RU2684289C1 - METHOD FOR OBTAINING COMPLEX OF TECHNETIUM-99m WITH RECOMBINANT PROTEIN-TARGETED MOLECULES FOR RADIONUCLIDE DIAGNOSIS OF ONCOLOGICAL DISEASES WITH HER-2/neu OVEREXPRESSION - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention refers to medicine, oncology and chemical engineering. Method of producing the technetium-99m complex with recombinant protein-targeted molecules for radionuclide diagnosis of oncological diseases with HER-2/neu overexpression consists in the fact, that at the first stage aquacarbonyl technetium-99m [Tc(HO)(CO)]is obtained, using sodium tetraborate sodium lyophilizate, decahydrate, sodium carbonate, disodium boron carbonate, which can also contain potassium sodium tartrate tetrahydrate or sodium citrate monohydrate, then lyophilisate is added with 1–3 ml sodium pertechnetate solution,Tc with activity of 0.74–3.7 GBq and heated in a boiling water bath for 20–30 minutes, cooled to room temperature and added with 1 M HCl to pH 7.4 or 300–900 mcl of 1 M sodium phosphate solution with pH 7.4, further, a chelating agent succinimid-1-yl 6-(bis(pyridin-2-ylmethyl)amino)hexanoate (DPAH) is added to DARPin in a phosphate buffer with pH 8.3-8.5 to obtain DPAH-DARPin, then aqua-carbonyl technetium-99 m [Tc(HO)(CO)]is bound with DARPin or DPAH-DARPin, for which to DARpin or DPAH-DARPin in amount of 100 mcg in a phosphate-buffer solution at pH 7.4 1 ml of aqua-carbonyl technetium-99m solution [Tc(HO)(CO)]is added, then incubated at 40 °C for 30–60 minutes, purification is carried out by gel filtration on a column.EFFECT: invention is aimed at radionuclide diagnosis of oncological diseases with HER-2/neu overexpression.1 cl, 12 ex

Description

The invention relates to medicine, oncology and pharmacology and can be used to obtain a complex of technetium-99m with recombinant targeted protein molecules for radionuclide diagnosis of cancer with overexpression of HER-2 / neu.

The main radioisotope in the diagnosis using radiopharmaceuticals is the technetium-99m isotope, which has the optimal half-life (6 hours) and the type of radiation (γ-radiation, energy 140 keV). One of the most studied and often mentioned in the literature tumor antigens is the surface receptor HER2 / neu, which is overexpressed in many human carcinomas (breast, lung, stomach, ovary, prostate cancer). A number of monoclonal antibodies have been developed for this antigen, suitable for use in diagnostic and therapeutic purposes. An alternative to targeted antibodies and their fragments are recombinant targeted protein molecules with antikyrin repeats (DARPin), which are specific for the cancer-associated HER2 / neu antigen [1-3].

Several methods are known for labeling targeted protein molecules (including antibodies, their fragments, affibody and DARPin) with a technetium-99m radionuclide. Labeling is considered to be the most effective when obtaining highly stable complexes of technetium-99m with targeted molecules containing chelate centers for metal binding, which are capable of forming fairly strong coordination bonds. Therefore, the main thing in the development of such radiopharmaceuticals is the selection of conditions for radiochemical synthesis for the formation of a durable complex with technetium-99m, resistant to dissociation in the blood and having a high yield. A promising complex for radiolabelling protein molecules is the technocetium aquacarbonyl [ ^99m Tc (H ₂ O) ₃ (СО) ₃ ] ⁺ , capable of forming strong, stable complexes with high yields with protein molecules having chelating centers for ^99m Tc (СО) ₃ - core [4, 5].

Targeted DARPin molecules have a hexahistidine moiety in their structure that can firmly bind aquacarbonyl technetium, as well as a successful introduction of a tridentant chelating agent based on ω-bis (pyridin-2-ylmethyl) amino) aliphatic acids that strongly bind avcacarbonyl to the DARPin structure. ].

Thus, the development of a method for producing a stable complex based on aquacarbonyl technetium-99m and targeted DARPin molecules for the radionuclide diagnosis of cancer with HER-2 / neu overexpression is relevant.

A known method of producing carbonyl complexes of short-lived technetium (I) and rhenium (I). The carbonyl complexes of short-lived technetium (I) and rhenium (I) of the general formula [Me (CO) ₃ (H ₂ O) ₃ ] ⁺ , where Me = ^99m Tc or ¹⁸⁶ Re, are obtained in one step. Potassium pertechnetate or perrenate is treated with a carbonylating agent in the presence of hydrohalic acid at a temperature of 200-220 ° C and a pressure of 100-160 atm. The disadvantage of this method is the use of high temperature and high pressure, which makes it inaccessible for use in clinics. And the method does not offer conditions for labeling targeted molecules of a protein nature [7].

A known method of producing a water-soluble carbonyl complex of short-lived technetium-99m, including the treatment of potassium pertechnetate K ^99m TcO _{4 with a} carbonylating agent in an aqueous solution at a temperature of 140-180 ° C and a pressure of 90-170 atm for 30-40 minutes Carbonylation is carried out in the presence of sodium iodide or potassium iodide and perchloric or sulfuric acids, and the resulting gaseous technetium-99m pentacarbonyl iodide is captured in an aqueous or aqueous-alcoholic solution in a receiving container. This method also differs by the application of conditions (temperature and pressure) unsuitable for routine radiopharmaceutical production in a clinic, and also the method does not affect the conditions for labeling targeted protein molecules [8].

A method is described for producing the [ ^99m Tc (CO) ₃ ] ⁺ -G3DARPin-His ₆ complex, which is characterized by a 3.5-fold higher level of absorption in tumor cells with HER2 / neu overexpression compared to HER2 / neu-negative cells in vivo [9 ]. However, the disadvantage of this complex is that the [ ^99m Tc (CO) ₃ ] ⁺ -G3DARPin-His ₆ complex in the body did not show the optimal tumor-liver to tumor-blood ratio. In this preparation method, direct binding of the technetium-99m aquacarbonyl complex ([ ^99m Tc (CO) ₃ ] ⁺ ) to G3DARPin-His ₆ is used using IsoLinc kits (Mallinckrodt-Tyco Med), which are difficult to access in Russia.

A known method of obtaining compounds of General formula: - [M (CO) ₃ (OH ₂ ) ₃ ] ⁺ , where M represents Mn, ^99m Tc, ¹⁸⁶ Re or ¹⁸⁸ Re, by the interaction of a metal in permellate form with carbon monoxide and a reducing agent, characterized a mixture of bases (including sodium carbonate), a reducing agent (including sodium borohydride), soluble in water, and, optionally, a stabilizing agent (including potassium sodium tartrate or sodium citrate), containing a solution of metal in permanganate, perchetate or perrenate in the presence of carbon monoxide and, optionally tionary in the presence of halide [10]. Also disclosed in this patent is a method for producing a labeled compound using the Fac- [M (CO) ₃ (OH ₂ ) ₃ ] ⁺ compound, a method for directly producing labeled compounds and a method for labeling substrates such as amino acids, peptides, proteins, sugars. The labeling method is not proposed in this patent for the targeted DARPin molecule directly or through a chelating agent, a tridentant ligand. The method of producing aquacarbonyl technetium is based on the use of toxic carbon monoxide gas, which is inconvenient for the production of radiopharmaceuticals in a clinic.

The closest to the proposed one can be considered a method of obtaining the complex [ ^99m Tc (CO) ₃ ] ⁺ -DARPin, which consists in the direct binding of [ ^99m Tc (CO) ₃ ] ⁺ with DARPin [11]. The patent provides a method for producing a therapeutic radioconjugate, a specifically binding substance with a short-lived alpha-emitting radioisotope for its delivery to pathological regions, while labeling of recombinant humanized mini-antibodies specific for the cancer-associated HER2 / neu antigen, a diagnostic gamma-emitting radioisotope, namely with a TC-99m aquacarbonyl complex, mini-antibodies are conjugated with human serum albumin, the resulting conjugate is purified, introduced into composition of the conjugate chelating agent DOTA (1,4,7,10-tetraazicyclode dodecane tetraacetic acid) or DTPA (diethylene triamine pentaacetic acid), the resulting conjugate is labeled with therapeutic radioisotopes, namely short-lived alpha-emitting radioisotopes, and the resulting preparation is purified.

In this method, to obtain the TC-99m aquacarbonyl complex, a mixture of 0.004 g (0.038 mmol) of Na ₂ CO ₃ and 0.005 g (0.13 mmol) of NaBH ₄ in an 8 ml tube with a screw cap with a rubber gasket is saturated for 10 minutes . Then, 3 ml of an eluate from a molybdenum generator containing up to 30 GBq Na [ ^99m TcO ₄ ] in a 0.9% aqueous NaCl solution was added via syringe and kept at a temperature of 75 ° C for 30 minutes. Cool and add 0.3 ml of 1M sodium phosphate, pH 7.4.

To 50 μl of mini-antibodies (concentration 2 mg / ml) in PBS buffer (7 mm NaH ₂ PO ₄ , 3 mm Na ₂ HPO ₄ , 350 mm NaCl, pH 7.4) add 25 μl of 0.5 M MES pH 6.2 and 25 μl obtained solution of [ ^99m Tc (CO) ₃ (OH ₂ ) ₃ ] ⁺ (~ 0.25 GBq) and incubated for 30 min at 37 ° C. The TC-99m aquacarbonyl complex specifically binds to the oligohistidine fragment of miniantibodies. To purify the mini-antibodies labeled with the TC-99m aquacarbonyl complex from the unbound radioisotope, gel filtration is performed on a Superdex-75 column in PBS buffer.

The disadvantages of this method is the need to use toxic CO gas in a cylinder, which must be saturated with the vial for 10 minutes, the radiochemical yield and purity are not indicated in the patent to evaluate the effectiveness of the proposed method under the stated conditions.

A new technical problem is to increase the selectivity of the method, increase product yields and increase versatility while simplifying the method.

To solve the problem, in a method for producing a complex of technetium-99m with recombinant targeted protein molecules for radionuclide diagnostics of oncological diseases with HER-2 / neu overexpression, including obtaining a complex with aquacarbonyl technetium-99m with targeted DARPin molecules, aquacarbonyl is obtained in the first stage 99m ^[99m Tc (H ₂ O) ₃ (CO) _3] ⁺ using sodium tetraborate decahydrate lyophilisate, sodium carbonate, disodium boranokarbonata, which may also contain sodium potassium tartrate tetrag fight or sodium citrate monohydrate, hereinafter, the lyophilisate is added to 3.1 ml of sodium pertechnetate solution, ^99m Tc with activity 0,74-3,7 GBq and heated in a boiling water bath for 20-30 minutes, cooled to room temperature and add 1M HCl to a pH of 7.4 or 300-900 μl of a 1M solution of sodium phosphate with a pH of 7.4, in the second stage, the chelating agent succinimid-1-yl 6- (bis (pyridin-2-ylmethyl) amino) hexanoate is added to DARPin in phosphate buffer (pH = 8.3-8.5) (DPAH-DARPin), in the third stage, the aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ^{+ is} coupled to DARPin or DPAH- DARPin, why to DARpin or DPAH-DARPin in an amount of 100 μg in a phosphate-buffered solution at pH = 7.4 add 1 ml of a solution of aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ , then incubate at 40 ° C for 30-60 minutes, purification is carried out by gel filtration on a NAP-5 column or similar.

A complex of technetium-99m with DARpin or DPAH-DARPin with a radiochemical purity of more than 99.0% is obtained.

Distinctive features showed in the inventive combination of new properties that are not explicitly derived from the prior art in this field and are not obvious to a specialist.

The proposed set of features is not described in the patent and scientific literature.

The method was tested with the subsequent identification of the obtained products.

The following are examples of specific methods of obtaining

Example 1. Obtaining aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺

A bottle with a capacity of about 10 ml containing lyophilisate 3.0 mg sodium tetraborate decahydrate, 8.0 mg sodium carbonate, 5.0 mg disodium boranocarbonate, was added 1 ml sodium pertechnetate solution, ^99m Tc with an activity of 0.74-3.7 GBq and heated in a boiling water bath for 20 minutes. Cool and 300 μl of a 1M sodium phosphate solution (pH 7.4) was added. The radiochemical purity (RCC) of the obtained complex was evaluated by thin layer chromatography (TLC) using PTSX-AF-A-UV plates (Sorbfil) in the MeOH / HCl system, (95: 5), R _f = 0.37. The RFC of the resulting complex was ≥95%.

Example 2. Obtaining aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺

The preparation was carried out according to the procedure described in example 1, with the difference that the lyophilisate also contained 9.0 mg of potassium sodium tartrate tetrahydrate. The RFC of the resulting complex was ≥95%.

Example 3. Obtaining aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺

The preparation was carried out according to the procedure described in example 1, with the difference that the lyophilisate also contained 9.0 mg of sodium citrate monohydrate. The RFC of the resulting complex was ≥95%.

Example 4. Method for the synthesis of succinimid-1-yl 6- (bis (pyridin-2-ylmethyl) amino) hexanoate (DPAH-NHS-ester)

The synthesis is carried out as described in the patent [6] using cyclohexanone. In the last step, 6- (di (pyridin-2-ylmethyl) amino) hexanoic acid is activated. For this, in a round bottom flask to a mixture of 6- (di (pyridin-2-ylmethyl) amino) hexanoic acid (as a hydrochloride salt) (1,200 g, 3.48 mmol) in 6 ml of tetrahydrofuran add triethylamine (0.510 ml, 3.48 mmol) and then the mixture was stirred for 30 min at room temperature. Then, N-hydroxysuccinimide (0.474 g, 4.2 mmol) and N, N-dicyclohexylcarbodiimide (0.867 g, 4.2 mmol) were added. Stirred for 48 hours at room temperature. The end of the reaction is determined by thin layer chromatography (eluent: ethyl acetate - ethanol = 10: 3). The chromatogram should have only one main spot with R _f 0.35. The precipitate of N, N-dicyclohexylurea is filtered off. Tetrahydrofuran was distilled off in the filtrate, 10 ml of water was added to the resulting oil, and extracted with methylene chloride (3 × 15 ml). The methylene chloride extract was dried over anhydrous sodium sulfate and the solvent was distilled off.

The resulting product was purified by silica gel column chromatography using hexane-ethyl acetate (1: 1) as the eluent, gradually increasing the gradient of the latter. The resulting light yellow oily mass was dried under vacuum. The estimated weight of the intermediate of succinimid-1-yl 6- (di (pyridin-2-ylmethyl) amino) hexanoate (DPAH-NHS-ester) is 0.874 g (62%).

Example 5. The procedure for modifying DARPin with an activated ester - succinimid-1-yl 6- (bis (pyridin-2-ylmethyl) amino) hexanoate

To 100 μl of a solution of DARPin (concentration 6 mg / ml) in 100 mmol of PBS, pH 8.0-8.5, add 10 μl of a solution of succinimid-1-yl 6- (bis (pyridin-2-ylmethyl) amino) hexanoate in acetonitrile (80-100 mcg). The sample is stirred and incubated for 4 hours at room temperature. Next, the sample is kept for 24 hours at a temperature of 5-10 ° C. Purification of the modified DPAH-DARPin product was performed by gel filtration using PD MiniTrap G-25 columns (GE Healthcare) calibrated with a buffer according to the manufacturer's instructions.

Example 6. The method of labeling with aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ targeted molecules DARPin

To 100 μl of a solution of DARPin (concentration 3 mg / ml) in buffer (100 mmol PBS, 350 mmol NaCl, pH = 7.4) add 1 ml of a solution of aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ obtained in example 1. The sample is mixed and incubated for 30 min at 40 ° C. Cleaning is carried out by gel filtration on a NAP-5 column according to the manufacturer's instructions. The RFC of the resulting complex is 99.6%.

Example 7. The method of labeling with aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ targeted molecules DARPin

The reaction mixture was prepared in the same manner as in Example 6, with the difference that 1 ml of a solution of aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ obtained in Example 1 was added.

2. The RFC of the resulting complex is 99.8%.

Example 8. The method of labeling with aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ targeted molecules DARPin

The reaction mixture was prepared in the same manner as in Example 6, with the difference that 1 ml of a solution of aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] obtained in Example was added.

3. The RFC of the resulting complex is 99.8%.

Example 9. The method of labeling with aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ targeted molecules DARPin

The reaction mixture was prepared in the same manner as in Example 6 with the difference that 1 ml of a solution of aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ obtained in Example 2 was added. The sample was stirred and incubated 20 min at 40 ° C. Cleaning is carried out by gel filtration on a NAP-5 column according to the manufacturer's instructions. The RFC of the resulting complex is 99.7%.

Example 10. The method of labeling with aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ targeted molecules DARPin

The reaction mixture was prepared in the same manner as in Example 9 with the difference that the sample was stirred and incubated for 40 min at 40 ° C. The RFC of the resulting complex is 99.8%.

Example 11. The method of labeling with aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ targeted molecules DARPin

The reaction mixture was prepared in the same manner as in Example 9 with the difference that the sample was stirred and incubated for 60 min at 40 ° C. The RFC of the resulting complex is 99.8%.

Example 12. The procedure for labeling with aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ DARPin target molecules modified with a DPAH-NHS-ester chelating agent K 100 μl DPAH-DARPin solution (concentration 3 mg / ml) in a buffer (100 mmol PBS, 350 mmol NaCl, pH = 7.4) add 1 ml of a solution of aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ , obtained according to example 2. The sample is mixed and incubated 30 min at 40 ° C. Cleaning is carried out by gel filtration on a NAP-5 column according to the manufacturer's instructions. The RFC of the resulting complex is 99.6%.

Justification of the regime

Obtaining aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ can be carried out in the presence of a co-ligand stabilizing [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ . As co-ligands, potassium sodium tartrate or sodium citrate was used (examples 1-3). In all cases, yields of [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ over 95% were observed.

The labeling of targeted DARPin molecules has been proposed by direct and indirect methods. In the direct method for labeling DARPin, a method for producing [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ without a stabilizing co-ligand is used (Example 6). The RFC of the obtained complex [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ with DARPin is 99.6%. Radiochemical yield of more than 80% before purification. In an indirect method for labeling DARPin, a method for producing [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ in the presence of a stabilizing co-ligand is used (Examples 7-8). The RFC of the obtained ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ complex with DARPin is 99.8%. Radiochemical yield of more than 85% before purification. The labeling reaction of ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ with DARPin was proposed to be carried out for 20-60 minutes at 40 ° C (examples 6, 9, 10). In all cases, the radiochemical yield is more than 80% before purification. The RFC of the obtained ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ complex with DARPin is 99.8%. The possibility of using a chelating agent DPAH-NHS-ester to modify DARPin has also been shown, with the labeling of modified DARPin-DPAH-DARPin showing high yields and RCP. The radiochemical yield of the ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ complex with DPAH-DARPin is about 90% prior to purification; the RFC of the resulting complex is 99.6%.

Sources of information taken into account when compiling the description:

1) DARPins: A new generation of protein Therapeutics / Michael T. Stumpp, H. Kaspar Binz and Patrick Amstutz // Drug Discovery Today. Volume 13, Numbers 15/16. August 2008.P. 695-701.

2) Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging / Robert Goldstein, Jane Sosabowski, Maria Livanos, Julius Leyton, Kim Vigor, Gaurav Bhavsar, Gabriela Nagy-Davidescu, Mohammed Rashid, Enrique Miranda, Jenny Yeung, Berend Tolner, Andreas Pluckthun, Stephen Mather, Tim Meyer, Kerry Chester // Eur. J. Nucl. Med. Mol. Imaging (2015) 42: 288-301.

3) Modern technologies for creating non-natural antibodies for clinical use / S.M. Deev, E.N. Lebedenko // Acta naturae. No. 1 2009, pp. 32-50.

4) Synthesis and Properties of Boranocarbonate: A convenient in situ CO source for the aqueous preparation of [99mTc (H ₂ O) ₃ (CO) ₃ ] + / Alberto, R. et al.,. // J. Am. Chem. Soc. 2001, 123 (13), 3135-3136.

5) Waibel, R. et al., Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc (I) - carbonyl complex. Nat. Biotechnol. 1999, 17 (9), 897-901.

6) Patent No. 2616974, 04/19/2017. Yusubov M.C., Larkina M.C., Podrezova E.V., Stasyuk E.S., Skuridin B.C. The method of obtaining ω-bis (pyridin-2-ylmethyl) amino) aliphatic acids - precursors with chelate centers for metal binding.

7) Patent 2125017, 02.07.1997. The method of obtaining water-soluble carbonyl complexes of short-lived technetium (I) and rhenium (I) / Gorshkov N.I., Lumpov A.A., Miroslavov A.E., Suglobov D.N.

8) Patent No. 2294897, 02.28.2005. A method of obtaining a water-soluble carbonyl complex of short-lived technetium-99m / Lumpov A.A., Gorshkov N.I., Suglobov D.N., Miroslavov A.E.

9) Hofstrom C, Altai M, Honarvar H, Strand J, Malmberg J, Hosseinimehr SJ, et al. HAHAHA, HEHEHE, HIHIHI, or HKHKHK: influence of position and composition of histidine containing tags on biodistribution of [(99m) Tc (CO) 3] (+) - labeled affibody molecules. J Med Chem. 2013; 56: 4966-74.

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Claims (1)

A method of producing a complex of technetium-99m with recombinant targeted protein molecules for radionuclide diagnostics of oncological diseases with HER-2 / neu overexpression, comprising obtaining a complex with aquacarbonyl technetium-99 m and targeted DARPin molecules, characterized in that aquacarbonyl technetium is produced in the first stage 99m ^[99m Tc (H ₂ O) ₃ (CO) _3] ⁺ using sodium tetraborate decahydrate lyophilisate, sodium carbonate, disodium boranokarbonata which can also comprise sodium or potassium tartrate tetrahydrate tension citrate monohydrate Ia, further to a lyophilisate is added 3.1 ml of sodium pertechnetate solution, ^99m Tc with activity 0,74-3,7 GBq and heated in a boiling water bath for 20-30 minutes, cooled to room temperature and 1M HCl was added to pH 7 , 4 or 300-900 μl of a 1M sodium phosphate solution with a pH of 7.4, then the chelating agent succinimid-1-yl 6- (bis (pyridin-2-ylmethyl) amino) hexanoate (DPAH) is added to DARPin in pH phosphate buffer 8.3-8.5 to obtain DPAH-DARPin, then the aquacarbonyl technetium-99 m [ ^99m Tc (H ₂ O) ₃ (СО) ₃ ] ^{+ is} coupled with DARPin or DPAH-DARPin, for which it is DARpin or DPAH- Darpin in 100 μg in a phosphate-buffered solution at pH 7.4 add 1 ml of a solution of aquacarbonyl technetium-99m [ ^99m Tc (H ₂ O) ₃ (CO) ₃ ] ⁺ , then incubate at 40 ° C for 30-60 minutes, purification is carried out by gel filtration on a column.