Development of a Diagnostic Programmed Cell Death 1-Ligand 1 Immunohistochemistry Assay for Nivolumab Therapy in Melanoma.

Nivolumab is a monoclonal antibody that blocks the interaction between programmed cell death 1 (PD1) and programmed cell death 1-ligand 1 (PD-L1), resulting in enhanced antitumor activity by the immune system. Nivolumab is currently approved by the US Food and Drug Administration (FDA) for melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, and urothelial carcinoma. PD-L1 IHC 28-8 pharmDx is FDA-approved as a complementary diagnostic for immunohistochemical (IHC) detection of PD-L1 in non-squamous NSCLC and melanoma. We report validation of PD-L1 IHC 28-8 pharmDx for PD-L1 detection on formalin-fixed, paraffin-embedded human melanoma specimens using Autostainer Link 48. A prevalence assessment of 104 melanoma specimens indicated that PD-L1 was detected across the full expression level range (0% to 100% of tumor cells). Assay robustness and precision studies were conducted at Agilent Technologies, with additional reproducibility studies performed at 3 external laboratories. Precision studies evaluated at ≥1% and ≥5% expression levels revealed a range of average negative agreement from 89.5%, 95% CI (83.2, 93.6) to 100%, 95% CI (97.3, 100), and average positive agreement from 85.5%, 95% CI (77.6, 90.9) to 100%, 95% CI (97.9, 100). For external reproducibility, precise results were obtained. These results demonstrate PD-L1 IHC 28-8 pharmDx is a precise, robust, and reproducible assay for determining PD-L1 expression in melanoma. This is the first PD-L1 IHC test to receive FDA approval as a complementary diagnostic in melanoma patients whereby positive PD-L1 expression is correlated with the magnitude of nivolumab treatment effect.

An Analytical Comparison of Dako 28-8 PharmDx Assay and an E1L3N Laboratory-Developed Test in the Immunohistochemical Detection of Programmed Death-Ligand 1.

AIM: Nivolumab, a fully human immunoglobulin G4 programmed death-1 (PD-1) immune checkpoint inhibitor antibody, has activity in melanoma, non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC), and Hodgkin lymphoma. Nivolumab is approved in the USA and EU for advanced melanoma, NSCLC, and RCC, and relapsed Hodgkin lymphoma in the USA. Programmed death-ligand 1 (PD-L1), a PD-1 ligand, is expressed on mononuclear leukocytes, myeloid cells, and tumor cells. PD-L1 is being investigated as a potential biomarker to predict the association of tumor PD-L1 expression with nivolumab efficacy. METHODS: Bristol-Myers Squibb and Dako previously reported on an automated PD-L1 immunohistochemical (IHC) assay that detects cell surface PD-L1 in formalin-fixed, paraffin-embedded, human tumor tissue specimens using Dako's Autostainer Link 48. The primary antibody for this assay is a rabbit monoclonal antihuman PD-L1 antibody, clone 28-8. Another rabbit monoclonal antihuman PD-L1 antibody, clone E1L3N, was compared with 28-8 for specificity and sensitivity using an identical detection method followed by vendor-recommended detection methods. RESULTS: Using PD-L1 null clones of L2987 and ES-2 tumor cell lines, both antibodies were specific for detection of PD-L1 on the plasma membrane, although E1L3N also stained cytoplasm in ES-2 knockout cells. Using the identical method, E1L3N was slightly more sensitive than 28-8 based on staining intensities. Using manufacturer-recommended detection methods and predefined scoring criteria for plasma membrane staining of tumor and immune cells, 28-8 demonstrated significantly improved detection compared with E1L3N. CONCLUSIONS: Epitope retrieval and highly sensitive detection reagents are key determinants in IHC detection of PD-L1.

Safety, correlative markers, and clinical results of adjuvant nivolumab in combination with vaccine in resected high-risk metastatic melanoma.

PURPOSE: The anti-programmed death-1 (PD-1) antibody nivolumab (BMS-936558) has clinical activity in patients with metastatic melanoma. Nivolumab plus vaccine was investigated as adjuvant therapy in resected stage IIIC and IV melanoma patients. EXPERIMENTAL DESIGN: HLA-A*0201 positive patients with HMB-45, NY-ESO-1, and/or MART-1 positive resected tumors received nivolumab (1 mg/kg, 3 mg/kg, or 10 mg/kg i.v.) with a multi-peptide vaccine (gp100, MART-1, and NY-ESO-1 with Montanide ISA 51 VG) every 2 weeks for 12 doses followed by nivolumab maintenance every 12 weeks for 8 doses. Primary objective was safety and determination of a maximum tolerated dose (MTD). Secondary objectives included relapse-free survival (RFS), overall survival (OS), and immunologic correlative studies. RESULTS: Thirty-three patients were enrolled. Median age was 47 years; 55% were male. Two patients had stage IIIC disease; 31 patients had stage IV disease. Median follow-up was 32.1 months. MTD was not reached. Most common related adverse events (>40%) were vaccine injection site reaction, fatigue, rash, pruritus, nausea, and arthralgias. Five related grade 3 adverse events [hypokalemia (1), rash (1), enteritis (1), and colitis (2)] were observed. Ten of 33 patients relapsed. Estimated median RFS was 47.1 months; median OS was not reached. Increases in CTLA-4(+)/CD4(+), CD25(+)Treg/CD4(+), and tetramer specific CD8(+) T-cell populations were observed with treatment (P < 0.05). Trends for lower baseline myeloid-derived suppressor cell and CD25(+)Treg/CD4(+) populations were seen in nonrelapsing patients; PD-L1 tumor status was not significantly associated with RFS. CONCLUSIONS: Nivolumab with vaccine is well tolerated as adjuvant therapy and demonstrates immunologic activity with promising survival in high-risk resected melanoma, justifying further study.

Safety, efficacy, and biomarkers of nivolumab with vaccine in ipilimumab-refractory or -naive melanoma.

PURPOSE: Nivolumab, a human immunoglobulin G4-blocking antibody against the T-cell programmed death-1 checkpoint protein, has activity against metastatic melanoma. Its safety, clinical efficacy, and correlative biomarkers were assessed with or without a peptide vaccine in ipilimumab-refractory and -naive melanoma. PATIENTS AND METHODS: In this phase I study, 90 patients with unresectable stage III or IV melanoma who were ipilimumab naive and had experienced progression after at least one prior therapy (cohorts 1 to 3, 34 patients) or experienced progression after prior ipilimumab (cohorts 4 to 6, 56 patients) received nivolumab at 1, 3, or 10 mg/kg every 2 weeks for 24 weeks, then every 12 weeks for up to 2 years, with or without a multipeptide vaccine. RESULTS: Nivolumab with vaccine was well tolerated and safe at all doses. The RECIST 1.1 response rate for both ipilimumab-refractory and -naive patients was 25%. Median duration of response was not reached at a median of 8.1 months of follow-up. High pretreatment NY-ESO-1 and MART-1-specific CD8(+) T cells were associated with progression of disease. At week 12, increased peripheral-blood T regulatory cells and decreased antigen-specific T cells were associated with progression. PD-L1 tumor staining was associated with responses to nivolumab, but negative staining did not rule out a response. Patients who experienced progression after nivolumab could respond to ipilimumab. CONCLUSION: In patients with ipilimumab-refractory or -naive melanoma, nivolumab at 3 mg/kg with or without peptide vaccine was well tolerated and induced responses lasting up to 140 weeks. Responses to nivolumab in ipilimumab-refractory patients or to ipilimumab in nivolumab-refractory patients support combination or sequencing of nivolumab and ipilimumab.

CD32B, the human inhibitory Fc-gamma receptor IIB, as a target for monoclonal antibody therapy of B-cell lymphoma.

Human CD32B (FcgammaRIIB), the low-affinity inhibitory receptor for IgG, is the predominant Fc receptor (FcR) present on B cells. Immunohistochemical and expression studies have identified CD32B expression in a variety of B-cell malignancies, suggesting that CD32B is a potential immunotherapeutic target for B-cell malignancies. A high-affinity monoclonal antibody (mAb 2B6), from a novel panel of anti-human CD32B-specific mAbs, was chimerized (ch2B6) and humanized (hu2B6-3.5). Both ch2B6 and hu2B6-3.5 were capable of directing cytotoxicity by peripheral blood mononuclear cells and monocyte-derived macrophages against B-lymphoma lines in vitro. In a human B-cell lymphoma mouse xenograft model, administration of ch2B6 or hu2B6-3.5 reduced tumor growth rate and improved tumor-free survival. Both the in vitro and in vivo activities of 2B6 required an intact Fc, suggesting an FcR-mediated mechanism of action. These data support the hypothesis that CD32B is a viable target for mAb treatment of B-cell lymphoproliferative disorders.