Patient-derived castration-resistant prostate cancer model revealed CTBP2 upregulation mediated by OCT1 and androgen receptor.

BACKGROUND: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. METHODS: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. RESULTS: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. CONCLUSIONS: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management.

Development of a chimeric cytokine receptor that captures IL-6 and enhances the antitumor response of CAR-T cells.

The efficacy of chimeric antigen receptor (CAR)-engineered T cell therapy is suboptimal in most cancers, necessitating further improvement in their therapeutic actions. However, enhancing antitumor T cell response inevitably confers an increased risk of cytokine release syndrome associated with monocyte-derived interleukin-6 (IL-6). Thus, an approach to simultaneously enhance therapeutic efficacy and safety is warranted. Here, we develop a chimeric cytokine receptor composed of the extracellular domains of GP130 and IL6RA linked to the transmembrane and cytoplasmic domain of IL-7R mutant that constitutively activates the JAK-STAT pathway (G6/7R or G6/7R-M452L). CAR-T cells with G6/7R efficiently absorb and degrade monocyte-derived IL-6 in vitro. The G6/7R-expressing CAR-T cells show superior expansion and persistence in vivo, resulting in durable antitumor response in both liquid and solid tumor mouse models. Our strategy can be widely applicable to CAR-T cell therapy to enhance its efficacy and safety, irrespective of the target antigen.

Acto3D: an open-source user-friendly volume rendering software for high-resolution 3D fluorescence imaging in biology.

Advances in fluorescence microscopy and tissue-clearing have revolutionised 3D imaging of fluorescently labelled tissues, organs and embryos. However, the complexity and high cost of existing software and computing solutions limit their widespread adoption, especially by researchers with limited resources. Here, we present Acto3D, an open-source software, designed to streamline the generation and analysis of high-resolution 3D images of targets labelled with multiple fluorescent probes. Acto3D provides an intuitive interface for easy 3D data import and visualisation. Although Acto3D offers straightforward 3D viewing, it performs all computations explicitly, giving users detailed control over the displayed images. Leveraging an integrated graphics processing unit, Acto3D deploys all pixel data to system memory, reducing visualisation latency. This approach facilitates accurate image reconstruction and efficient data processing in 3D, eliminating the need for expensive high-performance computers and dedicated graphics processing units. We have also introduced a method for efficiently extracting lumen structures in 3D. We have validated Acto3D by imaging mouse embryonic structures and by performing 3D reconstruction of pharyngeal arch arteries while preserving fluorescence information. Acto3D is a cost-effective and efficient platform for biological research.

High CD62L expression predicts the generation of chimeric antigen receptor T cells with potent effector functions.

Efficient generation of chimeric antigen receptor (CAR) T cells is highly influenced by the quality of apheresed T cells. Healthy donor-derived T cells usually proliferate better than patients-derived T cells and are precious resources to generate off-the-shelf CAR-T cells. However, relatively little is known about the determinants that affect the efficient generation of CAR-T cells from healthy donor-derived peripheral blood mononuclear cells (PBMC) compared with those from the patients' own PBMC. We here examined the efficiency of CAR-T cell generation from multiple healthy donor samples and analyzed its association with the phenotypic features of the starting peripheral blood T cells. We found that CD62L expression levels within CD8+ T cells were significantly correlated with CAR-T cell expansion. Moreover, high CD62L expression within naïve T cells was associated with the efficient expansion of T cells with a stem cell-like memory phenotype, an indicator of high-quality infusion products. Intriguingly, genetic disruption of CD62L significantly impaired CAR-T cell proliferation and cytokine production upon antigen stimulation. Conversely, ectopic expression of a shedding-resistant CD62L mutant augmented CAR-T cell effector functions compared to unmodified CAR-T cells, resulting in improved antitumor activity in vivo. Collectively, we identified surface expression of CD62L as a concise indicator of potent T cell proliferation. CD62L expression is also associated with functional properties of CAR-T cells. These findings are potentially applicable to selecting optimal donors to massively generate CAR-T cell products.

Role of RNA binding proteins of the Drosophila behavior and human splicing (DBHS) family in health and cancer.

RNA-binding proteins (RBPs) play crucial roles in the functions and homoeostasis of various tissues by regulating multiple events of RNA processing including RNA splicing, intracellular RNA transport, and mRNA translation. The Drosophila behavior and human splicing (DBHS) family proteins including PSF/SFPQ, NONO, and PSPC1 are ubiquitously expressed RBPs that contribute to the physiology of several tissues. In mammals, DBHS proteins have been reported to contribute to neurological diseases and play crucial roles in cancers, such as prostate, breast, and liver cancers, by regulating cancer-specific gene expression. Notably, in recent years, multiple small molecules targeting DBHS family proteins have been developed for application as cancer therapeutics. This review provides a recent overview of the functions of DBHS family in physiology and pathophysiology, and discusses the application of DBHS family proteins as promising diagnostic and therapeutic targets for cancers.

Steerable microcatheter for distal access of a giant cavernous carotid artery aneurysm during treatment with Pipeline Embolization Device: A case report and review of the literature.

Background: In the treatment of giant cerebral aneurysms with flow-diverting stents, access to the distal parent artery is critical but occasionally challenging. This article provides our experience with a novel steerable microcatheter in such a situation, as well as a review of the literature. Case Description: A 73-year-old woman presented with right ptosis and external ophthalmoplegia. Magnetic resonance angiography revealed a giant right cavernous internal carotid artery aneurysm. Endovascular treatment was planned with flow diversion, but distal access was not possible using the standard technique. A 2.4-Fr steerable microcatheter, Leonis Mova Selective, was implemented, and by bending the catheter tip toward the distal parent artery, a guidewire could be guided distally. After the catheter exchange, two flow-diverting stents were deployed successfully. Conclusion: Steerable microcatheters may provide an option in treatment with flow-diverting stents for giant cerebral aneurysms where access to the distal parent artery is compromised.

Delivery of a BET protein degrader via a CEACAM6-targeted antibody-drug conjugate inhibits tumour growth in pancreatic cancer models.

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody-drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

Gene editing technology to improve antitumor T-cell functions in adoptive immunotherapy.

Adoptive immunotherapy, in which tumor-reactive T cells are prepared in vitro for adoptive transfer to the patient, can induce an objective clinical response in specific types of cancer. In particular, chimeric antigen receptor (CAR)-redirected T-cell therapy has shown robust responses in hematologic malignancies. However, its efficacy against most of the other tumors is still insufficient, which remains an unmet medical need. Accumulating evidence suggests that modifying specific genes can enhance antitumor T-cell properties. Epigenetic factors have been particularly implicated in the remodeling of T-cell functions, including changes to dysfunctional states such as terminal differentiation and exhaustion. Genetic ablation of key epigenetic molecules prevents the dysfunctional reprogramming of T cells and preserves their functional properties.Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based gene editing is a valuable tool to enable efficient and specific gene editing in cultured T cells. A number of studies have already identified promising targets to improve the therapeutic efficacy of CAR-T cells using genome-wide or focused CRISPR screening. In this review, we will present recent representative findings on molecular insights into T-cell dysfunction and how genetic modification contributes to overcoming it. We will also discuss several technical advances to achieve efficient gene modification using the CRISPR and other novel platforms.

Exploring androgen receptor signaling pathway in prostate cancer: A path to new discoveries.

Androgen deprivation therapy has achieved significant success in treating prostate cancer through strategies centered on the androgen receptor. However, the emergence of castration-resistant prostate cancer highlights this therapy limitation, underscoring the need to elucidate the mechanisms of treatment resistance. This review aimed to focus on multifaceted resistance mechanisms, including androgen receptor overexpression, splice variants, missense mutations, the involvement of the glucocorticoid receptor, and alterations in coregulators and transcription factors, revealing their roles in castration-resistant prostate cancer progression. These mechanisms promote cell survival and proliferation, depending on the androgen receptor signaling pathway, leading to resistance to conventional therapies. Amplification and mutations in the androgen receptor gene facilitate selective adaptation in treatment-resistant cells, consequently diminishing therapeutic efficacy. Furthermore, the activation of glucocorticoid receptors and aberrant regulation of specific coregulators and transcription factors contribute to the activation of androgen receptor-independent signaling pathways, promoting cell survival and proliferation. These findings hold promise for identifying new targets for treating castration-resistant prostate cancer and developing personalized treatment strategies. The development of future therapies will hinge on precisely targeting the androgen receptor signaling pathway, necessitating a deeper understanding of the molecular targets unique to castration-resistant prostate cancer.

EBAG9-deficient mice display decreased bone mineral density with suppressed autophagy.

Estrogen receptor-binding fragment associated antigen 9 (EBAG9) exerts tumor-promoting effects by inducing immune escape. We focused on the physiological functions of EBAG9 by investigating the bone phenotypes of Ebag9-knockout mice. Female Ebag9-knockout mice have fragile bones with lower bone mineral density (BMD) compared with wild-type mice. Histomorphometric analyses demonstrated that lower BMD was mainly caused by decreased bone formation. Serum bone turnover markers showed that enhanced bone resorption also contributed to this phenotype. We revealed that EBAG9 promoted autophagy in both osteoblastic and osteoclastic lineages. In addition, the knockdown of Tm9sf1, a gene encoding a protein that functionally interacts with EBAG9, suppressed autophagy and osteoblastic differentiation of the murine preosteoblastic cell line MC3T3-E1. Finally, overexpression of TM9SF1 rescued the suppression of autophagy caused by the silencing of Ebag9. These results suggest that EBAG9 plays a physiological role in bone maintenance by promoting autophagy together with its interactor TM9SF1.

Optimal chest compression for cardiac arrest until the establishment of ECPR: Secondary analysis of the SAVE-J II study.

INTRODUCTION: The widespread incorporation of extracorporeal cardiopulmonary resuscitation (ECPR) for out-of-hospital cardiac arrest requires the delivery of effective and high-quality chest compressions prior to the initiation of ECPR. The aim of this study was to evaluate and compare the effectiveness of mechanical and manual chest compressions until the initiation of ECPR. METHODS: This study was a secondary analysis of the Japanese retrospective multicenter registry "Study of Advanced Life Support for Ventricular Fibrillation by Extracorporeal Circulation II (SAVE-J II)". Patients were divided into two groups, one receiving mechanical chest compressions and the other receiving manual chest compressions. The primary outcome measure was mortality at hospital discharge, while the secondary outcome was the cerebral performance category (CPC) score at discharge. RESULTS: Of the 2157 patients enrolled in the SAVE-J II trial, 453 patients (329 in the manual compression group and 124 in the mechanical compression group) were included in the final analysis. Univariate analysis showed a significantly higher mortality rate at hospital discharge in the mechanical compression group compared to the manual compression group (odds ratio [95% CI] = 2.32 [1.34-4.02], p = 0.0026). Multivariate analysis showed that mechanical chest compressions were an independent factor associated with increased mortality at hospital discharge (adjusted odds ratio [95% CI] = 2.00 [1.11-3.58], p = 0.02). There was no statistically significant difference in CPC between the two groups. CONCLUSION: For patients with out-of-hospital cardiopulmonary arrest who require ECPR, extreme caution should be used when performing mechanical chest compressions.

G-protein signaling of oxytocin receptor as a potential target for cabazitaxel-resistant prostate cancer.

Although the treatment armamentarium for patients with metastatic prostate cancer has improved recently, treatment options after progression on cabazitaxel (CBZ) are limited. To identify the mechanisms underlying CBZ resistance and therapeutic targets, we performed single-cell RNA sequencing of circulating tumor cells (CTCs) from patients with CBZ-resistant prostate cancer. Cells were clustered based on gene expression profiles. In silico screening was used to nominate candidate drugs for overcoming CBZ resistance in castration-resistant prostate cancer. CTCs were divided into three to four clusters, reflecting intrapatient tumor heterogeneity in refractory prostate cancer. Pathway analysis revealed that clusters in two cases showed up-regulation of the oxytocin (OXT) receptor-signaling pathway. Spatial gene expression analysis of CBZ-resistant prostate cancer tissues confirmed the heterogeneous expression of OXT-signaling molecules. Cloperastine (CLO) had significant antitumor activity against CBZ-resistant prostate cancer cells. Mass spectrometric phosphoproteome analysis revealed the suppression of OXT signaling specific to CBZ-resistant models. These results support the potential of CLO as a candidate drug for overcoming CBZ-resistant prostate cancer via the inhibition of OXT signaling.

Rupture Risk Factors and Strategies for Unruptured Distal Anterior Cerebral Artery Aneurysms.

BACKGROUND: Distal anterior cerebral artery (dACA) aneurysms are rare. Ruptured dACA aneurysms typically present with subarachnoid hemorrhage in conjunction with intracerebral hematoma and cause neurological deterioration. This study aimed to determine their risk of rupture and examine associated factors. METHODS: We retrospectively analyzed patients with dACA aneurysms to compare patient and aneurysm characteristics between ruptured and unruptured aneurysms. Clinical outcome was used the modified Rankin scale. Univariate analyses were performed to identify rupture risk factors. RESULTS: One hundred three patients with dACA aneurysms were examined (51 ruptured and 52 unruptured). The median aspect ratio of ruptured and unruptured aneurysms was 1.69 and 1.22, respectively (P < 0.01). The median maximum diameter of ruptured and unruptured aneurysms was 5.2 and 3.1 mm, respectively (P < 0.01). The median size ratio of ruptured and unruptured aneurysms was 3.32 and 2.17, respectively (P < 0.01). Maximum diameter was <5 mm in 45.2% of ruptured dACA aneurysms. dACA aneurysm, showing size ratio >2.4 and aspect ratio >1.4, had ruptured in 71.4% and 78.6%, respectively. We suggested that these are the threshold of size ratio and aspect ratio for rupture of dACA aneurysms. A total percentatge of 78.1% of aneurysms with aspect ratio >1.4 and size ratio >2.4 had ruptured. CONCLUSIONS: Distal anterior cerebral artery (dACA) aneurysms may rupture, even when small. We found a significant difference between ruptured and unruptured aneurysms with respect to maximum diameter, aspect ratio, and size ratio. Treatment for small aneurysms should be considered based on size ratio and aspect ratio, not just size.

Establishment of serological neutralizing tests using pseudotyped viruses for comprehensive detection of antibodies against all 18 lyssaviruses.

Rabies is a fatal zoonotic, neurological disease caused by rabies lyssavirus (RABV) and other lyssaviruses. In this study, we established novel serological neutralizing tests (NT) based on vesicular stomatitis virus pseudotypes possessing all 18 known lyssavirus glycoproteins. Applying this system to comparative NT against rabbit sera immunized with current RABV vaccines, we showed that the current RABV vaccines fail to elicit sufficient neutralizing antibodies against lyssaviruses other than to those in phylogroup I. Furthermore, comparative NT against rabbit antisera for 18 lyssavirus glycoproteins showed glycoproteins of some lyssaviruses elicited neutralizing antibodies against a broad range of lyssaviruses. This novel testing system will be useful to comprehensively detect antibodies against lyssaviruses and evaluate their cross-reactivities for developing a future broad-protective vaccine.

Recent insights on the clinical, pathological, and molecular features of intraductal carcinoma of the prostate.

Intraductal carcinoma of the prostate, a unique histopathologic entity that is often observed (especially in advanced prostate cancer), is characterized by the proliferation of malignant cells within normal acini or ducts surrounded by a basement membrane. Intraductal carcinoma of the prostate is almost invariably associated with an adjacent high-grade carcinoma and is occasionally observed as an isolated subtype. Intraductal carcinoma of the prostate has been demonstrated to be an independent poor prognostic factor for all stages of cancer, whether localized, de novo metastatic, or castration-resistant. It also has a characteristic genetic profile, including high genomic instability. Recognizing and differentiating it from other pathologies is therefore important in patient management, and morphological diagnostic criteria for intraductal carcinoma of the prostate have been established. This review summarizes and outlines the clinical and pathological features, differential diagnosis, molecular aspects, and management of intraductal carcinoma of the prostate, as described in previous studies. We also present a discussion and future perspectives regarding intraductal carcinoma of the prostate.

Stress granule-mediated RNA regulatory mechanism in Alzheimer's disease.

Living organisms experience a range of stresses. To cope effectively with these stresses, eukaryotic cells have evolved a sophisticated mechanism involving the formation of stress granules (SGs), which play a crucial role in protecting various types of RNA species under stress, such as mRNAs and long non-coding RNAs (lncRNAs). SGs are non-membranous cytoplasmic ribonucleoprotein (RNP) granules, and the RNAs they contain are translationally stalled. Importantly, SGs have been thought to contribute to the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD). SGs also contain multiple RNA-binding proteins (RBPs), several of which have been implicated in AD progression. SGs are transient structures that dissipate after stress relief. However, the chronic stresses associated with aging lead to the persistent formation of SGs and subsequently to solid-like pathological SGs, which could impair cellular RNA metabolism and also act as a nidus for the aberrant aggregation of AD-associated proteins. In this paper, we provide a comprehensive summary of the physical basis of SG-enriched RNAs and SG-resident RBPs. We then review the characteristics of AD-associated gene transcripts and their similarity to the SG-enriched RNAs. Furthermore, we summarize and discuss the functional implications of SGs in neuronal RNA metabolism and the aberrant aggregation of AD-associated proteins mediated by SG-resident RBPs in the context of AD pathogenesis. Geriatr Gerontol Int 2024; 24: 7-14.

Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells.

Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.

Identification of DNA damage response-related genes as biomarkers for castration-resistant prostate cancer.

Although hormone therapy is effective for the treatment of prostate cancer (Pca), many patients develop a lethal type of Pca called castration-resistant prostate cancer (CRPC). Dysregulation of DNA damage response (DDR)-related genes leads to Pca progression. Here, we explored DDR-related signals upregulated in CRPC tissues. We analyzed the gene expression profiles in our RNA-sequence (RNA-seq) dataset containing benign prostate, primary Pca, and CRPC samples. We identified six DDR-related genes (Ribonuclease H2 Subunit A (RNASEH2A), replication factor C subunit 2 (RFC2), RFC4, DNA Ligase 1 (LIG1), DNA polymerase D1 (POLD1), and DNA polymerase E4 (POLE4)) that were upregulated in CRPC compared with Pca tissues. By analyzing public databases and validation studies, we focused on RFC2 as a new biomarker. Functional analysis demonstrated that silencing of RFC2 expression inhibited cell proliferation and induced the expression of DNA damage and apoptosis markers in CRPC model cells. Furthermore, immunohistochemical (IHC) analysis revealed that high expression of RFC2 protein correlated with poor prognosis in patients with Pca and increased expression in CRPC tissues compared with localized Pca. Thus, our study suggests that six DDR-related genes would be important for Pca progression. RFC2 could be a useful biomarker associated with poor outcomes of patients with Pca.

Strategic Approach to Heterogeneity Analysis of Cutaneous Adnexal Carcinomas Using Computational Pathology and Genomics.

Cutaneous adnexal tumors are neoplasms that arise from skin appendages. Their morphologic diversity and phenotypic variability with rare progression to malignancy make them difficult to diagnose and classify, and there is currently no established treatment strategy. To overcome these difficulties, this study investigated the transcription factor SOX9 expression, morphology, and genetics of skin adnexal tumors for understanding their biology, especially their histogenesis. We showed that cutaneous adnexal tumors and their nontumor counterparts of skin and appendages exhibit expression patterns similar to that of SOX9. Its expression intensity and pattern, as well as histopathologic evaluation of tumors, were analyzed using digital images of 69 normal skin adnexal 9-type organs and 185 skin adnexal 29-type tumors as references. It was possible to distinguish basal cell carcinoma from squamous cell carcinoma, sebaceous carcinoma, and pilomatrixoma with significant differences, along with porocarcinoma from squamous cell carcinoma. Furthermore, unsupervised machine learning "computational pathology" was used to derive a multiregion whole-exome sequencing fusion method termed "genocomputed pathology." The genocomputed pathology of three representable adnexal carcinomas (porocarcinoma, hidradenocarcinoma, and spiradenocarcinoma) was evaluated for total nine cases. We showed that there was more heterogeneity than expected within the tumors as well as the coexistence of components lacking driver fusion genes. The presence or absence of potential driver genes, such as PIK3CA, YAP1, and PTEN, in each region was identified, highlighting a therapeutic strategy for cutaneous adnexal carcinoma encompassing heterogeneous tumors.

Long Term Follow-Up Study of a Randomized, Open-Label, Uncontrolled, Phase I/II Study to Assess the Safety and Immunogenicity of Intramuscular and Intradermal Doses of COVID-19 DNA Vaccine (AG0302-COVID19).

Pharmacological studies have demonstrated antibody production and infection prevention with an intradermal coronavirus disease 2019 (COVID-19) DNA vaccine (AG0302-COVID-19). This clinical trial aimed to investigate the safety and immunogenicity of high doses of AG0302-COVID19 when injected intramuscularly and intradermally. Healthy adults were randomly divided into three intramuscular vaccination groups (2 mg, three times at 2-week intervals; 4 mg, twice at 4-week intervals; and 8 mg, twice at 4-week intervals) and two intradermal groups (1 mg, three times at 2-week intervals or twice at 4-week intervals). After a one-year follow-up, no serious adverse events were related to AG0302-COVID-19. At Week 52, the changes in the geometric mean titer (GMT) ratios of the anti-S antibodies were 2.5, 2.4, and 3.2 in the 2, 4, and 8 mg intramuscular groups, respectively, and 3.2 and 5.1 in the three times and twice injected intradermal groups, respectively. The number of INF-γ-producing cells responsive to S protein increased after the first dose and was sustained for several months. AG0302-COVID-19 showed an acceptable safety profile, but the induction of a humoral immune response was insufficient to justify progressing to a Phase 3 program.