Takumi Kumai

EGFR inhibitors augment antitumour helper T-cell responses of HER family-specific immunotherapy

Background:Head and neck squamous cell carcinoma (HNSCC) is a major cause of cancer-related morbidity and mortality worldwide. Epidermal growth factor receptor (EGFR)-targeted therapy is an attractive strategy alternative to conventional cancer treatments for HNSCC, but its efficacy remains controversial. T-cell-based immunotherapy has been proposed as a novel therapeutic approach to improve the clinical outcome for HNSCC. In this study, we report human epidermal receptor (HER) family epitopes that induced CD4 T-cell responses to HNSCC. The results provide support for a novel strategy to treat HNSCC by combining EGFR-targeted therapy with T-cell-based immunotherapy.Methods:We evaluated the capacity of predicted CD4 T-cell peptide epitopes from EGFR to induce antitumour immune responses in vitro. In addition, EGFR inhibitors were evaluated for their ability to augment tumour MHC class II expression in HNSCC cell lines and subsequently increase T-cell recognition.Results:Among several predicted peptide epitopes, EGFR875–889 elicited CD4 T-cell responses that were restricted by HLA-DR4, DR15, or DR53 molecules, indicating that the peptide functions as a promiscuous T-cell epitope. The peptide-reactive T cells responded to autologous dendritic cells loaded with EGFR-expressing tumour cell lysates, indicating that these epitopes are naturally processed. In addition, the CD4 T cells were capable of directly recognising and killing HNSCC cells expressing EGFR and the appropriate HLA class II molecule. T cells reactive with the EGFR875–889 epitope could be detected in the blood of HNSCC patients. EGFR875–889-reactive CD4 T cells were also able to recognise several peptide analogues derived from homologous regions of EGFR family members, HER-2, HER-3 and c-MET. Finally, we examined the effects of EGFR tyrosine kinase inhibition or EGFR-blocking antibodies on CD4 T-cell tumour reactivity. Treatment of tumour cells with the EGFR inhibitors enhanced tumour recognition by EGFR875–889-reactive T cells presumably due to the upregulation of HLA-DR expression in the HNSCC cells.Conclusion:We identified novel CD4 T-cell EGFR epitopes and amongst these, EGFR875–889 functions as a promiscuous helper T-cell epitope that can elicit effective antitumour T-cell responses against tumours expressing HER family members and c-MET. These observations should facilitate the translation of T-cell-based immunotherapy into the clinic for the treatment of HNSCC and provide a rational basis for EGFR inhibition, immune-targeted combination therapy.

Peptide vaccines in cancer — old concept revisited

Synthetic peptide vaccines aim to elicit and expand tumor-specific T cells capable of controlling or eradicating the tumor. Despite the high expectations based on preclinical studies, the results of clinical trials using peptide vaccines have been disappointing. Thus, many researchers in the field have considered peptide vaccines as outdated and no longer viable for cancer therapy. However, recent progress in understanding the critical roles of immune adjuvants, modes of vaccine administration and T cell dynamics has lead to a rebirth of this approach and reconsidering the use of peptide vaccines for treating malignant disorders.

Six-transmembrane epithelial antigen of the prostate and enhancer of zeste homolog 2 as immunotherapeutic targets for lung cancer

BackgroundT-cell based immunotherapy for lung cancer (LC) could be a promising and novel therapeutic approach. Six-transmembrane epithelial antigen of the prostate (STEAP) and the polycomb group protein enhancer of zeste homolog 2 (EZH2) are highly expressed in LC and since the expression of molecules in normal tissue is significantly lower as compared to tumor cells, these proteins are considered as potential tumor-associated antigens (TAAs) for developing T-cell based immunotherapy.MethodsWe assessed the capacity of predicted CD4 T-cell epitopes from STEAP and EZH2 to induce anti-tumor immune responses to LC cell lines.ResultsOut of several predicted epitopes, two synthetic peptides, STEAP281-296 and EZH295-109, were effective in inducing CD4 T-cell responses that were restricted by HLA-DR1, DR15, or DR53 molecules, indicating that the peptides function as promiscuous T-cell epitopes. Moreover, STEAP281-296 and EZH295-109-reactive T-cells could directly recognize STEAP or EZH2 expressing LC cells in an HLA-DR restricted manner. In addition, some STEAP-reactive T-cells responded to STEAP+ tumor cell lysates presented by autologous dendric cells. Most significantly, both of these peptides were capable of stimulating in vitro T-cell responses in patients with LC.ConclusionsPeptides STEAP281-296 and EZH295-109 function as strong CD4 T-cell epitopes that can elicit effective anti-tumor T-cell responses against STEAP or EZH2 expressing LC. These observations may facilitate the translation of T-cell based immunotherapy into the clinic for the treatment of LC.

Optimization of Peptide Vaccines to Induce Robust Antitumor CD4 T-cell Responses

Previous work on peptide vaccines has focused primarily on boosting either antibody or CTL responses. Here, a vaccine strategy was optimized that elicited strong and effective CD4 T-cell responses with therapeutic antitumor effects in a murine melanoma model. Substantial evidence indicates that immunotherapy is a feasible and effective approach for the treatment of numerous types of cancer. Among various immunotherapy options, peptide vaccines to generate antitumor T cells appear as promising candidates, because of their cost effectiveness and ease of implementation. Nevertheless, most peptide vaccines are notorious for being weekly immunogenic and, thus, optimization of the vaccination strategy is essential to achieve therapeutic effectiveness. In addition, effective peptide vaccines must stimulate both CD8 cytotoxic and CD4 helper T lymphocytes. Our group has been successful in designing effective peptide vaccination strategies for inducing CD8 T-cell responses in mouse tumor models. Here, we describe a somewhat similar, but distinct, peptide vaccination strategy capable of generating vast CD4 T-cell responses by combining synthetic peptides with toll-like receptor (TLR) agonists and OX40/CD40 costimulation. This vaccination strategy was efficient in overcoming immune tolerance to a self-tumor–associated antigen and generated significant antitumor effects in a mouse model of malignant melanoma. The optimized peptide vaccine also allowed the expansion of adoptively transferred CD4 T cells without the need for lymphodepletion and IL2 administration, generating effective antimelanoma responses through the enhancement of proliferative and antiapoptotic activities of CD4 T cells. These results have practical implications in the design of more effective T-cell–based immunotherapies. Cancer Immunol Res; 5(1); 72–83. ©2016 AACR.

c-Met is a novel tumor associated antigen for T-cell based immunotherapy against NK/T cell lymphoma

Background: The expression of c-Met and its ligand HGF plays a critical role in cell proliferation and is involved in numerous malignancies. Because c-Met expression and its role in NK/T-cell lymphoma remain unclear, we studied the expression and function of c-Met in NK/T-cell lymphoma cells. In addition, we investigated the possibility that c-Met could function as a tumor-associated antigen for helper T lymphocytes (HTLs). Methods: We evaluated whether HGF and c-Met were expressed in NK/T-cell lymphoma and the capacity of predicted c-Met HTL epitopes to induce antitumor responses in vitro. In addition, c-Met inhibitor was evaluated for the ability to inhibit TGF-β production in tumor and subsequently increase HTL recognition. Results: c-Met and HGF were expressed in NK/T-cell lymphoma cell lines, nasal NK/T-cell lymphoma specimens and patient serum samples. Moreover, HGF was shown to promote NK/T cell lymphoma (NKTCL) proliferation in an autocrine manner. Furthermore, we have identified three novel c-Met HTL epitopes that were restricted by several HLA-DR molecules. Notably, peptide-induced HTL lines directly recognized and killed c-Met expressing NK/T-cell lymphomas and various epithelial solid tumors. The c-Met specific HTLs could also recognize dendritic cells (DCs) pulsed with c-Met expressing tumor cell lysates. In addition, we observed that c-Met inhibition augmented HTL recognition by decreasing TGF-β production by tumor cells. Lastly, autophagy partly regulated the HTL responses against tumors. Conclusions: We identified novel c-Met HTL epitopes that can elicit effective antitumor responses against tumors expressing c-Met. Our results provide the rationale of combining c-Met targeting therapy and immunotherapy for NKTCLs and epithelial tumors.

Programmed death-ligand 1 and its soluble form are highly expressed in nasal natural killer/T-cell lymphoma: a potential rationale for immunotherapy

Nasal natural killer/T-cell lymphoma (NNKTL) is an aggressive neoplasm with poor therapeutic responses and prognosis. The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway plays an important role in immune evasion of tumor cells through T-cell exhaustion. The aim of the present study was to examine the expression of PD-L1 and PD-1 molecules in NNKTL. We detected the expression of PD-L1 in biopsy samples from all of the NNKTL patients studied. PD-L1 was found on both malignant cells and tumor-infiltrating macrophages, while PD-1-positive mononuclear cells infiltrated the tumor tissues in 36% of patients. Most significantly, soluble PD-L1 (sPD-L1) was present in sera of NNKTL patients at higher levels as compared to healthy individuals and the levels of serum sPD-L1 in patients positively correlated with the expression of PD-L1 in lymphoma cells of tumor tissues. In addition, the high-sPD-L1 group of patients showed significantly worse prognosis than the low-sPD-L1 group. Furthermore, we confirmed that membrane and soluble PD-L1 was expressed on the surface and in the culture supernatant, respectively, of NNKTL cell lines. The expression of PD-L1 was observed in tumor tissues and sera from a murine xenograft model inoculated with an NNKTL cell line. Our results suggest that sPD-L1 could be a prognostic predictor for NNKTL and open up the possibility of immunotherapy of this lymphoma using PD-1/PD-L1 axis inhibitors.

Tumor-derived TGF-β and prostaglandin E2 attenuate anti-tumor immune responses in head and neck squamous cell carcinoma treated with EGFR inhibitor

BackgroundEGFR-targeted therapy is an attractive option for head and neck squamous cell carcinoma patients. We have recently reported the use of EGFR inhibitors as an adjunct treatment to enhance HLA-DR expression in tumor cells to improve cancer immunotherapy. Nevertheless, we observed that EGFR inhibitors resulted in decreased anti-tumor responses, regardless of upregulation of HLA-DR expression on the tumor cell. In this study, we specifically investigated the mechanisms by which EGFR inhibition modulated anti-tumor responses.MethodsAn EGFR inhibitor erlotinib was used to assess the modulation of anti-tumor responses by tumor antigen-specific helper T cells. We then examined whether administration of the EGFR inhibitor altered tumor cytokine profiles and expression of immune-related molecules on tumor cells.ResultsDespite the augmented HLA-DR expression on a gingival cancer cell line by EGFR inhibition, anti-tumor responses of EGFR reactive helper T cell clones against tumor cells were decreased. EGFR inhibition did not change the expression of CD80, CD86, or PD-L1 on the tumor cells. Conversely, production of transforming growth factor beta (TGF-β) and prostaglandin E2 was increased by EGFR inhibition, indicating that these immunosuppressive molecules were involved in diminishing tumor recognition by T cells. Significantly, attenuation of HTL responses against tumors after EGFR inhibition was reversed by the addition of anti-TGF-β antibody or COX2 inhibitors.ConclusionsTargeting TGF-β and prostaglandin E2 may allow for improved outcomes for cancer patients treated with combination immunotherapy and EGFR inhibitors.

Designing therapeutic cancer vaccines by mimicking viral infections

The design of efficacious and cost-effective therapeutic vaccines against cancer remains both a research priority and a challenge. For more than a decade, our laboratory has been involved in the development of synthetic peptide-based anti-cancer therapeutic vaccines. We first dedicated our efforts in the identification and validation of peptide epitopes for both CD8 and CD4 T cells from tumor-associated antigens (TAAs). Because of suboptimal immune responses and lack of therapeutic benefit of peptide vaccines containing these epitopes, we have focused our recent efforts in optimizing peptide vaccinations in mouse tumor models using numerous TAA epitopes. In this focused research review, we describe how after taking lessons from the immune system’s way of dealing with acute viral infections, we have designed peptide vaccination strategies capable of generating very high numbers of therapeutically effective CD8 T cells. We also discuss some of the remaining challenges to translate these findings into the clinical setting.

Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site

Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2′,5′)pA(3′,5′)p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45+ CD11bmid Ly6C+ cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8+ T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11, IFN-induced molecules, MX dynamin-like GTPase 1 (Mx1) and 2′-5′ oligoadenylate synthetase-like 1 (Oasl1), nitric oxide synthase 2 (Nos2), and interferon beta 1 (Ifnb1). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds.

CCL17 and CCL22/CCR4 signaling is a strong candidate for novel targeted therapy against nasal natural killer/T-cell lymphoma

Nasal natural killer/T-cell lymphoma (NNKTL) is associated with Epstein–Barr virus and has a poor prognosis because of local invasion and/or multiple dissemination. Various chemokines play a role in tumor proliferation and invasion, and chemokine receptors including the C-C chemokine receptor 4 (CCR4) are recognized as potential targets for treating hematologic malignancies. The aim of the present study was to determine whether specific chemokines are produced by NNKTL. We compared chemokine expression patterns in culture supernatants of NNKTL cell lines with those of other lymphoma or leukemia cell lines using chemokine protein array and ELISA. Chemokine (C-C motif) ligand (CCL) 17 and CCL22 were highly produced by NNKTL cell lines as compared to the other cell lines. In addition, CCL17 and CCL22 were readily observed in the sera of NNKTL patients. The levels of these chemokines were significantly higher in patients than in healthy controls. Furthermore, we detected the expression of CCR4 (the receptor for CCL17 and CCL22) on the surface of NNKTL cell lines and in tissues of NNKTL patients. Anti-CCR4 monoclonal antibody (mAb) efficiently induced antibody-dependent cellular cytotoxicity mediated by natural killer cells against NNKTL cell lines. Our results suggest that CCL17 and CCL22 may be important factors in the development of NNKTL and open up the possibility of immunotherapy of this lymphoma using anti-CCR4 mAb.