Purpose In a recent phase II study of onartuzumab (MetMAb), patients whose non–small cell lung cancer (NSCLC) tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit with onartuzumab plus erlotinib (O+E) versus erlotinib. We describe development and validation of a standardized MET IHC assay and, retrospectively, evaluate multiple biomarkers as predictors of patient benefit. Experimental Design Biomarkers related to MET and/or EGF receptor (EGFR) signaling were measured by IHC, FISH, quantitative reverse transcription PCR, mutation detection techniques, and ELISA. Results A positive correlation between IHC, Western blotting, and MET mRNA expression was observed in NSCLC cell lines/tissues. An IHC scoring system of MET expression taking proportional and intensity-based thresholds into consideration was applied in an analysis of the phase II study and resulted in the best differentiation of outcomes. Further analyses revealed a nonsignificant overall survival (OS) improvement with O+E in patients with high MET copy number (mean ≥5 copies/cell by FISH); however, benefit was maintained in “MET IHC-positive”/MET FISH-negative patients (HR, 0.37; P = 0.01). MET, EGFR, amphiregulin, epiregulin, or HGF mRNA expression did not predict a significant benefit with onartuzumab; a nonsignificant OS improvement was observed in patients with high tumor MET mRNA levels (HR, 0.59; P = 0.23). Patients with low baseline plasma hepatocyte growth factor (HGF) exhibited an HR for OS of 0.519 (P = 0.09) in favor of onartuzumab treatment. Conclusions MET IHC remains the most robust predictor of OS and progression-free survival benefit from O+E relative to all examined exploratory markers.

The objective of this study was to evaluate circulating hepatocyte growth factor (cHGF) as a pharmacodynamic biomarker of Met inhibition for onartuzumab (MetMAb, OA5D5v2) in a phase I trial in patients with advanced cancers and a phase II trial in non-small cell lung cancer (NSCLC). The phase I study was a dose escalation trial with onartuzumab administered i.v. once every three weeks. The phase II study was a randomized two-arm trial in which onartuzumab or placebo was administered in combination with erlotinib in 137 patients with second and third line (2/3L) NSCLC. cHGF levels were evaluated by ELISA at multiple time points over the treatment period. Onartuzumab administration resulted in an acute and sustained rise in cHGF in both the phase I and phase II studies. Elevation in cHGF was independent of dose or drug exposure and was restricted to onartuzumab treatment. Neither higher baseline nor elevated change in cHGF levels upon treatment could simply be attributed to tumor burden or number of liver metastasis. We have shown that elevated cHGF can consistently and reproducibly be measured as a pharmacodynamic biomarker of onartuzumab activity. The elevation in cHGF is independent of tumor type, dose administered, or dose duration. Although these studies were not powered to directly address the contribution of cHGF as a predictive, ontreatment, circulating biomarker, these data suggest that measurement of cHGF in future expanded studies is warranted.

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These assays highlight strategies and methodologies for a fit-for-purpose approach. Minimum qualification, full qualification and validation were chosen and supported programs at different stages of drug development.

Subcutaneous (SC) administration is a desired route for monoclonal antibodies (mAbs). However, formulating mAbs for small injection volumes at high concentrations with suitable stability and injectability is a significant challenge. Here, this work presents a platform technology that combines the stability of crystalline antibodies with injectability and tunability of soft hydrogel particles. Composite alginate hydrogel particles are generated via a gentle centrifugal encapsulation process which avoids use of chemical reactions or an external organic phase. Crystalline suspension of anti-programmed cell death protein 1 (PD-1) antibody (pembrolizumab) is utilized as a model therapeutic antibody. Crystalline forms of the mAb encapsuled in the hydrogel particles lead to stable, high concentration, and injectable formulations. Formulation concentrations as high as 315 mg mL −1 antibody are achieved with encapsulation efficiencies in the range of 89-97%, with no perceivable increase in the number of antibody aggregates. Bioanalytical studies confirm superior maintained quality of the antibody in comparison with formulation approaches involving organic phases and chemical reactions. This work illustrates tuning the alginate particles' disintegration by using partially oxide alginates. Crystalline mAb-laden particles are evaluated for their biocompatibility using cell-based in vitro assays. Furthermore, the pharmacokinetics (PK) of the subcutaneously delivered human anti-PD-1 mAb in crystalline antibody-laden alginate hydrogel particles in Wistar rats is evaluated.