Overview: For novel antibody discovery, we have developed methods that combine phage antibody display with laser capture microdissection to identify internalizing human scFvs that bind to live tumor cells and tumor cells in situ residing in their tissue microenvironment. In parallel, we have been identifying tumor cell surface antigens bound by these novel antibodies by screening yeast surface-displayed human cDNA libraries created in the lab, and by analyzing antibody immunoprecipitation products using mass spectrometry techniques. For therapeutic development, we are utilizing internalizing functions of these novel antibodies to achieve tumor-targeted intracellular delivery and arming the antibodies with effectors (drugs and siRNAs/miRNAs) to achieve targeted tumor killing.
Challenges and Approach
: Our goal is to develop monoclonal antibody therapy for cancer treatment. An antibody with therapeutic potential should either directly inhibit pathways critical for tumor growth/survival, engage the immune surveillance system to attack tumor cells, or serve as a targeting moiety to which effector molecules can be attached so as to create a targeted therapeutic (analogous to a guided missile). For the guided missile approach, the antibody should recognize either a tumor-specific or a tumor-associated cell surface antigen that is overexpressed by tumor cells relative to normal cells, allowing selective killing of tumors without causing significant damage to normal tissues. In addition, many effector molecules need to gain intracellular entry to exert their tumor killing function. Thus, the target antigen or epitope should be internalizing, allowing the targeting antibody to deliver effectors/payloads intracellularly to tumor cells. Finally, it is known that when removed from their original tissue environment, cultured tumor cells variably up- and down-regulate expression of cell surface molecules relative to tumor cells in situ. Thus a true targetable tumor antigen should be expressed by tumor cells residing in their tissue microenvironment as opposed to cultured cell lines. We call antigens with the aforementioned properties components of a “tumor-associated internalizing epitope space”, and hypothesize that these antigens are excellent targets for monoclonal antibody therapy development. Funded by the NIH/NCI, DoD and other agencies, we have taken a completely functional approach to dissect this previously uncharacterized tumor-associated internalizing epitope space, and have identified fully human monoclonal antibodies that target components of this epitope space and can be used to create novel anti-cancer therapeutics.
: We utilize antibody gene diversity libraries as a source of random shape repertoire to functionally probe the tumor specimen-associated internalizing epitope space. We have developed a novel laser capture microdissection (LCM)-based strategy that allows the selection of internalizing phage antibodies targeting tumor cells in situ residing in their natural stromal microenvironment. We use the high content screening (HCS) technique and time lapse confocal imaging to analyze tumor selectivity and internalization kinetics and pathways. We have identified a panel of novel internalizing human monoclonal antibodies that bind to true tumor epitopes expressed by tumor cells in situ, and demonstrated their tumor selective intracellular payload delivery in vitro and in vivo. Using these antibodies as capturing agents, we have identified target antigens by screening a yeast surface human cDNA display library created in the lab, and by affinity purification followed by mass spectrometry analysis. These novel antibody-antigen pairs are candidates for translational development of targeted cancer therapeutics.
: The overall translational strategy is to impart our tumor-targeting antibodies with tumor killing function, and develop accompanying biomarkers based on the antigen identified for patient stratification and treatment outcome monitoring. In other words, we are developing a personalized monoclonal antibody-based anti-cancer therapeutic. We will complete preclinical studies in the lab and collaborate with other parties for further translational development including cGMP, IND filing and clinical trials. Currently, we are pursuing several strategies to arm our internalizing antibodies. (1) We are developing antibody drug conjugates (ADCs) in collaboration with the pharmaceutical industry. (2) We are developing novel antibody-based systemic siRNA delivery vehicles to modulate genes critical for tumor growth and survival. (3) We are decorating the surface of nanoparticles encapsulating drugs or siRNA/miRNA to impart them with tumor targeting and intracellular delivery function in collaboration with laboratories skilled in nanotechnology development.
Current disease focus
: Although our approach is generally applicable and is not limited to particular tumor types, our current focus is on several incurable diseases with no good therapeutic options: prostate cancer with an emphasis on developing novel ADC for treating bone metastases, pancreatic cancer, mesothelioma, ovarian cancer, non small cell lung cancer and glioblastoma multiforme. The novel antibody/antigen pairs identified initially from studying these diseases could be relevant to other tumors as well.