PD-1 Blockade Treatment Research Articles

Dramatic Responses of Recurrent Upper Urinary Tract Urothelial Carcinoma Harboring FGFR3 and TP53 Activating Mutations to Pembrolizumab in Combination with Erdafitinib: A Case Report

BackgroundUpper tract urothelial carcinoma (UTUC) has a high recurrence rate and is likely refractory to systemic chemotherapy. The long-term outcomes and responses to immunotherapy and retreatment regimen after tumor recurrence for such cases had not yet been well-documented.Case PresentationHere we report a unique case of long-term follow-up with a 67-year-old woman, who was diagnosed with advanced UTUC, received radical nephroureterectomy with bladder cuff, and was refractory to chemotherapy with cisplatin and gemcitabine. Positive PD-L1 expression and somatic mutation of Ser249Cys in FGFR3 were identified in the tumor tissue. The patient then received pembrolizumab monotherapy and achieved complete response (CR) after 6 cycles of treatment. She discontinued pembrolizumab treatment thereafter but remained in CR for 3 years and 7 months until the recurrence of tumor in the right mid-ureter. The patient was then retreated with a combination of pembrolizumab and erdafitinib, and achieved CR again after the third cycle of treatment.ConclusionWe reported here a rare case of UTUC with concurrent pathogenic mutations in FGFR3 and TP53 with positive PD-L1 expression. The patient archived exceptional therapeutic responses to PD-1 blockade treatment and retreatment with combination of pembrolizumab and erdafitinib. Our results provide new insight into the duration of immunotherapy and the retreatment strategy after tumor recurrence based on individual genomic profiles.

Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.

Dynamic changes in circulating PD-1+CD8+ T lymphocytes for predicting treatment response to PD-1 blockade in patients with non-small-cell lung cancer.

The predictive value of immune monitoring with circulating CD8+ T lymphocytes for treatment response to programmed cell death protein 1 (PD-1) inhibitors has not been explored in non-small-cell lung cancer (NSCLC), prompting us to investigate whether dynamic changes in PD-1+CD8+ T lymphocytes have predictive value for durable clinical benefit (DCB) and survival after PD-1 blockade. Patients with recurrent and/or metastatic NSCLC treated with PD-1 inhibitors were enrolled (discovery cohort; n=94). Peripheral blood was obtained immediately before and after one cycle of treatment with PD-1 blockade. Phenotyping of circulating CD8+ T lymphocytes was conducted using multi-colour flow cytometry. Predictive values of dynamic changes in circulating PD-1+CD8+ T lymphocytes during the first cycle were validated in an independent cohort (validation cohort; n=54) of a prospective trial with a PD-1 inhibitor (NCT03486119). Circulating PD-1+CD8+ T lymphocytes were enriched with effector/memory populations with elevated expression of activation- and exhaustion-related markers. Reduction in the frequency of PD-1+ cells among CD8+ T lymphocytes after one cycle of treatment was associated with a higher probability of DCB and superior survival outcomes in the discovery cohort. Similar results were obtained in the analysis of tumour antigen NY-ESO-1-specific CD8+ T lymphocytes and the validation cohort. Mechanistically, PD-1 molecule expression on CD8+ T lymphocytes suppresses the effector functions of tumour antigen-specific CD8+ T lymphocytes. Dynamic changes in circulating PD-1+CD8+ T lymphocytes predict clinical, and survival benefit from PD-1 blockade treatment in NSCLC, providing a useful tool to identify patient subgroups who will optimally benefit from PD-1 inhibitors.

IMMU-11. DUAL TARGETING OF IL-6 AND CD40 OVERCOMES GLIOBLASTOMA RESISTANCE TO IMMUNE CHECKPOINT BLOCKADE

Abstract Glioblastoma (GBM) is refractory to current T cell-based immunotherapies such as checkpoint blockade. GBM is characterized by extensive infiltration of immunosuppressive macrophages (Mφs) that contribute to the treatment resistance. Here we develop a dual-targeting strategy to synergistically activate tumor-associated Mφs, which overcomes GBM resistance to therapeutic blockade of the PD1 and CTLA4 checkpoints. Consistent with a previously established role of IL-6 in alternative Mφ polarization, we show that targeting IL-6 by genetic ablation or pharmacological inhibition moderately improves T cell infiltration and enhances animal survival in a genetically engineered mouse GBM model. However, IL-6 inhibition does not synergize PD-1 and CTLA-4 blockade in GBM. Interestingly, we reveal that anti-IL-6 therapy reduces CD40 expression in GBM-associated Mφs. Our transcriptome analysis identifies a Stat3/HIF-1a-mediated axis, through which IL-6 regulates CD40 expression in Mφs. Finally, we show that combination of IL-6 blockade with CD40 stimulation robustly reverses Mφ-mediated tumor immunosuppression, enhances T cell infiltration, and sensitizes GBM to PD-1 and CTLA-4 blockade treatment, cumulating in inhibited tumor growth and extended animal survival. These findings illustrate a cellular mechanism that regulates Mφ-mediated tumor immunity, and suggest that dual-targeting IL-6 and CD40 may offer exciting opportunities for improving immunotherapy against GBM.

Combination of Photodynamic Therapy and a Flagellin-Adjuvanted Cancer Vaccine Potentiated the Anti-PD-1-Mediated Melanoma Suppression.

Immune checkpoint inhibitors become a standard therapy for malignant melanoma. As immune checkpoint inhibitor monotherapies proved to have limited efficacy in significant portion of patients, it is envisaged that combination with other therapeutic modalities may improve clinical outcomes. We investigated the effect of combining photodynamic therapy (PDT) and TLR5 agonist flagellin-adjuvanted tumor-specific peptide vaccination (FlaB-Vax) on the promotion of PD-1 blockade-mediated melanoma suppression using a mouse B16-F10 implantation model. Using a bilateral mouse melanoma cancer model, we evaluated the potentiation of PD-1 blockade by the combination of peritumoral FlaB-Vax delivery and PDT tumor ablation. A photosensitizing agent, pheophorbide A (PhA), was used for laser-triggered photodynamic destruction of the primary tumor. The effect of combination therapy in conjunction with PD-1 blockade was evaluated for tumor growth and survival. The effector cytokines that promote the activation of CD8+ T cells and antigen-presenting cells in tumor tissue and tumor-draining lymph nodes (TDLNs) were also assayed. PDT and FlaB-Vax combination therapy induced efficacious systemic antitumor immune responses for local and abscopal tumor control, with a significant increase in tumor-infiltrating effector memory CD8+ T cells and systemic IFNγ secretion. The combination of PDT and FlaB-Vax also enhanced the infiltration of tumor antigen-reactive CD8+ T cells and the accumulation of migratory CXCL10-secreting CD103+ dendritic cells (DCs) presumably contributing to tumor antigen cross-presentation in the tumor microenvironment (TME). The CD8+ T-cell-dependent therapeutic benefits of PDT combined with FlaB-Vax was significantly enhanced by a PD-1-targeting checkpoint inhibitor therapy. Conclusively, the combination of FlaB-Vax with PDT-mediated tumor ablation would serve a safe and feasible combinatorial therapy for enhancing PD-1 blockade treatment of malignant melanoma.

Abstract 917: Functional blocking of CD5 on T cells to enhance the efficacy of therapeutic PD-1 blockade in treatment of mouse 4T1 breast tumors

Abstract Blockade of programmed cell death protein 1 (PD-1) is approved for treatment of multiple human cancers and is the focus of multiple studies due to its key role in T cell function. However, only patients with ‘'hot'' tumors (i.e. those with increased numbers of tumor-infiltrating CD8+ T cells) respond well to the blockade. This suggests that new strategies to increase infiltration of CD8+ T cells into tumors will increase the number of tumor types and patients benefiting from anti-PD-1 therapy. CD5, a member of the scavenger receptor cysteine-rich (SRCR) superfamily, is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signaling and impair cytotoxic T lymphocyte (CTL) activation. CD5 knockout mice have increased anti-tumor immunity and reduced homograft tumor growth: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumor response and increase tumor-infiltrating CD8+ T cells. We report that in vivo administration of anti-CD5 blocking MAb treatment increased primary T cell activation in response to 4T1 tumor cell homografts and ex vivo activation as measured by increased CD69, Fas, Fas ligand, IFNγ, PD-1, and apoptosis. Further, anti-CD5 treatment enhanced the capacity of primary T cells to kill 4T1 tumor cells ex vivo. Mice receiving anti-CD5 and anti-PD-1 in combination exhibited increased overall survival compared to mice treated with either agent alone. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumor immunity and PD-1 blockade treatment. Citation Format: Faizah Alotaibi, Mateusz Rytelewski, Rene Figueredo, Ronak Zareardalan1, Saman Maleki Vareki, Xiufen Zheng, Meng Zhang, Peter Ferguson, Mikal El-Hajjar, Yousef Najajreh, Wei-ping Min, James Koropatnick. Functional blocking of CD5 on T cells to enhance the efficacy of therapeutic PD-1 blockade in treatment of mouse 4T1 breast tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 917.

Abstract 3865: A translational immuno-oncology platform to model T cell activation and Tex recovery in the tumor microenvironment

Abstract The tumor microenvironment (TME) is a complex network, consisting of malignant tumor cells, stromal and immune cells, blood vessels, extracellular matrix and soluble factors. The immune system plays an important role in combating tumor growth, and multiple studies associate immune infiltrate with beneficial outcome. Various tractable immuno-oncology targets have been identified that influence the behavior of immune cells in the TME. These include Cytotoxic CD8+ T cells; which can directly kill tumour cells and produce pro-inflammatory cytokines such as IFNγ. However, CD8+ T cell activity and survival can be suppressed by the TME and chronic exposure leads to the generation of exhausted T cells (Tex) which have a lowered proliferative capacity and diminished capacity for pro-inflammatory cytokine production. Conversely, a regulatory subset of CD4+ T cells (Tregs) act to skew the TME towards immunosuppression. Here we describe human and murine models to assess both relative T cell subset activation and whether T cell exhaustion can be broken. Screening assays using T cell stimulants such as SEB or IL-2 can be utilised to select candidate therapeutics based on functional target engagement. SEB stimulation of PBMC reveals a robust additive effect on T cell activation by established and emerging checkpoint inhibitors. Use of more complicated flow based readouts allows the discrimination of immune subset responses. IL-2, a key lymphocyte activator, drives pSTAT5 expression with differing potency in PBMC subsets (regulatory T cells, conventional CD4 cells, CD8 T cells, NKT cells and NK cells). The ability to target specific subset activation could expand anti-tumour responses. Persistent antigenic stimulation, such as chronic exposure to the TME (MHC I restricted peptides), can lead to a CD8 Tex cell phenotype. Tex cell generation and reversal can be modelled using CEFT peptide pool and monitored via the expression of markers such as LAG-3, TIM-3, TOX and IFNγ. We show here that CD8 Tex cells respond positively to PD-1 blockade treatment, as seen by a restoration in cellular proliferation and increased IFNγ production. Therapeutic candidates identified through in vitro lead optimization can be assessed for their ability to activate T cells via a murine pharmacodynamic (PD) model. Transgenic OT-I CD8 T cells expressing TCR recognising OVA peptide (SIINFEKL) were transferred into syngeneic mice with SIINFEKL challenge in the presence of test therapeutic. Flow cytometry was used to determine OT-I T cell proliferation, activation and cytokine release. As seen in vitro, OT-I cell activation was increased by CPI treatment. We show here the powerful combination of using sophisticated in vitro and in vivo models to determine the effect of therapeutic intervention in an immuno-oncology context. Citation Format: Robert Nunan, Hannah Gill, Ilona Aylott, Michelle Yap, Tommaso Iannitti, Louise Harvey, Rhiannon Jenkinson. A translational immuno-oncology platform to model T cell activation and Tex recovery in the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3865.

PD-1 checkpoint blockade in advanced melanoma patients: NK cells, monocytic subsets and host PD-L1 expression as predictive biomarker candidates

ABSTRACT Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival (OS) and a dramatic improvement in patient quality of life. Despite the success of this approach, the number of benefitting patients is limited and there is a need for predictive biomarkers as well as a deeper mechanistic analysis of the cellular populations involved in clinical responses. With the aim to find predictive biomarkers for PD-1 checkpoint blockade, an in-depth immune monitoring study was conducted in 36 advanced melanoma patients receiving pembrolizumab or nivolumab treatment at Karolinska University Hospital. Blood samples were collected before treatment and before administration of the second and fourth doses. Peripheral blood mononuclear cells were isolated and stained for flow cytometric analysis within 2 h of sample collection. Overall survival and progression-free survival (PFS) were inversely correlated with CD69 expression NK cells. In the myeloid compartment, high frequencies of non-classical monocytes and low frequencies of monocytic myeloid derived suppressor cells (MoMDSCs) correlated with response rates and OS. A deeper characterization of monocytic subsets showed that PD-L1 expression in MDSCs, non-classical and intermediate monocytes was significantly increased in patients with shorter PFS in addition to correlating inversely with OS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of activated NK cells and monocytic subsets are inversely correlated with survival and clinical benefit, suggesting that their role as predictive biomarkers should be further evaluated.

Abstract B043: Identification of secreted PD-L1 variants as a decoy of PD-L1 blockade antibody mediating the therapeutic resistance

Abstract Immune checkpoint blockade therapy targeting PD-L1/PD-1 have been widely used and shown remarkable clinical response in various cancers. However, therapeutic resistance after initial response is increasingly observed and the detailed mechanisms of resistance have yet to be fully understood. Recently, several mechanisms including loss-of-function alteration in genes related to antigen presentation and interferon-receptor signaling have been suggested to induce the resistance to PD-1 blockade treatment, yet few studies have focused on anti-PD-L1 (aPD-L1) blockade therapy. In current study, we performed whole exome sequencing and RNA-sequencing analysis in two non-small cell lung cancer (NSCLC) patients who were refractory during aPD-L1 therapy. We have identified the mutations in RNA splicing related gene, TARDBP/TDP-43, which were uncovered as hot spot mutations in Amyotrophic lateral sclerosis (ALS), and found two unique secreted PD-L1 variants, which lacked the transmembrane domain by aberrant RNA splicing. These secreted PD-L1 variants were shown to be stable by pulse chase assay, and worked as “decoys” of aPD-L1 antibody in the HLA-matched coculture system of iPSC-derived CD8 T cells and cancer cells. Moreover, expression of secreted mPD-L1 variant in mouse MC38 cancer cells conferred the resistance to PD-L1 blockade therapy, and soluble PD-L1 were time dependently accumulated in plasma using the MC38 syngeneic mice model. To further investigate the presence of secreted PD-L1 splicing variants in patients, we additionally analyzed 15 specimen who were resistant to anti-PD-L1 treatment by RNA-seq. We revealed that approximately 20% of therapeutic resistant patients harbored secreted PD-L1 variants and several new mutations in JAK1/2, the key mediator of interferon-receptor signaling, were found in a part of patients as well. Furthermore, we also identified the presence of secreted PD-L1 variants in the surgical specimens of squamous NSCLC patients without prior chemotherapy, targeted therapy, and /or immune-checkpoint inhibitor therapy. Consistent to the data, the plasma level of soluble PD-L1 in patients with secreted PD-L1 variants were high than those without the variants. Collectively, our results elucidated a novel resistant mechanism of PD-L1 blockade antibody mediated by secreted PD-L1 variants. The presence of sPD-L1 splicing variants or the level of soluble PD-L1 in plasma or pleural effusion may work as a biomarker to predict a patient’s response to PD-L1 blockade therapy. Citation Format: Bo Gong, Kazuma Kiyotani, Seiji Sakata, Ken Takahashi, Seiji Nagano, Shun Kumehara, Satoko Baba, Benjamin Besse, Noriko Yanagitani, Luc Friboulet, Makoto Nishio, Kengo Takeuchi, Hiroshi Kawamoto, Naoya Fujita, Ryohei Katayama. Identification of secreted PD-L1 variants as a decoy of PD-L1 blockade antibody mediating the therapeutic resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B043. doi:10.1158/1535-7163.TARG-19-B043

Single-Cell Deconvolution Identifies T-Cell Correlates of Response to PD-1 Blockade Treatment in AML

Background The combination of the αPD-1 (nivolumab) and hypomethylating agent azacytidine demonstrated encouraging response rate and overall survival in relapsed/refractory acute myeloid leukemia (AML) patients, compared to azacytidine alone1 (NCT02397720). However, the percentage of the patient who achieved an IWG 2016 response to such therapy was still limited (overall response rate = 33%), so it is desirable to have early predictive biomarkers to facilitate patient stratification and selection for future trials. A better understanding of T cells (primary targets of αPD-1 therapy) from bone marrow (BM) and peripheral blood (PB) in AML pre-therapy and on-therapy should yield valuable insights on the treatment-induced anti-tumor response. Methods We performed whole transcriptomic profiling (RNA-sequencing, RNA-seq) on T cells from a cohort of AML patients who were enrolled in the clinical trial mentioned above, and treated with azacytidine and nivolumab (Table 1). Sixty-four FACS-sorted patient-derived T cell samples from cryopreserved peripheral blood (PB) or bone marrow (BM) aspirates, which were collected pre-therapy (T0) and after the first round of treatment (end of cycle one, EOC1), were evaluated. By leveraging subset definitions based on scRNA-seq results from T cells of cancer patients, we implemented deconvolution of our bulk T-cell RNA-seq data to obtain the relative abundance of different T-cell subsets (in-silico dissection without physical isolation). Results We performed ex vivo cDNA library preparation on 2,000-100,000 sorted T cells and yielded a minimal of 17 million sequencing reads per BM T-cell library and 2.6 million sequencing reads per PBMC T-cell library. We compared the gene expression profile of peripheral blood T cells from AML patients (CD4: n = 16; CD8: n = 15) and healthy donors (HD, n = 8)2,3 to validate our methodology. The deconvolution results were consistent with previously published flow-cytometry data profiling cancer patients4,5 (Figure 1). In comparison with HDs, circulating CD4 T cells from AML patients consisted of a higher frequency of Treg (AML versus HD: 5.1% versus 0.6%, P<0.0001)4. Peripheral blood CD8 T cells from AML patients were with a significantly lower frequency of naïve (AML versus HD: 22.1% versus 55.9%, P<0.0001), a higher frequency of effector phenotype (AML versus HD: 35.6% versus 6.7%, P =0.0011), and an increasing frequency of exhausted phenotype (AML versus HD: 19.1% versus 0.0%, P<0.0001)5. Independent of the clinical responses attained on the azacytidine and nivolumab, comparison of the pre-therapy CD8 T cells from BM and PB from all AML patients using both gene set enrichment analysis and deconvolution indicated that the BM CD8 T cells were more activated/differentiated compared with PBMC CD8 T cells, likely reflective of an ongoing immune response against the AML. We also found treatment-induced gene expression profile changes in the AML circulating CD8 T cells, characterized by increased cell metabolism and cell proliferation. Deconvolution identified that pre-therapy relative abundance of exhausted (CD3+CD8+PD-1+CD45RO+) and effector (CD3+CD8+CD45RA+Tbet+PD-1lo) CD8 T cells (plasticity) could serve as subpopulations relevant for patient stratification (Figure 2). We further validated these results using CyTOF wherein these same subpopulations were differentially abundant between responders and non-responders at the pre-therapy time-point. Conclusions Collectively, the analyses of RNA-seq of CD8 T cells from AML patients on the azacytidine with nivolumab trial revealed that (1) the PD-1 blockade-based treatment-induced gene expression profiling changes (increased cell proliferation and metabolism) of CD8 T cells are detectable as early as EOC1 in the peripheral blood; (2) specific subpopulations of plastic CD8 T cells identified may have the potential to serve as an actionable biomarker to select AML patients most likely to benefit from such immune checkpoint therapies. These findings need to be confirmed in larger studies, planned or currently being conducted, with αPD-1 based therapies in AML and MDS.

EP1.01-55 Neoantigen Deletion Leads to Hyperprogressive Disease (HPD) in Non-Small Cell Lung Cancer (NSCLC) Treated with PD-1/PD-L1 Inhibitors

Hyperprogressive disease (HPD) is a distinct pattern of progression described in patients with cancer treated with PD-1/PD-L1 inhibitors. The incidence of HPD is reported to be 13.8% in advanced NSCLC. Recently multiple characteristics have been recognized to predict the risk of HPD, however the mechanism underlying have not been systemically studied. We report a Chinese patient who developed hyperprogressive disease after 4 cycles of PD-1 blockade treatment. Clinical and demographic data was collected from electronic records. Whole exome sequencing (WES) of the primary tumor was performed to identify tumor mutation burden (TMB) and tumor neoantigen burden (TNB) following the restricted pipeline. Liquid biopsy targeting ctDNA and T cell receptor (TCR) was constructed to monitor the efficacy of immunotherapy. A 70-year-old male with smoking history underwent chest CT scan in February 2018 and was found to have a right upper lobe mass with intrapulmonary metastasis in different lobes and multiple lymph nodes. Resection of right lower lung by video-assisted thoracic surgery (VATS) revealed poorly differentiated, squamous cell carcinoma. In July 2018, he progressed after two cycle of chemotherapy and radiation therapy targeting newly bone metastasis of lumbar spine, then he was screened to start Nivolumab at dose of 240mg daily/2 weeks. After four cycle of Nivolumab, he obtained PD and was assigned to palliative thoracic radiation therapy. The PD-L1 expression level of our patient was 1%≤TC<5%, next generation sequencing (NGS) didn’t show alteration in genes related to HPD, such as MDM2/MDM4 or EGFR. Blood sample retrieved before and after immunotherapy revealed the increase in bTMB (from 7.70 Muts/Mb to 18.00 Muts/Mb), however TNB truly expressed as neoantigens were rather lower compared to TMB, indicating the low immunogenicity of tumor. We also found a sharp fluctuate in the detection of driver genes mutations in ctDNA (TP53,CDKN2A,ERBB3,FBXW7), and the majority of high-frequency TCR dropped significantly during immunotharapy. It is reported that NSCLC with a high tumor mutation burden (TMB) may benefit from PD-1/PD-L1 inhibitors treatment, and TMB is considered as the surrogate of tumor neoantigen burden (TNB). However the deletion of neoantigen may occur at different level, including copy number variation at the DNA level, down-regulation of transcription level and inherited variation at the epigenetic level, resulting in the low immunogenicity of primary tumor. Deletion of neoantigen may provide a mechanism of immune evasion which leads to the resistance to PD-1/PD-L1 inhibitors. In this case, treatment of checkpoint inhibitors may activate certain signaling pathway of driver genes to support the hyperprogression disease (HPD). This case report focusing on neoantigen provides a new approach to predict the presence of hyperprogressive disease (HPD) before immunotherapy, and explains the potential mechanism of HPD.

Abstract 941: B cells are required to generate optimal antitumor immunity in response to PD-1 blockade treatment

Abstract Background: Melanoma is the most deadly form of skin cancer. Although, the immunogenic nature of melanoma makes this disease susceptible to immunotherapy, metastatic melanoma remains highly resistant to established immunotherapies like cancer vaccines. Anti-PD-1 antibodies alone showed great clinical activity but were still ineffective in many melanoma patients. These reports highlight the need to understand the molecular mechanism of immunotherapy induced anti-melanoma immune responses to develop new approaches for effective therapy against melanoma. B cells augment CD8+ T cell-mediated immunity during autoimmunity and allograft rejection, but the role of B cells in anti-tumor CD8+ T cell immunity and in cancer patient survival is controversial. Recently, we reported that intratumoral stimulation of TLR-7/8 in a mouse model of melanoma induced tumor regression and activation of B cells. B cells were required for tumor suppression in an antibody-independent manner. Also, we found that melanoma patients who responded to anti-PD-1 blockade therapy had significantly more B cells and CD8+ T cells in tumors than non-responders. Based on these evidences, we hypothesized that activated B cells induce and/or enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy. Methods: B16 melanoma mouse model was used. Wild type and B cell Knockout mice were used to determine treatment efficacy and generation of tumor-specific CD8+T cells in response to PD-L1 blockade and/or TLR-7/8 activation. TLR-7/8 agonist activated B cells were isolated and adoptively transferred into tumor-bearing B cell KO mice, which were treated with anti-PD-L1.Therapeutic efficacy and melanoma-specific CD8+ T cell immunity were determined. Results: We found that absence of B cells not only decreases the efficacy of TLR-7/8 agonist or anti-PDL-1 abs monotherapy but also TLR-7/8 agonist and anti-PD-L1 abs combination therapy. In addition, mice were unable to develop potent anti- melanoma CD8+T cell immunity in the absence of B cells in response to either mono or combination therapy. Interestingly, we reported prolonged survival of tumor bearing B cell KO mice which received TLR-7/8 agonist activated B cells compared to inactivated B cells recipients in response to anti-PD-L1 antibody therapy. Conclusions: These results suggest that activated B cells enhance anti-melanoma CD8+ T cell immunity in response to immunotherapy. We expect our findings to lead the field closer to development of biomarkers based on B cell activation that predict response to immunotherapy. Our findings may also facilitate design of more effective anti-melanoma immunotherapy. Citation Format: Shubhra Singh, Karishma Bavisi, Jason Roszik, Adi Diab, Richard E. Davis, Patrick Hwu, Willem W. Overwijk, Manisha Singh. B cells are required to generate optimal antitumor immunity in response to PD-1 blockade treatment [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 941.

De novo DNA methylation programs regulate T cell exhaustion and limit T cell-based immunotherapies

Abstract T cell-based immunotherapies have emerged as one of the most promising frontiers in the fight against cancer and chronic infections. However, it has become clear that prolonged exposure of T cells to their cognate antigen drives them toward a terminally differentiated state, limiting their capacity to mount an effector response. The commitment of T cells to this “exhausted” fate is currently a major barrier in the advancement of T cell immunotherapy efforts. To better understand mechanisms that reinforce the T cell exhaustion gene expression program, we investigated the role of de novo epigenetic programming in establishing exhausted T cells that are non-responsive during immune checkpoint blockade (ICB) therapy. Using mouse models of tumor and chronic viral infection, we observed that genetic deletion of the de novo DNA methyltransferase, Dnmt3a, in antigen-specific T cells that are chronically stimulated allowed them to remain highly functional despite expressing high levels of the checkpoint inhibitory receptor PD-1. Furthermore, PD-1 blockade treatment resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Building upon the findings from our murine studies, we have generated whole-genome DNA methylation profiles of human CD8 T cells from a wide array of differentiation states, and established an epigenetic-based human T cell multipotency index. Using this index, we report that tumor-associated PD-1hi CD8 T cells from pediatric solid tumors acquire an epigenetically reinforced terminal differentiation program. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key factor in limiting CD8 T cell-based immunotherapuetic approaches.

Quantitative Characterization of CD8+ T Cell Clustering and Spatial Heterogeneity in Solid Tumors.

Quantitative characterization of the tumor microenvironment, including its immuno-architecture, is important for developing quantitative diagnostic and predictive biomarkers, matching patients to the most appropriate treatments for precision medicine, and for providing quantitative data for building systems biology computational models able to predict tumor dynamics in the context of immune checkpoint blockade therapies. The intra- and inter-tumoral spatial heterogeneities are potentially key to the understanding of the dose-response relationships, but they also bring challenges to properly parameterizing and validating such models. In this study, we developed a workflow to detect CD8+ T cells from whole slide imaging data, and quantify the spatial heterogeneity using multiple metrics by applying spatial point pattern analysis and morphometric analysis. The results indicate a higher intra-tumoral heterogeneity compared with the heterogeneity across patients. By comparing the baseline metrics with PD-1 blockade treatment outcome, our results indicate that the number of high-density T cell clusters of both circular and elongated shapes are higher in patients who responded to the treatment. This methodology can be applied to quantitatively characterize the tumor microenvironment, including immuno-architecture, and its heterogeneity for different cancer types.

Enhancement of Antitumor Immunity Using Dendritic Cells Combined with Lenalidomide and Programmed Death Ligand-1 Blockade in Multiple Myeloma Mouse Model

Background: The therapeutic efficacy of dendritic cell (DC)-based immunotherapy may be potentiated in combination with other anticancer therapies that enhance DC function by modulating immune responses and the tumor microenvironment. In this study, we investigated the efficacy of DC vaccination in combination with lenalidomide and programmed death (PD)-1 blockade in a model of murine myeloma.Materials &Methods: MOPC-315 cell lines were injected subcutaneously to establish myeloma-bearing mice and the following five test groups were established: PBS control, DCs, DCs + lenalidomide, DCs + PD-1 blockade, and DCs + lenalidomide + PD-1 blockade. On day 0, mice were injected subcutaneously in the right flank with 5 × 105 MOPC-315 cells in a volume of 0.1 mL. After tumor growth, lenalidomide (0.5 mg/kg/day) was administrated orally once a day for 25 days with a 3-day break after the first 11-day dosing period. Each dose of DCs (1 × 106/mouse) was injected subcutaneously into the left flank of BALB/c mice in a volume of 0.1 mL PBS on days 11, 15, 25, and 29; anti-PD-1 (250 µg/mouse) was injected intraperitoneally in a 0.1 mL volume on the same days as DC vaccination.Results: This study showed that DC vaccination combined to the lenalidomide and PD-1 blockade regiment further inhibited MM tumor growth, consequently prolonging the survival of tumor-bearing mice. These effects were associated with a significant increase in IFN-γ-secreting splenocytes against MOPC-315 and YAC-1 cells, as well as the significantly increased the number of effector CD4+ T cells, CD8+ T cells, effector memory T cells, effector NK cells, and M1 macrophages while effectively discouraging suppressor cells, such as myeloid-derived suppressor cells (MDSCs), M2 macrophages, and regulatory T cells (Tregs) in the systemic immune compartment. These findings evidence the induction of systemic immune response potentially being able to eradicate disseminated diseases. In this study, DCs combined with lenalidomide and PD-1 blockade also heightened the anti-myeloma cell mediate immunity by inducing the Th1 polarization, as evidenced by the high-level production of IFN-γ in the spleen, and by suppressing Th2 immune responses, as evidenced by the low-level production of IL-10 and TGF-β in the spleen and tumor site. Tregs, MDSCs, and M2 macrophages are major elements molding the potent immunosuppressive environment in tumor tissues. The inhibition of Treg, MDSC, and M2 macrophage accumulation in the spleen should further contribute to effective anti-myeloma cell mediate immunity in the systemic immune compartment by reciprocally activating DCs or cytotoxic T lymphocytes.Conclusion: This study suggests that lenalidomide plus PD-1 blockade treatment synergistically enhances the efficacy of DC vaccination in a murine myeloma model by inhibiting the generation of immunosuppressive cells and the Th2 immune response and enhancing effector cells and the Th1 immune responses. We hereby propose a framework for a more efficacious DC-based vaccination strategy against MM with the combination of immunomodulatory drug lenalidomide and anti-PD-1 antibody. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract A26: De novo epigenetic programming restrains PD-1 blockade-mediated T cell rejuvenation

Abstract Immune checkpoint blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. Given that many of the impaired effector properties of terminally exhausted CD8 T cells appear to be heritably maintained even in the absence of antigen, we investigated the role of de novo DNA methylation programming as a cell-intrinsic mechanism for establishing the ICB-nonresponsive state of T-cell exhaustion. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells. Therapeutic approaches to reverse these programs can enhance ICB-mediated T cell rejuvenation and ultimately facilitate the control of chronic viral infections and tumor growth. These data establish de novo DNA methylation programming as a regulator of T cell exhaustion and barrier of ICB therapy. Citation Format: Hazem E. Ghoneim, Yiping Fan, Ardiana Moustaki, Hossam Abdelsamed, Pradyot Dash, Pranay Dogra, Robert Carter, Walid Awad, Geoff Neale, Paul G. Thomas, Ben Youngblood. De novo epigenetic programming restrains PD-1 blockade-mediated T cell rejuvenation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A26.

Abstract LB-116: Myeloid derived suppressor cells and NK cells are correlated with clinical benefit and survival in advanced melanoma patients treated with PD-1 blocking antibodies nivolumab and pembrolizumab

Abstract Blockade of the PD-1 receptor has revolutionized the treatment of metastatic melanoma, with significant increases in overall survival, and a dramatic improvement in patient quality of life. Despite the success of this therapeutic approach, the number of responding patients is limited and there is a need for predictive biomarkers and a deeper mechanistic analysis of the cellular populations involved in a clinical response. With this objective in mind, an in-depth immune monitoring study was conducted in advanced melanoma patients undergoing treatment with pembrolizumab or nivolumab. 29 patients receiving pembrolizumab (n=5) or nivolumab (n=24) treatment at Karolinska University Hospital were included in this study. Blood samples were collected from patients at the following time points: Before treatment and at the time of the second and fourth doses. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stained for flow cytometric analysis within two hours of sample collection. In the 29 patients included, median OS was 884 days from the start of treatment. Adverse events were observed in 18 patients (62%), including three grade 3-4 (10%). Patients were classified according to their response as PD (progressive disease, 45%), SD (stable disease, 3%) MR (mixed response, 7%) PR (partial response, 31%) and CR (complete response, 14%). For analytic purposes, patients were divided into two groups: 19 (59%) patients with clinical benefit (includes responders and patients with stable disease) and no clinical benefit (12 patients with progressive disease). Patients with clinical benefit had a median overall survival (MOS) of 1013 days, significantly longer than the 303 day MOS in the no clinical benefit group. After analyzing the patients PBMC with a broad flow cytometry panel that included major immune subsets (T cells, NK cells, dendritic cells and myeloid derived suppressor cells (MDSCs)), our results showed that, before treatment, the frequency of two populations is related to clinical benefit: On one hand, patients with clinical benefit had significantly lower frequencies of monocytic MDSCs. On the other hand, NK cell frequencies were higher in these patients. Furthermore, Kaplan Meier analysis showed that patients with high frequencies of NK cells had significantly longer MOS times when compared to patients with low NK cell frequencies. Similarly, patients with low frequencies of monocytic MDSCs, showed significantly better MOS. Our results suggest that cellular populations other than T cells can be critical in the outcome of PD-1 blockade treatment. Specifically, the frequencies of NK cells and monocytic MDSCs are correlated with survival and clinical benefit and their role as predictive biomarkers should be further evaluated. Citation Format: Yago Pico De Coaña, Maria Wolodarski, Irene van der Haar Avila, Giuseppe V. Masucci, Johan Hansson, Rolf Kiessling. Myeloid derived suppressor cells and NK cells are correlated with clinical benefit and survival in advanced melanoma patients treated with PD-1 blocking antibodies nivolumab and pembrolizumab [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-116.

Abstract SY07-02: Epigenetic regulation of T-cell exhaustion: Implications for cancer immunotherapy

Abstract Immune checkpoint blockade (ICB)-mediated rejuvenation of CD8 T-cell effector functions has emerged as one of the most promising frontiers for treating cancer and chronic infections. However, antigen-specific T cells that have experienced prolonged antigen exposure are often terminally differentiated, and have a limited capacity to mount an effector response during ICB treatment. Such exhaustion of effector potential is a major impediment of current T cell-based immunotherapy efforts. Using in vivo mouse models of tumor and chronic viral infection, we assessed the role of de novo epigenetic programming in establishing ICB-refractory exhausted T cells. We observed that genetic deletion of the de novo DNA methyltransferase, Dnmt3a, in T cells at the effector stage of an immune response to chronic lymphocytic choriomeningitis virus (LCMV) infection allowed antigen-specific T cells to remain highly functional despite expressing high levels of PD-1 and having prolonged exposure to antigen. Quite strikingly, PD-1 blockade treatment of chronically infected animals resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Whole-genome methylation profiling of WT and Dnmt3a-deficient LCMV-specific CD8 T cells identified de novo DNA methylation programs that are coupled to development of ICB-nonresponsive virus and tumor-specific T cells. Building upon these findings, we have identified de novo epigenetic programs acquired in human tumor-associated PD-1hi CD8 T cells. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key regulator in establishing ICB-refractory exhausted CD8 T cells and highlight epigenetic reprogramming of T cells as a novel approach to enhance T cell-based cancer therapies. Citation Format: Benjamin A. Youngblood, Hazem Ghoniem, Ardiana Moustaki, Hossam Abdelsamed, Yiping Fan, Paul Thomas, Elizabeth Stewart, Sara Federico. Epigenetic regulation of T-cell exhaustion: Implications for cancer immunotherapy [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 SY07-02.

De novo DNA methylation programs restrain T cell rejuvenation during immune checkpoint blockade therapy

Abstract Immune-checkpoint blockade (ICB)-mediated rejuvenation of CD8 T cell effector functions has emerged as one of the most promising frontiers for treating cancer and chronic infections. However, antigen-specific T cells that have experienced prolonged antigen exposure are often terminally differentiated, and have a limited capacity to mount an effector response during ICB treatment. Such exhaustion of effector potential is a major impediment of current T cell based immunotherapy efforts. Using in vivo mouse models of tumor and chronic viral infection, we assessed the role of de novo epigenetic programming in establishing ICB-refractory exhausted T cells. We observed that genetic deletion of Dnmt3a in T cells at the effector stage of an immune response to chronic lymphocytic choriomeningitis virus (LCMV) infection allowed antigen-specific T cells to remain highly functional despite expressing high levels of PD-1 and having prolonged exposure to antigen. Quite strikingly, PD-1 blockade treatment of chronically infected animals resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Whole-genome methylation profiling of WT and Dnmt3a-deficient LCMV-specific CD8 T cells identified de novo DNA methylation programs that are coupled to development of ICB-nonresponsive virus and tumor-specific T cells. Building upon these findings, we have identified de novo epigenetic programs acquired in human tumor-associated PD-1hi CD8 T cells. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key regulator in establishing ICB-refractory exhausted CD8 T cells and highlights epigenetic reprogramming of T cells as a novel approach to enhance T cell-based cancer therapies.

Development of thyroid dysfunction is associated with clinical response to PD-1 blockade treatment in patients with advanced non-small cell lung cancer

ABSTRACTPurpose: Drugs that blockade interaction between programmed cell-death protein 1 (PD-1) and its ligand (PD-L1) are promising. Immune-related adverse events (irAEs) might be associated with favorable clinical outcomes, and thyroid dysfunction is one of the most common irAE. We evaluated the association of thyroid dysfunction during PD-1 blockade with the treatment efficacy in patients with non-small cell lung cancer (NSCLC).Experimental Design: A total 58 patients with stage IV NSCLC treated with PD-1 blockade were enrolled. Patients were categorized into thyroid dysfunction and euthyroid groups. Overall survival (OS) and progression-free survival (PFS) of the two groups were compared. Patients, tumor, and medication factors were adjusted using Cox proportional hazard modeling. Objective response rate (RR) and durable control rate were assessed according to the severity of thyroid dysfunction.Results: OS [median 118.0 (73.0-267.0) vs. 71.0 (28.0-160.0) days, log-rank P = 0.025] and PFS [118.0 (73.0-267.0) vs. 61.0 (28.0-130.0), log-rank P = 0.014] were longer in the thyroid dysfunction group. After adjustment, thyroid dysfunction was an independent predictive factor for favorable outcome [adjusted HR = 0.11 (95% CI) 0.01-0.92 for overall death; 0.38 (0.17-0.85) for disease progression]. The severity of thyroid dysfunction was associated with durable control rate (P for trend = 0.008).Conclusions: Thyroid dysfunction during PD-1 blockade is associated with treatment response and could provide supplementary information for immune monitoring in patients with advanced NSCLC.