Tumor-selective antigen delivery to overcome antigen heterogeneity in targeted cellular immunotherapy

Abstract

however, they were found to have serious side effects and limitations due to high treatment resistance. Since the 2000s, immunotherapy using the immune system for anticancer treatment has been actively researched. In particular, since 2010, therapy using chimeric antigen receptor T (CAR-T) cells, which express chimeric antigen receptors through genetic engineering to recognize cancer cells, has been developed, showing high treatment efficacy of over 80% in patients with acute lymphoblastic leukemia. However, CAR-T cells, which demonstrated high anticancer efficacy, also have many limitations. For example, there is antigen-negative relapse, which is found in over 20% of treatment-failed patients. Moreover, CAR-T cells targeting solid tumors, unlike hematologic cancers, show low treatment efficacy and high relapse rates. Many researchers believe that the absence of antigens targeted by CAR-T cells is a common cause of these two limitations. To overcome these limitations, I developed multi-antigen-specific CAR-T cells that target multiple antigens simultaneously. However, in patients receiving treatment with multi-antigen-specific CAR-T cells, cancer relapse without expressing all the targeted antigens was observed, indicating that the limitations caused by antigen-negative relapse have not yet been overcome. To overcome these limitations of targeted immunotherapy, I aimed to develop a method of delivering specific antigens to cancer cells and design a treatment method using single-antigen-specific CAR-T cells to target the labeled antigens. In this study, I aimed to deliver antigens to the surface of cancer cells through genetic and chemical methodovvcd19s. To achieve genetic antigen delivery, I used oncolytic viruses to deliver the CD19 antigen. Experimental results confirmed the expression of the CD19 antigen in breast cancer-derived cell lines with high antigen heterogeneity, and the effective cytotoxicity of anti-CD19 CAR-T cells against CD19-expressing cancer cells was observed. Furthermore, in a breast cancer xenograft mouse model, CD19 expression was confirmed following the administration of oncolytic viruses. Additionally, to achieve cancer cell-specific CD19 antigen expression, I exchanged the promoter regulating CD19 antigen expression and confirmed cancer cell-specific antigen expression. Subsequently, in vivo experiments, the efficacy of anticancer treatment was observed in mice with oVVCD19 administration, regardless of the administration of anti-CD19 CAR-T cells, and mouse deaths due to virus toxicity were confirmed. Next, to label antigens on the surface of cancer cells chemically, I aimed to use metabolic glycan labeling with unnatural monosaccharides. The azido-conjugated mannosamine and BCN-FITC successfully labeled FITC on the surface of cancer cells, and the efficient cytotoxicity and cellular activity of anti-FITC CAR-T cells were observed. Moreover, in a xenograft mouse model injected with FITC-labeled cancer cells, the successful inhibition of cancer cell growth by anti-FITC CAR-T cells was confirmed. Furthermore, to confirm whether unnatural sugars can directly induce the activity of CAR-T cells, I used hapten conjugated unnatural sugar. The labeling of DNP antigens on the surface of cancer cells by Sia-DNP, a DNP conjugated sialic acid, was confirmed, and the cytotoxicity and activation of anti-DNP CAR-T cells were verified. Furthermore, successful DNP surface labeling with Sia-DNP and high anticancer efficacy by anti-DNP CAR-T cells were confirmed in animal experiments. Furthermore, I confirmed the applicability of this approach to cancer cells derived from other tissues, not limited to breast cancer, using prostate cancer cell lines.

Since ancient times, humanity has acknowledged the existence of cancer and has made efforts to develop various therapeutic agents to overcome it. After the 1900s, radiation therapy, chemotherapy, and targeted therapy were developed