Mapping Tumor Epitope Space by Direct Selection of Single-Chain Fv Antibody Libraries on Prostate Cancer Cells

Abstract

The identification of tumor-specific cell surface antigens is a critical step toward the development of targeted therapeutics for cancer. The epitope space at the tumor cell surface is highly complex, composed of proteins, carbohydrates, and other membrane-associated determinants including post-translational modification products, which are difficult to probe by approaches based on gene expression. This epitope space can be efficiently mapped by complementary monoclonal antibodies. By selecting human antibody gene diversity libraries directly on the surface of prostate cancer cells, we have taken a functional approach to identifying fully human, tumor-specific monoclonal antibodies without prior knowledge of their target antigens. Selection conditions have been optimized to favor tumor-specific antibody binding and internalization. To date, we have discovered >90 monoclonal antibodies that specifically bind and enter prostate cancer cells, with little or no binding to control cells. These antibodies are able to efficiently deliver intracellular payloads when attached to nanoparticles such as liposomes. In addition, a subset of the antibodies displayed intrinsic antiproliferative activity. These tumorspecific internalizing antibodies are likely to be useful for targeted therapeutics either alone or in combination with effector molecules. The antigens they bind constitute a tumor-specific internalizing epitope space that is likely to play a significant role in cancer cell homeostasis. Targeting components of this epitope space may facilitate development of immunotherapeutic and small molecule-based strategies as well as the use of other therapeutic agents that rely upon delivery to the interior of the tumor cell.

Targeted drug delivery to mesothelioma cells using functionally selected internalizing human single-chain antibodies

“…The library was created by subcloning human scFv gene repertoires from a naive phagemid (26) into a phage vector for multivalent display (25,27). The library was preincubated with control cells (BPH-1 and LP9/hTERT) at 4jC for 4 h to reduce binders to common cell surface antigens as described (17). The depleted library was further incubated with 10 6 M28 cells at 37jC for 1 h in medium containing 10% FCS, washed thrice with PBS, once with 100 mmol/L glycine/ 150 mmol/L NaCl (pH 2.8), lysed with 100 mmol/L triethylamine, neutralized with 1 mol/L Tris-HCl (pH 7.0), and used to infect log-phase TG1 and to produce polyclonal phage antibodies (17).…”

“…The library was preincubated with control cells (BPH-1 and LP9/hTERT) at 4jC for 4 h to reduce binders to common cell surface antigens as described (17). The depleted library was further incubated with 10 6 M28 cells at 37jC for 1 h in medium containing 10% FCS, washed thrice with PBS, once with 100 mmol/L glycine/ 150 mmol/L NaCl (pH 2.8), lysed with 100 mmol/L triethylamine, neutralized with 1 mol/L Tris-HCl (pH 7.0), and used to infect log-phase TG1 and to produce polyclonal phage antibodies (17). Polyclonal phage antibodies from the first round of selection were further selected on VAMT-1 cells (round 2) using procedures described above and used to produce polyclonal phage antibodies that were selected again on live M28 cells (round 3).…”

“…Polyclonal phage antibodies from the first round of selection were further selected on VAMT-1 cells (round 2) using procedures described above and used to produce polyclonal phage antibodies that were selected again on live M28 cells (round 3). Output of this round 3 selection was screened by FACS on M28 and VAMT-1 cells, respectively, to identify binders to both cell lines (17). ScFvs were sequenced to determine the number of unique clones as described (17).…”

“…Output of this round 3 selection was screened by FACS on M28 and VAMT-1 cells, respectively, to identify binders to both cell lines (17). ScFvs were sequenced to determine the number of unique clones as described (17).…”

“…Monoclonal antibodies are able to discern subtle differences in antigen structure and conformation and recognize antigenic determinants of diverse chemical composition with high affinity and specificity (15,16). As such, the tumor cell surface epitope space can be efficiently mapped by high-affinity interactions with monoclonal antibodies (17). Isolating these epitopes enables the antibodies to achieve specific binding to neoplastic cells, an ability that could be used in applications such as induction of antibodydependent cell cytotoxicity or inhibition of signaling pathways involved in tumor cell migration, growth, and survival.…”