immunogenicity assays
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Bioanalysis ◽  
2022 ◽  
Author(s):  
Caroline Kittinger ◽  
Jared Delmar ◽  
Lisa Hewitt ◽  
Rebecca Holcomb ◽  
Christopher Jones ◽  
...  

Development of biotherapeutics require pharmacokinetic/pharmacodynamic (PK/PD) and immunogenicity assays that are frequently in a ligand-binding assay (LBA) format. Conjugated critical reagents for LBAs are generated conjugation of the biotherapeutic drug or anti-drug molecule with a label. Since conjugated critical reagent quality impacts LBA performance, control of the generation process is essential. Our perspective is that process development methodologies should be integrated into critical reagent production to understand the impact of conjugation reactions, purification techniques and formulation conditions on the quality of the reagent. In this article, case studies highlight our approach to developing process conditions for different molecular classes of critical reagents including antibodies and a peptide. This development approach can be applied to the generation of future conjugated critical reagents.


2021 ◽  
Vol 23 (6) ◽  
Author(s):  
Andrew F. Dengler ◽  
Rachel Weiss ◽  
Tiffany Truong ◽  
Susan C. Irvin ◽  
Nidhi Gadhia ◽  
...  

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


2021 ◽  
Vol 12 ◽  
Author(s):  
Hans de Graaf ◽  
Ruth O. Payne ◽  
Iona Taylor ◽  
Kazutoyo Miura ◽  
Carol A. Long ◽  
...  

BackgroundTransmission blocking vaccines targeting the sexual-stages of the malaria parasite could play a major role to achieve elimination and eradication of malaria. The Plasmodium falciparum Pfs25 protein (Pfs25) is the most clinically advanced candidate sexual-stage antigen. IMX313, a complement inhibitor C4b-binding protein that forms heptamers with the antigen fused to it, improve antibody responses. This is the first time that viral vectors have been used to induce antibodies in humans against an antigen that is expressed only in the mosquito vector.MethodsClinical trial looking at safety and immunogenicity of two recombinant viral vectored vaccines encoding Pfs25-IMX313 in healthy malaria-naive adults. Replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding Pfs25-IMX313, were delivered by the intramuscular route in a heterologous prime-boost regimen using an 8-week interval. Safety data and samples for immunogenicity assays were taken at various time-points.ResultsThe reactogenicity of the vaccines was similar to that seen in previous trials using the same viral vectors encoding other antigens. The vaccines were immunogenic and induced both antibody and T cell responses against Pfs25, but significant transmission reducing activity (TRA) was not observed in most volunteers by standard membrane feeding assay.ConclusionBoth vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. However, the transmission reducing activity of the antibodies generated were weak, suggesting the need for an alternative vaccine formulation.Trial RegistrationClinicaltrials.gov NCT02532049.


Bioanalysis ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 1113-1121
Author(s):  
Daniel A Peterson ◽  
Thomas G Pottanat ◽  
Heather Denning ◽  
Nicoletta Bivi ◽  
John H Sloan ◽  
...  

Aim: We present a novel methodology to compare results between distinct immunogenicity assays, performed by two laboratories, for the same biotherapeutic. Materials & methods: Human serum pools from clinical trials were generated to provide representative immunogenicity titers. Pools were evaluated at two laboratories in a blinded fashion to assess the effect of assay format and laboratory change on clinical interpretation of immunogenicity results. Results: The laboratories validated two different assay formats and demonstrated comparable sensitivity and drug tolerance. Overall, the comparisons in assay format and laboratory ensured a comparable ability to detect treatment-emergent antidrug antibodies for a biotherapeutic. Conclusion: We have established an approach, using pooling of patient samples, that allows for the interlaboratory comparisons without creating duplicative results.


Bioanalysis ◽  
2021 ◽  
Author(s):  
Anna Laurén ◽  
Joanne Goodman ◽  
Jonas Blaes ◽  
John Cook ◽  
Kyra J Cowan ◽  
...  

Immunogenicity assays are required to evaluate anti-drug antibody (ADA) responses that can be generated against biotherapeutic modalities. Regulatory guidelines focus on clinical requirements, yet it has become apparent that industry has applied these clinical recommendations for immunogenicity assessment to nonclinical studies in varying degrees. ADAs are an anticipated outcome of dosing a humanized or fully human biotherapeutic into an animal. However, a nonclinical ADA response is rarely predictive of the immunogenic potential in humans. The addendum to ICH S6 recommends that immunogenicity should be explicitly examined where there is: evidence of altered pharmacodynamic activity; unexpected changes in exposure in the absence of a pharmacodynamic marker or evidence of immuno-mediated reactions. The European Bioanalytical Forum has extensively discussed and reached a consensus on a minimal strategic approach of when and what to include for nonclinical immunogenicity assessments. Additionally, this paper recommends a strategy for ADA assay validation and sample analysis for those cases when it is considered necessary to include an immunogenicity assessment in nonclinical toxicology studies.


2020 ◽  
Vol 22 (5) ◽  
Author(s):  
Matthew Alleyn ◽  
Kristin Closson ◽  
Adam Gentile ◽  
Nathan Gulbis ◽  
Christopher Taylor ◽  
...  

Abstract The use of biologic-based therapeutics has revolutionized our ability to treat complex diseases such as cancer- and autoimmune-related disorders. Biologic-based therapeutics are known to generate anti-drug immune responses or immunogenicity in clinical patients which can lead to altered pharmacokinetics, decreased drug efficacy, and unwanted adverse clinical events. Assays designed to detect and assess anti-drug immune responses are used to help monitor patients and improve drug safety. Utilizing a tiered approach, screening assays are developed first to identify patients that are potentially positive for anti-drug-specific antibodies. Patients that screen positive are subjected to additional tiers of testing that include a confirmation assay to confirm the presence of expected anti-drug-specific antibodies, a titer assay to assess relative levels of anti-drug-specific antibodies, and, depending on the drug’s mechanism of action or concerns of adverse clinical reactions, further characterization such as drug neutralization and anti-drug antibody isotyping. This tiered approach can prove to be detrimental to clinical samples from exposure to multiple cycles of testing, freeze thaws, and repeated handling by lab personnel. Multiplexing some of these assays together may streamline the characterization of anti-drug immune responses and help reduce the repeated usage of clinical samples. In this study, we combined a screening assay and anti-drug isotyping assays into one multiplexed assay using the Luminex® xMAP® Technology. The multiplexed assay was developed and validated to meet the FDA recommended guidelines for immunogenicity assessments. These results show that multiplexed assays perform comparably to industry standards. This study should encourage labs to explore the use of multiplexing immunogenicity assays to characterize anti-drug antibody responses quickly, with less repeat testing and reduced sample handling.


Bioanalysis ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 245-256
Author(s):  
Robert J Kubiak ◽  
Rosalinda HGP Arends ◽  
Nancy Lee ◽  
Meina Liang ◽  
Jianchun Zhang ◽  
...  

Aim: Competitive inhibition with excess unlabeled drug is used to confirm the presence of antidrug antibodies (ADA) in study samples. We evaluated specific and nonspecific responses from both drug-naive and drug-treated subjects to identify conditions required by the confirmatory assay to make accurate ADA classifications. Results: Nonspecific signal measured in drug-naive samples used to determine assay cut points was uniformly low and close to the screening cut point. Confirmatory assays performed on incurred study samples with nonspecific responses significantly above the level observed during cut point determination resulted in incorrect ADA classifications. Conclusion: Intensity of confirmatory response should be proportional to the screening response and therefore, to ensure accurate ADA classifications, the confirmatory responses cannot be considered as independent but need to be evaluated in relation to the screening responses.


2019 ◽  
Vol 22 (1) ◽  
Author(s):  
Francesca Civoli ◽  
Aparna Kasinath ◽  
Xiao-Yan Cai ◽  
Meenu Wadhwa ◽  
Andrew Exley ◽  
...  

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