Dr. Ho's laboratory studies cell surface proteins in broad scientific fields of molecular and cellular biology including receptor/ligand interactions, signaling pathways, antibody/protein engineering, structure and computational biology, and functional genomics. We also develop new antibody technologies. Some of our research has direct clinical application.

Developing glypicans as cancer therapeutic targets: structure and function

Antibody-based therapy has shown promising efficacy in hematologic tumors. However, this approach has shown limitations in most solid cancers. One of the critical factors for treating solid tumors is the identification of cancer-specific targets. Our long-term research interests lie primarily in the biology of cell surface receptors including glypicans (GPC3 and GPC2) and mesothelin for establishing them as cancer targets.

Mesothelin is a target candidate in many solid tumors. To understand its biological function, we collaborated with Byungkook Lee (NCI) to make a structure model and experimentally identified the functional binding domain for MUC16/CA125 around Y318 (residues 296-359; named IAB) consisting of 64 amino acids at the N-terminus of cell surface mesothelin. Our work supports the role of mesothelin and MUC16/CA125 as functional partners in the tumor microenvironment and cancer development.

Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) are important modulators of signal transduction pathways during development and disease. HSPGs mainly consist of glycosylphosphatidylinositol (GPI)-anchored glypicans and transmembrane syndecans. Much of our work has focused on fundamental aspects of glypicans in connections to Wnt/Yap signaling and cancer cell growth. Our goal is to investigate glypicans as new targets in cancer.  

We identified the Wnt functional binding sites on GPC3, one in the protein core and the other in heparan sulfate chains. First, we built a structure model of the GPC3/Wnt complex in collaboration with Byungkook Lee, then experimentally validated our model to determine the Wnt functional binding site on GPC3, providing evidence for GPC3 as a co-receptor for regulating Wnt. To explore the role of GPC3 in other signaling pathways, we conducted the original research that revealed the role of GPC3 in regulating Yap for liver cancer cell proliferation and discovered another signaling interaction between GPC3 and HGF in liver cancer. Recently, we identified FAT1, a potential cell surface receptor of Yap signaling in mammalian cells, was a novel GPC3 interacting protein in liver cancer cells. Furthermore, we found that our Wnt blocking single domain antibody (HN3) was capable of reaching the previously undescribed Wnt functional binding site in the N-lobe of the protein core of GPC3 and that F41 was the key residue in the hydrophobic groove for Wnt and HN3 binding. Additionally, we used our HS20 human antibody specific for the heparan sulfate chains of GPC3 to determine the Wnt binding motif which may function as a Wnt storage/transporter site on heparan sulfate chains. In collaboration with Jian Liu (University of North Carolina), we found that Wnt recognized a heparan sulfate structure containing IdoA2S and GlcNS6S, and that the 3-O-sulfation in GlcNS6S3S could enhance the binding of Wnt.

We started to apply what we learned from our GPC3 project to explore other glypicans in cancer. We found that GPC2 protein was expressed in nearly half of neuroblastoma cases and that GPC2 knockout inactivated Wnt/β-catenin signaling and reduced the expression of the target gene, N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. Furthermore, we conducted the proof-of-concept study that showed CAR T cells and immunotoxins targeting GPC2 could inhibit neuroblastoma growth in mouse models. As a result, we reported GPC2 to be a new therapeutic target in neuroblastoma.

Engineering antibody therapeutics for treating cancer: immunotoxins and CAR T cells

To overcome the limitations of antibody-based immunotherapy for solid tumors, we used multiple antibody technologies to generate large panels of human and xenogeneic antibodies and utilized multiple effector mechanisms to improve the anti-tumor activity of our tumor antigen-specific monoclonal antibodies. We have developed multiple strategies with different mechanisms of actions, such as immunotoxins, ADCs and CAR T cells. Our research program focuses on the development and implementation of antibody-based immunotherapeutic strategies for the treatment of solid tumors, including hepatocellular carcinoma (HCC), pancreatic cancer, neuroblastoma and mesothelioma. The strategies developed in our program may be applicable to other solid tumors.

We generated antibodies (HN3 and YP7) that bind glypican-3 (GPC3) on liver cancer cells. HN3 is a human single domain antibody isolated using phage display technology, which recognizes a cryptic Wnt binding site in the N-lobe of GPC3. YP7 is a monoclonal antibody isolated using mouse hybridoma technology, which binds an epitope at the C-lobe of GPC3 close to cell surface. Using these antibodies, we generated immunotoxins and ADCs to target GPC3-expressing cancers. We constructed a novel immunotoxin that can produce tumor regression via a dual mechanism of action. These immunotoxins inactivate cancer signaling via the antibody domain and inhibit protein synthesis via the toxin portion. This dual mechanism of action may be applicable to other antibody-toxin/drug conjugates for better anti-tumor efficacy. Immunogenicity and a short serum half-life may limit the ability of immunotoxins to transition to the clinic. To address these concerns, we engineered HN3-based immunotoxins in collaboration with Ira Pastan (NCI) to use various deimmunized Pseudomonas exotoxin (PE) domains and added an albumin binding single domain. We also designed CAR T cells using our GPC3 antibodies and demonstrated that humanized YP7 (hYP7) antibody derived CAR T cells had potent and persistent anti-tumor activity in orthotopic liver cancer mouse models. This was accomplished by using functional genomics sequencing and single cell-based T cell analysis.

Mesothelin is highly expressed in mesothelioma and various solid tumors. We previously isolated HN1, a human monoclonal antibody that disrupts the mesothelin-MUC16 interaction. Our recent research has been focused on the targeting of the membrane proximal region of mesothelin for enhanced anti-tumor activities. As a result, we isolated YP218, a unique rabbit monoclonal antibody targeting the C-terminal portion of mesothelin close to the tumor cell surface.

Developing antibody technology: single domain antibodies

We have developed a variety of antibody technologies to produce therapeutic antibodies. These include cell-based screening methodology for isolating antibodies using rabbit and mouse hybridomas, humanization of rabbit and mouse antibodies, and construction of phage display libraries for antibody discovery. Our antibody technologies, such as single domain antibody libraries, can be used to identify therapeutic and diagnostic antibodies for human diseases including cancer, infectious disease and neurological disease.

Single domain antibodies (also commonly called nanobodies) are known to bind restricted epitopes that may be inaccessible to conventional antibodies. To make a large single domain phage library, we established a method based on PCR-Extension Assembly and Self-Ligation (named ‘EASeL’) to construct a VNAR single domain antibody library from six nurse sharks (Ginglymostoma cirratum) in collaboration with Martin Flajnik (University of Maryland). We conducted next-generation sequencing analysis of 1.2 million shark VNAR single domains and showed that our shark single domain library is highly diverse. In addition, we applied the EASeL method to construct VHH single domain phage libraries from 20 camels (Camelus dromedaries). Single domain antibodies isolated from our phage libraries can have a high affinity (KD = 1 nM or less) for their tumor or viral antigens.

Antibody engineering is typically carried out by displaying antibody fragments on the surface of microorganisms (e.g. phage, bacteria and yeast). We established a new antibody engineering method known as 'mammalian cell display' that is adapted from yeast cell display. Using this approach, antibody fragments are expressed on human HEK-293 cells, and high affinity antigen binders are isolated from a combinatory library via flow cytometry.

Drug resistance is an important component of tumor biology that requires a complex cellular environment for study. We have established ex vivo tumor spheroid models using cell lines and primary patient cells in collaboration with Shuichi Takayama (University of Michigan) and V. Courtney Broaddus (UCSF). This allows us to study the molecular mechanisms of antibody drug resistance in a physiologically relevant cellular model. We have also used microarrays to profile gene expression in both spheroids and monolayers to identify new targets specific to the 3D biological structure of cancer.

Teaching Interests

BIOC301/302 - Biochemistry I/II