RECEPTOR-BINDING DOMAINS LIGANDS FOR THE DETECTION, DIAGNOSIS AND TREATMENT OF PANCREATIC CANCER

Abstract

Disclosed are methods for diagnosing pancreatic cancer, including measuring the expression level of ASCT1 and/or ASCT2 and/or XPR1. Also disclosed is a RBD ligand coupled to at least one contrast agent, that may be used as a probe for medical imaging.

Claims

1-15. (canceled)

16. Method for the detection of pancreatic cancer cells comprising detecting the expression of a cell surface nutrient receptor selected from ASCT1, ASCT2 and/or XPR1.

17. The method according to claim 16, for the diagnosis or monitoring of pancreatic cancer.

18. The method according to claim 16, being an in vivo method.

19. The method according to claim 16, for the in vivo diagnosis or monitoring of pancreatic cancer by medical imaging.

20. The method according to claim 16, for the in vivo diagnosis or monitoring of pancreatic cancer by magnetic resonance imaging (MRI), X-ray-based imaging techniques, computed tomography (CT), radiography, positron-emission tomography (PET), single photon emission tomography (SPECT), endoscopic ultrasound (EUS), magnetic resonance cholangiopancreatography, fluorimetry, fluoroscopy, fluorescence, or near-infrared (NIR) fluorescent imaging.

21. The method according to claim 16, wherein said means is a receptor-binding domain (RBD) ligand selected from the group comprising HERV-W.RBD, RD114.RBD, BaEV.RBD, SNV.RBD, SRV.RBD, MPMV.RBD, Xeno.RBD, variants and fragments thereof.

22. The method according to claim 16, wherein said means is a receptor-binding domain (RBD) ligand coupled with at least one contrast agent.

23. The method according to claim 16, wherein said means is a receptor-binding domain (RBD) ligand coupled with at least one contrast agent selected from a radiolabeled agent or a fluorescent agent.

24. The method according to claim 16, being an in vitro method for detecting pancreatic cancer cells, wherein said method comprises measuring the expression level of at least one cell surface nutrient transporter selected from ASCT1, ASCT2 and/or XPR1.

25. The method according to claim 16, being an in vitro method for detecting pancreatic cancer cells, wherein said method comprises measuring the expression level of at least one cell surface nutrient transporter selected from ASCT1, ASCT2 and/or XPR1 and comparing the measured expression level with a reference expression level.

26. The method according to claim 16, being an in vitro method for detecting pancreatic cancer cells, wherein said method comprises measuring the expression level of at least one cell surface nutrient transporter selected from ASCT1, ASCT2 and/or XPR1, and wherein said expression level is assessed at the protein level.

27. The method according to claim 16, being an in vitro method for detecting pancreatic cancer cells, wherein said method comprises measuring the expression level of at least one cell surface nutrient transporter selected from ASCT1, ASCT2 and/or XPR1, and wherein said expression level is assessed by detecting and quantifying said at least one cell surface nutrient transporter on the cell surface by detecting and/or quantifying binding of a ligand to said cell surface nutrient transporter.

28. The method according to claim 16, being an in vitro method for detecting pancreatic cancer cells, wherein said method comprises measuring the expression level of at least one cell surface nutrient transporter selected from ASCT1, ASCT2 and/or XPR1, and wherein said expression level is assessed by detecting and quantifying binding of an antibody or of a receptor-binding domain ligand comprising a part or the totality of a receptor-binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus to said cell surface nutrient transporter.

29. The method according to claim 16, being an in vitro method for detecting pancreatic cancer cells, wherein said method comprises measuring the expression level of at least one cell surface nutrient transporter selected from ASCT1, ASCT2 and/or XPR1, and wherein said expression level is assessed by detecting and quantifying binding of a receptor-binding domain ligand comprising a part or the totality of a receptor-binding domain (RBD) derived from the soluble part of a glycoprotein of an enveloped virus to said cell surface nutrient transporter, wherein said RBD ligand is HERV-W.RBD, RD114.RBD, BaEV.RBD, SNV.RBD, SRV.RBD, MPMV.RBD, or Xeno.RBD, variants or fragments thereof.

30. A receptor-binding domain (RBD) ligand coupled with at least one contrast agent, wherein said RBD is selected from the group HERV-W.RBD, RD114.RBD, BaEV.RBD, SNV.RBD, SRV.RBD, MPMV.RBD, Xeno.RBD, variants and fragments thereof.

31. The RBD ligand according to claim 30, being a probe for medical imaging.

32. A method for the treatment of pancreatic cancer in a subject comprising the administration to the subject of at least one RBD ligand selected from the group comprising HERV-W.RBD, RD114.RBD, BaEV.RBD, SNV.RBD, SRV.RBD, MPMV.RBD, Xeno.RBD, variants and fragments thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0303] FIG. 1 is a set of graphs showing flow cytometry analysis following the incubation of H2.RBD.GFP with MiaPaca2, Panc1wt, BxPC3 and CFPAC cell lines.

[0304] FIG. 2 is a set of graphs showing flow cytometry analysis following the incubation of Xeno.RBD.RFC with MiaPaca2, Panc1wt, BxPC3 and CFPAC cell lines.

[0305] FIG. 3 is a set of graphs showing flow cytometry analysis following the incubation of HERV-W.RBD.MFC with MiaPaca2, Panc1wt, BxPC3 and CFPAC cell lines.

[0306] FIG. 4 is a graph showing the recognition in vivo of different pancreatic tumor cell lines by I-125.HERV-W.RBD.MFC.

[0307] FIG. 5 is a graph showing the recognition in vivo of different pancreatic tumor cell lines by I-125.Xeno.RBD.RFC.

[0308] FIG. 6 is a graph showing the recognition in vivo of different pancreatic tumor cell lines by I-125.H2.RBD.GFP.

[0309] FIG. 7 is a set of images showing the recognition in vivo of a mouse xenografted with human pancreatic tumors or not (control) by I-125.HERV-W.RBD.MFC.

[0310] FIG. 8 is a set of images showing the recognition in vivo of a mouse xenografted with human pancreatic tumors or not (control) by I-125.Xeno.RBD.RFC.

[0311] FIG. 9 is a set of images showing the recognition in vivo of a mouse xenografted with human pancreatic tumors or not (control) by I-125.H2.RBD.GFP.

EXAMPLES

[0312] The present invention is further illustrated by the following examples.

Example 1

Materials and Methods

[0313] MiaPaca2 and Panc1wt cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS), BxPC3 and Capan1 cells were grown in RPMI supplemented with 10% fetal bovine serum (FBS) and CFPAC cells were grown in IMDM supplemented with 10% fetal bovine serum (FBS). All were incubated at 37? C. in a 5% CO2-95% air atmosphere.

[0314] Cells were cultured two days before at the concentration of 1*10.sup.6 per 10 cm plate then the supernatant was discarded and the plate rinsed with 5 mL of PBS and trypsinated. The cells were then resuspended in 100 ?L of PBA (PBS+2% FCS). Cells were then plated at the concentration of 1*10.sup.5 cells/point.

[0315] At this point, the work was performed at 4? C. Cells were centrifuged for 3 minutes at 1200 rpm and resuspended in 100 ?L of a solution comprising a RBD ligand (Table 1). The RBD ligand is incubated with the cells for 30 minutes at 37? C. (some RBD can also function in vitro at lower temperatures, down to 4? C.). The cells were then centrifuged and rinsed with PBA, then resuspended in 100 ?L anti-mouse A647 or anti-rabbit A647 ( 1/500 in PBA) for flow cytometry analysis.

TABLE-US-00001 TABLE1 listsoftheRBDligandusedfortheinvitro experiments. RBD Transporter H2.RBD.GFP GLUT-1 (SEQIDNO:32fusedtoGFP) XENO.RBD.RFC(SEQIDNO:8) XPR1 HERV-W.RBD.MFC(SEQIDNO:7) ASCT-1/ASCT-2

Results

[0316] We tested the capacity of RBD ligands of the invention to specifically recognize pancreatic cell lines in vitro. As shown in FIGS. 2 and 3, Xeno.RBD and HERV-W.RBD efficiently detect the pancreatic cell lines MiaPaca2, Panc1wt, BxPC3 and CFPAC. RBD ligands of the invention also specifically detect the Capan1 cell line (data not shown). On the contrary, H2.RBD does not allow the detection of these cell lines in vitro (FIG. 1).

Example 2

[0317] Materials and Methods

[0318] Radiolabeling of the RBD Ligand

[0319] I-125 was obtained from Perkin Elmer, and RBDs ligands were radiolabeled at the specific activity of 370 MBq/mg for SPECT imaging, using the IODO-GEN (Pierce Chemical Co.) method as previously described (Santoro et al. 2009. J. Nucl. Med. 50:2033-2041). The marked ligand used for the in vivo study are HERV-W.RBD.MFC (SEQ ID NO: 7); Xeno.RBD.RFC (SEQ ID NO: 8); and H2.RBD.GFP (SEQ ID NO: 32) with I-125 (10 ?Ci/?g).

Animals

[0320] All animal experiments were performed in compliance with the guidelines of the French government and the standards of Institut National de la Sant? et de la Recherche M?dicale for experimental animal studies (agreement D34-172-27).

[0321] Mice (5-week-old athymic FoxN1 mice) were obtained from Charles River/Harlan Laboratories and were acclimated for 1 week before experimental use. They were housed at 22? C. and 55% humidity with a light-dark cycle of 12 hours. Food and water were available ad libitum. The mice were force-fed with Lugol solution the day before imaging, and stable iodine was added to drinking water for the entire experimental period. [0322] Group 1 (6 mice): left side: CFPAC (injection of 5.10.sup.6 cells)/right side: HPAC (injection of 5.10.sup.6 cells); [0323] Group 2 (6 mice): left side: CFPAC (injection of 5.10.sup.6 cells); [0324] Group 3 (6 mice): control group.

[0325] RBD is injected intravenously at 50 ?g/mouse (radioactivity injected=500 ?Ci/mouse).

[0326] During acquisitions mice were under anesthetic gas isoflurane 2.5%.

SPECT-CT Imaging

[0327] Whole-body SPECT/CT images were acquired at various times after tail vein injection of 18 MBq radiolabeled I-125.RBD. Mice were anesthetized with 2% isoflurane and positioned on the bed of 4-head multiplexing multipinhole NanoSPECT camera (Bioscan Inc., Washington, USA).

[0328] Energy window was centered at 28 keV with ?20% width, acquisition times were defined to obtain 30 000 counts for each projection with 24 projections. Images and maximum intensity projections (MIPs) were reconstructed using the dedicated software Invivoscope? (Bioscan, Inc., Washington, USA) and Mediso InterViewXP? (Mediso, Budapest Hungary). Concurrent microCT whole-body images were performed for anatomic co-registration with SPECT data. Reconstructed data from SPECT and CT were visualized and co-registered using Invivoscope?.

Acquisitions with NanoSPECT-CT (Mediso):

[0329] SPECT-CT imaging was carried out 5 weeks post graft; [0330] 4 h, 24 h, 48 h, 72 h after injection of I-125.H2.RBD.GFP; [0331] 24 h, 48 h, 72 h, 96 h after injection of I-125.HERV-W.RBD.MFC and I-125.Xeno.RBD.RFC.

[0332] Image analysis was led with VivoQuant software and measures done counts/mm.sup.3.

Results

[0333] The animals were selected with homogeneous tumors; however, depending on the tumor cell type, the tumors did not grow at the same speed and therefore do not have the same volume between each group of animals and between tumor cell types at imaging.

[0334] To overcome the different tumor volumes in image analysis, the measured values correspond to the radioactivity measured per unit volume of tumor tissue and is expressed as counts/mm.sup.3 of tumor volume. Thus, a comparison of the radioactivity associated with RBD is possible, according to various tumor cell types without having a bias induced by the different volumes of the tumors.

[0335] The measured values for the control animals correspond to the radioactivity per unit volume in the whole body of the animal (counts/mm3). Areas not taken into account in all animals for analysis are the bladder due to the natural elimination (this may vary over time depending on the animal) and the tail as it is the injection site (residues can persist). The noise background is the natural and surrounding radioactivity detectors that can take into account independently of a radioactive source. The values measured in the range of the noise background are not significant.

[0336] FIGS. 4-5 and 7-8 demonstrate the recognition in vivo of different kind of pancreatic tumor cells by I-125.HERV-W.RBD.MFC and I-125.Xeno.RBD.RFC respectively. On the contrary, as demonstrated by FIGS. 6 and 9, I-125.H2.RBD.GFP does not allow efficient recognition of pancreatic cancer cells in vivo.