Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire.

Influenza vaccination in the elderly boosts antibodies against conserved viral proteins and egg-produced glycans.

Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age.

Animal immunization merges with innovative technologies: A new paradigm shift in antibody discovery.

Animal-derived antibody sources, particularly, transgenic mice that are engineered with human immunoglobulin loci, along with advanced antibody generation technology platforms have facilitated the discoveries of human antibody therapeutics. For example, isolation of antigen-specific B cells, microfluidics, and next-generation sequencing have emerged as powerful tools for identifying and developing monoclonal antibodies (mAbs). These technologies enable not only antibody drug discovery but also lead to the understanding of B cell biology, immune mechanisms and immunogenetics of antibodies.

A platform-agnostic, function first-based antibody discovery strategy using plasmid-free mammalian expression of antibodies.

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery.

Landscape of Non-canonical Cysteines in Human V Repertoire Revealed by Immunogenetic Analysis.

Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insights into B cell immunogenetics and paratope diversity. By analyzing large public NGS datasets, we map the landscape of non-canonical cysteines in human variable heavy-chain domains (Vs) at the repertoire level. We identify remarkable usage of non-canonical cysteines within the heavy-chain complementarity-determining region 3 (CDR-H3) and other CDRs and framework regions.

Mapping of a Novel H3-Specific Broadly Neutralizing Monoclonal Antibody Targeting the Hemagglutinin Globular Head Isolated from an Elite Influenza Virus-Immunized Donor Exhibiting Serological Breadth.

The discovery of potent and broadly protective influenza virus epitopes could lead to improved vaccines that are resistant to antigenic drift. Here, we describe human antibody C585, isolated from a vaccinee with remarkable serological breadth as measured by hemagglutinin inhibition (HAI). C585 binds and neutralizes multiple H3N2 strains isolated between 1968 and 2016, including strains that emerged up to 4 years after B cells were isolated from the vaccinated donor.

Rapid Elimination of Broadly Neutralizing Antibodies Correlates with Treatment Failure in the Acute Phase of Simian-Human Immunodeficiency Virus Infection.

Early human immunodeficiency virus type 1 (HIV-1) treatment during the acute period of infection can significantly limit the seeding of viral reservoirs and modify the course of disease. However, while a number of HIV-1 broadly neutralizing antibodies (bnAbs) have demonstrated remarkable efficacy as prophylaxis in macaques chronically infected with simian-human immunodeficiency virus (SHIV), intriguingly, their inhibitory effects were largely attenuated in the acute period of SHIV infection. To investigate the mechanism for the disparate performance of bnAbs in different periods of SHIV infection, we used LSEVh-LS-F, a bispecific bnAb targeting the CD4 binding site and CD4-induced epitopes, as a representative bnAb and assessed its potential therapeutic benefit in controlling virus replication in acutely or chronically SHIV-infected macaques.

A broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain III.

Dengue is the most widespread vector-borne viral disease caused by dengue virus (DENV) for which there are no safe, effective drugs approved for clinical use. Here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the DENV envelop protein domain III (DIII) combined with depletion by an entry defective DIII mutant, we identified a cross-reactive human monoclonal antibody (mAb), m366.6, which bound with high affinity to DENV DIII from all four DENV serotypes.

Antibody Aggregation: Insights from Sequence and Structure.

Monoclonal antibodies (mAbs) are the fastest-growing biological therapeutics with important applications ranging from cancers, autoimmunity diseases and metabolic disorders to emerging infectious diseases. Aggregation of mAbs continues to be a major problem in their developability. Antibody aggregation could be triggered by partial unfolding of its domains, leading to monomer-monomer association followed by nucleation and growth.

Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody.

The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (∼36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site.

Engineered antibody domains with significantly increased transcytosis and half-life in macaques mediated by FcRn.

Engineered antibody domains (eAds) are promising candidate therapeutics but their half-life is relatively short partly due to weak or absent binding to the neonatal Fc receptor (FcRn). We developed a novel approach to increase the eAd binding to FcRn based on a combination of structure-based design, computational modeling and phage display methodologies. By using this approach, we identified 2 IgG1 CH2-derived eAds fused to a short FcRn-binding motif derived from IgG1 CH3 that exhibited greatly enhanced FcRn binding with strict pH dependency.

Site-specific antibody-drug conjugation through an engineered glycotransferase and a chemically reactive sugar.

Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. Previously a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle was developed. Here, for the first time, we report the use of this method with some modifications to generate an ADC from a monoclonal antibody, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2.

The Antibody Germline/Maturation Hypothesis, Elicitation of Broadly Neutralizing Antibodies Against HIV-1 and Cord Blood IgM Repertoires.

We have previously observed that all known potent broadly neutralizing antibodies (bnAbs) against HIV-1 are highly divergent from their putative germline predecessors in contrast to bnAbs against viruses causing acute infections such as henipaviruses and SARS CoV, which are much less divergent from their germline counterparts. Consequently, we have hypothesized that germline antibodies may not bind to the HIV-1 envelope glycoprotein (Env) because they are so different compared to the highly somatically mutated HIV-1-specific bnAbs. We have further hypothesized that the immunogenicity of highly conserved epitopes on the HIV-1 envelope glycoproteins (Envs) may be reduced or eliminated by their very weak or absent interactions with germline antibodies and immune responses leading to the elicitation of bnAbs may not be initiated and/or sustained.

Engineered Fc based antibody domains and fragments as novel scaffolds.

Therapeutic monoclonal antibodies (mAbs) have been successful for the therapy of a number of diseases mostly cancer and immune disorders. However, the vast majority of mAbs approved for clinical use are full size, typically in IgG1 format. These mAbs may exhibit relatively poor tissue penetration and restricted epitope access due to their large size.

Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies.

The recently discovered Middle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans, with high mortality. Specific, highly effective therapeutics and vaccines against the MERS-CoV are urgently needed to save human lives and address the pandemic concerns. We identified three human monoclonal antibodies (MAbs), m336, m337, and m338, targeting the receptor (CD26/DPP4) binding domain (RBD) of the MERS-CoV spike glycoprotein from a very large naïve-antibody library (containing ∼10(11) antibodies).

Interactions of IgG1 CH2 and CH3 Domains with FcRn.

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small-size and other unique properties. However, when compared to full-size antibodies, most of the current antibody fragments of VH or VL display greatly reduced half-lives. A promising approach to overcome this problem is through the development of novel antibody fragments based on IgG Fc region, which contributes to the long half-life of IgG through its unique pH-dependent association with the neonatal Fc receptor (FcRn).

Conjugates of Small Molecule Drugs with Antibodies and Other Proteins.

Conjugates of small molecule drugs with antibodies (ADCs) and with other proteins (protein-drug conjugates, PDC) are used as a new class of targeted therapeutics combining the specificity of monoclonal antibodies (mAbs) and other proteins with potent cytotoxic activity of small molecule drugs for the treatment of cancer and other diseases. A(P)DCs have three major components, antibody (targeting protein), linker and payload, the cytotoxic drug. Recently, advances in identifying targets, selecting highly specific mAbs of preferred isotypes, optimizing linker technology and improving chemical methods for conjugation have led to the approval of two ADCs by Food and Drug Administration (FDA) and more than 30 ADCs in advanced clinical development.

Exceptionally potent and broadly cross-reactive, bispecific multivalent HIV-1 inhibitors based on single human CD4 and antibody domains.

Soluble forms of the human immunodeficiency virus type 1 (HIV-1) primary receptor CD4 (soluble CD4 [sCD4]) have been extensively characterized for a quarter of a century as promising HIV-1 inhibitors, but they have not been clinically successful. By combining a protein cavity-filling strategy and the power of library technology, we identified an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties, including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size.

Deep sequencing and Circos analyses of antibody libraries reveal antigen-driven selection of Ig VH genes during HIV-1 infection.

The vast diversity of antibody repertoires is largely attributed to heavy chain (V(H)) recombination of variable (V), diversity (D) and joining (J) gene segments. We used 454 sequencing information of the variable domains of the antibody heavy chain repertoires from neonates, normal adults and an HIV-1-infected individual, to analyze, with Circos software, the VDJ pairing patterns at birth, adulthood and a time-dependent response to HIV-1 infection. Our comparative analyses of the Ig VDJ repertoires from these libraries indicated that, from birth to adulthood, VDJ recombination patterns remain the same with some slight changes, whereas some V(H) families are selected and preferentially expressed after long-term infection with HIV-1.

Discovery of novel candidate therapeutics and diagnostics based on engineered human antibody domains.

The smallest independently folded antibody fragments, the domains, are emerging as promising scaffolds for candidate therapeutics and diagnostics that bind specifically targets of interest. The discovery of such binders is based on several technologies including structure-based design and generation of libraries of mutants displayed on phage or yeast, next-generation sequencing for diversity analysis, panning and screening of the libraries, affinity maturation of selected binders, and their expression, purification, and characterization for specific binding, function, and aggregation propensity. In this review, we describe these technologies as applied for the generation of engineered antibody domains (eAds), especially those derived from the human immunoglobulin heavy chain variable region (VH) and the second domain of IgG1 heavy chain constant region (CH2) as potential candidate therapeutics and diagnostics, and discuss examples of eAds against HIV-1 and cancer-related proteins.

Epitope mapping of M36, a human antibody domain with potent and broad HIV-1 inhibitory activity.

M36 is the first member of a novel class of potent HIV-1 entry inhibitors based on human engineered antibody domains (eAds). It exhibits broad inhibitory activity suggesting that its CD4-induced epitope is highly conserved. Here, we describe fine mapping of its epitope by using several approaches.

Highly efficient selection of epitope specific antibody through competitive yeast display library sorting.

Combinatory antibody library display technologies have been invented and successfully implemented for the selection and engineering of therapeutic antibodies. Precise targeting of important epitopes on the protein of interest is essential for such isolated antibodies to serve as effective modulators of molecular interactions. We developed a strategy to efficiently isolate antibodies against a specific epitope on a target protein from a yeast display antibody library using dengue virus envelope protein domain III as a model target.