The measurements of tumor size and weight change in mice were recorded twice a week. optimal efficacies. Along with the ADCs, two other forms of immunoconjugates (scFv-PE38KDEL and IgG1-AL1-PE38KDEL) were used to test the antibodies for delivering cytotoxic payloads to xenograft tumor models and to cultured cells experiments with the three forms of immunoconjugates revealed minimal off-target toxicities of the selected antibodies from the synthetic antibody libraries; the off-target toxicities of the control antibodies could have resulted from the antibodies propensity to target the liver in the animal models. Our ADC discovery platform and the knowledge gained from our tests on xenograft models with the three forms of immunoconjugates could be useful to anyone developing optimal ADC cancer therapeutics. Keywords: antibody-drug conjugate, immunotoxin, high through antibody screening, human gastric cancer, synthetic antibody library Introduction HER2-ECD (human epidermal growth factor receptor 2 C extracellular domain) is a prominent therapeutic target validated for treating HER2-positive breast and gastric cancer. More than 600 clinical studies currently registered in ClinicalTrials.gov are related to HER2-positive cancers, and about a quarter of these studies are involved with antibody-based therapeutics/diagnostics targeting HER2-ECD. In addition to breast cancer and gastric cancer, HER2 over-expression has been found in other diverse cancers,1,2 suggesting that a substantial number of cancer patients could potentially benefit from antibody therapeutics targeting HER2-ECD. Two HER2-ECD-targeting monoclonal antibodies are already approved for the APG-115 treatment of cancers in humans: 1) trastuzumab for HER2-positive breast, gastric and gastroesophageal APG-115 junction cancer; and 2) pertuzumab for HER2-positive breast cancer in metastatic or neoadjuvant setting.1,3 In addition, trastuzumab emtansine, an antibody-drug conjugate (ADC) composed of trastuzumab conjugated through a non-reducible Zfp622 linker to the tubulin inhibitor mertansine, is approved for treating HER2-positive metastatic breast cancer and has shown overall survival benefit and favorable safety profile over long follow-up durations.4 However, a recent report on the international randomized GATZBY Phase 2/3 trial indicates that trastuzumab emtansine is not superior to taxane in patients with previously treated HER2-positive advanced gastric cancer.5 While HER2-ECD remains a validated target in several ongoing and planned clinical gastric cancer studies,5,6 APG-115 HER2-specific therapeutic options for treating advanced gastric cancer are still limited. The aim of our study was to test ADCs designed to treat human HER2-positive gastric cancer. The treatment efficacies and off-target toxicities due to the antibodies as targeting modules were measured with the N87 model system, which has been a standard system as a HER2 over-expressing human gastric cancer cell line, and APG-115 xenograft model.2 Three HER2-ECD-positive single-chain variable fragment (scFv) candidates (GH2-20, GH2-61 and GH2-75) from the synthetic antibody libraries7,8 were selected through the high throughput antibody-based immunoconjugate discovery platform.9 Those with the highest potencies as targeting modules toward N87 cells were reformatted into human IgG1 antibodies and tested together with two positive control humanized IgG1 antibodies (trastuzumab and H32, which are humanized murine anti-HER2-ECD antibodies9) and one isotype negative control IgG antibody (nc). All these IgG1 antibodies were constructed with the same human IgG1 framework. The efficacies of these IgG1 antibodies as the targeting modules for delivering cytotoxic payloads to N87 xenograft tumor models and to cultured N87 cells were scrutinized with three forms of immunoconjugates in APG-115 this work: 1) ADC in the form of IgG1-vc-MMAE (monomethyl auristatin E linked to the IgG1 via valine-citrulline dipeptide cathepsin-cleavable linker);10 2) immunotoxin of a single polypeptide chain in the form of scFv-PE38KDEL fusion protein, where PE38KDEL is a truncated form of exotoxin (PE) A subunit toxin,11,12 as pioneered by Pastan and colleagues;13 and 3) immunotoxin in the form of IgG1-AL1-PE38KDEL, where the IgG1 is non-covalently linked to PE38KDEL through the adaptor-toxin fusion protein AL1-PE38KDEL.9 The AL1 fragment is composed of consecutive Protein A and Protein L separated by a polypeptide linker that enables Protein A and Protein L binding to the framework regions of the IgG1 simultaneously with nano-molar affinity.9 Since the IgG1 candidates were reformatted from the scFvs derived from the synthetic antibody libraries, which were designed using antibody-protein interaction principles from computational and experimental analyses8,14C17 and built on a single human variable domain antibody germline framework (IGKV1-NL1*01/IGHV3-23*04),7C9 the IgG1s bind to Protein A and Protein L through the variable domain framework.