Tumor-specific T-cell Responses Research Articles

CD11c-targeted Delivery of DNA to Dendritic Cells Leads to cGAS- and STING-dependent Maturation.

Immunotherapeutic activation of tumor-specific T cells has proven to be an interesting approach in anticancer treatment. Particularly, anti-CTLA-4 and anti-PD-1/PD-L1 treatment looks promising, and conceivably, even better clinical results might be obtained if such treatment could be combined with boosting the existing tumor-specific T-cell response. One way to achieve this could be by increasing the level of maturation of dendritic cells locally and in the draining lymph nodes. When exposed to cancer cells, dendritic cells may spontaneously mature because of danger-associated molecular patterns derived from the tumor cells. Double-stranded DNA play a particularly important role in the activation of the dendritic cells, through engagement of intracellular DNA-sensors, and signaling through the adaptor protein STING. In the present study, we have investigated the maturational response of human monocyte-derived dendritic cells (moDC) and human monocytic THP-1 cells to targeted and untargeted DNA. We used an anti-CD11c antibody conjugated with double-stranded DNA to analyze the maturation status of human moDCs, as well as maturation using a cGAS KO and STING KO THP-1 cell maturation model. We found that dendritic cells can mature after exposure to cytoplasmic double-stranded DNA delivered through CD11c-mediated endocytosis. Moreover, we show that THP-1 cells matured using IL-4, GM-CSF, and ionomycin upregulate DC-maturation markers after CD11c-targeted delivery of double-stranded DNA. This upregulation is completely abrogated in cGAS KO and STING KO cells.

T-cell Localization, Activation, and Clonal Expansion in Human Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to most therapies, including immune checkpoint blockade. To elucidate mechanisms of immunotherapy resistance, we assessed immune parameters in resected human PDA. We demonstrate significant interpatient variability in T-cell number, localization, and phenotype. CD8+ T cells, Foxp3+ regulatory T cells, and PD-1+ and PD-L1+ cells were preferentially enriched in tertiary lymphoid structures that were found in most tumors compared with stroma and tumor cell nests. Tumors containing more CD8+ T cells also had increased granulocytes, CD163+ (M2 immunosuppressive phenotype) macrophages, and FOXP3+ regulatory T cells. PD-L1 was rare on tumor cells, but was expressed by CD163+ macrophages and an additional stromal cell subset commonly found clustered together adjacent to tumor epithelium. The majority of tumoral CD8+ T cells did not express molecules suggestive of recent T-cell receptor (TCR) signaling. However, 41BB+PD-1+ T cells were still significantly enriched in tumors compared with circulation. Tumoral CD8+PD-1+ T cells commonly expressed additional inhibitory receptors, yet were mostly T-BEThi and EOMESlo, consistent with a less terminally exhausted state. Analysis of gene expression and rearranged TCR genes by deep sequencing suggested most patients have a limited tumor-reactive T-cell response. Multiplex immunohistochemistry revealed variable T-cell infiltration based on abundance and location, which may result in different mechanisms of immunotherapy resistance. Overall, the data support the need for therapies that either induce endogenous, or provide engineered, tumor-specific T-cell responses, and concurrently relieve suppressive mechanisms operative at the tumor site. Cancer Immunol Res; 5(11); 978-91. ©2017 AACR.

Neoadjuvant Interferons: Critical for Effective PD-1-Based Immunotherapy in TNBC.

The lack of targeted therapies available for triple-negative breast cancer (TNBC) patients who fail to respond to first-line chemotherapy has sparked interest in immunotherapeutic approaches. However, trials utilizing checkpoint inhibitors targeting the PD-1/PD-L1 axis in TNBC have had underwhelming responses. Here, we investigated the interplay between type I IFN signaling and the PD-1/PD-L1 axis and tested the impact of combining IFN inducers, as immune activators, with anti-PD-1, to induce an antimetastatic immune response. Using models of TNBC, we demonstrated an interplay between type I IFN signaling and tumor cell PD-L1 expression that affected therapeutic response. The data revealed that the type I IFN-inducer poly(I:C) was an effective immune activator and antimetastatic agent, functioning better than anti-PD-1, which was ineffective as a single agent. Poly(I:C) treatment induced PD-L1 expression on TNBC cells, and combined poly(I:C) and anti-PD-1 treatment prolonged metastasis-free survival in a neoadjuvant setting via the induction of a tumor-specific T-cell response. Use of this combination in a late treatment setting did not impact metastasis-free survival, indicating that timing was critical for immunotherapeutic benefit. Together, these data demonstrated anti-PD-1 as an ineffective single agent in preclinical models of TNBC. However, type I IFN inducers were effective immune activators, and neoadjuvant trials combining them with anti-PD-1 to induce a sustained antitumor immune response are warranted. Cancer Immunol Res; 5(10); 871-84. ©2017 AACR.

Highlighting Advances in the Treatment of Gliomas

Highlighting Advances in the Treatment of Gliomas

CD4+ T Cell and NK Cell Interplay Key to Regression of MHC Class Ilow Tumors upon TLR7/8 Agonist Therapy.

One of the next challenges in cancer immunotherapy is the resistance of tumors to T-cell-based treatments through loss of MHC class I. Here, we show that under these circumstances, the Toll-like receptor (TLR)-7/8 ligand imiquimod, but not the TLR3 ligand poly I:C or TLR9 ligand CpG, mediated an effective antitumor response. The rejection of these immune-escaped cancers was mediated by NK cells and CD4+ T cells, whereas activated CD8+ T cells were dispensable. Application of the innate immune stimulator at a distant site activated NK cells and thereby elicited tumor-specific T-cell responses in tumor-bearing mice. Mechanistically, imiquimod activated NK cells to kill tumor cells, resulting in release of tumor antigens and induction of tumor-specific CD4+ T cells. These T helper cells provoked a strong induction of CXCL9 and CXCL10 in the tumor environment. Simultaneously, imiquimod induced the expression of the cognate chemokine receptor CXCR3 on peripheral lymphocytes. This ignited intratumoral CD4+ T-cell infiltration and accumulation, which was critical for tumor rejection; CXCR3 blocking antibodies mitigated the clinical response. In the effector phase, NK cell recruitment to tumors and their activation depended on CD4+ T cells. Together, we have uncovered a potent immune axis of tumor-specific CD4+ T cells and NK cells that eliminates escaped MHC-Ilow tumors. Cancer Immunol Res; 5(8); 642-53. ©2017 AACR.

Rapid and Continued T-Cell Differentiation into Long-term Effector and Memory Stem Cells in Vaccinated Melanoma Patients.

Purpose: Patients with cancer benefit increasingly from T-cell-based therapies, such as adoptive T-cell transfer, checkpoint blockade, or vaccination. We have previously shown that serial vaccinations with Melan-AMART-126-35 peptide, CpG-B, and incomplete Freund adjuvant (IFA) generated robust tumor-specific CD8 T-cell responses in patients with melanoma. Here, we describe the detailed kinetics of early- and long-term establishment of T-cell frequency, differentiation (into memory and effector cells), polyfunctionality, and clonotype repertoire induced by vaccination.Experimental Design: Twenty-nine patients with melanoma were treated with multiple monthly subcutaneous vaccinations consisting of CpG-B, and either the native/EAA (n = 13) or the analogue/ELA (n = 16) Melan-AMART-126-35 peptide emulsified in IFA. Phenotypes and functionality of circulating Melan-A-specific CD8 T cells were assessed directly ex vivo by multiparameter flow cytometry, and TCR clonotypes were determined ex vivo by mRNA transcript analyses of individually sorted cells.Results: Our results highlight the determining impact of the initial vaccine injections on the rapid and strong induction of differentiated effector T cells in both patient cohorts. Moreover, long-term polyfunctional effector T-cell responses were associated with expansion of stem cell-like memory T cells over time along vaccination. Dominant TCR clonotypes emerged early and persisted throughout the entire period of observation. Interestingly, one highly dominant clonotype was found shared between memory and effector subsets.Conclusions: Peptide/CpG-B/IFA vaccination induced powerful long-term T-cell responses with robust effector cells and stem cell-like memory cells. These results support the further development of CpG-B-based cancer vaccines, either alone or as specific component of combination therapies. Clin Cancer Res; 23(13); 3285-96. ©2016 AACR.

Abstract 3088: Combination of 4SC-202 and IFN-γ restores mature APC phenotype in AML cells

Abstract Acute myeloid leukemia (AML) is characterized by a differentiation block resulting in accumulation of immature myeloid cells. Differentiation therapy, e.g. with all-trans retinoic acid (ATRA) has become a part of standard treatment for the acute promyelocytic leukemia variant of AML. Other agents like cytokines and HDAC inhibitors were shown to enhance differentiation of AML cells, which results in apoptosis induction or re-sensitization to ATRA. However, a differentiation therapy resulting in the restoration of antigen presenting function of AML cells would be a more desirable asset, since such cells will be able to induce or enhance immune response against residual malignant AML cells. Here, we analyzed the impact of 4SC-202 alone and in combination with different cytokines on differentiation of AML cell lines THP-1, HL-60 and MOLM-13 (AML-M4, -M2, and -M5 class, respectively). Myeloid lineage markers CD11b, CD86, CD14 and HLA-DR were analyzed by flow cytometry and quantitative PCR. Cytokine production was measured using Luminex multiplex system. Furthermore, chromatin immunoprecipitation (ChIP) was performed to assess the direct impact of 4SC-202 on promoter histone acetylation of differentiation genes. 4SC-202 is an orally available clinical stage epigenetic small molecule inhibitor which specifically targets histone deacetylases HDAC class I isoenzymes 1-3 as well as the lysine-specific demethylase LSD1 (KDM1A). 4SC-202 treatment of AML cell lines resulted in a dose-dependent increase of myeloid differentiation genes CD86 and ITGAM (=CD11b) on mRNA and protein level. On the chromatin level, 4SC-202 correspondently induced an upregulation of chromatin activation marks like acetylation of histone H3 lysine 9 and 27 (H3K9ac, H3K27ac) at the promoters of these genes. Interestingly, reference compounds like ATRA, bexarotene, GM-CSF and TNF-α were not as efficacious as 4SC-202 in the induction of differentiation markers in AML cells, whereas IFN-γ strongly induced MHC class II molecules HLA-DR and myeloid lineage marker CD14. Finally, the combination of 4SC-202 and IFN-γ resulted in a strong and complementary induction of differentiation and APC markers CD11b, CD14, CD86 and HLA-DR resulting in a CD86high/HLA-DRhigh phenotype which was able to produce pro-inflammatory cytokines. In summary, we demonstrate that 4SC-202 induces differentiation of AML cells of different subtypes and thus provide promising preclinical data for applying this novel epigenetic modulator for differentiation therapy of AML. Moreover, the mature APC phenotype induced by the combination of 4SC-202 and IFN-γ may further suggest an application in consolidation treatment of AML to enhance or even elicit a tumor-specific T-cell response against residual malignant AML cells preventing AML recurrence. Citation Format: Anne Catherine Bretz, Ulrike Parnitzke, Kerstin Kronthaler, Svetlana Hamm. Combination of 4SC-202 and IFN-γ restores mature APC phenotype in AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3088. doi:10.1158/1538-7445.AM2017-3088

Dendritic Cell Vaccination Enhances Immune Responses and Induces Regression of HER2pos DCIS Independent of Route: Results of Randomized Selection Design Trial.

Purpose: Vaccination with HER2 peptide-pulsed DC1s stimulates a HER2-specific T-cell response. This randomized trial aimed to establish safety and evaluate immune and clinical responses to vaccination via intralesional (IL), intranodal (IN), or both intralesional and intranodal (ILN) injection.Experimental Design: Fifty-four HER2pos patients [42 pure ductal carcinoma in situ (DCIS), 12 early invasive breast cancer (IBC)] were enrolled in a neoadjuvant HER2 peptide-pulsed DC1 vaccine trial. Patients were randomized to IL (n = 19), IN (n = 19), or ILN (n = 16) injection. Immune responses were measured in peripheral blood and sentinel lymph nodes by ELISPOT or in vitro sensitization assay. Pathologic response was assessed in resected surgical specimens.Results: Vaccination by all injection routes was well tolerated. There was no significant difference in immune response rates by vaccination route (IL 84.2% vs. IN 89.5% vs. ILN 66.7%; P = 0.30). The pathologic complete response (pCR) rate was higher in DCIS patients compared with IBC patients (28.6% vs. 8.3%). DCIS patients who achieved pCR (n = 12) and who did not achieve pCR (n = 30) had similar peripheral blood anti-HER2 immune responses. All patients who achieved pCR had an anti-HER2 CD4 immune response in the sentinel lymph node, and the quantified response was higher by response repertoire (P = 0.03) and cumulative response (P = 0.04).Conclusions: Anti-HER2 DC1 vaccination is a safe and immunogenic treatment to induce tumor-specific T-cell responses in HER2pos patients; immune and clinical responses were similar independent of vaccination route. The immune response in the sentinel lymph nodes, rather than in the peripheral blood, may serve as an endpoint more reflective of antitumor activity. Clin Cancer Res; 23(12); 2961-71. ©2016 AACR.

Synthetic Genomics makes anticancer DNA

Synthetic Genomics, the biotech firm founded by J. Craig Venter, has opened what it calls the first current Good Manufacturing Practice (cGMP) facility for making synthetic DNA. The company is working with Advaxis on an investigational cancer immunotherapy called ADXS-NEO that will enter a Phase I clinical trial this year. The patient-customized therapy is based on tumor-specific mutations that can trigger an immune response from the patient’s T cells. Advaxis uses Synthetic Genomics’ automated genomic workstation to produce DNA based on the mutations. It inserts the DNA sequence into bacteria to produce the protein that the mutation expresses. The proteins are then taken up by a patient’s antigen-presenting cells to activate a tumor-specific T-cell response.

Identification of T cell target antigens in glioblastoma stem-like cells using an integrated proteomics-based approach in patient specimens.

Glioblastoma (GBM) is a highly aggressive brain tumor and still remains incurable. Among others, an immature subpopulation of self-renewing and therapy-resistant tumor cells-often referred to as glioblastoma stem-like cells (GSCs)-has been shown to contribute to disease recurrence. To target these cells personalized immunotherapy has gained a lot of interest, e.g. by reactivating pre-existing anti-tumor immune responses against GSC antigens. To identify T cell targets commonly presented by GSCs and their differentiated counterpart, we used a proteomics-based separation of GSC proteins in combination with a T cell activation assay. Altogether, 713 proteins were identified by LC-ESI-MS/MS mass spectrometry. After a thorough filtering process, 32 proteins were chosen for further analyses. Immunogenicity of corresponding peptides was tested ex vivo. A considerable number of these antigens induced Tcell responses in GBM patients but not in healthy donors. Moreover, most of them were overexpressed in primary GBM and also highly expressed in recurrent GBM tissues. Interestingly, expression of the most frequent Tcell target antigens could also be confirmed in quiescent, slow-cycling GSCs isolated in high purity by the DEPArray technology. Finally, for a subset of these T cell target antigens, an association between expression levels and higher T cell infiltration as well as an increased expression of positive immune modulators was observed. In summary, we identified novel immunogenic proteins, which frequently induce tumor-specific Tcell responses in GBM patients and were also detected in vitro in therapy-resistant quiescent, slow-cycling GSCs. Stable expression of these T cell targets in primary and recurrent GBM support their suitability for future clinical use.

Abstract P2-04-27: CTLA-4 and PD-1 checkpoint inhibitors enhance individually tailored adaptive anti-tumor immune responses to overcome tumor immunosuppression and effectively treat triple-negative breast cancer

Abstract Despite a lack of unifying drivers in Triple-Negative Breast Cancer (TNBC), our lab and others have uncovered that these cancers have elevated expression of inflammatory genes and immunosuppressive molecules (i.e. PD-L1), as well as elevated numbers of infiltrating immune cells (including CD8+ T-cells and Foxp3+ T-regulatory cells) which suggests the therapeutic potential for single and combinations of checkpoint blockade antibodies. While early trials with PD-1 inhibitors have been encouraging for TNBC, only a fraction of treated patients respond to this therapy. To test and define the mechanisms that govern responses, we explored the utility and mechanistic basis of both PD-1 and CTLA-4 inhibition in generating tumor-specific immunity in an established murine model of TNBC. Consistent with patient samples, we found TNBC tumors from our model exhibited elevated PD-1+ expressing CD8+ T-cell infiltrates, Foxp3+ T-regulatory cell infiltrates (~66% of CD4+ TILs), as well as highly elevated tumor cell expression of PD-L1. We also found that while TNBC cells were easily killed by T-cell in vitro, TNBC tumors were highly immuno-suppressive and resistant to antigen-specific T-cell attack in vivo, even after adoptive transfer of up to 5x10E6 tumor-specific T-cells. However, we found that both CTLA-4 and PD-1 antibodies could curtail this immunosuppression to different degrees and through alternate mechanisms. Specifically, we found that CTLA-4 antibody mediated anti-tumor immunity through the elimination and blockade of Foxp3+ T-regulatory cells in the tumor microenvironment, which allow for potent T-cell expansion. Conversely, PD-1 antibodies elicited anti-tumor immunity through blockade of PDL1/PD1 signaling between tumor cells and T-cells in the TNBC tumor microenvironment that allowed for a more modest expansion of individually tailored T-cell specific clones in vivo. Strikingly, the combination of these antibodies and their alternate mechanisms of action resulted in greatly enhanced anti-tumor responses and led to regression of ~80% of tumors. This was accompanied by an augmented infiltration of T-cells into the tumor microenvironment and significantly enhanced systemic tumor-specific T-cell responses, which appear to be emergent properties of dual CTLA-4/PD-1 antibody treatment. However, we found that these treatments did not expand a common tumor-specific T-cell clone, despite adoptive transfer of identical tumor-specific immunodominant T-cells into mice after tumor implantation. Thus, despite our use of a highly homogeneous model utilizing genetically identical mice implanted with an identical tumor line bearing a unique tumor antigen under identical conditions, the tumor-specific T-cell responses were highly unique for each individual tumor. Collectively, our study suggest that dual blockade could be an effective therapeutic clinical strategy against TNBC and further suggest the utility of monitoring systemic immune response and TCR expansion of TILs as the most useful correlates in clinical studies utilizing CTLA-4 and PD-1 antibodies. Citation Format: Hartman ZC, Crosby EJ, Wei J-P, Yang X-Y, Lei G-J, Wang T, Liu C-X, Agarwal P, Morse MS, Lyerly HK. CTLA-4 and PD-1 checkpoint inhibitors enhance individually tailored adaptive anti-tumor immune responses to overcome tumor immunosuppression and effectively treat triple-negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-04-27.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Microwave ablation combined with OK-432 induces Th1-type response and specific antitumor immunity in a murine model of breast cancer

BackgroundMinimally invasive therapies, such as microwave ablation (MWA), are widely used for the treatment of solid tumors. Previous studies suggest that MWA is feasible for the treatment of small breast cancer, and thermal ablation may induce adaptive antitumor immunity. However, the induced immune responses are mostly weak, and the immunomodulation effects of MWA in breast cancer are unclear. Immunostimulant OK-432 can induce tumor-specific T-cell responses and may augment the immunity induced by MWA.MethodsWe treated 4T1 breast cancer bearing BALB/c mice with MWA, OK-432, MWA plus OK-432, or left without treatment. Survival time was evaluated with the Kaplan–Meyer method comparing survival curves by log-rank test. On day 25 after ablation, surviving mice received tumor rechallenge, and the rechallenged tumor volumes were calculated every 5 days. Immunohistochemistry and flow cytometry were used to evaluate the T-cell immune responses in ablated tissues and spleens. The tumor-specific immunity was assessed by enzyme-linked immunospot assays. Besides, the cytokine patterns were identified from enzyme-linked immunosorbent assay.ResultsMicrowave ablation plus OK-432 resulted in longer survival than single treatment and protect most surviving mice from tumor rechallenge. Both local and systemic T-cell responses were induced by MWA and were further enhanced by subsequent administration of OK-432. Moreover, the combination of MWA and OK-432 induced stronger tumor-specific immune responses than MWA alone. In addition, OK-432 and MWA synergistically promoted the production of Th1-type but not Th2-type cytokines, and polarized T-cell responses to Th1-dominant state.ConclusionsThe T-cell immune responses were activated by MWA in breast cancer. Furthermore, the combination of MWA and OK-432 induced Th1-type response and elicited specific antitumor immunity.

The Efficacy of Oncolytic Adenovirus Is Mediated by T-cell Responses against Virus and Tumor in Syrian Hamster Model.

Oncolytic adenoviruses (Ad) represent an innovative approach to cancer therapy. Its efficacy depends on multiple actions, including direct tumor lysis and stimulation of antiviral and antitumor immune responses. In this study, we investigated the roles of T-cell responses in oncolytic adenoviral therapy. An immunocompetent and viral replication-permissive Syrian hamster tumor model was used. The therapeutic mechanisms of oncolytic Ad were investigated by T-cell deletion, immunohistochemical staining, and CTL assay. Deletion of T cells with an anti-CD3 antibody completely demolished the antitumor efficacy of oncolytic Ad. Intratumoral injection of Ad induced strong virus- and tumor-specific T-cell responses, as well as antiviral antibody response. Both antiviral and antitumor T-cell responses contributed to the efficacy of oncolytic Ad. Deletion of T cells increased viral replication and extended the persistence of infectious virus within tumors but almost abrogated the antitumor efficacy. Preexisting antiviral immunity promoted the clearance of injected oncolytic Ad from tumors but had no effect on antitumor efficacy. Strikingly, the repeated treatment with oncolytic Ad has strong therapeutic effect on relapsed tumors or tumors insensitive to the primary viral therapy. These results demonstrate that T cell-mediated immune responses outweigh the direct oncolysis in mediating antitumor efficacy of oncolytic Ad. Our data have a high impact on redesigning the regimen of oncolytic Ad for cancer treatment. Clin Cancer Res; 23(1); 239-49. ©2016 AACR.

CTLA-4 Limits Anti-CD20–Mediated Tumor Regression

The inhibition of tumor growth by anti-CD20 antibody (Ab) treatment is mediated by Ab- and complement-dependent cytotoxicity in xenograft tumor models. In addition, anti-CD20 therapy for B-cell lymphoma can result in intrinsic and extrinsic tumor resistance to further Ab treatment. However, adaptive immune response-related resistance has not been well studied in anti-CD20-mediated tumor control, and adaptive immunity has long been underestimated. The purpose of this study was to explore whether T cells are involved in mediating the effects of anti-CD20 therapy and what factors contribute to adaptive immune response-related resistance. Using a syngeneic mouse B-cell lymphoma model, we investigated the role of CD8+ T cells in anti-CD20-mediated tumor regression. Furthermore, we revealed how the tumor-specific T-cell response was initiated by anti-CD20. Finally, we studied adaptive immune response-related resistance in advanced B-cell lymphoma. CD8+ T cells played an essential role in anti-CD20-mediated tumor regression. Mechanistically, anti-CD20 therapy promoted dendritic cell (DC)-mediated cross-presentation. Importantly, macrophages were also necessary for the increase in the tumor-specific CTL response after anti-CD20 treatment, via the production of type I IFN to activate DC function. Furthermore, adaptive resistance is gradually developed through the CTLA-4 pathway in Treg cells in larger lymphomas. Further blockade of CTLA-4 can synergize with anti-CD20 treatment in antitumor activities. The therapeutic function of anti-CD20 depends on tumor-specific CD8+ T-cell responses initiated by anti-CD20 through macrophages and DCs. CTLA-4 blockade can synergize with anti-CD20 to overcome adaptive immune response-related resistance in advanced B-cell lymphoma. Clin Cancer Res; 23(1); 193-203. ©2016 AACR.

Immunosuppressive Myeloid Cells’ Blockade in the Glioma Microenvironment Enhances the Efficacy of Immune-Stimulatory Gene Therapy

Survival of glioma (GBM) patients treated with the current standard of care remains dismal. Immunotherapeutic approaches that harness the cytotoxic and memory potential of the host immune system have shown great benefit in other cancers. GBMs have developed multiple strategies, including the accumulation of myeloid-derived suppressor cells (MDSCs) to induce immunosuppression. It is therefore imperative to develop multipronged approaches when aiming to generate a robust anti-tumor immune response. Herein, we tested whether combining MDSC depletion or checkpoint blockade would augment the efficacy of immune-stimulatory herpes simplex type-I thymidine kinase (TK) plus Fms-like tyrosine kinase ligand (Flt3L)-mediated immune stimulatory gene therapy. Our results show that MDSCs constitute >40% of the tumor-infiltrating immune cells. These cells express IL-4Rα, inducible nitric oxide synthase (iNOS), arginase, programmed death ligand 1 (PDL1), and CD80, molecules that are critically involved in antigen-specific Tcell suppression. Depletion of MDSCs strongly enhanced the TK/Flt3L gene therapy-induced tumor-specific CD8 Tcell response, which lead to increased median survival and percentage of long-term survivors. Also, combining PDL1 or CTLA-4 immune checkpoint blockade greatly improved the efficacy of TK/Flt3L gene therapy. Our results, therefore, indicate that blocking MDSC-mediated immunosuppression holds great promise for increasing the efficacy of gene therapy-mediated immunotherapies for GBM.

HLA Ligandome Analysis of Different Hematological Malignancies Identifies a Small Panel of "Pan-Leukemia"-Associated Antigens

Effective antigen-specific T-cell-based cancer immunotherapy requires exact knowledge of tumor-associated epitopes that can act as rejection antigens. While the current paradigm views mutation-derived neoantigens as the most promising targets, we have recently demonstrated that tumor-specific T-cell responses target panels of non-mutated tumor-associated self antigens in patients with hematological malignancies. Using the approach of direct HLA ligandome analysis by mass spectrometry, we were able to identify and characterize multiple immunogenic and naturally presented tumor-associated antigens for chronic lymphocytic leukemia (CLL, Kowalewski et. al., PNAS 2015), acute myeloid leukemia (AML, Berlin/Kowalewski et. al., Leukemia 2014), multiple myeloma (MM, Walz/Stickel et. al., Blood 2015) and chronic myeloid leukemia (CML, unpublished data).In this project we performed a comprehensive meta-analysis of our HLA ligandome data from different hematological malignancies (HM) to screen for the existence of "pan-leukemia" antigens for the broad application in T-cell based immunotherapy approaches in hematological malignancies.In a first step we performed unsupervised cluster analyses to identify similarities and differences in the HLA ligandome landscape of HM. To avoid skewed clustering due to HLA types of the samples, these analyses were performed specifically for the most common HLA allotypes in our datasets (A*02 (n=46 HM), A*03 (n=28 HM)). Distinct clustering was shown for the different entities (CLL, MM, CML, AML) as well as for the lymphoid versus myeloid malignancies on the HLA ligandome level. To identify leukemia-exclusive HLA ligands we compared the HLA ligandomes of CLL (HLA class I, n=35; HLA class II, n=30), AML (HLA class I, n=19; HLA class II, n=20), MM (HLA class I, n=15; HLA class II n=12) and CML (HLA class I, n=16; HLA class II n=15) with our normal tissue database including 153 HLA class I and 82 HLA class II ligandomes of various normal tissues (including normal blood, bone marrow and spleen). Cluster analysis of the leukemia-exclusive antigens showed identical clustering of the different entities and lymphoid/myeloid malignancies as shown before for the whole HLA ligandome and the respective source proteins. Overlap analysis revealed only 0.6% (16/2,716) and 0.3% (10/3,141) of the identified leukemia-exclusive HLA class I and class II antigens, respectively, to be represented across all analyzed hematological malignancies. These "pan-leukemia" antigens (n=26) include candidate antigens associated with T-cell activation (HSH2D), lymphoid development (IL2RF) and oncogenesis (LYN protooncogene, RAB5A). However, none of these "pan-leukemia" antigens shows frequent representation (>20%) across all 4 entities (CLL, AML, MM, CML). Furthermore, none of the "pan-leukemia" source proteins yielded corresponding peptides represented in all entities. To identify "pan-leukemia" HLA ligands, overlap analyses were performed in an allotype-specific fashion for the most frequent HLA allotypes (HLA-A*01, -A*02, -A*03, -A*24, -B*07, -B*08, -B*18) in our cohort. 0% (0/92) of HLA-A*01-, 1.6% (12/744) of HLA-A*02-, 1.4% (8/561) of HLA-A*03-, 0% (0/331) of HLA-A*24-, 0.1% (1/830) of HLA-B*07-, 0% (0/472) of HLA-B*08- and 0.8% (5/600) of the HLA-B*18-restricted peptides showed representation in all four entities. Out of these 26 "pan-leukemia" HLA ligands, only two (1 HLA-A*02-, 1 HLA-A*03-restricted peptide) showed frequent representation (>20%) in all entities. These peptides represent "pan-leukemia" targets that might be used for immunotherapeutic approaches in patients expressing the respective HLA allotype.Taken together, our approach of direct HLA ligandome analysis of hematological malignancies identified a small panel of "pan-leukemia"- proteins and peptides that show cancer-exclusive representation across all 4 included hematological malignancies. However, due to the low presentation frequencies of the candidate targets within the different entities, target discovery and compound development for the immunotherapy of HM may be more effectively achieved in an entity-specific or even patient-individualized manner. DisclosuresKowalewski:Immatics Biotechnologies GmbH: Employment. Schuster:Immatics Biotechnologies GmbH: Employment. Brümmendorf:Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Niederwieser:Novartis Oncology Europe: Research Funding, Speakers Bureau; Amgen: Speakers Bureau. Weisel:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Onyx: Consultancy; BMS: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Novartis: Honoraria.

Crystal clear: visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies.

Antibody-based PD-1/PD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre-existing T-cell function to modulate antitumor immunity. In this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-1/PD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural information will benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

Tailored Tumor Immunogenicity Reveals Regulation of CD4 and CD8 T Cell Responses against Cancer

CD4 and CD8 Tcells play a pivotal role in controlling tumor growth. However, the interplay of both cell types and their role in tumor suppression still remain elusive. In this study, we investigated the regulation of CD4 and CD8 Tcell responses to different classes of tumor-specific antigens in liver cancer mouse models. Tumors were induced in p19Arf-deficient mice by hydrodynamic injection of transposon plasmids encoding NrasG12V and pre-defined tumor antigens. This allowed for assessing the regulation oftumor-specific CD4 and CD8 Tcell responses. We showed that MHC class I tumor immunogenicity was essential to trigger tumor-directed CD4 Tcells. Tumor-specific CD8 Tcell responses arose independently of CD4 Tcells, but they required Th1-polarized CD4 Tcells for efficient tumor suppression. Our results further indicate that the immune system isincapable of eliciting sufficient numbers of Tcells directed against antigens derived from immunoedited tumors, which consequently leads to a lack ofT-cell-mediated tumor suppression in untreated hosts.

Abstract IA24: New frontiers in oncolytic virus therapy

Abstract After decades of research, oncolytic viruses have finally demonstrated clinical efficacy against human tumors. Talimogene laherparepvec is a first-in-class, injectable oncolytic virus derived from herpes simplex virus (HSV) type 1. In talimogene laherparepvec, the HSV-1 is modified through deletion of two non-essential viral genes. Deletion of the neurovirulence factor genes (ICP34.5) leads to preferential replication of the virus in tumor cells as compared with normal cells, while deletion of the ICP47 gene is designed to reduce virus-mediated suppression of antigen presentation. In addition, insertion of a GM-CSF cassette induces local GM-CSF production and is intended to recruit and activate antigen-presenting cells, ultimately stimulating systemic tumor-specific T-cell responses. Directly administered into accessible lesions, talimogene laherparepvec is therefore designed to have a dual mechanism of action, namely tumor lysis of injected lesions and the induction of a systemic antitumor immune response. In a randomized, phase III trial comparing talimogene laherparepvec therapy with subcutaneously administered GM-CSF in patients with injectable melanoma that was not surgically resectable, the durable response rate (objective response lasting continuously ≥ 6 months) was 16.3% in patients receiving talimogene laherparepvec and 2.1% in those receiving GM-CSF (1). However, the mechanism by which talimogene laherparepvec may induce responses in uninjected, distant lesions has not been fully elucidated. To investigate this question, we conducted preclinical experiments using various syngeneic models in which both injected and uninjected tumors in a single mouse could be evaluated. These studies indicated that, as anticipated, a high proportion of lesions that were directly injected with OncoVexmGM-CSF, an HSV-1 modified similarly to talimogene laherparepvec except that murine GM-CSF is used in lieu of human GM-CSF, underwent complete regression. A proportion of distant, uninjected tumors also regressed, and these lesions demonstrated significant immune cell infiltrates, with efficacy dependent on the generation of systemic antitumor CD8-positive T cells. There was no evidence of viral replication in any distant tumors, consistent with findings from other preclinical models. Ongoing biomarker studies in clinical trials are intended to confirm and extend these findings, including investigation of potential markers of response or resistance to talimogene laherparepvec monotherapy. In addition to its use as a single agent, there is interest in combining talimogene laherparepvec with other immunotherapeutic agents. Preclinical experiments using tumor cell lines both susceptible and resistant to talimogene laherparepvec monotherapy suggest that the addition of either anti-CTLA-4 or anti-PD-1 antibodies significantly increases the proportion of mice that are “cured.” Preliminary data from early-phase clinical trials in advanced melanoma demonstrate that talimogene laherparepvec can be safely combined with ipilimumab or pembrolizumab, with evidence of antitumor effect, including complete responses. A randomized, double-blind phase III trial of talimogene laherparepvec in combination with either pembrolizumab or placebo in patients with advanced melanoma is actively recruiting patients and will provide significant insight into the effectiveness of this combination. Based on its mechanism of action, talimogene laherparepvec may have utility in tumor types beyond melanoma. An ongoing phase I trial is examining the safety of talimogene laherparepvec when injected into hepatic metastases of various solid tumors, and, once an acceptable safety profile has been demonstrated and a dose established, combination trials with checkpoint inhibitors are planned. One key question is whether tumors that are generally resistant to checkpoint inhibition can be rendered sensitive with the addition of talimogene laherparepvec.