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Cancer vaccine immunotherapy facilitates the immune system's recognition of tumor-associated antigens, and the biomolecular design of these vaccines using nanoparticles is one important approach towards obtaining strong anti-tumor responses. Following activation of dendritic cells (DCs), a robust CD8+ T cell-mediated adaptive immune response is critical for tumor elimination. While the role of efficient antigen-presenting myeloid DCs (mDCs) is conventionally attributed towards vaccine efficacy, participation by highly cytokine-producing plasmacytoid DCs (pDCs) is less understood and is often overlooked. We examined vaccines based on the E2 protein nanoparticle platform that delivered encapsulated TLR9 agonist bacterial-like DNA (CpG1826 or CpG1018) or TLR7 agonist viral ssRNA to determine their efficacy over free agonists in activating both mDCs and pDCs for antigen presentation. Although mDCs were only activated by nanoparticle-encapsulated TLR9 agonists, pDCs were activated by all the individually tested constructs, and CpG1826 was shown to induce pDC cytokine production. Transfer of secreted factors from pDCs that were stimulated with a vaccine formulation comprising peptide antigen and CpG1826 enhanced mDC display of the antigen, particularly when delivered in nanoparticles. Only when treated with nanoparticle-conjugated vaccine could pDCs secrete factors to induce antigen display on naïve mDCs. These results reveal that pDCs can aid mDCs, highlighting the importance of activating both pDCs and mDCs in designing effective cancer vaccines, and demonstrate the advantage of using nanoparticle-based vaccine delivery.

PDF file - 43K, Antigen cascade.

PDF file - 90K, Consort diagram.

PDF file - 264K, Tumor growth rates following vaccine.

XLSX file - 32K, Adverse events.

PSA-TRICOM (PROSTVAC) is a novel vector-based vaccine designed to generate a robust immune response against prostate-specific antigen (PSA)-expressing tumor cells. The purpose of this report is to present an overview of both published studies and new data in the evaluation of immune responses to the PSA-TRICOM vaccine platform, currently in phase III testing. Of 104 patients tested for T-cell responses, 57% (59/104) demonstrated a ≥2-fold increase in PSA-specific T cells 4 weeks after vaccine (median 5-fold increase) compared with pre-vaccine, and 68% (19/28) of patients tested mounted post-vaccine immune responses to tumor-associated antigens not present in the vaccine (antigen spreading). The PSA-specific immune responses observed 28 days after vaccine (i.e., likely memory cells) are quantitatively similar to the levels of circulating T cells specific for influenza seen in the same patients. Measurements of systemic immune response to PSA may underestimate the true therapeutic immune response (as this does not account for cells that have trafficked to the tumor) and does not include antigen spreading. Furthermore, although the entire PSA gene is the vaccine, only one epitope of PSA is evaluated in the T-cell responses. Because this therapeutic vaccine is directed at generating a cellular/Th1 immune response (T-cell costimulatory molecules and use of a viral vector), it is not surprising that less than 0.6% of patients (2/349) tested have evidence of PSA antibody induction following vaccine. This suggests that post-vaccine PSA kinetics were not affected by PSA antibodies. An ongoing phase III study will evaluate the systemic immune responses and correlation with clinical outcomes. Cancer Immunol Res; 2(2); 133–41. ©2013 AACR.

PDF file - 62K, IL-5 following vaccine.

Background Sleep disturbances are associated with numerous mood disorders. Similarly, anxiety and depression are associated with modulation of the psychoneuroimmune (PNI) axis. This study hypothesized that changes in both monitored and self-reported measures of sleep would relate to changes in circulating cytokine levels in an emotionally distressed population of cervical cancer survivors. Methods Biospecimens, patient-reported outcome (PRO) measures, and actigraphy were collected from cervical cancer survivors enrolled in a biobehavioral clinical trial. Longitudinal changes over a 4-month period were examined. Sleep time measured by actigraphy and PRO were analyzed for correlative changes with emotional distress and serum cytokines (n = 71). Results Longitudinal change in the actigraph measure of sleep time was inversely associated with changes in depression and anxiety (test for linear trend, p = 0.02 and p = 0.05 respectively), as well as acute-phase response/pro-inflammatory cytokines (test for linear trend, p = 0.003, interleukin (IL)-2; 0.022, IL-1β; 0.0002, IL-6; and 0.049, tumor necrosis factor α). Conversely, changes in self-reported sleep problems were related to an increase in depression and anxiety (p = 0.001 and p = 0.01 respectively), the T helper 2 (Th2) cytokine IL-5 (p = 0.027), and the counter-regulatory cytokine IL-10 (0.016). Conclusion This study showed that an increase in sleep time or decrease in sleep problems corresponded with a reduction in self-reported emotional distress and attenuation of pro-inflammatory, Th2, and counter-regulatory cytokines. Our results support sleep measurement as a meaningful biobehavioral variable in cancer survivorship. This study also indicates that sleep investigators should be aware that choice of methodology might influence concordance with different classes of immune parameters.

Improvement in health behaviors following cancer diagnosis may contribute to better prognosis and well-being. This study examines the prevalence of health behaviors in cervical cancer survivors who have completed treatment, and associations between health behaviors and quality of life (QOL).We recruited 204 women who had completed treatment for cervical cancer to participate in a randomized counseling intervention. Participants provided information on health behaviors (smoking, physical activity, and alcohol consumption); QOL (Functional Assessment of Cancer Therapy-Cervical questionnaire); and depression (Patient-Reported Outcomes Measurement Information System), anxiety (Patient-Reported Outcomes Measurement Information System), and distress (Brief Symptom Inventory) at baseline (9-30 months after diagnosis) and subsequent to the intervention. Data were analyzed using multivariate general linear models.Participants ranged in age from 20 to 72 years at diagnosis (mean = 43 years), 41% were Hispanic, and 52% were non-Hispanic white. Three-fourths were stage 1 at diagnosis and 51% were treated with radiation with or without chemotherapy. At baseline, 15% of patients were current smokers, 4% reported alcohol consumption of >10 drinks per week, and 63% reported exercising

Immune checkpoint inhibition is a promising alternative treatment to standard chemotherapies; however, it fails to achieve long-term remission in a significant portion of patients. A previously developed protein nanoparticle-based platform (E2 nanoparticle) delivers cancer antigens to increase antigen-specific tumor responses. While prior work has focussed on prophylactic conditions, the objectives in this study are therapeutic. It is hypothesized that immune checkpoint inhibition, when augmented by antigen delivery using E2 nanoparticles containing CpG oligonucleotide 1826 (CpG) and a glycoprotein 100 (gp100) melanoma antigen epitope (CpG-gp-E2), would synergistically elicit antitumor responses. To identify a regimen primed for obtaining effective treatment results, immune benchmarks in the spleen and tumor are examined. Conditions that lead to significant immune activation, including increases in gp100-specific interferon gamma (IFN-𝜸), CD8 T cells in the spleen, tumor-infiltrating CD8 T cells, and survival time are identified. Based on the findings, the resulting combination of CpG-gp-E2 and anti-programmed cell death protein 1 (anti-PD-1) treatment in tumor-challenged mice yield significantly increased long-term survival; more than 50% of the mice treated with combination therapy were tumor-free, compared with 0% and ≈5% for CpG-gp-E2 and anti-PD-1 alone, respectively. Evidence of a durable antitumor response is also observed upon tumor rechallenge, pointing to long-lasting immune memory.

We have developed a novel, non-viral nanoparticle-based vaccine platform that has shown exciting promise for cancer antigen delivery to increase antigen-specific anti-tumor immune responses and significantly prolong survival in the aggressive B16-F10 melanoma model. Our non-viral protein nanoparticle has advantageous properties for antigen delivery and vaccine development. Nanoparticle studies have shown that there is an optimal size range for passive transport in the lymphatic system and uptake by antigen-presenting cells (approximately 20-45 nm); our particle is in this size range. Biodistribution studies of the nanoparticle have shown that they are effectively taken up by almost 50% of DCs within the draining lymph nodes. The structure of the nanoparticle allows for independent functionalization of its internal hollow cavity and external surface, enabling efficient and simultaneous adjuvant and antigen co-delivery. We, as have others, found that vaccine-elicited anti-tumor immune responses are insufficient to cure animals in this model.Immune checkpoint inhibition has provided significant clinical benefit and become an effective alternative treatment to standard chemotherapies in some tumor types. It is believed that immune checkpoint inhibition releases the brake on the existing anti-tumor immune response; however, even in the tumor types where there is clinical benefit, a significant proportion of patients fail to achieve long-term remission. We hypothesized that the combination of our anti-tumor protein nanoparticle vaccine ("E2"; to further prime the immune system to specific tumor-associated antigens) and immune checkpoint blockade will be synergistic and yield improved efficacy, even in the aggressive B16-F10 melanoma tumor model. Immunization with simultaneous delivery of CpG (adjuvant) and gp100 (melanoma MHC-I restricted antigen) within E2 nanoparticles results in significantly increased gp100-specific IFN-γ production by ELISpot, CD8 T cell count in the spleen, tumor-infiltrating CD8 T cell population, and survival time in the B16-F10 murine melanoma model. The combination of the CpG-gp-E2 nanoparticle with anti-PD-1 therapy significantly increased long-term survival; approximately 50% of the mice treated with combination therapy remained tumor-free for up to 60 days (cure) in mice that were inoculated with the poorly immunogenic B16-F10 syngeneic melanoma 24 hours prior to initiating treatment; this is compared with 0% and ≈5% for CpG-gp-E2 and anti-PD-1 treatments alone, respectively. The very limited efficacy of immune checkpoint inhibition in this model is well documented. Thus, this result is more significant. Evidence of a durable anti-tumor response was also observed upon tumor re-challenge. These investigations show that the combination of checkpoint inhibition with antigen delivery using a non-viral protein nanoparticle significantly improves the efficacy over vaccine or immune checkpoint blockade alone.To our knowledge, this is the first report demonstrating the synergistic effects of combination therapy of anti-PD-1 with protein-based nanoparticle vaccines. These data support the exceptional utility of our E2 nanoparticle-based vaccines, especially when combined with immune checkpoint blockade. Citation Format: Jo Anne Tucker, Medea Neek, Szu-Wen Wang, Edward L. Nelson. A novel protein nanoparticle antigen delivery platform combined with checkpoint inhibition has curative efficacy in the aggressive B16-F10 melanoma model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2223.

ObjectiveBenefits of social support (SS) during cancer survivorship are complex. This study examines change in SS over time in cervical cancer (CXCA) survivors who have completed definitive treatment and how changing SS impacts quality of life (QOL) and T-helper type 2 (Th2) cytokines.MethodsWe conducted a randomized trial in 204 CXCA survivors to test if psychosocial telephone counseling (PTC) could improve QOL compared to usual care (UC). Although PTC did not target SS, data were collected at baseline, 4 and 9months post-enrollment using the Medical Outcomes Survey Social Support scale. Biospecimens were collected to investigate associations with patient-reported outcomes. Data were analyzed using multivariate linear models and stepwise regression.ResultsParticipants' mean age was 43. PTC participants experienced increasing SS compared to UC at 4months (PTC-UC = 5.1; p = 0.055) and 9months (PTC-UC = 6.0; p = 0.046). Higher baseline SS and increasing SS were independently associated with improved QOL at 4 and 9months after adjusting for patient characteristics (p

Nanoparticles have attracted considerable interest as cancer vaccine delivery vehicles for inducing sufficient CD8+ T cell-mediated immune responses to overcome the low immunogenicity of the tumor microenvironment. Our studies described here are the first to examine the effects of clinically-tested human cancer-testis (CT) peptide epitopes within a synthetic nanoparticle. Specifically, we focused on two significant clinical CT targets, the HLA-A2 restricted epitopes of NY-ESO-1 and MAGE-A3, using a viral-mimetic packaging strategy. Our data shows that simultaneous delivery of a NY-ESO-1 epitope (SLLMWITQV) and CpG using the E2 subunit assembly of pyruvate dehydrogenase (E2 nanoparticle), resulted in a 25-fold increase in specific IFN-γ secretion in HLA-A2 transgenic mice. This translated to a 15-fold increase in lytic activity toward target cancer cells expressing the antigen. Immunization with a MAGE-A3 epitope (FLWGPRALV) delivered with CpG in E2 nanoparticles yielded an increase in specific IFN-γ secretion and cell lysis by 6-fold and 9-fold, respectively. Furthermore, combined delivery of NY-ESO-1 and MAGE-A3 antigens in E2 nanoparticles yielded an additive effect that increased lytic activity towards cells bearing NY-ESO-1+ and MAGE-A3+. Our investigations demonstrate that formulation of CT antigens within a nanoparticle can significantly enhance antigen-specific cell-mediated responses, and the combination of the two antigens in a vaccine can preserve the increased individual responses that are observed for each antigen alone.

Efficient delivery of antigens is of paramount concern in immunotherapies. We aimed to target antigen presenting cells (APCs) by conjugating CpG oligonucleotides to an E2 protein nanoparticle surface (CpG-PEG-E2). Compared to E2 alone, we observed ~4-fold increase of in vitro APC uptake of both CpG-PEG-E2 and E2 conjugated to non-CpG DNA. Furthermore, compared to E2-alone or E2 functionalized solely with polyethylene glycol (PEG), the CpG-PEG-E2 showed enhanced lymph node retention up to at least 48 hr and 2-fold increase in APC uptake in vivo, parameters which are advantageous for vaccine success. This suggests that enhanced APC uptake of nanoparticles mediated by oligonucleotide display may help overcome delivery barriers in vaccine development.

The transcription factor brachyury is a major driver of epithelial to mesenchymal transition (EMT) in human carcinoma cells. It is overexpressed in several human tumor types vs. normal adult tissues, except for testes and thyroid. Overexpression is associated with drug resistance and poor prognosis. Previous studies identified a brachyury HLA-A2 cytotoxic T-lymphocyte (CTL) epitope. The studies reported here describe an enhancer epitope of brachyury. Compared to the native epitope, the agonist epitope: (a) has enhanced binding to MHC class I, (b) increased the IFN-γ production from brachyury-specific T cells, (c) generated brachyury-specific T cells with greater levels of perforin and increased proliferation, (d) generated T cells more proficient at lysing human carcinoma cells endogenously expressing the native epitope, and (e) achieved greater brachyury-specific T-cell responses in vivo in HLA-A2 transgenic mice. These studies also report the generation of a heat-killed recombinant Saccharomyces cerevisiae (yeast) vector expressing the full-length brachyury gene encoding the agonist epitope. Compared to yeast-brachyury(native) devoid of the agonist epitope, the yeast-brachyury(agonist) enhanced the activation of brachyury-specific T cells, which efficiently lysed human carcinoma cells. In addition to providing the rationale for the recombinant yeast-brachyury(agonist) as a potential vaccine in cancer therapy, these studies also provide the rationale for the use of the agonist in (a) dendritic cell (DC) vaccines, (b) adjuvant or liposomal vaccines, (c) recombinant viral and/or bacterial vaccines, (d) protein/polypeptide vaccines, (e) activation of T cells ex vivo in adoptive therapy protocols, and (e) generation of genetically engineered targeted T cells.

Preclinical studies have demonstrated that the combination of systemic subcutaneous (s.c.) vaccination with intratumoral (i.t.) vaccination was superior in the induction of antitumor activity vs. vaccination with either route alone. A subsequent phase I study employing i.t.-s.c. vaccination was carried out in men with locally recurrent or progressive prostate cancer. rF-PSA-TRICOM (PROSTVAC) vaccine was administered intraprostatically and rV-PSA-TRICOM followed by rF-PSA-TRICOM vaccine was administered systemically. In that study no dose limiting toxicities were observed, 19/21 patients had stable or improved prostate-specific antigen (PSA) values and tumor-infiltrating lymphocytes (TILs) increased in post- vs. pre-treatment tumor biopsies, analyzed employing conventional immunohistochemistry (IHC). In the studies reported here, 31 phenotypes of peripheral blood mononuclear cells (PBMCs) were analyzed prevaccination and postvaccination as well as the functions of PBMC regulatory T cells (Tregs) and natural killer cells. A trend was observed in decreases in serum PSA with the reduction of circulating Tregs postvaccination. Digital IHC was employed prevaccination and postvaccination to measure CD4 and CD8 TILs, as well as Treg TILs by conventional IHC. Few correlations were observed with CD4, CD8 or Treg in TILs vs. PBMCs. However, patients with lower levels of CD4 TILs prevaccination showed the greatest increases in CD4 TILs postvaccine, while Treg TILs decreased postvaccine. There was also a strong correlation between decreases in serum PSA and increases in CD8 TILs postvaccine. These studies provide additional rationale for the use of i.t.-s.c. vaccinations and demonstrate a noncoordinate expression of specific immune subsets in PBMCs vs. tumor.

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor-induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome-wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration-resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin-2 (IL-2) and transforming growth factor-β (TGF-β) pathways. Studies revealed that the levels of expression of genes responsible for T-cell proliferation (C-FOS, C-JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4(+) CD25(high) CD127(-) Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.

The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8(+) T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimetic platform for cancer vaccine delivery. Simultaneous conjugation of a melanoma-associated gp100 epitope and CpG to the E2 nanoparticle (CpG-gp-E2) yielded an antigen-specific increase in the CD8(+) T cell proliferation index and IFN-γ secretion by 1.5-fold and 5-fold, respectively, compared to an unbound peptide and CpG formulation. Remarkably, a single nanoparticle immunization resulted in a 120-fold increase in the frequency of melanoma epitope-specific CD8(+) T cells in draining lymph nodes and a 30-fold increase in the spleen, relative to free peptide with free CpG. Furthermore, in the very aggressive B16 melanoma murine tumor model, prophylactic immunization with CpG-gp-E2 delayed the onset of tumor growth by approximately 5.5 days and increased animal survival time by approximately 40%, compared to PBS-treated animals. These results show that by combining optimal particle size and simultaneous co-delivery of molecular vaccine components, antigen-specific anti-tumor immune responses can be significantly increased.

Active immunotherapy is dependent on the ability of the immune system to recognize and respond to tumors. Despite overwhelming evidence to support a cell-mediated immune response to prostate cancer, it is insufficient to eradicate the disease. This is likely due to a high level of suppression at the tumor site from a variety of sources, including immunosuppressive cells. Immune cells entering the tumor microenvironment may be inhibited directly by the tumor, stromal cells or other immune cells that have been induced to adopt a suppressive phenotype. The resurgence of interest in immunotherapy following the approval of sipuleucel-T and ipilimumab by the Food and Drug Administration has brought about new strategies for overcoming tumor-mediated suppression and bolstering anti-tumor responses. Improved understanding of the immune response to prostate cancer can lead to new combination therapies, such as the use of vaccine with small molecule and checkpoint inhibitors or other immunotherapies.

Background One type of fecal occult blood test (FOBT), the unrehydrated guaiac fecal occult blood test (GT), is recommended by the United States Preventive Services Task Force and the Institute of Medicine for use in screening programs, but it has relatively low sensitivity as a single test for detecting advanced colonic neoplasms (cancer and adenomatous polyps ≥ 1 cm in diameter). Thus, improving the sensitivity of FOBT should make colon cancer screening programs that use these tests more effective. Methods We assessed prospectively the performance characteristics of two newer FOBTs in 5841 subjects at average risk for colorectal cancer in a large group – model managed care organization. The tests evaluated included a sensitive GT, a fecal immunochemical test (FIT), and the combination of both tests. Patients with positive and negative test results were advised to have colonoscopy and sigmoidoscopy, respectively. Sensitivity and specificity for detecting advanced neoplasms in the left colon within 2 years after the FOBT screening were evaluated for the two tests administered separately and in combination. Results A total of 139 patients were diagnosed with advanced colorectal neoplasms (n = 14 cancers, n = 128 adenomas) within the 2 years following their initial FOBT screening. Sensitivity for detecting cancer was 81.8% (95% confidence interval [CI] = 47.8% to 96.8%) for the FIT alone and 64.3% (95% CI = 35.6% to 86.0%) for the sensitive GT and the combination test. Sensitivity for detecting advanced colorectal adenomas was 41.3% (95% CI = 32.7% to 50.4%) for the sensitive GT, 29.5% (95% CI = 21.4% to 38.9%) for the FIT, and 22.8% (95% CI =16.1% to 31.3%) for the combination test. Specificity for detecting cancer and adenomas was 98.1% (95% CI = 97.7% to 98.4%) and 98.4% (95% CI = 98.0% to 98.7%), respectively, for the combination test; 96.9% (95% CI = 96.4% to 97.4%) and 97.3% (95% CI = 96.8% to 97.7%), respectively, for the FIT; and 90.1% (95% CI = 89.3% to 90.8%) and 90.6% (95% CI = 89.8% to 91.4%), respectively, for the sensitive GT. Conclusions The FIT has high sensitivity and specificity for detecting left-sided colorectal cancer, and it may be a useful

Purpose Survivors of cervical cancer experience quality-of-life (QOL) disruptions that persist years after treatment. This study examines the effect of a psychosocial telephone counseling (PTC) intervention on QOL domains and associations with biomarkers. Patients and Methods We conducted a randomized clinical trial in survivors of cervical cancer, who were ≥ 9 and less than 30 months from diagnosis (n = 204), to compare PTC to usual care (UC). PTC included five weekly sessions and a 1-month booster. Patient-reported outcomes (PROs) and biospecimens were collected at baseline and 4 and 9 months after enrollment. Changes in PROs over time and associations with longitudinal change in cytokines as categorical variables were analyzed using multivariable analysis of variance for repeated measures. Results Participant mean age was 43 years; 40% of women were Hispanic, and 51% were non-Hispanic white. Adjusting for age and baseline scores, participants receiving PTC had significantly improved depression and improved gynecologic and cancer-specific concerns at 4 months compared with UC participants (all P < .05); significant differences in gynecologic and cancer-specific concerns (P < .05) were sustained at 9 months. Longitudinal change in overall QOL and anxiety did not reach statistical significance. Participants with decreasing interleukin (IL) -4, IL-5, IL-10, and IL-13 had significantly greater improvement in QOL than those with increasing cytokine levels. Conclusion This trial confirms that PTC benefits mood and QOL cancer-specific and gynecologic concerns for a multiethnic underserved population of survivors of cancer. The improvement in PROs with decreases in T-helper type 2 and counter-regulatory cytokines supports a potential biobehavioral pathway relevant to cancer survivorship.

Vaccinia virus (VACV) is a useful model system for understanding the immune response to a complex pathogen. Proteome-wide Ab profiling studies reveal the humoral response to be strongly biased toward virion-associated Ags, and several membrane proteins induce Ab-mediated protection against VACV challenge in mice. Some studies have indicated that the CD4 response is also skewed toward proteins with virion association, whereas the CD8 response is more biased toward proteins with early expression. In this study, we have leveraged a VACV strain Western Reserve (VACV-WR) plasmid expression library, produced previously for proteome microarrays for Ab profiling, to make a solubilized full VACV-WR proteome for T cell Ag profiling. Splenocytes from VACV-WR–infected mice were assayed without prior expansion against the soluble proteome in assays for Th1 and Th2 signature cytokines. The response to infection was polarized toward a Th1 response, with the distribution of reactive T cell Ags comprising both early and late VACV proteins. Interestingly, the proportions of different functional subsets were similar to that present in the whole proteome. In contrast, the targets of Abs from the same mice were enriched for membrane and other virion components, as described previously. We conclude that a “nonbiasing” approach to T cell Ag discovery reveals a T cell Ag profile in VACV that is broader and less skewed to virion association than the Ab profile. The T cell Ag mapping method developed in the present study should be applicable to other organisms where expressible “ORFeome” libraries are also available, and it is readily scalable for larger pathogens.

We recently reported the clinical results of a Phase I trial combining ipilimumab with a vaccine containing transgenes for prostate-specific antigen (PSA) and for a triad of costimulatory molecules (PROSTVAC) in patients with metastatic castration-resistant prostate cancer. Thirty patients were treated with escalating ipilimumab and a fixed dose of vaccine. Of 24 chemotherapy-naïve patients, 58 % had a PSA decline. Combination therapy did not exacerbate the immune-related adverse events associated with ipilimumab. Here, we present updated survival data and an evaluation of 36 immune cell subsets pre- and post-therapy. Peripheral blood mononuclear cells were collected before therapy, at 13 days and at 70 days post-initiation of therapy, and phenotyped by flow cytometry for the subsets of T cells, regulatory T cells, natural killer cells, and myeloid-derived suppressor cells. Associations between overall survival (OS) and immune cell subsets prior to treatment, and the change in a given immune cell subset 70 days post-initiation of therapy, were evaluated. The median OS was 2.63 years (1.77-3.45). There were trends toward associations for longer OS and certain immune cell subsets before immunotherapy: lower PD-1(+)Tim-3(NEG)CD4EM (P = 0.005, adjusted P = 0.010), higher PD-1(NEG)Tim-3(+)CD8 (P = 0.002, adjusted P = 0.004), and a higher number of CTLA-4(NEG) Tregs (P = 0.005, adjusted P = 0.010). We also found that an increase in Tim-3(+) natural killer cells post- versus pre-vaccination associated with longer OS (P = 0.0074, adjusted P = 0.015). These results should be considered as hypothesis generating and should be further evaluated in larger immunotherapy trials.

PSA-TRICOM (PROSTVAC) is a novel vector-based vaccine designed to generate a robust immune response against prostate-specific antigen (PSA)-expressing tumor cells. The purpose of this report is to present an overview of both published studies and new data in the evaluation of immune responses to the PSA-TRICOM vaccine platform, currently in phase III testing. Of 104 patients tested for T-cell responses, 57% (59/104) demonstrated a ≥2-fold increase in PSA-specific T cells 4 weeks after vaccine (median 5-fold increase) compared with pre-vaccine, and 68% (19/28) of patients tested mounted post-vaccine immune responses to tumor-associated antigens not present in the vaccine (antigen spreading). The PSA-specific immune responses observed 28 days after vaccine (i.e., likely memory cells) are quantitatively similar to the levels of circulating T cells specific for influenza seen in the same patients. Measurements of systemic immune response to PSA may underestimate the true therapeutic immune response (as this does not account for cells that have trafficked to the tumor) and does not include antigen spreading. Furthermore, although the entire PSA gene is the vaccine, only one epitope of PSA is evaluated in the T-cell responses. Because this therapeutic vaccine is directed at generating a cellular/Th1 immune response (T-cell costimulatory molecules and use of a viral vector), it is not surprising that less than 0.6% of patients (2/349) tested have evidence of PSA antibody induction following vaccine. This suggests that post-vaccine PSA kinetics were not affected by PSA antibodies. An ongoing phase III study will evaluate the systemic immune responses and correlation with clinical outcomes. Cancer Immunol Res; 2(2); 133–41. ©2013 AACR.

Meeting abstracts The T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to

Preclinical studies have demonstrated that the combination of systemic subcutaneous (s.c.) vaccination with intratumoral (i.t.) vaccination was superior in the induction of antitumor activity vs. vaccination with either route alone. A subsequent phase I study employing i.t.-s.c. vaccination was carried out in men with locally recurrent or progressive prostate cancer. rF-PSA-TRICOM (PROSTVAC) vaccine was administered intraprostatically and rV-PSA-TRICOM followed by rF-PSA-TRICOM vaccine was administered systemically. In that study no dose limiting toxicities were observed, 19/21 patients had stable or improved prostate-specific antigen (PSA) values and tumor-infiltrating lymphocytes (TILs) increased in post- vs. pre-treatment tumor biopsies, analyzed employing conventional immunohistochemistry (IHC). In the studies reported here, 31 phenotypes of peripheral blood mononuclear cells (PBMCs) were analyzed prevaccination and postvaccination as well as the functions of PBMC regulatory T cells (Tregs) and natural killer cells. A trend was observed in decreases in serum PSA with the reduction of circulating Tregs postvaccination. Digital IHC was employed prevaccination and postvaccination to measure CD4 and CD8 TILs, as well as Treg TILs by conventional IHC. Few correlations were observed with CD4, CD8 or Treg in TILs vs. PBMCs. However, patients with lower levels of CD4 TILs prevaccination showed the greatest increases in CD4 TILs postvaccine, while Treg TILs decreased postvaccine. There was also a strong correlation between decreases in serum PSA and increases in CD8 TILs postvaccine. These studies provide additional rationale for the use of i.t.-s.c. vaccinations and demonstrate a noncoordinate expression of specific immune subsets in PBMCs vs. tumor.

Background. Talactoferrin alfa (talactoferrin), an agent with immune-stimulating properties, has demonstrated safety and preliminary efficacy in clinical trials. Methods. Ten patients (five males and five females) with stage IV non-small cell lung cancer (NSCLC) in a single-arm pilot study received orally administered talactoferrin (1.5 g, b.i.d.) for up to 24 weeks. Radiographic and immunologic studies were performed at baseline and at weeks 6 and 12. Circulating immune cells (natural killer cells [NKCs], CD4+, CD8+, and regulatory T cells) and systemic cytokine levels were measured to assess immune response. Results. Patients enrolled in the study had received a median of four prior chemotherapy regimens, and all patients were symptomatic. Talactoferrin was well tolerated, with no grade 3 or 4 toxicities. Median time to progression (TTP) and overall survival were 6 weeks and 14.5 weeks, respectively. The four patients with ≥9 weeks TTP had evidence of immunologic activity (three with increased NKC activity). Conclusions. The median of four previous chemotherapy regimens, with elevated levels of interleukin (IL) 6 and tumor necrosis factor-alfa in most patients, suggests these patients were poor candidates for immunotherapy.

245 Background: PSA-TRICOM is a vector-based therapeutic cancer vaccine designed to generate a targeted anti-tumor immune response against prostate-specific antigen (PSA)–expressing tumor cells. Early clinical trials have evaluated the immunologic impact of this vaccine and demonstrated promising clinical activity. PSA-TRICOM is being evaluated in a phase III trial in metastatic castration resistant prostate cancer (mCRPC). Methods: We recently conducted a broad overview of both published and new data which analyzed the immune responses to PSA-TRICOM. Immune responses included ELISPOT for antigen-specific immune response and flow-cytometry analysis of peripheral immune cells. Results: 104 patients (pts) with prostate cancer were tested for T-cell responses and 59 out of 104 (57%) demonstrated a greater than or equal to 2-fold increase in PSA-specific T cells 4 weeks after vaccine. The responders had a median 5-fold increase relative to pre-vaccine levels. For most pts PSA-specific immune responses (likely memory cells) seen 28 days following the most recent vaccine are quantitatively similar to levels of circulating influenza-specific T cells in the same pts. In addition, 19 out of 28 pts (68%) evaluated demonstrated immune responses to tumor-associated antigens not present in the vaccine (antigen spreading). Since PSA-TRICOM is designed to generate a cellular (TH1 immune response), it is not surprising that 2 out of 349 pts (

The MUC1 tumor-associated antigen is overexpressed in the majority of human carcinomas and several hematologic malignancies. Much attention has been paid to the hypoglycosylated variable number of tandem repeats (VNTR) region of the N-terminus of MUC1 as a vaccine target, and recombinant viral vector vaccines are also being evaluated that express the entire MUC1 transgene. While previous studies have described MUC1 as a tumor-associated tissue differentiation antigen, studies have now determined that the C-terminus of MUC1 (MUC1-C) is an oncoprotein, and its expression is an indication of poor prognosis in numerous tumor types. We report here the identification of nine potential CD8+ cytotoxic T lymphocyte epitopes of MUC1, seven in the C-terminus and two in the VNTR region, and have identified enhancer agonist peptides for each of these epitopes. These epitopes span HLA-A2, HLA-A3, and HLA-A24 major histocompatibility complex (MHC) class I alleles, which encompass the majority of the population. The agonist peptides, compared to the native peptides, more efficiently (a) generate T-cell lines from the peripheral blood mononuclear cells of cancer patients, (b) enhance the production of IFN-γ by peptide-activated human T cells, and (c) lyse human tumor cell targets in an MHC-restricted manner. The agonist epitopes described here can be incorporated into various vaccine platforms and for the ex vivo generation of human T cells. These studies provide the rationale for the T-cell-mediated targeting of the oncogenic MUC1-C, which has been shown to be an important factor in both drug resistance and poor prognosis for numerous tumor types.

Purpose: The T-box family transcription factor Brachyury is overexpressed in a variety of human carcinomas, including lung, breast, colon, ovarian and prostate. Brachyury has been shown to promote epithelial to mesenchymal transition (EMT) in tumor cells, a critical step in the path to metastasis. An HLA-A2 epitope of Brachyury has been shown to expand human T cells that are capable of lysing Brachyury-expressing tumor cells in an HLA-dependent manner. This study sought to define an agonist of this epitope in order to increase T cell activation and tumor lysis. Experimental Design: A novel agonist epitope of Brachyury was generated by residue substitution of the native epitope. Characterization of this epitope as an agonist included; comparison of HLA binding affinity and stability, interferon γ production by epitope-specific T cell lines, as well as Brachyury-and HLA-specific lysis of tumor cells. The presence of Brachyury-specific T cells that would recognize the agonist peptide, within the circulating PBMC of cancer patients, was determined by ELISPOT. Results: The agonist epitope was shown to bind HLA-A2 with higher affinity and stability than the native. T cell lines generated from both the native and agonist epitopes produced higher levels of interferon γ in response to stimulation with the agonist epitope. The agonist-specific T cell line was able to lyse a variety of Brachyury-expressing tumor cells more efficiently than the T cell line generated with the native epitope, and specificity of lysis was confirmed by cold-target inhibition and HLA-A2 blocking. Tetramer staining revealed Brachyury agonist-specific T cells in the PBMC of a prostate cancer patient after in vitro stimulation with the agonist peptide. PBMC from colon and ovarian cancer patients reacted to the agonist peptide in an interferon γ ELISPOT assay. Conclusions: An agonist epitope for Brachyury has been identified, which increased T cell activation and function as compared to the native epitope. T cells that react to the agonist peptide were detected in patients with carcinomas known to express high levels of Brachyury. This study supports the use of Brachyury as a cancer vaccine strategy, including the Brachyury agonist epitope. Citation Format: Jo A. Tucker, Diane J. Poole, Claudia Palena, Caroline Jochems, Jeffrey Schlom, Kwong Y. Tsang. Generation of human T cells directed against an agonist epitope of a transcription factor involved in epithelial to mesenchymal transition (EMT). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1260. doi:10.1158/1538-7445.AM2013-1260

Purpose: We recently published (Lancet Oncol. 2012;13(5):501-8) the clinical results of a trial combining ipilimumab (CTLA-4 blockade) with PSA-TRICOM, a viral vector vaccine containing transgenes for prostate-specific antigen (PSA) and three costimulatory molecules, in patients with metastatic castration-resistant prostate cancer. Thirty patients were treated with an escalating dose of ipilimumab, and fixed dose of vaccine. Of the 24 chemotherapy-naïve patients, 58% had a PSA decline from baseline. Combination therapy did not seem to exacerbate the immune-related adverse events associated with ipilimumab. Here, we evaluated the correlations of 30 immune cell subsets and clinical outcome in an effort to identify relevant biomarkers for immune monitoring of patients in clinical immunotherapy trials. Experimental Design: PBMCs were collected before therapy and at 70 days post initiation of therapy, and phenotyped by flow cytometry for T cells, regulatory T cells (Treg), natural killer (NK) cells and myeloid derived suppressor cells (MDSC). Inducible TH17 cells and intracellular IFNγ were evaluated after stimulation. Correlations of overall survival (OS) and immune cell subsets prior to treatment and 70 days post initiation of therapy, as well as OS and the change in a given immune cell subset were performed. Results: There were significant correlations with OS before therapy; Fewer monocytic MDSC correlated with longer survival (R= -0.72). There was a negative correlation with CD8+ central memory cells (CD8cm) (R= -0.64), and a positive correlation with the more activated PD1+ CD8cm subset (R= 0.67). At 70 days post initiation of therapy, high CD4cm (R= 0.54) and low CTLA-4+ Tregs (R= -0.53) correlated with longer survival. For correlations of OS and the change in a given immune cell subset, we found significant correlations for increased activated immature NK cells (R= 0.94) and decreased immature NK cells (R= -0.82). Additionally, a larger decrease in immunosuppressive cells such as CTLA-4+ Tregs and non-lineage MDSC after treatment correlated with increased OS (R= -0.37 and -0.83, respectively). Conclusions: The most striking result was the negative correlation at baseline of OS with the level of monocytic MDSC. Moreover, a decrease after therapy in the levels of CTLA-4+ Tregs and non-lineage MDSC correlated with increased OS. These results will be further evaluated in larger immunotherapy trials. Citation Format: Caroline Jochems, Jo A. Tucker, Ravi A. Madan, William L. Dahut, James L. Gulley, Jeffrey Schlom, Kwong-Yok Tsang. A combination trial of vaccine plus ipilimumab in patients with metastatic castration-resistant prostate cancer: immune correlates. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 498. doi:10.1158/1538-7445.AM2013-498

Purpose: Investigation of the effect of combination therapy for 5 cycles with bevacizumab, lenalidomide, docetaxel and prednisone (ART-P) on the immune response in patients with metastatic castration-resistant prostate cancer (mCRPC). The aim was to ascertain if treatment containing prednisone and docetaxel would impact immune responses, which could compromise the efficacy of subsequent immunotherapy. Experimental Design: We report here a study of immune responses in 12 patients enrolled in a phase II trial of ART-P at the National Cancer Institute (NCI). We compared peripheral blood mononuclear cells (PBMC) and serum samples collected at baseline and post 5 3-week cycles of ART-P treatment. PBMCs were analyzed using flow cytometry to characterize phenotypes of T-cells, regulatory T-cells, myeloid derived suppressor cells (MDSC) and natural killer (NK) cells, and T-cell proliferation as well as NK-cell functional activity were evaluated. Serum samples were analyzed for levels of cytokines, chemokines and vascular endothelial growth factor (VEGF) with ELISA assays. Results: The baseline characteristics for the 12 patients were: median age 65.7 years, Gleason score 8, and PSA 74 ng/ml. Greater than 50% decreases in PSA were seen in 9/12 patients, with a median decrease of 74%. Of the 12 patients 7 had a partial response, and 5 had stable disease by RECIST criteria. After 5 cycles of ART-P treatment we found no significant differences from baseline in T-cell proliferation and NK-cell function, or the frequencies of T-cells, regulatory T-cells, MDSC and NK-cells. There was also no change in the serum levels of cytokines and chemokines except for IL-6, which decreased. As expected, the serum levels of VEGF substantially decreased with therapy. Conclusions: Treatment of mCRPC patients with ART-P for 5 cycles did not alter the immune response in a way that would decrease the likelihood of successful immunotherapy, pre or post treatment with ART-P. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5379. doi:1538-7445.AM2012-5379

Immune suppression by regulatory T cells (Tregs) is associated with tumor evasion. An increase in the frequency and suppressive function of Tregs has been shown in a variety of solid tumors. The purpose of this study was to determine if the enhanced number and activity of these cells in prostate cancer patients could be due to tumor-induced changes in gene expression. CD4+CD25hiCD127lo Tregs were isolated from the peripheral blood of healthy donors and metastatic castration-resistant prostate cancer (mCRPC) patients. These samples were selected based on similar suppressive activity in a functional assay. A genome-wide expression array of 38, 500 genes was then performed to detect any effects in expression potentially induced by tumor. We found 384 genes had a three-fold or greater difference in expression between the groups. Differentially expressed genes were involved in cell cycle processes, cellular growth and proliferation, immune response, hematological system development and function, as well as the IL-2 and TGF-β pathways. Genes most up-regulated in the Tregs of mCRPC patients included C-FOS, C-JUN, DUSP1 and RGS1, which are crucial for regulation of T cell proliferation, activation and migration. We also observed increased expression of c-Jun protein in patient Tregs by flow cytometry. Patients had a significantly higher percentage of Tregs in their CD4+ population than the healthy donors, but interestingly, there was no difference in FoxP3 expression. These results indicate that tumor-derived factors may contribute to Treg mediated immune suppression by up-regulating genes that increase their proliferative and migratory capacities, allowing them to better home to the tumor and inhibit the host response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5404. doi:1538-7445.AM2012-5404