Jo A. Tucker

Identification and characterization of agonist epitopes of the MUC1-C oncoprotein

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.

Effect of Talactoferrin Alfa on the Immune System in Adults With Non-Small Cell Lung Cancer

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.

Identification by digital immunohistochemistry of intratumoral changes of immune infiltrates after vaccine in the absence of modifications of PBMC immune cell subsets

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.

Immunotherapy: Shifting the Balance of Cell-Mediated Immunity and Suppression in Human Prostate Cancer

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.

Generation of human T cells directed against an agonist epitope of Brachyury, a transcription factor involved in human tumor cell epithelial to mesenchymal transition (EMT)

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. A phase I clinical trial is ongoing at the NCI using a recombinant yeast Brachyury vaccine. We have previously demonstrated that agonist epitopes of tumorassociated antigens are more effective than native epitopes at activating antigen-specific T cell responses. The current study sought to identify an agonist of the Brachyury HLA-A2 epitope in order to increase T cell activation and tumor lysis.

Abstract 498: A combination trial of vaccine plus ipilimumab in patients with metastatic castration-resistant prostate cancer: immune correlates.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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-naive 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

Abstract 1260: Generation of human T cells directed against an agonist epitope of a transcription factor involved in epithelial to mesenchymal transition (EMT).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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

Abstract 5379: Combination treatment with Bevacizumab, Lenalidomide, Docetaxel and Prednisone (ART-P) does not impact the immune response in patients with metastatic castration-resistant prostate cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL 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

Abstract 5404: Up-regulation of proliferative and migratory genes in regulatory T cells from patients with metastatic castration-resistant prostate cancer

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 + CD25 hi CD127 lo 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