Radiotherapy Combination: Insight from Tumor Immune Microenvironment (TIME).

The view of Radiotherapy (RT) as a simple inducer of DNA damage resulting in tumor cell death has dramatically changed in recent years, and it is now widely accepted that RT can trigger an immune response which provides a sound basis for combining RT with immunotherapy. Given that, radiation can be delivered with different regimens, its effect on immune responses and Tumor Immune Microenvironment (TIME) may vary with dose and fractionation schedule. This fractional dose dependency may need to be more considered because of recent developments in RT delivery techniques making it possible to deliver precisely higher dosages per fraction (hypofractionation) while reducing exposure to normal tissues. Although combining radiotherapy with immunotherapy could be a promising strategy for synergistic enhancement of treatment efficacy, the selection of the best-matched combination of immunotherapy with each radiotherapy scheme remains to be addressed. Thus, for designing better therapeutic combinations, it is necessary to understand the immunological effects of RT. Here, we review the impact of conventional and different hypofractionation radiation schedules on the TIME. Subsequently, we highlight how knowing about these interactions may have implications for choosing a rational combination with targeted therapies.

The effects of Alpha-2a and Beta-1a Interferons on the Coronavirus disease 2019 prognosis.

BACKGROUND: The pandemic coronavirus disease 2019 (COVID-19) has been associated with substantial mortality worldwide. Efforts have continued to find an effective treatment for COVID-19. In vitro activity of interferon (IFN) subtypes has been shown against the SARS-CoV and MERS-CoV. Furthermore, the superiority of IFN-ß over IFN-α2b and IFN-α2a has been demonstrated in MERS treatment. Early studies showed a low plasma level of IFNs in the peripheral blood or lungs of patients with severe COVID-19. This study assessed the effects of IFN-alpha-2a and -beta-1a on the prognosis of patients with covid-19 infection. METHODS: We conducted a triple-blind randomized clinical trial on adult patients with moderate to severe COVID-19 from April 2021 to June 2021. The patients were diagnosed based on clinical and laboratory findings and randomly assigned into four groups (A, B, C, and D) using the envelope allocation method. Patients in group A received IFN ß-1a; group B received IFN ß-1a placebo; group C received IFN α-2a, and group D was treated with IFN α-2a placebo. All patients concomitantly received the national protocol medications as well. RESULTS: A total of 95 eligible patients were randomly assigned into groups. National Early Warning Score 2 (NEWS2) index showed significant differences between groups only on the first day of admission (p-value = 0.001). CT scan scores on the first and tenth days slightly improved, although they were not statistically significant. Duration of hospitalization and hospital discharge did not significantly differ among all treated groups (Table 1). Mortality rates showed no significant statistical difference between the groups. However, viral clearance significantly accelerated in the patients receiving IFN ß-1a or IFN α-2a (p < 0.05). CONCLUSION: It seems that IFN α-2a and IFN ß-1a are ineffective in treating COVID-19 patients. Further randomized clinical trials with large sample sizes are needed to estimate the effects of IFN α-2a or IFN ß-1a on the outcomes of COVID-19 disease.

Catalase-gold nanoaggregates manipulate the tumor microenvironment and enhance the effect of low-dose radiation therapy by reducing hypoxia.

Radiotherapy as a standard method for cancer treatment faces tumor recurrence and antitumoral unresponsiveness. Suppressive tumor microenvironment (TME) and hypoxia are significant challenges affecting efficacy of radiotherapy. Herein, a versatile method is introduced for the preparation of pH-sensitive catalase-gold cross-linked nanoaggregate (Au@CAT) having acceptable stability and selective activity in tumor microenvironment. Combining Au@CAT with low-dose radiotherapy enhanced radiotherapy effects via polarizing protumoral immune cells to the antitumoral landscape. This therapeutic approach also attenuated hypoxia, confirmed by downregulating hypoxia hallmarks, such as hypoxia-inducible factor α-subunits (HIF-α), vascular endothelial growth factor (VEGF), and EGF. Catalase stability against protease digestion was improved significantly in Au@CAT compared to the free catalase. Moreover, minimal toxicity of Au@CAT on normal cells and increased reactive oxygen species (ROS) were confirmed in vitro compared with radiotherapy. Using the nanoaggregates combined with radiotherapy led to a significant reduction of immunosuppressive infiltrating cells such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T-regs) compared to the other groups. While, this combined therapy could significantly increase the frequency of CD8+ cells as well as M1 to M2 macrophages (MQs) ratio. The combination therapy also reduced the tumor size and increased survival rate in mice models of colorectal cancer (CRC). Our results indicate that this innovative nanocomposite could be an excellent system for catalase delivery, manipulating the TME and providing a potential therapeutic strategy for treating CRC.

All about blinatumomab: the bispecific T cell engager immunotherapy for B cell acute lymphoblastic leukemia.

INTRODUCTION: B cell acute lymphoblastic leukemia-lymphoma (B-ALL) accounts for approximately 75% of ALL cases and is observed in children and adults. Recent advances in disease diagnosis, stratification and prognostication have led to a better characterization of different subgroups of ALL. Notwithstanding the significant improvement in the complete remission rate of B-ALL, patients with minimal residual disease (MRD) and relapsed/refractory (R/R) settings suffer from poor outcomes. HYPOTHESIS: However, novel therapies, such as agents targeting tyrosine kinases or the CD20 molecule, combination therapies and improved supportive care, have changed the treatment landscape of B-ALL. METHOD AND RESULTS: Meanwhile, blinatumomab has been FDA-approved for MRD-positive or R/R B-ALL patients. Blinatumomab is a bispecific T cell engager containing the CD3 and CD19 that recognize domains redirecting cytotoxic T cells to lyse B cells. Promising outcomes, including long-term overall survival and improved MRD-negative response rates, have been reported in patients who received this drug. Adding blinatumomab to new ALL regimens seems promising for achieving better outcomes in poor prognosis B-ALL patients. Nevertheless, the neurotoxicity and cytokine release syndrome are the two major adverse events following the blinatumomab therapy. CONCLUSION: This review summarizes the function and effectiveness of blinatumomab in R/R and MRD positive B-ALL patients. Furthermore, blinatumomab's positive and negative aspects as a novel therapy for B-ALL patients have been briefly discussed.

Dual blockage of both PD-L1 and CD47 enhances the therapeutic effect of oxaliplatin and FOLFOX in CT-26 mice tumor model.

Colorectal cancer is a poorly immunogenic. Such property can be reverted by using ICD. However, ICD inducers can also induce the expression of inhibitory checkpoint receptors CD47 and PD-L1 on tumor cells, making CRC tumors resistant to mainly CD8 T cell killing and macrophage-mediated phagocytosis. In this study, we examined the therapeutic effect of Oxaliplatin and FOLFOX regimen in combination with blocking antibodies against CD47 and PD-L1. FOLFOX and Oxaliplatin treatment lead to an increase in CD47 and PD-L1 expression on CT-26 cells invitro and invivo. Combining blocking antibodies against CD47 and PD-L1 with FOLFOX leads to a significant increase in survival and a decrease in tumor size. This triple combining regimen also leads to a significant decrease in Treg and MDSC and a significant increase in CD8 + INF-γ + lymphocytes and M1/M2 macrophage ratio in the tumor microenvironment. Our study showed triple combining therapy with FOLFOX, CD47 and PD-L1 is an effective treatment regimen in CT-26 mice tumor model and may consider as a potential to translate to the clinic.

NK cell therapy in relapsed refractory multiple myeloma.

Recent advances in adoptive cell therapy have considerably changed the paradigm of cancer immunotherapy. Although current immunotherapies could cure many patients with multiple myeloma (MM), relapsed/refractory MM (RR/MM) is still challenging in some cases. Natural killer (NK) cells are innate immune cells that exert effective cytotoxic activity against malignant cells like myeloma cells. In addition to their antitumor properties, NK cells do not induce graft versus host disease following transplantation. Therefore, they provide a promising approach to treating RR/MM patients. Currently, attempts have been made to produce large-scale and good manufacturing practices (GMP) of NK cells. Ex vivo expanded/activated NK cells derived from the own patient or allogenic donors are potential options for NK cell therapy in MM. Besides, novel cell-based products such as NK cell lines and chimeric antigen receptor (CAR)-NK cells may provide an off-the-shelf source for NK cell therapy. Here, we summarized NK cell activity in the MM microenvironment and focused on different NK cell therapy methods for MM patients.

Combining ablative radiotherapy and anti CD47 monoclonal antibody improves infiltration of immune cells in tumor microenvironments.

Radiotherapy as an anti-tumor treatment can stimulate the immune system. However, irradiated tumor cells express CD47 to escape the anti-tumor immune response. Anti- CD47 Immunotherapy is a possible way to tackle this problem. This study evaluated the effect of single high dose radiotherapy combined with an anti-CD47 monoclonal antibody (αCD47 mAb) in CT26 tumor-bearing BALB/c mice. We assessed the tumors volume and survival in mice 60 days after tumor implantation. Also, immune cell changes were analyzed by flow cytometry in tumors, lymph nodes, and spleen. Combination therapy enhanced the anti-tumor response in treated mice by increasing CD8+ T cells and M1 macrophages and decreasing M2 macrophages and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME). Also, our results showed that combination therapy increased survival time in mice compared to other groups. Furthermore, tumor volumes remarkably decreased in mice that received a single high dose RT plus αCD47 mAb. In conclusion, we showed that combining RT and αCD47 mAb improved the immune cell population in TME, regressed tumor growth, and increased survival in tumor-bearing mice.

Radiation dose and schedule influence the abscopal effect in a bilateral murine CT26 tumor model.

Radiotherapy (RT) can induce immune-mediated responses in local irradiated tumors, and non-irradiated distant metastasis is termed the abscopal effect. Here, we aimed to evaluate the impact of different RT doses and fractions on anti-tumor responses within local irradiated and distance non-irradiated tumor microenvironments. In mice bearing CT26 tumors, the primary tumor was irradiated with three different RT doses (16 Gy × 1F, 10 Gy × 2F, and 3 Gy × 10F) with the same biologically effective dose. Tumor volumes and immune cells changes were assessed in irradiated and non-irradiated tumors. Survival times were evaluated over 90 days. Only 16 Gy × 1F radiation increased CD8 + T cells number in the irradiated (p = 0.043) and non-irradiated (p = 0.047) tumors compared to the untreated group. A high frequency of tumor-associated macrophages-1 (TAM-1) and low TAM-2 was found in 16 Gy × 1F irradiated mice. Moreover, 16 Gy × 1F significantly induced interferon gamma (IFNγ)-producing CD8 + cells in the spleen compared to controls (p = 0.021). Hypofraction regimens (16 Gy × 1F, 10 Gy × 2F) caused a reduction in myeloid-derived suppressor cells in the irradiated tumors. We detected A modest growth delay in both flank tumors and long-term survival after hypofraction treatments (16 Gy × 1F, 10 Gy × 2F). A single high RT dose increased CD8 + cells number in irradiated (p = 0.000) and non-irradiated (p = 0.002) tumors approximal up to 2 points along with significant induction of IFN-γ production by CD8 + cells in the spleen when combined with anti- programmed death ligand-1 (PDL-1) (p = 0.000). Combination therapy was also associated with bilateral tumor growth control and increased life span in mice. Hypofractionated RT schedules, especially single high dose, seem the most effective regimen for inducing an abscopal effect. Immune checkpoint inhibitors could promote RT-induced systemic effects.

Association Between Human Leukocyte Antigens and Graft-Versus-Host Disease Occurrence in Allogeneic Hematopoietic Stem Cell Transplantation - A 10-Year Experience on Iranian Patients.

BACKGROUND: Human leukocyte antigen (HLA) molecules mediate critical roles in determining responsiveness or non-responsiveness of the immune system, especially in transplantation. Some studies have shown a possible association between certain HLA alleles and some allogeneic hematopoietic stem cell transplantation (allo-HSCT) outcomes such as acute/chronic graft-versus-host disease (aGVHD/cGVHD) and overall survival (OS). In the current study, we investigated any possible association of HLA subclasses and acute/chronic GVHD occurrence as well as OS in patients receiving HLA-matched sibling allo-HSCT. METHODS: We retrospectively evaluated the association of various HLA alleles with the incidence of aGVHD, cGVHD, and OS of 162 patients who received allo-HSCT from HLA-matched sibling between 2009-2018 at Taleghani hospital in Tehran. RESULTS: We found that the incidence of aGVHD grades II-IV was higher among patients who had HLA-B*07 (P=0.031) and HLA-DRB1*07 (P=0.052). The presence of HLA-A*01 was associated with 4.5-fold greater odds of incidence in the extensive-type of cGVHD (P=0.009). Furthermore, HLA-A*03 (P=0.089), HLA-B*13(P=0.013), HLA-B*40 (P=0.042), HLA-DRB1*02 (P=0.074), and HLA-DRB1*04 (P=0.039) were associated with a lower rate of OS. CONCLUSION: This study suggests that certain HLA alleles might influence the incidence and severity of acute or chronic GVHD in the context of HLA-matched sibling allo-HSCT. In addition, some specific HLA alleles help predict OS in allo-HSCT recipients. These results might be helpful in estimating the incidence of aGVHD, cGVHD, and OS as well as designing personalized therapy.

In-depth summary over cytomegalovirus infection in allogeneic hematopoietic stem cell transplantation recipients.

In this study, we reviewed various aspects of cytomegalovirus infection, including pathophysiology, diagnosis methods, and antiviral treatments. Background: Infections continue to be a major reason of complications like high non-relapse morbidity and mortality rate after allogenic hematopoietic stem cell transplantation. Cytomegalovirus is the most common infection in immunocompromised patients or those with graft-versus-host disease. The Latent-cytomegalovirus disease could increase the risk of reactivation in allogenic hematopoietic stem cell transplantation patients and lead to profound adverse effects on transplantation outcomes. Cytomegalovirus-specific CD4 + and CD8 + T cells reconstitution is crucial for protection against the virus reactivation. Different prophylactic, pre-emptive, and therapeutic anti-viral drugs are available to prevent cytomegalovirus infection/reactivation and treat resistant infections. Conclusion: Although there has been introduced various CMV antiviral treatment strategies like antiviral drugs, Vaccination, passive immunotherapies and adoptive transfer of CMV-specific T cells, further clinical trials are required to approve current therapies.

Pre-and Post-Transplant Serum Lactate Dehydrogenase Levels as a Predictive Marker for Patient Survival and Engraftment in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

BACKGROUND: The discovery of biomarkers to predict the development of complications associated with hematopoietic stem cell transplantation (HSCT) offers a potential avenue for the early identification and treatment of these life-threatening consequences. Serum lactate dehydrogenase (sLDH) has been identified as a potential biomarker for determining the outcome of allogenic HSCT (allo-HSCT). METHODS: A retrospective study was performed using data collected from 204 allo-HSCT recipient patients to examine the predictive value of sLDH levels pre- and post-allo-HSCT on patient survival, graft-versus-host-disease (GVHD) incidence, and time to platelet/white blood cells (WBC) engraftment. RESULTS: Our findings show that neither pre- (p= 0.61) nor post-transplantation (p= 0.55) sLDH levels were associated with GVHD incidence. However, elevated sLDH levels pre- and post-transplantation (≥ 386 and ≥ 409 IU/mL, respectively) were found to be adverse risk factors for patient survival (p= 0.16, p= 0.20, respectively). Furthermore, a median sLDH level ≥ 400 IU/mL from day +5 to day +15 post-transplantation had a significant positive association with enhanced time to platelet and white blood cell (WBC) engraftment, compared to patients with sLDH levels < 400 IU/mL (p< 0.001). CONCLUSION: Our data suggests that high sLDH levels pre- and post-allo-HSCT could be considered a predictor of poor patient survival. Furthermore, high levels of sLDH days 5-15 post-allo-HSCT could be associated with improved time to platelet and WBC engraftment; however, this appears to come at the cost of increased mortality risk.

Immunotherapy with adoptive cytomegalovirus-specific T cells transfer: Summarizing latest gene engineering techniques.

Cytomegalovirus (CMV) infection remains a major complication following allogeneic hematopoietic stem cell transplantation (HSCT). T cell response plays a critical role in inducing long-term immunity against CMV infection/reactivation that impairs during HSCT. Adoptive T cell therapy (ACT) via transferring CMV-specific T cells from a seropositive donor to the recipient can accelerate virus-specific immune reconstitution. ACT, as an alternative approach, can restore protective antiviral T cell immunity in patients. Different manufacturing protocols have been introduced to isolate and expand specific T cells for the ACT clinical setting. Nevertheless, HLA restriction, long-term manufacturing process, risk of alloreactivity, and CMV seropositive donor availability have limited ACT broad applicability. Genetic engineering has developed new strategies to produce TCR-modified T cells for diagnosis, prevention, and treatment of infectious disease. In this review, we presented current strategies required for ACT in posttransplant CMV infection. We also introduced novel gene-modified T cell discoveries in the context of ACT for CMV infection. It seems that these innovations are enabling to improvement and development of ACT utilization to combat posttransplant CMV infection.

Pre-Transplantation Serum Parathyroid Hormone Influences the Number of Mobilized CD34+ Hematopoietic Stem Cells in Autologous Hematopoietic Stem Cell Transplantation.

BACKGROUND: Parathyroid hormone (PTH) is a calcium homeostasis regulator and can affect bone marrow niche. PTH leads to the bone marrow stem cell niche expansion as well as the induction of stem cell mobilization from the bone marrow into peripheral blood. In this study, we evaluated the association between pre- transplantation serum PTH levels and the number of circulating CD34+ cells along with the platelets/white blood cells (Plt/WBC) engraftment in patients who underwent autologous Hematopoietic Stem Cell Transplantation. METHODS: Subjects for the study were 100 patients who received autologous hematopoietic stem cell transplantation (auto-HSCT), retrospectively. Serum levels of PTH, calcium, phosphorus, and alkaline phosphatase were measured before mobilization. Their impacts were measured on the number of mobilized CD34+ hematopoietic stem cells, and Plt/WBC engraftment. RESULTS: High levels of serum PTH (> 63.10 pg/mL) was significantly associated with higher number of CD34+ cells in peripheral blood after granulocyte- colony stimulating factor (G-CSF)-induced mobilization (p= 0.079*). Serum calcium at low levels were associated with higher number of circulating CD34+ cells post mobilization. Pre- transplantation serum levels of phosphorus and alkaline phosphatase on CD34+ numbers were not statistically significant. Serum Plt/WBC engraftment was not improved in presence of high levels of serum PTH. CONCLUSION: We suggested that serum PTH levels before transplantation could be influential in raising the number of circulating CD34+ hematopoietic stem cell after mobilization.

Serum Levels of Soluble Fas and Fas Ligand in Iranian Women with Pre-eclampsia.

BACKGROUND: The precise responsible mechanism of pre-eclampsia remains controversial however, recent data suggest a main role of the abnormal activation of the adaptive immune system and Apoptosis. In this study, we have measured serum levels of Fas/Fasl as two important members of extrinsic apoptotic pathway in patient with pre-eclampsia. METHODS: 207 participants including 99 pre-eclampsia patients and 108 age and sex-matched normal pregnant women were involved in the case-control study. Plasma sample from each participant was collected and stored at -20 °C until batch processing.Serum levels of Fas and Fas ligand were measured by ELISA for each participant including 99 pre-eclampsia patients and 108 normal pregnant women. Following a test of statistical normality, nonparametric data were analyzed by Mann-Whitney. RESULTS: sFas levels in case group was significantly higher than controls; 584 (397-892) pg/ml in cases opposed to 341 (213-602) pg/ml in controls (p value< 0.01). sFasL in pre-eclampsia women was a little lower than controls; 255 (173-318) pg/ml and in case group compared to 265.5 (184-381.5) pg/ml in controls. CONCLUSION: We have found the increased levels of sFas in patients with pre-eclampsia in compare with the healthy pregnant women. It seems that abnormality in sFAS is related with pre-eclampsia.

The expression of serotonin transporter protein in the skin of patients with chronic spontaneous urticaria and its relation with depression and anxiety.

Studies have indicated a possible role for serotonin transporter protein (SERT) in the pathophysiology of inflammatory skin disorders. This study was aimed to determine the expression of SERT in the skin of patients with chronic spontaneous urticaria (CSU) and its relation to depression and anxiety. In this case-control study, 30 CSU patients and 30 healthy controls were evaluated with skin biopsies to evaluate the expression of the SERT protein based on histopathologic findings. Beck depression and anxiety inventories were used to investigate depression and anxiety in the case group. Data were analyzed by SPSS software. P values < 0.05 were considered significant. The case group showed significantly higher percentage of stained cells (P < 0.0001) and intensity of SERT expression (P < 0.0001) compared with the control group. The patients with uncontrolled CSU showed significantly higher percentage (P < 0.002) and intensity (P < 0.006) of SERT expression, compared with those with controlled CSU. The intensity of SERT expression in CSU patients had no significant correlation with the severity of depression, but was significantly correlated with the severity of anxiety (r = 0.555; P = 0.001). The percentage of stained cells was significantly correlated with the severities of depression (r = - 0.433; P = 0.017) and anxiety (r = 0.528; P = 0.003). The SERT expression in patients with CSU was higher compared with controls, which can demonstrate the role of serotonin in the pathogenesis of this disease. This higher SERT expression is correlated with the severity of the disease.

Improved Immunogenicity Against a Her2/neu-Derived Peptide by Employment of a Pan HLA DR-Binding Epitope and CpG in a BALB/c Mice Model.

BACKGROUND: An efficient strategy to improve the immunogenicity of peptide vaccines is the use of a synthetic peptide containing cytotoxic T-lymphocyte (CTL) epitopes with T-helper (Th) inducing-epitopes. OBJECTIVE: Our purpose was to determine the use of human epidermal growth factor receptor-2 (Her2/neu)- specific CTL epitopes plus the pan HLA DR-binding epitope (PADRE) and CpG oligodeoxynucleotides (ODNs) to induce antitumor effects in vaccinated mice. METHOD: Female BALB/c mice were immunized subcutaneously with different vaccines. Three mice per group were euthanized to assess immune responses and the others were transplanted with TUBO cells. Enzyme-linked Immuno Spot assay (ELISpot) and flow cytometry studies were followed by tumor size and survival rate measurements in a TUBO tumor mice model. RESULTS: The results showed that mice vaccinated with the P5 peptide plus PADRE plus CpG produced higher antigen-specific CTL responses than mice vaccinated with the P5 peptide alone. Also, tumors in those mice grew more slowly and the survival rates were greater than mice in the other groups. CONCLUSION: We conclude that peptide vaccines containing epitopes that stimulate both CD4+ and CD8+ T-cells are effective at inducing anti-tumor immunity.

Relationship between Fas and Fas Ligand gene polymorphisms and pre-eclampsia.

AIM: In normal pregnancy, the Th1 subtype, responsible for the production of inflammatory cytokines, is reduced, and the Th2 subtype is increased to prohibit inflammation. In pre-eclampsia, the Th1 cell population is increased; thus, subsequent inflammation and trophoblast destruction occur. Polymorphisms in the Fas and Fas Ligand (FasL) promoter regions can influence Fas and FasL expression and accused to increase of Th1 subtype. METHODS: DNA samples from 153 pregnant women with pre-eclampsia and 140 controls were genotyped through polymerase chain reaction-restriction fragment length polymorphism. A Fisher's exact test was used to compare the distribution of individual polymorphisms. RESULTS: Fas-1377 AA, AG and GG genotypes were observed in 2.61%, 18.30% and 79.08% in the pre-eclampsia group opposed to 0%, 27.14% and 72.85% in the control group (P = 0.037), respectively. Fas-670 AA, AG and GG genotypes were observed in 37.9%, 41.8% and 20.3% of pre-eclampsia patients compared with 33.6%, 50.7% and 15.7% in healthy pregnant women (P = 0.291), respectively. No statically significant differences in the FasL-844 genotype were observed between the groups (P = 0.69). CONCLUSION: The Fas-1377G > A polymorphism is associated with a higher risk of pre-eclampsia.

Improving Multi-Epitope Long Peptide Vaccine Potency by Using a Strategy that Enhances CD4+ T Help in BALB/c Mice.

Peptide-based vaccines are attractive approaches for cancer immunotherapy; but the success of these vaccines in clinical trials have been limited. Our goal is to improve immune responses and anti-tumor effects against a synthetic, multi-epitope, long peptide from rat Her2/neu (rHer2/neu) using the help of CD4+ T cells and appropriate adjuvant in a mouse tumor model. Female BALB/c mice were vaccinated with P5+435 multi-epitope long peptide that presents epitopes for cytotoxic T lymphocytes (CTL) in combination with a universal Pan DR epitope (PADRE) or CpG-oligodeoxynucleotides (CpG-ODNs) as a Toll-like receptor agonist adjuvant. The results show that vaccination with the multi-epitope long peptide in combination with the PADRE peptide and CpG-ODN induced expansion of subpopulations of CD4+ and CD8+ cells producing IFN-γ, the average tumor size in the vaccinated mice was less than that of the other groups, and tumor growth was inhibited in 40% of the mice in the vaccinated group. The mean survival time was 82.6 ± 1.25 days in mice vaccinated with P5+435 + CpG+ PADRE. Our results demonstrate that inclusion of PADRE and CpG with the peptide vaccine enhanced significant tumor specific-immune responses in vaccinated mice.