Individual MAbs Research Articles

Multidomain Protein-Urea Interactions: Differences in Binding Behavior Lead to Different Destabilization Tendencies for Monoclonal Antibodies.

We study the influence of urea on the stability of monoclonal antibodies (mAbs) using molecular dynamics (MD) simulations in combination with differential scanning fluorimetry (DSF). We show that a denaturing cosolute such as urea binds strongly to the protein, which can lead to denaturation and enhanced aggregation behavior at high temperatures. The interaction between protein and urea crucially depends on the surface properties of the individual mAb domains and therefore affects the general binding to the protein differently. The study of these mechanisms for proteins with multiple domains, such as mAbs, encounters significant limitations in experimental analysis methods due to their complexity. Using computational and experimental methods, we are able to separate the protein-urea interaction by domain and show that Lennard-Jones interactions are mainly responsible for significant binding effects. Our results emphasize the potential of MD simulations in combination with Kirkwood-Buff theory to study the interactions between proteins with multiple domains and cosolutes as formulation excipients for drug discovery and development.

B-067 Human IgE Monoclonal Antibodies as Unique Tools for Allergy Diagnostics

Abstract Background Current blood-based tests for allergy diagnosis rely on human sera or plasma as the source of IgE antibody for method validation and calibration. Obtaining human samples with sufficiently high levels of IgE presents challenges for researchers and diagnostic companies. Here, we present a unique panel of allergen-specific human IgE monoclonal antibodies (mAb) that target inhalant, food and alpha-gal allergens and offer novel approaches for investigation of IgE responses and standardization of allergy diagnostic reagents and methods. Methods Human IgE mAb (n=30) targeting inhalant allergens from dust mite, cat and dog; food allergens from peanut, egg, milk, walnut and cashew, and galactose-α-1,3-galactose, were generated from B cells from symptomatic allergic patients with rhinitis, asthma, atopic dermatitis or red meat allergy using hybridoma technology. Total and allergen-specific IgE were quantified by ImmunoCAP (whole allergen ImmunoCAP were also performed). Allergen binding profiles and mAb titers were determined by two-site immunoassay (ELISA). Antibody purity was assessed using SDS-PAGE and Western Blot. Results SDS-PAGE showed single heavy and light chain bands under reducing conditions. Antibody recognition of target allergens was confirmed by Western Blot. Allergen-specific IgE mAb concentrations for inhalant allergens ranged: 9,000-37,000 kU/L for dust mite (n=3), 2,100-53,000 kU/L for cat (n=4), 5,300-21,000 for dog (n=2). Food allergens ranged: 16,300-49,400 kU/L for peanut (n=14), 15,700-81,000 for egg (n=2), 41,600 for milk (n=1), 7,100 for walnut (n=1) and 37,000 for cashew (n=1). Alpha-gal IgE monoclonals (n=2) ranged in concentration from 15,700-81,000 kU/L. The ratios of specific IgE to total IgE measured by ImmunoCAP vary by allergen and by individual human IgE mAb. Ratios for the inhalant mAb ranged from 0.04-1.05, for the food mAb the ratios ranged from 0.33-0.99. The two alpha-gal mAb showed relatively lower specific IgE levels (average ratio was 0.14; range=0.08-0.20). Conclusions Monoclonal IgE antibodies derived from patients presenting with allergic symptoms may provide a reliable replacement for human sera to investigate allergic disease. IgE mAb to inhalant and food allergens, and to the alpha-gal carbohydrate epitope, provide unique diagnostic markers and will offer improved accuracy for assessing patient allergy by standardizing diagnostic reagents and methods. Crystal structures of several of the human IgE mAb epitopes have recently been reported (Pomés et al, J Allergy Clin Immunol, 2024). The current panel of human IgE mAb can be expanded to include additional allergen targets, which may lead to the development of improved allergy therapeutics.

Charge Detection Mass Spectrometry Reveals Conformational Heterogeneity in Megadalton-Sized Monoclonal Antibody Aggregates.

Aggregation of protein-based therapeutics can occur during development, production, or storage and can lead to loss of efficacy and potential toxicity. Native mass spectrometry of a covalently linked pentameric monoclonal antibody complex with a mass of ∼800 kDa reveals several distinct conformations, smaller complexes, and abundant higher-order aggregates of the pentameric species. Charge detection mass spectrometry (CDMS) reveals individual oligomers up to the pentamer mAb trimer (15 individual mAb molecules; ∼2.4 MDa) whereas intermediate aggregates composed of 6-9 mAb molecules and aggregates larger than the pentameric dimer (1.6 MDa) were not detected/resolved by standard mass spectrometry, size exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), or by mass photometry. Conventional quadrupole time-of-flight mass spectrometry (QTOF MS), mass photometry, SEC, and CE-SDS did not resolve partially or more fully unfolded conformations of each oligomer that were readily identified using CDMS by their significantly higher extents of charging. Trends in the charge-state distributions of individual oligomers provides detailed insight into how the structures of compact and elongated mAb aggregates change as a function of aggregate size. These results demonstrate the advantages of CDMS for obtaining accurate masses and information about the conformations of large antibody aggregates despite extensive overlapping m/z values. These results open up the ability to investigate structural changes that occur in small, soluble oligomers during the earliest stages of aggregation for antibodies or other proteins.

Quantitative proteomics reveals cellular responses to individual mAb expression and tunicamycin in CHO cells

Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody–producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation.Key points• Proteome profiling of recombinant CHO cells under mild TM treatment.• Identified protein clusters are associated with the unfolded protein response (UPR).• The compared cell lines revealed noticeable disparities in protein expression levels.

Abstract 741: Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity

Abstract The expression of ILT (LILRB) family members and their major ligand HLA-G within solid tumors are associated with immune suppression and poor patient survival. Blocking multiple ILT family members might be more potent than targeting individual members by overcoming compensatory resistances within tumor microenvironment. Based on this hypothesis, we developed a monoclonal antibody (mAb) LITCHI 7 which reacts to three major ILT family members, ILT2, ILT4 and ILT5, and blocks ligand binding to these receptors. LITCHI 7 exhibits a broad binding capability to a wide range of human myeloid and T cell subsets that express ILT2, ILT4, and/or ILT5. Binding to monocyte-derived dendritic cells (moDC), LITCHI 7 released the inhibition on activating Fcγ receptor. On LPS-treated macrophages, LITCHI 7 enhanced inflammatory macrophage differentiation. The effect is primarily mediated by blocking ILT4 since ILT4 mono-specific mAb is as potent as cross-reactive LITHI 7 in modulating myeloid differentiation although both ILT2 and ILT5 are highly expressed by different myeloid subsets. The result was further confirmed by CRISPR Cas9 knock down of ILT2 or ILT5. In the absence of either ILT2 or ILT5, the accumulation of CD14+ monocytes and inflammatory macrophages were not changed during differentiation. While different ILT family members are generally enriched on myeloid cells, ILT2 is also expressed on NK cells and effector memory CD45RA+ T cells (Temra). Blocking ILT2 by LITCHI 7 enhanced TCR signaling when co-culturing the MART-1 specific T cell reporter line with MART-1/HLA-G positive melanoma cells. The effect of LITCHI 7 is mediated by ILT2 blockade since knocking out ILT2, but not ILT4 and ILT5, reduced accumulation of Temra in activated PBMC. To assess whether immune modulation of LITCHI 7 can be translated into anti-tumor activity by blocking different ILT family members, tumor killing assays were performed using innate and adaptive immune effector cells. LITCHI7 enhanced NK cell-mediated cytotoxicity on HLA-G+ tumor cells by blocking ILT2. Both ILT2 and ILT4 blockade play a role in macrophage phagocytosis on lymphoma cells since LITCHI 7 as well as individual anti-ILT2 or ILT-4 mAbs increased macrophage phagocytosis to a similar level. Finally, LITCHI 7 could boost in vitro T cell-mediated killing of CD19+ lymphoma by CD19 specific CAR-T cells in the presence of CD33+ myeloid cells. In summary, LITCHI 7 can enhance both innate and adaptive anti-tumor immunity through ILT2 or ILT4 blockade directly on effector cells or indirectly through myeloid cell reprogramming. As clinical trials are ongoing by targeting ILT2 and ILT4, blocking multiple ILT family members through a single cross-reactive mAb may provide broad anti-tumor effects and advantages when combining with additional oncology treatments. Citation Format: Shiming Ye, Dong Zhang, Brenal Singh, Jaishree Bankoti, Diane Cohen, Siusze Tan, Donghee Choi, Jonathan Hickson, Min-Zu Wu, Urvi Kolhatkar, Mikhail Binnewies, Wei Jin, Jason Hall, John Engelhardt, Alexander Shoemaker. Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 741.

Abstract 3136: Towards development of a novel biparatopic trop2-ADC

Abstract Background: Trophoblast cell surface antigen 2 (TROP-2) is a member of Tumor-associated Calcium Signal Transducer family (TACSTD2), which is a 35kDa transmembrane protein with 4 N-linked glycosylation sites. The overexpression of TROP2 in various cancers is associated with poor prognosis and increased risk of metastasis. Anti-TROP2 antibody drug conjugates (ADCs) are emerging as an innovative therapeutic intervention for cancer treatment. While these efforts were rewarded by the FDA approval of Trodelvy (Sacituzumab govitecan), a Trop2 targeted ADC, in 2020 for the TNBC patients, tumor heterogeneity could present one of the limitations for the Trop2-targeting therapy, leading to drug resistance. Here, we report our effort in developing a novel biparatopic TROP2 ADC which demonstrate superior cell killing activity compared with the monospecific TROP2 ADC, including certain resistant cell lines for the approved therapy. Methods: Various biparatopic Abs were engineered using two different mAbs targeting the two non-overlapping epitopes of Trop2. The physical stability and biological activities were characterized. Binding competition assay was utilized for epitope binning using SPR. Cell binding affinity was determined using flow cytometry. Cellular internalization activity was measured using Incucyte system, and tumor cell killing efficacy was evaluated in multiple cancer cell lines. CDX mouse models were utilized to investigate the TROP2-ADC in vivo efficacy. Results: We identified a biparatopic TROP2 mAb using two monospecific TROP2 mAbs with A recognizing the same epitope as Trodelvy, and B binding to a non-overlapping epitope vs Sacituzumab’s. mAb-B demonstrates superior ability to recognize a few resistance cell lines to mAb-A. The biparatopic TROP2 mAb-A/B displays a KD of 0.81 nM protein binding compared with 6.42 nM and 0.26 nM, respectively for mAb-A and mAb-B. Importantly, the biparatopic mAb-A/B demonstrates significantly faster cellular internalization activity compared to those of the individual mAbs, signifying the biparatopic scaffold could drive receptor clustering and subsequently enhance the internalization, trafficking to the lysosome and degradation of the target. As expected, the ADC of the biparatopic mAb-A/B conjugated with a TOPi payload shows cancer cell killing efficacy in both in vitro and in vivo experiments. Furthermore, we tested mAb-A, -B and biparatopic mAb-A/B for their binding affinity to 14 missense mutations in TROP2 ECD expressed in HEK293 cells. Interestingly, mAb-A showed decreased binding in 5 mutant cell lines, while mAb-B and biparatopic mAb-A/B maintains activity. Conclusion: We have engineered a novel TROP2 biparatopic antibody with unique biological features as well as superior in vitro and in vivo activities. Such a biparatopic ADC presents its potential to be a best-in-class ADC therapy by overcoming the limitation of the monospecific ADC therapy such as drug resistance. Citation Format: Yinan Wu, Brittany Jiang, Julliet Rivera, Fang Wang, Jing Zhou, Lin Liu, Silvia Hudson, Will Draper, Jennifer Li, Xiaoshan Min, Hang Chen, Zhulun Wang. Towards development of a novel biparatopic trop2-ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3136.

Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45.

Gamete surface protein P48/45 has been shown to be important for male gamete fertility and a strong candidate for the development of a malaria transmission-blocking vaccine (TBV). However, TBV development for Plasmodium vivax homolog Pvs48/45 has been slow because of a number of challenges: availability of conformationally suitable recombinant protein; the lack of an in vivo challenge model; and the inability to produce P. vivax gametocytes in culture to test transmission-blocking activity of antibodies. To support ongoing efforts to develop Pvs48/45 as a potential vaccine candidate, we initiated efforts to develop much needed reagents to move the field forward. We generated monoclonal antibodies (mAbs) directed against Pvs48/45 and characterized putative functional domains in Pvs48/45 using recombinant fragments corresponding to domains D1-D3 and their biological functionality through ex vivo direct membrane feeding assays (DMFAs) using P. vivax parasites from patients in a field setting in Brazil. While some mAbs partially blocked oocyst development in the DMFA, one mAb caused a significant enhancement of the infectivity of gametocytes in the mosquitoes. Individual mAbs exhibiting blocking and enhancing activities recognized non-overlapping epitopes in Pvs48/45. Further characterization of precise epitopes recognized by transmission-reducing and -enhancing antibodies will be crucial to design an effective immunogen with optimum transmission-reducing potential.

Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot®.

The affinity distribution of the antigen-specific memory Bcell (Bmem) repertoire in the body is a critical variable that defines an individual's ability to rapidly generate high-affinity protective antibody specificities. Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here, we introduce two variants of the Bcell ImmunoSpot® assay that are suitable for simultaneously assessing the affinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using relatively little labor and with high-throughput capacity. First, we experimentally validated that both ImmunoSpot® assay variants are suitable for establishing functional affinity hierarchies using Bcell hybridoma lines as model antibody-secreting cells (ASC), each producing mAb with known affinity for a defined antigen. We then leveraged both ImmunoSpot® variants for characterizing the affinity distribution of SARS-CoV-2 Spike-specific ASC in PBMC following COVID-19 mRNA vaccination. Such ImmunoSpot® assays promise to offer tremendous value for future Bcell immune monitoring efforts, owing to their ease of implementation, applicability to essentially any antigenic system, economy of PBMC utilization, high-throughput capacity, and suitability for regulated testing.

Nonspecificity fingerprints for clinical-stage antibodies in solution.

Monoclonal antibodies (mAbs) have successfully been developed for the treatment of a wide range of diseases. The clinical success of mAbs does not solely rely on optimal potency and safety but also require good biophysical properties to ensure a high developability potential. In particular, nonspecific interactions are a key developability parameter to monitor during discovery and development. Despite an increased focus on the detection of nonspecific interactions, their underlying physicochemical origins remain poorly understood. Here, we employ solution-based microfluidic technologies to characterize a set of clinical-stage mAbs and their interactions with commonly used nonspecificity ligands to generate nonspecificity fingerprints, providing quantitative data on the underlying physical chemistry. Furthermore, the solution-based analysis enables us to measure binding affinities directly, and we evaluate the contribution of avidity in nonspecific binding by mAbs. We find that avidity can increase the apparent affinity by two orders of magnitude. Notably, we find that a subset of these highly developed mAbs show nonspecific electrostatic interactions, even at physiological pH and ionic strength, and that they can form microscale particles with charge-complementary polymers. The group of mAb constructs flagged here for nonspecificity are among the worst performers in independent reports of surface and column-based screens. The solution measurements improve on the state-of-the-art by providing a stand-alone result for individual mAbs without the need to benchmark against cohort data. Based on our findings, we propose a quantitative solution-based nonspecificity score, which can be integrated in the development workflow for biological therapeutics and more widely in protein engineering.

Function-structure approach reveals novel insights on the interplay of Immunoglobulin G 1 proteoforms and Fc gamma receptor IIa allotypes.

Human Fc gamma receptor IIa (FcγRIIa) or CD32a has two major allotypes with a single amino acid difference at position 131 (histidine or arginine). Differences in FcγRIIa allotypes are known to impact immunological responses such as the clinical outcome of therapeutic monoclonal antibodies (mAbs). FcγRIIa is involved in antibody-dependent cellular phagocytosis (ADCP), which is an important contributor to the mechanism-of-action of mAbs by driving phagocytic clearance of cancer cells. Hence, understanding the impact of individual mAb proteoforms on the binding to FcγRIIa, and its different allotypes, is crucial for defining meaningful critical quality attributes (CQAs). Here, we report a function-structure based approach guided by novel FcγRIIa affinity chromatography-mass spectrometry (AC-MS) assays to assess individual IgG1 proteoforms. This allowed to unravel allotype-specific differences of IgG1 proteoforms on FcγRIIa binding. FcγRIIa AC-MS confirmed and refined structure-function relationships of IgG1 glycoform interactions. For example, the positive impact of afucosylation was higher than galactosylation for FcγRIIa Arg compared to FcγRIIa His. Moreover, we observed FcγRIIa allotype-opposing and IgG1 proteoform integrity-dependent differences in the binding response of stress-induced IgG1 proteoforms comprising asparagine 325 deamidation. The FcγRIIa-allotype dependent binding differences resolved by AC-MS were in line with functional ADCP-surrogate bioassay models. The molecular basis of the observed allotype specificity and proteoform selectivity upon asparagine 325 deamidation was elucidated using molecular dynamics. The observed differences were attributed to the contributions of an inter-molecular salt bridge between IgG1 and FcγRIIa Arg and the contribution of an intra-molecular hydrophobic pocket in IgG1. Our work highlights the unprecedented structural and functional resolution of AC-MS approaches along with predictive biological significance of observed affinity differences within relevant cell-based methods. This makes FcγRIIa AC-MS an invaluable tool to streamline the CQA assessment of therapeutic mAbs.

The dual action of human antibodies specific to Plasmodium falciparum PfRH5 and PfCyRPA: Blocking invasion and inactivating extracellular merozoites.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines.

CDX-585, a Bispecific Antibody with Dual Targeting of ILT4 and PD-1 Checkpoint Pathways

Immunoglobulin-like transcript 4 (ILT4) is an immunosuppressive molecule predominantly expressed on myeloid cells. Recent studies combining ILT4 suppression with programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) blockade have shown promising signs of activity in immune checkpoint inhibitor refractory patients. We theorized that coupling ILT4 and PD-1/PD-L1 blockade in a bispecific antibody (bsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced bridging of APCs to T cells. To test this approach, we developed CDX-585, a tetravalent ILT4xPD-1 IgG1-scFv bsAb from novel PD-1 and ILT-4 mAbs. CDX-585 is a potent antagonist of both PD-1 and ILT4. CDX-585 promotes M1 macrophage polarization and enhances pro-inflammatory cytokine secretion in response to lipopolysaccharide or CD40 agonist mAb treatment. In mixed lymphocyte reaction (MLR) assays, CDX-585 is more potent than the combination of parental antibodies. In a humanized NCG mouse SK-MEL-5 tumor model, CDX-585 exhibits greater antitumor activity than the combination of parental mAbs. A pilot study of CDX-585 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-585 effectively combines ILT4 and the PD-1 blockade into one molecule that is more potent than the combination of the parental antibodies, providing the rationale to advance this bsAb into clinical studies.

Characterizing the Plasma IgG Antibody Repertoire of Malaria-Naïve United Kingdom Adults Vaccinated with Novel Blood Stage Vaccine RH5.1/AS01 B

Abstract Plasmodium falciparum, the most lethal species of human malaria, is susceptible to vaccine-induced antibodies, yet current efforts are challenged by limited efficacy, lack of durability, and parasite evasion. A promising blood-stage vaccine antigen, Reticulocyte Binding Protein Homologue 5 (RH5), has low polymorphism frequencies and uses a non-redundant red blood cell (RBC) invasion pathway. The Draper Lab (Oxford University) clinically tested RH5.1, an engineered variant of RH5, in AS01 Badjuvant (GSK) designed to boost long-lasting, protective antibody titers. RH5-specific B cell receptors (BCRs) from United Kingdom malaria–naïve adult volunteers vaccinated with RH5.1/AS01 Bwere sequenced. Monoclonal antibodies (mAbs) were found to inhibit in vitrogrowth of RBC-invading parasite merozoites. Notably, the polyclonal plasma IgG of the volunteers exhibited high neutralizing potency, exceeding that of individual mAbs from the same donors, which raises the question: what is the disconnect between the BCR and circulating IgG repertoires? To examine this further, our lab completed BCR sequencing coupled with plasma IgG proteomics to characterize the plasma antibody repertoires of volunteers. Plasma mAbs were identified that bind to known growth inhibitory epitopes, including those that block binding between RH5 and its RBC coreceptor, CD147, alongside a population of non-neutralizing, synergizing mAbs. Additionally, non-neutralizing mAbs targeting cryptic epitopes on the N- and C-terminus were found at consistently high levels. This dynamic between neutralizing and non-neutralizing plasma mAbs suggests a role for non-neutralizing mAbs, which should be further explored in future RH5 vaccine engineering. Supported by grants from U.S. Agency for International Development (USAID) contract no. 7200AA20C00017 and the National Science Foundation Graduate Research Fellowship Program (NSF GRFP).

Evolving Real-World Effectiveness of Monoclonal Antibodies for Treatment of COVID-19

Treatment guidelines and U.S. Food and Drug Administration emergency use authorizations (EUAs) of monoclonal antibodies (mAbs) for treatment of high-risk outpatients with mild to moderate COVID-19 changed frequently as different SARS-CoV-2 variants emerged. To evaluate whether early outpatient treatment with mAbs, overall and by mAb product, presumed SARS-CoV-2 variant, and immunocompromised status, is associated with reduced risk for hospitalization or death at 28 days. Hypothetical pragmatic randomized trial from observational data comparing mAb-treated patients with a propensity score-matched, nontreated control group. Large U.S. health care system. High-risk outpatients eligible for mAb treatment under any EUA with a positive SARS-CoV-2 test result from 8 December 2020 to 31 August 2022. Single-dose intravenous mAb treatment with bamlanivimab, bamlanivimab-etesevimab, sotrovimab, bebtelovimab, or intravenous or subcutaneous casirivimab-imdevimab administered within 2 days of a positive SARS-CoV-2 test result. The primary outcome was hospitalization or death at 28 days among treated patients versus a nontreated control group (no treatment or treatment ≥3 days after SARS-CoV-2 test date). The risk for hospitalization or death at 28 days was 4.6% in 2571 treated patients and 7.6% in 5135 nontreated control patients (risk ratio [RR], 0.61 [95% CI, 0.50 to 0.74]). In sensitivity analyses, the corresponding RRs for 1- and 3-day treatment grace periods were 0.59 and 0.49, respectively. In subgroup analyses, those receiving mAbs when the Alpha and Delta variants were presumed to be predominant had estimated RRs of 0.55 and 0.53, respectively, compared with 0.71 for the Omicron variant period. Relative risk estimates for individual mAb products all suggested lower risk for hospitalization or death. Among immunocompromised patients, the RR was 0.45 (CI, 0.28 to 0.71). Observational study design, SARS-CoV-2 variant presumed by date rather than genotyping, no data on symptom severity, and partial data on vaccination status. Early mAb treatment among outpatients with COVID-19 is associated with lower risk for hospitalization or death for various mAb products and SARS-CoV-2 variants. None.

Molecular mechanism of antibody neutralization of coxsackievirus A16

Coxsackievirus A16 (CVA16) causes hand, foot and mouth disease in infants and young children. However, no vaccine or anti-viral agent is currently available for CVA16. Here, the functions and working mechanisms of two CVA16-specific neutralizing monoclonal antibodies (MAbs), 9B5 and 8C4, are comprehensively investigated. Both 9B5 and 8C4 display potent neutralization in vitro and prophylactic and therapeutic efficacy in a mouse model of CVA16 infection. Mechanistically, 9B5 exerts neutralization primarily through inhibiting CVA16 attachment to cell surface via blockade of CVA16 binding to its attachment receptor, heparan sulfate, whereas 8C4 functions mainly at the post-attachment stage of CVA16 entry by interfering with the interaction between CVA16 and its uncoating receptor SCARB2. Cryo-EM studies show that 9B5 and 8C4 target distinct epitopes located at the 5-fold and 3-fold protrusions of CVA16 capsids, respectively, and exhibit differential binding preference to three forms of naturally occurring CVA16 particles. Moreover, 9B5 and 8C4 are compatible in formulating an antibody cocktail which displays the ability to prevent virus escape seen with individual MAbs. Together, our work elucidates the functional and structural basis of CVA16 antibody-mediated neutralization and protection, providing important information for design and development of effective CVA16 vaccines and antibody therapies.

Development, validation, and implementation of a robust and quality control-friendly focused peptide mapping method for monitoring oxidation of co-formulated monoclonal antibodies.

Monoclonal antibody (mAb) coformulation containing two therapeutic proteins provides benefits of improved therapeutic efficacy and better patient compliance. Monitoring of the individual mAb stability in the coformulation is critical to ensure its quality and safety. Among post-translational modifications (PTMs), oxidation is often considered as one of the critical quality attributes (CQAs) as it potentially affects the structure and potency. Although hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC) have been used to monitor overall protein oxidation, mass spectrometry of peptide digests resolved by LC methods can afford superior selectivity and sensitivity for specific PTMs. With the advent of the Quadrupole Dalton (QDa) mass spectrometer as an affordable add-on detector, implementation of targeted oxidation assays in development and quality control (QC) laboratories is now feasible. In this study, as the first effort to implement MS-based methods for antibody coformulation in QC laboratories, we developed and validated a high-throughput and robust focused peptide mapping method using QDa for simultaneous site-specific monitoring of oxidation of methionine and tryptophan residues in heavy-chain (HC) complementary determining regions (CDRs) of two co-formulated mAbs. The method was validated in terms of accuracy, precision, linearity, range, quantitation limit (QL), specificity, and solution stability per recommendations in ICH Q2. The method robustness was systematically assessed involving multiple sample preparation and instrument method parameters. The method met the validation criteria in GMP laboratories with excellent robustness and was implemented in both GMP and development environments.

Characterization of Neutralizing Human Anti-Tetanus Monoclonal Antibodies Produced by Stable Cell Lines.

Tetanus toxin (TeNT) is produced by C. tetani, a spore-forming bacillus broadly spread in the environment. Although an inexpensive and safe vaccine is available, tetanus persists because of a lack of booster shots and variable responses to vaccines due to immunocompromised status or age-decreased immune surveillance. Tetanus is most prevalent in low- and medium-income countries, where it remains a health problem. Neutralizing monoclonal antibodies (mAbs) can prevent the severity of illness and death caused by C. tetani infection. We identified a panel of mAbs that bind to TeNT, some of which were investigated in a preclinical assay, showing that a trio of mAbs that bind to different sites of TeNT can neutralize the toxin and prevent symptoms and death in mice. We also identified two mAbs that can impair the binding of TeNT to the GT1b ganglioside receptor in neurons. In this work, to generate a series of cell lines, we constructed vectors containing sequences encoding heavy and light constant regions that can receive the paired variable regions resulting from PCRs of human B cells. In this way, we generated stable cell lines for five mAbs and compared and characterized the antibody produced in large quantities, enabling the characterization experiments. We present the results regarding the cell growth and viability in a fed-batch culture, titer measurement, and specific productivity estimation. The affinity of purified mAbs was analyzed by kinetics and under steady-state conditions, as three mAbs could not dissociate from TeNT within 36,000 s. The binding of mAbs to TeNT was confirmed by ELISA and inhibition of toxin binding to GT1b. The use of the mAbs mixture confirmed the individual mAb contribution to inhibition. We also analyzed the binding of mAbs to FcγR by surface plasmon resonance (SPR) and the glycan composition. Molecular docking analyses showed the binding site of an anti-tetanus mAb.

Formation of transient highly-charged mAb clusters strengthens interactions with host cell proteins and results in poor clearance of host cell proteins by protein A chromatography

Protein A chromatography with a high salt wash usually leads to robust clearance of host cell proteins (HCPs) in most recombinant monoclonal antibodies (mAbs), but a small subset of recalcitrant mAbs show significant HCP copurification. In this study, we carried out systematic studies using 4 different mAbs to explore the HCP copurification mechanism. HCP identification results revealed that the 3 high-HCP mAbs had many common HCPs which do not copurify with the low-HCP mAb, suggesting a similar mechanism is at play. Through wash evaluation, surface patch analysis, chain-swapping, domain evaluation, and structure-guided mutations, several charged residues in each mAb were found which correlated with HCP copurification. Surprisingly, these residues are also critical for self-association propensity. We observed an inverse correlation between diffusion interaction parameter and HCP copurification. Each of the high-HCP mAbs could form dynamic clusters consisting of 3∼6 mAb molecules. Therefore, a mAb cluster can exhibit higher net positive charges on the order of 3 to 6, compared with the individual mAb. In Protein A chromatography, high-HCP mAbs had elution tailing which contained high level of HCPs. Addition of Arginine-HCl or point mutations preventing cluster formation effectively reduced HCP copurification and elution tailing. Based on these results, we propose a novel HCP-copurification mechanism that formation of mAb clusters strengthens charge-charge interactions with HCPs and thus compromises HCP removal by Protein A chromatography. Besides arginine, histidine under acidic pH conditions prevented cluster formulation and resulted in effective HCP removal. Finally, structure-guided protein engineering and solution screening by using cluster size as indicator are useful tools for managing mAbs with high-HCP issues.

Susceptibility of SARS-CoV-2 Omicron Variants to Therapeutic Monoclonal Antibodies: Systematic Review and Meta-analysis.

ABSTRACTSARS-CoV-2 Omicron variants contain many mutations in its spike receptor-binding domain, the target of all authorized monoclonal antibodies (MAbs). Determining the extent to which Omicron variants reduced MAb susceptibility is critical to preventing and treating COVID-19. We systematically reviewed PubMed and three preprint servers, last updated 11 April 2022, for the in vitro activity of authorized MAbs against the Omicron variants. Fifty-one studies were eligible, including 50 containing Omicron BA.1 susceptibility data and 17 containing Omicron BA.2 susceptibility data. The first two authorized MAb combinations, bamlanivimab/etesevimab and casirivimab/imdevimab, were largely inactive against the Omicron BA.1 and BA.2 variants. In 34 studies, sotrovimab displayed a median 4.0-fold (interquartile range [IQR]: 2.6 to 6.9) reduction in activity against Omicron BA.1, and in 12 studies, it displayed a median 17-fold (IQR: 13 to 30) reduction in activity against Omicron BA.2. In 15 studies, the combination cilgavimab/tixagevimab displayed a median 86-fold (IQR: 27 to 151) reduction in activity against Omicron BA.1, and in six studies, it displayed a median 5.4-fold (IQR: 3.7 to 6.9) reduction in activity against Omicron BA.2. In eight studies against Omicron BA.1 and six studies against Omicron BA.2, bebtelovimab displayed no reduction in activity. Disparate results between assays were common. For authorized MAbs, 51/268 (19.0%) results for wild-type control variants and 78/348 (22.4%) results for Omicron BA.1 and BA.2 variants were more than 4-fold below or 4-fold above the median result for that MAb. Highly disparate results between published assays indicate a need for improved MAb susceptibility test standardization or interassay calibration.IMPORTANCE Monoclonal antibodies (MAbs) targeting the SARS-CoV-2 spike protein are among the most effective measures for preventing and treating COVID-19. However, SARS-CoV-2 Omicron variants contain many mutations in their spike receptor-binding domains, the target of all authorized MAbs. Therefore, determining the extent to which Omicron variants reduced MAb susceptibility is critical to preventing and treating COVID-19. We identified 51 studies that reported the in vitro susceptibility of the two main Omicron variants BA.1 and BA.2 to therapeutic MAbs in advanced clinical development, including eight authorized individual MAbs and three authorized MAb combinations. We estimated the degree to which different MAbs displayed reduced activity against Omicron variants. The marked loss of activity of many MAbs against Omicron variants underscores the importance of developing MAbs that target conserved regions of spike. Highly disparate results between assays indicate the need for improved MAb susceptibility test standardization.

Characterization of the Time-Domain Isotopic Beat Patterns of Monoclonal Antibodies in Fourier Transform Mass Spectrometry.

The time-domain transients in the Fourier transform mass spectrometry (FTMS) analysis of monoclonal antibodies (mAbs) are known to exhibit characteristic isotopic beat patterns. These patterns are defined by the isotopic distributions of all gaseous mAb ions present in the FTMS mass analyzer, originating from single or multiple charge states, and from single or multiple proteoforms. For an isolated charge state of a single proteoform, the mAb isotopic beat pattern resembles narrow splashes of signal amplitude (beats), spaced periodically in the time-domain transient, with broad (often exceeding 1 s) "valleys" between them. Here, we reinforce the importance of isotopic beat patterns for the accurate interpretation and presentation of FTMS data in the analysis of mAbs and other large biopolymers. An updated, mAb-grade version of the transient-mediated FTMS data simulation and visualization tool, FTMS Simulator is introduced and benchmarked. We then apply this tool to evaluate the charge-state dependent characteristics of isotopic beats in mAbs analyses with modern models of Orbitrap and ion cyclotron resonance (ICR) FTMS instruments, including detection of higher-order harmonics. We demonstrate the impact of the isotopic beat patterns on the analytical characteristics of the resulting mass spectra of individual and overlapping mAb proteoforms. The results reported here detail highly nonlinear dependences of resolution and signal-to-noise ratio on the time-domain transient period, absorption or magnitude mode spectra representation, and apodization functions. The provided description and the demonstrated ability to routinely conduct accurate simulations of FTMS data for large biopolymers should aid the end-users of Orbitrap and ICR FTMS instruments in the analysis of mAbs and other biopolymers, including viruses.