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As mesothelin is overexpressed in various types of cancer, it is an attractive target for therapeutic antibodies. T-cell bispecific antibodies bind to target cells and engage T cells via binding to CD3, resulting in target cell killing by T-cell activation. However, the affinity of the CD3-binding arm may influence CD3-mediated plasma clearance or antibody trapping in T-cell-containing tissues. This may then affect the biodistribution of bispecific antibodies. In this study, we used scFab and knob-into-hole technologies to construct novel IgG-based 1 + 1 MG1122-A and 2 + 1 MG1122-B bispecific antibodies against mesothelin and CD3&epsilon, MG1122-B was designed to be bivalent to mesothelin and monovalent to CD3&epsilon, using a 2 + 1 head-to-tail format. Activities of the two antibodies were evaluated in mesothelin-positive tumor cells in vitro and xenograft models in vivo. Although both antibodies exhibited target cell killing efficacy and produced regression of xenograft tumors with CD8+ T-cell infiltration, the antitumor efficacy of MG1122-B was significantly higher. MG1122-B may improve tumor targeting because of its bivalency for tumor antigen. It may also reduce systemic toxicity by limiting the activation of circulating T cells. Thus, MG1122-B may be useful for treating mesothelin-positive solid tumors.

The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor and plays an important role in carcinogenesis. In this study, the epidermal growth factor receptor binding peptide (EGBP) was identified using a phage display method and evaluated in U87MG cells in order to investigate the possibility to target the EGFR using an optical imaging system. Cyanine dye 5.5 (Cy5.5) was conjugated with EGBP-GGG-SC, EGBP-AOC-SC, and EGBP-AM2BA-SC. Cellular binding study of EGBP-Linker-Cy5.5 conjugates or (125)I-EGBP-Linker compounds was performed in U87MG cells. Optical imaging studies were performed in U87MG bearing mice. Three of seven clones from the 12-mer peptide library showed a specific binding affinity to rhEGFR, and they encoded the same 12 amino acid peptide sequence, FPMFNHWEQWPP. Confocal images show that the fluorescent signal of EGBP-Linker-Cy5.5 conjugates was decreased in the order: EGBP-AOC-Cy5.5≫EGBP-AM2BA-Cy5.5>EGBP-GGG-Cy5.5. EGBP-AOC-Cy5.5 appeared in cell cytoplasm and surface, and it was inhibited by free EGBP apparently. The cellular binding of EGBP-AOC-Cy5.5 exhibited a higher average radiance value than EGBP-GGG-Cy5.5 and EGBP-AM2BA-Cy5.5. Among various (125)I-EGBP-Linker compounds, EGBP-GGG showed a higher binding than other compounds. However, uptake of (125)I-EGBP-AOC was clearly inhibited by free EGBP in inhibition study. In an in vivo study, the fluorescent signal of EGBP-AOC-Cy5.5 treated mouse was mainly detected in the tumor and kidney. Among the three derivatives, EGBP-AOC-Cy5.5 was the optimized optical imaging agent for U87MG EGFR positive tumors in the animal model. This study demonstrated the EGBP-Linker-Cy5.5 conjugates may be useful as a potential EGFR target optical probe.

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.

Mesothelin (MSLN) is an antigen overexpressed in several malignancies, including mesothelioma and ovarian and pancreatic adenocarcinoma. It has been studied as a marker for diagnosis and a target for immunotherapy. Here, we adopted a bispecific antibody format for recruiting cytotoxic T cells to kill tumor cells. MG1122 is a novel, whole IgG-like bispecific antibody which recognizes CD3 on T cells and MSLN on tumor cells. MG1122 induced effective killing of MSLN-expressing tumor cells. This response was associated with robust activation of T cells as shown by nuclear factor of activated T cells (NFAT) activation, CD25 and CD69 upregulation and increased cytokine release. In addition, using a live cell analysis system, we found that levels of activated caspase 3/7 were amplified in the tumor cells by MG1122 treatment. Although MSLN is a surface antigen, it also exists as a secreted isoform, which can lead to tumor evasion against MSLN targeting antibodies. However, despite the high concentration of soluble MSLN, MG1122 still effectively bound and showed strong killing effects against MSLN-expressing tumor cells. Taken together, our MSLN/CD3 bispecific antibody, MG1122 offers a promising opportunity to redirect T cells to kill MSLN-expressing cancer cells. Citation Format: Yun-Jung Lee, Okjae Lim, Munkyung Kim, Jeewon Lee, Kisu Kim, Junhong Jung, SuA Lee, Sung Keun Kim, Haenaem Kwon, Yangmi Lim, Yong Yea Park, Jonghwa Won. MG1122, a whole IgG-like bispecific antibody targeting mesothelin and CD3, induces T cell-mediated killing of MSLN-expressing tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5768.

e14500Background: Engaging T cells to attack tumors is a new class of immunotherapy. Tumor-specific T cell-engaging antibodies have an ability to redirect and activate T cells to target tumor cells...

c-Met is a receptor tyrosine kinase involved in tumor cell growth, invasion, metastases and angiogenesis. Overexpression of c-Met is frequently observed in several tumor types. Here, we report the in vitro cell-binding properties and biodistribution and SPECT/CT imaging in glioma (U87MG) xenograft-bearing mice of (125)I-labeled c-Met-binding peptides (cMBPs) including analogs conjugated to amino acid and aliphatic carbon linkers. In vitro assays showed that the peptide without any linker and those with GGG and 8-aminooctanoic acid linkers had low cellular internalization and that IC(50) values of peptides were 1.5 microM, 65 nM and 85.3 nM, respectively. Biodistribution studies showed the GGG-containing peptide had higher tumor uptake and a higher tumor-to-blood activity concentration ratio than other receptor-binding ligands. SPECT/CT studies with a dedicated small-animal imaging system were performed in U87MG-bearing athymic mice. Although U87MG tumor xenografts could be visualized by SPECT/micro-CT using the various (125)I labeled cMBPs, image contrast and overall quality were unremarkable.