RU2537175C2 - Method for producing radioimmune preparation for diagnosing and treating oncological diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and represents a method for preparing a therapeutic radioconjugate of a specifically binding agent with a short-lived radionuclide for the delivery to pathological regions. Implementing the method involves labelling recombinant humanised mini-antibodies specific to the cancer-associated antigen HER2/neu, with the diagnostic gamma-ray radionuclide aquacarbonyl complex Tc-99m; the mini-antibodies are conjugated with human albumin; the prepared conjugate is purified and added with the chelating agent DOTA (1,4,7,10-tetraazacyclododecane tetraacetic acid) or DTPA (diethylene triamine pentaacetic acid); the prepared conjugate is labelled with the therapeutic alpha-ray radionuclides; and the produced preparation is purified.

EFFECT: producing the anti-cancer preparation possessing a low risk of the conflict with the immune system, simplifying the formulation and providing a demand for preparations with targeted agent delivery.

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Description

The invention relates to a technology for producing an antitumor preparation for detecting and destroying micrometastases, including at the cellular level, using γ-emitting radioisotopes, for example Tc-99m and short-lived α-emitting radioisotopes, for example, Bi-212, Bi-213, Pb- 212, Ra-223, Ac-225, mounted on a protein platform that provides specific binding to the target tumor cell.

The present invention can be used to obtain an antitumor preparation based on a nanoscale biocompatible conjugate of human serum albumin (HSA) and humanized mini-antibodies specific for the cancer-associated marker HER2 / neu. The drug is intended both for diagnosis and localization, and for the treatment of malignant neoplasms. The use of the drug for diagnosis is possible due to γ-emitting radioisotopes, for example, short-lived Tc-99m, attached to a protein conjugate that provides high-precision delivery. The therapeutic effect of the drug is achieved by atoms of α-emitting radioisotopes, for example Bi-212, Bi-213, Pb-212, Ra-223 or Ac-225, attached to the same conjugate.

State of the art

One of the most promising methods for the treatment of cancer is radioimmunotherapy using short-lived α-emitting radioisotopes (α-emitters) Bi-213; Bi-212, Pb-212, Ac-225 and other α-particles have high energy and short range in the substance, therefore, when a sufficient number of α-emitter atoms are localized in the immediate vicinity of the tumor cell, selective destruction of the tumor tissue is achieved with minimal damage to the surrounding tissues. To ensure selective localization of α-emitter atoms, antibodies to various tumor antigens (both native and in the form of fragments or modifications), as well as ligands capable of specifically binding to specific cell surface receptors, are most often used.

The main radioisotope used in the diagnosis of cancer is Тс-99m, which has an optimal half-life (6 hours) and type of radiation (γ-radiation, energy 140 keV). When using drugs for high-precision delivery of the Tc-99m radioisotope to the target, a significant decrease in its concentration is possible, which will ensure a reduction in the dose load on the patient and medical personnel.

One of the most studied and often mentioned in the literature tumor antigens is the surface receptor HER2 / neu. An increased level of expression of this antigen plays a key role in the pathogenesis of malignant breast tumors and a number of other forms of malignant neoplasms. A number of monoclonal antibodies have been developed for this antigen, suitable for use in diagnostic and therapeutic purposes.

Various methods are known for labeling protein molecules (including monoclonal antibodies) with radioisotopes. Labeling using bifunctional chelating agents, which are capable of forming fairly strong coordination bonds with a large number of cations, including cations of transition elements and lanthanides (Co, Cu, Y, In, Ac, Bi, etc.).

As chelators, DTPA (Diethylene triamine pentaacetic acid) and DOTA (1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ″ ″ - tetraacetic acid) and their derivatives are mainly used.

Various variants of the synthesis of the conjugate "antibody - chelator - radioisotope" are described in the literature. An example is the publication [Blend, M.J., Stastny, J.J., Swanson, S.M. and Breichbel, M.W. Labeling anti-HER2 / neu Monoclonal Antibodies with In-111 and Y-90 using a bifunctional DTPA Chelating agent. Cancer Biotherapy & Radiopharmaceuticals, 2003, vol. 18, no 3, pp 355-363], which primarily considers the conjugate of antibodies to HER2 / neu with DTPA, but also describes the method of conjugation with DOTA (in the form of p-SCN-bensyl- DOTA). The same data are contained in [Lewis M, Kao J, Anderson A-L, Shivley J, Raubitschek A. An improved method for conjugating monoclonal antibodies with N-hydroxysulfosuccinimidyl DOTA. Bioconj. Chem. 2001; 12: 320-324]. As a rule, along with native antibody molecules, the possibility of using any of their fragments and modifications that retain the ability to specifically bind to the antigen is mentioned.

Methods for the directed and controlled formation of covalent bonds between protein molecules, including those suitable for conjugation of HSA molecules and mini-antibodies, are described in [Hermanson G.T. 2008. Bioconjugate Techniques, 2nd Edition. Academic Press, Inc., pp. 1-1202]. US 2011/0286920 A1 [Jeong J.M., Lee M.C., Chung K.G. and Lee D.C. Complex of bifunctional chelating agent and mannosyl human serum albumin] describes a method for forming a complex between a modified HSA molecule and a bifunctional chelator for subsequent labeling with a radioisotope. In particular, the use of a DOTA chelator and a Bi-212 radioisotope are mentioned. However, the described complex did not include an element that provides specific binding to a tumor-specific antigen, which rendered the construct unsuitable for targeted delivery of an antitumor drug.

In patent WO 2012/089336 A1 [Caviglioli G., Parodi B., Cafaggi S., et al. A conjugate of human albumin and 2- (4-isothiocyanatobenzyl) -1,4,7,10-tetraazocyclododecane-1,4,7,10-tetraacetic acid useful for the localization of radionuclides for diagnostic and therapeutic purposes] a production method is also described conjugate of HSA with DOTA for the purpose of subsequent labeling with a radioactive isotope (including bismuth isotopes). However, for the synthesis of the described complex, the benzyl derivative DOTA (p-SCN-bensyl-DOTA) was used, in contrast to the hydroxysuccinylimide ester DOTA. In addition, the described complex also did not provide binding to a tumor-specific antigen.

Patent WO 2008/045604 [Yang D, Yu D. Efficient synthesis of chelators for nuclear imaging and radiotherapy: compositions and applications] also mentions antibody-chelator-TC-99m (for diagnostic purposes) and antibody-chelator designs -Bi-213 "(for therapeutic purposes). However, the HSA molecule was not used in the conjugate.

In patent US 2009/0136421 [Ranson M; Allen BJ and Bunn CL PAI-2 conjugates for the treatment and imaging of cancer] describes a construct that was proposed to label either Tc-99m (for diagnostic purposes) or Bi-213 (for therapeutic purposes). However, the binding of the construct to the tumor tissue was achieved not due to the antibody, but due to the PAI-2 molecule (plasmin activator inhibitor).

A series of works has been published in the press that describe the simultaneous labeling of the Trastuzumab molecule (a humanized monoclonal mouse antibody to HER2 / neu) with a fluorescent dye and a radioactive isotope (In-111 gamma emitter [Sampath L, Kwon S, Ke S, Wang W, Schiff R , Mawad ME, Sevick-Muraca EM. Dual-labeled trastuzumab-based imaging agent for the detection of human epidermal growth factor receptor 2 overexpression in breast cancer. J Nucl Med. 2007 Sep; 48 (9): 1501-10], or β-emitter Cu-64 [Sampath L, Kwon S, Hall MA, Price RE, Sevick-Muraca EM. Detection of Cancer Metastases with a Dual-labeled Near-Infrared / Positron Emission Tomography Imaging Agent. Transl Oncol. 2010 Oct 1; 3 (5): 307-217]). This approach made it possible to improve the diagnostic capabilities of the construct, but the possibility of its therapeutic use was not considered.

As a prototype, a patent was selected [US 6403771 B1, (ACTINIUM PHARMACEUTICALS, LIMITED (BM), 06/11/2002], which describes a method for producing a radio immunoconjugate for therapeutic use.

The radioimmunoconjugate contains an antibody or fragment thereof that provides specific binding to a tumor-specific antigen attached to a carrier molecule to which several atoms of α-emitting isotopes are attached. Bi-212 and Bi-213 are used as α emitters, and HSA is used as a carrier molecule. In this prototype, HSA is included in the conjugate in order to increase the number of sites for attachment of a chelating agent (respectively, to increase the number of isotope atoms per antibody molecule). It should be noted that HSA was mentioned as a possible but not necessary element of the radio immunoconjugate. As an example of a practical embodiment, said patent discloses molecules of a full-sized monoclonal antibody labeled with a radioactive isotope using a chelating agent, but without the participation of a carrier molecule. In the description of the synthesis, the main attention is paid to the choice of the radioisotope and the methodological aspects of labeling the conjugate.

The disadvantages of the prototype include:

- when describing the structure of the radiopharmaceutical, no attention is paid to solving the problem of the rapid removal of such radiopharmaceutical from the bloodstream, and, accordingly, a sharp decrease in its therapeutic effect if it contains fragments of antibodies with low molecular weight, such as mini-antibodies having a molecular weight of 30 kDa .

Disclosure of invention

The basis of the invention is the task of obtaining a therapeutic radioconjugate having at the same time a set of the following properties:

- a reduced risk of a radioimmunoconjugate conflict with the patient’s immune system;

- increased specificity;

- improved pharmacokinetic parameters;

- increased level of specific radioactivity (therapeutic activity);

- simplification and cheapening of the technology for its production and ensuring the growing demand for drugs targeted delivery of diagnostic and therapeutic agents for the treatment of cancer.

The problem is solved in that a method for producing a therapeutic radioconjugate, a specifically binding substance with a short-lived alpha-emitting radioisotope for its delivery to pathological areas, is proposed, while recombinant humanized mini-antibodies specific for the cancer-associated HER2 / neu antigen are diagnosed with diagnostic gamma-emitting with a radioisotope, namely, the TC-99m aquacarbonyl complex, conjugate mini-antibodies with human serum albumin, clean the floor the conjugate is introduced, the chelating agent DOTA (1,4,7,10 - tetraazicyclodecodane tetraacetic acid) or DTPA (diethylene triamine pentaacetic acid) is introduced into the conjugate, the resulting conjugate is labeled with therapeutic radioisotopes, namely, short-lived alpha-emitting and radioisotopes a drug.

In this case, short-lived α-emitting radioisotopes are used as a therapeutic radioisotope: Bi-212, and / or Bi-213, and / or Pb-212, and / or Ra-223, and / or Ac-225.

The binding of the diagnostic radioisotope in the conjugate is carried out using an oligohistidine chelator.

As a recombinant humanized antibody specific for the cancer-associated HER-2 / new antigen, a humanized mini-antibody scFv 4D5 is used.

The binding of the humanized mini-antibody to human serum albumin in the conjugate is carried out using an oligolysin chelator.

In addition, as a recombinant humanized mini-antibodies specific for the cancer-associated HER-2 / neu antigen, a humanized mini-antibody with oligolysin and oligohistidine fragments in the C-terminal part of the molecule is used, which are designed for specific conjugation of mini-antibodies with human serum albumin and diagnostic isotope, respectively.

In the described method, it is principally the use of humanized mini-antibodies. To date, in oncology practice, only two drugs have been used for radioimmunotherapy of tumors, both are used to treat non-Hodgkin's lymphoma: Bexxar © (GlaxoSmithkline, 2003), which is mouse IgG2a conjugated to medium energy beta-emitter I-131 (penetration radius 1 mm) and Zevalin © (Biogen IDEC, 2002) - mouse IgG1 conjugated to Y-90 high-energy β-emitter (penetration radius 11 mm) [D. Gusarov A brief overview of the biopharmaceutical market for the treatment of lymphatic cancer. Biopharmaceutical Journal. 2010.V.2. Number 4. p. 8-13]. The murine origin of these antibodies is often the cause of serious complications in their use, up to anaphylactic shock. The humanization of the components of preparations for radioimmunotherapy significantly reduces the risk of conflict with the patient’s immune system, while the use of murine or other xenogenic antibodies causes an immune response when the radioimmunoconjugate based on them is first introduced into the patient’s bloodstream, with the risk of anaphylactic shock when this radiopharmaceutical is reintroduced. Humanized antibodies are currently being produced using genetic engineering methods, replacing individual fragments of an antibody molecule with fully human sequences.

In contrast to the prototype, it is fundamentally the use of mini-antibodies and the use of full-sized antibodies is excluded. The main advantages of mini-antibodies are the lack of effector functions inherent in the Fc fragment of full-sized antibodies [Deyev S.M., Lebedenko E.N. Multivalency - the hallmark of antibodies used for optimization of tumor targeting by design. BioEssays. 2008. V.30. P.904-918].

Mini-antibodies are single polypeptide encoded by a single gene and comprise only one antigen binding site comprising the variable light domain (V _L) and heavy (V _H) chains joined flexible peptide linker. Mini-antibodies, unlike full-sized antibodies, lack the constant part, which contains the binding sites to the C1q complement system protein and Fc-fragment receptors (FcγR) and mediates the effector (secondary) functions of immunoglobulins (the ability to kill target cells by triggering antibody-dependent mechanisms cell cytotoxicity and complement dependent cytotoxicity). The absence of a constant part in mini-antibodies significantly improves their properties when used as a targeting component in the composition of radio immunoconjugates.

Despite the fact that obtaining humanized mini-antibodies is more difficult and the molecular weight is much lower than that of full-sized antibodies, their use in radioimmunotherapy has a number of significant advantages over full-sized molecules of monoclonal antibodies. One way to increase the molecular weight of radioimmunoconjugates using humanized mini-antibodies is to conjugate them with a molecule of an inert biocompatible carrier protein. In the described design, the conjugation of the molecules of humanized mini-antibodies is ensured by the HSA molecule (which is the fundamental difference from the prototype). Up to 40 secondary amino groups are located on the surface of the protein globule of the HSA molecule [Prasad, T.S.K. et al. (2009) Human Protein Reference Database - 2009 Update. Nucleic Acids Research. 37, D767-72], due to which the formation of covalent bonds with molecules of mini-antibodies is possible. In this prototype, the role of HSA in the pharmacokinetics of the resulting immunoconjugate was not specified in any way. In contrast to the prototype in this application, the optimization of the pharmacokinetics parameters was one of the reasons why HSA is a necessary structural element of the radio immunoconjugate.

To achieve a therapeutic effect, the radioimmunoconjugate molecule must be in the bloodstream for a sufficient time in order to reach the target organ and contact the surface of the tumor cell. The half-life of proteins through the kidneys correlates with their molecular weight: the glomerular filtration threshold is estimated at 60-65 kDa [Trejtnar, F., Laznicek, M. Analysis of renal handling of radiopharmaceuticals. The quarterly journal of nuclear medicine: official publication of the Italian Association of Nuclear Medicine (AIMN) [and] the International Association of Radiopharmacology (IAR) 2002; 46 (3): 181-94]. The molecular weight of the radioimmunoconjugate, consisting only of a mini-antibody, chelator and isotope, as a rule, does not exceed 50 kDa (on average about 30 kDa). The half-life of such molecules is estimated at 0.5-2 hours [Covell DG, Barbet J, Holton OD, Black CD, Parker RJ, Weinstein JN. Pharmacokinetics of monoclonal immunoglobulin G1, F (ab ′) 2, and Fab ′ in mice. Cancer Res. 1986 Aug; 46 (8): 3969-3978]. In this case, a significant portion of the conjugate will be excreted by the kidneys before it even has time to contact the target cells; instead of tumor tissue, kidney tissue will be exposed to radiation. The introduction of HSA (molecular weight 69 kDa) into the radioimmunoconjugate not only allows you to create a center of a nanoparticle around which conjugation of mini-antibody molecules occurs, but also increases the total molecular weight of the complex and its time in the bloodstream, respectively, reduces the radioactive effect on the kidneys. Since HSA is a natural human blood protein, there is practically no chance of an unwanted immune response to the administration of such a conjugate.

The drawing shows a diagram of the module for the synthesis of conjugate, where 1 is a bottle of eluent; 2 - radioisotope generator; 3, 11, 14 - waste collections; 4 - ionizing radiation detectors; 5 - syringe pump; 6 - column conditioning the eluate from the generator; 7 - reactor for preparing the reaction mixture; 8 - thermoreactor for the implementation of conjugation; 9 - gel filtration cartridge; 10 - a sterile bottle for the finished product; 12 - sterilizing filter; 13 - bottles for reagents.

An example embodiment of the invention

1. Materials used:

Recombinant mini-antibodies specific for the HER-2 antigen, clone 4D5, containing an oligo-histidine fragment in the C-terminal part of the molecule (hereinafter MA) (IBCh RAS), recombinant human serum albumin (HSA, Sigma, cat. No. A9731), Trout reagent (Pierce, cat. No. 26101), Sulfo-SMCC (Pierce, cat. No. 22322), DMSO (Sigma, cat. No. D8418), chelating agent DOTA-NHS (Macrocyclics, cat. No. B-280), chelating agent p -SCN-Bn-DTPA (Macrocyclics, cat. No. B-305), Chelex 100 (Sigma, cat. No. C7901), Triton X-100 (Sigma, cat. No. T9284).

Sigma or Merck salts, acids and alkalis of high purity are used to prepare buffer solutions. Dialysis is carried out in dialysis bags (Sigma, cat. No. D9277) and (Sigma, cat. No. D9777) according to the recommendations of the Manufacturer. In the work using deionized water with a conductivity of 18 MΩ. Measurement of optical densities is carried out on an SF-102 spectrophotometer (LLC NPO Interfotofizika).

Elution radioisotope generator Bi-212 or Bi-213 is carried out with hydrochloric acid ^{(TraceSELECT ® Ultra, for ultratrace analysis} , Cat. №96208-250) at a concentration of 0.3 mol / l. The pH adjustment of the mixture for the reaction of labeling the conjugate with α-emitting radioisotopes is carried out with a saturated solution of Na ₂ CO ₃ (Cat. No. m.1.06392.0500 EMSURE® ISO, for analysis). The complexation of Bi-212, Bi-213 ions is carried out with a 0.1 M solution of H ₃ Cit. (Cat. No m.1.00244.0500, for analysis). To carry out the labeling reaction, 100 ml of a conjugate solution prepared according to paragraphs are used. 2-8 with a concentration of 0.2 mg / ml in buffer solution No. 5 [0.02 M MES, 0.15 M NaCl, pH 6.2].

2. Obtaining recombinant humanized mini-antibodies specific for cancer-associated marker HER2 / neu (MA)

Freshly prepared competent cells of Escherichia coli SB536 strains are transformed with the pIG4D5 genetic construct and plated on Petri dishes with LB agar medium (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% bactoagar) with 2% glucose and ampicillin (final concentration 150 μg / ml) and incubated at 37 ° C overnight. The grown colonies are washed from a cup of 10 ml of LB liquid medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with ampicillin (final concentration 150 μg / ml) and transferred to 1 l of the same culture medium. Incubate with vigorous shaking at 37 ° C to an optical density of A550 equal to 0.8-1, then lower the temperature to 28 ° C and induce target genes by adding IPTG to a final concentration of 0.66 mM. Incubated overnight (14-16 hours) with vigorous shaking at 28 ° C.

The resulting culture is cooled in an ice bath, then the cells are separated from the culture fluid by centrifugation for 10 min at 4000-6000g 4 ° C. The supernatant is discarded. Cells are resuspended in an ice bath in TBS lysis buffer (30 mM Tris HCl, pH 7.6-7.8; 20 mM KH ₂ PO ₄ ; 150 mM NaCl), lysozyme is added to a concentration of 0.5 mg / ml and kept on ice with constant stirring for 40 min The resulting lysate is centrifuged for 30 minutes at 15,000 rpm; 4 ° C. The supernatant is discarded. The clarified lysate is filtered through a 0.22 μm millipore filter. To prevent hydrophobic interactions between proteins, 7/8 volumes of glycerol and 1/8 volume of 5M sodium chloride are added dropwise to the filtered clarified lysate. To reduce nonspecific sorption of proteins on an affinity carrier, imidazole is added to the lysate to a concentration of 10 mM and the pH is adjusted to 7.8 at 0-4 ° C with 1 M NaOH.

The prepared lysate was applied at a rate of 0.1-0.15 ml / min on a HisTrap FF column with a 5 ml Ni-NTA-Sepharose (GE Healthcare) equilibrated with LSW buffer (25 mM Tris, 20 mM KH ₂ PO ₄ ; 0.1 M NaCl; 10% glycerin; pH 7.8). The column is washed with 3 volumes of HSW buffer (25 mM Tris, 20 mM KH ₂ PO ₄ ; 0.5 M NaCl; 10% glycerol; pH 7.8) and 2 volumes of LSW buffer (25 mM Tris, 20 mM KH ₂ PO ₄ ; 0.1 M NaCl; 10% glycerol; pH 7.8) at a rate of 0.1-0.15 ml / min. To wash non-specifically bound proteins, the column is washed with 5 volumes of LSW buffer with the addition of 0.05% Tween20 at a rate of 0.03-0.04 ml / min, and then sequentially with 5 volumes of LSW buffer at the same speed.

MA was eluted with 10 volumes of an imidazole gradient column (0 □ 0.2 M) in LSW buffer (25 mM Tris, 20 mM KH ₂ PO ₄ ; 0.1 M NaCl; 10% glycerol; pH 7.8). The obtained fractions containing MA (~ 0.1 M imidazole) were combined, diluted 10 times to an imidazole concentration of 0.01 M. For further purification, the affinity chromatography step on HisTrap FF column with 5 ml Ni-NTA-Sepharose balanced (GE Healthcare), balanced LSW buffer (25 mM Tris, 20 mM KH ₂ PO ₄ ; 0.1 M NaCl; 10% glycerol; pH 7.8).

To change the buffer in the MA solution, NAP-5, PD-10 columns (GE Healthcare) are used. The resulting MAs contain oligolysin and oligohistidine fragments at the C-terminus of the molecule.

3. Labeling of MA with the Tc-99m aquacarbonyl complex

To obtain the Tc-99m aquacarbonyl complex, a mixture of 0.004 g (0.038 mmol) Na2CO3 and 0.005 g (0.13 mmol) NaBH4 in an 8 ml tube with a screw cap with a rubber gasket is saturated with CO for 10 min. Then, 3 ml of an eluate from a molybdenum generator containing up to 30 GBq.Na [99mTcO4] in a 0.9% aqueous NaCl solution was added via a syringe and kept at a temperature of 75 ° C for 30 min. Cool and add 0.3 ml of 1M Na phosphate, pH 7.4.

To 50 μl of MA (concentration 2 mg / ml) in PBS buffer (7 mM NaH2PO4, 3 mM Na2HPO4, 350 mM NaCl, pH 7.4), 25 μl of 0.5 M MES pH 6.2 and 25 μl of the resulting solution [99mTc (CO) 3 ( OH2) 3] + (~ 0.25 GBq) and incubated for 30 min at 37 ° C. The Tc-99m aquacarbonyl complex specifically binds to the MA oligohistidine fragment (specific activity ~ 2-2.5 GBq / mg protein). To purify MA labeled with a Tc-99m aquacarbonyl complex from an unbound radioisotope, gel filtration was performed on a Superdex-75 column in PBS buffer.

4. Preparation of CSA and MA for conjugation

The HSA protein is dissolved in buffer solution No. 1: [0.02 M NaHCO3, 0.15 M NaCl, pH 8.0], at a concentration of 10 mg / ml.

A solution of mini-antibodies (MA) in a buffer with a concentration of 12 mg / ml is dialyzed in 5 shifts against 1000x excess buffer solution No. 2 [0.02 M NaHCO3, 0.15 M NaCl, 0.3% Chelex-100, pH 8.0], 12 hours at + 4 ° C with stirring on a magnetic stirrer. Then, by dilution with buffer solution No. 1, the resulting MA solution was adjusted to a concentration of 5 mg / ml and used for conjugation.

For the introduction of SH-groups, Trout reagent is used in a 10-fold molar excess with respect to HSA molecules. The reaction was incubated for 1 h at room temperature with stirring, and then desalted on Zeba-spin columns (Pierce, Cat. No. 89894), washed with buffer No. 1 [0.02 M NaHCO3, 0.15 M NaCl, pH 8.0], according to the manufacturer's recommendations in order to remove excess unreacted reagent Trout. A solution of thiolated HSA protein is used for conjugation with mini antibodies.

Maleimide groups in the mini-antibodies are introduced using a 5-fold molar excess of sSMCC relative to the mini-antibodies. After incubating the reactions for 1 h at room temperature, the solutions are desalted on Zeba-spin columns (Pierce, Cat. No. 89890) washed with buffer [0.02 M NaHCO3, 0.15 M NaCl, pH 8.0] according to the manufacturer's recommendations in order to remove excess -sSMCC as well as reaction by-products.

5. Conjugation of MA with HSA

Thiolated using a 10-fold excess of Trout HSA reagent is mixed with a 5: 1 excess of activated sSMCC mini antibodies in a 1: 1 molar ratio. The mixture was incubated for 4 hours at room temperature. Monomeric mini-antibodies are practically absent in the reaction mixture; at the same time, a significant amount of free HSA molecules remains. The results of electrophoresis indicate that several molecules of mini-antibodies are attached to one molecule of HSA.

6. Purification of MA conjugate with HSA

Purification of the conjugates obtained from unconjugated HSA protein was carried out on a 3 ml Ni-NTA column (HisPur Ni-NTA Spin Columns, Pierce), balanced with buffer solution [0.02 M NaHCO3, 0.3 M NaCl, 0.02 M imidazole, 0.01% Triton X -100, pH 8.0]. The conjugates bound to the column elute with 3 ml of buffer solution [0.02 M NaHCO3, 0.3 M NaCl, 0.5 M imidazole, 0.01% Triton X-100, pH 8.0].

For further purification of conjugates from components with a molecular weight below 150 kDa, microconcentrators with a cutoff by molecular weight of 150 kDa (89922, Pierce, USA) using a buffer solution [0.02 M NaHCO3, 0.15 M NaCl pH 8.0], according to the instructions of the company, are used. manufacturer.

7. The inclusion of the chelating agent DOTA or DTPA in the conjugates of MA-HSA

A 40-fold excess of DOTA-NHS or p-SCN-Bn-DTPA is used to incorporate a chelating agent into the conjugates, assuming that the average molecular weight of the resulting conjugates is 150 kDa.

8. Determination of protein concentration in the resulting preparation

The protein concentration in the resulting preparations is determined by spectrophotometry at a wavelength of 280 nm using the following extinction coefficients:

HSA = 0.53

MA = 1.27

Conjugates MA-HSA, MA-HSA-DOTA and MA-HSA-DTPA = 0.93.

9. Electrophoresis of HSA conjugates with mini antibodies

Protein electrophoresis is carried out in a 4-12% gradient acrylamide gel (Life Technologies, Cat. No. NP 0322BOX) according to the recommendations of the manufacturer. As molecular weight markers, LMW low molecular weight markers are used (GE Healthcare, Cat. No. 17-0446-01).

10. Determination of the amount of chelating agent DOTA in the conjugates

DOTA titration in the conjugate was carried out according to the previously proposed method with slight changes (Dadachova et al, 1999). To do this, prepare the following solutions:

- Pb (II) -AA (III) solution: 0.15 M ammonium acetate, 10 μM Arsenazo III, 4.83 μM Pb (CH3CO2) 2 · 3H2O, pH 7.0. The lead salt is added to the solution from a concentrated solution of 0.01 M Pb (CH3CO2) 2 · 3H20 in 10 mM HCl;

- DOTA solution for titration: 0.1 mM DOTA-NHS solution in buffer solution No. 5 [0.02 M MES, 0.15 M NaCl, pH 6.2].

A DOTA titration solution (0 μl, 10 μl, 20 μl, etc. up to 50 μl) is added to 2 ml of a solution of Pb (II) -AA (III), stirred, allowed to stand at room temperature for 5 minutes, and the absorption of light obtained is measured solutions on an SF-102 spectrophotometer at a wavelength of 656 nm. A calibration line is constructed that shows the dependence of light absorption at a wavelength of 656 nm on the concentration of the DOTA solution added to it for titration.

A solution of HSA-MA-DOTA conjugates, 20 μl, is added to 2 ml of a solution of Pb (II) -AA (III), stirred, allowed to stand at room temperature for 5 minutes, and the light absorption of the obtained solutions is measured at a wavelength of 656 nm. All measurements are carried out 3 times and take the average value as the value.

The protein concentration in the obtained oligomeric constructs is 1 mg / ml; when 20 μl of MA-ChSA-DOTA conjugate solutions are added to 2 ml of a solution of Pb (II) -AA (III), the molar concentration of protein (weight average 150 kDa oligomer) in the mixture is :

C _oligomers = 0.067 (μM).

The number of DOTA molecules per oligomer molecule is calculated by the formula:

N = C _DOTA / C _oligomers .

The synthesis of the drug is carried out on the automated synthesis module shown in the drawing.

11. Elution of short-lived α-emitters from isotope generators (conditioning the eluate by fractionation)

Using a syringe pump 5, 1-2 ml of 0.3 M HCl are pumped from the bottle with eluent 1 through a radioisotope generator 2. The eluate is collected in a waste collector 3. Then, 0.5-1.5 eluent is pumped through a radioisotope generator 2. The presence of radioisotopes in the eluate is monitored by means of ionizing radiation sensors 4. The eluate is collected in a reaction mixture preparation reactor 7 for conditioning the solution before labeling, into which 10 μl of a 0.1 M H ₃ Cit complexing agent solution are previously added.

12. Elution of short-lived α-emitters from isotope generators (conditioning of the eluate by concentration)

Using a syringe pump 5, 4-6 ml of 0.3 M HCl are pumped from the bottle with eluent 1 through a radioisotope generator 2. The eluate is fed to the conditioning column of the eluate from generator 6 to concentrate and purify the eluate, while the target radioisotope is adsorbed on the column. The eluate at the outlet of the column is collected in a waste container 11.

0.5 to 0.8 ml of a 0.1 M solution of H ₃ Cit pH = 4 was passed through the conditioning column of the eluate from the generator 6. The eluate containing the target radioisotope is collected in a reaction mixture preparation reactor 7.

13. Labeling of short-lived α-emitting radioisotopes of conjugated antibodies

0.2-0.4 ml of a saturated Na ₂ CO ₃ solution is added to the reaction mixture preparation reactor 7, stirred for 3-4 minutes, and after the neutralization reaction is completed, the resulting mixture is transferred to a thermoreactor for conjugation 8. Previously, 100 μl are placed in a thermoreactor for conjugation 8 conjugated antibody buffer solution.

The solution in the thermoreactor for conjugation 8 is heated to a maximum of 40 ° C and stirred for 5-15 minutes.

14. The formation of the conditioned physico-chemical and biomedical form of the drug

After the completion of the labeling reaction, the solution is taken from the thermoreactor for conjugation 8 and fed to the gel filtration cartridge 9 for purification from unbound bismuth. At the output, the “dead” volume of the eluent is collected in the waste collector 14. Then, the buffer solution is passed through the gel filtration cartridge 9 and The “target” eluate is collected in a sterile product vial 10. After that, the gel filtration cartridge 9 is washed with a buffer solution and the tail eluate is collected in a waste collector 14.

If it is necessary to obtain a sterile product, the “target” eluate from the gel filtration cartridge 9 is passed through a sterilizing filter 12 and collected in a sterile drug vial 10. If necessary, to impart certain properties to the finished product, chemical components from reagent bottles can be added to the eluate 13.

Thus, the proposed method allows to obtain an antitumor drug for diagnosis and radioimmunotherapy, characterized by a set of simultaneously acting properties: low immunogenicity; increased specificity; improved pharmacokinetic parameters and an increased level of specific radioactivity (therapeutic activity). This method has potential therapeutic and commercial value.

Claims (6)

1. A method of obtaining a therapeutic radioconjugate, a specifically binding substance with a short-lived alpha-emitting radioisotope for its delivery to pathological areas, characterized in that they carry out the labeling of recombinant humanized mini-antibodies specific for the cancer-associated HER2 / neu antigen, a diagnostic gamma-emitting radioisotope, and it is the TC-99m aquacarbonyl complex that conjugates mini-antibodies with human serum albumin, purifies the resulting conjugate, and introduces in the conjugate, the chelating agent DOTA (1,4,7,10-tetraazicycloododecane tetraacetic acid) or DTPA (diethylene triamine pentaacetic acid) is used to label the resulting conjugate with therapeutic radioisotopes, namely, short-lived alpha-emitting radioisotopes and purify the resulting preparation. 2. The method according to claim 1, characterized in that as a therapeutic radioisotope using short-lived α-emitting radioisotopes: Bi-212, and / or Bi-213, and / or Pb-212, and / or Ra-223, and / or Ac-225. 3. The method according to claim 1, characterized in that the binding of the diagnostic radioisotope in the conjugate is carried out using an oligohistidine chelator. 4. The method according to claim 1, characterized in that as a recombinant humanized antibody specific for the cancer-associated antigen HER-2 / new, a humanized mini-antibody scFv 4D5 is used. 5. The method according to claim 1, characterized in that the binding of the humanized mini-antibodies to human serum albumin in the conjugate is carried out using an oligolysin chelator. 6. The method according to claim 1, characterized in that as a recombinant humanized mini-antibodies specific for the cancer-associated antigen HER-2 / neu, use a humanized mini-antibody with oligolysin and oligohistidine fragments in the C-terminal part of the molecule, which are intended for specific conjugation of mini-antibodies with human serum albumin and diagnostic isotope, respectively.

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