Makoto Azumi

Induction of EBV–Latent Membrane Protein 1–Specific MHC Class II–Restricted T-Cell Responses against Natural Killer Lymphoma Cells

EBV-encoded latent membrane protein 1 (LMP1) has oncogenic potential and is expressed in many EBV-associated malignancies. Although LMP1 is regarded as a potential tumor-associated antigen for immunotherapy and several LMP1-specific MHC class I-restricted CTL epitopes have been reported, little is known regarding MHC class II-restricted CD4 helper T-lymphocyte (HTL) epitopes for LMP1. The goal of the present studies was to determine whether MHC class II-restricted CD4 T-cell responses could be induced against the LMP1 antigen and to evaluate the antitumor effect of these responses. We have combined the use of a predictive MHC class II binding peptide algorithm with in vitro vaccination of CD4 T cells using candidate peptides to identify naturally processed epitopes derived from LMP1 that elicit immune responses against EBV-expressing tumor cells. Peptide LMP1(159-175) was effective in inducing HTL responses that were restricted by HLA-DR9, HLA-DR53, or HLA-DR15, indicating that this peptide behaves as a promiscuous T-cell epitope. Moreover, LMP1(159-175)-reactive HTL clones directly recognized EBV lymphoblastoid B cells, EBV-infected natural killer (NK)/T-lymphoma cells and naturally processed antigen in the form of LMP1+ tumor cell lysates presented by autologous dendritic cells. Because the newly identified epitope LMP1(159-175) overlaps with an HLA-A2-restricted CTL epitope (LMP1(159-167)), this peptide might have the ability to induce simultaneous CTL and HTL responses against LMP1. Overall, our data should be relevant for the design and optimization of T-cell epitope-based immunotherapy against various EBV-associated malignancies, including NK/T cell lymphomas.

Expression and functional role of MDL-1 (CLEC5A) in mouse myeloid lineage cells

Myeloid DNAX activation protein 12 (DAP12)‐associating lectin‐1 (MDL‐1), also known as C‐type lectin domain family 5, member A, is a type II transmembrane protein belonging to the C‐type lectin family and associates with DAP12 (also called KARAP or TYROBP). It has been reported that two isoforms of MDL‐1—long form (MDL‐1L) and short form (MDL‐1S)—exist in mice. Previously, we observed the marked induction of MDL‐1 mRNA expression during the pulmonary mycobacterial infection in mice. The data suggested that the MDL‐1‐expressing cells were involved in immune responses against mycobacterial infection; however, little is known about the function of MDL‐1 as yet. In this study, we demonstrated the significant protein expression of MDL‐1L and MDL‐1S in mouse neutrophils and macrophages. MDL‐1L was highly glycosylated by N‐linked glycan and sialic acid. Interestingly, the expression pattern of MDL‐1 was different between neutrophils and macrophages. MDL‐1 expression was notably induced during the differentiation of the mouse myeloid cell line 32Dcl3 into neutrophils. Additionally, we observed that MDL‐1 stimulation induced a significant amount of RANTES and macrophage‐derived chemokine production in 32Dcl3 cells in cooperation with signaling through TLR. MDL‐1 stimulation also up‐regulated CD11b expression and maintained cell survival. Our findings indicate that MDL‐1, therefore, plays an important role in immune defense as a result of an innate immunity, which involves neutrophils and macrophages.

Recognition of prostate and melanoma tumor cells by six-transmembrane epithelial antigen of prostate-specific helper T lymphocytes in a human leukocyte antigen class II-restricted manner.

The six-transmembrane epithelial antigen of prostate (STEAP) protein is an attractive candidate for T cell-based immunotherapy because it is overexpressed in prostate cancer and various other tumor types. Several peptide epitopes capable of stimulating CTLs that killed STEAP-expressing tumor cells have been described. Our goal was the identification of helper T lymphocyte (HTL) epitopes of STEAP for the optimization of T cell-based immunotherapies against STEAP-expressing malignancies. Candidate HTL epitopes for STEAP were predicted using in silico algorithms for HLA class II-binding peptides and were tested for their ability to elicit HTL responses by in vitro peptide vaccination of CD4 T lymphocytes from healthy individuals and prostate cancer patients. Two peptides (STEAP(102-116) and STEAP(192-206)) were effective in stimulating in vitro antitumor HTL responses in both normal individuals and prostate cancer patients. Notably, both STEAP HTL peptides behaved as promiscuous T-cell epitopes because they stimulated T cells in the context of more than one MHC class II allele. These newly described STEAP HTL epitopes could be of value for the design and optimization of T cell-based immunotherapy against STEAP-expressing tumors.

Six-Transmembrane Epithelial Antigen of the Prostate as an Immunotherapeutic Target for Renal Cell and Bladder Cancer

PURPOSE T-cell based immunotherapy for renal cell and bladder cancer is one of the most promising therapeutic approaches. STEAP is a novel cell surface protein that is over expressed in various cancers, including renal cell and bladder cancer. Recently we induced STEAP specific helper T lymphocytes that recognize the naturally processed STEAP peptide epitopes STEAP(102-116) and STEAP(192-206) arising from STEAP expressing tumor cells. Thus, STEAP may be a useful tumor associated antigen for designing T-cell based immunotherapy. We determined whether STEAP could induce anti-cellular immune responses to urological cancer. MATERIALS AND METHODS We selected 2 previously described STEAP derived epitope peptides, STEAP(102-116) and STEAP(192-206), and examined their ability to elicit helper T-lymphocyte responses by in vitro vaccination of CD4 T lymphocytes from healthy individuals and patients with cancer. RESULTS STEAP peptides induced helper T-lymphocyte responses using lymphocytes from healthy individuals that directly recognized STEAP expressing, DR positive renal cell and bladder cancer cells, and autologous dendritic cells pulsed with STEAP expressing tumor cell lysates in a major histocompatibility complex class II restricted manner. These peptides also stimulated T-cell responses in patients with renal cell or bladder cancer. Each STEAP peptides behaved as a promiscuous T-cell epitope, in that they stimulated T cells in the context of multiple major histocompatibility complex class II alleles. CONCLUSIONS Results show that STEAP helper T-lymphocyte epitopes could be used to optimize T-cell based immunotherapy against STEAP expressing renal cell and bladder cancer.

Focal adhesion kinase as an immunotherapeutic target

BackgroundFocal adhesion kinase (FAK) is a ubiquitously expressed non-receptor tyrosine kinase involved in cancer progression and metastasis that is found overexpressed in a large number of tumors such as breast, colon, prostate, melanoma, head and neck, lung and ovary. Thus, FAK could be an attractive tumor associated antigen (TAA) for developing immunotherapy against a broad type of malignancies. In this study, we determined whether predicted T cell epitopes from FAK would be able to induce anti-tumor immune cellular responses.MethodsTo validate FAK as a TAA recognized by CD4 helper T lymphocytes (HTL), we have combined the use of predictive peptide/MHC class II binding algorithms with in vitro vaccination of CD4 T lymphocytes from healthy individuals and melanoma patients.ResultsTwo synthetic peptides, FAK143–157 and FAK1,000–1,014, induced HTL responses that directly recognized FAK-expressing tumor cells and autologous dendritic cells pulsed with FAK-expressing tumor cell lysates in an HLA class II-restricted manner. Moreover, since the FAK peptides were recognized by melanoma patient’s CD4 T cells, this is indicative that T cell precursors reactive with FAK already exist in peripheral blood of these patients.ConclusionsOur results provide evidence that FAK functions as a TAA and describe peptide epitopes that may be used for designing T cell-based immunotherapy for FAK-expressing cancers, which could be used in combination with newly developed FAK inhibitors.

Long‐term persistence with mirabegron in a real‐world clinical setting

To examine the long‐term persistence rate with mirabegron in a real‐world clinical setting.

MP59-05 ANALYSIS OF FACTORS AFFECTING POST-OPERATIVE SPLIT RENAL FUNCTION AFTER PARTIAL NEPHRECTOMY USING R.E.N.A.L. NEPHROMETRY SCORE

RESULTS: 399 physicians completed the survey with 37% and 34% completing urologic oncology and endourology fellowships respectively. Answers to a question about the best oncologic treatment for 4-10cm RCC included: PN and RN are equal (56%), the best treatment is unknown (38%), and PN was inferior to RN (6%). Geographic location was the only predictor of response (p1⁄40.01). In case scenarios, the decision to offer PN rather than RN for a peripheral exophytic mass was influenced by comfort level with partial nephrectomy (p1⁄40.02). Open vs. MIS approach depends on comfort level and fellowship type. Oncology trained surgeons were more likely to offer open PN; OR 1.95, p1⁄40.003 and endourology fellowship graduates being more likely to offer MIS; OR 3.27, p<0.0001. Surgeon perception that they received adequate training in complex PN is predictive of offering PN for a centrally located RCC (p1⁄40.001). Academic practitioners are more likely to offer PN (p1⁄40.03). In addition, those completing training after 2001 are more likely to offer MIS (p1⁄40.02) and respondents who completed an oncology fellowship were more likely to offer PN to unhealthy patients (p1⁄40.03). CONCLUSIONS: Expert opinion about the best treatment for 410cm RCC varies significantly, with 70% of respondents willing to enroll patients in a randomized clinical trial comparing partial to radical nephrectomy in 4-10cm RCC.