Phase I Study of Fianlimab, a Human Lymphocyte Activation Gene-3 (LAG-3) Monoclonal Antibody, in Combination With Cemiplimab in Advanced Melanoma.

PURPOSE: Coblockade of lymphocyte activation gene-3 (LAG-3) and PD-1 receptors could provide significant clinical benefit for patients with advanced melanoma. Fianlimab and cemiplimab are high-affinity, human, hinge-stabilized IgG4 monoclonal antibodies, targeting LAG-3 and PD-1, respectively. We report results from a first-in-human phase-I study of fianlimab and cemiplimab safety and efficacy in various malignancies including advanced melanoma. METHODS: Patients with advanced melanoma were eligible for enrollment into four cohorts: three for patients without and one for patients with previous anti-PD-1 therapy in the advanced disease setting. Patients were treated with fianlimab 1,600 mg and cemiplimab 350 mg intravenously once every 3 weeks for up to 51 weeks, with an optional additional 51 weeks if clinically indicated. The primary end point was objective response rate (ORR) per RECIST 1.1 criteria. RESULTS: ORRs were 63% for patients with anti-PD-1-naïve melanoma (cohort-6; n = 40; median follow-up 20.8 months), 63% for patients with systemic treatment-naïve melanoma (cohort-15; n = 40; 11.5 months), and 56% for patients with previous neo/adjuvant treatment melanoma (cohort-16; n = 18, 9.7 months). At a median follow-up of 12.6 months for the combined cohorts (6 + 15 + 16), the ORR was 61.2% and the median progression-free survival (mPFS) 13.3 months (95% CI, 7.5 to not estimated [NE]). In patients (n = 13) with previous anti-PD-1 adjuvant therapy, ORR was 61.5% and mPFS 12 months (95% CI, 1.4 to NE). ORR in patients with previous anti-PD-1 therapy for advanced disease (n = 15) was 13.3% and mPFS 1.5 months (95% CI, 1.3 to 7.7). Treatment-emergent and treatment-related adverse events ≥grade 3 (G3) were observed in 44% and 22% of patients, respectively. Except for increased incidence of adrenal insufficiency (12%-G1-4, 4%-G3-4), no new safety signals were recorded. CONCLUSION: The current results show a promising benefit-risk profile of fianlimab/cemiplimab combination for patients with advanced melanoma, including those with previous anti-PD-1 therapy in the adjuvant, but not advanced, setting.

Molecular assessment of intratumoral immune cell subsets and potential mechanisms of resistance to odronextamab, a CD20×CD3 bispecific antibody, in patients with relapsed/refractory B-cell non-Hodgkin lymphoma.

BACKGROUND: Patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL) have a significant need for effective treatment options. Odronextamab is an Fc-silenced, human, CD20×CD3 bispecific antibody that targets CD20-expressing cells via T-cell-mediated cytotoxicity independent of T-cell/major histocompatibility complex interaction. Phase I results in patients with R/R B-NHL demonstrated that odronextamab monotherapy could achieve deep and durable responses with a generally manageable safety profile (ELM-1; NCT02290951). As part of a biomarker analysis of the same study, we investigated potential biomarkers and mechanisms of resistance to odronextamab. METHODS: Patients with R/R B-NHL enrolled in ELM-1 received one time per week doses of intravenous odronextamab for 4×21 day cycles, then doses every 2 weeks thereafter. Patient tumor biopsies were obtained at baseline, on-treatment, and at progression. Immune cell markers were analyzed by immunohistochemistry, flow cytometry, single-cell RNA sequencing, and whole genome sequencing. RESULTS: Baseline tumor biopsies showed that almost all patients had high proportions of B cells that expressed the CD20 target antigen, whereas expression of other B-cell surface antigens (CD19, CD22, CD79b) was more variable. Responses to odronextamab in patients with diffuse large B-cell lymphoma were not related to the relative level of baseline CD20 expression, cell of origin, or high-risk molecular subtype. A potential link was observed between greater tumor programmed cell death-ligand 1 expression and increased likelihood of response to odronextamab. Similarly, a trend was observed between clinical response and increased levels of CD8 T cells and regulatory T cells at baseline. We also identified an on-treatment pharmacodynamic shift in intratumoral immune cell subsets. Finally, loss of CD20 expression through inactivating gene mutations was identified as a potential mechanism of resistance in patients who were treated with odronextamab until progression, as highlighted in two detailed patient cases reported here. CONCLUSIONS: This biomarker analysis expands on clinical findings of odronextamab in patients with R/R B-NHL, providing verification of the suitability of CD20 as a therapeutic target, as well as evidence for potential mechanisms of action and resistance.

Safety and Immune Responses Following Anti-PD-1 Monoclonal Antibody Infusions in Healthy Persons With Human Immunodeficiency Virus on Antiretroviral Therapy.

Background: T cells in people with human immunodeficiency virus (HIV) demonstrate an exhausted phenotype, and HIV-specific CD4+ T cells expressing programmed cell death 1 (PD-1) are enriched for latent HIV, making antibody to PD-1 a potential strategy to target the latent reservoir. Methods: This was a phase 1/2, randomized (4:1), double-blind, placebo-controlled study in adults with suppressed HIV on antiretroviral therapy with CD4+ counts ≥350 cells/µL who received 2 infusions of cemiplimab versus placebo. The primary outcome was safety, defined as any grade 3 or higher adverse event (AE) or any immune-related AE (irAE). Changes in HIV-1-specific polyfunctional CD4+ and CD8+ T-cell responses were evaluated. Results: Five men were enrolled (median CD4+ count, 911 cells/µL; median age, 51 years); 2 received 1 dose of cemiplimab, 2 received 2 doses, and 1 received placebo. One participant had a probable irAE (thyroiditis, grade 2); another had a possible irAE (hepatitis, grade 3), both after a single low-dose (0.3 mg/kg) infusion. The Safety Monitoring Committee recommended no further enrollment or infusions. All 4 cemiplimab recipients were followed for 48 weeks. No other cemiplimab-related serious AEs, irAEs, or grade 3 or higher AEs occurred. One 2-dose recipient of cemiplimab had a 6.2-fold increase in polyfunctional, Gag-specific CD8+ T-cell frequency with supportive increases in plasma HIV RNA and decreases in total HIV DNA. Conclusions: One of 4 participants exhibited increased HIV-1-specific T-cell responses and transiently increased HIV-1 expression following 2 cemiplimab infusions. The occurrence of irAEs after a single, low dose may limit translating the promising therapeutic results of cemiplimab for cancer to immunotherapeutic and latency reversal strategies for HIV. Clinical Trials Registration. NCT03787095.

Cancer cell-derived type I interferons instruct tumor monocyte polarization.

Monocytes are highly plastic immune cells that modulate antitumor immunity. Therefore, identifying factors that regulate tumor monocyte functions is critical for developing effective immunotherapies. Here, we determine that endogenous cancer cell-derived type I interferons (IFNs) control monocyte functional polarization. Guided by single-cell transcriptomic profiling of human and mouse tumors, we devise a strategy to distinguish and separate immunostimulatory from immunosuppressive tumor monocytes by surface CD88 and Sca-1 expression. Leveraging this approach, we show that cGAS-STING-regulated cancer cell-derived IFNs polarize immunostimulatory monocytes associated with anti-PD-1 immunotherapy response in mice. We also demonstrate that immunosuppressive monocytes convert into immunostimulatory monocytes upon cancer cell-intrinsic cGAS-STING activation. Consistently, we find that human cancer cells can produce type I IFNs that polarize monocytes, and our immunostimulatory monocyte gene signature is enriched in patient tumors that respond to anti-PD-1 immunotherapy. Our work exposes a role for cancer cell-derived IFNs in licensing monocyte functions that influence immunotherapy outcomes.

CD22-targeted CD28 bispecific antibody enhances antitumor efficacy of odronextamab in refractory diffuse large B cell lymphoma models.

Although many patients with diffuse large B cell lymphoma (DLBCL) may achieve a complete response to frontline chemoimmunotherapy, patients with relapsed/refractory disease typically have poor outcomes. Odronextamab, a CD20xCD3 bispecific antibody that provides "signal 1" through the activation of the T cell receptor/CD3 complex, has exhibited early, promising activity for patients with highly refractory DLBCL in phase 1 trials. However, not all patients achieve complete responses, and many relapse, thus representing a high unmet medical need. Here, we investigated whether adding a costimulatory "signal 2" by engaging CD28 receptors on T cells could augment odronextamab activity. We demonstrate that REGN5837, a bispecific antibody that cross-links CD22-expressing tumor cells with CD28-expressing T cells, enhances odronextamab by potentiating T cell activation and cytolytic function. In preclinical DLBCL studies using human immune system-reconstituted animals, REGN5837 promotes the antitumor activity of odronextamab and induces intratumoral expansion of reprogrammable T cells while skewing away from a dysfunctional state. Although REGN5837 monotherapy shows limited activity and no toxicity in primate studies, it augments T cell activation when dosed in combination with odronextamab. In addition, analysis of non-Hodgkin lymphoma clinical samples reveals an increase in CD28+CD8+ T cells after odronextamab treatment, demonstrating the presence of a population that could potentially be targeted by REGN5837. Collectively, our data demonstrate that REGN5837 can markedly enhance the antitumor activity of odronextamab in preclinical NHL models, and the combination of these two bispecific antibodies may provide a chemotherapy-free approach for the treatment of DLBCL.

Complete responses to odronextamab in two patients with diffuse large B-cell lymphoma refractory to chimeric antigen receptor T-cell therapy.

Outcomes remain poor for patients with relapsed/refractory B-cell non-Hodgkin lymphoma (R/R B-NHL). While chimeric antigen receptor (CAR) T-cell therapy has revolutionised treatment, a significant proportion of patients relapse or fail to respond. Odronextamab is a CD20 × CD3 bispecific antibody that has demonstrated durable responses and a manageable safety profile in patients with R/R B-NHL in a first-in-human trial (NCT02290951). Here, we document two patients with diffuse large B-cell lymphoma refractory to CART-cell therapy. Both achieved complete responses that remain ongoing for ≥2 years following odronextamab. Neither patient experienced Grade ≥3 cytokine release syndrome or Grade ≥3 neurological adverse events during treatment.

Survival with Cemiplimab in Recurrent Cervical Cancer.

BACKGROUND: Patients with recurrent cervical cancer have a poor prognosis. Cemiplimab, the fully human programmed cell death 1 (PD-1)-blocking antibody approved to treat lung and skin cancers, has been shown to have preliminary clinical activity in this population. METHODS: In this phase 3 trial, we enrolled patients who had disease progression after first-line platinum-containing chemotherapy, regardless of their programmed cell death ligand 1 (PD-L1) status. Women were randomly assigned (1:1) to receive cemiplimab (350 mg every 3 weeks) or the investigator's choice of single-agent chemotherapy. The primary end point was overall survival. Progression-free survival and safety were also assessed. RESULTS: A total of 608 women were enrolled (304 in each group). In the overall trial population, median overall survival was longer in the cemiplimab group than in the chemotherapy group (12.0 months vs. 8.5 months; hazard ratio for death, 0.69; 95% confidence interval [CI], 0.56 to 0.84; two-sided P<0.001). The overall survival benefit was consistent in both histologic subgroups (squamous-cell carcinoma and adenocarcinoma [including adenosquamous carcinoma]). Progression-free survival was also longer in the cemiplimab group than in the chemotherapy group in the overall population (hazard ratio for disease progression or death, 0.75; 95% CI, 0.63 to 0.89; two-sided P<0.001). In the overall population, an objective response occurred in 16.4% (95% CI, 12.5 to 21.1) of the patients in the cemiplimab group, as compared with 6.3% (95% CI, 3.8 to 9.6) in the chemotherapy group. An objective response occurred in 18% (95% CI, 11 to 28) of the cemiplimab-treated patients with PD-L1 expression greater than or equal to 1% and in 11% (95% CI, 4 to 25) of those with PD-L1 expression of less than 1%. Overall, grade 3 or higher adverse events occurred in 45.0% of the patients who received cemiplimab and in 53.4% of those who received chemotherapy. CONCLUSIONS: Survival was significantly longer with cemiplimab than with single-agent chemotherapy among patients with recurrent cervical cancer after first-line platinum-containing chemotherapy. (Funded by Regeneron Pharmaceuticals and Sanofi; EMPOWER-Cervical 1/GOG-3016/ENGOT-cx9 ClinicalTrials.gov number, NCT03257267.).

Neoadjuvant cemiplimab for resectable hepatocellular carcinoma: a single-arm, open-label, phase 2 trial.

BACKGROUND: Surgical resection of early stage hepatocellular carcinoma is standard clinical practice; however, most tumours recur despite surgery, and no perioperative intervention has shown a survival benefit. Neoadjuvant immunotherapy has induced pathological responses in multiple tumour types and might decrease the risk of postoperative recurrence in hepatocellular carcinoma. We aimed to evaluate the clinical activity of neoadjuvant cemiplimab (an anti-PD-1) in patients with resectable hepatocellular carcinoma. METHODS: For this single-arm, open-label, phase 2 trial, patients with resectable hepatocellular carcinoma (stage Ib, II, and IIIb) were enrolled and received two cycles of neoadjuvant cemiplimab 350 mg intravenously every 3 weeks followed by surgical resection. Eligible patients were aged 18 years or older, had confirmed resectable hepatocellular carcinoma, an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate liver function. Patients were excluded if they had metastatic disease, if the surgery was not expected to be curative, if they had a known additional malignancy requiring active treatment, or if they required systemic steroid treatment or any other immunosuppressive therapy. After resection, patients received an additional eight cycles of cemiplimab 350 mg intravenously every 3 weeks in the adjuvant setting. The primary endpoint was significant tumour necrosis on pathological examination (defined as >70% necrosis of the resected tumour). Secondary endpoints included delay of surgery, the proportion of patients with an overall response, change in CD8+ T-cell density, and adverse events. Tumour necrosis and response were analysed in all patients who received at least one dose of cemiplimab and completed surgical resection; safety and other endpoints were analysed in the intention-to-treat population. Patients underwent pre-treatment biopsies and blood collection throughout treatment. This trial is registered with ClinicalTrials.gov (NCT03916627, Cohort B) and is ongoing. FINDINGS: Between Aug 5, 2019, and Nov 25, 2020, 21 patients were enrolled. All patients received neoadjuvant cemiplimab, and 20 patients underwent successful resection. Of the 20 patients with resected tumours, four (20%) had significant tumour necrosis. Three (15%) of 20 patients had a partial response, and all other patients maintained stable disease. 20 (95%) patients had a treatment-emergent adverse event of any grade during the neoadjuvant treatment period. The most common adverse events of any grade were increased aspartate aminotransferase (in four patients), increased blood creatine phosphokinase (in three), constipation (in three), and fatigue (in three). Seven patients had grade 3 adverse events, including increased blood creatine phosphokinase (in two patients) and hypoalbuminaemia (in one). No grade 4 or 5 events were observed. One patient developed pneumonitis, which led to a delay in surgery by 2 weeks. INTERPRETATION: This report is, to our knowledge, the largest clinical trial of a neoadjuvant anti-PD-1 monotherapy reported to date in hepatocellular carcinoma. The observed pathological responses to cemiplimab in this cohort support the design of larger trials to identify the optimal treatment duration and definitively establish the clinical benefit of preoperative PD-1 blockade in patients with hepatocellular carcinoma. FUNDING: Regeneron Pharmaceuticals.

Cemiplimab in locally advanced basal cell carcinoma after hedgehog inhibitor therapy: an open-label, multi-centre, single-arm, phase 2 trial.

BACKGROUND: Before February, 2021, there was no standard treatment regimen for locally advanced basal cell carcinoma after first-line hedgehog inhibitor (HHI) therapy. Cemiplimab, a PD-1 antibody, is approved for treatment of advanced cutaneous squamous cell carcinoma and has shown clinical activity as monotherapy in first-line non-small-cell lung cancer. Here, we present the primary analysis data of cemiplimab in patients with locally advanced basal cell carcinoma after HHI therapy. METHODS: We did an open-label, multicentre, single-arm, phase 2 trial across 38 outpatient clinics, primarily at academic medical centres, in Canada, Europe, and the USA. Eligible patients (aged ≥18 years and with an Eastern Cooperative Oncology Group performance status of 0 or 1) with a histologically confirmed diagnosis of metastatic basal cell carcinoma (group 1) or locally advanced basal cell carcinoma (group 2) who had progressed on or were intolerant to previous HHI therapy were enrolled. Patients were not candidates for further HHI therapy due to progression of disease on or intolerance to previous HHI therapy or having no better than stable disease after 9 months on HHI therapy. Patients received cemiplimab 350 mg intravenously every 3 weeks for up to 93 weeks or until progression or unacceptable toxicity. The primary endpoint was objective response by independent central review. Analyses were done as per the intention-to-treat principle. The safety analysis comprised all patients who received at least one dose of cemiplimab. The primary analysis is reported only for group 2; group 1 data have not reached maturity and will be reported when the timepoint, according to the statistical analysis plan, has been reached. This study is registered with ClinicalTrials.gov, NCT03132636, and is no longer recruiting new participants. FINDINGS: Between Nov 16, 2017, and Jan 7, 2019, 84 patients were enrolled and treated with cemiplimab. At data cutoff on Feb 17, 2020, median duration of follow-up was 15 months (IQR 8-18). An objective response per independent central review was observed in 26 (31%; 95% CI 21-42) of 84 patients, including two partial responses that emerged at tumour assessments before the data cutoff and were confirmed by tumour assessments done subsequent to the data cutoff. The best overall response was five (6%) patients with a complete response and 21 (25%) with a partial response. Grade 3-4 treatment-emergent adverse events occurred in 40 (48%) of 84 patients; the most common were hypertension (four [5%] of 84 patients) and colitis (four [5%]). Serious treatment-emergent adverse events occurred in 29 (35%) of 84 patients. There were no treatment-related deaths. INTERPRETATION: Cemiplimab exhibited clinically meaningful antitumour activity and an acceptable safety profile in patients with locally advanced basal cell carcinoma after HHI therapy. FUNDING: Regeneron Pharmaceuticals and Sanofi.

Phase 2 study of cemiplimab in patients with metastatic cutaneous squamous cell carcinoma: primary analysis of fixed-dosing, long-term outcome of weight-based dosing.

BACKGROUND: Cemiplimab, a high-affinity, potent human immunoglobulin G4 monoclonal antibody to programmed cell death-1 demonstrated antitumor activity in a Phase 1 advanced cutaneous squamous cell carcinoma (CSCC) expansion cohort (NCT02383212) and the pivotal Phase 2 study (NCT02760498). Here we report the primary analysis of fixed dose cemiplimab 350 mg intravenously every 3 weeks (Q3W) (Group 3) and provide a longer-term update after the primary analysis of weight-based cemiplimab 3 mg/kg intravenously every 2 weeks (Q2W) (Group 1) among metastatic CSCC (mCSCC) patients in the pivotal study (NCT02760498). METHODS: The primary objective for each group was objective response rate (ORR) per independent central review (ICR). Secondary endpoints included ORR by investigator review (INV), duration of response (DOR) per ICR and INV, and safety and tolerability. RESULTS: For Group 3 (n=56) and Group 1 (n=59), median follow-up was 8.1 (range, 0.6 to 14.1) and 16.5 (range, 1.1 to 26.6) months, respectively. ORR per ICR was 41.1% (95% CI, 28.1% to 55.0%) in Group 3, 49.2% (95% CI, 35.9% to 62.5%) in Group 1, and 45.2% (95% CI, 35.9% to 54.8%) in both groups combined. Per ICR, Kaplan-Meier estimate for DOR at 8 months was 95.0% (95% CI, 69.5% to 99. 3%) in responding patients in Group 3, and at 12 months was 88.9% (95% CI, 69.3% to 96.3%) in responding patients in Group 1. Per INV, ORR was 51.8% (95% CI, 38.0% to 65.3%) in Group 3, 49.2% (95% CI, 35.9% to 62.5%) in Group 1, and 50.4% (95% CI, 41.0% to 59.9%) in both groups combined. Overall, the most common adverse events regardless of attribution were fatigue (27.0%) and diarrhea (23.5%). CONCLUSION: In patients with mCSCC, cemiplimab 350 mg intravenously Q3W produced substantial antitumor activity with durable response and an acceptable safety profile. Follow-up data of cemiplimab 3 mg/kg intravenously Q2W demonstrate ongoing durability of responses. TRIAL REGISTRATION NUMBER: Clinicaltrials.gov, NCT02760498. Registered May 3, 2016, https://clinicaltrials.gov/ct2/show/NCT02760498.

Predictors of antibiotic utilization among intensive care unit patients.

Increased antibiotic utilization in hospital is linked to higher total treatment costs, together with increased length of stay, surgery and emergency admission. The aim of our retrospective cohort study was to investigate predictors of antibiotic utilization per single patient from an intensive care unit (ICU) of a tertiary care, university hospital in Serbia. Average utilization of antibiotics per patient was 23.9 ± 20.4 defined daily doses (DDDs). Diagnosis of systemic infection increased antibiotics utilization per patient for 10.0 DDDs, positive blood culture for 5.4 DDDs, isolation of Pseudomonas spp. for 19.5 DDDs, isolation of Acinetobacter spp. for 6.3 DDDs and injury for 7.3 DDDs per patient. Each new day of hospitalization and each additional drug prescribed increased utilization for further 0.3 DDDs and 1.2 DDDs, respectively. Appropriate and limited use of antibiotics in ICU is of key importance for preserving their effectiveness and decrease of bacterial resistance.

Cemiplimab in locally advanced cutaneous squamous cell carcinoma: results from an open-label, phase 2, single-arm trial.

BACKGROUND: Cemiplimab has shown substantial antitumour activity in patients with metastatic cutaneous squamous cell carcinoma. Patients with locally advanced cutaneous squamous cell carcinoma have poor prognosis with conventional systemic therapy. We present a primary analysis of the safety and antitumour activity of cemiplimab in patients with locally advanced cutaneous squamous cell carcinoma. METHODS: This pivotal open-label, phase 2, single-arm trial was done across 25 outpatient clinics, primarily at academic medical centres, in Australia, Germany, and the USA. Eligible patients (aged ≥18 years with histologically confirmed locally advanced cutaneous squamous cell carcinoma and an Eastern Cooperative Oncology Group performance status of 0-1) received cemiplimab 3 mg/kg intravenously over 30 min every 2 weeks for up to 96 weeks. Tumour measurements were done every 8 weeks. The primary endpoint was objective response, defined as the proportion of patients with complete or partial response, according to independent central review as per Response Evaluation Criteria in Solid Tumors version 1.1 for radiological scans and WHO criteria for medical photography. Data cutoff was Oct 10, 2018, when the fully enrolled cohort reached the prespecified timepoint for the primary analysis. Analyses were done as per the intention-to-treat principle. The safety analysis comprised all patients who received at least one dose of cemiplimab. This study is registered with ClinicalTrials.gov, number NCT02760498. FINDINGS: Between June 14, 2016, and April 25, 2018, 78 patients were enrolled and treated with cemiplimab. The median duration of study follow-up was 9·3 months (IQR 5·1-15·7) at the time of data cutoff. An objective response was observed in 34 (44%; 95% CI 32-55) of 78 patients. The best overall response was ten (13%) patients with a complete response and 24 (31%) with a partial response. Grade 3-4 treatment-emergent adverse events occurred in 34 (44%) of 78 patients; the most common were hypertension in six (8%) patients and pneumonia in four (5%). Serious treatment-emergent adverse events occurred in 23 (29%) of 78 patients. One treatment-related death was reported that occurred after onset of aspiration pneumonia. INTERPRETATION: Cemiplimab showed antitumour activity and an acceptable safety profile in patients with locally advanced cutaneous squamous cell carcinoma for whom there was no widely accepted standard of care. FUNDING: Regeneron Pharmaceuticals and Sanofi.

Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory phenotype.

Most patients with cancer do not develop durable antitumor responses after programmed cell death protein 1 (PD-1) or programmed cell death ligand 1(PD-L1) checkpoint inhibition monotherapy because of an ephemeral reversal of T cell dysfunction and failure to promote long-lasting immunological T cell memory. Activating costimulatory pathways to induce stronger T cell activation may improve the efficacy of checkpoint inhibition and lead to durable antitumor responses. We performed single-cell RNA sequencing of more than 2000 tumor-infiltrating CD8+ T cells in mice receiving both PD-1 and GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) antibodies and found that this combination synergistically enhanced the effector function of expanded CD8+ T cells by restoring the balance of key homeostatic regulators CD226 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), leading to a robust survival benefit. Combination therapy decreased CD8+ T cell dysfunction and induced a highly proliferative precursor effector memory T cell phenotype in a CD226-dependent manner. PD-1 inhibition rescued CD226 activity by preventing PD-1-Src homology region 2 (SHP2) dephosphophorylation of the CD226 intracellular domain, whereas GITR agonism decreased TIGIT expression. Unmasking the molecular pathways driving durable antitumor responses will be essential to the development of rational approaches to optimizing cancer immunotherapy.

Human Placenta-Derived Mesenchymal Stromal-Like Cells Enhance Angiogenesis via T Cell-Dependent Reprogramming of Macrophage Differentiation.

Peripheral arterial disease (PAD) is a leading cause of limb loss and mortality worldwide with limited treatment options. Mesenchymal stromal cell (MSC) therapy has demonstrated positive effects on angiogenesis in preclinical models and promising therapeutic efficacy signals in early stage clinical studies; however, the mechanisms underlying MSC-mediated angiogenesis remain largely undefined. Here, we investigated the mechanism of action of human placenta-derived MSC-like cells (PDA-002) in inducing angiogenesis using mice hind limb ischemia model. We showed that PDA-002 improved blood flow and promoted collateral vessel formation in the injured limb. Histological analysis demonstrated that PDA-002 increased M2-like macrophages in ischemic tissue. Analysis of the changes in functional T cell phenotype in the draining lymph nodes revealed that PDA-002 treatment was associated with the induction of cytokine and gene expression signatures of Th2 response. Angiogenic effect of PDA-002 was markedly reduced in Balb/c nude mice compared with wild type. This reduction in efficacy was reversed by T cell reconstitution, suggesting T cells are essential for PDA-002-mediated angiogenesis. Furthermore, effect of PDA-002 on macrophage differentiation was also T cell-dependent as a PDA-002-mediated M2-like macrophage skewing was only observed in wild type and T cell reconstituted nude mice, but not in nude mice. Finally, we showed that PDA-002-treated animals had enhanced angiogenic recovery in response to the second injury when PDA-002 no longer persisted in vivo. These results suggest that PDA-002 enhances angiogenesis through an immunomodulatory mechanism involving T cell-dependent reprogramming of macrophage differentiation toward M2-like phenotype. Stem Cells 2017;35:1603-1613.

Safety and efficacy of intramuscular human placenta-derived mesenchymal stromal-like cells (cenplacel [PDA-002]) in patients who have a diabetic foot ulcer with peripheral arterial disease.

The objective of this study was to examine the safety of cenplacel (PDA-002) in patients with peripheral arterial disease (PAD) and a diabetic foot ulcer (DFU). Cenplacel is a mesenchymal-like cell population derived from full-term human placenta. This phase 1, dose-escalation study investigated cenplacel in diabetic patients with chronic DFUs (Wagner grade 1 or grade 2) and PAD [ankle-brachial index (ABI) >0·5 and ≤0·9], enrolled sequentially into each of four dose cohorts (3 × 106 , 10 × 106 , 30 × 106 and 100 × 106 cells; administered intramuscularly on study days 1 and 8 in combination with standard of care). Overall, cenplacel was well tolerated in all 15 patients in the study. Before enrollment, nine patients had an ulcer for ≥6 months and 11 had an ABI of 0·7-0·85. No patient met dose-limiting toxicity criteria and no treatment-related serious adverse events were reported. There was preliminary evidence of ulcer healing in seven patients (five complete; two partial) within 3 months of cenplacel treatment, and circulating endothelial cell levels (a biomarker of vascular injury in PAD) were decreased within 1 month. Cenplacel was generally safe and well tolerated in patients with chronic DFUs and PAD. Outcomes from this study informed the doses, endpoints, biomarkers and patient population for an ongoing phase 2 trial.

Cyclin C regulates human hematopoietic stem/progenitor cell quiescence.

Hematopoietic stem cells (HSCs) can remain quiescent or they can enter the cell cycle, and either self-renew or differentiate. Although cyclin C and cyclin dependent kinase (cdk3) are essential for the transition from the G(0) to the G(1) phase of the cell cycle in human fibroblasts, the role of cyclin C in hematopoietic stem/progenitor cells (HSPCs) is not clear. We have identified an important role of cyclin C (CCNC) in regulating human HSPC quiescence, as knocking down CCNC expression in human cord blood CD34(+) cells resulted in a significant increase in quiescent cells that maintain CD34 expression. CCNC knockdown also promotes in vitro HSPC expansion and enhances their engraftment potential in sublethally irradiated immunodeficient mice. Our studies establish cyclin C as a critical regulator of the G(0)/G(1) transition of human HSPCs and suggest that modulating cyclin C levels may be useful for HSC expansion and more efficient engraftment.

Hedgehog signaling is dispensable for adult hematopoietic stem cell function.

The Hedgehog (Hh) signaling pathway is a developmentally conserved regulator of stem cell function. Several reports suggested that Hh signaling is an important regulator of hematopoietic stem cell (HSC) maintenance and differentiation. Here we test this hypothesis in vivo using both gain- and loss-of-function Hh genetic models. Surprisingly, our studies demonstrate that conditional Smoothened (Smo) deletion or overactivation has no significant effects on adult HSC self-renewal and function. Moreover, they indicate a lack of synergism between the Notch and Hh pathways in HSC function, as compound RBPJ and Smo deficiency does not affect hematopoiesis. In agreement with this notion, detailed genome-wide transcriptome analysis reveals that silencing of Hh signaling does not significantly alter the HSC-specific gene expression "signature." Our studies demonstrate that the Hh signaling pathway is dispensable for adult HSC function and suggest that Hh inhibition on leukemia-initiating cell maintenance can be targeted in future clinical trials.

Control of hematopoietic stem cell quiescence by the E3 ubiquitin ligase Fbw7.

Ubiquitination is a posttranslational mechanism that controls diverse cellular processes. We focus here on the ubiquitin ligase Fbw7, a recently identified hematopoietic tumor suppressor that can target for degradation several important oncogenes, including Notch1, c-Myc, and cyclin E. We have generated conditional Fbw7 knockout animals and inactivated the gene in hematopoietic stem cells (HSCs), progenitors, and their differentiated progeny. Deletion of Fbw7 specifically and rapidly affects hematopoiesis in a cell-autonomous manner. Fbw7(-/-) HSCs show defective maintenance of quiescence, leading to impaired self-renewal and a severe loss of competitive repopulating capacity. Furthermore, Fbw7(-/-) progenitors are unable to colonize the thymus, leading to a profound depletion of T cell progenitors. Deletion of Fbw7 in bone marrow (BM) stem cells and progenitors leads to the stabilization of c-Myc, a transcription factor previously implicated in HSC self-renewal. On the other hand, neither Notch1 nor cyclin E is visibly stabilized in the BM of Fbw7-deficient mice. Gene expression studies of Fbw7(-/-) HSCs and hematopoietic progenitors indicate that Fbw7 regulates, through the regulation of HSC cycle entry, the transcriptional "signature" that is associated with the quiescent, self-renewing HSC phenotype.

Methylation of RUNX1 by PRMT1 abrogates SIN3A binding and potentiates its transcriptional activity.

RUNX1/AML1 is required for the development of definitive hematopoiesis, and its activity is altered by mutations, deletions, and chromosome translocations in human acute leukemia. RUNX1 function can be regulated by post-translational modifications and protein-protein interactions. We show that RUNX1 is arginine-methylated in vivo by the arginine methyltransferase PRMT1, and that PRMT1 serves as a transcriptional coactivator for RUNX1 function. Using mass spectrometry, and a methyl-arginine-specific antibody, we identified two arginine residues (R206 and R210) within the region of RUNX1 that interact with the corepressor SIN3A and are methylated by PRMT1. PRMT1- dependent methylation of RUNX1 at these arginine residues abrogates its association with SIN3A, whereas shRNA against PRMT1 (or use of a methyltransferase inhibitor) enhances this association. We find arginine-methylated RUNX1 on the promoters of two bona fide RUNX1 target genes, CD41 and PU.1 and show that shRNA against PRMT1 or RUNX1 down-regulates their expression. These arginine methylation sites and the dynamic regulation of corepressor binding are lost in the leukemia-associated RUNX1-ETO fusion protein, which likely contributes to its dominant inhibitory activity.

Id1 restrains p21 expression to control endothelial progenitor cell formation.

Loss of Id1 in the bone marrow (BM) severely impairs tumor angiogenesis resulting in significant inhibition of tumor growth. This phenotype has been associated with the absence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of Id1 mutant mice. However, the manner in which Id1 loss in the BM controls EPC generation or mobilization is largely unknown. Using genetically modified mouse models we demonstrate here that the generation of EPCs in the BM depends on the ability of Id1 to restrain the expression of its target gene p21. Through a series of cellular and functional studies we show that the increased myeloid commitment of BM stem cells and the absence of EPCs in Id1 knockout mice are associated with elevated p21 expression. Genetic ablation of p21 rescues the EPC population in the Id1 null animals, re-establishing functional BM-derived angiogenesis and restoring normal tumor growth. These results demonstrate that the restraint of p21 expression by Id1 is one key element of its activity in facilitating the generation of EPCs in the BM and highlight the critical role these cells play in tumor angiogenesis.