Generation Of mAbs Research Articles

SARS-CoV-2-Reactive Mucosal B Cells in the Upper Respiratory Tract of Uninfected Individuals.

SARS-CoV-2 is a respiratory pathogen that can cause severe disease in at-risk populations but results in asymptomatic infections or a mild course of disease in the majority of cases. We report the identification of SARS-CoV-2-reactive B cells in human tonsillar tissue obtained from children who were negative for coronavirus disease 2019 prior to the pandemic and the generation of mAbs recognizing the SARS-CoV-2 Spike protein from these B cells. These Abs showed reduced binding to Spike proteins of SARS-CoV-2 variants and did not recognize Spike proteins of endemic coronaviruses, but subsets reacted with commensal microbiota and exhibited SARS-CoV-2-neutralizing potential. Our study demonstrates pre-existing SARS-CoV-2-reactive Abs in various B cell populations in the upper respiratory tract lymphoid tissue that may lead to the rapid engagement of the pathogen and contribute to prevent manifestations of symptomatic or severe disease.

A tale of the monoclonal anti-CD20 antibodies, in tribute to prof. Wacław Szybalski (1921-2020).

Technical advances that lead to the era of targeted therapeutics demanded several milestones that were reached in the second half of the previous century. Professor Wacław Szybalski was the first one to perform a stable gene transfer in eukaryotic cells. To do so, he used his own designed system consisting of HPRT-deficient cells and HAT selective medium. Moreover, the first-ever hybridoma cells were also constructed by Wacław Szybalski's team. These spectacular achievements made him not only a forerunner of gene therapy, but also became a foundation for immunotherapy, as hybridoma and their selection by the HPRT-HAT system turned into a crucial technical step during production of monoclonal antibodies (mAbs). Herein, we present a story of anti-CD20 mAbs, one of the most successful lines of anticancer drugs. When looking back into history, the prototypic mAb rituximab was considered the biggest step forward in the therapy of B-cell malignancies. Nowadays, the second and third generations of anti-CD20 mAbs are approved in clinical use and numerous breakthrough studies on immune effector mechanisms were conducted with the aforementioned immunotherapeutics as a model.

The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies.

Since the approval of the first monoclonal antibody (mAb) in 1986, a huge effort has been made to guarantee safety and efficacy of therapeutic mAbs. As of July 2021, 118 mAbs are approved for the European market for a broad range of clinical indications. In order to ensure clinical efficacy and safety aspects, (pre-)clinical experimental approaches evaluate the respective modes of action (MoA). In addition to antigen-specificity including binding affinity and -avidity, MoA comprise Fc-mediated effector functions such as antibody dependent cellular cytotoxicity (ADCC) and the closely related antibody dependent cellular phagocytosis (ADCP). For this reason, a variety of cell-based assays have been established investigating effector functions of therapeutic mAbs with different effector/target-cell combinations and several readouts including Fcγ receptor (FcγR)-mediated lysis, fluorescence, or luminescence. Optimized FcγR-mediated effector functions regarding clinical safety and efficacy are addressed with modification strategies such as point mutations, altered glycosylation patterns, combination of different Fc subclasses (cross isotypes), and Fc-truncation of the mAb. These strategies opened the field for a next generation of therapeutic mAbs. In conclusion, it is of major importance to consider FcγR-mediated effector functions for the efficacy of therapeutic mAbs.

Coadministration of Anti-Viral Monoclonal Antibodies With Routine Pediatric Vaccines and Implications for Nirsevimab Use: A White Paper.

Routine childhood vaccinations are key for the protection of children from a variety of serious and potentially fatal diseases. Current pediatric vaccine schedules mainly cover active vaccines. Active vaccination in infants is a highly effective approach against several infectious diseases; however, thus far, for some important viral pathogens, including respiratory syncytial virus (RSV), vaccine development and license by healthcare authorities have not been accomplished. Nirsevimab is a human-derived, highly potent monoclonal antibody (mAb) with an extended half-life for RSV prophylaxis in all infants. In this manuscript, we consider the potential implications for the introduction of an anti-viral mAb, such as nirsevimab, into the routine pediatric vaccine schedule, as well as considerations for coadministration. Specifically, we present evidence on the general mechanism of action of anti-viral mAbs and experience with palivizumab, the only approved mAb for the prevention of RSV infection in preterm infants, infants with chronic lung disease of prematurity and certain infants with hemodynamically significant heart disease. Palivizumab has been used for over two decades in infants who also receive routine vaccinations without any alerts concerning the safety and efficacy of coadministration. Immunization guidelines (Advisory Committee on Immunization Practices, Joint Committee on Vaccination and Immunization, National Advisory Committee on Immunization, Centers for Disease Control and Prevention, American Academy of Pediatrics, The Association of the Scientific Medical Societies in Germany) support coadministration of palivizumab with routine pediatric vaccines, noting that immunobiologics, such as palivizumab, do not interfere with the immune response to licensed live or inactivated active vaccines. Based on the mechanism of action of the new generation of anti-viral mAbs, such as nirsevimab, which is highly specific targeting viral antigenic sites, it is unlikely that it could interfere with the immune response to other vaccines. Taken together, we anticipate that nirsevimab could be concomitantly administered to infants with routine pediatric vaccines during the same clinic visit.

Toward Sézary Syndrome immunotherapy

Abstract Sézary syndrome (SS) is a leukemic form of cutaneous mature T-cell lymphoma characterized by circulating malignant CD4 T lymphocytes (Sezary cells). Patients with SS have a poor prognosis and current treatment options show high rates of relapse, morbidity or mortality. Thus, there is an unmet need for an efficient and safe treatment. Sézary cells have unique clonal potentially targetable epitopes, including their TCR, and TCR- and neoantigen-derived HLA-restricted peptides. Our general aim is to design a patient-tailored two-pronged strategy against SS. The specific aims are 1) to target SS clonal TCR B cell epitopes using mAb and/or CAR T cells, 2) to target SS HLA-restricted T-cell epitopes using TCR peptide- and/or neoantigen-specific human T cells, and 3) to validate efficacy in vitro and in mouse models. For the generation of mAb, apheresis-purified SS cells or SS TCR CDR3beta peptides were used for immunizations, and screening was done on SS vs non-SS CD4 cells as defined by flow cytometry using CD26 and/or PD-1. For in vitro expansion of SS peptide-specific T cells, SS patient-derived non-SS PBMC were stimulated in 96-well plates with IL-2 and pooled HLA class I+II SS peptides, 10 μM each, defined by SS WGS, WES and RNAseq-based predictions or peptidome studies. After one week, cells were exposed to autologous DC pre-loaded with peptide pools, and cytokine production was analyzed by flow cytometry. We have obtained preliminary data on aims 1 and 2 studying two SS patients with monoclonal T cell lymphomas, including potential mouse antibodies against a clonal SS TCR using cell and peptide immunization and T-cell hits that seem to be specific of a SS TCR HLA class-I-restricted CDR3beta sequence.

Monoclonal Antibodies for Prevention of Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of hospitalizations in infants worldwide. Palivizumab, a humanized monoclonal antibody against the RSV F protein, is the only licensed agent for prevention of severe RSV infection in high-risk infants. Palivizumab is administered intramuscularly, every month during the RSV season, usually 5 doses are required. In recent years, the resolution of the structure of the RSV F protein, with identification of potent neutralizing epitopes, and new technologies for production of monoclonal antibodies (mAbs) have facilitated the development of new alternative strategies for the prevention of RSV infections. One promising approach is a new generation of mAbs directed to new neutralizing epitopes and with prolonged half life. These enhanced mAbs are expected to provide adequate protection during the complete RSV season with a single intramuscular (IM) dose. The long-term goal of this approach is to provide passive immunization for the prevention of RSV lower respiratory tract infection to all infants (preterm and full term) in the first months of life before their initial exposure to RSV.

Rapid and efficient generation of T-cell receptor-like antibodies using chip-based single-cell analysis.

Generation of TCR-like monoclonal antibodies using conventional methods is markedly laborious and inefficient. We have proposed improvements of ISAAC (chip-based Ab-secreting cell [ASC] screening method), allows comprehensive analysis of ASCs at the single-cell level to obtain TCR-like antibodies; blocking procedure enables us to avoid the detection of non-TCR-like antibodies.

Tetramer Immunization and Selection Followed by CELLISA Screening to Generate Monoclonal Antibodies against the Mouse Cytomegalovirus m12 Immunoevasin.

The generation of reliable mAb of unique and desired specificities serves as a valuable technology to study protein expression and function. However, standard approaches to mAb generation usually involve large-scale protein purification and intensive screening. In this study, we describe an optimized high-throughput proof-of-principle method for the expanded generation, enrichment, and screening of mouse hybridomas secreting mAb specific for a protein of interest. Briefly, we demonstrate that small amounts of a biotinylated protein of interest can be used to generate tetramers for use as prime-boost immunogens, followed by selective enrichment of Ag-specific B cells by magnetic sorting using the same tetramers prior to hybridoma generation. This serves two purposes: 1) to effectively expand both low- and high-affinity B cells specific for the antigenic bait during immunization and 2) to minimize subsequent laborious hybridoma efforts by positive selection of Ag-specific, Ab-secreting cells prior to hybridoma fusion and validation screening. Finally, we employ a rapid and inexpensive screening technology, CELLISA, a high-throughput validation method that uses a chimeric Ag fused to the CD3ζ signaling domain expressed on enzyme-generating reporter cells; these reporters can detect specific mAb in hybridoma supernatants via plate-bound Ab-capture arrays, thereby easing screening. Using this strategy, we generated and characterized novel mouse mAb specific for a viral immunoevasin, the mouse CMV m12 protein, and suggest that these mAb may protect mice from CMV infection via passive immunity.

Generation and Characterization of Monoclonal Antibodies to the Ogawa Lipopolysaccharide of Vibrio cholerae O1 from Phage-Displayed Human Synthetic Fab Library.

Vibrio cholerae, cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, corepolysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2-O-methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. cholerae vaccines.

A Critical Review of Second-Generation Anti-EGFR Monoclonal Antibodies in Metastatic Colorectal Cancer.

Anti-EGFR monoclonal antibodies (mAbs) have become a relevant solution for the treatment of patients with metastatic colorectal cancer. Current anti-EGFR monoclonal antibodies face a series of problems, including resistance and non-durable response, and RAS and BRAF mutations serve as exclusion criteria for treatment with anti-EGFR mAbs. Advances in molecular tumor profiling and information on subsequent pathways responsible for disease progression and drug resistance helped develop a new generation of anti-EGFR mAbs. These second-generation mAbs have been developed to overcome existing resistance mechanisms and to limit common side effects. For the moment, existing literature suggests that these novel anti-EGFR mAbs are far from finding their way to clinical practice soon. In this review, we summarize and evaluate current data regarding ongoing research and completed clinical trials for different second-generation anti-EGFR monoclonal antibodies. Anti-EGFR mAbs exhibit efficacy in advanced colorectal cancer, but second-generation mAbs failed to prove their benefit in the treatment of metastatic colorectal cancer. Understanding the biological basis of primary and acquired drug resistance could allow scientists to design better clinical trials and develop improved second-generation mAbs.

Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker.

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.

Generation and reactivity analysis of human recombinant monoclonal antibodies directed against epitopes on HLA-DR.

In kidney transplantation, eplet mismatches between donor and recipient have been associated with de novo donor‐specific antibody development. Eplets are theoretically defined configurations of polymorphic amino acids and require experimental verification to establish whether they can be bound by alloantibodies. Human HLA‐specific monoclonal antibodies (mAbs) have been instrumental for this purpose but are largely lacking for HLA class II. In this study, we isolated single HLA‐DR‐specific memory B cells from peripheral blood of immunized individuals (n = 3) using HLA class II tetramers to generate recombinant human HLA‐DR antigen‐reactive mAbs (n = 5). Comparison of the amino acid composition of the reactive HLA alleles in relation to the antibody reactivity patterns led to identification of 3 configurations, 70Q 73A, 31F 32Y 37Y, and 14K 25Q recognized, respectively, by HLA‐DRB1*01:01, HLA‐DRB1*04:01, and HLA‐DRB1*07:01 antigen‐reactive mAbs. The first 2 correspond to eplets 70QA and 31FYY and can now be considered antibody verified. The latter indicates that eplet 25Q needs to be redefined before being considered as antibody verified. Generation and reactivity analysis of human HLA‐DR mAbs allowed for identification of amino acid configurations corresponding to known eplets, whereas the other patterns may be used to redefine eplets with similar, but not identical predicted amino acid composition.

Immunogenicity of Innovative and Biosimilar Monoclonal Antibodies.

The development of hybridoma technology for producing monoclonal antibodies (mAbs) by Kohler and Milstein (1975) counts as one of the major medical breakthroughs, opening up endless possibilities for research, diagnosis and for treatment of a whole variety of diseases. Therapeutic mAbs were introduced three decades ago. The first generation of therapeutic mAbs of murine origin showed high immunogenicity, which limited efficacy and was associated with severe infusion reactions. Subsequently chimeric, humanized, and fully human antibodies were introduced as therapeutics, these mAbs were considerably less immunogenic. Unexpectedly humanized mAbs generally show similar immunogenicity as chimeric antibodies; based on sequence homology chimeric mAbs are sometimes more “human” than humanized mAbs. With the introduction of the regulatory concept of similar biological medicines (biosimilars) a key concern is the similarity in terms of immunogenicity of these biosimilars with their originators. This review focuses briefly on the mechanisms of induction of immunogenicity by biopharmaceuticals, mAbs in particular, in relation to the target of the immune system.

Broad-spectrum monoclonal antibodies against chikungunya virus structural proteins: Promising candidates for antibody-based rapid diagnostic test development.

In response to the aggressive global spread of the mosquito-borne chikungunya virus (CHIKV), an accurate and accessible diagnostic tool is of high importance. CHIKV, an arthritogenic alphavirus, comprises three genotypes: East/Central/South African (ECSA), West African (WA), and Asian. A previous rapid immunochromatographic (IC) test detecting CHIKV E1 protein showed promising performance for detection of the ECSA genotype. Unfortunately, this kit exhibited lower capacity for detection of the Asian genotype, currently in circulation in the Americas, reflecting the low avidity of one of the monoclonal antibodies (mAbs) in this IC kit for the E1 protein of the Asian-genotype because of a variant amino acid sequence. To address this shortcoming, we set out to generate a new panel of broad-spectrum mouse anti-CHIKV mAbs using hybridoma technology. We report here the successful generation of mouse anti-CHIKV mAbs targeting CHIKV E1 and capsid proteins. These mAbs possessed broad reactivity to all three CHIKV genotypes, while most of the mAbs lacked cross-reactivity towards Sindbis, dengue, and Zika viruses. Two of the mAbs also lacked cross-reactivity towards other alphaviruses, including O'nyong-nyong, Ross River, Mayaro, Western Equine Encephalitis, Eastern Equine Encephalitis, and Venezuelan Equine Encephalitis viruses. In addition, another two mAbs cross-reacted weakly only with most closely related O'nyong-nyong virus. Effective diagnosis is one of the keys to disease control but to date, no antibody-based rapid IC platform for CHIKV is commercially available. Thus, the application of the mAbs characterized here in the rapid diagnostic IC kit for CHIKV detection is expected to be of great value for clinical diagnosis and surveillance purposes.

Antigen-Specific Human Monoclonal Antibodies from Transgenic Mice.

Due to the difficulties found when generating fully human monoclonal antibodies (mAbs) by the traditional method, several efforts have attempted to overcome these problems, with varying levels of success. One approach has been the development of transgenic mice carrying immunoglobulin (Ig) genes in germline configuration. The engineered mouse genome can undergo productive rearrangement in the B-cell population, with the generation of mouse B lymphocytes expressing human Ig (hIg) chains. To avoid the expression of mouse heavy or light chains, the endogenous mouse Ig (mIg) loci must be silenced by gene-targeting techniques. Subsequently, to obtain antigen-specific mAbs, conventional immunization protocols can be followed and the mAb technique used (fusion of activated B cells with mouse myeloma cells, screening, cloning, freezing, and testing) with these animThis chapter summarizes the most common chromatographic mAb andals expressing human Ig genes. This chapter describes the type of transgenic-knockout mice generated for various research groups, provides examples of human mAbs developed by research groups and companies, and includes protocols of immunization, generation, production, and purification of human mAbs from such mice. In addition, it also addresses the problems detected, and includes some of the methods that can be used to analyze functional activities with human mAbs.

An Efficient Method to Generate Monoclonal Antibodies from Human B Cells.

In the age of personalized medicine, an efficient method to generate monoclonal antibodies (mAbs) is essential for biomedical and immunotherapeutic research. Numerous aspects of basic B-cell biology can be studied at the monoclonal level, includingB-cell development, antibody responses to infection or vaccination, and autoimmune responses. Single-cell B-cell receptor cloning allows for the rapid generation of antigen-specific mAbs in a matter of several weeks. In this chapter, we provide an efficient method to generate mAbs from peripheral blood plasmablasts and memory B cells induced by infection and vaccination. Additionally, we provide a protocol on how to optimize single-cell B-cell sorting for both single-cell B-cell receptor cloning and single-cell RNA-sequencing, for the application of studying B-cell specificity and function (spec-seq). This protocol can be easily adapted for other B-cell populations, B cells in tissues, and B cells from other organisms.

Stimulating T Cells Against Cancer With Agonist Immunostimulatory Monoclonal Antibodies.

Elimination of cancer cells through antitumor immunity has been a long-sought after goal since Sir F. Macfarlane Burnet postulated the theory of immune surveillance against tumors in the 1950s. Finally, the use of immunotherapeutics against established cancer is becoming a reality in the past 5years. Most notable are the monoclonal antibodies (mAbs) directed against inhibitory T-cell receptors cytotoxic T lymphocyte antigen-4 and programmed death-1. The next generation of mAbs targeting T cells is designed to stimulate costimulatory receptors on T cells. Here we review the recent progress on these immunostimulatory agonist antibodies against the costimulatory receptors CD137, GITR, OX40, and CD27.

Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)

Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.

The structure of the C-terminal domain of the nucleoprotein from the Bundibugyo strain of the Ebola virus in complex with a pan-specific synthetic Fab.

The vast majority of platforms for the detection of viral or bacterial antigens rely on immunoassays, typically ELISA or sandwich ELISA, that are contingent on the availability of suitable monoclonal antibodies (mAbs). This is a major bottleneck, since the generation and production of mAbs is time-consuming and expensive. Synthetic antibody fragments (sFabs) generated by phage-display selection offer an alternative with many advantages over Fabs obtained from natural antibodies using hybridoma technology. Unlike mAbs, sFabs are generated using phage display, allowing selection for binding to specific strains or for pan-specificity, for identification of structural epitopes or unique protein conformations and even for complexes. Further, they can easily be produced in Escherichia coli in large quantities and engineered for purposes of detection technologies and other applications. Here, the use of phage-display selection to generate a pan-specific Fab (MJ20), based on a Herceptin Fab scaffold, with the ability to bind selectively and with high affinity to the C-terminal domains of the nucleoproteins (NPs) from all five known strains of the Ebola virus is reported. The high-resolution crystal structure of the complex of MJ20 with the antigen from the Bundibugyo strain of the Ebola virus reveals the basis for pan-specificity and illustrates how the phage-display technology can be used to manufacture suitable Fabs for use in diagnostic or therapeutic applications.