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Review
. 2020 Oct 15;12(10):658.
doi: 10.3390/toxins12100658.

Immunotoxin Screening System: A Rapid and Direct Approach to Obtain Functional Antibodies with Internalization Capacities

Shusei Hamamichi  1 , Takeshi Fukuhara  2   3   4 , Nobutaka Hattori  2   3   4
Affiliations
Review

Immunotoxin Screening System: A Rapid and Direct Approach to Obtain Functional Antibodies with Internalization Capacities

Shusei Hamamichi et al. Toxins (Basel). .

Abstract

Toxins, while harmful and potentially lethal, have been engineered to develop potent therapeutics including cytotoxins and immunotoxins (ITs), which are modalities with highly selective targeting capabilities. Currently, three cytotoxins and IT are FDA-approved for treatment of multiple forms of hematological cancer, and additional ITs are tested in the clinical trials or at the preclinical level. For next generation of ITs, as well as antibody-mediated drug delivery systems, specific targeting by monoclonal antibodies is critical to enhance efficacies and reduce side effects, and this methodological field remains open to discover potent therapeutic monoclonal antibodies. Here, we describe our application of engineered toxin termed a cell-based IT screening system. This unique screening strategy offers the following advantages: (1) identification of monoclonal antibodies that recognize cell-surface molecules, (2) selection of the antibodies that are internalized into the cells, (3) selection of the antibodies that induce cytotoxicity since they are linked with toxins, and (4) determination of state-specific activities of the antibodies by differential screening under multiple experimental conditions. Since the functional monoclonal antibodies with internalization capacities have been identified successfully, we have pursued their subsequent modifications beyond antibody drug conjugates, resulting in development of immunoliposomes. Collectively, this screening system by using engineered toxin is a versatile platform, which enables straight-forward and rapid selection for discovery of novel functional antibodies.

Keywords: DT3C; antibody drug conjugate; diphtheria toxin; drug delivery; immunoliposome; immunotoxin; monoclonal antibody.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mechanism of antibody: DT3C IT. (a) Formation of DT3C-mediated IT. DT3C consists of catalytic (Cat), translocation (T), and Fc-binding (3C) domains. The Fc-binding domain of DT3C specifically binds to an antibody. (b) Mechanism of IT-induced cytotoxicity. IT initially binds to an antigen expressed on the cell surface, and internalized into the cell where translocated terminus of DT3C is cleaved by the cellular furin protease, and catalytic domain of DT3C is released into the cytoplasm. Consequently, the catalytic domain ADP-ribosylates EF-2 and inhibits the protein translation machinery. (c) Cell-based IT screening system. Inside a well of the cell culture plate, antibodies secreted into the supernatant of the hybridoma library are pretreated with DT3C to form ITs. Subsequently, target cells are transferred into the well, and incubated. If the IT is bound to the target cells and internalized, then this leads to inhibition of protein translation machinery and ultimately cytotoxicity.
Figure 2
Figure 2
Cytotoxic activity of 6E1:DT3C IT. (a) Schematic diagram of IT assay. Purified 6E1 or control mouse IgG (mIgG) antibodies were pre-incubated with DT3C at 37 °C for 30 min to form ITs. After the IT formation, the indicated cells were seeded with various concentrations of ITs (n = 3 per treatment) and incubated at 37 °C for 3 days. Cell viability was measured by using WST-1 reagent. (b) Relative cell viability of A172, SH-SY5Y, and H4 cell lines after 6E1:DT3C treatment. The 6E1:DT3C IT induced cytotoxicity in all three cells lines tested whereby the logEC50s (ng/mL) were 4.59 (A172), 2.27 (SH-SY5Y), and 6.87 (H4). Assuming that 2 DT3Cs (75 kDa each) bind to one antibody, antibody:DT3C at 2000 ng/mL corresponds to 13 nM. Representative results of triplicate independent experiments. Data represent AVG ± SD.
Figure 3
Figure 3
Cellular uptake of 6E1-conjugated liposome. (a) Schematic diagram of cellular uptake procedure. Liposomes were initially generated through dissolution of lipids in ethanol, injection of the ethanol solution into aqueous buffer, and extrusion through polycarbonate membranes. Following the extrusion, the liposomes were conjugated with 6E1 or control mouse IgG (mIgG) antibodies, and fluorescently labeled with DiOC18(3). A172 cells and ILPs were simultaneously transferred to a 96-well plate. The cells were treated with the indicated phospholipid (PL) concentrations of ILPs (n = 3 per treatment), and incubated at 37 °C for 3 days. Phase contrast and fluorescent images were acquired by using IncuCyte ZOOM (Essen BioScience, Inc.; Ann Arbor, MI, USA). Green fluorescent areas were determined as percentage of green object confluency (GOC). (b) Enhanced uptake of 6E1-conjugated liposome. At 100 μM, 6E1-conjugated liposome was significantly taken up by the cells, and mIgG-conjugated liposome also demonstrated gradual increase of cellular uptake. At 30 μM, 6E1-conjugated liposome demonstrated recurrent increased cellular uptake while the control remained relatively low. At 10 μM, cellular uptake of both ILPs remained constantly similar. Representative results of triplicate independent experiments. Data represent AVG ± SD. (c) Ratio of GOC between 6E1-conjugated and control mIgG-conjugated liposomes. If there was no difference in cellular uptake between these ILPs, then the ratio would remain at 1 as indicated by dotted lines. The most enhanced difference was observed at 30 μM liposomal concentration wherein 6E1-conjugated liposome exhibited 5.01-fold (60 hrs) increase of the cellular uptake when compared to the control. Representative results of triplicate independent experiments.
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