Therapeutic mAbs Research Articles

NMR coupled with multivariate data analysis for monitoring the degradation of a formulated therapeutic monoclonal antibody.

The function of a protein is directly coupled to its higher-order structure (HOS). Deviations in this critical quality attribute (CQA) may be linked to a decrease in the efficacy and/or safety of the final therapeutic product. High-resolution nuclear magnetic resonance (NMR) spectroscopy has been recently highlighted for protein HOS characterization, thanks to its ability to capture small changes at the molecular and structural levels (primary, secondary, tertiary and quaternary). The present study was carried out to demonstrate the ability of NMR (1D 1H and 2D 1H-13C experiments) coupled with multivariate data analysis (PCA and PLS regression) to monitor the degradation of a formulated mAb-like protein and for the simultaneous quantification of related CQAs, i.e., potency, purity and impurities (aggregates and fragments). The results indicate that this approach could be applied to mitigate product quality risks during development, manufacturing and stability studies of mAb therapeutics.

Procollagen-lysine 2-oxoglutarate 5-dioxygenases are responsible for 5R-hydroxylysine modification of therapeutic T-cell bispecific monoclonal antibodies produced by Chinese hamster ovary cells.

We present a detailed mass spectrometric analysis of three 2 + 1 T-cell bispecific monoclonal antibodies (TCB mAbs), where an unexpected +15.9950Da mass shift in tryptic peptides was observed. This modification was attributed to the occurrence of 5R-hydroxylysine (Hyl) using a hybrid LC-MS/MS molecular characterization and CRISPR/Cas9 gene deletion approach. The modification was found at various sites within TCB mAbs, with a conspicuous hot spot motif mirroring a prior observation where Hyl was mapped to the CH1-VH Fab domain interface of IgGs. In contrast to the preceding report, our structural modeling analysis on TCB mAbs unveiled substantial differences in the orientation and flexibility of motifs in immediate proximity and across the artificial CH1-VL cross Fab interface and upstream elbow segment. Utilizing a hybrid database search, RNAseq, and a CRISPR/Cas9 knockout methodology in Chinese hamster ovary (CHO) production cell lines, procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) were conclusively identified as the catalyzing enzymes accountable for the 5R-Hyl modification in TCB mAbs. To quantitatively inhibit Hyl formation in TCB mAbs, the activity of all three Chinese hamster PLOD isoenzymes needs to be depleted via CRISPR/Cas9 gene knockout. Moreover, our investigation identified cell culture iron availability, process duration, and clonal variability in CHO cells as elements influencing the levels of Hyl formation in TCB mAbs. This research offers a solution for circumventing Hyl formation in therapeutic complex mAb formats, such as TCB mAbs, produced in CHO cell culture processes, thereby addressing potential technical and biological challenges associated with unintended Hyl modification.

Antitumor activity of PAbs generated by immunization with a novel HER3-targeting protein-based vaccine candidate in preclinical models.

Despite the cumulative evidence supporting HER3 as a target for antitumor therapies, no agents targeting HER3 have been approved for cancer treatment. Most of the agents evaluated in preclinical and clinical trials have been specific monoclonal antibodies (MAbs), with few examples of active immunotherapy directed against this receptor. However, some cancer vaccine formats may generate polyclonal antibodies (PAbs) that replicate the diverse effector mechanisms of MAbs, including ligand neutralization and receptor degradation. In this study, we developed a protein subunit-based monovalent vaccine candidate targeting the extracellular domain (ECD) of HER3. Immunization of mice with a formulation targeting murine ErbB3-ECD successfully overcome tolerance to this self-antigen, inducing high titers of ErbB3-specific PAbs. The antitumor potential of this formulation and the induced PAbs was demonstrated in vivo and in vitro in an ErbB3-overexpressing 3LL-D122-derived tumor model. The immunogenicity of the HER3-ECD-based vaccine candidate was confirmed by the induction of high titers of HER3-specific PAbs. Consistent with the initial results, HER3-ECD-targeting PAbs were cytotoxic in several human epithelial tumor cell lines and exerted antitumor effects in vivo. These results support the value of HER3 as a tumor antigen and the effector mechanisms of HER3-specific therapeutic MAbs, while suggesting the potential of the proposed vaccine candidate for the treatment of HER3-expressing carcinomas.

IDO2 Drives Autoantibody Production and Joint Inflammation in a Preclinical Model of Arthritis by Repressing Runx1 Function in B Cells.

The immunomodulatory enzyme IDO2 is an essential mediator of autoantibody production and joint inflammation in preclinical models of autoimmune arthritis. Although originally identified as a tryptophan-catabolizing enzyme, we recently discovered a previously unknown nonenzymatic pathway is essential for the proarthritic function of IDO2. We subsequently identified Runx1 (Runt-related transcription factor 1) as a potential component of the nonenzymatic pathway IDO2 uses to drive arthritis. In this study, we find that IDO2 directly binds Runx1 and inhibits its localization to the nucleus, implicating Runx1 as a downstream component of IDO2 function. To directly test whether Runx1 mediates the downstream pathway driving B cell activation in arthritis, we bred B cell conditional Runx1-deficient (CD19cre Runx1flox/flox) mice onto the KRN.g7 arthritis model in the presence or absence of IDO2. Runx1 loss did not affect arthritis in the presence of IDO2; however, deleting Runx1 reversed the antiarthritic effect of IDO2 loss in this model. Further studies demonstrated that the IDO2-Runx1 interaction could be blocked with a therapeutic anti-IDO2 mAb invitro and that Runx1 was required for IDO2 Ig's therapeutic effect invivo. Taken together, these data demonstrate that IDO2 mediates autoantibody production and joint inflammation by acting as a repressor of Runx1 function in B cells and implicate therapeutic targeting of IDO2-Runx1 binding as a strategy to inhibit autoimmune arthritis and other autoantibody-mediated diseases.

Development, physico-chemical characterization, and in vivo stability of a novel aglycosylated monoclonal antibody targeting FAM19A5

AbstractIntroducing aglycosylation into therapeutic monoclonal antibodies (mAbs) can prevent side effects associated with fragment crystallizable (Fc)-mediated effector functions. This modification induces structural changes in the heavy chain constant domain 2-constant domain 3 within Fc regions, which decreases antibody stability at acidic pH and high temperature. In this study, NS101, a novel aglycosylated mAb targeting family with sequence similarity 19, A5 (FAM19A5) for neurological diseases was evaluated with respect to its developability and in vivo stability as therapeutics. When recombinant CHO cells producing NS101 were cultivated using a fed-batch mode in a 500 L bioreactor, cell growth and mAb production profiles were consistent across three consecutive runs. NS101, thus produced, features an additional intra-disulfide bond in the heavy chain complementarity-determining region 3, contributing strong and sophisticated binding to the cryptic epitope. The melting temperature (Tm) of NS101 was lower than that of commercial glycosylated therapeutic mAbs, but NS101 showed better stability at 4 °C for 36 months. The binding affinity of NS101 to FAM19A5 and neonatal Fc receptor were comparable to those of glycosylated NS101. In addition, in three human cohort groups receiving 6, 12, and 24 mg/kg of NS101, the mean half-life was 22 days, and NS101 exhibited in vivo stability, considering that the half-lives of commercialized therapeutic mAbs and endogenous IgGs are 2–4 weeks and 21 days, respectively. Taken together, the results obtained here demonstrate that NS101, a novel aglycosylated mAb, has potential as a therapeutic agent for neurological diseases.

B-342 Teclistamab interference in serum protein electrophoresis and serum immunofixation electrophoresis in patients with recurrent or relapsed multiple myeloma

Abstract Background Serum protein electrophoresis (SPEP) and serum immunofixation electrophoresis (IFE) analyses are mainstays in the initial diagnosis, risk stratification, and evaluating treatment response in patients with multiple myeloma (MM) in accordance with the International Myeloma Working Group criteria. Published reports have demonstrated that therapeutic monoclonal antibodies (mAbs) used to treat MM (e.g. anti-CD38 IgG-kappa) are detectable by SPEP and IFE, which may interfere with accurate evaluation of complete response. Recently, Teclistamab (Tecvayli®; Janssen Biotech, Horsham Township, PA), a humanized bispecific IgG-lambda mAb targeting B-cell maturation antigen (BCMA) and CD3 on T cells was approved for treating patients with penta-refractory MM. In contrast with other IgG-kappa therapeutic mAbs, the degree to which Teclistamab may interfere with detection and quantification of monoclonal proteins is unknown. Therefore, we sought to assess the interference of Teclistamab therapy on routine SPEP and IFE analysis. Methods Patients treated with Teclistamab were identified by retrospective review of electronic medical records. Teclistamab (90 mg/mL) was spiked into a remnant patient sample with IgG lambda MM at a final concentration of 10 μg/mL, which corresponds to ½Cmax values in patients infused with Teclistamab. SPEP and IFE were performed using the Helena SPIFE Touch instrument. Computer-assisted densitometry was used to quantify monoclonal proteins in the gamma region of SPEP gels. Results In several patients with refractory MM treated with Teclistamab, IFE analysis revealed IgG lambda or lambda light chain monoclonal proteins, which were inconsistent with previously-identified monoclonal proteins (e.g. IgG kappa). In one patient diagnosed with IgG lambda MM, spiking 10 μg/mL Teclistamab increased M-spike abundance by approximately 0.15 g/dL when quantified by SPEP. Conclusions In patients with relapsed or refractory MM, treatment with Teclistamab may confound accurate clinical assessment of complete response by SPEP and IFE methods. Increased awareness of this preanalytical interference is necessary to guide clinical decision making.

Single-hit genome editing optimized for maturation in B cells redirects their specificity toward tumor antigens.

T-cell-based adoptive immunotherapy is a new pillar of cancer care. Tumor-redirected B cells could also contribute to therapy if their manipulation to rewire immunoglobulin (Ig) genes is mastered. We designed a single-chain Ig-encoding cassette ("scFull-Ig") that redirects antigen specificity when inserted at a single position of the IgH locus. This design, which places combined IgH and IgL variable genes downstream of a pVH promoter, nevertheless preserves all Ig functional domains and the intrinsic mechanisms that regulate expression from the IgM B cell receptor (BCR) expression to Ig secretion, somatic hypermutation and class switching. This single-locus editing provides an efficient and safe strategy to both disrupt endogenous Ig expression and encode a new Ig paratope. As a proof of concept, the functionality of scFull BCR and/or secreted Ig was validated against two different classical human tumor antigens, HER2 and hCD20. Once validated in cell lines, the strategy was extended to primary B cells, confirming the successful engineering of BCR and Ig expression and the ability of scFull-Ig to undergo further class switching. These results further pave the way for future B cell-based adoptive immunotherapy and strategies to express a therapeutic mAb with a variety of switched H-chains that provide complementary functions.

Facile Rebridging Conjugation Approach to Attain Monoclonal Antibody-Targeted Nanoparticles with Enhanced Antigen Binding and Payload Delivery.

Adopting conventional conjugation approaches to construct antibody-targeted nanoparticles (NPs) has demonstrated suboptimal control over the binding orientation and the structural stability of monoclonal antibodies (mAbs). Hitherto, the developed antibody-targeted NPs have shown proof of concept but lack product homogeneity, batch-to-batch reproducibility, and stability, precluding their advancement toward the clinic. To circumvent these limitations and advance toward clinical application, herein, a refined approach based on site-specific construction of mAb-immobilized NPs will be appraised. Initially, the conjugation of atezolizumab (anti-PDL1 antibody, Amab) with polymeric NPs was developed using bis-haloacetamide (BisHalide) rebridging chemistry, followed by click chemistry (NP-Fab BisHalide Ab and NP-Fc BisHalide Ab). For comparison purposes, mAb-immobilized NPs developed utilizing conventional conjugation methods, namely, N-hydroxysuccinimide (NHS) coupling and maleimide chemistry (NP-NHS Ab and NP-Mal Ab), were included. Next, flow cytometry and confocal microscopy experiments evaluated the actively targeted NPs (loaded with fluorescent dye) for cellular binding and uptake. Our results demonstrated the superior and selective binding and uptake of NP-Fab BisHalide Ab and NP-Fc BisHalide Ab into EMT6 cells by 19-fold and 13-fold, respectively. To evaluate the PDL1-dependent cell uptake and the selectivity of the treatments, a blocking step of the PDL1 receptor with Amab was performed prior to incubation with NP-Fab BisHalide Ab and NP-Fc BisHalide Ab. To our delight, the binding and uptake of fluorescent NPs were reduced significantly by 3-fold for NP-Fab BisHalide Ab, demonstrating the PDL1-mediated uptake. Moreover, NP-Fab BisHalide Ab and NP-Fc BisHalide Ab were entrapped with the paclitaxel payload, and their cytotoxicity was evaluated. They showed significant enhancements compared to free paclitaxel and NP-NHS Ab. Overall, this work will provide a facile conjugation method that could be implemented to actively target NPs with a plethora of therapeutic mAbs approved for various malignancies.

Applying UHPLC-HRAM MS/MS Method to Assess Host Cell Protein Clearance during the Purification Process Development of Therapeutic mAbs.

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample.

Therapeutic efficacy of JEW-M449, an anti-TCTP monoclonal antibody, administered via the nasal route in a BALB/c mouse model of ovalbumin-induced acute asthma

Numerous studies have highlighted the role of translationally controlled tumor protein (TCTP) as a key inflammatory mediator of asthma and allergies. Our previous study revealed that blocking the cytokine-like activity of TCTP using JEW-M449, an anti-TCTP monoclonal antibody (mAb), alleviated allergic inflammation in asthmatic mice. This study aimed to determine whether directly delivering JEW-M449 into the respiratory tract is a more effective way of mitigating airway inflammation in a mouse model of ovalbumin (OVA)-induced allergic airway inflammation than delivering this antibody via the intraperitoneal (IP) route. OVA-sensitized mice were intranasally administered JEW-M449 to enable its direct delivery to the respiratory tract before OVA challenge. We evaluated the changes in the levels of bronchoalveolar lavage fluid (BALF) cells, T helper type 2 (Th2) cytokines, OVA-specific immunoglobulin E (IgE), and histopathological alterations in the lung tissues. Intranasal (IN) administration of JEW-M449 significantly ameliorated the pathological changes associated with OVA-induced lung injury, including reduced inflammatory cell infiltration and mucus hypersecretion. Mice IN administered JEW-M449 also showed decreased OVA-mediated induction of Th2 cytokines in BALF and lung homogenates. Importantly, JEW-M449 delivered via the IN route reached the lung tissue more effectively and exerted superior anti-inflammatory effects in OVA-challenged mice than the IP-delivered JEW-M449. This study is the first to demonstrate the efficacy of directly delivering JEW-M449 anti-TCTP mAb into the respiratory tract to alleviate the asthma phenotype in a mouse model, thereby highlighting a potential delivery strategy for novel inhaled mAb therapeutics for human asthma.

Afucosylated anti-EBOV antibody MIL77-3 engages sGP to elicit NK cytotoxicity.

Zaire Ebolavirus (EBOV) is highly lethal and causes sporadic outbreaks. The passive administration of monoclonal antibodies (mAbs) represents a promising treatment regimen against EBOV. Mounting evidence has shown that the efficacy of a subset of therapeutic mAbs in vivo is intimately associated with its capacity to trigger NK activity, supporting glycomodification of Fc region of anti-EBOV mAbs as a putative strategy to enhance Fc-mediated immune effector function as well as protection in vivo. Our work here uncovers the potential harmful influence of this modification on host immune responses, especially for mAbs with cross-reactivity to secreted glycoproptein (sGP) (e.g., MIL77-3), and highlights it is necessary to evaluate the NK-stimulating activity of a fucosylated mAb engaged with sGP when a new candidate is developed.

In-Depth Characterization for Methionine Oxidization in Complementary Domain Region by Hydrophobic Interaction Chromatography.

The oxidation of the complementarity-determining region (CDR) in monoclonal antibodies (mAbs) is a critical quality attribute that can affect the clinical efficacy and safety of recombinant mAb therapeutics. In this study, a robust hydrophobic interaction chromatography (HIC) method was developed to quantify and characterize CDR oxidation variants in mAb-A by using a Proteomix Butyl-NP5 column. The HIC analysis revealed oxidation variants that eluted earlier than the main species with weaker hydrophobicity. It was found that Met105 in the CDR was more susceptible to oxidation. Additionally, it was noted that the oxidation of Met105 on a single heavy chain resulted in elution at a distinct position compared to the oxidation on two heavy chains. This observation led to the fractionation and enrichment of the oxidized variants for further evaluation of their biofunction. The study also demonstrated that the oxidation of Met105 did not impact the antigen-binding capacity but significantly reduced the PD-1/PD-L1 blockade activity of mAb-A. The HIC method, which was employed to quantify CDR oxidation, underwent validation and was subsequently utilized for stability studies as well as for assessing the similarity between mAb-A and its reference product.

Prediction of human serum concentration–time profiles of therapeutic monoclonal antibodies using common marmosets (Callithrix jacchus): initial assessment with canakinumab, adalimumab, and bevacizumab

Cynomolgus monkeys and human FcRn transgenic mice are generally used for pharmacokinetic predictions of therapeutic monoclonal antibodies (mAbs). In the present study, the application of the common marmoset, a small nonhuman primate, as a potential animal model for prediction was evaluated for the first time. Canakinumab, adalimumab, and bevacizumab, which exhibited linear pharmacokinetics in humans, were selected as the model compounds. Marmoset pharmacokinetic data were reportedly available only for canakinumab, and those for adalimumab and bevacizumab were acquired in-house. Four pharmacokinetic parameters for a two-compartment model (i.e. clearance and volume of distribution in the central and peripheral compartments) in marmosets were extrapolated to the values in humans with allometric scaling using the average exponents of the three mAbs. As a result, the observed human serum concentration–time curves of the three mAbs following intravenous administration and those of canakinumab and adalimumab following subcutaneous injections (with an assumed absorption rate constant and bioavailability) were reasonably predicted. Although further prediction studies using a sufficient number of other mAbs are necessary to evaluate the versatility of this model, the findings indicate that marmosets can be an alternative to preceding animals for human pharmacokinetic predictions of therapeutic mAbs.

Enhanced N-Glycan Profiling of Therapeutic Monoclonal Antibodies through the Application of Upper-Hinge Middle-Up Level LC-HRMS Analysis.

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up (UHMU)-level ultra-high-performance liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis to improve N-glycan profiling of mAbs. Two specific enzymes, known as IgG degradation enzymes (IGDEs), were used to selectively cleave therapeutic mAbs above the hinge region to separate antibody subunits for further Fc glycan analysis by means of the UHMU/LC-HRMS workflow. The complexity of the mass spectra of IGDEs-digested mAbs was significantly reduced compared to the intact MS level, enabling reliable assignment and relative quantitation of paired Fc glycoforms. The results of the UHMU/LC-HRMS analysis of nine approved therapeutics highlight the significance of this approach for in-depth glycoform profiling.

Monoclonal Antibodies and Antibody-drug Conjugates as Emerging Therapeutics for Breast Cancer Treatment.

When breast cells divide and multiply out of control, it is called breast cancer. Symptoms include lump formation in the breast, a change in the texture or color of the breast, or a discharge from the nipple. Local or systemic therapy is frequently used to treat breast cancer. Surgical and radiation procedures limited to the affected area are examples of local management. There has been significant worldwide progress in the development of monoclonal antibodies (mAbs) since 1986, when the first therapeutic mAb, Orthoclone OKT3, became commercially available. mAbs can resist the expansion of cancer cells by inducing the destruction of cellular membranes, blocking immune system inhibitors, and preventing the formation of new blood vessels. mAbs can also target growth factor receptors. Understanding the molecular pathways involved in tumor growth and its microenvironment is crucial for developing effective targeted cancer therapeutics. Due to their unique properties, mAbs have a wide range of clinical applications. Antibody-drug conjugates (ADCs) are drugs that improve the therapeutic index by combining an antigen-specific antibody with a payload. This review focuses on the therapeutic applications, mechanistic insights, characteristics, safety aspects, and adverse events of mAbs like trastuzumab, bevacizumab, pertuzumab, ertumaxomab, and atezolizumab in breast cancer treatment. The creation of novel technologies utilizing modified antibodies, such as fragments, conjugates, and multi-specific antibodies, must be a central focus of future studies. This review will help scientists working on developing mAbs to treat cancers more effectively.

Biophysical cartography of the native and human-engineered antibody landscapes quantifies the plasticity of antibody developability

Designing effective monoclonal antibody (mAb) therapeutics faces a multi-parameter optimization challenge known as “developability”, which reflects an antibody’s ability to progress through development stages based on its physicochemical properties. While natural antibodies may provide valuable guidance for mAb selection, we lack a comprehensive understanding of natural developability parameter (DP) plasticity (redundancy, predictability, sensitivity) and how the DP landscapes of human-engineered and natural antibodies relate to one another. These gaps hinder fundamental developability profile cartography. To chart natural and engineered DP landscapes, we computed 40 sequence- and 46 structure-based DPs of over two million native and human-engineered single-chain antibody sequences. We find lower redundancy among structure-based compared to sequence-based DPs. Sequence DP sensitivity to single amino acid substitutions varied by antibody region and DP, and structure DP values varied across the conformational ensemble of antibody structures. We show that sequence DPs are more predictable than structure-based ones across different machine-learning tasks and embeddings, indicating a constrained sequence-based design space. Human-engineered antibodies localize within the developability and sequence landscapes of natural antibodies, suggesting that human-engineered antibodies explore mere subspaces of the natural one. Our work quantifies the plasticity of antibody developability, providing a fundamental resource for multi-parameter therapeutic mAb design.

A generic anti-drug antibody assay for monoclonal antibody therapeutics with broad dynamic range eliminates the need for titer evaluation in preclinical studies

In preclinical protein therapeutic development studies, the emergence of anti-drug antibodies (ADA) can potentially impact drug pharmacokinetics and safety. While immunogenicity assessment is not mandatory in preclinical studies, banking samples can be valuable for interpreting unexpected pharmacological responses. Immunoassays that use generic reagents across different drug molecules can simplify ADA assessment and expedite sample evaluations. This work showcases the ability of the Gyrolab automated immunoassay platform to detect and quantify both drug-free and drug-bound (total) ADAs to monoclonal antibody (mAb) therapeutics in cynomolgus monkey preclinical studies. Compared to the previously reported total ADA ELISA, the Gyrolab assay exhibited a wider signal dynamic range and increased drug tolerance. Similar sensitivity, dynamic range and cut point factors were observed for four therapeutic mAbs of different isotypes using the Gyrolab assay. Here we present a comparison of ADA assays using bridging ELISA, total ADA ELISA and total ADA Gyrolab formats in a cynomolgus monkey study where the subjects were treated with a single dose of a mAb therapeutic. We demonstrate that the total ADA assays detected host ADA responses at earlier time points compared to the bridging ELISA. The Gyrolab assay has the best correlation between signal-to-noise (S/N) and titer over a wide ADA concentration range, highlighting the utility of Gyrolab in S/N reporting of ADA response to eliminate the need for secondary titer assays. Collectively, our results demonstrate that the generic ADA Gyrolab assay minimizes the necessity for extensive assay development and optimization for therapeutic mAbs, streamlining preclinical immunogenicity assessment to enable interpretation of pharmacological data.

Affinity-Resolved Size Exclusion Chromatography Coupled to Mass Spectrometry: A Novel Tool to Study the Attribute-and-Function Relationship in Therapeutic Monoclonal Antibodies.

Assessment of critical quality attributes (CQAs) is an important aspect during the development of therapeutic monoclonal antibodies (mAbs). Attributes that affect either the target binding or Fc receptor engagement may have direct impacts on the drug safety and efficacy and thus are considered as CQAs. Native size exclusion chromatography (SEC)-based competitive binding assay has recently been reported and demonstrated significant benefits compared to conventional approaches for CQA identification, owing to its faster turn-around and higher multiplexity. Expanding on the similar concept, we report the development of a novel affinity-resolved size exclusion chromatography-mass spectrometry (AR-SEC-MS) method for rapid CQA evaluation in therapeutic mAbs. This method features wide applicability, fast turn-around, high multiplexity, and easy implementation. Using the well-studied Fc gamma receptor III-A (FcγRIIIa) and Fc interaction as a model system, the effectiveness of this method in studying the attribute-and-function relationship was demonstrated. Further, two case studies were detailed to showcase the application of this method in assessing CQAs related to antibody target binding, which included unusual N-linked glycosylation in a bispecific antibody and Met oxidation in a monospecific antibody, both occurring within the complementarity-determining regions (CDRs).

Impact of isotype on the mechanism of action of agonist anti-OX40 antibodies in cancer: implications for therapeutic combinations

BackgroundOX40 has been widely studied as a target for immunotherapy with agonist antibodies taken forward into clinical trials for cancer where they are yet to show substantial efficacy. Here, we...

Immune-Mediated Liver Effects Associated With Administration of a Human Anti-IL-21 Receptor Antibody (ATR-107) in Rats.

The toxicity of ATR-107, a human anti-interleukin-21 receptor (IL-21R) monoclonal antibody (mAb), was evaluated in CD-1 mice and cynomolgus monkeys after single-dose intravenous (IV) administration, and in Sprague-Dawley (SD) rats and cynomolgus monkeys after weekly IV and subcutaneous (SC) administration in 13-week toxicity studies that included recovery. Adverse liver necrosis, diffuse bridging fibrosis, and higher liver enzymes occurred in rats in the low-dose IV group (10 mg/kg), but not at 50 or 250 mg/kg IV, and not following SC administration despite overlapping systemic ATR-107 exposures. Similar findings were not seen in mice or cynomolgus monkeys. A series of investigative rat toxicity studies showed liver findings only occurred after administration of at least 3 weekly doses, only occurred in rats that developed anti-drug antibodies (ADAs), and the incidence was associated with higher ADAs titers. However, the presence of ADAs did not always result in liver injury. Liver findings did not occur in nude rats, which had high ATR-107 exposures and no ADAs. These findings suggest an adaptive immune response with formation of ADAs was necessary for development of ATR-107-related liver findings, and that liver injury can occur in rats secondary to development of ADAs following repeated administration of a human therapeutic mAb.