Takuro Noguchi

Two Distinct Mechanisms of Augmented Antitumor Activity by Modulation of Immunostimulatory/Inhibitory Signals

Purpose: Blockade of CTL-associated antigen-4 (CTLA-4), an inhibitory immunomodulatory molecule on T cells, has been shown to enhance T-cell responses and induce tumor rejection, and a number of clinical trials with anti-CTLA-4 blocking monoclonal antibody (mAb) are under way. However, accumulating evidence indicates that anti-CTLA-4 mAb increases the number of CD4+CD25+Foxp3+ regulatory T cells (Treg) and that anti-CTLA4 mAb alone is often insufficient to reject established tumors in mice and humans. Thus, finding maneuvers to control Tregs and other immunosuppressive mechanisms remains a critical challenge. Experimental Design: The potential to enhance antitumor immune responses by combining anti-CTLA-4 mAb with anti–glucocorticoid-induced tumor necrosis factor receptor family related gene (GITR) mAb, a costimulatory molecule that abrogates directly/indirectly Treg-mediated immune suppression or anti-CD25 mAb that depletes Tregs was analyzed with two tumor models, CT26 (a murine colon carcinoma cell line) and CMS5a (a murine fibrosarcoma cell line). Results: Anti-CTLA-4/anti-GITR mAb combination treatment exhibited far stronger antitumor effects compared with either antibody alone. This strong antitumor effect was attributed to (a) increased numbers of CD8+ T cells infiltrating tumor sites in anti-CTLA-4 mAb–treated mice and (b) increased cytokine secretion and Treg resistance of tumor-specific CD8+ T cells with strongly upregulated CD25 expression in anti-GITR mAb–treated mice, indicating distinct quantitative/qualitative changes induced by modulating CTLA-4 and GITR signaling. Conclusions: This study shows that combined treatment with different immune modulators can augment antitumor immune responses and provides justification for exploring anti-CTLA-4/anti-GITR mAb combination treatment in the clinic. Clin Cancer Res; 16(10); 2781–91. ©2010 AACR.

Intracellular tumor-associated antigens represent effective targets for passive immunotherapy.

Monoclonal antibody (mAb) therapy against tumor antigens expressed on the tumor surface is associated with clinical benefit. However, many tumor antigens are intracellular molecules that generally would not be considered suitable targets for mAb therapy. In this study, we provide evidence challenging this view through an investigation of the efficacy of mAb directed against NY-ESO-1, a widely expressed immunogen in human tumors that is expressed intracellularly rather than on the surface of cells. On their own, NY-ESO-1 mAb could neither augment antigen-specific CD8(+) T-cell induction nor cause tumor eradication. To facilitate mAb access to intracellular target molecules, we combined anti-NY-ESO-1 mAb with anticancer drugs to accentuate the release of intracellular NY-ESO-1 from dying tumor cells. Strikingly, combination therapy induced a strong antitumor effect that was accompanied by the development of NY-ESO-1-specific effector/memory CD8(+) T cells that were not elicited by single treatments alone. The combinatorial effect was also associated with upregulation of maturation markers on dendritic cells, consistent with the organization of an effective antitumor T-cell response. Administration of Fc-depleted F(ab) mAb or combination treatment in Fcγ receptor-deficient host mice abolished the therapeutic effect. Together, our findings show that intracellular tumor antigens can be captured by mAbs and engaged in an efficient induction of CD8(+) T-cell responses, greatly expanding the possible use of mAb for passive cancer immunotherapy.

Peptide Vaccine Induces Enhanced Tumor Growth Associated with Apoptosis Induction in CD8+ T Cells

CD8+ CTLs play a critical role in antitumor immunity. However, vaccination with synthetic peptide containing CTL epitopes has not been generally effective in inducing protective antitumor immunity. In this study, we addressed the detailed mechanism(s) involved in this failure using a new tumor model of BALB/c transplanted tumors expressing NY-ESO-1, an extensively studied human cancer/testis Ag. Whereas peptide immunization with an H2-Dd–restricted CTL epitope derived from NY-ESO-1 (NY-ESO-1 p81–88) induced NY-ESO-181–88–specific CD8+ T cells in draining lymph nodes and spleens, tumor growth was significantly enhanced. Single-cell analysis of specific CD8+ T cells revealed that peptide immunization caused apoptosis of >80% of NY-ESO-181–88–specific CD8+ T cells at tumor sites and repetitive immunization further diminished the number of specific CD8+ T cells. This phenomenon was associated with elevated surface expression of Fas and programmed death-1. When peptide vaccination was combined with an adjuvant, a TLR9 ligand CpG, the elevated Fas and programmed death-1 expression and apoptosis induction were not observed, and vaccine with peptide and CpG was associated with strong tumor growth inhibition. Selection of appropriate adjuvants is essential for development of effective cancer vaccines, with protection of effector T cells from peptide vaccine-induced apoptosis being a prime objective.

Antibody-based therapy in colorectal cancer

Treatment in patients with nonresectable and resectable colorectal cancer at the advanced stage is challenging, therefore intensive strategies such as chemotherapy, signaling inhibitors and monoclonal antibodies (mAbs) to control the disease are required. mAbs are particularly promising tools owing to their target specificities and strong antitumor activities through multiple mechanisms, as shown by rituximab in B-cell non-Hodgkins lymphoma and trastuzumab in breast cancer. Three mAbs (cetuximab, bevacizumab and panitumumab) have been approved for the treatment of colorectal cancer in the USA and many other mAbs are being tested in clinical trials. The potential of antibody therapy is associated with several mechanisms including interference of vital signaling pathways targeted by the antibody and immune cytotoxicity selectively directed against tumor cells by tumor-bound antibody through the Fc portion of the antibody, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Moreover, recent experimental findings have shown that immune complexes formed by therapeutic mAbs with tumor-released antigens could augment the induction of tumor-specific cytotoxic CD8(+) T cells through activation of APCs. In addition, antibodies targeting immune checkpoints on hematopoietic cells have recently opened a new avenue for the treatment of cancer. In this review, we focus on mAb treatment in colorectal cancer and its immunological aspects.

Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8 + T cells

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8(+) T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8(+) T cells recognized D(d)-restricted 8mer peptides, NY-ESO-181-88. MAGE-A4-specific CD8(+) T cells recognized D(d)-restricted 9mer peptides, MAGE-A4265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8(+) T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.

Extrapleural hematoma as a complication following thoracotomy for pulmonary lobectomy

Extrapleural hematoma as a complication following thoracic surgery is rare. We report a case of an extrapleural hematoma following pleural lobectomy that resolved completely with nonsurgical treatment. A 63-year-old woman underwent left lower lobectomy for lung cancer through a left posterolateral thoracotomy. She had been prescribed the anticoagulant cilostazol to increase her heart rate for atrioventricular dissociation. Preoperatively, it was stopped, and a temporary pacemaker was placed to counteract bradycardia via the right jugular vein without complication. The chest tube was removed, and cilostazol was resumed on the third postoperative day. On day 7, she suddenly experienced left shoulder pain followed by hypotension, tachycardia, and anemia. Enhanced computed tomography (CT) revealed an extrapleural hematoma rather than a hemothorax. She became symptomatic after rapid infusion. The hematoma resolved without an invasive intervention. The CT results 189 days after the onset showed almost complete regression of the hematoma.

Abstract 4741: Therapeutic efficacy of a human-derived antibody against cancer-testis antigen NY-ESO-1

Rationale: It is accepted that the immune system can control cancer and a favorable clinical course correlates with high infiltration by CD8+ T cells in different cancer entities. Thus, boosting cancer-specific immunity is an interesting therapeutic modality. Cancer-testis antigens (CTA) are promising target antigens due to their tumor-restricted expression pattern and high immunogenicity. NY-ESO-1 is one of the best characterized CTA, and spontaneous anti-NY-ESO-1 humoral and cellular responses were described in patients with various cancers. We propose that antibodies (Abs) against intracellular CTA have therapeutic potential because they may facilitate the uptake and presentation of antigens by dendritic cells (DC), which supports cancer-specific T cell responses. This effect may be more pronounced when Abs are used in combination with therapies that result in death, and thus antigen release, of cancer cells. We report here the characterization of the first human anti-NY-ESO-1 monoclonal Ab. Methods: Human-derived monoclonal Ab 12D7 was obtained by molecular cloning from memory B cells of a melanoma patient, ZH-311. To characterize the binding of 12D7 we performed ELISA, Biacore analysis and tissue staining. In addition, we used 12D7 to immunoprecipitate native NY-ESO-1 from human melanoma cell line SK-MEL-37. As a proof of concept, we compared the activation of NY-ESO-1-specific CD8+ T cell clones by DC fed with NY-ESO-1 vs. NY-ESO-1/12D7 immune complexes (IC) in vitro. To determine the therapeutic effect in vivo, we treated BALB/c mice bearing syngeneic CT26/NY-ESO-1 colorectal tumors with 5-FU +/- 12D7. To investigate Ab accumulation in established tumors 12D7 was labeled with FITC and injected into mice 2 days after 5-FU treatment. Results: Human monoclonal 12D7 specifically bound to NY-ESO-1 with high affinity (pM range) and precipitated endogenous NY-ESO-1 from SK-MEL-37. The uptake and cross-presentation of NY-ESO-1 by DC was greatly enhanced when NY-ESO-1 was complexed with 12D7 before feeding. Importantly, uptake of NY-ESO-1/12D7 IC, but not NY-ESO-1, resulted in maturation of DCs as determined by upregulation of co-stimulatory molecules. Finally, 12D7 significantly improved the therapeutic efficacy of 5-FU treatment in established CT26/NY-ESO-1 tumors. Ab accumulation in CT26/NY-ESO-1 tumors was enhanced when mice were treated with 5-FU as compared with untreated mice. Conclusion: We propose that the release of intracellular tumor antigens by chemotherapy and the accumulation of Ab to such antigens results in the local formation of IC. These IC are efficiently cross-presented to tumor-specific CD8+ T cells by DCs and may induce in situ maturation of the latter, both of which support tumor-specific effector function and thus contributes to tumor control. Our results suggest that targeting intracellular antigens, more specifically CTA, with Abs is a useful strategy for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4741. doi:10.1158/1538-7445.AM2011-4741