Induction Of Cytotoxic T Lymphocytes Research Articles

A phase 3, randomized, double-blind, multicenter, placebo-controlled study of S-588410, a five-peptide cancer vaccine as an adjuvant therapy after curative resection in patients with esophageal squamous cell carcinoma.

S-588410, a cancer peptide vaccine (CPV), comprises five HLA-A*24:02-restricted peptides from five cancer-testis antigens. In a phase 2 study, S-588410 was well-tolerated and exhibited antitumor efficacy in patients with urothelial cancer. Therefore, we aimed to evaluate the efficacy, immune response, and safety of S-588410 in patients with completely resected esophageal squamous cell carcinoma (ESCC). This phase 3 study involved patients with HLA-A*24:02-positive and lymph node metastasis-positive ESCC who received neoadjuvant therapy followed by curative resection. After randomization, patients were administered S-588410 and placebo (both emulsified with Montanide™ ISA 51VG) subcutaneously. The primary endpoint was relapse-free survival (RFS). The secondary endpoints were overall survival (OS), cytotoxic T-lymphocyte (CTL) induction, and safety. Statistical significance was tested using the one-sided weighted log-rank test with the Fleming-Harrington class of weights. A total of 276 patients were randomized (N = 138/group). The median RFS was 84.3 and 84.1weeks in the S-588410 and placebo groups, respectively (P = 0.8156), whereas the median OS was 236.3weeks and not reached, respectively (P = 0.6533). CTL induction was observed in 132/134 (98.5%) patients who received S-588410 within 12weeks. Injection site reactions (137/140 patients [97.9%]) were the most frequent treatment-emergent adverse events in the S-588410 group. Prolonged survival was observed in S-588410-treated patients with upper thoracic ESCC, grade 3 injection-site reactions, or high CTL intensity. S-588410 induced immune response and had acceptable safety but failed to reach the primary endpoint. A high CTL induction rate and intensity may be critical for prolonging survival during future CPV development.

Abstract 6554: Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, with a very poor prognosis and limited therapeutic options. Repression of the MHC-I and reduced tumor infiltration of cytotoxic T-lymphocytes is often associated with immunotherapy resistance in SCLC. Lurbinectedin is FDA-approved as a second-line treatment for SCLC and we previously showed lurbinectedin as an effective therapeutic strategy in SCLC preclinical models. There are ongoing clinical trials combining immune checkpoint blockade with lurbinectedin in ES-SCLC. However, there is no insight into the effect of lurbinectedin on the immune microenvironment in SCLC. In this study, we evaluated the effect of lurbinectedin on the immune microenvironment and the anti-tumor effect with or without PD-L1 blockade. Results: To determine the effect of lurbinectedin in SCLC models, we treated immunocompetent flank RPP (Rb1, Trp53, and p130) and RPM (Rb1, Trp53, and MYCT58A) tumor-bearing mice with lurbinectedin (0.2 mg/kg, 1/7days) and/or anti-PD-L1 (300µg, 1/7days) for 3 weeks. Mice treated with anti-PD-L1 alone showed no anti-tumor response, and single-agent lurbinectedin caused a delay in tumor growth. However, 6 of the 10 mice treated with the combination of lurbinectedin and anti-PD-L1, had a complete response (100% reduction) and the other 4 had 90% tumor regression. Tumors were resected and analyzed by multicolor flow cytometry for changes in tumor-infiltrating lymphocytes (TILs). Furthermore, analysis of immune cells infiltrating into the tumors post- treatment showed significant induction of cytotoxic T-cells and a reduction of exhausted and regulatory T cells in the combination treatment arm. Similarly, pro-inflammatory M1 type macrophages and dendritic cells were increased, while immunosuppressive M2 type macrophages and MDSC cells were decreased. These effects are consistent with data showing that lurbinectedin treatment leads to an activation of the cGAS/STING pathway, type I/II interferons (IFNα/β), and pro-inflammatory chemokines (CCL5, CXCL10) in tumors. Interestingly, lurbinectedin treatment led to significant upregulation of mRNA and surface expression of MHC class-I genes (HLA-A/B/C) in vitro and in vivo. Finally, bulk RNA sequencing as well as RT-PCR of pre- and post-lurbinectedin treatment demonstrated an increase in DAMPs associated with immunogenic cell death. Conclusion: We provide the first mechanistic insight into the lurbinectedin-induced multimodal immune modulation in SCLC leading to a dramatic anti-tumor activity accompanied by the establishment of a strong anti-tumor immune microenvironment. Since lurbinectedin is already approved as a second-line agent in SCLC, our preclinical data provide a strong rationale for combining this regimen with inhibitors of the PD-L1 pathway and also highlight the immune subsets that the combination targets. Citation Format: Subhamoy Chakraborty, Yazhini Mahendravarman, Utsav Sen, Avisek Banerjee, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Lurbinectedin induces multimodal immune activation and augments the anti-tumor immune response in small-cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6554.

Intratumoral delivery of a highly active form of XCL1 enhances antitumor CTL responses through recruitment of CXCL9-expressing conventional type-1 dendritic cells.

Conventional type 1 dendritic cells (cDC1s) play a crucial role in antitumor immunity through the induction and activation of tumor-specific CD8+ cytotoxic T cells (CTLs). The chemokine XCL1 is a major chemotactic factor for cDC1s and its receptor XCR1 is selectively expressed on cDC1s. Here, we investigated the effect of intratumoral delivery of a highly active form of murine XCL1 (mXCL1-V21C/A59C) on cDC1-mediated antitumor immunity using a hydrophilic gel patch. The hydrophilic gel patch containing mXCL1-V21C/A59C increased cDC1 accumulation in the tumor masses and promoted their migration to the regional lymph nodes, resulting in enhanced induction of tumor-specific CTLs. Tumor-infiltrating cDC1s not only expressed XCR1 but also produced CXCL9, a ligand for CXCR3 which is highly expressed on CTLs and NK cells. Consequently, CTLs and NK cells were increased in the tumor masses of mice treated with mXCL1-V21C/A59C, while immunosuppressive cells such as monocyte-derived suppressive cells and regulatory T cells were decreased. We also confirmed that anti-CXCL9 treatment decreased the tumor infiltration of CTLs. The intratumoral delivery of mXCL1-V21C/A59C significantly decreased tumor growth and prolonged survival in E.G7-OVA and B16-F10 tumor-bearing mice. Furthermore, the antitumor effect of mXCL1-V21CA59C was enhanced in combination with anti-programmed cell death protein 1 treatment. Finally, using The Cancer Genome Atlas database, we found that XCL1 expression was positively correlated with tumor-infiltrating cDC1s and a better prognosis in melanoma patients. Collectively, our findings provide a novel therapeutic approach to enhance tumor-specific CTL responses through the selective recruitment of CXCL9-expressing cDC1s into the tumor masses.

Culture filtrate proteins from BCG act as adjuvants for cytotoxic T lymphocyte induction.

Mycobacterium bovis bacilli Calmette-Guerin (BCG) is a licensed vaccine against tuberculosis. It requires attenuated live bacteria to be effective, possibly because actively secreted proteins play a critical role in inducing anti-tuberculosis immunity. BCG also functions as an effective adjuvant. Moreover, the effects of BCG components as adjuvants are not important as those of attenuated live BCG, which is used in cancer immunotherapy. However, the BCG secreted proteins have not been paid attention in anticancer immunity. To understand mycobacterial secreted proteins' function, we investigate immune responses to BCG culture filtrate proteins (CFP). Here, CFP strongly induce both antigen-specific CD4+ T cells and specific CD8+ T cells, which may be functional cytotoxic T lymphocytes (CTLs). In this study, we clearly demonstrate that CFP acts as an adjuvant for CTL induction against specific co-administered proteins and propose CFP as a new protein adjuvant. The CTL response shows potent anticancer effects in mice. These findings could provide insight into the contribution of mycobacterial secreted proteins in both anticancer and antimycobacterial immunity.

IL18 Receptor Signaling Inhibits Intratumoral CD8+ T-Cell Migration in a Murine Pancreatic Cancer Model.

In pancreatic ductal adenocarcinoma (PDAC), the infiltration of CD8+ cytotoxic T cells (CTLs) is an important factor in determining prognosis. The migration pattern and interaction behavior of intratumoral CTLs are pivotal to tumor rejection. NLRP3-dependent proinflammatory cytokines IL-1β and IL-18 play a prominent role for CTL induction and differentiation. Here, we investigate the effects of T-cellular IL-1R and IL-18R signaling for intratumoral T-cell motility. Murine adenocarcinoma cell line Panc02 was stably transfected with ovalbumin (OVA) and fluorophore H2B-Cerulean to generate PancOVA H2B-Cerulean tumor cells. Dorsal skinfold chambers (DSFC) were installed on wild-type mice, and PancOVA H2B-Cerulean tumor cells were implanted into the chambers. PancOVA spheroids were formed using the Corning® Matrigel®-based 3D cell culture technique. CTLs were generated from OT-1 mice, Il1r-/- OT-1 mice, or Il18r-/- OT-1 mice and were marked with fluorophores. This was followed by the adoptive transfer of CTLs into tumor-bearing mice or the application into tumor spheroids. After visualization with multiphoton microscopy (MPM), Imaris software was used to perform T-cell tracking. Imaris analysis indicates a significantly higher accumulation of Il18r-/- CTLs in PancOVA tumors and a significant reduction in tumor volume compared to wild-type CTLs. Il18r-/- CTLs covered a longer distance (track displacement length) in comparison to wild-type (WT) CTLs, and had a higher average speed (mean track speed). The analysis of instantaneous velocity suggests a higher percentage of arrested tracks (arrests: <4 μm/min) for Il18r-/- CTLs. Our data indicate the contribution of IL-18R signaling to T-cell effector strength, warranting further investigation on phenomena such as intratumoral T-cell exhaustion.

A modulatory effect of L-arginine supplementation on anticancer effects of chemoimmunotherapy in colon cancer-bearing aged mice.

Myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) are increased in cancer-bearing aged hosts. Arginase-I in MDSCs degrades L-arginine, an amino acid required for T cell activation and proliferation. In this study, we compared the therapeutic efficacy of 5-fluorouracil (5-FU)/oxaliplatin (L-OHP) and cyclophosphamide (CP) between young and aged colon cancer-bearing mice. Therapy with 5-FU/L-OHP and CP significantly suppressed the in vivo growth of CT26 and MC38 colon carcinomas in syngeneic young mice, whereas this effect was attenuated in aged mice. L-arginine monotherapy showed no effect in aged mice. However, additional therapy with anti-programmed cell death (PD)-1 antibody and L-arginine supplementation boosted the effect of chemoimmunotherapy in aged mice, and some mice were cured. During all combination therapy, tumor-specific cytotoxic T lymphocytes (CTLs) were generated from mice with non-progressing tumor, but not from those with progressing tumor. Plasma L-arginine levels were lower in aged than young mice, and chemotherapy tended to decrease the plasma L-arginine levels in aged mice. Compared to young mice, CT26-bearing aged mice decreased arginase activity, arginase-I expression, and the proportion of monocytic MDSCs in tumor tissues, whereas contrasting results were observed in MC38-bearing aged mice. Importantly, the induction of tumor-specific CTLs was impaired at lower doses of L-arginine in vitro, and the infiltration of CTLs into CT26 tissues after chemoimmunotherapy was promoted by L-arginine administration in vivo. These results indicate that chemoimmunotherapy was less effective in cancer-bearing aged mice, but that L-arginine supplementation can modulate its therapeutic efficacy via its effect on tumor-specific CTLs.

Dendritic cell-mediated cross presentation of tumor-derived peptides is biased against plasma membrane proteins

BackgroundFor effective tumor elimination, cytotoxic CD8+ T cells must recognize tumor-derived antigens presented on class I major histocompatibility complex (MHC-I). Despite a general association between the expression of immunogenic antigens,...

A Phase 2 Study of S-588410 Maintenance Monotherapy for Platinum-Treated Advanced or Metastatic Urothelial Carcinoma.

Effective maintenance therapy for urothelial carcinoma (UC) is needed to delay progression after first-line chemotherapy. To evaluate S-588410, a cancer peptide vaccine containing five human leukocyte antigen (HLA)-A*24:02-restricted epitope peptides derived from five cancer-testis antigens (DEPDC1, MPHOSPH1, URLC10, CDCA1, and KOC1) in chemotherapy-treated, clinically stable patients with advanced or metastatic UC. This open-label, international, phase 2 trial enrolled patients with UC who had completed≥4 cycles of first-line platinum-containing chemotherapy without disease progression. Forty-five HLA-A*24:02-positive patients received subcutaneous injections of S-588410 (Montanide ISA 51 VG with 1 mg/mL of each peptide) weekly for 12 weeks then once every 2 weeks thereafter for up to 24 months. Thirty-six HLA-A*24:02-negative patients did not receive S-588410 (observation group). The primary endpoint was the rate of cytotoxic T-lymphocyte (CTL) induction against≥1 of the peptides at 12 weeks. The CTL induction rate in the S-588410 group was 93.3% (p < 0.0001, one-sided binomial test with a rate of≤50% as the null hypothesis). The antitumor response rate was 8.9% in the S-588410 group and 0% in the observation group; median progression-free survival was 18.1 versus 12.5 weeks and median overall survival was 71.0 versus 99.0 weeks, respectively. The most frequent treatment-emergent adverse event was injection-site reactions (47 events, grades 1-3) reported in 93.3% (n = 42/45) of participants. S-588410 demonstrated a high CTL induction rate, acceptable safety profile, and modest clinical response, as maintenance therapy in participants with advanced or metastatic UC who had received first-line platinum-based chemotherapy (EudraCT 2013-005274-22).

Antiviral cytotoxic T lymphocyte responses for long term prognosis of corneal infection by cytomegalovirus in immunocompetent subjects

Ocular cytomegalovirus (CMV) infections in immunocompetent individuals are rare, but its activation can cause chronic and relapsing inflammation in anterior segment of the eye resulting in loss of corneal clarity and glaucoma. Fifty five patients with anterior segment CMV infection were assessed for their clinical characteristics, and CMV corneal endotheliitis was found to cause significant loss of corneal endothelial cells. The disease duration with recurrences was significantly correlated with the maximum intraocular level of CMV DNA. To examine why CMV is activated in healthy immunocompetent individuals and causing corneal endothelial cell damage, assays of cytotoxic T cells (CTLs) which directly target infected corneal endothelial cells were performed for 9 HLA-matched CMV corneal endotheliitis patients (HLA-A*2402). When the cell loss was analyzed for associations with CTL responses, CMV-induced endothelial cell damage was mitigated by pp65-specific CTL induction. The recurrence-free time was also prolonged by pp65-specific CTL induction (hazard ratio (HR): 0.93, P = 0.01). In contrast, IE1-specific CTL was associated with endothelial cell damage and reduced the time for corneal transplantation (HR: 1.6, P = 0.003) and glaucoma surgery (HR: 1.5, P = 0.001). Collectively, induction of pp65-specific CTL was associated with improved visual prognosis. However, IE1-specific CTL without proper induction of pp65-specific CTL can cause pathological damage leading to the need of surgical interventions.

Multi-Level Computational Modeling of Anti-Cancer Dendritic Cell Vaccination Utilized to Select Molecular Targets for Therapy Optimization.

Dendritic cells (DCs) can be used for therapeutic vaccination against cancer. The success of this therapy depends on efficient tumor-antigen presentation to cytotoxic T lymphocytes (CTLs) and the induction of durable CTL responses by the DCs. Therefore, simulation of such a biological system by computational modeling is appealing because it can improve our understanding of the molecular mechanisms underlying CTL induction by DCs and help identify new strategies to improve therapeutic DC vaccination for cancer. Here, we developed a multi-level model accounting for the life cycle of DCs during anti-cancer immunotherapy. Specifically, the model is composed of three parts representing different stages of DC immunotherapy – the spreading and bio-distribution of intravenously injected DCs in human organs, the biochemical reactions regulating the DCs’ maturation and activation, and DC-mediated activation of CTLs. We calibrated the model using quantitative experimental data that account for the activation of key molecular circuits within DCs, the bio-distribution of DCs in the body, and the interaction between DCs and T cells. We showed how such a data-driven model can be exploited in combination with sensitivity analysis and model simulations to identify targets for enhancing anti-cancer DC vaccination. Since other previous works show how modeling improves therapy schedules and DC dosage, we here focused on the molecular optimization of the therapy. In line with this, we simulated the effect in DC vaccination of the concerted modulation of combined intracellular regulatory processes and proposed several possibilities that can enhance DC-mediated immunogenicity. Taken together, we present a comprehensive time-resolved multi-level model for studying DC vaccination in melanoma. Although the model is not intended for personalized patient therapy, it could be used as a tool for identifying molecular targets for optimizing DC-based therapy for cancer, which ultimately should be tested in in vitro and in vivo experiments.

Role of interferon regulatory factor 7 in corneal endothelial cells after HSV-1 infection

Viral infections of the cornea including herpes simplex virus 1 (HSV-1) cause visual morbidity, and the corneal endothelial cell damage leads to significant visual impairment. Interferon regulatory factor 7 (IRF7) has been identified as a significant regulator in corneal endothelial cells after an HSV-1 infection. To examine the role played by IRF7, the DNA binding domain (DBD) of IRF7 of human corneal endothelial cells (HCEn) was disrupted. An RNAi inhibition of IRF7 and IRF7 DBD disruption (IRF7 ∆DBD) led to an impairment of IFN-β production. Impaired IFN-β production by IRF7 ∆DBD was regained by IRF7 DNA transfection. Transcriptional network analysis indicated that IRF7 plays a role in antigen presentation function of corneal endothelial cells. When the antigen presentation activity of HCEn cells were examined for priming of memory CD8 T cells, IRF7 disruption abolished the anti-viral cytotoxic T lymphocyte (CTL) response which was dependent on the major histocompatibility complex (MHC) class I. To further examine the roles played by IRF7 in CTL induction as acquired immunity, the contribution of IRF7 to MHC class I-mediated antigen presentation was assessed. Analysis of IRF7 ∆DBD cells indicated that IRF7 played an unrecognized role in MHC class I induction, and the viral infection induced-MHC class I induction was abolished by IRF7 disruption. Collectively, the IRF7 in corneal endothelial cells not only contributed to type I IFN response, but also to the mediation of viral infection-induced MHC class I upregulation and priming of CD8 arm of acquired immunity.

Association of HLA-restricted HERV-derived peptide-related cytotoxic T-lymphocyte induction and sex bias in therapeutic responses in gastric cancer: Secondary Analysis of Data from 3 Randomized Trials and an Observational Study from 1977 to 2011

Association of HLA-restricted HERV-derived peptide-related cytotoxic T-lymphocyte induction and sex bias in therapeutic responses in gastric cancer: Secondary Analysis of Data from 3 Randomized Trials and an Observational Study from 1977 to 2011

Quality Verification with a Cluster-Controlled Manufacturing System to Generate Monocyte-Derived Dendritic Cells.

Dendritic cell (DC) vaccines for cancer immunotherapy have been actively developed to improve clinical efficacy. In our previous report, monocyte−derived DCs induced by interleukin (IL)−4 with a low−adherence dish (low−adherent IL-4−DCs: la−IL-4−DCs) improved the yield and viability, as well as relatively prolonged survival in vitro, compared to IL-4−DCs developed using an adherent culture protocol. However, la−IL-4−DCs exhibit remarkable cluster formation and display heterogeneous immature phenotypes. Therefore, cluster formation in la−IL-4−DCs needs to be optimized for the clinical development of DC vaccines. In this study, we examined the effects of cluster control in the generation of mature IL-4−DCs, using cell culture vessels and measuring spheroid formation, survival, cytokine secretion, and gene expression of IL-4−DCs. Mature IL-4−DCs in cell culture vessels (cluster−controlled IL-4−DCs: cc−IL-4−DCs) displayed increased levels of CD80, CD86, and CD40 compared with that of la−IL-4−DCs. cc−IL-4−DCs induced antigen−specific cytotoxic T lymphocytes (CTLs) with a human leukocyte antigen (HLA)−restricted melanoma antigen recognized by T cells 1 (MART−1) peptide. Additionally, cc−IL-4−DCs produced higher levels of IFN−γ, possessing the CTL induction. Furthermore, DNA microarrays revealed the upregulation of BCL2A1, a pro−survival gene. According to these findings, the cc−IL-4−DCs are useful for generating homogeneous and functional IL-4−DCs that would be expected to promote long−lasting effects in DC vaccines.

Effect of IL-15 addition on asbestos-induced suppression of human cytotoxic T lymphocyte induction

BackgroundAsbestos fibers possess tumorigenicity and are thought to cause mesothelioma. We have previously reported that exposure to asbestos fibers causes a reduction in antitumor immunity. Asbestos exposure in the mixed lymphocyte reaction (MLR) showed suppressed induction of cytotoxic T lymphocytes (CTLs), accompanied by a decrease in proliferation of CD8+ T cells. Recently, we reported that asbestos-induced suppression of CTL induction is not due to insufficient levels of interleukin-2 (IL-2). In this study, we continue to investigate the mechanism responsible for the effect of asbestos fibers on the differentiation of CTLs and focus on interleukin-15 (IL-15) which is known to be a regulator of T lymphocyte proliferation.MethodsFor MLR, human peripheral blood mononuclear cells (PBMCs) were cultured with irradiated allogenic PBMCs upon exposure to chrysotile B asbestos at 5 μg/ml for 7 days. After 2 days of culture, IL-15 was added at 1 ng/ml. After 7 days of MLR, PBMCs were collected and analyzed for phenotypic and functional markers of CD8+ T cells with fluorescence-labeled anti-CD3, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CD25, and anti-granzyme B antibodies using flow cytometry. To examine the effect of IL-15 on the expression level of intracellular granzyme B in proliferating and non-proliferating CD8+ lymphocytes, PBMCs were stained using carboxyfluorescein diacetate succinimidyl ester (CFSE) and then washed and used for the MLR.ResultsIL-15 addition partially reversed the decrease in CD3+CD8+ cell numbers and facilitated complete recovery of granzyme B+ cell percentages. IL-15 completely reversed the asbestos-induced decrease in percentage of granzyme B+ cells in both non-proliferating CFSE-positive and proliferating CFSE-negative CD8+ cells. The asbestos-induced decrease in the percentage of CD25+ and CD45RO+ cells in CD8+ lymphocytes was not reversed by IL-15.ConclusionThese findings indicate that CTLs induced upon exposure to asbestos possess dysfunctional machinery that can be partly compensated by IL-15 supplementation, and that IL-15 is more effective in the recovery of proliferation and granzyme B levels from asbestos-induced suppression of CTL induction compared with IL-2.

Phase II open-label study of S-588410 as maintenance monotherapy after first-line platinum-containing chemotherapy in patients with advanced or metastatic urothelial carcinoma.

440 Background: S-588410 is a cancer peptide vaccine composed of 5 human leukocyte antigen (HLA)-A*24:02-restricted epitope peptides derived from 5 cancer-testis antigens: DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1; all of which are highly expressed in urothelial carcinoma. This study aimed to evaluate the effect of S-588410 maintenance therapy on peptide-specific cytotoxic T-lymphocyte (CTL) induction in patients with advanced or metastatic urothelial carcinoma after first-line platinum-based chemotherapy. Methods: An open-label, multicenter phase II trial was performed across 62 sites in Japan, the United Kingdom, France and Bulgaria (EudraCT 2013-005274-22). Eligible patients had completed ≥4 cycles of first-line platinum-based chemotherapy without disease progression. HLA-A*24:02-positive patients received S-588410 (1 mg of each of 5 peptides mixed with Montanide ISA 51 VG) subcutaneously weekly for 12 weeks, then every 2 weeks for up to 2 years. HLA-A*24:02-negative patients were enrolled in an observation group and did not receive study drug. The primary endpoint for the S-588410 group was the CTL induction rate at 12 weeks, defined as the proportion of patients who showed increased CTL activity for ≥1 peptide. Secondary endpoints included CTL induction rate after 1 year, antitumor effect defined by immune-related response criteria, progression-free survival (PFS), overall survival (OS), and safety. Results: A total of 81 patients with platinum-sensitive advanced or metastatic urothelial carcinoma were enrolled (S-588410 group, n=45; observation group, n=36) between April 2014 and November 2017. Most patients were male and Asian with a mean age of 67 years. CTLs were induced in 42 (93.3%) patients who received S-588410 for 12 weeks (P&lt;0.0001, one-sided binomial test where the CTL induction rate is ≤50% as the null hypothesis). The CTL induction rate steadily increased to 95.6% within 48 weeks. CTL activity was high for the DEPDC1, MPHOSPH1 and URLC10 peptides. The response rate (immune-related complete response [CR] or partial response [PR]) was 8.9% (4/45 patients) in the S-588410 group and 0% in the observation group. Tumor imaging showed gradual (PR, n=3) and durable (CR, n=1) tumor shrinkage after ≥36 weeks in the S-588410 group. Median PFS was 18.1 weeks in the S-588410 group and 12.5 weeks in the observation group. Median OS was 71 and 99 weeks, respectively. The most frequent treatment-emergent adverse event was injection site reaction (42/45 patients [93.3%]; Grades 1–3). Pyrexia, rash and pruritus were also observed in the S-588410 group, but not the observation group. Conclusions: S-588410 showed a potent immune response and acceptable safety profile in patients with advanced or metastatic urothelial carcinoma, potentially offering a clinical benefit as post-chemotherapy maintenance therapy. Clinical trial information: EudraCT 2013-005274-22.

Immune Responses and Antitumor Effect through Delivering to Antigen Presenting Cells by Optimized Conjugates Consisting of CpG-DNA and Antigenic Peptide.

Immunotherapy using antigen-specific cytotoxic T lymphocytes (CTLs) has become one of the most attractive strategies for cancer treatment. For the induction of antigen-specific CTLs in vivo, the co-delivery of CpG-DNAs and antigens to the same antigen-presenting cells (APCs) is a promising strategy. In this study, we prepared conjugates consisting of 40mer of CpG-DNA (CpG40) and antigenic peptide (OVA257-264), which have the following distinctive features: (1) multiple CpG motifs in a molecule; (2) cleavage in the cytosol because of the disulfide bonding via cysteine residue between peptide and CpG-DNA; (3) conjugation designed to induce antigen presentation on MHC class I molecules. Immunization with the conjugate CpG40-C-OVA257-264 at the mouse tail base induced strong CTL activity at a very low peptide dose of 20 ng/head. It was found that the conjugates were internalized into C-type mannose receptor 1 (MRC1)-expressing cells in inguinal lymph nodes, indicating that the CpG portion in the conjugate acts as not only an adjuvant for the activation of TLR9 but also a carrier to APCs expressing MRC1. In a tumor-bearing mice model, mice immunized with CpG40-C-OVA257-264 conjugates exhibited long delays in tumor growth compared with those treated with PBS, OVA257-264 alone, or a mixture of CpG40 and OVA257-264. Therefore, CpG-C-peptide conjugates could be a new and effective platform for peptide vaccine for the treatment of cancers and infectious diseases.

Identification of two distinct populations of WT1-specific cytotoxic T lymphocytes in co-vaccination of WT1 killer and helper peptides.

Simultaneous induction of tumor antigen-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs) is required for an optimal anti-tumor immune response. WT1332, a 16-mer WT1-derived helper peptide, induce HTLs in an HLA class II-restricted manner and enhance the induction of WT1-specific CTLs in vitro. However, in vivo immune reaction to WT1332 vaccination in tumor-bearing patients remained unclear. Here, a striking difference in WT1-specific T cell responses was shown between WT1 CTL + WT1 helper peptide and WT1 CTL peptide vaccines in patients with recurrent glioma. WT1-specific CTLs were more strongly induced in the patients who were immunized with WT1 CTL + WT1 helper peptide vaccine, compared to those who were immunized with WT1 CTL vaccine alone. Importantly, a clear correlation was demonstrated between WT1-specific CTL and WT1332-specific HTL responses. Interestingly, two novel distinct populations of WT1-tetramerlow WT1-TCRlow CD5low and WT1-tetramerhigh WT1-TCRhigh CD5high CTLs were dominantly detected in WT1 CTL + WT1 helper peptide vaccine. Although natural WT1 peptide-reactive CTLs in the latter population were evidently less than those in the former population, the latter population showed natural WT1 peptide-specific proliferation capacity comparable to the former population, suggesting that the latter population highly expressing CD5, a marker of resistance to activation-induced cell death, should strongly expand and persist for a long time in patients. These results demonstrated the advantage of WT1 helper peptide vaccine for the enhancement of WT1-specific CTL induction by WT1 CTL peptide vaccine.

Transcutaneous immunization with a highly active form of XCL1 as a vaccine adjuvant using a hydrophilic gel patch elicits long-term CD8+ T cell responses

Memory CD8+ cytotoxic T-lymphocytes (CTLs) play a key role in protective immunity against infection and cancer. However, the induction of memory CTLs with currently available vaccines remains difficult. The chemokine receptor XCR1 is predominantly expressed on CD103+ cross-presenting dendritic cells (DCs). Recently, we have demonstrated that a high activity form of murine lymphotactin/XCL1 (mXCL1-V21C/A59C), a ligand of XCR1, can induce antigen-specific memory CTLs by increasing the accumulation of CD103+ DCs in the vaccination site and the regional lymph nodes. Here, we combined a hydrophilic gel patch as a transcutaneous delivery device and mXCL1-V21C/A59C as an adjuvant to further enhance memory CTL responses. The transcutaneous delivery of ovalbumin (OVA) and mXCL1-V21C/A59C by the hydrophilic gel patch increased CD103+ DCs in the vaccination site and the regional lymph nodes for a prolonged period of time compared with the intradermal injection of OVA and mXCL1-V21C/A59C. Furthermore, the hydrophilic gel patch containing OVA and mXCL1-V21C/A59C strongly induced OVA-specific memory CTLs and efficiently inhibited the growth of OVA-expressing tumors more than the intradermal injection of OVA and mXCL1-V21C/A59C. Collectively, this type of hydrophilic gel patch and a high activity form of XCL1 may provide a useful tool for the induction of memory CTL responses.

Immunization of Pigs with Recombinant Plasmids Containing Genes of Ubiquitinated p30, p54 and CD2v Proteins of African Swine Fever Virus

Abstract Three recombinant plasmid constructs, expressing chimeric proteins containing human ubiquitin fused to an ectodomain of one of the potentially protective proteins (p30, p54 and CD2v) of the attenuated MK-200 strain of African swine fever virus (ASFV), were created as potential inductors of specific antiviral cellular immunity. Three-time immunization of pigs with the mixture of these plasmids led to the formation of virus-specific cytotoxic T-lymphocytes (CTL), but did not induce production of virus-specific antibodies. After challenge with the homologous parental virulent ASFV strain M-78 at a dose of 103 HAD50, all five animals (four immunized pigs and one naïve) fell between the 4th and 7th days post infection. The obtained results demonstrated that induction of CTL did not protect pigs against challenge with the virulent ASFV. Balanced activation of CTL and antibody-mediated cellular mechanisms should be investigated.

ANGI-01 ALTERATION IN IMMUNE REGULATORY CELLS BEFORE AND AFTER TREATMENT BY STUPP REGIMEN WITH OR WITHOUT BEVACIZUMAB FOR GLIOBLASTOMA

BACKGROUNDIn our previous study, bevacizumab (Bev), a humanized anti- vascular endothelial growth factor monoclonal antibody, downregulated the expression of programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) immune checkpoint molecules, suppressed the infiltration of immunosuppressing cells such as regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), and induces cytotoxic T lymphocytes (CTL) infiltration. To explore the possibility that inhibition of immunosuppressive cell infiltration and induction of CTL were attributed to not only Bev alone but also radiation (RT) or temozolomide (TMZ), we re-evaluated those alterations in the tumor tissue obtained from patients before and after the treatment using Stupp regimen (RT concomitant with TMZ) without Bev therapy.MATERIALS & METHODSWe analyzed 10 tumor tissues from 5 patients with GBMs, which were paired samples of pre- and post- standard chemoradiotherapy (Stupp regimen: RT plus concomitant and adjuvant TMZ). Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded tissue of 10 tumors. The sections were stained with anti-Ki-67, anti-VEGF-A, anti-VEGFR1, anti-VEGFR2, anti-CD34, anti-HIF1 alpha, anti-CA9, anti-nestin, anti-PD-1, anti-PD-L1, anti-CD4, anti-CD8, anti-Foxp3, and anti-CD163 antibodies. All expressions were assessed by authors with blinded clinical information.RESULTSImmunohistochemical analyses demonstrated that the expression levels of immune regulatory molecules such as Foxp3, CD163, PD-1, PD-L1, CD4, and CD8 were not significantly changed after the treatment using the Stupp regimen, compared with combinational usage of Bev. In addition, expressions of VEGF/VEGFR, hypoxic markers, and stem cell marker were not altered before and after Stupp regimen, either. Bev persistently inhibited immune suppressive cells and immune checkpoint molecules via down-regulation of VEGF pathway. In contrast, Stupp regimen did not affect immune regulations and tumor microenvironment.CONCLUSIONThese results suggested that immunosupportive effect was caused by Bev administration, leading to the novel combinational treatment strategies, in addition to Stupp regimen.