding to LNCAP cells, with 50% saturation PSMA-sections is achieved at concentrations of 1 nm-120 nm.
4. An isolated monoclonal antibody or its antigennegative fragment according to claim 1, wherein the label is a particle which emits radioactive or fluorescent radiation.
5. An isolated monoclonal antibody or its antigennegative fragment according to claim 1, wherein the cytotoxic agent is toxic to cells by a substance selected from the group consisting of Taxol, a fragment of Pseudomonas exotoxin, cytochalasin B, gramicidin D, ethidium bromide, emetina, mitomycin, etoposide, anapsida, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and/or puromycin.
6. An isolated monoclonal antibody or its antigennegative fragment, which
a) bind with specific to prostate membrane antigen (PSMA) in its native form, present on the surface of tumor cells,
b) have the ability to undergo internalization is wholeway cell,
C) contact the LNCAP cells, but not in contact with cells that have lost expression specific to prostate membrane antigen, and
g) associated with a label or a cytotoxic agent and is distinguished by the fact that
d) include the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, or include the amino acid sequence of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, or CDRs, designated CDR H1, H2, H3, L1, L2 and L3, as shown on Fig, or the corresponding CDR regions, as shown in Fig.
7. Pharmaceutical composition for treating prostate cancer, comprising an effective amount of an isolated monoclonal antibody or its antigennegative fragment according to claim 1.
8. Pharmaceutical composition for treating prostate cancer, comprising an effective amount of an isolated monoclonal antibody or its antigennegative fragment according to claim 6.
9. Diagnostic kit for detecting prostate cancer, comprising an isolated monoclonal antibody or its antigennegative fragment according to claim 1.
10. Diagnostic kit for detecting prostate cancer, comprising an isolated monoclonal antibody or its antigennegative fragment according to claim 6.
11. The method of identification in vitro of tumor cells, wherein the cells are subjected to contact with isolera the data monoclonal antibody or its antigennegative fragment according to claim 1 and identify cells, which bind the specified isolated monoclonal antibody or its antigennegative fragment as tumor cells.
12. The method of identification in vitro of tumor cells, wherein the cells are subjected to contact with an isolated monoclonal antibody or its antigennegative fragment according to claim 6 and identify cells that bind the specified isolated monoclonal antibody or its antigennegative fragment as tumor cells.
13. Method for diagnostic identification of tumor cells, wherein the cells to be identified, exposed to contact with the isolated monoclonal antibody or its antigennegative fragment according to claim 1 and identify cells that bind the specified isolated monoclonal antibody or its antigennegative fragment as tumor cells.
14. The diagnostic method of identifying tumor cells, wherein the cells to be identified, exposed to contact with the isolated monoclonal antibody or its antigennegative fragment according to claim 6 and identify cells that bind the specified isolated monoclonal antibody or its antigennegative fragment as tumor cells.
15. Isolated polynucleotide, selected from the group status is the present of SEQ ID NO:8, 9, 17, 18, 19, 21, 23 and 24 and their corresponding combination to obtain monoclonal antibodies or their binding fragments according to claim 1 or 6.
SUBSTANCE: method for prediction of a risk of chronic dust bronchitis in coal mine workers consists in blood examination for genetic markers. It involves the genetic markers of systems: haptoglobin (HP), group-specific component (GC), fluorescent esterase (EsD), erythrocyte acid phosphatase (AcP). A prognostic coefficient (PC) score is calculated. If the marker 1-1 is found in the HP system, the PC is specified to be equal to (+1.5); the marker 1-2 makes the PC to be equal to (+0.1), while the marker 2-2 shows the PC (-1.1). If the marker 1-1 is found in the GC system, the PC is specified to be equal to (-1.9); the marker 1-2 makes the PC to be equal to (+1.7), while the marker 2-2 shows the PC (+5.5). If the marker 1-1 is found in the EsD system, the PC is specified to be equal to (-1.2); the marker 1-2 makes the PC to be equal to (+2.3), while the marker 2-2 shows the PC (+3). If the AcP system comprises the marker (aa), the PC is equal to (-5); the marker (ab) shows the PC equal to (0); the marker (bb) provides the PC to be equal to (+3.6); the marker (ac) - the PC equal to (+2.2), the marker (bc) - the PC equal to (-2.4), the marker (ee) - the PC equal to (-3.5). The PC for all the markers are summed up; total PC being (+5) and more enables predicting the propensity for chronic dust bronchitis, while total PC equal (-5) and less shows resistance to chronic dust bronchitis.
EFFECT: use of the declared method enables higher accuracy of prediction of chronic dust bronchitis in coal mine workers.
1 tbl, 4 ex
SUBSTANCE: positive portion of the panel comprises patient's plasma samples positive to RNA VHS and is presented in the form of 4 boxes accommodating NS5, NS4, NS3 and Core antigen antibodies respectively. A negative portion of the panel comprises donor's plasma samples containing other than HIV-1/2, HBV, HCV, syphilis, HBsAg antibodies, as well as from HCV-noninfected risk groups with additionally included heterophillic serums with rheumatoid factor (RF) and with autoimmune antibodies for strict control of specificity.
EFFECT: method improvement.
5 cl, 5 tbl, 1 dwg
SUBSTANCE: sequential staining by Romanowsky-Giemsa and conducting an immunocytochemistry reaction are used to analyse destructive processes in epitheliocytes nuclei, specifically changed lymphoid and epithelial cells, high colonisation activity of opportunistic and pathogenic microorganisms, and phagocytic activity. If observing brown-black inclusions in cells under optical microscopy ensured by specific staining of the viral antigens in the cell structures, mono- or mixed herpes viral infection Herpex simplex 1, 2, EBV, CMV are diagnosed.
EFFECT: using the technique enables higher accuracy of etiological diagnosis, expressivity and reduced injures of material sampling for research.
SUBSTANCE: recipient of a cadaver kidney graft is examined to compare recipient's and donor's HLA alloepitopes. The alloepitopes found in the donor, but not found in the recipient are recovered. It is followed by comparing the recipient's HLA-DR antigens and the recovered alloepitopes, the derived combinations are compared with the following combinations: A9C-DR7; A10C- DR3; A10C-DR6; A28C-DR1; Bw4-DR6; B8C-DR3; B12C-DR2; B27C-DR5. Detecting said combinations enables stating an adequacy of the preventive application of monoclonal antibodies blocking an alpha-chain of an interleukin-2 receptor molecule. The preventive application of the monoclonal antibodies blocking an alpha-chain of the interleukin-2 receptor molecule shall be indicated to the patients with said combinations.
EFFECT: improved outcome of the cadaver kidney grafting that is combined with reducing the price of the method.
1 tbl, 2 ex
SUBSTANCE: method is ensured by the double evaluation of total endotoxin (LPS) of gram-negative bacteria: prior to the beginning of the inhalation antibacterial therapy and one hour after the first antibiotic inhalation. It involves controlling the total endotoxin of the gram-negative bacteria in patient's blood serum by activated particle method. Double and higher endotoxin increase as compared with the reference testifies to a bactericidal action of the antibiotic and proves the effectiveness of the conducted antibacterial therapy.
EFFECT: using the method enables the early estimation of the effectiveness of the antibacterial therapy.
SUBSTANCE: invention describes a method for prediction of reduced transport function of band-3 protein of red-cell membranes of newborns delivered by mothers suffered aggravated herpes viral infection in the third trimester of pregnancy wherein newborn's peripheral blood is examined for the TNFa content; band-3 protein is detected in the newborn's red-cell membranes by disk electrophoresis in polyacryl amide gel (10%) with sodium dodecyl sulphate; a discriminant function is calculated by formula Df=(5.167×band-3)+(2.196×TNFα), wherein Df is the discriminant function, band-3 is red-cell membrane stromal protein, TNFα is newborn's blood interleukine, and if Df is more than 218.67, reduced transport anion function by protein band-3 through newborn's red-cell membranes is predicted.
EFFECT: invention provides detecting the effect of herpes viral infection on the content of protein band-3 in newborn's red-cell membranes.
SUBSTANCE: peripheral venous blood of the patients with autoimmune thyroiditis is examined for the absolute transferring receptor (CD71+) T-lymphocyte count and the interleukin-2 (IL-2) concentration. If the CD71+ T-lymphocyte count exceeds 0.3×109 cell/l, and the IL-2 concentration is less than 25 pg/ml, a favourable clinical course of the disease is predicted, while the CD71+ T-lymphocyte count less than 0.3×109 cell/l and the IL-2 concentration exceeding 25 pg/ml shows an unfavourable clinical course of autoimmune thyroiditis.
EFFECT: method provides more accurate and objective prediction with using the material, reagents and research methods widely applied in common clinical-laboratory practise.
SUBSTANCE: patient's peripheral blood is examined for the content of cytotoxic lymphocytes, %: (CD8/CD16), (CP8/GranzymeB), (CP16/GranzymeB), (CDS), (CD16/CD56) and (CD8/HLA DR). It is conmbined with determining cytotoxic activity (CTA), %. It is followed by calculating a RA activity index (AI) by formula: If observing the condition 11.8%<AI≤18.2%, a moderate degree of rheumatoid arthritis activity is diagnosed. If observing the condition AI>18.2%, a high degree of rheumatoid arthritis activity is diagnosed.
EFFECT: using the technique enables high-reliability differential diagnosis of the degree of rheumatoid arthritis activity.
SUBSTANCE: there are involved recording clinical and laboratory manifestations of a CNS injury during the first days of the disease that is followed by the calculation of total diagnostic coefficients related to the detected graduation levels of pathognomonic signs of the disease. Total (+)7 points and more is related to predicting the developing mixed tick-borne encephalitis and borreliosis infection. Total (-)8 points and less shows the developing potential monoinfection of tick-borne encephalitis. If deriving the intermediate values of total diagnostic coefficients when none of said limits is reached, the prognosis is uncertain.
EFFECT: using the method for stating basic tendencies of the developing pathological process at the early stages of the developing disease.
1 tbl, 2 ex
SUBSTANCE: with using indirect immunofluorescence and monoclonal antibodies, the patients suffering gastric ulcer (GU) are examined for an expression level of CD8, CDDR markers by peripheral blood lymphocytes with the CD8 level exceeding 24.5% and the CDDR level exceeding 15% enabling diagnosing a severe clinical course of GU.
EFFECT: use of the offered diagnostic technique provides high information value in diagnosing the severe clinical course of gastric ulcer, well-timed prediction of the severe clinical course of GU, instant diagnosing, provides well-timed and adequate complex of therapeutic measures.
1 tbl, 1 ex
SUBSTANCE: invention represents a human monoclonal antibody or its fragment which specifically binds with primate GM-CSF and neutralise it. There are also presented a polynucleotide molecule, a pharmaceutical composition, application of the monoclonal antibody.
EFFECT: invention may be used for neutralising higher or undesired activity of primate GM-CSF.
22 cl, 34 dwg, 5 tbl, 11 ex
SUBSTANCE: there are offered: recovered polynucleotides and polypeptides for binding human EGFR as a part of an antibody, a vector and a host cell for antibody expression, a method for producing an anti-EGFR antibody and an antibody fragment, the anti-EGFR antibody and the antibody fragment. There are discussed a composition containing the anti-EGFR antibody or its fragment, as well as applying the antibody and its fragment for treating EGFR-associated disorders. Besides, there are described versions of glycosylation of the anti-EGFR antibody or its fragment.
EFFECT: invention can find further application in therapy of various EGFR-mediated diseases.
30 cl, 6 ex, 32 dwg, 39 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology and biotechnology. There are offered versions of a TNF-alpha polypeptide wherein a number of heavy-chain single domain antibodies is equal to two. What is described is coding nucleic acid. There are disclosed versions of the compositions, a polypeptide-based kit, as well as versions of using it. What is described is a method for producing the polypeptide with using nucleic acid.
EFFECT: use of the invention provides the substantial growth of cytotoxicity lC50 (of the order of 10-9) that can find further application in therapy of TNFα-mediated disorders.
52 cl, 18 dwg, 11 tbl, 9 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: this invention relates to biotechnology and immunology. One proposes: JAM-A protein antibody or functional fragment thereof, hybridoma secreting such antibody, nucleic acid, expression vector and host cell as well as a method for the antibody and composition production. One considers application of the JAM-A protein antibody or functional fragment thereof.
EFFECT: invention usage ensures creation of new JAM-A protein antibodies which may be further applied in treatment or prevention of diseases related to proliferation of tumour cells extracting JAM-A protein.
34 cl, 31 dwg, 5 tbl, 19 ex
SUBSTANCE: polypeptide (versions) immunogenic with respect to meningococcal infections contains: an amino acid sequence at least 90 % identical to a sequence presented in the description (SEQ ID NO: 32), or said amino acid sequence, or a fragment of 80 sequenced amino acids of said sequence. What is described is an antibody which contacts with the polypeptide under the invention and which may be used as a drug. What is described is nucleic acid of the preset structure which codes the polypeptide or its versions and which may be used for treating or preventing a disease and/or an infection caused by Neisseria meningitides. The invention provides additional polypeptides applicable in advanced vaccines for preventing and/or treating meningococcal meningitis. The peptides can also find application in diagnosing of the disease and as targets of antibiotics.
EFFECT: higher clinical effectiveness for meningococcal meningitis.
19 cl, 2 tbl
FIELD: chemistry, pharmaceutics.
SUBSTANCE: present invention refers to biotechnology and immunology. There are offered: a monoclonal antibody or its fragment specifically bound with GDF8 and not bound with BMP11, a polynucleotide coding it, an expression vector and a host cell for antibody expression. There are studied: a method for producing the GDF8-specific antibody, a method for GDF8 presence test, a method of treating a GDF8-associated disorder.
EFFECT: use of the invention provides the new GDF8-specific antibodies that can find further application in the therapy of the GDF8-mediated diseases.
22 cl, 33 dwg, 8 tbl, 7 ex
SUBSTANCE: what is offered is an antibody or its antigen-binding fragment which specifically coupling hlL-4R with KD less than 200-pM measured with using surface plasmon resonance. What is described is a recovered nucleic acid molecule coding the antibody, and a based vector for producing the antibody. There are disclosed a host-vector system for producing the antibody or its antigen-binding fragment, and a method for producing the substances stated above with using such system. What is disclosed is using the antibody or antigen-binding fragment for preparing a drug for relieving (inhibiting) hlL-4R mediated diseases. What is disclosed is a composition on the basis of the antibody or antigen-binding fragment to be used in a method for treating a hlL-4R mediated disease or disorder in humans.
EFFECT: inventions can find application in therapy of the hlL-4R mediated diseases.
15 cl, 3 dwg, 5 tbl, 6 ex
FIELD: immunology and bioengineering.
SUBSTANCE: present invention refers to immunology and bioengineering. The variants of an antisubstance that is specific in relation to at least one globulomer Aβ(20-42) have been suggested. Each of the variants is characterized by the fact that it includes VH and VL parts; each of these parts contains three corresponding CDR. The antisubstance antigen-binding section has been revealed. They described: a coding nucleic acid and the vector that contains it, and a host cell that bears the vector that are used for the antisubstance production. The way of antisubstance production with the use of a cell has been discovered. The suggested inventions can find their application in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.
EFFECT: antosubstances that can be used in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.
10 cl, 28 dwg, 9 tbl, 12 ex
SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.
EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.
227 cl, 25 dwg, 16 tbl, 14 ex
SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.
EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.
43 cl, 20 dwg, 10 ex
SUBSTANCE: disclosed is an isolated thymic stromal lymphopoietin protein (TSLP) or antigenic fragment thereof for inducing immune response, as well as a nucleic acid molecule coding it.
EFFECT: invention can find further use in therapy of atopic diseases.
7 cl, 8 dwg, 2 tbl, 7 ex