ABSTRACT Antibody-drug conjugates (ADCs) rely on antibody-mediated internalization to deliver cytotoxic payloads into tumor cells. Therefore, quantitative assessment of antibody internalization is essential for ADC development, particularly during early antibody screening stages. However, conventional internalization assays, whether direct or indirect, often face challenges such as low throughput, reduced sensitivity, and limited target specificity due to spatial hindrance. Here, we introduce a versatile 3C peptide conjugate platform that utilizes the high-affinity binding of IgG by the C1-C3 domains of streptococcal protein G. This platform includes 3C-toxin for cytotoxicity-based internalization detection and 3C-pHAb for pH-sensitive fluorescent tracking. By simply incubating these reagents with antibodies, effective labeling is achieved without complex modifications, enabling sensitive and high-throughput evaluation of internalization. We validated the platform across multiple tumor-associated targets, including HER2, CDH6, LIV-1, LYPD3, and GPC3, demonstrating a strong correlation between 3C-based assays and the cytotoxic efficacy of corresponding ADCs. Notably, 3C-toxin showed superior target promiscuity compared to traditional DT3C methods, expanding applicability to a broader range of antigens. This platform provides a scalable solution for antibody internalization analysis, positioned to accelerate ADC discovery by providing reliable early-stage screening metrics.

ABSTRACT The PATHWAY anti-HER2/neu (4B5) Rabbit Monoclonal Primary Antibody immunohistochemistry (IHC) assay is used to identify patients eligible for anti-HER2 targeted therapy. Previous work has demonstrated that the 4B5 antibody can react with the related family member HER4, and the nuclear protein ZSCAN18, under experimental conditions. Here, we used surface plasmon resonance, automated capillary electrophoresis (ACE), and IHC to fully define the 4B5 epitope and binding characteristics. The key amino acids that contribute to antibody recognition were identified and characterized with the limits of detection determined for HER4 and ZSCAN18. Additionally, the protein target responsible for cytoplasmic and nuclear reactivity observed by 4B5 in non-breast tissue types, that is not attributable to either HER2, HER4, or ZSCAN18, was identified. By means of protein purification, mass spectrometry, ACE analysis and IHC, 4B5 was found to react with the cytosolic aldo-keto reductase family 1 members B1, B10 and B15 (AKR1B1, AKR1B10, AKR1B15). The binding characteristics and limits of detection for these off-target species were defined. In-silico docking analysis of computationally folded 4B5 paratope regions identified key differences in the 4B5 paratope interaction between a peptide containing the HER2 epitope versus that of a partially homologous peptide sequence common to the AKR1 family. Flexibility of the complementarity-determining region loops in 4B5 imparts paratope plasticity that enables interaction with AKR1B family proteins. This antibody off-target recognition can result in cytoplasmic and nuclear IHC staining.

ABSTRACT Co-formulated antibody cocktails are becoming an increasingly popular therapeutic class; however, they present analytical challenges over traditional single monoclonal antibody (mAb) formulations. One paramount concern is the formation of heteromeric species that have unknown impacts on safety and efficacy. Consequently, effective approaches for identifying and characterizing high-molecular weight (HMW) impurities are critical to the successful development of this therapeutic class. In this study, we used a multifaceted mass spectrometry approach to characterize a unique dimer species formed between two co-formulated mAbs under thermal stress, revealing an intriguing dimerization mechanism that is driven by complementarity-determining region clipping-induced domain swap. Size exclusion chromatography-mass spectrometry, complemented by post-column denaturation, was utilized at both intact and subunit levels to pinpoint the dimerization interface. Additionally, by probing the disulfide bond susceptibility changes via limited reduction and middle-down analysis, the structural changes of the involved domains were studied. These results highlight the critical role of sophisticated analytical methods in comprehending and addressing the complexities linked to co-formulated mAb cocktails.

ABSTRACT Antibody phage display, a biotechnological method for selecting fully human antibodies from diverse libraries, is firmly established in antibody drug discovery due to its speed and flexibility. In this study, we introduce Pioneer, one of the largest synthetic human antibody libraries developed to date, with approximately 2.2 × 1011 functional members. Pioneer utilizes SpyDisplay, a phage display selection system that relies on SpyTag-SpyCatcher protein ligation technology and enables rapid selection of high-affinity antibodies. The library’s antibodies are designed for favorable biophysical properties and developability and they can be rapidly labeled or converted into various formats for screening and functional assays. We validated the performance of Pioneer in a series of selection campaigns against diverse antigens. For antibodies against four targets, TIGIT, IL-6RA, C5aR and CXCR4, we performed in-depth functional and developability characterization. For all four targets, including the two challenging G-protein coupled receptors, we demonstrated that antibodies with parameters comparable to late-stage clinical candidates can be selected directly from the library. Our results highlight the effectiveness of the Pioneer library combined with SpyDisplay in generating therapeutic lead candidates with excellent affinity, specificity, and developability, offering a robust platform for rapid antibody discovery and development.

ABSTRACT Monoclonal antibodies are well established as promising treatment options for a broad range of patients with severe diseases. In some cases, the formation of anti-drug antibodies (ADA) may limit their clinical use and potentially affect safety and efficacy for patients. Despite extensive research, some factors contributing to the immunogenicity of therapeutic antibodies remain poorly understood. In particular, the immunogenicity potential associated with multivalent antibody formats targeting oligomeric protein antigens has thus far received insufficient attention. Large, target-related immune complexes (TRICs) may be formed that can trigger Fc-mediated downstream effects and have the potential to contribute to the development of an ADA response. Here, we present experimental evidence highlighting the roles of epitope, paratope, and binding geometry in defining the composition and size distribution of TRICs formed by IL-17A, a homodimeric cytokine, with four clinical anti-IL-17 antibodies, secukinumab (CosentyxⓇ), ixekizumab (TaltzⓇ), bimekizumab (BimzelxⓇ) and CJM112. Widely different ADA incidence rates have been reported for these antibodies. We found that all four antibodies formed closed-chain TRICs, each comprising two or more IgG molecules connected by an equivalent number of IL-17A homodimers. Secukinumab, the antibody with the lowest ADA incidence rate, uniquely exhibited primarily 2 + 2 closed-chain complexes. In contrast, CJM112 and bimekizumab showed higher amounts of 3 + 3 and 4 + 4 complexes. Additionally, CJM112, and to a greater extent, bimekizumab and ixekizumab, formed very high molecular weight TRICs. Our findings underscore the importance of conducting in-depth biophysical analyses of TRICs formed by therapeutic antibody candidates targeting multivalent protein antigens, to develop safer and more efficacious treatments.

ABSTRACT Next-generation antibodies include a growing number of bispecific and multispecific antibodies that are commonly used to redirect the immune system to fight cancer. Herein, we assessed the depth and breadth of epitope coverage as a proxy for functional diversity in human immune repertoires produced by two complementary in vivo platforms utilizing a common light chain, in a chicken (OmniClicTM) or rat (OmniFlic®) host species. We adopted NKp46 as a model to target antigen due to its use in emerging natural killer (NK) immune engagers that are being explored clinically as potentially safer alternatives to traditional CD3-based T cell engagers. To probe the epitope diversity of our antibody repertoires, we performed a detailed high throughput epitope binning study using surface plasmon resonance and corroborated our binning assignments with epitope mapping data deduced from hydrogen deuterium exchange mass spectrometry. Our results revealed broad epitope coverage and nuanced diversity both within and across repertoires, with few epitopes shared, suggesting that the complementary use of OmniClicTM and OmniFlic® produces more comprehensive coverage than either alone. Furthermore, our epitope binning assignments aligned with our complementarity-determining region-based sequence lineage assignments, enabling a direct comparison of sequence diversity across Clic and Flic repertoires despite their use of different scaffolds, a single functionally rearranged V(D)J scaffold versus multiple combinatorially assembled V(D)J scaffolds, respectively. The rich epitope diversity of both OmniClicTM and OmniFlic® yielded multiple candidates for functional NK activators, as determined in an antibody-dependent cellular cytotoxicity assay, demonstrating their value as building blocks in constructing optimized immune engagers.

ABSTRACT The emergence of anti-drug antibodies (ADAs) poses a major obstacle in the clinical development of therapeutic proteins (TPs) such as monoclonal antibodies and their derivatives. While standard multitiered ADA assays and neutralizing antibody assays offer valuable insights into the humoral immunogenicity risks of TPs, they are not sufficient to provide in-depth knowledge such as ADA epitope specificities. For complex multidomain biotherapeutics (MDBs), ADAs targeting individual domains can elicit distinct pharmacological effects. Therefore, it is crucial to implement straightforward and reliable methodologies to deconvolute ADA epitope profiles of MDBs. Herein, we report a case study using domain specificity analysis, linear peptide scanning and bioinformatic B cell epitope prediction to unveil the clinical ADA epitope landscape of TAK-186, a multidomain T cell engager that has been discontinued from clinical development. By applying this workflow, we observed strong domain specificity variability among patient samples. Furthermore, the data showed that many patients demonstrated evolved ADA epitope specificities throughout the course of the treatment. Several potential linear epitopes were identified subsequently through experimental and computational approaches. Overall, we presented in this study a practical strategy to elucidate and potentially mitigate the immunogenicity liabilities of complex biotherapeutics.

ABSTRACT The efficacy of therapeutic monoclonal antibodies (mAbs) often hinges on biodistribution to their site of action. However, traditional pharmacokinetic (PK) assessments – typically based on measuring plasma or total tissue concentrations – fail to reflect the interstitial concentrations that are most relevant for tissue targets. This study aimed to address this limitation by integrating experimentally determined vascular and interstitial volumes from tissues in SCID-beige mice with a comprehensive PK time-course and biodistribution analysis of four distinct anti-viral monoclonal antibodies (mAbs 1–4) with no endogenous mouse target. The biodistribution studies included 11 tissues, characterizing tissue and plasma concentrations over a 168-h time-course. Total and interstitial tissue concentrations were evaluated to better understand concentrations within the interstitial space compared to bulk tissue values. These data revealed significant tissue-specific partitioning, with fold-change analysis suggesting groupings correlating with capillary endothelium characteristics. A dynamic model was implemented for the estimation of antibody biodistribution coefficient (ABC) values at steady-state, partitioning ratio (PR) values at steady-state, and their associated equilibrium rate constants (t1/2eq, t’1/2eq) across 11 (ABC, t1/2eq) and 7 tissues (PR, t’1/2eq), respectively. Specifically, to understand non-binding, target-independent biodistribution, we combined data from mAbs 1, 2, and 3 to create a “typical mAb” (mAb 123) profile, from which these coefficients and ratios were derived. Analysis of mAb 4, a structurally similar IgG molecule with undesirable PK properties, enabled comparative insights into antibody distribution and kinetics. These studies provided a comprehensive dataset for understanding interstitial antibody PK, crucial for improving predictions of PK at the site-of-action and in vivo efficacy.

ABSTRACT N-glycosylation, a critical quality attribute of monoclonal antibodies, plays a pivotal role in regulating pharmacokinetics and pharmacodynamics through high-mannose (Man5) glycoform modulation. While our previous work demonstrated that N-acetyl-D-mannosamine (ManNAc) supplementation effectively reduces Man5 levels without compromising antibody yield or other critical quality attributes, the mechanistic basis remained unclear. This study systematically investigates ManNAc’s regulatory mechanism through a multi-parametric analysis. Cellular uptake studies revealed a 3-day latency period preceding Man5 reduction post-ManNAc administration. Subsequent transcriptional profiling showed no significant alterations in Man5-associated enzyme expression (Mgat1, Mgat2, Man2a1, SLC35A3), while metabolomic analysis demonstrated marked elevation of intracellular ManNAc, uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc), and cytidine-5’-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) levels. Mechanistic studies revealed two critical findings: (1) Chinese hamster ovary cells exhibit minimal endogenous N-acetyl-D-glucosamine-2-epimerase expression, and (2) CMP-Neu5Ac exerts potent inhibition on glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) activity in vitro, despite ManNAc’s lack of transcriptional regulation on GNE. We propose a metabolic flux redirection model, where ManNAc-derived CMP-Neu5Ac accumulation inhibits GNE activity, thereby shunting UDP-GlcNAc from sialic acid biosynthesis toward N-glycosylation pathways to reduce Man5 levels. This work not only identifies UDP-GlcNAc substrate limitation as a key constraint in antibody glycosylation but also establishes exogenous monosaccharide supplementation as a novel metabolic engineering strategy for Man5 optimization. These findings provide critical mechanistic insights for precision glycoengineering of therapeutic antibodies.

ABSTRACT The analytical comparability of biologic products and their biosimilars, including higher-order structure (HOS) assessment, ensures product quality and is required for regulatory approval. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to evaluate the HOS of Humira (adalimumab) and its biosimilars under normal and photo-stressed conditions. Under normal conditions, 1D and 2D NMR spectra showed strong structural similarity among all products. However, photo-stressed samples exhibited distinct structural differences, including increased methionine oxidation, and localized conformational changes, most notably in the reference product. These changes correlated with findings from size-exclusion chromatography, capillary isoelectric focusing, and mass spectrometry (MS), which revealed size and charge heterogeneity, as well as site-specific methionine oxidation in the heavy chains. The differences in photostability were found to be influenced by container closure systems (CCSs) and formulations. In contrast, circular dichroism spectral analysis showed minimal variation in secondary structures among stressed and unstressed samples. These results underscore the utility of NMR as a sensitive tool for comparative structural analysis of monoclonal antibodies and their biosimilars, particularly under stress conditions, and highlight the impact of formulation and CCS on product stability.