Protective Tumor Immunity Research Articles

Exploring the immunomodulatory potential of Brahmi (Bacopa monnieri) in the treatment of invasive ductal carcinoma.

Bacopa monnieri (L) Wettst, commonly known as Brahmi, stands as a medicinal plant integral to India's traditional medical system, Ayurveda, where it is recognized as a "medhya rasayana"-a botanical entity believed to enhance intellect and mental clarity. Its significant role in numerous Ayurvedic formulations designed to address conditions such as anxiety, memory loss, impaired cognition, and diminished concentration underscores its prominence. Beyond its application in cognitive health, Brahmi has historically been employed in Ayurvedic practices for the treatment of inflammatory diseases, including arthritis. In contemporary biomedical research, Bacopa monnieri can attenuate the release of pro-inflammatory cytokines TNF-α and IL-6 in animal models. However, there remains a paucity of information regarding Bacopa's potential as an anticancer agent, warranting further investigation in this domain. Based on previous findings with Brahmi (Bacopa monnieri), the current study aims to find out the role of Brahmi plant preparation (BPP) in immunomodulatory actions on IDC. Employing a specific BPP concentration, we conducted a comprehensive study using MTT assay, ELISA, DNA methylation analysis, Western blotting, ChIP, and mRNA profiling to assess BPP's immunomodulatory properties. Our research finding showed the role of BPP in augmenting the action of T helper 1 (TH1) cells which secreted interferon-γ (IFN-γ) which in turn activated cytotoxic T-lymphocytes (CTL) to kill the cells of IDC (*p < 0.05). Moreover, we found out that treatment with BPP not only increased the activities of tumor-suppressor genes (p53 and BRCA1) but also decreased the activities of oncogenes (Notch1 and DNAPKcs) in IDC (*p < 0.05). BPP had an immense significance in controlling the epigenetic dysregulation in IDC through the downregulation of Histone demethylation & Histone deacetylation and upregulation of Histone methylation and Histone acetylation (*p < 0.05). Our Chromatin immunoprecipitation (ChIP)-qPCR data showed BPP treatment increased percentage enrichment of STAT1 & BRCA1 (*p < 0.05) and decreased percentage enrichment of STAT3, STAT5 & NF ΚB (*p < 0.05) on both TBX21 and BRCA1 gene loci in IDC. In addition, BPP treatment reduced the hypermethylation of the BRCA1-associated-DNA, which is believed to be a major factor in IDC (*p < 0.05). BPP not only escalates the secretion of type 1 specific cytokines but also escalates tumor suppression and harmonizes various epigenetic regulators and transcription factors associated with Signal Transducer and Activator of Transcription (STAT) to evoke tumor protective immunity in IDC.

Diospyros malabarica fruit preparation mediates immunotherapeutic modulation and epigenetic regulation to evoke protection against non–small cell lung cancer (NSCLC)

Ethnopharmacological relevanceDiospyros malabarica is an ethnomedicinal plant with hypoglycaemic, anti-bacterial, and anti-cancer properties and it belongs to the Ebenaceae family which is well known for its medicinal uses since ancient times and application of its bark and unripened fruit has been significantly mentioned in Ayurvedic texts. The Diospyros malabarica species which is known as the Gaub in Hindi and Indian Persimmon in English is native to India, however, it is distributed throughout the tropics. Aim of the studyAs Diospyros malabarica fruit preparation (DFP) possesses medicinal values, the study aims to evaluate its role as natural, non-toxic, and cost-effective dendritic cells (DCs) maturing immunomodulatory agent and also as an epigenetic regulator to combat Non–small cell lung cancer (NSCLC) which is a type of lung cancer whose treatment options such as chemotherapy, radiation therapy, etc. are accompanied with some adverse side effects. Thus, immunotherapeutic strategies are in high demand to evoke tumor protective immunity against NSCLC without causing such side effects. Materials and methodsPeripheral Mononuclear Cells (PBMCs) derived monocytes of normal subjects and NSCLC patients were utilized to generate DCs matured with either LPS (LPSDC) or DFP (DFPDC). Mixed Lymphocyte Reaction (MLR) was carried out with the differentially matured DCs co-culturing T cells and cytotoxicity of lung cancer cells (A549) was measured through LDH release assay and cytokine profiling was carried out via ELISA respectively. PBMCs of normal subjects and NSCLC patients have transfected separately in vitrowith CRISPR-activation plasmid of p53 and CRISPR-Cas9 knockout plasmid of c-Myc to analyze epigenetic mechanism(s) in the presence and absence of DFP. ResultsDiospyros malabarica fruit preparation (DFP) treated DC upregulates the secretion of T helper (TH)1 cell specific cytokines (IFN-γ and IL-12) and signal transducer and activator of transcription molecules (STAT1 and STAT4). Furthermore, it also downregulates the secretion of TH2-specific cytokines (IL-4 and IL-10). Diospyros malabarica fruit preparation (DFP) enhances p53 expression by reducing methylation levels at the CpG island of the promoter region. Upon c-Myc knockout, epigenetic markers such as H3K4Me3, p53, H3K14Ac, BRCA1, and WASp were enhanced whereas H3K27Me3, JMJD3, and NOTCH1 were downregulated. ConclusionDiospyros malabarica fruit preparation (DFP) not only increases the expression of type 1 specific cytokines but also augments tumor suppression modulating various epigenetic markers to evoke tumor protective immunity without any toxic activities.

Neoantigen Synthetic Peptide Vaccine for Multiple Myeloma Elicits T Cell Immunity in a Pre-Clinical Model

Cancer immunotherapies such as immune checkpoint inhibitors (CI) are approved for a broad range of solid tumors and selected hematologic malignancies. Clinical response to CIs has been shown to be associated with mutation burden and is predicated on the presence of T cells specific to tumor-associated antigens. Therefore, increasing the frequency of such T cell responses should increase the efficacy of anti-tumor immunity. Individualized neoantigen peptide vaccines are a promising strategy to activate tumor-specific T cell immunity. Next generation sequencing of tumor specimens is now widely available and novel computational algorithms provide the means for the prediction and selection of neoantigens.We are conducting a preclinical study of a personalized vaccine in an animal model of multiple myeloma (MM). MM is an incurable cancer of plasma cells that resides in the bone marrow (BM) and causes lytic bone disease and other end-organ damage. MM is characterized by an intermediate level of somatic mutation that may yield mutational neoantigens for vaccine design. This investigation pursued two goals: first, to determine if neoantigens are immunogenic, and second, if they induce protective immunity against tumors. To address the immunogenicity of neoantigens, we used the MOPC.315.BM myeloma cell line in the immunocompetent BALB/c mouse background. MOPC.315.BM cells mimic human disease by homing to the BM and causing osteolytic lesions. We performed whole exome sequencing and RNAseq on MOPC.315.BM cells to identify expressed somatic mutations. Neoantigens were then selected based on predicted MHC class I and II binding and expression levels, and corresponding 27-mer peptides were synthesized and tested in vivo for their immunogenicity. Mice were immunized 3 times at day 0, 14 & 21 with pools of peptides in the presence of poly (I:C) adjuvant, and splenocytes were harvested at day 28 to measure peptide-specific T cell activation by IFNg ELISpot. Of the selected peptides, 25 of 66 induce high IFNg T cell responses in the spleen. Interestingly, 88% of these responses were CD4 T cells and 12% were CD8 T cells, consistent with other preclinical and clinical studies where neoantigens predicted to bind MHC-I molecules induced mainly CD4 T cell activation. Humoral immune response specific to the peptides were analyzed by ELISA and showed that 8 of the 25 peptides induce peptide-specific IgG antibodies in the serum.To determine whether neoantigens generate tumor protective immunity, we challenged vaccinated mice with the MOPC.315.BM cell line. We used a well-characterized antigen lambda 2315 idiotype (Id) produced by MOPC cells and known to activate CD4 T cells. Vaccines using only the Id were found to be weakly immunogenic and failed to elicit protective responses. We developed a new strategy of vaccination by adding a proprietary amino acid sequence comprising a high affinity HSP70 binding motif to the idiotype peptide. Mice vaccinated at days 0, 14 & 21 with the so-modified peptide and poly-(I:C) adjuvant had 4 times higher Id-specific IFNg CD4 T cell response in the spleen compared to mice vaccinated with the unmodified Id-peptide. When challenged with MOPC.315.BM cells after vaccination with the modified Id peptide, 60% of these mice maintained non-detectable Id level in their blood and resisted tumor growth for more than 70 days while unvaccinated mice had very high levels of circulating Id and died around 30 days after tumor injection. Id-mediated cytotoxicity is being investigated in vitro to complement these results. We are conducting tumor protection experiments with the 25 immunogenic peptides identified in the vaccination experiments described in the previous section.In summary, we showed that neoantigens derived from bioinformatic analysis of tumor genome sequencing are immunogenic and that they mainly activate CD4 T cells in this setting. Additionally, our vaccination strategy using the idiotype peptide showed that CD4 T cell immunity can be protective against tumor growth. We are currently investigating the potential of the neoantigens identified in the immunogenicity study to protect mice against tumor growth and eradicate pre-existing tumors. Our findings indicate that neoantigen vaccination is very promising and that future investigations combining this strategy with CIs and/or immunomodulatory agents may lead to the efficient generation of protective anti-tumor immunity. DisclosuresBekri:Agenus Inc: Research Funding. Cho:Ludwig Institute for Cancer Research: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Genentech: Other: advisory board, Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Agenus, Inc.: Research Funding.

Diospyros Peregrina Fruit Preparation Mediated Immunomodulation of Lymphocytes Isolated From The Blood of Breast Cancer Patients.

Breast cancer is an uncontrolled growth of epithelial cells. The loss of Breast Cancer gene1 (BRCA1) activity due to mutation or down-regulation of gene expression promotes tumorigenesis and increases the risk of breast cancer. We aimed to pulsate lymphocytes of breast cancer patients and normal individuals, using Diospyros peregrina fruit preparation (DFP) to study the cancer-protective immunity, and the signal transduction processes involved with it. We also investigated the role of DFP in the release of lymphocytic nitric oxide (NO), which is a key tumoricidal agent, known to regulate T-cell proliferation, cytokine production, cell signaling, and apoptosis. Using Ficoll-Hypaque gradient centrifugation, lymphocytes were isolated from the blood of 12 patients and 12 normal individuals. Cells were treated with or without DFP (2.5 µg/ml) for 48 hours. Both non-stimulated and stimulated cells were then subjected to MTT and NO release assay; following which qPCR was performed to estimate mRNA levels and percentage enrichment of certain genes. DFP stimulates lymphocytic proliferation (p=0.0118) and release of NO (p=0.01) significantly. DFP also noticeably enhances the expression of T helper (TH) cell 1 specific interferon-gamma (IFNG), interleukin 12 (IL12), T-Box Transcription Factor 21 (TBX21) and signal transducer and activator of transcription 1 (STAT1) genes. DFP treatment significantly increases tumor protective immunity by decreasing the expression levels of TH2 network-specific GATA3 and interleukin 4 (IL4) genes but increasing the expression levels of TH1 network-specific IFNG, IL12, TBX21, and STAT1 genes. DFP increases the expression levels of TH1 specific network genes which in turn help in evoking tumor protective immunity.

Integrin αvβ6–TGFβ–SOX4 Pathway Drives Immune Evasion in Triple-Negative Breast Cancer

Cancer immunotherapy shows limited efficacy against many solid tumors that originate from epithelial tissues, including triple-negative breast cancer (TNBC). We identify the SOX4 transcription factor as an important resistance mechanism to Tcell-mediated cytotoxicity for TNBC cells. Mechanistic studies demonstrate that inactivation of SOX4 in tumor cells increases the expression of genes in a number of innate and adaptive immune pathways important for protective tumor immunity. Expression of SOX4 is regulated by the integrin αvβ6 receptor on the surface of tumor cells, which activates TGFβ from a latent precursor. An integrin αvβ6/8-blocking monoclonal antibody (mAb) inhibits SOX4 expression and sensitizes TNBC cells to cytotoxic Tcells. This integrin mAb induces a substantial survival benefit in highly metastatic murine TNBC models poorly responsive to PD-1 blockade. Targeting of the integrin αvβ6-TGFβ-SOX4 pathway therefore provides therapeutic opportunities for TNBC and other highly aggressive human cancers of epithelial origin.

Overcoming resistance to STING agonist therapy to incite durable protective antitumor immunity

BackgroundActivating the Stimulator of Interferon Genes (STING) adaptor incites antitumor immunity against immunogenic tumors in mice, prompting clinical trials to test STING activators. However, STING signaling in the tumor microenvironment...

Isolation and Characterization of MAIT Cells from Tumor Tissues.

Mucosal-associated invariant T (MAIT) cell infiltration has been demonstrated in colorectal and hepatocellular carcinoma, and their ability to produce Th1- and Th17-associated cytokines, as well as their cytotoxic function, suggests that MAIT cells may have important functions in both reducing and promoting protective tumor immunity. Here, we describe enzymatic methods to isolate intraepithelial and lamina propria lymphocyte single cell suspensions from colon tissue and tumors containing viable MAIT cells, which can be used for further purification, flow cytometry analysis, or culture.

Abstract 3218: Anti-PD1 and -CTLA-4 combination in vivo enhances DC-based tumor-associated mitochondria antigen immunotherapy efficacy

Abstract Somatic mitochondria DNA (mtDNA) abnormalities have been identified in various types of human cancers. Previously, we generated tumor protective immunity under prophylactic and therapeutic approaches employing a murine kidney tumor model system and dendritic cell (DC)-based vaccination containing RENCA-derived mitochondria enriched lysates. Our results shown that tumor-associated mitochondria antigens (TAMAs) are targetable antigens for cancer immunotherapy (PMID: 26378078). Preliminary data also revealed increased endogenous PD-L1 mRNA expression levels in RENCA tumors of treated mice versus healthy kidney tissue. To investigate the impact of PD1/PD-L1 axis antagonism, Balb/C mice were challenged with RENCA cells at day 0 followed by TAMA-vaccination at day 3 and concurrent administration of anti-PD1 (aPD1) 5 times every 3 days. Interestingly, the aPD1-TAMA treatment resulted in reduced tumor growth, greater T CD4+ and T CD8+ cell infiltration, and lower intratumoral-MDSC as compared to the TAMA vaccine alone. In addition, we observed higher T cell activation from the aPD1-TAMA group upon co-culture with bone marrow-derived dendritic cells exposed to RENCA mitochondria lysate, suggesting that the inhibition of PD1 also enhanced the immunogenicity of the TAMA vaccine. Further analysis of the tumor environment revealed that the PD1-PD-L1 axis antagonism correlated with an increment of intratumoral CTLA4 expression and recruitment of T regulatory (Treg) cells. We introduced a third arm of treatment including a CTLA4 antagonist antibody in combination with aPD1 and TAMA vaccine in vivo. Strikingly, the tumor progression was greatly impacted causing tumor rejection in the majority of tumor-bearing animals, sustained tumor volume reduction in the others, a decrease in Treg cell recruitment, and concomitant increment of T CD8+ cell infiltration and activation. Altogether, the combination of check point inhibitors in vivo significantly improved the efficacy of the TAMA immunotherapy by remodeling the tumor microenvironment, reducing the immunosuppressive potential, and facilitating the infiltration of effector T cells into the tumor. Citation Format: Renzo F. Perales-Linares, Stefano Pierini, Mireia Uribe, Sergei Pustylnikov, Francesca Costabile, Silvia Beghi, Andrea Facciabene. Anti-PD1 and -CTLA-4 combination in vivo enhances DC-based tumor-associated mitochondria antigen immunotherapy efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3218.

Tissue-resident memory T cells mediate melanoma surveillance and are targets of immunotherapy

Abstract Non-recirculating tissue-resident memory T (TRM) cells reside permanently in epithelial tissues where they are known to protect against infection. TRM cells have now been identified in various human cancers where they correlate with improved patient outcomes, but the mechanisms through which they may mediate protective tumour immunity remain unclear. Using a novel mouse model of transplantable cutaneous melanoma, we show that tumour-specific TRM cells form spontaneously in the epithelial tumour microenvironment where they protect against cancer development independently of circulating memory T cells. A proportion of epicutaneously inoculated mice remained free of macroscopic tumours long after inoculation, and intravital imaging revealed that these mice harboured dormant melanoma cells in their skin that were dynamically surveyed by tumour-specific TRM cells. Depletion of TRM cells from these mice triggered melanoma escape, indicating that TRM cells suppress tumour progression by promoting cancer-immune equilibrium. Further, we found that pre-activated T cells transferred to tumour-bearing mice in a model of adoptive cell therapy gave rise to TRM cells that inhibited the growth of established melanoma. These tumour-specific TRM cells retained their capacity to execute effector functions and protect against tumour growth despite expression of various inhibitory molecules typically associated with T cell dysfunction. Combined, our findings reveal TRM cells to be durable mediators of anti-tumour immunity and suggest that targeting TRM cell responses could serve as a novel immunotherapeutic strategy to drive elimination of solid tumours.

CD4+ T cells induce rejection of urothelial tumors after immune checkpoint blockade.

Immune checkpoint blockade (ICB) provides clinical benefit to a minority of patients with urothelial carcinoma (UC). The role of CD4+ T cells in ICB-induced antitumor activity is not well defined; however, CD4+ T cells are speculated to play a supportive role in the development of CD8+ T cells that kill tumor cells after recognition of tumor antigens presented by MHC class I. To investigate the mechanisms of ICB-induced activity against UC, we developed mouse organoid-based transplantable models that have histologic and genetic similarity to human bladder cancer. We found that ICB can induce tumor rejection and protective immunity with these systems in a manner dependent on CD4+ T cells but not reliant on CD8+ T cells. Evaluation of tumor infiltrates and draining lymph nodes after ICB revealed expansion of IFN-γ-producing CD4+ T cells. Tumor cells in this system express MHC class I, MHC class II, and the IFN-γ receptor (Ifngr1), but none were necessary for ICB-induced tumor rejection. IFN-γ neutralization blocked ICB activity, and, in mice depleted of CD4+ T cells, IFN-γ ectopically expressed in the tumor microenvironment was sufficient to inhibit growth of tumors in which the epithelial compartment lacked Ifngr1. Our findings suggest unappreciated CD4+ T cell-dependent mechanisms of ICB activity, principally mediated through IFN-γ effects on the microenvironment.

IL-17 induces antitumor immunity by promoting beneficial neutrophil recruitment and activation in esophageal squamous cell carcinoma

ABSTRACTInterleukin (IL)-17 has been reported to play a controversial role in tumor immunity. Our previous studies showed that infiltration of IL-17-producing cells in esophageal squamous cell carcinoma (ESCC) induced tumor protective immunity by recruiting CD8+T lymphocytes, natural killer (NK) cells, and B lymphocytes into the tumor microenvironment. However, the mechanism of IL-17 regulation of tumor-associated neutrophils remains elusive in ESCC. In this study, we therefore evaluated the accumulation of myeloperoxidase (MPO)+ neutrophils and its association with IL-17-producing cells within ESCC tumor nests. We also investigated the effects of IL-17 on the recruitment and antitumor activity of neutrophils. MPO+ neutrophil infiltration was found to predict a favorable prognosis in ESCC patients and was positively correlated with IL-17+ cell density. IL-17 stimulated ESCC tumor cells to release more of the CXC chemokines CXCL2 and CXCL3, which are involved in neutrophil migration. Furthermore, IL-17 potentiates the direct killing capability of neutrophils by enhancing the production of cytotoxic molecules, including reactive oxygen species (ROS), MPO, TNF-related apoptosis-inducing ligand (TRAIL), and IFN-γ. Experiments in mice suggested that IL-17 alone might not affect tumor progression in the tumor-bearing host, but IL-17 can inhibit tumor growth by promoting beneficial neutrophil infiltration and activation at tumor sites. As emerging evidence indicates that targeting tumor-associated neutrophils is a strategy for antitumor therapy, our findings reveal a positive contribution of IL-17 to the modulation of neutrophil-mediated antitumor immunity in ESCC. This study provides further understanding of the mechanisms that selectively regulate functional activities of neutrophils, which may be critical for developing new tumor immunotherapy.

Prospects for combining targeted and conventional cancer therapy with immunotherapy.

Over the past 25 years, research in cancer therapeutics has largely focused on two distinct lines of enquiry. In one approach, efforts to understand the underlying cell-autonomous, genetic drivers of tumorigenesis have led to the development of clinically important targeted agents that result in profound, but often not durable, tumour responses in genetically defined patient populations. In the second parallel approach, exploration of the mechanisms of protective tumour immunity has provided several therapeutic strategies - most notably the 'immune checkpoint' antibodies that reverse the negative regulators of T cell function - that accomplish durable clinical responses in subsets of patients with various tumour types. The integration of these potentially complementary research fields provides new opportunities to improve cancer treatments. Targeted and immune-based therapies have already transformed the standard-of-care for several malignancies. However, additional insights into the effects of targeted therapies, along with conventional chemotherapy and radiation therapy, on the induction of antitumour immunity will help to advance the design of combination strategies that increase the rate of complete and durable clinical response in patients.

Iterative Design and in Vivo Evaluation of an Oncolytic Antilymphoma Peptide.

Oncolytic peptides represent a promising new strategy within the field of cancer immunotherapy. Here we describe the systematic design and evaluation of short antilymphoma peptides within this paradigm. The peptides were tested in vitro and in vivo to identify a lead compound for further evaluation as novel oncolytic immunotherapeutic. In vitro tests revealed peptides with high activity against several lymphoma types and low cytotoxicity toward normal cells. Treated lymphoma cells exhibited a reduced mitochondrial membrane potential that resulted in an irreversible disintegration of their plasma membranes. No caspase activation or ultrastructural features of apoptotic cell death were observed. One of these peptides, 11, was shown to induce complete tumor regression and protective immunity following intralesional treatment of murine A20 B-lymphomas. Due to its selectivity for lymphoma cells and its ability to induce tumor-specific immune responses, 11 has the potential to be used in intralesional treatment of accessible lymphoma tumors.

Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma.

The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-β1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-β1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth.

Abstract 262: Combination therapy of folate-targeted chemotherapeutics with anti-PD-1 antibody against folate receptor-positive tumors in immunocompetent murine models

Abstract The PD-1/PD-L1 (programmed cell death protein 1; programmed death-ligand 1) pathway plays a key role in immunological tolerance and autoimmunity. PD-1 is primarily expressed on activated T and B cells while its receptor, PD-L1, is expressed in a variety of cell types including resting lymphocytes, DCs, and macrophages. Recently, multiple human solid tumor types (melanoma, NSCLC, RCC, ovarian, and colorectal cancer) have been found to overexpress PD-L1 within its tumor microenvironment. More importantly, PD-1/PD-L1 blocking agents (pembrolizumab, nivolumab, lambrolizumab, MPDL3280A) have been found active as a single agent or in combination with chemotherapy and some have produced durable clinical responses. On the other hand, human epithelial tumors are also known to overexpress the high-affinity folate receptor (FR)-alpha that is capable of bringing folate-linked drugs into the cell cytosol via endocytosis. Using flow cytometry, we assessed PD-L1 expression in various FR-positive syngeneic mouse tumor models (M109, Renca, L1210A, 4T1-Cl2, ID8-Cl15). PD-L1 expression was found to vary significantly among different tumor types and could be found not only on CD45- malignant cells but also on tumor stromal cells such as F4/80+CD11b+ tumor-associated macrophages. Using a rat anti-mouse PD-1 antibody (clone RMP1-14), we studied in-vivo efficacy of a folate-targeted chemotherapy in combination with PD-1/PD-L1 blockage in PD-L1-expressing tumor models. In syngeneic models with high PD-L1 expression and tumor-infiltrating lymphocytes, a synergistic and curable anti-tumor effect was observed and the animals developed tumor-protective immunity against rechallenge. Thus, our preliminary results suggest that folate-targeted chemotherapeutics may also be enhanced by PD-1/PD-L1 blockage by means of overcoming tumor-mediated immune suppression Citation Format: Yingjuan June Lu, Leroy W. Wheeler, Vicky Cross, Elaine Westrick, Alex Lloyd, Christopher P. Leamon. Combination therapy of folate-targeted chemotherapeutics with anti-PD-1 antibody against folate receptor-positive tumors in immunocompetent murine models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 262.

IL-17A promotes migration and tumor killing capability of B cells in esophageal squamous cell carcinoma.

We have previously reported that the accumulation of IL-17-producing cells could mediate tumor protective immunity by promoting the migration of NK cells, T cells and dendritic cells in esophageal squamous cell carcinoma (ESCC) patients. However, there were no reports concerning the effect of IL-17A on tumor infiltrating B cells. In this study, we investigated the accumulation of CD20+ B cells in the ESCC tumor nests and further addressed the effect of IL-17A on the migration and cytotoxicity of B cells. There was positive correlation between the levels of CD20+ B cells and IL-17+ cells. IL-17A could promote the ESCC tumor cells to produce more chemokines CCL2, CCL20 and CXCL13, which were associated with the migration of B cells. In addition, IL-17A enhanced the IgG-mediated antibody and complement mediated cytotoxicity of B cells against tumor cells. IL-17A-stimulated B cells gained more effective direct killing capability through enhanced expression of Granzyme B and FasL. The effect of IL-17A on the migration and cytotoxicity of B cells was IL-17A pathway dependent, which could be inhibited by IL-17A inhibitor. This study provides further understanding of the roles of IL-17A in humoral response, which may contribute to the development of novel tumor immunotherapy strategy.

Malignancies of the skin and immunomodulatory antirheumatic therapy

For the clinical practice there is uncertainty as to what degree the therapeutic immunomodulation of rheumatoid arthritis (RA) is associated with a weakening of protective tumor immunity. Neoplasms of the skin in particular are known to exhibit increased incidence rates in association with therapeutic immunosuppression in transplantation medicine; however, the immunosuppression required for the prevention of allogenic graft rejection is much more intensive and thus not directly transferable to the potential risks for an onset or relapse of melanoma or non-melanoma skin cancers (NMSC), e. g. spinocellular and basal cell carcinomas in association with the antirheumatic treatment of RA. This review covers the association of RA and its pharmacotherapy with conventional synthetic (cs) and biologic disease-modifying antirheumatic drugs (DMARD) on the basis of a systematic literature search. The incidence rates of NMSC are twice as high in biologic-naive RA patients compared to the general population, whereas the respective incidence rates for melanoma do not differ. A biologic treatment with tumor necrosis factor (TNF) blockers compared with administration of csDMARD only has a minor, if any effect on the increase of NMSC risk but is associated with a trend towards an elevated incidence rate of new onset melanoma although significance level was not reached in all of the reviewed studies. The data on non-TNF blocking biologics is sparse. Accordingly, it is inappropriate to draw any strong conclusions on potentially associated skin cancer risks from the present lack of safety signals. The consideration of individual risk factors, recommendations on sufficient UV protection and regular skin monitoring may serve to improve the safety of DMARD therapy in RA.

Abstract IA25: Mechanisms of protective tumor immunity

Abstract Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. The pharmacologic inhibition of MFG-E8 function through genetic or engineering-based approaches blocks Treg induction, which intensifies vaccine-induced responses, leading to the regression of established tumors in mice. The clinical translation of these therapeutic strategies to Phase I testing in humans is underway. The detailed analysis of patients achieving sustained clinical benefits from irradiated, autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade also affords a rich opportunity to identify antigens associated with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. In this regard, we have identified antibodies against a panel of secreted and cell surface proteins that play important roles in tumor development and immune evasion. Isolation of the rare, antigen-specific memory B cells directed towards these important targets provides new opportunities for developing human monoclonal antibodies for cancer immunotherapy. Citation Format: Glenn Dranoff. Mechanisms of protective tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA25.

Anti-G-CSF treatment induces protective tumor immunity in mouse colon cancer by promoting protective NK cell, macrophage and T cell responses.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine that is highly expressed in human and mouse colorectal cancers (CRC). We previously reported that G-CSF stimulated human CRC cell growth and migration, therefore in this study we sought to examine the therapeutic potential of anti-G-CSF treatment for CRC. G-CSF is known to mobilize neutrophils, however its impact on other immune cells has not been well examined. Here, we investigated the effects of therapeutic anti-G-CSF treatment on CRC growth and anti-tumor immune responses. C57BL/6 mice treated with azoxymethane/dextran sodium sulfate (AOM/DSS) to induce neoplasms were administered anti-G-CSF or isotype control antibodies three times a week for three weeks. Animals treated with anti-G-CSF antibodies had a marked decrease in neoplasm number and size compared to the isotype control group. Colon neutrophil and macrophage frequency were unchanged, but the number of macrophages producing IL-10 were decreased while IL-12 producing macrophages were increased. NK cells were substantially increased in colons of anti-G-CSF treated mice, along with IFNγ producing CD4(+) and CD8(+) T cells. These studies are the first to indicate a crucial role for G-CSF inhibition in promoting protective anti-tumor immunity, and suggest that anti-G-CSF treatment is a potential therapeutic approach for CRC.

Amino-functionalized poly(L-lactide) lamellar single crystals as a valuable substrate for delivery of HPV16-E7 tumor antigen in vaccine development

BackgroundPoly(l-lactide) (PLLA) is a biodegradable polymer currently used in many biomedical applications, including the production of resorbable surgical devices, porous scaffolds for tissue engineering, nanoparticles and microparticles for the controlled release of drugs or antigens. The surfaces of lamellar PLLA single crystals (PLLAsc) were provided with amino groups by reaction with a multifunctional amine and used to adsorb an Escherichia coli-produced human papillomavirus (HPV)16-E7 protein to evaluate its possible use in antigen delivery for vaccine development.MethodsPLLA single crystals were made to react with tetraethylenepentamine to obtain amino-functionalized PLLA single crystals (APLLAsc). Pristine and amino-functionalized PLLAsc showed a two-dimensional microsized and one-dimensional nanosized lamellar morphology, with a lateral dimension of about 15–20 μm, a thickness of about 12 nm, and a surface specific area of about 130 m2/g. Both particles were characterized and loaded with HPV16-E7 before being administered to C57BL/6 mice for immunogenicity studies. The E7-specific humoral-mediated and cell-mediated immune response as well as tumor protective immunity were analyzed in mice challenged with TC-1 cancer cells.ResultsPristine and amino-functionalized PLLAsc adsorbed similar amounts of E7 protein, but in protein-release experiments E7-PLLAsc released a higher amount of protein than E7-APLLAsc. When the complexes were dried for observation by scanning electron microscopy, both samples showed a compact layer, but E7-APLLAsc showed greater roughness than E7-PLLAsc. Immunization experiments in mice showed that E7-APLLAsc induced a stronger E7-specific immune response when compared with E7-PLLAsc. Immunoglobulin G isotyping and interferon gamma analysis suggested a mixed Th1/Th2 immune response in both E7-PLLAsc-immunized and E7-APLLAsc-immunized mice. However, only the mice receiving E7-APLLAsc were fully protected from TC-1 tumor growth after three doses of vaccine.ConclusionOur results show that APLLA single crystals improve the immunogenicity of HPV16-E7 and indicate that E7-APLLAsc could be used for development of an HPV16 therapeutic vaccine against HPV16-related tumors.