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In adult cancer, immune signatures such as the T cell-inflamed gene expression profile (GEP) have been developed to predict which patients are likely to respond to immune checkpoint inhibitors (ICIs) beyond high tumor mutation burden (TMB) and PD-L1 expression. The GEP infers T cell infiltration and activation in the tumor microenvironment (TME) from transcriptomic data. However, it is not known whether tools such as GEP are applicable in pediatric cancer, as the TME in childhood cancers is largely unexplored and response to ICIs are rare. We have undertaken an integrated analysis of the pediatric TME using RNA-sequencing (RNA-seq) and immunohistochemistry (IHC). Our goal is to identify patients with T cell-inflamed or “hot” tumors who may benefit from ICIs. Through Australia's ZERO childhood cancer precision medicine program we performed RNA-seq on 347 high-risk pediatric cancers (estimated Citation Format: Chelsea Mayoh, Rachael L. Terry, Marie Wong, Loretta M. Lau, Dong Anh Khuong-Quang, Marion K. Mateos, Vanessa Tyrrell, Michelle Haber, David S. Ziegler, Mark J. Cowley, Joseph A. Trapani, Paul J. Neeson, Paul G. Ekert. The unexplored immune landscape of high-risk pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3044.

Adoptive immunotherapy utilizing chimeric antigen receptor (CAR) T cells has demonstrated high success rates in hematologic cancers, but results against solid malignancies have been limited to date, due in part to the immunosuppressive tumor microenvironment. Activation of the 4-1BB (CD137) pathway using an agonistic α-4-1BB antibody is known to provide strong costimulatory signals for augmenting and diversifying T-cell responses. We therefore hypothesized that a combination of α-4-1BB and CAR T-cell therapy would result in improved antitumor responses. Using a human-Her2 self-antigen mouse model, we report here that α-4-1BB significantly enhanced CAR T-cell efficacy directed against the Her2 antigen in two different established solid tumor settings. Treatment also increased the expression of IFNγ and the proliferation marker Ki67 in tumor-infiltrating CAR T cells when combined with α-4-1BB. Strikingly, α-4-1BB significantly reduced host immunosuppressive cells at the tumor site, including regulatory T cells and myeloid-derived suppressor cells, correlating with an increased therapeutic response. We conclude that α-4-1BB has a multifunctional role for enhancing CAR T-cell responses and that this combination therapy has high translational potential, given current phase I/II clinical trials with α-4-1BB against various types of cancer. Cancer Res; 77(6); 1296–309. ©2017 AACR.

Histone deacetylase inhibitors (HDACi) may engage host immunity as one basis for their antitumor effects. Herein, we demonstrate an application of this concept using the HDACi panobinostat to augment the antitumor efficacy of trastuzumab (anti-HER2) therapy, through both tumor cell autonomous and nonautonomous mechanisms. In HER2+ tumors that are inherently sensitive to the cytostatic effects of trastuzumab, cotreatment with panobinostat abrogated AKT signaling and triggered tumor regression in mice that lacked innate and/or adaptive immune effector cells. However, the cooperative ability of panobinostat and trastuzumab to harness host anticancer immune defenses was essential for their curative activity in trastuzumab-refractory HER2+ tumors. In trastuzumab-resistant HER2+ AU565pv xenografts and BT474 tumors expressing constitutively active AKT, panobinostat enhanced the antibody-dependent cell-mediated cytotoxicity function of trastuzumab. IFNγ–mediated, CXCR3-dependent increases in tumor-associated NK cells underpinned the combined curative activity of panobinostat and trastuzumab in these tumors. These data highlight the immune-enhancing effects of panobinostat and provide compelling evidence that this HDACi can license trastuzumab to evoke NK-cell–mediated responses capable of eradicating trastuzumab-refractory HER2+ tumors. Cancer Res; 77(10); 2594–606. ©2017 AACR.

Given that specific subsets of T helper 1 (Th1) and T helper 2 (Th2) CD4+ T cells have been shown to play key roles in tumor rejection models, we wanted to assess the contribution of either Th1 or Th2 CD4+ cell subtypes for redirected T-cell immunotherapy. In this study, we have developed a novel method involving retroviral transduction and in vitro T-cell polarization to generate gene-engineered mouse CD4+ Th1 and Th2 cells or T helper intermediate (Thi) cells expressing an anti–erbB2-CD28-ζ chimeric receptor. Gene-modified Th1 and Th2 polarized CD4+ cells were characterized by the preferential secretion of IFN-γ and interleukin-4, respectively, whereas Thi cells secreted both cytokines following receptor ligation. In adoptive transfer studies using an erbB2+ lung metastasis model, complete survival of mice was observed when transduced Th1, Th2, or Thi CD4+ cells were transferred in combination with an equivalent number of transduced CD8+ T cells. Tumor rejection was consistently associated with transduced T cells at the tumor site and interleukin-2 secretion. However, the surviving mice treated with gene-modified Th1 CD4+ cells were significantly more resistant to a subsequent challenge with a different erbB2+ tumor (4T1.2) implanted s.c. This result correlated with both increased expansion of Th1 CD4+ and CD8+ T cells in the blood and a greater number of these cells localizing to the tumor site following rechallenge. These data support the use of gene-modified CD4+ Th1 and CD8+ T cells for mediating a sustained antitumor response. [Cancer Res 2007;67(23):11428–37]

Adoptive immunotherapy involving genetic modification of T cells with antigen-specific, chimeric, single-chain receptors is a promising approach for the treatment of cancer. To determine whether gene-modified T cells could induce antitumor effects without associated autoimmune pathology, we assessed the ability of T cells expressing an anti-Her-2 chimeric receptor to eradicate tumor in Her-2 transgenic mice that express human Her-2 as a self-antigen in brain and mammary tissues. In adoptive transfer studies, we demonstrated significant improvement in the survival of mice bearing Her-2+ 24JK tumor following administration of anti-Her-2 T cells compared with control T cells. The incorporation of a lymphoablative step prior to adoptive transfer of anti-Her-2 T cells and administration of IL-2 were both found to further enhance survival. The reduction in tumor growth was also correlated with localization of transferred T cells at the tumor site. Furthermore, an antigen-specific recall response could be induced in long-term surviving mice following rechallenge with Her-2+ tumor. Importantly, antitumor effects were not associated with any autoimmune pathology in normal tissue expressing Her-2 antigen. This study highlights the therapeutic potential of using gene-engineered T cells as a safe and effective treatment of cancer. Cancer Res; 70(23); 9591–8. ©2010 AACR.

Histone deacetylase inhibitors (HDACIs) are a new class of chemotherapeutic drugs able to induce tumor cell apoptosis and/or cell cycle arrest; however, the molecular mechanisms underpinning their anticancer effects are poorly understood. Herein, we assessed the apoptotic pathways activated by three HDACIs, suberoylanilide hydroxamic acid, oxamflatin, and depsipeptide. We determined that all three drugs induced the accumulation of cells with a 4n DNA content and apoptosis mediated by the intrinsic apoptotic pathway. HDACI-induced mitochondrial membrane damage and apoptosis were inhibited by overexpression of Bcl-2, but not by the polycaspase inhibitor N-tert-butoxy-carbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk). Moreover, induction of a G(1)-S checkpoint through overexpression of p16(INK4A) or suppression of de novo protein synthesis also inhibited HDACI-induced cell death. Proteolytic cleavage of caspase-2, which is poorly inhibited by zVAD-fmk, was concomitant with HDACI-induced death; however, full processing of caspase-2 to the p19 active form was blocked by Bcl-2. Whereas all three drugs induce the activation of the proapoptotic Bcl-2 protein Bid upstream of mitochondrial membrane disruption, Bid cleavage in response to depsipeptide was significantly attenuated by zVAD-fmk. Suberoylanilide hydroxamic acid and oxamflatin could kill both P-glycoprotein (P-gp)(+) MDR cells and their P-gp(-) counterparts, whereas depsipeptide was shown to be a substrate for P-gp and was less effective in killing P-gp(+) cells. These data provide insight into the functional profile of three HDACIs and are important for the development of more rational approaches to chemotherapy, where information regarding the genetic profile of the tumor is matched with the functional profile of a given chemotherapeutic drug to promote favorable clinical responses.

The immunomodulatory (IMiD) drug Lenalidomide (Len) is known to stimulate natural killer (NK) cells and enhance anti-tumor responses. Len plus Dexamethasone (Dex) therapy (Len-Dex) is an effective treatment for multiple myeloma (MM). As part of a prospective clinical trial of monthly cycles of Len-Dex therapy we assessed NK cell number and function in 25 MM patients. Whilst NK numbers increased from a mean of 2×105/ml (baseline) to a mean of 3.9×105/ml (cycle 6) (p=0.05), the mean NK cell-mediated cytotoxicity in chromium assays against K562 significantly decreased from 48.9% to 27.6% respectively (p=0.0028). Further analysis showed that when NK cell cytotoxicity was assessed from MM patient peripheral blood mononuclear cells (PBMC) during therapy, no enhancement of cytotoxicity could be observed when cultured in vitro with Len. We hypothesized that Dex potently suppressed the Len-mediated enhancement of NK function. In vitro, Len increased normal donor (n=6) NK cytotoxicity from 38.5% (untreated) to 53.3%. Dex severely impaired NK cytotoxicity to Therefore, Len-mediates enhanced NK function through CD4+ T cell production of IL-2. The NK impairment observed in MM patients receiving Len-Dex is due to CD4+ T cell suppression. Our findings indicate that in order to harness both the anti-tumor and immunomodulatory capacity of Len the dose and timing of Dex therapy should be carefully reconsidered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5619.