Methods
Antibodies, reagents and cell culture
ABT-700, an anti-human c-Met antibody derived from the mAb 224G11 [
12] was produced in a stable CHO line. Fab and F(ab)’2 of mAb224G11 (ABT-700) were generated by digestion with papain or pepsin as described in the literature [
13]. Control human IgG was purchased from Sigma (I4506). 5D5 mouse anti-human c-Met antibody, the parental bivalent antibody from which the single armed antibody onartuzumab was derived, was purified from hybridoma supernatant (ATCC #HB11895). The anti-c-Met antibody, LY2875358, was expressed in and purified from HEK293 cells using amino acid sequences derived from published patent application US201012936. The c-Met tyrosine kinase inhibitor, PF-4217903, was purchased from Selleck (Catalog No.S1094). Recombinant human c-Met extracellular domain with a histidine tag (rh-c-Met ECD-6His) was expressed in and purified from HEK293 cells. HGF was purchased from R&D (rhHGF, #294-HGN/CF). The tumor cell lines A549 (ATCC #CCL-185), EBC1 (JCRB #0820), Hs746T (ATCC #HTB-135), and OE33 (Sigma #96070808) were maintained in DMEM (Gibco-Invitrogen cat. No. 11995) supplemented with 10 % fetal bovine serum (FBS) (HyClone SH30070.03). IM95 (JCRB #1075) were also maintained in DMEM, 10 % FBS with 10 mg/L insulin. SNU5 (ATCC #CRL-5973), NCI-H441 (ATCC #HTB-174), NCI-H1993 (ATCC #CRL-5909), MKN45 (JCRB 0245), SNU620 (KCLB #00620), and SNU638 (KCLB #00638) were cultured in RPMI-1640 (Gibco-Invitrogen, cat. No. 11875) supplemented with 10% FBS. MCF7 cells (ATCC HTB-22) were infected with control lentivirus or lentivirus containing human c-Met cDNA in pLVX-IRES-puro vector (Clontech). Stable clones overexpressing human c-Met protein indicated by Western Blot and FACS were isolated. These cells were grown in DMEM (Gibco-Invitrogen cat. No. 11995) supplemented with 10 % fetal bovine serum (FBS) (HyClone SH30070.03) and 2 μg/mL puromycin (Sigma). All cell lines were expanded in culture upon receipt and cryopreserved to provide cells at similar stage passages for all subsequent experiments. For cell lines not authenticated in the 6 months before use, c-Met expression was confirmed by FACS analysis. Information of additional cell lines is summarized in Additional file
1: Table S1.
Binding ELISA
96-well plates (Costar #3369) were coated with 100 μL/well of mouse anti-His antibody (Invitrogen #37-2900) at 1 μg/mL in PBS pH7.4 at 4 °C overnight, and then blocked using Superblock (Pierce, #37535) for one hour at room temperature. Plates were washed 4 times with PBST and then incubated with 100 μL of recombinant human c-Met extracellular domain (rh-c-Met ECD-6His) at 2 μg/mL in 10 % Superblock in PBST for 1 h at room temperature. Plates were washed 4 times with PBST and then incubated with ABT-700 or control human IgG in serial dilutions in 10 % Superblock in triplicate wells at room temperature for 1 h. Plates were washed 4 times with PBST and then incubated with 100 μL of 1:15,000 goat anti-human IgG-HRP (Thermo-scientific Pierce, Cat#31412) at room temperature for 1 h. Plates were washed 4 times in PBST and 100 μL of TMB (Pierce, #34028) was added to each well and incubated at room temperature until color developed (approximately 10 min). Reactions were stopped by addition of 2N sulfuric acid (Mallinckrodt chemicals, Cat#H381-05) and optical density (OD) was read at 450 nm.
FACS analysis
For cellular c-Met binding studies, cells were harvested from flasks when approximately 80 % confluent using Cell Dissociation Buffer (Invitrogen #13151-014 or #13150-016). Cell viability was checked by trypan blue staining to ensure >90 % live cells. Cells were washed once in PBS/1 % FBS (FACS buffer) then resuspended at 1.5-2.5 × 106 cells/mL in FACS buffer. Cells were added to a round bottom 96-well plate (BD Falcon #3910) at 100 μL/well. Ten μL of a 10x concentration of ABT-700 or controls in duplicate wells was added and plates were incubated at 4 °C for four hours. Wells were washed twice with FACS buffer then resuspended in 50 μL of 1:500 anti-human IgG Ab (AlexaFluor 488, Invitrogen #11013) diluted in FACS buffer. Plates were incubated at 4°C for one hour then washed twice with FACS buffer. Cells were then resuspended in 100 μL of PBS/1 % formaldehyde and analyzed on a Becton Dickinson LSRII flow cytometer. FACS binding studies were performed for each cell line in at least two independent experiments.
For Annexin V apoptosis detection, tumor cells were plated at 300,000 cells/ well in 12-well dishes in 2 ml serum-free media (RPMI, 0.1 % BSA). Cells were incubated overnight at 37 °C, 5 % CO2. Cells were treated with control hIgG (Sigma I4506) and ABT-700 at 10 μg/ml for 24 h. Cells were transferred from 12-well plate into 1.5 ml microcentrifuge tubes, pelleted, and washed with cold PBS. Cells were resuspended in 0.1 ml 1X Binding Buffer provided in kit (BD Pharmingen kit cat# 556547). 5 μl of FITC Annexin V and 5 μl propidium iodide (PI) were added and cells were incubated in the dark for 15 min. 400 μl of 1X Binding Buffer was added and cells were analyzed on a Becton Dickinson LSRII flow cytometer within one hour.
Determination of cellular c-Met phosphorylation and total level
A549 cells were plated at 40,000 per well in 96-well plate in growth media. Twenty four hours later, cells were pretreated with antibodies in duplicate wells for one hour at 37 °C, and then stimulated with HGF for 10 min at 37 °C. 1 nM (~100 ng/mL) HGF was used to stimulate c-Met as described in the literature [
11]. For SNU5 cells that have constitutively phosphorylated c-Met, the cells were plated at 20,000 per well in 96-well V-bottom plate in serum free medium. Twenty four hours later, cells were treated with antibodies in duplicate wells for six hours at 37 °C. Media were then removed and cells were lysed with 100 (for A549) or 150 (for SNU5) μL/well of Cell Lysis Buffer (Cell Signaling Technology #9803) supplemented with protease inhibitor tablet (Roche #11714900). ELISA capture plates were generated by pre-coating wells with 100 μL of an anti-c-Met antibody (R&D systems, # MAB3581) at 2 μg/mL) at 4 °C overnight, followed by blocking with 200 μL/well PBS/1 % BSA treatment for one hour at room temperature, and washed three times in PBST. Cell lysates were added to capture plates and incubated at 4°C overnight. Plates were washed 3 times in PBST, and incubated with anti-phospho-tyrosine 4G10-HRP conjugate (Millipore #16-105; 1:1000 diluted in PBST + 1 %BSA) for 2 h at room temperature. To determine total c-Met, secondary anti-c-Met HRP conjugate was used. Plates were washed 3 times in PBST and 100 μL of TMB was added to each well and incubated at room temperature until color developed. Reactions were stopped by addition of 100 μL/well 2N sulfuric acid, and the OD was read at 450 nm. These studies were performed for each cell line in at least two independent experiments.
Western blot analysis
Cells were plated at 300,000 per well in 12-well tissue culture plates and were incubated overnight in growth media. Cells were incubated with ABT-700 or control for the time points as indicated at 37 °C. Cells were then lysed with 100 μL/well of 2X LDS NuPAGE sample buffer (Invitrogen NP0007) with reducing reagent (Invitrogen NP0009). Cell lysates were resolved by SDS-PAGE using 4-12 % Bis-Tris NuPAGE gels (Invitrogen NP0322) and transferred to PVDF membranes (Millipore Immobilon-FL # IPFL07810). Blots were blocked with Odyssey Blocking Buffer (LI-COR # 927-40000) for one hour at room temperature, washed three times with PBST, and then incubated overnight with appropriate primary antibodies at 4 °C. Following overnight incubation with primary antibodies, blots were washed three times with PBST for ten minutes, and then incubated with either AlexaFluor680 goat anti-rabbit IgG (Invitrogen A21109, 1:10000) or goat anti-mouse IRDye 800CW (Odyssey # 926–32210, 1:5000) for one hour at room temperature. Blots were then washed three times with PBST, and visualized by scanning using an LI-COR Instrument Odyssey (Model # 9120). Primary antibodies included mouse anti-c-Met (Cell Signaling # 3148 or # 3127), rabbit anti-phospho-Y1234 c-Met (Cell Signaling Technology, # 3077), phosphor-PLCr (Cell Signaling #2821) , phosphor-Erk (Invitrogen #44680G), phosphor-Bad (Cell Signaling #9291), total bad (Cell Signaling #9292), Bim (Cell Signaling #2819), Bcl-xL (BD #51-6646GR), cytochrome C (BD Pharmingen Cat 556433), cleaved PARP (Epitomic #1074-1), and mouse anti-actin (Sigma, # A5441). At least two independent experiments were carried out for each cell line.
Cytochrome C release assay
SNU5 cells (4×106) were treated with control hIgG or ABT-700 for 24 h. Cells were washed in PBS, pelleted and resuspended in 50–100 μL Digitonin Lysis buffer (75 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mM EDTA, 350 μg/mL digitonin, and 250 mM sucrose) by pipetting up and down, and incubated for 30 s. Cells were pelleted at high speed in microcentrifuge for 1 min, supernatant (cytosolic fraction) was collected and 2x NuPAGE sample buffer was added for Western analysis. Pellets (organelle-containing membrane fraction) were washed in cold PBS 3 times, and lysed by sonication in 1x NuPAGE sample buffer for Western analysis. The study was performed in two independent experiments.
Proliferation assay
Tumor cells were plated in 96-well plate (Falcon 35–3075) in 180 μL growth media at 3000–5,000 cells/well. The cells were incubated overnight at 37 °C with 5 % CO2. On Day 2, dilutions of testing articles were added to the cell plate (20 μL/well) in triplicate wells. Untreated control wells (for 0 % control) and wells treated with 10 μM staurosporin (for 100 % kill) were included in each plate. The plates were incubated for 3–5 days at 37 °C with 5 % CO2. To quantify live cells, media was removed and 1x Cell Titer Aqueous One Solution (Promega, G3581) diluted in Opti-mem media (Invitrogen # 31985–070) was added to plates and the plates were then incubated for one hour at 37 °C. The OD at 490 nm was read on a M5 Spectramax plate reader (Molecular Probes). Percent inhibition was calculated based on 100 % kill and untreated control wells using the following formula: 100x (0 % control - treated)/(0 % control - 100 % kill). For cells grown in suspension such as SNU5, cells were plated in 96-well plate in 180 μL medium at 10,000 cells/well and incubated overnight at 37 °C with 5 % CO2. The same protocol as above was used for treatment and data processing except live cells were detected by adding forty μL of Cell Titer Aqueous One Solution to each well and the plates were then incubated for one hour at 37 °C. The experiments were repeated at least twice for each cell line.
Anti-tumor efficacy studies in vivo
Studies with gastric (SNU5 and SNU620), lung (EBC1, NCI-H441) and glioblastoma (U87MG) were carried out at Pierre Fabre. Animal Ethical Committee was registered under the CEA-CIPF-108 number. All experiments conformed to the United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia UKCCR for animal care and use. SCID mice were from Charles River Laboratories (L’Arbresle, France). Athymic Nude Mice were acquired from Harlan (Gannat, France). All animals were housed on a 12 h light/dark cycle, in sterilized filter-topped cages, in a temperature 22 +/−2 °C and in humidity (30 to 70 %) controlled room. Mice were maintained in sterile conditions with food and water provided ad libitum and manipulated according to French and European guidelines. Animals were examined before the initiation of experiments to ensure that they were healthy and acclimated to the laboratory environment.
Cells (5–10 × 106) were implanted s.c. into the right flank region of mice. Tumor bearing mice were size matched and randomized into study groups (n = 5 or 6 as shown in figure legends). Each experiment consisted of an ABT-700 dose evaluation injected i.p. compared to controls. Evaluation of the anti-tumor activity was determined by measuring tumor volume twice a week using the formula: π/6 × length × width × height.
For the ectopic EBC1 lung metastasis and survival model, mice were injected s.c. with 7x106 EBC1 cells on D0, and after 5 days, when tumor volume reached 60 mm3 to 80 mm3, mice were size matched and randomized into groups (n = 7) for treatment with ABT-700 or control administered by i.p. injections every 21 days. From D5 to D21, tumor volume was monitored twice a week with an electronic caliper. On day 21, subcutaneous primary tumors were resected from mice anesthetized with a Ketamine/Xylazine mixture (70/30) injected intra-muscularly. ABT-700 antitumor activity was monitored by following animal mortality.
Additional animal groups were introduced for the gastric xenograft model SNU5 in order to evaluate pharmacodynamic markers by immunohistochemistry as described in detail in Additional file
2: Supplementary methods.
Experiments with the gastric Hs746T s.c. xenograft model were conducted at AbbVie in compliance with AbbVie’s Institutional Animal Care and Use Committee and the National Institutes of Health Guide for Care and Use of Laboratory Animals guidelines in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. SCID mice were obtained from Charles River (Wilmington, MA). Mice were acclimated to the animal facilities for a period of at least one week prior to commencement of experiments.
Hs746T cells (2 × 106) were inoculated in the flanks of male SCID mice and tumor-bearing animals were size matched and randomly assigned to cohorts to a mean tumor volume of approximately 225 mm3 per group (N = 10) 15 days post inoculation of cells. Dosing for all agents was initiated on day 16. ABT-700 (10 mg/kg) was administered twice a week i.p. while docetaxel (7.5 mg/kg) was administered i.v. as a single dose. A human IgG control antibody was used as a negative control agent. Tumor dimensions were determined twice weekly and volume determined with the formula (L x W2)/2.
Fluorescence in situ hybridization (FISH) analysis
MET gene copy numbers in cell lines, tumor xenografts and human tumor tissue specimens was detected with a probe mix (Vysis LSI MET SpectrumOrange/Vysis CEP 7 SpectrumGreen, Abbott Molecular) using protocols described in detail in Additional file
2: Supplementary methods.
Statistical analysis
Results are expressed as the mean ± SEM. All data were analyzed with GraphPad Prism V6.05 (GraphPad Software, Inc., San Diego, CA). The difference in tumor growth between different groups was analyzed by two-way ANOVA Turkey’s multiple comparison tests. The survival data were analyzed using Log-rank (Mantel-Cox) test. A P value <0.05 was considered significant.
Discussion
MET amplification is associated with poor prognosis in a variety of cancers highlighting the significant unmet medical need of this patient population. We describe preclinical results that justify the clinical investigation of ABT-700 in cancer types with addiction to the
MET oncogene. In a phase 1 clinical trial in patients with advanced solid tumors [
25], ABT-700 was well tolerated and the monotherapy at the recommended dose of 15 mg/kg demonstrated anti-tumor activity in patients with
MET amplified solid tumors [
26]. By RECIST, 3 of 5 patients with tumors harboring
MET amplification, as determined by FISH, had a partial response (1 each ovarian, gastric and esophageal). Among these 3 patients, the duration of response was 19, 23, and 24 weeks, respectively [
26]. This study has been expanded to enroll additional patients with
MET amplification and to better define predictive biomarkers, safety and clinical benefit.
The tumor cell lines evaluated in our study herein that harbor
MET gene amplification, as defined by FISH analysis, and overexpress c-Met protein, are sensitive to ABT-700. Cancer cells addicted to constitutively activated c-Met undergo apoptosis upon exposure to ABT-700 monotherapy leading to tumor regression in preclinical animal models. Our data emphasizes the need for a patient selection strategy that harbor
MET amplification as they most likely would benefit from ABT-700 treatment. Ultimately, ABT-700 may have broader clinical utility as the prevalence of
MET amplified tumors increases with disease progression, recurrence and/or treatment regimens [
1‐
4]. Thus, acquiring fresh tumor biopsies for FISH analysis may be needed to identify patients with
MET amplification. Recently somatic splice site alterations at
MET exon 14 (METex14) that result in exon skipping and
MET activation were described in human cancers [
27]. Although we have not included METex14 analysis in our studies, it will be important to establish the clinical relevance of these genetic aberrations.
In addition to inhibiting tumors with MET gene amplification, ABT-700 also inhibits the subcutaneous xenograft growth of human tumor cell lines that have c-Met protein overexpression or autocrine HGF. Patients with tumors driven by HGF-dependent c-Met activation may benefit from combination of ABT-700 with chemotherapy. In this context, the demonstration of more robust and sustainable anti-tumor activity in animal models following the combination of ABT-700 with different standard of care cytoreductive chemotherapies provides the basis for exploration of effective combination therapy in the clinic.
ABT-700 is among the first reported bivalent anti-c-Met antibodies that lack agonistic activity [
12,
15]. By inhibiting both ligand-dependent and c-Met overexpression-induced signaling in broad types of cancer cells, ABT-700 differentiates from other therapeutic agents targeting the HGF/c-Met axis [
6,
7,
11] including the recently disclosed antagonistic IgG4 c-Met targeting antibody LY2875358 [
15]. ABT-700 binds a unique epitope on c-Met outside the Sema domain which 5D5 recognizes [
11,
12] and antagonizes c-Met signaling in a variety of cancer cells including SNU620 gastric cancers where LY2875358 demonstrated no in vivo antitumor activity. Since ABT-700 contains a humanized IgG1 heavy chain, antibody effector functions may also contribute to its antitumor activity. Results from in vitro ADCC functional assays indicate that ABT-700 can mediate the lysis of cellular targets by human natural killer cells (data not shown).
Competing interests
JW, LT, QZ, KV, AO, EB, MA, WP, PA, AB, LN and ER are employees of AbbVie. LG, AG and NC are employees of Pierre Fabre. MS, IS and EP are employees of Abbott. The design, study conduct, and financial support for this research were provided by AbbVie, Pierre Fabre & Abbott. AbbVie, Pierre Fabre and Abbott participated in the interpretation of data, review, and approval of the manuscript.
Authors’ contributions
Conception and design: JW, LG, ER. Development of methodology: JW, LG, LT, QZ, AG, KSV, AO, EB, MS, IS, MA, WP, PA. Acquisition of data (provided animals acquired and managed patients, provided facilities, etc.): LT, QZ, AG, KSV, AO, MS, IS, MA, WP, PA. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): JW, LG, LT, QZ, AG, KSV, AO, EB, MS, IS, EP, MA, WP, PA, AB, LN, NC and ER. Writing, review, and/or revision of the manuscript: JW, LG, LT, QZ, AG, KSV, AO, EB, MS, IS, EP, MA, WP, PA, AB, LN, NC and ER. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): JW, LG, LT, QZ, AG, KSV, AO, EB, MS, IS, EP, MA, WP, PA. Study supervision: JW, LG, EB, IS, EP, PA, AB, LN, NC and ER. All authors have read and approved the final version of the manuscript.