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Affinity-matured, functional anti-pathogen antibodies are present at low frequencies in natural human repertoires. These antibodies are often excellent candidates for therapeutic monoclonal antibodies. However, mining natural human antibody repertoires is a challenge. In this study, we demonstrate a new method that uses microfluidics, yeast display...
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... identified 247 anti-pathogen scFv sequences that were present at 0.1% frequency among populations of clones selected by FACS from vaccinated and non-vaccinated donor libraries (Supplementary Tables S2-S7). For each scFv, we sequenced the full-length V(D)J regions, including complementary-determining region 3 (CDR3), and computed various sequence analysis met- rics (V and J gene usage, divergence from germline, R/S ratio, and IgG isotype). ...
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... antigen-binding antibodies were first vali- dated for their ability to bind a pool of pneumococcal polysac- charides by ELISA. We found that all purified antibodies bound to the pneumococcal antigen pool with EC50s ranging from 0.001 nM to 5.3 nM (Supplementary Figure S6; Table 2). We then performed serotype-specific dot blots to determine the specificity of each mAb. ...
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... then performed serotype-specific dot blots to determine the specificity of each mAb. Most of the mAbs bound to one or two serotypes, whereas two antibodies (one from vaccinated donors and one from non-vaccinated donors) bound non-spe- cifically to most of the serotypes (Supplementary Figure S7; Table 2). ...
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... assays measure antibody-mediated bacterial kill- ing opsonization, which is the ability of the antibody to mark serotype-specific bacteria strains for ingestion/phagocytosis and subsequent killing. Most of the serotype-specific binders induced bacterial killing against the appropriate serotype (Table 2). Additional serotypes were tested to determine speci- ficity, and we generally found that the mAbs were serotype-spe- cific in their killing activity. ...
Citations
... This makes affinity maturation a key technique in protein engineering to improve affinity and binding interactions in vitro in order to optimize the therapeutic potential of antibodies [57]. Adler et al. observed that in vivo affinity-matured antibodies predominantly accumulate mutations within the CDR3 regions, with a total of 6-7 amino acid changes in the heavy and light chain [58]. However, during in vitro affinity maturation, libraries with a mutation rate of 1-2 are sufficient for isolating high-affinity binders [59]. ...
The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.
... Plasma hyperimmune globulin drugs have historically been used for emergency treatment during viral infection when other treatments are not available. For the severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2), serum/plasma units were collected from individuals who recovered from coronavirus disease 2019 (COVID- 19) and these drugs were used as the first line of treatment for infected individuals [1,2]. Hyperimmune globulins have significant advantages over monoclonal antibodies (mAbs); the use of purified plasma antibodies from convalescent donors or vaccinated animals has a demonstrated long history of successfully treating pathogens, including hepatitis B virus and rabies [3]. ...
... Deep antibody sequencing libraries were prepared and analyzed as described previously [12,19]. Libraries were sequenced on a MiSeq (Illumina, San Diego, CA, USA) using a 500 cycle MiSeq Reagent Kit v2, according to the manufacturer's instructions. ...
... A median of 140,738 sequence reads were obtained for each sequencing library (range: 104,305 to 960,104). Sequence analysis, including error correction, reading frame identification, and FR/CDR junction calls, was performed as previously described [19]. Clones were defined as sequences with a unique CDR3H amino acid sequence. ...
Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus.
... First, the V, D, and J gene segment usage (distribution) can be visualized by using a simple bar plot or scatter plot as well as the Ig class [101,102]. The sequence features, like nucleotide or amino acid motifs, can be shown with a logo plot [103]. Meanwhile, the distribution of CDR3 length, clonotype abundance, or amino acid charge can be displayed [68,77,[104][105][106][107]. ...
High-throughput sequencing for B cell receptor (BCR) repertoire provides useful insights for the adaptive immune system. With the continuous development of the BCR-seq technology, many efforts have been made to develop methods for analyzing the ever-increasing BCR repertoire data. In this review, we comprehensively outline different BCR repertoire library preparation protocols and summarize three major steps of BCR-seq data analysis, i. e., V(D)J sequence annotation, clonal phylogenetic inference, and BCR repertoire profiling and mining. Different from other reviews in this field, we emphasize background intuition and the statistical principle of each method to help biologists better understand it. Finally, we discuss data mining problems for BCR-seq data and with a highlight on recently emerging multiple-sample analysis.
... While theoretical diversity of the na ve combinatorial libraries is much higher than their achievable size, there is significantly less variety of the naturally paired VH VLs in the mammalian immune sys tem. A miniature phage library containing ∼10 6 naturally paired scFvs derived from cDNA of the individual B cells was constructed using a single cell microfluidics that allowed to mine for an extremely rear antibody variants as well as to display natural antibody paratopes from hun dreds of donors in one phage library [63]. This technique was advanced to high throughput single cell activity based screening and sequencing platform of thousands of antibodies from the mice immune cells and activated human memory cells [64]. ...
Functional Protein Engineering became the hallmark in biomolecule manipulation in the new millennium, building on and surpassing the underlying structural DNA manipulation and recombination techniques developed and employed in the last decades of 20th century. Because of their prominence in almost all biological processes, proteins represent extremely important targets for engineering enhanced or altered properties that can lead to improvements exploitable in healthcare, medicine, research, biotechnology, and industry. Synthetic protein structures and functions can now be designed on a computer and/or evolved using molecular display or directed evolution methods in the laboratory. This review will focus on the recent trends in protein engineering and the impact of this technology on recent progress in science, cancer- and immunotherapies, with the emphasis on the current achievements in basic protein research using synthetic antibody (sABs) produced by phage display pipeline in the Kossiakoff laboratory at the University of Chicago (KossLab). Finally, engineering of the highly specific binding modules, such as variants of Streptococcal protein G with ultra-high orthogonal affinity for natural and engineered antibody scaffolds, and their possible applications as a plug-and-play platform for research and immunotherapy will be described.
... expensive droplet-based microfluidics systems combined with the cost on regular maintenance and costly consumables). One example is to identify neutralizing mAbs against infectious agents, which have great therapeutic potentials (132,133). Currently, it is economically impractical to apply this technology to generate mAbs reagents that are sorely for basic immunology research in non-model species. However, this still represents an alternative option and can be taken into account if specific mAbs reagents against difficulty targets are urgently required to address interesting hypotheses. ...
Akin to their mammalian counterparts, teleost fish possess a complex assortment of highly specialized immune cells that are capable of unleashing potent innate immune responses to eradicate or mitigate incoming pathogens, and also differentiate into memory lymphocytes to provide long-term protection. Investigations into specific roles and functions of fish immune cells depend on the precise separation of each cell type. Commonly used techniques, for example, density gradient centrifugation, rely on immune cells to have differing sizes or densities and thus fail to separate between similar cell types (e.g. T and B lymphocytes). Furthermore, a continuously growing database of teleost genomic information has revealed an inventory of cellular markers, indicating the possible presence of immune cell subsets in teleost fish. This further complicates the interpretation of results if subsets of immune cells are not properly separated. Consequently, monoclonal antibodies (mAbs) against specific cellular markers are required to precisely identify and separate novel subsets of immune cells in fish. In the field of fish immunology, mAbs are largely generated using the hybridoma technology, resulting in the development of mAbs against specific cellular markers in different fish species. Nevertheless, this technology suffers from being labour-intensive, time-consuming and most importantly, the inevitable loss of diversities of antibodies during the fusion of antibody-expressing B lymphocytes and myeloma cells. In light of this, the focus of this review is to discuss the potential applications of fluorescence-activated cell sorting and droplet-based microfluidics, two emerging technologies capable of screening and identifying antigen-specific B lymphocytes in a high-throughput manner, in promoting the development of valuable reagents for fish immunology studies. Our main goal is to encourage the incorporation of alternative technologies into the field of fish immunology to promote the production of specific antibodies in a high-throughput and cost-effective way, which could better allow for the precise separation of fish immune cells and also facilitate the identification of novel immune cell subsets in teleost fish.
... The technology described here generates recombinant hyperimmune globulins for diverse conditions through high-throughput microfluidics, genomics and mammalian cell engineering. B cells from human donors or mice are run through a microfluidic platform, heavy and light chain immunoglobulin nucleic acid sequences are fused on a single-cell level to create antibody repertoires 18 , antibody repertoires are engineered into full-length expression constructs en masse, and then the full-length antibody expression constructs are stably introduced en masse into Chinese hamster ovary (CHO) cells in a site-directed manner. We applied our technology to develop 10 3 to 10 4 diverse recombinant hyperimmune globulin drug candidates to address unmet clinical needs for the COVID-19 pandemic, Zika virus disease, primary immune deficiency (PID) and transplant tolerance. ...
... Advanced molecular technology is required to capture a substantial fraction of a mammalian donor's diverse antibody repertoire. We reported methods for generating millions-diverse libraries of natively paired heavy and light chain immunoglobulin sequences in yeast 18 . That method used microfluidics to isolate millions of single B cells per hour into picoliter droplets for lysis, followed by overlap extension-reverse transcriptase-polymerase chain reaction (OE-RT-PCR), to generate libraries of natively paired single chain variable fragments (scFv). ...
Plasma-derived polyclonal antibody therapeutics, such as intravenous immunoglobulin, have multiple drawbacks, including low potency, impurities, insufficient supply and batch-to-batch variation. Here we describe a microfluidics and molecular genomics strategy for capturing diverse mammalian antibody repertoires to create recombinant multivalent hyperimmune globulins. Our method generates of diverse mixtures of thousands of recombinant antibodies, enriched for specificity and activity against therapeutic targets. Each hyperimmune globulin product comprised thousands to tens of thousands of antibodies derived from convalescent or vaccinated human donors or from immunized mice. Using this approach, we generated hyperimmune globulins with potent neutralizing activity against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in under 3 months, Fc-engineered hyperimmune globulins specific for Zika virus that lacked antibody-dependent enhancement of disease, and hyperimmune globulins specific for lung pathogens present in patients with primary immune deficiency. To address the limitations of rabbit-derived anti-thymocyte globulin, we generated a recombinant human version and demonstrated its efficacy in mice against graft-versus-host disease.
... Another point that should be considered is related to cost: the use of expensive sorting devices integrates an important part of the procedures to isolate antigen-specific single B lymphocytes from a polyclonal mixture. Alternatively, other strategies can be used, including antigen-coated magnetic beads (Adler et al., 2017), cell-based microarrays (Jin et al., 2011), and soft lithographic methods for micro engraving (Love et al., 2006). The downside? ...
The isolation of single monoclonal antibodies (mAbs) against a given antigen was only possible with the introduction of the hybridoma technology, which is based on the fusion of specific B lymphocytes with myeloma cells. Since then, several mAbs were described for therapeutic, diagnostic, and research purposes. Despite being an old technique with low complexity, hybridoma-based strategies have limitations that include the low efficiency on B lymphocyte-myeloma cell fusion step, and the need to use experimental animals. In face of that, several methods have been developed to improve mAb generation, ranging from changes in hybridoma technique to the advent of completely new technologies, such as the antibody phage display and the single B cell antibody ones. In this review, we discuss the hybridoma technology along with emerging mAb isolation approaches, taking into account their advantages and limitations. Finally, we explore the usefulness of the hybridoma technology nowadays.
... Similarly, pioneering work on single-molecule PCR amplification opened the era of emulsion PCR (ePCR) (11), which was successfully applied to amplifying complex gene libraries (12), PCR on microparticles in water-in-oil emulsions (13), and directed evolution driven by in vitro compartmentalization (14). The ePCR-based approaches were extensively adopted for different types of DNA libraries, such as random DNA libraries used in aptamer selection (SELEX) (15)(16)(17), antibody libraries (18)(19)(20), T cell receptor libraries (21), and libraries linking phylogeny with function on a single-cell level (22). ...
Significance
DNA libraries are predisposed to template mispairing during conventional “bulk” PCR, leading to the loss of unique sequences. The latter is facilitated by the nonuniform distribution of templates frequently observed in DNA libraries. These effects result in a prominent reduction of the original diversity. The encapsulation of DNA repertoires in liquid droplets abolishes the effects of mispairing in DNA libraries. The fundamental advantages of emulsion PCR (ePCR) over bulk PCR are illustrated by deep sequencing and mathematical modeling, which provide the general strategy for ePCR rationalization. The quasi single-molecule ePCR reveals total genetic information by counteracting the degeneration of DNA libraries’ diversity.
... Microfluidics uses in-situ PCR and can display libraries in the same run simultaneously. [119][120][121] Platforms like the Chromium Single Cell Immune Profiling Solution by 10X Genomics can provide pertinent information on TCR, BCR, cell surface protein expression, and RNA expression in the same cells. 122 In addition, the small volume provides increased sensitivity, decreases time and it improves the signal to noise ratio. ...
T cells recognize peptides bound to major histocompatibility complex (MHC) class I and class II molecules at the cell surface. This recognition is accomplished by the expression of T cell receptors (TCR) which are required to be diverse and adaptable in order to accommodate the various and vast number of antigens presented on the MHCs. Thus, determining TCR repertoires of effector T cells is necessary to understand the immunological process in responding to cancer progression, infection, and autoimmune development. Furthermore, understanding the TCR repertoires will provide a solid framework to predict and test the antigen which is more critical in autoimmunity. However, it has been a technical challenge to sequence the TCRs and provide a conceptual context in correlation to the vast number of TCR repertoires in the immunological system. The exploding field of single-cell sequencing has changed how the repertoires are being investigated and analyzed. In this review, we focus on the biology of TCRs, TCR signaling and its implication in autoimmunity. We discuss important methods in bulk sequencing of many cells. Lastly, we explore the most pertinent platforms in single-cell sequencing and its application in autoimmunity.
... Various screening approaches to investigating the antigen-specific cells have been undertaken in the last few years. Novel technological breakthroughs in microfluidics made it possible to select the immunoglobulins with distinct specificities from the Fab (antigen binding fragments) [1] or scFv (single-chain variable fragment) [2,3] libraries with natively paired heavy and light chains. Alternatively, the 10 × Genomics platform was used by Goldstein and colleagues for the high-throughput sequencing of the antigen-specific individual B-cell receptors (BCR), located directly on the living B lymphocytes, that recognize the desired protein [4]. ...
Effective and versatile screening of the peptide ligands capable of selectively binding to diverse receptors is in high demand for the state-of-the-art technologies in life sciences, including probing of specificity of the cell surface receptors and drug development. Complex microenvironment and structure of the surface receptors significantly reduce the possibility to determine their specificity, especially when in vitro conditions are utilized. Previously, we designed a publicly available platform for the ultra-high-throughput screening (uHTS) of the specificity of surface-exposed receptors of the living eukaryotic cells, which was done by consolidating the phage display and flow cytometry techniques. Here, we significantly improved this methodology and designed the fADL-1e-based phage vectors that do not require a helper hyperphage for the virion assembly. The enhanced screening procedure was tested on soluble human leukocyte antigen (HLA) class II molecules and transgenic antigen-specific B cells that express recombinant lymphoid B-cell receptor (BCR). Our data suggest that the improved vector system may be successfully used for the comprehensive search of the receptor ligands in either cell-based or surface-immobilized assays.
