Method of imaging of astroglial bank in diagnosing of high-grade gliomas

FIELD: medicine.

SUBSTANCE: delimitation of a high-grade glioma invasion is ensured by imaging of an astroglial bank surrounding the high-grade glioma. An immunogenic recombinant human GFAP is prepared and used to immunise a Balb/C mouse; spleen B-lymphocyte of this mouse are recovered and fused with myeloma cells of Sp 2/0-Ag14 mice; hybridomas are produced. Supernatants of the prepared hybridomas are tested by immunochemical techniques for the presence of anti-GFAP antibodies used to select a hybrid cell clone producing the anti-GFAP antibodies able to distinguish GFAP in vivo. The anti-GFAP antibodies are cleaned from the supernatant of the selected clone and covalently bound with liposomal nanocontainers containing a diagnostic mark. The antibodies of the selected hybrid cell clone is modified by g-amino groups of lysine residues and incubated with the stelths-liposome solution. The prepared nanosystem is introduced in a patient's vascular bed, and the astroglial bank is imaged by the arrangement of the diagnostic mark in cerebral tissues.

EFFECT: method allows preventing relapses of high-grade gliomas after their surgical management by choosing an optimal extent of a pending surgery ensured by delimitation of a tumour invasion by means of imaging of the astroglial bank.

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The invention relates to medicine, namely neurosurgery, can be used for in vivo diagnosis of poorly differentiated gliomas and prevention of postoperative recurrence.

In the structure neurooncological diseases 40-45% owned by the immature gliomas. The prognosis for patients with malignant gliomas is quite gloomy, despite considerable progress in modern neurooncological practice. The reason for this state of the problem is the inefficiency of the existing strategies for the treatment and diagnosis of the so vysokointensivnykh tumors. Quite often malignant glial cell are distributed throughout the brain. Active migration Gymnich cells excludes radical surgery of glioblastoma, resulting in a relapse of tumor growth after surgical treatment is inevitable, despite aggressive postoperative use of adjuvant radiotherapy and/or chemotherapy.

Thus, the ability to recognize brain tumor in early disease is a necessary condition to prevent mass of the impacts of invasive growing tumor to adjacent structures of the nervous tissue. Currently, however, modern imaging technologies are not sensitive enough to detect tumors at the Russian Academy of Sciences is her stage, especially for invasive visualization of the cords of tumor (Shaheen E. Lakhan and Lindsey Harle "Difficult diagnosis of brainstem glioblastoma multiforme in a woman: a case report and review of the literature J Med Case Reports. 2009; 3: 87).

In a period of active development of modern neuro-Oncology was conducted by a large number of studies in the treatment and diagnosis of the multiform glioblastoma. Actively developing areas such as immunotherapy, gene therapy and targeted drug delivery through the blood-brain barrier (BBB) (Gupta C. et al. "Monoclonal antibody 2C5-modified doxorubicin-loaded liposomes with significantly enhanced therapeutic activity against intracranial human brain U-87 MG tumor xenografts in nude mice" Cancer Immunol Immunother. 2007 Aug; 56(8):1215-23). It should be noted that significant progress in the treatment of a glioblastoma practically is not achieved, because still no developed access directly to tumor cells that migrated from the primary focus on perinatalny and perineurally ways. (Hefti M et al. "Multicentric tumor manifestations of high grade gliomas: independent proliferation or hallmark of extensive disease?", J.Cen Eur Neurosurg. 2010 Feb; 71(1):20-5.).

In the literature there is a publication which describes how the visualization of poorly differentiated gliomas method of magnetic resonance imaging (Thu MS et al. "lron Labeling and Pre-Clinical MRI Visualization of Therapeutic Human Neural Stem Cells in a Murine Glioma Model, J. PLoS One. 2009 Sep 29;4(9):e7218). All activities are conducted within the framework of the pilot experiment A Pilot Feasibility Study of Oral 5-Fluorocytosine and Genetically modified Neural Stem Cells Epressing Escherichia coli Cytosine Deaminase for Treatment of Recurrent High-Grade Gliomas and more focused on the study of therapeutic effect of neural stem cells.

The way the visual boundaries of tumor invasion, poorly differentiated gliomas for the purpose of diagnosis has a number of advantages, since a high frequency of postoperative recurrence of glioblastoma due to its invasive growth (Zalutsky M.R et al. "Dosimetry and Radiographic Analysis of 1311-Labeled Anti-Tenascin 81C6 Murine Monoclonal Antibody in Newly Diagnosed Patients with Malignant Gliomas: A Phase II Study", Journal of Nuclear Medicine Vol.46 No.6 1042-1051 2005). Tumor cells, quickly spreading on perinatalny and perineurally spaces, form a long, thin strands of invasion, macroscopically invisible, so that the tumor cannot be completely removed (Sanson M et al "FABP7 expression in glioblastomas: relation to prognosis, invasion and EGFR status", J Neurooncol. 2007 Sep; 84(3):245-8).

Thus, the complexity of the visualization of gliomas is the nature of the invasive tumor growth, in which her long strands are embedded deeply in the unaffected brain tissue. It is known that the boundaries of tumor invasion outlined astroglial shaft, repeating the configuration of borders of invasion (Chekhonin et al., 2008).

In the literature we have found ways of visualizing poorly-differentiated gliomas by astroglial shaft.

The objective of the invention is to develop a method of in vivo visualization of the boundaries of the invasion, poorly differentiated gliomas by astroglial shaft, maintainer of the tumor.

The technical result is to PR the prevention messages recurrence of poorly differentiated gliomas after their surgical treatment, by choosing the optimal amount of the forthcoming operation by determining the boundaries of tumor invasion through visualization astroglial the shaft of the tumor.

It is important to emphasize that the cytoplasmic GFAP protein in the normal functioning of the body inaccessible to circulating in the blood of antibodies. In the process of tumor growth, the number of reactive astrocytes increases as the invasion they collapse, accompanied by the release of GFAP in the extracellular space, where the protein is soon disposed in the blood and cerebrospinal fluid, where the level of growth of the tumor increases.

We found that GFAP reactive astrocytes merigliano shaft available for circulating in the blood nanosystems based anti-GFAP antibodies in poorly-differentiated gliomas. Received nanosystem able to penetrate through pathological, blood-brain barrier from the bloodstream in the region gliomas, which allowed us to visualize astroglial shaft during tumor invasion.

The invention consists in the following. Defining the boundaries of the invasion, poorly differentiated gliomas carried out by visualization astroglial shaft surrounding the immature glioma. Get immunogenic recombinant human GFAP. Subjected to immunization them mice of Balb/C Allocate-lymphocytes in the spleen of this mouse and merge with murine myeloma cells line Sp 2/0-Ag14. Get hybridoma. Test supernatant obtained hybrid immune is chemically for the presence of anti-GFAP antibodies. According to the results of immunochemical testing of them selected clone hybrid cells that produce anti-GFAP antibodies capable of recognizing GFAP in vivo. Of supernatant selected clones of hybrid cells secrete antibodies, and covalently associated with the liposomal nanocontainers containing the diagnostic label. For the conjugation of anti-GFAP antibodies with the liposomal nanocontainers antibodies selected hybridoma modify on the ε-amino lysine residues. Then incubated with a solution stelths-liposomes. Received nanosystem in the amount injected into the bloodstream of the patient. Visualization astroglial shaft for positioning a diagnostic label in brain tissue.

In the particular case, the received nanosysteme enter in the amount of not less than 500 μg/kg of patient's weight for protein.

In the particular case, for selection of clones producing anti-GFAP antibodies capable of recognizing GFAP in vivo method used immunochemical testing on a live culture of astrocytes and/or rats.

In the particular case, to increase the production of anti-GFAP antibodies conduct the introduction of selected clones of hybrid cells that produce anti-GFAP antibodies capable of recognizing GFAP in vivo, administered intraperitoneally to mice of Balb/C. Further, not less than 7 days, has been taken from them ascitic fluid. Of p is obtained ascites secrete antibodies by the method of immunoaffinity chromatography using GFAP, covalently bound CN-Br-separate.

Modification of the anti-GFAP antibody selected hybridoma on ε-amino lysine residues can be carried out, for example, using 2-aminosilane.

The invention is illustrated by the following figures.

Figure 1 shows a slice of the brain of the rat, where it was shown in vitro immunochemical glioma, surrounded astroglial shaft, where position 1 - astroglial shaft, position 2 - glioma 10 days after the implantation Gymnich cells in the rat brain.

Figure 2 presents the plot merigliano space, where there are invasive fibers (item 3) at lower magnification. Figure 3 presents the plot merigliano space surrounded by reactive astrocytes, at high magnification, where the position of the 4 - invasive heavy at high magnification, the position of the 5 - reactive astrocytes, spowoduje invasive heavy growing gliomas at high magnification.

Figure 4 presents the results of imaging of gliomas at astragalina shaft in vivo, where a is the visualization astroglial shaft in vivo at low magnification, and B - visualization astroglial shaft in vivo at high magnification.

The method is as follows.

To obtain immunogenic recombinant human GFAP can be used, for example, the technique in detail izlojena in the Ph.D. thesis of "Getting and immunokine is a mini-analysis of recombinant GFAP. Development of a quantitative enzyme immunoassay" (K.A. Pavlov, 2009).

A monoclonal anti-GFAP antibodies produced by hybridization of mouse myeloma line Sp 2/0-Ag14.1 with lymphocytes in the spleens of immune mice of Balb/c mice according to the method of Keller and Milstein (1976). Immunization conduct highly purified recombinant human GFAP. Subcutaneously three-point injected purified product GFAP (20 μg) with an equal volume of complete adjuvant's adjuvant at intervals of 1 every 10 days, with subsequent 2-month break. Then repeat the cycle immunization and on the 5-6th day after the second cycle of immunization injected intraperitoneally with 50 µg GFAP mixed with incomplete adjuvant's adjuvant. Efficacy assessed by the method of immunodiffusion and direct enzyme immunoassay. Hybridization is performed on the 3-4th day after the last injection of antigen in the ratio of myeloma cells and b lymphocytes 1:10. Hybrid clones are selected on selective medium containing gipoksantin, thymidine, aminopterin. The screening of hybridomas for the production of anti-GFAP antibodies spend methods sandwich option ELISA, immunohistochemistry and immunocytochemistry in vitro and in vivo. According to the results of immunochemical testing in vivo, in particular, on the living culture of astrocytes and/or rats are selected clone hybrid cells capable of recognizing GFAP in the native conformation. When ku is tipirovanii selected clones of hybrid cells in the peritoneal cavity of Balb/c mice (1-2 million left mouse), pre-sensitized by piers, formed ascitic tumor. Ascitic fluid (3-10 ml per mouse) receive in 7-10 days after inoculation of the cells. Purification of monoclonal antibodies from ascites performed using affinity chromatography on CN-Br-sepharose with immobilized protein GFAP according to the standard Protocol with elution by pH gradient.

Thus produced selected clone hybrid cells monoclonal antibodies are highly sensitive, specific and suitable for use in vivo.

Preparation of nanosystems consists of the preparation of liposomal nanocontainers with the diagnostic label and subsequent conjugation with anti-GFAP antibodies selected clones of hybrid cells.

Synthesis Paglierani liposomal nanocontainers or stelth-liposomes is carried out according to the usual method, modified in accordance with the objectives of a particular experiment (Kamps et al., J.Drug Target", 2000, 8(4), 235-245). The modification consists in the following.

The main components of stelth-liposomes are: lecithin from egg yolks of eggs, cholesterol and distearoylphosphatidylcholine (cells of the dspe), conjugated with PEG-2000 (peg 2000). To link to etiolirovannye antibodies to liposomes injected maleimide derived cells of the dspe-PEG-2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide-(polyethylene shall glycol)-2000)). In the molecule maleimide group attached to the external end of the chain PEG-2000, which allows "print" protein-vector outside the shielding layer of PEG.

With stelth-liposomes covalently bind any diagnostic label depending on the method of its detection, for example a fluorescent label, radioactive label (method scintigraphie), MRI-contrast(nanocontainers with contrast agent).

For the introduction of anti-GFAP antibody fragment, containing the SH group, antibodies modified by ε-amino lysine residues with 2-aminothieno in 0.1 mol/l sodium phosphate buffer at a concentration of 1 mg/ml in dark conditions for 1 h under argon at room temperature. After stopping the reaction etiolirovaniya from excess initialen released by gel filtration through Sephadex G-25.

Etiolirovannye anti-GFAP antibodies are incubated with the liposomal nanocontainers that already contains maleimide derived cells of the dspe-PEG-2000, which leads to the formation of a thioester link between SH-group MAT and maleimide fragment. This method allows efficient binding of antibodies to the surface stelth-liposomes.

After completion of the incubation separated from unbound antibodies and N-ethylmaleimide with gelfiltration on a column (1×17 cm) with Sephadex G-100, equilibrated with 0.1 mol/l sodium phosphate is a buffer (pH 7.4), at a flow rate of 25 ml/hour according to the testimony of spectrophotometry.

Received nanosysteme concentrated to the desired concentration and sterilized by the method of filtration through cellulose acetate filters (Millipore) with a hole diameter 0.22 μm in sterile conditions box.

For visualization of structures astroglial shaft in vivo obtained nanosysteme injected into the blood stream of the patient not less than 500 μg/kg of patient's weight, then intervals of time depending on the diagnostic method register the signal accumulation diagnostic labels, namely in the area of expression of GFAP reactive astrocytes accompanying the invasion of glioma.

The selective accumulation of monoclonal antibodies in the area of GFAP expression when administered intravenously can be measured by various methods depending on the type of diagnostic labels, in particular methods scintigraphy (radionuclide label), PET (short-lived isotopes), MRI (nanocontainers with contrast agent).

For evidence for the possibility of the proposed method with achieving the stated purpose and specified technical result is the following data.

EXAMPLE 1

Purified anti-GFAP antibodies from selected clones of hybrid cells that recognize GFAP in vivo, to determine the specificity to GFAP reactive Astros the tov, their functional activity and working concentration was tested on brain slices of rats with glioma C6. When immunofluorescence analysis on brain slices of rats with C6 glioma derived monoclonal antibodies clearly visualize reactive astrocytes around kentremendous tumors (Figure 1).

Anti-GFAP antibodies were dialyzed against 0.1 mol/l sodium phosphate buffer (pH 7.4) for 1 h under stirring, freeing from Tris.

Anti-GFAP antibodies modified by ε-amino lysine residues with 2-aminothieno in 0.1 mol/l sodium phosphate buffer at a concentration of 1 mg/ml for insertion of a fragment containing the SH group. The reaction etiolirovaniya were carried out in dark conditions for 1 h under argon at room temperature, stopped after separation of the excess 2-aminothieno from the protein by gel filtration through Sephadex G-25.

Simultaneously prepared liposomal nanocontainers. Lecithin, cholesterol, cells of the dspe-PEG-2000 maleimide derived and fluorescent label Dil dissolved in a mixture of chloroform-methanol (9:1) in the concentration of total lipids of 5-10 mg/ml in a molar ratio of 23:16:1.6:1:0.4. The mixture was dried on a rotary evaporator under reduced pressure. The dry lipid film was dissolved in absolute cyclohexane, frozen in liquid nitrogen, were liofilizovane, after which the mixture of lipids emulge is ovale in 0.1 M phosphate buffer. Gidratirovannuyu emulsion was treated in an ultrasonic disintegrator and 15-fold skipped through polycarbonate membrane filters with a pore size of 400, 200, 100 and 50 nm using a mini-extruder.

After that incubated etiolirovannye anti-GFAP antibodies with the liposomal nanocontainers that already contains maleimide derived cells of the dspe-PEG-2000 and diagnostic label Dil. Maleimide derivative leads to the formation of thioester communication with SH-group anti-GFAP antibodies. Department of liposomes conjugated with antibody from unbound antibodies and N-ethylmaleimide was performed using gelfiltration on a column (1×17 cm) with Sephadex G-100, equilibrated with 0.1 mol/l sodium phosphate buffer (pH 7.4), at a flow rate of 25 ml/hour. Control over the movement of the sample was carried out visually by the color of Dil. Sterilization of liposomes was performed by the method of filtration through cellulose acetate filters (Millipore) with a hole diameter 0.22 μm in sterile conditions box.

The efficiency of binding was assessed by the ratio of the concentration of protein in the fractions stelth-liposomes and unbound immunoglobulins. This indicator, with effective presence immunoglobulins was 70-80%.

Model of human glioblastoma in rats is induced glioma C6 (Auer RN.., et al A simple and reproducible experimental in vivo glioma model. Can J Neurol Sci 1981; 8:325-331). Based on morphology, the character with the positive growth and the spectrum of expressed proteins in glioma C6 is very close to the multiform glioblastoma person. Range expressed by C6 glioma cells proteins include proteins of the basal membrane proteins cell adhesion and tissue proteolytic enzymes, providing the degradation of the extracellular matrix during invasion, signaling proteins, growth factors and their receptors, supporting a high level of proliferation of tumor cells and stimulates angiogenesis (Grobben Century, De Deyn (2002) Rat C6 glioma as experimental model system for the study of glioblastoma growth and invasion. // Cell Tissue Res. 310, p.257-270).

For modeling glioblastomas have applied the technique of stereotactic implantation of pre-cultivated Gymnich cells in the striatum of the rat brain (Chekhonin, Becausev VP, Usubaliev G.M., 2007) (figure 1, 2, 3).

Five rats implanted by the method of stereotaxis glimne cell line C6 rat in an amount of 5·105. At 18 days of growth of glioma each rat under ketamine anesthesia in the blood system introduced 1 ml of the preparation obtained nanosystems containing 100 μg anti-GFAP antibodies at a ratio of 500 µg/kg for protein. Nanosystem based anti-GFAP antibodies circulate in the blood of each rat within 48 hours, after which each rat was subjected to perfusion with 4% solution of paraformaldehyde followed by decapitation and removing the brain. The extracted brain was subjected to additional fixation in 4% solution of paraformaldehyde at 4°C, after which lorazepam the microtome was prepared slices with a thickness of 100 microns. Slices with glioma was mounted on a glass slide with the subsequent application of the 80% glycerol in Tr-HCl buffer (pH 8.3) under the cover glass.

Evaluation of targeted delivery nanosystems based anti-GFAP antibodies to target in the organism of a living rat conducted using scanning laser confocal microscopy for fluorescent label Dil. Figure 4 around implanted C6 glioma cells (green fluorescence) are rendered reactive astrocytes that accompany tumor invasion (position a, position B).

Experimental immunochemical studies on living rats with implanted with C6 glioma showed promising applications received nanosystems for visualization astroglial shaft localization of diagnostic labels. In all cases, the 5 experimental animals rendered under the proposed method boundaries of tumoral invasion of glioma fully coincide with the boundaries of tumor invasion, defined by histological studies.

1. The method of determining the boundaries of the invasion, poorly differentiated gliomas, in which the boundaries of the infestation is determined by visualization astroglial shaft surrounding the immature glioma, which receive immunogenic recombinant GFAP person, they are subjected to immunization mice of Balb/C, isolated b-lymphocytes of the spleen that m is Shi and merge with murine myeloma cells line Sp 2/0-Agl4, get hybridoma, test supernatant obtained hybrid immunohistochemistry for the presence of anti-GFAP antibodies of them are selected clone hybrid cells that produce anti-GFAP antibodies capable of recognizing GFAP in vivo; clear anti-GFAP antibody from the supernatant of the selected clone and covalently associated with the liposomal nanocontainers containing the diagnostic label, while antibodies selected clones of hybrid cells modified by ε-amino groups of residues lysine and incubated with a solution stelths-liposomes obtained nanosysteme injected into the bloodstream of the patient and visualize astroglial shaft for positioning a diagnostic label in brain tissue.

2. The method according to claim 1, characterized in that the received nanosysteme enter in the amount of not less than 500 μg/kg of patient's weight for protein.

3. The method according to claim 1, characterized in that the selection of clones producing anti-GFAP antibodies capable of recognizing GFAP in vivo, carried out by the method of immunochemical testing on a live culture of astrocytes and/or rats.

4. The method according to claim 1, characterized in that to increase the production of anti-GFAP antibodies selected clone intraperitoneally to mice injected with cells of the selected clone, then not less than 7 days away from them ascitic fluid.

5. The method according to claim 1, characterized in that the purified anti-GFAP antibodies and the supernatant of the selected clone method immunoaffinity chromatography using GFAP, covalently bound CN-Br-separate.

6. The method according to any one of claims 1 to 5, characterized in that the anti-GFAP antibody selected clone modify on the ε-amino lysine residues with 2-aminosilane.



 

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