hOA-DN30: a highly effective humanized single-arm MET antibody inducing remission of ‘MET-addicted’ cancers

hOA-DN30 has been provided by Metis Precision Medicine B-Corp (Torino, Italy). Humanization of the DN30 mouse antibody, conversion into the ‘one-arm’ format, production, and purification of hOA-DN30 has been done by Fair Journey Biologics.

For affinity determination, the MET extracellular domain fused in frame with a human Fc domain (R&D System) was in the solid phase and pure MvDN30 or hOA-DN30 were in the liquid phase. ELISA signal was quantified by the multi-label reader VICTOR X4 (Perkin Elmer Instrument INC.).

For hOA-DN30 quantification in mouse serum, a goat anti-human IgG antibody (Sigma Aldrich) was in the solid phase. For hOA-DN30 quantification in monkey serum samples, neutravidin-coated plates were used to immobilize biotin-labeled goat anti-human IgG monkey adsorbed antibody (Southern Biotech). In both cases, pure hOA-DN30 and serum samples were in the liquid phase. The concentration of hOA-DN30 in serum was achieved by interpolating the optical density (OD) readings of the samples with the calibration curve fitted by a weighted (1/Y) 4 parameters regression function.

For dose-dependent experiments, cells were incubated in serum-free medium for 48 h with the indicated concentration of hOA-DN30. For time-dependent experiments, cells were incubated in serum-free medium in the presence of hOA-DN30 (1 μM) and samples were collected at the indicated time points. Proteins in cell extracts or in cell culture supernatants were resolved by SDS-PAGE and analyzed by Western blotting.

For analysis of MET recovery after shedding, cells were incubated in serum-free medium in the presence of hOA-DN30 (1 μM); after 48 h, cell monolayers were extensively washed and fresh serum-free medium was added. Cell culture supernatants and cell lysates were collected at different time points and analyzed as described above.

For inhibition of MET and downstream signalling pathway activation, cells were treated with the indicated concentrations of hOA-DN30 for 24 h in serum-free medium. Cell monolayers were lysed and analyzed as described above.

For scatter assay, cells were treated for 24 h with HGF (8 ng/ml, R&D System), hOA-DN30, or DN30 mAb (both 200 nM). Cell scattering was determined by optical microscopy.

For cell growth assay, cells were treated with increasing concentration of hOA-DN30, and viability was evaluated after 72 h by CellTiter-Glo luminescent cell viability assay (Promega Corp.), according to the manufacturer’s instructions. Chemiluminescence was detected with VICTOR X4.

For proliferation assay, cells were treated with hOA-DN30 (1 μM). After 48 h cellular DNA synthesis was determined measuring EdU incorporation using the Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to manufacturer’s instruction.

For cell death assay, cells were treated as described for cell growth assay. Cytotoxicity was evaluated after 72 h by CellTox™ Green Cytotoxicity Assay (Promega Corp., Madison, WI), according to the manufacturer’s instructions. Fluorescence was detected with VICTOR X4 and data normalized over CellTiter-Glo luminescence.

For determination of M192 tumor organoid viability, cells were incubated for 5 h with Alamar blue (Life Technologies). The signal measured in the cell culture supernatants by VICTOR X4 was set as time 0, then freshly prepared medium with 1 or 5 μM hOA-DN30 was added to the culture. Every 3 days, the medium was replaced with a fresh one containing the antibody. After 9 days, a new assessment of the Alamar blue signal was performed. Cell growth was measured calculating the ratio between day 9 and day 0 for each sample.

For determination of Antibody-Dependent Cellular Cytotoxicity (ADCC), cells were treated with increasing concentrations of hOA-DN30, and effector cells were added. After 6 h, ADCC activity has been measured by ADCC Reporter Bioassay (Promega Corp.) according to the manufacturer’s instructions.

All procedures in mice were performed according to protocols approved by the Ethical Committee for animal experimentation of the Candiolo Cancer Institute and by the Italian Ministry of Health.

To generate Cell Derived Xenografts (CDX), cancer cells were injected subcutaneously into the right posterior flank of adult NOD-SCID mice. To generate Patient Derived Xenografts (PDX), tumor materials derived from human gastric tumor specimens expanded for at least 2 generations in mice were implanted in a subcutaneous pocket generated in the flank of NOD-SCID mice as described in [31]. When tumors were established, animals were divided into experimental arms homogeneous for tumor size and randomly assigned to the different treatments. hOA-DN30 was administered by intravenous injection. Tumor size was evaluated periodically with a caliper. Tumor volume was calculated as described [30].

For pharmacokinetic (PK) evaluation in mice, adult male NOD/SCID mice bearing or not EBC-1 tumors were used. Animals were treated with 30 mg/kg of hOA-DN30 in a single intravenous administration. Animals were bled at different time points. hOA-DN30 levels in mouse serum were determined by ELISA assay (see above) by Accelera Srl. (Nerviano, Italy). PK analysis was performed according to standard non-compartmental and compartmental approach using Phoenix-WinNonlin package (v. 6.3, Pharsight Inc., Certara Company) by Accelera Srl.

For PK evaluation in Cynomolgus monkeys, the experiments have been performed by Accelera Srl. Three adult male monkeys were administered with hOA-DN30 as a single intravenous bolus at the dose of 11 mg/kg and animals were bled at different time points. hOA-DN30 concentrations in serum were determined by ELISA assay (see above). PK analysis was performed according to the standard non-compartmental approach as described above.

For pharmacodynamics (PD), the analysis was performed by Accelera Srl, applying an E_max (maximum kill rate) PK/PD model [32] to tumor volumes measured in mice treated with the indicated doses of hOA-DN30. The threshold systemic (plasma) concentration for tumour stabilization (C_τ) was calculated from the equilibrium status of the first differential equation of the model.

Determination of hOA-DN30 tolerability was performed by Accelera Srl. The antibody was administered intravenously according to ascending doses (30, 90, and 180 mg/kg) one week apart, or at the dose of 180 mg/mg at weekly intervals for two times to two adult Cynomolgus monkeys (one male, one female). Animal’s healthy status, clinical signs observation, body weights, and food consumption were evaluated periodically. Standard hematological and serum chemical parameters were determined. At necropsy, body weight measurement and macroscopic examinations were done.

All the studies performed by Accelera Srl. have been sponsored by Metis Precision Medicine B-Corp.

Averages, standard deviations, and P values obtained by Student’s t-Test were calculated using Microsoft Office Excel 2010 software (Microsoft Corporation). To calculate Kd and B_max, data from ELISA assay were analyzed and fitted according to nonlinear regression, one-site binding hyperbola curve, using GraphPad Prism software (GraphPad Software). To calculate IC₅₀, data from growth assays were analyzed and fitted according to a nonlinear regression, sigmoidal dose-response curve, using GraphPad Prism software. P values obtained by One-way or Two-way Anova were calculated using GraphPad Prism software.

The hOA-DN30 molecule was generated from the sequence of the DN30 mAb [33], modified to obtain a humanized antibody characterized by: (i) a monovalent structure, achieved by assembling three different polypeptides, i.e. one antibody light chain, one full-length IgG1 heavy chain, and one Fc region (Fig. 1A); (ii) introduction of specific amino acid modifications in the CH3 domains of the heavy chain and of the Fc domain, to produce the ‘knob into hole’ structure [34]; (iii) a high degree of homology/identity with human germline antibody sequences (91.75% and 93.4%, respectively). The recombinant purified product (Fig. 1B) has been tested for MET binding by ELISA assay. hOA-DN30 interacted with MET with high affinity; the calculated constant of dissociation (Kd) was similar to its parental reference molecule, the chimeric Fab fragment (MvDN30) [35] (Fig. 1C). No residual MET agonistic activity was observed by assessing hOA-DN30 ligand mimetic properties in scatter assay, a highly sensitive procedure to evaluate the induction of MET-mediated responses in the cells (Fig. 1D).

The ability of hOA-DN30 to promote MET receptor shedding and downregulation was evaluated in A549 NSCLC cells. hOA-DN30 efficiently induced MET shedding, resulting in a dose-dependent release of soluble MET ectodomains (ECDs) in the extracellular space, and in concomitant progressive elimination of MET receptor from the cells (Fig. 2A). At the highest dose tested (1 μM), MET depletion was detected starting from 1 h of antibody treatment, and after 4 days MET was erased from the cell, while accumulation of MET-ECDs in the extracellular environment was detectable after 8 h (Fig. 2B). After hOA-DN30 withdrawal, MET expression was recovered within 24 h (Fig. 2C). Kinetics of hOA-DN30-induced MET shedding and of MET expression recovery after antibody treatment were evaluated also in MET-amplified EBC-1 human NSCLC cells (Suppl. Fig. 1A and B). As a consequence of receptor over-expression due to gene amplification, human gastric carcinoma GTL-16 cells feature a constitutively active MET, highly phosphorylated in the absence of HGF [36]. Upon hOA-DN30 treatment, MET phosphorylation dropped-off according to the antibody given dose (Fig. 2D, left panels). In parallel, also the signalling pathway downstream MET was impaired, as assessed by phosphorylation levels of AKT and ERK, the two major nodes of the MET-triggered intracellular signalling cascade (Fig. 2D, right panels). Inhibition of MET and intracellular transducers activation was assessed also in EBC-1 cells (Suppl. Fig. 1C).

A panel of exponentially growing human carcinoma cells, characterized by a different MET gene copy number (Suppl. Table 1), were treated with increasing concentrations of hOA-DN30. The antibody inhibited the growth of all cancer cells carrying high MET gene amplification (Fig. 3A). The calculated IC₅₀ values were in the nanomolar range (Suppl. Fig. 2). Conversely, hOA-DN30 was not effective in cells carrying diploid or low amplified wild-type MET (Fig. 3A). To further analyze the mechanisms underlining the growth inhibition exerted by hOA-DN30, cell proliferation and cell death upon antibody treatment were evaluated in the highly MET-amplified cell lines. hOA-DN30 greatly affected cell proliferation, measured by the incorporation of EdU during the S phase of the cell cycle (Fig. 3B) and induced cell death (Fig. 3C). Moreover, dose-dependent inhibition of cell growth by hOA-DN30 was established in organoids derived from human tumor tissue expanded in mice after subcutaneous implantation of bioptic material derived from a colon cancer patient characterized by high MET gene amplification (PDX M192) [16], (Suppl. Fig. 3). To investigate if the antitumor activity of hOA-DN30 could be further enhanced by triggering Antibody-Dependent Cellular Cytotoxicity (ADCC), we performed an in vitro test incubating the highly MET-amplified EBC-1 and GTL-16 cells with the antibody, in the presence of effector cells. Data reported in Suppl. Fig. 4 show that hOA-DN30 did not exert this function. Finally, a preliminary study was conducted to assess the activity of hOA-DN30 in comparison to ABT-700, a MET antibody tested in the clinic (NCT01472016). This antibody interacts with the IPT-1 domain of MET and displays a mechanism of action different from hOA-DN30, as it inhibits both HGF- dependent and independent MET activation by impairing HGF binding and counteracting receptor dimerization [37, 38]. hOA-DN30 induced a higher maximal inhibition of MET-addicted EBC-1 cell growth compared to ABT-700 (Suppl. Fig. 5).

The ability of hOA-DN30 to inhibit tumor growth in vivo was tested on different MET-addicted Cell line Derived Xenograft (CDX) models, characterized by high levels of MET gene amplification (Suppl. Table 1). hOA-DN30 showed a therapeutic effect, inhibiting GTL-16 tumor growth with a dose-response behavior (29%, 83%, and 92% inhibition at 3.3, 10, and 60 mg/kg three times a week, respectively; Fig. 4A). Inhibition of cancer cell proliferation has been confirmed by Ki67 staining on the tumor masses upon sacrifice (Suppl. Fig. 6A). Dose-dependent down-modulation of the target in vivo was assessed by evaluation of MET phosphorylation (Fig. 4B) and MET protein levels (Suppl. Fig. 6B) within the tumors at sacrifice. Moreover, we proved the occurrence of MET shedding in vivo by measuring the amount of circulating human MET-ECD at different time points after hOA-DN30 treatment (Fig. 4C). Dose-dependent efficacy was also assessed in the EBC-1 CDX model. The hOA-DN30 dose of 30 mg/kg once a week was sufficient to induce complete tumor remission in all treated mice, and the effect persisted for a long time even after treatment discontinuation (Fig. 4D). Furthermore, EBC-1 tumors were strongly responsive to hOA-DN30 treatment, independently from the size of the tumor masses at first treatment (Suppl. Fig. 7A). To explore the impact of the treatment schedule on the therapeutic response to hOA-DN30, an equal total amount of antibody (2400 μg/mouse), fractionated in 2 or 12 administrations, was delivered to mice carrying EBC-1 tumors. Data reported in Fig. 4E show that few administrations at higher doses induced a better therapeutic response compared with frequent administrations at lower doses. On a third CDX model, SNU-5, hOA-DN30 was extremely effective, inducing complete tumor remission; moreover, upon antibody withdrawal, no tumor recurrence was observed (Fig. 4F). Anti-tumor efficacy was also observed against Hs746T, a gastric cancer CDX model featuring both MET-amplification and exon 14 skipping mutations (Suppl Fig. 7B). It is well known that the tumor microenvironment can favor tumor growth and can influence the response to anti-cancer molecules [39]. We previously reported that modifications in cancer cell metabolism instruct cancer-associated fibroblasts to increase HGF production, sustaining an adaptive resistance response [40]. To test if hOA-DN30 therapeutic activity could be impaired in the presence of HGF overexpression, tumors obtained by injection of EBC-1 cells featuring the above described adaptive resistance (EBC-1_Res) were treated with the antibody. Data reported in Suppl. Fig. 8 demonstrate that hOA-DN30 is active in breaking non-cell-autonomous resistance.

Finally, hOA-DN30 activity was evaluated against MET-amplified Patient Derived Xenografts (PDXs) of gastric cancer origin. MET gene copy number for each PDX is reported in Suppl. Table 1. In the GTR-561 model, characterized by a high MET gene copy number, tumors masses shrank very fast and completely disappeared. Upon suspension of the treatment, the antibody effect was extremely long-lasting; mice survived tumor-free for more than 90 days (Fig. 5A). A similar complete and durable tumor remission was observed also in SG-16, a second PDX model featuring high MET gene amplification (Fig. 5B). On the contrary, and in line with what was observed in vitro, the growth of PDXs characterized by a low grade of MET gene amplification was not impaired by hOA-DN30 treatment (Suppl. Fig. 9).

hOA-DN30 underwent preclinical studies to evaluate its pharmacokinetic (PK) properties in mice and in non-human primates. PK in mice was performed by administering a single dose (30 mg/kg) of the antibody by intravenous injection to immune-deficient mice bearing or not tumors. hOA-DN30 levels in serum samples are shown in (Fig. 6A). A PK profile was obtained by applying both non-compartmental and compartmental analysis. Results from the two different methods were in agreement (Suppl. Tables 2 and 3), showing low clearance (0.42 mL/h/kg), low volume of distribution (66–70 mL/kg), and prolonged half-life (approximately 5 days). Comparing data acquired from healthy mice with those obtained from tumor-bearing animals, the first decline phase was comparable between the two groups, as well as the volume of distribution, while differences were scored in terms of clearance (higher in mice with tumors), terminal half-life and mean residence time (both shorter in mice with tumors).

Data from the in vivo study performed on EBC-1 tumors treated with different doses of the antibody (see above Fig. 4D), were compared to the theoretical results obtained applying a PK/PD model developed by using an E_max (maximum kill rate) model [32]. The analysis showed that the model correctly describes tumor volume variations, in all the conditions tested (Fig. 6B). The mean values of the PK parameters obtained in tumor-bearing mice treated with 30 mg/kg of hOA-DN30 were used to simulate inside the PK/PD model the concentration-time profile of the antibody at the ascending doses of the compound. From this analysis, the in vivo serum concentration of hOA-DN30 maintaining tumor growth stabilization (Cτ) was estimated to be 72.9 μg/mL. Notably, administering the dose of 30 mg/kg, serum levels of the compound were above the Cτ threshold throughout the whole treatment (Suppl. Fig. 10).

The PK profile was also analyzed in non-human primates, by delivering hOA-DN30 as a single intravenous bolus at the dose of 11 mg/kg to male Cynomolgus monkeys. The serum concentration of the antibody declined according to a poly-exponential curve (Fig. 6C). The PK profile of the compound was characterized by low serum clearance (0.32 mL/h/kg), low volume of distribution (80 mL/kg), and long terminal half-life (8 days), in good agreement with data obtained in the mouse. PK parameters are reported in Suppl. Table 4.

To define the preclinical model/s suitable for studying antibody safety, we assessed hOA-DN30 binding properties versus MET derived from different species. hOA-DN30 cross-reacted with the MET receptor of human, rat, dog, and monkey origin, while the interaction with mouse MET was very weak (Suppl. Fig. 11A and B). Thus, the Cynomolgus monkey model was selected to perform a dose-escalation study. hOA-DN30, administered according to ascending doses (30, 90, and 180 mg/kg) once a week, was well tolerated. There were no significant toxicological observations or changes in hematology or blood chemistry analysis during the period of antibody administration. Moreover, no late-onset toxicity was observed during a two weeks dosing-free recovery period. Based on these data, we performed a second study in which the highest dose (180 mg/kg) was administered twice, at a weekly interval. Also during this study, no relevant change in body weight (Suppl. Fig. 12), food intake, clinical pathology, and gross examination were observed. Thus, the dose of 180 mg/kg was established as the no observed adverse effect level (NOAEL).