(19)
(11)EP 3 102 940 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
26.06.2019 Bulletin 2019/26

(21)Application number: 15746777.0

(22)Date of filing:  04.02.2015
(51)International Patent Classification (IPC): 
G01N 33/53(2006.01)
C07K 16/40(2006.01)
C07K 16/28(2006.01)
(86)International application number:
PCT/AU2015/050036
(87)International publication number:
WO 2015/117199 (13.08.2015 Gazette  2015/32)

(54)

ANTI-METALLOPROTEASE ANTIBODY FOR DIAGNOSIS AND TREATMENT OF CANCERS

ANTI-METALLOPROTEASE-ANTIKÖRPER ZUR DIAGNOSE UND BEHANDLUNG VON KREBS

ANTICORPS ANTI-MÉTALLOPROTÉASE POUR LE DIAGNOSTIC ET LE TRAITEMENT DE CANCERS


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 04.02.2014 US 201461935552 P

(43)Date of publication of application:
14.12.2016 Bulletin 2016/50

(73)Proprietors:
  • Monash University
    Melbourne, Victoria 3800 (AU)
  • Memorial Sloan Kettering Cancer Center
    New York, NY 10065 (US)
  • Ludwig Institute For Cancer Research Ltd.
    New York, NY 10017 (US)

(72)Inventors:
  • LACKMANN, Martin
    St Andrews Beach, Victoria 3941 (AU)
  • JANES, Peter W.
    Hawthorn East, Victoria 3123 (AU)
  • MUDIYANSELAGE, Lakmali Atapattu
    Hughesdale, Victoria 3166 (AU)
  • SCOTT, Andrew M.
    Kew East, Victoria 3102 (AU)
  • NIKOLOV, Dimitar B.
    New York, New York 10075 (US)
  • SAHA, Nayanendu
    Jackson Heights, New York 11372 (US)

(74)Representative: Schiweck Weinzierl Koch Patentanwälte Partnerschaft mbB 
Ganghoferstraße 68 B
80339 München
80339 München (DE)


(56)References cited: : 
WO-A1-2006/063415
US-A1- 2013 273 051
WO-A1-2012/148003
  
  • UWE LENDECKEL ET AL: "Increased expression of ADAM family members in human breast cancer and breast cancer cell lines", JOURNAL OF CANCER RESEARCH AND CLINICAL ONCOLOGY, SPRINGER INTERNATIONAL, BERLIN, DE, vol. 131, no. 1, 1 January 2005 (2005-01-01), pages 41-48, XP002675380, ISSN: 0171-5216, DOI: 10.1007/S00432-004-0619-Y [retrieved on 2004-09-30]
  • ATAPATTU, L. ET AL.: 'Antibodies binding the ADAM 10 substrate recognition domain inhibit Eph function' JOURNAL OF CELL SCIENCE vol. 125, 2012, pages 6084 - 6093, XP055218749
  • RAPPA, G. ET AL.: 'Biochemical and biological characterization of exosomes containing prominin-1/ CD 133' MOLECULAR CANCER vol. 12, page 62, XP021155432
  • HALL, J. ET AL.: 'A disintegrin and metalloproteinase 10 (ADAM10) regulates pancreatic cancer progression by accelerting tumorigenesis and metastasis' CANCER RESEARCH vol. 72, no. S UPPL, 2012, XP055218762 & DATABASE EMBASE Database accession no. 0051462581
  • RATH, B.H. ET AL.: 'Astrocytes Enhance the Invasion Potential of Glioblastoma Stem-Like Cells' PLOS ONE vol. 8, 2013, page E54752, XP055218759
  • DUFFY, M.J. ET AL.: 'The ADAMs family of proteases: new biomarkers and therapeutic targets for cancer?' CLINICAL PROTEOMICS vol. 8, page 9, XP021103846
  • BASS, R. ET AL.: 'ADAMs and protein disulfide isomsease: the key to reculated cell-surface protein ectodomain shedding?' BIOCHEMICAL JOURNAL vol. 429, 2010, pages E3 - E5, XP055218809
  • ATAPATTU, L. ET AL.: 'The role of proteases in regulting Eph/ephrin signaling' CELL ADHESION & MIGRATION vol. 8, 20 December 2014, pages 294 - 307, XP055218811
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

TECHNICAL FIELD



[0001] THIS INVENTION relates to an antibody to bind a proteolytically active form of an ADAM10 protease. More particularly, this invention relates to diagnosis and/or treatment of cancers using an antibody that binds a proteolytically active form of an ADAM10 protease that is expressed by tumour cells.

BACKGROUND



[0002] ADAM ('A Disintegrin And Metalloprotease') proteases catalyse the release of a range of cell surface proteins, activating receptor tyrosine kinase (RTK), Notch, cytokine-, chemokine- and adhesion signalling pathways important in normal and oncogenic development. Prominent oncogenic substrates include ligands and receptors in the Notch, erbB and Eph families, cytokines (TNFα, IL6), FASL, Slit, L-selectin and cadherins1, which are all shed by one of the two closely related and widely-expressed proteases ADAM10 and ADAM17 (TACE). These proteases are also frequently over-expressed in cancers, correlating with aberrant signalling and poor cancer prognosis in cancers of the colon, lung, stomach, uterus and ovary1,2, and as potent activators of key oncogenic pathways, recognized targets for multi-pathway inhibition3,4.

[0003] A study by Lendeckel et al. (J Cancer Res Clin Oncol, 2005, 131:41-48) also provides evidence that ADAMs, and in particular ADAM 9, 12, and 17, are upregulated in human breast cancer which indicates a putative role for these ADAMs in breast cancer.

[0004] ADAM10 in particular acts as principal sheddase for Notch5, Eph6,7 and certain EGFR ligands8 as well as E- and N-cadherin9. It's essential role for Notch function is highlighted by the resemblance of ADAM105 and Notch KO mice4, with embryonic lethal defects in somitogenesis, neurogenesis and vasculogenesis5. Notch signalling is triggered by binding of cell-surface-bound ligands, Delta-Like(1-4) or Jagged(1,2), to Notch receptors (Notchl-4), which initiates ADAM-mediated shedding of both ligand10 and receptor extracellular domains (ECD)11. Shedding of the notch ECD provides the signal for γ-secretases to cleave and release the Notch intracellular domain (NICD), acting as transcriptional activator for an extensive set of genes11, regulating cell proliferation, differentiation, EMT and cell survival. De-regulated Notch signalling promotes the progression of solid cancers12, particularly by driving angiogenesis13, while mutant activated Notch is a known cause in 50% of T-ALL. However, pan-specific γ-secretase inhibitors blocking NICD release14 cause severe intestinal toxicity, likely reflecting the diversity of γ-secretase targets15. Similarly, small-molecule inhibitors blocking the ADAM protease active site failed clinical development due to lack of specificity and off-target effects, reflecting the close structural homology of this site in all MMPs4,16.

[0005] A study by Atapattu et al. (J Cell Sci, 2012, 125:6084-6093) describes the antibody 8C7 which binds to the ADAM10 substrate binding pocket in the C domain.

[0006] US20130273051 discloses antibodies or antigen-binding fragments that selectively inhibit members of the metzincin protease family or subfamily, such as ADAMs, whereas WO2012/148003 discloses an antibody that binds to human receptor-type protein tyrosine phosphatase σ.

[0007] ADAMs are transmembrane proteins with an N-terminal pro-domain followed by metalloprotease (M), disintegrin (D), cysteine-rich (C), transmembrane and cytoplasmic domains3. WO2006/063415 discloses elucidation of the crystal structure of an ADAM10 substrate-recognition and proteinase-positioning module comprising the protein cysteine-rich and disintegrin domains. This patent further describes an acidic pocket within the cysteine-rich domain that forms a substrate-recognition site.

[0008] Intriguingly, their proteolytic specificity is not simply due to a typical substrate cleavage signature, but relies on non-catalytic interactions of the substrate with ADAM D+C domains that align the protease domain for effective cleavage6,17,18. In addition, emerging evidence suggests that adopting latent and active ECD conformations may regulate ADAM10 and 17 activities. This concept is based on the protein architecture revealed in crystal structures of the related snake venom metalloproteinases, adopting two different conformations that are stabilized by distinct disulphide connectivity of the D+C domains19,20: a closed conformation suggested to deny substrate access, and an open conformation that allows substrate access and promotes cleavage. ADAM10/17 ECDs indeed harbour a large number of disulfide bonds21, including the conserved thioredoxin CxxC motif typical for disulfide exchange reactions catalysed by protein disulfide isomerases (PDIs)22, and mild reducing or oxidising conditions and PDI treatment are known to alter ADAM17 activity23,24. Considering that reactive oxygen species (ROS) are frequently elevated in tumours due to RTK and pro-inflammatory signalling, and are known to activate ADAM10/1723,25, an effect on protease domain orientation may explain the well-documented conundrum that kinase-dependent cytosolic signalling regulates the activity of the extracellular ADAM protease domain3,26.

SUMMARY



[0009] The present invention is broadly directed to use of an antibody or antibody fragment, which specifically or selectively recognizes and binds a proteolytically active form of the ADAM10 protease. More particularly, the inventors have discovered that expression of proteolytically active ADAM10 protease is relatively increased in tumour cells, most prominently in certain cells that also express the putative tumour stem cell marker CD133. This proteolytically active ADAM10 protease expressed by tumour cells is typically, although not exclusively, a high molecular weight form of an ADAM10 protease. Accordingly, the antibody or antibody fragment may be used to detect tumour cells and also as a therapeutic agent for treating cancers, tumours and other malignancies.

[0010] In an embodiment the ADAM protease is ADAM10 protease. In one particular embodiment, the ADAM protease is ADAM10 and the antibody or antibody fragment is monoclonal antibody 8C7.

[0011] In a first aspect, the invention relates to an in vitro method of detecting a proteolytically active form of an ADAM10 protease, said method including the step of selectively binding an antibody or antibody fragment to the proteolytically active form of the ADAM10 protease to thereby detect the proteolytically active form of the ADAM10 protease, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645.

[0012] In a second aspect, the invention relates to an in vitro method of detecting a tumour cell, said method including the step of selectively binding an antibody or antibody fragment to the proteolytically active form of an ADAM10 protease expressed by the tumour cell to thereby detect the tumour cell, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645 and wherein the tumour cell is of a leukemia, lymphoma, lung cancer, colon cancer, adenoma, neuroblastoma, brain tumour, renal tumour, prostate cancer, sarcoma, breast cancer or melanoma.

[0013] Additionally, the invention relates to a kit for use according to the first and second aspect of the invention comprising an antibody or antibody fragment that specifically or selectively binds a proteolytically active form of an ADAM10 protease.

[0014] In a third aspect, the invention relates to an antibody or antibody fragment which specifically or selectively recognizes and binds a proteolytically active form of the ADAM10 protease to the ADAM10 protease expressed by a cell, for use in at least partly inhibiting a biological activity of the ADAM protease and/or one or more downstream signalling molecules in the cell, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645.

[0015] In one embodiment of the third aspect of the invention, the one or more downstream signalling molecules include one or more members of the Notch, erbB and Eph signalling pathways.

[0016] In a fourth aspect, the invention relates to an antibody or antibody fragment which specifically or selectively recognizes and binds a proteolytically active form of the ADAM10 protease for use in preventing or treating cancer in a mammal, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645.

[0017] Additionally, the present invention relates to the antibody or antibody fragment of the third or the fourth aspect of the invention, wherein the ADAM protease 10 is expressed by a tumour cell, wherein the tumour cell is of a leukemia, lymphoma, lung cancer, colon cancer, adenoma, neuroblastoma, brain tumour, renal tumour, prostate cancer, sarcoma, breast cancer or melanoma.

[0018] Further, the present invention relates to the in vitro method of the second aspect or the antibody or antibody fragment mentioned above, wherein the tumour cell expresses CD133.

[0019] Further, the present invention relates to the method of the first or the second aspect, the antibody or antibody fragment mentioned above, or the kit also being mentioned above, wherein the antibody or antibody fragment binds or interacts with one or more ADAM10 protease residues selected from the group consisting of: Arg557; Lys591; Pro628; Asp640; Val641; Phe642; Arg644, and Arg646, preferably wherein hydrogen bonds are formed between Asp640; Val 641; Phe 642; Arg644; and Arg646 and the antibody or antibody fragment.

[0020] Additionally, the present invention relates to the method of the first or the second aspect, the antibody or antibody fragment mentioned above, or the kit also being mentioned above, wherein the antibody or antibody fragment comprises at least a portion of at least one complementarity determining region (CDR) having a CDR amino acid sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2 or comprises at least a portion of at least one CDR having an amino acid sequence at least 85% identical to a CDR amino acid sequence in SEQ ID NO:1 and/or SEQ ID NO:2.

[0021] In a fifth aspect, the invention relates to a recombinant humanized antibody or antibody fragment which specifically or selectively binds a proteolytically active form of an ADAM10 protease and comprises at least one complementarity determining region (CDR) having an amino acid sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2 or comprises at least one CDR having an amino acid sequence at least 85% identical to a CDR amino acid sequence in SEQ ID NO:1 and/or SEQ ID NO: 2.

[0022] Throughout this specification, unless otherwise indicated, "comprise", "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers.

BRIEF DESCRIPTION OF THE DRAWINGS



[0023] 

Figure 1. Treatment with 8C7 mAb inhibits tumour growth. (A, B) Mice bearing human LIM1215 CRC tumours were treated with 8C7 mAb at indicated doses or reagent control (2 injections/week). Tumour volumes were measured twice weekly using a calliper (A), the tumour mass of resected tumours was recorded at the end of the experiments (B). (C) Tissue sections of resected tumours were analysed with anti-CD31 antibodies to stain endothelial cells, revealing significantly-reduced microvascular density in 8C7-treated tumours. (D) Cell death in tumours was assessed using TUNEL IHC staining of tumours, suggesting increased apoptosis after 8C7 treatment, especially within the vascularised tumour rim. (E) Western blot analysis of detergent lysates from resected tumour shows downregulation of ADAM10 and a number of its substrates, including Eph and Notch receptors in 8C7-treated tumours. Lysates from individual tumours in each group are shown, the bar graph illustrates mean and SD of densitometry measurements.

Figure 2. The mAb 8C7 preferentially targets CD133+/NICD+ tumour cells in the tumour rim. (A) Confocal microscopy of tumours from mice injected with Alexa647-labelled 8C7. 8C7 binding (green pseudocolour) is strongest in the tumour rim and near tumour vessels (labelled with rhodamine lectin, red). Blue, Hoechst stained nuclei. (B) Co-staining of tumours from 8C7-injected mice with antibodies against the tumour stem cell marker CD133, or against cleaved (active) Notch1 or Notch2 intracellular domains (NICD1, 2) (red). Purple, lectin labeled vessels; Blue, Hoechst stained nuclei. (C) Western blot for active Notch (NICD1) of tumour cell isolates that had been sorted for CD133 expression (CD1330, + or -). (D) Flow cytometry of tumour cell isolates from Alexa647-8C7-injected mice, stained for 8C7+/CD133+ cells. CD133 is present on a small cell population (0.94%), which are strongly positive for 8C7-bound ADAM10.

Figure 3. Treatment with 8C7 inhibits Notch signalling in LIM1215 tumours. (A) Protein extracts from LIM1215 tumours of 8C7-treated or control mice were analysed by Western blot with antibodies against NICD and the Notch target Hes1. Samples from individual tumours are shown. The diagram illustrates the mean and SD of the data quantitated by densitometry; *, p> 0.05. (B) RNA extracts from PBS, 8C7 or 8E11 control mAb-treated LIM1215 tumours were analysed by real time PCR for expression of the Notch targets Hes1 and Hey1. Data from n = 5 mice were analysed; *, p> 0.05. (C) CD133+ LIM1215 cells isolated from tumours were treated in culture with 8C7, control mAb 8E11, or gamma-secretase inhibitor (GSI). Lysates were analysed by Western blot for active Notch (NICD). A representative Western blot from 3 separate experiments is shown. The diagram illustrates the mean and SD of the data quantitated by densitometry. # indicates IgG bands from treatment mAb.

Figure 4. 8C7 preferentially recognises HMW ADAM10 present in tumours but not in normal mouse tissues. (A) Fluorescence microscopy of tissue sections from LIM1215 tumours and non-affected organs from mice injected with Alexa647-labelled anti-ADAMlO antibodies (green): 8C7 binds human and mouse ADAM1027, 4A11 binds only human ADAM10, m/h ADAM10 is a commercial mouse/human ADAM10-specific control antibody. Blue, Hoechst stained nuclei. (B) Western blot analysis of 8C7-bound ADAM10 recovered from indicated tissues of 8C7-treated, tumour-bearing mice by Protein A Sepharose (top). Lower panels show the overall ADAM10 expression, assessed by IP from tissue lysates with control anti-ADAM antibody, indicating prevalence of HMW ADAM10 in tumours. The protein band indicated with * in the spleen sample also appears in the blot from the PBS-treated mouse and reflects non-specific reactivity of the blotting antibody.

Figure 5. Recognition of the HMW form of ADAM10 by 8C7 is diagnostic for mouse and human tumours. (A) 8C7 Immunoprecipitates of ADAM10 from stomach tissues from GP130F knock-in mice developing spontaneous gastrointestinal tumours38,39 at 5 - 6 weeks of age. Samples from different part of the stomach are shown, F=Fundus, C=Corpus, A=Antrum, T=tumour. (B) Immunoprecipitation of ADAM10 from (snap) frozen human tumour (tum) or normal (norm) tissues with 8C7 or control ADAM10 mAb 4A11 and Western blot analysis with anti-ADAMlO pAbs. 8C7 preferentially binds a subset of the non-processed high MW ADAM10 that is prevalent in tumours. U, unprocessed; P, processed ADAM10.

Figure 6. 8C7 binds to the enzymatically active form of ADAM10. (A, B) Activity assays of ADAM10 IPs from LIM1215 cells (A) and human colorectal carcinoma (CRC) tumour tissue (B) shows that 8C7 selectively targets the HMW form of ADAM10. ADAM10 activity was measured with a fluorogenic peptide substrate (FRET assay, Es003, R&D Systems) or by cleavage of ephrin-A5 Fc as substrate. The protease activity relative to the protein level of ADAM10, as estimated from the Western blots of the IP is illustrated in the diagram. Mean and SEM of 3 independent assays are shown; unpaired t-test (two tailed) n=3; ***p<0.0001, **p<0.01, *p<0.05.

Figure 7. Protein X-ray crystal structure of the 8C7 F(ab')2 fragment binding the ADAM10 D+C domain.
(A) Crystal Structure of the mAb-8C7/ADAM10 complex. The heavy chain of the 8C7 mAb is in magenta and the light - in cyan. The disintegrin and cysteine-rich domains of ADAM10 are in green. Disulphide bridges in ADAM10 are drawn as sticks and colored in yellow. Glycosylation moieties are drawn as grey spheres. A calcium, bound in the ADAM disintegrin domain, is in blue. (B) The 8C7 (magenta/cyan)/ADAM10 (green) interface with the ADAM CxxCC motif residues coloured in red. (B) Two close-up views of the 8C7/ADAM10 interface. The right panel view is a 60-degree rotation from the left panel view. The heavy chain of the 8C7 mAb is in magenta, the light - in cyan, and ADAM10 is in green. The interacting residues are drawn as sticks and labeled on the right panel. The molecular surface of 8C7, which is in contact with ADAM10, is rendered in grey on the right panel.

Figure 8. 8C7 binding relies on an intact ADAM10 CXXC motif and is increased by cell exposure to ROS or RTK agonists. (A) Mutation of ADAM10 CxxC motif blocks binding of 8C7 but not of control mAb. Detergent cell lysates from ADAM10-/- MEFS, transfected with Wt or AXXA mutant ADAM10, were IPed with control anti-ADAM10 mAb (R&D Biosystems) or with 8C7 as indicated. Western blots were probed with anti-ADAMlO pAb. Densitometry was used to quantitate binding of the mutant protein relative to Wt ADAM10. (B) 8C7 binding to ADAM10 was tested in cells treated with reductant (DTT), oxidant (H2O2) or RTK stimulation (known to generate localised ROS). Binding of 8C7, but not control mAb 4A11, is significantly increased under oxidative,conditions and decreased under reducing conditions. Results from 3 independent assays were quantitated by densitometry, mean +/- SEM are shown, significance estimated by unpaired t-test (two tailed) n=3; ***p<0.0002, ** p<0.005. ADAM10 levels in 4A11 IPs are not significantly different.

Figure 9. ADAM10 contains labile PDI-susceptible cysteines in the CXXC motif that are implicated in the 8C7-binding epitope. (A) 8C7 and 4A11 IPs from LIM1215 cell lysates, were treated with Methyl-PEG 12- maleimide (MPM) to block free cysteines, then with PDI (5µg/ml) or DTT(20µM) followed by Maleimide-PEG2-biotin to detect labile cysteines. Biotinlyation and total ADAM10 levels were detected by Western blot using streptavidin-HRP antibody and α-ADAM10 pAb, respectively. (B) 4A11 and 8C7 IPs from LIM1215 cells treated in culture with 2.5mM MPM to block free cell-surface cysteines and with PDI/DTT to reduce labile disulfide bonds, followed by 2.5mM MBP to detect reduced cysteines. 8C7 fails to bind cell surface ADAM10 that has been modified by MBP, whereas MBP labeling of ADAM10 in 4A11 IPs slightly increases with PDI/DTT treatment. (C) 4A11 IPs from lysates of ADAM10-/- MEFs transfected with Wt ADAM10 and AxxA ADAM10 cDNAs were treated with PDI/DTT as above, and Western blots were probed using Streptavidin-HRP and α-ADAM10 pAbs. While PDI treatment of Wt ADAM10 results in increased biotinylation over time, there is little biotinylation in mutant AxxA ADAM10, suggesting the CxxC motif is labile.

Figure 10. Treatment with 8C7 attenuates tumour growth and down modulates levels of its major substrates, but does not affect overall mouse weights. (A) Weights of control and 8C7-treated LIM1215 bearing mice were measured over the time of treatment. (B) Tumour volumes of LIM1215 tumour-bearing mice were treated with PBS, 8C7 mAb or control mAb 8E11 growth rates calculated over the treatment time. (C) Western blot analysis of tumour lysates shows down-regulation of Jagged (both full length and cleaved forms) and TGFα, in 8C7-treated tumours. (D) Western blot analysis of tumour lysates shows down-regulation EGFR, phosphor-EGFR and EphA2 in 8C7-treated tumours.

Figure 11: Tumour microvessels stain with antibodies against Jagged-1. (A, B) Immunofluorescent confocal microscopy of tumours from mice injected with Alexa647-labelled 8C7 (cyan) and [Rhodamine]-RCA lectin (red). Sections were stained with antibodies against Jagged 1 (A), or 2 (B), (green). Blue, Hoechst stained nuclei.

Figure 12: 8C7 inhibits Jagged/Notch dependent lymphoma proliferation. A) Assay concept: lymphoma co-cultures with HUVECs, which had been transduced with the E4ORF1 gene auto-activating Akt (E4-HUVEC) to allow growth in serum- and cytokine-free culture: Lymphoma cells grow only with E4-HUVEC support; Jag1 knockdown (KD) in E4- HUVECs abolishes lymphoma propagation. B, C) mAb8C7 treatment inhibits the Jag1-dependent endothelial support of lymphoma proliferation.

Figure 13: The HMW form of ADAM10 is processed by Furin in-vitro and is present on the cell surface. (A) 8C7 and 4A11 IPs from LIM1215 lysates were treated with recombinant furin and reduced (Rd) samples analysed by Western blot with α-ADAM10 pAb and an ADAM10 pro-domain-specific antibody. Furin dose dependently cleaves the ADAM10 HMW (98kDa) band of both 8C7 and 4A11 IPs to release the prodomain and increase 64kDa ADAM10 levels. Detection of ADAM10-prodomain increases dose dependently (lower panels). The prodomain is more abundantly generated in 8C7 IPs despite of higher ADAM10 loading in 4A11 IPs. (B) Lysates from LIM1215 cells, incubated for 3 hours with 8C7 or 4A11 at 37°C or under indicated conditions preventing endocytosis. Protein-A beads were added to the cell lysates to pull down cell surface bound ADAM10. Western Blots were probed with α-ADAM10 pAB.

Figure 14: Processing by Furin does not affect ADAM10 activity. 8C7 IPs from whole cell LIM1215 lysates were left untreated (control) or treated with recombinant Furin for 1h, or 5h, as indicated, before assaying for ADAM10 sheddase activity. The activity (graph) was determined using the FRET assay with a fluorogenic peptide substrate (FRET assay, Es003, R&D Systems), expressed relative to the ADAM10 protein level determined by Western blot analysis under non-reducing (nRd) and reducing (Rd) conditions (as indicated). Despite complete conversion of the HMW form into the LMW form the ADAM10 activity remained unchanged compared to the control sample.

Figure 15. The CxxC-to-AxxA mutation does not affect ADAM10 localisation ADAM10/wt and ADAM10/AxxA cDNA was transfected into MEFADAM10-/- cells. Fixed and permeabilized cells were stained with primary Ab α-ADAM10 (R&D) and secondary antibody α-mouse-647. Wt and AxxA mutant ADAM10 display similar localization including both intracellular and cell surface staining.

Figure 16. 8C7-targeted tumour cells exhibit high levels of reactive oxygen species (ROS). Tumour cells from LIM1215 xenograft-bearing mice, injected with Alexa-labelled 8C7, were recovered and sorted for 8C7 positive or negative staining. Equal numbers of these cells were then compared for levels of ROS production using the Amplex red assay (Invitrogen).

Figure 17. 8C7 blocks tumour regrowth following chemotherapy. LIM1215 xenograft-bearing mice were treated with increasing doses of the chemotherapeutic irenotecan (once/week for 6 weeks, arrows), with or without 8C7 (twice weekly). A. Tumour volumes compared to control (PBS treated) mice. B. Change in tumour volume following last dose of chemotherapy. C. % of CD133+ cells in tumours recovered at end of experiment and analysed by flow cytometry.

Figure 18. 8C7 blocks spontaneous tumour growth in GP130F mice. Mice were treated with 8C7 or PBS from week 4 to 9 (2x/week), following which tumours were recovered and weighed.

Figure 19. A. 8C7-bound ADAM10 preferentially associates with notch and erbB receptors, and PDI. Associations are shown by co-immunoprecipitation and Western blots. Graphs show relative binding from densitometry analysis.

B. Comparison of 8C7/ADAM10D+C and snake venom metalloproteinases. 8C7/ADAM10D+C structure (right) with structures of full length snake venom metalloproteases (predicted to have similar overall structure). 8C7 binds in the region where the MP domain resides in the other structures.

Figure 20. Alamar blue growth assays on breast cancer cell lines. Breast cancer cell lines were treated with 8C7, metalloprotease inhibitor (GM6001) or EGFR kinase inhibitor (AG1478); on plastic (2D) or matrigel (3D). Time point day 4.


DETAILED DESCRIPTION



[0024] ADAM10 is a transmembrane metalloprotease frequently overexpressed in many tumours where it acts by releasing a range of cell surface proteins that promote tumour progression. By shedding ligands and receptors of the Notch, Eph, EGF, GP130/IL6 and chemokine families, ADAM10 activates key cancer signalling pathways and drug resistance mechanisms, but lack of active site inhibitors with sufficient specificity has hindered its clinical development to date. The ADAM10 protease comprises disintegrin and cysteine-rich domains having a substrate-binding pocket within the C domain that specifies ligand cleavage. The 8C7 monoclonal antibody specifically recognises the substrate binding domain and inhibits ADAM10-mediated cleavage of Eph receptor ligands (ephrins) and ephrin/Eph-dependent signalling and cell segregation in vitro. Analysis of the structure of 8C7 in complex with ADAM10 revealed binding to a critical cysteine-rich sequence motif that regulates ADAM10 function. 8C7 treatment inhibits growth of human colon tumour xenografts due to inhibition of Notch signalling and down-regulation of Notch, EGFR and Eph receptor levels. 8C7 specifically targets tumour cells, preferentially binding to cells that express the putative tumour stem cell marker CD133 and that display active Notch-signalling within tumours. More particularly, 8C7 selectively binds a proteolytically active form of ADAM10, which is diagnostic in mouse tumour models and in several human solid tumours. Accordingly, certain embodiments of the invention relate to 8C7 as an antibody suitable for human cancer therapeutic and diagnostic uses.

[0025] For the purposes of this invention, by "isolated" is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form.

[0026] By "protein" is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L- amino acids as are well understood in the art.

[0027] A "peptide" is a protein having no more than fifty (50) amino acids.

[0028] A "polypeptide" is a protein having more than fifty (50) amino acids.

[0029] As used herein, the abbreviation "ADAM" relates to a family of transmembrane metalloproteases that catalyse the release of a range of cell surface proteins, activating receptor tyrosine kinase (RTK), Notch, cytokine-, chemokine- and adhesion signalling pathways important in normal and oncogenic development. Prominent oncogenic substrates include ligands and receptors in the Notch, erbB and Eph families, cytokines (TNFα, IL6), FASL, Slit, L-selectin and cadherins. The amino acid residue numbering system used herein is based on ADAM sequences available under Genbank accession numbers: NM_001110 (human ADAM), NM_174496 (mouse ADAM), NM_007399 (bovine ADAM), NM_003183 (human ADAM17) and NM_009615 (mouse ADAM17).

[0030] As used herein an "antibody" is or comprises an immunoglobulin. The term "immunoglobulin" includes any antigen-binding protein product of a mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG and IgE and antigen-binding fragments thereof. Included in the term "immunoglobulin" are immunoglobulins that are chimeric or humanised or otherwise comprise altered or variant amino acid residues, sequences and/or glycosylation, whether naturally occurring or produced by human intervention (e.g. by recombinant DNA technology).

[0031] Antibody fragments include Fab and Fab'2 fragments, diabodies and single chain antibody fragments (e.g. scVs), although without limitation thereto. Typically, an antibody comprises respective light chain and heavy chain variable regions that each comprise CDR 1, 2 and 3 amino acid sequences The antibody or antibody fragment may comprise at least a portion of a CDR1, 2 and/or 3 amino acid sequence A preferred antibody fragment comprises at least one entire light chain variable region CDR and/or at least one entire heavy chain variable region CDR.

[0032] Antibodies and antibody fragments may be polyclonal or preferably monoclonal. Monoclonal antibodies may be produced using the standard method as for example, described in an article by Köhler & Milstein, 1975, Nature 256, 495, or by more recent modifications thereof as for example described in Chapter 2 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY, by immortalizing spleen or other antibody producing cells derived from a production species which has been inoculated with an ADAM protease or fragment thereof. It will also be appreciated that antibodies may be produced as recombinant synthetic antibodies or antibody fragments by expressing a nucleic acid encoding the antibody or antibody fragment in an appropriate host cell. Recombinant synthetic antibody or antibody fragment heavy and light chains may be co-expressed from different expression vectors in the same host cell or expressed as a single chain antibody in a host cell. Non-limiting examples of recombinant antibody expression and selection techniques are provided in Chapter 17 of Coligan et al., CURRENT PROTOCOLS IN IMMUNOLOGY and Zuberbuhler et al., 2009, Protein Engineering, Design & Selection 22 169.

[0033] Antibodies and antibody fragments may be modified so as to be administrable to one species having being produced in, or originating from, another species without eliciting a deleterious immune response to the "foreign" antibody. In the context of humans, this is "humanization" of the antibody produced in, or originating from, another species. Such methods are well known in the art and generally involve recombinant "grafting" of non-human antibody complementarity determining regions (CDRs) onto a human antibody scaffold or backbone.

[0034] In some embodiments, the antibody or antibody fragment is labeled.

[0035] The label may be selected from a group including a chromogen, a catalyst, biotin, digoxigenin, an enzyme, a fluorophore, a chemiluminescent molecule, a radioisotope, a drug or other chemotherapeutic agent, a magnetic bead and/or a direct visual label.

[0036] In the case of a direct visual label, use may be made of a colloidal metallic or non-metallic particle, a dye particle, an enzyme or a substrate, an organic polymer, a latex particle, a liposome, or other vesicle containing a signal producing substance and the like.

[0037] The fluorophore may be, for example, fluorescein isothiocyanate (FITC), Alexa dyes, tetramethylrhodamine isothiocyanate (TRITL), allophycocyanin (APC), Texas Red, Cy5, Cy3, or R-Phycoerythrin (RPE) as are well known in the art.

[0038] The enzyme may be horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase or glucose oxidase, although without limitation thereto.

[0039] Suitably, the antibody or antibody fragment specifically or selectively binds or interacts with a proteolytically active form of a human ADAM10 protease. Accordingly, the antibody or antibody fragment may be capable of distunguishing or discriminating between a proteolytically active form of an ADAM10 protease and a proteolytically inactive form of an ADAM10 protease.

[0040] One embodiment of an antibody or antibody fragment disclosed herein is mAb 8C7 which is a monoclonal antibody which specifically or selectively binds or interacts with a proteolytically active form of a human ADAM10 protease. By this is meant that 8C7 monoclonal antibody binds to, or interacts with, a proteolytically active form of an ADAM10 protease but does not bind to or interact with, or binds to or interacts with substantially less affinity, a proteolytically inactive form of an ADAM10 protease. Accordingly, the 8C7 monoclonal antibody may be capable of distinguishing or discriminating between a proteolytically active form of an ADAM10 protease and a proteolytically inactive form of an ADAM10 protease. In one form, the proteolytically active form of the ADAM10 protease may be characterized by the presence of a disulfide bond between: (i) a cysteine residue present in a loop that protrudes away from the ADAM10 cysteine-rich domain, which in ADAM10 comprises substrate-binding residues Glu573, Glu578, Glu589; and (ii) another cysteine in the amino acid sequence CXXC, such as C594 in the ADAM10 sequence C594HVCC598 (SEQ ID NO:3). The loop may comprise two intramolecular disulfide bonds: such as C594-C639 and C632-C645 in ADAM10.

[0041] In further embodiments, the antibody or antibody fragment may be characterized as binding or interacting with one or more human ADAM10 residues selected from the group consisting of: Arg557; Lys591; Pro628; Asp640; Val641; Phe642; Arg644, and Arg646, or the corresponding residues in other non-human mammalian ADAM proteases. Preferably, the binding or interaction includes hydrogen bonds formed between Asp640, Val 641, Phe 642, Arg644, and Arg646 of ADAM10 protease or the corresponding residues in other, non-human mammalian forms of ADAM proteases, and the antibody or antibody fragment.

[0042] Therefore, an antibody or antibody fragment useful according to the invention may broadly be defined as an antibody or antibody fragment characterized as having substantially the same or similar activity as 8C7 mAb in a mammal, with regard to specifically or selectively binding to, or interacting with, a proteolytically active form of an ADAM10 protease.

[0043] The 8C7 mAb comprises heavy and light chain variable region amino acid sequences as set forth below:

8C7 VH Protein Sequence:

8C7 VL Protein Sequence:



[0044] The CDR1, 2 and 3 amino acid sequences are underlined and numbered based on the Kabat numbering system.

[0045] The antibody or antibody fragment may comprise respective light chain and heavy chain variable regions that each comprise CDR 1, 2 and 3 amino acid sequences present in SEQ ID NO:1 and/or SEQ ID NO:2. This includes an amino acid sequence of at least a portion of an entire CDR1, 2 or 3, such as present in SEQ ID NO:1 and/or SEQ ID NO:2. By way of example, this may include at least 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 contiguous amino acids of an entire CDR1, 2 or 3, such as present in SEQ ID NO:1 and/or SEQ ID NO:2. A preferred antibody fragment comprises at least one entire light chain variable region CDR and/or at least one entire heavy chain variable region CDR set forth in SEQ ID NO:1 and/or SEQ ID NO:2.

[0046] In some embodiments, an antibody or antibody fragment may comprise an amino acid sequence at least 85%, at least 90% or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to at an amino acid sequence of least one immunoglobulin heavy chain and/or light chain CDR present in the amino acid sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2.

[0047] In some embodiments, the antibody or antibody fragment may comprise one, two or three immunoglobulin heavy chain and/or light chain CDRs present in the amino acid sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2, or one, two or three immunoglobulin heavy chain and/or light chain CDRs respectively at least 85%, at least 90% or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of a heavy chain and/or light chain CDR present in the amino acid sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2.

[0048] Terms used herein to describe sequence relationships between respective proteins include "comparison window", "sequence identity", "percentage of sequence identity" and "substantial identity". Because respective amino acid sequences may each comprise: (1) only one or more portions of a complete sequence that are shared by respective nucleic acids or proteins, and (2) one or more portions which are divergent between the nucleic acids or proteins, sequence comparisons are typically performed by comparing sequences over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically at least 6, 10, 12, 20 or more contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less (e.g. 5, 10 or 15%) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.

[0049] In one aspect the invention therefore provides an in vitro method of detecting a proteolytically active form of an ADAM10 protease, said method including the step of selectively binding an antibody or antibody fragment to the proteolytically active form of the ADAM10 protease to thereby detect the proteolytically active form of the ADAM10 protease, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645.

[0050] Suitably, the ADAM10 protease is expressed by a cell, including but not limited to lymphocytes, hepatocytes, kidney cells, epithelial cells, neural cells, vascular smooth muscle cells, intestinal epithelium and lung cells, inclusive of normal or non-malignant cells and tumour cells, although without limitation thereto.

[0051] In another aspect, the invention provides an in vitro method of detecting a tumour cell, said method including the step of selectively binding an antibody or antibody fragment to the proteolytically active form of an ADAM10 protease expressed by the tumour cell to thereby detect the tumour cell, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645 and wherein the tumour cell is of a leukemia, lymphoma, lung cancer, colon cancer, adenoma, neuroblastoma, brain tumour, renal tumour, prostate cancer, sarcoma, breast cancer or melanoma.

[0052] As generally used herein, the terms "cancer", "tumour", "malignant" and "malignancy" refer to diseases or conditions, or to cells or tissues associated with the diseases or conditions, characterized by aberrant or abnormal cell proliferation, differentiation and/or migration often accompanied by an aberrant or abnormal molecular phenotype that includes one or more genetic mutations or other genetic changes associated with oncogenesis, expression of tumour markers, loss of tumour suppressor expression or activity and/or aberrant or abnormal cell surface marker expression.

[0053] Some tumour cells typically express relatively increased levels of a proteolytically active form of an ADAM10 protease (e.g. compared to normal or non-malignant cells, whereby an antibody or antibody fragment such as 8C7 mAb is capable of distinguishing tumour cells from normal or non-malignant cells. Typically, the proteolytically active form of the ADAM10 protease expressed by tumour cells is a high molecular weight form of an ADAM10 protease which comprises the intramolecular disulfide bonds as hereinbefore described. ADAM10 proteases may exist in a high molecular weight form ("high molecular weight or HMW ADAM") or in a low molecular weight form ("LMW") as a result of release of an N-terminal prodomain by furin or other pro-protein convertases. Contrary to previous understanding, the inventors have shown that the HMW form of ADAM10 proteases is proteolytically active and that ADAM10 protease does not require release of the N-terminal prodomain to become proteolytically active.

[0054] In some embodiments, the tumour cells co-express relatively increased levels of a proteolytically active, HMW form of an ADAM10 protease and CD133. The tumour cells may further express one or more members of the Notch, Eph and/or erbB receptor families. Non-limiting examples of tumour cells include leukemias and lymphomas, lung cancer, colon cancer, adenomas, neuroblastomas, brain, renal and kidney tumours, prostate cancers, sarcomas and melanoma. For a more comprehensive review of potentially relevant tumours the skilled person is directed to Murphy, G., 2008, Nature Rev Cancer. 8 929-941, Mochizuki S & Okada Y, 2007, Cancer Sci 98, 621-628 Groth, C & Fortini ME, 2012, Semin Cell Dev Biol. 23:465-72.

[0055] It will therefore be understood that an antibody or antibody fragment disclosed herein may be used to detect tumour cells, to assist medical diagnosis of a cancer or cancer symptoms. Suitably, the in vitro method includes detecting the proteolytically active form of the ADAM10 protease expressed by tumour cells present in, or obtained from, a biological sample. In certain embodiments, the biological sample may be a pathology sample that comprises one or more fluids, cells, tissues, organs or organ samples obtained from a mammal. Non-limiting examples include blood, plasma, serum, lymphocytes, urine, faeces, amniotic fluid, cervical samples, cerebrospinal fluid, tissue biopsies, bone marrow and skin, although without limitation thereto.

[0056] Suitably, detection of the proteolytically active ADAM10 protease includes the step of forming a detectable complex between an antibody or antibody fragment and the proteolytically active ADAM10 protease. The complex so formed may be detected by any technique, assay or means known in the art including immunoblotting, immunohistochemistry, immunocytochemistry, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, and biosensor-based detection systems such as surface plasmon resonance, although without limitation thereto.

[0057] To facilitate detection the antibody may be directly labeled as hereinbefore described or a labeled secondary antibody may be used. The labels may be as hereinbefore described.

[0058] In some embodiments, detection methods may be performed in "high throughput" diagnostic tests or procedures such as performed by commercial pathology laboratories or in hospitals.

[0059] It will therefore be appreciated that another aspect of the invention provides a kit for detecting a proteolytically active form of an ADAM10 protease and/or a tumour cell as hereinbefore described, the kit comprising an antibody or antibody fragment as hereinbefore described.

[0060] The antibody or antibody fragment may be labeled with biotin, an enzyme, fluorophore or other label as previously described. Alternatively, the antibody or antibody fragment is unlabeled and a secondary antibody comprises a label. In embodiments where the antibody or antibody fragment or the secondary antibody is labeled with an enzyme, the kit may further comprise an appropriate substrate substrate such as diaminobanzidine (DAB), permanent red, 3-ethylbenzthiazoline sulfonic acid (ABTS), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), 3,3',5,5'-tetramethyl benzidine (TNB) and 4-chloro-1-naphthol (4-CN), although without limitation thereto. A non-limiting example of a chemiluminescent substrate is Luminol™, which is oxidized in the presence of HRP and hydrogen peroxide to form an excited state product (3-aminophthalate).

[0061] The kit may further comprise one or more reaction vessels (e.g multiwell plates, tubes etc), control antibodies and instructions for use.

[0062] Also disclosed herein is a method of inhibiting an ADAM protease and/or one or more downstream signalling molecules in a cell, including the step of binding an antibody or antibody fragment which selectively recognizes a proteolytically active form of the ADAM protease to the ADAM protease expressed by the cell, to thereby at least partly inhibit a biological activity of the ADAM protease and/or one or more downstream signalling molecules in the cell.

[0063] Preferably, the cell is a tumour cell.

[0064] In some embodiments, the tumour cell is of a leukemia, lymphoma, lung cancer, colon cancer, adenoma, neuroblastoma, brain tumour, renal tumour, prostate cancer, sarcoma or melanoma.

[0065] In certain embodiments, the tumour cell expresses CD133. The tumour cell that expresses CD133 may be a cancer stem cell (CSC).

[0066] As disclosed herein, tumour cells typically express relatively increased levels of a proteolytically active form of an ADAM10 protease (e.g. compared to normal or non-malignant cells). Typically, the proteolytically active form of the ADAM10 protease expressed by tumour cells is a high molecular weight form of an ADAM10 protease, although this form may additionally or alternatively comprise the intramolecular disulfide bonds referred to hereinbefore. Furthermore, treatment of human tumour-bearing mice with an antibody such as mAb 8C7 caused a significant, dose-dependent inhibition of tumour growth, with no discernible detrimental effects on the overall health of the mice. The treated tumours also displayed significantly reduced levels of downstream signalling molecules or effectors such as Notch, Eph, and erbB receptors and their ligands as well as MET, suggesting down-regulation of multiple oncogenic signalling pathways. This suggests that antibodies such as 8C7 may act, at least in part, by inhibiting the activation of the ADAM substrate, Notch. Furthermore, antibodies such as mAb 8C7 binding to ADAM10 prevent Notch cleavage and activation and largely blocked lymphoma proliferation, thereby demonstrating effective inhibition of Notch pathway activation. In certain embodiments, antibodies such as 8CT may target and/or deplete tumour cells that express CD133. The tumour cell that expresses CD133 may be a cancer stem cell (CSC).

[0067] Accordingly, it is proposed that, an antibody or antibody fragment disclosed herein may be suitable for administration to mammals, particularly humans, to treat or prevent cancer. As used herein, "treating", "treat" or "treatment" refers to a therapeutic intervention, course of action or protocol that at least ameliorates a symptom of cancer after the cancer and/or its symptoms have at least started to develop. As used herein, "preventing, "prevent" or "prevention" refers to therapeutic intervention, course of action or protocol initiated prior to the onset of cancer and/or a symptom of cancer so as to prevent, inhibit or delay or development or progression of the cancer or the symptom.

[0068] The antibody or antibody fragment suitably targets tumour cells that express proteolytically active ADAM10, as hereinbefore described. Accordingly, the antibody or antibody fragment may be sufficient to suitably inhibit the ability of proteolytically active ADAM10 to activate downstream substrates or effectors such as one or members of the Notch, erbB and Eph signalling pathways, although without limitation thereto.

[0069] In other embodiments, the antibody or antibody fragment may be coupled or conjugated to a cytotoxic agent that facilitates killing or disabling tumour cells that express proteolytically active ADAM10. The cytotoxic agent may be a radionuclide, a chemotherapeutic drug, a mutagen, a toxin, a mitosis inhibitor or other antiproliferative agent, a pro-apoptotic agent, a DNA intercalating agent or any other agent that assists or causes killing or disabling of tumour cells. Non-limiting examples of radionuclides include 211At, 212Bl, 213Bi, 125I, 111In, 193Pt and 67Ga, although without limitation thereto.

[0070] Chemotherapeutic drugs, mutagens, toxins, mitosis inhibitors, pro-apoptotic agents and DNA intercalating agents may include doxorubicin, N-acetyl-γ-calicheamicin, maytansinoids, taxoids, auristatins and duocarmycins, although without limitation thereto. Chemotherapeutic drugs, mutagens, toxins, mitosis inhibitors, pro-apoptotic agents and DNA intercalating agents may be coupled to the antibody or antibody fragment by a cleavable or non-cleavable linker to form an antibody-drug conjugate (ADC). Typically, the ADC is internalized by the tumour cell where the cleavable linker is cleaved to release the drug into the cell. In the case of non-cleavable linkers, these may be preferred where it is essential that the drug is entirely localized to the targeted tumour cell and there is no "leakage" of the drug from the targeted tumour cell into adjacent cells, tissues or fluids. In some embodiments, the chemotherapeutic drugs, mutagens, toxins, mitosis inhibitors, pro-apoptotic and DNA intercalating agents may be in the form of a pro-drug which is activated upon internalization inside a targeted tumour cell.

[0071] Also disclosed herein is the method and/or composition which may include a combination therapy where the antibody or antibody fragment disclosed herein (e.g 8C7 mAb) is combined with another therapy including but not limited to radiotherapy, chemotherapy, natural or holistic therapies or other immunotherapies that target cancers. It may also be disclosed that by way of example, the antibody or antibody fragment disclosed herein (e.g 8C7 mAb) may be administered in combination with another therapeutic agent such as one or more chemotherapeutic drugs, antibodies, mutagens, toxins, mitosis inhibitors, pro-apoptotic agents and/or DNA intercalating agents, such as hereinbefore described. In these embodiments, the antibody or antibody fragment and said another therapeutic agent are not coupled or conjugated, but are administered as separate molecules together or sequentially. A non-limiting example is combination therapy with irenotecan, a chemotherapeutic agent used to treat colorectal cancer.

[0072] Further, disclosed herein is that the antibody or antibody fragment and optionally, said another therapeutic agent, is administered to a mammal as a pharmaceutical composition comprising a pharmaceutically-acceptable carrier, diluent or excipient.

[0073] By "pharmaceutically-acceptable carrier, diluent or excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, liposomes and other lipid-based carriers, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water.

[0074] A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991), which is incorporated herein by reference.

[0075] Any safe route of administration may be employed for providing a patient with the composition of the disclosure. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of immunotherapeutic compositions, proteinaceous vaccines and nucleic acid vaccines.

[0076] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

[0077] Compositions of the present disclosure suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

[0078] The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present disclosure, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

[0079] The methods and compositions may be applicable to any mammal in which an ADAM10 protease is expressed by tumour cells.

[0080] In some embodiments, the tumours comprise tumour cells that co-express relatively increased levels of a proteolytically active form of an ADAM10 protease and CD133. The tumour cells may further express one or more members of the Notch, Eph and/or erbB receptor families. Non-limiting examples of cancers or tumours that may be treated or prevented include leukemias and lymphomas, lung cancer, colon cancer, adenomas, neuroblastomas, brain, renal and kidney tumours, prostate cancers, sarcomas and melanoma.

[0081] In particular embodiments, the term "mammal" includes but is not limited to humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs).

[0082] So that the present invention may be more readily understood and put into practical effect, the skilled person is referred to the following non-limiting examples.

EXAMPLES


INTRODUCTION



[0083] We previously defined the structure of the ADAM10 D+C domains and identified a substrate-binding pocket within the C domain that specifies ligand cleavage6. To further define the function of the substrate binding pocket, we raised antibodies against ADAM10, one of which, mAb8C7, specifically recognises the substrate binding domain, and inhibits ADAM10-mediated cleavage of Eph receptor ligands (ephrins) and ephrin/Eph-dependent signalling and cell segregation in vitro27. We thus sought to test 8C7 as a targeted therapeutic in tumour models. We find 8C7 treatment inhibits tumour growth of human colon xenografts, at least in part due to inhibition of Notch signalling, and down-regulation of Notch, EGFR and Eph receptor levels. In line with the critical role of Notch in maintenance of an undifferentiated cell phenotype, including that of tumour initiating colon cancer cells28,29, we find 8C7 selectively targets tumour cells expressing the putative cancer stem cell (CSC) marker CD133 and displaying active Notch-signalling within tumours30. CSCs are known to contribute to chemotherapy resistance, and accordingly, 8C7 reduces CSC levels and effectively inhibits tumour re-growth following chemotherapy. Importantly, we demonstrate that 8C7 selectively binds an active form of ADAM10, which we find to be over-expressed in mouse tumour models and in several human solid tumours, suggesting its potential as human cancer therapeutic and diagnostic.

MATERIALS & METHODS


Cell Culture and reagents



[0084] Human colorectal carcinoma cell line LIM1250 (obtained from the Ludwig Institute for Cancer Research, Melbourne, Australia) was maintained in RPMI 1640/10% FCS in 10% CO2/90% air. ADAM10-/- mouse embryonic fibroblasts5 were maintained in DMEM 10% FCS in 5% CO2/95% air atmosphere.

Mutagenesis



[0085] The sequence encoding full length human (OriGene) ADAM10 was subcloned into pcDNA3.1 vector (Invitrogen), including an N-terminal prolactin signal sequence and a C-terminal thrombin cleavage site. AxxA point mutation to human ADAM10-turbo-myc(OriGene) was introduced by site-directed mutagenesis (Quickchange XL, Stratagene) by introducing Alanines at Cys594 and Cys597 respectively. Mutants were transfected into MEF ADAM10-/- using X-treme transfection reagent (Roche) to confirm its expression by Western blot and Immunofluorence.

Immunoprecipitation and Western Blotting



[0086] Frozen human tissue samples were obtained from surgical biopsies between 1995 and 2007, held at the Austin Health Tissue Bank, under approval from the Monash University Human Research Ethics Committee. For protein analysis of tumours and tissues, 50mg tissue was homogenised in 1ml RIPA buffer (50mM Tris-HCL pH 7.4, 150mM NaCl, 1% Triton-X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, phosphatase and protease inhibitor cocktail); and protein concentration determined by BCA assay (Pierce). 20ug of total lysates or immunoprecipitates from 100ug of lysates were analysed by SDS-PAGE and Western blot. After various treatments, including exposure to 1mM DTT, 40mM H2O2, 1 µg/mL EGF or 1.5 µg/mL cross-linked EphrinA5-Fc{Lawrenson, 2002 #237}, and after 3 washes in PBS cell lysates were prepared in KALB lysis buffer (1.5 M NaCl, 0.5 M HEPES, 0.05 M EDTA, pH 7.4, 1% Triton X-100, phosphatase and protease inhibitor cocktail); loading levels for analysis were equalised on the basis of actin levels by performing Western blot analysis on cell lysates. Immunoprecipitation was done with mAb8C7 or with mAb4A11-coupled to Minileak beads (according to manufacturers' instructions, KEM EN TEC, Denmark), or with soluble 8C7 or anti-ADAM10 polyclonal antibodies (pAb) (Abcam pAb 39177) followed by Protein A Sepharose. ADAM10 was detected on Western blots with Abcam pAb 39177 and anti-rabbit-HRP secondary antibodies and visualized using an ECL substrate (Supersignal; Thermo Fisher Scientific). Samples reduced in SDS-samples buffer with 20 mM Dithiothreitol (5 min, 95°C) or left non-reduced were routinely analysed.

Maleimide-Biotinylation of free cysteine residues



[0087] LIM1215 colon carcinoma cells were starved overnight in serum free RPMI and used at 80% confluency. Cells were treated with 2.5mM Methyl-PEG12-maleimide (MPM, Thermo Scientific) in serum free RPMI for 30 min at 4° C to label any free cell surface protein cysteines. After three washes (PBS) cells were treated at room temperature with 5 µg/ml Protein Disulfide Isomerase (PDI, Sigma), or 10 µM DTT for indicated times. Following washes (PBS), free cysteines that formed after PDI treatment was reacted with Maleimide-PEG2-biotin (MPB) (Thermo Scientific) in serum free RPMI for 1 hour at 4° C. For analysis, washed (PBS) cells were lysed in KALB lysis buffer containing 5 mM N-ethylmaleimide prior to IP and Western blot analysis.

[0088] In-vitro biotinylation of 8C7 and 4A11 immunoprecipitates was performed in a similar manner using total washed IPs from LIM1215 cells.

Furin cleavage



[0089] mAb8C7 and 4A11 IPs from LIM1215 cell lysates were treated with furin (1 unit/ 100gl, New England Biolabs) in a buffer specified by the supplier (100 mM HEPES, pH 7.5 @25°C, 0.5% Triton X-100, 1 mM CaCl2) for 2 hours at 37° C. Samples were washed three times with PBS, prior to treatment with SDS-PAGE sample buffer (95°C, 5 min) and Western blot analysis.

Activity assay



[0090] ADAM10 was immunoprecipitated (IPed) by mixing 8C7 and 4A11 mAbs coupled to mini-leak beads with LIM1215 colon carcinoma cell lysates and human colorectal tumour lysates. IPs were treated with Mca-PLAQAV-Dpa-RSSSR-NH2 fluorogenic peptide substrate (R&D Systems, USA, cat N°, ES003), 10µM at 37 ° C for 1 hour, or using HPLC-purified monomeric ephrinA5-Fc{Vearing, 2005 #3050} at 50µg/mL at room temperature for 20 mins. Fluorescence substrate supernatants were analysed using a FLUOstar OPTIMA (BMG Labtech) plate reader at 320nm excitation and 405 emission wavelengths, cleavage of ephrin-A5 Fc analysed by Western blot analysis with α-ephrinA5 antibodies (R&D Systems, USA).

Tissue Immunohistochemistry and Immunofluorescence



[0091] Tissues were OCT-embedded (Tissue-TEK), sectioned (6-µm) and fixed (10 min, acetone). For IHC, Vector Labs ABC secondary antibody staining kit was used to detect the bound primary antibodies. Sections, counterstained with Haemotoxylin were imaged on an Olympus DotSlide system. For TUNEL, tumours were formalin fixed and paraffin-embedded. For IF, sections were incubated with directly conjugated primary or secondary antibodies, nuclei counterstained with Hoechst, and slides mounted with Fluoromount (Southern Biotech, Birmingham, AL USA) for imaging on a Leica confocal (SP5) microscope.

Flow cytometry



[0092] Single cell suspensions were made from LIM1215 tumours by digesting tumour pieces with Collagenase Type 3 / Deoxyribonuclease I (Worthington Biochemical Corp.) in HBBS (Gibco, Invitrogen, 1 h, 37°C), filtering through successive 40 µm and 20 µm sieves and treatment with Red Blood cell lysing buffer (Sigma-Aldrich). Cells were labelled with conjugated anti-CD 133 (Miltenyi Biotec) and FACS sorted on an Influx Cell Sorter (BD biosciences). Dead cells were detected with propidium iodide. For analysis of cell cultures, trypsinised cells were washed with FACS buffer (PBS, 1%FCS, 1mM EDTA), and labelled for 30 min at 4°C with fluorophore-conjugated mAbs as indicated, before analysis on a LSRII flow cytometer (Beckton Dickenson). Subsequent analysis was with FLOWJO software (TreeStar, CA).

Detection of reactive oxygen species (ROS) in tumour cell isolates



[0093] Tumours from mice injected with alexa-labelled 8C7 (100ug) were recovered and cell suspensions prepared as above. FACS sorted 8C7 bound/unbound tumour cells were tested using a reaction mix that included 50µM Amplex Red (Invitrogen) and 0.1U/ml HRP (Invitrogen) in Krebs Ringer Phosphate. 20µl of 8C7 bound tumour cells (a total of 5x104) were added to 100µl of the pre-warmed reaction mixture and incubated 1 hour at 37°C. A microplate reader (CLARIOstar, BMG Labtech) was used to measure fluorescence (Excitation 530-560nm and emission peak 590nm).

Xenograft experiments



[0094] Athymic nude mice (BALB/c, 4- to 6-week old; male) were from Animal Resources Centre, Canning Vale, WA, Australia. Animal procedures were conducted in accordance with guidelines of the Monash University Animal Ethics Committee. 7x106 LIM1250 cells in 200ul PBS/30% growth factor reduced Matrigel (BD Biosciences) were injected subcutaneously in the dorsal flanks of the mice. Once tumour volumes reached 75 - 150 mm3 (measured by callipers), mice were treated twice weekly by IP injection with either PBS, 8C7 or control antibody as indicated. Tumours and tissues were recovered for protein analysis, imaging by immunofluorescence microscopy or IHC, or flow cytometry.

Crystallization, Data Collection and Structure Determination


Expression and purification of ADAM10 disintegrin (D) and cysteine (C) rich domain



[0095] The sequence encoding bovine ADAM10 disintegrin and cysteine-rich domains (residues 455-646) (ADAM10: D+C) was subcloned as Fc fusion into a pcDNA3.1 vector (Invitrogen), including an N-terminal prolactin signal sequence and a C-terminal thrombin cleavage site followed by the Fc domain of human IgG, for expression in human embryonic kidney 293 (HEK293) cells. A stable cell line expressing ADAM10:D+C was generated. Roller bottles were used for large-scale expression of the protein. The initial purification was performed on a Protein-A-Sepharose (Amersham) column and the protein was eluted with 100mM glycine, 150mM NaCl pH 3.0. The Fc tag was removed by thrombin cleavage, and ADAM10:D+C was further purified to homogeneity by gel filtration chromatography (SD-200, buffer 20mM HEPES, 150mM NaCl, pH7.5). The protein eluted as a monomer (33 kDa). The two ADAM10:D+C bands on the SDS gels correspond to differently glycosylated forms of the recombinant protein and converge into a single band upon enzymatic deglycosylation.

Preparation of F(ab')2 fragment of the 8C7 monoclonal antibody



[0096] The F(ab')2 fragment was prepared by digesting the whole IgG (8C7) with pepsin at pH 3.0 (enzyme: substrate ratio 1:100). The reaction was terminated after 2 hrs incubation at room temperature by raising the pH to 8.0. The final purification was performed using gel filtration chromatography (SD-200, buffer 20 mM HEPES, 150mM NaCl, pH 7.5). The protein eluted as a monomer with molecular weight of approximately 110 kDa.

Crystallization and Structure Determination



[0097] For crystallization trials, ADAM10:D+C was mixed with F(ab')2 at 2:1 molar ratio (final concentration 20mg/ml) in a buffer containing 20mM HEPES, 150mM NaCl, and pH 7.5. The complex was crystallized in a hanging drop by vapor diffusion at room temperature against a reservoir containing 0.1M HEPES, 0.2M NaCl, and 1.6M ammonium sulfate pH 7.5. Sizable crystals, (space group P212121; Unit cell: 53.2, 141.7, 268.1, 90.0, 90.0, 90.0) grew after 2 months but could be reproduced in 2 to 3 days using the additive 30% 1, 4 Dioxane. The structure was determined at 2.75 A resolution using molecular replacement.

RESULTS


Systemic treatment with the anti-ADAM10 mAb 8C7 inhibits tumour growth, preferentially targeting Notch-active CD133+ tumour cells.



[0098] We sought to test the effect of anti-ADAM10 mAb8C7 on tumours which co-express high levels of the ADAM10-dependent Notch, Eph, and erbB receptor families. By screening a panel of human colorectal cancer (CRC) cell lines in which these pathways are commonly de-regulated31, we selected LIM1215 cells, which express high levels of EGFR, EphA2 and ADAM10, are known to be dependent on autocrine EGFR signalling in a mouse xenograft setting32, and also express high levels of Notch1 and 2, and the notch ligands Jagged 1 and 2.

[0099] Systemic treatment of mice bearing LIM1215 xenografts with mAb8C7 (8C7) caused a significant, dose-dependent inhibition of tumour growth, as measured by tumour volume and weight (Fig 1A,B), but with no discernible detrimental effects on overall health of the mice as measured by their weight (Fig 10A). In comparison, the control mAb8E11, raised alongside 8C7 but not recognising ADAM10, did not inhibit tumour growth (Fig 10B). The treated tumours also displayed less vascular staining (α-CD31, Fig 1C), and increased apoptosis (TUNEL staining, Fig 1D), suggesting inhibitory effects on blood supply. Western blot analysis of tumour protein extracts showed significantly reduced levels of notch, Eph, and erbB receptors and their ligands (Fig 1E, Fig 10C, D), as well as MET, suggesting down-regulation of multiple oncogenic signalling pathways. Considering the known role of Notch to promote neoangiogenesis, and to transcriptionally control33 EGFR34, Ephs and ephrins35,36 and its own expression, this suggests that 8C7 may act, at least in part, by inhibiting the activation of the prominent ADAM10 substrate, Notch.

[0100] To analyse 8C7 targeting within the tumour, we injected LIM tumour xenograft-bearing mice with Alexa647-labelled 8C7 and, to label the vasculature, three days later with Rhodamine-labelled Ricininus Communis Agglutinin ([Rhodamine] RCA) -lectin, before tumours were resected and sections analysed by confocal microscopy. We observed mAb8C7 binding to the tumour tissue, particularly within the outer margin and around blood vessels, visible by [Rhodamine] RCA-lectin staining (Fig 2A). In contrast there was detectable, but reduced mAb8C7-immuno reactivity within the bulk of the tumour.

[0101] Recently, colon carcinoma initiating cells, marked by expression of the putative stem cell marker CD133 and by active notch signalling, have been found, selectively associated with Notch ligand Jagged-1-expressing vascular endothelial cells, in colon tumour xenografts30. We therefore co-stained 8C7-injected LIM1215 colon tumour xenografts with antibodies against CD133 and against the Notch intracellular domain (NICD), generated during active Notch signalling by serial cleavage by α- and γ-secretases. Notably, mAb 8C7 preferentially targeted an anti-CD 133-stained cell population, in addition to co-staining anti-NICDl and NICD2 positive cells (Fig 2B), overall confirming activated Notch receptor signalling in these putative tumour-initiating cells. Interestingly, the lectin-labelled vessel cells co-stained with antibodies against Jagged1, consistent with reported endothelial expression of Jagged130, whereas Jagged2 was found on a separate cell population within the same microenvironment (Fig 11). FACS analysis of dissociated tumours from mice injected with Alexa647-8C7 showed significant tumour staining, whereby, in support of the imaging data, cells with brightest 8C7 staining were also CD133+ (Fig 2D). We also isolated CD133-enriched and depleted cell populations from tumours which, when compared for active NICD1 levels by Western blot, clearly confirmed high levels of Notch activity in the CD133-enriched population (Fig 2C).

mAb 8C7 binding to ADAM10 prevents Notch cleavage and activation



[0102] Since our anti-ADAM10 mAb 8C7 preferentially targeted cells with active Notch signalling and inhibited tumour growth, and we previously showed its ability to inhibit ADAM10-mediated Eph receptor signalling27, we sought to determine if 8C7 also inhibits Notch signalling. We treated mice bearing LIM1215 tumours for 3 weeks (when tumour growth rate was most affected) before recovering tumours and analysing by Western blot with anti-NICDl antibody. Comparison of 8C7 and control-treated tumours showed a significant decrease in levels of active Notch (Fig 3A). Furthermore the Notch target Hes1 was also substantially decreased in the tumours from treated mice, as confirmed by quantitative PCR (Fig 3B). We thus tested if this effect was due to direct inhibition of ADAM10-mediated Notch activation by 8C7, by in vitro treatment of CD133+-enriched tumour cell isolates with 8C7, the control mAb 8E11, or with γ-secretase inhibitor (GSI). 4h-treatment with 8C7, but not with the control mAb, decreased NICD1 levels significantly and comparable to inhibition with GSI (Fig 3C).

[0103] We also used a co-culture model in which Notch-dependent lymphoma survival and proliferation is afforded by contact with Jagged1-expressing vascular endothelial cell (HUVEC) feeder cells, which have been transduced with the adenoviral gene fragment E4ORF1 to drive Akt auto-activation and allow their serum-free propagation37 (Fig 12A). Treatment with mAb 8C7 largely blocked lymphoma proliferation in this setting (Fig 12B, C), demonstrating its effective inhibition of Notch pathway activation.

mAb 8C7 preferentially recognises a high molecular weight form of ADAM10 found predominantly in tumour tissues.



[0104] The preferential binding of 8C7 to tumour cells containing elevated Notch signalling prompted us to further investigate the selectivity of 8C7 in tumour-bearing mice. While we previously demonstrated 8C7 binding to mouse ADAM1027, comparison of tumour and organ tissues from mice, injected 3 days prior with Alexa647-labelled 8C7, showed robust staining in tumours, as compared to undetectable staining in all other organs tested, apart from the liver (Fig 4A). However, it is likely that the low liver staining is due to metastasis of the human tumour cells, previously described for this model, as a control antibody with specificity for human ADAM10 only (4A11) showed similar staining of the liver. By contrast, a control antibody recognising both, human and mouse ADAM10, strongly stained multiple mouse tissues (Fig. 4A, last column). We next analysed tumours and organs from mice treated long term with high (1mg) doses of 8C7, or untreated mice, by incubating detergent homogenates with Protein A Sepharose to recover 8C7 and bound ADAM10. ADAM10 was clearly detected in Protein A pull-downs from tumour tissue (Fig 4B, top), while other tissue showed much lower or undetectable levels. By contrast, immunoprecipitation with a control ADAM10 mAb suggested expression of ADAM10 in a range of tissue (apart from colon and intestine) at varying levels (Fig 4B, bottom). Interestingly, the control mAb detected ADAM10 in these non-tumour tissues predominately as a processed low molecular weight (LMW) form of approximately 64 kDa, whereas in tumours it also recognised the unprocessed high molecular weight (HMW) form, which is preferentially recognised by 8C7.

[0105] To assess if 8C7 also recognises the ADAM10 HMW form in other tumour models, we analysed stomach tissue from GP130F knock-in mice38,39, a transgenic mouse strain that spontaneously forms stomach adenomas in 5-6 weeks old mice. While at 3.5 weeks the HMW ADAM10 was barely detectable in stomach tissue, at 5 and 6 weeks the HMW form was clearly elevated, both in stomach adenomas and in associated stomach tissue, but not in similarly aged wild-type (Wt) mice (Fig 5A). We have also tested effects of 8C7 on tumour formation in the GP130F knock-in mice. Treatment of mice from around 4 weeks of age for 5 weeks caused a significant inhibition of tumour formation (Figure 18).

[0106] Considering the apparent diagnostic detection by 8C7 of the HMW form of ADAM10 in mouse tumours, we then went on to test human tumour tissues by immunoprecipitation (IP) with 8C7 or a control anti-human ADAM10 mAb (4A11). 4A11 IPs from colon tumour tissue showed a somewhat increased ratio of HMW/LMW ADAM10 compared to apparently normal colon tissue, while remarkably 8C7 specifically recognised the HMW form in human tumour tissue lysate (Fig 5B). Similarly, 8C7 also specifically bound HMW ADAM10 in lysates from breast, prostate and lung tumours, while 4A11 again bound both forms. Interestingly, 4A11 also detected some HMW ADAM10 in glioblastoma tissue whilst in benign brain tissues lower levels only the LMW form were detected, a finding verified in an independent set of non-tumour brain samples (not shown).

The HMW form of ADAM10 recognised by 8C7 represents the active protease.



[0107] The above data suggests that 8C7 preferentially targets an unprocessed form of ADAM10 that appears to be elevated in tumours, whereby conversion of full length unprocessed ADAM10 involves release by furin or other pro-protein convertases of the N-terminal prodomain, shown to control ADAM stability and protease activity40. We confirmed the HMW form as non-processed ADAM10, since incubation with furin resulted in conversion into LMW ADAM10 (Fig 13A). We confirmed by mass spectometry of SDS-PAGE-resolved HMW and LMW ADAM10 that Pro domain peptides are present only in the HMW band (not shown). Furthermore, we confirmed that the unprocessed form of ADAM10 is present on the cell surface, as suggested by its recognition by 8C7 in treated mice (above): by binding 8C7 to intact LIM1215 cells under culture conditions where endocytosis is inhibited (incubation on ice or in the presence of sucrose), we found a distinct enrichment of HMW ADAM10 in the Protein A-captured IP, as compared to control cells incubated at 37°C. Using the same protocol, the control anti-human ADAM10 mAb 4A11 bound similar amounts of processed and non-processed ADAM10, further confirming the presence also of HMW ADAM10 on the cell surface (Fig 13B).

[0108] Pro-domain processing is generally considered to generate the active form of ADAMs by releasing the Pro domain, which in turn can interact with and inhibit the mature metalloprotease, although for ADAM10 and 17 this does not occur via the cysteine switch mechanism of other metalloproteases40. Indeed, in the case of ADAM10 and 17, the Pro domain may have a necessary chaperone function, since addition of recombinant Pro domain rescues the activity of an inactive, prodomain-deleted form of ADAM1041, while ADAM17 can be activated on the cell surface without requiring removal of the Pro domain42. We therefore tested if 8C7 targeted ADAM10 represents an active or inactive population. MAb 8C7 and 4A11 IP from LIM1215 cells again showed preferential binding of 8C7 to ADAM10 enriched in the HMW form, while 4A11, similar to a commercial pAb against the cytoplasmic domain27, appeared to bind non-discriminately to both HMW and LMW bands (Fig 6A, top panels). To test for ADAM sheddase activity, we incubated the IPs with recombinant ephrin-A5/human Fc fusion protein, a known ADAM10 substrate6, or with a quenched fluorogenic peptide substrate, which fluoresces only when cleaved, and related the measured activity to the amount of ADAM10 determined by Western blot with a C-terminal-specific anti-ADAMlO antibody. Both assays revealed that 8C7 IPs contained much higher sheddase activity compared to 4A11 IPs (Fig 6), suggesting that 8C7 selectively targets an active form of ADAM10. Using the same assays to assess ADAM10 from human colon tumour tissue, in which 8C7 preferentially bound the HMW form, we confirmed that 8C7 IPs contained significantly higher sheddase activity than 4A11 IPs (Fig. 6B), indicating that 8C7 selectively recognises an active population of HMW ADAM10. A higher relative sheddase activity in 4A11 IPs in tumour versus LIM cell lysates likely reflects an overall greater proportion of active ADAM10 in tumour tissue, also captured by the 4A11 mAb, which does not discriminate between active and inactive ADAM10. Furthermore, since 8C7 preferentially binds unprocessed ADAM10, this suggests that processing is not required for ADAM10 activity, similar to ADAM1742. We verified this notion by comparing ADAM10 activity in 8C7 IPs, treated with or without furin, revealing no difference in sheddase activity in treated or untreated samples (Fig 14).

The 8C7 binding epitope implicates the ADAM10 thioredoxin CXXC motif, involved in regulating ADAM10 conformation and activity



[0109] We previously demonstrated that 8C7 binds the Cysteine-rich (C) domain of ADAM1027, and earlier had elucidated its protein structure in context with the adjacent Disintegrin (D) domain, revealing a continuous, elongated, slightly curved structure harbouring a negatively charged pocket mediating ADAM10-substrate recognition6. To define the exact binding site of 8C7 we determined the protein crystal structure of the ADAM10-D+C domain fragment in complex with a F(ab')2 fragment of 8C7 at 2.8 Å resolution (Table 1). A model of the structure of the complex is illustrated in Fig. 7, revealing an overall conformation very similar to that elucidated for the unbound ADAM10D+C6. The antibody complementary determining region (CDR) targets the C domain by interacting with Arg557, Asp589, Lys591, Pro628, Tyr 638, Cys 639. Asp640, Val 641, Phe 642, Arg644, and Arg646, including hydrogen bonds between Asp640, Val 641, Phe 642, Arg644, and Arg646 and 8C7 light chain CDR residues (Table 2). This epitope comprises a loop structure that protrudes away from the C-terminal part of the ADAM10 cystein-rich domain, harbouring the substrate-binding pocket encompassing residues Glu573, Glu578, Glu5896 and is stabilised by two intramolecular disulfide bonds, C594-C639 and C632-C645, one of which forms part of the ADAM10 sequence, C594HVCC598 (Fig 7A, B). Interestingly, this sequence represents a conserved thioredoxin CxxC motif, a consensus sequence for protein disulfide isomerase (PDI)-catalysed disulfide exchange reactions that is found in the analogous position of ADAM17. In the case of ADAM17, this motif is necessary for the modulation of protease activity by PDI24 and by redox changes. Thus, oxidising conditions were found to promote ADAM17 activity43, thought to be triggered by conformational changes resulting from disulfide isomerisation, a notion that was supported by the finding that PDI treatment alters ADAM17 recognition by conformation specific antibodies44.

[0110] In view of these findings, we tested if 8C7 binding to ADAM10 was dependent on modulation of the CxxC motif. Alanine replacement mutation to AxxA clearly ablated binding of 8C7, but not of control antibodies (Fig 8A), whereby comparable anti-ADAMlO cell surface staining of ADAM10-/- mouse embryonic stem cells (MEFs)5, transfected with either Wt or AXXA-mutant ADAM10, suggested that this mutation did not effect cell surface expression of ADAM10 (Fig 15). Treatment of LIM1215 cells with H2O2 significantly increased 8C7 binding to ADAM10 compared to the control mAb 4A11, while reducing conditions inhibited binding (Fig 8B). Similarly, stimulation of cells either with EGF to activate the EGF receptor (known to trigger local release of reactive oxygen species (ROS)45), or with ephrin-A5 (stimulating EphA receptor signalling), increased binding of 8C7 to ADAM10.

[0111] We also found 8C7-bound cells recovered from tumours showed markedly higher production of reactive oxygen species (ROS) compared to unbound (8C7 negative) cells (Fig 16). Tumours are high in ROS due to elevated RTK and pro-inflammatory signalling, and oxidative stress53,54. Interestingly, cancer stem cells (CSCs) are protected from toxic effects conferred by elevated ROS levels by expression of aldehyde dehydrogenase (ALDH), enabling them to maintain high levels of ROS55. Indeed, we also find 8C7 particularly targets cells co-staining with the CSC marker CD133 (Fig 2), consistent with 8C7 targeting CSCs producing high ROS levels.

[0112] Together these observations supported the notion that the preferential binding of 8C7 to the active conformation of ADAM10 may be modulated by disulfide rearrangement within the CXXC motif. We therefore assessed if ADAM10 does contain PDI-modulated cysteines, as recently suggested from MS analysis of labile cysteins in leukocyte cell surface proteins46. Following blocking of free cysteines with Methyl-PEG12-maleimide (MPM), 8C7 and 4A11 IPs from LIM1215 lysates were treated first with PDI, followed by Maleimide-PEG2-biotin (MPB) to label the free cysteines that had been generated via PDI activity. Western blot analysis of IPs using anti-ADAMlO antibodies and Streptavidin-HRP to detect cysteine-biotinylated proteins, revealed that PDI caused a marked increase in biotin labelled ADAM10, especially from 8C7 IPs (Fig 9A), suggesting that similar to ADAM1744, PDI preferentially acts on the active ADAM10 conformation. We performed the same experiments to also label ADAM10 on intact LIM1215 cells, which had been pretreated with the PDI inhibitor Bacitracin to limit endogenous PDI activity, and analysed 8C7 or 4A11 IPs from cell lysates by Western blot with Streptavidin-HRP or anti-ADAMlO antibodies to detect cysteine-biotinylated ADAM10. While Bacitracin treatment did not abrogate the presence of labile ADAM10 cysteins, it allowed detecting a moderate but distinct increase in MPB labelling of ADAM10 after PDI treatment (Fig 9B). Significantly, comparison of Western blot probed with Streptavidin or with anti-ADAMlO antibodies revealed that MPB labelling completely prevented 8C7, but not 4A11, from binding to ADAM10, suggesting that the C594-C639 interaction located within the 8C7 binding epitope indeed contains a labile cysteine, so that attachment of MPB to the released cysteine or the corresponding change in protein conformation ablated 8C7 binding. We assessed involvement of the C594HVCC598 motif in PDI-catalysed disulphide exchange by examining MPM labelling in ADAM10-/- MEFs transfected with Wt or A594HVA597 mutant ADAM10. Western blot analysis with Streptavidin-HRP and with anti-ADAM10 antibodies of IPs after serial treatment of cells with MPM, PDI and MPB shows that PDI-induced labelling with MPB is considerably reduced in IPs of mutant A594HVA597 ADAM10, strongly implicating this motif in PDI-facilitated disulfide exchange (Fig 9C).

[0113] We then investigated if the 8C7-targeted, active ADAM10 displays distinct protein interactions in cells compared to the overall interactions of ADAM10 bound by the non-selective 4A11 antibody. We used 8C7 and 4A11 immunoprecipitates from LIM1215 cell lysates, equalised for ADAM10 levels by Western blot, and resolved by SDS-PAGE and Coomassie Blue staining. Excised bands of various MW were then analysed by Mass Spectrometry to generate an unbiased comparison of 8C7- and 4A11-co-immunoprecipiated proteins. This revealed some marked differences, including preferential association of 8C7-bound ADAM10 with notch and other ADAM-regulated receptors. We confirmed this by co-immunoprecipitation and Western blot (Fig 19). Notably, 8C7 IPs were also enriched in PDIs, especially PDIA1 (over 50-fold more peptides detected), compared to 4A11 IPs (see also Fig 19). PDIA3, A4 and A6 were also detected. This confirms PDIs are indeed involved in regulating ADAM10 in cells, and that the conformation recognised by 8C7 is preferentially targeted by PDIs, as suggested by biotinylation experiments (Fig 9A).

[0114] Cancer stem-cells maintained by Notch signalling are thought to contribute to tumour chemoresistance, as well as metastasis and epithelial to mesenchymal transition (EMT)56,57. Since 8C7 particularly targets these cells, as indicated by co-staining with the stem cell marker CD133 and active Notch (NICD) (Fig 2), we therefore tested 8C7 treatment of LIM1215 xenografts in combination with irenotecan, a chemotherapeutic used clinically for CRC. Under irenotecan treatment tumours regressed, but relapsed after 6 weeks of injections. However, 8C7 treatment was able to block this relapse, indicating effective inhibition of cells remaining after chemotherapy, with 40% of tumours completely regressing (Figure 17A,B). Staining of CD133+ cells from remaining tumours showed a marked decrease in 8C7-treated mice compared to control (Figure 17C), consistent with 8C7 treatment targeting chemo-resistant tumour stem cells.

[0115] 8C7 clearly inhibits signalling by Notch (Fig 3), and also Eph receptors59, however the antibody does not interact directly with residues identified as contributing to substrate binding60, and indeed we find 8C7 does not inhibit substrate interaction. Rather 8C7-bound ADAM10 preferentially interacts with substrates, including notch and erbB receptors, suggesting the active conformation has a more accessible substrate-binding pocket (Fig 19A). To understand its possible mechanism of action, in the absence of a full length structure of ADAM10 we compared our 8C7-bound ADAM10 D+C structure with available full-length structures of snake venom proteases, which contain a similar overall M+D+C domain architecture and primary sequence cysteine patterns similar to that of the mammalian ADAMs. Interestingly, the MP domain generally resides within the concave site of the D+C region such that, assuming the mammalian ADAMs have a similar overall architecture, binding of 8C7 to the ADAM10 D+C region would displace the MP domain (Fig. 19B). This provides a potential molecular mechanism for ADAM10 activity inhibition by this antibody.

[0116] Lastly, the association of 8C7-bound ADAM10 with erbB receptors lead us to test effects of 8C7 on proliferation of breast cancer cells over-expressing these receptors, in which they drive proliferation. Indeed, 8C7 was able to inhibit proliferation of breast cancer cell lines SKBR3 (high erbB2) and MDA-MB-231 (high EGFR/erbB1), as well as LIM1215 colon cancer cells (Fig 20).

DISCUSSION



[0117] Emerging evidence suggests that ADAM10 and 17 activities may be regulated by adopting latent and active conformations. This suggestion was first based on crystal structures of the related snake venom metalloproteinases, which adopt two different conformations that are stabilized by distinct disulphide connectivity of the D domain19,20: a closed, C-shaped conformation seen in Atragin, that positions the active site cleft towards the substrate-binding C domain, suggested to deny substrate access; and an open, I-shaped conformation, exemplified by 'K-like', suggested to allow substrate access and promote cleavage20. The ADAM10D+C structure does not correlate directly with either form - while it shows sequence homology with Atragin it has a distinct disulfide bond pattern6. However, the C-terminal part of the D domain and the N-terminal part of the C domain of the ADAM10 structure resembles the analogous region of the snake venom ADAM homologue VAP1 (termed the Da and Cw domains), being super-imposable and having an analogous disulfide pattern19. The structure differs however in orientation of the C-terminal part of the C domain (the Ch domain) due to an additional sequence loop and differing disulfide bonds around it, leading to its reversed orientation relative to the Cw/Da domains. Notably, the distinct disulfide linked Cysteines include the redoxin CxxC motif, which lies directly adjacent to the loop. Thus, while the VAP1 structure is C-shaped, disulfide isomerisation of bonds within the CxxC motif would be expected to alter the conformation, and thus potentially substrate access and cleavage.

[0118] Prior evidence that disulfide isomerisation of ADAMs indeed occurs and modulates activity comes from studies on ADAM17, which show that its activity is inhibited by mutation of the analogous CxxC motif, and is regulated by modulating redox conditions43. Furthermore, these redox effects were shown to occur through modulation of protein disulfide isomerase (PDI) activity, where PDI modulated both activity of ADAM17 and its recognition by conformation-dependent antibodies44. Oxidative conditions were shown to favour activity, and suggested to inhibit PDI from catalysing a shift from an active to an inactive conformation.

[0119] We now describe a novel antibody, 8C7, which recognises a specific conformation of the ADAM10 substrate binding domain dependent on CxxC bonding. 8C7 binding is blocked by mutation of the CxxC motif, and is altered by modulating the redox environment. Moreover, biotin-labelling of labile cysteines with Maleimide-PEG2-biotin (MPB) inhibited binding of 8C7, but not a control ADAM10 mAb, suggesting 8C7 indeed binds labile, redox-modulated cysteines. Furthermore, our determination of the structure of 8C7 in complex with ADAM10 shows binding next to C639, disulfide-bonded to C594 in the CxxC motif. Our evidence suggests the 8C7-recognised conformation is active, since 8C7-immunoprecipitates of ADAM10 showed highly significant enrichment of protease activity. Also, oxidative conditions, known to enhance ADAM activity, favoured 8C7 binding. Experiments are underway to define the disulfide bonding pattern and structure of the presumed alternate, inactive ADAM10 C domain conformation. Interestingly, a recent NMR study shows two distinct, PDI-regulated conformations of bacterially expressed ADAM17, with distinct CxxC linkages, supporting the notion of CxxC isomerisation, although the activities of the two forms or their relevance for mammalian-expressed ADAM17 was not assessed47. The analogous changes in ADAM10 would correspond to the C594-C639 linkage in our 8C7-bound structure swapping to C594-C632, accommodated by a further change from C632-C645 to C639-C645.

[0120] Importantly, the apparent selectivity of 8C7 for active ADAM10 translates to selectivity for ADAM10 in tumours compared to other, normal tissues in both mouse models (CRC xenograft model and the GP130FF model of spontaneous gastric tumour formation), and in human tumour samples. This suggests 8C7 as a potentially powerful diagnostic for ADAM10 activity in tumours. Interestingly the 8C7-recognised form of ADAM10 that was specific to tumours was largely a HMW, unprocessed form, which we confirmed is present on the cell surface and is cleavable by furin. While the ADAM10 Pro domain is known to have an inhibitory function, it also has an essential chaperone function41, and ADAM10 Pro domain mutations which likely disrupt this function have recently been shown to attenuate ADAM10 activity in late onset Alzheimer's disease48. Moreover, reversible activation of unprocessed ADAM17 has recently been demonstrated42. Therefore, it is likely the unprocessed ADAM10 prevalent in tumours is similarly readily activated, and the high level of 8C7 binding indicates a high degree of activity in tumours. This activity is most likely supported by high levels of ROS in the tumour microenvironment49, favouring the active ADAM10 isomer.

[0121] As well as preferentially targeting tumours 8C7, significantly inhibited tumour growth. We previously found 8C7 inhibited ADAM10 mediated shedding of the Eph receptor ligand ephrin-A5, thus attenuating Eph receptor tyrosine phosphorylation and Eph-mediated cell segregation27. This is consistent with its targeting the substrate binding domain, which we previously showed is necessary for ephrin cleavage6. While the LIM1215 tumours expressed high levels of EphA2 we could not detect phosphorylation, consistent with known Eph kinase-independent function in tumours50-52, so a direct effect on Eph activity in tumours was not detectable. Rather we noted a dramatic down-regulation of EphA2 expression, and also of Notch receptors. A role for ADAM10 in Notch signalling is well established, and since Notch acts to modulate transcription of a variety of targets, including Eph receptors/ligands and Notch itself, we investigated effects of 8C7 on Notch activity. Treatment of tumour-bearing mice inhibited production of the cleaved active NICD in tumours, and there was also significant down-regulation of Notch-induced target genes (Hes1, Hey1). There was also decreased vascularity of 8C7-treated tumours consistent with inhibited Notch signalling. Treatment of isolated tumour cells also inhibited Notch activity, suggesting a direct effect on Notch. We further tested 8C7 in a separate in vitro assay of Notch-dependent lymphoma cell survival and proliferation, which confirmed a marked inhibition indicative of attenuated Notch signalling. Thus, 8C7-inhibition of tumour growth appears to be at least partially due to inhibition of Notch activity, although effects on other ADAM10-targets are also being investigated.

[0122] Notably, while 8C7 bound to the tumour mass, it was clearly most strongly bound to a distinct population of cells within tumours which were closely associated with blood vessels, and which expressed the putative tumour stem cell marker CD133. A recent study has described CD133+ cells in perivascular regions of human CRC, which display elevated Notch signalling due to ADAM17-mediated release of the ligand Jagged-1 from the endothelial cells30. In agreement we find 8C7-targeted CD133+ cells show high levels of NICD1 and 2, both by immunofluorescence staining of tumours and by analysis of CD133+ sorted cells by Western blot. Treatment of these cells in vitro with 8C7 also inhibited Notch activity and down-regulated Notch target genes. This suggests that 8C7 inhibition of ADAM10 activity is particularly acting on a subpopulation of tumour cells previously identified as having a stem cell phenotype. Since stem cells are believed to initiate and sustain tumour growth, and to mediate chemoresistance due to their slow cycling, the specific targeting of this population by 8C7, and its inhibitory effects on tumour growth, combined with its selectivity for active conformation of ADAM10 in tumours, indicate considerable potential for therapy. In support, we find 8C7 effectively blocks tumour regrowth following chemotherapy, and specifically depletes CD133+ cells.

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Table 1. Data collection and refinement statistics: crystal structure of the complex between ADAM10 D+C-domain and 8C7 F(Ab)2.
Adam/mAb 
Wavelength (Å) 0.9792
Resolution range (Å) 141.7 - 2.76 (2.91 - 2.76)
Space group P 21 21 21
Unit cell 53.326 141.679 268.08 90 90 90
Total reflections 234255
Unique reflections 52800
Multiplicity 4.4 (4.4)
Completeness (%) 98.63 (97.87)
Mean I/sigma(I) 10.32 (2.10)
Wilson B-factor 41.90
R-merge 0.125 (0.675)
R-work 0.2028 (0.3273)
R-free 0.2514 (0.3677)
Number of atoms 9942
macromolecules 9485
ligands 123
water 334
Protein residues 1252
RMS(bonds) 0.009
RMS(angles) 1.26
Ramachandran favored (%) 98
Ramachandran outliers (%) 0
Clashscore 7.55
Average B-factor 32.60
macromolecules 32.60
ligands 52.60
solvent 27.30


[0124] Statistics for the highest-resolution shell are shown in parentheses.
Table 2. Residue contacts in the complex between ADAM10 D+C-domain and 8C7 F(Ab)2.
Adam/Light Chain
Non-bonded contacts
 Atom no.Atom nameRes nameRes no.ChainAtom no.Atom nameRes nameRes no.ChainDistance
1. 12 CB VAL 451 A <--> 2056 CD1 ILE 72 B 3.72
2. 13 CG1 VAL 451 A <--> 2054 CG1 ILE 72 B 3.87
3. 13 CG1 VAL 451 A <--> 2056 CD1 ILE 72 B 3.58
4. 14 CG2 VAL 451 A <--> 2032 C ASP 70 B 3.80
5. 14 CG2 VAL 451 A <--> 2033 O ASP 70 B 3.62
6. 14 CG2 VAL 451 A <--> 2056 CD1 ILE 72 B 3.79
7. 782 CZ ARG 557 A <--> 2220 CD ARG 93 B 3.73
8. 782 CZ ARG 557 A <--> 2223 NH1 ARG 93 B 3.70
9. 783 NH1 ARG 557 A <--> 2223 NH1 ARG 93 B 3.60
10. 784 NH2 ARG 557 A <--> 2220 CD ARG 93 B 3.16
11. 1410 O VAL 641 A <--> 2231 CD1 TRP 94 B 3.11
12. 1410 O VAL 641 A <--> 2233 NE1 TRP 94 B 3.41
13. 1412 CG1 VAL 641 A <--> 2203 O SER 91 B 3.26
14. 1412 CG1 VAL 641 A <--> 2208 C ASN 92 B 3.82
15. 1412 CG1 VAL 641 A <--> 2209 O ASN 92 B 3.32
16. 1420 CD1 PHE 642 A <--> 2231 CD1 TRP 94 B 3.70
17. 1424 CZ PHE 642 A <--> 2255 CE2 PHE 96 B 3.84
18. 1424 CZ PHE 642 A <--> 2256 CZ PHE 96 B 3.84
Number of non-bonded contacts: 18
Adam/Heavy Chain
Hydrogen bonds
<-A TOM 1 -----><-ATOM 2 ----->  
 Atom no.Atom nameRes nameRes no.ChainAtom no.Atom nameRes nameRes no.ChainDistance
1. 1405 OD2 ASP 640 A <--> 3979 OH TYR 105 C 3.07
2. 1407 N VAL   A <--> 3951 O LEU 103 C 3.27
3. 1442 NH1 ARG   A <--> 3590 OD1 ASP 55 C 3.27
4. 1442 NH1 ARG   A <--> 3602 OD2 ASP 57 C 2.83
5. 1443 NH2 ARG   A <--> 3589 OD2 ASP 55 C 2.42
6. 1459 NH1 ARG   A <--> 3979 OH TYR 105 C 2.86
7. 1460 NH2 ARG   A <--> 3375 O ASN   C 3.26
                       
Non-bonded contacts
<-A TOM 1 -----> <-ATOM 2 ----->
 Atom no.Atom nameRes nameRes no.ChainAtom no.Atom nameRes nameRes no.ChainDistance
1. 1009 OD2 ASP 589 A <--> 3581 NH1 ARG 54 C 3.70
2. 1024 CG LYS 591 A <--> 3602 OD2 ASP 57 C 3.82
3. 1025 CD LYS 591 A <--> 3602 OD2 ASP 57 C 3.67
4. 1026 CE LYS 591 A <--> 3602 OD2 ASP 57 C 3.79
5. 1027 NZ LYS 591 A <--> 3601 OD1 ASP 57 C 3.73
6. 1311 C PRO 628 A <--> 3967 OH TYR 104 C 3.57
7. 1312 O PRO 628 A <--> 3967 OH TYR 104 C 3.46
8. 1313 CB PRO 628 A <--> 3964 CE2 TYR 104 C 3.59
9. 1313 CB PRO 628 A <--> 3966 CZ TYR 104 C 3.76
10. 1313 CB PRO 628 A <--> 3967 OH TYR 104 C 3.34
11. 1385 CB TYR 638 A <--> 3954 CD1 LEU 103 C 3.79
12. 1385 CB TYR 638 A <--> 3955 CD2 LEU 103 C 3.89
13. 1396 O CYS 639 A <--> 3954 CD1 LEU 103 C 3.76
14. 1400 CA ASP 640 A <--> 3951 O LEU 103 C 3.67
15. 1400 CA ASP 640 A <--> 3952 CB LEU 103 C 3.76
16. 1403 CB ASP 640 A <--> 3951 O LEU 103 C 3.36
17. 1403 CB ASP 640 A <--> 3977 CE2 TYR 105 C 3.76
18. 1403 CB ASP 640 A <--> 3979 OH TYR 105 C 3.50
19. 1404 CG ASP 640 A <--> 3979 OH TYR 105 C 3.62
20. 1405 OD2 ASP 640 A <--> 3979 OH TYR 105 C 3.07
21. 1407 N VAL 641 A <--> 3951 O LEU 103 C 3.27
22. 1412 CG1 VAL 641 A <--> 3962 CD2 TYR 104 C 3.57
23. 1413 CG2 VAL 641 A <--> 3951 O LEU 103 C 3.02
24. 1413 CG2 VAL 641 A <--> 3957 CA TYR 104 C 3.64
25. 1417 O PHE 642 A <--> 3615 CE LYS 59 C 3.22
26. 1417 O PHE 642 A <--> 3616 NZ LYS 59 C 3.47
27. 1418 CB PHE 642 A <--> 3401 CE2 TRP 33 C 3.67
28. 1418 CB PHE 642 A <--> 3403 CZ2 TRP 33 C 3.46
29. 1419 CG PHE 642 A <--> 3401 CE2 TRP 33 C 3.90
30. 1421 CD2 PHE 642 A <--> 3397 CG TRP 33 C 3.61
31. 1421 CD2 PHE 642 A <--> 3398 CD1 TRP 33 C 3.79
32. 1421 CD2 PHE 642 A <--> 3399 CD2 TRP 33 C 3.50
33. 1421 CD2 PHE 642 A <--> 3400 NE1 TRP 33 C 3.83
34. 1421 CD2 PHE 642 A <--> 3401 CE2 TRP 33 C 3.65
35. 1423 CE2 PHE 642 A <--> 3397 CG TRP 33 C 3.90
36. 1423 CE2 PHE 642 A <--> 3975 CD2 TYR 105 C 3.86
37. 1424 CZ PHE 642 A <--> 3421 OE1 GLN 35 C 3.83
38. 1438 CG ARG 644 A <--> 3403 CZ2 TRP 33 C 3.42
39. 1439 CD ARG 644 A <--> 3602 OD2 ASP 57 C 3.77
40. 1441 CZ ARG 644 A <--> 3559 CD2 TYR 52 C 3.71
41. 1441 CZ ARG 644 A <--> 3561 CE2 TYR 52 C 3.88
42. 1441 CZ ARG 644 A <--> 3589 OD2 ASP 55 C 3.15
43. 1442 NH1 ARG 644 A <--> 3559 CD2 TYR 52 C 3.53
44. 1442 NH1 ARG 644 A <--> 3588 CG ASP 55 C 3.56
45. 1442 NH1 ARG 644 A <--> 3590 OD1 ASP 55 C 3.27
46. 1442 NH1 ARG 644 A <--> 3589 OD2 ASP 55 C 3.09
47. 1442 NH1 ARG 644 A <--> 3599 CB ASP 57 C 3.24
48. 1442 NH1 ARG 644 A <--> 3600 CG ASP 57 C 3.47
49. 1442 NH1 ARG 644 A <--> 3602 OD2 ASP 57 C 2.83
50. 1443 NH2 ARG 644 A <--> 3559 CD2 TYR 52 C 3.87
51. 1443 NH2 ARG 644 A <--> 3561 CE2 TYR 52 C 3.79
52. 1443 NH2 ARG 644 A <--> 3588 CG ASP 55 C 3.47
53. 1443 NH2 ARG 644 A <--> 3590 OD1 ASP 55 C 3.87
54. 1443 NH2 ARG 644 A <--> 3589 OD2 ASP 55 C 2.42
55. 1455 CG ARG 646 A <--> 3979 OH TYR 105 C 3.53
56. 1456 CD ARG 646 A <--> 3979 OH TYR 105 C 3.66
57. 1457 NE ARG 646 A <--> 3564 OH TYR 52 C 3.90
58. 1458 CZ ARG 646 A <--> 3564 OH TYR 52 C 3.80
59. 1459 NH1 ARG 646 A <--> 3978 CZ TYR 105 C 3.81
60. 1459 NH1 ARG 646 A <--> 3979 OH TYR 105 C 2.86
61. 1460 NH2 ARG 646 A <--> 3375 O ASN 31 C 3.26
62. 1460 NH2 ARG 646 A <--> 3564 OH TYR 52 C 3.70
Number of hydrogen bonds: 7
Number of non-bonded contacts: 62



Claims

1. An in vitro method of detecting a proteolytically active form of an ADAM10 protease, said method including the step of selectively binding an antibody or antibody fragment to the proteolytically active form of the ADAM10 protease to thereby detect the proteolytically active form of the ADAM10 protease, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645.
 
2. An in vitro method of detecting a tumour cell, said method including the step of selectively binding an antibody or antibody fragment to the proteolytically active form of an ADAM10 protease expressed by the tumour cell to thereby detect the tumour cell, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645 and wherein the tumour cell is of a leukemia, lymphoma, lung cancer, colon cancer, adenoma, neuroblastoma, brain tumour, renal tumour, prostate cancer, sarcoma, breast cancer or melanoma.
 
3. A kit for use according to the method of any one of Claims 1-2, comprising an antibody or antibody fragment that specifically or selectively binds a proteolytically active form of an ADAM10 protease.
 
4. An antibody or antibody fragment which specifically or selectively recognizes and binds a proteolytically active form of the ADAM10 protease to the ADAM10 protease expressed by a cell, for use in at least partly inhibiting a biological activity of the ADAM protease and/or one or more downstream signalling molecules in the cell, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645.
 
5. The antibody or antibody fragment for use according to Claim 4, wherein the one or more downstream signalling molecules include one or more members of the Notch, erbB and Eph signalling pathways.
 
6. An antibody or antibody fragment which specifically or selectively recognizes and binds a proteolytically active form of the ADAM10 protease for use in preventing or treating cancer in a mammal, wherein the antibody or antibody fragment binds a region, portion and/or amino acid sequence of an ADAM10 protease that provides a switch between a proteolytically active and inactive form of an ADAM10 protease and wherein the region, portion or the amino acid sequence of the ADAM10 protease is of a loop which comprises a disulphide bond between a cysteine residue and an adjacent CXXC motif, preferably C594-C639 and C632-C645.
 
7. The antibody or antibody fragment for use according to Claim 4 or 6, wherein the ADAM protease10 is expressed by a tumour cell, wherein the tumour cell is of a leukemia, lymphoma, lung cancer, colon cancer, adenoma, neuroblastoma, brain tumour, renal tumour, prostate cancer, sarcoma, breast cancer or melanoma.
 
8. The method of Claim 2 or the antibody or antibody fragment for use according to Claim 7, wherein the tumour cell expresses CD133.
 
9. The method of Claim 1 or 2, the antibody or antibody fragment for use according to any one of Claims 4-8, or the kit according to Claim 3, wherein the antibody or antibody fragment binds or interacts with one or more ADAM10 protease residues selected from the group consisting of: Arg557; Lys591; Pro 628; Asp640; Val 641; Phe 642; Arg644, and Arg646, preferably wherein hydrogen bonds are formed between Asp640; Val 641; Phe 642; Arg644; and Arg646 and the antibody or antibody fragment.
 
10. The method of Claim 1 or 2, the antibody or antibody fragment for use according to any one of Claims 4-9 or the kit of Claim 3 or 9, wherein the antibody or antibody fragment comprises at least a portion of at least one complementarity determining region (CDR) having a CDR amino acid sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2 or comprises at least a portion of at least one CDR having an amino acid sequence at least 85% identical to a CDR amino acid sequence in SEQ ID NO:1 and/or SEQ ID NO:2.
 
11. A recombinant humanized antibody or antibody fragment which specifically or selectively binds a proteolytically active form of an ADAM10 protease and comprises at least one complementarity determining region (CDR) having an amino acid sequence set forth in SEQ ID NO:1 and/or SEQ ID NO:2 or comprises at least one CDR having an amino acid sequence at least 85% identical to a CDR amino acid sequence in SEQ ID NO:1 and/or SEQ ID NO:2.
 


Ansprüche

1. In vitro Verfahren zum Nachweisen einer proteolytisch aktiven Form einer ADAM10-Protease, wobei das Verfahren den Schritt des selektiven Bindens eines Antikörpers oder Antikörperfragments an die proteolytisch aktive Form der ADAM10-Protease umfasst, um dadurch die proteolytisch aktive Form der ADAM10-Protease nachzuweisen, wobei der Antikörper oder das Antikörperfragment einen Bereich, Teil und/oder Aminosäuresequenz einer ADAM10-Protease bindet, die einen Wechsel zwischen einer proteolytisch aktiven und inaktiven Form einer ADAM10-Protease bereitstellt und wobei der Bereich, Teil oder die Aminosäuresequenz der ADAM10-Protease von einer Schleife ist, die eine Disulfidbindung zwischen einem Cysteinrest und einem benachbarten CXXC-Motiv umfasst, vorzugsweise C594-C639 und C632-C645.
 
2. In vitro-Verfahren zum Nachweisen einer Tumorzelle, wobei das Verfahren den Schritt des selektiven Bindens eines Antikörpers oder Antikörperfragments an die proteolytisch aktive Form einer ADAM10-Protease umfasst, die von der Tumorzelle exprimiert wird, um dadurch die Tumorzelle nachzuweisen, wobei der Antikörper oder das Antikörperfragment einen Bereich, Teil und/oder Aminosäuresequenz einer ADAM10-Protease bindet, die einen Wechsel zwischen einer proteolytisch aktiven und inaktiven Form einer ADAM10-Protease bereitstellt, und wobei der Bereich, Teil oder die Aminosäuresequenz der ADAM10-Protease von einer Schleife ist, die eine Disulfidbindung zwischen einem Cysteinrest und einem benachbarten CXXC-Motiv umfasst, vorzugsweise C594-C639 und C632-C645, und wobei die Tumorzelle von einer Leukämie, Lymphom, Lungenkrebs , Darmkrebs, Adenom, Neuroblastom, Gehirntumor, Nierentumor, Prostatakrebs, Sarkom, Brustkrebs oder Melanom ist.
 
3. Kit zur Verwendung gemäß dem Verfahren nach einem der Ansprüche 1-2, umfassend einen Antikörper oder das Antikörperfragment, der spezifisch oder selektiv eine proteolytisch aktive Form einer ADAM10-Protease bindet.
 
4. Antikörper oder Antikörperfragment, der/das spezifisch oder selektiv eine proteolytisch aktive Form der ADAM10-Protease erkennt und an die durch eine Zelle exprimierte ADAM10-Protease bindet, zur Verwendung bei dem zumindest teilweisen Hemmen einer biologischen Aktivität der ADAM-Protease und/oder ein oder mehrerer Downstream-Signalmoleküle in der Zelle, wobei der Antikörper oder das Antikörperfragment einen Bereich, Teil und/oder Aminosäuresequenz einer ADAM10-Protease bindet, die einen Wechsel zwischen einer proteolytisch aktiven und inaktiven Form einer ADAM10-Protease bereitstellt und wobei der Bereich, Teil oder die Aminosäuresequenz der ADAM10-Protease von einer Schleife ist, die eine Disulfidbindung zwischen einem Cysteinrest und einem benachbarten CXXC-Motiv umfasst, vorzugsweise C594-C639 und C632-C645.
 
5. Antikörper oder Antikörperfragment zur Verwendung nach Anspruch 4, wobei die ein oder mehreren Downstream-Signalmoleküle ein oder mehrere Mitglieder der Notch-, erbB- und Eph-Signalwege umfassen.
 
6. Antikörper oder Antikörperfragment, der/das spezifisch oder selektiv eine proteolytisch aktive Form der ADAM10-Protease erkennt und bindet, zur Verwendung beim Verhindern oder Behandlen von Krebs in einem Säugetier, wobei der Antikörper oder das Antikörperfragment einen Bereich, Teil und/oder Aminosäuresequenz einer ADAm10-Protease bindet, die einen Wechsel zwischen einer proteolytisch aktiven und inaktiven Form einer ADAM10-Protease bereitstellt, und wobei der Bereich, Teil oder die Aminosäuresequenz der ADAM10-Protease von einer Schleife ist, die eine Disulfidbindung zwischen einem Cysteinrest und einem benachbarten CXXC-Motiv umfasst, vorzugsweise C594-C639 und C632-C645.
 
7. Antikörper oder Antikörperfragment zur Verwendung nach Anspruch 4 oder 6, wobei die ADAM-Protease10 von einer Tumorzelle exprimiert wird, wobei die Tumorzelle von einer Leukämie, Lymphom, Lungenkrebs, Darmkrebs, Adenom, Neuroblastom, Gehirntumor, Nierentumor, Prostatakrebs, Sarkom, Brustkrebs oder Melanom ist.
 
8. Verfahren nach Anspruch 2 oder der Antikörper oder das Antikörperfragment zur Verwendung nach Anspruch 7, wobei die Tumorzelle CD133 exprimiert.
 
9. Verfahren nach Anspruch 1 oder 2, der Antikörper oder das Antikörperfragment zur Verwendung nach einem der Ansprüche 4-8, oder der Kit nach Anspruch 3, wobei der Antikörper oder das Antikörperfragment an/mit ein oder mehreren ADAM10-Proteaseresten ausgewählt aus der Gruppe bestehend aus : Arg557; Lys591; Pro628; Asp640; Val641; Phe642; Arg644, und Arg646 bindet oder interagiert, vorzugsweise wobei Wasserstoffbrückenbindungen zwischen Asp640; Val641; Phe642; Arg644; und Arg646 und dem Antikörper oder Antikörperfragment gebildet werden.
 
10. Verfahren nach Anspruch 1 oder 2, der Antikörper oder das Antikörperfragment zur Verwendung nach einem der Ansprüche 4-9 oder der Kit nach Anspruch 3 oder 9, wobei der Antikörper oder das Antikörperfragment zumindest einen Teil zumindest einer Komplementarität bestimmenden Region (CDR) umfasst, die eine CDR Aminosäuresequenz wie in SEQ ID NO: 1 und/oder SEQ ID NO : 2 dargestellt, hat oder zumindest einen Teil zumindest einer CDR umfasst, die eine Aminosäuresequenz hat, die zumindest zu 85% zu einer CDR-Aminosäuresequenz in SEQ ID NO: 1 und/oder SEQ ID NO: 2 identisch ist.
 
11. Rekombinanter humanisierter Antikörper oder Antikörperfragment, der/das spezifisch oder selektiv eine proteolytisch aktive Form einer ADAM10-Protease bindet und zumindest eine Komplementarität bestimmende Region (CDR) umfasst, die eine Aminosäuresequenz wie in SEQ ID NO: 1 und/oder SEQ ID NO : 2 dargestellt, hat oder zumindest eine CDR mit einer Aminosäuresequenz umfasst, die zumindest zu 85% mit einer CDR-Aminosäuresequenz in SEQ ID NO: 1 und/oder SEQ ID NO: 2 identisch ist.
 


Revendications

1. Procédé in vitro de détection d'une forme d'une protéase ADAM10 protéolytiquement active, ledit procédé comprenant l'étape de lier sélectivement un anticorps ou un fragment d'anticorps à la forme de la protéase ADAM10 protéolytiquement active pour détecter ainsi la forme de la protéase ADAM10 protéolytiquement active, l'anticorps ou le fragment d'anticorps liant une région, une portion et/ou une séquence d'acides aminés d'une protéase ADAM10 qui fournit un commutateur entre une forme d'une protéase ADAM10 protéolytiquement active et inactive, et la région, la portion ou la séquence d'acides aminés de la protéase ADAM10 étant d'une boucle comportant une liaison disulfure entre un résidu de cystéine et un motif CXXC adjacent, de préférence C594-C639 et C632-C645.
 
2. Procédé in vitro de détection d'une cellule tumorale, ledit procédé comportant l'étape de lier sélectivement un anticorps ou un fragment d'anticorps à la forme d'une protéase ADAM10 protéolytiquement active exprimée par la cellule tumorale pour ainsi détecter la cellule tumorale, l'anticorps ou le fragment d'anticorps liant une région, une portion et/ou une séquence d'acides aminés d'une protéase ADAM10 qui fournit un commutateur entre une forme d'une protéase ADAM10 protéolytiquement active et inactive, et la région, la portion ou la séquence d'acides aminés de la protéase ADAM10 étant d'une boucle comportant une liaison disulfure entre un résidu de cystéine et un motif CXXC adjacent, de préférence C594-C639 et C632-C645, et la cellule tumorale étant d'une leucémie, d'un lymphome, d'un cancer du poumon, d'un cancer du côlon, d'un adénome, d'un neuroblastome, d'une tumeur cérébrale, d'une tumeur rénale, d'un cancer de la prostate, d'un sarcome, d'un cancer du sein ou d'une mélanome.
 
3. Kit pour utilisation selon le procédé de l'une quelconque des revendications 1 à 2 comportant un anticorps ou un fragment d'anticorps qui lie spécifiquement ou sélectivement une forme de la protéase ADAM10 protéolytiquement active.
 
4. Anticorps ou fragment d'anticorps qui reconnaît et lie spécifiquement ou sélectivement une forme de la protéase ADAM10 protéolytiquement active à la protéase ADAM10 exprimée par une cellule pour utilisation dans l'inhibition au moins partiellement d'une activité biologique de la protéase ADAM et/ou une ou plusieurs molécules de signalisation agissant en aval dans la cellule, l'anticorps ou le fragment d'anticorps liant une région, une portion et/ou une séquence d'acides aminés d'une protéase ADAM10 qui fournit un commutateur entre une forme d'une protéase ADAM10 protéolytiquement active et inactive, et la région, la portion ou la séquence d'acides aminés de la protéase ADAM10 étant d'une boucle comportant une liaison disulfure entre un résidu de cystéine et un motif CXXC adjacent, de préférence C594-C639 et C632-C645.
 
5. Anticorps ou fragment d'anticorps pour utilisation selon la revendication 4, l'une ou plusieurs molécules de signalisation agissant en aval comprenant un ou plusieurs membres de voies de signalisation de Notch, d'erbB et d'Eph.
 
6. Anticorps ou fragment d'anticorps qui reconnaît et lie spécifiquement ou sélectivement une forme de la protéase ADAM10 protéolytiquement active pour utilisation dans la prévention ou le traitement d'un cancer chez un mammifère, l'anticorps ou le fragment d'anticorps liant une région, une portion et/ou une séquence d'acides aminés d'une protéase ADAM10 qui fournit un commutateur entre une forme d'une protéase ADAM10 protéolytiquement active et inactive, et la région, la portion ou la séquence d'acides aminés de la protéase ADAM10 étant d'une boucle comportant une liaison disulfure entre un résidu de cystéine et un motif CXXC adjacent, de préférence C594-C639 et C632-C645.
 
7. Anticorps ou fragment d'anticorps pour utilisation selon la revendication 4 ou 6, la protéase ADAM10 étant exprimée par une cellule tumorale, la cellule tumorale étant d'une leucémie, d'un lymphome, d'un cancer du poumon, d'un cancer du côlon, d'un adénome, d'un neuroblastome, d'une tumeur cérébrale, d'une tumeur rénale, d'un cancer de la prostate, d'un sarcome, d'un cancer du sein ou d'une mélanome.
 
8. Procédé selon la revendication 2 ou l'anticorps ou le fragment d'anticorps pour utilisation selon la revendication 7, la cellule tumorale exprimant CD133.
 
9. Procédé selon la revendication 1 ou 2, l'anticorps ou le fragment d'anticorps pour utilisation selon l'une quelconque des revendications 4 à 8 ou le kit selon la revendication 3, l'anticorps ou le fragment d'anticorps liant ou interagissant avec un ou plusieurs résidus de la protéase ADAM10 choisis parmi le groupe constitué de: Arg557; Lys591 ; Pro628; Asp640; Val641; Phe642; Arg644 et Arg646, de préférence, des liaisons d'hydrogène étant formées entre Asp640; Val641; Phe642; Arg644 et Arg646 et l'anticorps ou le fragment d'anticorps.
 
10. Procédé selon la revendication 1 ou 2, l'anticorps ou le fragment d'anticorps pour utilisation selon l'une quelconque des revendications 4 à 9 ou le kit selon la revendication 3 ou 9, l'anticorps ou le fragment d'anticorps comportant au moins une portion d'au moins une région déterminant la complémentarité (CDR) ayant une séquence d'acides aminés CDR établie dans SEQ-ID N°: 1 et/ou SEQ-ID N°: 2 ou comportant au moins une portion d'au moins une CDR ayant une séquence d'acides aminés identique à au moins 85% à une séquence d'acides aminés CDR dans SEQ-ID N°: 1 et/ou SEQ-ID N°: 2.
 
11. Anticorps humanisé recombinant ou fragment d'anticorps qui lie spécifiquement ou sélectivement une forme d'une protéase ADAM10 protéolytiquement active et comprend au moins une région déterminant la complémentarité (CDR) ayant une séquence d'acides aminés établie dans SEQ-ID N°: 1 et/ou SEQ-ID N°: 2 ou comporte au moins une CDR ayant une séquence d'acides aminés identique à au moins 85% à une séquence d'acides aminés CDR dans SEQ-ID N°: 1 et/ou SEQ-ID N°: 2.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description