Application of blood microsampling in cynomolgus monkey and demonstration of equivalent monoclonal antibody PK parameters compared to conventional sampling.

PURPOSE: The purpose of this study was to evaluate the suitability of whole blood microsampling procedures in non-human primate (NHP) to support toxicokinetic assessments of biotherapeutics in non-human primates. METHOD: A one-month single dose intravenous pharmacokinetic (PK) study was performed in male cynomolgus monkeys with a human IgG1 control monoclonal antibody (mAb) as a surrogate monoclonal antibody biotherapeutic. In this study, both serum samples (conventional sample collection) and microsampling samples were collected. Microsampling samples were collected from two sites on cynomolgus monkey, with each site using two different devices for the whole blood collection. The drug concentrations from all sample types were determined using a quantitative ligand binding assay (LBA). The PK parameters obtained from microsampling samples and serum samples were examined using a standard PK analysis method. The comparability of key PK parameters from both sample types were analyzed statistically. RESULTS: Similar profiles of drug concentrations versus timepoints from all sampling procedures were observed. The correlations of PK concentration data obtained from serum and microsampling samples were ≥ 0.97 using Brand Alman Plot analysis. The key PK parameters obtained from microsampling samples were comparable to those obtained from serum samples (the % differences of mean PK parameters obtained from both sample types were within ±25%). CONCLUSION: This study confirmed that PK parameters obtained from samples using microsampling were comparable to that of serum samples in cynomolgus monkeys. Therefore, the microsampling procedure described can be used as a substitute for conventional sampling procedure to support PK/TK studies of biotherapeutics in non-clinical product developments.

Fit-for-Purpose Validation of a Ligand Binding Assay for Toxicokinetic Study Using Mouse Serial Sampling.

PURPOSE: The purpose of this study was to validate a ligand binding assay for the quantitation of a monoclonal antibody-based biotherapeutics (PF-57781346) in samples collected via capillary microsampling to support a regulated mouse toxicity study. METHOD: A quantitative ligand binding assay on the Gyrolab platform was developed to quantify PF-57781346 in blood samples derived from capillary mouse serial sampling. The method validation evaluated assay characteristics including accuracy and precision, influence of sample processing on drug quantitation, whole blood matrix selectivity, dilution linearity and the stability of the drug in the study sample matrix. RESULTS: The method validation demonstrated acceptable analytical characteristics. The whole blood selectivity testing demonstrated accuracy between -4.8% and 13.9% in 10 out of 10 individual whole blood samples, suggesting that drug quantitation from whole blood is not impacted by the serial sampling procedure. Short-term and long-term drug stability in study sample matrix were established to cover required stability for sample storage and analysis (accuracy between -7.3% and 6.1%). CONCLUSION: We reported a successful validation of a bioanalytical method that quantifies PF-55781346 in samples collected via capillary microsampling. The experience shared in this study could serve as a model process for bioanalytical method validation when capillary microsampling is used.

Preexisting antibody assays for gene therapy: Considerations on patient selection cutoffs and companion diagnostic requirements.

Recombinant adeno-associated virus (AAV) vectors are the leading delivery vehicle used for in vivo gene therapies. Anti-AAV antibodies (AAV Abs) can interact with the viral capsid component of an AAV-based gene therapy (GT). Therefore, patients with preexisting AAV Abs (seropositive patients) are often excluded from GT trials to prevent treatment of patients who are unlikely to benefit1 or may have a higher risk for adverse events outweighing treatment benefits. On the contrary, unnecessary exclusion of patients with high unmet medical need should be avoided. Instead, a risk-benefit assessment that weighs the potential risks due to seropositivity vs. severity of disease and available treatment options, should drive the decision if patient selection is required. Assays for patient selection must be validated according to their intended use following national regulations/standards for diagnostic assays in appropriate laboratories. In this review, we summarize the current process of patient selection, including assay cutoff criteria and related assay validation approaches. We further provide considerations on regulatory requirements for the development of in vitro diagnostic tests supporting market authorization of a corresponding GT.

Proposed mechanism of off-target toxicity for antibody-drug conjugates driven by mannose receptor uptake.

Antibody-drug conjugates (ADCs) are developed with the goal of increasing compound therapeutic index by specific and targeted delivery of a toxic payload to the site of action while considerably reducing damage to normal tissues. Yet, off-target hepatic toxicities have been reported for several ADC. Locations of these off-target toxicities coincide with the reported locations of cell surface mannose receptor (MR). The relative proportion of agalactosylated glycans on the Fc domain (G0F vs. G1F and G2F components) in monoclonal antibody (mAb)-based biotherapeutics is closer to some disease state IgG rather than to a normal serum-derived immunoglobulin. The lack of the terminal galactose on a G0F glycan creates an opportunity for the mAb to interact with soluble and cell surface MRs. MR is a known multi-domain lectin that specifically binds and internalizes glycoproteins and immune complexes with relatively high G0F content and has been found on the surface of various cell types, including immune cells of myeloid lineage, endothelial cells, and hepatic and splenic sinusoids. In this review paper it is proposed that the mechanism of the off-target toxicities for ADC biotherapeutics is at least in part driven by the carbohydrates, specifically agalactosylated glycans, such as G0F, their interactions with MR and resulting glycan-derived cellular uptake of ADCs. Several case studies are reviewed presenting corroborating information.

Considerations for the nonclinical safety evaluation of antibody drug conjugates for oncology.

Antibody drug conjugates (ADCs) include monoclonal antibodies that are linked to cytotoxic small molecules. A number of these agents are currently being developed as anti-cancer agents designed to improve the therapeutic index of the cytotoxin (i.e., cytotoxic small molecule or cytotoxic agent) by specifically delivering it to tumor cells. This paper presents primary considerations for the nonclinical safety evaluation of ADCs and includes strategies for the evaluation of the entire ADC or the various individual components (i.e., antibody, linker or the cytotoxin). Considerations are presented on how to design a nonclinical safety assessment program to identify the on- and off-target toxicities to enable first-in-human (FIH) studies. Specific discussions are also included that provide details as to the need and how to conduct the studies for evaluating ADCs in genetic toxicology, tissue cross-reactivity, safety pharmacology, carcinogenicity, developmental and reproductive toxicology, biotransformation, toxicokinetic monitoring, bioanalytical assays, immunogenicity testing, test article stability and the selection of the FIH dose. Given the complexity of these molecules and our evolving understanding of their properties, there is no single all-encompassing nonclinical strategy. Instead, each ADC should be evaluated on a case-by-case scientifically-based approach that is consistent with ICH and animal research guidelines.

Evolution of Antidrug Antibody Assays During the Development of Anti-Tissue Factor Pathway Inhibitor Monoclonal Antibody Marstacimab.

Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic coagulation pathway. In patients with hemophilia A or B, inhibition of TFPI is an alternative therapeutic approach that augments the extrinsic coagulation pathway. Marstacimab is an investigational fully human monoclonal antibody that binds and neutralizes TFPI and is being evaluated as a prophylactic treatment to prevent or reduce the frequency of bleeding episodes in patients with severe hemophilia A or B, with or without inhibitors (antibodies against coagulation factors). However, the efficacy, safety, and pharmacokinetics of marstacimab may be affected by the induction of antidrug antibody (ADA) responses. Here, we describe the evolution and validation of three quasi-quantitative electrochemiluminescence-based methods to detect marstacimab ADAs, starting from their use in a first-in-human phase 1 study to their use in phase 2 and 3 clinical studies of patients with severe hemophilia. For all three methods, validation criteria evaluated the performance of the assays in screening and confirmatory cut points, precision, selectivity, drug tolerance, target interference, and stability. Additional criteria for validation were dilution linearity (Methods 1 and 2) and low positive control concentration, prozone effect, plate homogeneity, and robustness (Method 3). The three methods met validation criteria and are a potentially valuable tool in detecting the induction of marstacimab ADAs during treatment in patients with hemophilia.

Evaluation of Cellular Immune Response to Adeno-Associated Virus-Based Gene Therapy.

The number of approved or investigational late phase viral vector gene therapies (GTx) has been rapidly growing. The adeno-associated virus vector (AAV) technology continues to be the most used GTx platform of choice. The presence of pre-existing anti-AAV immunity has been firmly established and is broadly viewed as a potential deterrent for successful AAV transduction with a possibility of negative impact on clinical efficacy and a connection to adverse events. Recommendations for the evaluation of humoral, including neutralizing and total antibody based, anti-AAV immune response have been presented elsewhere. This manuscript aims to cover considerations related to the assessment of anti-AAV cellular immune response, including review of correlations between humoral and cellular responses, potential value of cellular immunogenicity assessment, and commonly used analytical methodologies and parameters critical for monitoring assay performance. This manuscript was authored by a group of scientists involved in GTx development who represent several pharma and contract research organizations. It is our intent to provide recommendations and guidance to the industry sponsors, academic laboratories, and regulatory agencies working on AAV-based GTx viral vector modalities with the goal of achieving a more consistent approach to anti-AAV cellular immune response assessment.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

Determination of Anti-drug Antibody Affinity in Clinical Study Samples Provides a Tool for Evaluation of Immune Response Maturation.

Characterization of clinical anti-drug antibody (ADA) responses to biotherapeutics can be important to understanding the consequences of immunogenicity. ADA are expected to be polyclonal, with composition and affinities that evolve over time. Measuring ADA binding affinity can be complicated by the polyclonal nature of response, residual drug in sample, and low ADA levels. We developed a novel workflow to determine the apparent ADA affinity (KD) against a monoclonal antibody biotherapeutic, PF-06480605. An affinity capture elution pre-treatment step was used to isolate ADA and remove residual drug interference from samples. Solution-phase equilibrium incubation was performed using drug and sample ADA as variable and fixed binding interactants, respectively. Unbound ADA concentration was measured using a Singulex Erenna ligand-binding assay (LBA) method. Apparent ADA KD values were calculated using a custom R Shiny algorithm. KD values determined for ADA positive samples showed good correlation with other immunogenicity parameters, including titers and neutralizing antibody (NAb) activity with a general increase in affinity over time, indicative of a maturing immune response. Time of onset of high affinity responses (KD < 100 pM) varied between patients, ranging from 16 to 24 weeks. Antibody responses appeared monophasic at earlier time points, trending towards a biphasic response with a variable transition time and general increase in proportion of high affinity ADA over time. Herein, we provide a novel, sensitive bioanalytical method to determine the KD of ADA in clinical samples. The observed decrease in ADA KD is consistent with evidence of a maturing immune response.

When to Extend Monitoring of Anti-drug Antibodies for High-risk Biotherapeutics in Clinical Trials: an Opinion from the European Immunogenicity Platform.

The determination of a tailored anti-drug antibody (ADA) testing strategy is based on the immunogenicity risk assessment to allow a correlation of ADAs with changes to pharmacokinetics, efficacy, and safety. The clinical impact of ADA formation refines the immunogenicity risk assessment and defines appropriate risk mitigation strategies. Health agencies request for high-risk biotherapeutics to extend ADA monitoring for patients that developed an ADA response to the drug until ADAs return to baseline levels. However, there is no common understanding in which cases an extension of ADA follow-up sampling beyond the end of study (EOS) defined in the clinical study protocol is required. Here, the Immunogenicity Strategy Working Group of the European Immunogenicity Platform (EIP) provides recommendations on requirements for an extension of ADA follow-up sampling in clinical studies where there is a high risk of serious consequences from ADAs. The importance of ADA evaluation during a treatment-free period is recognized but the decision whether to extend ADA monitoring at a predefined EOS should be based on evaluation of ADA data in the context of corresponding clinical signals. If the clinical data set shows that safety consequences are minor, mitigated, or resolved, further ADA monitoring may not be required despite potentially detectable ADAs above baseline. Extended ADA monitoring should be centered on individual patient benefit.

2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccine Assays; Immunogenicity of Biotherapeutics and Novel Modalities; Integrated Summary of Immunogenicity Harmonization).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Feasibility of singlicate-based analysis in bridging ADA assay on Meso-Scale Discovery platform: comparison with duplicate analysis.

Aim: To investigate the feasibility of singlicate analysis in anti-drug antibody (ADA) assay by comparing performance characteristics for assays qualified in duplicate and singlicate formats. Materials & methods: We employed modeling to assess and quantify the impact of singlicate to cut point factor (CPF) in scenarios with the duplicate precision from 1-20% and the proportion of well-to-well variance to overall assay variance from 0.01-0.90. The impact to CPF by singlicate is marginal if the well-to-well coefficient of variation is <10% and represents <25% of the overall variability. Results & conclusion: The assay parameters including sensitivity, precision, selectivity, drug and target tolerance were comparable between singlicate and duplicate based assays. Our results suggested the minimal impact of singlicate analysis on ADA assay with good duplicate precision. The study provided additional supportive evidence that the singlicate-based analysis is feasible in ADA ligand binding assays.

Evaluation of the Humoral Response to Adeno-Associated Virus-Based Gene Therapy Modalities Using Total Antibody Assays.

The number of viral vector-based gene therapies (GTx) continues to grow with two products (Zolgensma® and Luxturna®) approved in the USA as of March 2021. To date, the most commonly used vectors are adeno-associated virus-based (AAV). The pre-existing humoral immunity against AAV (anti-AAV antibodies) has been well described and is expected as a consequence of prior AAV exposure. Anti-AAV antibodies may present an immune barrier to successful AAV transduction and hence negatively impact clinical efficacy and may also result in adverse events (AEs) due to the formation of large immune complexes. Patients may be screened for the presence of anti-AAV antibodies, including neutralizing (NAb) and total binding antibodies (TAb) prior to treatment with the GTx. Recommendations for the development and validation of anti-AAV NAb detection methods have been presented elsewhere. This manuscript covers considerations related to anti-AAV TAb-detecting protocols, including the advantages of the use of TAb methods, selection of assay controls and reagents, and parameters critical to monitoring assay performance. This manuscript was authored by a group of scientists involved in GTx development representing eleven organizations. It is our intent to provide recommendations and guidance to industry sponsors, academic laboratories, and regulatory agencies working on AAV-based GTx viral vector modalities with the goal of achieving a more consistent approach to anti-AAV TAb assessment. Graphical abstract.

2020 White Paper on Recent Issues in Bioanalysis: BMV of Hybrid Assays, Acoustic MS, HRMS, Data Integrity, Endogenous Compounds, Microsampling and Microbiome (Part 1 - Recommendations on Industry/Regulators Consensus on BMV of Biotherapeutics by LCMS, Advanced Application in Hybrid Assays, Regulatory Challenges in Mass Spec, Innovation in Small Molecules, Peptides and Oligos).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by Mass Spectrometry (hybrid assays, LCMS and HRMS) were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication covers the recommendations on (Part 1) Hybrid Assays, Innovation in Small Molecules, & Regulated Bioanalysis. Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation), Part 2B (Regulatory Input) and Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 5, and 6 (2021), respectively.

2020 White Paper on Recent Issues in Bioanalysis: Vaccine Assay Validation, qPCR Assay Validation, QC for CAR-T Flow Cytometry, NAb Assay Harmonization and ELISpot Validation (Part 3 - Recommendations on Immunogenicity Assay Strategies, NAb Assays, Biosimilars and FDA/EMA Immunogenicity Guidance/Guideline, Gene & Cell Therapy and Vaccine Assays).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14th WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Current Considerations for Immunoglobulin Isotype Characterization of Antibody Response against Biotherapeutics.

The ability of biotherapeutics to induce immune response in patients has been broadly accepted. Humoral immune response to biotherapeutics is expected to be polyclonal in nature with a high degree of diversity, including treatment-induced anti-drug antibodies (ADA) immunoglobulin isotype composition. Therapeutics with strong potential to induce immunity may produce a T cell-dependent response resulting in a gradual transition from initial IgM based to mature, IgG-based ADAs. Immunoglobulin class switch and transition to high affinity IgG1 and IgG4 antibodies were linked to a reduced drug efficacy, accelerated clearance, development of drug neutralizing antibodies, and modulation of hypersensitivity reaction rates. Examples presented herein demonstrate that understanding of isotype composition of ADA response can be highly important to predict future of disease progression. Isotype characterization of ADA response can be viewed highly useful, particularly for high immunogenicity risk biotherapeutics although may be less relevant or used as a research tool only for medium and low immunogenicity risk level therapeutics. Isotype-specific characteristics, methods of detection, and several case studies are presented herein.

Current Considerations on Characterization of Immune Response to Multi-Domain Biotherapeutics.

Compounds containing two or more structural domains with a distinct mode of action relevant to functionality have been defined as multi-domain biotherapeutics (MDBs). Several modalities, including endogenous protein fusions with an antibody Fc fragment or another polypeptide, bispecific antibodies, antibody-drug conjugates, as well as polyethylene glycol conjugates have been viewed as examples of MDBs. Similar to other biotherapeutics, MDBs have the potential to induce a host immune response, commonly detected in the form of anti-drug antibodies (ADAs). The need to characterize ADA specificity to a particular domain of the MDB has been identified as a potential regulatory requirement based on the compound nature of the drug and associated immunogenicity risk factors. MDB-related immunogenicity risk factors are discussed herein. The relative risk level of each of the immunogenicity factors was analyzed based on publicly available information. It is proposed that MDB-related immunogenicity risk factors can be divided into major and minor categories. Major risk category factors include (a) presence of immunogenic structural or linear epitopes of either non-human or human sequence origin and (b) significant homology of an MDB domain to an endogenous protein with a specific and unique function. Proposed minor risk category factors include (a) epitope spread, (b) repetitive antigenic structure of MDB, and (c) hapten-like effect due to chemical conjugation or fusion with a larger protein. Detailed modality-based information on several examples of MDBs is presented to support this proposal.

Development of a Highly Specific Anti-drug Antibody Assay in Support of a Nanoparticle-based Therapeutic.

PEGylated biotherapeutics can elicit anti-PEG (polyethylene glycol) immune responses in patients treated with this category of drugs. While anti-PEG antibody assays for this class of biotherapeutics have become a common element of the clinical immunogenicity testing strategy, the overall antibody incidence induced by the nanoparticle (NP) delivery system (such as ACCURINS®) has not been fully studied to date. To support the immunogenicity assessment of one of Pfizer's NP-based therapeutics, consisting of gedatolisib (GEDA) encapsulated in ACCURINS® (GEDA-NP), we developed an anti-GEDA-NP antibody (ADA) assay on the MSD platform for the detection of GEDA-NP induced ADA in human serum. The focus of our strategy was on developing a clinically relevant ADA assay and systematically addressing assay interference through rigorous assay optimization. Our efforts led to a fit-for-purpose assay for the detection of anti-GEDA-NP ADA in serum samples obtained from breast cancer patients. Results from method qualification indicated robust assay performance, as highlighted by inter and intra-assay precision within 25% CV for all controls, and reproducible response profiles across multiple runs during the assessment of assay cut points with breast cancer samples. The assay sensitivity was between 4.3 ng/mL and 123 ng/mL for surrogate positive controls of IgG and IgM isotypes, respectively. Additionally, assay interference from nonspecific matrix proteins and circulating drug was addressed, which ensured accurate assessment of ADA incidence that can be attributed to GEDA-NP.

Development of a Cell-Based Assay for the Detection of Neutralizing Antibodies to PF-06730512 Using Homogenous Time-Resolved Fluorescence.

The administration of biotherapeutics has the potential to induce potent immune responses. Among these responses, the production of anti-drug antibodies (ADA), including a subset of ADA referred to as neutralizing antibodies (NAb), is of heightened concern. Aside from their capacity to alter the pharmacological profile of a given biotherapeutic, NAb can also pose significant safety risks, especially in instances where an endogenous counterpart to the drug exists. As such, the inclusion of an assay to detect NAb in clinical samples is critical to the effectiveness of a tiered approach to immunogenicity assessment. PF-06730512 is a biotherapeutic protein being developed for the treatment of primary Focal Segmental Glomerulosclerosis (FSGS). To support the immunogenicity assessment of PF-06730512, a cell-based assay was developed for the detection of NAb in FSGS serum samples. Herein, we describe the development of the assay with a focus on the challenges faced, including drug and blood collection tube interferences in NAb detection. The outcome of our efforts was a robust assay capable of detecting 1 µg/mL of a NAb positive control in the presence of clinically relevant drug concentrations up to 30 µg/mL.

Recommendations for the Development of Cell-Based Anti-Viral Vector Neutralizing Antibody Assays.

Viral vector-based gene therapies (GTx) have received significant attention in the recent years and the number of ongoing GTx clinical trials is increasing. A platform of choice for many of these studies is adeno-associated virus (AAV). All humans may be exposed to natural AAV infections and could mount an immune response against the virus. Consequently, there can be a high prevalence of pre-existing anti-AAV immunity. This presents a potential limitation for AAV-based GTx due to the potential for AAV-specific antibodies to reduce the efficacy of the GTx. Therefore, appropriate assessment of potential subjects enrolled in these studies should include evaluation for the presence and degree of anti-AAV immunity, including anti-AAV neutralizing antibodies (NAb). Recommendations for the development and validation of cell-based anti-AAV NAb detection methods, including considerations related to selection of appropriate cell line, surrogate vector/reporter gene, assay matrix and controls, and methodologies for calculating assay cut-point are discussed herein. General recommendations for the key assay validation parameters are provided as well as considerations for the development of NAb diagnostic tests. This manuscript is produced by a group of scientists involved in GTx therapeutic development representing various companies. It is our intent to provide recommendations and guidance to industrial and academic laboratories working on viral vector based GTx modalities with the goal of achieving a more consistent approach to anti-AAV NAb assessment.