Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE.

Immunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G-Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

[Development of novel bispecific antibody therapy for multiple myeloma].

Over the past decade, new therapeutic modalities have markedly improved clinical outcomes for patients with multiple myeloma. Recently, immunotherapy using both bispecific antibodies (BsAb) and chimeric antigen receptor T cells (CAR-T cells) has induced further anti-myeloma responses. Different agents must be combined to overcome the heterogeneity of myeloma cell clones, and new modalities for the treatment of refractory myeloma must also be developed to strengthen therapeutic effects. We have developed a novel BiTE (bispecific T-cell engager)-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE is able to bind to both an Fc domain of a human immunoglobulin G monoclonal antibody (mAb) and the human CD3 molecule. This enables rapid generation of a mAb/B-BiTE complex and safely induces dual-lymphoid activation of both human T cells and NK cells against myeloma cells. Importantly, sequential immunotherapy using two different mAb/B-BiTE complexes can produce deep and durable anti-myeloma responses. To further advance treatment of multiple myeloma, it is important to determine how to combine and sequence immunotherapy with other agents while considering management of unique adverse events caused by activated immune cells.

[Recent progress of bispecific antibody-based therapy for hematological malignancies].

Based on the progress of gene-modification technologies, bispecific antibodies that possess antigen-binding sites with two different specificities have been developed. After the success of blinatumomab for treating refractory B-cell leukemia, series of clinical trials using bispecific antibodies for relapsed and refractory hematological malignancies are being conducted. Several bispecific antibodies target an antigen expressed by tumor cells and the CD3 molecule where binding of bispecific antibodies can generate artificial immunological synapses between tumor cells and human T cells. Therefore, fine tuning of binding affinity and/or structural conformation concomitant with bispecific antibodies may be required to induce antitumor effects and regulate immune-related adverse events, such as cytokine release syndrome. In the future, combination therapy of conventional chemotherapy and/or allogeneic stem-cell transplantation with bispecific antibody therapy will be necessary. Furthermore, molecular target therapy with bispecific antibody therapy is expected to pave the way for next-generation target therapy, resulting in the development of a further effective and safe treatment strategy for hematological malignancies.

[Development of innovative T-cell immunotherapy for hematological malignancies].

With the emergence of cancer immunotherapy, T cells have played important roles in inducing antitumor responses. Many types of antitumor receptors, which possess tumor-binding sites and T-cell activation sites, have been developed. For example, genetically engineered T-cell receptor, chimeric antigen receptor, and bispecific antibody can help us to educate and activate T cells specific for certain tumors. To generate optimal antitumor receptors, (1) selection/distribution of tumor antigens, (2) affinity/specificity and cross-reactivity of antitumor receptors, and (3) T-cell activation signals delivered from antitumor receptors should be considered. Accordingly, we explain how antitumor receptors recognize target antigens and summarize the mechanisms for on-target/off-target reactivity induced by T cells redirected with antitumor receptors. Furthermore, we discuss how antitumor receptors can be optimized for the development of next-generation cancer immunotherapy.

Induction of WT1-specific human CD8+ T cells from human HSCs in HLA class I Tg NOD/SCID/IL2rgKO mice.

Induction of specific immune response against therapy-resistant tumor cells can potentially improve clinical outcomes in malignancies. To optimize immunotherapy in the clinic, we aimed to create an in vivo model enabling us to analyze human cytotoxic T-lymphocyte (CTL) responses against human malignancies. To this end, we developed NOD/SCID/IL2rgKO (NSG) mice expressing the HLA class I molecules HLA-A*0201 and A*2402. In the bone marrow (BM) and spleen of HLA class I transgenic (Tg) NSG mice transplanted with cord blood hematopoietic stem cells (HSCs), we found human memory CD8(+) T cells and antigen-presenting cells. To evaluate antigen-specific human CTL responses, we immunized HLA class I Tg NSG mice using polyinosinic:polycytidylic acid mixed Wilms tumor 1 (WT1) peptides, with or without WT1 peptide-loaded autologous dendritic cells. After immunization, the frequencies of HLA-restricted WT1-specific CTLs increased significantly in the spleen. Next, we transplanted the WT1-specific T-cell receptor (WT1-TCR) gene-transduced human HSCs into HLA class I Tg NSG newborn mice. WT1 tetramer-positive CD8(+) T cells differentiated from WT1-TCR-transduced HSCs in the recipients' BM, spleen, and thymus. Upon stimulation with WT1 peptide in vitro, these CTLs produced interferon-γ and showed lytic activity against leukemia cells in an antigen-specific, HLA-restricted manner. HLA class I Tg NSG xenografts may serve as a preclinical model to develop effective immunotherapy against human malignancies.

Specific roles of each TCR hemichain in generating functional chain-centric TCR.

TCRα- and ß-chains cooperatively recognize peptide-MHC complexes. It has been shown that a "chain-centric" TCR hemichain can, by itself, dictate MHC-restricted Ag specificity without requiring major contributions from the paired TCR counterchain. Little is known, however, regarding the relative contributions and roles of chain-centric and its counter, non-chain-centric, hemichains in determining T cell avidity. We comprehensively analyzed a thymically unselected T cell repertoire generated by transducing the α-chain-centric HLA-A*02:01(A2)/MART127-35 TCRα, clone SIG35α, into A2-matched and unmatched postthymic T cells. Regardless of their HLA-A2 positivity, a substantial subset of peripheral T cells transduced with SIG35α gained reactivity for A2/MART127-35. Although the generated A2/MART127-35-specific T cells used various TRBV genes, TRBV27 predominated with >10(2) highly diverse and unique clonotypic CDR3ß sequences. T cells individually reconstituted with various A2/MART127-35 TRBV27 TCRß genes along with SIG35α possessed a wide range (>2 log orders) of avidity. Approximately half possessed avidity higher than T cells expressing clone DMF5, a naturally occurring A2/MART127-35 TCR with one of the highest affinities. Importantly, similar findings were recapitulated with other self-Ags. Our results indicate that, although a chain-centric TCR hemichain determines Ag specificity, the paired counterchain can regulate avidity over a broad range (>2 log orders) without compromising Ag specificity. TCR chain centricity can be exploited to generate a thymically unselected Ag-specific T cell repertoire, which can be used to isolate high-avidity antitumor T cells and their uniquely encoded TCRs rarely found in the periphery because of tolerance.

CDR3ß sequence motifs regulate autoreactivity of human invariant NKT cell receptors.

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid ligands presented by monomorphic CD1d. Human iNKT T cell receptor (TCR) is largely composed of invariant Vα24 (Vα24i) TCRα chain and semi-variant Vß11 TCRß chain, where complementarity-determining region (CDR)3ß is the sole variable region. One of the characteristic features of iNKT cells is that they retain autoreactivity even after the thymic selection. However, the molecular features of human iNKT TCR CDR3ß sequences that regulate autoreactivity remain unknown. Since the numbers of iNKT cells with detectable autoreactivity in peripheral blood is limited, we introduced the Vα24i gene into peripheral T cells and generated a de novo human iNKT TCR repertoire. By stimulating the transfected T cells with artificial antigen presenting cells (aAPCs) presenting self-ligands, we enriched strongly autoreactive iNKT TCRs and isolated a large panel of human iNKT TCRs with a broad range autoreactivity. From this panel of unique iNKT TCRs, we deciphered three CDR3ß sequence motifs frequently encoded by strongly-autoreactive iNKT TCRs: a VD region with 2 or more acidic amino acids, usage of the Jß2-5 allele, and a CDR3ß region of 13 amino acids in length. iNKT TCRs encoding 2 or 3 sequence motifs also exhibit higher autoreactivity than those encoding 0 or 1 motifs. These data facilitate our understanding of the molecular basis for human iNKT cell autoreactivity involved in immune responses associated with human disease.

Development of a novel redirected T-cell-based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia.

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/ß genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Aurora kinase A-specific T-cell receptor gene transfer redirects T lymphocytes to display effective antileukemia reactivity.

Aurora kinase A (AURKA) is overexpressed in leukemias. Previously, we demonstrated that AURKA-specific CD8(+) T cells specifically and selectively lysed leukemia cells, indicating that AURKA is an excellent target for immunotherapy. In this study, we examined the feasibility of adoptive therapy using redirected T cells expressing an HLA-A*0201-restricted AURKA(207-215)-specific T-cell receptor (TCR). Retrovirally transduced T cells recognized relevant peptide-pulsed but not control target cells. Furthermore, TCR-redirected CD8(+) T cells lysed AURKA-overexpressing human leukemic cells in an HLA-A*0201-restricted manner, but did not kill HLA-A*0201(+) normal cells, including hematopoietic progenitors. In addition, AURKA(207-215)-specific TCR-transduced CD4(+) T cells displayed target-responsive Th1 cytokine production. Finally, AURKA(207-215)-specific TCR-transduced CD8(+) T cells displayed antileukemia efficacy in a xenograft mouse model. Collectively, these data demonstrate the feasibility of redirected T cell-based AURKA-specific immunotherapy for the treatment of human leukemia.

Bispecific antibodies for multiple myeloma: past, present and future.

Despite the development of various therapeutic agents, multiple myeloma remains incurable. Recently, T-cell redirected immunotherapy has become a promising strategy for the treatment of refractory myeloma. Clinical trials using chimeric antigen receptor (CAR)-T cells and bispecific antibodies have demonstrated successful anti-myeloma responses in triple-class-refractory patients. However, unique and unwanted immune effects associated with on-target/off-target reactivity of activated immune cells need to be considered and properly managed. This review summarizes recent advances in bispecific antibodies for the treatment of refractory myeloma. It outlines the history of their development, along with a discussion of their mechanisms of action and their current and potential future role in myeloma therapy. As more evidence emerges to inform the timing of CAR-T-cell therapy, the results of clinical trials and off-the-shelf nature of bispecifics also suggest the timing of their treatment. These findings will promote further development and application of bispecifics for refractory myeloma in combination with other appropriate agents.

Dissection of the signal transduction machinery responsible for the lysyl oxidase-like 4-mediated increase in invasive motility in triple-negative breast cancer cells: mechanistic insight into the integrin-ß1-NF-κB-MMP9 axis.

Background: Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-ß1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-ß1 hastens the invasive outgrowth of TNBC cells at the molecular level. Methods: LOXL4-overexpressing stable clones were established from MDA-MB-231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. Results: Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-κB (NF-κB). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-ß activated kinase 1 (TAK1) were required for the activation of NF-κB through Iκß kinase kinase (IKKα/ß) phosphorylation. Conclusion: Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-ß1-TRAF4-TAK1-IKKα/ß-Iκßα-NF-κB-MMP9, is crucial for TNBC cell invasiveness.

Lysyl oxidase-like 4 promotes the invasiveness of triple-negative breast cancer cells by orchestrating the invasive machinery formed by annexin A2 and S100A11 on the cell surface.

Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-ß1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion.

Novel adoptive T-cell immunotherapy using a WT1-specific TCR vector encoding silencers for endogenous TCRs shows marked antileukemia reactivity and safety.

Adoptive T-cell therapy for malignancies using redirected T cells genetically engineered by tumor antigen-specific T-cell receptor (TCR) gene transfer is associated with mispairing between introduced and endogenous TCR chains with unknown specificity. Therefore, deterioration of antitumor reactivity and serious autoimmune reactivity are major concerns. To address this problem, we have recently established a novel retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). In this study, to test the clinical application of siTCR gene therapy for human leukemia, we examined in detail the efficacy and safety of WT1-siTCR-transduced T cells. Compared with conventional WT1-TCR (WT1-coTCR) gene-transduced T cells, these cells showed significant enhancement of antileukemia reactivity resulting from stronger expression of the introduced WT1-specific TCR with inhibition of endogenous TCRs. Notably, WT1-siTCR gene-transduced T cells were remarkably expandable after repetitive stimulation with WT1 peptide in vitro, without any deterioration of antigen specificity. WT1-siTCR gene-transduced T cells from leukemia patients successfully lysed autologous leukemia cells, but not normal hematopoietic progenitor cells. In a mouse xenograft model, adoptively transferred WT1-siTCR gene-transduced T cells exerted distinct antileukemia efficacy but did not inhibit human hematopoiesis. Our results suggest that gene-immunotherapy for leukemia using this WT1-siTCR system holds considerable promise.

Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice.

Whereas humanized mouse models have contributed significantly to human immunology research, human T cells developing in mouse thymic environment fail to demonstrate HLA-restricted function. To achieve HLA-restricted human immune response, we created an immune-compromised non-obese diabetic/SCID/IL2rg(null) strain (NSG) with homozygous expression of HLA class I heavy chain and light chain (NSG-HLA-A2/HHD). Transplantation of purified Lin-CD34+CD38- human hematopoietic stem cells into NSG-HLA-A2/HHD newborns resulted in the development of human CD4+ and CD8+ TCR alphabeta+ T cells and CD4-CD8- and CD8+ TCR gammadelta+ cells in recipient bone marrow and spleen. Human cytotoxic T lymphocytes (CTLs) become functionally mature, as evidenced by the production of granzyme corresponding to phenotypic transition from naïve to effector memory CTLs. In these recipients, human Th17 cells developed along with Th1 and Th2 cells. Epstein-Barr virus (EBV) infection in the humanized NSG-HLA-A2/HHD recipients resulted in the formation of lymphoproliferative lesions consisting mainly of human B cells with scattered human T cells. Human CTLs developing in the recipients recognized EBV-derived peptides in an HLA-restricted manner and exerted HLA-restricted cytotoxicity against EBV-infected human B cells. The HLA-expressing humanized mouse with functional HLA-restricted T cells and consistent representation of rare T-cell subsets overcomes a major constraint in human immunology, and serves as a useful model for investigation of human immune responses against pathogens and for the development of therapeutic strategies against human diseases.

Phosphorylated SARM1 is involved in the pathological process of rotenone-induced neurodegeneration.

Sterile alpha and Toll/interleukin receptor motif-containing protein 1 (SARM1) is a NAD+ hydrolase that plays a key role in axonal degeneration and neuronal cell death. We reported that c-Jun N-terminal kinase (JNK) activates SARM1 through phosphorylation at Ser-548. The importance of SARM1 phosphorylation in the pathological process of Parkinson's disease (PD) has not been determined. We thus conducted the present study by using rotenone (an inducer of PD-like pathology) and neurons derived from induced pluripotent stem cells (iPSCs) from healthy donors and a patient with familial PD PARK2 (FPD2). The results showed that compared to the healthy neurons, FPD2 neurons were more vulnerable to rotenone-induced stress and had higher levels of SARM1 phosphorylation. Similar cellular events were obtained when we used PARK2-knockdown neurons derived from healthy donor iPSCs. These events in both types of PD-model neurons were suppressed in neurons treated with JNK inhibitors, Ca2+-signal inhibitors, or by a SARM1-knockdown procedure. The degenerative events were enhanced in neurons overexpressing wild-type SARM1 and conversely suppressed in neurons overexpressing the SARM1-S548A mutant. We also detected elevated SARM1 phosphorylation in the midbrain of PD-model mice. The results indicate that phosphorylated SARM1 plays an important role in the pathological process of rotenone-induced neurodegeneration.

STAT1/3 signaling suppresses axon degeneration and neuronal cell death through regulation of NAD+-biosynthetic and consuming enzymes.

Nicotinamide adenine dinucleotide (NAD)+-biosynthetic and consuming enzymes are involved in various intracellular events through the regulation of NAD+ metabolism. Recently, it has become clear that alterations in the expression of NAD+-biosynthetic and consuming enzymes contribute to the axonal stability of neurons. We explored soluble bioactive factor(s) that alter the expression of NAD+-metabolizing enzymes and found that cytokine interferon (IFN)-γ increased the expression of nicotinamide nucleotide adenylyltransferase 2 (NMNAT2), an NAD+-biosynthetic enzyme. IFN-γ activated signal transducers and activators of transcription 1 and 3 (STAT1/3) followed by c-Jun N-terminal kinase (JNK) suppression. As a result, STAT1/3 increased the expression of NMNAT2 at both mRNA and protein levels in a dose- and time-dependent manner and, at the same time, suppressed activation of sterile alpha and Toll/interleukin receptor motif-containing 1 (SARM1), an NAD+-consuming enzyme, and increased intracellular NAD+ levels. We examined the protective effect of STAT1/3 signaling against vincristine-mediated cell injury as a model of chemotherapy-induced peripheral neuropathy (CIPN), in which axonal degeneration is involved in disease progression. We found that IFN-γ-mediated STAT1/3 activation inhibited vincristine-induced downregulation of NMNAT2 and upregulation of SARM1 phosphorylation, resulting in modest suppression of subsequent neurite degradation and cell death. These results indicate that STAT1/3 signaling induces NMNAT2 expression while simultaneously suppressing SARM1 phosphorylation, and that both these actions contribute to suppression of axonal degeneration and cell death.

CD21lo B Cells Could Be a Potential Predictor of Immune-Related Adverse Events in Renal Cell Carcinoma.

Immune checkpoint inhibitor (ICI) therapy increases the risk of immune-related adverse events (irAEs). In particular, combination checkpoint blockade (CCB) targeting inhibitory CTLA-4 and PD-1 receptors could lead to irAEs at a higher rate than ICI monotherapy. Management of irAEs is important while using ICIs. However, there are no reliable biomarkers for predicting irAEs. The aim of this study was to elucidate early B cell changes after CCB therapy in patients with renal cell carcinoma (RCC) and verify whether B cells can be a predictor of irAEs. This prospective cohort study was conducted with 23 Japanese patients with metastatic RCC. An increase in the proportion of CD21lo B cells and CD21lo memory B cells was confirmed following CCB therapy. Although there were no differences in clinical outcomes between irAE and no-irAE groups, the proportion of CD21lo B cells at baseline was lower in the irAE group, with a significant increase after the first cycle of CCB therapy. Further analysis revealed a moderate correlation between irAEs and CD21lo B cell levels at baseline (area under the curve: 0.83, cut-off: 3.13%, sensitivity: 92.3, specificity: 70.0). This finding indicates that patients with low baseline CD21lo B cell levels warrant closer monitoring for irAEs. The clinical registration number by the Certified Review Board of Ehime University is No. 1902011.

Gemcitabine enhances Wilms' tumor gene WT1 expression and sensitizes human pancreatic cancer cells with WT1-specific T-cell-mediated antitumor immune response.

Wilms' tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene-transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.