Expanding the landscape of antibody discovery.

Library:library screening technologies hold substantial promise for paired antibody:antigen discovery, but challenges have persisted. In this issue of Cell Reports Methods, Wagner et al. introduce a method that combines antibody-ribosome-mRNA complexes, antigen cell surface display, and single-cell RNA sequencing to successfully screen diverse antibody gene libraries against a library of viral receptor proteins.

Clonal Variant Analysis of Antibody Engineering Libraries.

In vitro antibody evolution is a powerful technique for improving monoclonal antibodies. This can be achieved by generating artificial diversity on an antibody template, which can be done using different in vitro diversification techniques. The resulting libraries consist of single- or multimutant variants of a defined antibody template that are screened for improved function using antibody display.

Clonal Lineage and Gene Diversity Analysis of Paired Antibody Heavy and Light Chains.

Antibodies consist of unique variable heavy (V) and variable light (V) chains, and both are required to fully characterize an antibody. Methods to detect paired heavy and light chain variable regions (V:V) using high-throughput sequencing (HTS) have recently enabled large-scale analysis of complete functional antibody responses. Here, we describe an HTS computational pipeline to analyze paired V:V antibody sequences and obtain a comprehensive profile of immune diversity landscapes, including gene usage, antibody isotypes, and clonal lineage analysis.

Beyond Single Clones: High-Throughput Sequencing in Antibody Discovery.

Antibody repertoire sequencing and display library screening are powerful approaches for antibody discovery and engineering that can connect DNA sequence with antibody function. Antibody display and screening studies have made a tremendous impact on immunology and biotechnology over the last decade, accelerated by technological advances in high-throughput DNA sequencing techniques. Indeed, bioinformatic analysis of antibody DNA library data has now taken a central role in modern antibody drug discovery, and is also critical for many ongoing studies of human immune development.

Antibody Data Analysis from Diverse Immune Libraries.

Antibody functional screening studies and next-generation sequencing require careful processing and interpretation of sequence data for optimal results. Here, we provide a detailed protocol for the functional analysis of antibody gene data, including antibody repertoire quantification and functional mapping of high-throughput screening data based on enrichment ratio values, which are a simple way to determine the enrichment of each sequenced antibody after sorting a display library against desired antigens. This protocol enables a user to apply a set of simple yet powerful bioinformatic tools for high-throughput analysis and interpretation of antibody data that is especially well suited for display library screening and for antibody discovery applications.

Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site.

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.

Cell activation-based screening of natively paired human T cell receptor repertoires.

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 TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR.

Bioinformatic Analysis of Natively Paired VH:VL Antibody Repertoires for Antibody Discovery.

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.

Highly protective antimalarial antibodies via precision library generation and yeast display screening.

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.

Quality Control: Chain Pairing Precision and Monitoring of Cross-Sample Contamination: A Method by the AIRR Community.

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.

Immortalization and functional screening of natively paired human T cell receptor repertoires.

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.

Optimized Production of Fc Fusion Proteins by Sortase Enzymatic Ligation.

Fc fusions are a growing class of drugs comprising an antibody Fc domain covalently linked to a protein or peptide and can pose manufacturing challenges. In this study we evaluated three synthetic approaches to generate Fc fusions, using Fc-insulin as a model drug candidate. Engineered human IgG1 was digested with HRV3C to produce an Fc fragment with a C-terminal sortase tag (Fc-LPETGGH).

Regulatory Approved Monoclonal Antibodies Contain Framework Mutations Predicted From Human Antibody Repertoires.

Monoclonal antibodies (mAbs) are an important class of therapeutics used to treat cancer, inflammation, and infectious diseases. Identifying highly developable mAb sequences could greatly reduce the time and cost required for therapeutic mAb development. Here, we present position-specific scoring matrices (PSSMs) for antibody framework mutations developed using baseline human antibody repertoire sequences.

Paired heavy- and light-chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses.

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.

Sequence-Signature Optimization Enables Improved Identification of Human HV6-1-Derived Class Antibodies That Neutralize Diverse Influenza A Viruses.

Sequence signatures of multidonor broadly neutralizing influenza antibodies can be used to quantify the prevalence of B cells with virus-neutralizing potential to accelerate development of broadly protective vaccine strategies. Antibodies of the same class share similar recognition modes and developmental pathways, and several antibody classes have been identified that neutralize diverse group 1- and group 2-influenza A viruses and have been observed in multiple human donors. One such multidonor antibody class, the HV6-1-derived class, targets the stem region of hemagglutinin with extraordinary neutralization breadth.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses.

An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate.

Mutational fitness landscapes reveal genetic and structural improvement pathways for a vaccine-elicited HIV-1 broadly neutralizing antibody.

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.