Fumito Ito

Anti-CD137 Monoclonal Antibody Administration Augments the Antitumor Efficacy of Dendritic Cell-Based Vaccines

In weakly and poorly immunogenic tumor models, we examined the effects of stimulating CD137 (4-1BB) in vivo by administering anti-CD137 monoclonal antibody after tumor lysate-pulsed dendritic cell (TP-DC) vaccination. TP-DC subcutaneous vaccination induced a transient up-regulation of CD137 on T cells and natural killer (NK) cells within vaccine-primed lymph nodes (VPLNs). In established pulmonary and subcutaneous tumor models, anti-CD137 synergistically enhanced tumor regression after TP-DC vaccination. In the subcutaneous tumor model, the combined therapy resulted in improved survival. Combined therapy also resulted in improved local control of subcutaneous tumor after surgical resection. Anti-CD137 polarized the cytokine release of VPLNs and spleen cells in response to tumor antigen toward a type 1 (interferon-γ) versus a type 2 (interleukin-4) profile. Cell depletion and the use of knockout animals identified that CD8+, CD4+, and NK cells were involved in the tumor rejection response and that CD8+ cells had the major effector role. Anti-CD137 administration resulted in increased proliferation of adoptively transferred OT-1 CD8+ T cells in the VPLNs of mice inoculated with B16-OVA TP-DCs. Polarization toward type 1 (interferon-γ) versus type 2 (interleukin-4) was also observed with the OT-1 cells from VPLNs and spleen cells after anti-CD137 injections. This polarization effect was abrogated by the in vivo depletion of NK cells. These findings indicate that the adjuvant effect of anti-CD137 given in conjunction with TP-DC vaccination is associated with the polarization of T effector cells toward a type 1 response to tumor antigen and is mediated via NK cells.

Antitumor reactivity of anti-CD3/anti-CD28 bead-activated lymphoid cells: Implications for cell therapy in a murine model

Ligation of TCR and CD28 expressed on T cells via mAbs results in activation of T cells capable of tumor destruction in adoptive immunotherapy. In a murine model, the authors examined in vitro activation conditions utilizing plate-immobilized and bead-conjugated mAbs that bind to CD3 and CD28. Bead-activated tumor-draining lymph node (TDLN) cells demonstrated superior cytokine (IFN-&ggr;, GM-CSF, IL-2, and IL-10) secretion and mediated tumor regression more efficiently compared with plate-activated cells. The bead-activated TDLN cells had a significantly higher percentage of CD4+ cells compared with plate-activated cells. On a per-cell basis, positively selected CD4+ cells activated with bead-coupled or plate immobilized mAbs mediated tumor-specific regression equally. Bead-activated CD4+ TDLN cells demonstrated significantly higher levels of tumor specific IL-2 secretion compared with plate-activated CD4+ cells that may provide helper function to CD8+ effector cells. The antitumor reactivity of bead-activated lymphoid cells depended upon their source. TDLN cells after bead activation were more potent than splenocytes from tumor-bearing hosts in mediating tumor regression in vivo. Bead-activated LN cells and splenocytes from nontumor-bearing hosts demonstrated nonspecific cytokine secretion and minimal efficacy in adoptive immunotherapy. At minimal doses of IL-2, the antitumor reactivity of bead-activated TDLN cells was significantly enhanced. Anti-CD3/anti-CD28 bead activation of tumor-primed T cells represents an efficient method to generate effector cells for immunotherapy.

Cancer Immunotherapy: Current Status and Future Directions

Better understanding of the underlying principles of tumor biology and immunology, enhanced by recent insights into the mechanisms of immune recognition, regulation, and tumor escape has provided new approaches for cancer immunotherapy. This article reviews the current status and future directions of cancer immunotherapy, with a focus on the recent encouraging results from immune-modulating antibodies and adoptive cell therapy.

Antitumor effector B cells directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10.

We have previously reported that adoptive transfer of tumor‐draining lymph node (TDLN) B cells confers tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer. In this study, we identified IL‐10‐producing cells within these B cells, and found that IL‐10 removal, either by using IL‐10−/− TDLN B cells or by systemic neutralization of IL‐10, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. Depletion of IL‐10 in B‐cell adoptive transfers significantly increased CTLs and B‐cell activity of PBMCs and splenic cells in the recipient. Activated TDLN B cells express Fas ligand, which was further enhanced by coculture of these TDLN B cells with 4T1 tumor cells. Effector B cells killed tumor cells directly in vitro in an antigen specific and Fas ligand‐dependent manner. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. These findings further define the biological function of antitumor effector B cells, which may offer alternative cellular therapies to cancer.

Immune Adjuvant Activity of Pre-Resectional Radiofrequency Ablation Protects against Local and Systemic Recurrence in Aggressive Murine Colorectal Cancer

Purpose While surgical resection is a cornerstone of cancer treatment, local and distant recurrences continue to adversely affect outcome in a significant proportion of patients. Evidence that an alternative debulking strategy involving radiofrequency ablation (RFA) induces antitumor immunity prompted the current investigation of the efficacy of performing RFA prior to surgical resection (pre-resectional RFA) in a preclinical mouse model. Experimental Design Therapeutic efficacy and systemic immune responses were assessed following pre-resectional RFA treatment of murine CT26 colon adenocarcinoma. Results Treatment with pre-resectional RFA significantly delayed tumor growth and improved overall survival compared to sham surgery, RFA, or resection alone. Mice in the pre-resectional RFA group that achieved a complete response demonstrated durable antitumor immunity upon tumor re-challenge. Failure to achieve a therapeutic benefit in immunodeficient mice confirmed that tumor control by pre-resectional RFA depends on an intact adaptive immune response rather than changes in physical parameters that make ablated tumors more amenable to a complete surgical excision. RFA causes a marked increase in intratumoral CD8+ T lymphocyte infiltration, thus substantially enhancing the ratio of CD8+ effector T cells: FoxP3+ regulatory T cells. Importantly, pre-resectional RFA significantly increases the number of antigen-specific CD8+ T cells within the tumor microenvironment and tumor-draining lymph node but had no impact on infiltration by myeloid-derived suppressor cells, M1 macrophages or M2 macrophages at tumor sites or in peripheral lymphoid organs (i.e., spleen). Finally, pre-resectional RFA of primary tumors delayed growth of distant tumors through a mechanism that depends on systemic CD8+ T cell-mediated antitumor immunity. Conclusion Improved survival and antitumor systemic immunity elicited by pre-resectional RFA support the translational potential of this neoadjuvant treatment for cancer patients with high-risk of local and systemic recurrence.

Adoptive Transfer of CD8+ T Cells Generated from Induced Pluripotent Stem Cells Triggers Regressions of Large Tumors Along with Immunological Memory

Current approaches to adoptive T-cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with useful in vivo characteristics. Theoretically, this limitation could be overcome by using induced pluripotent stem cells (iPSC) that could provide an unlimited source of autologous T cells. However, the therapeutic efficacy of iPSC-derived regenerated T cells remains to be demonstrated. Here, we report the first successful reprogramming of T-cell receptor (TCR) transgenic CD8(+) T cells into pluripotency. As part of the work, we established a syngeneic mouse model for evaluating in vitro and in vivo antitumor reactivity of regenerated T cells from iPSCs bearing a rearranged TCR of known antigen specificity. Stably TCR retained T-cell-derived iPSCs differentiated into CD4(+)CD8(+) T cells that expressed CD3 and the desired TCR in vitro Stimulation of iPSC-derived CD4(+)CD8(+) T cells with the cognate antigen in the presence of IL7 and IL15 followed by expansion with IL2, IL7, and IL15 generated large numbers of less-differentiated CD8(+) T cells with antigen-specific potent cytokine production and cytolytic capacity. Furthermore, adoptively transferred iPSC-derived CD8(+) T cells escaped immune rejection, mediated effective regression of large tumors, improved survival, and established antigen-specific immunological memory. Our findings illustrate the translational potential of iPSCs to provide an unlimited number of phenotypically defined, functional, and expandable autologous antigen-specific T cells with the characteristics needed to enable in vivo effectiveness. Cancer Res; 76(12); 3473-83. ©2016 AACR.

Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy.

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes.

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

Pre-resectional Radiofrequency Ablation as a Neoadjuvant in situ Tumor Vaccine

A lack of effective immune response against cancer is one of the major risk factors for developing local recurrence and distant metastases after curative resectional surgery. Prior studies revealed that systemic antitumor immunity is elicited by radiofrequency ablation (RFA) of tumor lesions, which is mainly considered a palliative procedure for unresectable tumors or for inoperable patients. Recently, we discovered an oncological benefit that depends on the adaptive arm of the antitumor immune response when RFA is performed in a neoadjuvant setting prior to surgical resection in preclinical murine models.

Abstract 5011: generation of rejuvenated murine antigen-specific T cells by reprogramming to pluripotency and redifferentiation

Background: Adoptive cell therapy with autologous antigen-specific T cells is a promising approach for treating patients with chronic viral infections and various cancers. A major limitation of this approach is the difficulty of obtaining sufficient numbers of T cells with the characteristics necessary for in vivo effectiveness. Less-differentiated T cells with long telomeres would be an ideal T-cell subset for adoptive T cell therapy; however, generating large numbers of these “young” T cells is problematic. This limitation of adoptive T cell therapy can be overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, and provide an unlimited source of autologous T cells for immunotherapy. However, in vivo antitumor reactivity of iPSC-rejuvenated T cells remains unknown due to the lack of animal model. Methods: To establish preclinical model of evaluating iPSC-rejuvenated antigen-specific T cells, we reprogrammed OT-1 T cell receptor (TCR) transgenic CD8 T cells using Sendai virus vectors encoding Oct3/4, Sox2, Klf4 and c-Myc transcription factors. Pluripotency and differentiation capacity of generated iPSCs were evaluated. Results: Twenty days after infection with Sendai virus vectors, we obtained murine embryonic stem cell-like colonies from OT-1 CD8 T cells. Immunofluorescence staining and RT-PCR analyses revealed pluripotency of established colonies. The iPSCs formed embryoid bodies in vitro, and upregulation of marker genes for all three germ layers was detected. The iPSCs differentiated into teratomas in vivo comprising all three embryonic germ layers in immune-deficient NOD/SCID mice. The iPSCs retained the same rearranged configuration of TCR chain genes as the original OT-1 T cells. Furthermore, OT-1 T cell-derived iPSCs differentiated into T cells on delta-like ligand 1 expressing OP9 (OP9/DL1) murine bone marrow stromal cells. Conclusions: Generation of rejuvenated T cells by reprogramming to pluripotency and redifferentiation is feasible. This type of approach may be useful for adoptive T cell therapy. We acknowledge DNAVEC Corporation for kindly providing Sendai virus vectors. This study was supported by the University of Michigan Start-up fund. Citation Format: Fumito Ito, Noemi Fusaki, Hidehito Saito. generation of rejuvenated murine antigen-specific T cells by reprogramming to pluripotency and redifferentiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2015-5011