Determining the Degree of Sulfo-tag Conjugation to AAV5 Vectors by LC-HRMS and Evaluating the Effects on Antibody Binding Affinity and Bridging Assay Sensitivity.

Pre-existing anti-AAV antibodies can be detected using ligand binding-based assay formats. One such format is the MSD-based bridging assay, which uses sulfo-tag-labeled AAV vectors as detection reagents. However, no method has been developed to accurately measure the degree of sulfo-tag labeling on AAV vectors. To fill this gap, we developed a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method to assess the degree of labeling (DoL) of sulfo-tag on AAV5 vectors, enabling the measurement of the DoL on AAV5 at six increasing levels of sulfo-tag challenge ratio. In addition, a Biacore-based assay was used to evaluate the binding affinity between an anti-AAV5 monoclonal antibody and various sulfo-tag labeled AAV5 vectors. The results indicated that increased DoL of sulfo-tag labeling on AAV5 did not compromise the binding affinity.Our study further employed the MSD-bridging assay to detect the binding Signal/Noise (S/N) ratios of four anti-AAV5 monoclonal antibodies (mAbs) to various sulfo-tag-labeled AAV5 vectors. The findings revealed a strong correlation between the degree of sulfo-tag labeling and both the S/N ratios and the sensitivity of MSD bridging assays. This result underscores the importance of optimizing the labeling of detection reagents to enhance assay sensitivity for detecting anti-AAV5 antibodies.

Industry Perspective on First-in-Human and Clinical Pharmacology Strategies to Support Clinical Development of T-Cell Engaging Bispecific Antibodies for Cancer Therapy.

T-cell-engaging bispecific antibodies (TCEs) that target tumor antigens and T cells have shown great promise in treating cancer, particularly in hematological indications. The clinical development of TCEs often involves a lengthy first-in-human (FIH) trial with many dose-escalation cohorts leading up to an early proof of concept (POC), enabling either a no-go decision or dose selection for further clinical development. Multiple factors related to the target, product, disease, and patient population influence the efficacy and safety of TCEs. The intricate mechanism of action limits the translatability of preclinical models to the clinic, thereby posing challenges to streamline clinical development. In addition, unlike traditional chemotherapy, the top dose and recommended phase II doses (RP2Ds) for TCEs in the clinic are often not guided by the maximum tolerated dose (MTD), but rather based on the integrated dose-response assessment of the benefit/risk profile. These uncertainties pose complex challenges for translational and clinical pharmacologists (PK/PD scientists), as well as clinicians, to design an efficient clinical study that guides development. To that end, experts in the field, under the umbrella of the American Association of Pharmaceutical Scientists, have reviewed learnings from published literature and currently marketed products to share perspectives on the FIH and clinical pharmacology strategies to support early clinical development of TCEs.

Applications of Organoids in Advancing Drug Discovery and Development.

Organoids are small, self-organizing three-dimensional cell cultures that are derived from stem cells or primary organs. These cultures replicate the complexity of an organ, which cannot be achieved by single-cell culture systems. Organoids can be used in testing of new drugs instead of animals. Development and validation of organoids is thus important to reduce the reliance on animals for drug testing. In this review, we have discussed the developmental and regulatory aspects of organoids and highlighted their importance in drug development. We have first summarized different types of culture-based organoid systems such as submerged Matrigel, micro-fluidic 3D cultures, inducible pluripotent stem cells, and air-liquid interface cultures. These systems help us understand the intricate interplay between cells and their surrounding milieu for identifying functions of target receptors, soluble factors, and spatial interactions. Further, we have discussed the advances in humanized severe-combined immunodeficiency mouse models and their applications in the pharmacology of immune-oncology. Since regulatory aspects are important in using organoids for drug development, we have summarized FDA and EMA regulations on organoid research to support pre-clinical studies. Finally, we have included some unique studies highlighting the use of organoids in studying infectious diseases, cancer, and fundamental biology. These studies also exemplify the latest technological advances in organoid development resulting in improved efficiency. Overall, this review comprehensively summarizes the applications of organoids in early drug development during discovery and pre-clinical studies.

Immunogenicity Risk Assessment of Process-Related Impurities in An Engineered T Cell Receptor Cellular Product.

Cell therapies such as genetically modified T cells have emerged as a promising and viable treatment for hematologic cancers and are being aggressively pursued for a wide range of diseases and conditions that were previously difficult to treat or had no cure. The process development requires genetic modifications to T cells to express a receptor (engineered T cell receptor (eTCR)) of specific binding qualities to the desired target. Protein reagents utilized during the cell therapy manufacturing process, to facilitate these genetic modifications, are often present as process-related impurities at residual levels in the final drug product and can represent a potential immunogenicity risk upon infusion. This manuscript presents a framework for the qualification of an assay for assessing the immunogenicity risk of AA6 and Cas9 residuals. The same framework applies for other residuals; however, AAV6 and Cas9 were selected as they were residuals from the manufacturing of an engineered T cell receptor cellular product in development. The manuscript: 1) elucidates theoretical risks, 2) summarizes analytical data collected during process development, 3) describes the qualification of an in vitro human PBMC cytokine release assay to assess immunogenicity risk from cellular product associated process residuals; 4) identifies a multiplexed inflammatory innate and adaptive cytokine panel with pre-defined criteria using relevant positive controls; and 5) discusses qualification challenges and potential solutions for establishing meaningful thresholds. The assessment is not only relevant to establishing safe exposure levels of these residuals but also in guiding risk assessment and CMC strategy during the conduct of clinical trials.

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.

A High Threshold of Biotherapeutic Aggregate Numbers is Needed to Induce an Immunogenic Response In Vitro, In Vivo, and in the Clinic.

BACKGROUND AND PURPOSE: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses. METHODS AND RESULTS: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials. CONCLUSION: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products.

An IQ Consortium Perspective on Best Practices for Bioanalytical and Immunogenicity Assessment Aspects of CAR-T and TCR-T Cellular Therapies Development.

CAR-T therapies have shown remarkable efficacy against hematological malignancies in the clinic over the last decade and new studies indicate that progress is being made to use these novel therapies to target solid tumors as well as treat autoimmune disease. Innovation in the field, including TCR-T, allogeneic or "off the shelf" CAR-T, and autoantigen/armored CAR-Ts are likely to increase the efficacy and applications of these therapies. The unique aspects of these cell-based therapeutics; patient-derived cells, intracellular expression, in vivo expansion, and phenotypic changes provide unique bioanalytical challenges to develop pharmacokinetic and immunogenicity assessments. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) Translational and ADME Sciences Leadership Group (TALG) has brought together a group of industry experts to discuss and consider these challenges. In this white paper, we present the IQ consortium perspective on the best practices and considerations for bioanalytical and immunogenicity aspects toward the optimal development of CAR-T and TCR-T cell therapies.

PK/PD and Bioanalytical Considerations of AAV-Based Gene Therapies: an IQ Consortium Industry Position Paper.

Interest and efforts to use recombinant adeno-associated viruses (AAV) as gene therapy delivery tools to treat disease have grown exponentially. However, gaps in understanding of the pharmacokinetics/pharmacodynamics (PK/PD) and disposition of this modality exist. This position paper comes from the Novel Modalities Working Group (WG), part of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ). The pan-industry WG effort focuses on the nonclinical PK and clinical pharmacology aspects of AAV gene therapy and related bioanalytical considerations.Traditional PK concepts are generally not applicable to AAV-based therapies due to the inherent complexity of a transgene-carrying viral vector, and the multiple steps and analytes involved in cell transduction and transgene-derived protein expression. Therefore, we explain PK concepts of biodistribution of AAV-based therapies and place key terminologies related to drug exposure and PD in the proper context. Factors affecting biodistribution are presented in detail, and guidelines are provided to design nonclinical studies to enable a stage-gated progression to Phase 1 testing. The nonclinical and clinical utility of transgene DNA, mRNA, and protein analytes are discussed with bioanalytical strategies to measure these analytes. The pros and cons of qPCR vs. ddPCR technologies for DNA/RNA measurement and qualitative vs. quantitative methods for transgene-derived protein are also presented. Last, best practices and recommendations for use of clinical and nonclinical data to project human dose and response are discussed. Together, the manuscript provides a holistic framework to discuss evolving concepts of PK/PD modeling, bioanalytical technologies, and clinical dose selection in gene therapy.

Best Practices and Considerations for Clinical Pharmacology and Pharmacometric Aspects for Optimal Development of CAR-T and TCR-T Cell Therapies: An Industry Perspective.

With the promise of a potentially "single dose curative" paradigm, CAR-T cell therapies have brought a paradigm shift in the treatment and management of hematological malignancies. Both CAR-T and TCR-T cell therapies have also made great progress toward the successful treatment of solid tumor indications. The field is rapidly evolving with recent advancements including the clinical development of "off-the-shelf" allogeneic CAR-T therapies that can overcome the long and difficult "vein-to-vein" wait time seen with autologous CAR-T therapies. There are unique clinical pharmacology, pharmacometric, bioanalytical, and immunogenicity considerations and challenges in the development of these CAR-T and TCR-T cell therapies. Hence, to help accelerate the development of these life-saving therapies for the patients with cancer, experts in this field came together under the umbrella of International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) to form a joint working group between the Clinical Pharmacology Leadership Group (CPLG) and the Translational and ADME Sciences Leadership Group (TALG). In this white paper, we present the IQ consortium perspective on the best practices and considerations for clinical pharmacology and pharmacometric aspects toward the optimal development of CAR-T and TCR-T cell therapies.

Bioanalytical Methods for Characterization of CAR-T Cellular Kinetics: Comparison of PCR Assays and Matrices.

Recently, multiple chimeric antigen receptor T-cell (CAR-T)-based therapies have been approved for treating hematological malignancies, targeting CD19 and B-cell maturation antigen. Unlike protein or antibody therapies, CAR-T therapies are "living cell" therapies whose pharmacokinetics are characterized by expansion, distribution, contraction, and persistence. Therefore, this unique modality requires a different approach for quantitation compared with conventional ligand binding assays implemented for most biologics. Cellular (flow cytometry) or molecular assays (polymerase chain reaction (PCR)) can be deployed with each having unique advantages and disadvantages. In this article, we describe the molecular assays utilized: quantitative PCR (qPCR), which was the initial platform used to estimate transgene copy numbers and more recently droplet digital PCR (ddPCR) which quantitates the absolute copy numbers of CAR transgene. The comparability of the two methods in patient samples and of each method across different matrices (isolated CD3+ T-cells or whole blood) was also performed. The results show a good correlation between qPCR and ddPCR for the amplification of same gene in clinical samples from a CAR-T therapy trial. In addition, our studies show that the qPCR-based amplification of transgene levels was well-correlated, independent of DNA sources (either CD3+ T-cells or whole blood). Our results also highlight that ddPCR can be a better platform for monitoring samples at the early phase of CAR-T dosing prior to expansion and during long-term monitoring as they can detect samples with very low copy numbers with high sensitivity, in addition to easier implementation and sample logistics.

Comparison of Pre-existing Anti-AAV8 Total Antibody Screening and Confirmatory Assays with a Cell-Based Neutralizing Assay in Normal Human Serum.

Pre-existing adeno-associated viruses (AAV) neutralizing antibodies (NAb) can prevent AAV vectors from transducing target tissues. The immune responses can include binding/total antibodies (TAb) and neutralizing antibodies (NAb). This study is aimed at comparing total antibody assay (TAb) and cell-based NAb assay against AAV8 to help inform the best assay format for patient exclusion criteria. We developed a chemiluminescence-based enzyme-linked immunosorbent assay to analyze AAV8 TAb in human serum. The specificity of AAV8 TAb was determined using a confirmatory assay. A COS-7-based assay was used to analyze anti-AAV8 NAbs. The TAb screening cut point factor was determined to be 2.65, and the confirmatory cut point (CCP) was 57.1%. The prevalence of AAV8 TAb in 84 normal subjects was 40%, of which 24% were NAb positive and 16% were NAb negative. All NAb-positive subjects were confirmed to be TAb-positive and also passed the CCP-positive criteria. All 16 NAb-negative subjects did not pass the CCP criterion for the positive specificity test. There was a high concordance between AAV8 TAb confirmatory assay and NAb assay. The confirmatory assay improved the specificity of the TAb screening test and confirmed neutralizing activity. We proposed a tiered assay approach, in which an anti-AAV8 screening assay should be followed by a confirmatory assay during pre-enrollment for patient exclusions for AAV8 gene therapy. This approach can be used in lieu of developing a NAb assay and can be also implemented as a companion diagnostic assay for post-marketing seroreactivity assessments due to ease of development and use.

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.

Immunogenicity assessment of AAV-based gene therapies: An IQ consortium industry white paper.

Immunogenicity has imposed a challenge to efficacy and safety evaluation of adeno-associated virus (AAV) vector-based gene therapies. Mild to severe adverse events observed in clinical development have been implicated with host immune responses against AAV gene therapies, resulting in comprehensive evaluation of immunogenicity during nonclinical and clinical studies mandated by health authorities. Immunogenicity of AAV gene therapies is complex due to the number of risk factors associated with product components and pre-existing immunity in human subjects. Different clinical mitigation strategies have been employed to alleviate treatment-induced or -boosted immunogenicity in order to achieve desired efficacy, reduce toxicity, or treat more patients who are seropositive to AAV vectors. In this review, the immunogenicity risk assessment, manifestation of immunogenicity and its impact in nonclinical and clinical studies, and various clinical mitigation strategies are summarized. Last, we present bioanalytical strategies, methodologies, and assay validation applied to appropriately monitor immunogenicity in AAV gene therapy-treated subjects.

Prevalence of Pre-Existing Neutralizing Antibodies Against Adeno-Associated Virus Serotypes 1, 2, 5, 6, 8, and 9 in Sera of Different Pig Strains.

Pre-existing neutralizing antibodies (NAb) to adeno-associated virus (AAV) may diminish the efficacy of AAV-based therapies depending on the titer. To support gene therapy studies in pigs, the seroprevalence of NAb to AAV1, 2, 5, 6, 8, and 9 serotypes were assessed in the sera of 3 different strains of pigs consisting of 60 Norsvin Topigs-20 strain, 22 Gottingen minipigs, and 40 Yucatan minipigs. Cell-based NAb assays were developed for various AAV serotypes. The sera were tested for NAb in a Lec-2 cell line for AAV9 vector and in a COS-7 cell line for the other AAV serotypes. In the 60 Topigs-20 strain 2 to 4 years of age, 100% were positive for AAV2 NAb, 45% positive for AAV6 NAb, and ∼20% positive for each of AAV1, 5, 8, and 9 NAb. These data showed that ∼80% of Norsvin Topigs-20 pigs evaluated were seronegative for pre-existing NAb to the AAV1, 5, 8, and 9 serotypes, respectively. In 22 Gottingen minipigs at 5-6 months of age, serum AAV serotype-specific NAb coexisted with that of various other AAV serotypes at 32% to 46% between two serotypes. These results suggested that coexisting NAb resulted either from multiple AAV serotype coinfection or from one (or more) serotypes that can crossreact with other AAV serotypes in some minipigs. Among the 40 Yucatan minipigs, 20 of the minipigs were <3 months old and were all negative for NAb against AAV5, 8, and 9, and only one of these 20 pigs was positive to AAV1 and 6. We further determined the titers in those positive pigs and found most Gottingen minipigs had low titer at 1:20, whereas some of Topigs-20 pigs had titers between 1:80 and 1:320, and some of Yucatan pigs had titers between 1:160 and 1:640. These results suggested that the majority of the pigs in the three strains would be amenable to gene therapy study using AAV1, AAV5, AAV8, and AAV9 and that prescreening on circulating AAV antibodies could be helpful before inclusion of pigs into studies.

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.

T-Cell Dependent Immunogenicity of Protein Therapeutics Pre-clinical Assessment and Mitigation-Updated Consensus and Review 2020.

Immune responses to protein and peptide drugs can alter or reduce their efficacy and may be associated with adverse effects. While anti-drug antibodies (ADA) are a standard clinical measure of protein therapeutic immunogenicity, T cell epitopes in the primary sequences of these drugs are the key drivers or modulators of ADA response, depending on the type of T cell response that is stimulated (e.g., T helper or Regulatory T cells, respectively). In a previous publication on T cell-dependent immunogenicity of biotherapeutics, we addressed mitigation efforts such as identifying and reducing the presence of T cell epitopes or T cell response to protein therapeutics prior to further development of the protein therapeutic for clinical use. Over the past 5 years, greater insight into the role of regulatory T cell epitopes and the conservation of T cell epitopes with self (beyond germline) has improved the preclinical assessment of immunogenic potential. In addition, impurities contained in therapeutic drug formulations such as host cell proteins have also attracted attention and become the focus of novel risk assessment methods. Target effects have come into focus, given the emergence of protein and peptide drugs that target immune receptors in immuno-oncology applications. Lastly, new modalities are entering the clinic, leading to the need to revise certain aspects of the preclinical immunogenicity assessment pathway. In addition to drugs that have multiple antibody-derived domains or non-antibody scaffolds, therapeutic drugs may now be introduced via viral vectors, cell-based constructs, or nucleic acid based therapeutics that may, in addition to delivering drug, also prime the immune system, driving immune response to the delivery vehicle as well as the encoded therapeutic, adding to the complexity of assessing immunogenicity risk. While it is challenging to keep pace with emerging methods for the preclinical assessment of protein therapeutics and new biologic therapeutic modalities, this collective compendium provides a guide to current best practices and new concepts in the field.

Assessment of romiplostim immunogenicity in adult patients in clinical trials and in a global postmarketing registry.

Antibodies to first-generation recombinant thrombopoietin (TPO) neutralized endogenous TPO and caused thrombocytopenia in some healthy subjects and chemotherapy patients. The second-generation TPO receptor agonist romiplostim, having no sequence homology to TPO, was developed to avoid immunogenicity. This analysis examined development of binding and neutralising antibodies to romiplostim or TPO among adults with immune thrombocytopenia (ITP) in 13 clinical trials and a global postmarketing registry. 60/961 (6·2%) patients from clinical trials developed anti-romiplostim-binding antibodies post-baseline. The first positive binding antibody was detected 14 weeks (median) after starting romiplostim, at median romiplostim dose of 2 µg/kg and median platelet count of 29.5 × 109 /l; most subjects had ≥98·5% of platelet assessments showing response. Neutralising antibodies to romiplostim developed in 0·4% of patients, but were unrelated to romiplostim dose and did not affect platelet count. Thirty-three patients (3·4%) developed anti-TPO-binding antibodies; none developed anti-TPO-neutralising antibodies. In the global postmarketing registry, 9/184 (4·9%) patients with spontaneously submitted samples had binding antibodies. One patient with loss of response had anti-romiplostim-neutralising antibodies (negative at follow-up). Collectively, anti-romiplostim-binding antibodies developed infrequently. In the few patients who developed neutralising antibodies to romiplostim, there was no cross-reactivity with TPO and no associated loss of platelet response.

Silicone Oil Particles in Prefilled Syringes With Human Monoclonal Antibody, Representative of Real-World Drug Products, Did Not Increase Immunogenicity in In Vivo and In Vitro Model Systems.

Silicone oil is a lubricant for prefilled syringes (PFS), a common primary container for biotherapeutics. Silicone oil particles (SiOP) shed from PFS are a concern for patients due to their potential for increased immunogenicity and therefore also of regulatory concern. To address the safety concern in a context of manufacturing and distribution of drug product (DP), SiOP was increased (up to ∼25,000 particles/mL) in PFS filled with mAb1, a fully human antibody drug, by simulated handling of DP mimicked by drop shock. These samples are characterized in a companion report (Jiao N et al. J Pharm Sci. 2020). The risk of immunogenicity was then assessed using in vitro and in vivo immune model systems. The impact of a common DP excipient, polysorbate 80, on both the formation and biological consequences of SiOP was also tested. SiOP was found associated with (1) minimal cytokine secretion from human peripheral blood mononuclear cells, (2) no response in cell lines that report NF-κB/AP-1 signaling, and (3) no antidrug antibodies or significant cytokine production in transgenic Xeno-het mice, whether or not mAb1 or polysorbate 80 was present. These results suggest that SiOP in mAb1, representative of real-world DP in PFS, poses no increased risk of immunogenicity.

Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation.

PURPOSE: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO). METHODS: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins. RESULTS: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified. CONCLUSION: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.

Considerations for optimization and validation of an in vitro PBMC derived T cell assay for immunogenicity prediction of biotherapeutics.

An immune response to a biotherapeutic can be induced when the therapeutic is processed and presented by antigen presenting cell to T helper cells. This study evaluates the performance of an in vitro assay that can elicit antigen specific effector T cell responses. Two biotherapeutics with known clinical immunogenicity [FPX1 and FPX2] were assessed for their ability to induce antigen-specific IFN-γ secreting T cells in peripheral blood mononuclear cells (PBMC). The 24 amino acid peptide component of FPX1 elicited an antigen-specific response in 16/34 (47%) individual naïve healthy donors. This in vitro effect was consistent with high rate of immunogenicity which was observed when this drug was administered in clinical trials. FPX2 did not induce antigen-specific T cells in vitro, which correlates with the low rate of development of anti-drug antibody responses to this molecule in the clinic. The assay has the potential to predict immunogenicity and help in the selection of biotherapeutics at the early development stage of a clinical candidate.