Placental and Fetal Effects of Onartuzumab, a Met/HGF Signaling Antagonist, When Administered to Pregnant Cynomolgus Monkeys

Placental and Fetal Effects of Onartuzumab, a Met/HGF Signaling Antagonist, When Administered to... Abstract Onartuzumab is an engineered single arm, monovalent monoclonal antibody that targets the MET receptor and prevents hepatocyte growth factor (HGF) signaling. Knockout mice have clearly demonstrated that HGF/MET signaling is developmentally critical. A pre- and postnatal development study (enhanced design) was conducted in cynomolgus monkeys to evaluate the potential developmental consequences following onartuzumab administration. Control or onartuzumab, at loading/maintenance doses of 75/50 mg/kg (low) or 100/100 mg/kg (high), was administered intravenously once weekly to 12 confirmed pregnant female cynomolgus monkeys per group from gestation day (GD) 20 through GD 174. Onartuzumab administration resulted in decreased gestation length, decreased birth weight, and increased fetal and perinatal mortality. A GD147 C-section was conducted for a subset of Control and High Dose monkeys, and identified placental infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate. These findings were limited to placentas from onartuzumab-treated animals. In addition, decreased cellularity of the hepatocytes with dilated hepatic sinusoids was inconsistently observed in the liver of a few fetal or infant monkeys that died in the perinatal period. Surviving offspring had some evidence of developmental delay compared with controls, but no overt teratogenicity. Overall, effects on the perinatal fetuses were consistent with those reported in knockout mice, but not as severe. Onartuzumab concentrations were low or below the level of detection in most offspring, with cord blood concentrations only 1%–2% of maternal levels on GD 147. Malperfusion secondary to onartuzumab-induced placental injury could explain the adverse pregnancy outcomes, fetal growth restriction and relatively low fetal exposures. onartuzumab, MET, ePPND, placenta, cynomolgus monkey Met is a receptor tyrosine kinase involved in critical cellular processes such as cell growth, differentiation, neovascularization and survival in normal and tumor cells (Birchmeier et al., 2003; Trusolino et al., 2010). Met signaling is activated through binding of hepatocyte growth factor (HGF, also known as scatter factor), the only known ligand for Met (Derksen et al., 2003). Met is frequently dysregulated in tumor cells via multiple mechanisms, particularly elevated expression, with or without gene mutations (Birchmeier et al., 2003; Kong-Beltran et al., 2006). The mitogenic, motogenic, and morphogenic cellular behavior activated by HGF/MET signaling is often referred to as “invasive growth” and is associated with the aggressive and metastatic potential of tumors. Dysregulation of the HGF/MET pathway in non-small cell lung cancer (NSCLC) occurs at the level of gene amplification, mutation, and over-expression of either MET or HGF (Landi et al., 2013; Sadiq and Salgia, 2013). The rate of gene amplification of MET in NSCLC vary between 2% and 20% (Beau-Faller et al., 2008; Cappuzzo et al., 2009; Onitsuka et al., 2010; Onozato et al., 2009). In NSCLC, rates of overexpression of MET vary between 25% and 75%, depending upon the sample set and nature of the diagnostic test utilized (Danilkovitch-Miagkova and Zbar, 2002; Ma et al., 2008; Olivero et al., 1996). Overexpression of MET or HGF have been associated with increased pathologic tumor stage and worse outcome in patients with NSCLC (Ichimura et al., 1996; Olivero et al., 1996; Siegfried et al., 1997; Spigel et al., 2012). HGF can also be overexpressed in NSCLC by the stroma where it can act to stimulate invasive growth and survival of NSCLC tumor cells. Onartuzumab (also known as MetMAb) is a unique humanized 1-armed (monovalent) antibody, using the “knobs into holes” technology that binds specifically to the cell surface Met receptor to prevent ligand binding and block downstream Met signaling and HGF-mediated activation (Carter et al., 1992; Merchant et al., 2013). Importantly, the monovalent design of onartuzumab inhibits HGF binding without inducing Met dimerization, which is considered a prerequisite for receptor activation (Birchmeier et al., 2003; Matsubara et al., 2010). Onartuzumab was developed as a potential therapy to be used in combination with the epidermal growth factor receptor tyrosine kinase inhibitor, Erlotinib, for the treatment of NSCLC (Spigel et al., 2012). Onartuzumab does not bind to rodent, rabbit, or canine MET. Onartuzumab blocks HGF binding to human c-Met with an inhibitory concentration (IC)50 of 1.8 nM and inhibits the subsequent induction of c-Met auto-phosphorylation and cell proliferation in many cancer cell lines. Moreover, onartuzumab binds cynomolgus monkey MET and human MET with comparable low nanomolar equilibrium dissociation constants (4.4 and 1.5 nM, respectively), and thus the nonclinical safety assessment of onartuzumab was conducted in cynomolgus monkeys (Merchant et al., 2013). Overall, onartuzumab was well tolerated in toxicity studies in cynomolgus monkeys when administered for up to 26 weeks and at up to 100 mg/kg. In the nonclinical safety studies there were no consistent toxicity findings attributed to onartuzumab, nor pharmacodynamic effects as a result of c-Met inhibition. Several published studies have described the lethal effects of homozygous (−/−) HGF or Met gene disruption during embryo-fetal development in mice (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995). Homozygous knockout (−/−) mouse pups died in utero between embryonic days 12.5 and 15.5 (E12.5–E15.5) and the absence of Met signaling revealed severe developmental abnormalities of the placenta, liver, neurons, and muscle in the limb, diaphragm, and tongue (Bladt et al., 1995; Ebens et al., 1996; Maina et al., 1997; Schmidt et al., 1995; Uehara et al., 1995). In contrast, heterozygous (+/−) pups did not appear to be adversely affected in that they were healthy and fertile, suggesting that complete Met inhibition may be required to result in a reproductive liability. Therefore, as part of the nonclinical safety assessment, an enhanced pre- and postnatal development (ePPND) study was conducted in cynomolgus monkeys to investigate potential effects of onartuzumab on pregnant/lactating females, embryo-fetal development and development of offspring up to postnatal day (PND) 91. Because adverse effects of onartuzumab on fetal development were considered likely, this study utilized a relatively small group size and a 3-month postnatal follow-up period to assess pregnancy outcomes. In addition, a subset of pregnancies (3 each from the control and high-dose groups) was terminated by cesarean section on gestation day (GD) 147 to assess the late gestation fetus, the late gestation placenta, and fetal onartuzumab concentrations via cord blood. MATERIALS AND METHODS This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Shin Nippon Biomedical Laboratories, Ltd. and was performed in accordance with the animal welfare bylaws of Shin Nippon Biomedical Laboratories, Ltd., Drug Safety Research Laboratories, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Animals and Husbandry This study was conducted under an IACUC-approved protocol in an AAALAC-approved facility. Experimentally naïve and sexually mature (at least 4 years old) female cynomolgus monkeys (Macaca fascicularis; country of origin, Cambodia) weighing between 2.6 and 4.5 kg were housed in stainless steel cages, and were acclimated for at least 4 weeks prior to mating. Monkeys were provided primate pellets (>100 g; Purina Mills LLC) once daily, had access to water ad libitum, and were provided additional supplements/treats at least 3 times per week. The room in which the monkeys were housed was set to maintain a temperature of 23°C–29°C, 30%–70% humidity, complete air exchange 15 times/h and 12-h light/dark cycles. Females that showed regular menstrual cycles were paired with males of proven fertility for 3 consecutive days, between the 12th and 14th days of the menstrual cycle (at estimated ovulation time). During the mating period, coitus was confirmed visually. The mid-point of the mating period was designated as GD 0. Females were housed individually during gestation, and with their respective offspring postpartum. Control and Test Articles Control article Onartuzumab vehicle; 10 mM histidine acetate, 120 mM sucrose, 0.04% polysorbate 20 (w/v), pH 5.4. Test article Onartuzumab, supplied as a solution at a nominal concentration of 60 mg/ml was diluted with Onartuzumab vehicle to the appropriate concentration prior to administration. Experimental Design On day 18 of presumed gestation, pregnancy status was diagnosed by an ultrasound examination. In total 36 pregnant females were sequentially assigned to 1 of 3 study groups based on the order of pregnancy diagnosis (see Table 1). Monkeys received the first administration (loading dose) of onartuzumab or vehicle control on GD 20 and subsequently received the maintenance dose once weekly via intravenous injection from GD 27 through parturition (up to GD174; maximum of 23 doses). The initial loading dose of 75 mg/kg or 100 mg/kg was administered to rapidly achieve saturating exposures, while the maintenance dose levels of 50 or 100 mg/kg were chosen so that the expected trough serum concentrations (Ctrough) of the low-dose group (group 2; 75/50 mg/kg) would achieve the predicted maximum serum concentrations (Cmax) at steady state observed for the median patient population at clinically tested dose levels. The Ctrough of the high-dose group (group 3; 100/100 mg/kg) would similarly achieve the Cmax at steady state observed for 90% of the clinical population. Weekly administration was designed to maintain steady state levels throughout the study and was based on the previously determined t1/2β values of 4–7 days in cynomolgus monkeys. Animals were examined at least twice daily for clinical signs of toxicity from GD 19 through lactational day (LD) 91. Pregnancy status was monitored via ultrasound on GDs 25, 32, 39, 46, 60, 74, 88, 102, 116, 130, 144 and 158. In utero examinations, including fetal growth and heart rate measurements were monitored via ultrasound at prespecified times throughout the pregnancy. Body weights were recorded weekly starting on GD20 and food consumption was recorded daily from GD20 to LD91. Blood samples for hematology and serum chemistry analysis were collected periodically throughout pregnancy and lactation, with additional blood samples collected from dams following an abortion, embryonic/fetal death, stillbirth, or offspring death. Maternal blood samples collected on GDs 20, 27, 34, 83, 86, 90, 118, and 153 and LDs 7, 28, 56, and 91 were assessed for onartuzumab concentrations. Additional maternal samples were collected on days 1 (or as soon as possible following confirmation of fetal or neonatal mortality), 28, 56 and 91 following confirmation of a fetal deal, stillbirth or offspring death, when applicable. Maternal blood collected on GDs 20, 34, and 118 and LDs 28 and 91 were assessed for the presence of antidrug antibodies (ADAs) directed against onartuzumab. Table 1. Study Design Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c a Vehicle and onartuzumab were administered IV in a volume of 2 ml/kg. b GD, gestation day. c Three dams were allocated to a GD 147 C-section rather than progressing through parturition. Table 1. Study Design Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c a Vehicle and onartuzumab were administered IV in a volume of 2 ml/kg. b GD, gestation day. c Three dams were allocated to a GD 147 C-section rather than progressing through parturition. For dams in which abortion or embryonic/fetal death was confirmed after GD80, the fetus was obtained via cesarean section and evaluated for external, visceral, or skeletal malformations to the extent possible. Although autolysis and/or postmortem trauma to these fetuses precluded comprehensive assessment, and a complete gross necropsy was not conducted, most fetuses were evaluable for a general histologic assessment of tissue composition and structures. Fetal tissues, including lungs, liver, heart, kidneys, spleen, mesenteric lymph nodes, thymus, and placenta were embedded in paraffin, sectioned, stained with hematoxylin-eosin, and examined microscopically. Stillborn offspring and offspring that died up to PND 30 were weighed, assessed for external morphological measurements and evaluated for external, visceral, and skeletal malformations. The lungs, liver, heart, kidneys, spleen, mesenteric lymph nodes, thymus, and placenta were embedded in paraffin, sectioned, stained with hematoxylin-eosin, and examined microscopically. Surviving offspring were monitored twice daily for clinical signs and body weights were measured weekly through PND 35 and every other week from PND 35 through PND 91. Skeletal examinations were conducted radiographically on PND 7, or once offspring weights exceeded 250 g. Morphological development, included head width, distance between the eyes, crown-rump length, tail length, chest circumference, fore and hind limb length and ano-genital distance were measured on PNDs 28 and 91. Blood from offspring was collected on PND 91 for hematology and serum chemistry, on PND 14, 28, 42, and 91 for toxicokinetic analysis of onartuzumab and on PND 42 and 91 for the presence of ADAs. Functional development, including pupillary reflex, Preyer’s reflex, grip strength and pain response were evaluated on PND 14. Offspring-dam neurobehavioral assessments were performed on PND 28 and 90. Ophthalmic and electrocardiographic examinations of offspring were conducted once between PND 80 and 85. Cesarean Section As a result of the increased incidence in abortions, fetal deaths, low birthweight, and offspring deaths in the onartuzumab-treated groups, investigative cesarean-sections were performed on GD 147 for 3 group 1 (control) and group 3 (high dose) pregnant monkeys. This design element was added relatively late in the study, and there was an inadequate number of group 2 (low dose 75/50 mg/kg) pregnant dams remaining for GD147 c-section. Fetal cord blood was collected to determine onartuzumab concentrations and presence of ADA. Amniotic fluid volume was measured at the time of c-section as an indirect measure of placental perfusion and developing fetal renal function; onartuzumab concentrations in amniotic fluid were not evaluated. The fetus and placenta were removed, weighed and examined externally and histologically as described earlier. Serum Onartuzumab and ADA Determination To determine onartuzumab exposure and appearance of ADAs in dams, blood was collected into serum separator tubes at several times throughout the dosing and lactational phases of the study. Blood was collected from dams as soon as possible and several timepoints following abortion, embryonic/fetal death, stillbirth, or offspring death. Similarly, blood was collected from surviving offspring on PNDs 14, 28, 42, and 91 and PNDs 42 and 91 for toxicokinetic and ADA analysis, respectively. The serum samples were assayed for onartuzumab and ADAs to onartuzumab in bridging ELISAs. For determination of onartuzumab concentrations, the assay used murine antionartuzumab complementarity determining region monoclonal antibodies in the capture phase, and F(ab′)2 fragmented, goat antihuman immunoglobulin (Ig)G Fc antibodies conjugated to horseradish peroxidase (HRP) were used for detection. The lower limit of quantification for this assay was 0.2 μg/ml. For detection of ADAs to onartuzumab, serum samples were incubated with equal concentrations of biotinylated onartuzumab and digoxigenin onartuzumab. Serum samples were then transferred to a high bind streptavidin coated plate. Following appropriate incubation, the plate was washed and mouse monoclonal anti-digoxin HRP was added for detection. Using a surrogate monoclonal antibody, the sensitivity of the assay was determined to be 2 ng/ml. The presence of circulating onartuzumab in a sample can interfere with the detection of ADAs, thereby decreasing the sensitivity of the assay. In the presence of up to 200 μg/ml of onartuzumab, the assay was able to detect 1000 ng/ml of the surrogate monoclonal antibody. Statistical Analysis All data were evaluated using MUSCOT statistical analysis software (Yukms Corp, Glenview, Illinois). Quantitative data were analyzed by Bartlett’s test for homogeneity of variance. When the variance was homogeneous, Dunnett’s test was applied to compare control and treatment groups. When variance was heterogenous by Barlett’s test, a Miller test was applied to compare control and treatment groups. For a comparison of data obtained following cesarian section on GD 147 in group 1 (control) and group 3 (high dose), the data were analyzed for homogeneity of variance by the F test. When the variance was homogeneous, student t test was applied and when the variance was heterogeneous, Aspim-Welch t test was applied. A probability value of p < .05 was considered statistically significant. RESULTS No Effects of Onartuzumab on Pregnant Dams Onartuzumab was administered by a slow IV bolus injection once weekly for up to 23 weeks to confirmed pregnant female cynomolgus monkeys (n = 12/group) during the period of organogenesis through parturition (ie, from GD 20 through GD 174 or birth). The dose levels tested were 0 mg/kg (vehicle control), 75/50 mg/kg (loading dose/maintenance dose; low dose), or 100/100 mg/kg (high dose). The dams were evaluated for onartuzumab-related changes in body weight, food consumption, physical examinations (body temperature and pulse oximetry), clinical pathology indices, including serum chemistry, hematology, urinalysis, serum chorionic gonadotropin. Weekly administration of onartuzumab up to 23 weeks was well tolerated in pregnant dams with no apparent clinical signs of maternal toxicity and no onartuzumab-related changes in any of the parameters measured. Effects of Onartuzumab on Pregnancy Outcome Onartuzumab-related effects on the fetus/neonate included increased rates of embryo-fetal death and early deliveries (see Tables 2 and 3). Of the 12 confirmed pregnancies in Group 1 (control) dams, 1 embryonic death (1 of 12; 8.3%) was detected in the first trimester (GD 39); in addition, C-sections were conducted on 3 group 1 (control) dams on GD 147, and all 3 fetuses appeared viable at delivery. The number of fetuses that survived to parturition was 8 of 9 (88.9%) with a mean gestational period of 162.3 ± 4.4 days. In contrast, prenatal embryo-fetal deaths occurred in 2 of 12 (16.7%) and 4 of 12 dams (33.3%) group 2 (low dose) dams in the first and second/third trimesters respectively, with no clear temporal pattern of effect during pregnancy (see Table 2). Furthermore 1 stillborn offspring (1 of 12; 8.3%) was delivered on GD 170. The rate of fetal survival to parturition in the group 2 dams given onartuzumab at the low dose (75/50) was 5 of 12 (41.7%) with a mean gestational period of 154.0 ± 13.1 days. Similarly, in group 3 dams (high-dose onartuzumab; 100/100), prenatal embryo-fetal deaths occurred in 2 of 12 (16.7%) in the first trimester and 2 of 9 (22.2%) in the second/third trimesters (Table 2); in addition C-sections were conducted on 3 pregnant dams on GD 147. The number of fetuses that survived to parturition in the group 3 dams was 5 of 9 (55.6%) with a mean gestational period of 148.8 ± 6.3 days. The incidences for abortion and fetal death in the onartuzumab-treated groups were considered high in the second and third trimesters as compared with historical control data from the testing facility (mean incidences for the second and third trimesters in controls are 1.1% [range 0.0%–12.5%] and 3.4% [range 0.0%–16.7%], respectively). Table 2. Summary of Pregnancy Outcome and Neonatal Survival Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e a n = 12 pregnant animals/group. b Onartuzumab administered intravenously (loading dose/maintenance dose) based on the most recent body weight measurement. c Data were calculated from the period of PND 0–7. d Data were calculated from the period PND 8–30. e Data of survival rate in neonates to PND 91. Abbreviation: NA, Not Available. Table 2. Summary of Pregnancy Outcome and Neonatal Survival Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e a n = 12 pregnant animals/group. b Onartuzumab administered intravenously (loading dose/maintenance dose) based on the most recent body weight measurement. c Data were calculated from the period of PND 0–7. d Data were calculated from the period PND 8–30. e Data of survival rate in neonates to PND 91. Abbreviation: NA, Not Available. Table 3. Summary of Mean Gestational Length and Neonatal Survival Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Abbreviations: GD, gestation day; PND, postnatal day. a On day 18 of presumed gestation, pregnancy status was diagnosed via ultrasound and 36 confirmed pregnant monkeys were assigned to 1 of 3 groups (n = 12/group). b Historical mean delivery day (range): GD 160.9 (143 − 175). c Overall neonatal survival through PND 91. d C-sections were performed on n = 3 control and high dose dams on GD 147 as part of a protocol amendment and are not included in this analysis. e p < .05 compared with control. Table 3. Summary of Mean Gestational Length and Neonatal Survival Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Abbreviations: GD, gestation day; PND, postnatal day. a On day 18 of presumed gestation, pregnancy status was diagnosed via ultrasound and 36 confirmed pregnant monkeys were assigned to 1 of 3 groups (n = 12/group). b Historical mean delivery day (range): GD 160.9 (143 − 175). c Overall neonatal survival through PND 91. d C-sections were performed on n = 3 control and high dose dams on GD 147 as part of a protocol amendment and are not included in this analysis. e p < .05 compared with control. Effects of Onartuzumab on Offspring Overall, onartuzumab administration to pregnant dams resulted in impaired growth of the fetus. This effect was apparent on GD 130, when the fetal head width and femoral length, determined via ultrasound, were significantly smaller in the high dose group as compared with controls. Of the fetuses that did survive to parturition there was an onartuzumab-dependent decrease in neonatal birth weight (see Table 4 and Figure 1). The mean birth weight of the offspring from Group 1 dams was 351 ± 41 g, which is consistent with the historical birth weights of cynomolgus monkeys at this facility (336.8 ± 34.5 g; unpublished data). In contrast, the birth weights of the offspring from dams given the low dose and high dose of onartuzumab during pregnancy were significantly lower; 250 ± 32 g and 210 ± 29 g, respectively. Furthermore, overall body weight of the surviving offspring from onartuzumab-treated dams remained lower than controls through the postnatal period until the day of necropsy (PND 91; see Figure 1). Table 4. Mean Body Weights at Birth and PND 91 Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) a Historical control mean birth weight at testing facility is 336.8 ± 34.5 gr. b p < .05 compared with control. Table 4. Mean Body Weights at Birth and PND 91 Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) a Historical control mean birth weight at testing facility is 336.8 ± 34.5 gr. b p < .05 compared with control. Figure 1. View largeDownload slide Mean body weight of offspring through PND 91. Numbers embedded in bar graphs represent the number of offspring/group at each timepoint. Of note, in the high dose group (100/100), 2 neonatal offspring died, 1 on PND 3 and 1 on PND 11, resulting in only n = 2 offspring from PND 14 through PND91 and therefore were not statistically compared with controls. Asterisk (*) indicates p < .05 compared with controls. Figure 1. View largeDownload slide Mean body weight of offspring through PND 91. Numbers embedded in bar graphs represent the number of offspring/group at each timepoint. Of note, in the high dose group (100/100), 2 neonatal offspring died, 1 on PND 3 and 1 on PND 11, resulting in only n = 2 offspring from PND 14 through PND91 and therefore were not statistically compared with controls. Asterisk (*) indicates p < .05 compared with controls. Consistent with the lower body weights, significantly lower values were noted in postnatal crown-rump length, tail length, and forelimb length on PNDs 28 and 91, and in hindlimb length on PND 28 in the low dose group when compared with the control group (data not shown). Trends toward low values were noted in hindlimb length on PND 91, and in chest circumference and ano-genital distance in male and females on PNDs 28 and 91 in the low dose group (data not shown). In the high dose group, a significantly lower value in chest circumference on PND 28, and trends toward low values in crown-rump length and hindlimb length on PNDs 28 and 91, and in forelimb length on PND 28 were noted when compared with the control group (data not shown). There were no onartuzumab-related skeletal abnormalities on PND7 and no effects on functional development measurement, including pupillary reflex, Preyer’s reflex, grip strength, or pain response on PND14. Similarly, no changes were noted in hematology, clinical chemistry, ophthalmology, or electrocardiography measurements in the offspring that survived to PND91. Cesarean Section GD147 Based on a higher than expected number of fetal and early postnatal losses of offspring from dams administered onartuzumab, it was decided to conduct cesarean sectioning on GD 147 for 3 dams each from the control and high dose groups to carefully evaluate pathological effects of onartuzumab on fetuses, including evaluation of the placenta, assessment of amniotic fluid volume, and collection of cord blood for concentration of onartuzumab. No statistically significant differences were noted in fetal body weight, placental weight, or amniotic fluid volume between the control group and high dose group; however, mean body weight in the high dose group (257.5 g) was slightly lower than the control group (281.7 g), and individual body weight of 1 fetus in the high dose group was extremely low for this gestational age (204.8 gr). No external or skeletal abnormalities were observed in any fetus. No visceral anomalies were identified with the exception of a small thymus noted for 1 high dose offspring. Significant decreases in absolute liver weight and high relative brain and epididymis (right and left) weights were noted in the high dose group when compared with the organ weights in the control group. These differences in organ weights were considered related to the general retardation of fetal growth, rather than a direct effect of onartuzumab on these specific tissues. One high dose fetus was substantially smaller, overall and in individual organ weights, than the other high-dose and control group GD147 fetuses; there were grossly evident indurated white foci noted in both the main and accessory placentae for this fetus. Offspring Necropsied at PND91 Retarded eruption of upper and lower first molar and canine teeth was observed bilaterally in 3 offspring and retarded eruption of upper first molar and canine teeth and lower canine tooth bilaterally in 1 offspring in the low dose group. In the high dose group, retarded eruption of upper and lower first molar and canine teeth was observed bilaterally in 1 offspring. In the control group, retarded eruption of upper and lower canine teeth was observed bilaterally in 1 offspring. Statistically significant decreases in absolute lung and spleen weights, and high relative brain and epididymis (left) weights were noted in the low dose group when compared with the organ weights in the control group. Statistically significant increases in relative kidney (left) weight were noted in the high dose group. These differences in organ weights were considered related to the general retardation of fetal growth and associated delay in postnatal growth, rather than a direct effect of onartuzumab on these specific tissues. Placental Evaluations Twelve placentae were examined grossly and histopathologically. These included 3 each from the control and high dose group cesarean sections on GD 147, which reflect the primary basis of comparison and evaluation in this study. In addition, 6 placentae were collected after abortion, fetal death, or early postnatal death, and also evaluated grossly and histologically. Although diffuse placental changes due to parturition and/or autolysis compromised full assessment of these 6 placentae, the findings attributed to onartuzumab were interpretable due to their multifocal distribution and similarities to the histopathology of the placentae from the c-section cohort. Placental weight evaluations were limited to the 6 placentae collected at c-section, and there were no differences in overall placental weights (main+accessory) between the high dose and control groups. However, the gross and histologic picture of the placentae from onartuzumab-treated dams was substantially different from that of the control group c-section placentae. Slight to marked infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate were observed in all examined placentae in the low dose and high dose groups. Increases in fibrin deposition in the chorionic plate, chorionic villus or decidual plate were observed in 1 placenta in the low dose group and 4 placentae in the high dose group (Figure 2). These placental findings corresponded to the gross observation of indurated white foci in 1 placenta in each of the low and high dose groups. Placental infarcts have not been reported as a background finding in cynomolgus monkeys, and were not observed in the 3 placentae obtained from the control group at GD147. Of note, the placenta is not routinely collected in an ePPND study, and in this study none of the placentae from offspring that survived until the PND 91 necropsy were assessed. Figure 2. View largeDownload slide Placental histopathology from fetuses delivered by cesarean section on GD 147. A, normal placenta from the control group. B, Scattered microinfarcts, hemorrhage and fibrin in a placenta from the high dose group. C, Area of severe compromise due to infarction, necrosis and fibrin in a placenta from the high dose group. All placentae were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Figure 2. View largeDownload slide Placental histopathology from fetuses delivered by cesarean section on GD 147. A, normal placenta from the control group. B, Scattered microinfarcts, hemorrhage and fibrin in a placenta from the high dose group. C, Area of severe compromise due to infarction, necrosis and fibrin in a placenta from the high dose group. All placentae were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Other Histopathology Findings In the liver, decreased cellularity of the hepatocytes with dilated sinusoids was observed in 1 fetus in the high dose group removed at cesarean sectioning on GD 147, and 1 offspring found dead in the early postnatal period from each of the low dose and high dose groups. Although consistent with the reported hepatic developmental abnormalities identified in mice defective for c-Met signaling, this finding was inconsistently distributed in the liver, with some lobes similar to controls (Figure 3). Also, although organ weights of fetuses removed at cesarean sectioning on GD 147 had significantly lower liver weight as compared with controls, this was primarily driven by the 1 liver with decreased hepatocyte cellularity. At the PND 91 necropsy, there were no histologic differences across groups for the liver. Figure 3. View largeDownload slide Liver histopathology from fetuses delivered by cesarean section on GD 147. A, normal late gestation fetal liver from the control group, highlighting typical degree of sinusoidal dilation and glycogen content. B, late gestation fetal liver from the high dose group demonstrating hepatic cord disarray, decreased cellularity and sinusoidal dilation. Fetal livers were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Figure 3. View largeDownload slide Liver histopathology from fetuses delivered by cesarean section on GD 147. A, normal late gestation fetal liver from the control group, highlighting typical degree of sinusoidal dilation and glycogen content. B, late gestation fetal liver from the high dose group demonstrating hepatic cord disarray, decreased cellularity and sinusoidal dilation. Fetal livers were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Another histologic finding attributed to onartuzumab administration was the presence of rare syncytial cells in the alveoli of the lung, which was observed in 2 offspring found dead by PND11, 2 fetuses removed at cesarean sectioning on GD 147 and 1 stillborn fetus. All affected fetuses/offspring were in the onartuzumab treated groups. Finally, there was a single offspring in the low dose group and necropsied on PND 91 with fibrous osteodystrophy (FOD). In this single offspring, all bones examined histologically were affected, including the sternum, femur, maxilla and mandible; however, there were no notable findings in the kidneys or parathyroid glands, and serum levels of calcium and phosphorous were normal at the time of necropsy. There was no evidence of a similar process in any of the other offspring from dams administered onartuzumab. Toxicokinetics and ADAs Exposure to onartuzumab increased with the increase in dose level from 75/50 to 100/100 mg/kg in pregnant cynomolgus monkeys. The increases in group mean Cmax and AUC83–90 values following the 10th weekly dose were generally dose proportional. Steady-state appeared to have been achieved by GD 34 (Table 5). The fetus-to-dam onartuzumab serum concentration ratios at the time of cesarean sectioning on GD 147 showed that onartuzumab was not extensively transferred to the fetuses (approximately 1%–2%; Table 6). Concentrations of onartuzumab in 3 of 4 surviving Group 2 (75/50) offspring were below the limit of quantitation on PND14. The remaining offspring had onartuzumab concentration of 4010 ng/ml. In the 2 surviving offspring from group 3 (100/100) dams, PND14 onartuzumab concentrations were 7980 and 527 ng/ml. Onartuzumab concentrations on PND 28/LD 28 in all offspring from group 2 and group 3 dams were below the limit of quantitation with the exception of 1 offspring from a group 2 dam (75/50), which had a serum concentration of 549 ng/ml and an offspring to dam ratio of <1%. ADAs to onartuzumab were not detected in any dams in the onartuzumab dose groups during the gestation period of the dosing phase, or in any animals in the control group. Additionally, ADAs to onartuzumab were not detected in the fetuses at the time of cesarean section, or in surviving offspring at or after PND 14. In total 5 of 12 dams (42%) in the low dose group and 6 of 12 dams (50%) in the high dose group had ADAs to onartuzumab detected on LDs 28 or 91 or after cesarean sectioning or fetal loss. Altogether, ADAs to onartuzumab were detected in 11 of 24 cynomolgus monkey dams (46%) given onartuzumab, but there did not appear to be any impact on the exposure of onartuzumab in the dams or their offspring due the formation of ADAs to onartuzumab. However, the incidence of ADAs was higher in dams with poor pregnancy outcomes as compared with dams with offspring surviving to PND 91 (Table 7). Table 5. Summary of the Group Mean Toxicokinetic Parameters for Onartuzumab in Dams Post First and Tenth Doses Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d a Group 2 animals received a loading dose of 75 mg/kg on GD20 and 50 mg/kg thereafter for the remainder of the study. Therefore 75 mg/kg was used to calculate Cmax first and Cmax tenth was calculated after 9 weekly injections of 50 mg/kg. b n = 12. c n = 9. d n = 10. e n = 8. Table 5. Summary of the Group Mean Toxicokinetic Parameters for Onartuzumab in Dams Post First and Tenth Doses Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d a Group 2 animals received a loading dose of 75 mg/kg on GD20 and 50 mg/kg thereafter for the remainder of the study. Therefore 75 mg/kg was used to calculate Cmax first and Cmax tenth was calculated after 9 weekly injections of 50 mg/kg. b n = 12. c n = 9. d n = 10. e n = 8. Table 6. Fetal Cord Blood and Fetus to Dam Ratio of Onartuzumab Serum Concentration at Cesarean Sectioning (GD147) Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Table 6. Fetal Cord Blood and Fetus to Dam Ratio of Onartuzumab Serum Concentration at Cesarean Sectioning (GD147) Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Table 7. Relationship Between Pregnancy Outcome and ADA Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 a Placenta collected for histopathology. Table 7. Relationship Between Pregnancy Outcome and ADA Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 a Placenta collected for histopathology. DISCUSSION This study demonstrates that administration of onartuzumab to cynomolgus monkeys once weekly via intravenous injection from GD 20 through parturition (up to GD174; maximum of 23 doses) resulted in a dose-dependent decrease in gestation length (early delivery) accompanied by low neonatal birth weight and decreased offspring survival in the absence of any notable maternal toxicity. Similarly, the mean body weight of fetuses removed on GD 147 was also slightly lower in the high dose group, and fetal growth retardation in the high dose group was indicated by in utero examination. These data are consistent with previous reports in humans, where an association between reduced HGF/MET signaling and developmental abnormalities has also been observed. Maternal serum HGF concentrations, but not umbilical cord HGF concentrations, were significantly decreased in pregnancies where infants were small for gestational age (SGA) when compared with pregnancies where infants were appropriate for gestational age (AGA) (Aoki et al., 1998). As HGF concentrations in the maternal serum are attributed to HGF released from the placenta into maternal circulation, but not the fetal circulation (Horibe et al., 1995), these results specifically implicate the inhibition of maternal MET signaling on fetal growth and development. However, the placental weight was also significantly lower in women of the SGA group when compared with the placental weight of women in the AGA group, whereas no overt difference in placental weights between control and onartuzumab-treated groups was identified from the GD147 C-sections. Given the pathology identified, including hemorrhage and fibrin in affected placentae, it is difficult to assess the placental weights in isolation. Results from this study are also consistent with previous reports that established the critical role of HGF/Met signaling pathway in embryogenesis and described embryo-fetal toxicity following Met pathway inhibition in rodents (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995). These reports described the lethal effects of hgf or met gene disruption during embryo-fetal development in mice. Although normal homozygous wildtype (+/+) and heterozygous (+/−) pups did not appear to be adversely affected, homozygous knockout (−/−) mouse pups died in utero between embryonic days 12.5 and 15.5 (E12.5−E15.5). Although the effects of onartuzumab administration during pregnancy were not as severe as those reported for hgf or met homozygous knockout phenotype in mice, our results clearly demonstrate that onartuzumab exhibits the expected pharmacological effect on fetuses at the doses tested in this study. These dose levels were chosen so that the expected trough serum concentrations (Ctrough) of the low-dose group (group 2; 75/50 mg/kg) would achieve the predicted maximum serum concentrations (Cmax) at steady state observed for the median patient population at clinically tested dose levels, and the Ctrough of the high-dose group (group 3; 100/100 mg/kg) would similarly achieve the Cmax at steady state observed for 90% of the clinical population. The principal histopathologic finding was the presence of increased fibrin and infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate of the placenta. Decreased cellularity of the hepatocytes with dilated sinusoids in the liver was also identified in 2 fetuses from onartuzumab-treated dams. These findings are consistent with previous reports that revealed that the absence of Met signaling resulted in severe developmental abnormalities of the placenta, liver, neurons, and muscle in the limb, diaphragm, and tongue in rodents (Bladt et al., 1995; Ebens et al., 1996; Maina et al., 1997; Schmidt et al., 1995; Uehara et al., 1995). However, the rodent placental finding is described as thinning, or atrophy, as compared with normal controls. In this study, the specific findings of placental infarcts, increased fibrin deposition, and hemorrhage are more consistent with placental pathology related to poor pregnancy outcomes in humans (Aurioles-Garibay et al., 2014; Bendon, 2012; Parks, 2015). Placental infarcts have not been previously reported for cynomolgus monkeys. The effects of onartuzumab on the placenta likely contributed to the poor pregnancy outcomes and developmental growth delays in this study. In humans, similar findings are associated with maternal malperfusion of the placenta (Parks, 2015). In this study, although only 3 high dose fetuses and placentae were evaluated on GD147, all 3 fetuses had both histologic placental pathology and very low concentrations of onartuzumab in their cord blood; therefore, we conclude that decreased fetal perfusion due to the placental injury likely resulted in the low fetal serum concentrations of onartuzumab. Of the high-dose fetuses evaluated, the one with evidence of onartuzumab-related liver pathology also had the highest concentration of onartuzumab detected in the fetal cord blood. The minimal fetal exposures achieved are attributed to the placental injury and may explain the limited extraplacental developmental effects on potential target tissues in the offspring of dams administered onartuzumab. The relationship of this pattern of defects is further supported by expression data showing MET in epithelial tissues and HGF in adjacent mesenchyme, and paracrine signaling is considered critical in these tissues (Sonnenberg et al., 1993). In met homozygous knockout (−/−) mice the liver defects were noted as early as E12.5 as indicated by a 40% reduction in liver volume and progressed by E14.5 when the liver was less than half of the normal weight. These gross findings correlated histologically with a severe decrease in cellularity within the liver parenchyma and enlarged sinusoidal spaces. The key placental defect noted in the absence of Met was an impairment of trophoblast cell development and a reduced number of trophoblast cells in the affected placentae, which resulted in an overall decrease in placental size. Morphologically similar placental and liver developmental abnormalities were also noted in hgf knockout pups (Schmidt et al., 1995; Uehara et al., 1995). In both hgf and met knockout mice, the combination of hepatic developmental effects and placental insufficiency contributed to the fetal mortality in these models, and emphasize a key role for HGF/MET signaling during organogenesis. Another histopathological finding that was noted in individual fetuses/offspring included syncytial cells in the alveoli of the lung, which was observed in 2 offspring found dead by PND11, 2 fetuses removed at cesarean sectioning on GD 147 and 1 stillborn fetus from onartuzumab-treated dams. The origin of the alveolar syncytial cells observed in this study is not fully clear, but their location in the lung, and the absence of other local inflammatory cells, suggests an amniotic origin. Due to the disruption of all layers of the placenta in some placentae from onartuzumab-treated animals, these cells may be placental syncytiotrophoblasts released into the amniotic fluid and aspirated prenatally. Similar findings have not been reported in investigations of HGF/Met signaling in lung. Likewise, in this study, there were no apparent effects of onartuzumab on fetal lung branching or alveolization, although potential for such effects was anticipated. In in vitro studies conducted with fetal rat lungs, HGF stimulates branching morphogenesis of both alveolar and bronchial epithelia and in vivo, HGF contributes to the growth and maturation of neonatal rat lungs (Ohmichi et al., 1998; Panos et al., 1996). Consistent with the association between HGF and lung development, preterm human infants with subsequent bronchcopulmonary dysplasia (BPD) had significantly lower tracheal aspirate HGF concentrations and required longer mechanical ventilation and higher supplemental oxygen when compared with preterm infants who survived without BPD (Lassus et al., 2003). No definitive onartuzumab-related changes were noted in external or skeletal findings, hematology, serum chemistry, functional development, ophthalmology, electrocardiography, or bone marrow examination in surviving offspring on PND91. However, retarded eruption of the upper and lower first molar and/or canine teeth was observed in 5 of 6 offspring in the onartuzumab groups, but only 1 of 8 offspring in the control group. No histopathological abnormalities were found in any of these teeth with retarded eruption. This potential developmental delay was attributed to gestational immaturity at the time of parturition and/or fetal growth retardation due to placental injury. Finally, a single offspring in the low-dose group had FOD affecting all bones evaluated at the PND 91 necropsy. Both Met and HGF are involved in the cross talk between osteoclasts and osteoblasts (Grano et al., 1996), although FOD has not specifically been identified in genetic models. Because this finding occurred in a single offspring, at the lower dose level, and can occur as a spontaneous developmental abnormality in animals, it was not attributed to onartuzumab administration. It was somewhat unusual in presentation in that there were no histologic abnormalities of the parathyroid glands or kidneys in this animal, and the circulating calcium and phosphorous levels were normal at the time of the necropsy. Although there are no reports of sporadic FOD in infant macaques, either in the historical database or in the literature, in humans there are examples of selective end-organ resistance to parathyroid hormone leading to osteodytrophy in the absence of overt renal effects or electrolyte abnormalities (Bastepe, 2013). This is just one example, but it highlights the overall challenges in interpreting results from a study when there is a rich literature highlighting potential developmental effects across multiple organ systems. Some of this has already been discussed with regard to the liver, lung, and musculoskeletal system. In addition, a potential role for HGF/Met signaling has been established for the structural and functional development of central nervous systems (Burdick et al., 2010; Campbell et al., 2006, 2009; Ieraci et al., 2002; Judson et al., 2011; Maina and Klein, 1999; Powell et al., 2001,, 2003; Sousa et al., 2009), and there is some in vitro evidence for a role in the structural development of the renal system (Pohl et al., 2000; Santos et al., 1994). For the nervous system, the evidence demonstrates that Met signaling contributes to the development of the cerebral cortex and cerebellum, and suggests that decreased pathway activity during critical periods of neurodevelopment pose another risk to the fetus that would be consistent with inhibition of the Met receptor during pregnancy. However, in this study there were no onartuzumab-related effects on the renal or nervous system tissues, or on neurobehavioral development, that was identified in offspring that survived to PND91. Given the low exposures achieved in the offspring, the absence of findings in this study does not preclude the potential developmental risk to the fetus during pregnancy. In humans, development of several organ systems, including the renal, musculoskeletal, and nervous systems, extends into late gestation and the perinatal period when fetal Ig concentrations are expected to be high. Placental transfer is expected to be similar to a bivalent IgG1 antibody since in vitro studies demonstrated that onartuzumab binds to human FcRn with a relative affinity similar to that of a bivalent, glycosylated IgG1 antibody (Merchant et al., 2013). Since direct effects of onartuzumab on maternal HGF/Met signaling at the placenta may occur much earlier in development, placental transfer of onartuzumab to the fetus may not be required to cause developmental and reproductive toxicity, which is consistent with the fact that the fetus to dam onartuzumab serum concentration ratios at the time of cesarean sectioning on GD 147 showed that only approximately 1%–2% of onartuzumab was transferred to the fetuses. This is in contrast to data with other monoclonal antibodies showing that fetal concentrations near the end of gestation are often comparable to, or even exceed, maternal concentrations (Bowman et al., 2013; DeSesso et al., 2012; Moffat et al., 2014; Pentšuk and van der Laan, 2009). In addition, the offspring to dam onartuzumab serum concentration ratios on PND 28/LD 28 were <1% in 1 offspring and were below or close to the limit of quantitation in all other surviving offspring at or after PND14. From the results described above, under the conditions of this study, no general toxicities in dams were observed in the onartuzumab-treated group at the low dose of 75/50 mg/kg (loading/maintenance dose) or the high dose of 100/100 mg/kg. At the same time, increased incidence of abortion and fetal death in the second and third trimesters, fetal growth retardation, shortened gestation length (early delivery), low fetal and neonatal birth weights, offspring deaths during early postnatal period, suppressed body weight of offspring through the postnatal period, and delayed morphological and behavioral development were observed in the onartuzumab-treated groups. Furthermore, histopathology revealed infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate of several placentae from the onartuzumab treated groups, and a few had and decreased cellularity of the hepatocytes with dilated sinusoids in fetuses and offspring. Accordingly, the above-mentioned findings in fetuses and offspring were considered to be associated with onartuzumab administration during pregnancy, and confirm that onartuzumab administration to pregnant women would pose a clear risk to both the developing placenta and fetus, and the mother’s ability to maintain the pregnancy. FUNDING This study was supported by Genentech, A member of the Roche Group. Rodney Prell, Noel Dybdal, Ihsan Nijem and Wendy Halpern are fulltime employees of Genentech, A member of the Roche Group. REFERENCES Aoki S. , Hata T. , Manabe A. , Miyazaki K. ( 1998 ). Decreased maternal circulating hepatocyte growth factor (HGF) concentrations in pregnancies with small for gestational age infants . Hum. Reprod. 13 , 2950 – 2953 . Google Scholar Crossref Search ADS PubMed Aurioles-Garibay A. , Hernandez-Andrade E. , Romero R. , Qureshi F. , Ahn H. , Jacques S. M. , Garcia M. , Yeo L. , Hassan S. S. ( 2014 ). Prenatal diagnosis of a placental infarction hematoma associated with fetal growth restriction, preeclampsia and fetal death: Clinicopathological correlation . Fetal Diagn. Ther. 36 , 154 – 161 . Google Scholar Crossref Search ADS PubMed Bastepe M. ( 2013 ). Genetics and epigenetics of parathyroid hormone resistance . Endocr. Dev. 24 , 11 – 24 . Google Scholar Crossref Search ADS PubMed Beau-Faller M. , Ruppert A. M. , Voegeli A. C. , Neuville A. , Meyer N. , Guerin E. , Legrain M. , Mennecier B. , Wihlm J. M. , Massard G. , et al. . ( 2008 ). MET gene copy number in non-small cell lung cancer: Molecular analysis in a targeted tyrosine kinase inhibitor naive cohort . J. Thorac. Oncol. 3 , 331 – 339 . Google Scholar Crossref Search ADS PubMed Bendon R. W. ( 2012 ). Nosology: Infarction hematoma, a placental infarction encasing a hematoma . Hum. Pathol. 43 , 761 – 763 . Google Scholar Crossref Search ADS PubMed Birchmeier C. , Birchmeier W. , Gherardi E. , Vande Woude G. F. ( 2003 ). Met, metastasis, motility and more . Nat. Rev. Mol. Cell. Biol. 4 , 915 – 925 . Google Scholar Crossref Search ADS PubMed Bladt F. , Riethmacher D. , Isenmann S. , Aguzzi A. , Birchmeier C. ( 1995 ). Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud . Nature 376 , 768 – 771 . Google Scholar Crossref Search ADS PubMed Bowman C. J. , Breslin W. J. , Connor A. V. , Martin P. L. , Moffat G. J. , Sivaraman L. , Tornesi M. B. , Chivers S. ( 2013 ). Placental transfer of Fc-containing biopharmaceuticals across species, an industry survey analysis . Birth Defects Res. 98 , 459 – 485 . Google Scholar Crossref Search ADS Burdick K. E. , DeRosse P. , Kane J. M. , Lencz T. , Malhotra A. K. ( 2010 ). Association of genetic variation in the MET proto-oncogene with schizophrenia and general cognitive ability . Am. J. Psychiatry 167 , 436 – 443 . Google Scholar Crossref Search ADS PubMed Campbell D. B. , Buie T. M. , Winter H. , Bauman M. , Sutcliffe J. S. , Perrin J. M. , Levitt P. ( 2009 ). Distinct genetic risk based on association of MET in families with co-occurring autism and gastrointestinal conditions . Pediatrics 123 , 1018 – 1024 . Google Scholar Crossref Search ADS PubMed Campbell D. B. , Sutcliffe J. S. , Ebert P. J. , Militerni R. , Bravaccio C. , Trillo S. , Elia M. , Schneider C. , Melmed R. , Sacco R. , et al. . ( 2006 ). A genetic variant that disrupts MET transcription is associated with autism . Proc. Natl. Acad. Sci. U.S.A . 103 , 16834 – 16839 . Google Scholar Crossref Search ADS PubMed Cappuzzo F. , Marchetti A. , Skokan M. , Rossi E. , Gajapathy S. , Felicioni L. , Del Grammastro M. , Sciarrotta M. G. , Buttitta F. , Incarbone M. , et al. . ( 2009 ). Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients . J. Clin. Oncol. 27 , 1667 – 1674 . Google Scholar Crossref Search ADS PubMed Carter P. , Kelley R. F. , Rodrigues M. L. , Snedecor B. , Covarrubias M. , Velligan M. D. , Wong W. L. , Rowland A. M. , Kotts C. E. , Carver M. E. and ( 1992 ). High level Escherichia coli expression and production of a bivalent humanized antibody fragment . Biotechnology (N Y) 10 , 163 – 167 . Google Scholar PubMed Danilkovitch-Miagkova A. , Zbar B. ( 2002 ). Dysregulation of Met receptor tyrosine kinase activity in invasive tumors . J. Clin. Invest. 109 , 863 – 867 . Google Scholar Crossref Search ADS PubMed Derksen P. W. , de Gorter D. J. , Meijer H. P. , Bende R. J. , van Dijk M. , Lokhorst H. M. , Bloem A. C. , Spaargaren M. , Pals S. T. ( 2003 ). The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma . Leukemia 17 , 764 – 774 . Google Scholar Crossref Search ADS PubMed DeSesso J. M. , Williams A. L. , Ahuja A. , Bowman C. J. , Hurtt M. E. ( 2012 ). The placenta, transfer of immunoglobulins, and safety assessment of biopharmaceuticals in pregnancy . Crit. Rev. Toxicol. 42 , 185 – 210 . Google Scholar Crossref Search ADS PubMed Ebens A. , Brose K. , Leonardo E. D. , Hanson M. G. Jr. , Bladt F. , Birchmeier C. , Barres B. A. , Tessier-Lavigne M. ( 1996 ). Hepatocyte growth factor/scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons . Neuron 17 , 1157 – 1172 . Google Scholar Crossref Search ADS PubMed Grano M. , Galimi F. , Zambonin G. , Colucci S. , Cottone E. , Zallone A. Z. , Comoglio P. M. ( 1996 ). Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro . Proc. Natl. Acad. Sci. U.S.A . 93 , 7644 – 7648 . Google Scholar Crossref Search ADS PubMed Horibe N. , Okamoto T. , Itakura A. , Nakanishi T. , Suzuki T. , Kazeto S. , Tomoda Y. ( 1995 ). Levels of hepatocyte growth factor in maternal serum and amniotic fluid . Am. J. Obstetr. Gynecol . 173 , 937 – 942 . Google Scholar Crossref Search ADS Ichimura E. , Maeshima A. , Nakajima T. , Nakamura T. ( 1996 ). Expression of c-met/HGF receptor in human non-small cell lung carcinomas in vitro and in vivo and its prognostic significance . Jpn. J. Cancer Res. 87 , 1063 – 1069 . Google Scholar Crossref Search ADS PubMed Ieraci A. , Forni P. E. , Ponzetto C. ( 2002 ). Viable hypomorphic signaling mutant of the Met receptor reveals a role for hepatocyte growth factor in postnatal cerebellar development . Proc. Natl. Acad. Sci. U.S.A . 99 , 15200 – 15205 . Google Scholar Crossref Search ADS PubMed Judson M. C. , Eagleson K. L. , Levitt P. ( 2011 ). A new synaptic player leading to autism risk: Met receptor tyrosine kinase . J. Neurodev. Disord. 3 , 282 – 292 . Google Scholar Crossref Search ADS PubMed Kong-Beltran M. , Seshagiri S. , Zha J. , Zhu W. , Bhawe K. , Mendoza N. , Holcomb T. , Pujara K. , Stinson J. , Fu L. , et al. . ( 2006 ). Somatic mutations lead to an oncogenic deletion of met in lung cancer . Cancer Res. 66 , 283 – 289 . Google Scholar Crossref Search ADS PubMed Landi L. , Minuti G. , D'Incecco A. , Salvini J. , Cappuzzo F. ( 2013 ). MET overexpression and gene amplification in NSCLC: A clinical perspective . Lung Cancer (Auckl.) 4 , 15 – 25 . 10.2147/LCTT.S35168. Google Scholar PubMed Lassus P. , Heikkila P. , Andersson L. C. , von Boguslawski K. , Andersson S. ( 2003 ). Lower concentration of pulmonary hepatocyte growth factor is associated with more severe lung disease in preterm infants . J. Pediatr. 143 , 199 – 202 . Google Scholar Crossref Search ADS PubMed Ma P. C. , Tretiakova M. S. , MacKinnon A. C. , Ramnath N. , Johnson C. , Dietrich S. , Seiwert T. , Christensen J. G. , Jagadeeswaran R. , Krausz T. , et al. . ( 2008 ). Expression and mutational analysis of MET in human solid cancers . Genes Chromosomes Cancer 47 , 1025 – 1037 . Google Scholar Crossref Search ADS PubMed Maina F. , Hilton M. C. , Ponzetto C. , Davies A. M. , Klein R. ( 1997 ). Met receptor signaling is required for sensory nerve development and HGF promotes axonal growth and survival of sensory neurons . Genes Dev. 11 , 3341 – 3350 . Google Scholar Crossref Search ADS PubMed Maina F. , Klein R. ( 1999 ). Hepatocyte growth factor, a versatile signal for developing neurons . Nat. Neurosci. 2 , 213 – 217 . Google Scholar Crossref Search ADS PubMed Matsubara D. , Ishikawa S. , Sachiko O. , Aburatani H. , Fukayama M. , Niki T. ( 2010 ). Co-activation of epidermal growth factor receptor and c-MET defines a distinct subset of lung adenocarcinomas . Am. J. Pathol. 177 , 2191 – 2204 . Google Scholar Crossref Search ADS PubMed Merchant M. , Ma X. , Maun H. R. , Zheng Z. , Peng J. , Romero M. , Huang A. , Yang N. Y. , Nishimura M. , Greve J. , et al. . ( 2013 ). Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent . Proc. Natl. Acad. Sci. U.S.A. 110 , E2987 – E2996 . Google Scholar Crossref Search ADS PubMed Moffat G. J. , Retter M. W. , Kwon G. , Loomis M. , Hock M. B. , Hall C. , Bussiere J. , Lewis E. M. , Chellman G. J. ( 2014 ). Placental transfer of a fully human IgG2 monoclonal antibody in the cynomolgus monkey, rat, and rabbit: A comparative assessment from during organogenesis to late gestation . Birth Defects Res. B Dev Reprod. Toxicol. 101 , 178 – 188 . Google Scholar Crossref Search ADS PubMed Ohmichi H. , Koshimizu U. , Matsumoto K. , Nakamura T. ( 1998 ). Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development . Development 125 , 1315 – 1324 . Google Scholar PubMed Olivero M. , Rizzo M. , Madeddu R. , Casadio C. , Pennacchietti S. , Nicotra M. R. , Prat M. , Maggi G. , Arena N. , Natali P. G. , et al. . ( 1996 ). Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas . Br. J. Cancer 74 , 1862 – 1868 . Google Scholar Crossref Search ADS PubMed Onitsuka T. , Uramoto H. , Ono K. , Takenoyama M. , Hanagiri T. , Oyama T. , Izumi H. , Kohno K. , Yasumoto K. ( 2010 ). Comprehensive molecular analyses of lung adenocarcinoma with regard to the epidermal growth factor receptor, K-ras, MET, and hepatocyte growth factor status . J. Thorac. Oncol. 5 , 591 – 596 . Google Scholar Crossref Search ADS PubMed Onozato R. , Kosaka T. , Kuwano H. , Sekido Y. , Yatabe Y. , Mitsudomi T. ( 2009 ). Activation of MET by gene amplification or by splice mutations deleting the juxtamembrane domain in primary resected lung cancers . J. Thorac. Oncol. 4 , 5 – 11 . Google Scholar Crossref Search ADS PubMed Panos R. J. , Patel R. , Bak P. M. ( 1996 ). Intratracheal administration of hepatocyte growth factor/scatter factor stimulates rat alveolar type II cell proliferation in vivo . Am. J. Respir. Cell Mol. Biology 15 , 574 – 581 . Google Scholar Crossref Search ADS Parks W. T. ( 2015 ). Placental hypoxia: The lesions of maternal malperfusion . Semin. Perinatol. 39 , 9 – 19 . Google Scholar Crossref Search ADS PubMed Pentšuk N. , van der Laan J. W. ( 2009 ). An interspecies comparison of placental antibody transfer: New insights into developmental toxicity testing of monoclonal antibodies . Birth Defects Res. B Dev. Reprod. Toxicol. 86 , 328 – 344 . Google Scholar Crossref Search ADS PubMed Pohl M. , Stuart R. O. , Sakurai H. , Nigam S. K. ( 2000 ). Branching morphogenesis during kidney development . Annu. Rev. Physiol. 62 , 595 – 620 . Google Scholar Crossref Search ADS PubMed Powell E. M. , Mars W. M. , Levitt P. ( 2001 ). Hepatocyte growth factor/scatter factor is a motogen for interneurons migrating from the ventral to dorsal telencephalon . Neuron 30 , 79 – 89 . Google Scholar Crossref Search ADS PubMed Powell E. M. , Muhlfriedel S. , Bolz J. , Levitt P. ( 2003 ). Differential regulation of thalamic and cortical axonal growth by hepatocyte growth factor/scatter factor . Dev. Neurosci. 25 , 197 – 206 . 72268. Google Scholar Crossref Search ADS PubMed Sadiq A. A. , Salgia R. ( 2013 ). MET as a possible target for non-small-cell lung cancer . J. Clin. Oncol. 31 , 1089 – 1096 . Google Scholar Crossref Search ADS PubMed Santos O. F. , Barros E. J. , Yang X. M. , Matsumoto K. , Nakamura T. , Park M. , Nigam S. K. ( 1994 ). Involvement of hepatocyte growth factor in kidney development . Dev. Biol. 163 , 525 – 529 . Google Scholar Crossref Search ADS PubMed Schmidt C. , Bladt F. , Goedecke S. , Brinkmann V. , Zschiesche W. , Sharpe M. , Gherardi E. , Birchmeler C. ( 1995 ). Scatter factor/hepatocyte growth factor is essential for liver development . Nature 373 , 699 – 702 . Google Scholar Crossref Search ADS PubMed Siegfried J. M. , Weissfeld L. A. , Singh-Kaw P. , Weyant R. J. , Testa J. R. , Landreneau R. J. ( 1997 ). Association of immunoreactive hepatocyte growth factor with poor survival in resectable non-small cell lung cancer . Cancer Res . 57 , 433 – 439 . Google Scholar PubMed Sonnenberg E. , Meyer D. , Weidner K. M. , Birchmeier C. ( 1993 ). Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development . J. Cell Biol. 123 , 223 – 235 . Google Scholar Crossref Search ADS PubMed Sousa I. , Clark T. G. , Toma C. , Kobayashi K. , Choma M. , Holt R. , Sykes N. H. , Lamb J. A. , Bailey A. J. , Battaglia A. , International Molecular Genetic Study of Autism, C ., et al. . ( 2009 ). MET and autism susceptibility: Family and case-control studies . Eur. J. Hum. Genet . 17 , 749 – 758 ., Google Scholar Crossref Search ADS PubMed Spigel D. R. , Edelman M. J. , Mok T. , O'Byrne K. , Paz-Ares L. , Yu W. , Rittweger K. , Thurm H. , MetLung Phase I. I. I. S. G. ( 2012 ). Treatment rationale study design for the metlung trial: A randomized, double-blind phase III study of onartuzumab (MetMAb) in combination with erlotinib versus erlotinib alone in patients who have received standard chemotherapy for stage IIIB or IV met-positive non-small-cell lung cancer . Clin. Lung Cancer 13 , 500 – 504 . Google Scholar Crossref Search ADS PubMed Trusolino L. , Bertotti A. , Comoglio P. M. ( 2010 ). MET signalling: Principles and functions in development, organ regeneration and cancer . Nat. Rev. Mol. Cell Biol. 11 , 834 – 848 . Google Scholar Crossref Search ADS PubMed Uehara Y. , Minowa O. , Mori C. , Shiota K. , Kuno J. , Noda T. , Kitamura N. ( 1995 ). Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor . Nature 373 , 702 – 705 . 10.1038/373702a0. Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

Placental and Fetal Effects of Onartuzumab, a Met/HGF Signaling Antagonist, When Administered to Pregnant Cynomolgus Monkeys

Toxicological Sciences , Volume 165 (1) – Sep 1, 2018

Loading next page...
 
/lp/ou_press/placental-and-fetal-effects-of-onartuzumab-a-met-hgf-signaling-ydZm2VQYGe
Publisher
Oxford University Press
Copyright
© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
ISSN
1096-6080
eISSN
1096-0929
D.O.I.
10.1093/toxsci/kfy141
Publisher site
See Article on Publisher Site

Abstract

Abstract Onartuzumab is an engineered single arm, monovalent monoclonal antibody that targets the MET receptor and prevents hepatocyte growth factor (HGF) signaling. Knockout mice have clearly demonstrated that HGF/MET signaling is developmentally critical. A pre- and postnatal development study (enhanced design) was conducted in cynomolgus monkeys to evaluate the potential developmental consequences following onartuzumab administration. Control or onartuzumab, at loading/maintenance doses of 75/50 mg/kg (low) or 100/100 mg/kg (high), was administered intravenously once weekly to 12 confirmed pregnant female cynomolgus monkeys per group from gestation day (GD) 20 through GD 174. Onartuzumab administration resulted in decreased gestation length, decreased birth weight, and increased fetal and perinatal mortality. A GD147 C-section was conducted for a subset of Control and High Dose monkeys, and identified placental infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate. These findings were limited to placentas from onartuzumab-treated animals. In addition, decreased cellularity of the hepatocytes with dilated hepatic sinusoids was inconsistently observed in the liver of a few fetal or infant monkeys that died in the perinatal period. Surviving offspring had some evidence of developmental delay compared with controls, but no overt teratogenicity. Overall, effects on the perinatal fetuses were consistent with those reported in knockout mice, but not as severe. Onartuzumab concentrations were low or below the level of detection in most offspring, with cord blood concentrations only 1%–2% of maternal levels on GD 147. Malperfusion secondary to onartuzumab-induced placental injury could explain the adverse pregnancy outcomes, fetal growth restriction and relatively low fetal exposures. onartuzumab, MET, ePPND, placenta, cynomolgus monkey Met is a receptor tyrosine kinase involved in critical cellular processes such as cell growth, differentiation, neovascularization and survival in normal and tumor cells (Birchmeier et al., 2003; Trusolino et al., 2010). Met signaling is activated through binding of hepatocyte growth factor (HGF, also known as scatter factor), the only known ligand for Met (Derksen et al., 2003). Met is frequently dysregulated in tumor cells via multiple mechanisms, particularly elevated expression, with or without gene mutations (Birchmeier et al., 2003; Kong-Beltran et al., 2006). The mitogenic, motogenic, and morphogenic cellular behavior activated by HGF/MET signaling is often referred to as “invasive growth” and is associated with the aggressive and metastatic potential of tumors. Dysregulation of the HGF/MET pathway in non-small cell lung cancer (NSCLC) occurs at the level of gene amplification, mutation, and over-expression of either MET or HGF (Landi et al., 2013; Sadiq and Salgia, 2013). The rate of gene amplification of MET in NSCLC vary between 2% and 20% (Beau-Faller et al., 2008; Cappuzzo et al., 2009; Onitsuka et al., 2010; Onozato et al., 2009). In NSCLC, rates of overexpression of MET vary between 25% and 75%, depending upon the sample set and nature of the diagnostic test utilized (Danilkovitch-Miagkova and Zbar, 2002; Ma et al., 2008; Olivero et al., 1996). Overexpression of MET or HGF have been associated with increased pathologic tumor stage and worse outcome in patients with NSCLC (Ichimura et al., 1996; Olivero et al., 1996; Siegfried et al., 1997; Spigel et al., 2012). HGF can also be overexpressed in NSCLC by the stroma where it can act to stimulate invasive growth and survival of NSCLC tumor cells. Onartuzumab (also known as MetMAb) is a unique humanized 1-armed (monovalent) antibody, using the “knobs into holes” technology that binds specifically to the cell surface Met receptor to prevent ligand binding and block downstream Met signaling and HGF-mediated activation (Carter et al., 1992; Merchant et al., 2013). Importantly, the monovalent design of onartuzumab inhibits HGF binding without inducing Met dimerization, which is considered a prerequisite for receptor activation (Birchmeier et al., 2003; Matsubara et al., 2010). Onartuzumab was developed as a potential therapy to be used in combination with the epidermal growth factor receptor tyrosine kinase inhibitor, Erlotinib, for the treatment of NSCLC (Spigel et al., 2012). Onartuzumab does not bind to rodent, rabbit, or canine MET. Onartuzumab blocks HGF binding to human c-Met with an inhibitory concentration (IC)50 of 1.8 nM and inhibits the subsequent induction of c-Met auto-phosphorylation and cell proliferation in many cancer cell lines. Moreover, onartuzumab binds cynomolgus monkey MET and human MET with comparable low nanomolar equilibrium dissociation constants (4.4 and 1.5 nM, respectively), and thus the nonclinical safety assessment of onartuzumab was conducted in cynomolgus monkeys (Merchant et al., 2013). Overall, onartuzumab was well tolerated in toxicity studies in cynomolgus monkeys when administered for up to 26 weeks and at up to 100 mg/kg. In the nonclinical safety studies there were no consistent toxicity findings attributed to onartuzumab, nor pharmacodynamic effects as a result of c-Met inhibition. Several published studies have described the lethal effects of homozygous (−/−) HGF or Met gene disruption during embryo-fetal development in mice (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995). Homozygous knockout (−/−) mouse pups died in utero between embryonic days 12.5 and 15.5 (E12.5–E15.5) and the absence of Met signaling revealed severe developmental abnormalities of the placenta, liver, neurons, and muscle in the limb, diaphragm, and tongue (Bladt et al., 1995; Ebens et al., 1996; Maina et al., 1997; Schmidt et al., 1995; Uehara et al., 1995). In contrast, heterozygous (+/−) pups did not appear to be adversely affected in that they were healthy and fertile, suggesting that complete Met inhibition may be required to result in a reproductive liability. Therefore, as part of the nonclinical safety assessment, an enhanced pre- and postnatal development (ePPND) study was conducted in cynomolgus monkeys to investigate potential effects of onartuzumab on pregnant/lactating females, embryo-fetal development and development of offspring up to postnatal day (PND) 91. Because adverse effects of onartuzumab on fetal development were considered likely, this study utilized a relatively small group size and a 3-month postnatal follow-up period to assess pregnancy outcomes. In addition, a subset of pregnancies (3 each from the control and high-dose groups) was terminated by cesarean section on gestation day (GD) 147 to assess the late gestation fetus, the late gestation placenta, and fetal onartuzumab concentrations via cord blood. MATERIALS AND METHODS This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Shin Nippon Biomedical Laboratories, Ltd. and was performed in accordance with the animal welfare bylaws of Shin Nippon Biomedical Laboratories, Ltd., Drug Safety Research Laboratories, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Animals and Husbandry This study was conducted under an IACUC-approved protocol in an AAALAC-approved facility. Experimentally naïve and sexually mature (at least 4 years old) female cynomolgus monkeys (Macaca fascicularis; country of origin, Cambodia) weighing between 2.6 and 4.5 kg were housed in stainless steel cages, and were acclimated for at least 4 weeks prior to mating. Monkeys were provided primate pellets (>100 g; Purina Mills LLC) once daily, had access to water ad libitum, and were provided additional supplements/treats at least 3 times per week. The room in which the monkeys were housed was set to maintain a temperature of 23°C–29°C, 30%–70% humidity, complete air exchange 15 times/h and 12-h light/dark cycles. Females that showed regular menstrual cycles were paired with males of proven fertility for 3 consecutive days, between the 12th and 14th days of the menstrual cycle (at estimated ovulation time). During the mating period, coitus was confirmed visually. The mid-point of the mating period was designated as GD 0. Females were housed individually during gestation, and with their respective offspring postpartum. Control and Test Articles Control article Onartuzumab vehicle; 10 mM histidine acetate, 120 mM sucrose, 0.04% polysorbate 20 (w/v), pH 5.4. Test article Onartuzumab, supplied as a solution at a nominal concentration of 60 mg/ml was diluted with Onartuzumab vehicle to the appropriate concentration prior to administration. Experimental Design On day 18 of presumed gestation, pregnancy status was diagnosed by an ultrasound examination. In total 36 pregnant females were sequentially assigned to 1 of 3 study groups based on the order of pregnancy diagnosis (see Table 1). Monkeys received the first administration (loading dose) of onartuzumab or vehicle control on GD 20 and subsequently received the maintenance dose once weekly via intravenous injection from GD 27 through parturition (up to GD174; maximum of 23 doses). The initial loading dose of 75 mg/kg or 100 mg/kg was administered to rapidly achieve saturating exposures, while the maintenance dose levels of 50 or 100 mg/kg were chosen so that the expected trough serum concentrations (Ctrough) of the low-dose group (group 2; 75/50 mg/kg) would achieve the predicted maximum serum concentrations (Cmax) at steady state observed for the median patient population at clinically tested dose levels. The Ctrough of the high-dose group (group 3; 100/100 mg/kg) would similarly achieve the Cmax at steady state observed for 90% of the clinical population. Weekly administration was designed to maintain steady state levels throughout the study and was based on the previously determined t1/2β values of 4–7 days in cynomolgus monkeys. Animals were examined at least twice daily for clinical signs of toxicity from GD 19 through lactational day (LD) 91. Pregnancy status was monitored via ultrasound on GDs 25, 32, 39, 46, 60, 74, 88, 102, 116, 130, 144 and 158. In utero examinations, including fetal growth and heart rate measurements were monitored via ultrasound at prespecified times throughout the pregnancy. Body weights were recorded weekly starting on GD20 and food consumption was recorded daily from GD20 to LD91. Blood samples for hematology and serum chemistry analysis were collected periodically throughout pregnancy and lactation, with additional blood samples collected from dams following an abortion, embryonic/fetal death, stillbirth, or offspring death. Maternal blood samples collected on GDs 20, 27, 34, 83, 86, 90, 118, and 153 and LDs 7, 28, 56, and 91 were assessed for onartuzumab concentrations. Additional maternal samples were collected on days 1 (or as soon as possible following confirmation of fetal or neonatal mortality), 28, 56 and 91 following confirmation of a fetal deal, stillbirth or offspring death, when applicable. Maternal blood collected on GDs 20, 34, and 118 and LDs 28 and 91 were assessed for the presence of antidrug antibodies (ADAs) directed against onartuzumab. Table 1. Study Design Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c a Vehicle and onartuzumab were administered IV in a volume of 2 ml/kg. b GD, gestation day. c Three dams were allocated to a GD 147 C-section rather than progressing through parturition. Table 1. Study Design Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c Group Treatment Loading Dose (mg/kg) on GD20a,b Maintenance Dose (mg/kg)a Number of Pregnant Animals 1 Vehicle 0 0 12c 2 Onartuzumab 75 50 12 3 Onartuzumab 100 100 12c a Vehicle and onartuzumab were administered IV in a volume of 2 ml/kg. b GD, gestation day. c Three dams were allocated to a GD 147 C-section rather than progressing through parturition. For dams in which abortion or embryonic/fetal death was confirmed after GD80, the fetus was obtained via cesarean section and evaluated for external, visceral, or skeletal malformations to the extent possible. Although autolysis and/or postmortem trauma to these fetuses precluded comprehensive assessment, and a complete gross necropsy was not conducted, most fetuses were evaluable for a general histologic assessment of tissue composition and structures. Fetal tissues, including lungs, liver, heart, kidneys, spleen, mesenteric lymph nodes, thymus, and placenta were embedded in paraffin, sectioned, stained with hematoxylin-eosin, and examined microscopically. Stillborn offspring and offspring that died up to PND 30 were weighed, assessed for external morphological measurements and evaluated for external, visceral, and skeletal malformations. The lungs, liver, heart, kidneys, spleen, mesenteric lymph nodes, thymus, and placenta were embedded in paraffin, sectioned, stained with hematoxylin-eosin, and examined microscopically. Surviving offspring were monitored twice daily for clinical signs and body weights were measured weekly through PND 35 and every other week from PND 35 through PND 91. Skeletal examinations were conducted radiographically on PND 7, or once offspring weights exceeded 250 g. Morphological development, included head width, distance between the eyes, crown-rump length, tail length, chest circumference, fore and hind limb length and ano-genital distance were measured on PNDs 28 and 91. Blood from offspring was collected on PND 91 for hematology and serum chemistry, on PND 14, 28, 42, and 91 for toxicokinetic analysis of onartuzumab and on PND 42 and 91 for the presence of ADAs. Functional development, including pupillary reflex, Preyer’s reflex, grip strength and pain response were evaluated on PND 14. Offspring-dam neurobehavioral assessments were performed on PND 28 and 90. Ophthalmic and electrocardiographic examinations of offspring were conducted once between PND 80 and 85. Cesarean Section As a result of the increased incidence in abortions, fetal deaths, low birthweight, and offspring deaths in the onartuzumab-treated groups, investigative cesarean-sections were performed on GD 147 for 3 group 1 (control) and group 3 (high dose) pregnant monkeys. This design element was added relatively late in the study, and there was an inadequate number of group 2 (low dose 75/50 mg/kg) pregnant dams remaining for GD147 c-section. Fetal cord blood was collected to determine onartuzumab concentrations and presence of ADA. Amniotic fluid volume was measured at the time of c-section as an indirect measure of placental perfusion and developing fetal renal function; onartuzumab concentrations in amniotic fluid were not evaluated. The fetus and placenta were removed, weighed and examined externally and histologically as described earlier. Serum Onartuzumab and ADA Determination To determine onartuzumab exposure and appearance of ADAs in dams, blood was collected into serum separator tubes at several times throughout the dosing and lactational phases of the study. Blood was collected from dams as soon as possible and several timepoints following abortion, embryonic/fetal death, stillbirth, or offspring death. Similarly, blood was collected from surviving offspring on PNDs 14, 28, 42, and 91 and PNDs 42 and 91 for toxicokinetic and ADA analysis, respectively. The serum samples were assayed for onartuzumab and ADAs to onartuzumab in bridging ELISAs. For determination of onartuzumab concentrations, the assay used murine antionartuzumab complementarity determining region monoclonal antibodies in the capture phase, and F(ab′)2 fragmented, goat antihuman immunoglobulin (Ig)G Fc antibodies conjugated to horseradish peroxidase (HRP) were used for detection. The lower limit of quantification for this assay was 0.2 μg/ml. For detection of ADAs to onartuzumab, serum samples were incubated with equal concentrations of biotinylated onartuzumab and digoxigenin onartuzumab. Serum samples were then transferred to a high bind streptavidin coated plate. Following appropriate incubation, the plate was washed and mouse monoclonal anti-digoxin HRP was added for detection. Using a surrogate monoclonal antibody, the sensitivity of the assay was determined to be 2 ng/ml. The presence of circulating onartuzumab in a sample can interfere with the detection of ADAs, thereby decreasing the sensitivity of the assay. In the presence of up to 200 μg/ml of onartuzumab, the assay was able to detect 1000 ng/ml of the surrogate monoclonal antibody. Statistical Analysis All data were evaluated using MUSCOT statistical analysis software (Yukms Corp, Glenview, Illinois). Quantitative data were analyzed by Bartlett’s test for homogeneity of variance. When the variance was homogeneous, Dunnett’s test was applied to compare control and treatment groups. When variance was heterogenous by Barlett’s test, a Miller test was applied to compare control and treatment groups. For a comparison of data obtained following cesarian section on GD 147 in group 1 (control) and group 3 (high dose), the data were analyzed for homogeneity of variance by the F test. When the variance was homogeneous, student t test was applied and when the variance was heterogeneous, Aspim-Welch t test was applied. A probability value of p < .05 was considered statistically significant. RESULTS No Effects of Onartuzumab on Pregnant Dams Onartuzumab was administered by a slow IV bolus injection once weekly for up to 23 weeks to confirmed pregnant female cynomolgus monkeys (n = 12/group) during the period of organogenesis through parturition (ie, from GD 20 through GD 174 or birth). The dose levels tested were 0 mg/kg (vehicle control), 75/50 mg/kg (loading dose/maintenance dose; low dose), or 100/100 mg/kg (high dose). The dams were evaluated for onartuzumab-related changes in body weight, food consumption, physical examinations (body temperature and pulse oximetry), clinical pathology indices, including serum chemistry, hematology, urinalysis, serum chorionic gonadotropin. Weekly administration of onartuzumab up to 23 weeks was well tolerated in pregnant dams with no apparent clinical signs of maternal toxicity and no onartuzumab-related changes in any of the parameters measured. Effects of Onartuzumab on Pregnancy Outcome Onartuzumab-related effects on the fetus/neonate included increased rates of embryo-fetal death and early deliveries (see Tables 2 and 3). Of the 12 confirmed pregnancies in Group 1 (control) dams, 1 embryonic death (1 of 12; 8.3%) was detected in the first trimester (GD 39); in addition, C-sections were conducted on 3 group 1 (control) dams on GD 147, and all 3 fetuses appeared viable at delivery. The number of fetuses that survived to parturition was 8 of 9 (88.9%) with a mean gestational period of 162.3 ± 4.4 days. In contrast, prenatal embryo-fetal deaths occurred in 2 of 12 (16.7%) and 4 of 12 dams (33.3%) group 2 (low dose) dams in the first and second/third trimesters respectively, with no clear temporal pattern of effect during pregnancy (see Table 2). Furthermore 1 stillborn offspring (1 of 12; 8.3%) was delivered on GD 170. The rate of fetal survival to parturition in the group 2 dams given onartuzumab at the low dose (75/50) was 5 of 12 (41.7%) with a mean gestational period of 154.0 ± 13.1 days. Similarly, in group 3 dams (high-dose onartuzumab; 100/100), prenatal embryo-fetal deaths occurred in 2 of 12 (16.7%) in the first trimester and 2 of 9 (22.2%) in the second/third trimesters (Table 2); in addition C-sections were conducted on 3 pregnant dams on GD 147. The number of fetuses that survived to parturition in the group 3 dams was 5 of 9 (55.6%) with a mean gestational period of 148.8 ± 6.3 days. The incidences for abortion and fetal death in the onartuzumab-treated groups were considered high in the second and third trimesters as compared with historical control data from the testing facility (mean incidences for the second and third trimesters in controls are 1.1% [range 0.0%–12.5%] and 3.4% [range 0.0%–16.7%], respectively). Table 2. Summary of Pregnancy Outcome and Neonatal Survival Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e a n = 12 pregnant animals/group. b Onartuzumab administered intravenously (loading dose/maintenance dose) based on the most recent body weight measurement. c Data were calculated from the period of PND 0–7. d Data were calculated from the period PND 8–30. e Data of survival rate in neonates to PND 91. Abbreviation: NA, Not Available. Table 2. Summary of Pregnancy Outcome and Neonatal Survival Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e Vehiclea Low (75/50)a,b High (100/100)a,b Testing Facility Historical Control (Range) Early losses (prior to GD50) 1 2 2 First trimester (0.0%–22.2%) Fetal death/abortion/stillborn (GD50–174) 0 2 0 Second trimester (0.0%–12.5%) 0 3 2 Third trimester (0.0%–16.7%) Scheduled C-section (GD147) 3 0 3 NA Neonatal death (PND 0–7) 0 1 2 (0.0%–7.7%)c Neonatal death (PND 8–30) 0 0 1 (0.00%–4.6%)d Scheduled necropsy (PND 91) 8 4 2 (91.2%–100.0%)e a n = 12 pregnant animals/group. b Onartuzumab administered intravenously (loading dose/maintenance dose) based on the most recent body weight measurement. c Data were calculated from the period of PND 0–7. d Data were calculated from the period PND 8–30. e Data of survival rate in neonates to PND 91. Abbreviation: NA, Not Available. Table 3. Summary of Mean Gestational Length and Neonatal Survival Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Abbreviations: GD, gestation day; PND, postnatal day. a On day 18 of presumed gestation, pregnancy status was diagnosed via ultrasound and 36 confirmed pregnant monkeys were assigned to 1 of 3 groups (n = 12/group). b Historical mean delivery day (range): GD 160.9 (143 − 175). c Overall neonatal survival through PND 91. d C-sections were performed on n = 3 control and high dose dams on GD 147 as part of a protocol amendment and are not included in this analysis. e p < .05 compared with control. Table 3. Summary of Mean Gestational Length and Neonatal Survival Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Groupa Dose Level (mg/kg) (Loading/Maintenance) Mean Gestational Delivery Day (Range)b No. of Live Births (%) Neonatal Survivalc (%) 1 0/0 GD 162.3 ± 4.4 (GD 154 − 168) 8/9 (88.9)d 8/8 (100) 2 75/50 GD 154.0 ± 13.1 (GD 141 − 170) 5/12 (41.7) 4/5 (80) 3 100/100 GD 148.8 ± 6.3e (GD 141 − 155) 5/9 (55.6)d 2/5 (40) Abbreviations: GD, gestation day; PND, postnatal day. a On day 18 of presumed gestation, pregnancy status was diagnosed via ultrasound and 36 confirmed pregnant monkeys were assigned to 1 of 3 groups (n = 12/group). b Historical mean delivery day (range): GD 160.9 (143 − 175). c Overall neonatal survival through PND 91. d C-sections were performed on n = 3 control and high dose dams on GD 147 as part of a protocol amendment and are not included in this analysis. e p < .05 compared with control. Effects of Onartuzumab on Offspring Overall, onartuzumab administration to pregnant dams resulted in impaired growth of the fetus. This effect was apparent on GD 130, when the fetal head width and femoral length, determined via ultrasound, were significantly smaller in the high dose group as compared with controls. Of the fetuses that did survive to parturition there was an onartuzumab-dependent decrease in neonatal birth weight (see Table 4 and Figure 1). The mean birth weight of the offspring from Group 1 dams was 351 ± 41 g, which is consistent with the historical birth weights of cynomolgus monkeys at this facility (336.8 ± 34.5 g; unpublished data). In contrast, the birth weights of the offspring from dams given the low dose and high dose of onartuzumab during pregnancy were significantly lower; 250 ± 32 g and 210 ± 29 g, respectively. Furthermore, overall body weight of the surviving offspring from onartuzumab-treated dams remained lower than controls through the postnatal period until the day of necropsy (PND 91; see Figure 1). Table 4. Mean Body Weights at Birth and PND 91 Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) a Historical control mean birth weight at testing facility is 336.8 ± 34.5 gr. b p < .05 compared with control. Table 4. Mean Body Weights at Birth and PND 91 Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) Group Mean Birth Weights ±SD (gr)a Mean Body Weights on PND 91 ± SD (gr) Postnatal Deaths Control 351 ± 41 (n = 8) 751 ± 123 (n = 8) 0 Low dose 250 ± 32b (n = 4) 533 ± 129b (n = 4) 0 High Dose 210 ± 29b (n = 4) 625 ± 21 (n = 2) 2 (PND3 and PND11) a Historical control mean birth weight at testing facility is 336.8 ± 34.5 gr. b p < .05 compared with control. Figure 1. View largeDownload slide Mean body weight of offspring through PND 91. Numbers embedded in bar graphs represent the number of offspring/group at each timepoint. Of note, in the high dose group (100/100), 2 neonatal offspring died, 1 on PND 3 and 1 on PND 11, resulting in only n = 2 offspring from PND 14 through PND91 and therefore were not statistically compared with controls. Asterisk (*) indicates p < .05 compared with controls. Figure 1. View largeDownload slide Mean body weight of offspring through PND 91. Numbers embedded in bar graphs represent the number of offspring/group at each timepoint. Of note, in the high dose group (100/100), 2 neonatal offspring died, 1 on PND 3 and 1 on PND 11, resulting in only n = 2 offspring from PND 14 through PND91 and therefore were not statistically compared with controls. Asterisk (*) indicates p < .05 compared with controls. Consistent with the lower body weights, significantly lower values were noted in postnatal crown-rump length, tail length, and forelimb length on PNDs 28 and 91, and in hindlimb length on PND 28 in the low dose group when compared with the control group (data not shown). Trends toward low values were noted in hindlimb length on PND 91, and in chest circumference and ano-genital distance in male and females on PNDs 28 and 91 in the low dose group (data not shown). In the high dose group, a significantly lower value in chest circumference on PND 28, and trends toward low values in crown-rump length and hindlimb length on PNDs 28 and 91, and in forelimb length on PND 28 were noted when compared with the control group (data not shown). There were no onartuzumab-related skeletal abnormalities on PND7 and no effects on functional development measurement, including pupillary reflex, Preyer’s reflex, grip strength, or pain response on PND14. Similarly, no changes were noted in hematology, clinical chemistry, ophthalmology, or electrocardiography measurements in the offspring that survived to PND91. Cesarean Section GD147 Based on a higher than expected number of fetal and early postnatal losses of offspring from dams administered onartuzumab, it was decided to conduct cesarean sectioning on GD 147 for 3 dams each from the control and high dose groups to carefully evaluate pathological effects of onartuzumab on fetuses, including evaluation of the placenta, assessment of amniotic fluid volume, and collection of cord blood for concentration of onartuzumab. No statistically significant differences were noted in fetal body weight, placental weight, or amniotic fluid volume between the control group and high dose group; however, mean body weight in the high dose group (257.5 g) was slightly lower than the control group (281.7 g), and individual body weight of 1 fetus in the high dose group was extremely low for this gestational age (204.8 gr). No external or skeletal abnormalities were observed in any fetus. No visceral anomalies were identified with the exception of a small thymus noted for 1 high dose offspring. Significant decreases in absolute liver weight and high relative brain and epididymis (right and left) weights were noted in the high dose group when compared with the organ weights in the control group. These differences in organ weights were considered related to the general retardation of fetal growth, rather than a direct effect of onartuzumab on these specific tissues. One high dose fetus was substantially smaller, overall and in individual organ weights, than the other high-dose and control group GD147 fetuses; there were grossly evident indurated white foci noted in both the main and accessory placentae for this fetus. Offspring Necropsied at PND91 Retarded eruption of upper and lower first molar and canine teeth was observed bilaterally in 3 offspring and retarded eruption of upper first molar and canine teeth and lower canine tooth bilaterally in 1 offspring in the low dose group. In the high dose group, retarded eruption of upper and lower first molar and canine teeth was observed bilaterally in 1 offspring. In the control group, retarded eruption of upper and lower canine teeth was observed bilaterally in 1 offspring. Statistically significant decreases in absolute lung and spleen weights, and high relative brain and epididymis (left) weights were noted in the low dose group when compared with the organ weights in the control group. Statistically significant increases in relative kidney (left) weight were noted in the high dose group. These differences in organ weights were considered related to the general retardation of fetal growth and associated delay in postnatal growth, rather than a direct effect of onartuzumab on these specific tissues. Placental Evaluations Twelve placentae were examined grossly and histopathologically. These included 3 each from the control and high dose group cesarean sections on GD 147, which reflect the primary basis of comparison and evaluation in this study. In addition, 6 placentae were collected after abortion, fetal death, or early postnatal death, and also evaluated grossly and histologically. Although diffuse placental changes due to parturition and/or autolysis compromised full assessment of these 6 placentae, the findings attributed to onartuzumab were interpretable due to their multifocal distribution and similarities to the histopathology of the placentae from the c-section cohort. Placental weight evaluations were limited to the 6 placentae collected at c-section, and there were no differences in overall placental weights (main+accessory) between the high dose and control groups. However, the gross and histologic picture of the placentae from onartuzumab-treated dams was substantially different from that of the control group c-section placentae. Slight to marked infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate were observed in all examined placentae in the low dose and high dose groups. Increases in fibrin deposition in the chorionic plate, chorionic villus or decidual plate were observed in 1 placenta in the low dose group and 4 placentae in the high dose group (Figure 2). These placental findings corresponded to the gross observation of indurated white foci in 1 placenta in each of the low and high dose groups. Placental infarcts have not been reported as a background finding in cynomolgus monkeys, and were not observed in the 3 placentae obtained from the control group at GD147. Of note, the placenta is not routinely collected in an ePPND study, and in this study none of the placentae from offspring that survived until the PND 91 necropsy were assessed. Figure 2. View largeDownload slide Placental histopathology from fetuses delivered by cesarean section on GD 147. A, normal placenta from the control group. B, Scattered microinfarcts, hemorrhage and fibrin in a placenta from the high dose group. C, Area of severe compromise due to infarction, necrosis and fibrin in a placenta from the high dose group. All placentae were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Figure 2. View largeDownload slide Placental histopathology from fetuses delivered by cesarean section on GD 147. A, normal placenta from the control group. B, Scattered microinfarcts, hemorrhage and fibrin in a placenta from the high dose group. C, Area of severe compromise due to infarction, necrosis and fibrin in a placenta from the high dose group. All placentae were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Other Histopathology Findings In the liver, decreased cellularity of the hepatocytes with dilated sinusoids was observed in 1 fetus in the high dose group removed at cesarean sectioning on GD 147, and 1 offspring found dead in the early postnatal period from each of the low dose and high dose groups. Although consistent with the reported hepatic developmental abnormalities identified in mice defective for c-Met signaling, this finding was inconsistently distributed in the liver, with some lobes similar to controls (Figure 3). Also, although organ weights of fetuses removed at cesarean sectioning on GD 147 had significantly lower liver weight as compared with controls, this was primarily driven by the 1 liver with decreased hepatocyte cellularity. At the PND 91 necropsy, there were no histologic differences across groups for the liver. Figure 3. View largeDownload slide Liver histopathology from fetuses delivered by cesarean section on GD 147. A, normal late gestation fetal liver from the control group, highlighting typical degree of sinusoidal dilation and glycogen content. B, late gestation fetal liver from the high dose group demonstrating hepatic cord disarray, decreased cellularity and sinusoidal dilation. Fetal livers were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Figure 3. View largeDownload slide Liver histopathology from fetuses delivered by cesarean section on GD 147. A, normal late gestation fetal liver from the control group, highlighting typical degree of sinusoidal dilation and glycogen content. B, late gestation fetal liver from the high dose group demonstrating hepatic cord disarray, decreased cellularity and sinusoidal dilation. Fetal livers were processed by immersion fixation in 10% neutral buffered formalin, followed by a series of alcohol dehydration steps, paraffin embedding, microtome sectioning at 4 μm onto glass slides, and staining with hematoxylin and eosin. Another histologic finding attributed to onartuzumab administration was the presence of rare syncytial cells in the alveoli of the lung, which was observed in 2 offspring found dead by PND11, 2 fetuses removed at cesarean sectioning on GD 147 and 1 stillborn fetus. All affected fetuses/offspring were in the onartuzumab treated groups. Finally, there was a single offspring in the low dose group and necropsied on PND 91 with fibrous osteodystrophy (FOD). In this single offspring, all bones examined histologically were affected, including the sternum, femur, maxilla and mandible; however, there were no notable findings in the kidneys or parathyroid glands, and serum levels of calcium and phosphorous were normal at the time of necropsy. There was no evidence of a similar process in any of the other offspring from dams administered onartuzumab. Toxicokinetics and ADAs Exposure to onartuzumab increased with the increase in dose level from 75/50 to 100/100 mg/kg in pregnant cynomolgus monkeys. The increases in group mean Cmax and AUC83–90 values following the 10th weekly dose were generally dose proportional. Steady-state appeared to have been achieved by GD 34 (Table 5). The fetus-to-dam onartuzumab serum concentration ratios at the time of cesarean sectioning on GD 147 showed that onartuzumab was not extensively transferred to the fetuses (approximately 1%–2%; Table 6). Concentrations of onartuzumab in 3 of 4 surviving Group 2 (75/50) offspring were below the limit of quantitation on PND14. The remaining offspring had onartuzumab concentration of 4010 ng/ml. In the 2 surviving offspring from group 3 (100/100) dams, PND14 onartuzumab concentrations were 7980 and 527 ng/ml. Onartuzumab concentrations on PND 28/LD 28 in all offspring from group 2 and group 3 dams were below the limit of quantitation with the exception of 1 offspring from a group 2 dam (75/50), which had a serum concentration of 549 ng/ml and an offspring to dam ratio of <1%. ADAs to onartuzumab were not detected in any dams in the onartuzumab dose groups during the gestation period of the dosing phase, or in any animals in the control group. Additionally, ADAs to onartuzumab were not detected in the fetuses at the time of cesarean section, or in surviving offspring at or after PND 14. In total 5 of 12 dams (42%) in the low dose group and 6 of 12 dams (50%) in the high dose group had ADAs to onartuzumab detected on LDs 28 or 91 or after cesarean sectioning or fetal loss. Altogether, ADAs to onartuzumab were detected in 11 of 24 cynomolgus monkey dams (46%) given onartuzumab, but there did not appear to be any impact on the exposure of onartuzumab in the dams or their offspring due the formation of ADAs to onartuzumab. However, the incidence of ADAs was higher in dams with poor pregnancy outcomes as compared with dams with offspring surviving to PND 91 (Table 7). Table 5. Summary of the Group Mean Toxicokinetic Parameters for Onartuzumab in Dams Post First and Tenth Doses Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d a Group 2 animals received a loading dose of 75 mg/kg on GD20 and 50 mg/kg thereafter for the remainder of the study. Therefore 75 mg/kg was used to calculate Cmax first and Cmax tenth was calculated after 9 weekly injections of 50 mg/kg. b n = 12. c n = 9. d n = 10. e n = 8. Table 5. Summary of the Group Mean Toxicokinetic Parameters for Onartuzumab in Dams Post First and Tenth Doses Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d Dose Group Dose Level (mg/kg) Cmax first (μg/ml) mean ± SD Cmax tenth (μg/ml) mean ± SD AUC83–91 (μg·day/ml) 2 75/50a 2610 ± 511b 2170 ± 199c 6600 ± 1300e 3 100 3290 ± 535b 4070 ± 473d 11000 ± 3940d a Group 2 animals received a loading dose of 75 mg/kg on GD20 and 50 mg/kg thereafter for the remainder of the study. Therefore 75 mg/kg was used to calculate Cmax first and Cmax tenth was calculated after 9 weekly injections of 50 mg/kg. b n = 12. c n = 9. d n = 10. e n = 8. Table 6. Fetal Cord Blood and Fetus to Dam Ratio of Onartuzumab Serum Concentration at Cesarean Sectioning (GD147) Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Table 6. Fetal Cord Blood and Fetus to Dam Ratio of Onartuzumab Serum Concentration at Cesarean Sectioning (GD147) Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Dose Group Dose Level (mg/kg) Cord Blood Concentration (μg/ml) Mean Concentration ± SD Fetus: Dam Ratio Mean Ratio ± SD 3 100 26.7 35.2 ±21.8 0.0161 0.0157 ± 0.0076 19.0 0.00798 60.0 0.0232 Table 7. Relationship Between Pregnancy Outcome and ADA Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 a Placenta collected for histopathology. Table 7. Relationship Between Pregnancy Outcome and ADA Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 Group Outcome ADA Group Outcome ADA Monkeys With Negative Pregnancy Outcomes Offspring Survived to Scheduled Necropsy Control Embryonic Death GD39 – Control Delivery GD168 – Necropsy PND91 75/50 Abortion GD32 – Control Delivery GD154 – Necropsy PND91 75/50 Stillbirth GD170 – Control Delivery GD163 – Necropsy PND91 75/50 Fetal Death GD130a + Control Delivery GD167 – Necropsy PND91 75/50 Fetal Death GD88a + Control Delivery GD164 – Necropsy PND91 75/50 Fetal Death GD70a + Control Delivery GD160 – Necropsy PND91 75/50 Fetal Death GD158a – Control Delivery GD162 Necropsy PND91 – 75/50 Abortion GD32 – Control Delivery GD160 Necropsy PND91 75/50 Delivery GD141 + 75/50 Delivery GD162 + Neonatal Death PND1 Necropsy PND91 100/100 Abortion GD25 – 75/50 Delivery GD154 – Necropsy PND91 100/100 Fetal Death GD116a + 75/50 Delivery GD136 – Necropsy PND91 100/100 Abortion GD126 + 75/50 Delivery GD161 – Necropsy PND91 100/100 Abortion GD32 – 100/100 Delivery GD 152 – Necropsy PND91 100/100 Delivery GD141 + 100/100 Delivery GD155 – Neonatal Death PND3 Necropsy PND91 100/100 Delivery GD143 Neonatal Death PND0a 100/100 Delivery GD153 + Neonatal Death PND11 a Placenta collected for histopathology. DISCUSSION This study demonstrates that administration of onartuzumab to cynomolgus monkeys once weekly via intravenous injection from GD 20 through parturition (up to GD174; maximum of 23 doses) resulted in a dose-dependent decrease in gestation length (early delivery) accompanied by low neonatal birth weight and decreased offspring survival in the absence of any notable maternal toxicity. Similarly, the mean body weight of fetuses removed on GD 147 was also slightly lower in the high dose group, and fetal growth retardation in the high dose group was indicated by in utero examination. These data are consistent with previous reports in humans, where an association between reduced HGF/MET signaling and developmental abnormalities has also been observed. Maternal serum HGF concentrations, but not umbilical cord HGF concentrations, were significantly decreased in pregnancies where infants were small for gestational age (SGA) when compared with pregnancies where infants were appropriate for gestational age (AGA) (Aoki et al., 1998). As HGF concentrations in the maternal serum are attributed to HGF released from the placenta into maternal circulation, but not the fetal circulation (Horibe et al., 1995), these results specifically implicate the inhibition of maternal MET signaling on fetal growth and development. However, the placental weight was also significantly lower in women of the SGA group when compared with the placental weight of women in the AGA group, whereas no overt difference in placental weights between control and onartuzumab-treated groups was identified from the GD147 C-sections. Given the pathology identified, including hemorrhage and fibrin in affected placentae, it is difficult to assess the placental weights in isolation. Results from this study are also consistent with previous reports that established the critical role of HGF/Met signaling pathway in embryogenesis and described embryo-fetal toxicity following Met pathway inhibition in rodents (Bladt et al., 1995; Schmidt et al., 1995; Uehara et al., 1995). These reports described the lethal effects of hgf or met gene disruption during embryo-fetal development in mice. Although normal homozygous wildtype (+/+) and heterozygous (+/−) pups did not appear to be adversely affected, homozygous knockout (−/−) mouse pups died in utero between embryonic days 12.5 and 15.5 (E12.5−E15.5). Although the effects of onartuzumab administration during pregnancy were not as severe as those reported for hgf or met homozygous knockout phenotype in mice, our results clearly demonstrate that onartuzumab exhibits the expected pharmacological effect on fetuses at the doses tested in this study. These dose levels were chosen so that the expected trough serum concentrations (Ctrough) of the low-dose group (group 2; 75/50 mg/kg) would achieve the predicted maximum serum concentrations (Cmax) at steady state observed for the median patient population at clinically tested dose levels, and the Ctrough of the high-dose group (group 3; 100/100 mg/kg) would similarly achieve the Cmax at steady state observed for 90% of the clinical population. The principal histopathologic finding was the presence of increased fibrin and infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate of the placenta. Decreased cellularity of the hepatocytes with dilated sinusoids in the liver was also identified in 2 fetuses from onartuzumab-treated dams. These findings are consistent with previous reports that revealed that the absence of Met signaling resulted in severe developmental abnormalities of the placenta, liver, neurons, and muscle in the limb, diaphragm, and tongue in rodents (Bladt et al., 1995; Ebens et al., 1996; Maina et al., 1997; Schmidt et al., 1995; Uehara et al., 1995). However, the rodent placental finding is described as thinning, or atrophy, as compared with normal controls. In this study, the specific findings of placental infarcts, increased fibrin deposition, and hemorrhage are more consistent with placental pathology related to poor pregnancy outcomes in humans (Aurioles-Garibay et al., 2014; Bendon, 2012; Parks, 2015). Placental infarcts have not been previously reported for cynomolgus monkeys. The effects of onartuzumab on the placenta likely contributed to the poor pregnancy outcomes and developmental growth delays in this study. In humans, similar findings are associated with maternal malperfusion of the placenta (Parks, 2015). In this study, although only 3 high dose fetuses and placentae were evaluated on GD147, all 3 fetuses had both histologic placental pathology and very low concentrations of onartuzumab in their cord blood; therefore, we conclude that decreased fetal perfusion due to the placental injury likely resulted in the low fetal serum concentrations of onartuzumab. Of the high-dose fetuses evaluated, the one with evidence of onartuzumab-related liver pathology also had the highest concentration of onartuzumab detected in the fetal cord blood. The minimal fetal exposures achieved are attributed to the placental injury and may explain the limited extraplacental developmental effects on potential target tissues in the offspring of dams administered onartuzumab. The relationship of this pattern of defects is further supported by expression data showing MET in epithelial tissues and HGF in adjacent mesenchyme, and paracrine signaling is considered critical in these tissues (Sonnenberg et al., 1993). In met homozygous knockout (−/−) mice the liver defects were noted as early as E12.5 as indicated by a 40% reduction in liver volume and progressed by E14.5 when the liver was less than half of the normal weight. These gross findings correlated histologically with a severe decrease in cellularity within the liver parenchyma and enlarged sinusoidal spaces. The key placental defect noted in the absence of Met was an impairment of trophoblast cell development and a reduced number of trophoblast cells in the affected placentae, which resulted in an overall decrease in placental size. Morphologically similar placental and liver developmental abnormalities were also noted in hgf knockout pups (Schmidt et al., 1995; Uehara et al., 1995). In both hgf and met knockout mice, the combination of hepatic developmental effects and placental insufficiency contributed to the fetal mortality in these models, and emphasize a key role for HGF/MET signaling during organogenesis. Another histopathological finding that was noted in individual fetuses/offspring included syncytial cells in the alveoli of the lung, which was observed in 2 offspring found dead by PND11, 2 fetuses removed at cesarean sectioning on GD 147 and 1 stillborn fetus from onartuzumab-treated dams. The origin of the alveolar syncytial cells observed in this study is not fully clear, but their location in the lung, and the absence of other local inflammatory cells, suggests an amniotic origin. Due to the disruption of all layers of the placenta in some placentae from onartuzumab-treated animals, these cells may be placental syncytiotrophoblasts released into the amniotic fluid and aspirated prenatally. Similar findings have not been reported in investigations of HGF/Met signaling in lung. Likewise, in this study, there were no apparent effects of onartuzumab on fetal lung branching or alveolization, although potential for such effects was anticipated. In in vitro studies conducted with fetal rat lungs, HGF stimulates branching morphogenesis of both alveolar and bronchial epithelia and in vivo, HGF contributes to the growth and maturation of neonatal rat lungs (Ohmichi et al., 1998; Panos et al., 1996). Consistent with the association between HGF and lung development, preterm human infants with subsequent bronchcopulmonary dysplasia (BPD) had significantly lower tracheal aspirate HGF concentrations and required longer mechanical ventilation and higher supplemental oxygen when compared with preterm infants who survived without BPD (Lassus et al., 2003). No definitive onartuzumab-related changes were noted in external or skeletal findings, hematology, serum chemistry, functional development, ophthalmology, electrocardiography, or bone marrow examination in surviving offspring on PND91. However, retarded eruption of the upper and lower first molar and/or canine teeth was observed in 5 of 6 offspring in the onartuzumab groups, but only 1 of 8 offspring in the control group. No histopathological abnormalities were found in any of these teeth with retarded eruption. This potential developmental delay was attributed to gestational immaturity at the time of parturition and/or fetal growth retardation due to placental injury. Finally, a single offspring in the low-dose group had FOD affecting all bones evaluated at the PND 91 necropsy. Both Met and HGF are involved in the cross talk between osteoclasts and osteoblasts (Grano et al., 1996), although FOD has not specifically been identified in genetic models. Because this finding occurred in a single offspring, at the lower dose level, and can occur as a spontaneous developmental abnormality in animals, it was not attributed to onartuzumab administration. It was somewhat unusual in presentation in that there were no histologic abnormalities of the parathyroid glands or kidneys in this animal, and the circulating calcium and phosphorous levels were normal at the time of the necropsy. Although there are no reports of sporadic FOD in infant macaques, either in the historical database or in the literature, in humans there are examples of selective end-organ resistance to parathyroid hormone leading to osteodytrophy in the absence of overt renal effects or electrolyte abnormalities (Bastepe, 2013). This is just one example, but it highlights the overall challenges in interpreting results from a study when there is a rich literature highlighting potential developmental effects across multiple organ systems. Some of this has already been discussed with regard to the liver, lung, and musculoskeletal system. In addition, a potential role for HGF/Met signaling has been established for the structural and functional development of central nervous systems (Burdick et al., 2010; Campbell et al., 2006, 2009; Ieraci et al., 2002; Judson et al., 2011; Maina and Klein, 1999; Powell et al., 2001,, 2003; Sousa et al., 2009), and there is some in vitro evidence for a role in the structural development of the renal system (Pohl et al., 2000; Santos et al., 1994). For the nervous system, the evidence demonstrates that Met signaling contributes to the development of the cerebral cortex and cerebellum, and suggests that decreased pathway activity during critical periods of neurodevelopment pose another risk to the fetus that would be consistent with inhibition of the Met receptor during pregnancy. However, in this study there were no onartuzumab-related effects on the renal or nervous system tissues, or on neurobehavioral development, that was identified in offspring that survived to PND91. Given the low exposures achieved in the offspring, the absence of findings in this study does not preclude the potential developmental risk to the fetus during pregnancy. In humans, development of several organ systems, including the renal, musculoskeletal, and nervous systems, extends into late gestation and the perinatal period when fetal Ig concentrations are expected to be high. Placental transfer is expected to be similar to a bivalent IgG1 antibody since in vitro studies demonstrated that onartuzumab binds to human FcRn with a relative affinity similar to that of a bivalent, glycosylated IgG1 antibody (Merchant et al., 2013). Since direct effects of onartuzumab on maternal HGF/Met signaling at the placenta may occur much earlier in development, placental transfer of onartuzumab to the fetus may not be required to cause developmental and reproductive toxicity, which is consistent with the fact that the fetus to dam onartuzumab serum concentration ratios at the time of cesarean sectioning on GD 147 showed that only approximately 1%–2% of onartuzumab was transferred to the fetuses. This is in contrast to data with other monoclonal antibodies showing that fetal concentrations near the end of gestation are often comparable to, or even exceed, maternal concentrations (Bowman et al., 2013; DeSesso et al., 2012; Moffat et al., 2014; Pentšuk and van der Laan, 2009). In addition, the offspring to dam onartuzumab serum concentration ratios on PND 28/LD 28 were <1% in 1 offspring and were below or close to the limit of quantitation in all other surviving offspring at or after PND14. From the results described above, under the conditions of this study, no general toxicities in dams were observed in the onartuzumab-treated group at the low dose of 75/50 mg/kg (loading/maintenance dose) or the high dose of 100/100 mg/kg. At the same time, increased incidence of abortion and fetal death in the second and third trimesters, fetal growth retardation, shortened gestation length (early delivery), low fetal and neonatal birth weights, offspring deaths during early postnatal period, suppressed body weight of offspring through the postnatal period, and delayed morphological and behavioral development were observed in the onartuzumab-treated groups. Furthermore, histopathology revealed infarcts with hemorrhage in the chorionic plate, chorionic villus and/or decidual plate of several placentae from the onartuzumab treated groups, and a few had and decreased cellularity of the hepatocytes with dilated sinusoids in fetuses and offspring. Accordingly, the above-mentioned findings in fetuses and offspring were considered to be associated with onartuzumab administration during pregnancy, and confirm that onartuzumab administration to pregnant women would pose a clear risk to both the developing placenta and fetus, and the mother’s ability to maintain the pregnancy. FUNDING This study was supported by Genentech, A member of the Roche Group. Rodney Prell, Noel Dybdal, Ihsan Nijem and Wendy Halpern are fulltime employees of Genentech, A member of the Roche Group. REFERENCES Aoki S. , Hata T. , Manabe A. , Miyazaki K. ( 1998 ). Decreased maternal circulating hepatocyte growth factor (HGF) concentrations in pregnancies with small for gestational age infants . Hum. Reprod. 13 , 2950 – 2953 . Google Scholar Crossref Search ADS PubMed Aurioles-Garibay A. , Hernandez-Andrade E. , Romero R. , Qureshi F. , Ahn H. , Jacques S. M. , Garcia M. , Yeo L. , Hassan S. S. ( 2014 ). Prenatal diagnosis of a placental infarction hematoma associated with fetal growth restriction, preeclampsia and fetal death: Clinicopathological correlation . Fetal Diagn. Ther. 36 , 154 – 161 . Google Scholar Crossref Search ADS PubMed Bastepe M. ( 2013 ). Genetics and epigenetics of parathyroid hormone resistance . Endocr. Dev. 24 , 11 – 24 . Google Scholar Crossref Search ADS PubMed Beau-Faller M. , Ruppert A. M. , Voegeli A. C. , Neuville A. , Meyer N. , Guerin E. , Legrain M. , Mennecier B. , Wihlm J. M. , Massard G. , et al. . ( 2008 ). MET gene copy number in non-small cell lung cancer: Molecular analysis in a targeted tyrosine kinase inhibitor naive cohort . J. Thorac. Oncol. 3 , 331 – 339 . Google Scholar Crossref Search ADS PubMed Bendon R. W. ( 2012 ). Nosology: Infarction hematoma, a placental infarction encasing a hematoma . Hum. Pathol. 43 , 761 – 763 . Google Scholar Crossref Search ADS PubMed Birchmeier C. , Birchmeier W. , Gherardi E. , Vande Woude G. F. ( 2003 ). Met, metastasis, motility and more . Nat. Rev. Mol. Cell. Biol. 4 , 915 – 925 . Google Scholar Crossref Search ADS PubMed Bladt F. , Riethmacher D. , Isenmann S. , Aguzzi A. , Birchmeier C. ( 1995 ). Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud . Nature 376 , 768 – 771 . Google Scholar Crossref Search ADS PubMed Bowman C. J. , Breslin W. J. , Connor A. V. , Martin P. L. , Moffat G. J. , Sivaraman L. , Tornesi M. B. , Chivers S. ( 2013 ). Placental transfer of Fc-containing biopharmaceuticals across species, an industry survey analysis . Birth Defects Res. 98 , 459 – 485 . Google Scholar Crossref Search ADS Burdick K. E. , DeRosse P. , Kane J. M. , Lencz T. , Malhotra A. K. ( 2010 ). Association of genetic variation in the MET proto-oncogene with schizophrenia and general cognitive ability . Am. J. Psychiatry 167 , 436 – 443 . Google Scholar Crossref Search ADS PubMed Campbell D. B. , Buie T. M. , Winter H. , Bauman M. , Sutcliffe J. S. , Perrin J. M. , Levitt P. ( 2009 ). Distinct genetic risk based on association of MET in families with co-occurring autism and gastrointestinal conditions . Pediatrics 123 , 1018 – 1024 . Google Scholar Crossref Search ADS PubMed Campbell D. B. , Sutcliffe J. S. , Ebert P. J. , Militerni R. , Bravaccio C. , Trillo S. , Elia M. , Schneider C. , Melmed R. , Sacco R. , et al. . ( 2006 ). A genetic variant that disrupts MET transcription is associated with autism . Proc. Natl. Acad. Sci. U.S.A . 103 , 16834 – 16839 . Google Scholar Crossref Search ADS PubMed Cappuzzo F. , Marchetti A. , Skokan M. , Rossi E. , Gajapathy S. , Felicioni L. , Del Grammastro M. , Sciarrotta M. G. , Buttitta F. , Incarbone M. , et al. . ( 2009 ). Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients . J. Clin. Oncol. 27 , 1667 – 1674 . Google Scholar Crossref Search ADS PubMed Carter P. , Kelley R. F. , Rodrigues M. L. , Snedecor B. , Covarrubias M. , Velligan M. D. , Wong W. L. , Rowland A. M. , Kotts C. E. , Carver M. E. and ( 1992 ). High level Escherichia coli expression and production of a bivalent humanized antibody fragment . Biotechnology (N Y) 10 , 163 – 167 . Google Scholar PubMed Danilkovitch-Miagkova A. , Zbar B. ( 2002 ). Dysregulation of Met receptor tyrosine kinase activity in invasive tumors . J. Clin. Invest. 109 , 863 – 867 . Google Scholar Crossref Search ADS PubMed Derksen P. W. , de Gorter D. J. , Meijer H. P. , Bende R. J. , van Dijk M. , Lokhorst H. M. , Bloem A. C. , Spaargaren M. , Pals S. T. ( 2003 ). The hepatocyte growth factor/Met pathway controls proliferation and apoptosis in multiple myeloma . Leukemia 17 , 764 – 774 . Google Scholar Crossref Search ADS PubMed DeSesso J. M. , Williams A. L. , Ahuja A. , Bowman C. J. , Hurtt M. E. ( 2012 ). The placenta, transfer of immunoglobulins, and safety assessment of biopharmaceuticals in pregnancy . Crit. Rev. Toxicol. 42 , 185 – 210 . Google Scholar Crossref Search ADS PubMed Ebens A. , Brose K. , Leonardo E. D. , Hanson M. G. Jr. , Bladt F. , Birchmeier C. , Barres B. A. , Tessier-Lavigne M. ( 1996 ). Hepatocyte growth factor/scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons . Neuron 17 , 1157 – 1172 . Google Scholar Crossref Search ADS PubMed Grano M. , Galimi F. , Zambonin G. , Colucci S. , Cottone E. , Zallone A. Z. , Comoglio P. M. ( 1996 ). Hepatocyte growth factor is a coupling factor for osteoclasts and osteoblasts in vitro . Proc. Natl. Acad. Sci. U.S.A . 93 , 7644 – 7648 . Google Scholar Crossref Search ADS PubMed Horibe N. , Okamoto T. , Itakura A. , Nakanishi T. , Suzuki T. , Kazeto S. , Tomoda Y. ( 1995 ). Levels of hepatocyte growth factor in maternal serum and amniotic fluid . Am. J. Obstetr. Gynecol . 173 , 937 – 942 . Google Scholar Crossref Search ADS Ichimura E. , Maeshima A. , Nakajima T. , Nakamura T. ( 1996 ). Expression of c-met/HGF receptor in human non-small cell lung carcinomas in vitro and in vivo and its prognostic significance . Jpn. J. Cancer Res. 87 , 1063 – 1069 . Google Scholar Crossref Search ADS PubMed Ieraci A. , Forni P. E. , Ponzetto C. ( 2002 ). Viable hypomorphic signaling mutant of the Met receptor reveals a role for hepatocyte growth factor in postnatal cerebellar development . Proc. Natl. Acad. Sci. U.S.A . 99 , 15200 – 15205 . Google Scholar Crossref Search ADS PubMed Judson M. C. , Eagleson K. L. , Levitt P. ( 2011 ). A new synaptic player leading to autism risk: Met receptor tyrosine kinase . J. Neurodev. Disord. 3 , 282 – 292 . Google Scholar Crossref Search ADS PubMed Kong-Beltran M. , Seshagiri S. , Zha J. , Zhu W. , Bhawe K. , Mendoza N. , Holcomb T. , Pujara K. , Stinson J. , Fu L. , et al. . ( 2006 ). Somatic mutations lead to an oncogenic deletion of met in lung cancer . Cancer Res. 66 , 283 – 289 . Google Scholar Crossref Search ADS PubMed Landi L. , Minuti G. , D'Incecco A. , Salvini J. , Cappuzzo F. ( 2013 ). MET overexpression and gene amplification in NSCLC: A clinical perspective . Lung Cancer (Auckl.) 4 , 15 – 25 . 10.2147/LCTT.S35168. Google Scholar PubMed Lassus P. , Heikkila P. , Andersson L. C. , von Boguslawski K. , Andersson S. ( 2003 ). Lower concentration of pulmonary hepatocyte growth factor is associated with more severe lung disease in preterm infants . J. Pediatr. 143 , 199 – 202 . Google Scholar Crossref Search ADS PubMed Ma P. C. , Tretiakova M. S. , MacKinnon A. C. , Ramnath N. , Johnson C. , Dietrich S. , Seiwert T. , Christensen J. G. , Jagadeeswaran R. , Krausz T. , et al. . ( 2008 ). Expression and mutational analysis of MET in human solid cancers . Genes Chromosomes Cancer 47 , 1025 – 1037 . Google Scholar Crossref Search ADS PubMed Maina F. , Hilton M. C. , Ponzetto C. , Davies A. M. , Klein R. ( 1997 ). Met receptor signaling is required for sensory nerve development and HGF promotes axonal growth and survival of sensory neurons . Genes Dev. 11 , 3341 – 3350 . Google Scholar Crossref Search ADS PubMed Maina F. , Klein R. ( 1999 ). Hepatocyte growth factor, a versatile signal for developing neurons . Nat. Neurosci. 2 , 213 – 217 . Google Scholar Crossref Search ADS PubMed Matsubara D. , Ishikawa S. , Sachiko O. , Aburatani H. , Fukayama M. , Niki T. ( 2010 ). Co-activation of epidermal growth factor receptor and c-MET defines a distinct subset of lung adenocarcinomas . Am. J. Pathol. 177 , 2191 – 2204 . Google Scholar Crossref Search ADS PubMed Merchant M. , Ma X. , Maun H. R. , Zheng Z. , Peng J. , Romero M. , Huang A. , Yang N. Y. , Nishimura M. , Greve J. , et al. . ( 2013 ). Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent . Proc. Natl. Acad. Sci. U.S.A. 110 , E2987 – E2996 . Google Scholar Crossref Search ADS PubMed Moffat G. J. , Retter M. W. , Kwon G. , Loomis M. , Hock M. B. , Hall C. , Bussiere J. , Lewis E. M. , Chellman G. J. ( 2014 ). Placental transfer of a fully human IgG2 monoclonal antibody in the cynomolgus monkey, rat, and rabbit: A comparative assessment from during organogenesis to late gestation . Birth Defects Res. B Dev Reprod. Toxicol. 101 , 178 – 188 . Google Scholar Crossref Search ADS PubMed Ohmichi H. , Koshimizu U. , Matsumoto K. , Nakamura T. ( 1998 ). Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development . Development 125 , 1315 – 1324 . Google Scholar PubMed Olivero M. , Rizzo M. , Madeddu R. , Casadio C. , Pennacchietti S. , Nicotra M. R. , Prat M. , Maggi G. , Arena N. , Natali P. G. , et al. . ( 1996 ). Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas . Br. J. Cancer 74 , 1862 – 1868 . Google Scholar Crossref Search ADS PubMed Onitsuka T. , Uramoto H. , Ono K. , Takenoyama M. , Hanagiri T. , Oyama T. , Izumi H. , Kohno K. , Yasumoto K. ( 2010 ). Comprehensive molecular analyses of lung adenocarcinoma with regard to the epidermal growth factor receptor, K-ras, MET, and hepatocyte growth factor status . J. Thorac. Oncol. 5 , 591 – 596 . Google Scholar Crossref Search ADS PubMed Onozato R. , Kosaka T. , Kuwano H. , Sekido Y. , Yatabe Y. , Mitsudomi T. ( 2009 ). Activation of MET by gene amplification or by splice mutations deleting the juxtamembrane domain in primary resected lung cancers . J. Thorac. Oncol. 4 , 5 – 11 . Google Scholar Crossref Search ADS PubMed Panos R. J. , Patel R. , Bak P. M. ( 1996 ). Intratracheal administration of hepatocyte growth factor/scatter factor stimulates rat alveolar type II cell proliferation in vivo . Am. J. Respir. Cell Mol. Biology 15 , 574 – 581 . Google Scholar Crossref Search ADS Parks W. T. ( 2015 ). Placental hypoxia: The lesions of maternal malperfusion . Semin. Perinatol. 39 , 9 – 19 . Google Scholar Crossref Search ADS PubMed Pentšuk N. , van der Laan J. W. ( 2009 ). An interspecies comparison of placental antibody transfer: New insights into developmental toxicity testing of monoclonal antibodies . Birth Defects Res. B Dev. Reprod. Toxicol. 86 , 328 – 344 . Google Scholar Crossref Search ADS PubMed Pohl M. , Stuart R. O. , Sakurai H. , Nigam S. K. ( 2000 ). Branching morphogenesis during kidney development . Annu. Rev. Physiol. 62 , 595 – 620 . Google Scholar Crossref Search ADS PubMed Powell E. M. , Mars W. M. , Levitt P. ( 2001 ). Hepatocyte growth factor/scatter factor is a motogen for interneurons migrating from the ventral to dorsal telencephalon . Neuron 30 , 79 – 89 . Google Scholar Crossref Search ADS PubMed Powell E. M. , Muhlfriedel S. , Bolz J. , Levitt P. ( 2003 ). Differential regulation of thalamic and cortical axonal growth by hepatocyte growth factor/scatter factor . Dev. Neurosci. 25 , 197 – 206 . 72268. Google Scholar Crossref Search ADS PubMed Sadiq A. A. , Salgia R. ( 2013 ). MET as a possible target for non-small-cell lung cancer . J. Clin. Oncol. 31 , 1089 – 1096 . Google Scholar Crossref Search ADS PubMed Santos O. F. , Barros E. J. , Yang X. M. , Matsumoto K. , Nakamura T. , Park M. , Nigam S. K. ( 1994 ). Involvement of hepatocyte growth factor in kidney development . Dev. Biol. 163 , 525 – 529 . Google Scholar Crossref Search ADS PubMed Schmidt C. , Bladt F. , Goedecke S. , Brinkmann V. , Zschiesche W. , Sharpe M. , Gherardi E. , Birchmeler C. ( 1995 ). Scatter factor/hepatocyte growth factor is essential for liver development . Nature 373 , 699 – 702 . Google Scholar Crossref Search ADS PubMed Siegfried J. M. , Weissfeld L. A. , Singh-Kaw P. , Weyant R. J. , Testa J. R. , Landreneau R. J. ( 1997 ). Association of immunoreactive hepatocyte growth factor with poor survival in resectable non-small cell lung cancer . Cancer Res . 57 , 433 – 439 . Google Scholar PubMed Sonnenberg E. , Meyer D. , Weidner K. M. , Birchmeier C. ( 1993 ). Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development . J. Cell Biol. 123 , 223 – 235 . Google Scholar Crossref Search ADS PubMed Sousa I. , Clark T. G. , Toma C. , Kobayashi K. , Choma M. , Holt R. , Sykes N. H. , Lamb J. A. , Bailey A. J. , Battaglia A. , International Molecular Genetic Study of Autism, C ., et al. . ( 2009 ). MET and autism susceptibility: Family and case-control studies . Eur. J. Hum. Genet . 17 , 749 – 758 ., Google Scholar Crossref Search ADS PubMed Spigel D. R. , Edelman M. J. , Mok T. , O'Byrne K. , Paz-Ares L. , Yu W. , Rittweger K. , Thurm H. , MetLung Phase I. I. I. S. G. ( 2012 ). Treatment rationale study design for the metlung trial: A randomized, double-blind phase III study of onartuzumab (MetMAb) in combination with erlotinib versus erlotinib alone in patients who have received standard chemotherapy for stage IIIB or IV met-positive non-small-cell lung cancer . Clin. Lung Cancer 13 , 500 – 504 . Google Scholar Crossref Search ADS PubMed Trusolino L. , Bertotti A. , Comoglio P. M. ( 2010 ). MET signalling: Principles and functions in development, organ regeneration and cancer . Nat. Rev. Mol. Cell Biol. 11 , 834 – 848 . Google Scholar Crossref Search ADS PubMed Uehara Y. , Minowa O. , Mori C. , Shiota K. , Kuno J. , Noda T. , Kitamura N. ( 1995 ). Placental defect and embryonic lethality in mice lacking hepatocyte growth factor/scatter factor . Nature 373 , 702 – 705 . 10.1038/373702a0. Google Scholar Crossref Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

Toxicological SciencesOxford University Press

Published: Sep 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off