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. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the 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. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galß1-4GalNAcß), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.

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. In this perspective article, we discuss the scientific merits of animal immunization combined with advanced methods for antibody generation as compared to animal-free alternatives through in-vitro-generated antibody libraries. The knowledge gained from animal-derived antibodies concerning the recombinational diversity, somatic hypermutation patterns, and physiochemical properties is found more valuable and prerequisite for developing in vitro libraries, as well as artificial intelligence/machine learning methods to discover safe and effective mAbs.

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. In this study, we describe a process to identify and rapidly re-format and express antibodies for functional characterization. We used two different approaches to isolate antibodies to five different targets: 1) flow cytometry to identify antigen-specific single B cells from the spleen of immunized human immunoglobulin transgenic mice; and 2) panning of phage libraries. PCR amplification allowed recovery of paired VH and VL sequences from 79% to 96% of antigen-specific B cells. All cognate VH and VL transcripts were formatted into transcription and translation compatible linear DNA expression cassettes (LEC) encoding whole IgG or Fab. Between 92% and 100% of paired VH and VL transcripts could be converted to LECs, and nearly 100% of them expressed as antibodies when transfected into Expi293F cells. The concentration of IgG in the cell culture supernatants ranged from 0.05 µg/ml to 145.8 µg/ml (mean = 18.4 µg/ml). Antigen-specific binding was displayed by 78-100% of antibodies. High throughput functional screening allowed the rapid identification of several functional antibodies. In summary, we describe a plasmid-free system for cloning and expressing antibodies isolated by different approaches, in any format of choice for deep functional screening that can be applied in any research setting during antibody discovery.

Landscape of Non-canonical Cysteines in Human VH 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 (VHs) 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. Furthermore, our study reveals the diversity and location of non-canonical cysteines and their associated motifs in human VHs, which are reminiscent of and more complex than those found in other non-human species such as chicken, camel, llama, shark, and cow. These results explain how non-canonical cysteines strategically occur in the human antibodyome to expand its paratope space. This study will guide the design of human antibodies harboring disulfide-stabilized long CDR-H3s to access difficult-to-target epitopes and influence a paradigm shift in developability involving non-canonical cysteines.

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. The crystal structure of C585 Fab in complex with the HA from A/Switzerland/9715293/2013 (H3N2) shows that the antibody binds to a novel and well-conserved epitope on the globular head of H3 HA and that it differs from other antibodies not only in its epitope but in its binding geometry and hypermutated framework 3 region, thereby explaining its breadth and ability to mediate hemagglutination inhibition across decades of H3N2 strains. The existence of epitopes such as the one elucidated by C585 has implications for rational vaccine design.IMPORTANCE Influenza viruses escape immunity through continuous antigenic changes that occur predominantly on the viral hemagglutinin (HA). Induction of broadly neutralizing antibodies (bnAbs) targeting conserved epitopes following vaccination is a goal of universal influenza vaccines and advantageous in protecting hosts against virus evolution and antigenic drift. To date, most of the discovered bnAbs bind either to conserved sites in the stem region or to the sialic acid-binding pocket. Generally, antibodies targeting the stem region offer broader breadth with low potency, while antibodies targeting the sialic acid-binding pocket cover narrower breadth but usually have higher potency. In this study, we identified a novel neutralizing epitope in the head region recognized by a broadly neutralizing human antibody against a broad range of H3N2 with high potency. This epitope may provide insights for future universal vaccine design.

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. We found that a single infusion of LSEVh-LS-F resulted in rapid decline of plasma viral loads to undetectable levels without emergence of viral resistance in the chronically infected macaques. In contrast, the inhibitory effect was robust but transient in the acutely infected macaques, despite the fact that all macaques had comparable plasma viral loads initially. Infusing multiple doses of LSEVh-LS-F did not extend its inhibitory duration. Furthermore, the pharmacokinetics of the infused LSEVh-LS-F in the acutely SHIV-infected macaques significantly differed from that in the uninfected or chronically infected macaques. Host SHIV-specific immune responses may play a role in the viremia-dependent pharmacokinetics. Our results highlight the correlation between the fast clearance of infused bnAbs and the treatment failure in the acute period of SHIV infection and may have important implications for the therapeutic use of bnAbs to treat acute HIV infections.IMPORTANCE Currently, there is no bnAb-based monotherapy that has been reported to clear the virus in the acute SHIV infection period. Since early HIV treatment is considered critical to restricting the establishment of viral reservoirs, investigation into the mechanism for treatment failure in acutely infected macaques would be important for the therapeutic use of bnAbs and eventually towards the functional cure of HIV/AIDS. Here we report the comparative study of the therapeutic efficacy of a bnAb in acutely and chronically SHIV-infected macaques. This study revealed the correlation between the fast clearance of infused bnAbs and treatment failure during the acute period of infection.

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. Immunogenetic analysis indicated that m366.6 is a germline-like mAb with very few somatic mutations from the closest VH and Vλ germline genes. Importantly, we demonstrated that it potently neutralized DENV both in vitro and in the mouse models of DENV infection without detectable antibody-dependent enhancement (ADE) effect. The epitope of m366.6 was mapped to the highly conserved regions on DIII, which may guide the design of effective dengue vaccine immunogens. Furthermore, as the first germline-like mAb derived from a naïve antibody library that could neutralize all four DENV serotypes, the m366.6 can be a tool for exploring mechanisms of DENV infection, and is a promising therapeutic candidate.

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. Although the aggregation propensities of antibodies and antibody-based proteins can be affected by the external experimental conditions, they are strongly dependent on the intrinsic antibody properties as determined by their sequences and structures. In this review, we describe how the unfolding and aggregation susceptibilities of IgG could be related to their cognate sequences and structures. The impact of antibody domain structures on thermostability and aggregation propensities, and effective strategies to reduce aggregation are discussed. Finally, the aggregation of antibody-drug conjugates (ADCs) as related to their sequence/structure, linker payload, conjugation chemistry and drug-antibody ratio (DAR) is reviewed.

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. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.

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. Importantly, the increased affinity resulted in significantly enhanced FcRn-mediated epithelial transcytosis and prolonged elimination half-life (mean 44.1 hours) in cynomolgus macaques. These results demonstrate for the first time that the half-life of isolated eAds can be prolonged (optimized) by increasing their binding to FcRn while maintaining their small size, which has important implications for development of therapeutics, including eAd-drug conjugates with enhanced penetration in solid tissues.

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. M860 bound to cell surface-associated HER2 with affinity comparable to that of Trastuzumab (Herceptin), but to a different epitope. The m860ADC was generated by enzymatically adding a reactive keto-galactose to m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F. It exhibited potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. This unique ADC may have utility as a potential therapeutic for HER2 positive cancers alone or in combination with other drugs. Our results also validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other disease markers.

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. Even if such responses are initiated, the maturation pathways are so extraordinarily complex that prolonged periods of time may be required for elicitation of bnAbs with defined unique sequences. We provided the initial evidence supporting this antibody germline/maturation hypothesis, which prompted a number of studies to design vaccine immunogens that could bind putative germline predecessors of known bnAbs and to explore complex B cell lineages. However, guiding the immune system through the exceptionally complex antibody maturation pathways to elicit known bnAbs remains a major challenge. Here, we discuss studies exploring the antibody germline/maturation hypothesis as related to elicitation of bnAbs against HIV-1 and present our recent data demonstrating the existence of germline-like precursors of VRC01 antibodies in a human cord blood IgM library.

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. A promising solution to this fundamental limitation is the engineering of smaller scaffolds based on the IgG1 Fc region. These scaffolds can be used for the generation of libraries of mutants from which high-affinity binders can be selected. Comprised of the CH2 and CH3 domains, the Fc region is important not only for the antibody effector function but also for its long half-life. This review focuses on engineered Fc based antibody fragments and domains including native (dimeric) Fc and monomeric Fc as well as CH2 and monomeric CH3, and their use as novel scaffolds and binders. The Fc based binders are promising candidate therapeutics with optimized half-life, enhanced tissue penetration and access to sterically restricted binding sites resulting in an increased therapeutic efficacy. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

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). They bound with high affinity: equilibrium dissociation constants for the three MAbs were equal to 4.2, 9.3, and 15 nM, respectively, as measured by Biacore for Fabs binding to RBD. The avidity for IgG1 m336, m337, and m338 was even higher: 99, 820, and 560 pM, respectively. The antibodies bound to overlapping epitopes that overlap the receptor binding site on the RBD as suggested by competition experiments and further supported by site-directed mutagenesis of the RBD and a docking model of the m336-RBD complex. The highest-affinity MAb, m336, neutralized both pseudotyped and live MERS-CoV with exceptional potency, 50% neutralization at 0.005 and 0.07 µg/ml, respectively, likely by competing with DPP4 for binding to the S glycoprotein. The exceptionally high neutralization activity of these antibodies and especially m336 suggests that they have great potential for prophylaxis and therapy of MERS-CoV infection in humans and as a tool for development of vaccine immunogens. The rapid identification (within several weeks) of potent MAbs suggests a possibility to use the new large antibody library and related methodology for a quick response to the public threat resulting from emerging coronaviruses. Importance: A novel human coronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV), was found to infect humans with a high mortality rate in 2012, just 1 decade after the appearance of the first highly pathogenic coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV). There are no effective therapeutics available. It is highly desirable to find an approach for rapidly developing potent therapeutics against MERS-CoV, which not only can be implemented for MERS treatment but also can help to develop a platform strategy to combat future emerging coronaviruses. We report here the identification of human monoclonal antibodies (MAbs) from a large nonimmune antibody library that target MERS-CoV. One of the antibodies, m336, neutralized the virus with exceptional potency. It therefore may have great potential as a candidate therapeutic and as a reagent to facilitate the development of vaccines against MERS-CoV.

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). The IgG Fc region comprises two CH2 and two CH3 domains. In this report, we present a comparative study of the FcRn binding capability of the CH2 and CH3 domains. The stability and aggregation resistance of these domains were also investigated and compared. We found that monomeric CH2 and CH3 domains exhibited the pH-dependent FcRn binding while the dimeric forms of CH2 and CH3 domains did not. Although all of these domains had high serum stability, they had aggregation tendencies as measured by dynamic light scattering. By providing a better understanding of the structure-activity relationship of the Fc fragment, these results guide further approaches to generate novel Fc-based small-size antibody fragments that possess pH-dependent FcRn binding capability, desired in vivo half-lives, and other favorable biophysical properties for their druggability.

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. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor, an antibody domain (m36.4) that targets the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested, with potencies about 10-, 50-, and 200-fold higher than those of the broadly neutralizing antibody VRC01, the U.S. FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity and a lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be useful for HIV-1 prevention and therapy, including eradication of the virus.

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.