Pre-vaccination Peripheral Blood Mononuclear Cells Research Articles

Abstract 6537: Single cell transcriptomics uncovers cellular and molecular differences in PBMCs of responders and non-responders to the MUC1 cancer vaccine given in the preventative setting

Abstract Introduction: A single arm trial (NCT007773097) and a double-blind, placebo controlled randomized trial (NCT02134925) were conducted in patients with newly diagnosed advanced colonic adenomas to test the safety and immunogenicity of the MUC1 antigen vaccine and its potential to prevent new adenoma formation. These are the first trials of a non-viral cancer vaccine administered in the absence of cancer. In both trials, the vaccine was safe and strongly immunogenic in 43% and 25% of participants (Responders), respectively. The lack of robust response in a significant number of participants suggested, for the first time, that even in a premalignant setting, the immune system may have already been exposed to regulatory influences that, in the case of the vaccine, determine who does and who does not respond. We hypothesized that there could be molecular and cellular differences in the immune competence between vaccine responders and non-responders, and that they could be identified by studying their pre-vaccination peripheral blood mononuclear cells (PBMCs). Methods: The two MUC1 vaccine trials are described in https://doi.org/10.1101/2022.10.05.22280474 and https://doi.org/10.1158%2F1940-6207.CAPR-12-0275. We performed single cell RNA-sequencing (scRNAseq) on banked pre-vaccination PBMCs from 16 Responders and 16 Non-Responders, determined by anti-MUC1 IgG response. Using differential gene expression (DGE), pathway enrichment, and network estimation analyses, we identified specific cell types, genes, and pathways that differ between responders and non-responders. Results: Pre-vaccination PBMCs from Responders contained a significantly higher percentage of CD4+ naive T cells, while Non-Responders showed significantly higher percentage of CD8+ T effector memory (TEM) cells and a higher percentage of CD16+ monocytes. DGE and gene interaction network analysis showed a higher level of expression of T cell activation genes, such as Fos and Jun, in the CD4+ naive T cells in Responders. Further network analysis showed that these genes were directly connected to response. We also found pre-vaccination specific gene ontology (GO) pathways for translational and transcriptional activity enriched in all cell types in Responders compared to Non-Responders. Conclusion: Our analyses identified candidate biomarkers that are predictive of a preventative cancer vaccine response. Thus, our results can be used for patient selection for vaccine administration. Furthermore, we identified cell type differences and transcriptional pathways that provide information of possible mechanisms of vaccine response. Citation Format: Daniel Y. Yuan, Michelle L. McKeague, Matthew T. Dracz, Olivera J. Finn, Panayiotis V. Benos. Single cell transcriptomics uncovers cellular and molecular differences in PBMCs of responders and non-responders to the MUC1 cancer vaccine given in the preventative setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6537.

IFNL3-adjuvanted HCV DNA vaccine reduces regulatory T cell frequency and increases virus-specific T cell responses

Although direct-acting antiviral (DAA) treatment results in a sustained virologic response (SVR) in most patients with chronic HCV infection, they are at risk of re-infection. Moreover, the immune system is not completely normalized even after SVR (e.g. increased regulatory T [Treg] cell frequency). We developed a DNA vaccine, GLS-6150, to prevent re-infection of patients with DAA-induced SVR and evaluated its safety and immunogenicity in individuals with chronic HCV infection. GLS-6150 consists of plasmids encoding HCV non-structural proteins (NS3-NS5A) and adjuvant IFNL3. The vaccine was administered 4 times at 4-weekly intervals to 3 groups (1, 3, or 6 mg/vaccination; n= 6 per group), followed by a 6 mg boost at 24 weeks (n= 14). Peripheral blood T cell responses were evaluated by interferon (IFN)-γ enzyme-linked immunospot assays, intracellular cytokine staining, and major histocompatibility complex class-I (MHC-I) dextramer staining. Treg cell frequency was assessed by flow cytometry. Severe adverse events or vaccine discontinuation were not reported. The IFN-γ spot-forming cells specific to NS3-NS5A were increased by GLS-6150. Both CD4+ and CD8+ T cells produced multiple cytokines. However, the frequency and phenotype of HCV-specific MHC-I dextramer+CD8+ T cells were not changed. Interestingly, the frequency of Treg cells, particularly activated Treg cells, was decreased by GLS-6150, as expected from previous reports that IFNL3 adjuvants decrease Treg cell frequency. Exvivo IFN-λ3 treatment reduced Treg frequency in pre-vaccination peripheral blood mononuclear cells. Finally, Treg cell frequency inversely correlated with HCV-specific, IFN-γ-producing T cell responses in the study participants. We demonstrate that GLS-6150 decreases Treg cell frequency and enhances HCV-specific T cell responses without significant side effects. A phase I clinical trial of GLS-6150 is currently underway in patients with DAA-induced SVR. NCT02027116. Although direct-acting antivirals (DAAs) are successfully used for the treatment of chronic hepatitis C virus (HCV) infection, a prophylactic HCV vaccine needs to be developed, especially for patients who achieve a sustained virologic response. In the current study, we show that a DNA vaccine (GLS-6150) was safe and increased HCV-specific T cell responses. A clinical trial is underway to test this vaccine in patients with a sustained virologic response following DAA therapy.

Association of haptoglobin genetic polymorphism with overall survival in advanced castration-resistant prostate cancer patients with personalized peptide vaccination

Although a peptide vaccination is potentially useful for cancers, there are some patients who do not show clinically beneficial response to this treatment. Previously, we have demonstrated gene expression profiling of pre-vaccination peripheral blood mononuclear cells in advanced castration-resistant prostate cancer patients with personalized peptide vaccination, and we have found some genes related with overall survival. In this study, we sequenced the upstream region of Haptoglobin (HP), which is the most significant q-value in a univariate Cox regression analysis (q-value=0.001) and highly expressed in patients with poor clinical response. Genetic polymorphism of the upstream of HP was identified and this polymorphism classifies patients with good or poor clinical response to a peptide vaccination. Our results suggested that mRNA level and genetic polymorphism of HP is related to the prognosis of advanced castration-resistant prostate cancer patients treated with peptide vaccination. Figure 1

A phase II study of personalized peptide vaccination combined with gemcitabine for non-resectable pancreatic cancer patients

We evaluated the safety of, and clinical and immune responses to personalized peptide vaccination with gemcitabine (GEM) as the first line therapy in patients with non-resectable pancreatic cancer. Pre-vaccination peripheral blood mononuclear cells (PBMCs) and plasma were prepared to examine cellular and humoral responses to 14 and 16 peptides in human leukocyte antigen (HLA)-A24+ or -A2+ patients, respectively. Only the reactive peptides (maximum of 4) were administered weekly at 3 mg/peptide. GEM was administered at 1000 mg/m(2) per week for 3 weeks, followed by 1 week of rest. Twenty-one patients with untreated and non-resectable pancreatic cancer were enrolled. The combination therapy was generally well tolerated. Boosting of cellular and humoral responses to the vaccinated peptides was observed in the post-vaccination (eighth) PBMCs and plasma from 14 of 18 and 13 of 18 patients tested, respectively. The best clinical responses were 7 cases of partial response, 9 cases of stable disease, and 5 cases of progressive disease. Median survival time of all 21 patients was 9.0 months (95% CI, 6-15.5 months) with a one year survival rate of 38%. Immune boosting in both cellular and humoral responses was well correlated with overall survival with a hazard ratio of 0.2 (95% CI, 0.06-0.73; log-rank p=0.0239). These results suggest a potential clinical benefit of this combination therapy for non-resectable pancreatic cancer patients as the first line therapy. Further exploration of this approach is warranted.

Immunologic and clinical effects of personalized selection of peptide vaccines in HLA-A2 positive patients with advanced cancer

3031 Background: Personalized selection of the right peptides for each patient could be a novel peptide-based immunotherapy for boosting anticancer immunity in many patients (pts). Here we describe immunologic and clinical effects of a personalized peptide vaccine for HLA-A2 positive advanced cancer pts who mostly failed the standard therapy. Methods: To evaluate safety and immune responses of vaccination, 74 HLA-A2 positive advanced cancer pts, including 27 hormone refractory prostate cancer (HRPC), 13 colon cancer (CC), 8 pancreatic cancer (PC), 6 gastric cancer (GC), 4 brain tumor (BT), and 16 other types of cancer (Others) pts were enrolled in a phase I/II clinical trial. Pre-vaccination peripheral blood mononuclear cells and/or plasma were measured for their CTL or IgG levels for each of the 14–20 candidate peptides which can induce HLA-A2-restricted CTL activity against cancer cells followed by weekly or bi-weekly subcutaneous administration of the top four peptides (3mg each) showing the strongest immune responses. The protocol was approved by each ethics committee. Results: Vaccination-related adverse events were mainly grade 1 or 2 skin reactions at the injection sites, and there were no severe adverse events except for rectal bleeding in a cervical cancer pt. In the post (6–8th)-vaccination samples, CTL responses were boosted in 46 of 70 pts tested, while IgG responses were boosted in 55 of 73 pts tested, respectively. Best clinical responses were 1 CR, 11 PR, 31 SD, and 31 PD. Objective responses were obtained in urologic and gynecologic cancers or brain tumors by vaccination in the thigh or upper back regions, respectively, suggesting that location of sentinel lymph node near to tumor site is a pivotal factor for tumor regression. Median survival times in all pts are 14.3 months: 25 months in HRPC, 9.7 in BT, 8 in GC, 7 in PC, 6 in CC, and 17 in Others, respectively. Conclusions: This personalized peptide vaccine could be recommended for further stages of clinical trials because of safety, boosting of immune responses, and potential clinical benefits. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GreenPeptide GreenPeptide GreenPeptide GreenPeptide

Phase I trial of personalized peptide vaccination for cytokine‐refractory metastatic renal cell carcinoma patients

The aim of this clinical trial was to investigate the toxicity and immunological responses of personalized peptide vaccination for cytokine-refractory metastatic renal cell carcinoma patients. Patients were confirmed to be human leukocyte antigen (HLA)-A24 or HLA-A2 positive and had histologically confirmed renal cell carcinoma. Ten patients were enrolled in the present study. The peptides to be administered were determined based on the presence of peptide-specific cytotoxic T lymphocyte precursors in peripheral blood mononuclear cells (PBMC) and peptide-specific IgG in the plasma of cancer patients. Patients received subcutaneous injections of four different peptides (3 mg/peptide) every 2 weeks. Vaccinations were well tolerated without any major adverse events. A minimal increase in peptide-specific interferon-gamma production in postvaccination PBMC was observed, regardless of higher levels of cytotoxic T lymphocyte activity in prevaccination PBMC. In contrast, an increase in peptide-specific IgG levels of postvaccination (sixth) plasma was observed in the majority of patients. After progression, five patients received interleukin-2 therapy and continuous vaccination, with survival of 31, 25, 23, 17, and 15 months, but interleukin-2 did not impede humoral responses boosted by the vaccination. These results encourage further clinical trials of personalized peptide vaccinations.

Immunologic Evaluation of Personalized Peptide Vaccination for Patients with Advanced Malignant Glioma

The primary goal of this phase I study was to assess the safety and immunologic responses of personalized peptide vaccination for patients with advanced malignant glioma. Twenty-five patients with advanced malignant glioma (8 grade 3 and 17 grade 4 gliomas) were evaluated in a phase I clinical study of a personalized peptide vaccination. For personalized peptide vaccination, prevaccination peripheral blood mononuclear cells and plasma were provided to examine cellular and humoral responses to 25 or 23 peptides in HLA-A24+ or HLA-A2+ patients, respectively; then, only the reactive peptides (maximum of four) were used for in vivo administration. The protocols were well tolerated with local redness and swelling at the injection site in most cases. Twenty-one patients received more than six vaccinations and were evaluated for both immunologic and clinical responses. Increases in cellular or humoral responses specific to at least one of the vaccinated peptides were observed in the postvaccination (sixth) samples from 14 or 11 of 21 patients, respectively. More importantly, significant levels of peptide-specific IgG were detected in the postvaccination tumor cavity or spinal fluid of all of the tested patients who showed favorable clinical responses. Clinical responses were 5 partial responses, 8 cases of stable disease, and 8 cases of progressive disease. The median overall survival for patients with recurrent glioblastoma multiforme in this study (n = 17) was 622 days. Personalized peptide vaccinations were recommended for the further clinical study to malignant glioma patients.

Immunological evaluation of personalized peptide vaccination for patients with pancreatic cancer

The prognosis of pancreatic cancer is extremely poor, and development of new treatment modalities is needed. One such treatment could be specific immunotherapy. To evaluate safety and immunological responses, we conducted a phase I study of personalized peptide vaccination for pancreatic cancer patients (n=11). Namely, pre-vaccination peripheral blood mononuclear cells were screened for their reactivity in vitro to each of 14 or 16 peptides in HLA-A24(+) or -A2(+) patients, and only the reactive peptides (maximum: 4) were vaccinated in vivo. This regimen was generally well tolerated, although inflammatory reactions at the injection site were observed in 7 patients. Delayed-type hypersensitivity to peptides used for vaccination was observed in 7 patients. Increased cellular and humoral immune responses to at least one of peptides used for vaccination were observed in the post-vaccination PBMCs and sera from 4 of 8 patients and 4 of 10 patients tested, respectively. The 6- and 12-month survival rates for patients who received >3 vaccinations (n=10) were 80% and 20%, respectively. Due to tolerability and capability of inducing specific immunity, further development of personalized peptide-based immunotherapy for pancreatic cancer patients is warranted.

Immunological evaluation of vaccination with pre-designated peptides frequently selected as vaccine candidates in an individualized peptide vaccination regimen

We previously conducted a trial of an 'individualized' regimen, in which cancer patients were vaccinated with peptides after the confirmation of pre-existing peptide-specific cytotoxic T lymphocyte (CTL) precursors. In this study, we performed a new trial of 'pre-designated' regimen, in which cancer patients were vaccinated with peptides that were frequently selected as vaccine candidates in the preceding individualized regimen. Eighteen cancer patients (10 with uterine cervical cancer and 8 with gastric cancer) were enrolled in the new regimen. The pre-designated regimen was well tolerated by all patients. Although peptide-specific CTL precursors and humoral responses increased in the majority of patients with the pre-designated regimen, neither of the responses correlated with clinical outcome. Three patients had long stable disease, and their pre-vaccination peripheral blood mononuclear cells contained peptide-specific CTL precursors reactive to more than 2 of 4 peptides. With the pre-designated regimen, the levels of pre-existing immunoglobulin G reactive to non-vaccinated peptides decreased in 5 of 15 patients with progressive disease, and their time to progression was very short, whereas such a decrease was rarely observed in the preceding individualized regimen. These results suggest that the pre-designated regimen can elicit a primary immune response, but may incidentally suppress pre-existing immune responses.

Detection of peptide-specific CTL-precursors in peripheral blood lymphocytes of cancer patients.

Development of therapeutic vaccines is one of the major areas of tumour immunotherapy today. However, clinical trials of peptide-based cancer vaccines have rarely resulted in tumour regression. This failure might be due to an insufficient induction of cytotoxic T lymphocytes in the current regimes, in which cytotoxic T lymphocytes-precursors in pre-vaccination peripheral blood mononuclear cells are not measured. Initiation of immune-boosting through vaccination could be better than that of immune-priming with regard to induction of prompt and strong immunity. If this is also the case for therapeutic vaccines, pre-vaccination measurement of peptide-specific cytotoxic T lymphocytes-precursors will be important. In the present study, we investigated whether cytotoxic T lymphocytes-precursors reacting to 28 kinds of peptides of vaccine candidates (13 and 15 peptides for HLA-A24+ and HLA-A2+ patients, respectively) were detectable in pre-vaccination peripheral blood mononuclear cells of 80 cancer patients. Peptide-specific cytotoxic T lymphocytes-precursors were found to be detectable in peripheral blood mononuclear cells of the majority of cancer patients (57 out of 80 cases, 71%). The mean numbers of positive peptides were 2.0 peptides per positive case. Peripheral blood mononuclear cells incubated with positive peptides, not with negative peptides, showed significant levels of HLA-class-I-restricted cytotoxicity to cancer cells. The profiles of positive peptides entirely varied among patients, and were not influenced by the cancer origin. These results may provide a scientific basis for the development of a new approach to cancer immunotherapy, e.g.) cytotoxic T lymphocytes-precursor-oriented peptide vaccine.British Journal of Cancer (2002) 87, 796–804. doi:10.1038/sj.bjc.6600548 www.bjcancer.com© 2002 Cancer Research UK

Detection of peptide-specific cytotoxic T-lymphocyte precursors used for specific immunotherapy of pancreatic cancer.

The prognosis of pancreatic cancer is extremely poor with a 5-year survival of approximately 3%. Thus, the development of new treatment modalities, including a specific immunotherapy, is required. Our study investigated whether cytotoxic T-lymphocyte (CTL) precursors reacting to peptides with vaccine candidates (13 peptides for HLA-A2+ or -A24+ patients, respectively) were detectable in the prevaccination peripheral blood mononuclear cells (PBMCs) of 15 pancreatic cancer patients. Peptide-specific CTL precursors were detectable in the majority (11 of 15, 73%) of patients, with a mean positive number of 1.5 peptides (ranging from 0-5 peptides) per patient. Positive peptide profiles varied among patients. These results may provide a scientific basis for a new kind of cancer immunotherapy, namely, a CTL precursor-oriented peptide vaccine, for pancreatic cancer patients.