Internalizing Cancer Antibodies from Phage Libraries Selected on Tumor Cells and Yeast-Displayed Tumor Antigens

Abstract

A number of approaches have been utilized to generate antibodies to cancer cell surface receptors which can be used as potential therapeutics. A number of these therapeutic approaches, including antibody-drug conjugates, immunotoxins, and targeted nucleic acid delivery, require antibodies that not only bind receptor, but that also undergo internalization into the cell upon binding. We previously reported the ability to generate cancer cell binding and internalizing antibodies directly from human phage antibody libraries selected for internalization into cancer cell lines. While a number of useful antibodies have been generated using this approach, limitations include the inability to direct the selections to specific antigens and identifying the antigen bound by the antibodies. Here we show that these limitation can be overcome by using yeast displayed antigens known to be associated with a cell type to select the phage antibody output after several rounds of selection on a mammalian cell line. We used this approach to generate several human phage antibodies to yeast displayed EphA2 and CD44. The antibodies bound both yeast displayed and mammalian cell surface antigen and were endocytosed upon binding to mammalian cells. This approach is generalizable to many mammalian cell surface proteins, results in the generation of functional internalizing antibodies, and does not require antigen expression and purification for antibody generation.

Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting

“…18 When profiled as a potential module for targeting nanoparticles, however, the scFv was unstable during the conjugation process, and, as a scFv, it bound poorly to protein A, with less than 20% being retained on the column. The scFv thus had good bioactivity, but was not viable as a therapeutic lead due to poor biophysical and manufacturing properties.…”

“…17 The originally identified antibody had strong internalization properties and biological activity in cell lines overexpressing EphA2. 18 Further characterization of the scFv revealed, however, that it had a low unfolding temperature and did not bind well to affinity resins, and thus could not be advanced as a therapeutic targeting moiety. Given that suitable biophysical and manufacturing properties were absolutely required, 2 sequential engineering campaigns were completed to derive a scFv with improved thermal stability and protein A binding.…”

Selecting an Optimal Antibody for Antibody- Drug Conjugate Therapy

“…Overexpression of the candidate gene on a test cell or reduction of the candidate gene expression via small interfering RNA (siRNA) can be used to confirm the identity of the candidate antigen. In one example, selection for phage internalizing in MDA-MB-231 breast cancer cells was followed by selection on yeast cells expressing either of two candidate proteins, leading to the isolation of antibodies recognizing the two candidates (Zhou et al 2010). The authors suggest that this method could be applied on a larger scale to isolate cell-binding antibodies to a large panel of candidate genes.…”

Discovery of Internalizing Antibodies to Tumor Antigens from Phage Libraries

“…In other instances, it has not proven possible to identify the antigen recognized by the internalizing antibody. To overcome this limitation, we recently reported the use of yeast displayed tumor antigens to direct selections to a specific tumor antigen (Figure 3) [37]. In this approach, phage antibodies are first selected for internalization on a tumor cell line known to express the antigen of interest, followed by selection for binding to the yeast displayed antigen.…”

“…In this approach, phage antibodies are first selected for internalization on a tumor cell line known to express the antigen of interest, followed by selection for binding to the yeast displayed antigen. Using this approach, we generated antibodies to EphA2 and CD44 that internalize into breast cancer cell lines [37]. …”