[] Checkpoint inhibitors. 1. Systemic immunotherapy of non-muscle invasive mouse bladder cancer with avelumab, an anti-PD-L1 immune checkpoint inhibitor. Bacillus Calmette-Guerin (BCG) is the standard of care for intravesical therapy for carcinoma in situ and non-muscle invasive, non-metastatic human urothelial carcinoma. In this study, an aggressive, bioluminescent orthotopic bladder cancer model, MB49 tumor cells transfected with luciferase (MB49luc), was used to study the antitumor effects of avelumab, an antibody to PD-L1. MB49luc murine tumor cells form multifocal tumors on the mucosal wall of the bladder reminiscent of non-muscle invasive, non-metastatic urothelial carcinomas. MB49luc bladder tumors are highly positive for the expression of PD-L1, and avelumab administration induced significant (P 0.05) antitumor effects. s. This bladder tumor model can be used to further identify host antitumor immune mechanisms and evaluate combinations of immune-based therapies for carcinoma in situ and non-muscle invasive, non-metastatic urothelial carcinoma, to provide the rationale for subsequent clinical studies. humans. [] High avidity NK cells. 1. An NK cell line (haNK) expressing high levels of granzyme and engineered to express the high affinity CD16 allele. Natural killer (NK) cells are known to play a role in mediating innate immunity, in enhancing adaptive immune responses, and have been implicated in mediating anti-tumor responses via antibody-dependent cell-mediated cytotoxicity (ADCC) by reactivity of CD16 with the Fc region of human IgG1 antibodies. The NK-92 cell line, derived from a lymphoma patient, has previously been well characterized and adoptive transfer of irradiated NK-92 cells has demonstrated safety and shown preliminary evidence of clinical benefit in cancer patients. The NK-92 cell line, devoid of CD16, has now been engineered to express the high affinity (ha) CD16 V158 FcgammaRIIIa receptor, as well as engineered to express IL-2; IL-2 has been shown to replenish the granular stock of NK cells, leading to enhanced perforin- and granzyme-mediated lysis of tumor cells. These studies show high levels of granzyme in haNK cells, and demonstrate the effects of irradiation of haNK cells on multiple phenotypic markers, viability, IL-2 production, and lysis of a spectrum of human tumor cells. Studies also compare endogenous irradiated haNK lysis of tumor cells with that of irradiated haNK-mediated ADCC using cetuximab, trastuzumab and pertuzumab monoclonal antibodies. These studies thus provide the rationale for the potential use of irradiated haNK cells in adoptive transfer studies for a range of human tumor types. Moreover, since only approximately 10% of humans are homozygous for the high affinity V CD16 allele, these studies also provide the rationale for the use of irradiated haNK cells in combination with IgG1 anti-tumor monoclonal antibodies. 2. In reported studies, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. [] Immunocytokines. 1. Enhanced antitumor effects by combining an IL-12/anti-DNA fusion protein with avelumab, an anti-PD-L1 antibody. The combined therapeutic potential of an immunocytokine designed to deliver IL-12 to the necrotic regions of solid tumors with an anti-PD-L1 antibody that disrupts the immunosuppressive PD-1/PD-L1 axis yielded a combinatorial benefit in multiple murine tumor models. The murine version of the immunocytokine, NHS-muIL12, consists of an antibody (NHS76) recognizing DNA/DNA-histone complexes, fused with two molecules of murine IL-12 (NHS-muIL12). By its recognition of exposed DNA, NHS-muIL12 targets IL-12 to the necrotic portions of tumors; it has a longer plasma half-life and better antitumor efficacy against murine tumors than recombinant murine IL-12. Concurrent therapy with NHS-muIL12 and an anti- PD-L1 antibody resulted in additive/synergistic antitumor effects in PD-L1-expressing subcutaneously transplanted tumors (MC38, MB49) and in an intravesical bladder tumor model (MB49). These results should provide the rationale to examine the combination of these agents in clinical studies. 2. We have examined the immunomodulatory and anti-tumor effects of the IL-15/Ra-Fc immunocytokine in tumor-free and highly metastatic tumor-bearing mice. Here, the IL-15/Ra-Fc immunocytokine significantly expanded NK and CD8+ T cells. In examining NK cell subsets, the greatest significant increase was in highly cytotoxic and migrating NK cells, leading to enhanced function on a per-cell basis. CD8+ T cell subset analysis determined that the IL-15/Ra-Fc immunocytokine significantly increased memory CD8+ T cells. In 4T1 breast tumor-bearing mice, the IL-15/Ra-Fc immunocytokine induced significant anti-tumor activity against spontaneous pulmonary metastases, depending on CD8+ T and NK cells, and resulting in prolonged survival. Similar anti-tumor activity was seen in the experimental pulmonary metastasis model of CT26 colon carcinoma cells, particularly when the IL-15/Ra-Fc immunocytokine was combined with a cocktail of checkpoint inhibitors, anti-CTLA-4 and anti-PD-L1. [] IDO inhibitor. Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase (IDO) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We have shown that peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-gamma, TNF-alpha, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines. [] Chordoma. 1. Chordoma, a rare bone tumor derived from the notochord, has been shown to be resistant to conventional therapies. The anti-PD-L1 monoclonal antibody avelumab is unique among checkpoint inhibitors in that it is a fully human IgG1 capable of mediating ADCC of PD-L1-expressing tumor cells. We examined four chordoma cell lines, first for expression of PD-L1, and in vitro for ADCC killing using NK cells and avelumab. PD-L1 expression was markedly upregulated by IFN-gamma in all four chordoma cell lines, which significantly increased sensitivity to ADCC. Brachyury is a transcription factor that is uniformly expressed in chordoma. Clinical trials are ongoing in which chordoma patients are treated with brachyury-specific vaccines. Co-incubating chordoma cells with brachyury-specific CD8+ T cells resulted in significant upregulation of PD-L1 on the tumor cells, mediated by the CD8+ T cells' IFN-gamma production, and increased sensitivity of chordoma cells to avelumab-mediated ADCC. Residential cancer stem cell subpopulations of chordoma cells were also killed by avelumab-mediated ADCC to the same degree as non-cancer stem cell populations. These findings suggest that as a monotherapy for chordoma, avelumab may enable endogenous NK cells, while in combination with T-cell immunotherapy, such as a vaccine, avelumab may enhance NK-cell killing of chordoma cells via ADCC.
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