Anti-Drug Antibodies (ADA) have been of increasing interest with the rise of immunotherapy treatment. Regulatory guidance recommends sponsors assess ADA during product development studies, including ADA incidence and the effect of ADA on efficacy, safety, and PK. They also caution against comparing ADA incidence between products. Recent literature described the effect of assessment schedules and other factors on incidence rates of ADA. Here we show the impact of different assessment schedules. We computed atezolizumab ADA incidence rates for 8 atezolizumab Phase III studies modeling reduced ADA assessment schedules. Atezolizumab studies included OAK, IMpower130, IMpower150, IMpower110, IMpower010, IMpower133, IMbrave150, IMspire150. All studies administered Q3W, and all but 3 of the studies collected ADA samples at Cycle (C) 1, 2, 3, 4, 8, 16, 24, 32, end of treatment and washout. Reduced ADA assessment schedules were chosen from publicly available ADA assessment schedules of other immune checkpoint inhibitors. Chosen schedules included assessments C 1,4 plus washout, similar to durvalumab ADA collection, and C 1, 2, 4, 8, 16, 24, 32, end of treatment plus washout, similar to nivolumab ADA collection. The calculated ADA incidence rates, computed assuming reduced ADA assessment schedules, were decreased across all atezolizumab studies, irrespective of the alternative incidence calculation schedule employed. When incidences were calculated using the most reduced ADA assessment schedule (C1, C4, washout), similar to durvalumab's collection, the rate for atezolizumab ADA decreased significantly, reaching a low of 2.5%. For example, the ADA rate in IMpower133 decreased from18.6% to 2.5% , and in IMpower010 from 30.4% to 15.5%. Across all eight studies, the computed incidence range decreased from 13.3%--36.4% to 2.5%--15.5%. Similarly, when an alternative schedule similar to nivolumab ADA collection schedule was used the ADA incidence rate for atezolizumab in IMpower130 decreased from 22.9% to 19.2%, and in IMspire150 from 13.3% to 13.0%. The computed incidence range across 8 studies decreased to 13.0%--34.7%. When reduced assessment schedules are used, transient ADA are less likely to be measured, resulting in a lower reported ADA rate. Conversely, frequent ADA sampling may result in an ADA incidence rate including positive transient cases that have little or no clinical significance. Our results highlight why ADA incidence should not be compared between different products. Richard S. Finn, Coen A. Bernaards, Steven J. Swanson, Barbara Gitlitz, Lily J. Nguyen, James Zanghi, Sophie Cousin. Impact of assessment schedules on atezolizumab anti-drug antibody incidence rates [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C038.

Immunogenicity of a therapeutic protein product may elicit an unintended immune response, and is a critical aspect evaluated in oncology clinical trials. The development of anti-drug antibodies (ADAs) can impact the pharmacokinetics, pharmacodynamics, efficacy, and safety of these therapies. We review the background and nomenclature of immunogenicity assessment in oncology studies and emphasize the complexities in ADA detection arising from assay sensitivity, drug interference, and notably, the frequency of patient sampling for ADA analysis. The applicability of common nomenclature, however, has limitations in the context of oncology. Of prime consideration for physicians is that the clinical impact of ADA is far more important than just their presence. Furthermore, the interpretation of immunogenicity data in oncology is complicated by patient-specific factors, concomitant treatments, and potential survivorship bias. Regulatory guidelines acknowledge these complexities, mandating specific statements on product labels cautioning against cross-trial comparisons of ADA incidence due to variations in assay methods and sampling schedules. Accurate interpretation of immunogenicity data, considering assay methodologies, study design, and sampling frequency, is crucial for clinicians to assess the clinical relevance of ADA findings and make informed treatment decisions for patients receiving therapeutic protein products in oncology. The focus should be on the clinical relevance of ADAs rather than simply their incidence.

Protein-based therapeutics may elicit undesired immune responses in a subset of patients, leading to the production of anti-drug antibodies (ADA). In some cases, ADAs have been reported to affect the pharmacokinetics, efficacy and/or safety of the drug. Accurate prediction of the ADA response can help drug developers identify the immunogenicity risk of the drug candidates, thereby allowing them to make the necessary modifications to mitigate the immunogenicity. In this study, we leveraged the rich clinical study data collected by Roche/Genentech to identify factors that impact drug immunogenicity. We focused on conventional monoclonal antibodies, but have included a variety of additional drug modalities in the analysis. We show that the clinical ADA incidences are associated with the mechanism of action of the drugs, the mechanism of action of comedications, the routes of drug administration and the diseases of the patient cohort. By combining these clinical factors with the in silico epitope prediction, we improved the prediction accuracy of drug immunogenicity in clinical trials (AUC of cross validation improved from 0.72 to 0.93).

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.

Therapeutic antibodies can elicit unwanted immune responses in a subset of patients, which leads to the production of anti-drug antibodies (ADA). Some of these ADAs have been reported to effect the pharmacokinetics, efficacy and/or safety of the therapeutic antibodies. The sequence diversity of antibodies are generated by VDJ recombination and mutagenesis. While the antibody generation process can create a large candidate pool for identifying high-affinity antibodies, it also could produce sequences that are foreign to the human immune system. However, it is not clear how VDJ recombination and mutagenesis impact the clinical ADA rate of therapeutic antibodies. In this study, we identified a positive correlation between the clinical ADA rate and the number of introduced mutations in the antibody sequences. We also found that the use of rare V alleles in human-origin antibody therapeutics is associated with higher risk of immunogenicity. The results suggest that antibody engineering projects should start with frameworks that contain commonly used V alleles and prioritize antibody candidates with low number of mutations to reduce the risk of immunogenicity.

Abstract Monocyte-derived dendritic cell (moDC)–based cancer therapies intended to elicit antitumor T-cell responses have limited efficacy in most clinical trials. However, potent and sustained antitumor activity in a limited number of patients highlights the therapeutic potential of moDCs. In vitro culture conditions used to generate moDCs can be inconsistent, and moDCs generated in vitro are less effective than natural DCs. On the basis of our study highlighting the ability for certain kinase inhibitors to enhance tumor antigenicity, we therefore screened kinase inhibitors for their ability to improve DC immunogenicity. We identified AKT inhibitor MK2206, DNA-PK inhibitor NU7441, and MEK inhibitor trametinib as the compounds most effective at modulating moDC immunogenicity. The combination of these drugs, referred to as MKNUTRA, enhanced moDC activity over treatment with individual drugs while exhibiting minimal toxicity. An evaluation of 335 activation and T-cell–suppressive surface proteins on moDCs revealed that MKNUTRA treatment more effectively matured cells and reduced the expression of tolerogenic proteins as compared with control moDCs. MKNUTRA treatment imparted to ICT107, a glioblastoma (GBM) DC-based vaccine that has completed phase II trials, an increased ability to stimulate patient-derived autologous CD8+ T cells against the brain tumor antigens IL13Rα2(345-354) and TRP2(180-188). In vivo, treating ICT107 with MKNUTRA, prior to injection into mice with an established GBM tumor, reduced tumor growth kinetics. This response was associated with an increased frequency of tumor-reactive lymphocytes within tumors and in peripheral tissues. These studies broaden the application of targeted anticancer drugs and highlight their ability to increase moDC immunogenicity.

212 Background: Timely and appropriate biomarker testing guides evidence-based treatment decision-making in advanced non-small cell lung cancer (aNSCLC). American Society of Clinical Oncology (ASCO) and National Comprehensive Cancer Network (NCCN) guidelines recommend that all treatment-eligible patients with non-squamous, or squamous histology in non-smokers undergo EGFR and ALK biomarker testing prior to initiating first line therapy. Genentech’s Learning and Clinical Integration team and Flatiron Health explored the frequency of EGFR/ALK testing and overall time between advanced disease diagnosis, results receipt and treatment initiation in clinical oncology practices. Methods: Structured and unstructured data were obtained from Flatiron’s electronic health record database. 6,991 patients from 166 clinics diagnosed after 1/1/14 with at least 2 visits before 8/31/15 were randomly selected from the Flatiron aNSCLC national cohort of > 25,000 patients. Dates of specimen collection, results receipt and treatment start were collected. Results: EGFR/ALK testing was conducted in 75% of non-squamous patients with wide variation across practices (< 20% to 100%). For squamous patients, 15% were tested overall, but with dramatic variation across practices (0% to 100%). For patients with a positive test result available prior to initiation of first line treatment, 79% of EGFR+ and 94% of ALK+ patients received the appropriate targeted therapy. However, for those patients tested after initiation of first line therapy, only 41% of EGFR+ and 65% of ALK+ patients received appropriate targeted first line therapy. EGFR/ALK tests results were received > 4 weeks from aNSCLC diagnosis in 32% and 34% of patients, respectively. Validation testing indicated that delays were attributed to non-lab factors, as test results were returned in < 2 weeks in 95% of cases. Conclusions: Wide variation in real-world practice illustrates the need to improve adherence to ASCO and NCCN biomarker testing guidelines. Educational intervention to improve quality of care in aNSCLC should focus on ensuring testing of almost all non-squamous patients, limiting testing to the non-smoking squamous cell population, and ensuring timely ordering of testing by clinicians.

2059 Background: A hallmark of glioblastoma is the high incidence of tumor recurrence, thought to be triggered by cancer stem cells. These tumorigenic cells are resistant to irradiation and chemotherapeutic agents. The target antigen, CD-133, was chosen because it has been reported as a cancer stem cell antigen overexpressed in glioblastoma tumors and associated with shorter survival. Recent clinical trials suggest that the mean overall survival for these patients is roughly 5-9 months, emphasizing the important unmet medical need in this disease requiring additional strategic approaches. Dendritic cell immunotherapies such as ICT-121 could provide benefit to patients by educating their immune systems to induce the formation of cytotoxic T cells that attack tumor cells bearing the target antigen. In addition to immediate attack on tumor cells present at dosing, a long-term memory response effective against tumor recurrence might be induced. Immunotherapy, such as ICT-121, that targets cancer stem cells could be an important treatment for this disease. Methods: This Phase I multi-center trial of ICT-121 targeting CD133 was designed to assess safety and tolerability (primary endpoint) and to monitor overall survival and progression-free survival (secondary endpoints). ICT-121 is comprised of autologous dendritic cells that are loaded with two HLA-A2 restricted epitopes of the CD133 antigen. CD133 is overexpressed on glioblastoma cancer stem cells. The HLA-A2 patients that had undergone resection for recurrence of glioblastoma were treated with ICT-121 once a week for 4 weeks during the induction phase and then once every 2 months during the maintenance phase until disease progression, death, ICT-121 depletion or discontinuation. Results: A total of 20 patients were treated and eight of these patients are still alive. Immune response data with cytokine mRNA expression demonstrated a response to the CD133 epitopes. A total of 20 patients were treated and eight of these patients are still alive. Conclusions: The results from this Phase I trial suggest that ICT-121 is both safe and well-tolerated with an immune response seen in a subset of patients. Clinical trial information: NCT02049489.

Detailed confirmatory testing and analysis verifies and strengthens the association between clinical outcomes and immune response of HLA-A2+ patients enrolled in a randomized phase 2 trial of ICT-107. 77 HLA-A2+ patients, randomized 2:1, received ICT-107 (autologous DCs incubated with 6 synthetic peptide CTL epitopes targeting GBM tumor/stem cell-associated antigens, including the four HLA-A2-restricted antigens HER-2, TRP-2, gp100, and IL-13Rα2) or matching control (un-incubated DC). Multimer testing was performed on a subset of these patients. The pioneering analysis heuristic of fusion metrics used in conjunction with Monte Carlo simulation was used to identify multimer immune responders. P-values between dependent variables and multiple overall survival (OS) or progression-free survival (PFS) metrics was performed using log-rank test and Fisher’s exact test. HLA-A2+ patients consistently continued to show evidence of immune response being associated with both OS and PFS. Multimer immune responders independently confirmed the ELISpot immune responder associations between assignment group (p=0.0308), and initial OS and PFS (p=0.0043 and 0.0352, respectively). Combining ELISpot and multimer responders strengthened or maintained associations with all OS and PFS metrics. Notable significant associations were determined when data was stratified by treatment group in both treatment and placebo subgroups, leading to speculation of the possible positive effects of DCs alone. This finding supports changing the placebo in the Phase III study from dendritic cells to PBMCs. The robust associations identified between OS and PFS with immunologic response, explored using both multimer and ELISpot analysis to determine immune response with fusion metrics in a Monte Carlo setting, provide support for the efficacy of ICT-107 to induce peptide-specific T cell responses in HLA-A2+ patients.

Dendritic Cells (DC) mediate anti-tumor immunity, and DC-based immunotherapies represent a promising way to fight cancer. However, questions about the optimal manufacturing process and delivery strategies still remain. So far, DC-based immunotherapies have only been tested for maturational state and expression of select CD markers. However, these characteristics do not establish successful peptide loading or actual presentation of peptides by HLA. To date, class I HLA presentation of peptides on loaded DC has only been confirmed using indirect immune monitoring techniques such as ELISPOT and tetramer staining. Here, we present the first quantitative assay designed for ICT-107, a DC immunotherapy being tested in glioblastoma, that directly assesses peptide presentation on pulsed DC. This assay may prove valuable for monitoring quality and potency of DC-based immunotherapies via a reliable, reproducible and straightforward method. We generated a T cell receptor mimic monoclonal antibody (TCRm), RL13A, specific for HLA-A∗01:01 in complex with MAGE-1 peptide (EADPTGHSY; “EAD9”). Next, mature DC from three different HLA-A1+ patients were pulsed with 20 μg/ml of EAD9 peptide. Loaded DC were then stained with PE-labeled RL13A. QuantiBRITE PE-Beads were used to establish a standard curve so that the number of HLA-A1/EAD9 complexes could subsequently be determined. Flow cytometric staining of peptide-pulsed DC with RL13A showed a clear shift in MFI compared to patient-matched, unpulsed DC. Using the QuantiBRITE PE-bead standard curve, the number of HLA-A1/EAD9 on the surface of ICT-107 was determined. All 3 lots of ICT-107 presented a comparable number of HLA-A1/EAD complexes (1200–2500 copies) on the surface. The data from ICT-107 provide proof of concept that an HLA-specific, quantitative assay can be used to directly confirm presentation of the peptides on DC-based immunotherapies. TCRm staining enables detection as well as quantification of HLA/peptide complexes presented by peptide-pulsed DC, and the assay can now be used to characterize the DC immunotherapy, ICT-107. Future applications for this assay include both the optimization of the manufacturing process and serving as a release assay for HLA-A1+ DC-based therapeutics containing the MAGE-1 EAD9 peptide. R. Buchli: Employee; Company/Organization; Pure MHC LLC. W.H. Hildebrand: Scientific/Medical Advisor; Company/Organization; Pure MHC LLC.

The administration of therapeutic proteins (also called biologics, biopharmaceuticals, biological products or biological medicinal products) and peptides often leads to the induction of immunogenicity. That is, the patient's immune system reacts to the administered therapeutic by producing anti-drug antibodies (ADAs) that neutralize the drug or accelerate clearance, leading… Download references

HLA antibodies of the IgM isotype have been considered as benign to transplant outcome. We report two cases of IgM-only AMR concurrent with rejection and after treatment in two kidney transplant patients in the absence of IgG anti-HLA antibodies. No immunoglobulin class switching of these antibodies was observed. Patients were screened for HLA antibodies of isotypes IgG, IgM and by the C1q assay utilizing the LabScreen Single Antigen assays by One Lambda (ThermoFisher, Canoga Park CA) on the Lumixex platform. The first patient, unsensitized, experienced a cellular and humoral rejection episode within 3 weeks of transplant, with DSA's to A25 and B7. Standard IgG screens were negative, but DSA was detected in the C1q and IgM screens. Three months post-transplant, a second biopsy showed continued AMR with DSA to only IgM. The rejection was successfully treated. Eight months post-transplant, the patient is stable but continues to make only IgM DSA. A second patient rejected a zero matched sibling graft soon after transplantation in 2007. By a CDC antibody screen, she had DSA to the donor's A1 and B51 antigens. The rejection was successfully treated. Six years later, a second rejection occurred with class II DSA detected to donor's DQ2 and DQ7 by the Single Antigen IgG assay. The rejection was successfully treated over the following nine months. One year after treatment, EDTA-enhanced IgG screens for class I and II were negative, but C1q and IgM screens detected DSA to donor B51 and DR11. Subsequent IgM screens also detected additional antibodies to donor A1 and DQA1*02:01. None of these antibodies have class-switched to IgG. These cases illustrate the value of adding IgM and/or C1q assays to antibody screening of rejection samples to detect DSA and follow treatment efficacy. These data strongly suggest that IgM HLA DSAs can have deleterious effects on transplanted kidneys.

All therapeutic proteins have the potential to induce anti-drug antibodies (ADA). Clinically relevant ADA can impact efficacy and/or safety of a biological therapeutic. Immunogenicity assessment strategy evaluates binding and neutralizing ADA, and the need for additional characterization (e.g., epitope, titer and so on) is determined using a risk-based approach. The choice of characterization assays depends on the type, application and immunogenicity of the therapeutic. ADA characterization can impact the interpretation of the risk profile of a given therapeutic, and offers insight into opportunities for risk mitigation and management. This article describes common ADA characterization methods. Strategic assessment and characterization of clinically relevant ADA are discussed, in order to support clinical options for safe and effective patient care and disease management.

Immunogenicity is a significant concern for biologic drugs as it can affect both safety and efficacy. To date, the descriptions of product immunogenicity have varied not only due to different degrees of understanding of product immunogenicity at the time of licensing but also due to an evolving lexicon that has generated some confusion in the field. In recent years, there has been growing consensus regarding the data needed to assess product immunogenicity. Harmonization of the strategy for the elucidation of product immunogenicity by drug developers, as well as the use of defined common terminology, can benefit medical practitioners, health regulatory agencies, and ultimately the patients. Clearly, understanding the incidence, kinetics and magnitude of anti-drug antibody (ADA), its neutralizing ability, cross-reactivity with endogenous molecules or other marketed biologic drugs, and related clinical impact may enhance clinical management of patients treated with biologic drugs. To that end, the authors present terms and definitions for describing and analyzing clinical immunogenicity data and suggest approaches to data presentation, emphasizing associations of ADA development with pharmacokinetics, efficacy, and safety that are necessary to assess the clinical relevance of immunogenicity.

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity. From the prolific discussions held during the workshop, specific recommendations are presented in this 2014 White Paper. As with the previous years' editions, this paper acts as a practical tool to help the bioanalytical community continue advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2014 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for Large molecules bioanalysis using LBA and Immunogenicity. Part 1 (Small molecules bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS, Electronic Laboratory Notebook and Regulatory Agencies' Input) were published in the Bioanalysis issues 6(22) and 6(23), respectively.

1290 Learning Objectives 1. Review the usual surgical approach to kidney transplantation. 2. Review what factors (donor and recipient) can increase the complication rate. 3. Review the various complications of kidney transplantation and their scintigraphic appearance. 4. Learn the time course of various complications. 5. Learn when SPECT/CT can be useful. 6. Understand the nephrology and transplant surgeon perspective. This multidisciplinary educational exhibit with specialists in transplant surgery, nephrology and nuclear medicine explores the spectrum of kidney transplant complications and their expected time course. A few illustrative cases from our institution will help to solidify knowledge of transplant complications and improve differential diagnoses. Utility of SPECT/CT is also reviewed. Clinical pearls and pitfalls from the involved specialties will also be shared.