Use Of Immune Checkpoint Blockade Research Articles

The emerging use of immune checkpoint blockade in the adjuvant setting for solid tumors: a review.

The use of immune checkpoint inhibitors has been approved in the advanced and metastatic setting for many types of solid tumors. Nonetheless, their role in the adjuvant setting is limited to the treatment of surgically resected melanoma. Ipilimumab was the first immune checkpoint inhibitor approved for this indication, followed by nivolumab and pembrolizumab. Many ongoing trials are evaluating these molecules in the postoperative setting, alone or in combination with other therapies. Preliminary results are promising regarding the treatment of other cutaneous tumors, lung cancers, head and neck squamous cell carcinomas, bladder cancer and renal cell carcinomas. Some data assessing their use for the adjuvant treatment of esophageal, colorectal, ovarian cancer and other solid tumors are similarly emerging.

Impact of combination therapy with anti-PD-1 blockade and a STAT3 inhibitor on the tumor-infiltrating lymphocyte status

Recently, clinical studies using anti-immune checkpoint molecule antibodies have been successful in solid tumors, such as melanoma and non-small cell lung cancers. However, pancreatic cancers are still intractable and difficult to treat once recurrence or metastasis occurs; thus, novel combined use of immune checkpoint blockade (ICB) with molecular targeted drugs is considered a therapeutic option.Previously, we developed a novel humanized MHC-double knockout (dKO) NOG mouse model and demonstrated that an anti-PD-1 antibody or a STAT3 inhibitor showed anti-tumor effects through an immunological mechanism.In the current study, using a humanized mouse model, we aimed to develop a combination therapy with an anti-PD-1 antibody and a STAT3 inhibitor (STX-0119) for use in vivo against pancreatic cancer. In an in vitro investigation, STX-0119 showed weak to moderate cytotoxic activity against several pancreatic cancer cell lines, which exhibited activated pSTAT3 and weak PD-L1 expression.However, unexpectedly, an in vivo study indicated that the combination of the anti-PD-1 antibody with STX-0119 remarkably reduced the anti-tumor effect and TIL numbers despite the effective anti-tumor activity against pancreatic cancer was produced individually by STX-0119 and the anti-PD-1 antibody.These results suggested that the combination of an anti-PD-1 antibody with specific signal inhibiting drugs should be carefully evaluated to avoid unexpected side effects, and such studies might contribute to the development of an effective combination regimen of ICB with cancer-targeting drugs such as tyrosine kinase inhibitors (TKIs).

Abstract LB-116: Combination of a cell cycle checkpoint kinase inhibitor and ionizing radiation leads to abscopal tumor response through increased CD8+ infiltration

Abstract Purpose/Objectives: The use of Immune checkpoint blockade across disease sites has been successful but many patients still remain unresponsive to immunotherapy. Therefore, it is imperative to Identify regimens to augment the efficacy of immunomodulatory agents. Recently it has been shown that mitotic cell cycle progression following ionizing radiation (IR)-induced double-stranded DNA breaks triggers micronuclei (MN) formation. By promoting inflammatory signaling through their repository role of cGAS that stimulates interferon-induced gene (STING) expression, MN can activate cell-intrinsic immune surveillance mechanisms. Our hypothesis was based on the rational that checkpoint kinase inhibitors abrogating the G2/M cell cycle checkpoint in the presence of IR-induced DNA damage will result in increased formation of MN and an augmented immune response. Materials/Methods: MCF10A and B16F10 cells were treated using IR ± AZD7762, a CHK1/2 inhibitor. Both cell lines were treated with 25nM of AZD7762 and received 17 and 8 Gy IR, respectively. Immunofluorescent studies were performed to assess the MN formation and cGAS co-localization. RT-qPCR was carried out to measure the IFN-β and CCL5 mRNA levels and Western blot evaluated phosphorylation of STAT1. Knockdown of TMEM173 (STING) in MCF10A - cells was used to assess IFN-β and CCL5 expression as a rescue experiment. Finally, a B16F10 melanoma tumor model was analyzed to evaluate the in vivo effects of the combinatorial treatment. Cells were injected in both right and left flank of the mice but only the right flank tumors irradiated with a single dose of 17Gy. Tumor tissues were collected and CD8+ T-cell infiltration was assessed. Results: MCF10A cells showed a statistical significant increase in MN formation (16%) in both 24 and 48 hours when treated with AZD7762+IR compared with IR (5%) or AZD7762 (8%) alone. IFN-β, CCL5 and p-STAT1 levels showed a similar trend as MN. When MCF10A cells with TMEM173 downregulation (siRNA) were treated with AZD7762+IR the expression of CCL5 was reversed confirming that the activation of the immune response is through the cGAS-STING pathway. The in vivo studies revealed that the combination of AZD7762+IR not only led to a significant impairment of the treated tumor growth but also and most importantly to an increased systemic immune response and a significant improved tumor control at the abscopal site. CD8+ T-cell tumor infiltration was found to be significantly higher in the combination group compared to single treatment in both treated and abscopal tumors. Conclusion: The combination of IR with agents targeting the G2/M checkpoint to drive mitotic progression was able to induce increased MN formation and interferon signaling in vitro and led to significant tumor growth delay in both irradiated and abscopal tumor sites. Citation Format: Ilias V. Karagounis, Hann-Hsiang Chao, Christoforos Tomas, Constantinos Koumenis, Amit Maity. Combination of a cell cycle checkpoint kinase inhibitor and ionizing radiation leads to abscopal tumor response through increased CD8+ infiltration [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 LB-116.

Abstract 2712: Effect of combined PD-1 and IL-6 blockade on K-ras mutant lung cancer

Abstract Lung cancer is the leading cause of cancer death and the second most common cancer diagnosed worldwide in both males and females. K-ras gene mutations are among the most common mutations encountered in non-small cell lung cancer (NSCLC), encompassing up to 30% of them, and are unfortunately associated with chemoresistance and poor prognosis. Recently, immunotherapy treatment of several cancers including NSCLC, particularly by targeting the co-inhibitory molecules program cell death protein 1 (PD-1) and program death-ligand 1 (PD-L1), have shown promising results given either in combination with standard chemotherapy or as neoadjuvant therapy. We have previously shown an important role for IL-6 in K-ras mutant lung tumorigenesis by reprogramming myeloid contexture in the lung tumor microenvironment (TME) and promoting tumor cell proliferation and angiogenesis through the activation of STAT3 pathway. Accordingly, we hypothesized that targeting both immunosuppressive TME and IL-6 driven pro-tumor immune response, through the use of immune checkpoint blockade (ICB), and inhibition of IL-6 would give us a synergistic/additive response and increases the tumor inhibitory effect of ICB. Briefly, starting at the age of 6 weeks, 4 cohorts of K-ras mutant mice (LSL-K-rasG12D/ CCSPCre) (CC-LR) were injected intraperitoneally with vehicle alone (PBS1x), anti-PD-1 (CD279) antibody alone (Clone: 29F.1A12- Bioxcell - BE0273-CUST) (200µg), anti-IL-6 antibody alone (Clone: MP5-20F3 - Bioxcell - BE0046-CUST) (20 mg/kg), or a combination of both. After 8 weeks of treatment, tumor burden and inflammation were studied and quantified. We found a significant (2-fold) decrease in tumor burden in the anti-PD1 alone treated group which was further reduced in the combined anti-PD-1 and anti-IL-6 treated group compared to age and sex matched vehicle treated group. There was also a significant difference in the lung macrophage infiltration in combined anti-PD1/anti-IL-6 treated group similar to what we previously observed in anti-IL-6 alone treated group. Our results suggest that immunomodulatory approaches by targeting cytokine networks like IL-6 blockade could be used to increase the efficacy of immunotherapy, i.e. PD-1 blockade, as a preventive and/or therapeutic strategy for K-ras mutant lung cancer. Citation Format: Marco A. Ramos-Castaneda, Neha Daga, Stephen Peng, Shanshan Deng, Walter Velazco, Mauricio Da Silva Caetano, Seyed Moghaddam. Effect of combined PD-1 and IL-6 blockade on K-ras mutant lung cancer [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 2712.

Costimulatory Molecules and Immune Checkpoints Are Differentially Expressed on Different Subsets of Dendritic Cells.

Dendritic cells (DCs) play a crucial role in initiating and shaping immune responses. The effects of DCs on adaptive immune responses depend partly on functional specialization of distinct DC subsets, and partly on the activation state of DCs, which is largely dictated by environmental signals. Fully activated immunostimulatory DCs express high levels of costimulatory molecules, produce pro-inflammatory cytokines, and stimulate T cell proliferation, whereas tolerogenic DCs express low levels of costimulatory molecules, produce immunomodulatory cytokines and impair T cell proliferation. Relevant to the increasing use of immune checkpoint blockade in cancer treatment, signals generated from inhibitory checkpoint molecules on DC surface may also contribute to the inhibitory properties of tolerogenic DCs. Yet, our knowledge on the expression of inhibitory molecules on human DC subsets is fragmentary. Therefore, in this study, we investigated the expression of three immune checkpoints on peripheral blood DC subsets, in basal conditions and upon exposure to pro-inflammatory and anti-inflammatory stimuli, by using a flow cytometric panel that allows a direct comparison of the activatory/inhibitory phenotype of DC-lineage and inflammatory DC subsets. We demonstrated that functionally distinct DC subsets are characterized by differential expression of activatory and inhibitory molecules, and that cDC1s in particular are endowed with a unique immune checkpoint repertoire characterized by high TIM-3 expression, scarce PD-L1 expression and lack of ILT2. Notably, this unique cDC1 repertoire was subverted in a group of patients with myelodysplastic syndromes included in the study. Applied to the characterization of DCs in the tumor microenvironment, this panel has the potential to provide valuable information to be used for investigating the role of DC subsets in cancer, guiding DC-targeting treatments, and possibly identifying predictive biomarkers for clinical response to cancer immunotherapy.

T cell receptor β-chain repertoire analysis to reveal potential predictive biomarker for the use of immune checkpoint blockade in patients with advanced solid tumors.

e14152 Background: Immune checkpoint blockade (ICB) targeted to CTLA-4, PD-1, and PD-L1 can promote antitumor T cell immunity and clinical responses. Tracking the dynamic changes of the T Cell Receptor (TCR) Immune Repertoire can reflect the evolution of the immune system in tumor immunotherapy patients, predict the immune response to ICB, and provide high-value information for clinical monitoring of immune function status of cancer patients to make effective clinical decisions. The purpose of this study was to explore the correlation between the peripheral blood TCR repertoire dynamic changes and the immune response of patients with anti-PD-1/PD-L1, and to accurately guide the individualized treatment of immunotherapy. Methods: We sequenced the TCRβ genes from T cells contained in peripheral blood mononuclear cells (PBMCs) from 24 advanced solid tumors patients (PD/7, PR/4, SD/13) treated with immune checkpoint blockade. Peripheral blood samples were collected from baseline, 8-week, and 16-week after treatment for each patient. bioinformatic analysis was used to detect the diversity and clonality changes of TCRβ-CDR3 sequence before and after treatment with ICB in patients, and provide auxiliary technology for further guiding clinical tumor immunotherapy. Results: The analysis result of Chao index showed that the TCR-CDR3 diversity in the PR group was significantly increased after treatment than SD or PD group. (pvalue = 0.001), and the number of CDR3 aa clonotypes also increased significantly (pvalue = 0.012). A total of 61 Vβ gene fragments and 15 Jβ gene fragments were uniquely identified during immunotherapy . There was a significant difference in the number of Vβ gene fragments between baseline samples and after treatment in PR group (pvalue = 0.042). Conclusions: In this project, PBMC samples was used to evaluate the changes of TCR repertoire between before and after treatment with immune checkpoint blockade in advanced solid tumor patients. The immune function status was significantly changed before and after treatment. Patients with PR had significantly increased diversity in TCR repertoire than patients with SD or PD. The results provide T cell receptor β-chain repertoire sequencing and analysis has potential to assess whether immune checkpoint blockade promotes global T cell expansion and/or loss of T cell clonotype diversity, and to identify predictive biomarker for the use of immune checkpoint blockade.

Palliative Radiation Therapy for Vertebral Metastases and Metastatic Cord Compression in Patients Treated With Anti-PD-1 Therapy.

Background: There is increasing use of immune checkpoint blockade (ICB) across multiple cancer types, including in patients at risk for vertebral metastases and cord compression. These patients are often treated with palliative radiotherapy (PRT); however, data evaluating the combination of PRT and ICB in patients with vertebral metastases is limited. Furthermore, patients with cord compression are generally excluded from prospective clinical trials. Therefore, we retrospectively evaluated outcomes following PRT and PD-1 inhibition in patients with vertebral metastases.Methods: We performed a retrospective chart review of 37 consecutive patients (total 57 lesions) treated with radiation for vertebral metastases who also received PD-1 inhibition. Patient, treatment and outcomes data were abstracted from the medical records.Results: Histologies included non-small cell lung cancer (n = 21), renal cell carcinoma (n = 9) and melanoma (n = 7). Out of 57 lesions,18 involved >1 segments of the vertebral column. There were isolated lesions in thoracic (16), lumbar (9), cervical (6), and sacral (8) vertebrae. Presenting symptoms included pain (19), numbness (10), and weakness (3). Eleven patients were asymptomatic. Radiologic cord compression was present in 12, epidural extension in 28 and compression fracture in 14. Eleven patients underwent surgical decompression prior to the onset of RT. Median radiation dose was 24 Gy (range 8–30 Gy). Stereotactic radiation was delivered in 4 patients; 33 patients received conformal RT. 21 patients received PD-1 inhibition after RT, 9 before RT and 7 with RT. Seven patients received concurrent CTLA-4 inhibitors with anti-PD-1 therapy.Treatment was in general well-tolerated. Toxicities included fatigue (6), transient pain flare (1), nausea/vomiting (1) and G1 skin changes (1). All patients reported some degree of pain relief. Numbness/weakness was improved in 6 of 13 patients with baseline symptoms (46%) and this was more likely in patients that received vertebral radiation after starting PD-1 inhibitors (71 vs. 17%, p = 0.04). Most patients (22 of 33 evaluable patients, 67%) had stability of irradiated lesions on subsequent follow up imaging performed at median of 30 days from RT, whereas 3 had a complete local response and 4 had a partial local response.Conclusions: We demonstrate that PRT administered to vertebral metastases was well-tolerated and effective in patients treated with PD-1 inhibitors. There was an encouraging rate of pain reduction and neurological improvement.

Immunotherapy Associated Pulmonary Toxicity: Biology Behind Clinical and Radiological Features.

The broader use of immune checkpoint blockade in clinical routine challenges clinicians in the diagnosis and management of side effects which are caused by inflammation generated by the activation of the immune response. Nearly all organs can be affected by immune-related toxicities. However, the most frequently reported are: fatigue, rash, pruritus, diarrhea, nausea/vomiting, arthralgia, decreased appetite and abdominal pain. Although these adverse events are usually mild, reversible and not frequent, an early diagnosis is crucial. Immune-related pulmonary toxicity was most frequently observed in trials of lung cancer and of melanoma patients treated with the combination of the anti-cytotoxic T lymphocyte antigen (CTLA)-4 and the anti-programmed cell death-1 (PD-1) antibodies. The most frequent immune-related adverse event in the lung is represented by pneumonitis due to the development of infiltrates in the interstitium and in the alveoli. Clinical symptoms and radiological patterns are the key elements to be considered for an early diagnosis, rendering the differential diagnosis crucial. Diagnosis of immune-related pneumonitis may imply the temporary or definitive suspension of immunotherapy, along with the start of immuno-suppressive treatments. The aim of this work is to summarize the biological bases, clinical and radiological findings of lung toxicity under immune checkpoint blockade, underlining the importance of multidisciplinary teams for an optimal early diagnosis of this side effect, with the aim to reach an improved patient care.

Unlocking the Potential of Immuno-Oncology in Advanced Cutaneous Squamous Cell Carcinoma: Role of Checkpoint Blockade

Dr Robert welcomed the delegates to the symposium and outlined the agenda for the meeting, before presenting the first session on understanding cutaneous squamous cell carcinoma (CSCC). The rising incidence of CSCC presents a major challenge for healthcare systems and the frequency and impact of progression to advanced disease is underestimated. The diverse range and potential complications of CSCC lesions require a multidisciplinary approach, in which dermatologists play an important role. The message that CSCC remains a disease of high unmet need was echoed by Dr Peris, who presented on existing treatment approaches for the management of advanced CSCC. In the absence of an established management pathway for patients with locally advanced or metastatic disease, clinicians must rely on limited or anecdotal evidence to inform treatment decisions. Conventional chemotherapy and targeted therapies produce variable responses that are often short-lived, demonstrating a need for more effective and tolerable systemic treatments. Guidelines recognise these limitations and do not make any firm recommendations for the treatment of advanced CSCC. The importance of a multidisciplinary approach was underlined by Dr Migden’s presentation on future novel therapeutic strategies in CSCC. Immunotherapy is an exciting frontier that is becoming increasingly relevant to many specialists, including dermatologists, with several ongoing trials of immune checkpoint inhibitors in patients with advanced CSCC. A strong rationale exists for immunotherapy in these patients and the current evidence base supports the use of immune checkpoint blockade as an alternative to cytotoxic chemotherapy and targeted agents. Dr Migden concluded the symposium with an interactive presentation of five case studies of successful checkpoint inhibitor treatment of locally advanced CSCC, emphasising the key role of dermatologists in a multidisciplinary team approach.

Unlocking the Potential of Immuno-Oncology in Advanced Cutaneous Squamous Cell Carcinoma: Role of Checkpoint Blockade

Dr Robert welcomed the delegates to the symposium and outlined the agenda for the meeting, before presenting the first session on understanding cutaneous squamous cell carcinoma (CSCC). The rising incidence of CSCC presents a major challenge for healthcare systems and the frequency and impact of progression to advanced disease is underestimated. The diverse range and potential complications of CSCC lesions require a multidisciplinary approach, in which dermatologists play an important role. The message that CSCC remains a disease of high unmet need was echoed by Dr Peris, who presented on existing treatment approaches for the management of advanced CSCC. In the absence of an established management pathway for patients with locally advanced or metastatic disease, clinicians must rely on limited or anecdotal evidence to inform treatment decisions. Conventional chemotherapy and targeted therapies produce variable responses that are often short-lived, demonstrating a need for more effective and tolerable systemic treatments. Guidelines recognise these limitations and do not make any firm recommendations for the treatment of advanced CSCC. The importance of a multidisciplinary approach was underlined by Dr Migden’s presentation on future novel therapeutic strategies in CSCC. Immunotherapy is an exciting frontier that is becoming increasingly relevant to many specialists, including dermatologists, with several ongoing trials of immune checkpoint inhibitors in patients with advanced CSCC. A strong rationale exists for immunotherapy in these patients and the current evidence base supports the use of immune checkpoint blockade as an alternative to cytotoxic chemotherapy and targeted agents. Dr Migden concluded the symposium with an interactive presentation of five case studies of successful checkpoint inhibitor treatment of locally advanced CSCC, emphasising the key role of dermatologists in a multidisciplinary team approach.

Kaposi\u2019s varicelliform eruption in a patient with metastatic melanoma and primary cutaneous anaplastic large cell lymphoma treated with talimogene laherparepvec and nivolumab

BackgroundImmune-directed therapies have become front-line therapy for melanoma and are transforming the management of advanced disease. In refractory cases, multi-modal immunoncology (IO) approaches are being utilized, including combining immune checkpoint blockade (ICB) with oncolytic herpes viruses. Talimogene laherparepvec (T-VEC) is the first genetically modified oncolytic viral therapy (OVT) approved for the treatment of recurrent and unresectable melanoma. The use of IO in patients with concomitant malignancies and/or compromised immune systems is limited due to systematic exclusion from clinical trials. For example, a single case report of a solid organ transplant patient successfully treated with T-VEC for metastatic melanoma has been reported. Furthermore, the use of ICB in T-cell malignancies is limited and paradoxical worsening has been described. To our knowledge, this is the first report of dual ICB/T-VEC being administered to a patient with concurrent primary cutaneous anaplastic large cell lymphoma (pcALCL) and melanoma.Case presentationHere we present the case of a patient with concomitant primary cutaneous ALCL and metastatic melanoma, progressing on anti-programmed death (PD)-1 therapy, who developed Kaposi’s varicelliform eruption after receiving the first dose of Talimogene laherparepvec.ConclusionThis case highlights the complexities of care of patients with coexistent cancers, demonstrates rapid progression of primary cutaneous ALCL on nivolumab and introduces a novel adverse effect of Talimogene laherparepvec.

Abstract B66: Targeted activation of innate immune adaptors MAVS and STING promotes antitumor responses in colorectal cancer models

Abstract While the use of immune checkpoint blockade (ICB) has gained significant traction as a viable therapy for certain cancers, many more cancers remain refractory to immunotherapies due to their highly immunosuppressive microenvironment. This is perhaps best exemplified in the largest group of cancers, colorectal carcinomas (CRCs) where ~90% of CRCs (non-microsatellite instable) are unresponsive to ICBs. Recent studies have indicated that this lack of response may be due to mutations and loss of genes that mediate the innate immune responses, such as MAVS (Mitochondrial Antiviral Signaling Protein) and STING (Stimulator of Interferon Genes). We hypothesized that delivery of active versions of these genes would elicit strong interferon signaling that could reverse the tumor immunosuppressive environment, to effectively create immune “hotspots” and allow for successful immune-mediated tumor regression. As a proof-of-concept, we developed doxycycline-inducible activated forms STING and MAVS in mouse CRC models (MC38 and CT26 lines), and found the expression of MAVS or STING resulted in robust activation of interferon-stimulated genes and other cytokines/chemokines. To determine if delivery of these genes could be achieved in vivo, we developed cell lines capable of resisting the anti-viral effect of interferon stimulation. This system allowed us to generate adenoviral vectors that express active forms of MAVS and more recently, STING. We found that Ad-MAVS strongly elicited interferon signaling in a variety of cell types and tumor lines, suggesting its effectiveness as an interferon stimulatory agent. To determine its anti-tumor effect, we intra-lesionally injected tumors and observed strongly inhibited tumor growth and enhanced T-cell infiltration in established tumor masses in vivo. This correlated with enhanced anti-tumor specific T cell responses, but not humoral responses. The induction of local and systemic immunity was confirmed in a bi-lateral treatment model that demonstrated the impact of systemic immunity by MAVS induction. Furthermore, we found that MAVS-induced tumor regression was lost in NOD/SCID mice, suggesting that the induction of tumor-specific T-cell adaptive immunity was critical to anti-tumor responses. Lastly, we found the activation of MAVS resulted in upregulated expression of PD-L1 on tumor cells, suggesting combinatorial treatments of checkpoint blockades therapy with targeted MAVS activation can result in synergistic effects. In conclusion, our data demonstrated the importance of innate immunity activation by MAVS or STING pathway in generating immune hotspots in colorectal tumor microenvironment, which promotes antitumor adaptive T cell responses and tumor growth regression. Citation Format: Li-Chung Tsao, Herbert Kim Lyerly, Zachary Hartman, Gabriel Deleon. Targeted activation of innate immune adaptors MAVS and STING promotes antitumor responses in colorectal cancer models [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 B66.

Immunotherapy of Hodgkin Lymphoma: Mobilizing the Patient's Immune Response.

Classic Hodgkin lymphoma has a unique tumor composition in that there is a paucity of malignant cells present, and most of the tumor consists of normal immune and stromal cells. Despite the presence of an immune infiltrate within the tumor microenvironment, the malignant cells effectively evade the immune system and appear to utilize the presence of immune cells to promote the growth and survival of Hodgkin-Reed-Sternberg cells. Hodgkin-Reed-Sternberg cells evade immune detection because of overexpression of programmed death 1 ligands, PD-L1 and PD-L2, which suppress T-cell activation, and loss of expression of major histocompatibility complex molecules that prevent effective immune recognition. Recognition of these immune defects has led to clinical use of immune checkpoint blockade in classic Hodgkin lymphoma. Clinical trials using antibodies that block programmed death 1/PD-L1 signaling have shown remarkable responses to therapy and have led to the approval of nivolumab and pembrolizumab for use in patients with relapsed and refractory disease. Trials are currently testing immune checkpoint blockade in earlier lines of therapy.

Abstract 2711: Development of a new innovative multifunctional immune checkpoint inhibitor

Abstract Blockade of PD-1, PD-L1 or CTLA-4 is an attractive strategy for Immuno-Oncology (I-O) therapy. Moreover, combination approaches, which inhibit non-redundant pathways of PD-1/PD-L1 and CTLA-4, have been reported to show enhanced anti-tumor activity as well as improved progression-free survival in clinical trials of melanoma. The use of immune checkpoint blockade (ICB) drugs, however, can lead to severe or life-threatening complications of autoimmune disorders such as diabetes, thyroiditis, pulmonary fibrosis and vitiligo. Therefore, new innovations are critical to overcome possibilities of unintended immunoactivation and minimize drug cross-reactions and complications. Here, we designed and synthesized a novel alkylating Pyrrole-Imidazole (PI) polyamide with indole-seco-CBI capable of disrupting expressions of human PD-1, PD-L1 and CTLA-4 genes (CCC07-01) by binding to a common motif. Our initial evaluation process confirmed that the induction of tumor- and tumor-environment-specific combinatorial ICB therapy was possible by only a single agent. CCC07-01 administration significantly suppressed PD-1, PD-L1 and CTLA-4 mRNA and protein expressions at low nanomolar doses (15, 10, 5 nM respectively) in several cell lines. Furthermore, logP characterization experiments found CCC07-01 to be sufficiently lipophilic that enhanced the molecule's capability to accumulate and be retained in tumor tissues. Additionally, elevated doses of CCC07-01 induced substantial cytotoxicity in SW480, RKO and A2058 cancer cells (IC50=27, 12 and 11 nM, respectively) but primary cells derived from normal human tissues were not affected. These results illuminated the therapeutic possibility of CCC07-01 through, at low doses, its anti-tumor effect in tumor microenvironments and generalized cytotoxicity at increased dosage levels. CCC07-01's enhanced retention in tumor may also contribute to lower risks of triggering adverse autoimmune events compared to other ICB approaches. Preliminary results from human RKO colon carcinoma xenograft NGS mice, following the engraftment of human HLA-matched peripheral blood mononuclear cells (PBMC), showed significant tumor reduction 14 days after the injection of CCC07-01 (2.3 mg/kg, p < 0.05) and displayed detectable amounts of predominant effector memory CD8 T cells in the spleen 14 days post-administration (9.2 mg/kg, p < 0.05). The approach of simultaneously targeting elements in immune checkpoints, as demonstrated by CCC07-01, can lead to new therapeutic opportunities and reduce healthcare costs by curbing undesired autoimmune complications. Together with Zenyaku Kogyo, we envision a breakthrough in the field of current antibody-based ICB therapy as we continue to evolve and improve the use of PI polyamides as a viable candidate for ICB. Citation Format: Hiroki Nagase, Keiko Fukushima, Asuka Hattori, Mayu Shinohara, Atsushi Takatori, Takayoshi Watanabe, Nobuko Koshikawa, Takahiro Inoue, Jason Lin, Yoshinao Shinozaki. Development of a new innovative multifunctional immune checkpoint inhibitor [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 2711.

Complimentary mechanisms of dual checkpoint blockade expand unique T-cell repertoires and activate adaptive anti-tumor immunity in triple-negative breast tumors

ABSTRACTTriple-negative breast cancer (TNBC) is an aggressive and molecularly diverse breast cancer subtype typified by the presence of p53 mutations (∼80%), elevated immune gene signatures and neoantigen expression, as well as the presence of tumor infiltrating lymphocytes (TILs). As these factors are hypothesized to be strong immunologic prerequisites for the use of immune checkpoint blockade (ICB) antibodies, multiple clinical trials testing single ICBs have advanced to Phase III, with early indications of heterogeneous response rates of <20% to anti-PD1 and anti-PDL1 ICB. While promising, these modest response rates highlight the need for mechanistic studies to understand how different ICBs function, how their combination impacts functionality and efficacy, as well as what immunologic parameters predict efficacy to different ICBs regimens in TNBC. To address these issues, we tested anti-PD1 and anti-CTLA4 in multiple models of TNBC and found that their combination profoundly enhanced the efficacy of either treatment alone. We demonstrate that this efficacy is due to anti-CTLA4-driven expansion of an individually unique T-cell receptor (TCR) repertoire whose functionality is enhanced by both intratumoral Treg suppression and anti-PD1 blockade of tumor expressed PDL1. Notably, the individuality of the TCR repertoire was observed regardless of whether the tumor cells expressed a nonself antigen (ovalbumin) or if tumor-specific transgenic T-cells were transferred prior to sequencing. However, responsiveness was strongly correlated with systemic measures of tumor-specific T-cell and B-cell responses, which along with systemic assessment of TCR expansion, may serve as the most useful predictors for clinical responsiveness in future clinical trials of TNBC utilizing anti-PD1/anti-CTLA4 ICB.

Immune related adverse events (irAEs): A unique profile based on tumor type.

e14606 Background: Immune checkpoint blockade(ICB) has revolutionized the treatment of a growing number of malignancies. Real world administration of oncolytics is often associated with increased adverse event rates versus what is demonstrated in clinical trials. Whether the tumor biology, site of disease burden or underlying organ dysfunction dictates the differing immune side effect profile in various malignancies remains to be understood. Methods: The incidence of grade 2-4 irAE was abstracted from medical records of all patients (pts) treated with ICB (ipilimumab and/or nivolumab) from 2011 to 2016 at a single institution. Results: Of the 126 pts reviewed, 82 pts had metastatic lung cancer, 31 pts had unresectable or metastatic melanoma, 13 pts had metastatic renal cancer(RCC). In the melanoma cohort, concurrent or sequential PD-1 and CTLA4 blockade was used in 10/31 pts. All of the lung cancer and RCC patients received anti-PD-1 alone. In the patients with lung cancer: pneumonitis was identified in 24%, SIRS in 16%, thrombosis in 11%, cerebritis in 9%, colitis in 4%, hepatitis in 4%, thyroiditis in 5%. In RCC, 8% pts experienced pneumonitis and 8% had thyroiditis. In the melanoma population, colitis was identified in 19%, SIRS in 10%, pneumonitis in 3%, thrombosis in 6%, adrenalitis in 6%, hepatitis in 3%. Colitis was seen in 2/13(15%) pts who got ipilimumab alone and 4/10(40%) pts who got both ipilimumab and nivolumab. Conclusions: Pneumonitis, SIRS and cerebritisappear to be the most prevalent irAEs in lung cancer as compared to melanoma or RCC. The incidence of pneumonitis was higher in RCC compared to melanoma. Colitis appears to have a higher incidence in melanoma, the incidence of which further increases when CTLA4 inhibitors are used in conjunction with anti-PD-1. The majority of the RCC patients tolerated ICB with no major irAEs. With the expanding use of ICB in advanced malignancies, increased awareness of these clinically significant and potentially serious irAEs is indispensable. Future trials designed to distinguish the incidence of irAEs in relation to specific tumor types would be informative. [Table: see text]

Association of diversity and composition of the gut microbiome with differential responses to PD-1 based therapy in patients with metastatic melanoma.

2 Background: Tremendous advances have been made in cancer therapy through the use of immune checkpoint blockade, although responses are not always durable. There is a growing appreciation of the role of the microbiome in cancer-related outcomes and recent evidence in murine models suggests that modulation of the gut microbiome may enhance responses to immune checkpoint blockade in melanoma. However this has not been investigated in patients. Here, we demonstrate that differential bacterial “signatures” exist in the gut microbiome of responders (R) and non-responders (NR) to anti-PD1 therapy at baseline, and that insights gained could be used to derive actionable strategies to enhance responses. Methods: We collected oral (n = 222) and gut microbiome (n = 113) samples on a large cohort of patients with metastatic melanoma (n = 228), with a majority treated with anti-PD1 therapy (n = 110). Patients were classified as either R or NR based on RECIST criteria, and 16S rRNA gene sequencing was performed to characterize the diversity and composition of the microbiomes. Immune profiling (via 7-marker IHC panel of CD3, CD8, PD-1, PD-L1, Granzyme B, RORγT and FoxP3) was performed in available tumors at baseline. Results: In these studies, we observed significant differences in the diversity and composition of the gut microbiome in R versus NR to PD1 blockade at baseline, but no clear differences in oral microbiomes. Specifically, R had a significantly higher alpha diversity compared to NR (p = 0.017). Notable differences were also seen in the composition of the gut microbiome of R versus NR. Immune profiling demonstrated significantly increased immune infiltrates in baseline tumor samples of R, with a positive correlation between CD8, CD3, PD1 and FoxP3 T-cell density and abundance of specific bacteria enriched in R (e.g. Faecalibacterium). Conclusions: Differences exist in the diversity and composition of the gut microbiome in R vs NR to anti-PD1 therapy. These results have far-reaching implications and suggest that modifications to the gut microbiome could potentially enhance therapeutic responses to immune checkpoint blockade.

Abstract B04: Intralesional vaccination with Ad-MAVS alters the immunosuppressive tumor microenvironment and elicits robust anti-tumor immunity in non-immunogenic cancers

Abstract Background and Hypothesis: While the use of immune checkpoint blockade has gained significant traction as a viable therapy for certain cancers, many tumors remain refractory to these immunotherapies due to their highly immunosuppressive microenvironment. As a novel approach to boost local anti-tumor immunity and sensitize cancers to these therapies, we constructed an adenoviral (Ad) vector to overexpress the innate signaling adaptor molecule, Mitochondrial Antiviral Signaling protein (MAVS). Overexpression of MAVS results in innate immune signaling downstream of the RIG-I like receptors (RLRs) resulting in the activation of multiple signaling pathways as well as the release of a Th1 cytokine milieu and type I interferons. We therefore hypothesized that adenoviral delivery of MAVS into the tumor microenvironment would produce robust inflammatory signaling which would alter the tumor microenvironment to both efficiently suppress tumor growth and stimulate anti-tumor immune responses. Methods: In order to determine the possible effect MAVS would have on different cell types within the tumor microenvironment we infected a range of primary and cancer cells types (Bone-marrow derived dendritic cells (BMDCs), primary fibroblasts, as well as 5 different murine tumor cell lines representing breast-4T1, colon-CT26CL25, bladder-MB49, melanoma-B16F10, and prostate-TRAMPC2) with Ad-MAVS or a control Ad. Innate immune responses were measured via luminex ELISA, microarray, and RT-qPCR. The impact on tumor growth was assessed by MTT and expression of PD-L1 by FACS. Lastly, we measured the anti-tumor effect and adaptive immune responses of animals treated with various combinations of Ad-MAVS, a control Ad, and PD-L1 targeting antibodies in vivo utilizing various immunocompetent and immune-compromised mouse models. Results: In vitro infection of BMDCs, MEFs, and murine tumor cell lines demonstrated that MAVS expression elicited widespread production of cytokines and chemokines indicative of a type I immune response (such as IL-12, TNF-alpha, RANTES, etc.) and interferon beta. These profound responses also triggered immune regulatory feedback expression of PD-L1, enabling their targeting by PD-L1 antibodies. Additionally, we found that Ad-MAVS infection of tumor cell lines significantly inhibited their proliferation in vitro, likely via interferon pathways. In vivo, treatment with Ad-MAVS also elicited robust stimulation of innate immune responses and numerous type I inflammatory cytokines. Moreover, in multiple types of tumor bearing animals, we found that a single intralesional treatment of Ad-MAVS significantly prolonged overall survival and was capable of eliciting systemic anti-tumor adaptive immunity to non-immunogenic tumors. Conclusion: Our study identifies that MAVS expression can elicit the production of a significant number of Th1-type cytokines, chemokines, and interferon-beta in multiple cell types within the tumor microenvironment. We also demonstrate that overexpression of MAVS results in the direct suppression of tumor cell growth and stimulation of tumor PD-L1 expression. These activities were also observed in vivo, as intralesional injection of Ad-MAVS elicited Th1 cytokines and anti-tumor immunity in multiple tumor types, which correlated with enhanced systemic T-cell responses to tumor-specific epitopes. Collectively, these data demonstrate that intralesion injection of Ad-MAVS alters the tumor microenvironment to stimulate anti-tumor immunity and potentially sensitizes non-immunogenic tumors to PD-L1 therapies. As such, Ad-MAVS represents a novel approach of stimulating anti-tumor immunity via activation of innate adaptor molecules to improve immunogenic anti-tumor responses and potentially the therapeutic efficacy of PD-L1 immune checkpoint blockade. Citation Format: Gabriel De Leon, Tao Wang, Pankaj Agarwal, H. Kim Lyerly, Zachary Hartman. Intralesional vaccination with Ad-MAVS alters the immunosuppressive tumor microenvironment and elicits robust anti-tumor immunity in non-immunogenic cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B04.

Abstract A60: Synergistic effect of targeted chemotherapy delivery using theranostic nanoparticles and PD-L1 blockade in an orthotopic mouse pancreatic cancer model

Abstract Pancreatic cancer is the fourth deadliest form of cancer with a dismal 5-year survival rate of less than 5%. As a result there is an urgent need for the development of more efficacious therapies for pancreatic cancer. In order to overcome the immune suppression that is prevalent in this disease, the use of immune checkpoint blockades, CTLA-4 and PD-L1, have been investigated. However, published reports have indicated that pancreatic cancer is generally non-responsive to these immunotherapies. Because it has been shown that combinatorial therapies consisting of immunotherapy and chemotherapy can act synergistically, we sought to investigate the efficacy of targeted delivery of the chemotherapeutic cisplatin using nanoparticle carriers along with PD-L1 blockade in a highly aggressive orthotopic model of pancreatic cancer derived from the PANC02 mouse cell line. Our lab has developed a receptor targeted magnetic iron oxide nanoparticle (IONP) platform for targeted tumor imaging and drug delivery and demonstrated its effect on tumor growth inhibition in breast, ovarian, and pancreatic cancer mouse models. Active targeting of IONPs requires conjugation of the recombinant amino terminal fragment (ATF) of the urokinase plasminogen activator (uPA) that will bind specifically to tumor cells and tumor associated stromal cells that express a high level of its receptor (uPAR). uPAR expression is associated with increased invasiveness and a poorer prognosis, which further makes this protein an ideal target. In the orthotopic PANC02 model, we found that intraperitoneal delivery of 100µg of PD-L1 blockade alone (clone 10F.9G2) twice per week for four injections reduced orthotopic tumor burden by ~30% relative to untreated mice. Whereas, the combination therapy with uPAR-targeted IONP-cisplatin (10mg/kg drug equivalent) decreased tumor burden by ~65%. Enhanced inhibition of ascites fluid accumulation was also observed following combinatorial therapy, 81% versus 55.6%, respectively. We observed that a high level of uPAR-targeted IONP-cisplatin accumulated in the orthotopic pancreatic tumor following intraperitoneal delivery by Prussian blue staining indicating the effective delivery of the chemotherapeutic drug to the tumor site which could mediate tumor growth inhibition. Additionally, this therapeutic synergy is likely due, in part, to the enhanced intratumoral infiltration of T cells following targeted IONP-drug treatment. Taken together, these findings indicate that the minimal therapeutic efficacy of immune checkpoint blockades such as PD-L1 in pancreatic cancer can be enhanced by the tumor-targeted delivery of chemotherapeutic drugs using theranostic nanoparticles and this combinatorial therapeutic regime warrants further investigation. Citation Format: Erica N. Bozeman, Ning Gao, Weiping Qian, Andrew Wang, Lily Yang. Synergistic effect of targeted chemotherapy delivery using theranostic nanoparticles and PD-L1 blockade in an orthotopic mouse pancreatic cancer model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A60.

Abstract IA31: Combining targeted therapy and immune checkpoint blockade to improve responses

Abstract There have been significant advances in treatment of metastatic melanoma over the past several years through the use of targeted therapy and immune checkpoint blockade. Oncogenic mutations in the BRAF gene occur in over half of melanoma tumors, and treatment with agents targeting this mutation results in objective responses in the majority of patients. However significant limitations exist as responses are not durable, with the majority of patients progressing on therapy within 6-10 months. Another area of significant progress involves the use of immune checkpoint blockade, such as with monoclonal antibodies blocking CTLA-4 and the PD-1 pathway. In contrast, treatment of patients with immune checkpoint inhibitors results in a lower response rate than those observed with targeted therapy, but ones that tend to be more durable. With these advances, we are posed with therapeutic dilemmas with regard to timing and sequence of therapy. Namely, there is significant debate as to whether to begin treatment with targeted therapy versus immune checkpoint blockade upfront, and at which point to change treatment strategy. There is growing evidence of potential synergy in combining treatment with BRAF-targeted therapy and immune checkpoint blockade, and this concept is being empirically tested in clinical trials. There a strong scientific rationale for combining these therapies, as treatment with BRAF inhibitors is associated with increased immunogenicity of melanoma tumors within 2 weeks of therapy - with increased melanoma antigens and CD8+ T cell infiltrate, and decreased immunosuppressive cytokines and VEGF. BRAF inhibitors may also directly activate T cells through paradoxical activation of the MAPK pathway. However there is a concurrent increase in expression of immunomodulatory molecules (such as PD-L1) within 2 weeks of therapy which is likely related to T cell infiltrate, though also suggest a possible immune mechanism of resistance. Clinical trials and studies in murine models are currently underway to explore synergy between these therapies, and insights gained will be discussed herein. It is becoming increasingly apparent that complexities exist regarding appropriate timing and sequence in these combination strategies to optimize responses and minimize toxicity. Ultimately, ideal approaches using combined BRAF-targeted therapy and immune checkpoint blockade will be built on a deep understanding of the molecular and immune effects of each of these therapies in isolation, as well as in combination. Citation Format: Jennifer A. Wargo. Combining targeted therapy and immune checkpoint blockade to improve responses. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA31.