Netrin-1 detection, companion test and therapy based on radiations

Abstract

The present invention is based on the finding that Netrin-(1) is retained in a stickier manner in the cell matrix at the cell periphery of the cancer cells, whereas Netrin-(1) is expressed in adults specifically in some tumors. It is also shown herein that Netrin-(1) is expressed very early during tumor formation. This makes Netrin-(1) an unexpected very specific target for imagery and/or targeted therapy. The present invention thus relates to compounds comprising an anti-Netrin-1 antibody, especially NP(137), a chelating moiety, optionally associated with a radioisotope, and their use either in imagery, diagnosis, especially companion diagnosis, or in targeted therapy. New diagnostic tests, which may be companion tests, and new cancer therapies, that may be combined to the companion test, are also proposed.

Claims

1.-17. (canceled)

18. A compound comprising: an anti-Netrin-1 antibody or antigen binding fragment thereof, and a chelating moiety bound to said antibody or fragment.

19. The compound of claim 18, wherein said chelating moiety is associated with a radioisotope.

20. The compound of claim 18, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising a variable domain VH comprising: a H-CDR1 having a sequence set forth as SEQ ID NO: 1; a H-CDR2 having a sequence set forth as SEQ ID NO: 2; a H-CDR3 having a sequence set forth as SEQ ID NO: 3; a variable domain VL comprising: a L-CDR1 having a sequence set forth as SEQ ID NO: 4; a L-CDR2 having a sequence YAS; a L-CDR3 having a sequence set forth as SEQ ID NO: 5; or a variable domain VH comprising: a H-CDR1 having a sequence set forth as SEQ ID NO: 22; a H-CDR2 having a sequence set forth as SEQ ID NO: 23; a H-CDR3 having a sequence set forth as SEQ ID NO: 24; a variable domain VL comprising: a L-CDR1 having a sequence set forth as SEQ ID NO: 25; a L-CDR2 having a sequence set forth as SEQ ID NO: 26; a L-CDR3 having a sequence set forth as SEQ ID NO: 5.

21. The compound of claim 18, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising a pair of VH and VL sequences selected from the following pairs: SEQ ID NO: 21 and 13, SEQ ID NO: 14 and 8, SEQ ID NO: 15 and 9, SEQ ID NO: 16 and 10, SEQ ID NO: 17 and 11, SEQ ID NO: 18 and 11, SEQ ID NO: 19 and 10, SEQ ID NO: 20 and 11, SEQ ID NO: 16 and 11, SEQ ID NO: 19 and 12, SEQ ID NO: 15 and 10.

22. The compound of claim 21, wherein the antibody further comprises a Human IgG1 Constant heavy chain (CH) and/or a Human IgG1 Constant light chain (CL).

23. The compound of claim 18, wherein the chelating moiety comprises NODAGA, NODAGA-NHS, DOTA, DOTA-NHS, p-SCN-Bn-NOTA, p-SCN-Bn-PCTA, p-SCN-Bn-oxo-DO3A, desferrioxamine-p-SCN, Diethylenetriamine Pentaacetic Acid (DTPA), or 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).

24. The compound of claim 19, wherein the radioisotope is .sup.68Ga, .sup.64Cu, .sup.89Zr, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.111In, .sup.99mTc, .sup.123I, .sup.177Lu, .sup.90Y, .sup.131I, .sup.213Bi, .sup.212Bi, .sup.211At or .sup.225Ac.

25. A method for imaging Netrin-1 presence or localization in a subject, comprising: a) administering to said subject a compound comprising: an anti-Netrin-1 antibody or antigen binding fragment thereof, a chelating moiety bound to said antibody or fragment, and a radioisotope associated to the chelating moiety, b) waiting from 4 to 172 h, obtaining binding of said compound by the netrin-1 sequestered in the extracellular matrix of the tumor; c) detecting or localizing said bound compound by in vivo imaging.

26. The method of claim 25, wherein localizing at step b) comprises highlighting the presence or accumulation of the compound in at least one body part.

27. The method of claim 26, wherein said body part is an organ or a tissue.

28. The method of claim 25, wherein the radioisotope is selected from the group consisting of .sup.68Ga, .sup.64Cu, .sup.89Zr, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.111In, .sup.99mTc, and .sup.123I.

29. A method of internal radiotherapy treatment of a netrin-1 expressing cancer in a patient having such cancer, comprising administering a sufficient amount of a compound comprising: an anti-Netrin-1 antibody or antigen binding fragment thereof, a chelating moiety bound to said antibody or fragment, and a radioisotope associated to the chelating moiety.

30. The method of claim 29, wherein the compound comprises a radioisotope selected from the group consisting of .sup.177Lu, .sup.90Y, .sup.131I, .sup.213Bi, .sup.212Bi, .sup.211At and .sup.225Ac.

31. A method of identifying and treating patients with a netrin-1 expressing cancer, comprising: a) administering to said subject a compound comprising: an anti-Netrin-1 antibody or antigen binding fragment thereof, a chelating moiety bound to said antibody or fragment, and a radioisotope associated to the chelating moiety; b) waiting from 4 to 172 h, obtaining binding of said compound by the netrin-1 sequestered in the extracellular matrix of the tumor; c) detecting said compound by in vivo imaging; d) visualizing localization of Netrin-1 presence or accumulation; e) treating said patient against the visualized cancer.

32. The method of claim 31 wherein the compound in a) comprises a radioisotope selected from the group consisting of .sup.68Ga, .sup.64Cu, .sup.89Zr, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.111In, .sup.99mTc, and .sup.123I.

33. The method of claim 31, wherein treating the patient in e) comprises administering to said patient an effective amount of a compound comprising: an anti-Netrin-1 antibody or antigen binding fragment thereof, a chelating moiety bound to said antibody or fragment, and a radioisotope associated to the chelating, wherein the radioisotope is selected from the group consisting of 1.sup.77Lu, .sup.90Y, .sup.131I, .sup.213Bi, .sup.212Bi, .sup.211At and .sup.225Ac.

34. The method of claim 31, wherein treating the patient in e) comprises administering to said patient an effective amount of a monoclonal antibody or an antigen-binding fragment thereof, comprising a variable domain VH comprising: a H-CDR1 having a sequence set forth as SEQ ID NO: 1; a H-CDR2 having a sequence set forth as SEQ ID NO: 2; a H-CDR3 having a sequence set forth as SEQ ID NO: 3; a variable domain VL comprising: a L-CDR1 having a sequence set forth as SEQ ID NO: 4; a L-CDR2 having a sequence YAS; a L-CDR3 having a sequence set forth as SEQ ID NO: 5; or a variable domain VH comprising: a H-CDR1 having a sequence set forth as SEQ ID NO: 22; a H-CDR2 having a sequence set forth as SEQ ID NO: 23; a H-CDR3 having a sequence set forth as SEQ ID NO: 24; a variable domain VL comprising: a L-CDR1 having a sequence set forth as SEQ ID NO: 25; a L-CDR2 having a sequence set forth as SEQ ID NO: 26; a L-CDR3 having a sequence set forth as SEQ ID NO: 5.

35. The method of claim 34, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising a pair of VH and VL sequences selected from the following pairs: SEQ ID NO: 21 and 13, SEQ ID NO: 14 and 8, SEQ ID NO: 15 and 9, SEQ ID NO: 16 and 10, SEQ ID NO: 17 and 11, SEQ ID NO: 18 and 11, SEQ ID NO: 19 and 10, SEQ ID NO: 20 and 11, SEQ ID NO: 16 and 11, SEQ ID NO: 19 and 12, SEQ ID NO: 15 and 10.

36. The method of claim 31, wherein in vivo imaging comprises PET or SPECT imaging.

37. A compound comprising: an anti-Netrin-1 antibody or antigen binding fragment thereof, and a chelating moiety bound to said antibody or fragment. wherein said chelating moiety is optionally associated with a radioisotope and wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof, comprising a variable domain VH comprising: a H-CDR1 having a sequence set forth as SEQ ID NO: 1; a H-CDR2 having a sequence set forth as SEQ ID NO: 2; a H-CDR3 having a sequence set forth as SEQ ID NO: 3; a variable domain VL comprising: a L-CDR1 having a sequence set forth as SEQ ID NO: 4; a L-CDR2 having a sequence YAS; a L-CDR3 having a sequence set forth as SEQ ID NO: 5; or a variable domain VH comprising: a H-CDR1 having a sequence set forth as SEQ ID NO: 22; a H-CDR2 having a sequence set forth as SEQ ID NO: 23; a H-CDR3 having a sequence set forth as SEQ ID NO: 24; a variable domain VL comprising: a L-CDR1 having a sequence set forth as SEQ ID NO: 25; a L-CDR2 having a sequence set forth as SEQ ID NO: 26; a L-CDR3 having a sequence set forth as SEQ ID NO: 5.

Description

[0164] The present invention will now be described in more detail using non-limiting examples referring to the drawing comprising:

[0165] FIG. 1: Netrin-1 binds and is retain within the extracellular matrix. Analysis of h-netrin-1 (human recombinant netrin-1) binding on extra cellular matrix components: recombinant mouse laminin (m-Laminin), recombinant human fibronectin (h-fibronectin) and recombinant human Vitronectin (h-Vitronectin) by bio-layer interferometry assays.

[0166] FIG. 2: Characterization of fragments conjugates. a. Chemical representation of DOTA (1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid)-HS ester and NODAGA (1,4,7-Triazacyclononane, 1-glutaric acid-5,7 acetic acid)-HS ester molecule used for chelating metals b. Representation of full anti-Netrin-1 (NP137), F(ab)2 and Fab conjugated to NODAGA or DOTA chelates. c. NP137), F(ab)2 and Fab conjugates have been produced by synthesis and enzymatic cleavage and subjected to electrophoresis in denaturing or non-denaturing conditions. d. Bio-layer interferometry analysis of NP137), F(ab)2 and Fab after chelating with NODAGA. Numbers indicates the concentration of NP137-NODAGA, F(ab)2-NODAGA and Fab-NODAGA.

[0167] FIG. 3: SPECT/Ct analysis and netrin-1 detection in tumors. a. Quantification of netrin-1 expression by Q-RT-PCR on 4T1 and 67NR cell lines. b. Maximum Intensity Projection of Tomographic scintigraphy and X-ray CT of the whole body of a Balb/c mouse bearing a 4T1 tumor (positive for Netrin-1), acquired from the left to the right at 4h, 24h; 48h and 72h after IV injection of NP137-NODAGA-.sup.111In. c. Maximum Intensity Projection of Tomographic scintigraphy and X-ray CT of the whole body of a Balb/c mouse bearing a 67NR (negative for Netrin-1) tumor, acquired from the left to the right at 24h; 48h and 72h after IV injection of NP137-NODAGA-.sup.111In.

[0168] FIG. 4: Measurement of radioactivity accumulation. Tumoral biodistribution ratio of a. .sup.111In-NODAGA-NP137-Fab, b .sup.111In-NODAGANP137-F(ab).sub.2 and c. -.sup.111In-NODAGA-NP137 in Balb/cJ mouse bearing 4T1 xenografts (positive for netrin-1) versus 67NR xenografts (negative for netrin-1) at 24h, 48h, 72h and 96h. Radioactivity incorporation is quantified by the percentage of the injected dose by gramme of tumor. d. Biodistribution properties of .sup.111In-NODAGA-NP137 in Balb/cJ mouse bearing 4T1 xenografts at 48h, 72h and 96h and measured for all organs. Radioactivity incorporation is quantified by the percentage of the injected dose by gram of organ.

[0169] FIG. 5: Measurement of radioactivity accumulation

[0170] a. Maximum Intensity Projection of Tomographic scintigraphy and X-ray CT of the whole body of a MMTV/neuT mouse tumor genetically modified to develop mammary tumors, acquired from the left to the right at 24h; 48h and 72h after injection of NP137-NODAGA-.sup.111In. b. Schematic representation and location of the 10 mammary glands in mice. c. Biodistribution properties of .sup.111In-NODAGA-NP137 in MMTV/NeuT mouse at 72h and measured for the tumors and all mice organs. Radioactivity incorporation is quantified by the percentage of the injected dose by gram of organ.

[0171] FIG. 6: A new anticancer therapy

[0172] a. Balb/cJ mice were engrafted with EMT6 cells by subcutaneous injection of 1 million cells. After 5 days, animals were treated by IV injection of PBS; DOTA-NP137 (anti-netrin1); DOTA-NP137-.sup.177Lu. n=9 animals/group for PBS and DOTA-NP137; n=12 animals/group for DOTA-NP137-.sup.177Lu; p<0.0001 between PBS and DOTA-NP137-.sup.177Lu and between DOTA-NP137 and DOTA-NP137-.sup.177Lu. b. DOTA-NP137-.sup.177Lu enhances mice survival engrafted with EMT6 cell line (see A). Kaplan-Meier survival curves analysis of mice survival treated or not with NP137. Mantel Cox test; n=9 animals/group for PBS and DOTA-NP137; n=12 animals/group for DOTA-NP137-.sup.177Lu; p<0.0001 between PBS and DOTA-NP137-.sup.177Lu and between DOTA-NP137 and DOTA-NP137-.sup.177Lu. c. Balb/c mice were engrafted with 4T1 cells by subcutaneous injection of 1 million cells. After 8 days, animals were treated by IV injection of PBS; DOTA-NP137 (anti-netrin-1); DOTA-NP137-.sup.177Lu. n=5 animals/group for PBS and DOTA-NP137; n=6 animals/group for DOTA-NP137-.sup.177Lu. d. DOTA-NP137-.sup.177Lu enhances mice survival engrafted with 4T1 cell line (see c). Kaplan-Meier survival curves analysis of mice survival treated or not with NP137. Mantel Cox test; n=5 animals/group for PBS and DOTA-NP137; n=6 animals/group for DOTA-NP137-.sup.177Lu; p<0.0001 between PBS and DOTA-NP137-.sup.177Lu and between DOTA-NP137 and DOTA-NP137-.sup.177Lu. e. NMRI nude mice were engrafted with SYO1 cells by subcutaneous injection of 5 million cells. After 8 days, animals were treated by IV injection of PBS; DOTA-NP137 (anti-netrin-1); DOTA-NP137-.sup.177Lu. n=9 animals/group for PBS and DOTA-NP137; n=12 animals/group for DOTA-NP137-.sup.177Lu; p<0.0001 between PBS and DOTA-NP137-.sup.177Lu and between DOTA-NP137 and DOTA-NP137-.sup.177Lu. f. NP137-.sup.177Lu survival of mice engrafted with H358 cells. Kaplan-Meier survival curves of mice treated or not with DOTA-NP137. Mantel Cox test; n=8 animals/group for PBS and DOTA-NP137; n=9 animals/group for NP137-.sup.177Lu; p=0.025 between PBS and NP137-.sup.177Lu and between DOTA-NP137 and NP137-.sup.177Lu.

[0173] FIG. 7: Quantification of netrin-1 in concentrated supernatants from cells treated or not with heparin.

[0174] FIG. 8: Maximum Intensity Projection of Tomographic scintigraphy and X-ray CT of the whole body of a NMRI nude mouse bearing a H358 (netrin-1-positive) tumor, acquired at 24 h, 48 h, 72 h and 96 h after IV injection of NP137-NODAGA-111 In.

MATERIALS AND METHODS

Tumor Cell Lines

[0175] 4T1 and 67NR murine mammary carcinoma cells were obtained from ATCC and cultured in RPMI-1640 (ATCC) medium supplemented with 10% foetal bovine serum (FBS, Gibco) and antibiotics (streptomycin and penicillin). EMT-6 murine mammary carcinoma cells were obtained from ATCC and cultured in Eagle Minimum Essential Medium (EMEM, ATCC) supplemented with 10% foetal bovine serum (FBS, Gibco) and antibiotics (streptomycin and penicillin). H358 human pulmonary adenocarcinoma H358 cells were obtained from ATCC and cultured in RPMI-1640 medium (ATCC) supplemented with 10% BBS (Gibco) and antibiotics. The cells were maintained in culture at 37? C. under a humidified atmosphere composed of 20% O.sub.2 and 5% CO.sub.2.

Western Blots:

[0176] Confluent cells were washed with cold PBS and discarded in lysis buffer (Tris 10 mM pH 7.6; SDS 5; Glycerol 10%; Triton X-100 1%, DTT 100 mM). After sonication, the proteins were assayed using the Pierce 660 nm protein assay reagent (Thermo Fisher Scientific) and after loading onto SDS 4-15% polyacrylamide gels (Bio-Rad) transferred to nitrocellulose membranes using the Trans-Blot Turbo Transfer (Bio-Rad). The membranes were blocked for one hour at room temperature with 5% fat-free milk powder for Netrin-1 and with 5% BSA. Staining was performed overnight with a primary antibody: Netrin-1 antibody (Ab126729, Abcam). After washing, the membranes were incubated with a secondary antibody, an anti-goat rabbit antibody coupled with HRP for 1 hour at room temperature. The West Dura (Pierce) chemiluminescence system was used to intensify the signal. Imaging was performed using Chemidoch Touch (Bio-Rad).

[0177] For the binding of netrin-1 in cellular matrix, 1?10.sup.6 cells were plated in a 100 mm.sup.3 culture dish. 24 h after, cells were treated with 200 pg/mL of heparin sodium salt from porcine intestinal mucosa (H3147-100KU, Sigma) diluted in 4 mL of medium without FBS. After one night of incubation, supernatant was collected. Centricons centrifugal filters were used to concentrate the protein in the collected supernatant. Pierce 660 nm protein assay reagent (22660, Thermofisher scientific) was then used to determine the concentration of protein, 30 pg of proteins was loaded on immunoblots.

In Vivo Preclinical Models:

[0178] The human monoclonal antibodies to NP137 (anti-netrin-1, HUM03) was kindly provided by Netris Pharma (Lyon, France). Female Balbc/J mice, 8-weeks old, were obtained from Janvier Laboratories (Le Genest-Saint-Isle, France). All syngeneic breast cancer cells 1?10.sup.6 EMT-6; 5?10.sup.5 4T1 and 1?10.sup.6 67-NR, were subcutaneously transplanted into the dorsal flank of 8-week-old female Balbc/J mice. The mice were maintained under specific pathogen-free conditions (Anican, LyonFrance and Imthernat facility, HCL Lyon, France) and stored in sterilized cages with filter lids. Their care and accommodation were in accordance with European and French institutional guidelines as defined by the local CECCAP Ethics Committee. The human cell lines H358 (1?10.sup.6 cells) or SKBR7 (2?10.sup.6 cells) were grafted onto 8 weeks female NMRI immunocompromised mice and maintained under the same conditions.

[0179] Tumor volume was assessed by measuring two perpendicular tumor diameters with a caliper three times a week. Individual tumor volumes were calculated as follows: V=(a*b2)/2. a being the largest diameter, b the smallest. When tumors reached a volume of 200-400 mm.sup.3, mice were randomly separated into groups of animals and subjected to treatment with either .sup.111In-NODAGA-NP137, .sup.111In-NODAGA-NP137-Fab, .sup.111In-NODAGA-NP137-F(ab).sub.2 or .sup.177Lu-DOTA-NP137 and submitted to imagery/therapy. For all experiments, the mice were anaesthetized using a gas protocol (isoflurane/oxygen (2.5%/2.5%).

Conjugation:

[0180] 1 mL of the anti-Netrin1 monoclonal antibody-NP137 (or its fragments as appropriate, same conditions for all of them) is added on an Amicon Ultra-15 50 k (UFC905096). Diafiltration against 0.1 M phosphate buffer (pH 8) containing 1.2 g/L of Chelex 100 is performed. This step is repeated seven times using 10 mL of 0.1 M phosphate buffer (pH 8) solution with a 25 minutes centrifugation at 4900 rpm between each wash. Anti-Netrin 1 antibody concentration is then calculated with a nanodrop. The concentration of the antibody is then adjusted in order to be at 50 ?M. Stock solution of DOTA-NHS ester/NODAGA (1,4,7-triazacyclononane, 1-glutaric acid-5,7 acetic acid)-HS ester (CheMatech (C084)) is dissolved in ultrapure water at a concentration of 10 mg/mL (=13.13 mM). 50 ?M of anti-Netrin1 antibody is combined with required DOTA-NHS of NODAGA-NHS solution at a ratio of 1:25. Reactions are conducted at room temperature for 4h and transferred to 4? C. for continuous end-over-end mixing overnight. Diafiltration against PBS (Chelex) is performed. This step is repeated seven times using 10 mL of PBS (Chelex) with a 25 minutes centrifugation at 4900 rpm between each wash. DOTA/NODAGA-Anti-Netrin 1 antibody concentration is then calculated with a nanodrop.

Radiolabeling:

[0181] NODAGA-NP137, NODAGA-NP137-Fab or NODAGA-NP137-F(ab).sub.2 (40-70 ?L, 5 mg/mL) were radiolabeled by adding 400 ?L of 100 mM acetate buffer pH5 and 40-400 MBq of high purity .sup.111In-chloride (Covidien, Petten, The Netherlands). The mixture was incubated for 30 minutes at 37? C. The reaction was stopped with 100 ?l of a 1 mM solution of DTPA. Free .sup.111In was removed using a PD-10 column. The column was first washed with 15 ml of 0.1 M acetate buffer, then the labelled mixture was loaded onto the column and eluted with the acetate buffer. .sup.111In-NODAGA-NP137, .sup.111In-NODAGA-NP137-Fab or .sup.111In-NODAGA-NP137-F(ab).sup.2 were first eluted. The radiochemical purity (RCP) of each 0.5 ml fraction was evaluated using ITLC-SG (Biodex, Tec-control black) and 50 mM citrate buffer (pH5) as the mobile phase. Radiolabeled NP-137 remained at the origin while unbound .sup.111In migrated with an Rf of 0.9-1. The highest radiochemical purity fractions were pooled.

[0182] For stability testing, aliquots of radiolabeled .sup.111In-NODAGA-NP137, .sup.111In-NODAGA-NP137-Fab or .sup.111In-NODAGA-NP137-F(ab).sub.2 were incubated at 37? C. in 2 mL phosphate buffered saline (pH 7.4) and the radiochemical purity (RCP) of the radiolabeled compounds was evaluated using ITLC-SG and 0.1 M citrate buffer pH5 as the mobile phase.

[0183] The same protocol may be applied for .sup.111In-DOTA-NP137, .sup.111In-DOTA-NP137-Fab or .sup.111In-DOTA-NP137-F(ab).sub.2.

[0184] The same protocol was used to produce .sup.177Lu-DOTA-NP137, with DOTA-NHS.

Biodistribution Studies

[0185] 1 to 10 MBq of radiolabeled .sup.111In-NODAGA-NP137, .sup.111In-NODAGA-NP137-Fab or .sup.111In-NODAGA-NP137-F(ab).sub.2 or .sup.111In DOTA-NP137 in a maximum volume of 100 ?L were injected intravenously into tumour-bearing mice (n=3 or 4 for each group). The mice were sacrificed at defined times: 4h, 24h, 48h, 72h and 96h after injection by cervical dislocation. Tissues of interest (blood, heart, lungs, spleen, kidneys, muscles, brain and skin) were removed, weighted and the radioactivity was counted for 5 min in a gamma scintillation counter (Wizard? gamma counter, Perkin Elmer, USA). Urine and faeces were collected in an individual metabolic cage for housing and counting. Tissue distribution was expressed as a percentage of the injected dose per gram (% ID/g). Renal and hepatobiliary elimination was expressed as cumulative radioactivity under the total activity injected.

Imaging:

[0186] The acquisitions were made using a Nano-SPECT/CT system for small animals (Bioscan, Washington, DC, USA). This system consists of four detectors (215?230 mm.sup.2 NaI, 33 PMTs) equipped with interchangeable multipinhole openings. The SPECT/CT acquisitions were performed after IV injection of 5-15 MBq (mega Becquerel) of radiolabeled molecule at different times: 24h, 48h, 72h and 96h. CT (55 kVp tube voltage, 500 ms exposure time and 180 projections) and SPECT/CT acquisitions were performed in tumour-bearing mice in a supine position, placed in a temperature-controlled bed (Minerve, Esternay, France), in order to maintain body temperature (set at 37? C.). The acquisition was performed for 40 minutes with two 15% windows centered on the two peaks 171 keV and 245 keV of .sup.1n. All image data were reconstructed and analyzed using the InVivo-Scope (Bioscan, Washington, DC, USA).

Generation of Fab and F(ab)2 fragments and Synthesis of DOTA and NODAGA-Immunoconjugates

[0187] Proteolytic fragments of NP137 were generated using Pierce? Fab and F(ab).sub.2 Preparation kits according to manufacturer's instructions. For conjugation of DOTA or NODAGA to surface lysine residues, NP137 and its fragments were conjugated at a molar ratio of 25:1 chelate:antibody with DOTA-NHS-ester or NODAGA-NHS ester (Chematech, Dijon, France) in metal-free buffers prepared using Chelex 100 resin. Briefly, 50 ?M antibodies were exchanged by diafiltration against 0.1 M phosphate buffer (pH8), then reacted with 1.25 mM DOTA-NHS-ester or NODAGA-NHS ester for 4h at 25? C. on a rotator. The reaction was transferred to 4? C. for continuous end-over-end mixing overnight. Excess chelator was removed by diafiltration against PBS. Immunoconjugates were stored at 4? C.

Antibodies Affinity Determination

[0188] The affinity of the antibody fragments for netrin-1 were determined by biolayer interferometry using the OctetRed96 system (ForteBio) at 30? C. with constant shaking at 1000 rpm in PBS, 0.02% Tween-20, 0.1% BSA (BB). Briefly, recombinant human netrin-1 (R&D)-coated HIS1 K biosensors were incubated with a concentration series of antibody or fragments, and association was observed for 5 min. Biosensors were then incubated in BB for a further 5 min to observe dissociation of the complex. Binding kinetics were evaluated with ForteBio Octet RED Evaluation software 6.1 using a 1:1 binding model to derive kon, koff, and KD values.

Results

Netrin-1 is a Poorly Diffusible Matrix Binding Protein:

[0189] Immunohistochemistry (IHC) has been the reference for the characterization of target expression in cancer for years. However, this strategy has recently been called into question by the recent data obtained with immune checkpoint inhibitors, as there is a strong discrepancy between target expression and response within the patients. Thus, patients responding to the PDL-1 antibody could be negative for the expression of PDL-1 in IHC and vice versa. It can be hypothesized that target expression is not stable over time, and that IHCs are made with paraffin blocks taken with the diagnosis of the primary tumor and that target expression is different in metastases. New diagnostic strategies are therefore to be developed to analyze target expression in real time on a whole-body scale, to highlight all the variations in protein expression within tumors and metastasis.

[0190] The present inventors found that netrin-1 is not diffusible in tumour cells as was thought when describing the axon guidance growth model. They obtained netrin-1 immunohistochemistry pictures in endometrium and ovary human tumors paraffin embedded tumor sections (not shown). Netrin-1 in the human tumor was found to be present in the basement membrane of the cells after IHC staining, suggesting accumulation within the cell extracellular matrix. To complete and confirm this new finding, we have characterized molecular partners of Netrin-1 within the matrix components. A screen of interaction of netrin-1 with matrix proteins using bio-Layer interferometry (BLI) assays was realized. As a result, netrin-1 is able to strongly bind to Fibronectin, Laminin and Vitronectin (FIG. 1).

[0191] Heparin blocks interaction of netrin-1 to the plastic material. While no netrin-1 was detected in the conditioned medium from netrin-1-expressing 4T1/EMT6 cells in non-heparin treated condition, when heparin was added, netrin-1 was detected (FIG. 7).

[0192] All these elements imply that netrin-1 is sequestered in the extracellular matrix of cancer cells rather than diffusible.

Characterization of a New Companion Test for Netrin-1 Real Time Detection:

[0193] Compounds were produced in which NP137 of fragments (Fab or F(ab)2) were fused to Indium 111 (11 In) to be detected by SPECT/Ct molecular imaging (FIG. 2a). The three molecules formed are believed to have different molecular activities in vivo, the complete antibodies have a longer half-life in the bloodstream and the Fab's are able to penetrate more rapidly into the tumor, the F(ab).sub.2 being an intermediate form (FIG. 2b). More precisely, the antibody or a fragment thereof has been conjugated to a metal chelator (DOTA or NODAGA) that binds to the lysine residues of the antibody or its fragment; and the chelator is associated or bound to the indium isotope, to complete the radiotracer. After steric exclusion chromatography purification, radiolabeled NP-137, Fab and F(ab).sub.2 were obtained with radiochemical purity (RCP) exceeding 98%. Radiochemical yields were 70% for .sup.111In-NODAGA-NP137, 60% for .sup.111In-NODAGA-NP137-Fab and 65% for .sup.111In-NODAGA-NP137-F(ab).sub.2. At 5 days after incubation, RCP was still greater than 95% in phosphate buffer saline (pH 7.4) indicating a suitable kinetic stability to perform in vitro and in vivo experiments. We show that this chemical modification, necessary to bind the isotope, did not disturb the ability of the three forms to bind Netrin-1 by bio-layer interferometry (FIG. 2d).

[0194] KD calculation after bio-layer interferometry analysis of the experiments presented in FIG. 2d show a high affinity KD as follows: [0195] NP137-NODAGA: 1.72 E-10 [0196] F(ab).sub.2-NODAGA: 1.51 E-10 [0197] Fab-NODAGA: 1.52 E-10.

[0198] To analyze the capacity of these molecules to detect Netrin-1 in vivo, we used two syngeneic models of tumor: 4T1 cells positive for Netrin-1 expression and 67NR negative for Netrin-1 as a negative control.

[0199] First, quantification of netrin-1 expression by Q-RT-PCR on 4T1 and 67NR cell lines was performed. Results on FIG. 3a confirm that there is netrin-1 expression only in the 4T1 cells.

[0200] Second, Maximum Intensity Projection of Tomographic scintigraphy and X-ray CT of the whole body of a Balb/cJ mice bearing a 4T1(positive for Netrin-1) tumor, acquired at 24h; 48h; 72h and 96h after IV injection of .sup.111In-NODAGA-NP137-F(ab).sub.2, .sup.111In-NODAGA-NP137-Fab, or .sup.111In-NODAGA-NP137 was obtained. Similarly, Maximum Intensity Projection of Tomographic scintigraphy and X-ray CT of the whole body of a Balb/c mouse bearing a 67NR (negative for Netrin-1) tumor, acquired at 24h; 48h and 72h after IV injection of .sup.111In-NODAGA-NP137-F(ab).sub.2, .sup.111In-NODAGA-NP137-Fab, or .sup.111In-NODAGA-NP137 was obtained.

[0201] A strong tumor intake is detectable in 4T1 tumors in .sup.111In-NODAGA-NP137 group (FIG. 3b), whereas slower intake was detectable in .sup.111In-NODAGA-NP137-F(ab).sub.2 group and in .sup.111In-NODAGA-NP137-Fab group (data not shown). Interestingly no intake could be detected for all molecules in 67NR bearing mice, implicating that tumoral intake is specific of tumoral Netrin-1 (compare FIGS. 3B and 3C). Localized tumor intake is also visible in H358 tumor at FIG. 8.

[0202] We performed ex vivo quantification of the intake ratio between 67NR and 4T1 cells implicating that the best specific incorporation is detected 48h after treatment (FIG. 4a-c). These data confirmed that tumor incorporation is better with full antibody than other forms.

[0203] Biodistribution properties of .sup.111In-NODAGA-NP137 in Balb/cJ mouse bearing 4T1 xenografts at 48h, 72h and 96h was measured for all organs. Radioactivity incorporation is quantified by the percentage of the injected dose by gram of organ. A strong tumor intake close to 25% of the injected dose (ID) was detected in the tumor after the indicated time laps. The incorporation within other organs did not reveal a specific bindings (FIG. 4d).

A New Diagnostic Tool

[0204] A further test was done in a transgenic model of mammary luminal breast cancer MMTV-NeuT (20), expressing endogenous levels netrin-1. FIG. 5a shows the evolution in time, from the left to the right at 24h, 48h and 72h after injection of .sup.111In-NODAGA-NP137.

[0205] A strong staining was detected in fat pad tissue, and this in the 10 mammary glands of the animals (see FIG. 5b).

[0206] It is very interesting to note that some tumors were visualized even before being detected by mammary palpation, which reinforces the relevance of this tracer as an early detecting tool for netrin-1 expressing tumors. This result was unexpected. A strong tumor intake close to 8% of the injected dose (ID) could be detectable after a tumor incorporation measurement, in all tumors arguing that .sup.111In NODAGA-NP137 could be a good diagnosis tool to describe the expression of Netrin-1 upon the appearance of a small lesion in term of tumor mass. (FIG. 5c).

[0207] FIG. 8 show the strong accumulation of NP137-NODAGA-.sup.111In in the netrin-1-positive human xenograft murine model H358 (human non-small cell lung cancer model). Similar results (not shown) were obtained with EMT6 (murine mammary carcinoma cell line. Tumoral uptakes in both models were more than 10% of ID/g (injected dose/weigh of organ in gram).

[0208] NP137-DOTA-.sup.177Lu a new theranostic compound to target resistant tumors.

[0209] We have developed an antibody fused to Lutecium 177 (called DOTA-NP137-.sup.177Lu on FIG. 6) that will emit beta radiation that effectively damages cancer cells. Lutecium will emit a strong dose of radiation in a range of 1.8 mm, with high specificity when coupled to a targeted therapy.

[0210] We first use this molecule to treat the 4T1 and the EMT6 cell lines. These cell lines are resistant to NP137 as a single agent and are known to belongs to the most aggressive preclinical models. Nevertheless, tumor growth decreased when treated with a single 10MBq dose of NP137-DOTA-.sup.177Lu compare to control groups with either PBS or DOTA-NP137 (FIGS. 6a, c and e). As a consequence, survival of mice was increased in the NP137-DOTA-.sup.177Lu treated group (FIGS. 6b and d). These results indicates that this new molecule could lead to antitumoral activities in vivo in tumor expressing netrin-1.

[0211] Therapeutic efficacy of NP137-.sup.177Lu was further assessed in the human lung cancer xenograft model H358 where the treatment again significantly reduced the rate of tumor growth corroborating a clear anti-tumoral effect (p<0.001) (FIG. 6f).

Discussion

[0212] In this study, we described a new companion test for the in vivo detection of netrin-1 by nuclear medicine SPECT/CT. Netrin-1 has been characterized as a therapeutic target in several types of cancer currently evaluated in clinical assays, but due to the lack of assay in a conventional test (i.e., serum detection with Elisa assays, mass spectrometry, robustness of pathology revealing netrin-1 in FFPE samples), we developed an innovative, simple and robust companion test to detect the high expression of netrin-1 in vivo in cancer cells. Based on our results, we can state that no or very-low a specific binding of .sup.111In-NODAGA-NP137 or .sup.111In-DOTA-NP137 detected in our preclinical models, as revealed by the RCP in the netrin-1 negative tumor model 67NR. These results show that netrin-1 is not widely expressed at the adult level, and show a high specificity for tumoral tissues. The targeting of the developmental Netrin-1 genes that are re-expressed during tumor formation therefore appears to be a key solution for improving the specificity of tumor imaging.

[0213] To identify the best molecule for SPECT or PET imaging, we designed three different agents based on the clinically used human anti-netrin-1 NP137 monoclonal antibody: NP137-IgG1 complete, NP137-F(ab)2 and NP137-Fab. All of these molecules can bind strongly to netrin-1. The best accumulation in vivo with tumour specificity was observed for the radiotracer containing full NP137-IgG1 and NP137-Fab; the best was the radiotracer containing the full antibody. Complete NP137-IgG1 showed the best accumulation within the tumour and the most promising results for transfer within the clinic. This transfer could be hypothesized with all the metals and compounds used for molecular imaging. In terms of more basic research, Netrin-1 has been described for years in neural development as a secreted molecule with a diffusible gradient. Netrin-1 is a ligand and as such is not a primary choice as the target of imagery or internal radiotherapy.

[0214] Moreover, and based on this demonstrated tumor incorporation we design a new molecule in which we fused NP137-DOTA with Lutecium 177 to form NP137-DOTA-.sup.177Lu. As a result, this molecule also accumulated specifically within the tumors expressing netrin-1. Lutecium 177 is a B-emitter able to deliver a strong dose of radiation in a range of 1.8 mm within the tumoral tissue. As consequence, we note a significant decrease in tumor growth correlated with a better survival on mice bearing tumor. It is remarkable to note that these tumor types were totally resistant to NP137 as a single agent. The survival studies demonstrate that treatment with this compound increased mice life two times with respect to control, in mice tumor models and in human tumor model. As NP137 has proven its safety as a single agent and doses .sup.177Lu are well characterized, so that this molecule can transferred to treat tumors in a simple manner.