The Diversity of Long-Term Persistent WT1-Specific Cytotoxic T Lymphocytes after Wilms' Tumor 1 Peptide Vaccination

[Introduction] Remarkable advances have been made in cancer immunotherapy, including the development of peptide-based cancer vaccines. Wilms' tumor 1 (WT1) is one of the cancer-testis antigens, and the WT1 gene is overexpressed in hematologic malignancies. Several clinical trials of WT1 peptide vaccines showed promising efficacy and high safety of the vaccines for hematologic malignancies. In these trials, immunological responses were assessed within 2 years after vaccination, and the transient WT1-specific immune response observed in many cases early after vaccination was confirmed. However, the long-term durability of the response of WT1-specific CD8+ cytotoxic T lymphocytes (CTLs) after peptide vaccine therapy and the T-cell receptor (TCR) diversity in those CTLs has not been clarified. More than 10 years ago, a patient with chronic myeloid leukemia (CML) received WT1 peptide vaccination after the failure of tyrosine kinase inhibitor therapy. After vaccination, WT1-specific CD8+ CTLs were observed. We continued the immunological assessment of the patient for more than 10 years after the WT1 peptide vaccination. Herein, we report our findings from the long-term monitoring of WT1-specific CTLs in the patient with CML and describe the results of our detailed analysis, including the functionality and clonality of the CTLs. [Methods] After obtaining written consent from a patient whose CML was difficult to control by imatinib, HLA-A*24:02-restricted modified-type WT1 peptide (WT1 peptide; 9 mer peptide of CYTWNGMNL) was administered to the patient. Post-vaccination, we followed up with the patient. Immune monitoring was performed using a WT1 tetramer assay after mixed lymphocyte peptide culture (MLPC assay). The limiting-dilution (mononuclear cells divided into 20 wells or more, equally containing 3 × 10 5 cells), 2-week cultures with WT1 peptide stimulation and counting of "positive wells" containing expanded WT1 tetramer+ CD8+ T cells were performed for the MLPC assay. The MLPC assay was used to detect functional WT1-specific CD8+ T cells that can expand in response to the WT1 peptide and estimate the frequency of these WT1-specific CD8+ T cells among all CD8+ T cells. For the functionality of WT1-specific CD8+ T cells, we evaluated WT1-specific cytotoxicity and cytokine production in the presence and absence of WT1 peptide pulse to T2A24 cells transfected with the GFP gene (T2A24-GFP). The phenotype and TCR of the WT1-specific CD8+ T cells expanded by MLPC were analyzed using flow cytometry and next-generation sequencing, respectively. [Results] After the WT1 peptide vaccination, the copy numbers of major bcr-abl transcripts gradually decreased, and a therapy free remission was achieved in the patient. No severe adverse effects were observed. The estimated frequency of WT1-specific CD8+ T cells peaked in the third year after vaccination (27 cells per 10 6 CD8+ T cells, 0.00027%) and then declined to 1 - 5 per 10 6 CD8+ T cells at 13 years after vaccination. The WT1-specific CD8+ T cells showed that WT1 peptide-specific cytotoxicity and WT1 peptide-specific IFN-γ release in vitro. These WT1-specific CTLs had different TCRs in each MLPC well. This result was confirmed by three independent analyses, and no common TCRs were detected. Twelve different TCRs were detected in the three analyses. [Conclusion] The WT1 peptide vaccine successfully generated long-lasting and diverse WT1-specific immune responses in a patient with CML. The WT1 peptide vaccine may be a efficient immune therapy for CML patients. Disclosures No relevant conflicts of interest to declare.

IMMU-07. A PHASE I STUDY OF MULTIPLE PEPTIDE TUMOR-ASSOCIATED ANTIGEN VACCINES IN NEWLY DIAGNOSED GLIOBLASTOMA

INTRODUCTION Targeting tumor-associated antigens (TAAs) via peptide vaccination has been tested against malignant glioma with minimal success. Poor responses are attributed to relatively low antigen level expression of the TAA and insufficient CD8+ T cell responses. Including a universal class II epitope that provides CD4+ T cell help towards CD8+ responses has been tested with the immunogenic tetanus toxoid epitope P30, but P30 has been employed as a separate peptide in this regard. The current study will employ targeting of three major glioma TAAs: EphA2, pp65 from Cytomegalovirus, and survivin. The ability to induce more potent TAA-specific immune responses will be tested using two novel strategies: P30-linked TAA peptides and a combinatorial vaccination of the linked peptides (P30-EPS). HYPOTHESES In Evaluation of Tumor Associated P30-Peptide Antigen I (ETAPA-I), P30-EPS is anticipated to have an acceptable toxicity profile. Multi-peptide vaccination is thought to bypass tumor heterogeneity and selection of antigen-negative clones, known as antigen escape. Moreover, the administration of EPS covalently linked to P30 is anticipated to increase the magnitude of antigen-specific immune responses and elicit both CD4- and CD8-mediated immune recognition. TRIAL DESIGN/OBJECTIVES A maximum of 24 patients with newly diagnosed, unmethylated WHO grade IV glioma will be treated. Following resection and standard of care chemoradiation, patients will be vaccinated serially during the priming phase (Day 1-22) and booster phase (Day 84 and 140). All P30-EPS vaccines during priming and boosting phases are co-administered with the adjuvant Hiltonol (Oncovir, poly-ICLC), and patients self-administer Hiltonol throughout the booster phase. The primary endpoint will evaluate the safety profile of P30-EPS. Secondary objectives include polyfunctional T-cell responses specific to EphA2, pp65 and survivin, TCR diversity, and survival. CONCLUSION We describe a study that employs known TAA targets of malignant glioma with the novel strategy of combinatorial class II-linked peptide vaccination.

Immunotherapy for recurrent glioblastoma: practical insights and challenging prospects

Glioblastoma (GB) is the most common high-grade intracranial malignant tumor with highly malignant biological behavior and a high recurrence rate. Although anti-PD-1/PD-L1 antibodies have achieved significant survival benefits in several kinds of solid tumors, the phase III clinical trial Checkmate 143 demonstrated that nivolumab, which targets PD-1, did not achieve survival benefits compared with bevacizumab in recurrent glioblastoma (rGB) patients. Nevertheless, neoadjuvant anti-PD-1 therapy followed by surgery and adjuvant anti-PD-1 therapy could effectively activate local and systemic immune responses and significantly improve the OS of rGB patients. Furthermore, several studies have also confirmed the progress made in applying tumor-specific peptide vaccination or chimeric antigen receptor-T (CAR-T) cell therapy to treat rGB patients, and successes with antibodies targeting other inhibitory checkpoints or costimulatory molecules have also been reported. These successes inspired us to explore candidate combination treatments based on anti-PD-1/PD-L1 antibodies. However, effective predictive biomarkers for clinical efficacy are urgently needed to avoid economic waste and treatment delay. Attempts to prolong the CAR-T cell lifespan and increase T cell infiltration through engineering techniques are addressing the challenge of strengthening T cell function. In this review, we describe the immunosuppressive molecular characteristics of rGB; clinical trials exploring anti-PD-1/PD-L1 therapy, tumor-specific peptide vaccination, and CAR-T cell therapy; candidate combination strategies; and issues related to strengthening T cell function.

Peptide Vaccination against Cytomegalovirus Induces Specific T Cell Response in Responses in CMV Seronegative End-Stage Renal Disease Patients

Introduction: Cytomegalovirus (CMV) reactivation occurs in seronegative patients after solid organ transplantation (SOT) particularly from seropositive donors and can be lethal. Generation of CMV-specific T cells helps to prevent CMV reactivation. Therefore, we initiated a clinical phase I CMVpp65 peptide vaccination trial for seronegative end-stage renal disease patients waiting for kidney transplantation. Methods: The highly immunogenic nonamer peptide NLVPMVATV derived from CMV phosphoprotein 65(CMVpp65) in a water-in-oil emulsion (Montanide™) plus imiquimod (Aldara™) as an adjuvant was administered subcutaneously four times biweekly. Clinical course as well as immunological responses were monitored using IFN-γ ELISpot assays and flow cytometry for CMV-specific CD8+ T cells. Results: Peptide vaccination was well tolerated, and no drug-related serious adverse events were detected except for Grade I–II local skin reactions. Five of the 10 patients (50%) mounted any immune response (responders) and 40% of the patients presented CMV-specific CD8+ T cell responses elicited by these prophylactic vaccinations. No responders experienced CMV reactivation in the 18 months post-transplantation, while all non-responders reactivated. Conclusion: CMVpp65 peptide vaccination was safe, well tolerated, and clinically encouraging in seronegative end-stage renal disease patients waiting for kidney transplantation. Further studies with larger patient cohorts are planned.

The peptide vaccine of the future

The approach of peptide-based anti-cancer vaccination has proven the ability to induce cancer-specific immune responses in multiple studies for various cancer entities. However, clinical responses remain so far limited to single patients and broad clinical applicability was not achieved. Therefore, further efforts are required to improve peptide vaccination in order to integrate this low side effect therapy into the clinical routine of cancer therapy. To design clinically effective peptide vaccines in the future, different issues have to be addressed and optimized comprising antigen target selection as well as choice of optimal adjuvants and vaccination schedules. Furthermore, the combination of peptide-based vaccines with other immuno- and molecular targeted therapies as well as the development of predictive biomarkers could further improve efficacy. In this review, current approaches in the development of peptide-based vaccines and critical implications for optimal vaccine design are discussed.

Identification of HLA-A*02:01-restricted candidate epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2 that may be natural targets of CD8+ T cell recognition in vivo

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-γ-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three response patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines. IMPORTANCE For the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three reaction patterns, suggesting the existence of an immunodominance hierarchy following peptide vaccination, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.