There are no adequate or well-controlled studies in pregnant women, and animal reproduction studies are not always predictive of human response. Therefore, Herceptin should be used during pregnancy only if the potential benefit to the mother outweighs the potential risk to the fetus. In the post-marketing setting, oligohydramnios (decreased amniotic fluid) has been reported in women who received Herceptin during pregnancy, either in combination with chemotherapy or as a single agent. Given the limited number of reported cases, the high background rate of occurrence of oligohydramnios, the lack of clear temporal relationships between drug use and clinical findings, and the lack of supportive findings in animal studies, an association between Herceptin and oligohydramnios has not been established. Herceptin appears to be relatively nonimmunogenic. Only 1 of 903 patients evaluated developed neutralizing antibodies to Herceptin. The development of anti-Herceptin antibodies in this patient was not associated with clinical signs or symptoms. CONFIDENTIAL Page 9 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 1.5 CLINCAL PHARMACOKINETICS OF TRASTUZUMAB A Phase I single dose study (H0407g) of intravenous trastuzumab infusions ranging from 10-500 mg resulted in dose-dependent pharmacokinetics (PK) with serum clearance of trastuzumab decreasing with an increasing dose at doses <250 mg. PK modeling of trastuzumab concentration-time data from 7 patients that were administered doses of 250 mg and 500 mg had in a mean half-life of 5.8 days (range 1-32 days). Additionally, PK modeling showed that weekly trastuzumab doses ≥250 mg resulted in serum trough levels of >20 µg/mL that was above the minimum effective concentration observed in preclinical xenograft studies in tumor-bearing mice. The Phase I data supported the weekly dosing schedule that was implemented in all subsequent Phase II and Phase III clinical trials. A weight-based dose schedule was adopted after two Phase II trials (H0551g and H0552g) suggested that inter-subject variability in trastuzumab PK was related to body weight. These findings resulted in a trastuzumab dose schedule of a 4 mg/kg loading dose followed by a weekly 2 mg/kg maintenance dose utilized in the two pivotal Phase III trials (H0648g and H0649g) that were the basis of the BLA filing and subsequent FDA approval of trastuzumab for HER2+ metastatic breast cancer. The trastuzumab PK data from studies H0407g (Phase I), H0551g (Phase II), and H0649 (pivotal) have been subsequently reanalyzed by a population PK approach using nonlinear mixed effect modeling (NONMEM) {ref: Lu et al.}. A linear two-compartment model best described the concentration-time data, and accounted for the accumulation of trastuzumab serum concentrations seen in the Phase II and Phase III clinical studies. A covariate analysis was conducted using the subjects from these single agent studies to evaluate the effect of pathophysiologic covariates (e.g. age, weight, shed antigen) on the PK parameter estimates. The covariates, that significantly influenced clearance, were the level of shed antigen and the number of metastatic sites. Volume of distribution was significantly influenced by weight and shed antigen level. Additionally, data from the Phase III study, H0648g, were added to assess the influence of concomitant chemotherapy on trastuzumab PK. Importantly, chemotherapy (AC or paclitaxel) did not significantly alter trastuzumab PK. The estimated half-life of trastuzumab based on the final model was 28.5 days. Analysis of data obtained from two Phase II studies which utilized a loading dose of 8 mg/kg trastuzumab followed by a 6 mg/kg maintenance dose administered every 3 weeks (q3 week) as a single-agent (Baselga et al. 1998), and in combination with paclitaxel (175 mg/m2) (Leyland-Jones et al. 2000), confirmed that a two-compartment model best describes the PK of trastuzumab. Model-independent analysis of the of the data obtained in these studies gives comparable PK parameter estimates to those obtained by the population PK model, thus confirming the validity of the population PK model. In addition, the population PK model adequately predicted trastuzumab serum concentrations obtained independently in these studies. After two treatment cycles, trastuzumab exposure were similar to those measured in the once weekly dosing regimen used in the pivotal trials. Trough levels were in excess of the targeted serum concentrations established from preclinical xenograft models, and as expected, peak levels were greater than those observed upon weekly administration. The apparent half-life of trastuzumab in these CONFIDENTIAL Page 10 of 102 Protocol: HERCEPTIN® + E75/GM-CSF Version 3.1 ML25749 05 March 2017 studies was determined to be approximately 21 days, and the PK was supportive of a q3 week dosing schedule. The efficacy and safety results from these Phase II studies with q3 week dosing do not appear to be different from those with weekly dose-schedules (Slamon et al.