Development of antibody engineering-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 15 years, Dr Mitchell Ho's laboratory at the National Cancer Institute (NCI) has developed GPC3 and other glypicans such as GPC1 and GPC2 as a new family of cancer therapeutic targets and generated novel antibody and cell-based immunotherapeutic technologies for treating solid tumors including liver cancer, pediatric cancers, and other solid tumors. In FY2023, we continued to optimize the CAR-T cells targeting GPC3 for treating hepatocellular carcinoma (HCC), a major form of liver cancer. In our previous study (Li et al. Gastroenterology, 2020), we found CAR-T cells targeting a membrane-proximal site recognized by YP7 antibody were more potent than the CAR-T cells targeting a membrane distal site recognized by HN3, indicating current CAR formats are not tailored toward targeting membrane distal epitopes. In FY2023, using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced beta-catenin levels in HCC cells. Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane-proximal epitope, leads to robust T-cell signaling and induces swift and durable eradication of HCC tumors. We published this work in FY2023 in Hepatology Communications (Aarti Kolluri, Dan Li, Nan Li, Zhijian Duan, Lewis R Roberts, Mitchell Ho. Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC. Hepatol Commun. 2023 Jan 18;7(2):e0022. doi: 10.1097/HC9.0000000000000022. eCollection 2023 Feb 1.). In addition to GPC3 and GPC2, in FY2023 Dr. Mitchell Ho's lab developed novel CAR T cells targeting GPC1, an oncofetal antigen expressed in pancreatic cancer. We reported the generation of dromedary camel VHH nanobody (D4)-based CAR T cells targeting GPC1 and the optimization of the hinge (H) and transmembrane domain (TM) to improve activity. We found that a structurally rigid IgG4H and CD28TM domain brought the two D4 fragments in proximity, driving CAR dimerization and leading to enhanced T-cell signaling and tumor regression in pancreatic cancer models with low antigen density in female mice. Furthermore, single-cell-based proteomic and transcriptomic analysis of D4-IgG4H-CD28TM CAR T cells revealed specific genes (e.g., HMGB1) associated with high T-cell polyfunctionality. Our study demonstrates the potential of VHH-based CAR T for pancreatic cancer therapy and provides an engineering strategy for developing potent CAR T cells targeting membrane-distal epitopes. This work was published in Nature Communications in April 2023 (Nan Li, Alex Quan, Dan Li, Jiajia Pan, Hua Ren, Gerard Hoeltzel, Natalia de Val, Dana Ashworth, Weiming Ni, Jing Zhou, Sean Mackay, Stephen M Hewitt, Raul Cachau, Mitchell Ho. The IgG4 hinge with CD28 transmembrane domain improves VHH-based CAR T cells targeting a membrane-distal epitope of GPC1 in pancreatic cancer. Nat Commun. 2023 Apr 8;14(1):1986. doi: 10.1038/s41467-023-37616-4. To pursue the clinical development of CAR-T cell therapy, we have collaborated with Carol Thiele, Rosandra Kaplan, and Rosa Nguyen in the Pediatric Oncology Branch at the CCR, NCI to produce clinical-ready CAR-T cells for an upcoming clinical trial treating neuroblastoma in the NIH Clinical Center. In a major effort through our collaboration, we demonstrated that our single-chain variable fragment targeting GPC2, CT3, integrated into a CAR vector with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain (CT3.28H) elicited the best preclinical anti-tumor activity compared with other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. We concluded that given the robust preclinical activity of CT3.28H, these results form a promising basis for further clinical testing in children with neuroblastoma. The work was published in the Journal of Immunotherapy of Cancer in FY2023 (Sun et al. 2023). We have developed GPC3 (hYP7), GPC2 (CT3) and mesothelin (hYP218) CAR T cells for clinical trials at the NIH for treating liver cancer, mesothelioma, and neuroblastoma. Dr. Mitchell Ho has obtained funds from the Cancer Moonshot program and the NCI CCR to support these clinical trials for treating cancer patients at the NIH.