Shigehisa Kitano

Humoral immune responses in patients vaccinated with 1–146 HER2 protein complexed with cholesteryl pullulan nanogel

The CHP‐HER2 vaccine, comprising truncated 146HER2 protein complexed with nanogels of cholesteryl pullulan (CHP), is a novel protein antigen vaccine that elicits 146HER2‐specific CD8+ and CD4+ T‐cell immune responses in patients with HER2‐expressing tumors. We analyzed the humoral responses in patients vaccinated with CHP‐HER2 and those with CHP‐HER2 plus granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). The vaccine was injected subcutaneously at a dose of 300 µg protein. Nine patients received the vaccine alone over the first four injections, followed by CHP‐HER2 with GM‐CSF or OK‐432, whereas six received CHP‐HER2 plus GM‐CSF from the first cycle. 146HER2‐specific IgG antibodies were induced in 14 patients, who were negative at baseline. The antibodies became detectable after the second or third vaccination and reached plateau levels after the third or fourth cycle in patients vaccinated with CHP‐HER2 plus GM‐CSF. In contrast, the antibodies appeared only after the third to sixth vaccination and the plateau appeared after the fourth to eighth cycle in patients vaccinated with the CHP‐HER2 vaccine alone over the first four cycles. The antibodies induced by the vaccine were not reactive with HER2 antigen expressed on the cell surface in any of the patients. Epitope analysis using overlapping peptides revealed a single region in the 146HER2 protein, amino acids 127–146, in eight patients who were initially vaccinated with CHP‐HER2 alone. Similarly, the same HER2 region was recognized dominantly in patients vaccinated with GM‐CSF. Our results indicate that CHP‐HER2 induced HER2‐specific humoral responses in patients with HER2‐expressing tumors and that GM‐CSF seems to accelerate the responses. (Cancer Sci 2008; 99: 601–607)

Computational Algorithm-Driven Evaluation of Monocytic Myeloid-Derived Suppressor Cell Frequency for Prediction of Clinical Outcomes

Kitano and colleagues developed an algorithm to determine the frequency of monocytic MDSC (m-MDSC), performed a retrospective analysis of samples from patients treated with ipilimumab, and found m-MDSC frequencies inversely correlated with clinical response and CD8+ T-cell expansion following treatment. Evaluation of myeloid-derived suppressor cells (MDSC), a cell type implicated in T-cell suppression, may inform immune status. However, a uniform methodology is necessary for prospective testing as a biomarker. We report the use of a computational algorithm-driven analysis of whole blood and cryopreserved samples for monocytic MDSC (m-MDSC) quantity that removes variables related to blood processing and user definitions. Applying these methods to samples from patients with melanoma identifies differing frequency distribution of m-MDSC relative to that in healthy donors. Patients with a pretreatment m-MDSC frequency outside a preliminary definition of healthy donor range (<14.9%) were significantly more likely to achieve prolonged overall survival following treatment with ipilimumab, an antibody that promotes T-cell activation and proliferation. m-MDSC frequencies were inversely correlated with peripheral CD8+ T-cell expansion following ipilimumab. Algorithm-driven analysis may enable not only development of a novel pretreatment biomarker for ipilimumab therapy, but also prospective validation of peripheral blood m-MDSCs as a biomarker in multiple disease settings. Cancer Immunol Res; 2(8); 812–21. ©2014 AACR.

HER2-Specific T-Cell Immune Responses in Patients Vaccinated with Truncated HER2 Protein Complexed with Nanogels of Cholesteryl Pullulan

Purpose: We developed a complex of tumor antigen protein with a novel nanoparticle antigen delivery system of cholesteryl pullulan (CHP). To target HER2 antigen, we prepared truncated HER2 protein 1-146 (146HER2) complexed with CHP, the CHP-HER2 vaccine. We designed a clinical study to assess the safety of the vaccine and HER2-specific T-cell immune responses measured by the newly developed enzyme-linked immunospot assay with mRNA-transduced phytohemagglutinin-stimulated CD4+ T cells in HLA-A2402-positive patients with therapy-refractory HER2-expressing cancers. Experimental Design: Nine patients with various types of solid tumors were enrolled. Each patient was s.c. vaccinated biweekly with 300 μg of CHP-HER2 vaccine for three times followed by booster doses. HER2-specific T-cell responses were evaluated by enzyme-linked immunospot assay by targeting autologous phytohemagglutinin-stimulated CD4+ T cells transduced with 146HER2-encoding mRNA to cover both identified peptides and unknown epitopes for MHC class I and class II that might exist in the sequence of the vaccine protein. Results: CHP-HER2 vaccine was well tolerated; the only adverse effect was grade 1 transient skin reaction at the sites of vaccination. HER2-specific CD8+ and/or CD4+ T-cell immune responses were detected in five patients who received four to eight vaccinations, among whom both T-cell responses were detected in these patients. In four patients with CD8+ T-cell responses, two patients reacted to previously identified HER263-71 peptide and the other two reacted only to 146HER2 mRNA-transduced cells. Conclusions: CHP-HER2 vaccine was safe and induced HER2-specific CD8+ and/or CD4+ T-cell immune responses.

Paradoxical activation of T cells via augmented ERK signaling mediated by a RAF inhibitor

Callahan and colleagues show that RAF inhibitors can potentiate T-cell activation by increasing T-cell expansion and ERK signaling, and when combined with CTLA-4 blockade show superior tumor control in two transplantable mouse tumor models. RAF inhibitors selectively block extracellular signal–regulated kinase (ERK) signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T-cell activation, based on the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T-cell activation and signaling in vitro and in vivo. We observe that T-cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with cytotoxic T-lymphocyte antigen 4 (CTLA-4) blockade in vivo potentiates T-cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Finally, this combination demonstrates superior antitumor activity, compared with either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T-cell function by potentiating T-cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models. Cancer Immunol Res; 2(1); 70–79. ©2013 AACR.

Granzyme B is a novel interleukin-18 converting enzyme

BACKGROUND Granzyme B (GrB) is recognized to induce apoptosis; however, little is known about its possible role in other biological events. IL-18, a potent inflammatory cytokine, is produced as an inactive precursor (proIL-18). Several cells, including monocytes/macrophage lineage and non-hematopoietic cells such as keratinocytes, produce proIL-18. ProIL-18 requires appropriate processing to become active. Caspase-1 is the authentic IL-18 processing enzyme and is essential for IL-18 release from monocyte/macrophage lineage cells. However, caspase-1 is absent in non-hematopoietic cells, suggesting that there is another candidate to cleave proIL-18 except for caspase-1. OBJECTIVE GrB can invade and be active in cytoplasm of non-hematopoietic cells via perforin, therefore we investigated whether GrB converts proIL-18 into the biologically active form. METHODS Recombinant proIL-18 (rproIL-18) was produced and purified for protease reaction with GrB; this incubate was evaluated by immunoblotting. Biological activity of the proteolytic fragment cleaved by GrB was determined by IFN-gamma assay using KG-1 cells. IFN-gamma induction was also analyzed between extracts from GrB(+)/caspase-1(-) human CD8+ T cells and proIL-18 from normal human keratinocytes (NHK). RESULTS The proteolytic fragment that GrB cleaved proIL-18 had the same sequence and biological activity compared with mature IL-18 cleaved by caspase-1. Culture extracts from CD8+ T cells was able to cleave proIL-18 into authentic mature IL-18. IFN-gamma induction was also detected in NHK treated with CD8+ T cells. CONCLUSION GrB is a potent IL-18 converting enzyme and suggest that GrB secreted by CTLs and/or NK cells may initiate IL-18 release from target cells, leading to the development of inflammation.

Determination of Cellularly Processed HLA-A2402-Restricted Novel CTL Epitopes Derived from Two Cancer Germ Line Genes, MAGE-A4 and SAGE

Purpose: For identification of CTL epitopes useful for cancer vaccines, it is crucial to determine whether cognate epitopes are presented on the cell surface of target cancer cells through natural processing of endogenous proteins. For this purpose, we tried to use the cellular machinery of both mice and human to define naturally processed CTL epitopes derived from two “cancer germ line” genes, MAGE-A4 and SAGE. Experimental Design: We vaccinated newly produced HLA-A2402 transgenic mice with DNA plasmids encoding target antigens. Following screening of synthesized peptides by splenic CD8+ T cells of vaccinated mice, we selected candidate epitopes bound to HLA-A2402. We then examined whether human CD8+ T cells sensitized with autologous CD4+ PHA blasts transduced by mRNA for the cognate antigens could react with these selected peptides in an HLA-A2402-restricted manner. Results: After DNA vaccination, murine CD8+ T cells recognizing MAGE-A4143-151 or SAGE715-723 in an HLA-A2402-restricted manner became detectable. Human CTLs specific for these two peptides were generated after sensitization of HLA-A2402-positive CD8+ T cells with autologous CD4+ PHA blasts transduced with respective mRNA. CTL clones were cytotoxic toward tumor cell lines expressing HLA-A2402 and cognate genes. Taken together, these CTL epitopes defined in HLA-A24 transgenic mice are also processed and expressed with HLA-A2402 in human cells. The presence of SAGE715-723-specific precursors was observed in HLA-A2402-positive healthy individuals. Conclusions: Two novel HLA-A2402-restricted CTL epitopes, MAGE-A4143-151 and SAGE715-723, were identified. Our approach assisted by cellular machinery of both mice and human could be widely applicable to identify naturally processed CTL epitopes.

Nivolumab for advanced melanoma: pretreatment prognostic factors and early outcome markers during therapy

Background An anti-programmed cell death protein 1 monoclonal antibody, nivolumab, is one of the most effective drugs for advanced melanoma. Tumor cell-derived or immune cell-derived markers and clinical predictors such as serum lactate dehydrogenase (LDH) and cutaneous adverse events, have already been described as prognostic factors for advanced melanoma treated with nivolumab. We sought to identify further clinical predictors that can be determined in routine clinical practice. Methods We retrospectively analyzed clinical findings of 98 consecutive patients with unresectable stage III or IV melanoma treated with nivolumab, at the National Cancer Center Hospital or at Keio University Hospital, in Tokyo, Japan, between July 2014 and July 2016. These patients had been administered nivolumab at a dose of 2mg/kg every 3 weeks. Results As for pretreatment prognostic factors, ECOG performance status (PS) ≥1, maximum tumor diameters of ≥30mm, elevated LDH and elevated C-reactive protein were significantly associated with poor overall survival (OS) (hazard ratio [HR] 0.29 [P<0.001], HR 0.40 [p=0.003], HR 0.29 [P<0.001], HR 0.42 [P=0.004], respectively) on univariate analysis. Among these factors, PS and LDH were identified as independent variables by multivariate analysis. As for early markers examined during therapy, patients with absolute lymphocyte count (ALC) ≥ 1000/μl (Week3: HR 0.40 [P=0.004], Week6: HR 0.33 [P=0.001]) and absolute neutrophil count (ANC) <4000/μl (Week3: HR 0.46 [P=0.014], Week6: HR 0.51 [P=0.046]) had significantly better OS. Conclusion ALC≥1000/μl and ANC<4000/μl during treatment appear to be early markers associated with OS. Nivolumab might have minimal efficacy in patients with a massive tumor burden.

Clinical Development of Immune Checkpoint Inhibitors

Recent progress in cancer immunotherapy has been remarkable. Most striking are the clinical development and approval of immunomodulators, also known as immune checkpoint inhibitors. These monoclonal antibodies (mAb) are directed to immune checkpoint molecules, which are expressed on immune cells and mediate signals to attenuate excessive immune reactions. Although mAbs targeting tumor associated antigens, such as anti-CD20 mAb and anti-Her2 mAb, directly recognize tumor cells and induce cell death, immune checkpoint inhibitors restore and augment the antitumor immune activities of cytotoxic T cells by blocking immune checkpoint molecules on T cells or their ligands on antigen presenting and tumor cells. Based on preclinical data, many clinical trials have demonstrated the acceptable safety profiles and efficacies of immune checkpoint inhibitors in a variety of cancers. The first in class approved immune checkpoint inhibitor is ipilimumab, an anti-CTLA-4 (cytotoxic T lymphocyte antigen-4) mAb. Two pivotal phase III randomized controlled trials demonstrated a survival benefit in patients with metastatic melanoma. In 2011, the US Food and Drug Administration (FDA) approved ipilimumab for metastatic melanoma. Several clinical trials have since investigated new agents, alone and in combination, for various cancers. In this review, we discuss the current development status of and future challenges in utilizing immune checkpoint inhibitors.

Comparison of tumor-infiltrating lymphocytes between primary and metastatic tumors in breast cancer patients

The presence of tumor‐infiltrating lymphocytes (TILs) is associated with favorable long‐term outcome in breast cancer. However, little is known about changes in TILs during metastatic progression. To confirm our hypothesis that malignant tumors escape from the host immune system during metastasis, we evaluated the percentage of TILs in paired samples of primary and metastatic breast tumors. We retrospectively identified 25 patients with human epidermal growth factor receptor‐2 (HER2+, n = 14) and triple negative (TN, n = 11) early breast cancer diagnosed between 1990 and 2009 at Tokai University Hospital (Isehara, Japan) and who subsequently experienced regional or distant recurrence confirmed by tumor biopsy/resection. Hematoxylin–eosin‐stained slides of these paired samples were evaluated for stromal TILs. Immunohistochemical staining was carried out using primary antibodies against CD4, CD8, Foxp3, programmed cell death ligand 1 (PD‐L1), PD‐L2, and HLA class I for characterizing the TILs and breast tumors. The percentage of TILs in the primary tumors was significantly higher (average 34.6%) than that in metastatic tumors (average 15.7%) (paired t‐test, P = 0.004) and that of CD8+ and CD4+ T cells significantly decreased from primary to metastatic tumors (paired t‐test, P = 0.008 and P = 0.026, respectively). The PD‐L1, PD‐L2, and HLA class I antibody expression changed from positive to negative and vice versa from the primary to the metastatic tumors. Tumors at first metastatic recurrence in HER2+ and TN breast cancers have a lower percentage of TILs and CD8+ and CD4+ T cells compared to primary tumors, which indicates that immune escape plays a role in tumor progression.

Comprehensive immunohistochemical analysis of tumor microenvironment immune status in esophageal squamous cell carcinoma.

Immunotherapy with anti-PD-1 antibody preliminarily showed promising efficacy for treating esophageal squamous cell carcinoma (ESCC). Herein, we used tissue microarrays and immunohistochemically analyzed PD-L1 and various tumor infiltrating immune cells (TIICs) in specimens from 196 ESCC patients who had undergone curative resection without preoperative therapy. PD-L1 expressions in tumor cells (TCs) and TIICs, as well as infiltration of lymphocytes (CD4+, CD8+, FOXP3+, and PD- 1+) and macrophages (CD68+ and CD204+), were evaluated. PD-L1 was expressed in TCs of 18.4% and in TIICs of 83.3% of these patients. PD-L1 expressions in TCs and TIICs were associated with significant infiltration of various TIIC types, especially CD8+ cells. PD-L1 expressions in both TCs and TIICs were significantly associated with favorable overall survival, and combining their levels enhanced prognostic accuracy. Prognostic impacts of PD-L1 expressions in TCs and TIICs, abundant PD-1+ cell infiltration, a high CD8+/FOXP3+ ratio, and the CD8+/CD204+ ratio remained significant after adjusting for clinicopathological factors. In conclusion, PD-L1 expression reflects anti-tumor immunity, and PD-1/PD-L1 expression and the ratio of infiltrating effector to immune suppressor cells have prognostic value. Therapeutic strategies inhibiting the PD-1/PD-L1 signal and immune suppressor cells are anticipated in ESCC patients.