Kosuke Ichikawa

Clinical responses to EGFR-tyrosine kinase inhibitor retreatment in non-small cell lung cancer patients who benefited from prior effective gefitinib therapy: a retrospective analysis

BackgroundGefitinib was the first epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of advanced non-small cell lung cancer (NSCLC). Few treatment options are available for NSCLC patients who have responded to gefitinib treatment and demonstrated tumor progression. The present study was conducted to evaluate the efficacy and toxicity of the 2nd EGFR-TKI administration.MethodsWe retrospectively analyzed 11 patients who had obtained a partial response (PR) or stable disease (SD) with gefitinib treatment and were re-treated with EGFR-TKI after failure of the initial gefitinib treatment.ResultsThree patients (27%) were treated with gefitinib as the 2nd EGFR-TKI, and 8 patients (73%) received erlotinib. Only one patient (9%) showed PR, 7 (64%) achieved SD, and 3 (27%) had progressive disease. The disease control rate was 73% (95% CI, 43% - 91%) and the median progression-free survival was 3.4 months (95% CI, 2 - 5.2). The median overall survival from the beginning of the 2nd EGFR-TKI and from diagnosis were 7.3 months (95% CI, 2.7 - 13) and 36.7 months (95% CI, 23.6 - 43.9), respectively. No statistical differences in PFS or OS were observed between gefitinib and erlotinib as the 2nd EGFR-TKI (PFS, P = 0.23 and OS, P = 0.052). The toxicities associated with the 2nd EGFR-TKI were generally acceptable and comparable to those observed for the initial gefitinib therapy.ConclusionsOur results indicate that a 2nd EGFR-TKI treatment can be an effective treatment option for gefitinib responders.

Vaccination with CD133+ melanoma induces specific Th17 and Th1 cell–mediated antitumor reactivity against parental tumor

Accumulating evidence suggests that cancer cells possess a small subpopulation that survives during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular-targeting therapy. CD133 is a putative marker that distinguishes a minor subpopulation from normal differentiated tumor cells in many cancers. Although it is necessary to eradicate all cancer cells to obtain a cure, effective treatment to eliminate the CD133+ treatment–tolerant cells has not been elucidated. In this study, we demonstrated that a CD133+ subpopulation in murine melanoma is immunogenic and that effector T cells specific for the CD133+ melanoma cells mediated potent antitumor reactivity, curing the mice of the parental melanoma. CD133+ melanoma antigens preferentially induced type 17 T helper (Th17) cells and Th1 cells but not Th2 cells. CD133+ melanoma cell–specific CD4+ T-cell treatment eradicated not only CD133+ tumor cells but also CD133− tumor cells while inducing long-lasting accumulation of lymphocytes and dendritic cells with upregulated MHC class II in tumor tissues. Further, the treatment prevented regulatory T-cell induction. These results indicate that T-cell immunotherapy is a promising treatment option to eradicate CD133+ drug-tolerant cells to obtain a cure for cancer.

Critical Roles of Chemoresistant Effector and Regulatory T Cells in Antitumor Immunity after Lymphodepleting Chemotherapy.

Antitumor immunity is augmented by cytotoxic lymphodepletion therapies. Adoptively transferred naive and effector T cells proliferate extensively and show enhanced antitumor effects in lymphopenic recipients. Although the impact of lymphodepletion on transferred donor T cells has been well evaluated, its influence on recipient T cells is largely unknown. The current study demonstrates that both regulatory T cells (Tregs) and effector CD8+ T cells from lymphopenic recipients play critical roles in the development of antitumor immunity after lymphodepletion. Cyclophosphamide (CPA) treatment depleted lymphocytes more efficiently than other cytotoxic agents; however, the percentage of CD4+CD25+ Foxp3+ Tregs was significantly increased in CPA-treated lymphopenic mice. Depletion of these chemoresistant Tregs following CPA treatment and transfer of naive CD4+ T cells augmented the antitumor immunity and significantly suppressed tumor progression. Further analyses revealed that recipient CD8+ T cells were responsible for this augmentation. Using Rag2−/− mice or depletion of recipient CD8+ T cells after CPA treatment abrogated the augmentation of antitumor effects in CPA-treated reconstituted mice. The transfer of donor CD4+ T cells enhanced the proliferation of CD8+ T cells and the priming of tumor-specific CD8+ T cells originating from the lymphopenic recipients. These results highlight the importance of the recipient cells surviving cytotoxic regimens in cancer immunotherapies.

Epitope diversification driven by non-tumor epitope-specific Th1 and Th17 mediates potent antitumor reactivity

MHC class I-restricted peptide-based vaccination therapies have been conducted to treat cancer patients, because CD8⁺ CTL can efficiently induce apoptosis of tumor cells in an MHC class I-restricted epitope-specific manner. Interestingly, clinical responders are known to demonstrate reactivity to epitopes other than those used for vaccination; however, the mechanism underlying how antitumor T cells with diverse specificity are induced is unclear. In this study, we demonstrated that dendritic cells (DCs) that engulfed apoptotic tumor cells in the presence of non-tumor MHC class II-restricted epitope peptides, OVA(323-339), efficiently presented tumor-associated antigens upon effector-dominant CD4⁺ T cell balance against regulatory T cells (Treg) for the OVA(323-339) epitope. Th1 and Th17 induced tumor-associated antigens presentation of DC, while Th2 ameliorated tumor-antigen presentation for CD8⁺ T cells. Blocking experiments with anti-IL-23p19 antibody and anti-IL-23 receptor indicated that an autocrine mechanism of IL-23 likely mediated the diverted tumor-associated antigens presentation of DC. Tumor-associated antigens presentation of DC induced by OVA(323-339) epitope-specific CD4⁺ T cells resulted in facilitated antitumor immunity in both priming and effector phase in vivo. Notably, this immunotherapy did not require pretreatment to reduce Treg induced by tumor. This strategy may have clinical implications for designing effective antitumor immunotherapies.

A Phase II Study of Irinotecan for Patients with Previously Treated Small-Cell Lung Cancer

Objective: Chemotherapy with irinotecan plus cisplatin has shown promise in chemo-naïve small-cell lung cancer (SCLC) patients. However, irinotecan treatment for relapsed or refractory SCLC has not been adequately evaluated. This phase II study evaluated the appropriate treatment schedule of irinotecan as a single agent. This study was designed to determine the antitumor activity, toxicity, and survival in previously treated SCLC patients. Methods: Previously treated SCLC patients with at least one platinum-based regimen received irinotecan (100 mg/m2) on days 1 and 8, every 3 weeks, until disease progression. The assessment of the response rate was the primary endpoint. Results: Thirty patients were enrolled, with an objective response rate of 41.3% (95% confidence interval [CI] 25.5–59.3), and a disease control rate of 69%. Median progression-free and overall survival was 4.1 months (95% CI, 2.2–5.4) and 10.4 months (95% CI, 8.1–14), respectively. The grade 3/4 hematological toxicities were neutropenia (36.7%), thrombocytopenia (3.3%), anemia (13.3%), and febrile neutropenia (6.6%). There were no grade 4 nonhematological toxicities. Frequent grade 3 nonhematological toxicities included diarrhea (10%), anorexia (6.6%), and hyponatremia (6.6%). Conclusions: This phase II study showed a high objective response rate and long survival. Irinotecan monotherapy schedule used was well tolerated, and could be an active treatment option for these patients.

Transfer of in vitro-expanded naïve T cells after lymphodepletion enhances antitumor immunity through the induction of polyclonal antitumor effector T cells

The adoptive transfer of effector T cells combined with lymphodepletion has demonstrated promising antitumor effects in mice and humans, although the availability of tumor-specific T cells is limited. We and others have also demonstrated that the transfer of polyclonal naïve T cells induces tumor-specific effector T cells and enhances antitumor immunity after lymphodepletion. Because tumors have been demonstrated to induce immunosuppressive networks and regulate the function of T cells, obtaining a sufficient number of fully functional naïve T cells that are able to differentiate into tumor-specific effector T cells remains difficult. To establish culture methods to obtain a large number of polyclonal T cells that are capable of differentiating into tumor-specific effector T cells, naïve T cells were activated with anti-CD3 mAbs in vitro. These cells were stimulated with IL-2 and IL-7 for the CD8 subset or with IL-7 and IL-23 for the CD4 subset. Transfer of these hyperexpanded T cells after lymphodepletion showed significant antitumor efficacy, and tumor-specific effector T cells were primed from these expanded T cells in tumor-bearing hosts. Moreover, these ex vivo—expanded T cells maintained T cell receptor diversity and showed long-term persistence of memory against specific tumors. Further analyses revealed that combination therapy consisting of vaccination with dendritic cells that were co-cultured with irradiated whole tumor cells and the transfer of ex vivo—expanded T cells significantly enhanced antitumor immunity. These results indicate that the transfer of ex vivo—expanded polyclonal T cells can be combined with other immunotherapies and augment antitumor effects.

Abstract 5654: Lymphodepletion induces T cell homeostatic proliferation and augments antitumor effects of PD-1/PD-L1 blockade therapy

Programmed death receptor-1 (PD-1)/programmed death receptor ligand-1 (PD-L1) blockade therapy has demonstrated to enhance antitumor immunity. Recent evidence has shown that anti-PD-1/PD-L1 therapy restores the function of exhausted effector T cells and augments antitumor immune responses. The expression of PD-L1 has been best studied as a biomarker for the anti-PD-1/PD-L1 therapy. Because PD-L1 expression on tumor cells is induced by IFN-γ which is mainly produced by effector T cells, induction of antitumor effector T cells in cancer patients could be an important for the anti-PD-1/PD-L1 therapy. Indeed, previous studies have suggested that the increase of tumor-infiltrating lymphocytes is the predictive biomarker for PD-1/PD-L1 blockade therapy. We and others have demonstrated that lymphodepletive therapies, such as chemotherapy and radiotherapy, induce tumor-specific effector T cells from naive T cells. Naive T cells rapidly proliferate and elicit memory-like functions after lymphodepletion (homeostatic proliferation). These findings indicate that lymphodepletive regimens could augment the antitumor efficacies of the anti-PD-1/PD-L1 therapy. In the current study, we transferred naive T cells into mice after whole-body irradiation or chemotherapy. These mice were inoculated s.c. with PD-L1+ B16F10 melanoma cells or PD-L1- MCA205 fibrosarcoma and then were treated with anti-PD-1 mAbs. Lymphodepletion by whole body irradiation, but not cytotoxic agents following the transfer of naive T cells significantly enhanced antitumor immunity of anti-PD-1 mAbs. This augmentation required both of CD4+ and CD8+ T cells, but not NK cells. Further experiments revealed that immune cells infiltrating MCA205 expressed PD-L1. Transfer of naive T cells from IFN-γ knockout mice abrogated the enhancement of antitumor effects of anti-PD-1 mAb therapy by lymphodepletion. These results suggest that lymphodepletion induces homeostatic proliferation of T cells and augments antitumor effects of PD-1/PD-L1 blockade therapy. Citation Format: Masashi Arita, Satoshi Watanabe, Takahashi Miho, Miyuki Sato, Aya Ohtsubo, Kosuke Ichikawa, Rie Kondo, Tetsuya Abe, Junta Tanaka, Toshiyuki Koya, Toshiaki Kikuchi. Lymphodepletion induces T cell homeostatic proliferation and augments antitumor effects of PD-1/PD-L1 blockade therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5654. doi:10.1158/1538-7445.AM2017-5654

Abstract 1550: Successful treatment of advanced tumors with chemo-immunotherapy: a combination of cyclophosphamide and inhibition of chemo-resistant immune suppressor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL It is well known that naive T cells transferred into lymphopenic hosts develop into memory like T cells and acquire some effector functions. We and others have been shown that transfer of naive T cells following sublethal whole body irradiation augmented antitumor immunity and inhibited tumor progression. Further analyses showed that antitumor effector T cells were primed from transferred naive T cells. Although the exact mechanism underlying this enhancement of antitumor immune responses remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. Combination of lymphodepletion by cytotoxic regimens, such as chemotherapy or radiotherapy, and transfer of naive T cells seems to be a promising strategy. Although whole body irradiation induces lymphopenia to tumor-bearing animals and enhances antitumor immunity, it has not been routinely used in clinical settings. To test whether cytotoxic agents deplete lymphocytes and enhance antitumor immune responses, we infused cyclophosphamide (CPA), fludarabine, cisplatin and etoposide at the sublethal doses to mice. CPA treatment depleted 95% of lymphocytes in mice, and enhanced antitumor effects of transferred naive T cells. Other cytotoxic drugs failed to augment antitumor immunity in combination with transfer of T cells. Previously, we have demonstrated that CD4+CD25+Foxp3+ regulatory T cells (Treg) and CD11b+Gr-1+ myeloid-derived suppressor cells (MDSC) inhibited priming of antitumor effector T cells. Depletion of these suppressor cells increased the number of tumor-specific T cells and augmented antitumor immune responses. To examine the effect of sublethal doses of cyclophosphamide on immune suppressor cells, we injected cyclophosphamide into mice and harvested lymph-nodes and spleens for FACS analyses. Unexpectedly, we found a significant increase in the frequency of Treg and MDSC after CPA treatment. Magnetically isolated Treg and MDSC from cyclophosphamide treated mice suppressed tumor-specific responses of effector T cells in vitro. Depletion of Treg with anti-CD25 monoclonal antibodies following CPA administration and transfer of naive T cells increased the number of antitumor effector T cells. Further, the combination of Treg depletion, CPA treatment and transfer of naive T cells succeeded to cure 20-day established skin tumors in mice. We have also been testing whether inhibition of MDSC after CPA treatment augments antitumor immunity. Our results showed that CPA treatment efficiently depletes lymphocytes and enhanced antitumor immune responses despite an increase of immune suppressor cells. Further inhibition of suppressor cells after CPA administration successfully treated advanced tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1550. doi:1538-7445.AM2012-1550

Abstract 2685: Irradiation and reconstitution with in vitro-expanded polyclonal T cells from naïve mice augments antitumor response

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Transfer of naive T cells into lymphopenic tumor-bearing hosts induces antitumor effector T cells effectively and inhibits tumor progression. Although the exact mechanism underlying this augmentation of antitumor immune responses remains unclear, possible explanations have been proposed; depletion of suppressor cells, improvement of tumor-antigen presentation, and elimination of lymphocytes competing activation cytokines. Thus, the combination of lymphodepletion by cytotoxic regimens, such as chemotherapy or radiotherapy, and transfer of naive T cells seems to be an attractive approach. Because of difficulty to acquire enough number of naive T cells from cancer patients, it is necessary to establish a method for obtaining the large number of naive T cells. The current study seeks to determine whether lymphodepletion and transfer of in vitro expanded T cells enhances antitumor immunity. We stimulated CD4+ and CD8+ T cells from naive C57/BL6 mice with homeostatic cytokines. At day9 of culture, their number increased 4 to 10-fold. FACS analysis demonstrated TCR diversity of stimulated T cells. Resulted cells were injected into mice harboring MCA205 fibrosarcoma after sublethal irradiation (500 rad). As well as transfer of fresh naive T cells, transfer of stimulated T cells strongly inhibited tumor-progression. To confirm whether in vitro stimulated T cells become antitumor effector T cells and retarded tumor growth, GFP+ T cells were expanded in vitro and transferred into irradiated wild mice. On the same day, mice were inoculated s.c. with tumor cells. Twelve days later, tumor-draining LNs were harvested and cells were stimulated with the anti-CD3/IL-2 method. Activated tumor-draining LN cells were analyzed for secretion of IFN-γ after specific tumor stimulation. Intracellular IFN-γ staining revealed that the majority of effector T cells were GFP+ donor cells. We next examined whether dendritic cell vaccination enhances antitumor immune responses of in vitro stimulated T cells. Bone marrow dendritic cells were co-cultured with irradiated tumor cells and were injected into mice reconstituted with in vitro stimulated T cells. Compared with transfer of T cells alone, the combination of vaccination and transfer of T cells significantly delayed tumor progression. We report in this study that even after nonspecific stimulation, naive T cells retain their ability to respond to tumor-antigens when they are transferred into lymphopenic hosts. Irradiation and reconstitution with in vitro stimulated T cells can be useful conditioning for cancer immunotherapy, especially for vaccination with tumor antigens. 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 2685. doi:10.1158/1538-7445.AM2011-2685

Abstract 2694: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked is a CD133+ tumor-specific protein and induces antitumor immunity

Cancer cells that exclusively maintain the ability of self-renewal and differentiation are termed cancer stem cells (CSCs). It is still controversial if the classical CSC hierarchy exists in all of solid tumors, however, accumulating evidence suggests that heterogeneity within cancer cells exists and that cancer survives as the cells with CSC features during potentially lethal stresses, including chemotherapy, radiation treatment, and molecular targeting therapy. Although it is necessary to eradicate CSCs to obtain cure of cancer, effective treatment has not been elucidated. Interestingly, most of the identified immunogenic tumor antigens are cancer/testis (CT) antigens. One of the reasons why CT antigens are immunogenic is that they are unlikely to maintain peripheral tolerance, owing to restricted expression in the testis and in immortal malignant cells, because Tregs are maintained with antigen stimulation by dendritic cells (DCs) that acquire dying cells in the steady state. Since CSCs are highly immortal, it is possible that they possess immunogenic antigens that are not expressed in differentiated cancer cells or normal epithelial cells, and that these antigens may be ideal therapeutic targets for cancer treatment. Recently it was reported that CT antigens are mainly expressed in CSCs. We isolated CD133+ tumor cells, which possessed CSC properties, from B16 melanoma cells. We found that CSC-specific LN T cells primed by the CD133+ tumor vaccine mediated potent antitumor therapeutic efficacy by eradicating CSCs in tumor, thereby curing parental melanomas that comprised 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 2694. doi:10.1158/1538-7445.AM2011-2694