BackgroundImmunotherapy against solid tumors has long been hampered by the development of immunosuppressive tumor microenvironment, and the lack of a specific tumor-associated antigen that could be targeted in different kinds of solid tumors. Human leukocyte antigen G (HLA-G) is an immune checkpoint protein (ICP) that is neoexpressed in most tumor cells as a way to evade immune attack and has been recently demonstrated as a useful target for chimeric antigen receptor (CAR)-T therapy of leukemia by in vitro studies. Here, we design and test for targeting HLA-G in solid tumors using a CAR strategy.MethodsWe developed a novel CAR strategy using natural killer (NK) cell as effector cells, featuring enhanced cytolytic effect via DAP12-based intracellular signal amplification. A single-chain variable fragment (scFv) against HLA-G is designed as the targeting moiety, and the construct is tested both in vitro and in vivo on four different solid tumor models. We also evaluated the synergy of this anti-HLA-G CAR-NK strategy with low-dose chemotherapy as combination therapy.ResultsHLA-G CAR-transduced NK cells present effective cytolysis of breast, brain, pancreatic, and ovarian cancer cells in vitro, as well as reduced xenograft tumor growth with extended median survival in orthotopic mouse models. In tumor coculture assays, the anti-HLA-G scFv moiety promotes Syk/Zap70 activation of NK cells, suggesting reversal of the HLA-G-mediated immunosuppression and hence restoration of native NK cytolytic functions. Tumor expression of HLA-G can be further induced using low-dose chemotherapy, which when combined with anti-HLA-G CAR-NK results in extensive tumor ablation both in vitro and in vivo. This upregulation of tumor HLA-G involves inhibition of DNMT1 and demethylation of transporter associated with antigen processing 1 promoter.ConclusionsOur novel CAR-NK strategy exploits the dual nature of HLA-G as both a tumor-associated neoantigen and an ICP to counteract tumor spread. Further ablation of tumors can be boosted when combined with administration of chemotherapeutic agents in clinical use. The readiness of this novel strategy envisions a wide applicability in treating solid tumors.
The present study reports the case of a 24-year-old female affected with primary Sjögren's syndrome (pSS), who presented with mucosa-associated lymphoid tissue (MALT) lymphoma of the submandibular gland. Reports of such cases, particularly in young patients, are very rare. The patient, who presented no oral or ocular symptoms prior to the development of the mass, underwent surgical ablation of the gland, and MALT lymphoma was diagnosed by histopathology. Since MALT lymphoma in the submandibular gland is rarely observed in otherwise healthy young females, a rheumatologist and an oncologist were consulted. Following a number of immunological tests, the results of the Schirmer's and Saxon tests were negative. However, the antinuclear antibody test revealed a speckled appearance, and there was also strong positivity for the serological markers of Sjögren's syndrome. Consequently, pSS was diagnosed, despite the fact that the patient did not fulfill all the diagnostic criteria for the disease. Therefore, MALT lymphoma in a single salivary gland should be used as a differential diagnosis for Sjögren's syndrome in young asymptomatic patients. Additionally, a multidisciplinary team is required for the treatment and management of these patients.
Human leukocyte antigen G (HLA-G) is a molecule within the tumor microenvironment (TME) that modulates the innate and adaptive immune systems by interacting with inhibitory receptors on the surface of immune cells and thus functions as an immune checkpoint. It could be potentially expressed by all tumor types but is not expressed by either healthy tissues surrounding the tumor cells or vital normal tissues. Chimeric antigen receptors (CARs) for adoptive cell therapy (ACT) have been successful in clinical trials against hematologic cancers; however, challenges have been encountered when applying this approach to the treatment of solid tumors. These obstacles are mainly due to the lack of a ubiquitous tumor-associated antigen (TAA) across different tumor types without “on-target/off-tumor” reactivity and the immunosuppressive nonphysical TME. To address these issues, we developed a novel switch HLA-G CAR carrying an inducible Caspase9 (iC9) suicide gene system that binds to HLA-G1~G7 isoforms and expressed this CAR in natural killer (NK) cells. We tested these HLA-G CAR-NK cells in a variety of adult cancer models and discovered that they mediate significant tumor cytolysis in triple-negative breast cancer (TNBC), glioblastoma (GBM), pancreatic (PA) cancer, and ovarian (OV) cancer in vitro and in TNBC and GBM xenograft models in vivo. Coculturing HLA-G CAR-NK cells with vital normal cell lines did not cause cell damage. We further discovered that surface-exposed HLA-G is chemoinducible, which in turn increases tumor sensitivity to both HLA-G CAR effector-mediated antitumor responses and tumor-infiltrative NK cells. The underlying mechanism of tumor and HLA-G CAR NK cell interaction may be through upregulation of membranous HLA-G via demethylation of the TAP-1 promoter or enhanced activity of the TAP1/signal peptide peptidase (SPP) pathways. In conclusion, HLA-G CAR-NK cells could be an option for treating solid tumors of different cell types, and a regimen comprising chemotherapy followed by HLA-G CAR immunotherapy may improve responses compared to those achieved with CAR-T cell immunotherapy alone. Citation Format: Chia-Ing Jan, Shi-Wei Huang, Peter Canoll, Yu-Chuan Lin, Hsin-Man Lu, Shao-Chih Chio, Der-Yang Cho. Human leukocyte antigen G as a novel target for switch-based chimeric antigen receptor natural killer cell therapy of solid cancers [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A61.
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