Steven L. Highfill

Disruption of CXCR2-Mediated MDSC Tumor Trafficking Enhances Anti-PD1 Efficacy

Preventing trafficking of myeloid-derived suppressor cells to the tumor site enhances the efficiency of checkpoint blockade. Cross-Checking Cancer Immunological checkpoint blockades are a promising approach in cancer immunotherapy—pushing the immune response to the tumor past tolerance into attack mode. Now, Highfill et al. show that inhibiting myeloid-derived suppressor cells by preventing their trafficking into the tumor site can enhance the efficiency of checkpoint blockade. They found in a mouse model of rhabdomyosarcoma that blocking a chemokine receptor on myeloid-derived suppressor cells in conjunction with antibodies to the checkpoint molecule programmed death 1 induced substantial antitumor effects. Moreover, patients with rhabdomyosarcoma that had elevated levels of ligands for this chemokine receptor had poorer prognosis. These data suggest that combining these therapies may increase the likelihood of patient response, checking cancer into the boards. Suppression of the host’s immune system plays a major role in cancer progression. Tumor signaling of programmed death 1 (PD1) on T cells and expansion of myeloid-derived suppressor cells (MDSCs) are major mechanisms of tumor immune escape. We sought to target these pathways in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma of childhood. Murine RMS showed high surface expression of PD-L1, and anti-PD1 prevented tumor growth if initiated early after tumor inoculation; however, delayed anti-PD1 had limited benefit. RMS induced robust expansion of CXCR2+CD11b+Ly6Ghi MDSCs, and CXCR2 deficiency prevented CD11b+Ly6Ghi MDSC trafficking to the tumor. When tumor trafficking of MDSCs was inhibited by CXCR2 deficiency, or after anti-CXCR2 monoclonal antibody therapy, delayed anti-PD1 treatment induced significant antitumor effects. Thus, CXCR2+CD11b+Ly6Ghi MDSCs mediate local immunosuppression, which limits the efficacy of checkpoint blockade in murine RMS. Human pediatric sarcomas also produce CXCR2 ligands, including CXCL8. Patients with metastatic pediatric sarcomas display elevated serum CXCR2 ligands, and elevated CXCL8 is associated with diminished survival in this population. We conclude that accumulation of MDSCs in the tumor bed limits the efficacy of checkpoint blockade in cancer. We also identify CXCR2 as a novel target for modulating tumor immune escape and present evidence that CXCR2+CD11b+Ly6Ghi MDSCs are an important suppressive myeloid subset in pediatric sarcomas. These findings present a translatable strategy to improve the efficacy of checkpoint blockade by preventing trafficking of MDSCs to the tumor site.

Reduction of MDSCs with All-trans Retinoic Acid Improves CAR Therapy Efficacy for Sarcomas.

The efficacy of chimeric antigen receptor (CAR)–modified T cells against solid tumors is not proven. Retinoids are clinically accessible and were found to modulate tumor myeloid-derived suppressor cells, enhancing the efficacy of CAR therapies targeting solid tumors. Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2+ sarcoma and neuroblastoma cell lines in vitro. However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2+ sarcoma, despite effectively controlling GD2+ neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro. Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869–80. ©2016 AACR.

Activation of Hematopoietic Stem/Progenitor Cells Promotes Immunosuppression Within the Pre-metastatic Niche

Metastatic tumors have been shown to establish microenvironments in distant tissues that are permissive to disseminated tumor cells. Hematopoietic cells contribute to this microenvironment, yet the precise initiating events responsible for establishing the pre-metastatic niche remain unclear. Here, we tracked the developmental fate of hematopoietic stem and progenitor cells (HSPC) in tumor-bearing mice. We show that a distant primary tumor drives the expansion of HSPCs within the bone marrow and their mobilization to the bloodstream. Treatment of purified HSPCs cultured ex vivo with tumor-conditioned media induced their proliferation as well as their differentiation into immunosuppressive myeloid cells. We furthered tracked purified HSPCs in vivo and found they differentiated into myeloid-derived suppressor cells in early metastatic sites of tumor-bearing mice. The number of CD11b(+)Ly6g(+) cells in metastatic sites was significantly increased by HSPC mobilization and decreased if tumor-mediated mobilization was inhibited. Moreover, pharmacologic mobilization of HSPCs increased metastasis, whereas depletion of Gr1(+) cells abrogated the metastasis-promoting effects of HSPC mobilization. Finally, we detected elevated levels of HSPCs in the circulation of newly diagnosed cancer patients, which correlated with increased risk for metastatic progression. Taken together, our results highlight bone marrow activation as one of the earliest steps of the metastatic process and identify circulating HSPCs as potential clinical indicators of metastatic niche formation.

Neutralization of murine myeloid-derived suppressor cells enhances the efficacy of a chimeric antigen receptor T-cells directed against pediatric solid tumors

Genetically engineered T-cells that express chimeric antigen receptors (CARs) to directly target tumor-expressed antigens have shown remarkable activity in clinical trials for hematologic malignancies, but remain unproven for the treatment of solid tumors. We find that in addition to its well known high expression in neuroblastoma, the disialoganglioside GD2 is also highly expressed on pediatric sarcomas. Some human sarcoma cell lines have GD2 expression levels equivalent to that of the prototypical neuroblastoma cell line LAN5, and immunohistochemical staining of primary human tumor tissue samples taken from essentially all patients with metastatic osteosarcoma and a subset of patients with alveolar rhabdomyosarcoma demonstrate robust expression of GD2 on the cell surface. Based on these results, we have developed models to test the ability of GD2-CAR T-cells to target and lyse human sarcomas in vitro and in vivo. We find that GD2-CAR modified T-cells induce specific lysis of GD2-expressing solid tumors in vitro even at effector:target ratios as low as 1:1, but fail to induce a significant response in vivo using the same human cell lines. Interestingly, we discovered that the pediatric sarcoma xenografts induce a large expansion of murine CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) that inhibit human T-cell responses in vitro. These results lead us to adopt a combinatorial therapeutic strategy in which we first neutralized the suppressive potential of MDSC by administration of all-trans retinoic acid (ATRA) followed by GD2-CAR therapy. This combinatorial approach results in significant improvements in both overall survival and tumor growth. Given that retinoids are already available in the clinic, these results suggest that the effectiveness of CAR T-cell therapy for solid tumors could be enhanced by coadministration of retinoids to modulate the myeloid derived suppressor cells.

A pan-inhibitor of DASH family enzymes induces immune-mediated regression of murine sarcoma and is a potent adjuvant to dendritic cell vaccination and adoptive T-cell therapy

Multimodality therapy consisting of surgery, chemotherapy, and radiation will fail in approximately 40% of patients with pediatric sarcomas and result in substantial long-term morbidity in those who are cured. Immunotherapeutic regimens for the treatment of solid tumors typically generate antigen-specific responses too weak to overcome considerable tumor burden and tumor suppressive mechanisms and are in need of adjuvant assistance. Previous work suggests that inhibitors of DASH (dipeptidyl peptidase IV activity and/or structural homologs) enzymes can mediate tumor regression by immune-mediated mechanisms. Herein, we demonstrate that the DASH inhibitor, ARI-4175, can induce regression and eradication of well-established solid tumors, both as a single agent and as an adjuvant to a dendritic cell (DC) vaccine and adoptive cell therapy (ACT) in mice implanted with the M3-9-M rhabdomyosarcoma cell line. Treatment with effective doses of ARI-4175 correlated with recruitment of myeloid (CD11b+) cells, particularly myeloid DCs, to secondary lymphoid tissues and with reduced frequency of intratumoral monocytic (CD11b+Ly6-ChiLy6-Glo) myeloid-derived suppressor cells. In immunocompetent mice, combining ARI-4175 with a DC vaccine or ACT with tumor-primed T cells produced significant improvements in tumor responses against well-established M3-9-M tumors. In M3-9-M-bearing immunodeficient (Rag1−/−) mice, ACT combined with ARI-4175 produced greater tumor responses and significantly improved survival compared with either treatment alone. These studies warrant the clinical investigation of ARI-4175 for treatment of sarcomas and other malignancies, particularly as an adjuvant to tumor vaccines and ACT.

Nfatc2 and Tob1 Have Non-Overlapping Function in T Cell Negative Regulation and Tumorigenesis

Nfatc2 and Tob1 are intrinsic negative regulators of T cell activation. Nfatc2-deficient and Tob1-deficient T cells show reduced thresholds of activation; however, whether these factors have independent or overlapping roles in negative regulation of T cell responses has not been previously examined. Here, we show that Nfatc2 knockout (KO) but not Tob1 KO mice have age-associated accumulation of persistently activated T cells in vivo and expansion of the CD44+ memory cell compartment and age-associated lymphocytic infiltrates in visceral organs, without significant changes in numbers of CD4+CD25+Foxp3+ regulatory T cells (Treg). In vitro, CD4+CD25− “conventional” T cells (Tconvs) from both KO strains showed greater proliferation than wild type (WT) Tconvs. However, while Tregs from Nfatc2 KO mice retained normal suppressive function, Tregs from Tob1 KOs had enhanced suppressive activity. Nfatc2 KO Tconvs expanded somewhat more rapidly than WT Tconvs under conditions of homeostatic proliferation, but their accelerated growth capacity was negated, at least acutely, in a lymphoreplete environment. Finally, Nfatc2 KO mice developed a previously uncharacterized increase in B-cell malignancies, which was not accelerated by the absence of Tob1. The data thus support the prevailing hypothesis that Nfatc2 and Tob1 are non-redundant regulators of lymphocyte homeostasis.

Abstract LB-284: Bone marrow-derived progenitor cells develop into myeloid-derived suppressor cells at metastatic sites.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The ability of tumor cells to metastasize to distant tissues is the most lethal aspect of cancer. Prior to detectable metastasis tumors elicit distant changes within the bone marrow and future sites of metastasis, including formation of the pre-metastatic niche. We have found that prior to detectable spontaneous metastasis, a primary tumor causes enhanced production and mobilization of progenitor cells from the bone marrow into the blood. We find in cancer patients that increased circulating progenitors are associated with metastatic disease progression. By utilizing bone marrow transplant models and a novel ex vivo lung culture, we demonstrate that bone marrow-derived progenitor cells accumulate at the primary tumor as well as metastatic lesions. As our cancer models approach detectable spontaneous metastases, we observe a loss of progenitor cells in pre-metastatic sites and an increase in myeloid-derived suppressor cells (MDSCs). We further traced adoptively transferred bone marrow-derived progenitors in tumor-bearing mice and demonstrate that these cells contribute to the MDSC population. Together, these data suggest that circulating progenitor cells provide a prognostic tool to predict metastatic dissemination. Furthermore, these cells likely contribute to metastatic progression by developing into MDSCs within the primary tumor and at metastatic sites. Citation Format: Amber Giles, Yorleny Vicioso, Christianne Persenaire, Miki Kasai, Steven Highfill, Arnulfo Mendoza, Rosandra Kaplan. Bone marrow-derived progenitor cells develop into myeloid-derived suppressor cells at metastatic sites. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-284. doi:10.1158/1538-7445.AM2013-LB-284

A Phase 1 trial of autologous monocytes stimulated ex vivo with Sylatron ® (Peginterferon alfa-2b) and Actimmune ® (Interferon gamma-1b) for intra-peritoneal administration in recurrent ovarian cancer

BackgroundOvarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both cytostatic and cytotoxic properties, while monocytes stimulated with interferons have potent cytotoxic properties. Due to the highly immune suppressive properties of ovarian cancer, ex vivo stimulation of autologous patient monocytes with interferons and infusion of all three agents intraperitoneally (IP) can provide a strong pro-inflammatory environment at the site of disease to kill malignant cells.MethodsPatient monocytes are isolated through counterflow elutriation and stimulated ex vivo with interferons and infused IP through a semi-permanent catheter. We have designed a standard 3 + 3 dose escalation study to explore the highest tolerated dose of interferons and monocytes infused IP in patients with chemotherapy resistant ovarian cancer. Secondary outcome measurements of changes in the peripheral blood immune compartment and plasma cytokines will be studied for correlations of response.DiscussionWe have developed a novel immunotherapy focused on the innate immune system for the treatment of ovarian cancer. We have combined the use of autologous monocytes and interferons alpha and gamma for local–regional administration directly into the peritoneal cavity. This therapy is highly unique in that it is the first study of its type using only components of the innate immune system for the locoregional delivery consisting of autologous monocytes and dual interferons alpha and gamma.Trial Registration ClinicalTrials.gov Identifier: NCT02948426, registered on October 28, 2016. https://clinicaltrials.gov/ct2/show/NCT02948426

Plasma from some cancer patients inhibits adenoviral Ad5f35 vector transduction of dendritic cells

BACKGROUND Pooled AB serum is often used as a media supplement for cell culture but it has the potential to transmit infectious diseases. To avoid this risk, we used autologous plasma as a media supplement for manufacturing dendritic cells (DCs) for cancer immunotherapy. We noticed inconsistencies in the DCs and investigated their nature and cause. METHODS Adenovirus human epidural growth factor receptor 2 (adHER2/neu) DCs for 21 patients were manufactured from autologous peripheral blood monocytes that were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 3 days, transduced with Ad5f35HER2ECTM and then treated with lipopolysaccharide and interferon (IFN)-γ for 1 day. The cells were cultured in RPMI-1640 supplemented with either 10% heat inactivated autologous or AB plasma. RESULTS Twenty-eight adHER2/neu DCs were manufactured for 21 patients using autologous plasma and 68 were manufactured for 20 of those patients using AB plasma. The expression of human epidural growth factor receptor 2 (HER2/neu) was less for DCs manufactured with autologous plasma (70.3 ± 33.3% versus 86.1 ± 22.8%; P <0.01). Manufacturing adHER2/neu DCs using monocytes from three healthy subjects and plasma from one patient with low HER2/neu expression (18%) resulted in low HER2/neu expression by all three DCs (13%, 16% and 23%). Analysis of the levels of 1322 proteins in eight plasma samples associated with low HER2/neu expression and in 12 associated with high HER2/neu expression revealed that the levels of 14 predicted HER2/neu transduction efficiency. CONCLUSION The manufacture of adHER2/neu DC using autologous plasma as a media supplement resulted in inconsistent HER2/neu expression. It is likely that variability in the levels of multiple proteins in autologous plasma contributed to low HER2/neu expression.

Bone marrow-derived progenitor cells develop into myeloid-derived suppressor cells at metastatic sites

The ability of tumor cells to metastasize to distant tissues is the most lethal aspect of cancer. Prior to detectable metastasis tumors elicit distant changes within the bone marrow and future sites of metastasis, including formation of the pre-metastatic niche. We have found that prior to detectable spontaneous metastasis, a primary tumor causes enhanced production and mobilization of progenitor cells from the bone marrow into the blood. We find in cancer patients that increased circulating progenitors are associated with metastatic disease progression. By utilizing bone marrow transplant models and a novel ex vivo lung culture, we demonstrate that bone marrow-derived progenitor cells accumulate at the primary tumor as well as metastatic lesions. Blood and bone marrow of tumor-bearing mice are able to form significantly more myeloid colonies in a CFU assay. As our cancer models approach detectable spontaneous metastases, we observe a loss of progenitor cells in pre-metastatic sites and a two- to four- fold increase in myeloid-derived suppressor cells (MDSCs). We further traced adoptively transferred bone marrow-derived progenitors in tumor-bearing mice and demonstrate that these cells contribute to the MDSC population. The MDSCs identified by immunophenotyping by flow cytometry are immunosuppressive and capable of suppressing T cell proliferation in response to anti-CD3/anti-CD28 bead stimulation. Together, these data suggest that myeloid-derived suppressor cells contribute to metastatic progression at distant sites. Further, bone marrow-derived progenitor cells can provide a prognostic marker for tumor progression and metastatic risk.