RADIO-PHARMACEUTICAL COMPLEXES

Abstract

The invention provides a method for the formation of a tissue-targeting thorium complex, said method comprising; a) forming an octadentate chelator comprising four hydroxypyridinone (HOPO) moieties, substituted in the N-position with a C.sub.1-C.sub.3alkyl group, and a coupling moiety terminating in a carboxylic acid group; b) coupling said octadentate chelator to at least one tissue-targeting peptide or protein comprising at least one amine moiety by means of at least one amide-coupling reagent whereby to generate a tissue-targeting chelator; and c) contacting said tissue-targeting chelator with an aqueous solution comprising an ion of at least one alpha-emitting thorium isotope. A method of treatment of a neoplastic or hyperplastic disease comprising administration of such a tissue-targeting thorium complex, as well as the complex and corresponding pharmaceutical formulations are also provided.

Claims

1. A method for the formation of a tissue-targeting thorium complex, said method comprising: a) forming an octadentate chelator comprising four hydroxypyridinone (HOPO) moieties, substituted in the N-position with a C.sub.1-C.sub.3 alkyl group, and a coupling moiety terminating in a carboxylic acid group; b) coupling said octadentate chelator to at least one tissue-targeting peptide or protein comprising at least one amine moiety by means of at least one amide-coupling reagent whereby to generate a tissue-targeting chelator; and c) contacting said tissue-targeting chelator with an aqueous solution comprising an ion of at least one alpha-emitting thorium isotope.

2. The method of claim 1 wherein step b) is conducted in aqueous solution.

3. The method of claim 1 wherein said amide-coupling reagent is functional in aqueous solution.

4. The method of claim 1 wherein said amide-coupling reagent is a carbodiimide coupling reagent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N′-diisopropyl-carbodiimide (DIC) or N,N′-dicyclohexylcarbodiimide (DCC).

5. The method of claim 1 wherein step b) is conducted in aqueous solution at pH between 4 and 9.

6. The method of claim 1 wherein step b) is conducted between 15 and 50° C. for 5 to 120 minutes.

7. The method of claim 1 wherein step c) is conducted between 15 and 50° C. for 1 to 60 minutes.

8. The method of claim 1 wherein said octadentate chelator comprises four 3,2-HOPO moieties.

9. The method of claim 1 wherein said octadentate chelator is selected from formulae (VIb) and (VII): ##STR00033## wherein R.sub.C is a linker moiety terminating in a carboxylic acid moiety, such as [—CH.sub.2-Ph-N(H)—C(═O)—CH.sub.2—CH.sub.2—C(═O)OH], [—CH.sub.2—CH.sub.2—N(H)—C(═O)—(CH.sub.2—CH.sub.2—O).sub.1-3—CH.sub.2—CH.sub.2—C(═O)OH] or [—(CH.sub.2).sub.1-3-Ph-N(H)—C(═O)—(CH.sub.2).sub.1-5—C(═O)OH], wherein Ph is a phenylene group, preferably a para-phenylene group.

10. The method of claim 1 wherein said tissue-targeting moiety is a monoclonal or polyclonal antibody, an antibody fragment (such as Fab, F(ab′).sub.2, Fab′ or scFv), or a construct of such antibodies and/or fragments.

11. The method of claim 1 wherein said tissue-targeting moiety has binding affinity for the CD22 receptor, FGFR2, Mesothelin, HER-2, PSMA or CD33.

12. A tissue-targeting thorium complex formed or formable by the method of claim 1.

13. The tissue-targeting thorium complex of claim 12 comprising four 3,2-HOPO moieties.

14. The tissue-targeting thorium complex of claim 12 having binding affinity for the CD22 receptor, FGFR2, Mesothelin, HER-2, PSMA or CD33.

15. The tissue-targeting thorium complex of claim 12 comprising the 4+ ion of an alpha-emitting thorium radionuclide such as .sup.227Th.

16. The tissue-targeting thorium complex of claim 12 comprising an octadentate chelator of formula (VIb) or (VII): ##STR00034## wherein R.sub.C is a coupling moiety joined by an amide group to a tissue targeting moiety, preferably AGC0019.

17. The tissue-targeting thorium complex of claim 12 comprising a tissue targeting moiety selected from a monoclonal or polyclonal antibody, an antibody fragment (such as Fab, F(ab′).sub.2, Fab′ or scFv), or a construct of such antibodies and/or fragments.

18. The tissue-targeting thorium complex of claim 12 comprising a tissue targeting moiety comprising at least one peptide chain having at least 90% sequence similarity with at least one of the following sequences: Light Chain: TABLE-US-00005 (SEQ ID NO: 1) DIQLTQSPSSLAVSAGENVTMSCKSSQSVLYSANHKNYLAWYQQKPGQSP KLLIYWASTRESGVPDRFTGSGSGTDFTLTISRVQVEDLAIYYCHQYLSS WTFGGGTKLEIKR (SEQ ID NO: 2) DIQLTQSPSSLASAAVEDRTMSCKSSQSVLYSANHKNYLAWYQQKPGQKA KLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCHQYLSS WTFGGGTKLEIKR Heavy Chain: (SEQ ID NO: 3) QVQLQESGAELSKPGASVKMSCKASGYTFTSYWLHWIKQRPGQGLEWIGY INPRNDYTEYNQNFKDKATLTADKSSSTAYMQLSSLTSEDSAVYYCARRD ITTFYWGQGTTLTVSS  (SEQ ID NO: 4) QVQLQQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGY INPRNDYTEYNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRD ITTFYWGQGTTVTVSS (SEQ ID NO: 5) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQAPGQGLEWIGY INPRNDYTEYNQNFKDKATITADESTNTAYMELSSLRSEDTAFYFCARRD ITTFYWGQGTTVTVSS.

19. A pharmaceutical formulation comprising at least one tissue-targeting thorium complex as claimed in claim 12.

20. The pharmaceutical formulation of claim 19 further comprising citrate buffer.

21. The pharmaceutical formulation of claim 19 further comprising p-aminobutyric acid (PABA), and optionally EDTA and/or at least one polysorbate,

22. Use of a tissue-targeting thorium complex as claimed in claim 12 or a pharmaceutical formulation thereof in the manufacture of a medicament for the treatment of hyperplastic or neoplastic disease.

23. Use as claimed in claim 22 wherein said disease is a carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed type cancer including Non-Hodgkin's Lymphoma or B-cell neoplasms, breast, endometrial, gastric, acute myeloid leukemia, prostate or brain, mesothelioma, ovarian, lung or pancreatic cancer

24. A method of treatment of a human or non-human animal (particularly one in need thereof) comprising administration of at least one tissue-targeting thorium complex as claimed in claim 12 or at least one pharmaceutical formulation thereof.

25. The method of claim 24 for the treatment of hyperplastic or neoplastic disease, such as a carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed type cancer, including Non-Hodgkin's Lymphoma or B-cell neoplasms, breast, endometrial, gastric, acute myeloid leukemia, prostate or brain, mesothelioma, ovarian, lung or pancreatic cancer

26. A tissue-targeting thorium complex as claimed in claim 12 or a pharmaceutical formulation thereof for use in the treatment of hyperplastic and/or neoplastic disease such as a carcinoma, sarcoma, myeloma, leukemia, lymphoma or mixed type cancer including Non-Hodgkin's Lymphoma or B-cell neoplasms, breast, endometrial, gastric, acute myeloid leukemia, prostate or brain, mesothelioma, ovarian, lung or pancreatic cancer.

27. A kit for use in a method according to claim 1, said kit comprising: i) an octadentate chelator comprising four hydroxypyridinone (HOPO) moieties, substituted in the N-position with a C.sub.1-C.sub.3 alkyl group, and coupling moiety terminating in a carboxylic acid group; ii) at least one tissue-targeting peptide or protein comprising at least one amine moiety; iii) at least one amide-coupling reagent; and iv) optionally and preferably an alpha-emitting thorium radionuclide, such as .sup.227Th.

Description

BRIEF SUMMARY OF THE FIGURES

[0152] FIG. 1: Data demonstrating the stabilising effect of EDTA/PABA on the non-radioactive antibody conjugate AGC1118 in solution.

[0153] FIG. 2: Effect on hydrogen peroxide levels of different buffers containing antibody HOPO conjugates irradiated with 10 kGy of radiation.

[0154] FIG. 3: Radiostabilizing effect of .sup.227Th-AGC1118 (IRF assay) with a specific activity up to ca 8000 Bq/μg.

[0155] FIG. 4: Cytotoxicity of .sup.227Th-AGC1118 against Ramos with different total activity (4 hours incubation time) (see Example 3)

[0156] FIG. 5: .sup.227Th-AGC0718 induces target-specific cell killing of CD33-positive cells in vitro (see Example 4)

[0157] FIG. 6: Cell cytotoxicity of .sup.227Th-AGC0118 at high (20 kBq/μg) and low (7.4 kBq/μg) specific activity. Negative control was a low-binding peptide-albumin complex with same dose range, same incubation time and days before readout (see Example 5).

[0158] FIG. 7: .sup.227Th-AGC2518 induces target-specific cell killing of FGFR2-positive cells in vitro (see Example 6).

[0159] FIG. 8: .sup.227Th-AGC2418 induces target-specific cell killing of Mesothelin-positive cells in vitro (see Example 7).

[0160] FIG. 9: .sup.227Th-AGC1018 induces target-specific and dose dependent cell killing of PSMA-positive LNCaP cells in vitro (see Example 9).

[0161] The invention will now be illustrated by the following non-limiting examples. All compounds exemplified in the examples form preferred embodiments of the invention (including preferred intermediates and precursors) and may be used individually or in any combination in any aspect where context allows. Thus, for example, each and all of compounds 2 to 4 of Example 2, compound 10 of Example 3 and compound 7 of Example 4 form preferred embodiments of their various types.

EXAMPLE 1

Synthesis of Compound of Formula (VIII)

[0162] ##STR00015##

Example 1 a

Synthesis of Dimethyl 2-(4-nitrobenzyl) malonate

[0163] ##STR00016##

[0164] Sodium hydride (60% dispersion, 11.55 g, 289 mmol) was suspended in 450 mL tetrahydrofuran (THF) at 0° C. Dimethyl malonate (40.0 mL, 350 mmol) was added drop wise over approximately 30 minutes. The reaction mixture was stirred for 30 minutes at 0° C. 4-Nitrobenzyl bromide (50.0 g, 231 mmol) dissolved in 150 mL THF was added drop wise over approximately 30 minutes at 0° C., followed by two hours at ambient temperature.

[0165] 500 mL ethyl acetate (EtOAc) and 250 mL NH.sub.4Cl (aq, sat) was added before the solution was filtered. The phases were separated. The aqueous phase was extracted with 2*250 mL EtOAc. The organic phases were combined, washed with 250 mL brine, dried over Na.sub.2SO.sub.4, filtered and the solvents were removed under reduced pressure.

[0166] 300 mL heptane and 300 mL methyl tert-butyl ether (MTBE) was added to the residue and heated to 60° C. The solution was filtered. The filtrate was placed in the freezer overnight and filtered. The filter cake was washed with 200 mL heptane and dried under reduced pressure, giving the title compound as an off-white solid.

[0167] Yield: 42.03 g, 157.3 mmol, 68%.

[0168] 1H-NMR (400 MHz, CDCl3): 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, 8.7 Hz).

Example 1 b

Synthesis of 2-(4-Nitrobenzyl)propane-1,3-diol

[0169] ##STR00017##

[0170] Dimethyl 2-(4-nitrobenzyl) malonate (28.0 g, 104.8 mmol) was dissolved in 560 mL THF at 0° C. Diisobutylaluminium hydride (DIBAL-H) (1M in hexanes, 420 mL, 420 mmol) was added drop wise at 0° C. over approximately 30 minutes. The reaction mixture was stirred for two hours at 0° C.

[0171] 20 mL water was added drop wise to the reaction mixture at 0° C. 20 mL NaOH (aq, 15%) was added drop wise to the reaction mixture at 0° C. followed by drop wise addition of 20 mL water to the reaction mixture. The mixture was stirred at 0° C. for 20 minutes before addition of approximately 150 g MgSO4. The mixture was stirred at room temperature for 30 minutes before it was filtered on a Büchner funnel. The filter cake was washed with 500 mL EtOAc. The filter cake was removed and stirred with 800 mL EtOAc and 200 mL MeOH for approximately 30 minutes before the solution was filtered. The filtrates were combined and dried under reduced pressure.

[0172] DFC on silica using a gradient of EtOAc in heptane, followed by a gradient of MeOH in EtOAc gave the title compound as a pale yellow solid.

[0173] Yield: 15.38 g, 72.8 mmol, 69%.

[0174] 1H-NMR (400 MHz, CDCl3): 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-Nitrobenzyl)propane-1,3-diyl dimethanesulfonate

[0175] ##STR00018##

[0176] 2-(4-nitrobenzyl)propane-1,3-diol (15.3 g, 72.4 mmol) was dissolved in 150 mL CH.sub.2Cl.sub.2 at 0° C. Triethylamine (23 mL, 165 mmol) was added, followed by methanesulfonyl chloride (12 mL, 155 mmol) drop wise over approximately 15 minutes, followed by stirring at ambient temperature for one hour.

[0177] 500 mL CH.sub.2Cl.sub.2 was added, and the mixture was washed with 2*250 mL NaHCO.sub.3 (aq, sat), 125 mL HCl (aq, 0.1 M) and 250 mL brine. The organic phase was dried over Na.sub.2SO.sub.4, filtered and dried under reduced pressure, giving the title compound as an orange solid.

[0178] Yield: 25.80 g, 70.2 mmol, 97%.

[0179] 1H-NMR (400 MHz, CDCl3): 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 1 d

Synthesis of Di-tert-butyl(azanediylbis(ethane-2,1-diyl))dicarbamate

[0180] ##STR00019##

[0181] Imidazole (78.3 g, 1.15 mol) was suspended in 500 mL CH.sub.2Cl.sub.2 at room temperature. Di-tert-butyl dicarbonate (Boc.sub.2O) (262.0 g, 1.2 mol) was added portion wise. The reaction mixture was stirred for one hour at room temperature. The reaction mixture was washed with 3*750 mL water, dried over Na.sub.2SO.sub.4, filtered and the volatiles were removed under reduced pressure.

[0182] The residue was dissolved in 250 mL toluene and diethylenetriamine (59.5 mL, 550 mmol) was added. The reaction mixture was stirred for two hours at 60° C.

[0183] 1 L CH.sub.2Cl.sub.2 was added, and the organic phase was washed with 2*250 mL water. The organic phase was dried over Na.sub.2SO.sub.4, filtered and reduced under reduced pressure.

[0184] DFC on silica using a gradient of methanol (MeOH) in CH.sub.2Cl.sub.2 with triethylamine gave the title compound as a colorless solid.

[0185] Yield: 102 g, 336 mmol, 61%.

[0186] .sup.1H-NMR (400 MHz, CDCl3): 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-nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(ethane-2,1-diyl))tetracarbamate

[0187] ##STR00020##

[0188] 2-(4-Nitrobenzyl)propane-1,3-diyl dimethanesulfonate (26.0 g, 71 mmol) and di-tert-butyl(azanediylbis(ethane-2,1-diyl))dicarbamate (76.0 g, 250 mmol) were dissolved in 700 mL acetonitrile. N,N-diisopropylethylamine (43 mL, 250 mmol) was added. The reaction mixture was stirred for 4 days at reflux.

[0189] The volatiles were removed under reduced pressure.

[0190] DFC on silica using a gradient of EtOAc in heptane gave the tile compound as pale yellow solid foam.

[0191] Yield: 27.2 g, 34.8 mmol, 49%.

[0192] .sup.1H-NMR (400 MHz, CDCl3): 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 N.SUP.1.,N.SUP.1′.-(2-(4-nitrobenzyl)propane-1,3-diyl)bis(N.SUP.1.-(2-aminoethyl)ethane-1,2-diamine), AGC0020

[0193] ##STR00021##

[0194] Tetra-tert-butyl (((2-(4-nitrobenzyl)propane-1,3-diyl)bis(azanetriyl))tetrakis(ethane-2,1-diyl))tetracarbamate (29.0 g, 37.1 mmol) was dissolved in 950 mL MeOH and 50 mL water. Acetyl chloride (50 mL, 0.7 mol) was added drop wise over approximately 20 minutes at 30° C. The reaction mixture was stirred overnight.

[0195] The volatiles were removed under reduced pressure and the residue was dissolved in 250 mL water. 500 mL CH.sub.2Cl.sub.2 was added, followed by 175 mL NaOH (aq, 5M, saturated with NaCl). The phases were separated, and the aqueous phase was extracted with 4*250 mL CH.sub.2Cl.sub.2. The organic phases were combined, dried over Na.sub.2SO.sub.4, filtered and dried under reduced pressure, giving the title compound as viscous red brown oil.

[0196] Yield: 11.20 g, 29.3 mmol, 79%. Purity (HPLC FIG. 9): 99.3%.

[0197] .sup.1H-NMR (300 MHz, CDCl.sub.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).

[0198] .sup.13C-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

[0199] ##STR00022##

[0200] 2-pyrrolidinone (76 mL, 1 mol) and diethyl oxalate (140 mL, 1.03 mol) was dissolved in 1 L toluene at room temperature. Potassium ethoxide (EtOK) (24% in EtOH, 415 mL, 1.06 mol) was added, and the reaction mixture was heated to 90° C.

[0201] 200 mL EtOH was added portion wise 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 (5M, aq) was added slowly while stirring. 200 mL brine and 200 mL toluene was added, and the phases were separated.

[0202] The aqueous phase was extracted with 2×400 mL CHCl.sub.3. The combined organic phases were dried (Na.sub.2SO.sub.4), filtered and reduced in vacuo. The residue was recrystallized from EtOAc, giving the title compound as a pale yellow solid.

[0203] Yield: 132.7 g, 0.72 mol, 72%.

Example 1 h

Synthesis of Ethyl 3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylate

[0204] ##STR00023##

[0205] {Ethyl 5-hydroxy-6-oxo-1,2,3,6-tetrahydropyridine-4-carboxylate} (23.00 g, 124.2 mmol) was dissolved in 150 mL p-xylene and Palladium on carbon (10%, 5.75 g) was added. The reaction mixture was stirred at reflux over night. After cooling to room temperature, the reaction mixture was diluted with 300 mL MeOH and filtered through a short pad of Celite®. The pad was washed with 300 mL MeOH. The solvents were removed in vacuo, giving the title compound as a pale red-brownish solid.

[0206] Yield: 19.63 g, 107.1 mmol, 86%. MS (ESI, pos): 206.1[M+Na].sup.+, 389.1 [2M+Na].sup.+

Example 1 i

Synthesis of Ethyl 3-methoxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate

[0207] ##STR00024##

[0208] {ethyl 3-hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylate} (119.2 g, 0.65 mol) was dissolved in 600 mL dimethyl sulfoxide (DMSO) and 1.8 L acetone at room temperature. K.sub.2CO.sub.3 (179.7 g, 1.3 mol) was added. Methyl iodide (MeI) (162 mL, 321 mmol) dissolved in 600 mL acetone was added drop wise over approximately 1 hour at room temperature.

[0209] 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 at reflux overnight. The reaction mixture was reduced under reduced pressure and 2.5 L EtOAc was added.

[0210] The mixture was filtered and reduced under reduced pressure. Purification by dry flash chromatography (DFC) on SiO.sub.2 using a gradient of EtOAc in heptane gave the title compound.

[0211] Yield: 56.1 g, 210.1 mmol, 32%. MS (ESI, pos): 234.1[M+Na].sup.+, 445.1 [2M+Na].sup.+

Example 1 j

Synthesis of Ethyl 3-(benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate

[0212] ##STR00025##

[0213] {ethyl 3-methoxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylate} (5.93 g, 28.1 mmol) was dissolved in 80 mL dichlormethane (DCM) at −78° C. and BBr.sub.3 (5.3 mL, 56.2 mmol) dissolved in 20 mL DCM was added drop wise. The reaction mixture was stirred for 1 hour at −78° C. before heating the reaction to 0° C. The reaction was quenched by drop wise addition of 25 mL tert-butyl methyl ether (tert-BuOMe) and 25 mL MeOH. The volatiles were removed in vacuo. The residue was dissolved in 90 mL DCM and 10 mL MeOH and filtered through a short pad of SiO.sub.2. The pad was washed with 200 mL 10% MeOH in DCM. The volatiles were removed in vacuo. The residue was dissolved in 400 mL acetone. K.sub.2CO.sub.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 at reflux overnight. The reaction mixture was diluted with 200 mL EtOAc and washed with 3×50 mL water and 50 mL brine. The combined aqueous phases were extracted with 2×50 mL EtOAc. The combined organic phases were dried (Na.sub.2SO.sub.4), filtered, and the volatiles were removed in vacuo and purified by dry flash chromatography on SiO.sub.2 using EtOAc (40-70%) in heptanes as the eluent to give the title compound.

[0214] Yield: 5.21 g, 18.1 mmol, 65%. MS (ESI, pos): 310.2[M+Na].sup.+, 597.4 [2M+Na].sup.+

Example 1 k

Synthesis of 3-(Benzyloxy)-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxylic acid

[0215] ##STR00026##

[0216] {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 (5M, aq) was added. The reaction mixture was stirred for 2 hours at room temperature before the reaction mixture was concentrated to approximately ⅓ in vacuo. The residue was diluted with 150 mL water and acidified to pH 2 using hydrogen chloride (HCl) (5M, aq). The precipitate was filtered and dried in vacuo, giving the title compound as a colorless solid. Yield: 22.52 g, 86.9 mmol, 89%.

Example 1 l

Synthesis of 3-(Benzyloxy)-1-methyl-4-(2-thioxothiazolidine-3-carbonyl)pyridine-2(1H)-one (AGC0021)

[0217] ##STR00027##

[0218] {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-thiazoline-2-thiol (1.94 g, 16.3 mmol) was dissolved in 50 mL DCM. N,N′-Dicyclohexylcarbodiimide (DCC) (3.36 g, 16.3 mmol) was added. The reaction mixture was stirred over night. The reaction was filtered, the solids washed with DCM and the filtrate was reduced in vacuo. The resulting yellow solid was recrystallized from isopropanol/DCM, giving AGC0021. Yield: 4.65 g, 12.9 mmol, 87%. MS(ESI, pos): 383[M+Na].sup.+, 743 [2M+Na].sup.+

Example 1 m

Synthesis of AGC0023

[0219] ##STR00028##

[0220] AGC0020 (8.98 g; 23.5 mmol) was dissolved in CH.sub.2Cl.sub.2 (600 mL). AGC0021 (37.43 g; 103.8 mmol) was added. The reaction was stirred for 20 hours at room temperature. The reaction mixture was concentrated under reduced pressure.

[0221] DFC on SiO.sub.2 using a gradient of methanol in a 1:1 mixture of EtOAc and CH.sub.2Cl.sub.2 yielded AGC0023 as a solid foam.

[0222] Average yield: 26.95 g, 20.0 mmol, 85%.

Example 1 n

Synthesis of AGC0024

[0223] ##STR00029##

[0224] AGC0023 (26.95 g; 20.0 mmol) was dissolved in ethanol (EtOH) (675 mL). Iron (20.76 g; 0.37 mol) and NH.sub.4Cl (26.99 g; 0.50 mol) were added, followed by water (67 mL). The reaction mixture was stirred at 70° C. for two hours. More iron (6.75 g; 121 mmol) was added, and the reaction mixture was stirred for one hour at 74° C. More iron (6.76 g; 121 mmol) was added, and the reaction mixture was stirred for one hour at 74° C. The reaction mixture was cooled before the reaction mixture was reduced under reduced pressure.

[0225] DFC on SiO.sub.2 using a gradient of methanol in CH.sub.2Cl.sub.2 yielded AGC0024 as a solid foam.

[0226] Yield 18.64 g, 14.2 mmol, 71%.

Example 1 o

Synthesis of AGC0025

[0227] ##STR00030##

[0228] AGC0024 (18.64 g; 14.2 mmol) was dissolved in CH.sub.2Cl.sub.2 (750 mL) and cooled to 0° C. BBr.sub.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) while stirring at 0° C. The volatiles were removed under reduced pressure. HCl (1.25M in EtOH, 320 mL) was added to the residue. The flask was then spun using a rotary evaporator at atmospheric pressure and ambient temperature for 15 minutes before the volatiles were removed under reduced pressure.

[0229] DFC on non-endcapped C.sub.18 silica using a gradient of acetonitrile (ACN) in water yielded AGC0025 as a slightly orange glassy solid.

[0230] Yield 13.27 g, 13.9 mmol, 98%.

Example 1 p

Synthesis of AGC0019

[0231] ##STR00031##

[0232] 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 reduced under reduced pressure. DFC on non-endcapped 018 silica using a gradient of ACN in water yielded a greenish glassy solid.

[0233] The solid was dissolved in MeOH (62 mL) and water (10.6 mL) at 40° C. The solution was added drop wise to EtOAc (750 mL) under sonication. The precipitate was filtered, washed with EtOAc and dried under reduced pressure, giving AGC0019 as an off-white solid with a greenish tinge.

[0234] Yield: 9.20 g, 8.7 mmol, 78%. H-NMR (400 MHz, DMSO-d.sub.6), .sup.13C-NMR (100 MHz, DMSO-d.sub.6).

EXAMPLE 2

Isolation of Pure Thorium-227

[0235] Thorium-227 is isolated from an actinium-227 generator. Actinium-227 was produced through thermal neutron irradiation of Radium-226 followed by the decay of Radium-227 (t½=42.2 m) to Actinium-227. Thorium-227 was selectively retained from an Actinium-227 decay mixture in 8 M HNO.sub.3 solution by anion exchange chromatography. A column of 2 mm internal diameter, length 30 mm, containing 70 mg of AG® 1-X8 resin (200-400 mesh, nitrate form) was used. After Actinium-227, Radium-223 and daughters had eluted from the column, Thorium-227 was extracted from the column with 12 M HCl. The eluate containing Thorium-227 was evaporated to dryness and the residue resuspended in 0.01 M HCl prior to labelling step.

EXAMPLE 3

Cytotoxicity of .SUP.227.Th-AGC1118 Against Ramos

Example 3 a

Generation of the Anti-CD22 Monoclonal Antibody (AGC1100)

[0236] The sequence of the monoclonal antibody (mAb) hLL2, also called epratuzumab, here denoted AGC1100, was constructed as described in Leung, Goldenberg, Dion, Pellegrini, Shevitz, Shih, and Hansen: Molecular Immunology 32: 1413-27, 1995.

[0237] The mAb used in the current examples was produced by Immunomedics Inc, New Jersey, USA. Production of this mAb could for example be done in Chinese hamster ovarian suspension (CHO—S) cells, transfected with a plasmid encoding the genes encoding the light and the heavy chain. First stable clones would be selected for using standard procedures. Following approximately 14 days in a single-use bioreactor, the monoclonal antibody may be harvested after filtration of the supernatant. AGC1100 would be further purified by protein A affinity chromatography (MabSelect SuRe, Atoll, Weingarten/Germany), followed by an ion exchange step. A third purification step based on electrostatic and hydrophobicity could be used to remove aggregates and potentially remaining impurities. The identity of AGC1100 would be confirmed by isoelectric focusing, SDS-PAGE analysis, N-terminal sequencing and LC/MS analysis. Sample purity would be further analyzed by size-exclusion chromatography (SEC).

Example 3 b) Coupling of mAb AGC1100 (Epratuzumab) with the Chelator AGC0019 (Compound of Formula (VIII)) to Give Conjugate AGC1118

[0238] ##STR00032##

[0239] Prior to conjugation, phosphate buffer pH 7.5 was added to the antibody solution (AGC1100) to increase the buffering capacity of the solution. The amount of AGC1100 (mAb) in the vessel was determined.

[0240] The 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.

[0241] A 1/1/3 molar equivalent solution of chelator/N-hydroxysuccinimide (NHS)/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was prepared to activate the chelator. For conjugation to the antibody a molar ratio of 7.5/7.5/22.5/1 (chelator/NHS/EDC/mAb) of the activated chelator was charged to mAb. After 20-40 minutes, the conjugation reaction was quenched with 12% v/v 0.3M Citric acid to adjust pH to 5.5.

[0242] The solution was then buffer exchanged into 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 diafiltration the solution was discharged to a formulation container. The product was formulated with 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 to obtain 2.6 mg/mL AGC1118 in 30 mM citrate, 70 mM NaCl, 2 mM EDTA, 0.5 mg/mL pABA 0.1% w/v PS80, pH 5.5. Finally, the solution was filtered through a 0.2 μm filter into sterile bottles prior to storage.

Example 3 c

Preparation of a Dose on .SUP.227.Th-AGC1118 Injection

[0243] A vial of 20 MBq thorium-227 chloride film was dissolved in 2 ml 8M HNO.sub.3 solution and left for 15 minutes before withdrawing the solution for application to an anion exchange column for removal of radium-223 that had grown in over time. The column was washed with 3 ml 8M HNO.sub.3 and 1 ml water prior to elution of thorium-227 with 3 ml 3M HCl. The eluted activity of thorium-227 was measured and a dose of 10 MBq transferred to an empty 10 ml glass vial. The acid was then evaporated using a vacuum pump and having the vial in a heating block (set to 120° C.) for 30-60 minutes. After reaching room temperature, 6 ml AGC1118 conjugate 2.5 mg/ml was added for radiolabelling. The vial was gently mixed and left for 15 minutes at room temperature. The solution was then sterile filtered into a sterile vial and sample withdrawn for iTLC analysis to determine RCP before use.

Example 3 d

Cytotoxicity of .SUP.227.Th-AGC1118 Against Ramos with Different Total Activity and Specific Activity

[0244] In this study doses of .sup.227Th-AGC1118 were tested by varying total activity and specific activity with 4 hours incubation time. This study was run in a 96 well plate format at specific activity at 10/50 kBq/μg and total activity at 5, 10, 20 and 40 kBq/ml. Ramos cells were cultured in RPMI1640-medium with 10% FBS and 1% Pencillin/Streptomycin (Passage 22). Cells were transferred to a centrifuge tube and centrifuged at 300 G for 5 minutes and suspend in 5 mL medium before counting on 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/well) in a 96 well plate (80.000 cells/well). CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used for measuring cell viability. See FIG. 4.

EXAMPLE 4

Cytotoxicity of .SUP.227.Th-AGC0718 Against HL-60

Example 4 a

Generation of the Anti-CD33 Monoclonal Antibody (AGC0700)

[0245] The sequence of the monoclonal antibody (mAb) HuM195/Iintuzumab, here denoted as AGC0700, was retrieved from the literature as described in (1) and (2). Manufacturing of AGC0700 was conducted at the facilities of CobraBiologics (Södertälje, Sweden). Briefly, the amino acid sequences of heavy- and light-chains were back-translated into DNA sequence using Vector NTI® Software (Invitrogen/Life-Technologies Ltd., Paisley, United Kingdom). The codon for the C-terminal lysine (Lys) was omitted from the IgG1 heavy chain gene to facilitate precise determination of the conjugate to antibody ratio (CAR) as outlined in Example 2. The resulting DNA sequence was codon optimized for expression in mammalian cells and synthesized by GeneArt (GeneArt/Life-Technologies Ltd., Paisley, United Kingdom) and further cloned into an expression vector by CobraBiologics (Södertälje, Sweden). Chinese hamster ovarian suspension (CHO—S) cells were stably transfected with the plasmid encoding the V.sub.H- and V.sub.L-domains of AGC0700 and grown in presence of standard CD-CHO medium (Invitrogen/Life-Technologies Ltd., Paisley, United Kingdom), supplemented with puromycin (12.5 mg/I; Sigma Aldrich). Stable clones, expressing AGC0700, were selected via limiting dilution over 25 generations. Clone stability was assessed by measuring protein titers from supernatants. A cell bank of the most stable clone was established and cryo-preserved.

[0246] Expression of the mAb was carried out at 37° C. for approximately 14 days in a single-use bioreactor at a 250 L scale. The monoclonal antibody was harvested after filtration of the supernatant. AC0700 was further purified via a protein A affinity column (MabSelect SuRe, Atoll, Weingarten/Germany), followed by one anion (QFF-Sepharose; GE Healthcare)—and a cation (PorosXS; Invitrogen/Life-Technologies Ltd.)—exchange chromatography to increase purity and final yield. The identity of AGC0700 was confirmed by isoelectric focusing and SDS-PAGE analysis. Activity of purified AGC0700 was analyzed in a binding ELISA to immobilized CD33-Fc target (Novoprotein). Sample purity was analyzed by size-exclusion chromatography (SEC).

REFERENCES

[0247] (1) Scheinberg D A. “Therapeutic uses of the hypervariable region of monoclonal antibody M195 and constructs thereof. U.S. Pat. No. 6,007,814 (1999 Dec. 28). [0248] (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 Chelator AGC0019 (Compound of Formula (VIII)) to Give Conjugate AGC0718

[0249] Conjugations were performed as described in example 3 with minor exceptions.

[0250] Prior to conjugation, phosphate buffer pH 7.5 was added to the antibody solution (AGC0700) to increase the buffering capacity of the solution. The amount of AGC0700 (mAb) in the vessel was determined.

[0251] The 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.

[0252] A 1/1/3 molar equivalent solution of chelator/NHS/EDC was prepared to activate the chelator. For conjugation to the antibody a molar ratio of 20/20/60/1 (chelator/NHS/EDC/mAb) of the activated chelator was charged to mAb. After 40-60 minutes, the conjugation reaction was quenched with 12% v/v 0.3M Citric acid to adjust pH to 5.5.

[0253] The solution was then buffer exchanged into 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 diafiltration the solution was discharged to a formulation container. The product was formulated with TFF buffer (30 mM Citrate, 154 mM NaCl, 2 mM EDTA, 2 mg/ml pABA, pH 5.5) to obtain 2.5 mg/mL AGC0718 in 30 mM citrate, 154 mM NaCl, 2 mM EDTA, 2 mg/mL pABA, pH 5.5. Finally, the solution was filtered through a 0.2 μm filter into sterile bottles prior to storage.

Example 4 c

Preparation of a Dose on .SUP.227.Th-AGC0718 Injection

[0254] A vial of 20 MBq thorium-227 chloride film was dissolved in 2 ml 8M HNO3 solution and left for 15 minutes before withdrawing the solution for application to an anion exchange column for removal of radium-223 that had grown in over time. The column was washed with 3 ml 8M HNO3 and 1 ml water prior to elution of thorium-227 with 3 ml 3M HCl. The eluted activity of thorium-227 was measured and a dose of 10 MBq transferred to an empty 10 ml glass vial. The acid was then evaporated using a vacuum pump and having the vial in a heating block (set to 120° C.) for 30-60 minutes. After reaching room temperature, 6 ml AGC0718 conjugate 2.5 mg/ml was added for radiolabelling. The vial was gently mixed and left for 15 minutes at room temperature. The solution was then sterile filtered into a sterile vial and sample withdrawn for iTLC analysis to determine RCP before use.

Example 4 d

Cytotoxicity of .SUP.227.Th-AGC0718 Against HL-60 with Different Total Activity

[0255] To demonstrate cell toxicity of .sup.227Th-AGC0718 after binding to CD33+-cells, in vitro cell toxicity assays were performed. For this purpose, the human myelogenic leukemic HL-60 cell line, as well as a CD33-negative B-cell line (Ramos), were exposed to .sup.227Th-AGC0718. Total activities of 2 and 20 kBq/ml were tested at a specific activity of 44 kBq/μg. All experimental procedures are described in RD2013.093. Briefly, 50 000 human HL-60 cells/ml in IMDM-medium were prepared with 10% FBS and 1% Penicillin/Streptomycin and seeded at a density of 100.000 cells/well in a 24 well plate. Cells were incubated for 4 h at 37° C. with activities of 0 to 20 kBq/ml of .sup.227Th-AGC0718. A respective .sup.227Th-isotype control conjugate sample as well as ab unlabelled AGC0718 sample were prepared in parallel as respective controls. Cells were washed afterwards with fresh medium and seeded into a new 24-well culture plate.

[0256] At different time points, cells were harvested and the viability was measured using the CellTiterGlo kit (Promega). The viability was expressed in % by setting the positive control (untreated cells) to 100%. See FIG. 5.

EXAMPLE 5

Cytotoxicity of .SUP.227.Th-AGC0118 Against SKOV-3

Example 5 a

Generation of AGC0100 (Trastuzumab)

[0257] Trastuzumab monoclonal antibody (here denoted as AGC0100) was purchased from Roche and dissolved to a concentration of 10 mg/ml in PBS (Dulbecco BIOCHROM).

Example 5 b

Coupling of mAb AGC0100 (Trastuzumab) with the Chelator AGC0019 (Compound of Formula (VIII)) to Give Conjugate AGC0118

[0258] Conjugations were performed as described in example 3 with minor modifications and. TFF purification of final conjugated mAb was replaced by gelfiltration column chromatography.

[0259] 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 in example 3 b). The molar ratio of chelator/NHS/EDC during activation was 1/1/3. A molar ratio of 8/8/25/1 corresponding to chelator/NHS/EDC/mAb and 30-40 min conjugation time, resulted in a CAR (chelator to antibody ratio) of 0.7-0.9 for conjugated AGC0118. The reaction was quenched by the addition of 12% v/v 0.3M citric acid to final pH of 5.5.

[0260] Purification and buffer exchange of AGC0118 conjugates into 30 mM Citrate pH 5.5, 154 mM NaCl were performed by gelfiltration on a Superdex 200 (GE Healthcare) column connected to an AKTA system (GE Healthcare). The protein concentration at Abs 280 nm was measured before the product was formulated with buffer (to obtain 2.5 mg/mL AGC0118 in 30 mM citrate, 154 mM NaCl, 2 mM EDTA, 2 mg/mL pABA, pH 5.5). Finally, the solution was filtered through a 0.2 μm filter into sterile bottles prior to storage.

Example 5 c

Preparation of a Dose on .SUP.227.Th-AGC0118 Injection

Labelling was Performed as Previously Described:

[0261] A vial of 20 MBq thorium-227 chloride film was dissolved in 2 ml 8M HNO3 solution and left for 15 minutes before withdrawing the solution for application to an anion exchange column for removal of radium-223 that had grown in over time. The column was washed with 3 ml 8M HNO3 and 1 ml water prior to elution of thorium-227 with 3 ml 3M HCl. The eluted activity of thorium-227 was measured and a dose of 10 MBq transferred to an empty 10 ml glass vial. The acid was then evaporated using a vacuum pump and having the vial in a heating block (set to 120° C.) for 30-60 minutes. After reaching room temperature, 6 ml AGC0118 conjugate 2.5 mg/ml was added for radiolabelling. The vial was gently mixed and left for 15 minutes at room temperature. The solution was then sterile filtered into a sterile vial and sample withdrawn for iTLC analysis to determine RCP before use.

Example 5 d

Cytotoxicity of .SUP.227.Th-AGC0118 Against SKOV-3 with Different Total Activity

[0262] Cell cytotoxicity was tested to various doses of .sup.227Th-AGC0118 by varying the total activity added to wells during 4 hours incubation time. SKOV-3 cells were seeded 10000 per well in a 96 well plate the day before experiment. A series of total activities 5, 10, 20 and 40 kBq/ml of chelated .sup.227Th-AGC0118, at specific activity 20 kBq/μg, were added to the cells at day 1. Remaining non-bound .sup.227Th-AGC0118 were removed by multi array pipette, followed by one additional wash with medium and subsequently fresh culture medium, after the end of incubation period. SKOV-3 cells were cultured in Mc-Coy medium with 10% FBS and 1% Penicillin/Streptomycin. Serum-free medium replaced the culture medium during the incubation with .sup.227Th-AGC0118. At day four the CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used for measuring cell viability. See FIG. 6.

EXAMPLE 6

Cytotoxicity of .SUP.227.Th-AGC2518 Against NCI-H716

Example 6 a

Generation of the FGFR2 Monoclonal Antibody (BAY1179470; AGC2500)

[0263] The generation of the monoclonal antibody BAY 1179470, here further referred to AGC2500, is described in detail in WO2013076186A1. Briefly, the antibody was retrieved upon biopanning on FGFR2 antigen. The resulting human IgG1 antibody was expressed in CHO cells and purified using a protein A affinity column (MAb Select Sure), followed by size-exclusion chromatography to isolate monomeric fractions. The antibody was formulated into PBS, pH 7.4. Analytical SEC demonstrated homogeneity >99%.

Example 6 b

Coupling of mAb AGC2500 with the Chelator AGC0019 (Compound of Formula (VIII)) to Give Conjugate AGC2518

[0264] The antibody-containing solution was adjusted to pH 7.5. The 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 chelator/NHS/EDC was prepared to activate the chelator. For conjugation to the antibody a molar ratio of 10/10/30/1 (chelator/NHS/EDC/mAb) of the activated chelator was charged to mAb. After 30 minutes, the conjugation reaction was quenched with 12% v/v 0.3M Citric acid to adjust pH to 5.5. The reaction sample was further loaded on to a HiLoad 16/600 Superdex 200 (prep-grade) column to isolate monomeric fractions with 30 mM Citrate, 70 mM NaCl, pH 5.5 as mobile phase. At the end of the chromatography, the antibody conjugate AGC2518 was concentrated to 2.5 mg/ml in 30 mM Citrate, 70 mM NaCl, 2 mM EDTA and 0.5 mg/ml pABA. All procedures are described in RD.2014.092, Journal No. 211/149, 140619 AEF.

Example 6 c

Preparation of a Dose on .SUP.227.Th-AGC2518 Injection

[0265] A vial of 20 MBq thorium-227 chloride film was dissolved in 2 ml 8M HNO3 solution and left for 15 minutes before withdrawing the solution for application to an anion exchange column for removal of radium-223 that had grown in over time. The column was washed with 3 ml 8M HNO3 and 1 ml water prior to elution of thorium-227 with 3 ml 3M HCl. The eluted activity of thorium-227 was measured and a dose of 10 MBq transferred to an empty 10 ml glass vial. The acid was then evaporated using a vacuum pump and having the vial in a heating block (set to 120° C.) for 30-60 minutes. After reaching room temperature, 6 ml AGC2518 conjugate 2.5 mg/ml was added for radiolabelling. The vial was gently mixed and left for 15 minutes at room temperature. The solution was then sterile filtered into a sterile vial and sample withdrawn for iTLC analysis to determine RCP before use.

Example 6 d

Cytotoxicity of .SUP.227.Th-AGC2518 Against NCI-H716 Cells with Different Total Activities

[0266] To demonstrate cell toxicity of .sup.227Th-AGC2518 after binding to FGFR2+-cells, in vitro cell toxicity assays were performed. For this purpose, the human colorectal cancer cell line NCI-H716 was exposed to .sup.227Th-AGC2518. Total activities of 2, 10, 20 and 40 kBq/ml were tested at a specific activity of 2 kBq/μg. An unrelated isotype control was prepared similar in parallel. All experimental procedures are described in RD2014.138. Briefly, 400 000 human NCI-H716 cells/ml in RPMI 1640-medium were prepared with 10% FBS and 1% Penicillin/Streptomycin and seeded at a density of 80.000 cells/well in a 96 well plate. Cells were incubated for 30 min at 37° C. with activities of 0 to 40 kBq/ml of .sup.227Th-AGC2518 and a respective .sup.227Th-isotype control conjugate sample. Cells were washed afterwards with fresh medium and seeded into a new 96-well culture plate. After 5 and 7 days, cells were harvested and the viability was measured using the CellTiterGlo kit (Promega). The viability was expressed in % by setting the positive control (untreated cells) to 100%. See FIG. 7.

EXAMPLE 7

Cytotoxicity of .SUP.227.Th-AGC2418 Against HT29 Cells

Example 7 a

Generation of the Mesothelin Monoclonal Antibody (BAY 86-1903; AGC2400)

[0267] The generation of the monoclonal antibody BAY 86-1903, here further referred to AGC2400, is described in detail in WO2009068204. Briefly, the antibody was retrieved upon biopanning on Mesothelin antigen. The resulting human IgG1 antibody was expressed in CHO cells and purified using a protein A affinity column (MAb Select Sure), followed by aggregate removal using a HIC column (Toyopearl Butyl 600M). The antibody was formulated into PBS, pH 7.5.

Example 7 b

Coupling of mAb AGC2400 with the Chelator AGC0019 (Compound of Formula (VIII)) to Give Conjugate AGC2418

[0268] The antibody-containing solution was adjusted to pH 7.5. The 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 chelator/NHS/EDC was prepared to activate the chelator. For conjugation to the antibody a molar ratio of 16.5/16.5/49.5/1 (chelator/NHS/EDC/mAb) of the activated chelator was charged to mAb. After 30 minutes, the conjugation reaction was quenched with 12% v/v 0.3M Citric acid to adjust pH to 5.5. The reaction sample was further loaded on to a HiLoad 16/600 Superdex 200 (prep-grade) column to isolate monomeric fractions with 30 mM Citrate, 70 mM NaCl, pH 5.5 as mobile phase. At the end of the chromatography, the antibody conjugate AGC2418 was concentrated to 2.5 mg/ml in 30 mM Citrate, 70 mM NaCl, 2 mM EDTA and 0.5 mg/ml pABA. All procedures are described in RD.2014.111, Journal No. 211/160, 140814 AEF.

Example 7 c

Preparation of a Dose on .SUP.227.Th-AGC2418

[0269] A vial of 20 MBq thorium-227 chloride film was dissolved in 2 ml 8M HNO3 solution and left for 15 minutes before withdrawing the solution for application to an anion exchange column for removal of radium-223 that had grown in over time. The column was washed with 3 ml 8M HNO3 and 1 ml water prior to elution of thorium-227 with 3 ml 3M HCl. The eluted activity of thorium-227 was measured and a dose of 10 MBq transferred to an empty 10 ml glass vial. The acid was then evaporated using a vacuum pump and having the vial in a heating block (set to 120° C.) for 30-60 minutes. After reaching room temperature, 6 ml AGC2418 conjugate 2.5 mg/ml was added for radiolabelling. The vial was gently mixed and left for 15 minutes at room temperature. The solution was then sterile filtered into a sterile vial and sample withdrawn for iTLC analysis to determine RCP before use.

Example 7 d

Cytotoxicity of .SUP.227.Th-AGC2418 Against HT29 Cells, Overexpressing Mesothelin Antigen, with Different Total Activities

[0270] To demonstrate cell toxicity of .sup.227Th-AGC2418 after binding to Mesothelin+-cells, in vitro cell toxicity assays were performed. For this purpose, the human colorectal cancer cell line HT29, transfected with the Mesothelin antigen, was exposed to .sup.227Th-AGC2418. Total activities were titrated over 12 points in a threefold dilution, starting at 5 kBq/ml at a specific activity of 10 kBq/μg. An unrelated isotype control was prepared similar in parallel. All experimental procedures are described in RD2014.154. Briefly, 200 000 human HT29 cells, transfected with Mesothelin antigen, cells/ml in RPMI 1640-medium were prepared with 10% FBS, 1% Penicillin/Streptomycin, 1% NaHCO3, 600 μg/ml Hygromycin B and seeded at a density of 40.000 cells/well in a 96 well plate. Cells were incubated for 6 days at 37° C. with activities of 0 to 40 kBq/ml of .sup.227Th-AGC2418 and a respective .sup.227Th-isotype control conjugate sample. At Day 6, cells were harvested and the viability was measured using the CellTiterGlo kit (Promega). The viability was expressed in % by setting the positive control (untreated cells) to 100%.

EXAMPLE 8

Comparison of Stability of Amide and Isothiocyanate-Linked Conjugates

[0271] AGC1118 and the corresponding conjugate having an isothiocyanate coupling moiety (AGC1115) were stored in aqueous solution at 40° C. for 11 days. Samples were taken periodically.

TABLE-US-00004 40° C. samples normalized to each 4° C. sampling point AGC1118 AGC1115 CAR (% norm) CAR (% norm) Day 0 100 100 Day 5 105 92 Day 11 103 88

[0272] It can be seen from the above table that no measurable decrease in conjugate concentration was seen for the amide-coupled conjugate. In contrast, the isothiocyanate conjugate decreased by 8% after 5 days and by 12% after 11 days.

EXAMPLE 9

Example 9 a

Generation of the PSMA Monoclonal Antibody (AGC1000)

[0273] The PSMA monoclonal antibody, hereinafter referred to as AGC1000, was purchased from Progenics, USA.

Example 9 b

Coupling of mAb AGC1000 with the Chelator AGC0019 (Compound of Formula (VIII)) to Give Conjugate AGC1018

[0274] The antibody-containing solution was adjusted to pH 7.5. The chelator AGC0019 was dissolved in 1:1, DMA:0.1M MES buffer pH 5.5. NHS and EDC were dissolved in 0.1M MES buffer pH 5.5. A 1/1/2 molar equivalent solution of chelator/NHS/EDC was prepared to activate the chelator. For conjugation to the antibody a molar ratio of 20/20/40/1 (chelator/NHS/EDC/mAb) of the activated chelator was charged to mAb in 4 portions with 10 minutes between each portion. After 50 minutes, the conjugation reaction was quenched with 12% v/v 1M TRIS pH 7.3. The conjugate was purified and buffer exchanged by tangential flow filtration (TFF). The formulation buffer was 30 mM Citrate, 70 mM NaCl, 2 mM EDTA, 0.5 mg/ml pABA, pH 5.5. At the end of diafiltration the solution was discharged to a bulk container and the concentration was adjusted to 2.7 mg/ml. Finally, the bulk solution was filtered through a 0.2 μm sterile filter and transferred to sterile vials for storage at −20° C.

Example 9 c

Preparation of a Dose of .SUP.227.Th-AGC1018 Injection

[0275] A vial of approx. 50 MBq Th-227 chloride film was dissolved in 2 ml 8M HNO3 solution and left for 15 minutes before withdrawing the solution for application to an anion exchange column for removal of radium-223 that had grown in over time. The column was washed with 3 ml 8M HNO.sub.3 and 1 ml water prior to elution of Th-227 with 3 ml 3M HCl. The HCl eluate was evaporated using a vacuum pump and a heating block set to 100° C. for 60-90 minutes. The activity of the dried Th-227 was measured in a dose calibrator. The dry Th-227 was dissolved in 0.05M HCl to give a concentration of 0.5 MBq/μl. For radiolabelling, the conjugate AGC1018 was diluted in formulation buffer in order to achieve 25 μg mAb in 200 μl. To the AGC1018 solution, 1 MBq Th-227 was mixed and the exact Th-227 activity measured on a Germanium detector. Chelation was allowed for 30-60 minutes at room temperature before sterile filtration into a sterile vial. A sample was withdrawn for iTLC analysis to determine RCP before use.

Example 9 d

Cytotoxicity of .SUP.227.Th-AGC1018 Against PSMA Expressing LNCaP Cells

[0276] To demonstrate cell toxicity of .sup.227Th-AGC1018 after binding to PSMA positive cells, in vitro cell toxicity assays were performed. For this purpose, the human prostate cancer cell line LNCaP was exposed to .sup.227Th-AGC1018. Total activities were titrated over 12 points in a threefold dilution, starting at 20 kBq/ml at a specific activity of 40 kBq/μg. An unrelated isotype control was prepared in parallel. All experimental procedures are described in archive RD.2015.101. Briefly, human LNCaP cells were cultured in RPMI 1640-medium supplemented with 10% FBS and 1% Penicillin/Streptomycin. Cells were seeded at a density of 2500 cells/well in a 96 well plate. 24 hours after seeding (Day 1), the cells were exposed to .sup.227Th-AGC1018 and .sup.227Th-isotype control at total activities ranging from 0 to 20 kBq/ml for 5 days at 37° C. At Day 6, cells were harvested and the viability was measured using the CellTiterGlo kit (Promega). The viability was expressed in % by setting the positive control (untreated cells) to 100%.