Recombinant mAb Research Articles

Новая коронавирусная инфекция COVID-19 и состояние гуморального иммунного ответа у пациентов с гематологическими злокачественными опухолями

AIM. To study the baseline condition of the humoral component in the adaptive immune system and its changes in patients with hematologic malignancies who received the standard chemotherapy with and without monoclonal antibodies (mAb) after a COVID-19 infection. MATERIALS & METHODS. The study enrolled 51 patients with hematologic malignancies (AL, NHL, cHL, MM, CMPN). They were treated at the NN Burdenko Main Military Clinical Hospital. Patients were aged 24–84 years (median 50.6 years); there were 14 women and 37 men. The control group consisted of 16 healthy medical professionals working at the hospital who had had COVID-19. The main group included a subgroup of patients (n = 21) treated with mAb chemotherapy. In all patients, blood serum was tested for anti-SARS-CoV-2 antibodies of different classes. RESULTS. Significant differences were identified while assessing the level of IgG antibodies to SARS-CoV-2 S1-protein in lymphoma and multiple myeloma patients (median 431 BAU/mL vs. 667 BAU/mL; p < 0.05) as well as in patients with lymphomas and chronic myeloproliferative neoplasms (median 431 BAU/mL vs. 705 BAU/mL; p < 0.05). The juxtaposition of the control (n = 16) and main (n = 51) groups showed that their median levels of anti-SARS-CoV-2 IgG antibodies did not differ and accounted for 15.7 units. In the groups of patients with (n = 21) and without (n = 30) mAb chemotherapy, by the semi-quantitative method, the median levels of IgM/IgG antibodies to the S1-protein receptor-binding domain and SARS-CoV-2 nucleocapsid proteins accounted for 10.1 units vs. 16.1 units (p < 0.05). In the same groups of patients, the quantitative method yielded the median levels of anti-SARS-CoV-2 S1-protein IgG antibodies of 433 BAU/mL and 595 BAU/mL, respectively (p < 0.05). CONCLUSION. Patients with hematologic malignancies show a decline in the levels of anti-SARS-CoV-2 S-protein IgG antibodies. Non-recipients of mAb chemotherapy have a higher level of anti-SARS-CoV-2 IgG antibodies. Patients with an indication for mAb chemotherapy (rituximab and obinutuzumab) need to be protected from COVID-19 by means of epidemiological preventive measures. One of them is anti-SARS-CoV-2 vaccination prior to mAb chemotherapy onset or post-exposure prophylaxis with recombinant humanized IgG mAbs tixagevimab + cilgavimab.

Application of Recombinant Monoclonal Antibodies from Transgenic Chicken Bioreactors in Enzyme-Linked Immunosorbent Assay.

Transgenic chicken bioreactors can efficiently produce egg whites containing large quantities of recombinant proteins. We previously developed transgenic chickens that produce recombinant monoclonal antibodies (mAbs) against epidermal growth factor receptor 2 (HER2). However, the practical applications of mAbs derived from transgenic eggs have not yet been examined. Therefore, we aimed to evaluate whether these recombinant mAbs can be used in enzyme-linked immunosorbent assay (ELISA). Recombinant HER2 mAbs from transgenic eggs were dissolved in phosphate-buffered saline and applied directly to 96-well microplates as immobilized antibodies without purification. The performance of ELISA using the unpurified recombinant HER2 mAbs from transgenic eggs was comparable to that of ELISA using commercially available purified recombinant HER2 mAbs. Moreover, ELISA using unpurified recombinant HER2 mAbs from transgenic eggs demonstrated high antigen specificity and was successfully applied to samples from cultured cell lysates derived from HER2-positive and HER2-negative cell lines. The unpurified recombinant HER2 mAbs from transgenic eggs were also efficiently used as immobilized antibodies in paper-based ELISA. In conclusion, our findings suggest that recombinant mAbs from transgenic eggs have the potential to be used to develop economic ELISA devices. To the best of our knowledge, this study is the first to use recombinant HER2 mAbs from transgenic eggs in ELISA.

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.

Preclinical efficacy of peanut-specific IgG4 antibody therapeutic IGNX001

BackgroundExisting therapeutic strategies are challenged by long times to achieve effect and often require frequent administration. Peanut allergic individuals would benefit from a therapeutic that provides rapid protection against accidental exposure within days of administration while carrying little risk of adverse reactions. ObjectiveGuided by the repertoire of human IgE monoclonal antibodies (mAbs) from allergic individuals, we sought to develop a treatment approach leveraging the known protective effects of allergen-specific IgG4 antibodies. MethodsWe applied our single-cell RNA sequencing SEQ SIFTER™ platform to whole blood samples from peanut allergic individuals to discover IgE mAbs. These were then class-switched by replacing the IgE constant region with IgG4 while retaining the allergen-specific variable regions. In vitro mast cell activation tests (MATs), basophil activation tests (BATs), enzyme-linked immunosorbent assays (ELISAs), and an in vivo peanut allergy mouse model were used to evaluate the specificity, affinity, and activity of these recombinant IgG4 mAbs. ResultsWe determined that human peanut-specific IgE mAbs predominantly target immunodominant epitopes on Ara h 2 and Ara h 6 and that recombinant IgG4 mAbs effectively blocked these epitopes. IGNX001, a mixture of two such high-affinity IgG4 mAbs, provided robust protection against peanut-mediated mast cell activation in vitro as well as against anaphylaxis upon intragastric peanut challenge in a peanut allergy mouse model. ConclusionWe developed a peanut-specific IgG4 antibody therapeutic with convincing preclinical efficacy starting from a large repertoire of human monoclonal IgE antibodies from demographically and geographically diverse individuals. These results warrant further clinical investigation of IGNX001 and underscore the opportunity for the application of this therapeutic development strategy in other food and environmental allergies.

Monoclonal Antibodies for Rift Valley Fever Virus Nucleocapsid: Application in IgG/IgM ELISA for Sero-Diagnosis.

Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley fever (RVF). Currently, RVFV has spread from Africa to Asia, and due to its ability to cause high mortality rates, it has significantly impacted human health and economic development in many societies. Highly specific and sensitive systems for sero-diagnosis of RVFV infection are needed for clinical use. BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of IgG and IgM using the new MAbs were established and evaluated. Serum samples from 96 volunteers and 93 patients of suspected RVF from Kenya were tested compared with the ELISA systems based on inactivated viruses and the rabbit polyclonal antibody. Three monoclonal antibodies against rRVFV-N protein were established. The performance of the MAb-based sandwich IgG ELISA and the IgM capture ELISA perfectly matched the ELISA systems using the inactivated virus or the polyclonal antibody. Recombinant RVFV-N protein-specific MAbs were developed and they offer useful tools for RVFV studies. The MAb-based ELISA systems for detecting IgG and IgM offer safe and useful options for diagnosing RVFV infections in humans.

Isolation and characterization of rabies monoclonal antibody charge variants.

Current postexposure prophylaxis of rabies includes vaccines, human rabies immunoglobulin (RIG), equine RIG, and recombinant monoclonal antibodies (mAb). In the manufacturing of rabies recombinant mAb, charge variants are the most common source of heterogeneity. Charge variants of rabies mAb were isolated by salt gradient cation exchange chromatography (CEX) to separate acidic and basic and main charge variants. Separated variants were further extensively characterized using orthogonal analytical techniques, which include secondary and tertiary structure determination by far and near ultraviolet circular dichroism spectroscopy. Charge and size heterogeneity were evaluated using CEX, isoelectric focusing (IEF), capillary-IEF, size exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and western blotting. Antigen binding affinity was assessed by enzyme linked immuno-sorbent assay and rapid florescence foci inhibition test. Results from structural and physicochemical characterizations concluded that charge variants are formed due to posttranslational modification demonstrating that the charge heterogeneity, these charge variants did neither show any considerable physicochemical change nor affect its biological function. This study shows that charge variants are effective components of mAb and there is no need of deliberate removal, until biological functions of rabies mAb will get affected.

Abstract CT132: A Phase I/IIa clinical trial (TranStar101) to evaluate the safety, tolerability and pharmacokinetics of OSEMITAMAB administered as monotherapy or in combination with nivolumab or standard of care in patients with locally advanced or metastatic solid tumors

Abstract Introduction: TST001/osemitamab is a novel, recombinant humanized IgG1 mAb with improved CLDN18.2 binding affinity and enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and can result in more efficient cytotoxicity of tumors expressing human CLDN18.2 across a broad range of levels including low to medium expression. Methods: This is an open-label, multi-center phase I/IIa first in human (FIH) study of osemitamab administered as monotherapy or in combination with nivolumab or standard of care for patients with locally advanced or metastatic solid tumors. The study includes two parts: Part A (completed), a dose escalation of osemitamab as a monotherapy in solid tumors, osemitamab intravenous infusion at dose from 1mg/kg to 10mg/kg and once every two weeks (Q2W) or once every three weeks (Q3W) were evaluated; Part B (ongoing) includes expansion cohorts of osemitamab in combination with other therapies (cohort A and B). Cohort A includes combination of osemitamab, nivolumab, and mFOLFOX6 as the 1st line treatment for G/GEJ cancer. Cohort B includes osemitamab in combination with nivolumab in advanced pre-treated G/GEJ cancer. Results: As of Nov. 30, 2023, 68 patients have been treated: 38 patients were enrolled in the Part A dose escalation,13 in Part B Cohort A, and 17 in Part B Cohort B. The population (N=68) consisted of 59% males, median age 62 years and 50% with G/GEJ cancer. Doses evaluated are 3 to 10mg/kg Q3W and 1 to 6 mg/kg Q2W in part A. One dose limiting toxicity of Grade 3 infusion related reaction was reported at the 1mg/kg dose Q2W. The MTD was not reached. The recommended phase 2 dose was determined as 6mg/kg Q3W or 4mg/kg Q2W based on the totality of available safety, efficacy, PK/PD data including information from another phase I/II study (TranStar102). The most common TRAEs (occurred in ≥ 20% subjects) were nausea (73.5%), vomiting (48.5%), fatigue (30.9%). TRAE of Grade ≥ 3 across all tested doses (3-10 mg/kg) that occurred in ≥3% of patients included nausea, vomiting, hypoalbuminemia, hypertension, infusion related reaction, and lymphocyte count increased. Within the dose ranges studied, PK exposures increased as dose increased. The mean CLss value of osemitamab ranged from 9.4 to 13.7 mL/day/kg across different dose levels, without a clear dose relationship. Mild accumulation was observed following Q2W and Q3W dosing. Conclusion: Osemitamab as monotherapy or in combination with nivolumab or mFOLFOX plus nivolumab is well tolerated and safe with a PK profile consistent with other studies (TranStar 102 study -NCT04495296). TranStar 101 is currently ongoing. More information will be shared. Clinical trial information: NCT04396821. Study Sponsor: Suzhou Transcenta Therapeutics Co., Ltd. Citation Format: Yelena Janjigian, Anthony Tolcher, Rutika Mehta, Michael Cecchini, Brian Van Tine, Madappa Kundranda, Alese Olatunji, Manish R. Patel, Jordan Berlin, Caio Max Sao Pedro Rocha-Lima, Dulabh Monga, Ben George, Aaron Scott, Zhenzhong Xia, Mohamed Elsafy, Erikca Jones, Zhenling Yao, Chuan Qi, Caroline Germa, Nash Gabrail. A Phase I/IIa clinical trial (TranStar101) to evaluate the safety, tolerability and pharmacokinetics of OSEMITAMAB administered as monotherapy or in combination with nivolumab or standard of care in patients with locally advanced or metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT132.

Combinatory glycoengineering of monoclonal antibodies and its application in cancer therapy: a narrative review.

The application of immunotherapy, especially in terms of recombinant monoclonal antibodies (mAbs), is a revolution in the pharmaceutical industry field. Glycosylation plays an essential role in the structures and functions of mAbs, which must be carefully monitored and designed throughout their entire lifespan to ensure safety and efficacy. This review aimed to summarize the current status of the glycoengineering of mAbs for providing reference for the pharmaceutical industry. The application of glycoengineering of mAbs in cancer therapy will also be discussed in this article. We searched the PubMed/MEDLINE database to identify studies published between 1970 and 2023 that investigated glycoengineering of recombinant mAbs and its applications. The major findings of these studies were summarized. This article reviews the relationship between glycosylation profiles and antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), shelf-life, and other properties of mAbs. Furthermore, the rational design of glycosylation profiles provides solutions to the unmet needs of new drug development and biosimilar manufacturers. This review also emphatically describes various feasible strategies to optimize the glycosylation pattern in biomanufacturing reported in recent years, especially the approaches of combinatory glycoengineering explored in this field. Finally, we share examples of glycoengineering of mAbs applied in cancer therapy. Glycan modifications have been achieved through genetic and metabolic glycoengineering. A better understanding of the interplay between cells and the exogenous environment help the combinatorial strategy for glycoengineering in precise control mode. Furthermore, new high-tech approaches for glycoengineering are leading biological engineering and biotherapeutics to a new stage.

Antibody verification of HLA class I and class II eplets by human monoclonal HLA antibodies.

In solid organ transplantation, formation of de novo donor-specific HLA antibodies is induced by mismatched eplets on donor HLA molecules. While several studies have shown a strong correlation between the number of eplet mismatches and inferior outcomes, not every eplet mismatch is immunogenic. Eplets are theoretically defined entities, necessitating formal proof that they can be recognised and bound by antibodies. This antibody verification is pivotal to ensure that clinically relevant eplets are considered in studies on molecular matching. Recombinant human HLA-specific monoclonal antibodies (mAbs) were generated from HLA-reactive B cell clones isolated from HLA immunised individuals using recombinant HLA molecules. Subsequently, the reactivity patterns of the mAbs obtained from single antigen bead assay were analysed using HLA-EMMA software to identify single or configurations of solvent accessible amino acids uniquely present on the reactive HLA alleles and were mapped to eplets. Two HLA class I and seven HLA class II-specific human mAbs were generated from four individuals. Extensive mAb reactivity analysis, led to antibody verification of three HLA-DR-specific eplets, and conversion of five eplets (one HLA-A, one HLA-B, two HLA-DR, and one HLA-DP), from provisionally verified to truly antibody-verified. Finally, one HLA-DQ-specific eplet was upgraded from level A2 to level A1 verification evidence. The generation of recombinant human HLA-specific mAbs with different specificities contributes significantly to the antibody verification of eplets and therefore is instrumental for implementation of eplet matching in the clinical setting.

Rapid generation of human recombinant monoclonal antibodies from antibody-secreting cells using ferrofluid-based technology.

Monoclonal antibodies (mAbs) are one of the most important classes of biologics with high therapeutic and diagnostic value, but traditional methods for mAbs generation, such as hybridoma screening and phage display, have limitations, including low efficiency and loss of natural chain pairing. To overcome these challenges, novel single B cell antibody technologies have emerged, but they also have limitations such as in vitro differentiation of memory B cells and expensive cell sorters. In this study, we present a rapid and efficient workflow for obtaining human recombinant monoclonal antibodies directly from single antigen-specific antibody secreting cells (ASCs) in the peripheral blood of convalescent COVID-19 patients using ferrofluid technology. This process allows the identification and expression of recombinant antigen-specific mAbs in less than 10 days, using RT-PCR to generate linear Ig heavy and light chain gene expression cassettes, called "minigenes", for rapid expression of recombinant antibodies without cloning procedures. This approach has several advantages. First, it saves time and resources by eliminating the need for in vitro differentiation. It also allows individual antigen-specific ASCs to be screened for effector function prior to recombinant antibody cloning, enabling the selection of mAbs with desired characteristics and functional activity. In addition, the method allows comprehensive analysis of variable region repertoires in combination with functional assays to evaluate the specificity and function of the generated antigen-specific antibodies. Our approach, which rapidly generates recombinant monoclonal antibodies from single antigen-specific ASCs, could help to identify functional antibodies and deepen our understanding of antibody dynamics in the immune response through combined antibody repertoire sequence analysis and functional reactivity testing.

Performance of optimized prototype LUMIPULSE G immunoassays for plasma pTau181 and pTau217

AbstractBackgroundThere is a need for automated random access plasma phosphorylated Tau (pTau) methods that reflect AD pathology with consistent performance. LUMIPULSE (LP) assays could offer a solution. Initial prototype pTau181 and pTau217 LP assays showed lower clinical performance versus Quanterix Homebrew assays (Poster #79533 – P4‐06). This new study presents optimized pTau181 and pTau217 LP assays, showing data on their analytical and clinical performance compared to the initial prototype method (Ref) and an in‐house pTau181 Quanterix Homebrew assay.MethodPlasma pTau181 and pTau217 LP assays were developed using recombinant pTau181 mAb RD‐070, resp. pTau217 mAb RD‐085, combined with an ALP‐Fab conjugate from recombinant N‐terminal Tau mAb RD‐073. In the optimized automated protocol, an assay specific diluent (ASD) is added (20%) to the sample to increase signal specificity. Analytical and clinical performance of the reference versus ASD‐treated prototypes to distinguish AD from controls was evaluated using 40 CSF‐confirmed AD‐dementia and 40 age‐matched healthy volunteer plasma samples.ResultAnalytical performance: Both optimized pTau181 and pTau217 LP tests demonstrated high precision comparable to their reference assays (inter‐run %CV on 3 neat plasma samples < 7.4). LLOQ was not affected by ASD‐treatment. Clinical performance: pTau181 LP assays demonstrated comparable AUC and median fold change with (AUC 0.828, fold change 1.9) and without ASD (AUC 0.823, fold change 1.5) while the SIMOA assay demonstrated a higher AUC (0.866) and fold change (2.9). For pTau217, the performance improved from an initial AUC of 0.918 to 0.935, and a fold change of 3.72 instead of initially 2.76. (Figure 1). All assays correlated strongly (p<0.00001) (Table 1); highest correlations among them were observed with the pTau217 ASD‐treated assay.ConclusionUsing an assay specific diluent resulted in improved clinical performance for the pTau217 prototype LUMIPULSE assay, in line with other published pTau217 assays. We did not achieve improved performance of the pTau181 LP assay with the ASD addition. Most important to emphasize, a performant pTau217 assay prototype is under development on an fully automated, scalable platform. Next steps include verification of performance in additional well‐characterized AD cohorts and characterization of lot consistency and robustness of the pTau181 and pTau217 N‐terminal LUMIPULSE assays.

High-volume hybridoma sequencing on the NeuroMabSeq platform enables efficient generation of recombinant monoclonal antibodies and scFvs for neuroscience research

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

B-294 Delineating the Diversity of Recombinant Monoclonal Antibodies Isolated From Alpaca Polyclonal Mixture

Abstract Introduction Polyclonal antibodies are a mixture of antibodies produced by plasma B-cells and are often obtained by injecting a specific antigen into a host system. They are commonly used for research and diagnostic purposes and are inexpensive and relatively quick to produce. But they lack batch-to-batch reproducibility making them less reliable than recombinant monoclonal antibodies (mAbs). Our team designed a polyclonal sequencing method to sequence the mAbs directly from the immunoserum and then used recombinant technology to reliably express the mAbs. The diversity of the epitopes of these recombinant mAbs are then examined with Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS). Methods In this study, we used a novel mass-spectrometry-based approach to do de novo sequencing of the Alpaca IgGs directly from the immunoserum without analyzing the RNA-seq data. Using our established REpAb sequencing platform and a machine learning-based bioinformatic analysis, we successfully compiled complete amino acid sequences of individual alpaca IgG clones found in the original polyclonal mixture. Using this approach, we isolated ten unique sequences from the polyclonal mixture and expressed them recombinantly. In addition, we performed Hydrogen-deuterium exchange Mass spectrometry experiments on 4 of the ten mAbs to identify their binding sites. For HDX analysis, the antigen digestion was performed using Pepsin/protease XIII column, and the peptides were identified using Waters SELECT SERIES Cyclic IMS Mass spectrometer. Preliminary data The four mAbs which showed strong binding to the antigen were chosen to characterize further using HDX-MS experiments. The preliminary MS/MS experiments provided us with approximately 90% of the amino acid sequence coverage of the antigen. Interestingly, the HDX-MS results showed that different mAbs bind to different antigen regions, which is evident from the deuterium exchange. Overall, our results showed that the monoclonal antibodies isolated from the polyclonal mixture bind to different antigen regions and can represent the diversity in the polyclonal mixture. Novel aspect De novo sequencing functional mAbs from immunoserum and studying their epitope diversities with HDX-MS may help discover novel therapeutics antibodies.

Generation of recombinant mAbs to vaccinia virus displaying high affinity and potent neutralization.

Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.

High throughput purification of monoclonal recombinant antibodies using a Protein-A coated membrane plate system

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.

Novel neutralizing mouse-human chimeric monoclonal antibodies against the SARS-CoV-2 receptor binding domain.

Introduction. Neutralizing antibodies have been widely used for the prophylaxis and treatment of COVID-19.Hypothesis. The major target for these neutralizing antibodies is the receptor-binding domain (RBD) of the viral spike protein.Aim. In the present study, we developed and characterized three neutralizing chimeric mouse-human mAbs for potential therapeutic purposes.Methodology. Light and heavy chain variable region genes of three mouse mAbs (m4E8, m3B6, and m1D1) were amplified and ligated to human Cγ1 and Cκ constant region genes by PCR. After cloning into a dual promoter mammalian expression vector, the final constructs were transiently expressed in DG-44 cells and the purified chimeric antibodies were characterized by ELISA and Western blotting. The neutralizing potency of the chimeric mAbs was determined by three different virus neutralization tests including sVNT, pVNT, and cVNT.Results. All three recombinant chimeric mAbs display human constant regions and are able to specifically bind to the RBD of SARS-CoV-2 with affinities comparable to the parental mAbs. Western blot analysis showed similar epitope specificity profiles for both the chimeric and the parental mouse mAbs. The results of virus neutralization tests (sVNT, pVNT, and cVNT) indicate that c4E8 had the most potent neutralizing activity with IC50 values of 1.772, 0.009, and 0.01 µg ml-1, respectively. All chimeric and mouse mAbs displayed a similar pattern of reactivity with the spike protein of the SARS-CoV-2 variants of concern (VOC) tested, including alpha, delta, and wild-type.Conclusion. The chimeric mAbs displayed neutralizing potency similar to the parental mouse mAbs and are potentially valuable tools for disease control.

Development of a recombinant hepatitis B immunoglobulin derived from B cells collected from healthy individuals administered with hepatitis B virus vaccines: A feasibility study.

In Japan, plasma with a high concentration of Hepatitis B Virus (HBV) antibodies for hepatitis B immunoglobulin (HBIG) is almost entirely imported. We aimed to produce recombinant HBIG by isolating immunoglobulin cDNAs against the HBV surface antigen (HBsAg). B cells expressing HBsAg antibodies were obtained from blood center personnel who had been administered HB vaccine booster and then isolated by either an Epstein-Barr virus hybridoma or an antigen-specific memory B cell sorting method. Each cDNA of the heavy and light chains of the target antibody was cloned into an IgG1 expression vector and transfected into Expi293F cells to produce a recombinant monoclonal antibody (mAb), which was screened by ELISA and in vitro HBV neutralizing assays. The cross-reactivity of the mAbs to normal human molecules was evaluated by ELISA and immunohistochemistry. Antibody cDNAs were cloned from 11 hybridoma cell lines and 204 HBsAg-bound memory B cells. Three of the resulting recombinant mAbs showed stronger neutralizing activity in vitro than the currently used HBIG. All three bind to the conformational epitope(s) of HBsAg but not to human DNA or cells. We successfully isolated HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV. To obtain an alternative source for HBIG, HBV-neutralizing monoclonal antibodies from B cells collected from healthy plasma donors boosted against the HBV may be useful.

Generation and Next-Generation Sequencing-Based Characterization of a Large Human Combinatorial Antibody Library.

Antibody phage display is a key technology for the discovery and development of target-specific monoclonal antibodies (mAbs) for use in research, diagnostics, and therapy. The construction of a high-quality antibody library, with larger and more diverse antibody repertoires, is essential for the successful development of phage display-derived mAbs. In this study, a large human combinatorial single-chain variable fragment library (1.5 × 1011 colonies) was constructed from Epstein-Barr virus-infected human peripheral blood mononuclear cells stimulated with a combination of two of the activators of human B cells, the Toll-like receptor 7/8 agonist R848 and interleukin-2. Next-generation sequencing analysis with approximately 1.9 × 106 and 2.7 × 106 full-length sequences of heavy chain variable (VH) and κ light chain variable (Vκ) domains, respectively, revealed that the library consists of unique VH (approximately 94%) and Vκ (approximately 91%) sequences with greater diversity than germline sequences. Lastly, multiple unique mAbs with high affinity and broad cross-species reactivity could be isolated from the library against two therapeutically relevant target antigens, validating the library quality. These findings suggest that the novel antibody library we have developed may be useful for the rapid development of target-specific phage display-derived recombinant human mAbs for use in therapeutic and diagnostic applications.

Nirsevimab: review of pharmacology, antiviral activity and emerging clinical experience for respiratory syncytial virus infection in infants.

Respiratory syncytial virus (RSV) is a leading cause of hospitalization and infant mortality worldwide. There are currently no approved vaccines against RSV, and immunoprophylaxis with the mAb palivizumab is limited to extremely vulnerable infants in resource-rich settings due to its high cost and the need for monthly injections throughout the RSV season. Nirsevimab (formerly MEDI8897) is a highly potent, long-acting, human, recombinant mAb that received approval for the prevention of RSV infection in newborns and infants during their first RSV season from the EMA and the UK's Medicines and Healthcare products Regulatory Agency in November 2022 based on positive results in Phase 2b and 3 clinical trials. Nirsevimab targets the highly conserved site Ø of the prefusion conformation of the RSV fusion (F) protein and contains a triple amino acid substitution in the Fc domain that extends its half-life, allowing for a single dose to cover a typical RSV season in regions with temperate climates. In this article I review key attributes of nirsevimab with an emphasis on pharmacology, pharmacokinetics, antiviral activity, and the potential for resistance and escape variants. I also summarize current progress in clinical trials and consider future research priorities.

Trastuzumab Charge Variants: a Study on Physicochemical and Pharmacokinetic Properties.

Post-translational modifications in bioprocessing and storage of recombinant mAbs are the main sources of charge variants. While the profile of these kinds of variants is considered an important attribute for the therapeutic mAbs, there is controversy about their direct role in safety and efficacy. In this study, the physicochemical and pharmacokinetic (PK) properties of the separated charge variants belonging to a trastuzumab potential biosimilar, were examined. The acidic peaks, basic peaks, and main variants of trastuzumab were separated and enriched by semi-preparative weak cation exchange. A panel of analytical techniques was utilized to characterize the physicochemical properties of these variants. The binding affinity to HER2 and FcγRs and the PK parameters were evaluated for each variant. Based on the results, the charge variants of the proposed biosimilar had no significant influence on the examined efficacy and PK parameters. During the development and production of biosimilar monoclonal antibodies, evaluating the effect of their charge variants on efficacy and PK parameters is needed.