High-content Analysis of Antibody Phage-display Library Selection Outputs Identifies Tumor Selective Macropinocytosis-dependent Rapidly Internalizing Antibodies

Abstract

Many forms of antibody-based targeted therapeutics, including antibody drug conjugates, utilize the internalizing function of the targeting antibody to gain intracellular entry into tumor cells. Ideal antibodies for developing such therapeutics should be capable of both tumor-selective binding and efficient endocytosis. The macropinocytosis pathway is capable of both rapid and bulk endocytosis, and recent studies have demonstrated that it is selectively up-regulated by cancer cells. We hypothesize that receptor-dependent macropinocytosis can be achieved using tumor-targeting antibodies that internalize via the macropinocytosis pathway, improving potency and selectivity of the antibody-based targeted therapeutic. Although phage antibody display libraries have been utilized to find antibodies that bind and internalize to target cells, no methods have been described to screen for antibodies that internalize specifically via macropinocytosis. We hereby describe a novel screening strategy to identify phage antibodies that bind and rapidly enter tumor cells via macropinocytosis. We utilized an automated microscopic imaging-based, High Content Analysis platform to identify novel internalizing phage antibodies that colocalize with macropinocytic markers from antibody libraries that we have generated previously by laser capture microdissection-based selection, which are enriched for internalizing antibodies binding to tumor cells in situ residing in their tissue microenvironment (Ruan, W., Sassoon, A., An, F., Simko, J. P., and Liu, B. (2006) Identification of clinically significant tumor antigens by selecting phage antibody library on tumor cells in situ using laser capture microdissection. Mol. Cell. Proteomics. 5, 2364–2373). Full-length human IgG molecules derived from macropinocytosing phage antibodies retained the ability to internalize via macropinocytosis, validating our screening strategy. The target antigen for a cross-species binding antibody with a highly active macropinocytosis activity was identified as ephrin type-A receptor 2. Antibody-toxin conjugates created using this macropinocytosing IgG were capable of potent and receptor-dependent killing of a panel of EphA2-positive tumor cell lines in vitro. These studies identify novel methods to screen for and validate antibodies capable of receptor-dependent macropinocytosis, allowing further exploration of this highly efficient and tumor-selective internalization pathway for targeted therapy development.

Therapeutic potential of targeting the Eph/ephrin signaling complex

“…as an anti-EphA2/anti-CD3 bispecific construct to kill EphA2-expressing tumor cells in vitro and in mice (Hammond et al, 2007). Another anti-EphA2 scFv Ab was shown to destroy prostate cancer cells, after being converted to a full-length IgG (Ha et al, 2014). Recently, yet another anti-EphA2 scFv fragment was obtained from a large human synthetic Ab library by a selection against the ligand-binding domain of the receptor.…”

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies

“…The resulting yeast antibody display libraries will be subjected to FACS-based selection against fluorescence-labeled antigens or ligands to identify high affinity binders. The figure is adapted from our original drawings in [13] and [16]…”

“…Phage antibody display libraries are widely used to generate monoclonal antibodies with affinity for target antigens, and purified recombinant protein antigens and living cells are frequently used as the selection targets for phage antibody display libraries [1–13]. Although very large libraries can be generated and screened in phage antibody display-based selections, it is difficult to fine-tune the selections to select for specific binding properties such as affinity.…”

“…Additionally, while selection of phage antibody display libraries on living cells yields antibodies that bind to native epitopes on the cell surface, it is difficult to direct the selections towards a specific target. One method of addressing these limitations is to generate yeast antibody display libraries from pre-enriched phage antibody display outputs selected on recombinant antigens, live cells, or tissues [7, 10–13]. Yeast surface display [14] has been widely utilized to screen large libraries for proteins or protein fragments with specific binding properties [15–21], and protocols for the construction and application of yeast surface display libraries have been previously described [21–23].…”

ALPPL2 is a highly specific and targetable tumor cell surface antigen

“…The beads with antigens captured were loaded onto a SDS-PAGE gel (4-20% gradient polyacrylamide) (Thermo Fisher Scientific) in duplicates, one for Gelcode staining (Thermo Fisher Scientific), and the other Western blot analysis to locate the membrane protein band by streptavidin-conjugated with horseradish peroxidase (HRP) (Jackson ImmunoResearch Laboratories) and ECL substrate (Thermo Fisher Scientific).Bands on the Gelcode-stained gel corresponding to positions on the Western blot were excised, in-gel digested with trypsin and analyzed by tandem mass spectrometry (MS/MS, Mass Spectrometry Facility, University of Minnesota). Spectra were searched using Protein Prospector against the SwissProt database(Ha et al, 2014).…”