RU2708458C2 - Method for labeling activated lymphocytes in vitro with a complex compound - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to a method for labeling activated lymphocytes in vitro with a complex compound. Method involves incubation of activated lymphocytes with RFP ^99mTc-THEOXYM in amount of 2 ml with activity of 350–500 MBq, with periodic shaking during 20 minutes, after incubation, in a test tube with cell mass phosphate-salt buffer is added and total volume is increased to 10 ml, then test tube is centrifuged for 5–6 minutes at 1,500–2,000 rpm, after that supernatant fluid is completely removed, labeled ^99mTc-theoxyme cell conglomerate is resuspended in 1 ml of a phosphate-salt buffer. Viability of labeled activated lymphocytes in vitro by the disclosed method remains higher than 95 %.

EFFECT: ready cell preparation may be administered intraperitoneally paravertebrally at the level of vanes or in the lumbar region and examined pathways of labeled cell migration in vivo by SPECT/CT.

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Description

The invention relates to medicine, namely to oncoimmunology and radionuclide diagnostics, and can find application for monitoring cell distribution during cell immunotherapy for cancer patients. The labeling method can be universal for various types of activated autologous and allogeneic cells (cytotoxic T-lymphocytes, natural killer cells (NK) and dendritic cells).

Over the past decades, significant progress has been made in the field of immunotherapy, numerous immunotherapeutic strategies have been developed that have demonstrated their safety and effectiveness in a number of clinical trials. Immunotherapy currently includes the following areas: creation of control point inhibitors, natural antibodies and conjugated with chemotherapeutic drugs or radioactive particles, antitumor vaccines, as well as adaptive cell therapy. The most promising area is the use of innate immunity effect cells, in particular NK and T-lymphocytes, the antitumor potential of which is enhanced by in vitro cultivation in the presence of cytokines. This method was developed [1] and successfully implemented in clinical practice [2]; it showed relatively high therapeutic efficacy in an integrated approach. However, at present there is no consensus on the key components of such therapy, and questions still arise: where, how, and how many cells must be introduced to the patient in order for such therapy to be an objective success. Also, migration routes of activated human lymphocytes with intradermal administration have not been studied.

Known methods for the topical diagnosis of inflammatory heart diseases (RU 2136218 C1, RU 2244515 C1), which are based on the same principle of intravenous administration of autoleukocytes labeled with ^99m Tc-hexamethylene propyleneaminoxime.

However, in the known methods there is no quality control of the efficiency of labeling cells of autologous leukocytes.

A known method for identifying foci of inflammation using multiple organ scintigraphy (RU 2648877 C1), which is based on the use of autoleukocytes labeled with ^99m Tc-teoksimom or ^99m Tc-tseretekom for intravenous administration to the patient.

The disadvantage of this method is the use of Gemacon-type packets, which makes it difficult or even impossible to completely separate the cell mass from the supernatant containing free radiopharmaceutical, which may affect the reliability of the results during SPECT studies.

A known method for assessing the functional state of the spleen (RU 2152168 C1). A dynamic gamma scintigraphy is carried out with ⁹⁹ TC labeled red blood cells damaged by heat. Determine the time of onset of maximum activity of γ-radiation in the spleen (T _max ), splenic-hepatic index (SPI). Sequestration function of the spleen is evaluated by the activity-time curve. The amount of functioning red pulp of the spleen is determined by STI. When T _max equal to 90 ± 30 min, and the IPI equal to 94%, sequestration function of the red pulp is estimated normal.

There is also known a method for the diagnosis of focal lesions of the spleen (RU 2190959 C2). A radionuclide study is carried out using ^99m Tc labeled and heat-damaged red blood cells. A radiopharmaceutical with a radioactivity of 20 mCi is used. 2.5 hours after its administration, single-photon emission computed tomography is performed, and focal lesions of the spleen are determined when registration of defects in accumulation or foci of hyperfixation of the radiopharmaceutical on SPECT sections.

However, in the known methods, the labeling of cells is incorrect: intravenous administration of a transport substance without a radionuclide, followed by blood sampling and incubation with the radionuclide, which may affect the reliability of the SPECT study.

The prototype of the proposed technical solution is a method for labeling mononuclear cells of red bone marrow using ^99m TC-examethasime (RU 2343933 C1). The cell suspension is incubated with 4 ml of ^99m Tc-examethasime for 10 minutes at room temperature, followed by washing, centrifugation for 5 minutes at 150g, after which the supernatant is collected and resuspended to obtain ^99m Tc-examethasime-labeled red bone marrow mononuclear cells while incubation is carried out with 1500-2000 mBq ^99m Tc-examethasime, washing is carried out with 10 ml of isotonic sodium chloride solution, and resuspension in 10 ml of isotonic sodium chloride solution.

The disadvantage of this method is the use of radiopharmaceuticals with high specific activity, which is not economically feasible, since the cells have a finite saturation with the drug and an increase in activity does not affect this property; also, all actions to wash and resuspend the cell mass were carried out using physiological saline, which negatively affects the functional activity of cells.

The technical result of the invention is the production of radiolabeled activated lymphocyte culture with high viability and efficiency of cell labeling.

The technical result is achieved by the fact that, as in the known method, they carry out the labeling of activated cells in vitro.

A feature of the proposed method is that the cells are incubated with ^99m Tc-teoxim in a volume of 1.5-2 ml, an activity of 350-500 MBq, with periodic shaking of the cell suspension for 20 minutes, then phosphate-buffered saline is added to the labeled cell substance volume of 7-10 ml and centrifuged for 6 minutes at 2000 revolutions / min with a centrifuge rotor baketnym is withdrawn supernatant containing a free ^99m Tc-teoksim derived cell conglomeration resuspended in 1 ml of phosphate-buffered saline, is then determined using astvora trypan blue viability of labeled lymphocytes in the Goryaev chamber tagging evaluated quality by measuring the activity of a cell mass obtained by the radiometer and give scintigrams tubes labeled cell mass. Moreover, autologous or allogeneic lymphocytes and dendritic cells are labeled.

The invention is illustrated by a detailed description, clinical example and illustrations, which depict:

FIG. 1 - Test tube with labeled lymphocytes: A - precipitated labeled lymphocytes; B - scintigram of labeled cell mass;

FIG. 2 - Patient body scintigraphy K.: B - the injection site of the labeled cell preparation, G - lymphocytes from the injection site migrated to the right axillary lymph node, D - allergic test using pure radiopharmaceutical ^99m Tc-teoxime;

FIG. 3 - SPECT / CT study of migration pathways of activated K. ^99m -labeled Tc-teoxim lymphocytes of patient K.: D - labeled activated lymphocytes migrated to the right axillary lymph node.

The method is as follows.

Under sterile conditions of a laminar box, the amount of activated lymphocytes necessary for administration to a patient (5-30 * 10 ⁶ cells / ml) is collected from culture bottles with a pipette, and placed in a sterile tube with a conical bottom. Activated lymphocytes are precipitated by centrifugation, after which the supernatant is removed with a pipette and the cells are resuspended in a volume of 1 ml.

1. An eluate in a volume of 2 ml with an activity of 350-500 MBq, obtained from a ^99m Tc technetium generator, is introduced under aseptic conditions into the TEOXIM bottle using a syringe, the contents of the bottle are mixed by shaking until the drug is completely dissolved.

2. A ^99m Tc-TEOXIM is introduced into the cell suspension with a sterile syringe. Then the lymphocytes are incubated (with periodic shaking) for 20 minutes.

3. After incubation, phosphate-buffered saline is added to the test tube with cell mass, and the total volume is adjusted to 10 ml. Then the tube is installed in a centrifuge with a bucket rotor and centrifuged for 5-6 minutes at 1500-2000 rpm, after which the supernatant is completely removed.

4. ^99m Tc-teoxim-labeled cell conglomerate is suspended in 1 ml of phosphate buffered saline.

5. Determine with the help of a trypan blue solution the vitality of labeled lymphocytes in the Goryaev chamber or on an automatic cell counter.

6. Measure the activity of the obtained cell mass in a radiometer.

7. Receive a scintigraphic image of the tube with labeled cell mass (Fig. 1).

Clinical example.

Patient K., 1965 ave., Diagnosis: cancer of the left breast (T3N0M1), previous treatment: surgical, PCT. She entered the Department of Clinical Immunology of the MRRC. them. A.F. Adaptive cell immunotherapy.

To isolate a population of mononuclear cells, peripheral blood obtained by puncture of the ulnar vein was used. Isolated lymphocytes were cultured for 3 days in a medium supplemented with IL-2, IL-15. After completion of the cultivation process, activated lymphocytes in the amount of 10 million cells were obtained. Then, ^99m Tc-TEOXIM with a volume of 2 ml and an activity of 350 MBq was added to the obtained cell mass, incubated for 20 min. After incubation, phosphate-buffered saline was added to the test tube with cell mass, bringing the total volume to 10 ml, centrifuged for 6 minutes at 2000 rpm in a centrifuge with a bucket rotor. Then, the supernatant with free radiopharmaceutical was completely removed. Cells were resuspended in 1 ml. phosphate buffered saline. Using the trypan blue dye in the Goryaev chamber, cell viability was determined, which amounted to 96.4%. The cell mass activity was measured using a RIS-A1 “Dose Calibrator” radiometer - 29.0 MBq. Activated lymphocytes labeled with ^99m Tc-teoxim were injected intracutaneously to the patient paravertebrally at the level of the scapula using an insulin syringe.

5 days before the introduction of the labeled activated lymphocytes, the patient was injected intracutaneously with the free ^99m Tc-TEOXIM in the same places, on the scintigrams the accumulation of the drug was observed only at the injection sites, as well as physiological accumulation (kidneys, bladder, stomach).

The migration pathways of labeled activated lymphocytes were visualized immediately after administration and 4 hours after cells were introduced on a SPECT / CT combined Discovery NM / CT 670 system. Image analysis showed that ^99m Tc-texim-labeled activated lymphocytes from the injection site migrated to the right axillary lymph node ( Fig. 2 and Fig. 3).

Using the proposed method in a clinic allows one to obtain activated lymphocytes labeled with ^99m Tc-teoxim and then study their migration in vivo by SPECT / CT during adaptive cell immunotherapy. The labeled culture viability index with this labeling method is at least 95%, and the methods for instrumental verification of labeling efficiency (measuring the activity of the actual labeled cell mass and its scintigraphy) indicate that it was the cells that captured ^99m Tc-teoxime, and in the free form of the radiopharmaceutical the obtained cell preparation does not contain, which is important for the exclusion of false positive results of SPECT studies. The method was tested on five patients with various nosological forms of oncological pathology. In the analysis, a regularity was observed for all clinical cases - labeled activated lymphocytes from the injection sites directed to the lymph nodes, some of which are located near the area of the main oncological pathology. This fact means that the main goal of adoptive cell biotherapy is achieved - the activation of a specific antitumor response of the immune system.

Claims (2)

1. The method of labeling activated lymphocytes in vitro with a complex compound, including the labeling of activated cells in vitro, characterized in that 5-30 * 10 ⁶ cells / ml, necessary for administration to the patient, are collected from culture bottles and placed into a sterile tube, activated lymphocytes precipitated by centrifugation, after which the supernatant was removed and the cells were resuspended in a volume of 1 ml, then an eluate in a volume of 2 ml with an activity of 350-500 MBq obtained from a ^99m Tc technetium generator was introduced using a syringe TSA in a vial with TEOXIM and mix by shaking until the drug is completely dissolved, then ^99m Tc-TEOXIM is added to the cell suspension and then the lymphocytes are incubated with periodic shaking for 20 minutes, after incubation, phosphate-buffered saline is added to the tube with cell mass and the total is adjusted volume up to 10 ml, then the tube is centrifuged for 5-6 minutes at 1500-2000 rpm, after which the supernatant is completely removed, ^99m Tc-toxime-labeled cell conglomerate is resuspended in 1 ml of phosphate-buffered saline and Using a trypan blue solution, the viability of labeled lymphocytes is determined in the Goryaev chamber or on an automatic cell counter, the activity of the obtained cell mass is measured in a radiometer, and a scintigraphic image of the tube with labeled cell mass is obtained. 2. The method according to p. 1, characterized in that they carry out the labeling of autologous or allogeneic lymphocytes and dendritic cells.

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