EA043221B1 - RADIOPHARMACEUTICAL COMPLEXES - Google Patents

Description

The invention relates to a complex of tissue-targeted thorium-227 with a certain octadentate ligand, as well as to a method for the formation of this thorium complex, to a pharmaceutical formulation containing the specified thorium complex, the use of a thorium complex and a pharmaceutical formulation for the treatment of hyperplastic or neoplastic diseases, and to a method for the treatment of hyperplastic or neoplastic diseases, including the introduction of this complex or the specified pharmaceutical formulation.

Prerequisites for the invention

For the successful treatment of various diseases in mammals, methods of specific cell destruction are of great importance. Typical examples of such treatment are the treatment of malignant diseases such as sarcomas and carcinomas. However, the selective removal of certain cell types may also play a key role in the treatment of other diseases, especially hyperplastic and neoplastic diseases.

Currently, the most common methods of selective treatment are surgery, chemotherapy and external radiation. Targeted radionuclide therapy is, however, an emerging and promising field that can deliver highly cytotoxic radiation to cells specifically associated with a particular disease. The most common forms of radiopharmaceuticals currently approved for human treatment use radionuclides that emit beta or gamma particles. However, for use in therapy, alpha particle emitting radionuclides are also of interest because of their specific cell killing potential.

The radiation range of typical alpha-emitting radionuclides in the physiological environment is typically less than 100 µm, which is only a few cell diameters. Because of this, such sources of radiation are suitable for the treatment of tumors, including micrometastases, since the range of action can reach the nearest cells in the tumor, however, only a small amount of the emitted energy will go beyond the target cells, provided appropriate targeting. Thus, it is not necessary to target every cell, but damage to surrounding healthy tissue can be minimized (see Feinendegen et al., Radiat Res 148:195-201 (1997)). In contrast, the range of action of beta particles in an aquatic environment is 1 mm or more (see Wilbur, Antibody Immunocon Radiopharm 4: 85-96 (1991)).

The energy of alpha radiation is high compared to the energy of beta particles, gamma radiation and X-rays, as a rule, it is 5-8 MeV, or 5-10 times higher than the energy of beta particles and 20 or more times higher than the energy of gamma rays. Thus, such exposure to a large amount of energy at a very short distance provides an extremely high linear energy transfer (LET) of α radiation, a high relative biological effectiveness (RBE) and a low oxygen amplification factor (OER) compared to gamma and beta radiation ( see Hall, Radiobiology for the radiologist, fifth edition, Lippincott Williams & Wilkins, Philadelphia PA, USA, 2000). This explains the exceptional cytotoxicity of alpha-emitting radionuclides, but also imposes stringent requirements on the biological targeting of such isotopes, as well as providing the necessary level of control and depth of study of the distribution of alpha-emitting radionuclides to prevent unacceptable side effects.

In table. 1 below shows the physical decay properties of alpha-emitting radionuclides currently proposed in the literature as radionuclides with potential therapeutic efficacy.

Table 1

Candidate nuclide Tl/2* Clinical studies regarding ²²⁵ Ac 10.0 days leukemia ²ⁿ At 7.2 hours glioblastoma ² 13 _Bi 46 minutes leukemia ²²³ Ra 11.4 days skeletal metastasis ²²⁴ Ra 3.66 days ankylosing spondylitis

* Half life.

Until now, with respect to radioimmunotherapy, the focus has been on ²¹¹ At, ²¹³ Bi and ²²⁵ Ac, and these three nuclides have been studied in clinical immunotherapy studies.

Some of the proposed radionuclides are perishable, ie. their half-life is less than 12 hours. Because of this short half-life, commercial production and distribution of these radionuclides is difficult. The introduction of a nuclide with a short half-life also increases the dose of radiation received by the body until the drug reaches the target site.

The recoil energy of alpha radiation in many cases will lead to the release of daughter nuclides from the decay site of the parent nuclide. Such a recoil energy is sufficient for many daughter nuclei to leave the chemical environment in which the parent nuclide was located, for example, if the parent nuclide was present in a complex with a ligand, such as a chelating agent. This will happen even if the daughter nuclide is chemically compatible, i.e. can form complexes with the same ligand. Similarly, if the daughter nuclide is a gas, in particular an inert gas such as radon, or if the daughter nuclide is chemically incompatible with the ligand, such a release effect will be even greater. If the half-lives of the daughter nuclides are more than a few seconds, they can diffuse and enter the circulatory system, since they are not retained by the substance that was part of the complex that retained the parent nuclide. These free radioactive progeny radionuclides can also cause unwanted systemic toxicity.

Several years ago, a proposal was put forward to use thorium-227 (T _1/2 = 18.7 days) under the control of the daughter isotope ²²³ Ra (see WO 01/60417 and WO 02/05859). Such an application was possible in a situation where a carrier system was used, which made it possible to keep the daughter nuclides in a closed environment. In one case, the radionuclide is located in the liposome, and the significant size of the liposome (compared to the distance of the recoil energy) allows you to keep the daughter nuclides in the liposome. In the second case, osteotropic complexes of the radionuclide are used, which are embedded in the bone matrix and thus limit the release of daughter nuclides. These methods are particularly preferred, however, in some circumstances the use of liposomes is undesirable, and there are many soft tissues in which radionuclides cannot be surrounded by a mineralized matrix to retain progeny isotopes.

Relatively recently, it has been found that the toxicity of ²²³ Ra daughter nuclei that are released after ²²⁷ Th decay is better tolerated by the mammalian body than expected from previous studies using similar nuclei. In the absence of specific means to retain the daughter radium, which is formed during the decay of -227, in accordance with the description above, it is clear from the publicly available information on the toxicity of radium that the use of thorium-227 as a medicinal agent is impossible, since the use of doses necessary to achieve a therapeutic effect from the decay of thorium-227 would result in a highly toxic and possibly lethal dose of radiation from the decay of radium daughter nuclides, thus there is no therapeutic window.

WO 04/091668 describes that it has surprisingly been found that there is a therapeutic window in which a therapeutically effective amount of a targeted thorium227 radionuclide can be administered to a patient (typically a mammal) without producing enough radium-223 to to cause unacceptable myelotoxicity. Thus, it can be used to treat and prevent all types of diseases in both bone and soft tissue areas of the body.

In view of the above developments, it is now possible to use thorium-227 alpha emitting nuclei in endoradionuclide therapy without lethal myelotoxicity resulting from ²²³ Ra production. Despite this, the therapeutic window is relatively narrow and it is undesirable under any circumstances to administer more alpha-emitting radioisotope to a patient than necessary. Therefore, the utility of this new therapeutic window would be greatly enhanced if alpha-emitting thorium-227 were used as part of a complex and targeted with a high degree of reliability.

Since the decay of radionuclides is constantly taking place, the time that is spent between the release of the material and the administration of the drug to the patient is of extreme importance. In addition, it would be extremely useful if the alpha-emitting thorium nuclei were complex, targeted and/or applied in a form that would not take much time and would not be difficult to obtain, preferably, would be carried out with using a small number of steps, short incubation periods with multiple steps, and/or temperatures that do not irreversibly affect the properties of the target compound. In addition, processes that can be carried out in solvents that do not require removal prior to use (especially aqueous solutions) have the advantage that a solvent evaporation step or a dialysis step is eliminated.

In addition, it would be extremely beneficial if a thorium-labeled drug with improved stability could be developed. This is critical to meeting quality standards for product stability while providing a logistical loop for delivering a dose of drug to a patient. Thus, formulations with minimal radiolysis over a period of 1-4 days are preferred.

It has previously been shown that octadentate chelating agents containing hydroxypyridinone groups can be used to coordinate alpha-emitting thorium-277, for subsequent attachment to the target moiety (WO 2011098611). Octadentate chelators containing four 3,2-hydroxypyridinone groups have been described with linker groups attached to an amine-based scaffold with a separate reactive group used for conjugation with

- 2 043221 targeted molecule. The preferred structures of the previous invention contained 3,2-hydroxypyridinone groups and used the isothiocyanate moiety as the preferred chemical structure to couple to the antibody component as shown in the ALGDD-NCS junction. Isothiocyanate is widely used to label proteins through amine groups. The isothiocyanate group reacts with the amino terminus and primary amines in proteins and can be used to label many proteins, including antibodies. Although the thiourea bond formed in these conjugates is fairly stable, it has been described that antibody conjugates derived from fluorescent isothiocyanates degrade over time. [Banks P.R., Paquette D.M., Bioconjug Chern (1995) 6:447-458]. Thiourea formed by the reaction of fluorescein isothiocyanate with amines under basic conditions is also susceptible to conversion to guanidine [Dubey I, Pratviel G, Meunier BJournal: Bioconjug Chern (1998) 9:627-632]. Because of the long half-life of thorium-227 (18.7 days) coupled to a monoclonal antibody with a long biological half-life, it is desirable to use more stable binding moieties to produce conjugates that are more chemically stable in vivo and upon storage.

The most relevant previous work on hydroxypyridinone ligand conjugation was published in WO 2013/167754. It describes ligands having a water-soluble moiety with hydroxyalkyl functionality. Due to the high reactivity of the hydroxyl groups of this class of chelates, activation as an activated ester is not possible, since in this case many parallel reactions occur, resulting in a mixture of products through esterification reactions. Thus, according to WO 2013/167754, ligands must be coupled to the tissue-targeted protein via alternative chemical structures such as isothiocyanate to produce a less stable thiourea conjugate as described above. In addition, WO 2013167755 and WO 2013167756 describe hydroxyalkyl/isothiocyanate conjugates useful for targeting CD33 and CD22 antibodies, respectively.

The inventors of the present invention have found that by forming a tissue-targeted complex by binding a specific chelator to the appropriate targeting moieties, and then adding alpha-particle emitting 4+ thorium ion ²²⁷ Th on the fly, under non-stringent conditions, a complex can be obtained with the binding moiety, which remains more stable during storage of the complex and when it is administered to the patient.

Brief summary of the invention

Thus, according to a first aspect, the present invention provides a tissue-targeting thorium complex, which is a compound comprising

wherein said tissue-targeted Y fragment is a monoclonal antibody selected from hLL2 (epratuzumab), HM195 (lintuzumab), trastuzumab, BAY 1179470 and BAY 86-1903 (mesothelium); and 4+ ion of the thorium radionuclide ²²⁷ Th emitting alpha particles.

In a preferred embodiment of the tissue-targeted thorium complex, said tissue-targeted monoclonal antibody comprises at least one peptide chain comprising an amino acid sequence with at least 90% sequence identity with at least one of the following: sequences:

Light chain:

DIQLTQSPSSLAVSAGENVTMSCKSSQSVLYSANHKNYLAWYQQKPGQ SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVQVEDLAIYYCHQYLSSWTF GGGTKLEIKR (SeqIDl)

DIQLTQ SPS SLAS AAVEDRTMSCKS SQ SVLYS ANHKNYL AWYQQKPGQ KAKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSSWTF GGGTKLEIKR (SeqID2)

Heavy chain:

QVQLQESGAELSKPGASVKMSCKASGYTFTSYWLHWIKQRPGQGLEWI GYINPRNDYTEYNQNFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARRDI TTFYWGQGTTLTVSS (SeqID3)

QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEW IGYINPRNDYTEYNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRDIT TFYWGQGTTVTVSS (SeqID4)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEW

IGYINPRNDYTEYNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRDIT

TFYWGQGTTVTVSS(SeqID5).

In accordance with the second aspect of the present invention, a method for forming

-3 043221 tissue-targeted thorium complex, said method comprising the following steps:

a) formation of an octadentate chelator AGC0019 having the following structure:

b) connecting said octadentate chelator to at least one tissue-targeted moiety containing at least one amine moiety with at least one amide-binding reagent selected from N-hydroxymaleimide, carbodiimide, and/ or azodicarboxylate activating reagents, wherein a tissue-targeted chelator is obtained, said tissue-targeted fragment being a monoclonal antibody selected from hLL2 (epratuzumab), HuM195 (lintuzumab), trastuzumab, BAY 1179470 and BAY 86-1903 (mesothelin); And

c) bringing said tissue-targeted chelator into contact with an aqueous solution containing a 4+ ion of the thorium radionuclide ²²⁷ Th emitting alpha particles.

In such complexes (and in all aspects of the present invention), the thorium ion complex is typically formed by a ligand containing the appropriate octadentate hydroxypyridinone, which in turn is attached to the tissue-targeted moiety via an amide bond.

This method is a method for synthesizing a 3,2hydroxypyridinone octadentate chelate comprising a reactive carboxylate function that can be activated as an active ester (such as an N-hydroxysuccinimide ester (NHS ester)) either in situ or by synthesis and isolation of the most active ester.

The resulting NHS ester can be used in a simple conjugation step to produce a wide variety of chelated protein forms. In addition, highly stable antibody conjugates are already labeled with thorium-227. This can take place at or near normal temperature, typically with high radiochemical yields and purity.

The method according to the invention is preferably carried out in an aqueous solution, and in accordance with one embodiment of the invention, it can be carried out in the absence or substantially in the absence (at a content of less than 1% by volume) of any organic solvent.

The tissue-targeted fragments are a monoclonal antibody selected from hLL2 (epratuzumab), HuM195 (lintuzumab), trastuzumab, BAY 1179470 and BAY 86-1903 (mesothelin).

The tissue-targeted complexes of the present invention may be formulated into medicaments suitable for administration to human and non-human animal patients.

Thus, according to a further aspect of the invention, methods are provided for preparing a pharmaceutical formulation, comprising preparing a tissue-targeted complex as described herein by adding at least one pharmaceutical carrier and/or excipient. Suitable carriers and excipients include buffers, chelating agents, stabilizing agents, and other appropriate components known to those skilled in the art and described in accordance with any aspect herein.

According to a first aspect of the invention, a tissue-targeted thorium complex is provided. Such a complex will have the characteristics described in the text of this document, in particular, the preferred characteristics described in this document. The specified complex can be obtained using any of the methods described in this document. Thus, at least one tissue-targeted thorium complex can be prepared using such methods in accordance with any aspect or embodiment of the invention as described herein.

In accordance with another aspect of the present invention provides a pharmaceutical formulation containing any of the complexes described herein. The specified preparative form can be obtained using any of the methods described herein, and may contain at least one buffer, stabilizer and/or auxiliary substance. The buffer and stabilizer can be chosen to help protect the tissue-targeted complex from radiolysis. In accordance with one embodiment of the invention, the radiolysis of the complex in the formulation is minimal, even several days after the production of the formulation. This is a significant advantage as it eliminates potential product quality and logistical issues in the supply of medicines, which are key to implementation and practical application.

- 4 043221 of this technology.

The present invention has proven useful in the preparation of a variety of thorium-labeled antibody conjugates for targeting sites of biological interest in the body, such as tumor-associated receptors.

Detailed description of the invention

In the context of the present invention, the term tissue targeting, as used herein, means that the appropriate substance (particularly in the form of a tissue-targeting complex as described herein) is intended to localize (and in particular to localize the interconnected complex thorium) preferably to at least one tissue site where its presence is desired (eg, to deliver radioactive decay). Thus, the tissue-targeted moieties or moieties serve to better localize the respective substance in at least one site of the patient's body after it has been administered to such a patient, compared to a concentration of the same complex without such a targeted moiety. In this case, such a targeted fragment is preferably chosen to specifically bind to cell surface receptors associated with cancer cells or other receptors associated with the tumor microenvironment.

Several targets are known to be associated with hyperplastic and neoplastic diseases. Such targets include certain receptors, cell surface proteins, transmembrane proteins, and proteins/peptides that reside in the extracellular matrix in the vicinity of the affected cells. Examples of cell surface receptors and antigens that can be associated with neoplastic diseases include CD22, CD33, FGFR2 (CD332), PSMA, HER2, mesothelin, etc. The tissue-targeted fragment (eg, peptide or protein) has specificity for at least one antigen or receptor selected from CD22, CD33, FGFR2 (CD332), PSMA, HER2, and mesothelin.

CD22 or differentiation cluster 22 is a molecule belonging to the SIGLEC family of lectins (SIGLEC = sialic acid binding immunoglobulin-like lectins).

CD33 or Siglec-3 is a transmembrane receptor expressed on myeloid cell lines.

FGFR2 is a receptor for fibroblast growth factor. It is a protein that is encoded in humans by the FGFR2 gene, which is located on chromosome 10.

HER2 is a member of the human epidermal growth factor receptor (HER/EGFR/ERBB) family.

Prostate specific membrane antigen (PSMA) is an enzyme encoded in humans by the FOLH1 (folate hydrolase 1) gene.

Mesothelin, also known as MSLN, is a protein that is encoded in humans by the MSLN gene.

In this case, especially preferred tissue-targeted binding fragments are selected from fragments that bind specifically to the CD22 receptor. This can be expressed in the fact that, for example, such a fragment has a higher binding affinity (50 times or more) for cells expressing CD22 compared to cells that do not express CD22 (for example, at least 100 times higher affinity binding, preferably at least 300 times higher binding affinity). There is reason to believe that the expression and/or overexpression of CD22 occurs in cells with certain stages of the disease (as indicated herein); thus, a binding fragment specific for CD22 may act to target the complex to such cells. Similarly, a tissue-targeted fragment can bind to cell surface markers (eg, CD22 receptors) that are present in the cell in the vicinity of diseased cells. Compared to the surface of healthy cells, the expression of CD22 cell surface markers may be more potent on the surface of diseased cells or more potent on the cell surface during periods of growth or replication compared to the dormant phase. In one embodiment, a tissue-targeted CD22-specific binding moiety can be used in combination with another binding moiety for a disease-specific cell surface marker, resulting in a double binding complex. Tissue-targeted binding fragments for CD-22 are typically peptides or proteins.

Various aspects of the invention as described in this document relate to the treatment of diseases, in particular, they are intended for selective targeting in the affected tissue, and may also relate to complexes, conjugates, drugs, formulations, drug kits that are used to implement methods in accordance with the invention. In all aspects, the affected tissue may be in a single site of the body (eg, in the case of a localized solid tumor) or may be in different sites (eg, when multiple joints are affected in arthritis, or in the case of dispersed malignant tumors or metastatic cancer).

The affected tissue to be targeted may be in soft tissues, in

- 5 043221 calcified tissues or in several areas, which may all be in soft tissues, in calcified tissues or may include at least one area of soft tissues and/or at least one area of calcified tissues. In accordance with one of the embodiments of the invention, targeting is carried out in at least one area of the soft tissues. The sites targeted and the sites of disease onset may be the same or may be different (eg, in the case of specific targeting to metastases). In the case of targeting at multiple sites, targeting may be at the site of origin of the disease or multiple secondary sites.

As used herein, the term soft tissue refers to tissues that do not have a hard, mineralized matrix. In particular, when used throughout this document, the term soft tissue can mean any tissue that is not a skeletal tissue. Accordingly, as used herein, the term soft tissue disease means a disease occurring in soft tissues as defined herein. In particular, the present invention may be used to treat a variety of cancers, thus the term soft tissue disease includes carcinomas, sarcomas, myelomas, leukemias, lymphomas, and mixed type cancers that occur in any soft (i.e., non-mineralized) tissues, as well as other non-cancerous diseases of such tissues. Soft tissue malignancies include solid tumors originating in soft tissues, as well as metastatic and micrometastatic tumors. Indeed, soft tissue diseases can include a primary solid soft tissue tumor and at least one metastatic soft tissue tumor in the same patient. Alternatively, the soft tissue disease may be only the primary tumor or only metastases when the primary tumor is in the skeletal tissues. In particular, diseases that can be treated in accordance with all aspects of the present invention, or in which tissue is targeted with a thorium complex in accordance with all aspects of the present invention, are hematological tumors, in particular neoplastic diseases of lymphocytes, for example, lymphomas and lymphoid leukemias, including non-Hodgkin's lymphoma, B-cell neoplasms, or B-cell lymphomas. Similarly, diseases that can be treated in accordance with all aspects of the present invention, or in which tissue is targeted with a thorium complex in accordance with all aspects of the present invention, are any neoplastic diseases of the bone marrow, spine (in particular, spinal cord), lymph nodes and/or blood cells.

Some examples of B-cell tumors that can be treated in accordance with all aspects of the present invention, or in which tissue is targeted with a thorium complex in accordance with aspects of the present invention, include the following diseases: chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (eg, Waldenström's macroglobulinemia), splenic marginal zone lymphoma, plasma cell tumors (eg, plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin storage diseases, heavy chain diseases), extranodal marginal zone B-cell lymphoma (MALT-lymphoma) type), nodular B-cell lymphoma from cells of the marginal zone (ULKKZ), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, large B-cell lymphoma of the mediastinum (thymus), intravascular large B-cell lymphoma, primary lymphoma of serous cavities and lymph oma/leukemia Burkitt.

Some examples of tumors that can be treated with an FGFR2 targeting agent of the present invention include tumors in which mutational events are associated with tumor initiation and progression, including breast, endometrial, and gastrointestinal cancers.

Some examples of tumors of myeloid origin that can be treated with the CD33 targeting agent of the present invention include acute myeloid leukemia (AML).

Other examples of tumors that can be treated with the prostate specific membrane antigen (PSMA) targeting agent of the present invention include prostate and brain cancers.

Other examples of tumors that can be treated with the epidermal growth factor receptor type 2 (HER-2) targeting agent of the present invention include breast cancers.

Other examples of tumors that can be treated with the mesothelin targeting agent of the present invention include cancers such as mesothelioma, ovarian, lung and pancreatic cancer.

The main factor affecting the effectiveness of the present invention is the stability of antibody conjugates during storage for certain periods. Thus, the stability of non-radioactive conjugates of antibodies and the final drug product labeled with thorium should be

- 6 043221 comply with the strict criteria that are required in the production and distribution of radiopharmaceutical products. Surprisingly, the formulation described herein containing the tissue-targeted moiety was found to exhibit excellent storage stability. This is true even at high temperatures, which are usually used in accelerated stability studies.

In accordance with one embodiment of the invention, which is applicable to all combined aspects of the invention, the tissue-targeting complex may be dissolved in an appropriate buffer. In particular, it has been found that when using a citrate buffer, an unusually stable formulation is obtained. Preferably a citrate buffer is used in the range of 1-100 mM (pH value 4-7), in particular in the range of 10-50 mM, most preferably a citrate buffer in the range of 20-40 mM.

In accordance with yet another embodiment of the invention, which is applicable to all combined aspects of the invention, the tissue-targeting complex can be dissolved in an appropriate buffer containing para-aminobutyric acid (PABA). The preferred combination is citrate buffer (preferably at the concentrations described herein) in combination with PABA. Preferred concentrations for PABA for use in accordance with any aspect of the present invention, including in combination with other agents, are about 0.005-5 mg/ml, preferably 0.01-1 mg/ml, more preferably 0.01-1 mg /ml Concentrations in the range of 0.1-0.5 mg/ml are most preferred.

In accordance with another embodiment of the invention, which is applicable to all combined aspects of the invention, the tissue-targeting complex can be dissolved in an appropriate buffer containing ethylenediaminetetraacetic acid (EDTA). The preferred combination is the use of citrate buffered EDTA. A particularly preferred combination is the use of citrate buffered EDTA in the presence of PABA. It is preferred in such combinations that citrate, PABA and EDTA (as the case may be) be present in the concentration ranges and preferred concentration ranges indicated herein. Preferred concentrations for EDTA for use in accordance with any aspect of the present invention, including in combination with other agents, are about 0.02-200 mM, preferably 0.2-20 mM, more preferably 0.05-8 mM.

In accordance with another embodiment of the invention, which is applicable to all combined aspects of the invention, the tissue-targeting complex can be dissolved in an appropriate buffer containing at least one polysorbate (PEG grafted ester of sorbitol and fatty acid).

Preferred polysorbates include polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 80 (polyoxyethylene (20) sorbitan monolaurate), and mixtures thereof. The most preferred polysorbate is polysorbate 80 (P80). Preferred concentrations for polysorbate (in particular, for the preferred polysorbates specified herein) for use in accordance with any aspect of the present invention, including in combination with other agents, are approximately 0.001-10% w/v, preferably 0 01-1% w/v, more preferably 0.02-0.5% w/v.

Despite the fact that PABA has already been described as a radiostabilizer (see US 4880615 A), the positive effect of PABA in the present invention was observed during storage of a non-radioactive conjugate. This stabilizing effect in the absence of radiolysis is a particularly unexpected advantage, since the synthesis of the tissue-targeted chelator will typically occur substantially prior to contact with the thorium ion. Thus, a tissue-targeted chelator can be prepared from one hour to three years prior to contact with the thorium ion and will preferably be kept in contact with PABA for at least part of this period. In other words, steps a) and b) of the present invention can be carried out from one hour to three years before step c) and between steps b) and c), the tissue-targeted chelator can be stored in contact with PABA, in particular in a buffer, for example, in citrate buffer and, in some cases, together with EDTA and/or polysorbate. Preferred types and concentrations of materials are specified in this document. Thus, PABA is a particularly preferred component of the formulations of the invention and can provide long-term stability for a tissue-targeted chelator and/or for a tissue-targeted thorium complex. In FIG. 1 shows the effect of PABA in the system of the present invention.

The use of a citrate buffer as described herein provides unexpected stability benefits for the tissue-targeted thorium complex in the formulations of the present invention. The study of irradiation on the effect of buffer solutions in the production of hydrogen peroxide was carried out by the authors of the present invention, and unexpected results were obtained. It is known that hydrogen peroxide is formed as a result of water radiolysis and contributes to the chemical modification of protein conjugates in solution. Thus, the formation of hydrogen peroxide has an undesirable effect on the purity and stability of the product.

- 7 043221

In FIG. 2 shows that unexpectedly lower levels of hydrogen peroxide were observed when measured in solutions of the conjugate of the HOPO antibody of the present invention, which were irradiated with Co-60 (10 kGy) in citrate buffer compared to all other test buffers. Thus, the formulation of the present invention preferably contains a citrate buffer as described herein.

In addition, the authors of the present invention found unexpected results in relation to the combined effect of certain components in the formulations of the present invention. Again, this refers to the stability of the radiolabeled conjugate. The aim of the study was to evaluate the stability of the ²²⁷ Th-AGC1118 conjugate (see below) during storage. An infrared fluorescence binding assay was performed using ²²⁷ Th-AGC1118 at a specific activity of approximately 8000 Bq/μg. Five different storage solutions were prepared for ²²⁷ Th-AGC1118 using 30-100 mM citrate buffer, or 30 mM citrate buffer supplemented with 0.02, 0.2 or 2 mg/mL rABA, pH 5.5. In FIG. 3 shows a significant positive effect on radiostability of the formulations of the present invention, in particular when combined with citrate and/or PABA in the ranges indicated herein. When conducting the above study, it was found that citrate is the most effective buffer; surprisingly, it was also found that such an effect was enhanced by the addition of PABA.

A key component of the methods, complexes and formulations of the present invention is an octadentate chelator moiety. The most relevant work on the formation of complexes of thorium ions with hydroxypyridinone ligands was published as WO 2011/098611, which describes the relative ease of complexing thorium ions with octadentate NOPO-containing ligands.

Previously known chelators for thorium also include polyaminopolyacid chelators that include a straight, cyclic or branched polyazaalkane backbone with acidic (eg, carboxyalkyl) groups attached to nitrogen atoms in the backbone. Examples of such chelators include DOTA derivatives such as p-isothiocyanatobenzyl-1,4,7,10tetraazacyclodecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA) and DTPA derivatives such as p-isothiocyanatobenzyl -diethylenetriaminepentaacetic acid (p-SCN-Bz-DTPA), DOTA derivatives are cyclic chelators, DTPA derivatives are linear chelators.

Examples of 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid derivatives have been given previously, but standard methods cannot simply be used to chelate thorium to DOTA derivatives. When the DOTA derivative is heated using a metal, the chelate is efficiently produced, but often in low yield. During this procedure, irreversible denaturation of at least a portion of the ligand may occur. In addition, due to the relatively high susceptibility to irreversible denaturation, it is generally necessary to avoid attaching the targeted moiety until all heating steps have been completed. This requires an additional chemical step (with all the necessary processing and separation), which must be carried out during the decay period of the thorium isotope emitting alpha particles. Obviously, it is preferable not to process the material emitting alpha particles in this way and not to produce the corresponding waste in excess of the required level. In addition, during the time it takes to obtain the conjugate, part of the thorium is lost, the decay of which will occur during such a preparation period.

A key aspect of the present invention in all respects is the use of an appropriate octadentate ligand, in particular an appropriate octadentate hydroxypyridinone-containing ligand comprising four HOPO moieties. Such ligands typically contain at least four chelating groups, each independently of the other having the following substituted pyridine (I) structure:

(1) where R ¹ may be an alkyl group such as straight or branched C1-C5 alkyl groups including methyl, ethyl, n- or isopropyl and n-, sec-iso- or tert-butyl. Preferably, R ¹ may be C1-C ₃ , in particular methyl. In accordance with the present invention, a methyl substituent is present on the nitrogen atom of all four fragments of formula (I).

Alkyl groups referred to throughout this document are generally C1- _C8 straight or branched chain alkyl groups such as methyl, ethyl, or isopropyl, n-, iso-t- or sec-butyl, etc. .d.

In some previously published inventions such as WO 2013/167756, WO 2013/167755 and

- 8 043221

WO 2013/167754, the group corresponding to R ¹ was previously a solvent group such as hydroxy or hydroxyalkyl (for example, -CH ₂ OH, -CH ₂ -CH ₂ OH, -CH ₂ -CH ₂ -CH ₂ OH and t .d.). This has certain advantages in terms of higher solubility, however, such chelators are difficult to attach to targeted moieties using amide bonds due to reactivity at the R ¹ position. Thus, in the present invention, R ¹ is not hydroxyl or hydroxyalkyl.

In formula (I), the R2- ^R6 groups can be independently selected from H, OH, ═O, a connecting moiety and a linker moiety. Preferably exactly one of the R2-R ⁶ groups will be ═O and one of the R2-R ⁶ groups will be OH. The remaining three groups from the R2-R ⁶ groups may be H, however, at least one of the R2-R ⁶ groups will be a linker moiety and/or a bridging moiety. The connecting moiety is described hereinafter, however, it ends with a carboxylic acid to attach an amide bond to the targeted moiety. Such a connecting moiety may be attached directly to the ring in one of the R ² R ⁶ groups, however, more preferably it is attached to a linker moiety, which itself will represent one of the R 2 -R ⁶ groups.

In accordance with the present invention, all four fragments of the octadentate ligand, as shown above, which are part of the complex, are 3,2-HOPO fragments.

Appropriate chelating moieties may be formed using methods known to those skilled in the art, including those described in US 5,624,901 (eg, Examples 1 and 2) and WO 2008/063721 (which are incorporated herein by reference).

Chelating groups include groups of formula (II) below

(SCH

In the above formula (II), the =O moiety is an oxo group attached to any carbon atom on the pyridine ring, -OH is a hydroxy moiety attached to any carbon atom on the pyridine ring, a -RL is a linker moiety, which attaches a hydroxypyridinone fragment to other fragments that make up the complex to form a common octadentate ligand. Any linker moiety as described herein can be used as R _L including short hydrocarbyl groups such as C ₁ -C ₈ hydrocarbyl including C ₁ -C ₈ alkyl, alkenyl or alkynyl groups including methyl, ethyl , propyl, butyl, pentyl and/or hexyl groups of all topologies. _RL can be attached to the ring of formula (II) through any carbon atom in the pyridine ring. The _RL groups can then, in turn, be linked directly to another chelating moiety, to another linker group, and/or to a central atom or group, such as a ring or other matrix. Linkers, chelating groups and, if necessary, template fragments are chosen to form the appropriate octadentate ligand.

RC is a connecting fragment, as described below. Suitable moieties include hydrocarbyl groups such as alkyl or alkenyl groups ending in a carboxylic acid group. The present inventors have found that when using a linker moiety with a carboxylic acid to form an amide, for example using the methods of the present invention, a more stable conjugation between the chelator and the tissue-targeted moiety is provided.

In accordance with the present invention, the -OH and =O moieties of formula II are located on adjacent atoms of the pyridine ring, thus a 3,2-hydroxypyridinone derivative is very useful. The RN group is a methyl substituent.

Thus, in accordance with the present invention, four fragments of 3,2-hydroxypyridinone are present in the structure of the octadentate ligand.

Therefore, the chelating groups are the groups of formula (IIa) CH ₃

^r _l (Ua)

As used herein, the term linker moiety (R _L in formula (II) and formula (IIa)) means a chemical building block that serves to attach at least two chelating groups in octadentate ligands, which are key components in various aspects of the present invention. Linker moieties can also be attached to a bridging moiety that serves to attach an octadentate ligand to a tissue-targeted moiety. Typically, each chelating group (for example, the above

- 9 043221 nye group of formula (I) and/or (II) and/or (IIa)) is bidentate, thus, there are four chelating HOPO groups in the ligand. Such chelating groups are linked together via their linker moieties and attached to the tissue-targeting moiety (in the method of the present invention) via a link moiety. Thus, a linker moiety (eg, the RL group in formula (II)) may be shared by several chelating groups of formula (I) and/or (II). Linker fragments can also serve as an attachment point between the octadentate ligand included in the complex and the target fragment. In such a case, at least one linker fragment is attached to the connecting fragment ( _RC in formula (II)). Suitable linker moieties include short hydrocarbyl groups such as C ₁ -C ₁₂ hydrocarbyl including C ₁ -C ₁₂ alkyl, alkenyl or alkynyl groups including methyl, ethyl, propyl, butyl, pentyl and/or hexyl groups of all topologies. Other groups that may be present on linker moieties (RL) include any sufficiently stable functional groups such as aryl groups (eg phenyl groups), amides, amines (especially secondary or tertiary) and/or ethers. RC fragments may also contain alkyl and/or aryl sections and, if necessary, groups such as amine, amide and ether linkages. As a rule, all components of the connecting fragment must be resistant to the storage conditions in which the complex is located. Such conditions include alpha radiolysis, thus labile functional groups are not preferred.

The connecting moiety may contain a terminal carboxylic acid, at least one alkyl moiety (eg, a methyl or ethyl moiety), at least one amide moiety, and at least one aryl moiety (eg, a phenyl group). The connecting moiety can be attached to one or more linker moieties of the octadentate ligand via a carbon-carbon, amide, amine and/or ether bond.

The junction moiety ( _RC ) that attaches the octadentate ligand to the target moiety can be selected from the following:

[-CH ₂ -Ph-N(H)-C(=O)-CH ₂ -CH ₂ -C(=O)OH],

[ _-CH2- CH2-N(H)-C(=O)-(CH2-CH2-O)i. ₃ -CH2-CH ₂ -C(=O)OH] or [-[CH ₂ ]i.3-Ar-N(H)-C(=O)-[CH ₂ ]i.5-C(=O )OH], where Ar is an aromatic group, such as a substituted or unsubstituted phenylene group, and Ph is a phenylene group, preferably a para-phenylene group.

Linker fragments may be or contain any other sufficiently stable chemical bonds, including groups of esters, ethers, amines and/or amides. The total number of atoms attached to two chelating moieties (counted by the shorter path if more than one path exists) will generally be limited to maintain the proper arrangement of the chelating moieties for complex formation. Thus, the linker moieties will be selected such that the number of atoms between the chelating moieties is no more than 15, preferably 1-12 atoms, more preferably 1-10 atoms. If the linker moiety is attached directly to two chelating moieties, then the length of the linker will typically be 1-12 atoms, preferably 2-10 atoms (eg ethyl, propyl, n-butyl, etc.). If the linker moiety is attached to a central template (see below), then each linker may be shorter than two separate linkers that are attached to the chelating moieties. In this case, the preferred length of the linker may be 1-8 atoms, preferably 1-6 atoms (relevant groups are methyl, ethyl and propyl, as well as groups having an ester, ether or amide bond at one end or both ends).

In addition to the linker moiety, which primarily serves to connect the various chelating groups of the octadentate ligand to each other and/or to attach them to the central matrix, the octadentate ligand also contains a linking moiety ( _RC ) with a terminal carboxylic acid. The function of the connecting moiety is to attach the octadentate ligand to the target moiety via a stable covalent bond, in particular via an amide. Preferably, the linking moieties are covalently attached to the chelating groups, either by direct covalent attachment to one of the chelating groups or, more commonly, by attaching to a linker moiety or matrix. If two or more connecting fragments are used, each of them can be attached to any of the available sites, for example, on a matrix, linker or chelating group.

A connector fragment may have the following structure:

R ⁷ -X where R ⁷ means a bridge fragment, that is, an element selected from substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and X is the target moiety to which the amide or carboxylic acid or equivalent is attached

- 10 043221 tape functional group. Preferred bridging moieties include all of the groups listed herein as appropriate linker moieties.

Preferred targeting moieties include all moieties described herein, and preferred reactive X groups include any groups capable of acting as a carboxylic acid in forming an amide covalent bond to the targeted moiety, including, for example, -COOH, -SH, -NHR and groups where R from NHR can be H or any of the short hydrocarbyl groups described herein. Particularly preferred groups for attachment to the target moiety include the epsilon-amines of lysine residues. Among others, examples of suitable reactive X groups include N-hydroxysuccimidyl ethers, imidoesters, acyl halides, N-maleimides, and alpha-haloacetyl.

In accordance with the present invention, the selected R ⁷ bridging moiety is the corresponding substituted aryl, and the selected bridging moiety (RC) that attaches the octadentate ligand to the target moiety is [-C(=O)-CH ₂ CH ₂ -X-] where the free carboxylate group on the HOPO ligand is activated in situ as N-hydroxysuccinimide ester in aqueous solution immediately prior to conjugation to the targeted moiety.

The connecting fragment is preferably attached in such a way that the resulting conjugated octadentate ligand can be used in the formation of stable metal ion complexes. Thus, the linking moiety preferably binds to the linker, template, or chelating moiety at a site where it does not significantly interfere with complex formation. Such a site is preferably located on the linker or matrix, more preferably at a position remote from the surface attached to the target.

Each moiety of formula (I) or (II) or (IIa) in an octadentate ligand may be attached to the remainder of the ligand via an appropriate linker group, as described herein, in any appropriate topology. For example, the four groups of formula (I) and/or (II) and/or (IIa) may be linked by their linker groups to the backbone to form a straight ligand, or may be bridged by a linker group to form an oligomeric type structure, which may be unbranched or cyclic. Alternatively, ligand moieties of formulas (I) and/or (II) and/or (IIa) can be attached in a cross or star topography to the central atom or group, with each of the moieties attached via a linker (e.g. via RL fragment). Linker (RL) moieties may be attached exclusively via carbon-carbon bonds, or may be linked to each other or to other chelating groups, to a backbone, matrix, linking moiety, or other linker, via any sufficiently stable functional group, including amine, amide, ether or a thioether bond.

The star arrangement is shown in formula (III) below where all group and position designations are as described above, and T additionally means the central atom or group of the matrix, for example, a carbon atom, a hydrocarbyl chain (for example, any of the chains described above), aliphatic or aromatic a ring (including heterocyclic rings) or a fused ring system. The simplest matrix is a single carbon atom, which is then attached to each of the chelating moieties via their connecting groups. In addition, longer chains are also possible, such as ethyl or propyl, with two chelating moieties attached to each end of the matrix. Obviously, any sufficiently stable bonds can be used to connect the matrix and linker fragments, including carbon-carbon bonds, esters, ethers, amines, amides, thioether or disulfide bonds.

Obviously, in the structures of formula (II), (III), (IV) and (IVb), positions on the pyridine ring that are not otherwise substituted (for example, by a linker or connecting moiety) may, as appropriate, carry substituents, described for R1-R ⁵ in formula (I). In particular, small alkyl substituents, such as methyl, ethyl or propyl groups, may be present at any position.

In addition, the octadentate ligand will additionally contain at least one compound

- 11 043221 thread fragment as described above. It may be any appropriate structure, including any structures specified herein, and together with the targeted fragment, it will end in the final complex or carboxylic acid in accordance with the methods of the present invention.

The connecting moiety can be attached to any appropriate point on the linker, template, or chelating moiety, such as points a, b and/or c, as indicated in formula (III). Any sufficiently stable bonds can be used to attach the connecting moiety, for example, carbon-carbon bonds, esters, ethers, amines, amides, thioether or disulfide bonds. Likewise, groups capable of forming any such bonds with the target moiety can be used as the functional end of the connecting moiety, so that the specified moiety will end with such groups when attached to the target moiety.

An alternative framework type structure is shown by formula (IV) below

C .......R _B ........

(IV) where all group and position designations are as described above, and RB additionally means a framework fragment, which will generally have a similar structure and function to any of the linker fragments described herein, thus, if the context allows, any the definition of a linker moiety can be applied to a scaffold moiety. The corresponding framework fragments form a scaffold to which chelating fragments are attached using their linker groups. Usually three or four wireframe fragments are required. As a rule, three fragments are required for a linear framework, or four fragments if the framework is cyclic. Particularly preferred backbone moieties include short hydrocarbon chains (such as, for example, as described herein), optionally having a heteroatom or functional moiety at one or both ends. In this regard, amine and amide groups are particularly suitable.

The connecting moiety can be attached to any appropriate point on the linker, backbone or chelating moiety, for example, to points a, b and/or c, as indicated in formula (IV). Any sufficiently stable bonds can be used to attach the connecting moiety, for example, carbon-carbon bonds, esters, ethers, amines, amides, thioether or disulfide bonds. Likewise, groups capable of forming any such bonds with the target moiety can be used as the functional end of the connecting moiety, so that the specified moiety will end with such groups when attached to the target moiety.

An example of a framework-type octadentate ligand with four chelating 3,2-HOPO moieties attached to the framework via an amide linker group would be a ligand of the following formula (V):

Rc

O O O O (V)

Obviously, the linking fragment _RC can be attached at any appropriate point on this molecule, for example, to one of the secondary amine groups or to a branch point on any of the alkyl groups of the framework. A preferred site for the _RC group is shown in formula (V). _RC will terminate in a carboxylic acid or will be attached via an amide bond to a tissue-targeted moiety in appropriate aspects of the invention. All small alkyl groups, such as the backbone propylene group or the n-substituting ethylene group, may be substituted with other small alkylenes, for example any of the alkylenes described herein (of such alkylenes, methylene, ethylene, propylene and butylene are particularly suitable).

Examples of matrix-type octadentate ligands, each having four chelating 3,2-HOPO moieties attached by ethylamide groups to ethyl and propyl diamine, respectively, would be the ligands of the following formula (VI):

- 12 043221

Obviously, any of the alkyl groups shown in formula (VI) as ethylene moieties may be independently substituted with other small alkylene groups such as methylene, propylene or n-butylene. Symmetry should preferably be maintained, so the central propylene C3 chain is preferred while the remaining ethylene groups remain, or the two ethylenes attaching HOPO moieties to one or both of the central tertiary amines may be replaced by methylene or propylene.

Formula (VIb) shows a possible position for the RC bridging moiety to be present in formula (VI) at any suitable position, eg a -CH- group.

As stated above, an octadentate ligand will typically include a bridging moiety that can attach to the rest of the ligand at any point. A suitable point for attaching a connecting fragment is shown below in the formula (VIb)

where RC means any appropriate connecting moiety, in particular for attachment to a tissue-targeting group via an amide group. As the RC group in formula (VIb) and throughout this document, a short hydrocarbyl group, e.g. fabric fragment.

Matrix examples also include other matrices in which the RC connecting group is covalently bonded to the nitrogen atom in the amino framework as shown in formula (VII)

Octadentate ligands having suitable ligand attachment sites include the ligands of formulas (VIII) and (IX) below

- 13 043221

The synthesis of compound (VIII) is described hereinafter, it follows the synthesis route, which is also described below in this document.

AGC0019 and compounds of formulas (VI), (VIb), (VII), (VIII) and (IX) are octadentate chelators with linker moieties ending in carboxylic acid groups. The octadentate ligands shown in these structures as well as the linker moieties shown are also examples of their type and can be combined in any combination. Such combinations will be apparent to those skilled in the art.

Step a) of the method of the present invention may be carried out using any suitable synthetic route. Typically, this will involve attaching the four corresponding HOPO moieties (eg moieties of formulas (I) and/or (II) and/or (IIa)) via a linker group to the junction moiety, optionally via a template. All of these groups are described in this document; in this context, preferred embodiments of the invention are equally preferred. The connection between the HOPO fragments, linkers, connecting fragment and, if necessary, the matrix, as a rule, is carried out using a stable group, such as an amide, amine, ether or carbon-carbon bond. Methods for synthesizing such bonds, as well as any necessary protective strategies, are well known to those skilled in the art of synthetic chemistry. Some specific examples of synthetic methods are given below in the Examples section. The following are specific examples of such methods, however, the illustrated synthetic methods can also be used by those skilled in the art in a general context. Thus, the methods illustrated in the Examples section are intended to be general descriptions and, where the context permits, may be applied to all aspects and embodiments of the invention.

Preferably, alpha emitting thorium and an octadentate ligand complexes according to all aspects of the present invention can be prepared without heating above 60°C (e.g. without heating above 50°C), preferably without heating above 38 °C, most preferably without heating to a temperature above 25°C (for example, obtained at a temperature in the range of 20-38°C). Typical temperature ranges are, for example, 15-50°C or 20-40°C. The complexation reaction (part c)) according to the method of the present invention) can be carried out for any suitable period, however, such a period is preferably 1-120 minutes, preferably 1-60 minutes, more preferably 5-30 minutes.

In addition, it is preferred that the conjugate of the targeting moiety and the octadentate ligand be prepared prior to the addition of the alpha emitting thorium isotope ( ²²⁷ Th ⁴⁺ ion). Thus, the products of the invention are or can be prepared by complexation using an alpha emitting thorium isotope ( ^{227Th 4+} ion) with an octadentate ligand conjugate and a tissue-targeted moiety (tissue-targeted chelator).

Various types of targeted compounds can be coupled to thorium (thorium-227) via an octadentate chelator (including a bridging moiety as described herein). The targeted moiety may be selected from known targeting groups which include monoclonal or polyclonal antibodies, growth factors, peptides, hormones and hormone analogs, folate derivatives, biotin, avidin and streptavidin or analogs thereof. Other possible targeting groups include appropriate functionalized RNAs, DNAs or fragments thereof (eg, aptamers), oligonucleotides, carbohydrates, lipids, or compounds obtained by combining such groups with or without proteins, etc. As mentioned above, PEG fragments can be included to increase biological retention time and/or to reduce immunostimulation.

In general, as used herein, tissue-targeted fragments may be peptides or proteins, namely structures that are primarily formed from an amide backbone between amino acid components, with or without secondary and tertiary structural features.

In accordance with one embodiment of the invention, the tissue-targeted fragment may exclude antibodies and antibody fragments conjugated to osteotropes, liposomes, and folates.

In accordance with the present invention, the ²²⁷ Th complex can be formed using targeted complexing agents that are or can be attached via an amide bond to tissue-targeted moieties as described herein. Typically, targeted moieties have a molecular weight in the range of 100 g/mol to several million g/mol (in particular 100 g/mol to 1 million g/mol) and preferably have affinity directly for the disease-associated receptor and /or include an appropriate pre-introduced binding fragment (for example, biotin or avidin) associated with a molecule that targets the disease, before the introduction of ²²⁷ Th.

Appropriate targeting fragments may include poly- and oligopeptides, proteins, DNA and RNA fragments, aptamers, etc., preferably a protein, such as avidin, streptavidin, a polyclonal or monoclonal antibody (including IgG and IgM type antibodies), or a mixture of proteins or protein fragments or constructs. Particularly preferred may be antibodies, antibody constructs, antibody fragments (e.g., Fab fragments, or any fragments comprising at least one antigen-binding region, fragment constructs (e.g., single-chain antibodies), or mixtures thereof. Suitable fragments, in particular, include Fab, F(ab') ₂ , Fab' and/or scFv Antibody constructs can be any of the antibody constructs or fragments specified herein.

In accordance with one embodiment of the invention relating to targeting, which is applicable to all aspects of the invention, the selected specific binding fragment (tissue-targeted fragment) can be targeted to the CD22 receptor. Such a tissue-targeted fragment may be a peptide having sequence similarity or identity to at least one of the following sequences.

Light chain:

mouse

DIQLTQSPSSLAVSAGENVTMSCKSSQSVLYSANHKNYLAWYQQ KPGQSP Humanized ------------SA-V-DR-----------------------------KA

Mouse KLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVOVEDLAIYYCHQ YLSS Humanized ---------------S—S---------F—SL-P--I-T--------Mouse WTFGGGTKLEIKR (SeqIDl) Humanized -------------(SeqID2)

Heavy chain:

mouse

OVOLOESGAELSKPGASVKMSCKASGYTFTSYWLHWIKORPGOG

LEWIG Humanized 1 Q—VK—S—V----------------VR-A--------H'ised2 —VQ—VK—S—V-- --------------VR-A--------Mouse

YINPRNDYTEYNONFKDKATLTADKSSSTAYMQLSSLTSEDSAVY

YCAR

H'isedl --------------------1—E-TN—-E—-R—T-F-F—

H'ised2 --------------------I—E-TN—-E—-R—T-F-FMouse RDITTFYWGQGTTLTVSS (SeqID3)

H'isedl -------------V— (SeqID4)

H'ised2 -------------V— (SeqID5)

In the above sequences - in humanized (H'ised) sequences means that the residue is unchanged relative to the mouse sequence.

In the above sequences (SeqID1-5), the regions in bold are assumed to be key specific binding sites (CDRs), the underlined regions are regions of secondary binding importance, and the non-highlighted regions are structural regions rather than specific binding regions. .

The tissue-targeted fragment may have a sequence with significant sequence identity or similarity to at least one of the SeqID1-5 sequences. Significant sequence identity/similarity means that the sequence has at least 80% sequence identity relative to the full length of the sequence and/or at least 90% sequence identity with specific binding sites (areas in bold and, in some cases, the underlined sections above). Preferred sequence similarity, or more preferably sequence identity, may be at least 92%, 95%, 97%, 98%, or 99% for regions in bold, and preferably also for the entire length of the sequence. Sequence identity and/or similarity can be determined using the BestFit program of the Genetics Computer Group, version 10, University of Wisconsin. The program uses the local Smith-Waterman algorithm with the following default parameters: deletion penalty = 8, deletion continuation penalty = 2, average pairing = 2.912, average pairing error = 2.003.

A tissue-targeted fragment may contain more than one peptide sequence; in this case, at least one, preferably all, sequences can (independently of each other) meet the above criteria for sequence similarity, preferably sequence identity, to any of the SeqID1-5 sequences.

A tissue-targeted fragment may have a binding affinity for CD22, and in accordance with one embodiment of the invention, may also have a sequence with about 40 or less variations relative to the entire domain (preferably 0 to 30 variations). Such variations may be due to insertions, deletions and/or substitutions and may be contiguous or non-contiguous with respect to SeqID1-5. Substitutions or insertions are typically substitutions or insertions of at least one of the 20 amino acids of the genetic code, with the substitution

- 15 043221 nia, as a rule, are conservative.

In accordance with a second embodiment of the invention relating to targeting, which is applicable to all aspects of the invention, the selected specific binding fragment (tissue-targeted fragment) can be targeted to the CD33 receptor. Such a tissue-targeted fragment may be a monoclonal antibody and may be selected from lintuzumab and lintuzumab with an additional lysine residue at the C-terminus.

In accordance with a third embodiment of the invention relating to targeting, which is applicable to all aspects of the invention, the selected specific binding fragment (tissue-targeted fragment) can be targeted to the HER-2 antigen. The tissue-targeted fragment may be a monoclonal antibody, and is preferably trastuzumab.

Examples of other relevant antibody sequences for targeting FGFR2, mesothelin and PSMA are shown in the Examples section. However, those skilled in the art will recognize that any known form of protein that targets a disease-specific target and that contains a lysine residue in the sequence is potentially possible in the methods of the present invention, and therefore in all other aspects.

Of key importance with regard to the alpha emitting component of thorium is that it has recently been found that certain alpha radioactive isotopes of thorium (eg, ²²⁷ Th) can be administered in amounts that are both therapeutically effective and do not cause intolerable myelotoxicity. In accordance with all aspects of the present invention, the isotope of thorium is thorium-227 ( ²²⁷ Th). As used throughout this document, the term non-intolerant myelotoxicity means (which is especially important) that the amount of radium-223, which is obtained as a result of the decay of the administered radioisotope of thorium-227, is generally insufficient to be the direct cause of death of the patient. However, those skilled in the art will appreciate that the degree of bone marrow damage (and potential for death) that would be an acceptable side effect of such treatment will vary greatly depending on the type of disease being treated, the goals of the treatment regimen, and the disease prognosis for the patient. While the patient according to the present invention is preferably a human, the present invention can also be used to treat other mammals, in particular companion animals such as dogs; thus, the degree of bone marrow damage also depends on the biological species of the patient. The acceptable degree of bone marrow damage will generally be higher in the treatment of malignant diseases compared to benign diseases. One of the best known parameters for measuring the degree of myelotoxicity is the level of neutrophils; in accordance with the present invention, the amount of ²²³ Ra that does not cause intolerable myelotoxicity will generally be such a controlled amount that the proportion of neutrophils at the lowest point (maximum decrease in level) is at least 10% of the pre-treatment neutrophil level. Preferably, the amount of ²²³ Ra that does not cause intolerable myelotoxicity will be such that the proportion of neutrophils is at least 20% at the maximum reduction in level, more preferably at least 30%. The most preferred at the maximum decrease in the level is the proportion of neutrophils, constituting at least 40%.

In addition, compounds containing radioactive thorium (eg, ²²⁷ Th) may be used at high dosage regimens when the myelotoxicity of the resulting radium (eg, ²²³ Ra) would otherwise be intolerable, and when stem cell transplantation or a similar reconstitution method is used in parallel. In such cases, the number of neutrophils may decrease to less than 10% at the maximum decrease in the level, and in exceptional cases, to 5% or, if necessary, to a level of less than 5%, provided that appropriate precautions have been taken, and subsequently was stem cell transplantation. Such techniques are well known to those skilled in the art.

An isotope of thorium of particular interest in accordance with the present invention is thorium-227, and if the context permits, thorium-227 is an isotope in any reference to thorium herein. Thorium-227 is relatively easy to obtain, it can be obtained directly from neutron-irradiated ²²⁶ Ra, which will contain the parent nuclide ²²⁷ Th, i.e. ²²⁷ Ac (T _1/2 = 22 years). Actinium-227 can be quite simply separated from the ²²⁶ Ra target and used as a ²²⁷ Th generator. If necessary, this process can be carried out on an industrial scale, thus avoiding drug delivery problems that occur with most other alpha-emitting nuclides that are considered candidates for use in molecularly targeted therapy.

As a result of the decay of thorium-227, radium-223 is obtained. In this case, the half-life of the primary daughter radionuclide is 11.4 days. From a source of pure ²²⁷ Th, only moderate amounts of radium are obtained during the first few days. However, the potential toxicity of ²²³ Ra is higher than the toxicity of ²²⁷ Th, since after the emission of alpha particles of ²²³ Ra for several minutes, radiation of alpha particles from three daughter radionuclides with a short half-life period follows (see Table 2 below, which shows the decay chain for thorium-227). table 2

Nuclide Type of radiation Average particle energy (MeV) Half life ^227Th os 6.02 18.72 days ²²³ Ra OS 5.78 11.43 days ²¹⁹ Rn OS 6.88 3.96 seconds ₂ 15po OS 7.53 1.78 ms ^2nPb β 0.45 36.1 minutes ^2U Bi 6.67 2.17 minutes 207y| β 1.42 4.77 minutes ²⁰⁷ _pb stable

One of the reasons that thorium-227 (T _1/2 = 18.7 days) has not been widely considered for use in alpha therapy is that its decay produces harmful products.

Thus, in order to distinguish with thorium complexes, where the natural isotope of thorium with the highest enrichment factor is used, i.e. thorium-232 (half-life: 1010 years, essentially non-radioactive), it should be understood that the thorium complexes and compositions based on them described in the present invention include an alpha particle emitting isotope of thorium (i.e., at least one isotope of thorium with a half-life of less than 103 years, such as thorium-227) with an enrichment factor greater than natural, for example, at least 20% more. This does not affect the definition of the method of the invention, according to which a therapeutically effective amount of radioactive thorium, such as thorium-227, is explicitly required, but preferably it affects all aspects of the invention.

In accordance with all aspects of the present invention, the alpha emitting thorium ion is the thorium-227 ion. Thorium 4+ ion is the ion for use in the complexes of the present invention. Accordingly, the 4+ ion of thorium-227 is used.

Thorium-227 can be used in amounts sufficient to provide the desired therapeutic effect without producing such an amount of radium-223 that it could cause unacceptable suppression of bone marrow function. It is necessary that the daughter isotopes remain within the target site so that an additional therapeutic effect can be achieved as a result of their decay. However, it is not necessary to control the breakdown products of thorium to achieve a beneficial therapeutic effect without causing unacceptable myelotoxicity.

Assuming that the effect of destruction of tumor cells will be provided mainly by thorium-227, and not its daughter nuclides, the probable therapeutic dose of this isotope can be established by comparison with other nuclides that emit alpha particles. For example, for astatine-211, therapeutic doses in animals would typically be 2-10 MBq/kg. Adjusted for the length of the half-life and energy, the appropriate dosage for thorium-227 would be at least 36-200 kBq/kg body weight. This value will be set as the lower limit of the amount of ²²⁷ Th that can be administered to achieve the intended therapeutic effect. This calculation takes into account the retention of astatine and thorium. However, it is clear that thorium's half-life of 18.7 days results in greater excretion of this isotope before decay. Therefore, such a calculated dosage will generally be considered the minimum effective amount. The therapeutic dose, expressed in terms of fully retained ²²⁷ Th (i.e. ²²⁷ Th that is not excreted from the body), will typically be at least 18 or 25 kBq/kg, preferably at least 36 kBq/kg, more preferably at least 75 kBq/kg, eg 100 kBq/kg or more. It is expected that large amounts of thorium will have a great therapeutic effect, however, such amounts cannot be used if this would lead to unacceptable side effects. Similarly, if thorium is administered in a form with a short biological half-life (i.e., the half-life of the drug containing thorium being eliminated from the body), then large amounts of the radioisotope are needed to achieve a therapeutic effect, since a significant amount of thorium will be eliminated from the body before its collapse. However, this will also lead to a corresponding decrease in the amount of radium-223 produced. The above amounts of thorium-227, which are applied at full isotope retention, can easily be correlated with equivalent doses with shorter half-lives. Specialists can make such calculations, these calculations are given in document WO 04/091668 (for example, in the text of examples 1 and 2).

If a radioactively labeled compound releases daughter nuclides, it is important, as appropriate, to know what happens to each of these radioactive daughter nuclides. For ²²⁷ Th, the main daughter product is ²²³ Ra, which is being clinically evaluated for its osteotropic properties. Radium-223 is extremely rapidly cleared from the blood and concentrated in the skeleton or excreted from the body through the intestines or through the kidneys (see Larsen, J Nucl Med 43 (5, Supplement): 160P (2002)). Therefore, radium-223, which is formed in vivo as a result of the decay of ²²⁷ Th,

- 17 043221 slightly affects healthy soft tissues. In a Muller study at Int. J. Radiat. Biol. 20:233-243 (1971) regarding the distribution ^{of 227} Th as a dissolved citrate salt, it was found that ²²³ Ra formed from ²²⁷ Th in soft tissues is redistributed to bone or excreted from the body. Thus, with respect to the dosage of thorium, the known toxicity of alpha-emitting radium, in particular bone marrow toxicity, is problematic.

In fact, in WO 04/091668, it was found for the first time that a tolerable dose of ²²³ Ra of at least 200 kBq/kg can be administered to a human. These data are given in the referenced publication. Thus, it has surprisingly been found that there is a therapeutic window within which a therapeutically effective amount of ²²⁷ Th (eg, a dose in excess of 36 kBq/kg) can be administered to a mammalian patient without the risk of intolerable or lethal myelotoxicity to the patient. However, it is extremely important to make optimal use of such a therapeutic window, hence it is important that the radioactive thorium complex be obtained quickly and efficiently, and high affinity must also be ensured so that the maximum dose is delivered to the target site.

The amount of ²²³ Ra that is produced from the breakdown of ²²⁷ Th in the drug will depend on the biological half-life of the radiolabeled compound. The ideal situation would be to use a complex with rapid tumor absorption, including internalization into tumor cells, stable retention in the tumor, and a short biological half-life in healthy tissues. However, complexes with less than ideal biological half-lives can be used provided that the dose of ²²³ Ra is kept at an acceptable level. The amount of radium-223 that is formed in vivo will depend on the amount of thorium administered and the biological retention time of the thorium complex. In each particular case, a specialist can easily calculate the resulting amount of radium-223. The maximum input amount of ²²⁷ Th will be determined by the amount of radium that is formed in vivo; such an amount must be less than the amount that will cause an unacceptable level of side effects, in particular myelotoxicity. Such an amount will typically be less than 300 kBq/kg, in particular less than 200 kBq/kg, more preferably less than 170 kBq/kg (eg less than 130 kBq/kg). The minimum effective dose will be determined by the cytotoxicity of thorium, the sensitivity of the affected tissue to alpha radiation, and the degree to which thorium can effectively be complexed, retained in such a complex, and delivered by the targeted complex (in this case, the complex is a combination of a ligand and a targeted complex). fragment).

According to the method of the invention, the thorium complex is preferably administered at a dosage of thorium-227 of 18-400 kBq/kg body weight, preferably 36-200 kBq/kg, (e.g. 50200 kBq/kg), more preferably 75-170 kBq/kg. kg, in particular, 100-130 kBq/kg. Accordingly, for a single administration, the approximate dose can be calculated by multiplying the indicated values by the corresponding body weight in the range, for example, 30-150 kg, preferably 40100 kg, for example, the dose may be from 540 to 4000 kBq, etc.) . Moreover, it is desirable that the dose of thorium, the agent included in the complex and the route of administration are such that the dose of radium-223 generated in vivo is less than 300 kBq/kg, more preferably less than 200 kBq/kg, even more preferably less than 150 kBq/kg, especially less than 100 kBq/kg. Again, by multiplying the indicated ranges by the indicated body mass values, the radiation exposure dose value for ²²³ Ra can be obtained. The above levels refer to a fully retained dose of ²²⁷ Th, however, such doses can be applied taking into account some of the ²²⁷ Th that is excreted from the body before decay.

In the event that the biological half-life of the ²²⁷ Th complex is short compared to the physical half-life (for example, if it is less than 7 days, in particular less than 3 days), administration of significantly higher doses will be required to provide an equivalent retention dose. Thus, for example, a fully retained dose of 150 kBq/kg is equivalent to a complex with a half-life of 5 days with an administration dose of 711 kBq/kg. The equivalent administration dose for the respective retention doses can be calculated from the rate of biological clearance of the complex using methods known to those skilled in the art.

Since the decay of one ²²⁷ Th nucleus produces a ²²³ Ra atom, the retention and therapeutic activity ^{of 227} Th will be directly related to the dose of ²²³ Ra to which the patient is exposed. The calculation of the resulting amount of ²²³ Ra in each particular case can be carried out using known methods.

Thus, in a preferred embodiment, the present invention provides a method for the treatment of said diseases in a mammal (as described herein), said method comprising administering to said patient a therapeutically effective amount of at least one tissue-targeted thorium complex according to as described in this document.

Obviously, it is preferable to minimize patient exposure to the ²²³ Ra daughter isotope, except when its properties are put to good use. In particular, the amount of radium-223,

- 18 043221 formed in vivo will typically be greater than 40 kBq/kg, eg greater than 60 kBq/kg. In some cases, the amount of radium-223 generated in vivo must be greater than 80 kBq/kg, for example greater than 100 or 115 kBq/kg.

For conjugates labeled with thorium-227, in appropriate carrier solutions, intravenous, intracavitary (eg, intraperitoneal), subcutaneous, oral, or topical routes of administration may be used, they may be administered as a single dose or in accordance with a dosage regimen that provides for the introduction of parts. Preferably, the targeting moiety-conjugated complexes are administered as solutions parenterally (eg, by transdermal administration), especially using an intravenous or intracavitary route of administration. Preferably, the compositions of the present invention are formulated with a sterile parenteral solution.

The thorium-227 in the methods and products of the present invention may be used alone or in combination with other therapies, including surgery, radiotherapy, chemotherapy, other radionuclides, or tissue temperature control, etc. This constitutes another, preferred embodiment of the method according to the invention, accordingly, the formulations/drugs may contain at least one additional therapeutically active agent, for example, another radioactive agent or a chemotherapeutic agent.

In accordance with one particularly preferred embodiment of the invention, the patient is treated in parallel with stem cells and/or other supportive therapy to reduce myelotoxicity caused by exposure to radium-223.

The thorium (Thorium-227) labeled molecules of the invention can be used to treat cancerous and non-cancer diseases by targeting receptors associated with the disease. Typically, ²²⁷ Th will be used in this manner in radioimmunotherapy based on the binding of a ²²⁷ Th chelator to an antibody, antibody fragment, or antibody construct or antibody fragment for the treatment of cancer and non-cancer diseases. The use of ²²⁷ Th in the methods and medicaments of the present invention is particularly useful in the treatment of all forms of cancer, including carcinomas, sarcomas, lymphomas and leukemias, in particular in the treatment of lung, breast, prostate, bladder, kidney, stomach, prostate, esophagus, brain, ovaries, uterus, oral cancer, colorectal cancer, melanoma, multiple myeloma, and non-Hodgkin's lymphoma.

In accordance with another embodiment of the invention, the treatment of a patient with both soft tissue disease and skeletal disease can be carried out using ²²⁷ Th and ²²³ Ra, which is formed in vivo after administration of thorium. According to such a particularly preferred aspect, an additional therapeutic component of the treatment is provided by the use of a non-tolerably myelotoxic amount of ²²³ Ra when the target is skeletal disease. According to this therapeutic method, ²²⁷ Th is ^generally used to treat primary and/or metastatic soft ^tissue cancer by targeting the appropriate areas of the body; patient. Such a skeletal disease may be skeletal bone metastases resulting from a primary soft tissue cancer, or it may be a primary disease in which the soft tissue is treated to counteract the metastatic cancer. In some cases, soft tissue and skeletal disease may be unrelated (eg, adjunctive treatment of skeletal disease in a patient with rheumatic soft tissue disease).

Conditions for which these methods, uses, and other aspects of the present invention are particularly useful include neoplastic and hyperplastic diseases such as carcinoma, sarcoma, myeloma, leukemia, lymphoma, or mixed cancers, including non-Hodgkin's lymphoma or B-cell neoplasms, cancer breast, endometrial cancer, stomach cancer, acute myeloid leukemia, prostate or brain cancer, mesothelioma cancer, ovarian, lung or pancreatic cancer.

Below are some examples of synthesis methods. The steps described for these synthetic methods can be applied in various embodiments of the present invention. Step a), for example, can be carried out using the intermediate connection AGC0021, which is described below for many or all embodiments of the invention.

Synthesis of key intermediate product AGC0020.

K,K,K',K'-Tetrakis(2-aminoethyl)-2-(4-nitrobenzyl)propane-1,3-diamine

- 19 043221

a) dimethylmalonate, sodium hydride, THF, b) DIBAL-G, THF, c) MsCl, Nets, CH2CI2,

d) imidazole, Boc ₂ O, CH2CI2, toluene, f) DIPEA, acetonitrile, f) MeOH, water, AcCl

Synthesis of the key intermediate AGC0021.

3-(Benzyloxy)-1-methyl-4-[(2-thioxo-1,3-thiazolidin-3-yl)carbonyl]pyridin-2(1Н)-one

d

I

a) diethyl oxolate, potassium ethoxide, toluene EtOH, b) Pd / C, p-xylene, c) Mel, K ₂ CO3, DMSO, acetone,

HS

d) i) VVgz, DCM, ii) BnBr, K ₂ CO ₃ . K1, acetone, f) NaOH, water, MeOH, f) , DCC, DMAP, DCM

Synthesis of a chelate of a compound of formula (VIII).

4-{[4-(3-[bis(2-{[(3-Hidroxy-1-methyl-2-oxo-1,2-dihydropyridin-4-yl)carbonyl]amino}ethyl)amino]-2 - {[bis (2- {[(3 - hydroxy-1 -methyl-2-oxo-1,2-dihydropyridin-4-yl) carbonyl] amino } ethyl) amino] methyl} propyl) phenyl] amino } -4 -oxobutanoic acid

In the process for preparing the complexes of the present invention, it is preferred that the coupling reaction between the octadentate chelator and the tissue-targeted moiety be carried out in an aqueous solution. This has several advantages. First of all, the manufacturer does not need to remove all the solvent to an acceptable level and obtain the appropriate certification confirming such removal. Second, it reduces waste and, most importantly, speeds up production by eliminating the separation or solvent removal step. In the context of the production of radiopharmaceutical products in accordance with the present invention, it is important that the synthesis is carried out in the shortest possible time due to the constant decay of the radioisotope, since valuable material is consumed during the time taken to obtain them, as well as extraneous daughter isotopes are formed.

Suitable aqueous solutions include purified water and buffers, such as any of the various buffers known to those skilled in the art. Commonly known aqueous buffers are acetate, citrate, phosphate (eg, phosphate buffered saline, PBS) and sulfonate buffers (eg, MES).

- 20 043221

In accordance with one embodiment of the invention, the method includes obtaining a first aqueous solution of a ligand containing hydroxypyridinone octadentate (as described herein) and a second aqueous solution of a tissue-targeted moiety (as described herein) and contacting said first and said second aqueous solution.

Appropriate linking moieties are described in detail above, and all groups and moieties described herein as linking and/or linking moieties are suitable for linking a targeted moiety to a ligand. Some preferred connecting groups include amide groups, ester groups, ethers and amine connecting groups. Esters and amides can be formed by activation of ester groups from a carboxylic acid. Such a carboxylic acid may be present on the targeting moiety, on the linking moiety and/or on the ligand moiety, and will typically react with an alcohol or amine to form an ester or amide. Such methods are known to those skilled in the art and may use known activating agents, including N-hydroxymaleimide, carbodiimide, and/or azodicarboxylate activating agents such as DCC, DIC, EDC, DEAD, DIAD, etc.

According to the invention, an octadentate chelator comprising said four hydroxypyridinone moieties (HOPO) substituted at the N-position with a C ₁ -alkyl group and a linking moiety ending in a carboxylic acid group can be activated using at least one coupling agent (for example , any reagent described herein) and an activating agent such as N-hydroxysuccinimide (NHS), resulting in an NHS ester of an octadentate chelator. Separation of such an activated ester (eg, NHS) may be performed, or it may be used without separation to couple to any tissue-targeted moiety with a free amine group (such as a lysine side chain). Other activated esters are known to those skilled in the art and can be any effective leaving group ester such as fluoro groups, tosylates, mesylates, iodides, and the like. However, NHS esters are preferred.

The coupling reaction is preferably carried out over a relatively short period and at approximately ordinary temperature. Typical times for a 1-step or 2-step coupling reaction will be about 1-240 minutes, preferably 5-120 minutes, more preferably 10-60 minutes. Typical temperatures for the coupling reaction will be 0-90°C, preferably 15-50°C, more preferably 20-40°C. Suitable temperatures are about 25°C or about 38°C.

The coupling of the octadentate chelator to the targeted moiety is generally carried out under conditions that do not adversely affect (or at least do not irreversibly affect) the binding capacity of the targeted moiety. Since binding moieties are typically peptide or protein based, relatively mild conditions are required to avoid denaturation or loss of secondary/tertiary structure. Aqueous conditions (as described herein in all contexts) are preferred, preferably avoiding extreme pH and/or redox conditions. Thus, step b) can be carried out at a pH value in the range of 3-10, preferably 4-9, more preferably 4.5-8. The preferred conditions are redox neutral or slightly reducing to avoid oxidation in air.

A preferred tissue-targeted chelator that can be used in accordance with all aspects of the invention is AGC0018 as described herein. Complexes of AGC0018 with ²²⁷ Th ions constitute a preferred embodiment of the complexes and associated formulations, uses, methods of the invention, etc. Other embodiments of the invention in all such aspects of the invention include ²²⁷ Th-complexes of AGC0019 conjugated to tissue targeting fragments (as described herein), including monoclonal antibodies with binding affinity for any CD22 receptor, FGFR2, mesothelin, HER- 2, PSMA or CD33.

Brief description of the figures

Fig. 1 - Data showing the stabilizing effect of EDTA/PABA on non-radioactive AGC1118 antibody conjugate in solution.

Fig. 2 is the effect on hydrogen peroxide levels of various buffers containing HOPO antibody conjugates irradiated with 10 kGy radiation.

Fig. 3 - radio stabilizing effect of ²²⁷ Th-AGC1118 (analysis by infrared fluorescence) with a specific activity up to approximately 8000 Bq/μg.

Fig. 4 Cytotoxicity of ²²⁷ Th-AGC1118 against Ramos with other total activity (incubation time: 4 h) (see example 3).

Fig. ^5-227 Th-AGC0718 induces selective killing of CD33 positive cells in vitro (see example 4).

- 21 043221

Fig. 6 - cytotoxicity of ²²⁷ Th-AGC0118 at high (20 kBq/µg) and low (7.4 kBq/µg) specific activity. The negative control was a weakly bound peptide-albumin complex with the same dosage, incubation time and number of days to data sampling (see Example 5).

Fig. ^7-227 Th-AGC2518 induces selective killing of FGFR2 positive cells in vitro (see Example 6).

Fig. ^8-227 Th-AGC2418 induces selective killing of mesothelin-positive cells in vitro (see example 7).

Fig. ^9-227 Th-AGC1018 induces selective and dose dependent cell killing of PSMA positive LNCaP cells in vitro (see Example 9).

The present invention is further illustrated by the following non-limiting examples. All compounds exemplified in the Examples section constitute preferred embodiments of the invention (including preferred intermediates and precursors) and may be used singly or in combination in any aspect as the context permits. Thus, for example, individually and collectively, compounds 2-4 according to example 2, compound 10 according to example 3, and compound 7 according to example 4 constitute various types of preferred embodiments of the invention.

Example 1 Synthesis of compounds of formula (VIII)

Example 1 a). Synthesis of dimethyl 2-(4-nitrobesyl)malonate

Sodium hydride (60% dispersion, 11.55 g, 289 mmol) was suspended in 450 ml of tetrahydrofuran (THF) at 0°C. Dimethylmalonate (40.0 mL, 350 mmol) was added dropwise over about 30 minutes. The reaction mixture was stirred for 30 min at 0°C. 4-Nitrobenzyl bromide (50.0 g, 231 mmol) dissolved in 150 ml THF was added dropwise over about 30 minutes at 0° C. and then over two hours at ambient temperature.

Before the solution was filtered, 500 ml of ethyl acetate (EtOAc) and 250 ml of NH ₄ Cl (aq., sat.) were added. The phases have been separated. The aqueous phase was extracted with 2x250 ml EtOAc. The organic phases were combined, washed with 250 ml of brine, dried over Na ₂ SO ₄ , filtered and the solvents were removed under reduced pressure.

300 ml of heptane and 300 ml of methyl tert-butyl ether (MTBE) were added to the residue and heated to 60°C. The solution was filtered, the filtrate was placed in a refrigerator overnight, then filtered. The filter cake was washed with 200 ml of heptane and dried under reduced pressure to give the main compound as an off-white solid.

Yield: 42.03 g, 157.3 mmol, 68%.

L-NMR (400 MHz, CDCls): 3.30 (d, 2H, 7.8 Hz), 3.68 (t, 1H, 7.8 Hz), 3.70 (s, 6H), 7.36 (d, 2H, 8.7 Hz), 8.13 (d , 2H, 8.7Hz).

Example 1 b). Synthesis of 2-(4-nitrobesyl)propane-1,3-diol

Dimethyl 2-(4-nitrobenzyl)malonate (28.0 g, 104.8 mmol) was dissolved in 560 ml THF at 0°C. Diisobutylaluminum hydride (DIBAL-H) (1 M in hexanes, 420 ml, 420 mmol) was added dropwise at 0° C. over approximately 30 minutes. The reaction mixture was stirred at 0° C. for two hours. To the reaction mixture was added dropwise 20 ml of water at 0°C. 20 ml of NaOH (aq., 15%) was added dropwise to the reaction mixture at 0°C, and then 20 ml of water was added dropwise to the reaction mixture. The mixture was stirred at 0°C for 20 min before adding approximately 150 g of MgSO ₄ . The mixture was stirred at room temperature for 30 minutes before being filtered using a Buchner funnel. The filter cake was washed with 500 ml EtOAc. Before the solution was filtered, the filter cake was removed and stirred with 800 ml EtOAc and 200 ml MeOH for approximately 30 minutes. The filtrates were combined and dried under reduced pressure. Dry flash chromatography on silica gel using a gradient of EtOAc in heptane followed by a gradient of MeOH in EtOAc gave the title compound as a pale yellow solid.

Yield: 15.38 g, 72.8 mmol, 69%.

- 22 043221 ¹ H-NMR (400 MHz, CDC13): 1.97-2.13 (m, 3H), 2.79 (d, 2H, 7.6 Hz), 3.60-3.73 (m, 2H), 3.76-3.83 (m, 2H) , 7.36 (d, 2H, 8.4 Hz), 8.14 (d, 2H, 8.4 Hz).

Example 1 c). Synthesis of 2-(4-nitrobesyl)propane-1,3-diyldimethanesulfonate no ₂ ό

Y^OMs

MsCr

2-(4-Nitrobesyl)propane-1,3-diol (15.3 g, 72.4 mmol) was dissolved in 150 ml CH ₂ Cl ₂ at 0°C. Triethylamine (23 ml, 165 mmol) was added, then methanesulfonyl chloride (12 ml, 155 mmol) was added dropwise over about 15 minutes, then the mixture was stirred at ordinary temperature for one hour. 500 ml CH ₂ Cl ₂ was added and the mixture was washed with 2x250 ml NaHCO ₃ (aq., sat.), 125 ml HCl (aq., 0.1M) and 250 ml brine. The organic phase was dried over Na ₂ SO ₄ , filtered and dried under reduced pressure to give the main compound as an orange solid.

Yield: 25.80 g, 70.2 mmol, 97%.

'H-NMR (400 MHz, CDCl ₃ ): 2.44-2.58 (m, 1H), 2.87 (d, 2H, 7.7 Hz), 3.03 (s, 6H), 4.17 (dd, 2H, 10.3, 6.0 Hz), 4.26 (dd, 2H, 10.3, 4.4 Hz), 7.38 (d, 2H, 8.6 Hz), 8.19 (d, 2H, 8.6 Hz).

Example 1d). Synthesis of di-tert-butyl(azandiylbis(ethane-2,1-diyl))dicarbamate

I O ui ο ι

Η Η

Imidazole (78.3 g, 1.15 mol) was suspended in 500 ml CH ₂ Cl ₂ at room temperature. Di-t-butyl dicarbonate (Boc ₂ O) (262.0 g, 1.2 mol) was added in portions. The reaction mixture was stirred for one hour at room temperature. The reaction mixture was washed at 3x750 ml, dried over Na ₂ SO ₄ , filtered and the volatiles were removed under reduced pressure. The residue was dissolved in 250 ml of toluene and diethylenetriamine (59.5 ml, 550 mmol) was added. The reaction mixture was stirred at 60° C. for two hours.

Added 1 l CH ₂ Cl ₂ and the organic phase was washed with 2x250 ml of water. The organic phase was dried over Na ₂ SO ₄ , filtered and recovered under reduced pressure.

Dry flash chromatography on silica gel using a gradient of methanol (MeOH) in CH2Cl2 with triethylamine gave the title compound as a colorless solid.

Yield: 102 g, 336 mmol, 61%.

'H-NMR (400 MHz, CDCl ₃ ): 1.41 (s, 18H), 1.58 (bs, 1H), 2.66-2.77 (m, 4H), 3.13-3.26 (m, 4H), 4.96 (bs, 2H) .

Example 1 e). Synthesis of tetra-tert-butyl (((2-(4-nitrobesyl)propane-1,3-diyl)-bis-(azantriyl))tetrakis(ethane-2,1-diyl))tetracarbamate

2-(4-Nitrobenzyl) propane-1,3-diyldimethanesulfonate (26.0 g, 71 mmol) and di-tert-butyl (azandiylbis(ethane-2,1-diyl)) dicarbamate (76.0 g, 250 mmol ) was dissolved in 700 ml of acetonitrile. Ν,N-diisopropylethylamine (43 ml, 250 mmol) was added. The reaction mixture was stirred for 4 days at reflux.

The volatiles were removed under reduced pressure.

Dry flash chromatography on silica gel using a gradient of EtOAc in heptane gave the main compound as a dense pale yellow foam.

Yield: 27.2 g, 34.8 mmol, 49%.

1Н-NMR (400 MHz, CDCl ₃ ): 1.40 (s, 36H), 1.91-2.17 (m, 3H), 2.27-2.54 (m, 10H), 2.61-2.89 (m, 2H), 2.98-3.26 (m , 8H), 5.26 (bs, 4H), 7.34 (d, 2H, 8.5 Hz), 8.11 (d, 2H, 8.5 Hz).

Example 1 f). Synthesis of N1,N1-(2-(4-nitrobesyl)propane-1,3-diyl)-bis-(N ¹ -(2-aminoethyl)ethane-1,2diamine), AGC0020

- 23 043221

Tetra-tert-butyl (((2-(4-nitrobenzyl)propane-1,3-diyl)-bis-(azantriyl))tetrakis(ethane-2,1-diyl))tetracarbamate (29.0 g, 37, 1 mmol) was dissolved in 950 ml of MeOH and 50 ml of water. Acetyl chloride (50 ml, 0.7 mol) was added dropwise over about 20 minutes at 30°C. The reaction mixture was stirred overnight.

The volatiles were removed under reduced pressure and the residue was dissolved in 250 ml of water. 500 ml CH ₂ Cl ₂ was added, followed by 175 ml NaOH (aq., 5 M, saturated NaCl). The phases were separated and the aqueous phase was extracted with 4x250 ml CH ₂ Cl ₂ . The organic phases were combined, dried over Na ₂ SO ₄ , filtered and dried under reduced pressure to give the main compound as a viscous red-brown oil.

Yield: 11.20 g, 29.3 mmol, 79%. Purity (HPLC Fig. 9): 99.3%.

¹ H-NMR (300 MHz, CDCl ₃ ): 1.55 (bs, 8H), 2.03 (dt, 1H, 6.6, 13.3 Hz), 2.15 (dd, 2H, 12.7, 6.6), 2.34-2.47 (m, 10H) , 2.64-2.77 (m, 10H), 7.32 (d, 2H, 8.7 Hz), 8.10 (d, 2H, 8.7 Hz).

13С-NMR (75 MHz, CDCl3): 37.9, 38.5, 39.9, 58.0, 58.7, 123.7, 130.0, 146.5, 149.5.

Example 1 g). Synthesis of ethyl 5-hydroxy-6-oxo-1,2,3,6-tetrahydropyridine-4-carboxylate

2-Pyrrolidinone (76 ml, 1 mol) and diethyl oxalate (140 ml, 1.03 mol) were dissolved in 1 L of toluene at room temperature. Added potassium ethoxide (EtOK) (24% in ethanol, 415 ml, 1.06 mol) and the reaction mixture was heated to 90°C. 200 ml EtOH was added in portions during the first hour of the reaction due to thickening of the reaction mixture. The reaction mixture was stirred overnight and cooled to room temperature. 210 ml HCl (5 M, aq.) was added slowly with stirring.

200 ml of brine and 200 ml of toluene were added and the phases were separated. The aqueous phase was extracted using 2x400 ml CHCl ₃ . The mixed organic phases were dried (Na ₂ SO ₄ ), filtered and recovered under vacuum. The residue was recrystallized from EtOAc to give the main compound as a pale yellow solid.

Yield: 132.7 g, 0.72 mol, 72%.

Example 1 h). Synthesis of ethyl 3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylate

{Ethyl 5-hydroxy-6-oxo-1,2,3,6-tetrahydropyridine-4-carboxylate} (23.00 g, 124.2 mmol) was dissolved in 150 mL of p-xylene and carbon supported palladium catalyst ( 10%, 5.75 g). The reaction mixture was stirred over night at the boil under reflux. After cooling to room temperature, the reaction mixture was diluted with 300 ml of MeOH and filtered through a short pad of Celite®. The support was washed with 300 ml of MeOH. The solvents were removed in vacuo to give the main compound as a pale reddish brownish solid.

Yield: 19.63 g, 107.1 mmol, 86%. MS (ESI, pos): 206.1 [M+Na]+, 389.1 [2M+Na] ⁺ .

Example 1 i). Synthesis of ethyl 3-methoxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate

{Ethyl 3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylate} (119.2 g, 0.65 mol) was dissolved in 600 ml of dimethyl sulfoxide (DMSO) and 1.8 L of acetone at room temperature. K ₂ CO ₃ (179.7 g, 1.3 mol) was added. Methyl iodide (MeI) (162 mL, 321 mmol) dissolved in 600 mL of acetone was added dropwise over about 1 hour at room temperature.

The reaction mixture was stirred for an additional two hours at room temperature before MeI (162 ml, 2.6 mol) was added. The reaction mixture was stirred over night at the boil under reflux. The reaction mixture was restored under reduced pressure and 2.5 L of EtOAc was added.

The mixture was filtered and reconstituted under reduced pressure. Purification by dry flash chromatography (DFC) on SiO ₂ using an EtOAc gradient in heptane gave the main compound.

Yield: 56.1 g, 210.1 mmol, 32%. MS (ESI, pos): 234.1 [M+Na] ⁺ , 445.1 [2M+Na] ⁺ .

- 24 043221

Example 1 j). Synthesis of ethyl 3-(benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate

{Ethyl 3-methoxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate} (5.93 g, 28.1 mmol) was dissolved in 80 ml dichloromethane (DCM) at -78°C and BBr ₃ (5.3 ml, 56.2 mmol) dissolved in 20 ml DCM was added dropwise. The reaction mixture was stirred for 1 h at -78°C before heating the reaction to 0°C. The reaction was quenched by adding dropwise 25 ml of tert-butyl methyl ether (tert-BuOMe) and 25 ml of MeOH. The volatiles were removed in vacuo. The residue was dissolved in 90 yl DCM and 10 ml MeOH and filtered through a short pad of SiO ₂ . The plate was washed with 200 ml of 10% MeOH in DCM. The volatiles were removed in vacuo. The residue was dissolved in 400 ml of acetone. K ₂ CO ₃ (11.65 g, 84.3 mmol), KI (1.39 g, 8.4 mmol) and benzyl bromide (BnBr) (9.2 ml, 84.3 mmol) were added. The reaction mixture was stirred over night at the boil under reflux. The reaction mixture was diluted with 200 ml EtOAc and washed with 3x50 ml water and 50 ml brine. The combined aqueous phases were extracted with 2x50 ml EtOAc. The combined organic phases were dried (Na ₂ SO ₄ ), filtered and the volatiles removed in vacuo and purified by dry flash chromatography on SiO ₂ using EtOAc (40-70%) in heptanes as eluent to give the main compound.

Yield: 5.21 g, 18.1 mmol, 65%. MS (ESI, pos): 310.2 [M+Na]+, 597.4 [2M+Na]+.

Example 1k). Synthesis of 3-(benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylic acid

{Ethyl 3-(benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate} (27.90 g, 97.1 mmol) was dissolved in 250 ml MeOH and 60 ml NaOH (5 M , water). The reaction mixture was stirred at room temperature for 2 hours before the reaction mixture was concentrated to about 1/3 in vacuo. The residue was diluted with 150 ml of water and acidified to pH 2 using hydrogen chloride (HCl) (5 M, aq.). The precipitate was filtered and dried in vacuo to give the main compound as a colorless solid. Yield: 22.52 g, 86.9 mmol, 89%.

Example 1l). Synthesis of 3-(benzyloxy)-1-methyl-4-(2-thioxothiazolidine-3-carbonyl)pyridin-2(1H)-one (AGC0021)

{3-(Benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylic acid} (3.84 g, 14.8 mmol), 4-dimethylaminopyridine (DMAP) (196 mg, 1, 6 mmol) and 2-thiazolin-2-thiol (1.94 g, 16.3 mmol) were dissolved in 50 ml of DCM. N,N'-dicyclohexylcarbodiimide (DCC) (3.36 g, 16.3 mmol) was added. The reaction mixture was stirred overnight. The reaction was filtered, the solids were washed with DCM and the filtrate was recovered in vacuo. The resulting yellow solid was recrystallized from isopropanol/DCM to give AGC0021. Yield: 4.65 g, 12.9 mmol, 87%. MS(ESI, pos): 383 [M+Na] ⁺ , 743 [2M+Na] ⁺ .

Example 1 m). Synthesis of AGC0023

AGC0020 (8.98 g; 23.5 mmol) was dissolved in CH ₂ Cl ₂ (600 ml). AGC0021 (37.43 g, 103.8 mol) was added. The reaction mixture was stirred for 20 hours at room temperature. The reaction mixture was concentrated under reduced pressure.

- 25 043221

Dry flash chromatography on SiO ₂ using a gradient of methanol in 1:1 EtOAc and CH ₂ Cl ₂ gave AGC0023 as a dense foam.

Average yield: 26.95 g, 20.0 mmol, 85%.

Example 1n). Synthesis of AGC0024

AGC0023 (26.95 g; 20.0 mmol) was dissolved in ethanol (EtOH) (675 ml). Iron (20.76 g; 0.37 mol) and NH4Cl (26.99 g; 0.50 mol) were added followed by water (67 ml). The reaction mixture was stirred at 70° C. for two hours. Added additional amount of iron (6.75 g; 121 mmol) and the reaction mixture was stirred for one hour at 74°C. Added additional amount (6.76 g; 121 mmol) and the reaction mixture was stirred for one hour at 74°C. The reaction mixture was cooled before the reaction mixture was restored under reduced pressure.

Dry flash chromatography on SiO ₂ using a gradient of methanol in CH ₂ Cl ₂ gave AGC0024 as a dense foam. Yield: 18.64 g, 14.2 mmol, 71%.

Example 1 o). Synthesis of AGC0025

AGC0024 (18.64 g; 14.2 mmol) was dissolved in CH ₂ Cl ₂ (750 ml) and cooled to 0°C. BBr ₃ (50 g; 0.20 mol) was added and the reaction mixture was stirred for 75 minutes. The reaction was quenched by careful addition of methanol (MeOH) (130 ml) with stirring at 0°C. The volatiles were removed under reduced pressure. HCl (1.25 M in EtOH, 320 ml) was added to the residue. The flask was then centrifuged using a rotary evaporator at atmospheric pressure and at ambient temperature for 15 minutes before the volatiles were removed under reduced pressure.

Dry flash chromatography on non-endcapped C ₁₈ silica gel using a gradient of acetonitrile (ACN) in water gave AGC0025 as a slightly orange glassy solid.

Yield: 13.27 g, 13.9 mmol, 98%.

Example 1 p). Synthesis of AGC0019

AGC0025 (10.63 g; 11.1 mmol) was dissolved in ACN (204 ml) and water (61 ml) at room temperature. Succinic anhydride (2.17 g; 21.7 mmol) was added and the reaction mixture was stirred for two hours. The reaction mixture was restored under reduced pressure. Dry flash chromatography on non-endcapped C ₁₈ silica gel using an ACN gradient in water gave a greenish glassy solid. The solid was dissolved in MeOH (62 ml) and water (10.6 ml) at 40°C. The solution was added dropwise to EtOAc (750 ml) under sonication. The precipitate was filtered, washed with EtOAc and dried under reduced pressure to give AGC0019 as an off-white solid with a greenish tinge.

Yield: 9.20 g, 8.7 mmol, 78%.

1H-NMR (400 MHz, DMSO-d ₆ ), 13C-NMR (100 MHz, DMSO-d ₆ ).

Example 2. Isolation of pure thorium-227.

Thorium-227 is isolated from an actinium-227 generator. Actinium-227 was obtained by irradiating radium-226 with thermal neutrons, followed by the decay of radium-227 (t _1/2 =42.2 min) to actinium-227.

- 26 043221

Thorium-227 was selectively retained from the decomposition mixture of actinium-227 in 8 M HNO3 solution by anion-exchange chromatography. A column was used with an internal diameter of 2 mm, a length of 30 mm, containing 70 mg of resin AG®1-X8 (200-400 mesh, nitrate form). After actinium-227, radium-223 and daughter radionuclides were eluted from the column, thorium-227 was extracted from the column using 12 M HCl. The eluate containing thorium-227 was evaporated to dryness and the residue was taken up in 0.01 M HCl prior to the labeling step.

Example 3 Cytotoxicity of ²²⁷ Th-AGC1118 against Ramos.

Example 3 a). Preparation of anti-CD22 monoclonal antibody (AGC1100).

The sequence of the monoclonal antibody (mAb) hLL2, also referred to as epratuzumab, herein AGC1100, was constructed as described in Leung, Goldenberg, Dion, Pellegrini, Shevitz, Shih, and Hansen: Molecular Immunology 32: 1413-27, 1995. mAb manufactured by Immunomedics Inc, New Jersey, USA was used in the present examples. The production of this mAb can, for example, be carried out in Chinese hamster ovary cells in suspension (CHO-S) transfected with a plasmid encoding the genes encoding the light and heavy chains. The first stable clones are selected to use standard procedures. After approximately 14 days, the monoclonal antibody can be harvested in a disposable bioreactor after filtering the supernatant. Further purification of AGC1100 was carried out by protein A binding affinity chromatography (MabSelect SuRe, Atoll, Weingarten/Germany) followed by an ion exchange step. A third purification step can be used to remove aggregates and possibly remaining impurities based on electrostatic and hydrophobic properties. The identity of AGC1100 was confirmed by isoelectric focusing, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE analysis), N-terminal sequencing, and liquid chromatography with mass spectrometry (LC/MS). Further analysis of the purity of the sample was performed by size exclusion chromatography (SEC).

Example 3 b). Coupling of mAb AGC1100 (epratuzumab) with AGC0019 chelator (compound of formula (VIII)) to produce AGC1118 conjugate

AGC0019 AGC0018

Before conjugation, a phosphate buffer (pH 7.5) was added to the antibody solution (AGC1100) to increase the buffering capacity of the solution. The amount of AGC1100 (monoclonal antibody, mAb) in the vessel was determined.

Chelator AGC0019 was dissolved in 1:1, DMA: 0.1 M MES buffer, pH 5.4. NHS and EDC were dissolved in 0.1 M MES buffer, pH 5.4.

A 1/1/3 molar equivalent solution of the chelator/N-hydroxysuccinimide (NHS)/1-ethyl3-(3-dimethylaminopropyl)cabodiimide (EDC) was prepared to activate the chelator. For antibody conjugation, an activated chelator in a molar ratio of 7.5/7.5/22.5/1 (chelator/NHS/EDC/mAb) was added to the mAb. After 20-40 min, the conjugation reaction was quenched with 12% (v/v) 0.3 M citric acid to adjust the pH to 5.5.

The solution was then buffered with 30 mM citrate, 70 mM NaCl, 2 mM EDTA, 0.5 mg/mL pABA, pH 5.5 (TFF buffer) by tangential flow filtration at constant volume. At the end of the diafiltration, the solution was poured into the formulation container. TFF buffer (30 mM citrate, 70 mM NaCl, 2 mM EDTA, 0.5 mg/mL pABA, pH 5.5) and 7% (w/v) polysorbate 80 were added to give the product to give 2.6 mg/ml AGC1118 in 30 mM citrate, 70 mM NaCl, 2 mM EDTA, 0.5 mg/ml pAVA 0.1% (w/v) PS80, pH 5.5. Finally, the solution was filtered through a 0.2 pm filter into sterile flasks before storage.

Example 3 c). Receiving an injection dose of ²²⁷ Th-AGC1118.

The contents of the test tube with 20 MBq of thorium-227 chloride film were dissolved in 2 ml of 8 M HNO3 solution and left for 15 min before the solution was withdrawn and applied to an anion exchange column to remove the radium-223 formed during this time. The column was washed with 3 ml 8 M HNO3 and 1 ml water before eluting thorium-227 with 3 ml 3 M HCl. The elution activity of thorium-227 was measured, and a dose of 10 MBq was transferred to an empty 10 ml glass tube. Then vaporized ki

- 27 043221 slot by using a vacuum pump and placing the tube in a heating block (set at 120°C) for 30-60 min. After reaching room temperature, 6 ml of AGC1118 conjugate 2.5 mg/ml was added to introduce radioactive isotopes. The contents of the tube were gently mixed and left for 15 min at room temperature. Then, the solution was sterilized filtered into a sterile tube, and a sample was taken for iTLC (instant thin layer chromatography) analysis to determine radiochemical purity before use.

Example 3d). Cytotoxicity of ²²⁷ Th-AGC1118 against Ramos with different general activity and specific activity.

This study tested doses of ²²⁷ Th-AGC1118 by varying total activity and specific activity, incubation time was 4 h. , 20 and 40 kBq/ml.

Ramos cells were cultured in RPMI1640 medium with 10% FBS and 1% penicillin/streptomycin (Passage 22). The cells were transferred to a centrifuge tube, centrifuged at 300 g for 5 min and suspended in 5 ml medium before counting using a Z2 Coulter counter. The cell suspension was diluted with medium to a cell concentration of 400,000 cells/ml and transferred to 48 wells (200 μl per well) in a 96-well plate (80,000 cells per well). The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to measure cell viability, see FIG. 4.

Example 4 Cytotoxicity of ²²⁷ Th-AGC0718 against HL-60.

Example 4 a). Preparation of anti-CD33 monoclonal antibody (AGC0700).

The sequence of the monoclonal antibody (mAb) HuM195/lintuzumab, herein referred to as AGC0700, was obtained as described in the literature according to (1) and (2). The AGC0700 was manufactured by CobraBiologics (Södertälje, Sweden). Briefly, heavy and light chain amino acid sequences were reverse-translated into DNA using Vector NTI® Software (Invitrogen/LifeTechnologies Ltd., Paisley, UK). determination of the conjugate to antibody ratio (CAR) as described in Example 2. The resulting DNA sequence was codon-optimized for expression in mammalian cells and synthesized by GeneArt (GeneArt/Life-Technologies Ltd., Paisley, UK) and then cloned into the CobraBiologics expression vector (Södertälje, Sweden) Chinese hamster ovary cells in suspension (CHO-S) were stably transfected with a plasmid encoding the VH and VL domains of AGC0700 and grown in the presence of standard CD-CHO medium (Invitrogen/Life-Technologies Ltd., Paisley, UK). ) with the addition of puromycin (12.5 mg/l, Sigma Aldrich) Stable clones expressing AGC0700 were selected by limiting dilution over 25 generations. Clone stability was assessed by measuring protein titers from the supernatant. A cell bank of the most stable clone was obtained, and its cryopreservation was carried out.

mAb expression was carried out at 37° C. for approximately 14 days in a 250 L scale disposable bioreactor. The monoclonal antibody was harvested after filtering the supernatant. Further purification of AC0700 was performed using a Protein A binding affinity chromatography column (MabSelect SuRe, Atoll, Weingarten/Germany), followed by a single anion exchange (QFF-Sepharose; GE Healthcare) and cation exchange chromatography (PorosXS; Invitrogen/LifeTechnologies Ltd.) to enhance purity and final yield. The identity of AGC0700 was confirmed by isoelectric focusing and SDS-PAGE analysis. Analysis of the activity of purified AGC0700 was carried out by ELISA with binding to the immobilized target CD33-Fc (Novoprotein). Sample purity analysis was performed by size exclusion chromatography (SEC).

Bibliography:

(1) Scheinberg D.A. Therapeutic uses of the hypervariable region of monoclonal antibody M195 and constructs thereof. U.S. Patent Application 6007814 (1999 Dec 28).

(2) Co M.S. et al.; J Immunol. 1992 Feb 15; 148(4): 1149-54. Chimeric and humanized antibodies with specificity for the CD33 antigen.

Example 4 b). Coupling of mAb AGC0700 (lintuzumab) with the AGC0019 chelator (compound of formula (VIII)) to produce an AGC0718 conjugate.

Conjugation was carried out as described in example 3 with minor differences.

Before conjugation, a phosphate buffer (pH 7.5) was added to the antibody solution (AGC0700) to increase the buffering capacity of the solution. The amount of AGC0700 (monoclonal antibody, mAb) in the vessel was determined.

Chelator AGC0019 was dissolved in 1:1, DMA: 0.1 M MES buffer, pH 5.4. NHS and EDC were dissolved in 0.1 M MES buffer, pH 5.4.

Received 1/1/3 molar equivalent solution of chelator/NHS/EDC to activate the chelator. For conjugation with an antibody, an activated chelator in a molar ratio of 20/20/60/1

- 28 043221 (chelator/NHS/EDC/mAb) added to mAb. After 40-60 min, the conjugation reaction was quenched with 12% (v/v) 0.3 M citric acid to adjust the pH to 5.5.

The solution was then buffer exchanged with 30 mM citrate, 154 mM NaCl, 2 mM EDTA, 2 mg/ml pABA, pH 5.5 (TFF buffer) by tangential flow filtration at constant volume. At the end of the diafiltration, the solution was poured into the formulation container. TFF buffer (30 mM citrate, 154 mM NaCl, 2 mM EDTA, 2 mg/mL pABA, pH 5.5) was added to obtain the product to give 2.5 mg/mL AGC0718 in 30 mM citrate, 154 mM NaCl, 2 mM EDTA, 2 mg/ml rAVA, pH 5.5. Finally, the solution was filtered through a 0.2 μm filter into sterile flasks before storage.

Example 4 c). Receiving an injection dose of ²²⁷ Th-AGC0718.

The contents of the test tube with 20 MBq of thorium-227 chloride film were dissolved in 2 ml of 8 M HNO3 solution and left for 15 min before the solution was withdrawn and applied to an anion exchange column to remove the radium-223 formed during this time. The column was washed with 3 ml 8 M HNO3 and 1 ml water before eluting thorium-227 with 3 ml 3 M HCl. The elution activity of thorium-227 was measured, and a dose of 10 MBq was transferred to an empty 10 ml glass tube. The acid was then evaporated by using a vacuum pump and placing the tube in a heating block (set at 120°C) for 30-60 minutes. After reaching room temperature, 6 ml of AGC0718 conjugate 2.5 mg/ml was added to introduce radioactive isotopes. The contents of the tube were gently mixed and left for 15 min at room temperature. Then, the solution was sterilized filtered into a sterile tube, and a sample was taken for iTLC (instant thin layer chromatography) analysis to determine radiochemical purity before use.

Example 4d). Cytotoxicity of ²²⁷ Th-AGC0718 against HL-60 with different overall activity.

To demonstrate the cytotoxicity of ²²⁷ Th-AGC0718 after binding to CD33+ cells, an in vitro cytotoxicity assay was performed. For this, the human myeloid leukemia cell line HL-60 as well as the CD33-negative B cell line (Ramos) were exposed to ²²⁷ Th-AGC0718. Produced testing of the total activity of 2 and 20 kBq/ml with a specific activity of 44 kBq/mcg. All experimental procedures are described in RD2013.093. Briefly, 50,000 human HL-60 cells/ml were generated in Iscove's Modified Dulbecco's Medium (IMDM) using 10% FBS and 1% penicillin/streptomycin; seeding was done at a density of 100,000 cells per well in a 24-well plate. Cells were incubated for 4 h at 37°C with an activity of 0 to 20 kBq/ml ^{of 227} Th-AGC0718. In parallel, a corresponding isotype control ²²⁷ Th conjugate sample was obtained as appropriate controls, as well as an unlabeled AGC0718 sample. The cells were then washed with fresh medium and seeded in a new 24-well culture plate.

Cell harvest was performed at various time points and cell viability measurements were made using the CellTiterGlo kit (Promega). Viability was expressed as a percentage, while the viability of the positive control (untreated cells) was taken as 100%; see fig. 5.

Example 5 Cytotoxicity of ²²⁷ Th-AGC0118 against SKOV-3.

Example 5 a). Getting AGC0100 (trastuzumab).

Trastuzumab, a monoclonal antibody (herein referred to as AGC0100) was purchased from Roche; it was dissolved to a concentration of 10 mg/ml in PBS (Dulbecco BIOCHROM).

Example 5 b). Coupling of the AGC0100 mAb (trastuzumab) with the AGC0019 chelator (compound of formula (VIII)) to produce an AGC0118 conjugate.

Conjugation was carried out as described in example 3 with minor modifications. Purification of the final mAb conjugate with TFF was replaced by gel filtration column chromatography.

To trastuzumab in PBS was added 11% 1 M phosphate buffer, pH 7.4. Chelator (AGC0019) NHS and EDC were dissolved in the same solutions as described for example 3 b). Upon activation, the molar ratio of chelator/NHS/EDC was 1/1/3. With a molar ratio of 8/8/25/1 for the chelator/NHS/EDC/mAb mixture and with a conjugation time of 30-40 min, a CAR (chelator to antibody ratio) of 0.7-0.9 for conjugated AGC0118 was obtained. The reaction was quenched by adding 12% (v/v) 0.3M citric acid to a final pH of 5.5.

Purification and replacement of AGC0118 conjugate buffer in 30 mM citrate, pH 5.5, 154 mM NaCl was performed by gel filtration on a Superdex 200 column (GE Healthcare) in combination with an AKTA system (GE Healthcare). The protein concentration was measured at Abs 280 nm prior to adding buffer to the product (to obtain 2.5 mg/ml AGC0118 in 30 mM citrate, 154 mM NaCl, 2 mM EDTA, 2 mg/ml rAVA, pH 5.5). Finally, the solution was filtered through a 0.2 μm filter into sterile flasks before storage.

Example 5 c). Receiving an injection dose of ²²⁷ Th-AGC0118.

Labeling was performed as described above.

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The content of the tube with 20 MBq of thorium-227 chloride film was dissolved in 2 ml of 8 M HNO ₃ solution and left for 15 min. before the solution was recovered and applied to an anion exchange column to remove the radium-223 generated during this time. The column was washed with 3 ml 8 M HNO ₃ and 1 ml water before eluting thorium-227 with 3 ml 3 M HCl. The elution activity of thorium-227 was measured, and a dose of 10 MBq was transferred to an empty 10 ml glass tube. The acid was then evaporated by using a vacuum pump and placing the tube in a heating block (set at 120°C) for 30-60 minutes. After reaching room temperature, 6 ml of AGC0118 conjugate 2.5 mg/ml was added to introduce radioactive isotopes. The contents of the tube were gently mixed and left for 15 min at room temperature. Then, the solution was sterilized filtered into a sterile tube, and a sample was taken for iTLC (instant thin layer chromatography) analysis to determine radiochemical purity before use.

Example 5d). Cytotoxicity of ²²⁷ Th-AGC0118 against SKOV-3 with other overall activity.

Cytotoxicity testing was performed with various doses of ²²⁷ Th-AGC0118 by varying the total activity with the addition to the wells during incubation for 4 hours. The seeding density of SKOV-3 cells was 10,000 cells per well in a 96-well plate the day before the experiment. On day 1, chelating ²²⁷ Th-AGC0118 was sequentially added to the cells with a total activity of 5, 10, 20 and 40 kBq/ml, with a specific activity of 20 kBq/μg. The remaining unbound ²²⁷ Th-AGC0118 was removed using a multichannel pipette, followed by an additional wash with medium and fresh culture medium after the end of the incubation period. SKOV-3 cells were cultured in McCoy's medium with 10% FBS and 1% penicillin/streptomycin. During incubation with ²²⁷ ThAGC0118, the culture medium was replaced with medium without serum. On the fourth day, the CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to measure cell viability; see fig. 6.

Example 6 Cytotoxicity of ²²⁷ Th-AGC2518 against NCI-H716.

Example 6 a). Preparation of FGFR2 monoclonal antibody (BAY1179470; AGC2500).

The preparation of the monoclonal antibody BAY 1179470, hereinafter referred to as AGC2500, is described in detail in WO 2013076186A1. Briefly, the antibody was obtained after biopanning on the FGFR2 antigen. The resulting human IgG1 antibody was expressed in Chinese Hamster Ovary (CHO) cells and purified using a protein A binding affinity chromatography column (MAb Select Sure), followed by size exclusion chromatography to isolate monomeric fractions. The antibody was combined with PBS, pH 7.4. Homogeneity determined by size exclusion chromatography was > 99%.

Example 6 b). Coupling of mAb AGC2500 with AGC0019 chelator (compound of formula (VIII)) to obtain AGC2518 conjugate.

The pH value of the antibody-containing solution was adjusted to pH 7.5. Chelator AGC0019 was dissolved in 1:1, DMA: 0.1 M MES buffer, pH 5.4. NHS and EDC were dissolved in 0.1 M MES buffer, pH 5.4. Received 1/1/3 molar equivalent solution of chelator/NHS/EDC to activate the chelator. For antibody conjugation, a 10/10/30/1 molar activated chelator (chelator/NHS/EDC/mAb) was added to the mAb. After 30 min, the conjugation reaction was quenched with 12% (v/v) 0.3 M citric acid to adjust the pH to 5.5. The reaction sample was then loaded onto a HiLoad 16/600 Superdex 200 (pre-grade) column for isolation of monomer fractions using 30 mM citrate, 70 mM NaCl, pH 5.5 as the mobile phase. At the end of the chromatography, the AGC2518 antibody conjugate was concentrated to 2.5 mg/ml in 30 mM citrate, 70 mM NaCl, 2 mM EDTA and 0.5 mg/ml rAVA. All procedures are described in RD.2014.092, Journal No. 211/149, 140619 AEF.

Example 6 c). Receiving an injection dose of ²²⁷ Th-AGC2518.

The contents of the test tube with 20 MBq of thorium-227 chloride film were dissolved in 2 ml of 8 M HNO3 solution and left for 15 min before the solution was withdrawn and applied to an anion exchange column to remove the radium-223 formed during this time. The column was washed with 3 ml 8 M HNO ₃ and 1 ml water before eluting thorium-227 with 3 ml 3 M HCl. The elution activity of thorium-227 was measured, and a dose of 10 MBq was transferred to an empty 10 ml glass tube. The acid was then evaporated by using a vacuum pump and placing the tube in a heating block (set at 120°C) for 30-60 minutes. After reaching room temperature, 6 ml of AGC2518 conjugate 2.5 mg/ml was added to introduce radioactive isotopes. The contents of the tube were gently mixed and left for 15 min at room temperature. Then, the solution was sterilized filtered into a sterile tube, and a sample was taken for iTLC (instant thin layer chromatography) analysis to determine radiochemical purity before use.

Example 6d). Cytotoxicity of ²²⁷ Th-AGC2518 against NCI-H716 cells with other overall activity.

To demonstrate the cytotoxicity of ²²⁷ Th-AGC2518 after binding to FGFR2+ cells, an in vitro cytotoxicity assay was performed. For this, a human colorectal cancer cell line

- 30 043221

NCI-H716 was exposed to ²²⁷ Th-AGC2518. The total activity was tested at 2, 10, 20 and 40 kBq/ml with a specific activity of 2 kBq/μg. In parallel, an unrelated isotype control was obtained. All experimental procedures are described in RD2014.138. Briefly, 400,000 human NCI-H716 cells/ml were generated in RPMI 1640 medium using 10% FBS and 1% penicillin/streptomycin; seeding was done at a density of 80,000 cells per well in a 96-well plate. Cells were incubated for 30 min at 37°C with 0 to 40 kBq/ml activity of ²²⁷ Th-AGC2518 and the corresponding isotype control ²²⁷ Th conjugate sample. The cells were then washed with fresh medium and seeded in a new 96-well culture plate. Cell spores were performed at five and seven days and cell viability measurements were made using the CellTiterGlo kit (Promega). Viability was expressed as a percentage, with the viability of the positive control (untreated cells) being taken as 100%, see FIG. 7.

Example 7 Cytotoxicity of ²²⁷ Th-AGC2418 against HT29 cells.

Example 7 a). Preparation of mesothelin monoclonal antibody (BAY 86-1903; AGC2400).

The preparation of the monoclonal antibody BAY 86-1903, hereinafter referred to as AGC2400, is described in detail in WO 2009068204. Briefly, the antibody was obtained after biopanning on the mesothelin antigen. The resulting human IgG1 antibody was expressed in Chinese Hamster Ovary (CHO) cells and purified using a Protein A binding affinity chromatography column (MAb Select Sure), followed by removal of aggregates using a hydrophobic chromatography column (Toyopearl Butyl 600M). The antibody was combined with PBS, pH 7.5.

Example 7 b). Coupling of mAb AGC2400 with chelator AGC0019 (compound of formula (VIII)) to obtain an AGC2418 conjugate.

The pH value of the antibody-containing solution was adjusted to pH 7.5. Chelator AGC0019 was dissolved in 1:1, DMA: 0.1 M MES buffer, pH 5.4. NHS and EDC were dissolved in 0.1 M MES buffer, pH 5.4. Received 1/1/3 molar equivalent solution of chelator/NHS/EDC to activate the chelator. For antibody conjugation, an activated chelator in a molar ratio of 16.5/16.5/49.5/1 (chelator/NHS/EDC/mAb) was added to the mAb. After 30 min, the conjugation reaction was quenched with 12% (v/v) 0.3 M citric acid to adjust the pH to 5.5. The reaction sample was then loaded onto a HiLoad 16/600 Superdex 200 (pre-grade) column for isolation of monomer fractions using 30 mM citrate, 70 mM NaCl, pH 5.5 as the mobile phase. At the end of the chromatography, the AGC2418 antibody conjugate was concentrated to 2.5 mg/ml in 30 mM citrate, 70 mM NaCl, 2 mM EDTA and 0.5 mg/ml rAVA. All procedures are described in RD.2014.111, Journal No. 211/160, 140814 AEF.

Example 7 c). Receiving a dose of ²²⁷ Th-AGC2418.

The contents of the test tube with 20 MBq of thorium-227 chloride film were dissolved in 2 ml of 8 M HNO3 solution and left for 15 min before the solution was withdrawn and applied to an anion exchange column to remove the radium-223 formed during this time. The column was washed with 3 ml 8 M HNO3 and 1 ml water before eluting thorium-227 with 3 ml 3 M HCl. The elution activity of thorium-227 was measured, and a dose of 10 MBq was transferred to an empty 10 ml glass tube. The acid was then evaporated by using a vacuum pump and placing the tube in a heating block (set at 120°C) for 30-60 minutes. After reaching room temperature, 6 ml of AGC2418 conjugate 2.5 mg/ml was added to introduce radioactive isotopes. The contents of the tube were gently mixed and left for 15 min at room temperature. Then, the solution was sterilized filtered into a sterile tube, and a sample was taken for iTLC (instant thin layer chromatography) analysis to determine radiochemical purity before use.

Example 7d). Cytotoxicity of ²²⁷ Th-AGC2418 against mesothelin antigen overexpressing HT29 cells with different overall activity.

To demonstrate the cytotoxicity of ²²⁷ Th-AGC2418 after binding to mesothelin+ cells, an in vitro cytotoxicity assay was performed. For this, a human colorectal cancer cell line HT29 transfected with mesothelin antigen was exposed to ²²⁷ Th-AGC2418. Total activity was tested at 12 points in a three-fold dilution, starting at 5 kBq/ml with a specific activity of 10 kBq/μg. In parallel, an unrelated isotype control was obtained. All experimental procedures are described in RD2014.154. Briefly, 200,000 human HT29 cells/ml were generated in RPMI 1640 medium using 10% FBS and 1% penicillin/streptomycin 1% NaHCO ₃ , 600 μg/ml hygromycin B; seeding was done at a density of 40,000 cells per well in a 96-well plate. Cells were incubated for 6 days at 37° C. with 0 to 40 kBq/ml activity of ²²⁷ Th-AGC2418 and the corresponding isotype control ²²⁷ Th conjugate sample. Cell harvest was performed on the sixth day and cell viability measurements were made using the CellTiterGlo kit (Promega). Viability was expressed as a percentage, while the viability of the positive control (untreated cells) was taken as 100%.

Example 8

Stability comparison of amide and isocyanate-linked conjugates AGC1118 and the corresponding isocyanate-linking conjugate (AGC1115) were stored in aqueous solution at 40°C for 11 days. Samples were taken periodically.

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40°C samples normalized to 4°C sampling point

AGC1118 AGC1115 CAR (% normal) CAR (% normal) Day 0 100 100 Day 5 105 92 Day 11 103 88

It can be seen from the table above that no measurable decrease in the concentration of the conjugate was observed for the amide-bound conjugate. In contrast, the concentration of the isocyanate conjugate decreased by 8% after 5 days and by 12% after 11 days.

Example 9

Example 9 a). Obtaining PSMA monoclonal antibody (AGC1000).

The PSMA monoclonal antibody, hereinafter referred to as AGC1000, was purchased from Progenics, USA.

Example 9 b). Coupling of mAb AGC1000 with AGC0019 chelator (compound of formula (VIII)) to obtain an AGC1018 conjugate.

The pH value of the antibody-containing solution was adjusted to pH 7.5. Chelator AGC0019 was dissolved in 1:1, DMA: 0.1 M MES buffer, pH 5.5. NHS and EDC were dissolved in 0.1 M MES buffer, pH 5.5. Received 1/1/2 molar equivalent solution of chelator/NHS/EDC to activate the chelator. For antibody conjugation, a 20/20/40/1 molar activated chelator (chelator/NHS/EDC/mAb) was added to the mAb in 4 portions with 10 min between each portion. After 50 min, the conjugation reaction was quenched with 12% (v/v) 1 M TRIS, pH 7.3. The conjugate was purified and buffer exchanged by tangential flow filtration (TFF). Prescription buffer: 30 mM citrate, 70 mM NaCl, 2 mM EDTA, 0.5 mg/ml pABA, pH 5.5. At the end of the diafiltration, the solution was poured into a container and the concentration was adjusted to 2.7 mg/ml. Finally, the main volume of the solution was filtered through a sterile filter with a pore size of 0.2 μm and transferred to sterile tubes for storage at -20°C.

Example 9 c). Receiving an injection dose of ²²⁷ Th-AGC1018.

The contents of a tube with approximately 50 MBq of thorium-227 chloride film were dissolved in 2 ml of 8 M HNO ₃ solution and left for 15 min before the solution was withdrawn and applied to an anion exchange column to remove the radium-223 formed during this time. The column was washed with 3 ml 8 M HNO ₃ and 1 ml water before eluting thorium-227 with 3 ml 3 M HCl. The HCl eluate was then evaporated using a vacuum pump and a heating block (set at 100° C.) for 60-90 minutes. The activity of dried Th-227 was measured in a dose calibrator. Dry Th-227 was dissolved in 0.05 M HCl to give a concentration of 0.5 MBq/µl. For the introduction of radioactive isotopes, the AGC1018 conjugate was diluted in prescription buffer to obtain 25 μg mAb in 200 μl. 1 MBq of Th-227 was mixed with the AGC1018 solution, and the activity of Th-227 was accurately measured using a germanium detector. Chelating was carried out for 30-60 min at room temperature before sterilizing filtration into a sterile tube. A sample was taken for iTLC (instant thin layer chromatography) analysis to determine radiochemical purity before use.

Example 9d). Cytotoxicity of ²²⁷ Th-AGC1018 against PSMA-expressing LNCaP cells.

To demonstrate the cytotoxicity of ²²⁷ Th-AGC1018 after binding to PSMA positive cells, an in vitro cytotoxicity assay was performed. For this, the human prostate cancer cell line LNCaP was exposed to ²²⁷ Th-AGC1018. Total activity was tested at 12 points in a three-fold dilution, starting at 20 kBq/ml with a specific activity of 40 kBq/μg. In parallel, an unrelated isotype control was obtained. All experimental procedures are described in the archive RD.2015.101.

Briefly, human LNCaP cells were cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. Seeding density was 2500 cells per well in a 96-well plate. 24 hours after seeding (day 1), cells were exposed to the isotype control ²²⁷ Th-AGC1018 and ²²⁷ Th with a total activity in the range of 0-20 kBq/ml for 5 days at 37°C. Cell harvest was performed on the sixth day and cell viability measurements were made using the CellTiterGlo kit (Promega). Viability was expressed as a percentage, while the viability of the positive control (untreated cells) was taken as 100%.

Claims (22)

1. A tissue-targeted thorium complex, which is a compound comprising wherein said tissue-targeted Y fragment is a monoclonal antibody selected from hLL2 (epratuzumab), HuM195 (lintuzumab), trastuzumab, BAY 1179470 and BAY 86-1903 (mesothelin); And 4+ ion of thorium radionuclide ²²⁷ Th emitting alpha particles. 2. A tissue-targeted thorium complex according to claim 1, comprising said tissue-targeting monoclonal antibody comprising at least one peptide chain comprising an amino acid sequence with at least 90% sequence identity with at least one of the following sequences: Light chain: DIQLTQSPSSLAVSAGENVTMSCKSSQSVLYSANHKNYLAWYQQKPGQ SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVQVEDLAIYYCHQYL SSWTFGGGTKLEIKR (SeqIDl) DIQLTQSPSSLASAAVEDRTMSCKSSQSVLYSANHKNYLAWYQQKPGQ KAKLLIYWASTRESGVP SRF SGSGSGTDFTFTIS SLQPEDIATYYCHQYL SSWTFGGGTKLEIKR (SeqID2) Heavy chain: QVQLQESGAELSKPGASVKMSCKASGYTFTSYWLHWIKQRPGQGLEWI GYINPRNDYTEYNONFKDKATLTADKSS ST AYMQLSSLTSED S AVYYC ARRDITTFYWGQGTTTLTVSS (SeqID3) QyQLQQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEW IGYINPRNDYTEYNONFKDKATITADESTNTAYMELSSLRSEDTAFYFC ARRDITTFYWGQGTTVTVS S (SeqID4) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEW IGYINPRNDYTEYNONFKDKATITADESTNTAYMELSSLRSEDTAFYFC ARRDITTFYWGQGTTVTVS S (SeqID5). 3. A method for forming a tissue-targeted thorium complex, said method comprising the following steps: a) formation of an octadentate chelator AGC0019 having the following structure: b) coupling said octadentate chelator to at least one tissue-targeted moiety containing at least one amine moiety with at least one amide binding agent selected from N-hydroxymaleimide, carbodiimide and/or azodicarboxylate activating reagents, wherein a tissue-targeted chelator is obtained, wherein said tissue-targeted fragment is a monoclonal antibody selected from hLL2 (epratuzumab), HuM195 (lintuzumab), trastuzumab, BAY 1179470 and BAY 86-1903 (mesothelin); And c) bringing said tissue-targeted chelator into contact with an aqueous solution containing a 4+ ion of the alpha particle-emitting thorium radionuclide ²²⁷ Th. 4. Method according to claim 3, characterized in that step b) is carried out in an aqueous solution. 5. The method according to claim 3 or 4, wherein said amide binding agent is a carbodiimide binding agent selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-diisopropylcarbodiimide (DIC) or N,N'-dicyclohexylcarbodiimide (DCC). 6. Method according to any one of claims 3 to 5, characterized in that step b) is carried out in an aqueous solution at a pH value in the range of 4-9. 7. Method according to any one of claims 3-6, characterized in that step b) is carried out at a temperature of 1550°C for 5-120 minutes. 8. The method according to any one of claims 3 to 7, characterized in that step c) is carried out at a temperature of 1550°C for 1-60 minutes. - 33 043221 9. A pharmaceutical formulation for the treatment of hyperplastic or neoplastic diseases, comprising at least one tissue-targeting thorium complex according to claim 1 or 2 in a therapeutically effective amount. 10. Pharmaceutical formulation according to claim 9, additionally containing a citrate buffer. 11. Pharmaceutical formulation according to claim 9 or 10, additionally containing para-aminobutyric acid (PABA) and, if necessary, EDTA (ethylenediaminetetraacetic acid) and/or at least one polysorbate. 12. The use of a tissue-targeted thorium complex according to claim 1 or 2 for the manufacture of a medicament for the treatment of hyperplastic or neoplastic diseases. 13. The use of a pharmaceutical formulation according to any one of claims 9 to 11 for the manufacture of a medicament for the treatment of hyperplastic or neoplastic diseases. 14. Use according to claim 12 or 13, characterized in that said disease is carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed cancer, breast cancer, endometrial cancer, gastric cancer, acute myeloid leukemia, prostate or brain cancer , mesothelioma, ovarian, lung or pancreatic cancer. 15. Use according to claim 14, wherein the mixed cancer is non-Hodgkin's lymphoma or B-cell neoplasms. 16. A method of treating hyperplastic or neoplastic diseases in a human or animal, comprising administering at least one tissue-targeting thorium complex according to claim 1 or 2 in a therapeutically effective amount, or at least one pharmaceutical formulation according to any one of claims 9 -eleven. 17. The method of claim 16 wherein the hyperplastic or neoplastic disease is selected from carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed type cancer, breast cancer, endometrial cancer, gastric cancer, acute myeloid leukemia, prostate or brain cancer, mesothelioma , ovarian, lung, or pancreatic cancer. 18. The method of claim 17, wherein the mixed cancer is non-Hodgkin's lymphoma or B-cell neoplasms. 19. The use of a tissue-targeted thorium complex according to claim 1 or 2 for the treatment of hyperplastic and/or neoplastic disease. 20. The use of a pharmaceutical formulation according to any one of claims 9 to 11 for the treatment of a hyperplastic and/or neoplastic disease. 21. Use according to claim 19 or 20, wherein the hyperplastic and/or neoplastic diseases are selected from carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed type cancer, breast cancer, endometrial cancer, gastric cancer, acute myeloid leukemia, prostate cancer or brain, mesothelioma, ovarian, lung, or pancreatic cancer. 22. Use according to claim 21, wherein the mixed cancer is non-Hodgkin's lymphoma or B-cell neoplasms.