Development, Validation and Application of a Bridging ELISA for Detection of Antibodies against GQ1001 in Cynomolgus Monkey Serum.

Immunogenicity is a major issue associated with the PK, efficacy, and safety evaluation of therapeutic protein products during pre-clinical and clinical studies. A multi-tiered approach consisting of screening, confirmatory, and titration assays has been widely adopted for anti-drug antibody testing. GQ1001, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently linked to a cytotoxin of DM1, possesses a novel format of antibody-drug conjugates. In this study, we reported the development, validation, and application of an acid-dissociation bridging enzyme-linked immunosorbent assay for the detection of antibodies against GQ1001 in cynomolgus monkey serum. The sensitivity of the screening assay was 126.141 ng/mL in undiluted serum. The screening assay and confirmatory assay were neither affected by the naïve monkey serum nor by 2% and 5% (v/v) erythrocyte hemolysates. Moreover, the assay was not subject to interference by 2500 ng/mL of human IgG1 in the samples. Drug interference at low positive control (150 ng/mL) and high positive control (8000 ng/mL) of anti-GQ1001 antibodies was not observed when GQ1001 concentrations were below 3.125 µg/mL and 100 µg/mL, respectively. Furthermore, no hook effect was observed for the positive antibodies in the concentration range of 8 to 64 µg/mL. The validated assay was, thereafter, successfully applied to a single-dose toxicity study of GQ1001. Anti-drug antibody positive rates among dosing animals and testing samples were reported, and no significant impact was found on toxicokinetic outcomes.

Development of a bridging ELISA for detection of antibodies against ZV0203 in cynomolgus monkey serum.

Immunogenicity has been a major concern in the safety evaluation of therapeutic proteins. The assessment of the unwanted immunogenicity of the therapeutic proteins performed in animals prior to clinical trials has been a regulatory requirement. In preclinical studies of therapeutic proteins, cynomolgus monkeys are usually the most relevant animal species. ZV0203, a recombinant humanized anti-human epidermal growth factor receptor 2 monoclonal antibody covalently bound to a cytotoxic drug (Duo-5), possesses a novel format of antibody drug conjugates. In this study, we reported the development, validation, and application of a bridging enzyme-linked immunosorbent assay for the detection of antibodies against ZV0203 in cynomolgus monkey serum. Drug interference at low positive control (18.0 ng/mL) and high positive control (130 ng/mL) of anti-ZV0203 antibodies was not observed when ZV0203 concentration is below 1.74 µg/mL and 1.49 µg/mL, respectively. In addition, no interference was found from mouse IgG1, but interference was observed with human IgG1. No effect of hemolysis was found on the analysis results of the testing samples present in 100% pooled rabbit serum containing 2% (V/V) erythrocyte hemolysates. Besides, spiked anti-ZV0203 antibody in rabbit serum was stable after 5 freeze/thaw cycles. The results showed that the method is suitable for the detection of anti-ZV0203 antibodies in cynomolgus monkey serum. The assay was also successfully applied in the repeated dose study of ZV0203.

Development and validation of an automated assay for anti-drug-antibodies in rat serum.

The potential immunogenicity of therapeutic human and humanized monoclonal antibodies (mAb) is a significant concern, and so preclinical testing of therapeutic mAbs routinely includes assessment of anti-drug antibody (ADA) induction. Here, we report the development of automated screening and confirmatory bridging ELISAs for the detection of rat antibodies against DH1042, an engineered human mAb for the SARS-CoV-2 receptor-binding domain. The assays were evaluated for specificity, sensitivity, selectivity, absence of a prozone effect, linearity, intra- and inter- assay precision, and robustness, and found to be suitable for purpose. The assays were then used to evaluate anti-DH1042 antibodies in the sera of rats dosed with lipid-nanoparticle (LNP)-encapsulated mRNA encoding DH1042. Rats received two doses of 0.1, 0.4 or 0.6 mg/kg/dose LNP-mRNA 8 days apart. Twenty-one days after the second dose, 50-100% of rats had developed confirmed anti-DH1042 ADA depending on dose level. No animals in the control group developed anti-DH1042 ADA. These assays reflect new applications for a non-specialized laboratory automation platform, and the methodologies and approaches reported here provide a template that can be adapted for the automated detection and confirmation of ADA in preclinical testing of other biologics.

Dual-target Bridging ELISA for Bispecific Antibodies.

Bispecific antibodies (BsAbs) are typically monoclonal antibody (mAb)-derived molecular entities engineered to bind to two distinct targets, including two antigens or two epitopes on the same antigen. When compared to parental monoclonal antibodies or combinational therapies, the generated BsAbs have the ability to bridge the two targets and thus may offer additional clinical benefits. Characterizing BsAbs' ability to bind to both targets simultaneously is critical for their biotherapeutic development. A range of bi-functional quantitative bridging assays to enable target-specific capture and detection of binding properties include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and cell-based flow cytometry. Developing suitable and robust cell-based bioassays is more challenging than non-cell-based binding assays because cell-based assays with complex matrices can be inherently variable and often lack precision. Compared to SPR, ELISA has a rapid setup and readily available method, being widely and extensively applied in almost every laboratory. Here, we describe a dual-target bridging ELISA assay that characterizes the ability of a HER2(human epidermal growth factor receptor 2)/PD-L1(programmed cell death ligand 1) BsAb in binding to both HER2 and PD-L1 simultaneously, a prerequisite for its envisioned mode of action. Graphical abstract.

Characterization of robust immune responses to a bispecific antibody, a novel class of antibody therapeutics.

Background: Anti-A/B is a bispecific monoclonal antibody that blocks activities of soluble targets A and B. Robust immune responses were observed in a multiple-dose cynomolgus monkey toxicology study, negatively impacting the toxicokinetics/pharmacodynamics profile of anti-A/B in some animals. This was unexpected as similar findings were not observed in the two previously studied parental molecules. Methodology & Results: This paper discusses our characterization strategy for evaluating the immunogenic domain(s) of anti-A/B and our mitigation plan to monitor immunogenicity in the first-in-human clinical study. The characterization results from the cynomolgus monkey and Phase I studies are discussed. Conclusion: The characterization strategy discussed informed understanding of immunogenicity results and clinical impact, which can be broadly applied to other molecules with multiple-binding domains.

Detection and kinetics of persistent neutralizing anti-interferon-beta antibodies in patients with multiple sclerosis. Results from the ABIRISK prospective cohort study.

Two validated assays, a bridging ELISA and a luciferase-based bioassay, were compared for detection of anti-drug antibodies (ADA) against interferon-beta (IFN-ß) in patients with multiple sclerosis. Serum samples were tested from patients enrolled in a prospective study of 18 months. In contrast to the ELISA, when IFN-ß-specific rabbit polyclonal and human monoclonal antibodies were tested, the bioassay was the more sensitive to detect IFN-ß ADA in patients' sera. For clinical samples, selection of method of ELISA should be evaluated prior to the use of a multi-tiered approach. A titer threshold value is reported that may be used as a predictor for persistently positive neutralizing ADA.

A simple and cost-effective assay for measuring anti-drug antibody in human patients treated with Adalimumab.

It has been reported that 90% of the anti-drug antibody (ADA) to Adalimumab in human patients bound to the TNF-binding area, resulted in the annual loss of responses to Adalimumab up to 24%. It is of urgency to develop a cost-effective and easy-to-use ADA diagnostic kit for diagnosis of potential drug-resistance in patients treated with Adalimumab in clinic hospitals to avoid the tremendous economic and human costs to patients and health-care providers. In this study, we reported the generations of mouse monoclonal and monkey polyclonal antibodies against Adalimumab as assay standards and positive quality controls respectively. A Bridging ELISA assay was successfully developed with a limit of detection (LOD) between 22-80ng/ml. The preliminary validation of assay was carried out first with 50 normal human sera, further validated by screening the ADA in 192 serum samples from monkeys treated with or without Adalimumab. Our data showed that the Bridging ELISA kit is very sensitive, highly specific and ready for study in human clinic trials.

Immunogenicity screening assay development for a novel human-mouse chimeric anti-CD147 monoclonal antibody (Metuzumab).

The clinical effect of patient immune responses to therapeutic antibodies affect product safety and efficacy, which makes the development of valid, sensitive immune assays a key aspect of antibody drug development. In this paper, we reported the generations of mouse monoclonal and Cynomolgus monkey polyclonal antibodies against the anti-CD147 antibody (Metuzumab) as the internal standards and the positive controls. Seven mouse monoclonal antibodies were shown to recognize both (Fab)2 and full length of Metuzumab, but not the control normal human IgGs, and monoclonal anti-Metuzumab, Clone 2D9 was chosen to be used as the internal standard for anti-Metuzumab study. A Bridging ELISA assay was developed by coating the wells with the antibody drug, and the anti-drug antibody (ADA) in the animal sera were detected by enzyme-labeled antibody. Its limit of detection (LOD) was determined to be 0.39ng/ml of anti-Metuzumab antibody (ADA) with linear range between 0.39-50ng/ml and R(2)=0.994. For normal monkey sera, a minimal dilution was determined to be 1:80. However, very different from peptide or other protein drugs, strong interferences from the residual antibody drugs were observed from most of the testing monkey sera in the preclinical study. It was experimentally determined that the concentration of the residual antibody drug in the assay have to be lower than 1µg/ml, so the assays were carried out at 1:100 dilution of the monkey sera. In the pre-clinical study, 32 monkeys were treated with escalating doses of Metuzumab between 0, 10, 50, 200mg/kg for 13 times over 13weeks of time period. 16 of them were terminated right after the last injection, while the other 16 were rested for additional 4weeks before termination. Afraid to miss any positive response to antibody drug, sera samples were collected at six time points, including 2-, 6- and 10-weeks post 1st dose, prior to last dose, and 2-, 4-weeks into recovery. The highest positive rates were seen with the Medium- and High-dose group 2-weeks post the first injection, 6 out 8 monkeys in the High-dose were positive for free ADA. However, no significant pathologic and clinic adversary effect was observed in those monkeys.