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SCLC is a highly aggressive tumor with a 5-year survival rate of less than 6%. A heterogeneous disease, SCLC is classified into four subtypes that include tumors with neuroendocrine and non-neuroendocrine features. Immune checkpoint blockade has been recently added for the frontline treatment of SCLC; however, this therapy has only led to modest clinical improvements. The lack of clinical benefit in a cancer type known to have a high tumor mutational burden has been attributed to poor T-cell infiltration and low expression of MHC-class I in most SCLC tumors. In an attempt to devise a more effective immunotherapeutic regimen, this study investigated an alternate approach on the basis of the use of the clinical-stage interleukin-15 superagonist, N-803.Preclinical models of SCLC spanning all molecular subtypes were used to evaluate the susceptibility of SCLC to natural killer (NK)-mediated lysis in vitro, including NK cells activated by N-803. Antitumor activity of N-803 was evaluated in vivo with a xenograft model of SCLC.In vitro and in vivo data revealed differences in susceptibility of SCLC subtypes to lysis by NK cells and that NK cells activated by N-803 effectively lyse SCLC tumor cells across all variant subtypes, regardless of their expression of MHC-class I.These findings highlight the potential of a novel immune-based intervention using a cytokine-based therapeutic option for the treatment of SCLC. We hypothesize that N-803 may provide benefit to most patients with SCLC, including those with immunologically cold tumors lacking MHC expression.

PDF file - 1861K, Murine T cells express internal and cell surface CTLA-4 at 72 hrs. after activation.

Tumor cells use various methods of immunosuppression to overcome antitumor immunity. One such method is that of programmed death ligand-1 (PD-L1 or B7-H1), which upon binding its receptor PD-1 on T cells triggers apoptotic death of the activated T cells. Overexpression of the costimulatory molecule CD80 on PD-L1+ tumor cells, or inclusion of a soluble form of CD80 (CD80-Fc), maintains the activation of PD-1+–activated T cells. Using T cells from CD28-deficient mice and antibodies to block CD28 on human T cells, we now report that a soluble form of CD80 mediates this effect by simultaneously neutralizing PD-1–PD-L1-mediated immunosuppression and by providing CD80–CD28 costimulation, and is more effective than antibodies to PD-L1 or PD-1 in maintaining IFNγ production by PD-1+ activated T cells. Therefore, soluble CD80 may be a more effective therapeutic than these checkpoint antibodies for facilitating the development and maintenance of antitumor immunity because it has the dual functions of preventing PD-L1–mediated immunosuppression and simultaneously delivering the second signal for T-cell activation. Cancer Immunol Res; 2(7); 610–5. ©2014 AACR.

Supplementary figures 1 & 2

Supplemental Figure S7. SX-682 treatment does not alter PMN-MDSC suppressive capacity

Purpose:Natural killer (NK)-cell–based immunotherapy may overcome obstacles to effective T-cell–based immunotherapy such as the presence of genomic alterations in IFN response genes and antigen presentation machinery. All immunotherapy approaches may be abrogated by the presence of an immunosuppressive tumor microenvironment present in many solid tumor types, including head and neck squamous cell carcinoma (HNSCC). Here, we studied the role of myeloid-derived suppressor cells (MDSC) in suppressing NK-cell function in HNSCC.Experimental Design:The ability of peripheral and tumor-infiltrating MDSC from mice bearing murine oral cancer 2 (MOC2) non-T-cell–inflamed tumors and from patients with HNSCC to suppress NK-cell function was studied with real-time impedance and ELISpot assays. The therapeutic efficacy of SX-682, a small-molecule inhibitor of CXCR1 and CXCR2, was assessed in combination with adoptively transferred NK cells.Results:Mice bearing MOC2 tumors pathologically accumulate peripheral CXCR2+ neutrophilic-MDSC (PMN-MDSC) that traffic into tumors and suppress NK-cell function through TGFβ and production of H2O2. Inhibition of MDSC trafficking with orally bioavailable SX-682 significantly abrogated tumor MDSC accumulation and enhanced the tumor infiltration, activation, and therapeutic efficacy of adoptively transferred murine NK cells. Patients with HNSCC harbor significant levels of circulating and tumor-infiltrating CXCR1/2+ CD15+ PMN-MDSC and CD14+ monocytic-MDSC. Tumor MDSC exhibited greater immunosuppression than those in circulation. HNSCC tumor MDSC immunosuppression was mediated by multiple, independent, cell-specific mechanisms including TGFβ and nitric oxide.Conclusions:The clinical study of CXCR1/2 inhibitors in combination with adoptively transferred NK cells is warranted.

Methods not essential to the main conclusions but important for critical review of methods used for this work

Supplementary Figure 1 Morphology of PIN in B13-MYC mice. Supplementary Figure 2 Phenotypic analysis of PIN in a B13-MYC mouse (ventral lobe, 52 weeks old). Supplementary Figure 3 Additional phenotypic analysis of B-MYC/Pten mouse PIN (anterior lobe, 8 weeks). Supplementary Figure 4 Invasive adenocarcinoma lesion in a 14 week old B-MYC/Pten animal. Supplementary Figure 5 Chromogranin (a) staining in a primary tumor and Foxa2 (b) staining in a lymph node metastasis from a 24 week-old B-MYC/Pten animal shows focal scattered tumor cells staining positively (arrows). Supplementary Figure 6 Morphological features of castrated B-MYC/Pten animals. Supplementary Figure 7. Genes within regions of copy number gains and losses are enriched in specific gene ontology (GO) gene sets. Supplementary Table 1. Prostate Phenotype in B-MYC/PtenFl/Fl mice and wild type (FVB/N) mice. Supplementary Table 2. Metastatic Spread in B-MYC/PtenFl/Fl mice and wild type (FVB/N) mice. Supplementary Table 3. Prostate Phenotype in B-MYC/PtenFl/+ mice and wild type mice. Supplementary Table 4. Effects of androgen withdrawal/castration in B-MYC/PtenFl/Fl mice and wild type (FVB/N) mice. Supplementary Table 5. Metastatic Spread in B-MYC/PtenFl/Fl mice after androgen withdrawal. Supplementary Table 6. Somatic copy number gains and losses in each sample. Supplementary Table 7. Regions of Copy Number alterations in each sample. Supplementary Table 8. Regions with recurrent gains and losses in at least two mice. Supplementary Table 9. Regions of high copy number gain and low copy number loss in any tumor specimen.

Chronic inflammation often precedes malignant transformation and later drives tumor progression. Likewise, subversion of the immune system plays a role in tumor progression, with tumoral immune escape now well recognized as a crucial hallmark of cancer. Myeloid-derived suppressor cells (MDSC) are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. Thus, MDSCs may define an element of the pathogenic inflammatory processes that drives immune escape. The secreted alarmin HMGB1 is a proinflammatory partner, inducer, and chaperone for many proinflammatory molecules that MDSCs develop. Therefore, in this study, we examined HMGB1 as a potential regulator of MDSCs. In murine tumor systems, HMGB1 was ubiquitous in the tumor microenvironment, activating the NF-κB signal transduction pathway in MDSCs and regulating their quantity and quality. We found that HMGB1 promotes the development of MDSCs from bone marrow progenitor cells, contributing to their ability to suppress antigen-driven activation of CD4+ and CD8+ T cells. Furthermore, HMGB1 increased MDSC-mediated production of IL-10, enhanced crosstalk between MDSCs and macrophages, and facilitated the ability of MDSCs to downregulate expression of the T-cell homing receptor L-selectin. Overall, our results revealed a pivotal role for HMGB1 in the development and cancerous contributions of MDSCs. Cancer Res; 74(20); 5723–33. ©2014 AACR.

Supplementary Figure S3. HMGB1 produced by tumor cells promotes tumor growth and metastasis.

Supplementary Table and Figure Legends. Legend for Supplementary Tables S1-S2 and Supplementary Figures S1-S4.

Supplementary Table S1. Quantitation of HMGB1 secreted by cultured tumor cells, ex vivo macrophages and MDSC, in vivo grown tumors, and cultures of MDSC differentiated from bone marrow progenitor cells.

Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations (CNA) are found in most aggressive primary human prostate cancers, and the overall number of such changes is increased in metastases. Chromosome 10q23 deletions, encompassing PTEN, and amplification of 8q24, harboring MYC, are frequently observed, and the presence of both together portends a high risk of prostate cancer-specific mortality. In extant genetically engineered mouse prostate cancer models (GEMM), isolated MYC overexpression or targeted Pten loss can each produce early prostate adenocarcinomas, but are not sufficient to induce genetic instability or metastases with high penetrance. Although a previous study showed that combining Pten loss with focal MYC overexpression in a small fraction of prostatic epithelial cells exhibits cooperativity in GEMMs, additional targeted Tp53 disruption was required for formation of metastases. We hypothesized that driving combined MYC overexpression and Pten loss using recently characterized Hoxb13 transcriptional control elements that are active in prostate luminal epithelial cells would induce the development of genomic instability and aggressive disease with metastatic potential. Neoplastic lesions that developed with either MYC activation alone (Hoxb13-MYC) or Pten loss alone (Hoxb13-Cre∣PtenFl/Fl) failed to progress beyond prostatic intraepithelial neoplasia and did not harbor genomic CNAs. By contrast, mice with both alterations (Hoxb13-MYC∣Hoxb13-Cre∣PtenFl/Fl, hereafter, BMPC mice) developed lethal adenocarcinoma with distant metastases and widespread genome CNAs that were independent of forced disruption of Tp53 and telomere shortening. BMPC cancers lacked neuroendocrine or sarcomatoid differentiation, features uncommon in human disease but common in other models of prostate cancer that metastasize. These data show that combined MYC activation and Pten loss driven by the Hoxb13 regulatory locus synergize to induce genomic instability and aggressive prostate cancer that phenocopies the human disease at the histologic and genomic levels. Cancer Res; 76(2); 283–92. ©2015 AACR.

BackgroundImmune checkpoint blockade (ICB) has achieved unprecedented success in treating multiple cancer types. However, clinical benefit remains modest for most patients with solid malignancies due to primary or acquired resistance. Tumor-intrinsic loss of major histocompatibility complex class I (MHC-I) and aberrations in antigen processing machinery (APM) and interferon gamma (IFN-γ) pathways have been shown to play an important role in ICB resistance. While a plethora of combination treatments are being investigated to overcome ICB resistance, there are few identified preclinical models of solid tumors harboring these deficiencies to explore therapeutic interventions that can bypass ICB resistance. Here, we investigated the combination of the epigenetic modulator entinostat and the tumor-targeted immunocytokine NHS-IL12 in three different murine tumor models resistant to αPD-1/αPD-L1 (anti-programmed cell death protein 1/anti-programmed death ligand 1) and harboring MHC-I, APM, and IFN-γ response deficiencies and differing tumor mutational burden (TMB).MethodsEntinostat and NHS-IL12 were administered to mice bearing TC-1/a9 (lung, HPV16 E6/E7+), CMT.64 lung, or RVP3 sarcoma tumors. Antitumor efficacy and survival were monitored. Comprehensive tumor microenvironment (TME) and spleen analysis of immune cells, cytokines, and chemokines was performed. Additionally, whole transcriptomic analysis was carried out on TC-1/a9 tumors. Cancer Genome Atlas (TCGA) datasets were analyzed for translational relevance.ResultsWe demonstrate that the combination of entinostat and NHS-IL12 therapy elicits potent antitumor activity and survival benefit through prolonged activation and tumor infiltration of cytotoxic CD8+T cells, across αPD-1/αPD-L1 refractory tumors irrespective of TMB, including in the IFN-γ signaling-impaired RVP3 tumor model. The combination therapy promoted M1-like macrophages and activated antigen-presenting cells while decreasing M2-like macrophages and regulatory T cells in a tumor-dependent manner. This was associated with increased levels of IFN-γ, IL-12, chemokine (C-X-C motif) ligand 9 (CXCL9), and CXCL13 in the TME. Further, the combination therapy synergized to promote MHC-I and APM upregulation, and enrichment of JAK/STAT (janus kinase/signal transducers and activators of transcription), IFN-γ-response and antigen processing-associated pathways. A biomarker signature of the mechanism involved in these studies is associated with patients’ overall survival across multiple tumor types.ConclusionsOur findings provide a rationale for combining the tumor-targeting NHS-IL12 with the histone deacetylase inhibitor entinostat in the clinical setting for patients unresponsive to αPD-1/αPD-L1 and/or with innate deficiencies in tumor MHC-I, APM expression, and IFN-γ signaling.

BackgroundSmall cell lung cancer (SCLC) is a highly aggressive tumor with a 5-year survival rate of less than 5%. Traditionally characterized as a neuroendocrine (NE) cancer, several subtypes have now been identified which vary in their phenotypic and transcriptional profiles. Classical NE tumors are molecularly defined as ASCL1+ or NEUROD1+ and exhibit an epithelial phenotype, expressing cytokeratin and E-cadherin (E-Cad). In contrast, non-classical variants express POU2F3 or YAP1 and are enriched in mesenchymal features, such as high levels of vimentin (Vim). Prior studies describe that non-NE variants of SCLC are less susceptible to chemotherapy and may arise via therapeutic selection. With the addition of immune checkpoint blockade to first-line chemotherapy for the treatment of advanced SCLC, understanding whether SCLC variants respond differently to immunotherapy is crucial.MethodsWe utilized a range of pre-clinical models to investigate whether molecular and phenotypic variants of SCLC differ in their susceptibility to immune-mediated lysis. Following extensive characterization at the RNA and protein levels for expression of ASCL1, NEUROD1, POU2F3, YAP1, epithelial E-Cad, mesenchymal Vim, and other markers of cell phenotype, a panel of cells including each variant subtype were selected for further study.ResultsUpon exposure to healthy donor effector NK cells, the more epithelial cells were highly susceptible to NK-mediated cytotoxicity while all mesenchymal SCLC cells remained highly refractory to NK-mediated lysis. This prompted us to investigate immunotherapy approaches such as the addition of N803, a mutant IL-15 superagonist, to improve the activation and proliferation of NK cells. In a xenograft model utilizing the mesenchymal YAP1+ H841 cell line subcutaneously implanted into nude mice devoid of all immune cells except for NK cells, we observed that the weekly administration of N803 resulted in a significant increase in the number of activated NK cells within the spleens of treated mice. Additionally, NK cells from treated mice produced significantly higher levels of IFN-gamma and granzyme B, resulting in a significant decrease in overall tumor burden.ConclusionsOur data indicates that N803-activated NK cells effectively mediate lysis of SCLC across all variant types, including those previously completely refractory to traditional NK cell lysis. These results highlight the potential of N803 as a novel immune-based intervention for the treatment of all variants of SCLC.Ethics ApprovalPBMCs were obtained from healthy donors at the NIH Clinical Center Blood Bank (NCT00001846). All animal studies were approved and conducted in accordance with an IACUC-approved animal protocol (LTIB-57) with the approval the NIH/NCI Institutional Animal Care and Use Committee.

BackgroundBackground: Treatment of patients with castration-resistant prostate cancer (CRPC) includes the use of next-generation hormonal therapies such as abiraterone or enzalutamide. Although these agents extend survival, a significant proportion of patients exhibit primary or acquired resistance to treatment. In recent years, immune checkpoint blockade has led to remarkable responses in patients with several tumor types, however, CRPC has remained resistant to immunotherapy. Previous studies have demonstrated that different tumor variants could emerge along the progression of prostate cancer, including tumors undergoing phenotypic plasticity in the context of an epithelial-mesenchymal transition. Our laboratory and others have shown that phenotypic plasticity is a driver of resistance to immunotherapy. Based on this knowledge, we investigated whether changes in tumor phenotype could affect the response of CRPC to immune-based therapies, and ways this can be mitigated.MethodsThe androgen sensitive LNCAP prostate cancer cell line was used to derive LNCAP cells resistant to enzalutamide (LNCAP-EnzaR) or abiraterone (LNCAP-AbiR). Resistant cell lines and parental LNCAP cells were comparatively evaluated for features of EMT and neuroendocrine phenotype via RT-PCR, ELISA, western blot, immunofluorescence, and RNAseq. Changes in the susceptibility to NK-cell mediated cytotoxicity were evaluated with NK cells isolated from peripheral blood from healthy donors. LNCAP-EnzaR cells were also grown in vivo in NSG MHC-deficient mice, and tumors were characterized for phenotypic markers and potential therapeutic targets.ResultsAcquisition of resistance to both enzalutamide and abiraterone was associated with a significant increase in mesenchymal tumor features, including high levels of vimentin and fibronectin, and the loss of epithelial features and cell-to-cell attachments. LNCAP-Enza-R and LNCAP-AbiR cells showed a significant reduction (up to 90%) in susceptibility to NK-cell mediated cytotoxicity and antibody-dependent cell cytotoxicity (ADCC), compared with parental cells. These results prompted us to investigate approaches to improve immune-mediated lysis, including inhibition of estrogen receptor 1 (ESR1), which was identified as highly upregulated in LNCAP-EnzaR cells via RNAseq analysis. In a xenograft model of LNCAP-EnzaR cells, we corroborated the maintenance of tumor phenotypic plasticity and the expression of actionable targets.ConclusionsOur data indicates that acquisition of resistance to androgen receptor inhibition is associated with marked reduction of susceptibility to immune attack, and the acquisition of tumor phenotypic plasticity. Future studies will investigate approaches that revert tumor plasticity, including blockade of ESR1, TGF-beta or IL-8, for potential improvement of tumor susceptibility to immune attack in CRPC.Ethics ApprovalPBMCs were obtained from healthy donors at the NIH Clinical Center Blood Bank (NCT00001846). All animal studies were approved and conducted in accordance with an IACUC-approved animal protocol (LTIB-57) with the approval the NIH/NCI Institutional Animal Care and Use Committee.

Simple Summary Multiple types of primary tumors and metastases that present with very little if any immune cell infiltration (so-called immunologically “cold” tumors) do not respond to current immunotherapies. In this study, we show that recently developed intratumoral application-based immunotherapy using mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA therapy) efficiently suppresses tumor growth in a murine bilateral pheochromocytoma model. Moreover, MBTA therapy increases the recruitment of innate immune cells followed by adaptive immune cells not only to primary (injected) tumors but also distal (non-injected) tumors. We also demonstrated that after successful MBTA therapy of subcutaneous pheochromocytoma, long-term immunological memory is driven by CD4+ T cells. Taken together, this study helps to better understand the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination. Abstract Immunotherapy has become an essential component in cancer treatment. However, the majority of solid metastatic cancers, such as pheochromocytoma, are resistant to this approach. Therefore, understanding immune cell composition in primary and distant metastatic tumors is important for therapeutic intervention and diagnostics. Combined mannan-BAM, TLR ligand, and anti-CD40 antibody-based intratumoral immunotherapy (MBTA therapy) previously resulted in the complete eradication of murine subcutaneous pheochromocytoma and demonstrated a systemic antitumor immune response in a metastatic model. Here, we further evaluated this systemic effect using a bilateral pheochromocytoma model, performing MBTA therapy through injection into the primary tumor and using distant (non-injected) tumors to monitor size changes and detailed immune cell infiltration. MBTA therapy suppressed the growth of not only injected but also distal tumors and prolonged MBTA-treated mice survival. Our flow cytometry analysis showed that MBTA therapy led to increased recruitment of innate and adaptive immune cells in both tumors and the spleen. Moreover, adoptive CD4+ T cell transfer from successfully MBTA-treated mice (i.e., subcutaneous pheochromocytoma) demonstrates the importance of these cells in long-term immunological memory. In summary, this study unravels further details on the systemic effect of MBTA therapy and its use for tumor and metastasis reduction or even elimination.

Objective (1) To examine the prevalence of college-aged students on campus who had a history of concussion(s) or traumatic brain injury (TBI) and associated symptoms and (2) to evaluate the rate of campus service utilization among students who had a TBI. Setting A representative sampling, randomly selected campus-wide e-mail was sent out with a Qualtrics online survey. Participants College-aged students (N = 466, n = 429) at a northeastern US university responded and consented to participate in the online survey. Design Descriptive study using an online survey to evaluate the prevalence of TBI and subsequent utility of campus services. Main outcome measures The survey assessed concussion history and the diagnosing clinician, activity in which it occurred, level of consciousness, associated symptoms, and utilization of campus services for academic accommodation. Results Prevalence of concussion/TBI in the study population (51.7%) is notably higher than previous reporting in adult and college populations (∼10%-25%). Campus disability and accommodative services were underutilized (10%-13%) and were accessed more readily by the female population. In addition, there were reportable increases in symptom prevalence and service utilization with a greater number of previous concussions. Conclusions Awareness and utilization of accommodative services, especially with a history of concussions, need to be improved in order to ensure academic and occupational success of students.

Combination of immune checkpoint blockade (ICB) with chemotherapy is becoming more commonly used for the treatment of various cancer types, including advanced non-small cell lung cancer, and triple negative breast cancer (TNBC) in patients who have PD-L1 positive tumors, among others. Although chemotherapy-ICB modalities have been shown to improve clinical responses over those observed with ICB alone, long-term disease control is achieved only in a small subset of patients. To provide a rational basis for combination therapy, and to potentially develop more effective approaches, in this study we investigated the impact of chemotherapy on the tumor microenvironment (TME) in the context of PD-1/PD-L1 axis blockade. ICB refractory murine models were utilized, including the TNBC 4T1 model and the Lewis Lung Carcinoma (LLC) model. Following treatments with anti-PD-L1, docetaxel, or the combination anti-PD-L1 plus docetaxel, tumors were collected 4 days after the last treatment to evaluate early changes in the TME. Analyses included the characterization of tumor immune infiltrates via flow cytometry, RNA expression profiling via RT-PCR, RNA in situ hybridization, and immunohistochemistry for detection of immune infiltrates, cancer-associated fibroblasts, and phospho-Smad2 signaling. RNA analysis of 4T1 and LLC tumors revealed a significant upregulation of several transforming growth factor-beta (TGF-beta) downstream target genes in the anti-PD-L1 monotherapy group. This increase was associated with enhanced expression of TGF-beta in the TME, as determined by RNA in situ hybridization, together with enhanced TGF-beta signaling denoted by increased expression of phospho-Smad2. Tumors treated with anti-PD-L1 monotherapy also exhibited increased numbers of alpha-smooth muscle actin positive fibroblasts in the TME. The effects observed with anti-PD-L1 as a monotherapy were not observed in tumors treated with docetaxel monotherapy or in those treated with the combination docetaxel plus anti-PD-L1 therapy, suggesting that upregulation of immunosuppressive TGF-beta signaling in the TME could be prevented by the addition of chemotherapy. Ongoing and future studies are aimed at understanding the mechanisms involved in the upregulation of TGF-beta signaling in the TME in response to anti-PD-L1 therapy in non-responsive tumors, the effect of various chemotherapies in this phenomenon, and the study of additional tumor models for optimization of chemotherapy-ICB modalities. Citation Format: Masafumi Iida, Lucas A Horn, Haiyan Qin, Kristen Fousek, Jeffrey Schlom, Claudia Palena. Chemotherapy reduces TGF-beta signaling triggered by blockade of the PD-1/PD-L1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6100.

Simple Summary Innovative strategies to reduce immune suppression and activate tumor-specific immunity are needed to help patients who do not respond or become resistant to immune checkpoint blockade therapies. In this study, we demonstrate that the addition of a cancer vaccine targeting a tumor-associated antigen to a checkpoint inhibitor-based immunotherapy induces greater numbers of proliferative, activated, and cytotoxic tumor-infiltrating T cells, leading to improved antitumor activity in tumors otherwise resistant to immunotherapy. Our results provide the rationale for the addition of cancer vaccines in combination immunotherapy approaches being evaluated in the clinic. Abstract Resistance to immune checkpoint blockade therapy has spurred the development of novel combinations of drugs tailored to specific cancer types, including non-inflamed tumors with low T-cell infiltration. Cancer vaccines can potentially be utilized as part of these combination immunotherapies to enhance antitumor efficacy through the expansion of tumor-reactive T cells. Utilizing murine models of colon and mammary carcinoma, here we investigated the effect of adding a recombinant adenovirus-based vaccine targeting tumor-associated antigens with an IL-15 super agonist adjuvant to a multimodal regimen consisting of a bifunctional anti-PD-L1/TGF-βRII agent along with a CXCR1/2 inhibitor. We demonstrate that the addition of vaccine induced a greater tumor infiltration with T cells highly positive for markers of proliferation and cytotoxicity. In addition to this enhancement of cytotoxic T cells, combination therapy showed a restructured tumor microenvironment with reduced Tregs and CD11b+Ly6G+ myeloid cells. Tumor-infiltrating immune cells exhibited an upregulation of gene signatures characteristic of a Th1 response and presented with a more diverse T-cell receptor (TCR) repertoire. These results provide the rationale for the addition of vaccine-to-immune checkpoint blockade-based therapies being tested in the clinic.

Background LAIR-1 is an immune inhibitory receptor expressed on several immune cell types including activated T cells, B cells, NK cells, macrophages, and dendritic cells. The ligands for LAIR-1 contain collagen-like domains which are commonly found in extracellular matrix collagens and complement component C1q. In numerous cancer types, including gastric, colon, ovarian, bladder, and others, upregulation of collagens has been shown to enhance tumor growth, metastases, and invasion while actively suppressing antitumor immunity. Although humans produce a natural, soluble decoy, LAIR-2, that competes with LAIR-1 for binding of collagen domains, excess LAIR ligands in the tumor often result in an immune suppressive environment. Methods Here, we report on a novel immunotherapy approach which combined NC410, a LAIR-2-Fc fusion protein capable of blocking LAIR-1 signaling, and bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β ‘trap’) fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking programmed death ligand 1 (anti–PD-L1). Results We demonstrate that the combination of NC410 and bintrafusp alfa more effectively controls in vivo tumor growth of the collagen rich MC38 colon carcinoma compared to either monotherapy. We hypothesize that this potent anti-tumor immune response is propagated through the synergy of activated tumor infiltrating lymphocytes and a repolarization of macrophages towards a tumoricidal phenotype. MC38 tumors treated with the combination of NC410/Bintrafusp alfa contained higher numbers of infiltrating CD4+ and CD8+ T cells and higher numbers of CD38+ and MHCII+ M1 polarized macrophages. Conclusions This study highlights the synergy of reshaping the large suppressive myeloid cell populations often present in tumors with activation of adaptive T-cell immune responses dampened by checkpoint inhibition. The results also provide the rationale for the future evaluation of this combination therapy in the clinic. Acknowledgements Bintrafusp alfa was kindly provided by EMD Serono under a CRADA with the NCI. Trial Registration N/A Ethics Approval Mice were maintained under pathogen-free conditions in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines. All animal studies were approved by the NIH Intramural Animal Care and Use Committee under protocol LTIB-038. Consent N/A

BackgroundDespite the success of immune checkpoint blockade therapy in the treatment of certain cancer types, only a small percentage of patients with solid malignancies achieve a durable response. Consequently, there is a need to develop novel approaches that could overcome mechanisms of tumor resistance to checkpoint inhibition. Emerging evidence has implicated the phenomenon of cancer plasticity or acquisition of mesenchymal features by epithelial tumor cells, as an immune resistance mechanism.MethodsTwo soluble factors that mediate tumor cell plasticity in the context of epithelial-mesenchymal transition are interleukin 8 (IL-8) and transforming growth factor beta (TGF-β). In an attempt to overcome escape mechanisms mediated by these cytokines, here we investigated the use of a small molecule inhibitor of the IL-8 receptors CXCR1/2, and a bifunctional agent that simultaneously blocks programmed death ligand 1 (PD-L1) and traps soluble TGF-β.ResultsWe demonstrate that simultaneous inhibition of CXCR1/2, TGF-β, and PD-L1 signaling synergizes to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells, significantly enhancing T-cell infiltration and activation in tumors, and leading to improved antitumor activity.ConclusionsThis study highlights the potential benefit of combined blockade of CXCR1/2 and TGF-β signaling for modulation of tumor plasticity and potential enhancement of tumor responses to PD-L1 blockade. The data provide rationale for the evaluation of this novel approach in the clinic.

The lack of serial biopsies in patients with a range of carcinomas has been one obstacle in our understanding of the mechanism of action of immuno-oncology agents as well as the elucidation of mechanisms of resistance to these novel therapeutics. While much information can be obtained from studies conducted with syngeneic mouse models, these models have limitations, including that both tumor and immune cells being targeted are murine and that many of the immuno-oncology agents being evaluated are human proteins, and thus multiple administrations are hampered by host xenogeneic responses. Some of these limitations are being overcome by the use of humanized mouse models where human peripheral blood mononuclear cells (PBMC) are engrafted into immunosuppressed mouse strains. Bintrafusp alfa (M7824) is an innovative first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII to function as a TGF-β “trap” fused to a human IgG1 antibody blocking PD-L1. A phase I clinical trial of bintrafusp alfa showed promising anti-tumor efficacy in heavily pretreated advanced solid tumors, and multiple clinical studies are currently ongoing. There is still much to learn regarding the mechanism of action of bintrafusp alfa, including its effects on both human immune cells in the periphery and in the tumor microenvironment (TME), and any temporal effects upon multiple administrations. By using the NSG-β2m−/− mouse strain humanized with PBMC, we demonstrate here for the first time: (a) the effects of bintrafusp alfa administration on human immune cells in the periphery vs. the TME using three different human xenograft models; (b) temporal effects upon multiple administrations of bintrafusp alfa; (c) phenotypic changes induced in the TME, and (d) variations observed in the use of multiple different PBMC donors. Also discussed are the similarities and differences in the data thus far obtained employing murine syngeneic models, from clinical trials, and in the use of this humanized mouse model. The results described here may guide the future use of this agent or similar immunotherapy agents as monotherapies or in combination therapy studies.

Immunotherapy has become an essential component of cancer treatment, however, a majority of patients with solid metastatic cancers, such as pheochromocytoma (PHEO), do not respond to this type of therapy. Recently, we developed an intratumoral (i.t.) immunotherapy based on the unique combination of TLR ligands, anti-CD40 antibodies, and mannan, which is artificially bound to tumor cells via an anchor (MBTA therapy). This therapy resulted in the complete eradication of aggressive subcutaneous PHEO in 67% of mice and demonstrated a systemic antitumor immune response and regression of non-treated lesions in the metastatic model (1). To further evaluate this systemic effect generated during MBTA therapy, we established a murine bilateral PHEO model, where MBTA therapy was i.t. injected into one tumor, and the distant (non-treated) tumor was monitored for changes in size and immune cell infiltration. The growth of both MBTA-treated and distant tumors was reduced compared to that of the control. Interestingly, survival of the MBTA-treated mice was twice as long compared to the control mice. Moreover, we have made several unique observations during the experiments which were focused on the tumor microenvironment. Flow cytometry analysis revealed the ability of MBTA therapy to significantly increase the infiltration of innate immune cells (monocytes, DCs, macrophages, NK cells) not only in MBTA-treated tumors, but also in distal tumors, despite the fact that MBTA therapy was designed to elicit only local inflammation. An analysis of the macrophage phenotype revealed a switch from protumor M2 to antitumor M1 macrophages in both tumors during the entire MBTA therapy treatment. Analysis of splenic adaptive immune cells revealed that naïve CD4+ or CD8+ T cells differentiated into central memory cells and effector memory cells. CD4+ and CD8+ T cells were elevated in MBTA-treated and distant tumors with a significantly higher frequency of CD8+ effector memory T cells. Moreover, the adoptive transfer of CD4+ and CD8+ T cells revealed that immune memory, after tumor rechallenging, was driven by CD4+ T cells. Collectively, these results illustrate the ability of MBTA therapy to activate both parts of the immune system and render a systemic antitumor response against non-treated metastases. We believe that our results could lead to the use of MBTA therapy in patients with aggressive, metastatic lesions. Reference: Caisova et al., Cancers (Basel), 2019. 11(5).

BackgroundLeukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immune inhibitory receptor that binds collagen-like domains commonly found in extracellular matrix (ECM) collagens and complement component C1q. LAIR-1 is expressed on several immune cell types including activated T cells, B cells, NK cells, dendritic cells, and macrophages. Numerous cancer types including gastric, colon, ovarian, bladder, and others, upregulate collagens which enhances tumor growth, metastases, and invasion while actively suppressing antitumor immunity. While a soluble decoy, LAIR-2, is expressed in humans and competes with LAIR-1 for binding of collagen domains, excess LAIR ligands in the tumor often result in an immune suppressive environment.MethodsHere, we report on a novel immunotherapy approach combining NC410, a novel fusion protein consisting of two LAIR-2 molecules grafted on to an IgG1 antibody backbone, capable of targeting the tumor ECM and blocking LAIR-1 signaling; and bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the human transforming growth factor β receptor II (TGF-βRII or TGF-β ”trap”) fused via a flexible linker to the C-terminus of each heavy chain of an IgG1 antibody blocking programmed death ligand 1 (anti–PD-L1).ResultsWe have demonstrated that the combination of NC410 and bintrafusp alfa more effectively controls in vivo tumor growth of the collagen rich MC38 colon and EMT6 mammary carcinomas compared to either monotherapy. We demonstrate that this potent anti-tumor immune response is propagated through the synergy of activated tumor infiltrating lymphocytes and a repolarization of myeloid cells in the tumor microenvironment. MC38 tumors treated with the combination of NC410 plus bintrafusp alfa contained higher numbers of infiltrating T cells, NK cells, and M1 polarized macrophages.ConclusionsThis study highlights the synergy of reshaping the large suppressive myeloid cell populations often present in tumors with activation of adaptive T-cell immune responses dampened by checkpoint inhibition. The results also provide the rationale for the future evaluation of this combination therapy in the clinic.

// Lucas A. Horn 1,* , Nicholas G. Ciavattone 2,* , Ryan Atkinson 1 , Netsanet Woldergerima 1 , Julia Wolf 1 , Virginia K. Clements 1 , Pratima Sinha 1 , Munanchu Poudel 1 and Suzanne Ostrand-Rosenberg 1 1 Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA 2 Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland, Baltimore, MD, USA * These authors contributed equally to this project Correspondence to: Suzanne Ostrand-Rosenberg, email: // Keywords : tumor-induced immune suppression, checkpoint inhibitor blockade, T cell activation, solid tumors, cancer immunotherapy Received : April 21, 2017 Accepted : July 26, 2017 Published : August 03, 2017 Abstract Bi-specific T cell engagers (BiTEs) activate T cells through CD3 and target activated T cells to tumor-expressed antigens. BiTEs have shown therapeutic efficacy in patients with liquid tumors; however, they do not benefit all patients. Anti-tumor immunity is limited by Programmed Death 1 (PD1) pathway-mediated immune suppression, and patients who do not benefit from existing BiTES may be non-responders because their T cells are anergized via the PD1 pathway. We have designed a BiTE that activates and targets both T cells and NKT cells to PDL1 + cells. In vitro studies demonstrate that the CD3xPDL1 BiTE simultaneously binds to both CD3 and PDL1, and activates healthy donor CD4 + and CD8 + T cells and NKT cells that are specifically cytotoxic for PDL1 + tumor cells. Cancer patients’ PBMC are also activated and cytotoxic, despite the presence of myeloid-derived suppressor cells. The CD3xPDL1 BiTE significantly extends the survival time and maintains activated immune cell levels in humanized NSG mice reconstituted with human PBMC and carrying established human melanoma tumors. These studies suggest that the CD3xPDL1 BiTE may be efficacious for patients with PDL1 + solid tumors, in combination with other immunotherapies that do not specifically neutralize PD1 pathway-mediated immune suppression.

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they are significant contributors to the immune suppressive tumor microenvironment (TME). The TME is a hostile locale due to deficiencies in oxygen (hypoxia) and nutrients, and the presence of reactive oxygen species (ROS). The survival of tumor cells within the TME is partially governed by two mechanisms: (i) Activation of the transcription factor Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) which turns on genes that attenuate oxidative stress; and (ii) The presence of High Mobility Group Box Protein-1 (HMGB1), a damage-associated molecular pattern molecule (DAMP) that induces autophagy and protects against apoptosis. Because Nrf2 and HMGB1 promote tumor cell survival, we speculated that Nrf2 and HMGB1 may facilitate MDSC survival. We tested this hypothesis using Nrf2(+/+) and Nrf2(−/−) BALB/c and C57BL/6 mice and pharmacological inhibitors of HMGB1. In vitro and in vivo studies demonstrated that Nrf2 increased the suppressive potency and quantity of tumor-infiltrating MDSC by up-regulating MDSC production of H(2)O(2) and decreasing MDSC apoptosis. Decreased apoptosis was accompanied by a decrease in the production of MDSC, demonstrating that MDSC levels are homeostatically regulated. Pharmacological inhibition of autophagy increased MDSC apoptosis indicating that autophagy increases MDSC half-life. Inhibition of HMGB1 also increased MDSC apoptosis and reduced MDSC autophagy. These results combined with our previous findings that HMGB1 drives the accumulation of MDSC, demonstrate that HMGB1 maintains MDSC viability by inducing autophagy. Collectively, these findings identify Nrf2 and HMGB1 as important factors that enable MDSC to survive in the TME.

Recruitment of myeloid-derived suppressor cells (MDSCs) into tumors induces local immunosuppression in carcinomas. Here, we assessed whether SX-682, an orally bioavailable small-molecule inhibitor of CXCR1 and CXCR2, could block tumor MDSC recruitment and enhance T cell activation and antitumor immunity following multiple forms of immunotherapy. CXCR2(+) neutrophilic MDSCs (PMN-MDSCs) were the most abundant myeloid cell subset within oral and lung syngeneic carcinomas. PMN-MDSCs demonstrated greater suppression of tumor-infiltrating lymphocyte killing of targets compared with macrophages. SX-682 significantly inhibited trafficking of PMN-MDSCs without altering CXCR2 ligand expression. Trafficking of CXCR1(+) macrophages was unaltered, possibly due to coexpression of CSF1R. Reduced PMN-MDSC tumor infiltration correlated with enhanced accumulation of endogenous or adoptively transferred T cells. Accordingly, tumor growth inhibition or the rate of established tumor rejection following programed death–axis (PD-axis) immune checkpoint blockade or adoptive cell transfer of engineered T cells was enhanced in combination with SX-682. Despite CXCR1/2 expression on tumor cells, SX-682 appeared to have little direct antitumor effect on these carcinoma models. These data suggest that tumor-infiltrating CXCR2(+) PMN-MDSCs may prevent optimal responses following both PD-axis immune checkpoint blockade and adoptive T cell transfer therapy. Abrogation of PMN-MDSC trafficking with SX-682 enhances T cell–based immunotherapeutic efficacy and may be of benefit to patients with MDSC-infiltrated cancers.

Tumor progression relies on the ability of cancer cells to effectively invade surrounding tissues and propagate. Among the many mechanisms that contribute to tumor progression is the epithelial-to-mesenchymal transition (EMT), a phenotypic plasticity phenomenon that increases the cancer cells' motility and invasiveness and influences their surrounding microenvironment by promoting the secretion of a variety of soluble factors. One such factor is IL-8, a chemokine with multiple pro-tumorigenic roles within the tumor microenvironment (TME), including stimulating proliferation or transformation of tumor cells into a migratory or mesenchymal phenotype. Further, IL-8 can increase tumor angiogenesis or recruit larger numbers of immunosuppressive cells to the tumor. Prognostically, observations in many tumor types show that patients with higher levels of IL-8 at baseline experience worse clinical outcomes. Additionally, studies have shown that the chemokine directly contributes to the development of resistance to both chemotherapy and molecularly targeted agents. More recently, clinical studies evaluating levels of IL-8 in patients receiving immune checkpoint inhibition (ICI) therapy deduced that myeloid tumor infiltration driven by IL-8 contributes to resistance to ICI agents and that peripheral IL-8 can predict outcomes to ICI therapy. Further, pre-clinical data demonstrate that targeting IL-8 or its receptors enables improved tumor killing by immune cells, and treatment strategies combining blockade of the IL-8/IL-8R axis with ICI ultimately improve anti-tumor efficacy. Based on these results and the prognostic capacity of IL-8, there are a number of ongoing clinical trials evaluating the addition of IL-8 targeting strategies to immune-based therapies.

Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations (CNA) are found in most aggressive primary human prostate cancers, and the overall number of such changes is increased in metastases. Chromosome 10q23 deletions, encompassing PTEN, and amplification of 8q24, harboring MYC, are frequently observed, and the presence of both together portends a high risk of prostate cancer-specific mortality. In extant genetically engineered mouse prostate cancer models (GEMM), isolated MYC overexpression or targeted Pten loss can each produce early prostate adenocarcinomas, but are not sufficient to induce genetic instability or metastases with high penetrance. Although a previous study showed that combining Pten loss with focal MYC overexpression in a small fraction of prostatic epithelial cells exhibits cooperativity in GEMMs, additional targeted Tp53 disruption was required for formation of metastases. We hypothesized that driving combined MYC overexpression and Pten loss using recently characterized Hoxb13 transcriptional control elements that are active in prostate luminal epithelial cells would induce the development of genomic instability and aggressive disease with metastatic potential. Neoplastic lesions that developed with either MYC activation alone (Hoxb13-MYC) or Pten loss alone (Hoxb13-Cre∣PtenFl/Fl) failed to progress beyond prostatic intraepithelial neoplasia and did not harbor genomic CNAs. By contrast, mice with both alterations (Hoxb13-MYC∣Hoxb13-Cre∣PtenFl/Fl, hereafter, BMPC mice) developed lethal adenocarcinoma with distant metastases and widespread genome CNAs that were independent of forced disruption of Tp53 and telomere shortening. BMPC cancers lacked neuroendocrine or sarcomatoid differentiation, features uncommon in human disease but common in other models of prostate cancer that metastasize. These data show that combined MYC activation and Pten loss driven by the Hoxb13 regulatory locus synergize to induce genomic instability and aggressive prostate cancer that phenocopies the human disease at the histologic and genomic levels. Cancer Res; 76(2); 283–92. ©2015 AACR.

Considerando a constante evolução do futebol, o surgimento dos gestores de clubes de futebol e o tema da profissionalização da arbitragem como pontos centrais, e partindo do pressuposto de que a imagem dos árbitros de futebol sempre foi alvo de desconfiança e suspeição no que concerne as suas condutas, este estudo tem como objetivo verificar se a profissionalização da arbitragem poderia influenciar na imagem dos árbitros na ótica dos profissionais ligados aos clubes de futebol no RS. Evidenciou-se que o preconceito vinculado à imagem dos árbitros parece não estar relacionado aos indivíduos que exercem a atividade, mas na atividade exercida por eles, ou seja, ao estereótipo vinculado aos árbitros de futebol. ABSTRACT Referee’s professionalization and its influence on referee’s image: a study through the view of professionals involved to the football management in Rio Grande do Sul Given the constant evolution of football, the emergence of managers in football clubs and the issue of referee’s professionalization as main points, and assuming that the image of football referees has always been the target of distrust and suspicious regarding their conducts, this study aims to check if the referee’s professionalization could influence on the referee’s image in the view of professionals involved to the football management in Rio Grande do Sul. It was evident that the prejudice linked to the referee’s image seems not to be related to the individuals performing activity, but the activity exerted by them, more specifically, to the stereotype linked to Football referees.

Tumor cells utilize the secretion of proinflammatory cytokines and chemokines and the surface expression of inhibitory ligands to promote an immune-suppressive tumor microenvironment that facilitates tumor growth and metastasis. The overexpression of IL-8, TGF-β, PD-L1, the IL-8 receptors CXCR1 and CXCR2, as well as numerous other factors by various solid and hematologic cancers are major obstacles in the promotion of a strong and effective antitumor immune response. Both IL-8 and TGF-β have been identified as key potentiators of tumor cell plasticity and the acquisition of mesenchymal features by epithelial cancer cells, i.e., mesenchymalization, which increases metastatic dissemination. Both IL-8 and TGF-β can also promote tumor angiogenesis, myeloid-derived suppressor cell (MDSC) function in the tumor microenvironment, and resistance to immune-mediated killing. Additionally, tumor cell expression of PD-L1 inhibits effective T-cell responses at the tumor site. Here we report the use of a systematic and rational multimodal cancer immunotherapy approach that combines induction of T cell potentiation through an IL-15 super agonist and adenoviral cancer vaccine with inhibition of three immunosuppressive pathways through blockade of IL-8, TGF-β, and PD-L1. Combination of the novel small-molecule inhibitor SX-682 that irreversibly binds and blocks both CXCR1 and CXCR2 with an adenoviral-based cancer vaccine, N-IL15 (previously designated ALT-803), and the anti-PD-L1/TGFβRII agent, M7824, significantly delayed tumor growth in murine models of breast and colon cancer. Mechanistic studies demonstrated that this multimodal combination therapy was able to markedly increase tumor infiltration with CD4+ and CD8+ T cells, including central memory and effector memory subsets, and to decrease infiltration with Ly6G+/Ly6C- MDSC. Studies with SX-682 as a monotherapy suggest that CXCR1/2 inhibition reduces migration rates of Ly6G+/Ly6C- MDSC (CXCR2+). Studies with models of claudin-low, mesenchymal-enriched, human triple-negative breast cancer demonstrated that CXCR1/2 inhibition was also effective at promoting a switch from a mesenchymal to an epithelial tumor phenotype, while simultaneously enhancing the tumor cells’ susceptibility to immune-mediated cytotoxicity. Our results provide rationale for the future evaluation of this multimodal therapy in the clinic. Citation Format: Lucas Horn, Duane Hamilton, Dean Maeda, John Zebala, John Lee, Shahrooz Rabizadeh, Frank Jones, Patrick Soon-Shiong, Zhen Su, Jeffrey Schlom, Claudia Palena. Multimodal cancer immunotherapy combining IL-8 inhibition, adenovirus vaccine, IL-15 super agonist, and anti-PD-L1/TGFβRII agent reduces mesenchymalization and enhances anti-tumor efficacy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B04.

The occurrence of phenotypic plasticity in carcinomas, which results in the acquisition of mesenchymal features by epithelial cancer cells, associates with a poor prognosis and has been described as a mechanism of primary resistance and failure to treatment with checkpoint blockade immunotherapy targeting the PD-1/PD-L1 pathway. IL-8 and TGF-β are two of the factors that mediate phenotypic plasticity in epithelial tumors to promote an immune-suppressive microenvironment. Here we report on an immunotherapy approach aimed at overcoming escape mechanisms mediated by IL-8 and TGF-β accumulation in the tumor microenvironment via the use of two clinical-stage agents: SX-682, a dual CXCR1/2 small-molecule inhibitor, with the bifunctional anti-PD-L1/TGFβRII agent, bintrafusp alfa (M7824) that simultaneously blocks PD-L1 while trapping soluble TGF-β. We demonstrate that the concurrent inhibitions of CXCR1/CXCR2, TGF-β, and PD-L1 signaling synergize to reduce mesenchymal tumor features in murine models of breast and lung cancer, and to markedly increase expression of tumor epithelial E-cadherin while reducing infiltration with suppressive granulocytic myeloid-derived suppressor cells (G-MDSC), significantly enhancing CD4+ and CD8+ T-cell infiltration and activation in tumors, and leading to improved antitumor activity. This combination approach was effective in tumor models with varying immune cell infiltrates, degrees of plasticity, and responses to immunotherapy. The results of these studies provide the rationale for the future evaluation of this multimodal therapy in the clinic. Citation Format: Lucas A. Horn, Jeffrey Riskin, Heidi A. Hempel, Hanne Lind, Kristen Fousek, Duane H. Hamilton, Kristen K. McCampbell, Jeffrey Schlom, Claudia Palena. Simultaneous inhibition of CXCR1/CXCR2, TGF-β, and PD-L1 overcomes immune resistance driven by tumor plasticity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B43.

Cancer immunotherapies aimed at neutralizing the programmed death-1 (PD-1) immune suppressive pathway have yielded significant therapeutic efficacy in a subset of cancer patients. However, only a subset of patients responds to antibody therapy with either anti-PD-1 or anti-PD-L1 antibodies. These patients appear to have so-called “hot” tumors containing tumor-reactive T cells. Therefore, checkpoint blockade therapy may be effective in a larger percentage of cancer patients if combined with therapeutics that also activate tumor-reactive T cells. Radiotherapy (RT) is a prime candidate for combination therapy because it facilitates activation of both local antitumor immunity and antitumor immunity at non-radiated, distant sites (abscopal response). However, RT also promotes tumor cell expression of PD-L1 and facilitates the development of myeloid-derived suppressor cells (MDSC), a population of immune suppressive cells that also suppress through PD-L1. This article will review how RT induces MDSC, and then describe two novel therapeutics that are designed to simultaneously activate tumor-reactive T cells and neutralize PD-1-mediated immune suppression. One therapeutic, a CD3xPD-L1 bispecific T cell engager (BiTE), activates and targets cytotoxic T and NKT cells to kill PD-L1+ tumor cells, despite the presence of MDSC. The BiTE significantly extends the survival time of humanized NSG mice reconstituted with human PBMC and carrying established metastatic human melanoma tumors. The second therapeutic is a soluble form of the costimulatory molecule CD80 (sCD80). In addition to costimulating through CD28, sCD80 inhibits PD-1 suppression by binding to PD-L1 and sterically blocking PD-L1/PD-1 signaling. sCD80 increases tumor-infiltrating T cells and significantly extends survival time of mice carrying established, syngeneic tumors. sCD80 does not suppress T cell function via CTLA-4. These studies suggest that the CD3xPD-L1 BiTE and sCD80 may be efficacious therapeutics either as monotherapies or in combination with other therapies such as radiation therapy for the treatment of cancer.

Multiple cytokines, chemokines, and growth factors play critical roles as mediators of paracrine signals between the tumor and cellular components of the tumor microenvironment (TME), ultimately leading to tumor growth, survival, and progression. One of the soluble factors that has a multifaceted role in cancer progression is the chemokine IL-8 (CXCL8), which signals through the receptors CXCR1 and CXCR2.Abnormally high expression of IL-8 is found in multiple cancer types, including in triple negative breast, colorectal, lung, and head and neck tumors, among others. IL-8 can foster tumor progression by various means, including (a) promoting the trafficking of immune-suppressive cells to the tumor microenvironment, mainly granulocytic myeloid-suppressor cells (G-MDSCs), which exert a suppressive role on anti-tumor immunity; (b) promoting phenotypic plasticity in carcinoma cells via changes from an epithelial into a mesenchymal-like state; and (c) increasing the survival of cancer stem-like cell populations in the tumor. Tumor phenotypic plasticity has been linked to advanced tumor stage, presence of metastases, poor prognosis in numerous cancer types, and tumor resistance to anti-cancer therapies, including chemotherapy and small molecule-targeted therapies. Recent work has also shown that upregulation of IL-8 and enhanced mesenchymal tumor features are enriched in tumors that are resistant to checkpoint inhibitor therapy. In agreement, our laboratory has demonstrated a role for IL-8 expression in tumor resistance to lysis by immune effector cells. The multifaceted roles of IL-8 in cancer make it a promising therapeutic target. Blockade of IL-8 signaling may be achieved by direct neutralization of IL-8 via a monoclonal antibody or via inhibition of its receptors. Our laboratory has now demonstrated a role for IL-8 in the context of acquired resistance to EGFR inhibition and in the context of acquired resistance to chemotherapies in preclinical models of lung cancer. Alleviation of IL-8 signaling via a neutralizing antibody was shown to reduce mesenchymal features of the resistant cells, and markedly enhanced their sensitivity to therapies. Our group has also shown that neutralization of IL-8 via a monoclonal anti-IL8 antibody is able to revert mesenchymal features in claudin-low, triple negative tumor models, resulting in enhanced immune-mediated lysis with NK and antigen-specific T cells in vitro and reduced tumor infiltration with G-MDSC and improved responses to chemotherapy in vivo. Current work in our laboratory is evaluating inhibition of the CXCR1/2 receptors via the use of the small molecule inhibitor SX-682, as an alternative approach to abrogate signaling mediated not only by IL-8 but also by all other chemokines of the same family that bind to these receptors. In addition to IL-8, the pleiotropic cytokine TGF-β is a driver of phenotypic plasticity and cancer progression and has been implicated as a driver of T-cell exclusion from tumors. Our current experiments are exploring the hypothesis that by reducing mesenchymal features in the tumor via inhibition of IL-8 and/or TGF-β signaling we could enhance the anti-tumor efficacy of the PD-1/PD-L1 axis blockade. Citation Format: Lucas A Horn, Hanne Lind, Heidi Hempel, Kristen Fousek, Claudia Palena. IL-8 in tumor progression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN08-01. doi:10.1158/1535-7163.TARG-19-CN08-01

Tumor cells use various immune-suppressive strategies to overcome antitumor immunity. One such method is tumor expression of programmed death ligand-1 (PD-L1), which triggers apoptotic death or anergy upon binding programmed death-1 (PD-1) on T cells. Our previous in vitro cellular studies with human and mouse PD-L1+ tumor cells demonstrated that a soluble form of the costimulatory molecule CD80 prevented PD-L1–mediated immune suppression and restored T-cell activation by binding PD-L1 and blocking interaction with PD-1. We now report that in vivo treatment of established syngeneic PD-L1+ CT26 colon carcinoma and B16F10 melanoma tumors with CD80-Fc delays tumor growth and promotes tumor-infiltrating T cells. Studies with PD-1−/− and CD28−/− mice demonstrate that soluble CD80 acts in vivo by simultaneously neutralizing PD-1 suppression and activating through CD28. We also report that soluble CD80 mediates its effects by activating transcription factors EGR1-4, NF-κB, and MAPK, downstream signaling components of the CD28 and T-cell receptor pathways. Soluble CD80 binds to CTLA-4 on activated human peripheral blood mononuclear cells. However, increasing quantities of CTLA-4 antagonist antibodies do not increase T-cell activation. These results indicate that soluble CD80 does not suppress T-cell function through CTLA-4 and suggest that CTLA-4 acts as a decoy receptor for CD80, rather than functioning as a suppressive signaling receptor. Collectively, these studies demonstrate that soluble CD80 has therapeutic efficacy in vivo in mouse tumor systems and that its effects are due to its ability to inhibit PD-1–mediated suppression while concurrently activating T cells through CD28. Cancer Immunol Res; 6(1); 59–68. ©2017 AACR.

Programmed death ligand 1 (PD-L1, also known as B7 homolog 1 or CD274) is a major obstacle to antitumor immunity because it tolerizes/anergizes tumor-reactive T cells by binding to its receptor programmed death-1 (CD279), renders tumor cells resistant to CD8+ T cell– and FasL-mediated lysis, and tolerizes T cells by reverse signaling through T cell–expressed CD80. PD-L1 is abundant in the tumor microenvironment, where it is expressed by many malignant cells, as well as by immune cells and vascular endothelial cells. The critical role of PD-L1 in obstructing antitumor immunity has been demonstrated in multiple animal models and in recent clinical trials. This article reviews the mechanisms by which PD-L1 impairs antitumor immunity and discusses established and experimental strategies for maintaining T cell activation in the presence of PD-L1–expressing cells in the tumor microenvironment.

Tumor cells employ various methods of immune suppression to overcome antitumor immunity. One such method is that of programmed death ligand-1 (PD-L1 or B7-H1), which upon binding its receptor programmed death-1 (PD-1) on T cells triggers apoptotic death of the activated T cells. Over-expression of the costimulatory molecule CD80 on PD-L1+ tumor cells, or inclusion of a soluble form of CD80 (CD80-Fc) maintains the activation of PD-1+ activated T cells. Using T cells from CD28-deficient mice and antibodies to block CD28 on human T cells, we now report that a soluble form of CD80 mediates this effect by simultaneously neutralizing the PD-1/PD-L1-mediated immune suppression and by providing CD80-CD28 costimulation. In summary, PD-L1 is a potent mediator of immune suppression that inhibits antitumor immunity in many cancer patients. In this study, we show that a soluble form of the costimulatory molecule CD80 increases the production of IFNγ by PD-1+ activated T cell more effectively than antibodies to PD-1 or PD-L1. Therefore, soluble CD80 may be a more effective therapeutic than these checkpoint antibodies for facilitating the development and maintenance of antitumor immunity because it has the dual functions of preventing PD-L1-mediated immune suppression and simultaneously delivering the second signal for T-cell activation.

Most multicellular organisms show a physiological decline in immune function with age. However, little is known about the mechanisms underlying these changes. We examined Drosophila melanogaster, an important model for identifying genes affecting innate immunity and senescence, to explore the role of phagocytosis in age-related immune dysfunction. We characterized the localized response of immune cells at the dorsal vessel to bacterial infection in 1-week- and 5-week-old flies. We developed a quantitative phagocytosis assay for adult Drosophila and utilized this to characterize the effect of age on phagocytosis in transgenic and natural variant lines. We showed that genes necessary for bacterial engulfment in other contexts are also required in adult flies. We found that blood cells from young and old flies initially engulf bacteria equally well, while cells from older flies accumulate phagocytic vesicles and thus are less capable of destroying pathogens. Our results have broad implications for understanding how the breakdown in cellular processes influences immune function with age.

Myeloid-derived suppressor cells (MDSCs) are present in most cancer patients and experimental animals where they exert a profound immune suppression and are a significant obstacle to immunotherapy. IFN-γ and IL-4 receptor alpha (IL-4Rα) have been implicated as essential molecules for MDSC development and immunosuppressive function. If IFN-γ and IL-4Rα are critical regulators of MDSCs, then they are potential targets for preventing MDSC accumulation or inhibiting MDSC function. Because data supporting a role for IFN-γ and IL-4Rα are not definitive, we have examined MDSCs induced in IFN-γ-deficient, IFN-γR-deficient, and IL-4Rα-deficient mice carrying three C57BL/6-derived (B16 melanoma, MC38 colon carcinoma, and 3LL lung adenocarcinoma), and three BALB/c-derived (4T1 and TS/A mammary carcinomas, and CT26 colon carcinoma) tumors. We report that although MDSCs express functional IFN-γR and IL-4Rα, and have the potential to signal through the STAT1 and STAT6 pathways, respectively, neither IFN-γ nor IL-4Rα impacts the phenotype, accumulation, or T-cell suppressive potency of MDSCs, although IFN-γ and IL-4Rα modestly alter MDSC-macrophage IL-10 crosstalk. Therefore, neither IFN-γ nor IL-4Rα is a key regulator of MDSCs and targeting these molecules is unlikely to significantly alter MDSC accumulation or function.

Life history theory hypothesizes that genetically based variation in life history traits results from alleles that alter age-specific patterns of energy allocation among the competing demands of reproduction, storage, and maintenance. Despite the important role that alleles with age-specific effects must play in life history evolution, few naturally occurring alleles with age-specific effects on life history traits have been identified. A recent mapping study identified S6 kinase (S6k) as a candidate gene affecting lipid storage in Drosophila. S6k is in the target of rapamycin pathway, which regulates cell growth in response to nutrient availability and has also been implicated to influence many life history traits from fecundity to life span. In this article, we used quantitative complementation tests to examine the effect of allelic variation at S6k on a range of phenotypes associated with metabolism and fitness in an age-, diet-, and sex-specific manner. We found that alleles of S6k have pleiotropic effects on total protein levels, glycogen storage, life span, and the immune response and demonstrate that these allelic effects are age, diet, and sex specific. As many of the genes in the target of rapamycin pathway are evolutionarily conserved, our data suggest that genes in this pathway could play a pivotal role in life history evolution in a wide range of taxa.

Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by driving them into a proautophagic state.