Development of antibody engineering-based cancer therapies

Abstract public domain (NIH RePORTER)

Heparan sulfate proteoglycans (HSPGs) regulate numerous cell surface signaling events and act as 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. Over the past 16 years, Dr. Mitchell Ho's laboratory at the National Cancer Institute (NCI) has developed GPC3 and other glypicans such as GPC1 and GPC2 into a new family of cancer therapeutic targets. The lab has also generated novel antibody and cell-based immunotherapeutic technologies for treating solid tumors, including liver cancer, pediatric cancers, and other solid tumors. CAR-T therapy has achieved remarkable remission in patients with B-cell malignancies; however, its efficacy in treating solid tumors remains limited. In FY2024, we further advanced CAR-T cell therapy for solid tumors by investigating a combination therapy approach using an engineered long-acting interleukin (IL)-7 (rhIL-7-hyFc or NT-I7) alongside CAR-T cells targeting three antigens: glypican-2 (GPC2), glypican-3 (GPC3), and mesothelin (MSLN). This approach was tested against various solid tumor types, including liver cancer, neuroblastoma, ovarian cancer, and pancreatic cancer in mice. This work was conducted through a CRADA collaboration with NeoImmuneTech, Inc. for cancer drug development. We assessed the anticancer activity of CAR-T cells targeting GPC2, GPC3, and MSLN in combination with NT-I7. Xenograft tumor models, including an orthotopic liver cancer model, were established using NOD scid gamma mice engrafted with cell lines from hepatocellular carcinoma, neuroblastoma, ovarian cancer, and pancreatic cancer. Mice were monitored through bioluminescence in vivo tumor imaging and tumor volume measurements with a caliper. We evaluated immunophenotyping of CAR-T cells stimulated by NT-I7 for memory markers, exhaustion markers, and T-cell signaling molecules using flow cytometry and western blotting. Compared with the IL-2 combination, NT-I7 demonstrated enhanced tumor regression, improved T-cell expansion, reduced expression of exhaustion markers (programmed cell death protein 1 or PD-1 and lymphocyte-activation gene 3 or LAG-3), and increased generation of stem cell-like memory CAR-T cells. The STAT5 pathway was shown to be downstream of NT-I7 signaling, mediated by increased IL-7 receptor expression in CAR-T cells. Furthermore, NT-I7 combined with CAR-T cells improved efficacy against tumors with low antigen density in mice, presenting a potential option for patients with heterogeneous antigenic profiles. Our study provides a rationale for NT-I7 plus CAR-T cell combination therapy for solid tumors in humans and was published in the Journal of Immunotherapy of Cancer (Li et al., 2024). Rational design of chimeric antigen receptor T (CAR-T) cells based on the recognition of antigenic epitopes that evoke the most potent CAR activation is crucial for optimizing immune therapy. In solid tumors, in addition to GPC1, GPC2, GPC3, and mesothelin, the B7-H3 transmembrane protein is an emerging target with two distinct epitope motifs, IgC and IgV, in its ectodomain. In FY2024, we generated dromedary camel nanobodies targeting B7-H3 and demonstrated that CAR-T cells based on nanobodies recognizing the IgC domain (but not the IgV domain) exhibited potent antitumor activity against large tumors in female mice. These CAR-T cells were characterized by highly activated T cell signaling and significant tumor infiltration. Single-cell transcriptome RNA sequencing, coupled with functional T-cell proteomics analysis, identified key upregulated genes potentially critical for the persistence of polyfunctional CAR-T cells in mice. Our results emphasize the importance of specific target antigen epitopes in optimizing CAR-T activity and provide a nanobody-based B7-H3 CAR-T product for use in solid tumor therapy.

Abstract public domain (NIH RePORTER)

Our study was published in Nature Communications (Li et al., 2023). We have developed GPC3 (hYP7), GPC2 (CT3), and mesothelin (hYP218) CAR-T cells for clinical trials at the NIH Clinical Center, targeting liver cancer, mesothelioma, and neuroblastoma. Dr. Mitchell Ho has received funding from the Cancer Moonshot program and the NCI CCR Intramural Research Program/NCI CCR Office of Translational Resources to support these new clinical trials for treating cancer patients at the NIH. In addition to his own laboratory work, Dr. Ho has actively collaborated with labs both inside and outside the NIH to develop novel cancer drugs and has published research papers in FY2024 (Nguyen et al., Clinical Cancer Research 2024; Bell et al., Nature Biomedical Engineering 2024).