Expression Of OX40 Research Articles

Abstract 3222: The sharing of T cell clones in peripheral CD8+PD-1+ T cells with TILs is a novel biomarker predicting the efficacy of anti-PD-L1 therapy

Abstract Background: The optimal biomarker to select suitable patients for immune checkpoint blockades (ICBs) therapy remains to be well established. The dynamic variations of molecular alterations during ICBs treatment need to be better understood. Here,we explored a novel biomarkerbased on T cell receptor (TCR) repertoire (TcR) and traced the variations during PD-L1 blockade delivery.Methods:Patients treated with anti-PD-L1 clinical trials were enrolled in this pilot study (n=21). Next-generation sequencing (NGS) was performed to assess molecular tumor burden index (mTBI) based on a 1021 gene panel, for genomic DNA from tumor tissue and cfDNA. Multiple PCR and NGS on the CDR3 region of TCR beta chain was used to reflect the distribution of T cell clones in TILs and peripheral CD8+PD1+ T cells. We applied gene altation data combined with each patient’s major histocompatibility complex (MHC) class I haplotype in a neoantigen prediction platform that evaluates binding affinities of somatic peptides to class I MHC, antigen processing, and self-similarity. Neoantigen peptides were synthesis and used to stimulate Peripheral blood mononuclear cell(PBMC)from the same patient. The expression of OX-40 and 4-1BB were anylisied by flow cytometry to determine activity change of stimulated T cells. Results: A number of T cell clones (1-24) in TILs were shared in peripheral CD8+PD1+ T cells. The total ratio of shared clones to top 100 clones in TILs ranged from 0.00% to 31.35%, while the mean ratio of each shared clone was 1.24% (0.00% to 3.18%). Patients responding to anti-PD-L1 therapy showed higher mean ratio of shared clonesthan non-responding ones (1.76% vs. 0.85%; P= 0.012), our further analysis showed the progression free survival (PFS) is also longer in patients with higher mean ratio of shared clones (P=0.022). Intriguingly, the ratio of shared clones to total CD8+PD1+ T cell clones of peripheral blood showed synchronized change consistent with mTBI and tumor burden. PBMC stimulated by neoantigen peptide expressed higher OX-40 and 4-1BB than control. Further TCR sequencing performed on stimulated T cells showed consistency with shared clone at baseline. Conclusion: The high mean ratio of shared clones in TILs is a potential and promising biomarker to predict the efficacy of ICBs therapy and reflect the target T lymphocytes variations during the treatment, which warrants the further validation in large cohort. Note: This abstract was not presented at the meeting. Citation Format: Jiefei Han, Zhijie Wang, Yuqi Wang, Si Chen, Hua Bai, Jianchun Duan, Jie Wang. The sharing of T cell clones in peripheral CD8+PD-1+ T cells with TILs is a novel biomarker predicting the efficacy of anti-PD-L1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3222.

Immune recurrence score using 7 immunoregulatory protein expressions can predict recurrence in stage I\u2013III breast cancer patients

BackgroundImmune cells in the tumour microenvironment play an essential role in tumorigenesis. This study aimed to evaluate the immunoregulatory protein expression of breast cancer and reveal their prognostic role.MethodsExpression of 10 immune markers (PD-1/PD-L1/PD-L2/IDO/TIM-3/OX40/OX40L/B7-H2/ B7-H3/B7-H4) with known/possible clinical relevance was identified in stromal tumour-infiltrating lymphocytes or tumour tissue of stage I–III breast cancer patients.ResultsA total of 392 patients, including 271(69.1%) luminal A, 36(9.2%) luminal B, 32(8.2%) HER2-positive and 53(13.5%) triple negative disease, were included. Expression of PD-1 and PD-L1 was higher in HER2-positive and triple negative disease. By contrast, expression of TIM-3, OX40 and OX40L were higher in luminal disease. We devised an immune recurrence score (IRS) using seven markers with prognostic value (B7-H2/B7-H3/B7-H4/OX40/OX40L/PD-L1/PD-L2). Patients were classified as high-risk (7.9%), intermediate-risk (67.6%), or low-risk (24.5%). In the multivariate analysis, IRS low-risk (adjusted HR 0.14, p = 0.001) and intermediate-risk (adjusted HR 0.32, p = 0.002) had significantly lower risk of recurrence compared with high-risk. The prognostic role of IRS was maintained in both luminal A and non-luminal A patients.ConclusionsThis study identified immunoregulatory protein expression of breast cancer patients using 10 immune markers. In addition, we devised an IRS which may predict recurrence in stage I-III breast cancer patients.

Tumor mutation burden and PD-L1 expression in SDH/FH mutated solid tumors.

1524 Background: Succinate Dehydrogenases and Fumarate Hydratase (SDH/FH) deficient tumors are characterized by succinate/fumarate accumulation and resultant pesudohypoxia that drives malignant transformation. This state of pseudohypoxia leads to dysregulation of PD-1 receptor-ligand signaling. In this study, we explored tumor mutation burden (TMB), gene expression of PD-L1, and expression of other immune checkpoint- associated genes in a diverse cohort of human tumors harboring SDH A, B, C, D and FH mutations. Methods: Retrospective analysis was performed on whole exome sequencing (WES; ~150x coverage) and whole transcriptomic RNAseq (~200x106 reads per tumor) data from NantHealth to identify tumors harboring SDHx and/or FH mutations. WES was performed on tumor tissue and matched normal for each patient to assess TMB. TMB was measured by counting all somatic-specific non-synonymous exonic mutations, with > 200 mutations qualified as TMB-high. Immune checkpoint therapy-related gene expression was evaluated for PDL1, CTLA4 , IDO, LAG3, FOXP3, PDL2, TIGIT, TIM3 and OX40 . Results: Among tumor samples from 3377 patients analyzed, 42 patients were found to harbor potentially-pathogenic & pathogenic mutations in the SDHA, B, C, D and FH genes. The most common tumor types with SDH/FH mutations were lung (n = 7), breast (n = 6), and colon cancer (n = 6). Our analysis revealed that TMB was positively correlated with the presence of SDH/FH mutations (p < 0.001). High PD-L1 expression was also significantly correlated with the presence of SDH/FH mutation (p < 0.05), while CTLA4, IDO, LAG3, FOXP3, and OX40 expression was significantly higher in SDH/FH mutated samples (p < 0.05). Conclusions: We report for the first time an association between increased TMB and increased PD-L1 expression in a variety of SDH/FH mutated tumors. These key parameters, imply that a higher TMB may drive the evolutionary pressure to select clones with a PDL1 high phenotype. This observation supports a potential therapeutic role for inhibition of PD-1/PD-L1 pathway in these tumors.

Expression of OX40 Gene and its Serum Levels in Neuromyelitis Optica Patients.

Neuromyelitis optica (NMO), also known as Devic's disease, is an autoimmune disorder of the central nervous system (CNS) in which immune system cells and antibodies primarily attack the optic nerves and the spinal cord. OX40 (CD134) is a tumor necrosis factor (TNF)-receptor family member expressed primarily on activated CD4+ and CD8+ T-cells. In an autoimmune disease, OX40 is typically up-regulated at sites of inflammation, and increases in the number of peripheral CD4+ T-cells expressing OX40. OX40 and its ligand OX40L are key TNF members that augment T-cell expansion, cytokine production, and promote T-cell survival. The aim of this study was to evaluate and compare of OX40 gene expression and its serum levels in patients with NMO and healthy controls. Twenty sex-/age-matched healthy controls (HC) (median age = 32 years, 15 females/5 males) were engaged for the present study. Expression of OX40 at the transcript level and serum protein levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assays, respectively. The results indicated OX40 expression in patients was significantly lower than in healthy controls (p = 0.001). However, the serum level of OX40 was not significantly different between groups (p = 0.37). In addition, the results indicated that CD134 expression was not age-related (p = 0.041). Overall, this study suggests to us that OX40 levels are not a suitable marker for diagnosis or treatment of NMO.

Increased Expressions of OX40 and OX40 Ligand in Patients with Primary Immune Thrombocytopenia.

Background OX40, which is also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4), and its ligand (OX40L) play a critical role in the pathogenesis of autoimmune diseases. Immune thrombocytopenia (ITP), a hemorrhagic autoimmune disorder, is characterized by low platelet counts that are predominantly caused by antiplatelet autoantibodies. In this study, we firstly investigated the clinical significance of OX40 and OX40L expression in the pathogenesis of ITP in patients. Methods Fifty-four newly diagnosed ITP patients and 24 healthy controls (HCs) were enrolled in this study. The percentage of OX40+CD4+T cells among CD4+T cells was analyzed by flow cytometry, and the expression levels of OX40 and OX40L mRNA were analyzed by quantitative real-time PCR. Plasma soluble OX40L (sOX40L) levels were analyzed by ELISA, and plasma levels of antiplatelet autoantibodies were analyzed by a solid-phase technique. Results Compared with HCs, the frequencies of OX40+CD4+T cells were significantly increased in ITP patients, particularly in patients with positive antiplatelet autoantibodies compared to those with negative antiplatelet autoantibodies. The elevated frequencies of OX40+CD4+T cells were negatively correlated with low platelet counts in patients with positive antiplatelet autoantibodies. Plasma sOX40L levels in ITP patients were significantly greater than those in HCs and increased in patients with positive antiplatelet autoantibodies compared to those with negative antiplatelet autoantibodies. Plasma sOX40L levels were negatively correlated with low platelet counts in patients with positive antiplatelet autoantibodies. Additionally, the mRNA expression levels of OX40 and OX40L in PBMCs from ITP patients were also notably greater than those from HCs, and the expression levels of OX40 and OX40L were significantly different in ITP patients with positive and negative antiplatelet autoantibodies. Conclusion These data indicated that increased expression levels of OX40 and OX40L were involved in the pathogenesis of ITP, and OX40 and OX40L may be valuable therapeutic targets for ITP.

Relative Expression of OX40, OX40L mRNA, and OX40L Serum Levels in Women with Recurrent Spontaneous Abortion

ABSTRACTThis study determined the roles of OX40 and OX40L in women with recurrent spontaneous abortion (RSA). We compared the expression of OX40 and OX40L genes in peripheral blood mRNA levels and serum levels of OX40L in women with a history of RSA to the control group. In this case-control study, 40 women with a history of RSA (case group), and 40 others with no history of abortion (control group) were investigated. The expressions of OX40 mRNA and OX40L mRNA were determined in the two groups using the quantitative polymerase chain reaction. Also, the enzyme-linked immunosorbent assay was used to determine the levels of serum OX40L in the two groups. There were no significant differences in the maternal age of women in the two groups (30.1 ± 4.28 years in the case vs. 30.03 ± 4.23 years in the control group). There was no difference in terms of the levels of OX40 and OX40L mRNA between the groups (p = 0.08 and p = 0.56, respectively). In addition, there was no significant correlation between the expression of OX40 and OX40L mRNA levels with age or the number of abortions. The correlation between OX40 and OX40L mRNA levels was not significant. RSA history group turned to show a higher level of serum OX40L than the control group (p = 0.03). In conclusion, our findings demonstrated that the expression of OX40 mRNA and OX40L mRNA was similar between women with a history of RSA and the control group. The elevation of serum OX40L level may be considered as a risk factor for RSA.

Abstract P2-07-09: Immune recurrence score (IRS) using 7 immunoregulatory protein expression can predict recurrence in stage I-III breast cancer patients

Abstract Background: Immune cells in the tumor microenvironment play an essential role in tumor progression and regression. However, immunologic characteristics and their prognostic role have not been clearly identified in breast cancer patients. This study aimed to evaluate the immunologic characteristics and their prognostic role in breast cancer patients. Methods: This study enrolled pathologically proven stage I-III breast cancer patients. We performed immunohistochemical staining in stromal tumor-infiltrating lymphocytes (TILs) using 10 immune markers (PD-1, PD-L1, PD-L2, IDO, TIM3, OX40, OX40L, B7-H2, B7-H3, B7-H4) with known/possible clinical relevance. Expression of PD-1, PD-L1, and PD-L2 was also measured in adjacent tumor tissue. Intensity and proportion of the staining was measured for each immune marker. The intensity of immunohistochemical staining (IS, intensity score) was graded as follows: 0 (negative), 1 (weak), 2 (moderate), 3 (strong). The proportion of staining (PS, proportion score) was graded as follows: 0 (stain under <1%), 1 (1% to 5%), 2 (5% to 10%), 3 (10 to 25%), 4 (25% to 50%), Grade 5 (> 50%). Immune markers were defined as positive with one of the following; IS 1 with PS over 3, IS 2 with PS over 2, IS 3 with PS over 1. Results: A total of 392 patients, 271 (69.1%) luminal A, 36 (9.2%) luminal B, 32 (8.2%) HER2-positive, and 53 (13.5%) triple negative breast cancers were included. In total, PD-1 was expressed by stromal TILs in 130 (33.2%) patients, PD-L1 in 47 (12.0%), PD-L2 in 109 (27.8%), B7-H2 in 225 (57.4%), B7-H3 in 227 (57.9%), B7-H4 in 106 (27.0%), TIM3 in 111 (28.3%), IDO in 96 (24.5%), OX40 in 137 (34.9%), and OX40L in 165 (42.1%). In addition, PD-L1 was expressed in 15 (3.8%) tumor tissue, PD-L2 in 237 (60.5%), and PD-1 was not expressed in tumor tissue. Each breast cancer subtype showed different immunologic characteristics. Expression of PD-1 (stromal TILs) and PD-L1 (Tumor and stromal TILS) was higher in HER2-positive and triple negative breast cancer. By contrast, expression of TIM-3, OX40, and OX40L by stromal TILs were higher in luminal A and luminal B breast cancer. In the univariate analysis, expression of B7-H3 was associated with worse DFS and expression of OX40 and B7-H4 was associated with favorable DFS. Expression of PD-L1, PD-L2, OX40L, and B7-H2 had a tendency of favorable DFS. We devised an immune recurrence score (IRS) using 7 markers with prognostic value (B7-H2, B7-H3, B7-H4, OX40, OX40L, PD-L1, and PD-L2). Patients were classified as high-risk (31 patients, 7.9%), intermediate-risk (265, 67.6%), or low-risk (96, 24.5%) according to 7 immune marker expression. In the multivariate analysis, IRS low-risk (adjusted HR, 0.14; 95% CI, 0.04 – 0.45; p = 0.001) and intermediate-risk (adjusted HR, 0.32; 95% CI, 0.16 - 0.656.42; p = 0.002) had significantly lower risk of recurrence compared with high-risk. In the subgroup analysis, the prognostic role of IRS were maintained in both luminal A patients and non-luminal A patients. Conclusions: This study identified immunologic characteristics of breast cancer patients using 10 immune markers. In addition, we devised an IRS with 7 immune markers which may predict recurrence in stage I-III breast cancer patients. Citation Format: Lee D-W, Ryu HS, Lee K-H, Jin M-S, Woo G-U, Suh KJ, Yang Y, Kim H-J, Lee H-B, Kim T-Y, Moon H-G, Han W, Park IA, Noh D-Y, Im S-A. Immune recurrence score (IRS) using 7 immunoregulatory protein expression can predict recurrence in stage I-III breast cancer patients [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-07-09.

Biomarkers of immunotherapy response in rectal adenocarcinoma treated with neoadjuvant chemoradiotherapy.

593 Background: Immunotherapy (IO) in combination with chemoradiotherapy (CRT) in the neoadjuvant treatment for locally advanced rectal adenocarcinoma (RAC) is an area of active clinical research. There are no well-defined biomarkers in RAC predicting response to neoadjuvant therapy. Increase in tumor PD-L1 expression and tumor infiltrating lymphocytes (TIL) has been observed after neoadjuvant CRT which potentially enhances response to IO. We report herein expression of PD-L1 and CD8+ TIL and other biomarkers of immune activation including CXCL9, TIM3 (HAVCR2), IDO1, IFNG, IL17RE, LAG3 and OX40 (TNFRSF4) pre and post neoadjuvant CRT in RAC. Methods: We retrospectively evaluated 38 RAC patients from 2007-2016 treated with neoadjuvant CRT using 5FU based therapy. Pre and post CRT tissue samples were stained with VENTANA PD-L1 (SP263) rabbit monoclonal antibody to quantify PD-L1 expression. CD8+ TIL were recorded over one high power field (hpf) (40x objective) in the area of densest infiltrate. Additional biomarkers CXCL9, TIM3 (HAVCR2), IDO1, IFNG, IL17RE, LAG3 and OX40 (TNFRSF4) were assayed with RT-PCR. Independent sample and paired sample t-tests were used for statistical analysis of difference in biomarker expression. Relevant clinical endpoints including local relapse, distant metastases and survival were collected. Results: Median age was 60 years (range 32–87). Median follow up was 16.7 months (range 2.6-120.1). Median duration from completion of CRT to resection was 73 days (range 44–315). PD-L1 expression increased from 10% to 23% in pre-CRT versus post-CRT patients. CD8+ TIL increased in 30/39 (83%) patients with an average increase from 66.48 to 129.20 CD8+ TIL/hpf in the pre-CRT versus post-CRT setting. Using RT-PCR, a statistically significant increase in the expression of CXCL9, IFNG and OX40 (TNFRSF4) was found pre-CRT versus post-CRT using paired samples t-test. Conclusions: Neoadjuvant CRT for RAC increased PD-L1 expression and CD8+ TIL along with an increased expression of CXCL9, IFNG and OX40 (TNFRSF4). These data suggest a novel role for IO in neoadjuvant treatment of rectal cancer. Clinical outcome data on these patients will be presented.

Abstract B128: Timing and sequence of CpG and anti-OX40 is critical for in situ vaccination

Abstract Immunotherapy seeks to enhance or manipulate host antitumor immunity by various approaches, among them, in situ vaccination. In situ vaccination is a local intervention in which immune enhancing agents are injected locally into one site of tumor, triggering a T-cell immune response locally that then travels to attack cancer throughout the body. We have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting systemic immune response, if any, is detected by the regression of the distant, untreated tumor. In this test for abscopal therapeutic effects, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG)—a TLR9 ligand—and agonist anti-OX40 antibody has provided the most impressive results. This combination lead to durable disease control and long-term treatment-free survival in multiple mouse models of cancer. CpG induced myeloid cells to secrete cytokines, which subsequently induced OX40 expression on T-cells. Thus, we hypothesized that administration sequence and timing may affect the antitumor responses of in situ vaccination. In order to screen for the best sequence and timing we implanted A20 lymphoma tumors bilaterally in opposite sides of the abdomen of Balb/C mice. After tumors were established, one tumor was injected at the selected sequence and timing with the test agents and the resulting immune response was monitored by the measuring growth of the distant, untreated tumor. The systemic antitumor response required the presence of both CD4+ and CD8+ T-cells, as mice treated with the corresponding depleting antibodies were unable to control tumor growth. Even a single injection of low-dose CpG (50µg) and anti-OX40 (8µg) resulted in a fully protective systemic immune response. In addition, the cured animals were protected from rechallenge with the same A20 tumor but not unrelated tumors. Decreasing the dose even further to 10µg CpG and 1µg anti-OX40 partially preserved the therapeutic response with a long-term survival of 60%. Concurrent administration of CpG and anti-OX40 resulted in eradication of both local and distant disease. Sequential administration of CpG followed by anti-OX40 preserved the therapeutic efficacy. However, sequential administration of anti-OX40 followed by CpG significantly attenuated the therapeutic effect. While CpG followed by a 24- or 48-hour-delayed anti-OX40 treatment preserved the therapeutic efficacy of concurrent therapy, 72h delay in anti-OX40 administration resulted in reduced therapeutic effect. These data demonstrate the importance of the administration sequence for fully protective and curative antitumor immune responses. Our data suggest that the anti-OX40 antibody should be administered at the same time as CpG or somewhat delayed but not the other way around. The combination of anti-OX40 and CpG is currently studied in a phase I trial. Our results here impact the planning of future clinical trials of in situ vaccination with these two agents. Citation Format: Idit Sagiv-Barfi, Debra K. Czerwinski, Ronald Levy. Timing and sequence of CpG and anti-OX40 is critical for in situ vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B128.

Quantitative Multiplex Immunohistochemistry Identifies Immunosuppression in the AML Bone Marrow and NK-Cells As Prognostic Biomarker in Intermediate-Risk Patients

BACKGROUNDA complex interaction between blasts and surrounding cells in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment sustains blast proliferation and confers chemoresistance. T- and NK-cells have been shown to be dysfunctional in AML, which might be associated with immune evasion and poor prognosis. Here, we present a comprehensive analysis of the immune contexture of the AML BM at diagnosis and study its interaction with clinicopathological variables.METHODSDiagnostic BM biopsies (n=69) were collected from AML patients treated in the Helsinki University Hospital during 2005-2017 and age and gender-matched controls (n=12) to construct tissue microarrays (TMA). Using 8-plex immunohistochemistry (mIHC) and computerized image analysis, we determined cell abundance and immunophenotypic states of millions of immune cells. Immunoprofiles were integrated with a total of 120 clinicopathological variables including cytogenetics and molecular genetics, ELN (European Leukemia Net) risk classification, disease burden parameters, and patient demographics.RESULTSUnsupervised hierarchical clustering of the immunologic contexture defined by mIHC analysis grouped AML patients distinctly from control subjects (Fig 1a). By extracting significant differences (Mann-Whitney U test, q<0.05) and annotating immunologic markers as either anti-cancer or immunosuppression drivers based on literature, we observed an interesting polarization of increased immunosuppression in AML compared to control BM (Fig 1b). In AML patients, lower fraction of granzyme B expressing (GrB+) cells was noted both in CD3+CD4+ helper T-cells (10.9% vs 24.3%, q=0.002, in AML vs control BM) and CD3+CD8+ cytotoxic T-cells (23.5% vs 33.5%, q=0.02). Moreover, we observed pronounced T-cell inhibition features, such as higher proportion of regulatory T-cells (1.5% vs 0.0% FOXP3+ of helper T-cells, q<0.001), and lower expression of class I HLA in BM cells (89.1% vs 100.0%, q<0.001). Putative exhausted PD1+ T-cells were also markedly enriched in the AML BM (15.7% vs 1.7% PD1+ of helper T-cells and 13.2% vs 2.0% PD1+ of cytotoxic T-cells, q<0.001).Among the high interpatient heterogeneity, we discovered two main immune profiles. Cluster 1 was characterized with higher proportion (Log2 fold change >0.5, q<0.05) of cytotoxic T-cells and expression of CD57, CD27, and CD25 in T-cells, as well as higher expression of PD-L1 in the BM. Moreover, lower expression (Log2 fold change <0.5, q<0.05) of OX40 and CD45RO in T-cells and proportion of CD11c+BDCA1+ (type 1 myeloid dendritic cells) were observed. Patients of Cluster 1 were associated with longer event-free survival (EFS; HR 1.9, p=0.049) as well as lower age (median 53.6 vs 64.4 years, p=0.001). No connection between immunologic clusters and FLT3 or NPM1 genotype, complex karyotype, ELN risk class, blast proportion or leukocyte count was found.To support clinical decision-making in ELN 2017 intermediate-risk patients, we developed a risk stratification model focusing on this particular subgroup (n=28) using L1-penalized Cox regression. Patient age over 60 years (HR 8.1, CI95% 2.5-26.6 p<0.001) and low proportion of CD45+CD2+CD3- NK-cells (HR 0.92, CI95% 0.85-0.99 p=0.03) predicted worse EFS.In intermediate-risk patients (n=68) of a separate validation cohort (n=145) analyzed with flow cytometry, low NK cell proportion and high age predicted worse EFS (HR 2.7 CI95% 1.6-4.6 p<0.001) and OS (HR 3.9 CI95% 2.1-7.3 p<0.001) after adjusting with induction therapy protocol. Lower NK-cell proportion was associated with FLT3-ITD genotype (0.45% vs 1.1% NK-cells/all cells in FLT3-ITD+ vs FLT3-ITD- AML patients, p=0.01), and higher than median PB leukocyte (WBC) count (0.50% vs 1.9% NK-cells/all cells in patients with PB WBC ≥7.8x10E9/L vs <7.8x10E9/L, p<0.001).CONCLUSIONSUsing TMA cytometrics with mIHC and automated image analysis for detailed characterization of the immune contexture, we discovered pronounced immune exhaustion and suppression in AML BM. An aging-related immune profile was identified and was associated with poor prognosis. Furthermore, survival prediction of intermediate-risk patients might be enhanced by considering patient age and NK-cell proportion. Taken together, immunophenotyping of AML patients might improve risk stratification and identify a subgroup benefiting from immunomodulatory treatments. [Display omitted] DisclosuresPallaud:Novartis: Employment. Marques Ramos:Novartis: Employment. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Ariad: Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

In Situ Vaccination with Cpg and Anti-OX40 Antibody: Preclinical Optimization for Clinical Translation

In-situ vaccination is a local intervention in which immune enhancing agents are injected locally into one site of tumor, triggering a T cell immune response locally that then travels to attack cancer throughout the body.We have employed a preclinical strategy whereby the same syngeneic tumor is implanted at two separate sites in the body. One tumor is then injected with the test agents and the resulting systemic immune response, if any, is detected by the regression of the distant, untreated tumor.In this test for abscopal therapeutic effects, the combination of unmethylated CG-enriched oligodeoxynucleotide (CpG) - a TLR9 ligand - and agonist anti-OX40 antibody has provided impressive results. This combination lead to durable disease control and long-term treatment-free survival in multiple mouse models of cancer. CpG induced myeloid cells to secrete cytokines, which subsequently induced OX40 expression on T cells.Thus, we hypothesized that administration sequence and timing may affect the anti-tumor responses of in-situ vaccination.In order to screen for the best sequence and timing we implanted A20 lymphoma tumors bilaterally in opposite sides of the abdomen of Balb/C mice. After tumors were established, one tumor was injected at the selected sequence and timing with the test agents and the resulting immune response was monitored by the measuring growth of the distant, untreated tumor.As opposed to our usual schedule of three injections, even a single injection of CpG (50µg) and anti-OX40 (8µg) resulted in a fully protective systemic immune response. In addition, the cured animals were protected from re-challenge with the same A20 tumor but not unrelated tumors.Decreasing the dose even further to 10µg CpG and 1µg anti-OX40 partially preserved the therapeutic response with a long-term survival of 60%. Concurrent administration of CpG and anti-OX40 resulted in eradication of both local and distant disease. Sequential administration of CpG followed by anti-OX40 preserved the therapeutic efficacy. However, the opposite order of anti-OX40 followed by CpG significantly attenuated the therapeutic effect. While CpG followed by a 24- or 48-hour-delayed anti-OX40 treatment preserved the therapeutic efficacy, a 72h delay in anti-OX40 administration resulted in reduced therapeutic effect.These data demonstrate the importance of the administration sequence for fully protective anti-tumor immune responses. Our data suggest that the anti-OX40 antibody should be administered at the same time as CpG or with only a slight delay but not in the reverse order.Low-dose radiotherapy (2×2 Gy) is an effective treatment for patients with indolent non-Hodgkin's lymphoma. This treatment results in high response rates at the treated site. Since immune infiltrating cells in the tumor microenvironment are essential for in situ vaccination of CpG and anti-OX40 we aimed to assess the effect of adding radiation in our pre-clinical models of lymphoma. We found that the addition of 2x2 Gy radiation did not interfere with the induction of a protective immune response by of CpG and anti-OX40.Given the effectiveness of low dose radiotherapy for local control and its lack of interference with the immune related abscopal response in the pre-clinical model, we are including radiation in our current clinical trials. In addition, we have incorporated our findings in the preclinical model regarding dosing and scheduling of CpG and anti-OX40 antibody to the design of our current in situ vaccination lymphoma clinical trial. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Tracking T Cell Activation By OX40 Immuno-PET: A Novel Strategy for Imaging of Graft Versus Host Disease

BACKGROUNDGraft versus host disease (GvHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) mediated by donor immune cells reacting against host tissues. GvHD diagnosis is often challenging and superior non-invasive imaging strategies specifically detecting early GvHD are critically needed to improve clinical care of HCT recipients. Positron emission tomography (PET) imaging for GvHD diagnosis employing conventional tracers (18F-FDG) have largely been confounding, mainly due to their low specificity. Monitoring T cell activation and expansion using T-cell targeted PET tracers seems a more promising approach (Ronald et al., Cancer Res 77(11) 2893, 2017). We recently reported a novel immuno-PET tracer (64Cu-DOTA-mAbOX40) that enables non-invasive imaging of activated murine T cells expressing the cell surface activation marker OX40 (Alam et al., JCI 128(6) 2569, 2018). In the present work, we evaluated the utility of this immuno-PET strategy to image activated T cells in a major MHC-mismatch mouse model of acute GvHD.METHODSBalb/C (H-2Kd) recipients were irradiated with 8.8 Gy and on the same day received intravenously (i.v.) 5 x 10e6 T-cell depleted bone marrow (BM) cells with or without 1 x 10e6 CD4 and CD8 T cells positively selected from C57BL/6 (H-2Kb) mice. Severity of GvHD was assessed by clinical GvHD scoring. Flow cytometry of lymphoid organs from BM control and GvHD mice was performed at day 7 after HCT to determine OX40 protein expression on immune cells. For imaging studies, anti-OX40 monoclonal antibody (mAb) specific for murine OX40 (clone: OX86, BioXcell) was conjugated to DOTA chelate. The conjugate was evaluated by mass spectrometry (an average ratio of 1.4 DOTAs per mAb was obtained) and subsequently radiolabeled with 64CuCl2 (final specific activity 10-15μCi/μg and radiochemical purity >99%). Mice were tail-vein injected with 64Cu-DOTA-mAbOX40 (100 µCi, i.v.) at day 7 after HCT and PET-CT imaging performed 24 hours after injection. Immediately following PET-CT mice were euthanized and radioactivity measured in dissected weighed tissues using a gamma-counter.RESULTSFlow cytometry analysis of OX40 expression in lymphoid organs isolated at day 7 after HCT revealed significantly higher proportions and absolute numbers of OX40 expressing cells in the spleen and cervical lymph nodes (LN) isolated from mice that received BM + T cells (GvHD group) compared with mice having received BM cells alone (p<0.05). In vivo OX40-ImmunoPET performed at day 8 after HCT revealed increased radiotracer uptake in spleen (p < 0.0001), mesenteric LN (p < 0.01) and the abdominal region (p < 0.001) of mice with GvHD compared with BM control mice (Fig. 1A and B). Interestingly, 64Cu-DOTA-mAbOX40 uptake in spleen, mesenteric LN and abdominal region positively correlated with the GvHD score [spleen, r=0.6, p=0.0018; mesenteric LN, r=0.42, p=0.042; abdomen, r=0.77, p < 0.0001]. Biodistribution analysis using gamma counting of tissues confirmed the PET results showing the same trends; significantly increased uptake in GvHD mice compared with BM controls in spleen (p < 0.01), cervical LN (p < 0.01), mesenteric LN (p < 0.01) and GvHD target organs e.g. small intestine (p < 0.05), colon (p < 0.05) and skin (p < 0.01). Importantly, outcome analysis of GvHD mice receiving tracer doses of OX40 mAb at day 7 after HCT did not reveal any significant worsening of GvHD in terms of survival, body weight loss or GvHD score, compared with mice receiving the appropriate isotype control, supporting the safety of this OX40-targeted imaging approach.CONCLUSIONThe OX40 immuno-PET tracer enabled specific imaging of alloreactive OX40+ activated T cells in a murine model of acute GvHD. Efforts are ongoing to develop a humanized version of the 64Cu-DOTA-mAbOX40 tracer that will provide a readily translatable tool for GvHD diagnosis in the clinical setting.FIGURE 1. 64Cu-DOTA-AbOX40 PET-CT imaging in a mouse model of acute GvHD. (A) Representative day 8 64Cu-DOTA-AbOX40 PET-CT images in BM controls or GvHD group. H, heart (including cardiac muscle and blood); Li, liver; Sp, spleen; Bl, blood vessels and venous sinuses; BM, bone marrow; Ab, abdomen. (B) Quantitative region of interest PET image analysis of indicated organs in BM controls (n=12, blue filled boxes) or GvHD mice (n=12, red filled boxes). Outliers are represented as dots. [Mann-Whitney test , ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05]. [Display omitted] DisclosuresGambhir:CellSight Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Potent Activation of Human T Cells by mRNA Encoding Constitutively Active CD40

New strategies for augmenting the actual performance of therapeutic T cells in vivo are needed for improving clinical outcome of adoptive cell therapy. Cumulative findings suggest that CD40 plays an intrinsic role in T cell costimulation. Recently, we demonstrated the ability of truncated, auto-oligomerizing CD40 derivatives to induce strong activation of APCs in a ligand-independent manner. We reasoned that constitutively active CD40 (caCD40) can similarly exert enhancing effects on human antitumor T cells. To test this assumption, we transfected human T cells with in vitro-transcribed caCD40 mRNA. In polyclonal T cells, caCD40 triggered IFN-γ secretion and upregulated CD25 and 4-1BB. In antimelanoma tumor-infiltrating lymphocytes (TILs), caCD40 induced massive production of IFN-γ, exerting a pronounced synergistic effect when coexpressed with constitutively active TLR4 devoid of its extracellular ligand binding. In unselected "young" TILs, caCD40 reproducibly increased surface expression of CD25, OX40, 4-1BB, CD127, and CD28. Three days post-mRNA electroporation of CD8 TILs, caCD40 elevated IFN-γ and TNF-α production and cytolytic activity in the presence of autologous but not HLA-I-mismatched melanoma. Enhanced killing of autologous melanoma by young TILs was observed 4 d posttransfection. These findings suggest that caCD40 can function as a potent T cell adjuvant and provide essential guidelines for similar manipulation of other key members of the TNFR family.

Radiation Followed by OX40 Stimulation Drives Local and Abscopal Antitumor Effects in an Anti-PD1-Resistant Lung Tumor Model.

Purpose: Radiation is used extensively to treat localized cancer, but improved understanding of its effects on the immune system has increased interest in its potential systemic (abscopal) effects, particularly in combination with checkpoint inhibitors such as anti-PD1. The majority of patients either do not respond or develop resistance to monotherapy over time. Here, we investigated the efficacy of OX40 (CD134) stimulation as an alternative immunotherapeutic approach in combination with radiotherapy (XRT) in a murine model of anti-PD1-resistant lung tumors.Experimental Design: We established a bilateral tumor model in 129Sv/Ev mice using an anti-PD1-resistant lung tumor cell line. Primary tumors were treated with intratumoral injection of an OX40 agonist antibody, given as adjuvant therapy after XRT (36 Gy in three 12-Gy fractions), whereas secondary tumors were left untreated to investigate abscopal outcomes.Results: The combination of XRT followed by OX40 stimulation effectively inhibited local and systemic antitumor growth, limited lung metastases, and improved survival rates. This treatment regimen augmented CD4+ and CD8+ T-cell expansion. XRT induced the expression of OX40 on T cells in tumors and spleens and increased the percentages of splenic CD103+ dendritic cells.Conclusions: Our data extend the benefits of radiation to systemic disease control, especially when combined with anti-OX40 agonist to promote immunologically mediated abscopal effects. Moreover, this study provides a rational treatment approach and sequence to overcome anti-PD1-resistant poorly immunogenic tumors. Clin Cancer Res; 24(22); 5735-43. ©2018 AACR.

Expression of the Immune Checkpoint Modulator OX40 in Acute Lymphoblastic Leukemia Is Associated with BCR-ABL Positivity

OX40 and its ligand are members of the TNF/TNF receptor superfamily, which includes various molecules influencing cellular signaling and function of both tumor and immune cells. The ability of OX40 to promote proliferation and differentiation of activated T cells fueled present attempts to modulate this immune checkpoint to reinforce antitumor immunity. While we recently found evidence for the involvement of OX40 in pathophysiology of acute myeloid leukemia including natural killer (NK) cell immunosurveillance, less is known on its role in acute lymphoblastic leukemia (ALL). In the present study, OX40 expression on ALL cells was significantly associated with positivity for the adverse risk factor BCR-ABL. In line, signaling via OX40 increased metabolic activity of primary ALL cells and resulted in release of cytokines involved in disease pathophysiology. Furthermore, interaction of ALL-expressed OX40 with its cognate ligand on NK cells stimulated ALL cell lysis. The data presented thus not only identify the yet unknown involvement of OX40/OX40L in ALL pathophysiology and NK cell immunosurveillance but also point to the necessity to thoroughly consider the consequences of modulating the OX40/OX40L molecule system beyond its effects on T cells when developing OX40-targeting approaches for cancer immunotherapy.

P3.04-15 IDO and Tim3 Gene Expression is Correlated in NSCLC Patients with Low PDL1 Gene Expression

Tumor cells evade immunosurveillance by expression of immune checkpoint proteins PD-L1, IDO, and TIM3, even when tumor-infiltrating lymphocytes (TILs) are present. Combination of checkpoint inhibitors Pembrolizumab(PD-1i) and Epacadostat (IDOi) failed to show synergy in melanoma. Yet there are several candidate targets for checkpoint inhibition and rational selection of which combinations to explore is lacking. In particular, which checkpoints to target when PD-L1 is not expressed is largely unexplored. The purpose of this study was to assess the expression of 12 immune checkpoint genes in lung cancer patients with high and low expression of PD-L1. Retrospective review of deep whole transcriptomic sequencing (RNA-Seq) (∼200x106 reads per tumor) on 1467 unselected clinical cases from NantHealth. 112 NSCLC cases were categorized as PD-L1-low and PD-L1-high by median splitting. Expression and co-expression of 12 checkpoint markers (CTLA4,IDO,FOXP3,LAG3,TIGIT,VEGFA,VEGFC,VEGFB,PDL2,PDL1,OX40, and TIM3) were analyzed for tissue-specific enrichment and within PD-L1-defined categories. Immune-cell infiltration was estimated using RNA deconvolution based on known immune cell marker genes (Bindea et al 2013). CTLA, TIGIT and FOXP3 showed high correlations across all 112 samples as well within the PD-L1 low and high expression groups and differential expression across the two groups, with higher expression of the markers among the PD-L1 high expression group versus the low group. Within the PD-L1 high expression group, the expression of markers LAG3, TIM3, OX40, FOXP3, CTLA4 and TIGIT was both higher and more highly correlated than in the PD-L1 low group. Within the PD-L1-low category, IDO1 and TIM3 have relatively high expression and are highly correlated with each other (R=0.72, p=3.85x10ˆ-10). PD-L1 is a primary driver of immune suppression, however when PD-L1 is low there may be some differential role for IDO or TIM3. Combination IDOi and Tim3i should be considered in PD-L1 low patients.

Follicular T Helper Cell Signatures in Primary Biliary Cholangitis and Primary Sclerosing Cholangitis.

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are the most common cholestatic liver diseases. While PBC is generally accepted to be an autoimmune disorder characterized by pathognomonic autoantibodies against mitochondrial antigens, the pathogenesis of PSC is less precisely defined; however, some degree of altered immunity toward autoantigens has been suggested. Follicular T helper (Tfh) cells, a distinct clusters of differentiation (CD)4 T‐cell subset specialized in facilitating antibody responses, have been shown to contribute to humoral autoimmunity in various disorders; yet, there is only limited information on possible alterations of Tfh cells in the context of cholestatic liver diseases. Thus, we addressed this important question by analyzing the frequency, activation status, and function of Tfh cells and frequencies of regulatory follicular T helper (Tfr) cells in well‐defined cohorts of patients with PBC and patients with PSC. Interestingly, we observed a significant increase in circulating chemokine (C‐X‐C motif) receptor 5 (CXCR5)+programmed death 1 (PD‐1) +CD4+ Tfh cells in patients with PBC but not in those with PSC. Although the frequency of potentially pathogenic chemokine (C‐C motif) receptor 7 (CCR7)lowCXCR5+PD‐1+CD4+ Tfh cells was increased in both disorders compared to healthy donors, the increase was significantly more pronounced in PBC. Furthermore, in patients with PBC, Tfh cells displayed stronger expression of the activation markers OX40 and inducible costimulator of T cells, correlated with anti‐anti‐mitochondrial antibody M2 and immunoglobulin M titers, and were most significantly increased in patients with cirrhosis. Tfr cell numbers were similarly increased; however, Tfh/Tfr ratios were unaltered in PSC and PBC. These alterations did not correlate with increased secretion of the Tfh signature cytokine interleukin‐21 in sorted CD4 T cells. Conclusion: Significant alterations occur in the Tfh cell compartment in cholestatic liver diseases, suggesting that Tfh cells influence the pathogenesis of PBC and to a lesser extend PSC.

HIV-Infected Subjects With Poor CD4 T-Cell Recovery Despite Effective Therapy Express High Levels of OX40 and α4β7 on CD4 T-Cells Prior Therapy Initiation.

HIV-infected subjects with suboptimal CD4 restoration despite suppressive combined antiretroviral treatment (cART) (immunodiscordant subjects) have been classically characterized after a variable period of time under cART. Recently, we have reported that an increased frequency of proliferating CD4 T-cells in these subjects is already present before the cART onset. The potential contribution of peripheral compensatory homeostatic proliferation (HP) is yet unknown. We aimed to analyze the expression of HP-related cellular markers on CD4 T-cells of immunodiscordant subjects before cART. We analyzed the expression of OX40 and α4β7 on peripheral CD4 T-cells from immunodiscordant and control subjects (n = 21 each group) before cART initiation, and also on available follow-up samples (after 24 month of suppressive cART). Additionally, we tested the expression of these markers in an in vitro system for the study of human HP processes. Immunodiscordant subjects showed increased levels of OX40 and α4β7 on CD4 T-cells before cART initiation. While the cART tended to reduce these levels, immunodiscordant subjects still maintained comparatively higher levels of OX40 and α4β7 after 24 months under suppressive cART. These HP-related markers were upregulated in vitro during the human HP, especially during the fast HP. Our results are compatible with exacerbated HP processes in immunodiscordant subjects, already before the cART onset.

The In Vitro Impact of Glycyrrhizic Acid on CD4+ T Lymphocytes through OX40 Receptor in the Patients with Allergic Rhinitis.

Glycyrrhizic acid (GA), the major bioactive component of glycyrrhiza, possesses anti-inflammatory, anti-allergic, and immunomodulatory activities. This study aimed to investigate the in vitro anti-allergic effect of GA through the OX40 receptor in patients with allergic rhinitis. Purified naive CD4+ T cells of patients with allergic rhinitis (n = 12) were activated with anti-CD3/anti-CD28 with and without anti-OX40 agonist mAbs and then treated with 50, 100, and 200μM GA and 0.1μM dexamethasone. Cells were incubated (72h) to measure cell proliferation. Expression of OX40 in anti-OX40 mAb stimulated CD4+ T cells was evaluated by flow cytometry. mRNA expression of the OX40 receptor and T-bet, GATA-3, and forkhead box P3 (FoxP3) transcriptional factors were measured by a quantitative polymerase chain reaction. The levels of interleukin (IL)-4, IL-10, and interferon-γ (IFN-γ) were also measured. GA inhibited significantly the augmented T cell proliferation induced with anti-OX40 mAb. Protein and gene expression of OX40 was also decreased significantly. Dexamethasone and GA inhibited T-bet and GATA-3 genes expression, but this inhibition was only significant for GATA-3. In contrast, enhanced gene expression of FoxP3 was seen using 200μM GA and dexamethasone. The levels of IL-4, IL-10, and IFN-γ decreased after treatment with both dexamethasone and GA, but the ratio of IFN-γ/IL-4 (Th1/Th2 balance) increased significantly due to 200μM GA treatment. This study suggests that GA may have a therapeutic effect on allergic rhinitis, partly by modulation of the Th1/Th2 balance through suppression of OX40 and increasing the activity of regulatory T cells.

Abstract LB-127: From bench to bedside: Exploring OX40 receptor modulation in a phase 1/2a study of the OX40 costimulatory agonist BMS-986178 ± nivolumab (NIVO) or ipilimumab (IPI) in patients with advanced solid tumors

Abstract Background: Activation of OX40, a costimulatory protein in the tumor necrosis factor receptor super family, enhances T effector cell activation and inhibits T regulatory (Treg) cell-mediated suppression. Preclinical data showed that combination of an OX40 agonist with checkpoint blockade (anti-PD-1 or anti-CTLA-4) enhanced antitumor activity vs checkpoint blockade alone. BMS-986178 is a fully human IgG1 agonist monoclonal antibody that binds with high affinity to OX40 and was well tolerated ± NIVO (anti-PD-1; Olszanski AJ, et al. J Immunother Cancer 2017;5(suppl2) [abstract O17]). The relationship between receptor modulation and the activity of agonist OX40 agents in the clinic is not clear. Here we present findings on the regulation of OX40 receptor expression and T-cell activation by BMS-986178 ± NIVO or IPI (anti–CTLA-4) from the phase 1/2a study in patients with advanced solid tumors (NCT02737475). Methods: Patients with ≥ 1 prior therapy were treated in this open-label, dose-escalation and -expansion study. During escalation, patients received BMS-986178 monotherapy 20-320 mg IV Q2W, BMS-986178 20-320 mg + NIVO 240 mg IV Q2W, or BMS-986178 20-320 mg + IPI 1 mg/kg IV Q3W. Pharmacokinetics (PK) and systemic pharmacodynamics (PD) were evaluated, including analysis of OX40 receptor occupancy (RO), CD4+ T cell- and Treg-cell surface OX40 expression, soluble OX40 (sOX40) levels, cytokines, and proliferation of effector memory T cells in circulation. Results: BMS-986178 PK was linear from 20 to 320 mg when administered as monotherapy (n = 20), + NIVO (n = 38), or + IPI (n = 32). Moreover, BMS-986178 ± NIVO or IPI stimulated the production of IFN-γ and increased proliferating (Ki-67+) effector memory T cells. Concomitant downregulation of OX40 expression on the cell surface of CD4+ T cells and Tregs was observed as OX40 RO approached saturation at BMS-986178 doses of ≥ 40 mg. Furthermore, BMS-986178 doses of 20-160 mg ± NIVO or IPI increased sOX40 levels, while BMS-986178 doses of ≥ 160 mg ± NIVO or IPI led to sOX40 plateau. Conclusions: Clinical PK/PD findings showed efficient BMS-986178 target engagement and confirmed preclinical observations that BMS-986178 can modulate RO and OX40/sOX40 expression. Furthermore, peripheral T-cell activation was observed in patients treated with BMS-986178 ± NIVO or IPI. Coupled with our preclinical observations, these findings highlight a complex dose-response relationship between BMS-986178 receptor saturation, receptor modulation, and induction of soluble receptor. These data support further clinical investigation of a broader dose range and optimal schedules for BMS-986178 ± NIVO or IPI. Citation Format: Rui Wang, Yan Feng, Ed Hilt, Xiling Yuan, Chan Gao, Xiao Shao, Yongliang Sun, Michael D'silva, Katherine Yang, Becky Penhallow, Goce Bogdanoski, Rajesh Anand, Irene Pak, Danielle Greenawalt, Anke Klippel, Nataly Manjarrez-Orduno, Robert Neely, Michael Quigley, Michael Hedrick, Praveen Aanur, Z Cao. From bench to bedside: Exploring OX40 receptor modulation in a phase 1/2a study of the OX40 costimulatory agonist BMS-986178 ± nivolumab (NIVO) or ipilimumab (IPI) in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-127.