Impacts of cachexia progression in addition to serum IgG and blood lymphocytes on serum nivolumab in advanced cancer patients.

PURPOSE: Serum nivolumab concentrations exhibit a large variation in cancer patients. Cancer cachexia inducing systemic inflammation promotes the elimination of endogenous proteins, while its association with serum nivolumab remains unclear. The present study aimed to evaluate the impacts of cachexia progression in addition to blood components on serum nivolumab in cancer patients. METHODS: Thirty-eight non-small-cell lung cancer or renal cell cancer patients receiving biweekly intravenous nivolumab were enrolled. Blood samples were collected just before dosing at the 7th administration of nivolumab or later. Serum nivolumab together with serum proteins, inflammatory markers, and peripheral blood leukocytes were determined. Cancer cachexia was classified using the Glasgow Prognostic Score (GPS). Immune-related adverse events (irAEs) were monitored during the study period. RESULTS: Cancer patients had a large variation in serum nivolumab concentrations (interquartile range, 12-21 µg/mL per mg/kg). The serum nivolumab concentration was positively correlated with serum albumin, while negatively associated with serum globulin and immunoglobulin G (IgG). A negative correlation was observed between serum nivolumab and blood lymphocytes. Regarding cachexia progression, the patients with GPS 2 had a higher serum interleukin-6 concentration and a lower serum nivolumab concentration than those with GPS 0 or 1. The GPS, serum IgG, and blood lymphocytes were identified as independent variables for serum nivolumab. The incidence of irAEs was not associated with the nivolumab dose or serum nivolumab. CONCLUSION: Cachexia progression had a negative impact on serum nivolumab in cancer patients. The interindividual variation in serum nivolumab was characterized by cachexia progression in addition to blood components.

Bioanalytical Challenges due to Prior Checkpoint Inhibitor Exposure: Interference and Mitigation in Drug Concentration and Immunogenicity Assays.

Monoclonal antibodies (mAbs) are a leading class of biotherapeutics. In oncology, patients often fail on early lines of biologic therapy to a specific target. Some patients may then enroll in a new clinical trial with a mAb specific for the same target. Therefore, immunoassays designed to quantify the current mAb therapy or assess immunogenicity to the drug may be susceptible to cross-reactivity or interference with residual prior biologics. The impact of two approved anti-PD-1 mAbs, pembrolizumab and nivolumab, was tested in several immunoassays for cemiplimab, another approved anti-PD-1 mAb. The methods included a target-capture drug concentration assay, a bridging anti-drug antibody (ADA) assay and a competitive ligand-binding neutralizing antibody (NAb) assay. We also tested bioanalytical strategies to mitigate cross-reactivity or interference in these assays from other anti-PD-1 biologics. Both pembrolizumab and nivolumab cross-reacted in the cemiplimab drug concentration assay. This was mitigated by addition of antibodies specific to pembrolizumab or nivolumab. ADA specific for pembrolizumab and nivolumab did not interfere in the cemiplimab ADA assay. However, pembrolizumab and nivolumab generated a false-positive response in a target-capture NAb assay. Our results demonstrate that similar exogenous pre-existing anti-PD-1 mAbs (biotherapeutics) such as pembrolizumab and nivolumab are detected and accurately quantified in the cemiplimab drug concentration assay. However, once steady state is achieved for the new therapy, prior biologics would likely not be detected. Cross-reactivity and interference in immunoassays from previous treatment with class-specific biotherapeutic(s) pose significant bioanalytical challenges, especially in immuno-oncology.

Use of an alternative signature peptide during development of a LC-MS/MS assay of plasma nivolumab levels applicable for multiple species.

Recently, immune checkpoint inhibitors, including anti-programmed cell death protein 1 (PD-1) antibodies, have dramatically changed treatment strategies for several cancers. In pharmacokinetic/pharmacodynamic studies, experiments using a variety of animal species are assumed. We have identified optimal multiple reaction monitoring transitions for signature candidate peptides of nivolumab in human, mouse, and rat plasma and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify nivolumab (an anti-PD-1 antibody) using trastuzumab as the internal standard. Calibration curves were linear in the range of 1-200 µg/mL. The intra- and inter-day precision and accuracy in human plasma fulfilled Food and Drug Administration guideline criteria for bioanalytical validation. There was no need to change the measurement method in mouse plasma. On the other hand, in rat plasma, an interference peak was observed at a retention time similar to that of the surrogate peptide ASGITFSNSGMHWVR (550.75 > 661.50) employed in human and mouse plasma. Therefore, we confirmed that ASQSVSSYLAWYQQKPGQAPR (785.0 > 940.2) can be used as an alternate nivolumab surrogate peptide in rat plasma at the same concentration range as used in human and mouse plasma. Using our method, the concentration range and a gradual increase in trough value were confirmed in clinical samples from two antibody-treated patients, including one with gastric cancer and one with non-small-cell lung cancer. The time course and blood concentration transition also were evaluated in nivolumab administration experiments in mouse and rat. The present study showed that the selection of the optimal peptide is essential for accurate LC-MS/MS measurement of nivolumab concentration in human, mouse, and rat plasma. The method developed here is expected to be of use in non-clinical and clinical pharmacokinetic studies.

Therapeutic drug monitoring of nivolumab in routine clinical practice. A pilot study.

OBJECTIVE: A review of the literature about the anti-programmed death 1 monoclonal antibody nivolumab permits to verify the existence  of several issues still unresolved about their dosing schedule. The aim of the present work was to explore possibilities of nivolumab treatment  personalization through therapeutic drug monitoring, in order to  improve their effectiveness and efficiency. METHOD: Observational, prospective study carried out from May 2017  through June 2019 in patients with different tumor diagnoses treated with nivolumab. Blood samples were obtained in the routine  clinical practice, once nivolumab steady state was reached. Serum  nivolumab levels were determined by means of quantitative ELISA. The  standard schedule of 3 mg/kg every two weeks (Q2W) was modified in  some patients due to different circumstances, and resulting serum  concentrations were compared with those from the non-modified  patients and the published data. RESULTS: Blood samples from 19 patients in treatment with nivolumab were analyzed. A total of 39 samples of nivolumab were  analyzed between 6th and 27th cycles. The standard schedule of 3  mg/kg every two weeks was modified in 12/19 (60%) patients, with  intervals of 3, 4, 5, 6 or 7 weeks, once the steady state was reached.  No statistically significant differences were detected when comparing  every two weeks and every four week intervals. When the intervals  were six or seven weeks, mean plasma concentration showed a  statistically significant difference compared with every two weeks. CONCLUSIONS: Current data contribute to confirm former suspects about the possibilities of exploring new scenarios to improve and  personalize nivolumab dosage. Additional studies to confirm it in bigger  series and correlate it with clinical results, and to better define the role  of therapeutic drug monitoring in the treatment, are warranted, not only by financial concerns but also for improving quality of life of patients  and clinical management aspects.

Objetivo: Una revisión de la literatura sobre nivolumab permite  verificar la existencia de diversos aspectos sin resolver sobre su  intervalo de dosificación. El objetivo del presente estudio ha sido  explorar las posibilidades de personalización del tratamiento con  nivolumab mediante la monitorización terapéutica de sus  concentraciones séricas para mejorar su efectividad y eficiencia.Método: Estudio observacional, prospectivo, realizado entre mayo de 2017 y junio de 2019 en pacientes tratados con nivolumab que  estaban diagnosticados de diferentes tumores. Se obtuvieron muestras  de sangre en la práctica clínica habitual, una vez alcanzado el estado de  equilibrio de nivolumab. Las concentraciones séricas de nivolumab  fueron determinadas mediante ELISA cuantitativo. La pauta posológica  habitual de 3 mg/kg cada dos semanas tuvo que ser modificada en  algunos pacientes debido a diferentes circunstancias, y las  concentraciones séricas resultantes se compararon con las  correspondientes a los pacientes en los que no se modificó y con datos  publicados.Resultados: Se analizaron muestras de 19 pacientes que recibieron inicialmente 3 mg/kg de nivolumab cada dos semanas. Se  analizó un total de 39 muestras, entre los ciclos 6 y 27. La pauta  habitual se modificó, una vez alcanzado el estado de equilibrio, en  12/19 (60%) pacientes, en los que se amplió el intervalo a 3, 4, 5, 6 o 7 semanas. No se encontraron diferencias estadísticamente significativas  al comparar la administración cada dos semanas y cada cuatro  semanas. Cuando los intervalos fueron de seis o siete semanas, la  concentración sérica media mostró una diferencia estadísticamente  significativa en comparación con la administración cada dos semanas.Conclusiones: La información recogida parece confirmar la necesidad de explorar nuevos escenarios para personalizar la  dosificación de nivolumab. Se necesitan estudios adicionales en series  de mayor tamaño para confirmar esta información, correlacionarla con  los resultados clínicos y definir mejor el papel de la monitorización  terapéutica, no solo por motivos económicos, sino también para mejorar  la calidad de vida de los pacientes y facilitar la administración  clínica del tratamiento.

A Machine-Learning Approach to Identify a Prognostic Cytokine Signature That Is Associated With Nivolumab Clearance in Patients With Advanced Melanoma.

Lower clearance of immune checkpoint inhibitors is a predictor of improved overall survival (OS) in patients with advanced cancer. We investigated a novel approach using machine learning to identify a baseline composite cytokine signature via clearance, which, in turn, could be associated with OS in advanced melanoma. Peripheral nivolumab clearance and cytokine data from patients treated with nivolumab in two phase III studies (n = 468 (pooled)) and another phase III study (n = 158) were used for machine-learning model development and validation, respectively. Random forest (Boruta) algorithm was used for feature selection and classification of nivolumab clearance. The 16 top-ranking baseline inflammatory cytokines reflecting immune-cell modulation were selected as a composite signature to predict nivolumab clearance (area under the curve (AUC) = 0.75; accuracy = 0.7). Predicted clearance (high vs. low) via the cytokine signature was significantly associated with OS across all three studies (P < 0.01), regardless of treatment (nivolumab vs. chemotherapy).

Intracranial antitumor responses of nivolumab and ipilimumab: a pharmacodynamic and pharmacokinetic perspective, a scoping systematic review.

BACKGROUND: Recently, two phase II trials showed intracranial activity of the immune checkpoint inhibitors nivolumab and ipilimumab in patients with melanoma brain metastases. However, it is generally assumed that large molecules like monoclonal antibodies nivolumab and ipilimumab cannot penetrate and pass an intact blood brain barrier (BBB). In this systematic review we provide a pharmacodynamic and pharmacokinetic consideration of the clinical activity of the immune checkpoint inhibitors nivolumab and ipilimumab in melanoma brain metastases. METHODS: Pubmed was systematically searched for prospective phase II and III studies on nivolumab and ipilimumab in melanoma brain metastases and cerebrospinal fluid (CSF) levels of nivolumab and ipilimumab. Results were discussed and a perspective on the pharmacodynamics and pharmacokinetics for the intracranial activity of these agents was given. RESULTS: Two phase II studies with the combination nivolumab and ipilimumab and one phase II study with ipilimumab monotherapy in melanoma brain metastases were included in this review. One article reported drug levels of nivolumab in CSF. Intracranial responses were achieved in 16 of 35 patients (46%; 95% confidence interval (CI) 29-63) in a phase II study cohort treated with nivolumab and ipilimumab. In a second phase II study in 94 patients, the rate of intracranial clinical benefit was 57% (95% CI 47-68). The CSF/serum ratio of nivolumab was 0.88-1.9% in a cohort of metastatic melanoma patients treated with nivolumab 1-3 mg/kg. Nivolumab concentrations ranged from 35 to 150 ng/ml in CSF of these patients, which is in the range of the half maximal effective concentration (EC50) of 0.64 nM. CONCLUSIONS: Ipilimumab and nivolumab are active in melanoma brain metastases. Nivolumab penetrates into the CSF. Based on the described findings the general consensus that monoclonal antibodies do not penetrate into the central nervous system (CNS) and cannot have a direct intracranial effect needs to be reconsidered.

Efficacy of nivolumab four-weekly dosing schedule based on body weight.

Nivolumab has received regulatory approval to be given by weight-based or flat dosing every two weeks or by flat dosing every four weeks. However, flat dosing would lead to unnecessarily high doses for patients with lower body weight, increasing the drug usage and probability of toxicity. We review the rationale of using a four-weekly hybrid dosing strategy using weight-based and flat-dosing regimens adopted by some jurisdictions.

Correlation between nivolumab exposure and treatment outcomes in non-small-cell lung cancer.

INTRODUCTION: Nivolumab treatment is subject to large interpatient variability in both efficacy and toxicity, which may partly be explained by differences in nivolumab exposure. Exposure-response relationships in regular healthcare have not been extensively investigated for nivolumab. Therefore, we aimed to identify possible exposure-response relationships in nivolumab-treated patients with non-small-cell lung cancer (NSCLC). METHODS: Patients with NSCLC who started second-line nivolumab therapy (3 mg/kg Q2W) between May 5th 2016 and August 1st 2017, and from whom serial blood samples, toxicity data and outcome data were prospectively collected, were included. Follow-up was carried out until November 1st 2017. Patients were classified according to the best overall response (BOR) based on the Response Evaluation Criteria in Solid Tumours, v1.1, and toxicities according to the Common Terminology Criteria for Adverse Events. Nivolumab trough concentrations were measured after 2, 4 and 10 weeks of treatment, excluding dose delays, and calculated geometric means were tested versus BOR or toxicity using analysis of variance and an independent samples t-test, respectively. Overall survival (OS) and progression-free survival were compared between high and low trough concentration groups. RESULTS: Seventy-six patients were evaluable for analyses. Responders (n = 15) had higher mean trough concentrations than patients with progression (n = 33): 47% higher after 2 weeks (p = 0.001), 53% higher after 4 weeks (p = 0.008) and 73% higher after 10 weeks (p = 0.002). Higher trough concentrations were associated with longer OS (p = 0.001). CONCLUSIONS: This study shows that patients with NSCLC with a response to nivolumab had a higher nivolumab exposure than patients with progression, indicating a potential exposure-response relationship. Further clinical research should focus on clarifying these exposure-response relationships.

An LC-MS/MS Method for Absolute Quantification of Nivolumab in Human Plasma: Application to Clinical Therapeutic Drug Monitoring.

BACKGROUND: Nivolumab is a fully humanized IgG4 monoclonal antibody that targets the programmed death-1 (PD-1) receptor, disrupting PD-1-mediated signaling and restoring antitumor immunity. The objective of this study was to develop a nivolumab quantification method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and to evaluate its application in clinical therapeutic drug monitoring. METHODS: Nivolumab was purified from human plasma using rProtein A resin and then digested with trypsin. The ASGITFSNSGMHWVR peptide (multiple reaction monitoring transition: m/z 550.6→661.4) was detected as a surrogate peptide of nivolumab by triple quadrupole mass spectrometry. Plasma samples (126) were collected from 14 patients with non-small cell lung cancer who were undergoing clinical dosing regimen with nivolumab. The pharmacokinetic data were analyzed using Phoenix NLME software (Version 7.0, Certara, St. Louis, MO) based on a previously reported population pharmacokinetics (PPK) model of nivolumab. RESULTS: Nivolumab was selectively detected in human plasma and the linear range was 5-200 mcg/mL (R = 0.99). The accuracy and intraday and interday imprecision were within ±15% of the quality control values of 5 (lower limit of quantification), 10 (low), 80 (medium), and 160 (high) mcg/mL. The nivolumab concentrations measured using LC-MS/MS were consistent with those of previously reported PPK models, and the pharmacokinetic parameters could be adequately predicted from a single trough concentration using a Bayesian approach. CONCLUSIONS: An absolute quantification method for nivolumab using LC-MS/MS was successfully developed and validated. Combined with PPK analysis, this method should be useful for the therapeutic drug monitoring of nivolumab in clinical practice.

Development of an Enzyme-Linked Immune Sorbent Assay to Measure Nivolumab and Pembrolizumab Serum Concentrations.

BACKGROUND: Treatment with monoclonal antibodies (mAbs) against programmed cell death protein 1 receptor is subject to high variation in treatment outcome among cancer patients. For these agents, no exposure-response (ER) relationships have been investigated in routine health care settings. However, ER relationships have been identified for several other mAbs used in oncology. Methods to conveniently measure serum concentrations of anti-programmed cell death protein 1 mAbs in routine health care may clarify possible ER relationships. Therefore, the authors aimed to develop an enzyme-linked immune sorbent assay (ELISA) for the measurement of both nivolumab and pembrolizumab serum concentrations of treated cancer patients. METHODS: Optimal capture antigen and detection antibody concentrations were selected based on titrations. Nivolumab calibration standards ranging from 0.2 to 300 ng/mL were tested in duplicate. Accuracy was assessed in 2 recovery experiments. Intra- and interassay variations were assessed on 3 different days by 2 independent technicians. The developed ELISA was also set up for pembrolizumab calibration curves. Cross-reactivity of nivolumab measurements with ipilimumab was assessed. Of one nivolumab treated patient, serum concentrations in follow up samples were measured and presented. RESULTS: Nivolumab calibration standards of 0.20-25 ng/mL were used. Nivolumab trough concentrations after 1 cycle in 8 patients ranged from 17.3 to 31.1 mcg/mL. The range of accuracy was 84%-105%, whereas intra- and interassay variations showed a coefficient of variation of 5.5% and 10.1%, respectively. No cross-reactivity with ipilimumab was detected. Pembrolizumab trough concentrations (n = 8) ranged from 9.1 to 19.7 mcg/mL after 1 infusion. CONCLUSIONS: The in-house-developed ELISA provides the opportunity to measure both nivolumab and pembrolizumab serum concentrations. This may help identify possible ER relationships in treated cancer patients and may potentially lead to dose adjustments in the future.