PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by ...melanoma driver mutations and modified by oncogenic signaling inhibition.
Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated.
No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1-exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines.
In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1-exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies.
Background In melanoma, dysregulation of the MAPK pathway, usually via BRAF.sup.V600 or NRAS.sup.Q61 somatic mutations, leads to constitutive ERK signaling. While BRAF inhibitors are initially ...effective for BRAF-mutant melanoma, no FDA-approved targeted therapies exist for BRAF-inhibitor-resistant BRAF.sup.V600, NRAS mutant, or wild-type melanoma. Methods The 50% inhibitory concentration (IC50) of SCH772984, a novel inhibitor of ERK1/2, was determined in a panel of 50 melanoma cell lines. Effects on MAPK and AKT signaling by western blotting and cell cycle by flow cytometry were determined. Results Sensitivity fell into three groups: sensitive, 50% inhibitory concentration (IC.sub.50) < 1 muM; intermediately sensitive, IC.sub.50 1-2 muM; and resistant, >2 muM. Fifteen of 21 (71%) BRAF mutants, including 4 with innate vemurafenib resistance, were sensitive to SCH772984. All three (100%) BRAF/NRAS double mutants, 11 of 14 (78%) NRAS mutants and 5 of 7 (71%) wild-type melanomas were sensitive. Among BRAF.sup.V600 mutants with in vitro acquired resistance to vemurafenib, those with MAPK pathway reactivation as the mechanism of resistance were sensitive to SCH772984. SCH772984 caused G1 arrest and induced apoptosis. Conclusions Combining vemurafenib and SCH722984 in BRAF mutant melanoma was synergistic in a majority of cell lines and significantly delayed the onset of acquired resistance in long term in vitro assays. Therefore, SCH772984 may be clinically applicable as a treatment for non-BRAF mutant melanoma or in BRAF-mutant melanoma with innate or acquired resistance, alone or in combination with BRAF inhibitors. Keywords: ERK inhibitor, BRAF inhibitor, Melanoma, Targeted therapy, Acquired resistance
Therapeutic uses of asparaginases (ASNase) have been shown to elicit immune responses resulting in the development of potentially life-threatening human anti-bacterial antibodies (Ab). A robust ...screening enzyme-linked immunosorbent assay (ELISA) to detect binding Ab(+) against ASNase has been developed and validated for therapeutic monitoring to support clinical trials. Recently, a protein chip bioassay (Biacore) was developed for the Ab of these proteins. These methods were compared.
A Biacore T-100 analyzer using a protein bioassay and an ELISA assay were used to determine the IgG immmuboglobulin Ab against ASNase in sera from 84 acute lymphoblastic leukemia (ALL) patients plus 6 controls (n=121 samples). These samples were characterized for anti-ASNase Ab neutralizing activity. Human E. coli ASNase, pegaspargase and Erwinase proteins were covalently coupled to the carboxy-methylated dextran matrix of a CM5 sensor chip (surface plasmon resonance, SPR). In the course of a nested experimental design, a wide range of human sera from patients who had obvious clinical allergic reactions after either native or pegaspargase treatments were tested. The data were fitted by a parametric logistic equation (+/-95% confidence interval, CI), which ranged from <3.0% to <14%.
The specificity of Ab(+) was evaluated using "spiked" human IgG antibodies. Both assays provide near excellent linearity and sensitivity of response (<0.8 to <500 ratio and 1-3000 resonance units RU) of anti-ASNase Ab in human sera with low variance. The bioassay method was ten times more sensitive than the ELISA Ab assay. The lowest limit of quantification of Ab(+) ratio for the SPR assay was 0.6 whereas the upper limit of quantification was 3000 RU. The SPR assay results were in excellent accord with both the Ab(-) and the Ab(+). Ab(-) by the ELISA method (<1.003 ratio) was related to a mean RU value of 8.1. Despite the narrow range of ambiguity around the 1.1 Ab(+) ratio values, the majority of the specimens (93.2%) were determined to be Ab(+) by either ELISA or SPR determination.
The vast majority (81/84 = 96.4%) of the IgG Ab(+) were neutralizing. The SPR Ab determination technique is reliable, accurate and more sensitive than the ELISA method.
Asparaginases are the cornerstone therapy of many successful combination regimens for the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children and adolescents. ...Currently, two asparaginase formulations are available in the US, native Escherichia coli asparaginase (ASNase) and pegaspargase. A third formulation native Erwinia asparaginase (Erwinase, ERW) has recently been made available under a licensing exception for personal use. We report here the development and validation process of ERW pharmacoanalytical assays and the results in a few patients.
We developed and systematically validated the ERW enzyme activity and ERW concentration, anti-ERW antibody and related assays. Pharmacokinetic and pharmacodynamic (PK-PD) studies were performed in a limited number of patients who received 6,000 IU/m2 x 3 per week x 2 courses, and 4 patients who received 25,000 IU/m2 x 3 per week x 2 courses of ERW.
The linearity and range of the Erwinase calibration lines for the pharmacoanalytical assays were excellent. The accuracy and precision were better than the FDA limit allows for oncology biological products (<30%) coefficient of variation (%CV) and related parameters in the quantification of ERW concentration. The validation of these parameters was equal to or better than during the assay development. PK-PD analyses of ERW in a few patients yielded an average half-life of elimination of 15.8+/-1.64 hours. There was an excellent PD response post ERW administration resulting in an ERW concentration-dependent asparagine (ASN, <0.5 microM) and glutamine (GLN, <50 microM) deamination. Pharmacodynamic correlations demonstrated that 0.1 to 0.2 IU/ml of ERW in serum were sufficient for 90% GLN and/or ASN deamination for up to 2 weeks. No anti-ERW antibody Ab(+) was seen among those few patients. None of the other 5 patients had an adverse event. Based on these post hoc results, simulations on various doses and schedules of this drug have been made.
The pharmacoanalytical assays were excellent tools to evaluate the PK and PD data of ERW in pediatric patients with HR ALL. However, this initial PK-PD evidence needs further validation in future clinical trials. Insights into the PD contributions of ERW in anti-E. coli ASNase Ab(+) patients will guide us in optimal design and use of ERW as part of combination chemotherapy regimens in future clinical trials.
Keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cuSCCs) develop in 15–30% of patients with BRAFV600E metastatic melanoma treated with BRAF inhibitors (BRAFi). These lesions resemble ...mouse skin tumors induced by the two-stage DMBA/TPA skin carcinogenesis protocol; in this protocol BRAFi accelerates tumor induction. Since prior studies demonstrated cyclooxygenase 2 (COX-2) is necessary for DMBA/TPA tumor induction, we hypothesized that COX-2 inhibition might prevent BRAFi-accelerated skin tumors. Celecoxib, a COX-2 inhibitor, significantly delayed tumor acceleration by the BRAFi inhibitor PLX7420 and decreased tumor number by 90%. Tumor gene expression profiling demonstrated that celecoxib partially reversed the PLX4720-induced gene signature. In PDV cuSCC cells, vemurafenib (a clinically approved BRAFi) increased ERK phosphorylation and soft agar colony formation; both responses were greatly decreased by celecoxib. In clinical trials trametinib, a MEK inhibitor (MEKi) increases BRAFi therapy efficacy in BRAFV600E melanomas and reduces BRAFi-induced KA and cuSCC frequency. Trametinib also reduced vemurafenib-induced PDV soft agar colonies, but less efficiently than celecoxib. The trametinb/celecoxib combination was more effective than either inhibitor alone. In conclusion, celecoxib suppressed both BRAFi-accelerated skin tumors and soft-agar colonies, warranting its testing as a chemopreventive agent for non-melanoma skin lesions in patients treated with BRAFi alone or in combination with MEKi.
•Celecoxib delays squamous cell carcinoma (SCC) tumor acceleration by PLX7420.•It also decreases SCC tumor number by 90%.•It partially reversed the PLX4720-induced gene signature.•Trametinb/celecoxib combination is more effective than either inhibitor alone.
A significant barrier to effective immune clearance of cancer is loss of antitumor cytotoxic T cell activity. Antibodies to block pro-apoptotic/downmodulatory signals to T cells are currently being ...tested. Because invariant natural killer T cells (iNKT) can regulate the balance of Th1/Th2 cellular immune responses, we characterized the frequencies of circulating iNKT cell subsets in 21 patients with melanoma who received the anti-CTLA4 monoclonal antibody tremelimumab alone and 8 patients who received the antibody in combination with MART-126-35 peptide-pulsed dendritic cells (MART-1/DC). Blood T cell phenotypes and functionality were characterized by flow cytometry before and after treatment. iNKT cells exhibited the central memory phenotype and showed polyfunctional cytokine production. In the combination treatment group, high frequencies of pro-inflammatory Th1 iNKT CD8(+) cells correlated with positive clinical responses. These results indicate that iNKT cells play a critical role in regulating effective antitumor T cell activity.
Tumor responses to programmed cell death protein 1 (PD-1) blockade therapy are mediated by T cells, which we characterized in 102 tumor biopsies obtained from 53 patients treated with pembrolizumab, ...an antibody to PD-1. Biopsies were dissociated, and single-cell infiltrates were analyzed by multicolor flow cytometry using two computational approaches to resolve the leukocyte phenotypes at the single-cell level. There was a statistically significant increase in the frequency of T cells in patients who responded to therapy. The frequency of intratumoral B cells and monocytic myeloid-derived suppressor cells significantly increased in patients' biopsies taken on treatment. The percentage of cells with a regulatory T-cell phenotype, monocytes, and natural killer cells did not change while on PD-1 blockade therapy. CD8(+) memory T cells were the most prominent phenotype that expanded intratumorally on therapy. However, the frequency of CD4(+) effector memory T cells significantly decreased on treatment, whereas CD4(+) effector T cells significantly increased in nonresponding tumors on therapy. In peripheral blood, an unusual population of blood cells expressing CD56 was detected in two patients with regressing melanoma. In conclusion, PD-1 blockade increases the frequency of T cells, B cells, and myeloid-derived suppressor cells in tumors, with the CD8(+) effector memory T-cell subset being the major T-cell phenotype expanded in patients with a response to therapy.
It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes ...and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy.
A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation.
A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1-specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1-specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells.
Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses.
Adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) is a promising cancer treatment. Here, we investigate the in vivo functional activity ...and dynamics of the transferred cells by analyzing samples from 3 representative patients with melanoma enrolled in a clinical trial of ACT with TCR transgenic T cells targeted against the melanosomal antigen MART-1. The analyses included evaluating 19 secreted proteins from individual cells from phenotypically defined T-cell subpopulations, as well as the enumeration of T cells with TCR antigen specificity for 36 melanoma antigens. These analyses revealed the coordinated functional dynamics of the adoptively transferred, as well as endogenous, T cells, and the importance of highly functional T cells in dominating the antitumor immune response. This study highlights the need to develop approaches to maintaining antitumor T-cell functionality with the aim of increasing the long-term efficacy of TCR-engineered ACT immunotherapy.
A longitudinal functional study of adoptively transferred TCR–engineered lymphocytes yielded revealing snapshots for understanding the changes of antitumor responses over time in ACT immunotherapy of patients with advanced melanoma.
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