Development of new antibody-based cancer therapies

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Heparan sulfate proteoglycans (HSPGs) regulate numerous cell surface signaling events. They are extracellular modulators of signal transduction pathways during development and diseases such as cancer. HSPGs are cell-surface proteins that mainly consist of glycosylphosphatidylinositol (GPI)-anchored glypicans and transmembrane syndecans. In the last over 14 years, Dr Mitchell Ho's laboratory at the National Cancer Institute (NCI) has studied GPC3 and other glypicans as a new family of cancer targets and developed novel antibody and cell-based immunotherapeutic technologies for treating solid tumors including liver cancer, pediatric cancers and other solid tumors. In FY2022, we created novel immunotoxins targeting GPC1 in pancreatic cancer and showed immunotoxins inhibited pancreatic cancer in mice and published our preclinical studies in Molecular Cancer Therapeutics [Pan et al. Molecular Cancer Therapeutics, 2022]. GPC1 is a cell surface proteoglycan that is upregulated in multiple types of human cancers including pancreatic cancer. We investigated whether GPC1 could be a target of antibody-toxin fusion proteins (i.e., immunotoxins) for treating pancreatic cancer. We constructed a panel of GPC1-targeted immunotoxins derived from a functional domain of Pseudomonas exotoxin A. An albumin-binding domain was also introduced into the anti-GPC1 immunotoxin to improve serum half-life. Small-molecule screening was performed to identify irinotecan that shows synergistic efficacy with the immunotoxin. We showed that GPC1 was internalized upon antibody binding. Anti-GPC1 immunotoxins alone inhibited tumor growth in a pancreatic cancer xenograft model. The immunotoxin treatment reduced active beta-catenin expression in tumor cells. Furthermore, immunotoxins containing an albumin-binding domain in combination with irinotecan caused pancreatic tumor regression. GPC1 expression was reduced by the immunotoxin treatment due to the degradation of the internalized GPC1 and its short cellular turnover rate. Our data indicate that the GPC1-targeted immunotoxin inhibits pancreatic tumor growth via degradation of internalized GPC1, downregulation of Wnt signaling, and inhibition of protein synthesis. The anti-GPC1 immunotoxin in combination with irinotecan thus provides a potential new treatment strategy for patients with pancreatic tumors. We also summarized our work in studying GPC1 as an immunotherapeutic target in pancreatic cancer in a review article published in the American Journal of Physiology Cell Physiology [Pan and Ho, Am J Physiol Cell Physiol. 2021]. In FY2022, we also developed novel PD-L1 targeted shark VNAR single-domain-based CAR-T cell strategy for treating breast cancer and liver cancer [Li et al. Molecular Therapy Oncolytics 2022]. Chimeric antigen receptor (CAR)-T cell therapy shows excellent potency against hematological malignancies, but it remains challenging to treat solid tumors, mainly because of a lack of appropriate antigenic targets and an immunosuppressive tumor microenvironment (TME). The checkpoint molecule programmed death-ligand 1 (PD-L1) is widely overexpressed in multiple tumor types, and the programmed death-ligand 1 (PD-1)/PD-L1 interaction is a crucial mediator of immunosuppression in the TME. In this new study, we constructed a semi-synthetic shark VNAR phage library and isolated anti-PD-L1 single-domain antibodies. Among these VNARs, B2 showed cross-reactivity to human, mouse, and canine PD-L1, and it partially blocked the interaction of human PD-1 with PD-L1. CAR (B2) T cells specifically lysed human breast cancer and liver cancer cells by targeting constitutive and inducible expression of PD-L1 and hindered tumor metastasis. Combination of PD-L1 CAR (B2) T cells with CAR T cells targeted by GPC3 (a liver cancer-specific antigen) regresses liver tumors in mice. We concluded that PD-L1-targeted shark VNAR single-domain-based CAR-T therapy is a novel strategy to treat breast and liver cancer. This study provides a rationale for potential use of PD-L1 CAR-T cells as a monotherapy or in combination with a tumor-specific therapy in clinical studies. Besides cell-based therapy, we also developed bispecific antibody hYP7-OKT3-hFc targeting GPC3 and showed its potent activity in mice [Chen et al. Mol Cancer Ther 2022]. We also published three protocols regarding the construction and production of CAR-T cells and immunotoxins for cancer therapy in STAR Protocols (Cell Press) and Methods in Molecular Biology [Li et al. STAR Protocols 2021; Li and Ho, Methods in Molecular Biology 2022; Fleming and Ho, Methods in Molecular Biology 2022]. We shared our protocols and methodologies in the scientific community to help advance the CAR-T field quickly. To develop CAR-T for treating other solid tumors, we collaborated with Dr. Ira Pastan's lab and Dr. Raffit Hassan's lab to develop novel CAR-T cell therapies targeting mesothelin [Tomar et al., Mol Cancer Ther 2022; Liu et al., PNAS, 2022] and with Javed Khan's lab and Brad St Croix's lab to develop novel bicistronic CAR against GPC2 or CD276 in neuroblastoma [Tian et al., J Clin Invest. 2022]. We developed hYP218 (against membrane-proximal epitope) CAR T cells [Tomar et al. Mol Cancer Ther 2022]. Our results show that hYP218 CAR T cells, targeting mesothelin epitope close to cell membrane, are very effective against mesothelin-positive tumors and are associated with increased persistence and tumor infiltration. These results support its clinical development to treat patients with mesothelin-expressing cancers. In another study led by Dr. Pastan's lab and our lab, we have identified shed mesothelin as a major obstacle to successful antibody therapies and prepared a monoclonal antibody that inhibits shedding and makes very active CAR T cells whose activity is not blocked by shed mesothelin and merits further preclinical development [Liu et al., PNAS, 2022]. CAR-T cell therapies targeting single antigens perform poorly in clinical trials for solid tumors due to heterogenous expression of tumor-associated antigens (TAAs), limited T-cell persistence and exhaustion. A project led by Dr. Khan's lab, Dr. St Croix's lab and our lab aimed to identify optimal CARs against GPC2 or CD276 (B7-H3), which were highly but heterogeneously expressed in neuroblastoma (NB), a lethal extracranial solid tumor of childhood. We made a bicistronic "OR" CAR (BiCisCAR). BiCisCAR T-cells effectively eliminated tumor cells expressing GPC2 or CD276. For clinical development, we have developed GPC3 (hYP7), GPC2 (CT3) and mesothelin (hYP218) CAR T cells for the clinical trials at the NIH for treating liver cancer, mesothelioma, and neuroblastoma as supported by the Cancer Moonshot program and the NCI CCR.