Patent Application
20090298079
This application claims priority to European Application No. 08103958.8, filed 14 May 2008, the entire contents of which is hereby incorporated by reference.
The present invention relates to the area of the hepatocyte growth factor receptor (HGFR) protein. More specifically, the present invention relates to the identification of the high affinity binding site of HGFR for its ligand, the hepatocyte growth factor (HGF), and methods for the identification of antagonists of HGFR targeting the high affinity binding site of HGFR.
The hepatocyte growth factor receptor (also known as Met) is a tyrosine kinase and is the product of the c-met proto-oncogene. It consists of a 50 kDa α-subunit and of a 145 kDa β-subunit, which are linked by a disulfide bond, the α-subunit being completely extracellular, while the β-subunit includes (from N- to C-terminus) an extracellular region, a transmembrane domain and a cytoplasmic tyrosine kinase domain. The mature α/β hetero-dimeric receptor is generated by proteolytic processing and terminal glycosilation from a 170 kDa single-chain precursor.
HGF, also known as Scatter Factor, is a heparin-binding glycoprotein with a broad spectrum of biological activities including cell proliferation, motility, survival and morphogenesis. It is synthesized and secreted as an inactive single chain precursor (pro-HGF) that is stored into the extracellular matrix due to its high affinity for proteoglycans. Pro-HGF undergoes proteolytic cleavage at residues R494-V495 to give rise to the biologically active form, a disulfide-linked α/β hetero-dimer, where the α-chain consists of an N-terminal domain followed by four kringle domains and the β-chain shares structural homology with the chymotrypsin family of serine proteases. The β-chain, however, lacks proteolytic activity since two of the three critical residues that form the catalytic triad typical of serine proteases are not conserved in HGF. Despite its inability to signal, pro-HGF binds to Met at high affinity and displaces active HGF.
Recently, a number of structure-function studies have shed some light onto the interactions between the extracellular portion of Met and HGF.
The Met extracellular region has a modular structure, which encompasses three functional domains: the Sema domain (present also in Semaphorins and plexins) which spans the first 500 residues at the N-terminus of the protein and has a seven-bladed β-propeller structure, the PST domain (also found in Plexins, Semaphorins and Integrins) which covers about 50 residues and contains four conserved disulfide bonds, and additional 400 residues which link the PSI domain to the trans-membrane helix and are occupied by four IPT domains (Immunoglobulin-related domains present in Plexins and Transcription factors).
HGF is a bivalent ligand, containing a high affinity binding site for Met in the α-chain and a low affinity binding site in the β-chain. Cooperation between the α- and the β-chain is required for the biological activity of HGF; while the α-chain, and more precisely the N-domain and the first kringle, is sufficient for Met binding, the β-chain is necessary for Met activation.
Resolution of the crystal structure of the SEMA and PSI domains of Met in complex with the β-chain of HGF (see i.a. WO-A-2005/108424) revealed that the low affinity binding site for HGF is located in blades 2-3 of the β-propeller, and that the portion of HGF-β that binds to Met is the same region that serine proteases use to bind to their substrates or inhibitors. Importantly, determination of HGF β-chain crystal structure at 2.53 Å resolution and specific mutagenesis analysis unveiled that the residues involved in Met binding in the activation pocket of HGF β-chain get exposed only following proteolytic conversion of pro-HGF, thus explaining why pro-HGF binds to Met at high affinity without activating it. While the low affinity interaction between the β-chain of HGF and the Sema domain of Met is well characterized both structurally and functionally, at the moment it is not clear what region of Met binds to the α-chain of HGF at high affinity. Thus, the main mechanism by which HGF activates Met still remains poorly understood. This is somehow surprising when considering the great biological and therapeutic importance of this pathway.
HGF-Met signaling is essential during embryogenesis and in tissue regeneration in the adult life. Importantly, deregulated HGF Met signaling plays a key role in tumorigenesis and metastasis. Inappropriate Met activation by different mechanisms including autocrine HGF stimulation, receptor overexpression, gene amplification and point mutation is described in a wide variety of human malignancies and correlates with poor prognosis. These findings resulted in a growing interest in the HGF-Met pathway as a target for cancer therapy, leading to the development of a variety of Met/HGF inhibitors. These include small molecule compounds targeting Met kinase activity, neutralizing anti Met or anti-HGF antibodies, decoy receptors and HGF-derived factors. Nevertheless, whether such molecules target the high affinity binding site for HGF and which is the exact molecular mechanism at the basis of HGF binding to Met at high affinity, remains still unknown. This may prevent from isolating more selective therapeutic agents, with increased sensitivity and fewer side effects. The exact knowledge of the Met high affinity binding site for HGF will certainly help to design highly specific antagonists of Met.
The need is therefore felt for improved solutions enabling the identification of Met antagonists with increased sensitivity and fewer side effects for the development of more effective therapeutic strategies for the treatment of cancers.
The object of this disclosure is providing such improved solutions.
According to the present invention such objects are achieved thanks to the solution having the characteristics referred to specifically in the ensuing claims. The claims form integral part of the technical teaching herein provided in relation to the present invention.
Thus, object of the present disclosure is the identification of the HGF high affinity binding site to the hepatocyte growth factor receptor (HGFR), i.e. the extracellular IPT-3 and IPT-4 domains of HGFR. A further object of the present invention is to provide means for the identification of antagonists of HGFR targeting the high affinity biding site of HGFR for HGF for the development of new therapeutic strategies for the treatment of cancers.
In an embodiment, the present invention provides for the use of a polypeptide comprising, or a polynucleotide encoding at least the extracellular IPT-3 and IPT-4 domains of hepatocyte growth factor receptor for the screening and/or development of pharmacologically active agents useful in the treatment of cancer, in particular a cancer with dysregulation of hepatocyte growth factor receptor activity.
In an embodiment, the pharmacologically active agent is a hepatocyte growth factor receptor inhibitor and/or antagonist and can be selected among small molecule inhibitors, aptamers, antisense nucleotides, RNA-based inhibitors, siRNAs, antibodies, peptides, dominant negative factors.
In a further embodiment, the present invention concerns a method to detect the ability of a test agent to act as an antagonist/inhibitor of hepatocyte growth factor receptor useful in the treatment of cancer, preferably a cancer with dysregulation of hepatocyte growth factor receptor activity, comprising the steps of:
(a) putting in contact a test agent with at least the extracellular IPT-3 and IPT-4 domains of hepatocyte growth factor receptor, or cells expressing at least the extracellular IPT-3 and IPT-4 domains of hepatocyte growth factor receptor,
(b) measuring hepatocyte growth factor receptor activity, function, stability and/or expression, and
(c) selecting the agent that reduces hepatocyte growth factor receptor activity, function, stability and/or expression.
In a still further embodiment, the present invention concerns the use of the extracellular IPT-3 and IPT-4 domains of hepatocyte growth factor receptor as a medicament for treating cancer.
The present invention will now be described in detail in relation to some preferred embodiments by way of non-limiting examples with reference to the annexed drawings, wherein:
The data presented in the present disclosure suggest that the α-chain of HGF binds to the IPT region of Met at high affinity, and that it does so independently of proteolytic processing of the ligand. They also suggest that HGF binding to IPT in the context of a trans-membrane Met lacking the Sema domain is sufficient for transmitting the signal for receptor activation to the cytoplasmic kinase domain, although without distinction between the inactive and active form of the ligand. Finally, they provide evidence that engineered proteins derived from the IPT region and Sema domain of Met are capable of neutralizing the pro-invasive activity of HGF both in vitro and in vivo.
It has been known for long time that HGF is a bivalent factor. Early protein engineering studies identified a high affinity Met-binding site in the N domain and first kringle of HGF. Subsequently, combined biochemical and biological analysis demonstrated that the HGF serine protease-like domain (β-chain), while not necessary for binding, plays a key role in mediating receptor activation. More recently, detailed crystallographic and mutagenesis data have thoroughly characterized both structurally and functionally the low affinity Met-binding site on the β-chain of HGF and its interaction with the Sema domain of Met. The interface between the α-chain of HGF and Met had remained however elusive. Small angle X-ray scattering and cryo-electron microscopy studies suggested the presence of contacts among the N-terminal and first kringle domain of HGF and the Sema domain of Met. However, plasmon resonance analysis revealed that this interaction has a very low affinity (about 2 times lower than that of HGF-β for Sema and 100 times lower than that of HGF-α for the intact receptor). Since this weak interaction cannot account per se for the tight bond between HGF and Met, the high affinity HGF-binding site on Met had still to be identified.
The results presented here contribute to fill this gap and suggest that this long sought-after HGF-binding site lies in the IPT region of Met and more precisely in the last two immunoglobulin like domains close to the cell membrane. Several distinct experimental evidences provided in the present disclosure suggest that this is the case. Firstly, a soluble, deleted Met receptor containing nothing but the four IPT domains (IPT) binds to HGF with substantially the same affinity as the entire extra-cellular portion of Met. Conversely, Sema displays very low affinity towards HGF. Secondly, IPT binds to active HGF, pro-HGF or HGF-A with unchanged strength. Thirdly, deletion of IPT 1 and IPT 2 does not affect the affinity of IPT for any form of HGF. Fourthly, an engineered Met receptor carrying a large deletion in its ectodomain corresponding to the Sema domain, the PSI module and the first two immunoglobulin-like domains (MetΔ25-741) retains the ability to bind to HGF and to transduce the signal for kinase activation to the inside of the cell, although it cannot distinguish between active HGF and Pro-HGF. Finally, a dimeric form of HGF NK1, which is known to contain the minimal Met binding domain of HGF-α, is capable of eliciting activation of MetΔ25-741 as efficiently as if not more powerfully than HGF, thus identifying in IPT 3-4 the HGF NK1-binding site.
While these data point at a key role of IPT in HGF binding, it is noteworthy that two previous structure/function studies on the extracellular portion of met failed to identify any ligand binding site in this region. A first draft of the Met ectodomain map suggested that the Sema domain is necessary and sufficient for HGF binding based on ELISA assays. A second study analyzed the role of the Sema domain in receptor dimerization and suggested that an engineered form of the Met extracellular portion containing a deletion in the Sema domain was not capable of co-precipitating HGF.
The present disclosure further demonstrates that cooperation between Sema and IPT is observed also when the extracellular portion of met is used as a biotechnological tool to inhibit HGF-induced invasive growth. In the in vitro analysis and in mouse xenografts both the IPT and Sema-PSI soluble proteins displayed a significant inhibitory effect. However, none of them could achieve the powerful inhibition displayed by the full Met ectodomain, which contains both the low affinity and high affinity HGF-binding site. This implies that both of these interactions contribute to controlling Met activity. While the HGF-β-Sema contact had already been identified as a target for therapy, the results presented here unveil a second interface that offers opportunities for pharmacological intervention. Recombinant proteins or antibodies that bind to the IPT region in place of bona fide HGF have an application as highly competitive inhibitors of Met for the treatment of HGF/Met-dependent cancers.
Materials and Methods
Protein Engineering
Soluble or trans-membrane receptors and engineered ligands described in this work have been generated by standard PCR and genetic engineering techniques. All factors conserve the leader sequence of their parental protein at the N-terminus. The amino acid (aa) sequences of soluble Met proteins (Gene Bank N. X54559) correspond to aa 1-24 (signal peptide) plus: Decoy Met, aa 25-932; Sema, aa 25-515; Sema-PSI, aa 25-562; PSIIPT, aa 516-932; IPT, aa 563-932; IPT Δ1, aa 657-932; IPT Δ1-2, aa 742-932; IPT-3, aa 742-838; IPT-4, aa 839-932. At the C-terminus of each molecule a double FLAG (SDYKDDDDK—SEQ ID No.:19) or single MYC (EQKLISEEDLN—SEQ ID No.:20) epitope sequence and a poly-histidine tag (HHHHHHH—SEQ ID No.:21) were added for protein detection and purification. The transmembrane engineered MetΔ25-741 is identical to wild-type Met except for the deleted region (aa 25-741). The amino acid sequences of engineered HGF proteins (Gene Bank N. M73239) correspond to aa 1-31 (signal peptide) plus: HGF, aa 32-728; HGF-α, aa 32-473; HGF NK1, aa 32-205; HGF-β, aa 495-728. The above MYC or FLAG epitope and poly-histidine tag were added at the C-terminus. Uncleavable HGF has been described before (Mazzone, M. et al. (2004) J Clin Invesn. 114(10), 1418-1432). NK1-NK1 is a dimeric form of HEGF NK1 consisting of the same N-terminal region HGF repeated in tandem (aa 1-205 directly linked to aa 32-205 without spacer). The cDNAs encoding all engineered proteins were subcloned into the lentiviral transfer vector pRRLsin.PPT.CMV.eGFP.Wpre (SEQ ID No.:1) in place of the gfp cDNA as disclosed in Follenzi, A. et al. (2000) Nat Genet. 25(2), 217-222. The GFP coding sequence was replaced by the following cDNAs: decoy met FLAG.his (SEQ ID No.:2), Sema FLAG.his (SEQ ID No.:3), Sema-PSI FLAG.his (SEQ ID No.:4), PST-IPT FLAG.his (SEQ ID No.:5), IPT FLAG.his (SEQ ID No.:6), IPT Δ1 FLAG.his (SEQ ID No.:7), IPT Δ1-2 FLAG.his (SEQ ID No.:8), IPT 3 FLAG.his (SEQ ID No.:9), IPT 4 FLAG.his (SEQ ID No.:10), Met Δ25-741 (SEQ ID No.:11), HGF MYC his (SEQ ID No.:12), HGF-α MYC his (SEQ ID No.:13), HGF-NK1 MYC his (SEQ ID No.:14), HGF-β MYC his (SEQ ID No.:15), uncleavable HGF MYC his (SEQ ID No.:16), NK1-NK1 his (SEQ ID No.:17), angiostatin NYC his (SEQ ID No.:18).
Enzyme-Linked Immunosorbant Assays
All engineered receptors and factors were collected from the conditioned medium of lentiviral vector-transduced MDA-MB-345 human melanoma cells in the absence of serum. Factor purification was performed by immobilized-metal affinity chromatography as previously described in Michieli P. et al. (2002) Nat Biotechnol. 20(5), 488-495. Conversion of pro-HGF into active HGF was performed by incubating purified pro-HGF (maximal concentration 100 ng/μl) with 2-10% FBS (Sigma, St. Louis, Mo.) at 37° C. for 24 hours. Factor conversion was analyzed by Western blotting using anti-HGF antibodies (R&D Systems, Minneapolis, Minn.). Uncleavable HGF subjected to the same incubation with FBS was used as pro-HGF in all assays that compared active HGF with unprocessed HGF. Binding of engineered ligands to soluble receptors was measured by ELISA using FLAG-tagged soluble receptors in solid phase and MYC-tagged engineered ligands in liquid phase. A fixed concentration of purified soluble receptor (100 ng/well) was adsorbed to 96-well ELISA plates. Protein-coated plates were incubated with increasing concentrations of engineered ligands, and binding was revealed using biotinylated anti-HGF antibodies (R&D Systems, Minneapolis, Minn.) or anti-MYC antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.). Binding data were analyzed and fit using Prism software (Graph Pad Software, San Diego, Calif.).
Cell Culture
MDA-MB-435 human melanoma cells were purchased from the Georgetown University Tissue Culture Shared Resource (Washington, District of Columbia). Cells were maintained in DMEM supplemented with 10% FBS (Sigma). TOV-112D human ovarian carcinoma cells were obtained from ATCC (Rockville, Md.; ATCC N. CRL-11731) and were cultured using a 1:1 mixture of MCDB 105 Medium and Medium 199 supplemented with 15% BBS (all from Sigma). A549 human lung carcinoma cells were also obtained from ATCC (ATCC N. CCL-185) and maintained in RPMI supplemented with 10% FBS.
Lentiviral Vectors
Vector stocks were produced by transient transfection of 293T cells as previously described in Follenzi, A. et al. (2000) Nat Genet. 25 (2), 217-222. Briefly, the plasmid DNA mix for transfection was prepared as follows: ENV plasmid (VSV-G), 9 μg; PACKAGING plasmid pMDLg/pRRE 16.2 μg; REV plasmid, 6.25 μg; TRANSFER VECTOR (plasmid #2-18), 37.5 μg. The plasmids were diluted in a solution of TE/CaCl2, to which a HBS solution was added while vortexing at maximum speed. The DNA/CaCl2/HBS mix was immediately added drop-wise to the cell plates that were then incubated at 37° C. After 14-16 hours the culture medium was replaced with a fresh one. Cell culture supernatants containing vector particles were collected about 36 hours after medium changing. After collection, the supernatants were filtered through 0.2 μm pore membranes and stored at −80° C. Viral p24 antigen concentration was determined by the HIV-1 p24 core profile ELISA kit (NEN Life Science Products, Boston, Mass.) according to the manufacturer's instructions. Cells were transduced in six-well plates (105 cells/well in 2 ml of medium) using 40 ng/ml of p24 in the presence of 8 μg/ml polybrene (Sigma) as described in Vigna, E. and Naldini, L. (2000) J Gene Med. 2(5), 308-316. Medium was changed about 18 hours after transduction. Cell growth and protein production was monitored over time. Transduced cell lines were then seeded in 15 cm plates, grown to 80% confluence and incubated in medium without serum. After 72 hours, the supernatants containing the recombinant soluble proteins were collected, filtered and purified by affinity chromatography or stored at −30° C.
Immuno-Precipitation and Western Blot Analysis
Cell lysis, immuno-precipitation and Western blot analysis were performed using extraction buffer (EB) as described in Longati, P. et al. (1994) Oncogene 9(1), 49-57. Signal was detected using ECL system (Amersham Biosciences, Piscataway, N.J.) according to the manufacturer's instructions. Anti-Met antibodies for immunoprecipitation have been described by Ruco, L. P. et al. (1996) J Pathol. 180(3), 266-270 and were purchased from UBI (Lake Placid, N.Y.). Anti-Met antibodies for Western blot were purchased from Santa Cruz. Anti-FLAG antibodies were obtained from Sigma. Met phosphorylation analysis in lentiviral vector transduced MDA-MB-435 cells was performed as previously described in Michieli, P. et al. (2004) Cancer Cell 6(1), 61-73.
HGF Cross-Linking and Met Activation Analysis
Lentiviral vector-transduced TOV-112D cells expressing MetΔ25-741 were subjected to surface biotinylation analysis using an ECL™ Surface Biotinylation Module kit (Amersham Biosciences) according to the manufacturer's instructions. Chemical cross-linking was performed as previously described in Mazzone, M. et al. (2004) J Clin Invest. 114(10), 1418-1432. Briefly, cells were deprived of serum growth factors for 3 days and then incubated with 1 μM HGF for 3 hours. Cell lysates were immuno-precipitated using antibodies directed against the C-terminal portion of Met as disclosed in Ruco, L. P. et al. (1996) J Pathol. 180(3), 266-270, resolved by SDS-PAGE using a 3-10% polyacrylamide gradient and analyzed by Western blotting using anti-HGF antibodies (R&D). For receptor activation analysis, TOV-112D cells expressing MetΔ25-741 were deprived of serum growth factors for 3 days and then stimulated with 1 nM HGF, Uncleavable HGF, HGF Nk1 or NK1-NK1 for 10 minutes. Cells were lysed using EB as described in Longati, P. et al. (1994) Oncogene 9(1), 49-57. Cellular proteins were immuno-precipitated with anti-met antibodies as above and analyzed by Western blotting using anti-phosphotyrosine antibodies (UBI). The same blots were re-probed with anti-Met antibodies (Ruco, L. P. et al. (1996) J Pathol. 180(3), 266-270).
Biological Assays
Collagen invasion assays using MDA MB-435 cells were performed using preformed spheroids as described in Michieli, P. et al. (2004) Cancer Cell 6(1), 61-73, Briefly, spheroids were generated by incubating cells overnight (700 cells/well) in non-adherent 96-well plates (Greiner, Frickenhausen, Germany) in the presence of 0.24 g/ml methylcellulose (Sigma). Spheroids were embedded into a collagen matrix containing 1.3 mg/ml type I collagen from rat tail (BD Biosciences, Bedford, Mass.) and 10% FBS using 96-well plates (40 spheroids/well), Embedded spheroids were cultured at 37° C. for 24 hours, and then stimulated with 30 ng/ml HGF (R&D) or no factor for additional 24 hours. The number of tubules sprouting from each spheroid was scored by microscopy. At least 12 spheroids per experimental point were analyzed.
Tumorigenesis Assays
Lentiviral vector-transduced MDA-MB-435 tumor cells (3·106 cells/mouse) in 0.2 ml of DMEM were injected subcutaneously into the right posterior flank of six-week old immunodeficient nu−/− female mice on Swiss CD-1 background (Charles River Laboratories, Calco, Italy). Tumor size was evaluated every 2 days using a caliper. Tumor volume was calculated using the formula V=4/3πx2y/2 where x is the minor tumor axis and y the major tumor axis. A mass of 15 mm3-corresponding approximately to the initial volume occupied by injected cells was chosen as threshold for tumor positivity. Mice whose tumors were below this threshold were considered tumor-free. After approximately 4 weeks, mice were euthanized and tumors were extracted for analysis. Animals were subjected to autopsy. Tumors and lungs were embedded in paraffin and processed for histology. Micrometastasis analysis was performed by microscopy on serial lung sections stained with hematoxylin and eosin. Tumor sections were stained with hematoxylin and eosin and analyzed by an independent pathologist not informed of sample identity. Transgene expression was determined on tumor sections by immuno-histochemistry using anti-FLAG antibodies (Sigma). Sections were counterstained with Meyer hematoxylin (Sigma). Tumor angiogenesis was analyzed by immuno-histochemistry using anti-Von Willebrand factor antibodies (DAKO, Glostrup, Denmark). Sections were counterstained as above. Vessel density was assessed by microscopy. At least 12 fields per animal were analyzed. All animal procedures were approved by the Ethical Commission of the University of Turin, Italy, and by the Italian Ministry of Health.
Statistical Analysis
Statistical significance was determined using a two-tail homoscedastic Student's t-Test (array 1, control group; array 2, experimental group). For all data analyzed, a significance threshold of p<0.05 was assumed, In all figures, values are expressed as mean±standard deviation, and statistical significance is indicated by a single (p<0.05) or double (p<0.01) asterisk.
Results
Engineering of HGF/Met Functional Domains
A schematic representation of the functional domains contained in Met and HGF is shown in
ELISA Analysis of Met-HGF Interactions
The ability of Met ectodomains to interact with HGF was tested in ELISA binding assays. Soluble receptors (Decoy Met, Sema-PSI, Sema, PSI-IPT, IPT) were immobilized in solid-phase and exposed to increasing concentrations of active HGF. Binding was revealed using biotinylated anti-HGF antibodies. Non-specific HGF binding was determined using bovine serum albumin (BSA) in solid phase instead of soluble Met domains Binding affinity was determined by nonlinear regression analysis as described in Materials and Methods. In these conditions, decoy Met bound to HGF with a KD of approximately 0.2-0.3 nM. Consistent with previous measurements, Sema-PSI and Sema bound to HGF with an affinity at least one log lower compared to decoy Met. Surprisingly, both PSI-IPT and IPT bound to HGF very efficiently, with almost the same affinity as decoy Met (
To determine the affinity of each Met module for pro-HGF, HGF-α, HGF NK1 and HGF-β and to compare it with that for active HGF, engineered receptors were immobilized in solid phase and exposed to increasing concentrations of MYC-tagged ligands. Binding was revealed using anti-MYC antibodies. Non-specific binding was determined using the kringle-containing protein angiostatin (AS) also tagged with a MYC epitope—in liquid phase. Pro-HGF, HGF α-chain and HGF NK1, which represents the minimal Met-binding module of HGF α-chain, bound to Decoy Met with a 3-, 4- and 10-time reduced affinity compared to active HGF, respectively (
The α-Chain of HGF Binds to IPT Domains 3 and 4 with High Affinity
The IPT region of Met extends for about 400 amino acids and contains four IPT domains. To more finely map the IPT-HGF interface, a series of IPT variants that were deleted in one or more domains were engineered (
In fact, IPT Δ1-2, a protein corresponding to the last two immunoglobulin-like domains of Met, bound to the α-chain of HGF with equal if not higher strength than IPT. However, further deletion of either the third or fourth immunoglobulin-like domain did almost completely impair HGF-α binding. Similar results were obtained using active HGF or pro-HGF instead of HGF-α. These data suggest that the last two immunoglobulin-like domains of Met, that lie close to the trans-membrane helix in the context of a bona fide Met, are sufficient for binding the α-chain of HGF at high affinity.
IPT Domains 3 and 4 are Sufficient for Binding to HGF in Living Cells.
To determine whether HGF could bind to IPT 3 and 4 in the context of a membrane-anchored receptor, a Met protein carrying a large deletion in the extracellular region was engineered. Amino acids 25-741, corresponding to the Sema domain (aa 25-515), the PSI domain (aa 516-562) and the first two IPT domains (IPT 1 and 2, aa 563-741) were deleted, generating a recombinant receptor TO containing IPT domains 3 and 4, the transmembrane helix and the full cytoplasmic region (
To examine whether MetΔ25-741 could bind to HGF, lentiviral vector-transduced cells were incubated in the presence or absence of recombinant HGF and subsequently treated them with the cross-linking agent BS3. Cell lysates were immuno-precipitated with an antibody raised against the C-terminal portion of Met, resolved by SDS PAGE and analyzed by western blotting using anti-HGF biotinylated antibodies. As a control, the same analysis was performed on wildtype TOV-112D cells. Immunoblots showed a distinct band with a molecular weight of approximately 180 kD in the lane corresponding to cells expressing MetΔ25-741 treated with HGF but not in lanes corresponding to the same cells without HGF or to wild-type TOV-112D cells, either in the presence or absence of the ligand (
HGF Binding to IPT Domains 3 and 4 Results in Met Activation in Living Cells.
The inventors next tested whether HGF binding to MetΔ25-741 could induce Met kinase activation. To this end, lentiviral vector transduced TOV-112D cells were stimulated with pro-HGF or active HGF, and cell lysates were immunoprecipitated with anti-Met antibodies as above. Receptor activation was determined by Western blot analysis using anti-phosphotyrosine antibodies. The same blots were re-probed with anti-Met antibodies to normalize the amount of receptor immuno-precipitated. Remarkably, both pro-HGF and active HGF were capable of inducing robust phosphorylation of MetΔ25-741 (
Soluble IPT Inhibits HGF-Induced Invasive Growth In Vitro.
In a previous study, it has been demonstrated that the extracellular portion of Met expressed as a soluble protein (Decoy Met) inhibits HGF-induced invasive growth both in vitro and in mouse models of cancer (Michieli P. et al. (2004) Cancer Cell 6(1), 61-73). Recombinant soluble Sema-PSI was also shown to inhibit both ligand-dependent and -independent Met phosphorylation (Kong-Beltran M. et al. (2004) Cancer Cell 6 (1), 75-84). Based on these results, it has been tested whether soluble IPT displayed HGF/Met antagonistic activity in living cells. MDA-MB-435 human melanoma cells, which express Met and are an established model system for analysis of HGF-mediated invasive growth, were transduced with lentiviral vectors encoding soluble Decoy Met, Sema-PSI or IPT. Cells transduced with an empty vector were used as control. Lentiviral vector-transduced cells secreting comparable levels of soluble factors (approximately 50 pmol/106 cells/24 hours) were serum-starved for several days, allowing the recombinant factors to accumulate in the medium, and then stimulated with recombinant HGF. Met tyrosine phosphorylation was determined by immuno-blotting with anti-phosphotyrosine antibodies as described above. As shown in
To test the inhibitory potential of Met ectodomains in a more biological setting, the same cells were employed to perform an HGF-dependent branching morphogenesis assay. Preformed cell spheroids were seeded in a three dimensional collagen matrix and then stimulated with recombinant HGF to form tubular structures. Branching was quantified by scoring the mean number of tubules sprouting from each colony. As shown in
Soluble IPT Displays Anti-Tumor and Anti-Metastatic Activity in Mice.
The above results prompted the present inventors to explore the therapeutic potential of soluble IPT in mouse models of cancer. Lentiviral vector-transduced MDA-MB-435 melanoma cells were injected subcutaneosly into CD-1 nu−/− mice, and tumor growth was monitored over time. After approximately three weeks, tumors were extracted for analysis, and mice were subjected to autopsy. In a Kaplan-Meier-like analysis, where the percentage of tumor-free animals is plotted against time and tumor latency is quantified calculating the median in days, all engineered soluble receptors delayed the appearance of experimental tumors. However, IPT was slightly more effective than Sema-PSI and decoy Met was more potent than either IPT or Sema-PSI (
As HGF is a potent pro-angiogenic factor, it has been determined whether inhibition of HGF/Met in tumors resulted in impairment of angiogenesis. Tumor sections were analyzed by immuno-histochemistry using antibodies against Von Willebrand factor, and vessel density was assessed by microscopy (
The identification of the high affinity HGF binding site on the HGFR provided in the present disclosure allows designing of novel procedures leading to the generation of more specific inhibitors/antagonists of HGF and of HGFR. The following are non-limiting examples of novel methods for the identification of inhibitors/antagonists of HGF/HGFR targeting the high affinity binding site of HGFR or utilizing the high affinity binding site of HGFR as a tool to generate novel inhibitors/antagonists.
Development of a Monoclonal Antibody that Binds to Extracellular IPT-3 and IPT-4 Domains of HGF Preventing HGF Binding
Given that interaction of HGF with the extracellular IPT-3 and IPT-4 domains is essential for high affinity HGF binding, one can generate specific monoclonal antibodies that bind to IPT-3 and IPT-4 and compete with HGF for HGFR binding. This can be achieved by several strategies.
(A) A recombinant protein or peptide derived from IPT-3 and IPT-4 is generated by standard genetic engineering technology or chemical synthesis. This protein or peptide is injected into an appropriate laboratory animal (usually a mouse or a rat) to give rise to an immune reaction. Splenocytes are then isolated from the immunized animal and fused to a myeloma cell line, and antibody-producing hybridoma clones are selected by standard monoclonal antibody technology. Antibodies directed against IPT-3 and IPT-4 are then screened by an ELISA method similar to the ones described in this disclosure that utilizes recombinant IPT-3 and IPT-4 in solid phase and hybridoma-produced antibodies in liquid phase. Binding is revealed using anti-mouse immuno globulin antibodies that are available commercially. Alternatively, antibodies are screened for their ability to displace recombinant HGF (in liquid phase) from IPT-3 and IPT-4 (in solid phase), or for their ability to immuno-precipitate recombinant IPT-3 and IPT-4 proteins.
(B) A polynucleotide sequence coding for IPT-3 and IPT-4 inserted in an appropriate expression vector is injected directly into a laboratory animal to give rise to an immune response against the gene product. Antibodies directed against IPT-3 and IPT-4 are then isolated and screened as described above.
(C) A polynucleotide sequence coding for IPT-3 and IPT-4 inserted in an appropriate expression vector is transferred into a mammalian cell line not expressing HGFR to obtain expression of IPT-3 and IPT-4 on the cell surface. Cells expressing IPT-3 and IPT-4 are then injected into a laboratory animal to give rise to an immune response, and antibodies directed against IPT-3 and IPT-4 are isolated and screened as described above.
(D) A library of native antibodies generated by standard genetic engineering techniques (for example by the technology known as phage display) from mammalian lymphocytes (preferably human, for example from lymphocytes infiltrating a tumor expressing HGFR) is screened using recombinant IPT-3 and IPT 4 proteins. Positive clones (i.e. those clones that bind to IPT-3 and IPT-4 at high affinity) are then isolated, expanded, and the antibody characterized biochemically.
(E) Human memory B cells are isolated from the peripheral blood of a patient harboring a tumor expressing HGFR as disclosed by several studies including Traggiai E. et al. (2004) Nat Med. 10(8), 871-875. Once cultures of immortalized memory B cells are established, a skilled artisan in the field can screen for cells secreting an antibody directed against IPT-3 and IPT-4 using the methods described here above in (A). Having identified such antibody-producing cells, the desired antibody can be cloned by polymerase chain reaction and standard genetic engineering procedures.
Identification of a Test Compound that Binds to Extracellular IPT-3 and IPT-4 Domains of HGFR Inhibiting HGFR Activity
With a different approach, it is possible to isolate test compounds of diverse origin that bind to the high affinity HGF binding site of HGFR and interfere with HGF-induced HGFR activation. This can be achieved by several strategies.
(A) Using ELISA assays similar to those described in this disclosure, a skilled artisan in the field can screen a compound library (including but not limited to a synthetic chemical library, a natural compound library, a small molecule library, a peptide library) for agents that displace HGF interaction with IPT-3 and IPT-4. In this kind of assays, recombinant IPT 3 and IPT-4 protein is immobilized in solid phase and incubated with a fixed amount of HGF in liquid phase. Following exposure to library compounds, HGF binding is measured with commercially available anti-HGF antibodies.
(B) Using a cell line expressing an engineered form of the HGFR containing nothing but the IPT-3 and IPT-4 domains in the extracellular part similar to that described in this disclosure (MetΔ25-741), a skilled artisan in the field can screen a compound library (including but not limited to a synthetic chemical library, a natural compound library, a small molecule library, a peptide library) for agents that displace HGF interaction with IPT-3 and IPT-4 or inhibit HGF-induced HGFR activation. This can be achieved by putting said engineered cells in contact with the library and then measuring HGF-induced HGFR phosphorylation as described in the present study, or by other means that reveal HGFR activation including a scatter assay, a reconstituted matrix invasion assay, a branching morphogenesis assay, a cell survival assay or other in vitro biological tests as described in Michieli, P. et al. (2004) Cancer Cell 6(1), 61-73.
(C) Using the engineered cell line described above, a skilled artisan in the field can screen a genetic library (including but not limited to a cDNA expression library, a short hairpin RNA library, an antisense DNA library, a random nucleotide library) for polynucleotides or gene products that displace HGF interaction with IPT-3 and IPT 4 or inhibit HGF-induced HGFR activation. This can be achieved by transfecting, transducing or anyway introducing the nucleotide library into said cells and then testing the ability of HGF to activate the deleted form of HGFR expressed by the same cells. HGFR activation is measured as described in (B).
Exemplary Functional Assays that Measure the Biological Activity of Compounds that Bind to Extracellular IPT-3 and IPT-4 Domains of HGFR
Whatever strategy is employed to generate anti-IPT antibodies or IPT-binding compounds, the final product (i.e. monoclonal antibodies directed against IPT-3 and IPT-4 or natural or synthetic compounds that bind to IPT-3 and IPT-4) is then subjected to biological assays aimed at determining whether these agents have the ability to interfere with HGFR activity. These assays can be performed in vitro using cultured mammalian cells or in vivo using laboratory animals.
(A) Scatter assay. Epithelial cells growing in compact colonies in a Petri dish and expressing HGFR are induced to ‘scatter’ by stimulation with HGF. As a result of HGF stimulation, cells in the Petri dish appear more separated and dispersed. This assay can be performed in the presence of several test compounds. Among the compounds tested, an HGF/HGFR inhibitor/antagonist can be identified by the absence of a scattered phenotype in response to HGF stimulation.
(B) Cell migration assay. Cells expressing HGFR have the ability to migrate towards an HGF gradient. In other words, cells are attracted by chemotaxis towards higher concentrations of HGF. This ability can be exploited to screen for HGF inhibitors in a Boyden chamber assay. Cells are seeded in the first chamber and HGF is applied in the second chamber that is separated from the first by a porous membrane. Cells migrate through the membrane and reach the second chamber were HGF is more concentrated. Agents that inhibit this process are identified as HGF/HGFR inhibitors/antagonists.
(C) Transwell™ migration assay or reconstituted matrix invasion assay. This is a variation of the assay described in (B) in which the porous membrane is covered with a layer of collagen, Matrigel™, or other reconstituted organic matrices. Cells have to digest the organic matrix in order to migrate through it. This is a more rigorous assay that measures invasion rather than simple migration of cells.
(D) Collagen invasion assay or branching morphogenesis assay. In this assay, mammalian cells expressing HGFR (preferably epithelial cells or carcinoma cells) are seeded in a tridimensional layer of collagen and then allowed to grow until they form spheroids of approximately 1,000 cells each. Alternatively, spheroids can be pre-formed ahead by incubating cells overnight in non-adherent 96-well plates in the presence of methylcellulose as disclosed in Michieli, P. et al. (2004) Cancer Cell 6(1), 61-73. Once spheroids are embedded in the collagen layer, they are stimulated with HGF and incubated at 37° C. This results in the sprouting of hollow tubules from the spheroids; each tubule is formed by several cells organized in a tubular structure and polarized so that there is a side of the cell that sees the lumen and a second side that sees the medium outside. As the assay goes on tubules tend to branch and to form a more complex architecture. This assay is highly specific for HGF. Agents that inhibit this process have a high probability to represent very specific HGF/HGFR antagonists.
(E) Mitogenic assay. HGF has the ability to induce DNA replication and cell division in some cell expressing HGFR. The most responsive cells are certainly primary hepatocytes, usually mouse or rat. To test HGF-induced DNA replication, cells are deprived of serum growth factors and then stimulated with increasing concentrations of HGF. Shortly after, radioactive thymidine is added and the cells are incubated at 37° C. for approximately one day. Following extensive washing and fixation, radioactive thymidine incorporated into cell DNA is measured by liquid scintillation counting or other standard methods that allow radiation quantification.
(F) Survival assay. HGF has the ability to protect HGFR-expressing cells against apoptosis or programmed cell death. This can be exploited to measure HGFR activity in a survival assay, Cells are preincubated with HGF and the test compound (potential HGFR inhibitor), and then subjected to an apoptotic stimulus such as a toxic drug, absence of adherence, hypoxia, heat shock, radiation, or DNA damage. After an appropriate time interval, cell death is measured by standard methods including TUNEL (Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling), nucleosomes, DNA ladder, caspase activity, vital dye staining or any variation thereof.
(G) Mouse tumorigenesis assay. In this kind of assay, the activity of a potential HGF/HGFR inhibitor is tested directly in a laboratory animal, preferably a mouse or a rat. There are several methods for obtaining a tumor in a mouse. The most utilized strategy is to create a xenograft, i.e. to transplant tumor cells (usually of human origin) into an animal recipient (usually an immunodeficient mouse). Cells can be implanted subcutaneously (a quick and simple method to obtain an experimental tumor) or orthotopically, i.e. in the same organ where the tumor cell has been isolated (e.g. a breast carcinoma into the mammary fat pad, a colon carcinoma into the intestine mucosa, a hepatocarcinoma into the liver parenchyma and so on). Whatever the method employed is, injection of tumor cells into a laboratory animal gives rise to an experimental tumor. This tumor-bearing animal can now be used to evaluate the anti-tumor potential of test compounds. Anti-HGF/HGFR antibodies or compounds can be delivered to an animal carrying a tumor lesion with dysregulated HGF/HGFR signaling by the most appropriate existing method including intravenous injection, intraperitoneal injection, osmotic pump, oral administration, suppository, gene therapy protocol, local administration and so on. After an appropriate treatment period, the animal is euthanized and its tumor and organs are explanted for analysis.
(H) Mouse metastogenesis assay. Experimental metastases can be induced in a mouse by systemic injection of tumor cells. These get entrapped in the lung capillaries and subsequently extravasate to give rise to pulmonary metastases. These can be measured upon autopsy by several approaches including microscopy, histological analysis, immuno-histochemical methods, whole-body imaging. The test compounds are delivered as described in (G).
(I) Gene therapy protocol. In case the HGF/HGFR inhibitor is an antibody, a recombinant protein, a peptide or a small interfering RNA, delivery to the tumor-bearing animal can be achieved by a gene therapy approach. This consists in introducing the desired polynucleotide into an appropriate delivery vector that can be chosen among lentiviral vectors, adenoviral vectors, retroviral vectors, naked DNA, or any variation thereof. The vector preparation can be delivered sytemically or locally to the tumor depending on the tumor site, on the vector or on the tumor histotype. The biological effects of gene therapy are analyzed as described for other compounds in (G).
| Number | Date | Country | Kind |
|---|---|---|---|
| 08103958.8 | May 2008 | EP | regional |
