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Background The aim of this study is to profile the cytokines and immune cells of body fluid from metastatic gastric cancer (mGC), and evaluate the potential role as a prognostic factor and the feasibility as a predictive biomarker or monitoring source for immune checkpoint inhibitor. Methods Body fluid including ascites and pleural fluid were obtained from 55 mGC patients and 24 matched blood. VEGF-A, IL-10, and TGF-β1 were measured and immune cells were profiled by fluorescence assisted cell sorting (FACS). Results VEGF-A and IL-10 were significantly higher in body fluid than in plasma of mGC. Proportion of T lymphocytes with CD69 or PD-1, memory T cell marked with CD45RO, and number of Foxp3+ T regulatory cells (Tregs) were significantly higher in body fluid than those in blood of mGC. Proportion of CD8 T lymphocyte with memory marker (CD45RO) and activation marker (HLA-DR), CD3 T lymphocyte with PD-1, and number of FoxP3+ Tregs were identified as independent prognostic factors. When patients were classified by molecular subgroups of primary tumor, VEGF-A was significantly higher in genomically stable (GS)-like group than that in chromosomal instability (CIN)-like group while PD-L1 positive tumor cells (%) showed opposite results. Monitoring immune dynamics using body fluid was also feasible. Early activated T cell marked with CD25 was significantly increased in chemotherapy treated group. Conclusions By analyzing cytokines and proportion of immune cells in body fluid, prognosis of patients with mGC can be predicted. Immune monitoring using body fluid may provide more effective treatment for patients with mGC. Electronic supplementary material The online version of this article (10.1186/s40425-019-0708-8) contains supplementary material, which is available to authorized users.

ObjectiveMucosal-associated invariant T (MAIT) cells are innate T cells with immunoregulatory activity and were recently found to be associated with various tumor types. The role of intrasinusoidal MAIT cells in hepatocellular carcinoma (HCC) has not been fully characterized.DesignPeripheral blood samples were obtained from patients with HCC and healthy controls. Liver-associated mononuclear cells (LMCs) were collected from liver perfusions of donors and patients with HCC undergoing liver transplantation. Blood and liver perfusates from patients with HCC were analyzed by flow cytometry for CD3 +CD161+Vα7.2+MAIT cell frequency, phenotype, and function.ResultsThere were fewer MAIT cells in the peripheral blood and liver of patients with HCC than in the healthy controls. Interferon-γ (IFN-γ) production by these cells was also reduced. Peripheral MAIT cells showed upregulation of HLA-DR (Human Leukocyte Antigen DR） and the inhibitory molecule PD-1 (Programmed Cell Death Protein 1), but no significant differences in upregulation were found in intrasinusoidal MAIT cells. MAIT cells were significantly enriched in the liver relative to that in the peripheral blood of patients with HCC. High levels of activation markers and exhaustion markers including HLA-DR, CD69, and PD-1 were observed in LMCs of patients with HCC but not in the peripheral blood. Single-cell RNA sequencing revealed that intrasinusoidal MAIT cells exhibited distinct features in patients with HCC and the controls.ConclusionOur study showed that alterations in MAIT cells are associated with HCC. The distinct activity and function of MAIT cells in the peripheral blood and liver of patients with HCC might suggest a potential role of these cells in disease pathogenesis.

BackgroundImmTAC molecules are bispecific fusion proteins consisting of an affinity-enhanced T cell receptor fused to an anti-CD3 effector that can redirect T cells to target cells. Tebentafusp, a gp100-directed ImmTAC, has demonstrated survival benefit in metastatic uveal melanoma [1]. We previously reported that a tumor microenvironment with a high immunosuppressive CD163+ tumor-associated macrophage (TAM): CD3 T cell ratio was associated with reduced benefit from tebentafusp [2]. Here, we explored whether IL-2 could potentiate T cells to overcome inhibition of ImmTAC-mediated killing by TAM-like M2 macrophages.MethodsTumor biopsies from a Phase 2 trial of metastatic uveal melanoma HLA-A*02:01+ patients treated with tebentafusp (NCT02570308) were used to quantify CD163+ TAMs and CD3+ T cells by immunohistochemistry (N=107) and to measure gene expression by bulk RNAseq (N=70). Pro-inflammatory M1 and TAM-like M2 macrophages were generated in vitro from healthy donors (N=5) and their effect on ImmTAC-mediated T cell activation and tumor killing was assessed against THP-1 tumor cells. T cells were untreated or pre-treated for 4 days with commercially sourced IL-2 or IL-15.ResultsIn vitro, ImmTAC-mediated T cell killing of tumor cells was reduced by 85±5% in the presence of TAM-like M2 but not M1 macrophages. Consistent with this finding, below median CD163:CD3 ratio was associated with greater tumor shrinkage (TS) (odds ratio OR=2.9, p=0.014) and longer overall survival (OS) (hazard ratio HR=0.4, p=0.001) in tebentafusp-treated patients. We next explored in vitro whether the T cell activating cytokines IL-2 and IL-15 could overcome TAM-mediated inhibition of T cell redirection by ImmTAC. At clinically relevant doses, IL-2 but not IL-15 treatment resulted in dose dependent restoration of ImmTAC-mediated T cell killing of tumor cells in the presence of TAM-like M2 macrophages—60% and 83% restoration of killing at 50 and 150 U/ml of IL-2, respectively. Consistent with this observation, increased expression of IL2RB (HR 0.3, pConclusionsLow CD163+ TAM to CD3 T cell ratio and high IL2RB/G expression in tumors at baseline were associated with longer OS and greater TS in tebentafusp-treated metastatic uveal melanoma patients in a Phase 2 trial. In vitro, TAM-like M2 macrophages suppressed ImmTAC-mediated T cell killing of tumor cells, an effect abrogated by IL-2. These observations provide strong rationale for combining IL-2 biased to IL2RB/G with ImmTAC molecules to enhance benefit in tumors with high levels of TAMs.Trial RegistrationNCT02570308ReferencesPiperno-Neumann S, Hassel JC, Rutkowski P et al. Abstract CT002: Phase 3 randomized trial comparing tebentafusp with investigator’s choice in first line metastatic uveal melanoma. Cancer Res. 2021; 81 (13 Supplement) CT002.Hassel JC, Benlahrech A, Stanhope S, et al. Abstract 1673: Uveal melanoma study patients with low CD163:CD3 ratio in tumor biopsy and low serum IL-6 showed enhanced tumor shrinkage (TS) and overall survival (OS) on tebentafusp. Cancer Res. 2021; 81 (13 Supplement) 1673.Ethics ApprovalThe Oxford A REC approved protocol 13/SC/0226 was used to obtain written consent for all blood donations and was fully approved by the National Research Ethics Committee South Central.

BackgroundTriple negative breast cancer (TNBC) is a subtype of breast cancers with poor prognosis and targeted drug therapies are limited. To develop novel and efficacious therapies for TNBC, we developed a bispecific antibody F7AK3 that recognizes both trophoblast cell surface antigen 2 (TROP2) and CD3 and evaluated its antitumor activities both in vitro and in vivo.MethodsThe binding affinities of F7AK3 to the two targets, TROP2 and CD3, were evaluated by surface plasmon resonance. Binding of F7AK3 to TNBC cells and T cells were evaluated by flow cytometry. Immunofluorescent staining was performed to demonstrate the interactions between T cells with TNBC cells. The cytotoxicity of T cells against TNBC cell lines and primary tumor cells mediated by F7AK3 were determined in vitro. In vivo antitumor activity of F7AK3 was investigated in a xenograft TNBC tumor model, using immunodeficient mice that were reconstituted with human peripheral blood mononuclear cells.ResultsWe demonstrated that F7AK3 binds specifically to human TROP2 and CD3 antigens, as well as TNBC cell lines and primary tumor cells. Human T cells can only be activated by F7AK3 in the presence of target tumor cells. F7AK3 recruits T cells to TROP2+ tumor cells in vitro and into tumor tissues in vivo. Antitumor growth activity of F7AK3 is observed in a xenograft TNBC tumor model.ConclusionThis study showed the antitumor potential of an anti-TROP2xCD3 bispecific antibody F7AK3 to TNBC tumor cells both in vitro and in vivo. These data demonstrate that F7AK3 has the potential to treat TNBC patients, which warrants further preclinical and clinical evaluation of the F7AK3 in advanced or metastatic TNBC patients.

Despite unique genetic alterations within brain metastases (BrMs) and an immunologically distinct surrounding microenvironment, the composition and functional properties of tumor-infiltrating lymphocytes within BrM remain largely unexplored. In particular, the expression of coinhibitory receptors, such as programmed cell death 1 (PD-1), T cell immunoglobulin mucin receptor 3 (TIM-3), and lymphocyte activation gene 3 (LAG-3), within BrMs is unknown. Using multiplexed quantitative immunofluorescence (QIF), this study evaluates the localized expression of PD-L1, level and functional profile of major T cell subsets, and coinhibitory receptors within lung cancer-associated BrMs and primary lung tumors. Clinicopathologically annotated samples from 95 patients with lung cancer between 2002 and 2015 were represented in a tissue microarray format. Spatially resolved and multiplexed QIF was used to evaluate PD-L1 protein, phenotype markers for major T cell subsets (CD3, CD4, CD8, and FOXP3), cell-localized activation and proliferation markers (granzyme B and Ki67), and coinhibitory receptors (PD-1, LAG-3, and TIM-3). The signal for each marker was measured in marker-selected tissue compartments, and associations between marker levels, tumor location, and major clinicopathological variables were studied. In total, 41 primary lung tumors and 65 BrMs were analyzed, including paired samples from 11 patients. Levels of tumor PD-L1 expression were comparable between BrMs and primary lung tumors. BrMs had significantly lower levels of all T cell subsets relative to primary lung tumors, and T cells in BrMs displayed lower levels of granzyme B than primary lesions. PD-1, TIM-3, and LAG-3 levels in CD3+ T-cells were also significantly lower in BrMs. Marker expression in patients with paired samples from BrMs and primary lung tumors showed comparable results. High CD3+ T-cells, as well as high levels of TIM-3 and LAG-3 in CD3+ T-cells, were associated with longer overall survival in BrMs but not primary lung tumors. Lung cancer-associated BrMs display lower T cell infiltration, markers of cytolytic function, and immune regulatory signals than primary lung tumors. Despite these differences, high TIM-3 and high LAG-3 expressions in CD3+ T-cells were associated with longer survival. These features are accompanied by comparable levels of PD-L1 protein expression compared with primary lung tumors. These results highlight unique aspects of the tumor immune microenvironment within the brain and provide further support for intracranially focused therapies.

BackgroundBispecific T cell engagers represent the majority of bispecific antibodies (BsAbs) entering the clinic to treat metastatic cancer. The ability to apply these agents safely and efficaciously in the clinic, particularly for solid tumors, has been challenging. Many preclinical studies have evaluated parameters related to the activity of T cell engaging BsAbs, but many questions remain.Main bodyThis study investigates the impact of affinity of T cell engaging BsAbs with regards to potency, efficacy, and induction of immunomodulatory receptors/ligands using HER-2/CD3 BsAbs as a model system. We show that an IgG BsAb can be as efficacious as a smaller BsAb format both in vitro and in vivo. We uncover a dichotomous relationship between tumor-associated antigen (TAA) affinity and CD3 affinity requirements for cells that express high versus low levels of TAA. HER-2 affinity directly correlated with the CD3 engager lysis potency of HER-2/CD3 BsAbs when HER-2 receptor numbers are high (~200 K/cell), while the CD3 affinity did not impact potency until its binding affinity was extremely low (3 mg/kg. This data confirms that low CD3 affinity provides an opportunity for improved safety and dosing for T cell engaging BsAbs. T cell redirection also led to upregulation of Programmed cell death 1 (PD-1) and 4-1BB, but not CTLA-4 on T cells, and to Programmed death-ligand 1 (PD-L1) upregulation on HER-2HI SKOV3 tumor cells, but not on HER-2LO OVCAR3 tumor cells. Using this information, we combined anti-PD-1 or anti-4-1BB monoclonal antibodies with the HER-2/CD3 BsAb in vivo and demonstrated significantly increased efficacy against HER-2HI SKOV3 tumors via both combinations.ConclusionsOverall, these studies provide an informational dive into the optimization process of CD3 engaging BsAbs for solid tumors indicating that a reduced affinity for CD3 may enable a better therapeutic index with a greater selectivity for the target tumor and a reduced cytokine release syndrome. These studies also provide an additional argument for combining T cell checkpoint inhibition and co-stimulation to achieve optimal efficacy.

BackgroundPoor infiltration and limited activation of transferred T cells are fundamental factors impeding the development of adoptive cell immunotherapy in solid tumors. A tumor-penetrating peptide iRGD has been widely used to deliver drugs deep into tumor tissues. CD3-targeting bispecific antibodies represent a promising immunotherapy which recruits and activates T cells.MethodsT-cell penetration was demonstrated in tumor spheroids using confocal microscope, and in xenografted tumors by histology and in vivo real-time fluorescence imaging. Activation and cytotoxicity of T cells were assessed by flow cytometry and confocal microscope. Bioluminescence imaging was used to evaluate in vivo antitumor effects, and transmission electron microscopy was used for mechanistic studies.ResultsWe generated a novel bifunctional agent iRGD-anti-CD3 which could immobilize iRGD on the surface of T cells through CD3 engaging. We found that iRGD-anti-CD3 modification not only facilitated T-cell infiltration in 3D tumor spheroids and xenografted tumor nodules but also induced T-cell activation and cytotoxicity against target cancer cells. T cells modified with iRGD-anti-CD3 significantly inhibited tumor growth and prolonged survival in several xenograft mouse models, which was further enhanced by the combination of programmed cell death protein 1 (PD-1) blockade. Mechanistic studies revealed that iRGD-anti-CD3 initiated a transport pathway called vesiculovacuolar organelles in the endothelial cytoplasm to promote T-cell extravasation.ConclusionAltogether, we show that iRGD-anti-CD3 modification is an innovative and bifunctional strategy to overcome major bottlenecks in adoptive cell therapy. Moreover, we demonstrate that combination with PD-1 blockade can further improve antitumor efficacy of iRGD-anti-CD3-modified T cells.

BackgroundImmunotherapies may prolong the survival of patients with small-cell lung cancer (SCLC) to some extent. The role of forkhead box protein P3 (FOXP3) in tumor microenvironment (TME) remains controversial. We aimed to examine FOXP3-related expression characteristics and prognostic values and to develop a clinically relevant predictive system for SCLC.MethodsWe enrolled 102 patients with histologically confirmed SCLC at stages I–III. Through immunohistochemistry, we determined the expression pattern of FOXP3 and its association with other immune biomarkers. By machine learning and statistical analysis, we constructed effective immune risk score models. Furthermore, we examined FOXP3-related enrichment pathways and TME traits in distinct cohorts.ResultsIn SCLC, FOXP3 level was significantly associated with status of programmed death-ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), CD4, CD8, and CD3 (p=0.002, p=0.001, p=0.002, p=0.030, and pCD28, basic excision repair genes MUTYH, POLD1, POLD2, and oxidative phosphorylation related gene cytochrome c oxidase subunit 8A with FOXP3 expression were revealed. Moreover, we found low-immune risk score group had statistically higher activated CD4+ memory T cells (p=0.014) and plasma cells (p=0.049) than the high-risk group. The heterogeneity of tumor-infiltrating immune cells might represent a promising feature for risk prediction in SCLC.ConclusionFOXP3 interacts closely with immune biomarkers on tumor-infiltrating cells in TME. This study highlighted the crucial prognostic value and promising clinical applications of FOXP3 in SCLC.

Background Interleukin(IL)-30/IL-27p28 production by Prostate Cancer (PC) Stem-Like Cells (SLCs) has proven, in murine models, to be critical to tumor onset and progression. In PC patients, IL-30 expression by leukocytes infiltrating PC and draining lymph nodes correlates with advanced disease grade and stage. Here, we set out to dissect the role of host immune cell-derived IL-30 in PC growth and patient outcome. Methods PC-SLCs were implanted in wild type (WT) and IL-30 conditional knockout (IL-30KO) mice. Histopathological and cytofluorimetric analyses of murine tumors and lymphoid tissues prompted analyses of patients’ PC samples and follow-ups. Results Implantation of PC-SLCs in IL-30KO mice, gave rise to slow growing tumors characterized by apoptotic events associated with CD4+T lymphocyte infiltrates and lack of CD4+Foxp3+ T regulatory cells (Tregs). IL-30 knockdown in PC-SLCs reduced cancer cell proliferation, vascularization and intra-tumoral Indoleamine 2,3-Dioxygenase (IDO)+CD11b+Gr-1+ myeloid-derived cells (MDCs) and led to a significant delay in tumor growth and increase in survival. IL-30-silenced tumors developed in IL-30KO mice, IL-30−/−tumors, lacked vascular supply and displayed frequent apoptotic cancer cells entrapped by perforin+TRAIL+CD3+Tlymphocytes, most of which had a CD4+T phenotype, whereas IL-10+TGFβ+Foxp3+Tregs were lacking. IL-30 silencing in PC-SLCs prevented lung metastasis in 73% of tumor-bearing WT mice and up to 80% in tumor-bearing IL-30KO mice. In patients with high-grade and locally advanced PC, those with IL-30−/−tumors, showed distinct intra-tumoral cytotoxic granule-associated RNA binding protein (TIA-1)+CD4+Tlymphocyte infiltrate, rare Foxp3+Tregs and a lower biochemical recurrence rate compared to patients with IL-30+/+tumors in which IL-30 is expressed in both tumor cells and infiltrating leukocytes. Conclusion The lack of host leukocyte-derived IL-30 inhibits Tregs expansion, promotes intra-tumoral infiltration of CD4+T lymphocytes and cancer cell apoptosis. Concomitant lack of MDC influx, obtained by IL-30 silencing in PC-SLCs, boosts cytotoxic T lymphocyte activation and cancer cell apoptosis resulting in a synergistic tumor suppression with the prospective benefit of better survival for patients with advanced disease. Electronic supplementary material The online version of this article (10.1186/s40425-019-0668-z) contains supplementary material, which is available to authorized users.

Background We previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFβ blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFβ blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP). Methods The original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression. Results At baseline, a significantly higher level of expression (p

Background The tumor microenvironment (TME) is a complex mixture of tumor epithelium, stroma and immune cells, and the immune component of the TME is highly prognostic for tumor progression and patient outcome. In lung cancer, anti-PD-1 therapy significantly improves patient survival through activation of T cell cytotoxicity against tumor cells. Direct contact between CD8+ T cells and target cells is necessary for CD8+ T cell activity, indicating that spatial organization of immune cells within the TME reflects a critical process in anti-tumor immunity. Current immunohistochemistry (IHC) imaging techniques identify immune cell numbers and densities, but lack assessment of cell–cell spatial relationships (or “cell sociology”). Immune functionality, however, is often dictated by cell-to-cell contact and cannot be resolved by simple metrics of cell density (for example, number of cells per mm2). To address this issue, we developed a Hyperspectral Cell Sociology technology platform for the analysis of cell–cell interactions in multi-channel IHC-stained tissue. Methods Tissue sections of primary tumors from lung adenocarcinoma patients with known clinical outcome were stained using multiplex IHC for CD3, CD8, and CD79a, and hyperspectral image analysis determined the phenotype of all cells. A Voronoi diagram for each cell was used to approximate cell boundaries, and the cell type of all neighboring cells was identified and quantified. Monte Carlo analysis was used to assess whether cell sociology patterns were likely due to random distributions of the cells. Results High density of intra-tumoral CD8+ T cells was significantly associated with non-recurrence of tumors. A cell sociology pattern of CD8+ T cells surrounded by tumor cells was more significantly associated with non-recurrence compared to CD8+ T cell density alone. CD3+ CD8- T cells surrounded by tumor cells was also associated with non-recurrence, but at a similar significance as cell density alone. Cell sociology metrics improved recurrence classifications of 12 patients. Monte Carlo re-sampling analysis determined that these cell sociology patterns were non-random. Conclusion Hyperspectral Cell Sociology expands our understanding of the complex interplay between tumor cells and immune infiltrate. This technology could improve predictions of responses to immunotherapy and lead to a deeper understanding of anti-tumor immunity. Electronic supplementary material The online version of this article (10.1186/s40425-018-0488-6) contains supplementary material, which is available to authorized users.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with short overall survival; the standard of care (SoC) is chemotherapy. Immunotherapies in development aim to remodel the stroma by depleting immunosuppressive cell types or using T-cell redirection to kill tumor cells. To date, none of these methods have improved overall survival beyond SoC. Next generation immunotherapies that employ histopathology and molecular subtyping1 for target and patient selection may succeed. Here we leverage a spatial transcriptomics platform (Nanostring Digital Spatial Profiling, DSP) to reveal molecular signaling in tumoral and stromal cells in 57 PDAC patients using tumor microarrays (TMAs). This approach is rapid and clinically relevant as molecular and histology data can be easily bridged.MethodsTMAs generated from surgical resection tissue were commercially sourced. DSP was performed using the CTA RNA panel (1,800 target genes) using PanCK fluorescence for tumor/stroma segmentation. In parallel, slides were chromogenically stained for T-cells (CD3) and macrophages (CD68/CD163). Differential gene expression, gene signature and gene co-expression network analysis was performed using linear models in R.2 3ResultsDifferential gene expression analysis and correlation to IHC confirmed the DSP platform successfully profiled tumor and stromal compartments (figure 1). Immune cell signatures4 and pathway analysis revealed a heterogenous stromal environment. Using a fibroblast gene signature derived from single-cell RNAseq5 we found fibroblast density was positively correlated to PDGFR signaling and MHC-II expression but negatively correlated to B, CD4+ T and neutrophil cell levels (figure 2a). This finding supports the idea that atypical antigen presentation in cancer associated fibroblasts (CAFs) may be exploitable for immunotherapies.6 We constructed a co-expression network from in-situ stromal gene expression and used it to identify receptors coordinately expressed with the immunosuppressive macrophage marker CSF1R as a bispecific antibody partner (figure 2b).7 Classical macrophage markers were identified but also receptors with lesser-known functions in macrophages (TIM3/HAVCR2, FPR3, MS4A6A, LILRB4). Surveying target pairs in this method allows rapid, patient-specific confirmation in serial TMA sections with singleplex IHC or RNAscope.Abstact 928 Figure 1Segmentation strategy and validation of DSP (A) PanCK, CD68 and CD3 staining from two representative tumor cores; (B, C) correlation of gene transcripts in stroma to cell counts from chromogenic staining; (D) heatmap of selected genes differentially expressed in tumor and stroma (n=57 patients).Abstract 928 Figure 2Exploration of the stromal compartment in PDAC TMAs. (A) Heatmap of selected cell type and gene signatures from gene expression in the stroma, color represents single sample enrichment score using GSVA method; (B) a gene co-expression subnetwork in the stroma centered on CSF1R, edge thickness represents strength of correlation, green nodes have evidence for cell surface expression based on proteomic profiling.7ConclusionsIn this study we were able to recapitulate known PDAC biology using very small samples of primary tumors. The combination of TMAs and DSP enables a rapid validation of targets and hypothesis generation for bispecific parings. Further analysis of untreated (n=14) and post-adjuvant chemotherapy (n=26) patients using RNA DSP, IHC and bulk RNAseq is under way. Results from this cohort will enable an integrated histopathology and molecular approach to developing next-generation immunotherapies.ReferencesCollisson EA, Bailey P, Chang DK, Biankin AV. Molecular subtypes of pancreatic cancer. Nat Rev Gastroenterol Hepatol 2019 April;16(4):207–220.Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK (2015). “limma powers differential expression analyses for RNA-sequencing and microarray studies.” Nucleic Acids Research 43(7):e47.Hänzelmann S, Castelo R, Guinney J (2013). “GSVA: gene set variation analysis for microarray and RNA-Seq data.” BMC Bioinformatics 14,7.Charoentong P, Finotello F, Angelova M, Mayer C, Efremova M, Rieder D, Hackl H, Trajanoski Z. Pan-cancer immunogenomic analyses reveal genotype-immunophenotype relationships and predictors of response to checkpoint blockade. Cell Rep 2017 January 3;18(1):248–262.Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, Rotem A, Rodman C, Lian C, Murphy G, Fallahi-Sichani M, Dutton-Regester K, Lin JR, Cohen O, Shah P, Lu D, Genshaft AS, Hughes TK, Ziegler CG, Kazer SW, Gaillard A, Kolb KE, Villani AC, Johannessen CM, Andreev AY, Van Allen EM, Bertagnolli M, Sorger PK, Sullivan RJ, Flaherty KT, Frederick DT, Jané-Valbuena J, Yoon CH, Rozenblatt-Rosen O, Shalek AK, Regev A, Garraway LA. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 2016 April 8;352(6282):189–96.Elyada E, Bolisetty M, Laise P, Flynn WF, Courtois ET, Burkhart RA, Teinor JA, Belleau P, Biffi G, Lucito MS, Sivajothi S, Armstrong TD, Engle DD, Yu KH, Hao Y, Wolfgang CL, Park Y, Preall J, Jaffee EM, Califano A, Robson P, Tuveson DA. Cross-species single-cell analysis of pancreatic ductal adenocarcinoma reveals antigen-presenting cancer-associated fibroblasts. Cancer Discov 2019 August;9(8):1102–1123. Bausch-Fluck D, Hofmann A, Bock T, Frei AP, Cerciello F, Jacobs A, Moest H, Omasits U, Gundry RL, Yoon C, Schiess R, Schmidt A, Mirkowska P, Härtlová A, Van Eyk JE, Bourquin JP, Aebersold R, Boheler KR, Zandstra P, Wollscheid B. A mass spectrometric-derived cell surface protein atlas. PLoS One 2015 April 20;10(3):e0121314.Ethics ApprovalSpecimens were harvested from unused tissue after a surgical tumor resection procedure. A discrete legal consent form from both hospital and individuals was obtained by the commercial tissue vendor BioMax US for all samples analyzed in this abstract. All human tissues are collected under HIPPA approved protocols.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Background The pancreatic cancer vaccine, GVAX, induces novel lymphoid aggregates in the otherwise immune quiescent pancreatic ductal adenocarcinoma (PDAC). GVAX also upregulates the PD-1/PD-L1 pathway, and a pre-clinical model demonstrated the anti-tumor effects of combination GVAX and anti-PD-1 antibody therapy (GVAX/αPD-1). Resistance to GVAX was associated with an immune-suppressive myeloid cell infiltration, which may limit further therapeutic gains of GVAX/αPD-1 therapy. The expression of CSF-1R, a receptor important for myeloid cell migration, differentiation and survival, and the effect of its therapeutic blockade in the context of GVAX in PDAC has not been investigated. Methods Lymphoid aggregates appreciated in 24 surgically resected PDAC from patients who received one dose of neoadjuvant GVAX were analyzed with multiplex immunohistochemistry. Flow cytometry analysis of tumor infiltrating T-cells in a murine model of PDAC was performed to investigate the therapeutic effects and mechanism of anti-CSF-1R/anti-PD-1/GVAX combination immunotherapy. Results High CSF-1R expression in resected PDAC from patients who received neoadjuvant GVAX was associated with a higher myeloid to lymphoid cell ratio (p

Background Immune mobilising TCRs against cancer (ImmTAC) are a novel class of soluble bispecifics which bind both CD3 on T cells and specific peptide/MHC antigen on tumour cells.1 Four ImmTAC molecules are currently in clinical trials, one of these being tebentafusp which targets a gp100-derived HLA-A*02:01 peptide. Tebentafusp has demonstrated monotherapy activity in cutaneous and uveal melanoma.2 3 Here, we explored approaches to enhance ImmTAC-mediated T cell activation and anti-tumour activity. We hypothesised that the cytokine IL-2, which is approved for treatment of cutaneous melanoma and renal cell cancer, might augment ImmTAC-mediated T cell responses.4 Methods Tumour cell killing was determined by IncuCyte assay. Cell lines, healthy donor PBMC, tumour cell populations and cytokines including IL-2 (Proleukin, Novartis) were sourced commercially. ImmTAC molecules recognising peptides from either gp100 or the tumour antigen PRAME5 were used for in vitro assays. Surface marker expression was determined by flow cytometry. Results Clinically relevant IL-2 levels (60 to 600U/ml) improved both the speed (maximum killing after ~15 hours following 60 U/ml IL-2 pre-treatment versus ~30 hours without IL-2) and potency (mean EC50 13.6pM with IL-2 versus 81.2pM in absence of supplementation) of antigen-specific ImmTAC-dependent tumour killing in vitro by either PBMC or purified T cell populations. Addition of IL-2 was sufficient to improve killing responses when either T cell numbers or target peptide:HLA levels were limiting, and IL-2-cultured TILs exhibited ImmTAC-dependent killing. Dose-dependent cytokine enhancement of ImmTAC-mediated killing response was also consistent with increased surface CD3 and CD69 expression on T cell populations. Notably, weak PBMC donor populations with minimal intrinsic killing activity could be induced to provide robust ImmTAC-dependent tumour cell killing responses by physiological IL-2 treatment. Conclusions These results suggest that the anti-tumour activity of ImmTAC molecules could be enhanced by combination treatment with the cytokine IL-2. Ethics Approval The study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226 References Lowe, et al. Cancer Treat Rev 2019. Vol.77, p35–432. Middleton, et al. ( 2019) J. Clin. Oncol. 37, no. 15_suppl (May 20, 2019). Shoustari, et al. SITC poster 2019, J. Immunother Cancer 2019;7. Tang and Harding Cytokine: X 2019 Moureau, et al. ( 2020) AACR poster

Background A cancer lesion may avoid detection by the immune system through a variety of immunosupressive mechanisms involving myeloid-derived suppressor cells and regulatory T-cells. One such mechanism recently discovered is the immuno¬suppressive effect of a specific peptide, i.e., P3028, produced through degradation of albumin. In this study, involving biopsies obtained from patients with tonsillar cancer (TC), P3028 is assessed in relation to overall immune phenotype, as indicated by presence and distribution of CD8+ T-cells as well as to other specific immune cells. Methods Immunohistochemistry was performed on fresh frozen biopsies. CD8+ T-cells were used to classify the cancer lesions into immune phenotypes: ‘inflamed’ (lympho¬cytes infiltrating cancer cell areas), ‘immune excluded’ (lymphocytes in surrounding stroma, but few within cancer cell areas) and ‘desert’ (few lymphocytes in cancer cell areas and in surrounding stroma). P3028 was graded (semi-quantitatively) as high or low. Quantitative flow cytometry was performed focusing on CD8+, CD3+, and CD4+ T-cells, macrophages and dendritic cells. The study was approved by the Swedish Ethical Review Authority (no. 2017/580). Written informed consent was obtained from the patients. A copy of the written consent is available for review by the Editor of this journal. Results Based on immunohistochemistry focusing on presence and distribution of CD8+ T-cells, most TC lesions were found to be of an ‘inflamed’ immune phenotype. This particular phenotype also featured low expression of immunosuppressive peptide P3028 (cf. other immune phenotypes). Flow cytometry verified that ‘immune excluded’ and ‘inflamed’ cancer lesions were associated with high levels of CD8+ T-cells (cf. desert lesions). The presence of CD3+ and CD4+ T-cells as well as macrophages and dendritic cells in relation to immune phenotypes were indicated. Conclusions TC lesions may be classified into ‘inflamed’, ‘immune excluded’, and ‘desert’ phenotypes based on presence and distribution of CD8+ T-cells. Other immune cells may be associated with these immune phenotypes, including CD3+ and CD4+ T-cells, macro¬phages, and dendritic cells. P3028 is present in TC lesions: low levels of this immunosuppressive peptide are observed in the ‘inflamed’ phenotype. Arguably, P3028 prevents successful recruitment of immune cells in TC. Inferentially, presence and distribution of P3028 may be considered as a prognostic marker as well as a treatment target of this condition. Ethics Approval The study was approved by the Swedish Ethical Review Authority (no. 2017/580). Consent Written informed consent was obtained from the patients. A copy of the written consent is available for review by the Editor of this journal.

Background Two main approaches for T cell-based therapies involve molecular T cell redirection by CD3 bispecific molecules such as bispecific T-cell engagers (BiTE) and cellular T cell redirection by genetic modification of T cells with chimeric antigen receptors (CAR) or transgenic T cell receptors (TCR). BiTEs redirect the cytotoxic activity of endogenous polyclonal T cells by simultaneously engaging CD3 on T cells and tumor antigens on target cells. BiTE potency studies have relied on primary cells, which measure target cell killing through redirected T cell cytotoxicity (RTCC) or cytokine release. However, these primary cell-based assays suffer from high donor-to-donor variability, as well as lengthy and hard to implement protocols Methods We have recently developed a new RTCC assay and cytokine immunoassays that are simple, sensitive and can quantitatively measure the potency of BiTEs and similar biologics. In this assay, preactivated cytotoxic T cells and target cells (both in cryopreserved thaw-and-use format) stably expressing a HaloTag-HiBiT fusion protein are co-incubated with a BiTE, which results in lysis of the target cells and subsequent release of the Halotag-HiBiT protein. These HiBiT proteins then bind to extracellular LgBiT provided in the detection reagent and form functional NanoLuc Luciferase to generate luminescence. Results The assay is homogenous, highly sensitive, and has a robust assay window. Use of CAR-T has demonstrated promising results in treating leukemia, while the development of TCR-engineered T cells that can recognize intracellular tumor antigens, is still in early stages. To facilitate the screening and characterization of new transgenic TCRs, we used CRISPR/Cas9 to develop two TCRas-null reporter T cell lines, which are CD4+ or CD8+. Reintroduction of peptide-specific TCR a and s chains into TCRas-null reporter T cell lines results in peptide-dependent TCR activation and luciferase reporter expression. The select expression of CD4 or CD8 in the TCRas-null reporter T cell lines can enable the development of transgenic TCRs for both MHCI- and MHCII-restricted tumor antigen targets. Conclusions Together, these bioluminescent bioassays represent a new set of tools for the discovery and development of T cell-based immunotherapies.

Background Immunotherapy has changed the standard of care for multiple cancers; however, its efficacy is limited. Chemotherapy and radiation had little effect in pancreatic ductal adenocarcinoma (PDAC) outcome1 in patients with metastatic disease, hence the urgency for new effective courses of treatment. Increasing evidence suggests mucosal-associated invariant T-cells (MAIT) play a role in anti-cancer T-cell responses, by recognizing transformed cells or bacterial products. MAIT respond towards microbial antigens and vitamin derivatives, produce pro-inflammatory cytokines2 3 and have been found present in primary and metastatic cancer lesions.3 4 Long-term survival PDAC patients present a unique microbiome pattern. In contrast, some microbial species may promote oncogenesis.5 6The focus of this project is the characterization of MAIT as immune effector cells in PDAC specimens. Methods We performed a retrospective analysis of long-term survivors (LTS) and short-term survivors (STS) patients with pancreatic cancer associating clinical endpoints with the presence of MAIT infiltration in the tumor tissue using immunofluorescence staining for MR1 (MHC class I-related gene, a MAIT ligand receptor), CD3 and TCR Vα7.2 (frequently reported chain in MAIT). Tumor infiltrating lymphocytes (TILs) were expanded and tested for recognition of microbial products presented to TILs or to PBMCs defined by cytokine production (ELISA), cytotoxicity (CD107a induction assay), CD69 or 4-1BB upregulation (flow cytometry). Reactive MAIT will be molecularly defined by deep TCR (T-cell receptor) sequencing which allows to ‘back-trace’ MR1 reactive TIL in the tumor specimen. The complex interaction of microbial antigen presentation from freshly harvested tumor specimens to TILs is being optimized for Nanolive technology that allows to follow live cell interactions for several days. Results TIL reactivity directed against microbial products from different bacterial species was detected by IFN-γ production and CD69 upregulation in responder TILs. A broader panel of TILs is currently being tested against bacterial species. TCRs will undergo laser microdissection for subsequent TCR repertoire sequencing. A more pronounced MAIT infiltration in close vicinity to tumor cells in LTS compared to STS is being studied, further supporting the anti-tumor role of MAIT. Conclusions MAIT cells may exhibit anti-tumor properties, based on cytokine production and cellular marker activation. TCRs directed against cancer cells can serve as viable blueprints to engage with MR1 on PDAC recognizing tumor-associated targets or microbial products that elicit IFN-γ production. This allows to explore MAIT TCRs for adoptive therapies or distinct microbial species that drive clinically relevant responses. Acknowledgements The authors would like to thank to Champalimaud Foundation Biobank and Vivarium Facility at Champalimaud Foundation. Ethics Approval This study was approved by the Champalimaud Foundation Ethics Committee and by Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon. Consent For each patient, written informed consent and approval by the Ethical Committee of the Champalimaud Foundation will be obtained. The study will be in compliance with the Declaration of Helsinki. References Sideras, K. et al. Role of the immune system in pancreatic cancer progression and immune modulating treatment strategies. Cancer Treat. Rev 2014;40: 513–522. Toubal A, Nel I, Lotersztajn S & Lehuen A. Mucosal-associated invariant T cells and disease. Nat Rev. Immunol 2019;19:643–657. Lukasik Z, Elewaut D & Venken K. Mait cells come to the rescue in cancer immunotherapy?Cancers (Basel). 12, 1–19 ( 2020). Vacchini A, Chancellor A., Spagnuolo J., Mori L. & De Libero G. MR1-restricted t cells are unprecedented cancer fighters. Front Immunol 2020;11:1–8. Aykut B, et al. The fungal mycobiome promotes pancreatic oncogenesis via activation of MBL. Nature 2019:574;264–267. Pushalkar S, et al. The pancreatic cancer microbiome promotes oncogenesis by induction of innate and adaptive immune suppression. Cancer Discov 2018;8:403–416.

Background An intriguing phenomenon in many solid tumors is the de novo lymphoid neogenesis of tertiary lymphoid structure (TLS). However, it remains unclear whether spontaneous TLS restricts or promotes tumor progression. MDSCs are major contributors to tumor evasion of adaptive immune responses. We hypothesize that MDSCs repel TLS as a histological pattern in bladder cancers. Methods To perform a spatial analysis of MDSDs and other immune cell phenotypes in the bladder tumor microenvironment we used MultiOmyx™, an immunofluorescence (IF) assay utilizing a pair of directly conjugated Cyanine dye-labeled (Cy3, Cy5) antibodies per round of staining. Using a 14-marker panel and proprietary cell segmentation and classification algorithms developed at NeoGenomics we have analyzed the presence of TLS (positive for CD20, CD3, and PNAd), followed by a spatial analysis of MDSCs, T cell subtypes, and M1/M2-type tumor-associated macrophages (TAMs) in relation to the TLS in 25 FFPE samples from patients with bladder cancer. Results To test our initial hypothesis that MDSCs repel TLS in bladder cancer, we quantitated the presence of immune cell phenotypes inside the TLS, and in tissue regions near and far from the TLS. As expected we found a significantly higher density of B cells and T cells present inside the TLS compared to either near or far from the TLS. However, when quantitating MDSCs we found a linear decrease related to their proximity to the TLS, with a 25% lower density in the region near the TLS, and a 46% decrease in the regions far from the TLS. This pattern was not observed for TAMs that were found at the same density inside, near, or far from the TLS, confirming that this pattern is MDSC specific. We are currently performing further analysis of MDSCs co-expressed with either CXCR2 or iNOS for additional answers to the possible mechanisms behind this finding. Conclusions The finding on the co-abundance of MDSCs and T/B cells following the distance from TLS suggests that MDSCs may repel TLS-derived T and B cells through cell-cell interaction via physical proximity. This study has important implications for bladder cancer immunotherapy.

Background The development of immunotherapies holds great promise for the treatment of refractory infections and cancer. Current approaches, although effective in many settings, have limitations that prevent their widespread use. Hence, several aspects require improvements, including the re-wiring of T-cell fates and function. T-cell dysfunction is central to the persistence of several chronic viral infections and the progression of malignancies. Upon activation, T cells can follow several paths of differentiation, leading to terminal effector differentiation and/or exhaustion which are widely recognized as dysfunctional features limiting human immune competence. Furthermore, dysfunctional features induced during laboratory-based manipulations of T-cell products prior to adoptive cell transfer has a determining effect on outcomes. Similarly, repeated antigen encounters after transfer in vivo favors the development of T-cell dysfunction. However, the nature and underlying mechanisms of T-cell dysfunction are still incompletely understood. Methods Combining genomics, phenotypic and functional analyses in various physiologically and clinically relevant settings, we investigated the key factors leading to T-cell dysfunction. Specifically, we evaluated the impact of repeated stimulations using CD3/CD28-coated beads or antigen-loaded dendritic cells in human T-cell long term cultures, and BCMA-expressing cells for anti-BCMA CAR T cells. We also examined mouse antigen-specific T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection as well as datasets obtained from circulating T cells from acute myeloid leukemia (AML) patients. Results We identified telomere-independent cellular senescence as a central aspect of exhausted PD-1-expressing T cells following repeated stimulations. Mechanistically, it is associated the induction of p16INK4a. Additionally, we found that cellular senescence features are partly regulated by the activation of caspase-8, through a non-apoptotic function of this molecule not previously described in T cells. Conclusions We thus conclude that caspase-8 may regulate the balance between apoptosis and proliferation by protecting T cells from cellular senescence. Senescence-associated mechanisms may be seen as key players in T-cell dysfunction occurring following repeated stimulations and as such should be considered as novel immune checkpoints impeding the success of T-cell adoptive immunotherapy in humans. Ethics Approval This study was approved by the local Maisonneuve-Rosemont Hospital research ethics authorities and participants’ informed consent was obtained (CER2020-2141 and CER13030).

Background Metastatic castration-resistant prostate cancer (mCRPC) and Ewing sarcoma (EWS) are diseases for which immune therapies could potentially provide benefit. STEAP1 (Six Transmembrane Epithelial Antigen of the Prostate 1) is a cell surface protein with elevated expression in mCRPC 1 and EWS.2 Methods We designed AMG 509, a novel, half-life extended, STEAP1 x CD3 XmAb® 2+1 bispecific antibody to induce T cell-mediated cytotoxicity against STEAP1-expressing cancer cells. AMG 509 contains two identical anti-STEAP1 Fab domains, an anti-CD3 scFv domain, and an effectorless Fc domain that extends serum half-life. We characterized STEAP1 expression in normal and tumor tissues by immunohistochemistry, and we assessed the pharmacological properties of AMG 509 including binding, T cell-mediated redirected lysis, and in vivo antitumor activity. Results We detected high STEAP1 surface expression on 80% of primary prostate tumors (n=88), 89% of mCRPC lesions (n=114), including 84% of mCRPC bone metastases (n=31), and 63% of EWS samples (n=35). In contrast, in normal tissues (n=72), low STEAP1 expression was detected in only six other tissues, including the normal prostate. AMG 509 bound to recombinant human CD3e with a KD of 27.6 nM, and it bound specifically to 293T cells transfected with human STEAP1 with an EC50 of 3.8 nM. AMG 509 triggered potent T cell-redirected lysis of STEAP1-positive cancer cells, with a median EC50 of 37 pM across 19 cancer cell lines that endogenously express various levels of STEAP1. AMG 509-mediated cytotoxicity was specific, as it showed no activity against prostate cancer cells in which STEAP1 was knocked out. AMG 509 was 65-fold more potent in inducing the redirected lysis of prostate cancer cells in vitro than an XmAb® molecule with a single anti-STEAP1 Fab domain. AMG 509 had greater cytotoxic activity against high STEAP1-expressing cancer cells than against low STEAP1-expressing cancer cells, and it had minimal activity against normal cells. This preferential killing of high STEAP1-expressing cells is likely driven by the avidity conferred by the dual STEAP1-binding domains, a feature that may help reduce off-target effects in the clinic. In vivo, AMG 509 induced robust anti-tumor activity in prostate cancer and EWS mouse xenograft models, with concomitant CD8+ T-cell activation and expansion in tumors. Conclusions AMG 509 is a specific, first-in-class T cell-recruiting antibody with avidity-driven activity against STEAP1-positive malignancies. AMG 509 is currently being evaluated for safety, pharmacokinetics, and efficacy in a phase 1, first-in-human study in patients with mCRPC (NCT04221542). Acknowledgements The authors acknowledge Micah Robinson, PhD of Amgen Inc. for medical writing support. Trial Registration ClinicalTrials. gov Identifier: NCT04221542 Ethics Approval All animal experimental protocols were approved by an Institutional Animal Care and Use Committee (IACUC protocol number 2015-01243) and were conducted in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) (Amgen) or the standards of the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (IACUC protocol number 15015x) in a facility certified with an Office of Laboratory Animal Welfare (OLAW) (UTHSA). References Grunewald TGP, Ranft A, Esposito I, Silva-Buttkus P da, Aichler M, Baumhoer D, Schaefer KL, Ottaviano L, Poremba C, Jundt G, Jurgens H, Dirksen U, Richter GHS, Burdach S. High STEAP1 expression is associated with improved outcome of Ewing’s sarcoma patients. Ann Oncol 2012; 23:2185–2190. Hubert RS, Vivanco I, Chen E, Rastegar S, Leong K, Mitchell SC, Madraswala R, Zhou Y, Kuo J, Raitano AB, Jakobvits A, Saffran SC, Afar DE. STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA 1999;96:14523–14528.

Background CAR T therapy has achieved remarkable results in the treatment of hematological tumors such as leukemia, lymphoma, and multiple myeloma. However, there remains challenges in treating solid tumors. These challenges include physical barriers, tumor microenvironment immunosuppression, tumor heterogeneity and target specificity. Especially, due to tumor microenvironmental barriers, CAR T cells are not effectively exposed to tumor antigens and cannot activate co-stimulation signals on CAR molecules, thus conventional CAR T cell therapy has thus far shown weak cell expansion in solid tumor patients, achieved little or no therapeutic responses. Here, we developed CAR T cells based on a novel CoupledCAR® technology to overcome the lack of persistence of solid tumor CAR T cells in vivo. Methods We designed a ‘CoupledCAR’ lentivirus vector containing a single-chain variable fragment (scFv) targeting human TSHR. The lentivirus was produced by transfecting HEK-293T cells with ‘CoupledCAR’ lentiviral vectors and viral packaging plasmids. Patient‘s CD3 T cells were cultured in X-VIVO medium containing 125U/mL 1interleukin-2 (IL-2), and transduced with ‘CoupledCAR’ lentivirus at certain MOI. Transduction efficiency and was evaluated at 7 to 9 days after ‘CoupledCAR’ lentivirus transduction, and quality controls for fungi, bacteria, mycoplasma, chlamydia, and endotoxin were performed. After infusion, serial peripheral blood samples were collected, and the expansion and the cytokine release of CART cells were detected by FACS and QPCR. The evaluation of response level for patients were performed at month 1,month 3,and month 6 by PET/CT. Results We used prostatic acid phosphatase (PAP) as an exemplary CAR target for prostate cancer and demonstrated that our CoupledCAR® significantly enhanced the expansion of PAP CAR T cells in vitro and in vivo. Further, we observed that this expansion showed more memory-like phenotypes, and caused little exhaustion of PAP CAR T cells. Also, we find coupled solid tumor CAR T cells have stronger tumor killing ability. We demonstrated this simple expansion to enable the persistence of solid tumor CAR T cells and can be further applied to other kinds of T cell therapy like TCR T and TILs. Conclusions We developed a novel platform technology (CoupledCAR®) that allows solid tumor CAR T cells to rapidly expand. This initial CAR T cell expansion enabled enhanced trafficking and infiltration of the tumor tissue whereby further cell expansion occurred and thereby achieved tumor clearance. We have carried clinical trials and obtained early promising clinical data. We will further verify the safety and efficacy of this technology in the treatment of different kinds of solid tumors in the clinic research.

Background Immuno-intervention through targeting of activating and inhibitory immune checkpoints (ICP), has shown promising results in the clinic over the last years. To facilitate these researches, mouse models expressing humanized ICP instead of their mouse counterparts were developed. Herein, we describe a novel CD28 humanized mouse model (hCD28 model), designed to test compounds targeting human CD28 (hCD28). Methods Human and mouse CD28 (mCD28) have different signalling responses, with hCD28 being known for inducing higher levels of pro-inflammatory cytokines upon stimulation with ligands/superagonists. This can be explained by the expression of CD28i, a hCD28 amplifier isoform which is not found in mouse. Additionally, evidences suggested that the different signalling between human and mCD28 relies on one amino acid change in the intracellular domain (ICD).1 Because the hCD28 model was developed to assess hCD28-targeting therapeutics, we decided to keep the expression of both canonical and CD28i isoforms to avoid undermining the biological effects of the testing antibodies. Although keeping the human ICD could favour the evaluation of cytokine production and therefore the safety of the test therapeutics, we decided to keep the mouse ICD to enable a proper interaction of CD28 with its signalling partners, allowing a physiological stimulation of CD28 in efficacy studies. Results hCD28 mice express hCD28 on T cells and the frequency of CD3 T cells is comparable in both WT and hCD28 mice. Stimulation of hCD28 mice-isolated T cells with hCD28 ligands and agonist antibodies resulted in T cell proliferation and cytokine production, suggesting that hCD28 is functional in mouse cells. MC38 uptake rate and kinetic of growth were comparable in WT and hCD28 mice, suggesting no major defect in the immune response in the hCD28 mice. Importantly, splenocytes and tumor draining lymph nodes cells isolated from tumor-bearing hCD28 mice showed higher production of IL-2 and IFN-gamma upon in vitro re-challenged with MC38 when compared to WT cells. Since the frequency of CD3 cells (Treg, CD4+ and CD8+) is comparable to WT mice, this could be explained by the expression of the amplifier CD28i isoform, which is absent in WT mice. Conclusions The hCD28 model described here supports the efficacy assessment of hCD28-targeting biologics, enabling PK/PD studies as hCD28 expression levels and pattern are physiological. However, after careful consideration of the CD28 biology, we decided to keep the mouse ICD, although it triggers lower pro-inflammatory cytokine production than CD28 human ICD. As such, this model is not suitable for toxicology/safety studies. Reference Porciello N, Grazioli P, Campese AF, et al. A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nat Commun 2018; 9:1–16.

Background T cells in the tumor micro-environment require TCR/MHC engagement and co-stimulatory receptor engagement to achieve complete activation. Solid tumors often lack expression of CD28 ligands, so we hypothesized that activation of CD28 signaling could be beneficial in solid tumors. We designed tumor-associated-antigen (TAA) x CD28 bispecific antibodies that conditionally costimulate CD28 only in the presence of TAA and TCR engagement. Clinical application of this class of antibodies has potential to enhance activity of either anti-PD(L)1 antibodies or TAA x CD3 T cell engagers. Methods We designed a stability and affinity optimized anti-CD28 antibody that can be paired with TAA of choice to engage CD28 monovalently using Xencor’s XmAb 2+1 and 1+1 platforms. In vitro T cell activation with these bispecifics was measured by T cell proliferation, cytokine production, and cytotoxicity, in co-cultures of human cancer cell lines mixed with primary human CD3-stimulated T cells. In vitro activity was validated in a CMV recall assay measuring CMV+ T cell proliferation of CMV+ PBMC co-cultured with cancer cell lines ectopically treated with pp65-derived NLV-peptide. In vivo anti-tumor and T cell proliferative activity of B7H3 x CD28 bispecific antibodies were determined in tumor-bearing huPBMC-NSG mice treated simultaneously with TAA x CD3 bispecific antibody. In vivo activity of PDL1 x CD28 antibodies was determined with hCD28 KI mice inoculated with MC38 tumors expressing hPDL1-antigen. Finally, safety and tolerability of B7H3 x CD28 and PDL1 x CD28 was determined in cynomolgus monkeys. Results B7H3 x CD28 and PDL1 x CD28 antibodies enhanced T cell degranulation, cytokine secretion, and cancer cell cytotoxicity in concert with CD3 stimulation only in the presence of target antigen. B7H3 x CD28, alone or in combination with anti-PD1 antibody, enhanced proliferation of CMV+ T cells recognizing cancer cells loaded with pp65-derived NLV peptide. PDL1 x CD28 also enhanced CMV+ cell expansion but did not synergize with anti-PD1 antibody treatment. B7H3 x CD28 significantly enhanced in vivo anti-tumor activity of TAA x CD3 antibodies while also promoting greater T cell expansion. In hCD28 mice inoculated with MC38 tumors expressing hPDL1, PDL1 x CD28 antibody inhibited tumor growth greater than an anti-PDL1 antibody alone. B7H3 x CD28 and PDL1 x CD28 were well tolerated in cynomolgus monkeys. Conclusions B7H3 x CD28 and PDL1 x CD28 bispecific antibodies show promising anti-tumor activity and warrant further development.

Background RO7122290 (RO) is a bispecific antibody-like fusion protein that simultaneously targets FAP, abundantly expressed by cancer-associated fibroblasts in most solid tumors, and 4-1BB, transiently expressed on activated T cells. Pre-clinical experiments revealed strong intra-tumoral CD8+ T cells infiltration in FAP-positive tumors, cytokines induction and significant anti-tumor activity mediated by RO (signal 2 of T cell activation), upon TCR/CD3 engagement (signal 1) or in combination with atezolizumab (ATZ). In this first-in-human study, the pharmacodynamic (PD) effects of RO were assessed, both as single agent (SA) (Part A) and in combination with ATZ (Part B). Methods Pts with advanced and/or metastatic solid tumors were eligible for this ongoing Phase 1/1b trial (EUDRACT 2017-003961-83). RO was administered intravenously, weekly (QW) at escalating dose levels (DLs). In Part A, 62 pts were treated at 13 DLs of RO, dose range 5–2000 mg. In Part B, 39 pts were treated at 8 DLs of RO, dose range 45–2000 mg, with ATZ 1200 mg Q3W. Secondary biomarker objective was characterization of PD effects in tumor tissue and blood. The endpoints were change from baseline in intra-tumoral density (cell/mm2) and proliferation (Ki67) of CD8+ T cells measured by immunohistochemistry (IHC), and change in activation (4-1BB) and proliferation (Ki67) of peripheral CD8+ T cells measured by flow cytometry. Exploratory objectives were characterization of PD effects in plasma/serum and measurement of intra-tumoral immune activation. The endpoints were change in peripheral cytokines (TNF-alfa, IFN-gamma, IL-6) and soluble(s) factors (sCD25, s4-1BB) concentration measured by ELISA, and intra-tumoral changes in gene expression measured by RNAseq. Results In the periphery, we observed transient expression of 4-1BB on CD4+ and CD8+ T cells, along with secretion of s4-1BB and inflammatory cytokines, suggesting 4-1BB targeting and potent T cell activation. The concomitant induction of proliferating T cells indicated the potential association to priming and formation of tumor-reactive T cells. In the tumor, we detected increased CD8+ T cells infiltration and proliferation, in both Parts. Proliferating CD8+ T cells increased in both tumor nests and surrounding stroma, with a preferential accumulation in the latter. RNAseq analysis revealed induction of 4-1BB, PD-1 and IFN-gamma, indicating intra-tumoral T cells activation in Part B. Conclusions PD activity was consistent with the postulated MoA, confirming RO to be a potent tumor-targeted 4-1BB agonist in the clinical setting. Our observations suggest that RO can synergize with endogenous T cell receptor stimulation, both as SA and in combination with ATZ.

Background Lung cancer, frequently presents with advanced stage disease with approximately 15–30% of patients first diagnosed by a malignant pleural effusion (MPE).Currently, we have limited understanding of the cellular complex immune landscape compositions of MPE and how this cellular composition impacts response to therapy. Therefore, in this pilot study, we aimed to characterize the cellular composition of MPE in patients with metastatic lung adenocarcinoma (LADC). Methods A custom multiplex immunofluorescence (mIF) panel was designed and optimized using the Opal™ 7 color Kit (Akoya Biosciences) against six immune markers including cytokeratins (CK), PD-L1, PD-1, CD3 CD8, and CD68 (figure 1). We selected 4 MPE cases from LADC patients to validate this mIF panel. Regions of interest (ROI) were scanned in high magnification using the multispectral microscopy Vectra Polaris (Akoya Biosciences) to capture the multiplex immune cell phenotypes and to be analyzed by the image analysis InForm software. Results The median number of cells observed was 4,883.5 (range 1773–8292 cells).The median cells expressing CK was 15% (including tumor and mesothelial cells), CD3+ T-cell was 38%, cytotoxic T-cells CD3+CD8+ was 3%, and macrophages CD68+ was 14% (table1). The median number of CK+ cells expression PD-L1 was 1%. Additionally, the median number of CD3 T-cells expressing PD-1 or PD-L1 was in total 1%. Interestingly, with didn’t see macrophages CD68+ expressing PD-L1 in this small cohort. Furthermore, an exploratory observation showed that patients with high percentage of cytotoxic T-cells CD3+CD8+ and high percentage of macrophages CD68+ had better overall survival (table 2). Conclusions In our cohort of MPE, we were able to assess, with extraordinary fidelity according to the antibodies included in the panel, several cell phenotypes, showing that we successfully multiplexed these biomarkers using mIF. These results demonstrate the practical scalability of this method for studying different aspects of cytological material and the data generated with the image analysis can be used to explore prognosis and potential therapeutic response in the future. References American Thoracic S. Management of malignant pleural effusions. Am J Respir Crit Care Med 2000;162(5):1987–2001. Parra ER, Uraoka N, Jiang M, Cook P, Gibbons D, Forget M-A, et al. Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues. Scientific reports 2017;7(1):13380. Nieto JC, Zamora C, Porcel JM, Mulet M, Pajares V, Munoz-Fernandez AM, et al. Migrated T lymphocytes into malignant pleural effusions: an indicator of good prognosis in lung adenocarcinoma patients. Sci Rep 2019;9(1):2996.

Background Neuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells. Methods We evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule. Results AMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study. Conclusions AMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET. Ethics Approval All in vivo work was conducted under IACUC-approved protocol #2009-00046.

Background The effective treatment of solid tumors remains an unmet medical need. Several concepts exist to treat malignancies, including antibody-drug or -immunotoxin conjugates, immune checkpoint inhibition, CAR- T cells, as well as bispecific T cell engagers. CD3-based T cell engagers are highly potent therapeutic molecules with T cell cytotoxicity activities in the picomolar range. Alongside this highly potent anti-tumor activity is the risk of on-target off-tumor effects due to low levels of expression of the target antigen in normal tissue, as has been observed for the tumor-associated antigen mesothelin (MSLN). Methods Low-affinity antibody fragments to the tumor-associated antigen MSLN were generated, and a multispecific MATCH4 molecule was constructed containing bivalent low-affinity MSLN binding domains, a CD3 binding domain, and a serum albumin-binding domain for half-life extension. This molecule was tested in a cytotoxicity assay using human PBMCs co-cultured with H226 or MeT-5A cells, which express high or low levels of MSLN, respectively. The MeT-5A line, derived from mesothelial cells in the pleural fluid of non-cancerous individuals, represents normal MSLN-expressing cells. Soluble MSLN was added to determine effects on cytotoxicity. In vivo xenograft mouse studies were conducted using a tumor cell/PBMC co-implantation model, followed by regular dosing with molecules of interest. Results Here we report the design and the promising preclinical activity of the MATCH4 molecule in vitro and in vivo. We demonstrate that the low-affinity bivalent MSLN T cell engager has increased in vitro potency in T cell activation and tumor cell killing, as compared to a high-affinity monovalent counterpart on high MSLN expressing cells. We also demonstrate that the activity on low MSLN expressing cells is reduced for the low-affinity bivalent compared to the high affinity monovalent molecule. Because soluble MSLN is shed from cancer cells into cancer patient serum, we also demonstrate that up to 500 ng/mL of soluble MSLN does not interfere with the cytotoxic activity of the low affinity bivalent T cell engager, compared to the effects of soluble MSLN on a high affinity monovalent T cell engager. Importantly, we demonstrate in vivo that the low-affinity bivalent molecule significantly inhibits tumor growth in a dose-dependent manner. Conclusions Collectively, these data demonstrate anti-tumor efficacy by this novel multispecific low affinity bivalent T cell engager. These data indicate the potential of this molecule to increase the therapeutic window by reducing safety concerns on normal tissue where MSLN expression is low, and yet increase cytotoxicity to MSLN-expressing cancer cells.

Background Bispecific T cell engagers (BiTE) is a fast-growing class of immunotherapies. They are bispecific antibody that bind to T cell-surface protein (for example, CD3e) and a specific tumor associate antigen (TAA) on tumor cells, by which to redirect T cells against tumor cells in a MHC-independent manner. A successful example in the clinical is Blinatumomab, a BiTE antibody against CD3/CD19 approved in 2014 to treat acute lymphoblastic leukemia. Currently, many CD3-based BiTE are in clinical trials, including BCMAxCD3, Her2xCD3, CEAxCD3, and PSMAxCD3. To evaluate the efficacy of BiTE in vitro, human peripheral blood monocyte cells (hPBMC) are commonly being used as a source of T cells to co-culture with tumor cells. The disadvantage of using hPBMC is donor-to-donor variability and the availability of the original donor if a study needs to be repeated. Methods To overcome this, we proposed to replace hPBMC with T cells from human CD3e (hCD3) genetically engineered mouse models mice (GEMM) for in in vitro coculture assay. T cells were isolated from hCD3 GEMM mice using negative selection mouse T cell isolation kit. Conventional tumor cell lines or luciferase-engineered patient-derived-xenograft (PDX)-derived organoids (PDXO) expressing specific antigens are co-cultured with hCD3 T cells in 96-well plates in the presence of BiTE antibody. Results We measured the killing of tumor cells using either flow cytometry or luciferase activity as readouts. To analyze tumor-reactivity of T cells to cancer cell line or organoids, IFN-gamma in the culture medium was measured and activation markers on T cells was assessed. Conclusions Our data showed the feasibility of using humanized mice T cells as a replacement for hPBMCs to assess BiTE antibody in vitro. We are further validating the application of murine hCD3 T cells for in vivo models to test bispecific T cell engagers.

Background Elevated levels of Prostaglandin E2 (PGE2), an eicosanoid notably synthesized by the cyclooxygenase-2 (COX-2), exert strong immunosuppressive effects in the tumor microenvironment. COX-2-positive solid tumors have the ability to use this pathway as a resistance mechanism, especially to escape from the host immune system, thus limiting the anti-tumor effects of immune checkpoint inhibitors (ICI). These immunosuppressive effects are largely mediated by the EP4 receptor, expressed on multiple immune cells. Methods A novel series of EP4 receptor antagonists has been developed, with improved pharmacokinetic properties when compared to the EP4 receptor antagonists currently being evaluated in clinical trials. An intensive lead optimization program led to the identification of DT095895, a small molecule development candidate with a ‘best-in class’ potential. DT095895 was assessed in multiple syngeneic mouse tumor models selected for their COX-2 expression profile. Results DT095895 preclinical package will be presented in the poster. Efficacy was seen both in a monotherapy setting, as well as in combination with an ICI. Additionally, a specific biomarker program was implemented and validated in order to show target engagement. A phospho-flow murine whole blood assay was set-up to assess the ability of DT095895 to inhibit CREB phosphorylation induced by a selective EP4 receptor agonist in CD3+ cells. This biomarker was further developed for human whole blood to support Phase 1 and clinical trials studies. Conclusions DT095895 is a selective EP4 receptor antagonist and demonstrates strong anti-tumor effects in multiple syngeneic mouse tumor models, both as a monotherapy and in combination with ICI, through the inhibition of the PGE2-induced immunosuppression. DT095895 progresses in regulatory development.

BackgroundThe positive role of CD8+ tumor-infiltrating lymphocytes (TIL) in patients with colorectal cancer (CRC) has been well described but the prognostic value of CD4 T cell subsets remained to be investigated. In this study, we expanded TIL from surgically resected liver metastases of patients with CRC and characterized the phenotype and the prognostic value of expanded-CD4 T cells.MethodsLiver metastases were surgically resected from 23 patients with CRC. Tumors were enzymatically digested and cultured in high dose of interleukin-2 for up to 5 weeks. T cell phenotype and reactivity of cultured-T cells were measured by flow cytometry and correlated with patients’ clinical outcomes.ResultsWe successfully expanded 21 over 23 TIL from liver metastases of patients with CRC. Interestingly, we distinguished two subsets of expanded T cells based on T cell immunoglobulin mucin domain-containing protein 3 (TIM-3) expression. Medians fold expansion of expanded T cells after rapid expansion protocol was higher in CD3+TIM-3low cultures. In an attempt to investigate the correlation between the phenotype of expanded CD4 T cells and clinical outcomes, we observed on one hand that the level of Tregs in culture as well as the expression of both PD1 and TIM-3 by expanded T cells was not correlated to the clinical outcomes. Interestingly, on the other hand, cultures containing high levels of Th17 cells were associated with a poor prognosis (p=0.0007).ConclusionsOur data confirmed the presence of Th17 cells in expanded T cells from liver metastases. Among CD4 T cell characteristics investigated, TIM-3 but not programmed cell death protein 1 predicted the expansion capacity of TIL while only the Th17 phenotype showed correlation with patients’ survival, suggesting a particular role of this T cell subset in CRC immune contexture.Trial registration numberNCT02817178.

BackgroundOX40 (CD134) is a costimulatory molecule of the tumor necrosis factor receptor superfamily that is currently being investigated as a target for cancer immunotherapy. However, despite promising results in murine tumor models, the clinical efficacy of agonistic αOX40 antibodies in the treatment of patients with cancer has fallen short of the high expectation in earlier-stage trials.MethodsUsing lymphocytes from resected tumor, tumor-free (TF) tissue and peripheral blood mononuclear cells (PBMC) of 96 patients with hepatocellular and colorectal cancers, we determined OX40 expression and the in vitro T-cell agonistic activity of OX40-targeting compounds. RNA-Seq was used to evaluate OX40-mediated transcriptional changes in CD4+ and CD8+ human tumor-infiltrating lymphocytes (TILs).ResultsHere, we show that OX40 was overexpressed on tumor-infiltrating CD4+ T cells compared with blood and TF tissue-derived T cells. In contrast to a clinical candidate αOX40 antibody, treatment with an Fc-engineered αOX40 antibody (αOX40_v12) with selectively enhanced FcγRIIB affinity, stimulated in vitro CD4+ and CD8+ TIL expansion, as well as cytokine and chemokine secretions. The activity of αOX40_v12 was dependent on FcγRIIB engagement and intrinsic CD3/CD28 signals. The transcriptional landscape of CD4+ and CD8+ TILs shifted toward a prosurvival, inflammatory and chemotactic profile on treatment with αOX40_v12.ConclusionsOX40 is overexpressed on CD4+ TILs and thus represents a promising target for immunotherapy. Targeting OX40 with currently used agonistic antibodies may be inefficient due to lack of OX40 multimerization. Thus, Fc engineering is a powerful tool in enhancing the agonistic activity of αOX40 antibody and may shape the future design of antibody-mediated αOX40 immunotherapy.

BackgroundWe have previously reported that the upregulation of galectin-9 (Gal-9) on CD4+ and CD8+ T cells in HIV patients was associated with impaired T cell effector functions. Gal-9 is a ligand for T cell immunoglobulin and mucin domain-3, and its expression on T cells in cancer has not been investigated. Therefore, we aimed to investigate the expression level and effects of Gal-9 on T cell functions in patients with virus-associated solid tumors (VASTs).Methods40 patients with VASTs through a non-randomized and biomarker-driven phase II LATENT trial were investigated. Peripheral blood mononuclear cells and tumor biopsies were obtained and subjected to immunophenotyping. In this trial, the effects of oral valproate and avelumab (anti-PD-L1) was investigated in regards to the expression of Gal-9 on T cells.ResultsWe report the upregulation of Gal-9 expression by peripheral and tumor-infiltrating CD4+ and CD8+ T lymphocytes in patients with VASTs. Our results indicate that Gal-9 expression is associated with dysfunctional T cell effector functions in the periphery and tumor microenvironment (TME). Coexpression of Gal-9 with PD-1 or T cell immunoglobulin and ITIM domain (TIGIT) exhibited a synergistic inhibitory effect and enhanced an exhausted T cell phenotype. Besides, responding patients to treatment had lower Gal-9 mRNA expression in the TME. Translocation of Gal-9 from the cytosol to the cell membrane of T cells following stimulation suggests persistent T cell receptor (TCR) stimulation as a potential contributing factor in Gal-9 upregulation in patients with VASTs. Moreover, partial colocalization of Gal-9 with CD3 on T cells likely impacts the initiation of signal transduction via TCR as shown by the upregulation of ZAP70 in Gal-9+ T cells. Also, we found an expansion of Gal-9+ but not TIGIT+ NK cells in patients with VASTs; however, dichotomous to TIGIT+ NK cells, Gal-9+ NK cells exhibited impaired cytotoxic molecules but higher Interferon gamma (IFN-γ) expression.ConclusionOur data indicate that higher Gal-9-expressing CD8+ T cells were associated with poor prognosis following immunotherapy with anti-Programmed death-ligand 1 (PD-L1) (avelumab) in our patients’ cohort. Therefore, for the very first time to our knowledge, we report Gal-9 as a novel marker of T cell exhaustion and the potential target of immunotherapy in patients with VASTs.

BackgroundIt is now recognized that many anticancer treatments positively modulate the antitumor immune response. Clinical and experimental studies have shown that inhibitors of the classical renin–angiotensin system (RAS) reduce tumor progression and are associated with better outcomes in patients with colorectal cancer. RAS components are expressed by most immune cells and adult hematopoietic cells, thus are potential targets for modulating tumor-infiltrating immune cells and can provide a mechanism of tumor control by the renin–angiotensin system inhibitors (RASi).AimTo investigate the effects of the RASi captopril on tumor T lymphocyte distribution in a mouse model of colorectal liver metastases.MethodsLiver metastases were established in a mouse model using an autologous colorectal cancer cell line. RASi (captopril 750 mg/kg) or carrier (saline) was administered to the mice daily via intraperitoneal injection, from day 1 post-tumor induction to endpoint (day 15 or 21 post-tumor induction). At the endpoint, tumor growth was determined, and lymphocyte infiltration and composition in the tumor and liver tissues were analyzed by flow cytometry and immunohistochemistry (IHC).ResultsCaptopril significantly decreased tumor viability and impaired metastatic growth. Analysis of infiltrating T cells into liver parenchyma and tumor tissues by IHC and flow cytometry showed that captopril significantly increased the infiltration of CD3+ T cells into both tissues at day 15 following tumor induction. Phenotypical analysis of CD45+ CD3+ T cells indicated that the major contributing phenotype to this influx is a CD4 and CD8 double-negative T cell (DNT) subtype, while CD4+ T cells decreased and CD8+ T cells remained unchanged. Captopril treatment also increased the expression of checkpoint receptor PD-1 on CD8+and DNT subsets .ConclusionCaptopril treatment modulates the immune response by increasing the infiltration and altering the phenotypical composition of T lymphocytes and may be a contributing mechanism for tumor control.

BackgroundFirst-in-human (FIH) clinical trials require careful selection of a safe yet biologically relevant starting dose. Typically, such starting doses are selected based on toxicity studies in a pharmacologically relevant animal model. However, with the advent of target-specific and highly active immunotherapeutics, both the Food and Drug Administration and the European Medicines Agency have provided guidance that recommend determining a safe starting dose based on a minimum anticipated biological effect level (MABEL) approach.MethodsWe recently developed a T cell activating bispecific antibody that effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma in mice (hEGFRvIII:CD3 bi-scFv). hEGFRvIII:CD3 bi-scFv is comprized of two single chain antibody fragments (bi-scFvs) that bind mutant epidermal growth factor receptor variant III (EGFRvIII), a mutation frequently seen in malignant glioma, and human CD3ε on T cells, respectively. In order to establish a FIH dose, we used a MABEL approach to select a safe starting dose for hEGFRvIII:CD3 bi-scFv, based on a combination of in vitro data, in vivo animal studies, and theoretical human receptor occupancy modeling.ResultsUsing the most conservative approach to the MABEL assessment, a dose of 57.4 ng hEGFRvIII:CD3 bi-scFv/kg body weight was selected as a safe starting dose for a FIH clinical study.ConclusionsThe comparison of our MABEL-based starting dose to our in vivo efficacious dose and the theoretical human receptor occupancy strongly supports that our human starting dose of 57.4 ng hEGFRvIII:CD3 bi-scFv/patient kg will be safe.

Affinity-optimized T cell receptor (TCR)-engineered lymphocytes targeting tumor antigens can mediate potent antitumor responses in cancer patients, but also bear substantial risks for off-target toxicities. Most preclinical studies have focused on T cell responses to antigen-specific stimulation. In contrast, little is known on the regulation of T cell responsiveness through continuous TCR triggering and consequent tonic signaling. Here, we addressed the question whether increasing the TCR affinity can lead to chronic interactions occurring directly between TCRs and MHC-(self) molecules, which may modulate the overall functional potency of tumor-redirected CD8 T cells. For this purpose, we developed two complementary human CD8 T cell models (i.e. HLA-A2 knock-in and knock-out) engineered with incremental-affinity TCRs to the HLA-A2/NY-ESO-1 tumor antigen. The impact of HLA-A2 recognition, depending on TCR affinity, was assessed at the levels of the TCR/CD3 complex, regulatory receptors, and signaling, under steady-state conditions and in kinetic studies. The quality of CD8 T cell responses was further evaluated by gene expression and multiplex cytokine profiling, as well as real-time quantitative cell killing, combined with co-culture assays. We found that HLA-A2 per se (in absence of cognate peptide) can trigger chronic activation followed by a tolerance-like state of tumor-redirected CD8 T cells with increased-affinity TCRs. HLA-A2 pos but not HLA-A2 neg T cells displayed an activation phenotype, associated with enhanced upregulation of c-CBL and multiple inhibitory receptors. T cell activation preceded TCR/CD3 downmodulation, impaired TCR signaling and functional hyporesponsiveness. This stepwise activation-to-hyporesponsive state was dependent on TCR affinity and already detectable at the upper end of the physiological affinity range (K D ≤ 1 μM). Similar findings were made when affinity-increased HLA-A2 neg CD8 T cells were chronically exposed to HLA-A2 pos -expressing target cells. Our observations indicate that sustained interactions between affinity-increased TCR and self-MHC can directly adjust the functional potential of T cells, even in the absence of antigen-specific stimulation. The observed tolerance-like state depends on TCR affinity and has therefore potential implications for the design of affinity-improved TCRs for adoptive T cell therapy, as several engineered TCRs currently used in clinical trials share similar affinity properties.

BackgroundBispecific T-cell engagers are an established therapeutic strategy for the treatment of hematologic malignancies but face several challenges when it comes to their application for the treatment of solid tumors, including on-target off-tumor adverse events. Employing an avidity-mediated specificity gain by introducing an additional binding moiety for the tumor-associated antigen can be achieved using formats with a 2+1 stoichiometry.MethodsBesides biochemical characterization and validation of target cell binding to cancer cells with different HER3 expression, we used in vitro co-culture assays with human peripheral blood mononuclear cells (PBMCs) and HER3-expressing target cells to determine T-cell activation, T-cell proliferation and PBMC-mediated cancer cell lysis of HER3-positive cell lines by the trivalent, bispecific antibodies.ResultsIn this study, we developed trivalent, bispecific antibodies comprising a silenced Fc region for T-cell retargeting to HER3-expressing tumor cells, combining a bivalent single-chain diabody (scDb) fused to a first heterodimerizing Fc chain with either an Fab or scFv fused to a second heterodimerizing Fc chain. All these HER3-targeting T-cell engagers comprising two binding sites for HER3 and one binding site for CD3 mediated target cell killing. However, format and orientation of binding sites influenced efficacy of target cell binding, target cell-dependent T-cell activation and T-cell-mediated target cell killing. Beneficial effects were seen when the CD3 binding site was located in the scDb moiety. These molecules showed efficient killing of medium HER3-expressing cancer cells with very low induction of cytokine release, while sparing target cells with low or undetectable HER3 expression.ConclusionOur study demonstrates that these trivalent, bispecific antibodies represent formats with superior interdomain spacing resulting in efficient target cell killing and a potential advantageous safety profile due to very low cytokine release.

BackgroundReceptor tyrosine kinase-like orphan receptor 1 (ROR1) is expressed on a variety of difficult to treat solid and hematological malignancies. Several therapeutic concepts targeting ROR1 are currently in clinical studies, including antibody-drug conjugates (ADCs), chimeric antigen receptor engineered T cells, as well as a bispecific T cell engager. In contrast to ADCs, T cell engagers have the capacity to induce tumor cell depletion irrespective of tumor cell mitotic activity. For the therapy of ROR1 expressing tumors, we engineered a T cell engager with prolonged half-life to support convenient administration schemes.MethodsNM32-2668, a ROR1-targeting T cell engager with prolonged serum half-life was engineered by joining three humanized rabbit antibody variable region (Fv) fragments specific for ROR1, CD3ɛ, and serum albumin, into our tri-specific scMATCHTM3 format. Each Fv fragment was stabilized using the ʎ-capTM technology. NM32-2668 was tested in assays for specific tumor lysis, induction of T cell proliferation, and cytokine release. These studies were performed using human T cells co-cultured with tumor cell lines and human tumor samples expressing various levels of ROR1. In vivo xenograft mouse studies were conducted using a human mantle cell lymphoma model in NCG mice engrafted with human PBMCs.ResultsHere we report the design and the promising preclinical activity of the scMATCHTM3 ROR1/CD3/hSA T cell engager NM32-2668 in vitro and in vivo. Importantly, we demonstrate potent and specific cytotoxic activity in the sub-nanomolar range on tumor cell lines expressing different levels of ROR1. NM32-2668 also mediates ROR1 dependent T cell activation and cytokine release. We observe robust tumor cell killing activity of NM32-2668 over an extended time period and at multiple ratios of effectors to targets in a real time imaging-based cytotoxicity assay. This molecule also mediates T cell proliferation in response to target cell binding. NM32-2668 mediates in vitro lysis of CLL patient tumor cells, T cell activation, and cytokine release, with minimal IL-6 involvement. In an in vivo mantle cell lymphoma model (Jeko-1) engrafted with human PBMCs, we observe tumor regression and eradication.ConclusionsCollectively, these data demonstrate robust anti-tumor efficacy by NM32-2668, a scMATCHTM3 ROR1/CD3/hSA. Our results demonstrate that NM32-2668 promotes ROR1 dependent T cell activation and proliferation, as well as T cell-mediated tumor cell lysis. The activity of NM32-2668 has the potential to provide significant benefit to patients with ROR1+ malignancies on a convenient dosing schedule. We intend to rapidly progress NM32-2668 to clinical development.

BackgroundWe previously described the capacity of RO7122290 (RO) - a FAP-targeted 4-1BB bispecific antibody - to induce CD8+ T cell infiltration and activation in the tumor (Moreno V. et al, SITC 2020). Aiming to compare pharmacodynamic (PD) changes in tumor nests and stroma, paired tumor biopsies from patients treated with RO (Part A) and RO + atezolizumab (Part B) were analysed by digital spatial profiling (DSP, Nanostring).MethodsSeven paired (baseline and on-treatment) FFPE tumor tissue biopsies (three from Part A, four from Part B) obtained from an ongoing Phase 1/1b trial (EUDRACT 2017-003961-83) were assessed for mRNA and protein expression. Biopsies were taken from six different tumor types at different RO doses. Up to twelve Regions of Interest (ROIs) were collected per slide and the morphology markers PanCK, CD8, CD3 and DAPI were applied. The ROIs were further annotated in tumor nests and stroma segments based on PanCK staining. The immune-oncology 58-plex protein and 78-plex mRNA expression panels (Nanostring) were used to profile all samples. Data were normalized according to Nanostring guidelines and filtered based on relevance (absolute log2 fold change > 1) and significance (FDR < 0.05, p-value).ResultsThe level of CD8+ T cell infiltration measured by spatial profiling correlated with the level measured by IHC, in both tumor nests and stroma. The activation markers 4-1BB and PD-1 were upregulated, confirming the PD effect already measured by mRNA sequencing. We also identified novel protein markers - CD40, PD-L1 and IDO1 - being upregulated after treatment. Spatial regulation differed among the markers with 4-1BB, PD-1 and CD40 upregulated only in the stroma, PD-L1 and IDO1 upregulated in the tumor nests and in the stroma. IDO1 induction is particularly relevant, since this protein is known to attenuate 4-1BB-mediated effector responses. Conventional IHC analysis performed on 14 paired biopsies confirmed IDO1 being upregulated in 11 out of 14 cases and revealed dendritic cells, macrophages and stromal cells to express IDO1. Importantly, IDO1 upregulation was observed in both Part A (3 out of 3) and Part B (8 out of 11).ConclusionsSpatial profiling allowed us to identify key markers that are spatially regulated after treatment and to gain new insights on the MoA of RO. The induction of IDO1 by RO confirms the dual immunoregulatory nature of 4-1BB signaling and highlights IDO1 as a potential resistance mechanism for RO in the clinical setting, both as single agent and in combination with atezolizumab.Trial RegistrationEUDRACT Number: 2017-003961-83; Protocol Number: BP40087ReferencesMoreno V. et al, Pharmacodynamic assessment of a novel FAP-targeted 4–1BB agonist, administered as single agent and in combination with atezolizumab to patients with advanced solid tumors, Nov 1 2020, Journal for ImmunoTherapy of Cancer, presented at SITC 2020

BackgroundPseudomyxoma peritonei (PMP) is a rare, slow-growing abdominal cancer with no efficacious treatment options in non-resectable and recurrent cases. Otherwise, rare activating mutations in the GNAS oncogene are remarkably frequent in PMP and the mutated gene product, guanine nucleotide-binding protein α subunit (Gsα), is a potential tumor neoantigen, presenting an opportunity for targeting by a therapeutic cancer vaccine.MethodsTumor and blood samples were collected from 25 patients undergoing surgery for PMP (NCT02073500). GNAS mutation analysis was performed by next-generation targeted sequencing or digital droplet PCR. Responses to stimulation with Gsα mutated (point mutations R201H and R201C) 30 mer peptides were analyzed in peripheral blood T cells derived from patients with PMP and healthy donors. Fresh PMP tumor samples were analyzed by mass cytometry using a panel of 35 extracellular markers, and cellular subpopulations were clustered and visualized using the visual stochastic network embedding analysis tool.ResultsGNAS mutations were detected in 22/25 tumor samples (88%; R201H and R201C mutations detected in 16 and 6 cases, respectively). Strong T cell proliferation against Gsα mutated peptides was observed in 18/24 patients with PMP. Mass cytometry analysis of tumor revealed infiltration of CD3 +T cells in most samples, with variable CD4+:CD8 + ratios. A large proportion of T cells expressed immune checkpoint molecules, in particular programmed death receptor-1 and T cell immunoreceptor with Ig and ITIM, indicating that these T cells were antigen experienced.ConclusionThese findings point to the existence of a pre-existing immunity in patients with PMP towards mutated Gsα, which has been insufficient to control tumor growth, possibly because of inhibition of antitumor T cells by upregulation of immune checkpoint molecules. The results form a rationale for exploring peptide vaccination with Gsα peptides in combination with immune checkpoint inhibiton as a possible curative treatment for PMP and other GNAS mutated cancers.

BackgroundImmunotherapy has revolutionized therapeutic patterns of small cell lung cancer (SCLC). Human leukocyte antigen class II (HLA class II) is related to antitumor immunity. However, the implications of HLA class II in SCLC remain incompletely understood.Materials and methodsWe investigated the expression patterns of HLA class II on tumor cells and tumor-infiltrating lymphocytes (TILs) by immunohistochemistry staining and its association with clinical parameters, immune markers, and recurrence-free survival (RFS) in 102 patients with stage I–III SCLC with radical surgery. Additionally, an HLA class II-based immune risk model was established by least absolute shrinkage and selection operator regression. With bioinformatics methods, we investigated HLA class II-related enrichment pathways and immune infiltration landscape in SCLC.ResultsHLA class II on tumor cells and TILs was positively expressed in 9 (8.8%) and 45 (44.1%) patients with SCLC, respectively. HLA class II on TILs was negatively associated with lymph node metastasis and positively correlated with programmed death-ligand 1 (PD-L1) on TILs (pConclusionsHLA class II plays a crucial role in tumor immune microenvironment and recurrence prediction. This work demonstrates the prognostic and clinical values of HLA class II in patients with SCLC.

BackgroundResistance to an immune checkpoint inhibitor (ICI) is a major obstacle in cancer immunotherapy. The causes of ICI resistance include major histocompatibility complex (MHC)/histocompatibility locus antigen (HLA) class I loss, neoantigen loss, and incomplete antigen presentation. Elimination by natural killer (NK) cells would be expected to be an effective strategy for the treatment of these ICI-resistant tumors. We previously demonstrated that a lipid nanoparticle containing a stimulator of an interferon gene (STING) agonist (STING-LNP) efficiently induced antitumor activity via the activation of NK cells. Thus, we evaluated the potential of reducing ICI resistance by STING-LNPs.MethodsLung metastasis of a B16-F10 mouse melanoma was used as an anti-programmed cell death 1 (anti-PD-1)-resistant mouse model. The mice were intravenously injected with the STING-LNP and the mechanism responsible for the improvement of anti-PD-1 resistance by the STING-LNPs was analyzed by RT-qPCR and flow cytometry. The dynamics of STING-LNP were also investigated.ResultsAlthough anti-PD-1 monotherapy failed to induce an antitumor effect, the combination of the STING-LNP and anti-PD-1 exerted a synergistic antitumor effect. Our results indicate that the STING-LNP treatment significantly increased the expression of CD3, CD4, NK1.1, PD-1 and interferon (IFN)-γ in lung metastases. This change appears to be initiated by the type I IFN produced by liver macrophages that contain the internalized STING-LNPs, leading to the systemic activation of NK cells that express PD-1. The activated NK cells appeared to produce IFN-γ, resulting in an increase in the expression of the PD ligand 1 (PD-L1) in cancer cells, thus leading to a synergistic antitumor effect when anti-PD-1 is administered.ConclusionsWe provide a demonstration to show that a STING-LNP treatment can overcome PD-1 resistance in a B16-F10 lung metastasis model. The mechanism responsible for this indicates that NK cells are activated by stimulating the STING pathway which, in turn, induced the expression of PD-L1 on cancer cells. Based on the findings reported herein, the STING-LNP represents a promising candidate for use in combination therapy with anti-PD-1-resistant tumors.

Background Myeloid-derived suppressor cells (MDSC) have been found to play an important role in limiting immune responses in cancer. Higher circulating MDSC levels have been associated with greater tumor burden, poorer response to immunotherapy, and poorer survival. Optimal measurement of MDSC levels could provide clinicians with a useful prognostic and/or management tool. Methods A whole blood (WB) nine color, 11 parameter flow cytometric assay was designed, utilizing fluorescently-labeled antibodies against CD45, CD3, CD19, CD20, CD56, CD16, HLA-DR, CD33, CD11b, CD14 and CD15, and BD Trucount beads for quantitation. Total MDSC were defined as CD45 + CD3−CD19−CD20−CD56−CD16−HLA-DR−CD33 + CD11b + cells, while the monocytic (M-MDSC) and polymorphonuclear subsets were defined as CD14+ or CD15+, respectively. Results A novel gating strategy was devised to eliminate granulocytes and improve consistency in gating. Several pre-analytical variables were found to significantly affect MDSC quantitation, including collection tube type and time elapsed between blood collection and testing. Total and M-MDSC levels were a mean of 63% and 73% greater, respectively, with K2EDTA compared to Na+heparin collection tubes (N = 5). In addition, time elapsed at room temperature prior to cell labeling affected MDSC quantitation; by 24 h after blood collection, total and M-MDSC levels were a mean of 26% and 57% lower compared to testing as soon as possible after collection (N = 6). Refrigeration of samples at 4 °C ameliorated time-dependent effects at both 4 and 8 h, but not 24 h after blood collection. To establish normal ranges for this assay, MDSC levels were quantified in 67 healthy subjects (30 male, 37 female) ages 20–93. No significant differences in total or M-MDSC levels were detected for ages ≤60 compared to > 60 (p = 0.5 and p = 0.8, respectively). Finally, assay results demonstrated significantly higher MDSC levels among patients with hepatocellular carcinoma (N = 55) compared to age-matched healthy controls (N = 27) for total and M-MDSC (p = 0.006 and 0.004, respectively). Conclusions MDSC are a heterogenous group of cells, and their quantitation in WB can be affected by a number of pre-analytical variables. Consideration of these factors, and measurement using a material type that has not been manipulated, such as whole blood, is likely to yield the most accurate results. Electronic supplementary material The online version of this article (10.1186/s40425-019-0674-1) contains supplementary material, which is available to authorized users.

BackgroundImmune checkpoint inhibitors, the most widespread class of immunotherapies, have demonstrated unique response patterns that are not always adequately captured by traditional response criteria such as the Response Evaluation Criteria in Solid Tumors or even immune-specific response criteria. These response metrics rely on monitoring tumor growth, but an increase in tumor size and/or appearance after starting immunotherapy does not always represent tumor progression, but also can be a result of T cell infiltration and thus positive treatment response. Therefore, non-invasive and longitudinal monitoring of T cell infiltration are needed to assess the effects of immunotherapies such as checkpoint inhibitors. Here, we proposed an innovative concept that a sufficiently large influx of tumor infiltrating T cells, which have a smaller diameter than cancer cells, will change the diameter distribution and decrease the average size of cells within a volume to a degree that can be quantified by non-invasive MRI.MethodsWe validated our hypothesis by studying tumor response to combination immune-checkpoint blockade (ICB) of anti-PD-1 and anti-CTLA4 in a mouse model of colon adenocarcinoma (MC38). The response was monitored longitudinally using Imaging Microstructural Parameters Using Limited Spectrally Edited Diffusion (IMPULSED), a diffusion MRI-based method which has been previously shown to non-invasively map changes in intracellular structure and cell sizes with the spatial resolution of MRI, in cell cultures and in animal models. Tumors were collected for immunohistochemical and flow cytometry analyzes immediately after the last imaging session.ResultsImmunohistochemical analysis revealed that increased T cell infiltration of the tumors results in a decrease in mean cell size (eg, a 10% increase of CD3+ T cell fraction results a ~1 µm decrease in the mean cell size). IMPULSED showed that the ICB responders, mice with tumor volumes were less than 250 mm3 or had tumors with stable or decreased volumes, had significantly smaller mean cell sizes than both Control IgG-treated tumors and ICB non-responder tumors.ConclusionsIMPULSED-derived cell size could potentially serve as an imaging marker for differentiating responsive and non-responsive tumors after checkpoint inhibitor therapies, a current clinical challenge that is not solved by simply monitoring tumor growth.

BackgroundPeritoneal carcinomatosis is a hallmark of advanced peritoneal tumor progression, particularly for tubal/ovarian high-grade serous carcinomas (HGSCs). Patients with peritoneal carcinomatosis have poor survival rates and are difficult to treat clinically due to widespread tumor dissemination in the peritoneal cavity.MethodsWe developed a clinically relevant, genetically induced, peritoneal carcinomatosis model that recapitulates the histological morphology and immunosuppressive state of the tumor microenvironment of metastatic peritoneal HGSCs by intraperitoneally injecting shp53, AKT, c-Myc, luciferase and sleeping beauty transposase, followed by electroporation (EP) in the peritoneal cavity of immunocompetent mice (intraperitoneal (IP)/EP mice).ResultsSimilar to the spread of human ovarian cancers, IP/EP mice displayed multiple tumor nodules attached to the surface of the abdomen. Histopathological analysis indicated that these tumors were epithelial in origin. These IP/EP mice also displayed a loss of CD3+T cell infiltration in tumors, highly expressed inhibitory checkpoint molecules in tumor-infiltrating and global CD4+and CD8+T cells, and increased levels of transforming growth factor-β in the ascites, all of which contribute to the promotion of tumor growth.ConclusionsOverall, our tumor model recapitulates clinical peritoneal HGSC metastasis, which makes it ideal for preclinical drug screening, testing of immunotherapy-based therapeutics and studying of the tumor biology of peritoneal carcinomatosis.

Meeting abstracts Immunological status in surgically resected specimens affects the outcome of ‘operable’ colorectal cancer patients. For example, a high number of tumor infiltrating lymphocytes, CD3+ cells, CD8+ cells, or CD45RO+ cells in a tumor is associated with improved prognosis. However

Background Adoptive T cell therapy (ACT) has shown great promise in melanoma, with over 50 % response rate in patients where autologous tumor-reactive tumor-infiltrating lymphocytes (TIL) can be cultured and expanded. A major limitation of ACT is the inability to generate or expand autologous tumor-reactive TIL in 25–45 % of patients tested. Methods that successfully identify tumors that are not suitable for TIL generation by standard methods would eliminate the costs of fruitless expansion and enable these patients to receive alternate therapy immediately. Methods Multispectral fluorescent immunohistochemistry with a panel including CD3, CD8, FoxP3, CD163, PD-L1 was used to analyze the tumor microenvironment in 17 patients with melanoma among our 36-patient cohort to predict successful TIL generation. Additionally, we compared tumor fragments and enzymatic digestion of tumor samples for efficiency in generating tumor-reactive TIL. Results Tumor-reactive TIL were generated from 21/36 (58 %) of melanomas and for 12/13 (92 %) tumors where both enzymatic and fragment methods were compared. TIL generation was successful in 10/13 enzymatic preparations and in 10/13 fragment cultures; combination of both methods resulted in successful generation of autologous tumor-reactive TIL in 12/13 patients. In 17 patients for whom tissue blocks were available, IHC analysis identified that while the presence of CD8+ T cells alone was insufficient to predict successful TIL generation, the CD8+ to FoxP3+ ratio was predictive with a positive-predictive value (PPV) of 91 % and negative-predictive value (NPV) of 86 %. Incorporation of CD163+ macrophage numbers and CD8:PD-L1 ratio did not improve the PPV. However, the NPV could be improved to 100 % by including the ratio of CD8+:PD-L1+ expressing cells. Conclusion This is the first study to apply 7-color multispectral immunohistochemistry to analyze the immune environment of tumors from patients with melanoma. Assessment of the data using unsupervised hierarchical clustering identified tumors from which we were unable to generate TIL. If substantiated, this immune profile could be applied to select patients for TIL generation. Additionally, this biomarker profile may also indicate a pre-existing immune response, and serve as a predictive biomarker of patients who will respond to checkpoint blockade. We postulate that expanding the spectrum of inhibitory cells and molecules assessed using this technique could guide combination immunotherapy treatments and improve response rates. Electronic supplementary material The online version of this article (doi:10.1186/s40425-015-0091-z) contains supplementary material, which is available to authorized users.

Background Ovarian cancer is immunogenic and residual tumor volume after surgery is known to be prognostic. Ovarian cancer often follows a recurring-remitting course and microscopic disease states may present ideal opportunities for immune therapies. We sought to establish the immune profile of a murine model of ovarian cancer that allows in vivo tumor imaging and the quantitation of microscopic disease. Results and Discussion Baseline imaging and weight measurements were taken within 1 and 2 weeks after intraperitoneal tumor injection, respectively. Significantly higher photons per second from baseline imaging were first observed 5 weeks after tumor cell injection (p