Landscape of helper and regulatory antitumour CD4+ T cells in melanoma.

Within the tumour microenvironment, CD4+ T cells can promote or suppress antitumour responses through the recognition of antigens presented by human leukocyte antigen (HLA) class II molecules1,2, but how cancers co-opt these physiologic processes to achieve immune evasion remains incompletely understood. Here we performed in-depth analysis of the phenotype and tumour specificity of CD4+ T cells infiltrating human melanoma specimens, finding that exhausted cytotoxic CD4+ T cells could be directly induced by melanoma cells through recognition of HLA class II-restricted neoantigens, and also HLA class I-restricted tumour-associated antigens. CD4+ T regulatory (TReg) cells could be indirectly elicited through presentation of tumour antigens via antigen-presenting cells. Notably, numerous tumour-reactive CD4+ TReg clones were stimulated directly by HLA class II-positive melanoma and demonstrated specificity for melanoma neoantigens. This phenomenon was observed in the presence of an extremely high tumour neoantigen load, which we confirmed to be associated with HLA class II positivity through the analysis of 116 melanoma specimens. Our data reveal the landscape of infiltrating CD4+ T cells in melanoma and point to the presentation of HLA class II-restricted neoantigens and direct engagement of immunosuppressive CD4+ TReg cells as a mechanism of immune evasion that is favoured in HLA class II-positive melanoma.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients: A proof-of-concept study.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients. Data showed decreased glycolytic activity in red blood cells of 2/3 of patients with lower-risk MDS. These results highlight a potential effect of the PK activator in this setting.

HLA-A*03 and response to immune checkpoint blockade in cancer: an epidemiological biomarker study.

BACKGROUND: Predictive biomarkers could allow more precise use of immune checkpoint inhibitors (ICIs) in treating advanced cancers. Given the central role of HLA molecules in immunity, variation at the HLA loci could differentially affect the response to ICIs. The aim of this epidemiological study was to determine the effect of HLA-A*03 as a biomarker for predicting response to immunotherapy. METHODS: In this epidemiological study, we investigated the clinical outcomes (overall survival, progression free survival, and objective response rate) after treatment for advanced cancer in eight cohorts of patients: three observational cohorts of patients with various types of advanced tumours (the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets [MSK-IMPACT] cohort, the Dana-Farber Cancer Institute [DFCI] Profile cohort, and The Cancer Genome Atlas) and five clinical trials of patients with advanced bladder cancer (JAVELIN Solid Tumour) or renal cell carcinoma (CheckMate-009, CheckMate-010, CheckMate-025, and JAVELIN Renal 101). In total, these cohorts included 3335 patients treated with various ICI agents (anti-PD-1, anti-PD-L1, and anti-CTLA-4 inhibitors) and 10 917 patients treated with non-ICI cancer-directed therapeutic approaches. We initially modelled the association of HLA amino-acid variation with overall survival in the MSK-IMPACT discovery cohort, followed by a detailed analysis of the association between HLA-A*03 and clinical outcomes in MSK-IMPACT, with replication in the additional cohorts (two further observational cohorts and five clinical trials). FINDINGS: HLA-A*03 was associated in an additive manner with reduced overall survival after ICI treatment in the MSK-IMPACT cohort (HR 1·48 per HLA-A*03 allele [95% CI 1·20-1·82], p=0·00022), the validation DFCI Profile cohort (HR 1·22 per HLA-A*03 allele, 1·05-1·42; p=0·0097), and in the JAVELIN Solid Tumour clinical trial for bladder cancer (HR 1·36 per HLA-A*03 allele, 1·01-1·85; p=0·047). The HLA-A*03 effect was observed across ICI agents and tumour types, but not in patients treated with alternative therapies. Patients with HLA-A*03 had shorter progression-free survival in the pooled patient population from the three CheckMate clinical trials of nivolumab for renal cell carcinoma (HR 1·31, 1·01-1·71; p=0·044), but not in those receiving control (everolimus) therapies. Objective responses were observed in none of eight HLA-A*03 homozygotes in the ICI group (compared with 59 [26·6%] of 222 HLA-A*03 non-carriers and 13 (17·1%) of 76 HLA-A*03 heterozygotes). HLA-A*03 was associated with shorter progression-free survival in patients receiving ICI in the JAVELIN Renal 101 randomised clinical trial for renal cell carcinoma (avelumab plus axitinib; HR 1·59 per HLA-A*03 allele, 1·16-2·16; p=0·0036), but not in those receiving control (sunitinib) therapy. Objective responses were recorded in one (12·5%) of eight HLA-A*03 homozygotes in the ICI group (compared with 162 [63·8%] of 254 HLA-A*03 non-carriers and 40 [55·6%] of 72 HLA-A*03 heterozygotes). HLA-A*03 was associated with impaired outcome in meta-analysis of all 3335 patients treated with ICI at genome-wide significance (p=2·01 × 10-8) with no evidence of heterogeneity in effect (I2 0%, 95% CI 0-0·76) INTERPRETATION: HLA-A*03 is a predictive biomarker of poor response to ICI. Further evaluation of HLA-A*03 is warranted in randomised trials. HLA-A*03 carriage could be considered in decisions to initiate ICI in patients with cancer. FUNDING: National Institutes of Health, Merck KGaA, and Pfizer.

Nivolumab plus Ipilimumab versus Sunitinib in Advanced Renal-Cell Carcinoma.

BACKGROUND: Nivolumab plus ipilimumab produced objective responses in patients with advanced renal-cell carcinoma in a pilot study. This phase 3 trial compared nivolumab plus ipilimumab with sunitinib for previously untreated clear-cell advanced renal-cell carcinoma. METHODS: We randomly assigned adults in a 1:1 ratio to receive either nivolumab (3 mg per kilogram of body weight) plus ipilimumab (1 mg per kilogram) intravenously every 3 weeks for four doses, followed by nivolumab (3 mg per kilogram) every 2 weeks, or sunitinib (50 mg) orally once daily for 4 weeks (6-week cycle). The coprimary end points were overall survival (alpha level, 0.04), objective response rate (alpha level, 0.001), and progression-free survival (alpha level, 0.009) among patients with intermediate or poor prognostic risk. RESULTS: A total of 1096 patients were assigned to receive nivolumab plus ipilimumab (550 patients) or sunitinib (546 patients); 425 and 422, respectively, had intermediate or poor risk. At a median follow-up of 25.2 months in intermediate- and poor-risk patients, the 18-month overall survival rate was 75% (95% confidence interval [CI], 70 to 78) with nivolumab plus ipilimumab and 60% (95% CI, 55 to 65) with sunitinib; the median overall survival was not reached with nivolumab plus ipilimumab versus 26.0 months with sunitinib (hazard ratio for death, 0.63; P<0.001). The objective response rate was 42% versus 27% (P<0.001), and the complete response rate was 9% versus 1%. The median progression-free survival was 11.6 months and 8.4 months, respectively (hazard ratio for disease progression or death, 0.82; P=0.03, not significant per the prespecified 0.009 threshold). Treatment-related adverse events occurred in 509 of 547 patients (93%) in the nivolumab-plus-ipilimumab group and 521 of 535 patients (97%) in the sunitinib group; grade 3 or 4 events occurred in 250 patients (46%) and 335 patients (63%), respectively. Treatment-related adverse events leading to discontinuation occurred in 22% and 12% of the patients in the respective groups. CONCLUSIONS: Overall survival and objective response rates were significantly higher with nivolumab plus ipilimumab than with sunitinib among intermediate- and poor-risk patients with previously untreated advanced renal-cell carcinoma. (Funded by Bristol-Myers Squibb and Ono Pharmaceutical; CheckMate 214 ClinicalTrials.gov number, NCT02231749 .).

Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.

Daclatasvir plus sofosbuvir for previously treated or untreated chronic HCV infection.

BACKGROUND: All-oral combination therapy is desirable for patients with chronic hepatitis C virus (HCV) infection. We evaluated daclatasvir (an HCV NS5A replication complex inhibitor) plus sofosbuvir (a nucleotide analogue HCV NS5B polymerase inhibitor) in patients infected with HCV genotype 1, 2, or 3. METHODS: In this open-label study, we initially randomly assigned 44 previously untreated patients with HCV genotype 1 infection and 44 patients infected with HCV genotype 2 or 3 to daclatasvir at a dose of 60 mg orally once daily plus sofosbuvir at a dose of 400 mg orally once daily, with or without ribavirin, for 24 weeks. The study was expanded to include 123 additional patients with genotype 1 infection who were randomly assigned to daclatasvir plus sofosbuvir, with or without ribavirin, for 12 weeks (82 previously untreated patients) or 24 weeks (41 patients who had previous virologic failure with telaprevir or boceprevir plus peginterferon alfa-ribavirin). The primary end point was a sustained virologic response (an HCV RNA level of <25 IU per milliliter) at week 12 after the end of therapy. RESULTS: Overall, 211 patients received treatment. Among patients with genotype 1 infection, 98% of 126 previously untreated patients and 98% of 41 patients who did not have a sustained virologic response with HCV protease inhibitors had a sustained virologic response at week 12 after the end of therapy. A total of 92% of 26 patients with genotype 2 infection and 89% of 18 patients with genotype 3 infection had a sustained virologic response at week 12. High rates of sustained virologic response at week 12 were observed among patients with HCV subtypes 1a and 1b (98% and 100%, respectively) and those with CC and non-CC IL28B genotypes (93% and 98%, respectively), as well as among patients who received ribavirin and those who did not (94% and 98%, respectively). The most common adverse events were fatigue, headache, and nausea. CONCLUSIONS: Once-daily oral daclatasvir plus sofosbuvir was associated with high rates of sustained virologic response among patients infected with HCV genotype 1, 2, or 3, including patients with no response to prior therapy with telaprevir or boceprevir. (Funded by Bristol-Myers Squibb and Pharmasset (Gilead); A1444040 ClinicalTrials.gov number, NCT01359644.).

Peginterferon lambda for the treatment of HBeAg-positive chronic hepatitis B: A randomized phase 2b study (LIRA-B).

BACKGROUND & AIMS: Peginterferon lambda-1a (lambda) is a Type-III interferon, which, like alfa interferons, has antiviral activity in vitro against hepatitis B virus (HBV) and hepatitis C virus (HCV); however, lambda has a more limited extra-hepatic receptor distribution. This phase 2b study (LIRA-B) evaluated lambda in patients with chronic HBV infection. METHODS: Adult HBeAg+ interferon-naive patients were randomized (1:1) to weekly lambda (180 µg) or peginterferon alfa-2a (alfa) for 48 weeks. The primary efficacy endpoint was HBeAg seroconversion at week 24 post-treatment; lambda non-inferiority was demonstrated if the 80% confidence interval (80% CI) lower bound was >-15%. RESULTS: Baseline characteristics were balanced across groups (lambda N=80; alfa N=83). Early on-treatment declines in HBV-DNA and qHBsAg through week 24 were greater with lambda. HBeAg seroconversion rates were comparable for lambda and alfa at week 48 (17.5% vs. 16.9%, respectively); however lambda non-inferiority was not met at week 24 post-treatment (13.8% vs. 30.1%, respectively; lambda vs. alfa 80% CI lower bound -24%). Results for other key secondary endpoints (virologic, serologic, biochemical) and post hoc combined endpoints (HBV-DNA <2000 IU/ml plus HBeAg seroconversion or ALT normalization) mostly favored alfa. Overall adverse events (AE), serious AE, and AE-discontinuation rates were comparable between arms but AE-spectra differed (more cytopenias, flu-like, and musculoskeletal symptoms observed with alfa, more ALT flares and bilirubin elevations seen with lambda). Most on-treatment flares occurred early (weeks 4-12), associated with HBV-DNA decline; all post-treatment flares were preceded by HBV-DNA rise. CONCLUSIONS: On-treatment, lambda showed greater early effects on HBV-DNA and qHBsAg, and comparable serologic/virologic responses at end-of-treatment. However, post-treatment, alfa-associated HBeAg seroconversion rates were higher, and key secondary results mostly favored alfa. ClinicalTrials.gov number: NCT01204762.

Daclatasvir plus peginterferon alfa and ribavirin for treatment-naive chronic hepatitis C genotype 1 or 4 infection: a randomised study.

OBJECTIVE: To evaluate the safety and efficacy of daclatasvir, an HCV NS5A inhibitor with pangenotypic activity, administered with peginterferon-alfa-2a/ribavirin. DESIGN: In this Phase 2b double-blind, placebo-controlled study, treatment-naive adults with HCV genotype 1 (N=365) or 4 (N=30) infection were randomly assigned (2:2:1) to daclatasvir 20 mg or 60 mg, or placebo once daily plus weekly peginterferon-alfa-2a and twice-daily ribavirin. Daclatasvir recipients achieving protocol-defined response (PDR; HCV-RNA

Preliminary study of two antiviral agents for hepatitis C genotype 1.

BACKGROUND: Patients with chronic hepatitis C virus (HCV) infection who have not had a response to therapy with peginterferon and ribavirin may benefit from the addition of multiple direct-acting antiviral agents to their treatment regimen. METHODS: This open-label, phase 2a study included an exploratory cohort of 21 patients with chronic HCV genotype 1 infection who had not had a response to previous therapy (i.e., had not had ≥2 log(10) decline in HCV RNA after ≥12 weeks of treatment with peginterferon and ribavirin). We randomly assigned patients to receive the NS5A replication complex inhibitor daclatasvir (60 mg once daily) and the NS3 protease inhibitor asunaprevir (600 mg twice daily) alone (group A, 11 patients) or in combination with peginterferon alfa-2a and ribavirin (group B, 10 patients) for 24 weeks. The primary end point was the percentage of patients with a sustained virologic response 12 weeks after the end of the treatment period. RESULTS: A total of 4 patients in group A (36%; 2 of 9 with HCV genotype 1a and 2 of 2 with genotype 1b) had a sustained virologic response at 12 weeks after treatment and also at 24 weeks after treatment.. Six patients (all with HCV genotype 1a) had viral breakthrough while receiving therapy, and resistance mutations to both antiviral agents were found in all cases; 1 patient had a viral response at the end of treatment but had a relapse after the treatment period. All 10 patients in group B had a sustained virologic response at 12 weeks after treatment, and 9 had a sustained virologic response at 24 weeks after treatment. Diarrhea was the most common adverse event in both groups. Six patients had transient elevations of alanine aminotransferase levels to more than 3 times the upper limit of the normal range. CONCLUSIONS: This preliminary study involving patients with HCV genotype 1 infection who had not had a response to prior therapy showed that a sustained virologic response can be achieved with two direct-acting antiviral agents only. In addition, a high rate of sustained virologic response was achieved when the two direct-acting antiviral agents were combined with peginterferon alfa-2a and ribavirin. (Funded by Bristol-Myers Squibb; ClinicalTrials.gov number, NCT01012895.).

Efficacy of an interferon- and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 in treatment-naive patients with HCV genotype 1 infection.

BACKGROUND & AIMS: The combination of peginterferon and ribavirin with telaprevir or boceprevir is the standard treatment of hepatitis C virus (HCV) genotype 1 infection. However, these drugs are not well tolerated because of their side effects and suboptimal virologic responses. In a phase 2a, open-label study, we examined the safety and efficacy of an interferon-free, ribavirin-free regimen of direct-acting antivirals, comprising daclatasvir (an NS5A replication complex inhibitor), asunaprevir (an NS3 protease inhibitor), and BMS-791325 (a non-nucleoside NS5B inhibitor), in patients with chronic HCV infection. METHODS: We analyzed data from 66 treatment-naive patients with HCV genotype 1 infection without cirrhosis who were assigned randomly to groups given daclatasvir (60 mg, once daily), asunaprevir (200 mg, twice daily), and BMS-791325 (75 or 150 mg, twice daily) for 12 or 24 weeks. The primary end point was an HCV-RNA level less than 25 IU/mL at 12 weeks after treatment (sustained virologic response at 12 weeks [SVR12]). RESULTS: In 64 patients, HCV-RNA levels were less than 25 IU/mL by week 4 of treatment (including 48 of 49 patients with HCV genotype 1a infection and 45 of 46 patients with the non-CC interleukin 28B genotype). Sixty-one patients (92%) achieved SVR12, based on a modified intention-to-treat analysis. Virologic responses were similar between 12 and 24 weeks of treatment. During the study, 2 patients experienced viral breakthrough and 1 patient relapsed. There were no grade 3-4 increases in levels of alanine or aspartate aminotransferases or bilirubin; there were no deaths or discontinuations resulting from serious adverse events or adverse events related to the treatment regimen. The most common adverse events were headache, asthenia, and gastrointestinal symptoms. CONCLUSIONS: In a phase 2a study, the all-oral, interferon-free, and ribavirin-free regimen of daclatasvir, asunaprevir, and BMS-791325 was well tolerated and achieved high rates of SVR12 in patients with HCV genotype 1 infection. Further studies of this regimen are warranted. ClinicalTrials.gov, number NCT01455090.

Randomized trial of daclatasvir and asunaprevir with or without PegIFN/RBV for hepatitis C virus genotype 1 null responders.

BACKGROUND & AIMS: Patients with chronic hepatitis C virus (HCV) infection and prior null response (<2 log HCV RNA decline after ⩾ 12 weeks of PegIFN/RBV) have limited options. We evaluated daclatasvir plus once- or twice-daily asunaprevir in non-cirrhotic genotype 1 null responders. METHODS: In this randomized, phase 2a, open-label, 24-week treatment study, 101 patients received daclatasvir (60 mg) once-daily. In addition, 38 genotype 1b patients received asunaprevir (200mg) twice- (DUAL A1) or once-daily (DUAL A2); 36 genotype 1a and 5 genotype 1b patients received asunaprevir twice- (QUAD B1) or once-daily (QUAD B2) plus PegIFN/RBV; and 18 genotype 1a and 4 genotype 1b patients received asunaprevir twice-daily plus ribavirin (TRIPLE B3). The primary endpoint was undetectable HCV RNA 12 weeks post-treatment (sustained virologic response, SVR12). RESULTS: Across all groups, mean HCV RNA was ⩾ 6 log IU/ml, and 99% of patients had a non-CC IL28B genotype. SVR12 rates were 78% (A1), 65% (A2), 95% (B1), and 95% (B2). In B3, most genotype 1a patients experienced virologic breakthrough. The most common adverse events were headache, diarrhea, and asthenia. Grade 3-4 aminotransferase elevations were infrequent and not treatment-limiting. CONCLUSIONS: In genotype 1 null responders, daclatasvir plus twice-daily asunaprevir DUAL therapy is effective for most genotype 1b patients, and daclatasvir, asunaprevir, and PegIFN/RBV QUAD therapy is effective for nearly all genotype 1a and 1b patients; but neither DUAL nor TRIPLE therapy is effective for genotype 1a patients. Interferon-free regimens including daclatasvir and twice-daily asunaprevir for genotype 1 null responders should be tailored to subtype.

Mitapivat improves ineffective erythropoiesis and iron overload in adult patients with pyruvate kinase deficiency.

ABSTRACT: Pyruvate kinase (PK) deficiency is a rare, hereditary disease characterized by chronic hemolytic anemia. Iron overload is a common complication regardless of age, genotype, or transfusion history. Mitapivat, an oral, allosteric PK activator, improves anemia and hemolysis in adult patients with PK deficiency. Mitapivat's impact on iron overload and ineffective erythropoiesis was evaluated in adults with PK deficiency who were not regularly transfused in the phase 3 ACTIVATE trial and long-term extension (LTE) (#NCT03548220/#NCT03853798). Patients in the LTE received mitapivat throughout ACTIVATE/LTE (baseline to week 96; mitapivat-to-mitapivat [M/M] arm) or switched from placebo (baseline to week 24) to mitapivat (week 24 to week 96; placebo-to-mitapivat [P/M] arm). Changes from baseline in markers of iron overload and erythropoiesis were assessed to week 96. Improvements in hepcidin (mean, 4770.0 ng/L; 95% confidence interval [CI], -1532.3 to 11 072.3), erythroferrone (mean, -9834.9 ng/L; 95% CI, -14 328.4 to -5341.3), soluble transferrin receptor (mean, -56.0 nmol/L; 95% CI, -84.8 to -27.2), and erythropoietin (mean, -32.85 IU/L; 95% CI, -54.65 to -11.06) were observed in the M/M arm (n = 40) from baseline to week 24, sustained to week 96. No improvements were observed in the P/M arm (n = 40) to week 24; however, upon transitioning to mitapivat, improvements similar to those observed in the M/M arm were seen. Mean changes from baseline in liver iron concentration by magnetic resonance imaging at week 96 in the M/M arm and the P/M arm were -2.0 mg Fe/g dry weight (dw; 95% CI, -4.8 to -0.8) and -1.8 mg Fe/g dw (95% CI, -4.4 to 0.80), respectively. Mitapivat is the first disease-modifying pharmacotherapy shown to have beneficial effects on iron overload and ineffective erythropoiesis in patients with PK deficiency. This trial was registered at www.ClinicalTrials.gov as #NCT03548220 (ACTIVATE) and #NCT03853798 (LTE).

Pharmacodynamics, safety, and pharmacokinetics of BMS-663068, an oral HIV-1 attachment inhibitor in HIV-1-infected subjects.

BACKGROUND: BMS-663068 is a prodrug of the small-molecule inhibitor BMS-626529, which inhibits human immunodeficiency virus type 1 (HIV-1) infection by binding to gp120 and interfering with the attachment of virus to CD4+ T-cells. METHODS: Fifty HIV-1-infected subjects were randomized to 1 of 5 regimen groups (600 mg BMS-663068 plus 100 mg ritonavir every 12 hours [Q12H], 1200 mg BMS-663068 plus 100 mg ritonavir every bedtime, 1200 mg BMS-663068 plus 100 mg ritonavir Q12H, 1200 mg BMS-663068 Q12H plus 100 mg ritonavir every morning, or 1200 mg BMS-663068 Q12H) for 8 days in this open-label, multiple-dose, parallel study. The study assessed the pharmacodynamics, pharmacokinetics, and safety of BMS-663068. RESULTS: The maximum median decrease in plasma HIV-1 RNA load from baseline ranged from 1.21 to 1.73 log(10) copies/mL. Plasma concentrations of BMS-626529 were not associated with an antiviral response, while low baseline inhibitory concentrations and the minimum and average steady-state BMS-626529 plasma concentrations, when adjusted by the baseline protein binding-adjusted 90% inhibitory concentration (inhibitory quotient), were linked with antiviral response. BMS-663068 was generally well tolerated. CONCLUSIONS: Administration of BMS-663068 for 8 days with or without ritonavir resulted in substantial declines in plasma HIV-1 RNA levels and was generally well tolerated. Longer-term clinical trials of BMS-663068 as part of combination antiretroviral therapy are warranted. Clinical Trials Registration.NCT01009814.

Disease-free Survival Analysis for Patients with High-risk Muscle-invasive Urothelial Carcinoma from the Randomized CheckMate 274 Trial by PD-L1 Combined Positive Score and Tumor Cell Score.

BACKGROUND: The CheckMate 274 trial demonstrated improved disease-free survival (DFS) with adjuvant nivolumab versus placebo in patients with muscle-invasive urothelial carcinoma at high risk of recurrence after radical surgery in both the intent-to-treat population and the subset with tumor programmed death ligand 1 (PD-L1) expression ≥1%. OBJECTIVE: To analyze DFS by combined positive score (CPS), which is based on PD-L1 expression in both tumor and immune cells. DESIGN, SETTING, AND PARTICIPANTS: We randomized a total of 709 patients 1:1 to nivolumab 240 mg or placebo every 2 wk intravenously for ≤1 yr of adjuvant treatment. INTERVENTION: Nivolumab 240 mg. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary endpoints were DFS in the intent-to-treat population and patients with tumor PD-L1 expression ≥1% using the tumor cell (TC) score. CPS was determined retrospectively from previously stained slides. Tumor samples with both quantifiable CPS and TC were analyzed. RESULTS AND LIMITATIONS: Of 629 patients evaluable for CPS and TC, 557 (89%) had CPS ≥1, 72 (11%) had CPS <1, 249 (40%) had TC ≥1%, and 380 (60%) had TC <1%. Among patients with TC <1%, 81% (n = 309) had CPS ≥1. DFS was improved with nivolumab versus placebo for patients with TC ≥1% (hazard ratio [HR] 0.50, 95% confidence interval [CI] 0.35-0.71), those with CPS ≥1 (HR 0.62, 95% CI 0.49-0.78), and patients with both TC <1% and CPS ≥1 (HR 0.73, 95% CI 0.54-0.99). CONCLUSION: More patients had CPS ≥1 than TC ≥1%, and most patients who had TC <1% had CPS ≥1. In addition, patients with CPS ≥1 experienced improved DFS with nivolumab. These results may, in part, explain the mechanisms underlying a benefit with adjuvant nivolumab even in patients who had both TC <1% and CPS ≥1. PATIENT SUMMARY: We studied survival time without cancer recurrence (disease-free survival; DFS) for patients treated with nivolumab versus placebo after surgery to remove the bladder or components of the urinary tract for bladder cancer in the CheckMate 274 trial. We assessed the impact of levels of the protein PD-L1 expressed either on tumor cells (tumor cell score; TC) or on both tumor cells and immune cells surrounding the tumor (combined positive score; CPS). DFS was impoved with nivolumab versus placebo for patients with TC ≥1%, CPS ≥1, and for patients with both TC <1% and CPS ≥1. This analysis may help physicians understand which patients would benefit most from treatment with nivolumab.

In vitro antiviral characteristics of HIV-1 attachment inhibitor BMS-626529, the active component of the prodrug BMS-663068.

BMS-663068 is the phosphonooxymethyl prodrug of BMS-626529, a novel small-molecule attachment inhibitor that targets HIV-1 gp120 and prevents its binding to CD4(+) T cells. The activity of BMS-626529 is virus dependent, due to heterogeneity within gp120. In order to better understand the anti-HIV-1 spectrum of BMS-626529 against HIV-1, in vitro activities against a wide variety of laboratory strains and clinical isolates were determined. BMS-626529 had half-maximal effective concentration (EC(50)) values of <10 nM against the vast majority of viral isolates; however, susceptibility varied by >6 log(10), with half-maximal effective concentration values in the low pM range against the most susceptible viruses. The in vitro antiviral activity of BMS-626529 was generally not associated with either tropism or subtype, with few exceptions. Measurement of the binding affinity of BMS-626529 for purified gp120 suggests that a contributory factor to its inhibitory potency may be a relatively long dissociative half-life. Finally, in two-drug combination studies, BMS-626529 demonstrated additive or synergistic interactions with antiretroviral drugs of different mechanistic classes. These results suggest that BMS-626529 should be active against the majority of HIV-1 viruses and support the continued clinical development of the compound.

Baseline Serum Autoantibody Signatures Predict Recurrence and Toxicity in Melanoma Patients Receiving Adjuvant Immune Checkpoint Blockade.

PURPOSE: Adjuvant immunotherapy produces durable benefit for patients with resected melanoma, but many develop recurrence and/or immune-related adverse events (irAE). We investigated whether baseline serum autoantibody (autoAb) signatures predicted recurrence and severe toxicity in patients treated with adjuvant nivolumab, ipilimumab, or ipilimumab plus nivolumab. EXPERIMENTAL DESIGN: This study included 950 patients: 565 from CheckMate 238 (408 ipilimumab versus 157 nivolumab) and 385 from CheckMate 915 (190 nivolumab versus 195 ipilimumab plus nivolumab). Serum autoAbs were profiled using the HuProt Human Proteome Microarray v4.0 (CDI Laboratories, Mayaguez, PR). Analysis of baseline differentially expressed autoAbs was followed by recurrence and severe toxicity signature building for each regimen, testing of the signatures, and additional independent validation for nivolumab using patients from CheckMate 915. RESULTS: In the nivolumab independent validation cohort, high recurrence score predicted significantly worse recurrence-free survival [RFS; adjusted HR (aHR), 3.60; 95% confidence interval (CI), 1.98-6.55], and outperformed a model composed of clinical variables including PD-L1 expression (P < 0.001). Severe toxicity score was a significant predictor of severe irAEs (aHR, 13.53; 95% CI, 2.59-86.65). In the ipilimumab test cohort, high recurrence score was associated with significantly worse RFS (aHR, 3.21; 95% CI, 1.38-7.45) and severe toxicity score significantly predicted severe irAEs (aHR, 11.04; 95% CI, 3.84-37.25). In the ipilimumab plus nivolumab test cohort, high autoAb recurrence score was associated with significantly worse RFS (aHR, 6.45; 95% CI, 1.48-28.02), and high severe toxicity score was significantly associated with severe irAEs (aHR, 23.44; 95% CI, 4.10-212.50). CONCLUSIONS: Baseline serum autoAb signatures predicted recurrence and severe toxicity in patients treated with adjuvant immunotherapy. Prospective testing of the signatures that include datasets with longer follow-up and rare but more severe toxicities will help determine their generalizability and potential clinical utility. See related commentary by Hassel and Luke, p. 3914.

Deep learning reveals predictive sequence concepts within immune repertoires to immunotherapy.

T cell receptor (TCR) sequencing has been used to characterize the immune response to cancer. However, most analyses have been restricted to quantitative measures such as clonality that do not leverage the complementarity-determining region 3 (CDR3) sequence. We use DeepTCR, a framework of deep learning algorithms, to reveal sequence concepts that are predictive of response to immunotherapy. We demonstrate that DeepTCR can predict response and use the model to infer the antigenic specificities of the predictive signature and their unique dynamics during therapy. The predictive signature of nonresponse is associated with high frequencies of TCRs predicted to recognize tumor-specific antigens, and these tumor-specific TCRs undergo a higher degree of dynamic changes on therapy in nonresponders versus responders. These results are consistent with a biological model where the hallmark of nonresponders is an accumulation of tumor-specific T cells that undergo turnover on therapy, possibly because of the dysfunctional state of these T cells in nonresponders.

Soluble PD-L1 as an early marker of progressive disease on nivolumab.

BACKGROUND: Soluble PD-L1 (sPD-L1) has been associated with worse prognosis in numerous solid tumors. We determined sPD-L1 levels before and during nivolumab treatment in two prospective clinical trials of metastatic clear cell renal cell carcinoma (RCC) and melanoma patients, and investigated its relationship to clinical factors, biomarkers, and outcome. METHODS: Using a new Single Molecule Array assay, serum sPD-L1 level were determined in RCC (CheckMate 009, n=91) and melanoma (CheckMate 038-Part 1, n=78) prior to, and at two time points on treatment. Gene expression data was obtained from biopsies taken prior to, and at day 28 on treatment. Results were integrated with clinical variables, tumor PD-L1 status from immuno-histochemistry, and genomic mutation status. RESULTS: In RCC patients, sPD-L1 levels were higher in patients with progressive disease as their best response. For both RCC and melanoma patients, progressive or stable disease was associated with an increase in sPD-L1 on nivolumab therapy, whereas mean sPD-L1 levels did not change or declined in patients with objective responses. By categorizing RCC patients into transcriptomic molecular subtypes, we identified a subgroup where the associations between sPD-L1 and progressive disease were particularly evident. In baseline biopsies, we identified six biological processes that were associated with sPD-L1 level in both RCC and melanoma: higher sPD-L1 is associated with lower tumor expression of the Hallmark gene sets 'hypoxia', 'fatty acid metabolism', 'glycolysis', 'MTORC1 signaling' and 'androgen response', and with higher expression of 'KRAS signaling_Down'. CONCLUSION: Baseline and on-therapy sPD-L1 levels in RCC have the potential to predict progressive disease on PD-1 inhibitor nivolumab. In a hypothesis-generating analysis of tumor gene expression, high baseline sPD-L1 is associated with a tumor metabolic state reflecting potentially targetable processes in both melanoma and RCC. In both trials, we observed associations between change in sPD-L1 on treatment and outcome metrics. sPD-L1 levels may further refine a nivolumab-refractory subtype of RCC within transcriptionally based subtypes of RCC.

Biomarker analysis from CheckMate 214: nivolumab plus ipilimumab versus sunitinib in renal cell carcinoma.

BACKGROUND: The phase 3 CheckMate 214 trial demonstrated higher response rates and improved overall survival with nivolumab plus ipilimumab versus sunitinib in first-line therapy for advanced clear-cell renal cell carcinoma (RCC). An unmet need exists to identify patients with RCC who are most likely to benefit from treatment with nivolumab plus ipilimumab. METHODS: In exploratory analyses, pretreatment levels of programmed death ligand 1 were assessed by immunohistochemistry. Genomic and transcriptomic biomarkers (including tumor mutational burden and gene expression signatures) were also investigated. RESULTS: Biomarkers previously associated with benefit from immune checkpoint inhibitor-containing regimens in RCC were not predictive for survival in patients with RCC treated with nivolumab plus ipilimumab. Analysis of gene expression identified an association between an inflammatory response and progression-free survival with nivolumab plus ipilimumab. CONCLUSIONS: The exploratory analyses reveal relationships between molecular biomarkers and provide supportive data on how the inflammation status of the tumor microenvironment may be important for identifying predictive biomarkers of response and survival with combination immunotherapy in patients with RCC. Further validation may help to provide biomarker-driven precision treatment for patients with RCC.

Molecular correlates of response to nivolumab at baseline and on treatment in patients with RCC.

BACKGROUND: Nivolumab is an immune checkpoint inhibitor targeting the programmed death-1 receptor that improves survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). In contrast to other tumor types that respond to immunotherapy, factors such as programmed death ligand-1 (PD-L1) status and tumor mutational burden show limited predictive utility in ccRCC. To address this gap, we report here the first molecular characterization of nivolumab response using paired index lesions, before and during treatment of metastatic ccRCC. METHODS: We analyzed gene expression and T-cell receptor (TCR) clonality using lesion-paired biopsies provided in the CheckMate 009 trial and integrated the results with their PD-L1/CD4/CD8 status, genomic mutation status and serum cytokine assays. Statistical tests included linear mixed models, logistic regression models, Fisher's exact test, and Kruskal-Wallis rank-sum test. RESULTS: We identified transcripts related to response, both at baseline and on therapy, including several that are amenable to peripheral bioassays or to therapeutic intervention. At both timepoints, response was positively associated with T-cell infiltration but not associated with TCR clonality, and some non-Responders were highly infiltrated. Lower baseline T-cell infiltration correlated with elevated transcription of Wnt/ß-catenin signaling components and hypoxia-regulated genes, including the Treg chemoattractant CCL28. On treatment, analysis of the non-responding patients whose tumors were highly T-cell infiltrated suggests association of the RIG-I-MDA5 pathway in their nivolumab resistance. We also analyzed our data using previous transcriptional classifications of ccRCC and found they concordantly identified a molecular subtype that has enhanced nivolumab response but is sunitinib-resistant. CONCLUSION: Our study describes molecular characteristics of response and resistance to nivolumab in patients with metastatic ccRCC, potentially impacting patient selection and first-line treatment decisions. TRIAL REGISTRATION NUMBER: NCT01358721.