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Figure S1 Sequence alignments of human and mouse TENB2. Human (NM_016192.2) and mouse (NM_019790.4) TENB2 (TmeFF2) protein sequences from the NCBI database were aligned using GSeqweb (Genentech, Inc.) for human and mouse TENB2 similarity analysis.

Figure S2 Flow cytometric analysis of anti-TENB2 binding to HEK cells (HEK293, obtained from ATCC) that were stably transfected with human or mouse TENB2. Cross-reactivity of anti-TENB2 to human and mouse TENB2 was evaluated in HEK cells (HEK293, obtained from ATCC) that were stably transfected with human or mouse TENB2. Cells were selected and maintained using G418 antibiotic solution (Invitrogen) at 400 μg·mL−1. Cells were grown to 90% confluence and removed from plates using Cell Dissociation Buffer (Invitrogen). Cells were washed and resuspended in fluorescence-activated cell-sorting (FACS) buffer (PBS with 1% BSA) and incubated for 45 min with humanized anti-TENB2 (ranging from 0.008 to 18 μg·mL−1) followed by a 30 min incubation with anti-human secondary antibody conjugated to phycoerythrin (anti-PE). Analysis was performed with a FACS Caliber flow cytometer (BD Biosciences).

Figure S3 Mass spectrometry data for anti-TENB2-MMAE (ADC) before (top) and after (bottom) DOTA conjugation. An aliquot of each protein sample was injected on a PLRP-S column [1000 Å, 50 × 2.1 mm, Varian (formerly Polymer Laboratories), Amherst, MA] maintained at 75°C with a linear gradient from 34–45% acetonitrile in 0.01% formic acid and water. The column eluent was directed by electrospray ionization into an Agilent 6220 time-of-flight mass spectrometer (Agilent Technologies, Santa Clara, CA) with fragmentor, capillary, skimmer and octupole RF voltages, respectively, at 415, 5500, 120 and 300 V. Data analysis was performed using the maximum entropy deconvolution algorithm within the Agilent Mass Hunter Qualitative Analysis software.

Figure S4 Size-exclusion HPLC radiochromatogram of an aliquot of the radioimmunoconjugate, 111In-anti-TENB2-MMAE (ADC), used for a tracer in tissue distribution and imaging studies. Size-exclusion high performance liquid chromatography (HPLC) characterization of all radioimmunoconjugates was carried out on a Phenomenex™ BioSep-SEC-S 3000, 300 × 7.8 mm, 5 μm column.

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