Checkpoint Inhibitor Antibody Research Articles

Highlights of This Issue

Replication stress is emerging as an important mechanism for inhibitors of the DNA damage response. In this study, Parsels and colleagues defined the contribution of replication stress resulting from nucleotide depletion and PARP1 trapping to radiosensitization by the combination of WEE1 and PARP inhibitors, respectively, in KRAS-mutant NSCLC. They found that the enhanced radiosensitization from combined WEE1 and PARP inhibitors required PARP1 trapping, a dominant mechanism relative to nucleotide depletion. This study highlights the importance of replication stress to the therapeutic activity of WEE1 and PARP inhibitors and that PARP trapping potency is an important consideration in the choice of PARP inhibitor for combination therapy.YAP1, a target of the Hippo pathway, is regarded as an oncogene in most human cancers. However, YAP1 co-operates with p73 in hematopoietic cells and plays a tumor suppressor role. Maruyama and colleagues conducted a novel transcription-based functional screening and identified candidate YAP1 activators (n = 47) from a small-molecule chemical compound library (n = 18,606). These compounds were characterized to determine whether this assay provides bona fide YAP1 activators. Importantly, one YAP1 activator induced apoptosis in human multiple myeloma and chronic myeloid leukemia cells. Hence, YAP1 activation may be useful at suppressing hematological cancers.Immune checkpoint inhibitor antibodies that block PD-L1/PD-1 interactions are promising therapeutic agents; however, only a small percentage of cancer patients benefit from these agents. Small-molecule modulators of immune checkpoints and tumor microenvironment (TME) offer several advantages that complement current therapies, and may expand the population that responds to PD-L1/PD-1 targeted therapies. Herein, a novel mechanism is discovered by which small-molecule modulators of Sigma1, a unique ligand-operated integral membrane scaffolding protein, can be used to trigger PD-L1 degradation in cancer cells by selective autophagy. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies regulating tumor microenvironment.Precision oncology is currently limited in scope when using genomic biomarkers to guide prescription of molecularly targeted medications. In this study, Ding and colleagues applied state-of-the-art machine learning algorithms to create drug sensitivity prediction models using data from large pharmacogenomics studies, e.g., the Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE). The resulting models can accurately predict the efficacy of a wide array of molecularly targeted and non-specific chemotherapy drugs. Translated to the clinic, similar models have the potential to improve treatment outcomes for a significant population of patients currently receiving ineffective therapy.

Unconventional Approaches to Modulating the Immunogenicity of Tumor Cells.

For several years, it has been known that histone deacetylase inhibitors have the potential to alter the immunogenicity of tumor cells exposed to checkpoint inhibitory immunotherapy antibodies. HDAC inhibitors can rapidly reduce expression of PD-L1 and increase expression of MHCA in various tumor types that subsequently facilitate the antitumor actions of checkpoint inhibitors. Recently, we have discovered that drug combinations which cause a rapid and intense autophagosome formation also can modulate the expression of HDAC proteins that control tumor cell immunogenicity via their regulation of PD-L1 and MHCA. These drug combinations, in particular those using the irreversible ERBB1/2/4 inhibitor neratinib, can result in parallel in the internalization of growth factor receptors as well as fellow-traveler proteins such as mutant K-RAS and mutant N-RAS into autophagosomes. The drug-induced autophagosomes contain HDAC proteins/signaling proteins whose expression is subsequently reduced by lysosomal degradation processes. These findings argue that cancer therapies which strongly promote autophagosome formation and autophagic flux may facilitate the subsequent use of additional antitumor modalities using checkpoint inhibitor antibodies.

P2.07-026 Nivolumab in Non-Small Cell Lung Cancer (NSCLC): Facing the Reality

Nivolumab, a human IgG4 programmed death (PD)-1 immune checkpoint inhibitor antibody, is approved in Israel and other parts of the world, for the treatment of patients with advanced non-small cell lung cancer (NSCLC) and disease progression during or after platinum-based chemotherapy, without a need to determine the level of PD-L1 expression in the tumor. In this series, we retrospectively analyzed the results of treating patients with NSCLC by nivolumab. This series represents real life results, out of a clinical trial, with broad inclusion criteria.

P2.02-072 Reliability of Small Biopsy Samples for Tumor PD-L1 Expression in Non-Small-Cell Lung Cancer

Programmed death 1 immune checkpoint inhibitor antibody has been effective in patients with PD-L1 positive advanced NSCLC. However, surgically resected specimens or core-needle biopsy samples were used to estimate drug potency in past clinical trials. The aim of this study is to prospectively investigate small sample reliability for NSCLC to determine the PD-L1 expression status. We prospectively enrolled patients who underwent diagnostic biopsy by any procedures (under rigid bronchoscopy, flexible bronchoscopy, CT & US-guided core-needle, excisional method) from March 2017 to March 2018 (ongoing study). Pathologically confirmed non-small cell lung cancer (NSCLC), PD-L1 expression was evaluated in our institution using companion diagnostic PD-L1 immunohistochemistry：PD-L1 IHC 22C3 pharmDx (Daco) with autostainer Link 48, detecting a driver mutation in parallel. We evaluated: 1) the total number of tumor cells and sample size; 2) compared PD-L1 expression for each procedure using tumor proportion score: TPS (<1%, 1～49%, 50%≤), 3) the concordance rate of PD-L1 expression status by biopsy and surgical materials; and 4) the efficacy of administration for PD-1 immune checkpoint inhibitor antibody. During the first three months 44 cases of PD-L1 expression were evaluated. 33 cases were sampled by bronchoscopy (6 under rigid scope with BF 1T260, 17 TBBs using 1T260/P260F in 4/13 cases, 10 TBNAs), and 7 cases were CT-guided core-needle biopsy. The TPS (<1%, 1～49%, 50%≤) was 8/6/10 for TBB, 3/4/2 for TBNA, and 4/0/3 for CT-guided. Four cases harboring EGFR mutation showed a lower PD-L1 expression (<10%). Utilizing smaller samples to evaluate PD-L1 expression, and the frequency of TPS were comparable to past clinical trials using larger samples. Smaller samples might be an accurate alternative to assess PD-L1 expression. Current data will be updated at the conference.

442P - Safety of nivolumab in patients with advanced or metastatic non-small cell lung cancer who have received at least one prior systemic regimens: Expanded access program in Korea (ONO-4538-X04)

Background: Nivolumab, a fully human programmed death PD-1 immune checkpoint inhibitor antibody, has been approved in Korea for the treatment of advanced non–small-cell lung cancer (NSCLC) that has progressed on or after platinum-based chemotherapy. We have assessed the safety of nivolumab in 299 patients with previously treated, advanced or metastatic NSCLC who were enrolled in the NSCLC expanded access program (EAP) in Korea. Methods: Our EAP included subjects with histologically documented NSCLC who have progressed after systemic treatment with a minimum of 1 prior systemic treatment for stage IIIB/IV disease. Unlike Checkmate-017 and 057, our EAP also included subjects with ECOG performance status (PS) of 2 and with two or more prior systemic treatments (heavily treated patients). Subjects were treated with 3 mg/kg of nivolumab IV every 2 weeks for a maximum of 24 months. Each 14-day dosing period constituted a single cycle. Patients included in the analysis had received ≥ 1 dose of nivolumab and were monitored for adverse events (AEs) using NCI CTC version 4.0. Results: As of September 1, 2017 (DLP: data lock point), 333 NSCLC patients participated from 36 hospitals in Korea. Out of 333, 299 patients received nivolumab and 206 (68.9%) were male. 21 (7.0%) were current smokers and 169 (56.5%) were former smokers. 86 (28.8%) had squamous, 212 (70.9%) had non-squamous and 1 (0.3%) had adenosquamous histology. 49 (16.4%) had PS 0, 212 (70.9%) had PS 1 and 38 (12.7%) had PS 2. 116 patients (38.8%) were heavily treated before (3rd line or more). At DLP, 40 patients were still receiving nivolumab treatments, whereas 259 patients had discontinued the treatment. Respiratory complication was the most frequent (39.6% any grade and 7.7% grade 3 or greater) treatment-emergent AE (TEAE). There were 118 patients (39.5%) who reported any treatment related adverse events (TRAEs) and 24 patients (8.0%) reported grade 3 or greater (table). 18 patients (6.0%) discontinued due to AEs.Table: 442P Treatment related adverse events reported in > 1% of patientsEventsAll GradesGrade 3 to 4Rash26 (8.7%)2 (0.7%)Pruritus14 (4.7%)0Fatique13 (4.3%)1 (0.3%)Diarrhea11 (3.7%)0Nausea8 (2.7%)1 (0.3%)Decreased appetite8 (2.7%)0Hypothyroidism8 (2.7%)0Pneumonia7 (2.3%)2 (0.7%)Pneumonitis5 (1.7%)2 (0.7%)Dyspnea5 (1.7%)1 (0.3%)Hyperthyroidism5 (1.7%)0Asthenia4 (1.3%)0Pyrexia4 (1.3%)1 (0.3%)Constipation4 (1.3%)0Oedema peripheral4 (1.3%)0 Open table in a new tab Conclusions: The results demonstrate that Korean EAP participants had safety profile similar to those previously reported in Checkmate-017 and Checkmate-057 studies, which strongly support the practical use of nivolumab for Korean NSCLC patients. Clinical trial indentification: ONO-4538-X04 Legal entity responsible for the study: Ono Pharmaceutical Funding: Ono pharmaceutical Disclosure: All authors have declared no conflicts of interest.

286P - Nivolumab in renal cell carcinoma patients in India

Background: Nivolumab is a human IgG4 programmed death-1 immune checkpoint inhibitor antibody approved for use in multiple cancers like lung, melanoma, head and neck and renal cell cancers. Immunotherapy with Nivolumab is approved for use as second line therapy in Renal cell cancers (RCC), and has recently become available for use in India. There is no data about use of Nivolumab in Indian patients. Methods: We retrospectively reviewed RCC patients who received treatment with Nivolumab in a tertiary hospital in the second and later lines setting. The objective was to study efficacy and safety of Nivolumab. Results: 7 patients received Nivolumab at 3mg/kg dose, every 2 weeks, between May 2016 and June 2017. Median age of patients was 53 years (range 42-66 years). ECOG status at presentation ranged between 1-3. All patients had received oral TKIs as first line therapy- 3 patients received Sunitinib, 4 patients received Pazopanib. 2 patients also received Everolimus as 2nd line therapy. Response was assessed by RECIST criteria at 2 months. 2 patients (28.6%) achieved partial response, 2 had stable disease (28.6%) and 3 patients (42.8%) had progressive disease. The 2 patients who had partial response are continuing treatment for 13 months and 11 months without any progression. The median progression free survival (PFS) is 4 months. Adverse events occurred in 3 patients (42%). The common adverse events were fatigue in 28.5% (grade 1) and skin rash in 14.2% (grade 1). There were no grade 3,4 adverse events and no treatment related discontinuation. Conclusions: Nivolumab is an effective second line regimen in selected patients with metastatic RCC. Long-term responses have been seen in responding patients. Immunotherapy with Nivolumab is well tolerated, and would need to be studied in more patients with longer followup to know the PFS and Overall survival (OS) benefit. Legal entity responsible for the study: Dr Amit Rauthan Funding: None Disclosure: A. Rauthan: Advisory board- Roche, Merck, BMS All other authors have declared no conflicts of interest.

P2.07-039 Nivolumab Experience in Patients with Previously Treated Advanced Non Small Cell Lung Cancer (NSCLC) in Toledo, Spain

Nivolumab is a fully human IgG4 programmed death-1 (PD-1), an immune checkpoint inhibitor antibody and it has demonstrated durable responses and tolerability in heavily pretreated patients with advanced NSCLC. This is an observational study to describe our experience with Nivolumab in previously treated patients with advanced NSCLC .

Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy

Despite its success in several clinical trials, cancer immunotherapy remains limited by the rarity of targetable tumor-specific antigens, tumor-mediated immune suppression, and toxicity triggered by systemic delivery of potent immunomodulators. Here, we present a proof-of-concept immunomodulatory gene circuit platform that enables tumor-specific expression of immunostimulators, which could potentially overcome these limitations. Our design comprised de novo synthetic cancer-specific promoters and, to enhance specificity, an RNA-based AND gate that generates combinatorial immunomodulatory outputs only when both promoters are mutually active. These outputs included an immunogenic cell-surface protein, a cytokine, a chemokine, and a checkpoint inhibitor antibody. The circuits triggered selective Tcell-mediated killing of cancer cells, but not of normal cells, invitro. In invivo efficacy assays, lentiviral circuit delivery mediated significant tumor reduction and prolonged mouse survival. Our design could be adapted to drive additional immunomodulators, sense other cancers, and potentially treat other diseases that require precise immunological programming.

Skin Cancers in Organ Transplant Recipients.

Long-term utilization of immunosuppression in organ transplant recipients (OTRs) leads to decreased immune-mediated tumor surveillance and development of malignant tumors. A delicate balance needs to be maintained in the intensity of immunosuppression to keep the risk of malignancy low without jeopardizing life-saving graft function. OTRs are prone to developing skin cancers that exhibit unique epidemiologic, pathophysiologic, and prognostic characteristics. In this review, we discuss the most commonly reported skin cancers in OTRs: squamous cell carcinoma (SCC), basal cell carcinoma (BCC), Kaposi sarcoma, Merkel cell carcinoma, and malignant melanoma (MM). Tumors in this high-risk population are aggressive and may respond poorly to standard therapies; however, new targeted therapies are promising. Checkpoint inhibitor antibodies have been used for treatment of cutaneous SCC, Merkel cell carcinoma, and MM; epidermal growth factor receptor inhibitors have been used for cutaneous SCC; hedgehog pathway inhibitors have been used for BCC; and BRAF and MEK inhibitors are being used increasingly in the management of MM. Guidelines for dermatologic screening are variable and primarily based on expert opinion. Prospective evidence-based trials by multidisciplinary groups are needed to better define surveillance schedules for pre- and posttransplant cutaneous malignancies.

1056P - Estimated costs of treatment-related adverse events (TRAEs) for recurrent or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN) in the checkmate 141 trial

Background: Nivolumab, a programmed death-1 (PD-1) immune checkpoint inhibitor antibody, is approved in the United States and the European Union for treatment of SCCHN progressing on or after platinum-based chemotherapy. In the phase 3 CheckMate 141 trial, nivolumab significantly improved overall survival vs investigator’s choice (IC) of standard, single-agent systemic therapy (methotrexate, docetaxel, or cetuximab) in patients with R/M SCCHN. This study assessed the estimated costs of managing grade 3–4 TRAEs requiring treatment in CheckMate 141. Methods: The frequency, grade, and attribution of TRAEs for which treatment was received were extracted from CheckMate 141 patient-level safety data. Grade 3–4 TRAE treatment costs were estimated based on principle diagnosis codes of the International Classification of Disease, 9th Revision in the Healthcare Cost and Utilization Project National Inpatient Sample data (2012–2014), adjusted to reflect 2013-equivalent US costs. Results: Among the 347 patients in the safety population, 236 received nivolumab and 111 received IC. A total of 88 grade 3–4 TRAEs requiring treatment were observed: 28 among patients receiving nivolumab and 60 among patients receiving IC. The cost of managing TRAEs per treated patient was 4.5 times higher in the IC arm ($4913) than in the nivolumab arm ($1072). Patients receiving docetaxel and methotrexate had the highest incidence of TRAEs and estimated TRAE management costs (Table).Table1056PNivolumab (n = 236)IC (combined) (n = 111)ICCetuximab (n = 13)Docetaxel (n = 52)Methotrexate (n = 46)Number of grade 3–4 TRAEs requiring treatment (%)28/88 (31.8)60/88 (68.2)2/60 (3.3)36/60 (60.0)22/60 (36.7)Total estimated cost of managing grade 3–4 TRAEs, $253,067545,37417,855333,307194,211Cost of managing grade 3–4 TRAEs, mean per treated patient, $10724913137364104222 Open table in a new tab Conclusions: Patients with platinum-refractory R/M SCCHN treated with nivolumab had fewer grade 3–4 TRAEs, lower estimated total costs of managing TRAEs, and reduced TRAE costs per treated patient compared with standard, single-agent systemic therapy. Clinical trial identification: NCT02105636 Legal entity responsible for the study: Bristol-Myers Squibb Funding: Bristol-Myers Squibb Disclosure: S. Bobiak: Former Bristol-Myers Squibb employee (at the time the submitted work was started); Spouse is current employee of Bristol-Myers Squibb. J.W. Shaw, M. Contente, B. Korytowsky: Bristol-Myers Squibb employee and shareholder. D.D. Stenehjem: Consulting fees from Bristol-Myers Squibb; Unrestricted research grant (unrelated to work regarding the content of this abstract) from Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

Concurrent PD-1 Blockade Negates the Effects of OX40 Agonist Antibody in Combination Immunotherapy through Inducing T-cell Apoptosis.

Combination therapies that depend on checkpoint inhibitor antibodies (Abs) such as for PD-1 or its ligand (PD-L1) together with immune stimulatory agonist Abs like anti-OX40 are being tested in the clinic to achieve improved antitumor effects. Here, we studied the potential therapeutic and immune effects of one such combination: Ab to PD-1 with agonist Ab to OX40/vaccine. We tested the antitumor effects of different treatment sequencing of this combination. We report that simultaneous addition of anti-PD-1 to anti-OX40 negated the antitumor effects of OX40 Ab. Antigen-specific CD8+ T-cell infiltration into the tumor was diminished, the resultant antitumor response weakened, and survival reduced. Although we observed an increase in IFNγ-producing E7-specifc CD8+ T cells in the spleens of mice treated with the combination of PD-1 blockade with anti-OX40/vaccine, these cells underwent apoptosis both in the periphery and the tumor. These results indicate that anti-PD-1 added at the initiation of therapy exhibits a detrimental effect on the positive outcome of anti-OX40 agonist Ab. These findings have important implications on the design of combination immunotherapy for cancer, demonstrating the need to test treatment combination and sequencing before moving to the clinic. Cancer Immunol Res; 5(9); 755-66. ©2017 AACR.

Treg depletion potentiates checkpoint inhibition in claudin-low breast cancer.

Claudin-low breast cancer is an aggressive subtype that confers poor prognosis and is found largely within the clinical triple-negative group of breast cancer patients. Here, we have shown that intrinsic and immune cell gene signatures distinguish the claudin-low subtype clinically as well as in mouse models of other breast cancer subtypes. Despite adaptive immune cell infiltration in claudin-low tumors, treatment with immune checkpoint inhibitory antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death receptor 1 (PD-1) were ineffective in controlling tumor growth. CD4+FoxP3+ Tregs represented a large proportion of the tumor-infiltrating lymphocytes (TILs) in claudin-low tumors, and Tregs isolated from tumor-bearing mice were able to suppress effector T cell responses. Tregs in the tumor microenvironment highly expressed PD-1 and were recruited partly through tumor generation of the chemokine CXCL12. Antitumor efficacy required stringent Treg depletion combined with checkpoint inhibition; delays in tumor growth were not observed using therapies that modestly diminished the number of Tregs in the tumor microenvironment. This study provides evidence that the recruitment of Tregs to the tumor microenvironment inhibits an effective antitumor immune response and highlights early Treg recruitment as a possible mechanism for the lack of response to immune checkpoint blockade antibodies in specific subtypes of cancer that are heavily infiltrated with adaptive immune cells.

New Approaches to Melanoma Treatment: Checkpoint Inhibition with Novel Targeted Therapy

Melanoma is the most dangerous type of skin cancer, largely due to its propensity for recurrence and metastasis, even after removal of malignant tissue. When melanoma reaches advanced stages, the disease becomes refractory to many types of therapy, which has created a need for novel therapeutic strategies to combat the disease. Our group focuses on the oncogenic function of a neuronal receptor, metabotropic glutamate receptor 1 (mGluR1). When mGluR1 is aberrantly expressed in melanocytes, elevated levels of extracellular glutamate mediate the constitutive activation of the receptor to promote cell proliferation. We are exploring the potential synergistic efficacy when combining a glutamatergic signaling inhibitor with a checkpoint inhibitor antibody

BRAF inhibitors: resistance and the promise of combination treatments for melanoma.

Identification of mutations in the gene encoding the serine/threonine-protein kinase, BRAF, and constitutive activation of the mitogen-activated protein kinase (MAPK) pathway in around 50% of malignant melanomas have led to the development and regulatory approval of targeted pathway inhibitor drugs. A proportion of patients are intrinsically resistant to BRAF inhibitors, and most patients who initially respond, acquire resistance within months. In this review, we discuss pathway inhibitors and their mechanisms of resistance, and we focus on numerous efforts to improve clinical benefits through combining agents with disparate modes of action, including combinations with checkpoint inhibitor antibodies. We discuss the merits of combination strategies based on enhancing immune responses or overcoming tumor-associated immune escape mechanisms. Emerging insights into mechanisms of action, resistance pathways and their impact on host-tumor relationships will inform the design of optimal combinations therapies to improve outcomes for patients who currently do not benefit from recent treatment breakthroughs.

Abstract 5594: High throughput combinatorial profiling of checkpoint inhibitor antibodies on the iQue® Screener PLUS

Abstract Checkpoint inhibitors have become valuable immuno-modulatory targets in the advancement of cancer treatment. Looking for the synergy between new checkpoint inhibitor antibodies and known inhibitors is an important aspect of this research. The iQue Screener PLUS platform is a powerful tool to simultaneously assess these interactions in a single well of a microtiter plate. ForeCyt® software provides plate-level analytics and high content visualization to generate deep insight rapidly. Using a mixed lymphocyte reaction (MLR) model, we profiled potential synergies of several known checkpoint inhibitors antibodies. Responses of PD-1, PD-L1, and CD73 inhibitors both individually, and in combination with CTLA-4 inhibitors, were assessed for proliferation, viability and cytokine secretion simultaneously in the same well. MultiCyt® cell-based and bead-based reagents were used for this analysis. Synergies ranging from 2-10 fold increase over CTLA-4 alone were observed in the secretion of TNFα and IL-1β. Results were obtained and analysis completed in a 384-well plate in 30 minutes. In conclusion, this study highlights the power of the iQue Screener PLUS platform to rapidly characterize multiple endpoints and the ForeCyt software to provide high content visualization that reveals actionable insights. Citation Format: Tom Hasaka. High throughput combinatorial profiling of checkpoint inhibitor antibodies on the iQue® Screener PLUS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5594. doi:10.1158/1538-7445.AM2017-5594

Abstract 5571: Immune-checkpoint inhibition via enzyme-mediated degradation of kynurenine

Abstract Tryptophan oxidation and the ensuing production of Kynurenine (Kyn) and downstream products have been established to play an important role in immunosuppression within the tumor microenvironment (TME). The rate limiting step in Tryptophan oxidation is catalyzed by the IFN-γ inducible IDO-1 enzyme which is upregulated in numerous cancers and also, in some instances via the ectopic expression of TDO, which is normally expressed primarily in the liver. Currently, IDO-1 small molecule inhibitors in combination with checkpoint inhibitor antibodies are being evaluated in &amp;gt;12 clinical trials. However, whether the immune-suppressive effects of Tryptophan catabolism results from its depletion in the TME, or from the accumulation of Kynurenine, is not known. To distinguish between the effects of Trp depletion and Kyn accumulation in the TME we used an engineered Kynureninase (KYNase) enzyme that selectively degrades Kyn into immunologically inert and non-toxic metabolites. Peritumoral injection of PEGylated KYNase completely depletes serum and tumor Kyn levels for up to 72 hrs, while leaving Trp concentrations in both compartments unaffected. In the IDO-1 expressing CT26 colon carcinoma model KYNase treatment as monotherapy resulted in 16% complete and durable responses, accompanied by long-term immunity to tumor re-challenge. KYNase had no effect on tumor growth in IDO-/- mice, nor in RAG-/- or in CD8+ T cell depleted mice. Analysis of the TME demonstrated KYNase treatment resulted in increased accumulation of CD8+ cells, with a greater proportion expressing Granzyme B, undergoing proliferation and permeating the tumor interior. This effect was specific to the TME, as no detectable changes to the immune compartment occurred in other organs examined, nor overt signs of autoimmunity and toxicity. The increase in CD8+ cells was consistent with in vitro data showing that elevated concentration of Kyn directly induces apoptosis of activated CD8+ T cells partially through IL-2 suppression. No changes to the Treg percentages and phenotypes in the TME were observed, indicating that the Treg compartment is not impacted by treatment with KYNase. Cytokine analysis of tumor digests revealed increased IL-2 concentrations, as well as IFNγ and IL-9, in tumors after KYNase treatment. Furthermore, ex vivo stimulation of TIL after KYNase treatment demonstrated that the increase in cytokines in the TME is not solely due to an increase in TIL, but also an increased effector cytokine capacity on a per cell basis. Administration of KYNase together with either αCTLA4 or αPD1 synergistically elicited complete and durable regression of multiple established tumor models. In summary, our data support the hypothesis that Kyn accumulation in the TME rather than Trp depletion is the dominant IDO-mediated immune suppressive mechanism, and that enzyme-mediated Kyn depletion is a promising cancer immunotherapeutic approach. Citation Format: Todd A. Triplett, Kendra Triplett, Everett Stone, Michelle Zhang, Mark Manfredi, Candice Lamb, Yuri Tanno, Lauren Ehrlich, George Georgiou. Immune-checkpoint inhibition via enzyme-mediated degradation of kynurenine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5571. doi:10.1158/1538-7445.AM2017-5571

Abstract 4622: Characterization of sulfatide reactive type II NKT cells from mouse lung

Abstract The immune system plays a major role in the elimination of tumors. CD8 T cell infiltration is known to be a good prognostic indicator. The development of therapies based on checkpoint inhibitor antibodies was an important breakthrough in increasing survival by limiting the exhaustion of cytotoxic cells and increasing tumor cell eradication. However, the immune system also contains regulatory cells that protect the organism from inappropriate activation of immune cells against self-antigens. Thus, the fact that tumor cells are autologous leads regulatory cells to inhibit the activation of anti-tumor cytotoxic cells and thus increase tumor escape. Therefore, understanding the function and the activation of regulatory cells might help to develop therapies to limit the activation of regulatory cells in order to increase tumor clearance. Natural killer T (NKT) cells are lymphocytes with features of natural killer (NK) and of T cells placing them at the interface of innate and adaptive immunity. Like NK cells, they rapidly produce cytokines after stimulation, orienting the immune response. As T cells, they express a T cell receptor (TCR) that allows the recognition of specific lipids presented by the non-classical MHC-I molecule CD1d. According to their TCR usage, two populations of NKT cells are described: type I and type II. All type I NKT cells express a semi-invariant TCR (Valpha24Jalpha18 in humans, Valpha14Jalpah18 in mice) that recognizes α-galactosylceramide (αGalCer). They can be identified with the αGalCer-loaded CD1d tetramer. In contrast, type II NKT cells express a more diverse TCR repertoire. There is no currently identified lipid antigen recognized by all type II NKT cells, making their identification more difficult. A fraction of them recognize sulfatide. By using sulfatide-loaded CD1d-tetramers, we observed for the first time that sulfatide-reactive type II NKT cells were enriched in the lung, a major site of tumor metastasis. Moreover, we previously showed that in vivo stimulation of type II NKT cells with sulfatide increased the number of tumor nodules in the lung. An in-depth phenotype analysis revealed that they were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells are either CD4+CD8- or double negative. Type II NKT cells do not express PLZF, the master regulator of NKT cell development and exist in PLZF-/- mice contrary to type I NKT cells. We showed that type II NKT cells also expressed markers of myeloid cells, c-Kit, CD11b and Ly6C even though histological analysis revealed lymphocytic morphology. Interestingly, at steady state, type II NKT cells expressed granzyme A but not granzyme B or perforin. The in vivo injection of sulfatide increased the expression of the activation markers CD69 and CD44 as well as granzyme A. Since the regulatory functions of type II NKT cells have been shown to be critical in tumor immunity, the detailed characterization of these cells could help to develop a new immunotherapy for cancer. Citation Format: Lise Pasquet, Shingo Kato, Tony Adams, Susan Sharrow, Theresa Davies-Hill, Elaine Jaffe, Xia Zheng, Motoshi Suzuki, Damian Kovalovsky, Jay Berzofsky, Masaki Terabe. Characterization of sulfatide reactive type II NKT cells from mouse lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2017-4622

Abstract CT077: Five-year follow-up from the CA209-003 study of nivolumab in previously treated advanced non-small cell lung cancer (NSCLC): Clinical characteristics of long-term survivors

Abstract Introduction: Prior to the introduction of immunotherapies, treatment options were limited for patients (pts) with NSCLC who progressed after first-line platinum doublet chemotherapy. The majority of pts with advanced disease died within 1 y of diagnosis, and 5-y survival for metastatic NSCLC was ~1%. Nivolumab, a programmed death-1 (PD-1) immune checkpoint inhibitor antibody, showed encouraging activity in pts with heavily pretreated advanced NSCLC in a phase 1 dose-escalation cohort expansion trial (CA209-003; NCT00730639). Based on improved overall survival (OS) versus docetaxel in 2 phase 3 studies in previously treated advanced NSCLC (CheckMate 017 and 057), nivolumab was approved in this population. Reports of long-term efficacy and safety with immune checkpoint inhibitors are limited. Here we report updated results from CA209-003 based on ~5 y of follow-up, representing the longest survival follow-up for an immune checkpoint inhibitor in advanced NSCLC to date. Methods: Pts with heavily pretreated (1-5 prior systemic regimens) advanced NSCLC received nivolumab (1, 3, or 10 mg/kg) every 2 wk in 8-wk cycles for up to 96 wk. The primary objective was safety and tolerability; secondary objectives included objective response rate and duration of response. OS from the time of first dose was an exploratory objective. The minimum follow-up for the current analysis was 58.25 mo. Results: At database lock, the Kaplan-Meier-estimated 5-y OS rate in all pts (N = 129) was 16% (95% confidence interval [CI]: 10, 23). OS rates at 5 y were similar in pts with squamous (SQ; n = 54; 16% [95% CI: 8, 28]) and non-SQ (n = 74; 15% [95% CI: 8, 25]) NSCLC (excludes 1 pt with unknown histology). Of the 16 pts who survived ≥5 y (median age: 61.5 y [range: 44, 80]), 9 pts were male and 12 were current smokers at baseline (2 former smokers; 2 unknown). In 10 evaluable pts, PD-1 ligand 1 (PD-L1) expression was ≥1% in 7 pts (≥50% in 5) and &amp;lt;1% in 3 pts. Nine of the 16 pts completed the maximum number of nivolumab cycles per protocol; the remainder discontinued due to adverse events (n = 4), disease progression (n = 2), and initiation of a new treatment regimen (n = 1). Twelve of the 16 pts achieved a partial response, and 2 pts each had stable disease (SD) and progressive disease (PD) as best overall response (BOR) to nivolumab. At 5 y, per investigator, 12 pts had received no further therapy after stopping nivolumab treatment and were without evidence of PD. Further details of the disease and treatment course in select pts will be presented. Conclusions: Nivolumab resulted in durable OS in a notable proportion of pts with pretreated advanced NSCLC, as demonstrated by a 5-y OS rate of 16%. Long-term survivors had diverse baseline and on-treatment characteristics including histology, PD-L1 expression level (including pts with &amp;lt;1% PD-L1), and BOR to nivolumab (including pts with SD/PD). Citation Format: Julie Brahmer, Leora Horn, David Jackman, David Spigel, Scott Antonia, Matthew Hellmann, John Powderly, Rebecca Heist, Lecia Sequist, David C. Smith, Philip Leming, William J. Geese, Dennis Yoon, Ang Li, Scott Gettinger. Five-year follow-up from the CA209-003 study of nivolumab in previously treated advanced non-small cell lung cancer (NSCLC): Clinical characteristics of long-term survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT077. doi:10.1158/1538-7445.AM2017-CT077

Abstract LB-200: A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody

Abstract Background. It is generally accepted that immune reactions against cancer cells can be induced by immunotherapy. Here, we investigated the possibility that an antibody response has contributed to the success of the immunotherapy of a cancer patient. Methods. A patient with metastatic melanoma was successfully treated by adoptive transfer of ex vivo expanded autologous tumor reactive T cells1. This patient is still tumor free 9 years after treatment. Peripheral blood memory B cells were immortalized by ectopic Bcl-6 and Bcl-xL expression2 and analyzed for the presence of tumor-reactive B cells. Results. We isolated one B cell clone that produced an antibody, named AT1412, recognizing a novel cell surface epitope on the tetraspanin CD9 which is broadly expressed and involved in multiple cellular activities including proliferation and adherence. AT1412 bound more strongly to melanoma cells than to melanocytes and other healthy tissues indicating that the AT1412 epitope is overexpressed on tumor cells. In addition, AT1412 strongly reacted with other tumor types including colon, pancreas and, breast cancer. Further analysis revealed that AT1412 favors binding to a clustered state of CD9. CD9 clusters are dependent on palmitoylation and known to be present on metastatic cells3. Previously published high affinity mouse antibodies targeting human CD9 were found to trigger aggregation of platelets and thus unfit for therapeutic use4. In sharp contrast, AT1412 does not induce platelet aggregation. To address whether AT1412 fails to aggregate platelets because of its low affinity for CD9 we generated high affinity variants of AT1412 using our highly efficient affinity maturation platform. We obtained AT1412 variants with affinities up to 250 fold higher than the wild type AT1412 comparable to that of mouse anti-human CD9 antibodies. These high affinity variants still did not aggregate platelets indicating that the absence of platelet aggregation by AT1412 is due to the spatial arrangement of the AT1412 epitope present on CD9 and not its low affinity. The antibody was able to reduce growth of melanoma tumors and block metastasis in melanoma xenografted mice. A striking synergistic inhibition of tumor growth was observed in mice that were treated with our CD9 antibody in the presence of a clinically successful anti-PD1 checkpoint inhibitor antibody. Discussion. These data suggest that the antibody contributed to the success of the immunotherapy in this patient. This antibody could act together with tumor-reactive T cells in eradicating circulating tumor cells and/or preventing settlement of metastatic tumor cells. Importantly, no antibody-related adverse effects were observed during and after treatment of this patient indicating that the antibody is safe for use in humans. 1. Verdegaal, Cancer Immunol Immunother, 2011 2. Kwakkenbos, Nat Med, 2010 3. Yang, JBC, 2006 4. Boucheix, FEBS Letter, 1983 Citation Format: Remko Schotte, Pauline van Helden, Daniel Go, Christien Fatmawati, Els Verdegaal, Camile Bru, Julien Villaudy, Koen Wagner, Sjoerd van der Burg, Hergen Spits, Wouter Pos. A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2017-LB-200

Abstract CT070: A open-label phase 3b/4 safety trial of flat-dose nivolumab in combination with ipilimumab in patients with advanced non-small cell lung cancer (NSCLC)

Abstract Background: Nivolumab and ipilimumab are immune checkpoint inhibitor antibodies with distinct but complementary mechanisms of action, and their combination may improve clinical outcomes compared with single-agent therapy. Nivolumab plus ipilimumab is approved for the first-line treatment of metastatic melanoma and has shown encouraging clinical activity in other tumor types, including NSCLC. In CheckMate 012, a multi-cohort phase 1 trial that evaluated nivolumab as monotherapy or in combination with other agents in chemotherapy-naïve patients with NSCLC, weight-based administration of nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg) yielded objective response rates (ORR) of up to 47%, depending on the dosing schedule. The discontinuation rates attributable to treatment-related adverse events (AEs) in patients treated with nivolumab plus ipilimumab were similar to nivolumab monotherapy, and AEs were consistent with the known safety profiles of the individual agents. Pharmacokinetic, safety, and efficacy data indicate comparable safety and efficacy profiles for 240 mg flat-dose nivolumab and 3 mg/kg nivolumab. This open-label phase 3b/4 study (ClinicalTrials.gov identifier: NCT02869789) will characterize the safety of flat-dose nivolumab combined with ipilimumab in patients with advanced NSCLC. Moreover, this study will evaluate this combination in special patient populations that are typically excluded from NSCLC trials. Methods: Adult patients with stage IV/recurrent NSCLC and no prior systemic anticancer therapy, including epidermal growth factor receptor and anaplastic lymphoma kinase inhibitors (cohort A), or with stage IIIb/IV NSCLC and recurrence or progression during or after one prior platinum doublet chemotherapy regimen (cohort B) will be enrolled, with a planned sample size of 400 patients per cohort. Patients are required to have assessment of programmed death-1 ligand 1 expression, Eastern Cooperative Oncology Group performance status (ECOG PS) 0–1, and no untreated brain metastases, carcinomatous meningitis, autoimmune disease, or active malignancy requiring concurrent intervention. A third cohort (A1) of approximately 200 patients with no prior systemic therapy will have either ECOG PS 2 or one or more of the following: asymptomatic untreated brain metastases, renal or hepatic dysfunction, and/or HIV. All patients will receive flat-dose nivolumab (240 mg every 2 weeks) combined with weight-based ipilimumab (1 mg/kg every 6 weeks). The primary endpoint is a descriptive assessment of the number and percentage of patients with high-grade treatment-related select and immune-mediated AEs in cohorts A and B. Secondary efficacy endpoints include progression-free survival, ORR, duration of response, and patient-reported outcomes based on the Functional Assessment of Cancer Therapy-Lung (FACT-L). Citation Format: Rathi N. Pillai, Bradley Lash, Istvan Albert, Gabrielle Gagnon, Carl Chakmakjian, Neal Ready, Wenhua Hu, Lee Krug, Sutapa Mukhopadhyay, Luis Paz-Ares. A open-label phase 3b/4 safety trial of flat-dose nivolumab in combination with ipilimumab in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT070. doi:10.1158/1538-7445.AM2017-CT070