177-LU LABELED ACTIVE SITE INHIBITED FACTOR VII

Abstract

There is provided a 177-Lu labelled peptide for site-specific targeting of TF thereby enabling treatment of a cancer disease associated with high TF expression; e.g. treatment of cancer by administering to a patient an effective amount of the 177-Lu labelled peptide.

Claims

1.17. (canceled)

18. A therapeutic agent for targeted radionuclide therapy of Tissue Factor expressing cancers, the therapeutic agent comprising a 177-Lu labelled Tissue Factor binding peptide conjugate, wherein the peptide is an active site inhibited factor Vlia (FVIIai), and is coupled to 177-Lu by a chelating agent selected from the group consisting of DTPA, DOTA, NOTA, NODAGA, CB-TE2A, H4octapa, or p-SCN-Bn-H4octapa.

19. The therapeutic agent of claim 18, wherein the chelating agent is selected from the group consisting of DTPA and DOTA.

20. The therapeutic agent of claim 18, wherein the chelating agent is DTPA.

21. The therapeutic agent of claim 18 for use as a medicament.

22. The therapeutic agent of claim 18 for use in the treatment of cancer.

23. The therapeutic agent of claim 18 for use in the treatment of cancer selected from breast, gastric, esophageal, liver, colorectal and pancreatic cancer.

24. The therapeutic agent of claim 18, wherein the FVIIai comprises an amino acid sequence as set forth in SEQ ID NO: 1 with at least one amino acid unit thereof modified.

25. The therapeutic agent of claim 24, wherein the FVIIai comprises an amino acid sequence as set forth in SEQ ID NO: 1 with amino acid units Ser344, Asp242, and His193 thereof modified.

26. A method of treatment of a cancer disease associated with high TF expression, the method comprising administering to a patient a 177-Lu labelled Tissue Factor binding peptide conjugate, wherein the peptide is an active site inhibited factor Vlia (FVIIai), and is coupled to 177-Lu by a chelating agent selected from the group consisting of DTPA, DOTA, NOTA, NODAGA, CB-TE2A, H4octapa, or p-SCN-Bn-H4octapa.

27. The method according to claim 26, wherein the cancer disease is selected from breast cancer, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, and pancreatic cancer.

28. The method of claim 26, wherein the FVIIai comprises an amino acid sequence as set forth in SEQ ID NO: 1 with at least one amino acid unit thereof modified.

29. The method of claim 26, wherein the FVIIai comprises an amino acid sequence as set forth in SEQ ID NO: 1 with amino acid units Ser344, Asp242, and His193 thereof modified.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 shows .sup.177Lu-FVIIai biodistribution.

[0029] FIG. 2 shows 64Cu-FVIIai and 177Lu-FVIIai as a theranostic pair. The diagnostic agent (.sup.64Cu-NOTA-FVIIai) has a similar biodistribution, thus highlighting the potential of using .sup.64Cu-NOTA-FVIIai and .sup.177Lu-FVIIai as a theranostic pair.

[0030] FIG. 3 shows .sup.177Lu-FVIIai dose escalation.

[0031] FIG. 4 shows competition with non-labeled FVIIai. Blocking with FVIIai result in markedly reduced tumor accumulation measured ex vivo by biodistribution. The blocking in the tumors (arrows) is also evident on the SPECT/CT images on the right hand side.

[0032] FIG. 5 shows tumor growth delay. One treatment with 177Lu-FVIIai results in a tumor growth delay in an animal model with tissue factor positive tumors. Additional data is pending.

[0033] FIG. 6 is the sequence listing of SEQ ID NO: 1 (the amino acid sequence of native human coagulation Factor VII).

DETAILED DESCRIPTION OF THE INVENTION

[0034] The terms “variant” or “variants”, as used herein, is intended to designate human Factor VII having the sequence of SEQ ID NO: 1, wherein one or more amino acids of the parent protein have been substituted by another amino acid and/or wherein one or more amino acids of the parent protein have been deleted and/or wherein one or more amino acids have been inserted in protein and/or wherein one or more amino acids have been added to the parent protein. Such addition can take place either at the N-terminal end or at the C-terminal end of the parent protein or both. In one embodiment of the invention the variant has a total amont of amino acid substitutions and/or additions and/or deletions independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The activation of factor VII to factor VIIa, involves the hydrolysis of a single peptide bond between Arg152 and Ile153, resulting in a two-chain molecule consisting of a light chain of 152 amino acid residues and a heavy chain of 254 amino acid residues held together by a single disulfide bond.

[0035] Preferably, the FVIIa of SEQ ID NO: 1 is active site inhibited factor VIIa (FVIIai) and modified in such a way that it is catalytically inactive, such as having the amino acid modification comprised of Ser344, Asp242, and His193.

[0036] By “catalytically inactivated in the active site of the FVIIa polypeptide” is meant that a FVIIa inhibitor is bound to the FVIIa polypeptide and decreases or prevents the FVIIa catalyzed conversion of FX to FXa. A FVIIa inhibitor may be identified as a substance, which reduces the amidolytic activity by at least 50% at a concentration of the substance at 400 μM in the FVIIa amidolytic assay described by Persson et al. (Persson et al., J. Biol. Chem. 272: 19919-19924 (1997)). Preferred are substances reducing the amidolytic activity by at least 50% at a concentration of the substance at 300 μM; more preferred are substances reducing the amidolytic activity by at least 50% at a concentration of the substance at 200 μM.

[0037] The “FVIIa inhibitor” may be selected from any one of several groups of FVIIa directed inhibitors. Such inhibitors are broadly categorised for the purpose of the present invention into i) inhibitors which reversibly bind to FVIIa and are cleavable by FVIIa, ii) inhibitors which reversibly bind to FVIIa but cannot be cleaved, and iii) inhibitors which irreversibly bind to FVIIa. For a review of inhibitors of serine proteases see Proteinase Inhibitors (Research Monographs in cell and Tissue Physiology; v. 12) Elsevier Science Publishing Co., Inc., New York (1990).

[0038] The FVIIa inhibitor moiety may also be an irreversible FVIIa serine protease inhibitor. Such irreversible active site inhibitors generally form covalent bonds with the protease active site. Such irreversible inhibitors include, but are not limited to, general serine protease inhibitors such as peptide chloromethylketones (see, Williams et al., J. Biol. Chem. 264:7536-7540 (1989)) or peptidyl cloromethanes; azapeptides; acylating agents such as various guanidinobenzoate derivatives and the 3-alkoxy-4-chloroisocoumarins; sulphonyl fluorides such as phenylmethylsulphonylfluoride (PMSF); diisopropylfluorophosphate (DFP); tosylpropylchloromethyl ketone (TPCK); tosyllysylchloromethyl ketone (TLCK); nitrophenyl-sulphonates and related compounds; heterocyclic protease inhibitors such as isocoumarines, and coumarins.

[0039] Referring to FIG. 1 there is shown ex vivo biodistribution of 177Lu-DTPA-FVIIai performed at 1, 4, 24, 72 168 and 312 hours after intra venous administration of 2 MBq 177Lu-DTPA-FVIIai in NMRI nude mice bearing subcutaneous BxPC-3 pancreatic adenocarcinoma xenograft tumors. The uptake in the tumors was 1.16±0.04, 25 1.97±0.18, 1.95±0.07, 1.01±0.06, 0.31±0.02 percent injected dose per gram (%ID/g) at 1, 4, 24, 72 and 168 hours post-injection. The delivered radiation dose is estimated to 0.12 Gy/MBq resulting in a delivered dose of 1.82 Gy to the tumors for a 15MBq dose.

[0040] Referring to FIG. 2 there is shown a comparison of the ex vivo biodistribution of 30 177Lu-DTPA-FVIIai (top) and 64Cu-NOTA-FVIIai (bottom) shows a comparable biodistiribution. This illustrates the potential of using 64Cu-NOTA-FVIIai and 177Lu-DTPA-FVIIai as a theranostic pair for diagnostic and treatment of TF positive tumors.

[0041] Referring to FIG. 3 there is shown the result of a dose escalation study. No saturation of the delivered fraction of 177Lu-DTPA-FVIIai is observed when increasing the dose from 1 MBq to 30 MBq per animal. This illustrates the possibility of increasing the delivered radiation dose to the tumors by increasing the injected dose.

[0042] In FIG. 4 a competition study with pre-injection of an excess amount of unlabeled FVIIai was performed in mice bearing BxPC-3 tumors. The tumor uptake measured ex vivo (top) was significantly reduced in mice preinjected with FVIIai, compared to mice injected with 177Lu-DTPA-FVIIai only (2.5±0.16%ID/g to 1.7±0.05%ID/g; p<0.05). 177Lu-DTPA-FVIIai SPECT/CT imaging (bottom) of mice with (right) or without (left) pre-injection of excess FVIIai. The competiton with FVIIai visually reduced the tumor uptake. Together the ex vivo and in vivo data from the competition experiment confirm the TF mediated uptake of 177Lu-DTPA-FVIIai in the tumors.

[0043] The efficacy of tissue factor targeted radionuclide therapy using 177Lu-DTPA-FVliai is shown in FIG. 5. The effect of 177Lu-DTPA-FVIIai on the growth of subcutaneous BxPC-3 tumors was evaluated until day 8 in a pilot study (top). A significant inhibition of tumor growth was observed on day 8 (169±10% versus 270±21%; p=0.0002). The long term effect of a single injection of 15 MBq 177Lu-DTPA-FVIIai on BxPC-3 tumor growth was evaluated in a separate experiment (bottom). A significant inhibition of tumor growth was observed on day 19 (426±45% versus 614±49%; p=0.02).