Yusuke Nakamura’s research while affiliated with National Institute of Biomedical Innovation, Health and Nutrition and other places

Background Existing circulating cell-free DNA (cfDNA) assays are primarily centralized, requiring specialized sample handling and transportation. Implementing a flexible, decentralized sequencing system at point of care, with minimal technical oversight, can enhance turnaround times and patient access to genomic profiling. In this study, we aimed to evaluate the clinical feasibility of an automated and decentralized cfDNA next-generation sequencing (NGS) assay for identifying actionable alterations in advanced solid tumors. Methods Genomic profiling of plasma cfDNA from 298 patients with advanced solid tumors was conducted using an automated NGS assay. We assessed concordance of tumor mutations detected in plasma cfDNA and patient-matched tumor tissues analyzed by an FDA-approved assay, investigating clinical factors influencing circulating tumor DNA (ctDNA) aberration (mut-ctDNA) detection sensitivity. Results Sequencing success rates for cfDNA genomic profiling was significantly higher than archived tumor tissue (99% vs 96%). Mut-ctDNA detection rates ranged from 20% to 67% across different solid tumors. Targetable or resistance alterations were found in 18% of the patients. About 72% of the patients showed concordant alterations from tissue and plasma. The level of concordance was associated with the cancer type, tumor burden, and metastatic location. Notably, 63 plasma-only alterations were identified in 18% of patients and were more frequently observed in those with prior targeted treatments (24%) compared to chemotherapy (10%). Conclusions This study underscores the clinical feasibility of an automated, decentralized cfDNA genomic profiling approach. It emphasizes the importance of considering confounding clinical factors when selecting plasma- or tissue-based profiling assay. Such an approach holds promise for enhancing patient access to timely genomic profiling and targeted therapy selection.

Background The development of T-cell receptor (TCR)-based T-cell therapies is hampered by the difficulties in identifying therapeutically effective tumor-specific TCRs from the natural repertoire of a patient’s cancer-specific T cells. Methods Here, we mimic experimentally near-patient conditions to analyze the T-cell repertoire in euthymic tumor-bearing mice responding to the H-2K b -presented neoantigen p68 S551F (mp68). We temporarily separated the time point of mp68 expression from that of cancer cell transplantation to exclude the influence of injection-induced inflammation on T-cell priming. Thus, the mp68-specific T-cell response could only develop after the acute inflammatory phase had subsided. Results We found that mp68-specific TCRs isolated from either tumor-infiltrating T cells or spleens of mice immunized with mp68-expressing cancer cells are diverse and not inherently therapeutic when introduced into peripheral T cells and used for adoptive therapy of established tumors. While measuring short-term T-cell responses in vitro was unreliable for some TCRs in predicting their therapeutic failure, assessing the persistence of cancer cell destruction by TCR-modified T cells in long-term cultures accurately predicted therapeutic outcomes. A tumor-derived TCR with optimal function was also correctly identified with this approach when analyzing human TCRs that recognize the HLA-A2-presented neoantigen CDK4 R24L . Conclusions We show that a neoantigen-directed T-cell response in tumor-bearing hosts comprises a diverse repertoire. Infiltration and expansion of certain T-cell clonotypes in the tumor do not necessarily correlate with therapeutic efficacy of their TCRs in adoptive therapy. We propose that analysis of persistent rather than immediate responses of TCR-modified T cells in vitro serves as a reliable parameter to identify TCRs that are therapeutically effective in vivo.

T cells play a pivotal role in cancer immunotherapy by recognizing neoantigens presented on HLA molecules, which are specifically expressed by cancer cells. While neoantigens are generally unique to individual cancers, shared antigens that are common in a subset of cancer patients, represent promising immunotherapeutic targets. Since frameshift insertions or deletions (indels) are reported to generate highly immunogenic neoantigens through novel open reading frames. However, few studies have explored shared neoantigens from frameshift indels, which are shred among multiple cancer patients. In this study, we analyzed frameshift neoantigens from frameshift indels in TCGA exome sequencing data, identifying 760 recurrent frameshift mutation clusters (FSCs) that shared frameshifted open reading frames and premature stop codons. Among these, we focused on two FSCs in the APC gene, which generate potential neoantigens (AS3 and AS10) presented the HLA-A24 molecule. We successfully identified the T cell receptor (TCR) sequences for APC frameshift neoantigens from healthy donor’s blood. Genetically engineered T cells expressing these TCRs demonstrated recognition and cytotoxic activity against target cells presenting these APC frameshift neoantigens. Additionally, we explored the development of an antibody targeting a complex formed by the APC shared neoantigen AS10 and the HLA-A24 molecule. Using phage display library screening, we isolated single-chain variable fragments (scFvs) that specifically recognized the AS10-HLA-A24 complex. A bispecific antibody (BsAb) was then designed to connect T cells via an anti-CD3 scFv to the AS10 neoantigen on HLA-A24. We found that the AS10 BsAb specifically recognized both the AS10-HLA-A24 complex and CD3 protein, and showed significant IFN-γ release and cytotoxicity against target cells expressing frameshift neoantigens. By focusing on recurrent frameshift mutations and shared neoantigens, this study provides compelling evidence for the development of more effective and broadly applicable immunotherapeutic strategies. Citation Format Kazuma Kiyotani, Peng Zhao, Xiaojing Wu, Yusuke Nakamura. T cell receptors and antibodies targeting shared indel-derived neoantigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3409.

Introduction Pancreatic cancer shows very poor prognosis and high resistance to conventional standard chemotherapy and immunotherapy; therefore, the development of new breakthrough therapies is highly desirable. Method We retrospectively evaluated the safety and efficacy of neoantigen peptide-pulsed dendritic cell (Neo-P DC) vaccine therapy after surgical treatment of pancreatic cancer. Result The result showed induction of neoantigen-specific T cells in 13 (81.3%) of the 16 patients who received Neo-P DC vaccines. In survival analysis of the nine patients who received Neo-P DC vaccines after recurrence, longer overall survival was observed in patients with neoantigen-specific T cell induction than those without T cell induction. Notably, only one of the seven patients who received Neo-P DC vaccines as adjuvant setting developed recurrence, and no patient died during median follow-up 61 months after surgery (range, 25-70 months). Furthermore, TCR repertoire analyses were performed in a case treated with Neo-P DC vaccine combined with long and short peptides, and one significantly dominant clone induced by the long peptide was detected among CD4⁺ T cell populations. Discussion The present study suggests the feasibility and efficacy of Neo-P DC vaccine therapy after surgical treatment of pancreatic cancer in both postoperative recurrence cases and adjuvant setting. A case analysis suggests the importance of combination with long peptides targeting CD4⁺ T cell.

The Ryukyu archipelago is located southwest of the Japanese islands, and people originally from this region, the Ryukyu population, have a unique genetic background distinct from that of other populations, including people from mainland Japan. However, few genetic studies have focused on the Ryukyu population. In this study, we performed genome-wide association studies (GWAS) on the serum levels of alanine aminotransferase (ALT, n = 15,224), aspartate aminotransferase (AST, n = 15,203), and gamma-glutamyl transferase (GGT, n = 14,496) in the Ryukyu population. We found 13 loci with a genome-wide significant association (P < 5 × 10-8), three for ALT, four for AST, and six for GGT, including one novel locus associated with ALT: rs117595134-A in HMMR/HMMR-AS1, ß = - 0.131, standard error = 0.024, P = 4.90 × 10-8. Rs117595134-A is common in the Japanese population but is not observed in other ethnic populations in the 1000 genomes database. Additionally, 77 of 80 loci derived from Korean GWAS and 541 of 716 loci from European GWAS showed the same directions of effect (P = 1.41 × 10-19, P = 2.50 × 10-44, binomial test), indicating that most of susceptibility loci are shared between the Ryukyu population and other ethnic populations.

Background/Objectives: Neoantigens have attracted attention as ideal therapeutic targets for anti-tumour immunotherapy because the T cells that respond to neoantigens are not affected by central immune tolerance. Recent findings have revealed that the activation of CD4-positive T cells plays a central role in antitumor immunity, and thus targeting human leukocyte antigen (HLA) class II-restricted neoantigens, which are targets of CD4-positive T cells, is of significance. However, there are very few detailed reports of neoantigen vaccine therapies that use an HLA class II-restricted long peptide. In the present study, we retrospectively analysed the ability of HLA class II-restricted neoantigen-pulsed dendritic cell vaccines to induce immune response in five breast cancer patients. Methods: We performed whole exome and RNA sequencing of breast cancer tissues and neoantigen prediction using an in silico pipeline. We then administered dendritic cells pulsed with synthesized an HLA class II-restricted long peptide containing an epitope with high affinity to HLA class I in the lymph node. Results: ELISPOT analysis confirmed that a T-cell response specific for the HLA class II-restricted neoantigen was induced in all cases. TCR repertoire analysis of peripheral blood mononuclear cells before and after treatment in three patients showed increases of specific T-cell clones in two of the three patients. Importantly, no recurrence was observed in all patients. Conclusions: Our analysis demonstrated the immunological efficacy of the HLA class II-restricted neoantigen peptide dendritic cell vaccine against breast cancer and provides useful information for the development of neoantigen vaccine therapy for breast cancer.

Background Even when tumors are infiltrated by T cells, progressive growth eventually kills the host. The use of immune checkpoint inhibitors (ICI) or adoptive transfer of tumor-infiltrating lymphocytes (TILs) have led to advanced immunotherapies which can be successful in some patients with certain types of cancers. Even though these immunotherapies have revolutionized modern cancer treatments, achieving tumor eradication remains a major challenge.1 2 When ICI and TIL therapies are effective, cancer-specific neoantigens seem to be the important immunological targets.3 4 An alternative approach isolates T cell receptors (TCRs) from neoantigen-specific T cells for adoptive transfer of TCR-engineered peripheral blood T cells (TCR-T cell therapy).5 6 We wanted to determine the requirements needed for TCR-T cell therapy to eradicate established solid tumors. Methods We used the UV-induced, primary and autochthonous cancer cell line 6132A and its well-studied neoantigen mL9.⁷ We isolated a TCR from mL9-specific CD4⁺ T cells (CD4TCR) and another TCR from 6132A-specific CD8⁺ T cells (CD8TCR) generated from the spleen of 6132A–immune mice. We cloned the CD4TCR and the CD8TCR into retroviral vectors and evaluated in vitro and in vivo responses of TCR-engineered CD4⁺ and CD8⁺ T cells. Results The CD4TCR recognized the immunosuppressive tumor stroma and caused destruction followed by long-term growth arrest of tumors formed by the MHC class II negative 6132A cancer cells. However, the CD4TCR failed to eradicate the tumors completely. Remarkably, the CD8TCR was ineffective on its own, yet large unmanipulated solid tumors were eradicated when the CD8TCR was combined with the CD4TCR. Thus, direct cancer cell recognition by the CD8TCR together with stromal recognition by the CD4TCR was needed for effective therapy. As underlying mechanism, in vitro analyses suggest a four-cell type interaction between CD4⁺ and CD8⁺ T cells together with stromal APCs and cancer cells leading to effective elimination of cancer cells.⁸ Conclusions CD8TCRs eradicated long-established solid tumors expressing unmanipulated mutant neoantigens only when combined with a CD4TCR. In vitro experiments suggest a four-cell interaction being essential for cancer cell eradication. References • Haslam A, V Prasad. Estimation of the percentage of us patients with cancer who are eligible for and respond to checkpoint inhibitor immunotherapy drugs. JAMA Netw Open, 2019; 2 (5):e192535. • Rosenberg SA, NP Restifo. Adoptive cell transfer as personalized immunotherapy for human cancer. Science, 2015; 348 (6230):62–8. • Tran E, PF Robbins, SA Rosenberg. ‘Final common pathway’ of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol, 2017; 18 (3):255–262. • Gubin MM, et al. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature, 2014; 515 (7528):577–81. • Kim SP, et al. Adoptive cellular therapy with autologous tumor-infiltrating lymphocytes and T-cell receptor-engineered T cells targeting common p53 neoantigens in human solid tumors. Cancer Immunol Res, 2022:OF1-OF15. • Leidner R, et al. Neoantigen T-cell receptor gene therapy in pancreatic cancer. N Engl J Med, 2022; 386 (22):2112–2119. • Monach PA, et al. A unique tumor antigen produced by a single amino acid substitution. Immunity, 1995; 2 (1):45–59. • Wolf SP, et al. One CD4+TCR and One CD8+TCR targeting autochthonous neoantigens are essential and sufficient for tumor eradication. Clin Cancer Res 2024; 30 (8):1642–1654.