MAPPs for the identification of immunogenic hotspots of biotherapeutics; an overview of the technology and its application to the biopharmaceutical arena.

INTRODUCTION: Anti-drug antibody (ADA) responses are becoming an increasing concern as more highly engineered and sophisticated biotherapeutics enter the clinic. An arsenal of tools has been developed to identify potential T cell epitopes that may drive unwanted immunological responses to protein therapeutics; one such tool is termed 'Major Histocompatibility Complex-Associated Peptide Proteomics' (MAPPs). This review highlights the evolution of this MHC II profiling technology, its technological advantages and limitations, and its utility in helping to de-risk the immunogenicity of biotherapeutics. Areas covered: A comprehensive literature review was performed along with discussions with key leaders in the field of MAPPs to summarize the importance of monitoring potential immunogenicity of clinical molecules. Herein we also describe how MAPPs has been applied specifically for monitoring MHC II peptides derived from biotherapeutics. Expert commentary: Given the importance of this growing field we discuss the complementary tools used in conjunction with MAPPs and review case studies where this approach has informed clinical studies and in some cases allowed re-engineering of the biotherapeutic moiety to a less immunogenic format.

Determination of critical quality attributes for monoclonal antibodies using quality by design principles.

Quality by design (QbD) is a global regulatory initiative with the goal of enhancing pharmaceutical development through the proactive design of pharmaceutical manufacturing process and controls to consistently deliver the intended performance of the product. The principles of pharmaceutical development relevant to QbD are described in the ICH guidance documents (ICHQ8-11). An integrated set of risk assessments and their related elements developed at Roche/Genentech were designed to provide an overview of product and process knowledge for the production of a recombinant monoclonal antibody. This chapter describes the identification of critical quality attributes (CQAs) as an important first step for QbD development of biopharmaceuticals. A systematic scientific based risk ranking and filtering approach allows a thorough understanding of quality attributes and an assignment of criticality for their impact on drug safety and efficacy. To illustrate the application of the approach and tools, a few examples from monoclonal antibodies are shown. The identification of CQAs is a continuous process and will further drive the structure and function characterization of therapeutic proteins.

Host cell proteins in biotechnology-derived products: A risk assessment framework.

To manufacture biotechnology products, mammalian or bacterial cells are engineered for the production of recombinant therapeutic human proteins including monoclonal antibodies. Host cells synthesize an entire repertoire of proteins which are essential for their own function and survival. Biotechnology manufacturing processes are designed to produce recombinant therapeutics with a very high degree of purity. While there is typically a low residual level of host cell protein in the final drug product, under some circumstances a host cell protein(s) may copurify with the therapeutic protein and, if it is not detected and removed, it may become an unintended component of the final product. The purpose of this article is to enumerate and discuss factors to be considered in an assessment of risk of residual host cell protein(s) detected and identified in the drug product. The consideration of these factors and their relative ranking will lead to an overall risk assessment that informs decision-making around how to control the levels of host cell proteins.

Association of endogenous anti-interferon-α autoantibodies with decreased interferon-pathway and disease activity in patients with systemic lupus erythematosus.

OBJECTIVE: Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE. METHODS: Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays. RESULTS: AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN(low) and IFN(high) , that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFN(high) patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro. CONCLUSION: Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.

Allelic variation in HLA-DRB1 is associated with development of antidrug antibodies in cancer patients treated with atezolizumab that are neutralizing in vitro.

The treatment of diseases with biologic agents can result in the formation of antidrug antibodies (ADA). Although drivers for ADA formation are unknown, a role for antigen presentation is likely, and variation in human leukocyte antigen (HLA) genes has been shown to be associated with occurrence of ADA for several biologics. Here, we performed an HLA-wide association study in 1982 patients treated with the anti-PD-L1 antibody atezolizumab across eight clinical trials. On average, 29.8% of patients were ADA-positive (N = 591, range of 13.5%-38.4% per study) and 14.6% of patients were positive for ADA that were neutralizing in vitro (neutralizing antibodies [NAb], N = 278, range of 6.4%-21.9% per study). In a meta-analysis of logistic regression coefficients, we found statistically significant associations between HLA class II alleles and ADA status. The top-associated alleles were HLA-DRB1*01:01 in a comparison of ADA-positive versus ADA-negative patients (p = 3.4 × 10-5 , odds ratio [OR] 1.96, 95% confidence interval [95% CI] 1.64-2.28) and HLA-DQA1*01:01 when comparing NAb-positive with ADA-negative patients (p = 2.8 × 10-7 , OR 2.31, 95% CI 1.98-2.66). Both alleles occur together on a common HLA haplotype, and analyses considering only NAb-negative, ADA-positive patients did not yield significant results, suggesting that the genetic association is mainly driven by NAb status. In conclusion, our study showed that HLA class II genotype is associated with the risk of developing ADA, and specifically NAb, in patients treated with atezolizumab, but the effect estimates suggest that immunogenetic factors are not sufficient as clinically meaningful predictors.

Evaluation of atezolizumab immunogenicity: Efficacy and safety (Part 2).

Antibody therapeutics can be associated with unwanted immune responses resulting in the development of anti-drug antibodies (ADA). Optimal methods to evaluate the potential effects of ADA on clinical outcomes in oncology are not well established. In this study, we assessed efficacy and safety, based on ADA status, in patients from over 10 clinical trials that evaluated the immune checkpoint inhibitor atezolizumab as a single agent or as combination therapy for several types of advanced cancers. ADA can only be observed post randomization, and imbalances in baseline prognostic factors can confound the interpretation of ADA impact. We applied methodology to account for the confounding effects of baseline clinical characteristics and survivorship bias on efficacy. Adjusted meta-analyses revealed that despite numerical differences in overall survival and progression-free survival between ADA-positive and ADA-negative patients from some studies, ADA-positive patients from studies with an overall treatment effect derived benefit from atezolizumab, compared with their adjusted controls. Based on large, pooled populations from atezolizumab monotherapy or combination studies, unadjusted descriptive analyses did not identify a clear relationship between ADA status and frequency or severity of adverse events. Data also suggested that any ADA impact is not driven by neutralizing activity. Collectively, this exploratory analysis suggests that the potential for ADA development should not impact treatment decisions with atezolizumab.

Evaluation of atezolizumab immunogenicity: Clinical pharmacology (part 1).

Baseline patient characteristics and prognostic factors are important considerations in oncology when evaluating the impact of immunogenicity on pharmacokinetics (PK) and efficacy. Here, we assessed the impact of anti-drug antibodies (ADA) on the PK of the immune checkpoint inhibitor atezolizumab (an anti-PD-L1 monoclonal antibody). We evaluated data from ≈ 4500 patients from 12 clinical trials across different tumor types, treatment settings, and dosing regimens. In our dataset, ~ 30% of patients (range, 13-54%) developed treatment-emergent ADA, and in vitro neutralizing antibodies (NAb) were seen in ~ 50% of ADA-positive (+) patients. Pooled time course data showed a trend toward lower atezolizumab exposure in ADA+ patients, which was more pronounced in ADA+/NAb+ patients. However, the atezolizumab concentration distributions overlapped, and drug concentrations exceeded 6 µg/ml, the target concentration required for receptor saturation, in greater than 95% of patients. Patients had sufficient exposure regardless of ADA status. The dose selected to allow for dosing over effects from ADA resulted in a flat exposure-response relationship. Analysis of study results by ADA titer showed that exposure and overall survival were not affected in a clinically meaningful way. High tumor burden, low albumin, and high CRP at baseline showed the greatest association with ADA development but not with subsequent NAb development. These imbalanced factors at baseline can confound analysis of ADA impact. ADA increases atezolizumab clearance minimally (9%), and its impact on exposure based on the totality of the clinical pharmacology assessment does not appear to be clinically meaningful.

Real-world data prognostic model of overall survival in patients with advanced NSCLC receiving anti-PD-1/PD-L1 immune checkpoint inhibitors as second-line monotherapy.

BACKGROUND AND AIM: The objective of this retrospective, observational, noninterventional cohort study was to investigate prognostic factors of overall survival (OS) in patients with advanced non-small cell lung cancer (aNSCLC) and to develop a novel prognostic model. METHODS: A total of 4049 patients with aNSCLC diagnosed between January 2011 and February 2020 who received atezolizumab, nivolumab, or pembrolizumab as second-line monotherapy were selected from a real-world deidentified database to build the cohort. Patients could not have received first-line treatment with clinical study drug(s) nor immune checkpoint inhibitors including anti-programmed cell death 1 (PD-1)/programmed death-ligand 1 (PD-L1), and anti-cytotoxic T-lymphocyte-associated protein 4 therapies. RESULTS: Patients had a median age of 69 years; 45% were female, 75% White, 70% had stage IV at initial diagnosis, and 70% had nonsquamous histology. A Cox proportional hazards model with lasso regularization was used to build a prognostic model for OS using 18 baseline demographic and clinical factors based on the real-world data cohort. The risk-increasing prognostic factors were abnormally low albumin and chloride levels, Eastern Cooperative Oncology Group performance status score ≥ 2, and abnormally high levels of alkaline phosphatase and white blood cells. The risk-decreasing prognostic factors were PD-L1 positivity, longer time from advanced diagnosis to start of first-line therapy, and higher systolic blood pressure. The performance of the model was validated using data from the OAK trial, and the c-index for the OAK trial validation cohort was 0.65 and 0.67 for the real-world data cohort. CONCLUSIONS: Based on baseline demographic and clinical factors from a real-world setting, this prognostic model was developed to discriminate the risk of death in patients with aNSCLC treated with checkpoint inhibitors as second-line monotherapy, and it performed well in the real-world data and clinical trial cohorts.

Time-dependent population PK models of single-agent atezolizumab in patients with cancer.

PURPOSE: The time-varying clearance (CL) of the PD-L1 inhibitor atezolizumab was assessed on a population of 1519 cancer patients (primarily with non-small-cell lung cancer or metastatic urothelial carcinoma) from three clinical studies. METHODS: The first step was to identify the baseline covariates affecting atezolizumab CL without including time-varying components (stationary covariate model). Two time-varying models were then investigated: (1) a model allowing baseline covariates to vary over time (time-varying covariate model), (2) a model with empirical time-varying Emax CL function. RESULTS: The final stationary covariate model included main effects of body weight, albumin levels, tumor size, anti-drug antibodies (ADA) and gender on atezolizumab CL. Both time-varying models resulted in a clear improvement of the data fit and visual predictive checks over the stationary model. The time-varying covariate model provided the best fit of the data. In this model, the main driver for change in CL over time was variations in albumin level with an increase in serum albumin (improvement in a patient's status) mirroring a decrease in CL. Time-varying ADAs had a small impact (9% increase in CL). None of the covariates impacted atezolizumab CL by more than ± 30% from median. The estimated maximum decrease in CL with time was 22% with the Emax model. CONCLUSION: The overall impact of covariates on atezolizumab CL did not warrant any change in atezolizumab dosing recommendations. The results support the hypothesis that variation in atezolizumab CL over time is associated with patients' disease status, as shown with other checkpoint inhibitors.

Molecular Interaction Characterization Strategies for the Development of New Biotherapeutic Antibody Modalities.

The characterization of target binding interactions is critical at each stage of antibody therapeutic development. During early development, it is important to design fit-for-purpose in vitro molecular interaction characterization (MIC) assays that accurately determine the binding kinetics and the affinity of therapeutic antibodies for their targets. Such information enables PK/PD (pharmacokinetics/pharmacodynamics) modeling, estimation of dosing regimens, and assessment of potency. While binding kinetics and affinities seem to be readily obtained, there is little discussion in the literature on how the information should be generated and used in a systematic manner along with other approaches to enable key drug development decisions. The introduction of new antibody modalities poses unique challenges to the development of MIC assays and further increases the need to discuss the impact of developing context-appropriate MIC assays to enable key decision making for these programs. In this paper, we discuss for the first time the challenges encountered when developing MIC assays supporting new antibody modalities. Additionally, through the presentation of several real case studies, we provide strategies to overcome these challenges to enable investigational new drug (IND) filings.

Changes in Manufacturing Processes of Biologic Therapies Can Alter the Immunogenicity Profile of the Product.

Manufacturing process changes may alter the characteristics of a protein therapeutic. In 2009, somatropin (version 1.0), a recombinant human growth hormone therapeutic, underwent a manufacturing update (version 1.1). The immunogenicity of somatropin version 1.1 as a daily subcutaneous injection was evaluated in 2014 in a prospective, open-label, single-arm clinical study of treatment-naive pediatric patients with idiopathic human growth hormone deficiency for 1 year. The primary end point was the proportion of patients who developed antidrug antibodies (ADAs) after treatment. Eighty-two patients were enrolled. The mean (SD) treatment duration was 347 (53) days. The incidence of ADAs was 3.7%. No neutralizing antibodies were observed in the three patients with ADA-positive samples. Two patients (2.6%) had growth attenuation, but they were not ADA positive. The manufacturing changes for somatropin version 1.1 resulted in a similar safety and efficacy profile compared with somatropin version 1.0 and a different immunogenicity profile with a lower incidence of ADAs.

Quantitative determination of humanized monoclonal antibody rhuMAb2H7 in cynomolgus monkey serum using a Generic Immunoglobulin Pharmacokinetic (GRIP) assay.

Preclinical pharmacokinetic (PK) assays are important to help evaluate the safety and efficacy of a potential biotherapeutic before clinical studies. The assay typically requires a biotherapeutic-specific reagent to minimize matrix effects especially when the host species are non-human primates such as cynomolgus monkeys and the biotherapeutic is a humanized monoclonal antibody (MAb). Recombinant humanized mAb 2H7 (rhuMAb2H7) binds to the extracellular domain of CD20 that is expressed on B cells and results in B cell depletion. It is currently being evaluated for its therapeutic potential in rheumatoid arthritis (RA) in clinical studies. During the early development of rhuMAb2H7, a cynomolgus monkey PK assay was needed to help assess the pharmacokinetic parameters of rhuMAb2H7 in a pilot cynomolgus monkey study. However, development of a cynomolgus monkey PK assay was challenging due to lack of rhuMAb2H7-specific reagents. Here we describe an alternative method for detection of rhuMAb2H7 in cynomolgus monkey serum using polyclonal antibodies against human IgGs. This assay quantifies rhuMAb2H7 in 10% cynomolgus monkey serum with high sensitivity, accuracy, and precision. This assay successfully supported the rhuMAb2H7 development, and has the potential to be used to quantify other humanized MAb biotherapeutics in serum from a variety of non-human species.

Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products.

The appropriate evaluation of the immunogenicity of biopharmaceuticals is of major importance for their successful development and licensure. Antibodies elicited by these products in many cases cause no detectable clinical effects in humans. However, antibodies to some therapeutic proteins have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In addition, antibodies can affect drug efficacy. In non-clinical studies, anti-drug antibodies (ADA) can complicate interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, it is important to develop testing strategies that provide valid assessments of antibody responses in both non-clinical and clinical studies. This document provides recommendations for antibody testing strategies stemming from the experience of contributing authors. The recommendations are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry. The strategies proposed are also expected to contribute to better understanding of antibody responses and to further advance immunogenicity evaluation.

Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products.

Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.

Impact of SPR biosensor assay configuration on antibody: Neonatal Fc receptor binding data.

Binding interactions with the neonatal Fc receptor (FcRn) are one determinant of pharmacokinetic properties of recombinant human monoclonal antibody (rhumAb) therapeutics, and a conserved binding motif in the crystallizable fragment (Fc) region of IgG molecules interacts with FcRn. Surface plasmon resonance (SPR) biosensor assays are often used to characterize interactions between FcRn and rhumAb therapeutics. In such assays, generally either the rhumAb (format 1) or the FcRn protein (format 2) is immobilized on a biosensor chip. However, because evidence suggests that, in some cases, the variable domains of a rhumAb may also affect FcRn binding, we evaluated the effect of SPR assay configuration on binding data. We sought to assess FcRn binding properties of 2 rhumAbs (rhumAb1 and rhumAb2) to FcRn proteins using these 2 biosensor assay formats. The two rhumAbs have greater than 99% sequence identity in the Fc domain but differ in their Fab regions. rhumAb2 contains a positively charged patch in the variable domain that is absent in rhumAb1. Our results showed that binding of rhumAb1 to FcRn was independent of biosensor assay configuration, while binding of rhumAb2 to FcRn was highly SPR assay configuration dependent. Further investigations revealed that the format dependency of rhumAb2-FcRn binding is linked to the basic residues that form a positively charged patch in the variable domain of rhumAb2. Our work highlights the importance of analyzing rhumAb-FcRn binding interactions using 2 alternate SPR biosensor assay configurations. This approach may also provide a simple way to identify the potential for non-Fc-driven FcRn binding interactions in otherwise typical IgGs.

Advancements in Understanding Immunogenicity of Biotherapeutics in the Intraocular Space.

Therapeutic breakthroughs in a number of retinal degenerative diseases have come about through the development of biotherapeutics administered directly into the eye. As a consequence of their use, we have gained more insight into the immune privileged status of the eye and the various considerations that development, manufacturing, and use of these drugs require. It has been observed that therapeutic proteins injected into the vitreous can elicit an immune response resulting in the production of anti-drug antibodies (ADAs) which can have clinical consequences. This review includes discussion of the anatomy, physiology, and specific area of the eye that are targeted for drug administration. The various immunologic mechanisms involved in the immune responses to intraocularly administered protein are discussed. This review entails discussion on chemistry, manufacturing, and control (CMC) and formulation-related issues that may influence the risk of immunogenicity. Based on the available immunogenicity profile of the marketed intraocular drugs and their reported adverse events, the animal models and the translational gap from animals to human are discussed. Thus, the objective of this review article is to assess the factors that influence immunogenicity in relation to intraocular administration and the steps taken for mitigating immunogenicity risks.

Strategies to compare clinical antitherapeutic antibody data when changing assay platforms: a case study.

Zhihua Julia Qiu has over 20 years post PhD experience in academic institutes, pharmaceutical industry and biotechnology startup settings; focused on novel therapeutics discovery and development and diagnostic tools. She is currently a Scientist in the Bioanalytical Sciences department at Genentech; responsible for developing, evaluating and implementing Bioanalytical strategy to support protein therapeutics development. That includes assay development and validation to evaluate PK, antitherapeutic antibodies as well as biomarkers in both nonclinical and clinical studies for Immunology and Oncology indications. In addition, she has led the evaluation of multiple novel technology platforms and transitioning assay platform to enable continuous support for the development of protein therapeutics and antibody-drug conjugates.

Evaluation of total IGF-I assay methods using samples from Type I and Type II diabetic patients.

Measurements of circulating insulin-like growth factor-I (IGF-I) levels are an important part of many studies on growth and development. Circulating IGF-I levels are growth hormone (GH) dependent and are also impacted by age, gender, nutritional status and disease. Moreover, IGF-I is the main pharmacodynamic marker of GH activity. The majority of circulating IGF-I is associated with high affinity insulin-like growth factor-binding proteins (IGFBPs), making accurate and precise measurements of total IGF-I concentrations in biological matrices technically challenging. Many total IGF-I assay methods combine an immunoassay with a sample preparation method aimed at removing IGFBPs. However, not all sample preparation methods efficiently remove all IGFBPs or BP fragments (BPFs), and there is currently no reference method for IGF-I measurement against which these IGF-I assays can be calibrated. We have evaluated a number of IGF-I immunoassays and sample preparation methods using plasma samples from normal donors and from donors with Type I and Type II diabetes mellitus. In order to eliminate the variability between assays due to differences in assay standardization, we used the same preparation of highly pure, fully characterized IGF-I as the standard for all assays. We found that the data produced by many of the IGF-I assay methods showed good agreement when IGF-I levels in samples from normal individuals were measured. However, we found that these agreements were quite poor when IGF-I levels in samples from diabetics were measured. This was true of methods that claimed to physically separate IGFBPs from IGF-I either by acid/ethanol extraction or by acid chromatography. Several methods have recently been developed that physically separate IGF-I from IGFBPs followed by a chemical displacer to displace any residual BPs or BPFs from IGF-I. We found that the data generated by these displacement methods showed good agreement when assaying samples from diabetic as well as normal donors. There is considerable discussion in the literature as to whether individuals with diabetes have normal circulating levels of IGF-I. Many of the published studies are based on assays that may not accurately measure IGF-I levels due to problems with assay standardization and/or with assay methodology. Displacement methods may enable us to more accurately measure IGF-I levels in diabetes.

New FDA draft guidance on immunogenicity.

A "Late Breaking" session was held on May 20 at the 2013 American Association of Pharmaceutical Scientists-National Biotech Conference (AAPS-NBC) to discuss the US Food and Drug Administration's (FDA) 2013 draft guidance on Immunogenicity Assessment for Therapeutic Protein Products. The session was initiated by a presentation from the FDA which highlighted several key aspects of the 2013 draft guidance pertaining to immunogenicity risk, the potential impact on patient safety and product efficacy, and risk mitigation. This was followed by an open discussion on the draft guidance which enabled delegates from biopharmaceutical companies to engage the FDA on topics that had emerged from their review of the draft guidance. The multidisciplinary audience fostered an environment that was conducive to scientific discussion on a broad range of topics such as clinical impact, immune mitigation strategies, immune prediction and the role of formulation, excipients, aggregates, and degradation products in immunogenicity. This meeting report highlights several key aspects of the 2013 draft guidance together with related dialog from the session.

Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.