ABSTRACT
The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.
Subject(s)
HIV Infections , HIV-1 , Humans , HIV-1/genetics , HIV Antibodies , Antibodies, Neutralizing , Peptides , Amino Acid Sequence , Vaccines, Subunit , Neutralization Tests , env Gene Products, Human Immunodeficiency VirusABSTRACT
Molecular characterization of antibody immunity and human antibody discovery is mainly carried out using peripheral memory B cells, and occasionally plasmablasts, that express B cell receptors (BCRs) on their cell surface. Despite the importance of plasma cells (PCs) as the dominant source of circulating antibodies in serum, PCs are rarely utilized because they do not express surface BCRs and cannot be analyzed using antigen-based fluorescence-activated cell sorting. Here, we studied the antibodies encoded by the entire mature B cell populations, including PCs, and compared the antibody repertoires of bone marrow and spleen compartments elicited by immunization in a human immunoglobulin transgenic mouse strain. To circumvent prior technical limitations for analysis of plasma cells, we applied single-cell antibody heavy and light chain gene capture from the entire mature B cell repertoires followed by yeast display functional analysis using a cytokine as a model immunogen. We performed affinity-based sorting of antibody yeast display libraries and large-scale next-generation sequencing analyses to follow antibody lineage performance, with experimental validation of 76 monoclonal antibodies against the cytokine antigen that identified three antibodies with exquisite double-digit picomolar binding affinity. We observed that spleen B cell populations generated higher affinity antibodies compared to bone marrow PCs and that antigen-specific splenic B cells had higher average levels of somatic hypermutation. A degree of clonal overlap was also observed between bone marrow and spleen antibody repertoires, indicating common origins of certain clones across lymphoid compartments. These data demonstrate a new capacity to functionally analyze antigen-specific B cell populations of different lymphoid organs, including PCs, for high-affinity antibody discovery and detailed fundamental studies of antibody immunity.
Subject(s)
Bone Marrow , Plasma Cells , Mice , Animals , Humans , Mice, Transgenic , Spleen , Saccharomyces cerevisiae , Antibodies, Monoclonal , Receptors, Antigen, B-Cell/genetics , Antibody Formation , CytokinesABSTRACT
Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRß (TCRα:ß) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:ß genes from individual cells, ensuring fidelity using a suppression PCR. We then screened TCRα:ß libraries expressed in an immortalized cell line using peptide-pulsed antigen-presenting cells and sequenced activated clones to identify the cognate TCRs. Our results validated an experimental pipeline that allows large-scale repertoire datasets to be annotated with functional specificity information, facilitating the discovery of therapeutically relevant TCRs.
Subject(s)
Receptors, Antigen, T-Cell , T-Lymphocytes , Humans , Receptors, Antigen, T-Cell, alpha-beta/genetics , Cloning, Molecular , Antigens , Peptides/geneticsABSTRACT
Next-generation DNA sequencing (NGS) of human antibody repertoires has been extensively implemented to discover novel antibody drugs, to analyze B-cell developmental features, and to investigate antibody responses to infectious diseases and vaccination. Because the antibody repertoire encoded by human B cells is highly diverse, NGS analyses of antibody genes have provided a new window into understanding antibody responses for basic immunology, biopharmaceutical drug discovery, and immunotherapy. However, many antibody discovery protocols analyze the heavy and light chains separately due to the short-read nature of most NGS technologies, whereas paired heavy and light chain data are required for complete antibody characterization. Here, we describe a computational workflow to process millions of paired antibody heavy and light chain DNA sequence reads using the Illumina MiSeq 2x300 NGS platform. In this workflow, we describe raw NGS read processing and initial quality filtering, the annotation and assembly of antibody clonotypes relating to paired heavy and light chain antibody lineages, and the generation of complete heavy+light consensus sequences for the downstream cloning and expression of human antibody proteins.
Subject(s)
Antibodies , Computational Biology , Humans , Computational Biology/methods , Immunoglobulin Light Chains/genetics , High-Throughput Nucleotide Sequencing/methodsABSTRACT
Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged continuously, challenging the effectiveness of vaccines, diagnostics, and treatments. Moreover, the possibility of the appearance of a new betacoronavirus with high transmissibility and high fatality is reason for concern. In this study, we used a natively paired yeast display technology, combined with next-generation sequencing (NGS) and massive bioinformatic analysis to perform a comprehensive study of subdomain specificity of natural human antibodies from two convalescent donors. Using this screening technology, we mapped the cross-reactive responses of antibodies generated by the two donors against SARS-CoV-2 variants and other betacoronaviruses. We tested the neutralization potency of a set of the cross-reactive antibodies generated in this study and observed that most of the antibodies produced by these patients were non-neutralizing. We performed a comparison of the specific and non-specific antibodies by somatic hypermutation in a repertoire-scale for the two individuals and observed that the degree of somatic hypermutation was unique for each patient. The data from this study provide functional insights into cross-reactive antibodies that can assist in the development of strategies against emerging SARS-CoV-2 variants and divergent betacoronaviruses.
Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Viral , Humans , Membrane Glycoproteins , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Envelope ProteinsABSTRACT
A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.
ABSTRACT
The monoclonal antibody CIS43 targets the Plasmodium falciparum circumsporozoite protein (PfCSP) and prevents malaria infection in humans for up to 9 mo following a single intravenous administration. To enhance the potency and clinical utility of CIS43, we used iterative site-saturation mutagenesis and DNA shuffling to screen precise gene-variant yeast display libraries for improved PfCSP antigen recognition. We identified several mutations that improved recognition, predominately in framework regions, and combined these to produce a panel of antibody variants. The most improved antibody, CIS43_Var10, had three mutations and showed approximately sixfold enhanced protective potency in vivo compared to CIS43. Co-crystal and cryo-electron microscopy structures of CIS43_Var10 with the peptide epitope or with PfCSP, respectively, revealed functional roles for each of these mutations. The unbiased site-directed mutagenesis and screening pipeline described here represent a powerful approach to enhance protective potency and to enable broader clinical use of antimalarial antibodies.
Subject(s)
Antimalarials , Malaria Vaccines , Antibodies, Protozoan , Antimalarials/pharmacology , Cryoelectron Microscopy , Humans , Plasmodium falciparum , Protozoan Proteins , Saccharomyces cerevisiae/geneticsABSTRACT
New approaches in high-throughput analysis of immune receptor repertoires are enabling major advances in immunology and for the discovery of precision immunotherapeutics. Commensurate with growth of the field, there has been an increased need for the establishment of techniques for quality control of immune receptor data. Our laboratory has standardized the use of multiple quality control techniques in immunoglobulin (IG) and T-cell receptor (TR) sequencing experiments to ensure quality control throughout diverse experimental conditions. These quality control methods can also validate the development of new technological approaches and accelerate the training of laboratory personnel. This chapter describes multiple quality control techniques, including split-replicate cell preparations that enable repeat analyses and bioinformatic methods to quantify and ensure high sample quality. We hope that these quality control approaches can accelerate the technical adoption and validated use of unpaired and natively paired immune receptor data.
Subject(s)
High-Throughput Nucleotide Sequencing , Research Design , Computational Biology/methods , High-Throughput Nucleotide Sequencing/methods , Immunoglobulins/genetics , Receptors, Antigen, T-Cell/geneticsABSTRACT
Functional analyses of the T cell receptor (TCR) landscape can reveal critical information about protection from disease and molecular responses to vaccines. However, it has proven difficult to combine advanced next-generation sequencing technologies with methods to decode the peptide-major histocompatibility complex (pMHC) specificity of individual TCRs. We developed a new high-throughput approach to enable repertoire-scale functional evaluations of natively paired TCRs. In particular, we leveraged the immortalized nature of physically linked TCRα:ß amplicon libraries to analyze binding against multiple recombinant pMHCs on a repertoire scale, and to exemplify the utility of this approach, we also performed affinity-based functional mapping in conjunction with quantitative next-generation sequencing to track antigen-specific TCRs. These data successfully validated a new immortalization and screening platform to facilitate detailed molecular analyses of disease-relevant antigen interactions with human TCRs.
Subject(s)
Receptors, Antigen, T-Cell, alpha-beta , Receptors, Antigen, T-Cell , Antigens , Humans , Peptides/chemistry , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
Antiviral monoclonal antibody (mAb) discovery enables the development of antibody-based antiviral therapeutics. Traditional antiviral mAb discovery relies on affinity between antibody and a viral antigen to discover potent neutralizing antibodies, but these approaches are inefficient because many high affinity mAbs have no neutralizing activity. We sought to determine whether screening for anti-SARS-CoV-2 mAbs at reduced pH could provide more efficient neutralizing antibody discovery. We mined the antibody response of a convalescent COVID-19 patient at both physiological pH (7.4) and reduced pH (4.5), revealing that SARS-CoV-2 neutralizing antibodies were preferentially enriched in pH 4.5 yeast display sorts. Structural analysis revealed that a potent new antibody called LP5 targets the SARS-CoV-2 N-terminal domain supersite via a unique binding recognition mode. Our data combine with evidence from prior studies to support antibody screening at pH 4.5 to accelerate antiviral neutralizing antibody discovery.
ABSTRACT
Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be â¼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.
Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Aged , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/ultrastructure , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation , B-Lymphocytes/immunology , Binding Sites , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/ultrastructure , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Immunoglobulin Light Chains/ultrastructure , Male , Protein Binding , Protein Interaction Domains and Motifs , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Vero CellsABSTRACT
The re-emergence of Zika virus (ZIKV) caused widespread infections that were linked to Guillain-Barré syndrome in adults and congenital malformation in fetuses, and epidemiological data suggest that ZIKV infection can induce protective antibody responses. A more detailed understanding of anti-ZIKV antibody responses may lead to enhanced antibody discovery and improved vaccine designs against ZIKV and related flaviviruses. Here, we applied recently-invented library-scale antibody screening technologies to determine comprehensive functional molecular and genetic profiles of naturally elicited human anti-ZIKV antibodies in three convalescent individuals. We leveraged natively paired antibody yeast display and NGS to predict antibody cross-reactivities and coarse-grain antibody affinities, to perform in-depth immune profiling of IgM, IgG, and IgA antibody repertoires in peripheral blood, and to reveal virus maturation state-dependent antibody interactions. Repertoire-scale comparison of ZIKV VLP-specific and non-specific antibodies in the same individuals also showed that mean antibody somatic hypermutation levels were substantially influenced by donor-intrinsic characteristics. These data provide insights into antiviral antibody responses to ZIKV disease and outline systems-level strategies to track human antibody immune responses to emergent viral infections.
Subject(s)
Antibodies, Viral/immunology , Antibody Formation/immunology , Host-Pathogen Interactions/immunology , Zika Virus Infection/immunology , Zika Virus Infection/virology , Zika Virus/immunology , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Antibodies, Viral/genetics , Antibody Formation/genetics , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Computational Biology/methods , Flow Cytometry , High-Throughput Nucleotide Sequencing , Humans , Neutralization Tests , Peptide LibraryABSTRACT
Vaccine-based elicitation of broadly neutralizing antibodies holds great promise for preventing HIV-1 transmission. However, the key biophysical markers of improved antibody recognition remain uncertain in the diverse landscape of potential antibody mutation pathways, and a more complete understanding of anti-HIV-1 fusion peptide (FP) antibody development will accelerate rational vaccine designs. Here we survey the mutational landscape of the vaccine-elicited anti-FP antibody, vFP16.02, to determine the genetic, structural, and functional features associated with antibody improvement or fitness. Using site-saturation mutagenesis and yeast display functional screening, we found that 1% of possible single mutations improved HIV-1 envelope trimer (Env) affinity, but generally comprised rare somatic hypermutations that may not arise frequently in vivo. We observed that many single mutations in the vFP16.02 Fab could enhance affinity >1,000-fold against soluble FP, although affinity improvements against the HIV-1 trimer were more measured and rare. The most potent variants enhanced affinity to both soluble FP and Env, had mutations concentrated in antibody framework regions, and achieved up to 37% neutralization breadth compared to 28% neutralization of the template antibody. Altered heavy- and light-chain interface angles and conformational dynamics, as well as reduced Fab thermal stability, were associated with improved HIV-1 neutralization breadth and potency. We also observed parallel sets of mutations that enhanced viral neutralization through similar structural mechanisms. These data provide a quantitative understanding of the mutational landscape for vaccine-elicited FP-directed broadly neutralizing antibody and demonstrate that numerous antigen-distal framework mutations can improve antibody function by enhancing affinity simultaneously toward HIV-1 Env and FP.
Subject(s)
AIDS Vaccines/immunology , Broadly Neutralizing Antibodies/immunology , HIV Antibodies/immunology , HIV-1/immunology , Mutation , env Gene Products, Human Immunodeficiency Virus/immunology , AIDS Vaccines/genetics , Broadly Neutralizing Antibodies/genetics , HIV Antibodies/genetics , HIV-1/genetics , Humans , env Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
Understanding protective mechanisms of antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We discovered a new antibody, 910-30, that targets the SARS-CoV-2 ACE2 receptor binding site as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. We performed sequence and structural analyses to explore how antibody features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer revealed its binding interactions and ability to disassemble spike. Despite heavy chain sequence similarity, biophysical analyses of IGHV3-53/3-66 antibodies highlighted the importance of native heavy:light pairings for ACE2 binding competition and for SARS-CoV-2 neutralization. We defined paired heavy:light sequence signatures and determined antibody precursor prevalence to be ~1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These data reveal key structural and functional neutralization features in the IGHV3-53/3-66 public antibody class to accelerate antibody-based medical interventions against SARS-CoV-2. HIGHLIGHTS: A molecular study of IGHV3-53/3-66 public antibody responses reveals critical heavy and light chain features for potent neutralizationCryo-EM analyses detail the structure of a novel public antibody class member, antibody 910-30, in complex with SARS-CoV-2 spike trimerCryo-EM data reveal that 910-30 can both bind assembled trimer and can disassemble the SARS-CoV-2 spikeSequence-structure-function signatures defined for IGHV3-53/3-66 class antibodies including both heavy and light chainsIGHV3-53/3-66 class precursors have a prevalence of 1:44,000 B cells in healthy human antibody repertoires.
ABSTRACT
Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from â¼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/immunology , Molecular Probes/immunology , Pneumonia, Viral/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Antibody Specificity/immunology , Binding Sites, Antibody/immunology , Biotinylation , COVID-19 , Cryoelectron Microscopy , Humans , Pandemics , Peptidyl-Dipeptidase A/metabolism , Receptors, Virus/metabolismABSTRACT
Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes. Funding: Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID). Support for this work was also provided by COVID-19 Fast Grants, the Jack Ma Foundation, the Self Graduate Fellowship Program, and NIH grants DP5OD023118, R21AI143407, and R21AI144408. Some of this work was performed at the Columbia University Cryo-EM Center at the Zuckerman Institute, and some at the Simons Electron Microscopy Center (SEMC) and National Center for Cryo-EM Access and Training (NCCAT) located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR, and the NIH National Institute of General Medical Sciences (GM103310). Conflict of Interest: The authors declare that they have no conflict of interest. Ethical Approval: Peripheral blood mononuclear cells (PBMCs) for B cell sorting were obtained from a convalescent SARS-CoV-2 patient (collected 75 days post symptom onset under an IRB approved clinical trial protocol, VRC 200 - ClinicalTrials.gov Identifier: NCT00067054) and a healthy control donor from the NIH blood bank pre-SARS-CoV-2 pandemic.
ABSTRACT
Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ~0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike-ectodomain probes.
ABSTRACT
Yeast display has become an important tool for modern biotechnology with many advantages for eukaryotic protein engineering. Antibody-based peptide interactions are often used to quantify yeast surface expression (e.g., by fusing a target protein to a FLAG, Myc, polyhistidine, or other peptide tag). However, antibody-antigen interactions require high stability for accurate quantification, and conventional tag systems based on such interactions may not be compatible with a low pH environment. In this study, a SNAP tag was introduced to a yeast display platform to circumvent disadvantages of conventional antibody display tags at low pH. SNAP forms a covalent bond with its small-molecule substrate, enabling precise and pH-independent protein display tagging. We compared the SNAP tag to conventional antibody-based peptide fusion and to direct fluorescent domain fusion using antibody fragment crystallizable (Fc) gene libraries as a case study in low pH protein engineering. Our results demonstrated that covalent SNAP tags can effectively quantify protein-surface expression at low pH, enabling the enrichment of Fc variants with increased affinity at pH 6.0 to the neonatal Fc receptor (FcRn). Incorporation of a covalent SNAP tag thus overcomes disadvantages of conventional antibody-based expression tags and enables protein-engineering applications outside of physiological pH.
Subject(s)
Cell Surface Display Techniques/methods , Recombinant Fusion Proteins , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Humans , Hydrogen-Ion Concentration , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/metabolism , Receptors, Fc/genetics , Receptors, Fc/metabolism , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Yeasts/geneticsABSTRACT
Emulsion-based techniques have dramatically advanced our understanding of single-cell biology and complex single-cell features over the past two decades. Most approaches for precise single cell isolation rely on microfluidics, which has proven highly effective but requires substantial investment in equipment and expertise that can be difficult to access for researchers that specialize in other areas of bioengineering and molecular biotechnology. Inspired by the robust droplet generation technologies in modern flow cytometry instrumentation, here we established a new platform for high-throughput isolation of single cells within droplets of tunable sizes by combining flow focusing with ultrasonic vibration for rapid and effective droplet formation. Application of ultrasonic pressure waves to the flowing jet provided enhanced control of emulsion droplet size, permitting capture of 25,000 to 50,000 single cells per minute. As an example application, we applied this new droplet generation platform to sequence the antibody variable region heavy and light chain pairings (VH:VL) from large repertoires of single B cells. We demonstrated the recovery of > 40,000 paired CDRH3:CDRL3 antibody clusters from a single individual, validating that these droplet systems can enable the genetic analysis of very large single-cell populations. These accessible new technologies will allow rapid, large-scale, and precise single-cell analyses for a broad range of bioengineering and molecular biotechnology applications.