Cross-presentation of disialoganglioside GD3 to natural killer T cells.

GD3, a ganglioside expressed on human melanoma, can be recognized by the humoral immune system. In this paper, we demonstrate that immunizing mice with the human melanoma cell line SK-MEL-28 (GD3+ GM2- CD1-) or with syngeneic APCs loaded with GD3 can induce a GD3-reactive natural killer T (NKT) cell response. GD3-reactive NKT cells were detected among splenocytes of immunized mice at frequencies of approximately 1:2000 both by ELISPOT and GD3-loaded mouse CD1d tetramer analysis. GD3-reactive NKT cells did not react with GM2, a closely related ganglioside, and were not detectable in unimmunized mice. GD3-reactive NKT cells initially produced IL-4 and IFN-gamma followed by IL-10. They were CD1d restricted in that reactivity was abrogated when APCs were blocked with anti-CD1d monoclonal antibody before being loaded with GD3 or when APCs from CD1d knockout mice were used. Because SK-MEL-28 does not express any isoform of human CD1, GD3 must be cross-presented by murine APCs in vivo. This is the first analysis of a natural ligand for mouse NKT cells and the first definitive paper of cross-presentation to NKT cells. This could be a mechanism for NKT cell recognition of tumor gangliosides in CD1- tumors.

Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.

GD3, a ganglioside expressed on melanoma, is the only tumour-associated glycolipid described to date that can induce a CD1d-restricted natural killer T (NKT)-cell response. We analysed the fine specificity of GD3-reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3-reactive NKT cells. One population was CD4+ CD8- and was specific for GD3; the other population was CD4- CD8- and cross-reacted with GM3 in a CD1d-restricted manner, but did not cross-react with GM2, GD2, or lactosylceramide. This indicated that the T-cell receptors reacting with GD3 recognize glucose-galactose linked to at least one N-acetyl-neuraminic acid but will not accommodate a terminal N-acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT-cell response. Coimmunization of GM3-loaded antigen-presenting cells (APCs) with GD3-loaded APCs suppressed the NKT-cell response to GD3 in a CD1d-restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2-4 days. In vitro, GM3-loaded APCs also suppressed the interleukin-4 response, but not the interferon-gamma response, of NKT cells to alpha-galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2-like NKT-cell responses.

PD-L1 Expression and Survival among Patients with Advanced Non-Small Cell Lung Cancer Treated with Chemotherapy.

BACKGROUND: Recent clinical trial results have suggested that programmed cell death ligand 1 (PD-L1) expression measured by immunohistochemistry may predict response to anti-programmed cell death 1 (PD-1) therapy. Results on the association between PD-L1 expression and survival among patients with advanced non-small cell lung cancer (NSCLC) treated with chemotherapy are inconsistent. MATERIAL AND METHODS: We evaluated the relationship between PD-L1 expression and overall survival (OS) among 204 patients with advanced NSCLC treated at Aarhus University Hospital, Aarhus, Denmark, from 2007 to 2012. PD-L1 expression was measured using a prototype immunohistochemistry assay with the anti-PD-L1 22C3 antibody (Merck). PD-L1 strong positivity and weak positivity were defined to be traceable to the clinical trial version of the assay. RESULTS: Twenty-five percent of patients had PD-L1 strong-positive tumors, and 50% had PD-L1 weak-positive tumors. No statistically significant association was found between PD-L1 expression and survival; adjusted hazard ratio of 1.34 (95% confidence interval, 0.88-2.03; median OS, 9.0 months) for the PD-L1 strong-positive group and 1.07 (0.74-1.55; median OS, 9.8 months) for the PD-L1 weak-positive group compared with the PD-L1-negative group (median OS, 7.5 months). No association was seen between PD-L1 expression and OS when PD-L1 expression levels were stratified by median or tertiles. CONCLUSIONS: In concordance with previous studies, we found PD-L1 measured by immunohistochemistry to be frequently expressed in patients with advanced NSCLC. However, PD-L1 expression is not a strong prognostic marker in patients with advanced NSCLC treated with chemotherapy.

Development of a Prototype Immunohistochemistry Assay to Measure Programmed Death Ligand-1 Expression in Tumor Tissue.

CONTEXT: - With the abundance of therapeutics targeted against programmed death receptor-1 and its ligand (PD-L1) that are currently approved or in clinical development, there is interest in identifying those patients most likely to respond to these drugs. Expression of PD-L1 may be an indicator of an initial and robust inflammatory response to the presence of tumor cells. Therefore, tumors that express PD-L1 may be the most likely to respond to therapies that interrupt the negative feedback mechanism that leads to PD-L1 upregulation. OBJECTIVE: - To develop a prototype immunohistochemistry assay using the anti-PD-L1 antibody clone 22C3. DESIGN: - The assay was developed and optimized using commercially available reagents and archival tumor-bank tissue. RESULTS: - The optimized immunohistochemistry method had high precision and reproducibility. Using the prototype assay in 142 non-small cell lung cancer and 79 melanoma archival tumor-bank tissue samples, PD-L1 staining was observed at the plasma membrane of nucleated tumor and nontumor cells and, in some cases, as a distinct lichenoid pattern at the tumor-stroma border. Using a preliminary scoring method, 56% (80 of 142) of non-small cell lung cancer and 53% (42 of 79) of melanoma samples were defined as PD-L1+ based on a modified H-score of 1 or more or the presence of a distinctive staining pattern at the tumor-stroma interface. CONCLUSIONS: - The immunohistochemistry assay using the anti-PD-L1 antibody 22C3 merits further investigation in clinical trials and prevalence assessments to further understand the prognostic and predictive value of PD-L1 expression in cancer.

Prognostic Significance of PD-L1 in Patients with Non-Small Cell Lung Cancer: A Large Cohort Study of Surgically Resected Cases.

INTRODUCTION: The aim of our analysis was to evaluate the prognostic effect of programmed cell death ligand-1 (PD-L1) expression in patients with non-small cell lung cancer (NSCLC). METHODS: PD-L1 expression among 1070 surgically resected NSCLC specimens was evaluated by immunohistochemical analysis. Data were analyzed using Cox proportional hazard models adjusting for age, sex, smoking status, histologic type, stage, and performance status. RESULTS: Sixty-eight patients (6%) were strongly PD-L1 positive and 410 (38%) were weakly PD-L1 positive. A significantly higher prevalence of PD-L1 positivity was observed among patients with squamous cell carcinoma and among stage IIIB and IV patients. PD-L1 expression may be associated with poorer overall survival, with an adjusted hazard ratio of 1.56 (95% confidence interval [CI]: 1.08-2.26, p = 0.02) for strong PD-L1 positivity, 1.18 (95% CI: 0.96-1.46; p = 0.12) for weak PD-L1 positivity, and 1.23 (95% CI: 1.00-1.51; p = 0.05) for the combined strongly and weakly positive groups compared with PD-L1 negativity. Negative prognostic effect of PD-L1 expression was not statistically significant after adjustment for postoperative chemotherapy or radiotherapy. Similar results were observed for progression-free survival. Among stage I patients, the disease recurrence rate was higher in the PD-L1-positive versus in the PD-L1-negative group (48% versus 27%, p < 0.001), with an adjusted hazard ratio for disease-free survival of 2.01 (95% CI, 1.08-3.73; p = 0.03) for strong PD-L1 positivity and 1.57 (95% CI, 1.17-2.11; p = 0.003) for weak PD-L1 positivity compared with PD-L1 negativity. CONCLUSIONS: Tumor PD-L1 expression may be associated with poor prognosis in patients with NSCLC, although its significance weakens when postoperative therapy is considered.

Sequential immunization of melanoma patients with GD3 ganglioside vaccine and anti-idiotypic monoclonal antibody that mimics GD3 ganglioside.

GD3 ganglioside is an attractive target for immunotherapy of melanoma because it is abundantly expressed on all melanomas but not expressed on most normal tissues. Although GD3 has proven to be one of the least immunogenic gangliosides, our recent studies showed that anti-GD3 antibodies can be induced in patients immunized either with GD3-lactone-KLH (GD3-L-KLH) plus QS-21 adjuvant or with BEC2 anti-idiotypic monoclonal antibody vaccine, which mimics GD3, plus Bacillus Calmette-Guérin. We compared the immunogenicity of these two vaccines and tested whether one vaccine could prime an antibody response to the other. This is the first clinical trial immunizing patients with both antigen and anti-idiotypic monoclonal antibody vaccine. Twenty-four melanoma patients were randomized to be immunized with either BEC2 followed by GD3-L-KLH or in the opposite order. Our prior study suggested that a 25-microg dose of BEC2 was more immunogenic than our standard dose of 2.5 mg and therefore was used in this trial. Overall, 10 of 24 patients (42%) developed anti-GD3 antibodies detectable by ELISA, five in each cohort. All antibody responses were in response to the GD3-L-KLH vaccine. We found no evidence of priming by either vaccine. Antibody responses did not correlate with survival outcomes. Cellular responses were detected by enzyme-linked immunospot against BEC2, Bacillus Calmette-Guérin, and KLH, but not against GD3. We confirmed that GD3-L-KLH vaccine induces anti-GD3 antibodies, but we were unable to confirm our previous finding that a 25-microg dose of BEC2 is immunogenic. Future multivalent ganglioside vaccines should include the GD3-L-KLH vaccine.

Phase I Study of Pembrolizumab (MK-3475; Anti-PD-1 Monoclonal Antibody) in Patients with Advanced Solid Tumors.

PURPOSE: This phase I study evaluated the safety, maximum tolerated dose, antitumor activity, and pharmacokinetics and pharmacodynamics of pembrolizumab in patients with advanced solid tumors. EXPERIMENTAL DESIGN: In a 3 + 3 dose escalation study, 10 patients received pembrolizumab 1, 3, or 10 mg/kg intravenously every 2 weeks until progression or intolerable toxicity. Seven additional patients received 10 mg/kg every 2 weeks. Thirteen patients participated in a 3-week intrapatient dose escalation (dose range, 0.005-10 mg/kg) followed by 2 or 10 mg/kg every 3 weeks. Tumor response was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. RESULTS: No dose-limiting toxicities were observed. Maximum administered dose was 10 mg/kg every 2 weeks. One patient with melanoma and one with Merkel cell carcinoma experienced complete responses of 57 and 56+ weeks' duration, respectively. Three patients with melanoma experienced partial responses. Fifteen patients with various malignancies experienced stable disease. One patient died of cryptococcal infection 92 days after pembrolizumab discontinuation, following prolonged corticosteroid use for grade 2 gastritis considered drug related. Pembrolizumab exhibited pharmacokinetic characteristics typical of humanized monoclonal antibodies. Maximum serum target engagement was reached with trough levels of doses greater than or equal to 1 mg/kg every 3 weeks. Mechanism-based translational models with a focus on intratumor exposure prediction suggested robust clinical activity would be observed at doses ≥2 mg/kg every 3 weeks. CONCLUSIONS: Pembrolizumab was well tolerated and associated with durable antitumor activity in multiple solid tumors. The lowest dose with full potential for antitumor activity was 2 mg/kg every 3 weeks.

Pharmacokinetics, pharmacodynamics, and immunogenicity of belatacept in adult kidney transplant recipients.

BACKGROUND AND OBJECTIVES: Belatacept is a first-in-class, selective co-stimulation blocker recently approved for the prophylaxis of organ rejection in adult kidney transplant recipients. The objective of this study was to report the pharmacokinetics, pharmacodynamics, and immunogenicity of belatacept. METHODS: The pharmacokinetics, pharmacodynamics (CD86 receptor occupancy), and immunogenicity of belatacept were studied in de novo adult kidney transplant recipients in phase II and III clinical studies. RESULTS: Following multiple doses of 5 or 10 mg/kg, the geometric mean (percentage coefficient of variation) maximum serum concentration and area under the serum concentration-time curve over one dosing interval of belatacept were 136 (20%) and 238 (27%) µg/mL, and 13,587 (27%) and 21,241 (35%) µg·h/mL, respectively. The median belatacept elimination half-life was 8-9 days. Belatacept exhibited concentration-dependent binding to CD86 receptors. The pre-dose CD86 receptor occupancy by belatacept decreased from 94 to 65% between day 5 and 1 year post-transplant, with corresponding pre-dose trough serum concentrations of belatacept decreasing from ~35 to 4 µg/mL during this period. The cumulative incidence of developing anti-belatacept antibodies was 5.3% up to 3 years post-transplant and had no impact on belatacept exposure. CONCLUSIONS: Belatacept in adult kidney transplant demonstrated linear pharmacokinetics with low variability, concentration-dependent pharmacodynamics, and a low incidence of anti-drug antibodies.

Ipilimumab increases activated T cells and enhances humoral immunity in patients with advanced melanoma.

Ipilimumab, a fully human monoclonal antibody, which blocks cytotoxic T-lymphocyte antigen-4, has demonstrated an improvement in overall survival in 2 phase III trials of patients with advanced melanoma. To gain an understanding of its mechanism of action, the effects of ipilimumab on T-cell populations and on humoral immune responses were studied in patients with advanced melanoma from 2 phase II trials. Antibody levels against 5 tumor antigens were assessed at baseline and up to 12 weeks after ipilimumab treatment. Serologic reactivity to the cancer-testis antigen NY-ESO-1 increased by at least 5-fold at week 12 of treatment in 10% to 13% of patients. Increased antibody levels were also observed to the tumor antigens Melan-A, MAGE-A4, SSX2, and p53. Immunocompetence was evaluated with tetanus boosters administered before ipilimumab and pneumococcal and influenza vaccines given 5 days after ipilimumab treatment. At week 7, most patients who received ipilimumab and vaccine showed greater humoral responses relative to baseline titers. For peripheral T-cell populations, statistically significant increases in the percent of activated (HLA-DR) CD4 and CD8 T cells with concomitant decreases in naive CD4 and CD8 T cells were observed after ipilimumab treatment. These changes were evident by week 4 of treatment. Increases were also observed in central memory, effector memory, and activated ICOS CD4 T cells, but not in ICOS CD8 T cells or in FoxP3 CD4 regulatory T cells. These results suggest that ipilimumab can enhance immune responses mediated by different T-cell populations, and humoral immunity, in melanoma patients.

Recommendations for the validation of flow cytometric testing during drug development: II assays.

Flow cytometry-based assays serve as valuable tools for various aspects of the drug development process ranging from target discovery and characterization to evaluation of responses in a clinical setting. The integrity of the samples and the appropriate selection and characterization of the reagents used in these assays are in themselves challenging. These concerns taken together with the flow-based technology makes the validation of flow cytometry assays a challenging effort. Therefore, apart from summarizing the role of flow cytometry technology in various stages of drug development, this manuscript focuses on recommendations for the validation of methods applying flow cytometry. Information is also provided on the relevant validation parameters for different types of flow cytometry assays to guide the users of this platform. Together, the recommendations and the information on regulatory guidelines provided in this manuscript represent the consensus of all the authors and can assist the flow cytometry user in implementing the appropriate method validation strategies.

Development and validation of flow cytometry methods for pharmacodynamic clinical biomarkers.

Flow cytometry is a powerful analytical tool for the analysis of multiple biological parameters of individual cells or particles within heterogeneous cell populations. It has been widely used in biomedical research to perform immunophenotyping, cell counting and numerous cell function assessments, such as intracellular cytokine production, protein phosphorylation, cell proliferation and apoptosis. The implementation of standardized flow cytometry-based biomarker assays in clinical trials remains a challenge due to the limited stability of clinical specimens and the technical variations between instruments. To ensure data quality, it is crucial to develop robust assays for clinical applications. In this review, we summarize current practice in developing, validating and implementing flow cytometry assays to evaluate biomarkers in clinical research.