Mining human antibody repertoires

Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties.Key words: human monoclonal antibodies, B cells, hybridoma technology, display technologies, antibody libraries, antibody engineering     

A novel synthetic na?ve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.Key words: human antibody library, phage display, oncofetal fibronectin, vascular tumor targeting, scFv antibody fragments, chelating recombinant antibody (CRAb)     

Recent advances in the generation of human monoclonal antibody

The use of monoclonal antibodies (mAbs) has now gained a niche as an epochal breakthrough in medicine. Engineered antibodies (Abs) currently account for over 30% of biopharmaceuticals in clinical trials. Several methods to generate human mAbs have evolved, such as (1) immortalization of antigen-specific human B cell hybridoma technology, (2) generation of chimeric and humanized antibody (Ab) from mouse Ab by genetic engineering, (3) acquisition of antigen-specific human B cells by the phage display method, and (4) development of transgenic mice for producing human mAbs. Besides these technologies, we have independently developed a method to generate human mAbs by combining the method of in vitro immunization using peripheral blood mononuclear cells and the phage display method. In this paper, we review the developments in these technologies for generating human mAbs.     

B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies

The recent development of screening strategies based on the generation and display of large libraries of antibody fragments has allowed considerable advances for the in vitro isolation of monoclonal antibodies (mAbs). We previously developed a technology referred to as the 'ADLib (Autonomously Diversifying Library) system', which allows the rapid screening and isolation in vitro of antigen-specific monoclonal antibodies (mAbs) from libraries of immunoglobulin M (IgM) displayed by the chicken B-cell line DT40. Here, we report a novel application of the ADLib system to the production of chimeric human mAbs. We have designed gene knock-in constructs to generate DT40 strains that coexpress chimeric human IgG and chicken IgM via B-cell-specific RNA alternative splicing. We demonstrate that the application of the ADLib system to these strains allows the one-step selection of antigen-specific human chimeric IgG. In addition, the production of chimeric IgG can be selectively increased when we modulate RNA processing by overexpressing the polyadenylation factor CstF-64. This method provides a new way to efficiently design mAbs suitable for a wide range of purposes including antibody therapy.     

A novel synthetic naïve human antibody library allows the isolation of antibodies against a new epitope of oncofetal fibronectin

Human monoclonal antibodies (mAbs) can routinely be isolated from phage display libraries against virtually any protein available in sufficient purity and quantity, but library design can influence epitope coverage on the target antigen. Here we describe the construction of a novel synthetic human antibody phage display library that incorporates hydrophilic or charged residues at position 52 of the CDR2 loop of the variable heavy chain domain, instead of the serine residue found in the corresponding germline gene. The novel library was used to isolate human mAbs to various antigens, including the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis. In particular, the mAb 2H7 was proven to bind to a novel epitope on EDA, which does not overlap with the one recognized by the clinical-stage F8 antibody. F8 and 2H7 were used for the construction of chelating recombinant antibodies (CRAbs), whose tumor-targeting properties were assessed in vivo in biodistribution studies in mice bearing F9 teratocarcinoma, revealing a preferential accumulation at the tumor site.     

单个B细胞抗体制备技术及应用

单克隆抗体是现代生命科学研究的重要工具,为许多领域的发展作出了不可估量的贡献。随着PCR技术和单克隆抗体技术的发展和成熟,单个B细胞抗体制备技术迅速兴起。该技术能够对单个的抗原特异性B细胞进行抗体基因的体外克隆和表达,保证了轻重链可变区的天然配对,相较于传统的抗体制备技术具有效率高、全人源、基因多样性更丰富等优势。单个B细胞抗体制备技术已成为制备全人抗体的热门方法,同时也促进了包括抗体发生成熟、疫苗保护机制、疫苗开发、肿瘤及自身免疫疾病等免疫学相关研究。文中就单个B细胞抗体制备技术的过程及应用作简要综述。     

Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

The ability of monoclonal antibodies (mAbs) to target specific antigens with high precision has led to an increasing demand to generate them for therapeutic use in many disease areas. Historically, the discovery of therapeutic mAbs has relied upon the immunization of mammals and various in vitro display technologies. While the routine immunization of rodents yields clones that are stable in serum and have been selected against vast arrays of endogenous, non-target self-antigens, it is often difficult to obtain species cross-reactive mAbs owing to the generally high sequence similarity shared across human antigens and their mammalian orthologs. In vitro display technologies bypass this limitation, but lack an in vivo screening mechanism, and thus may potentially generate mAbs with undesirable binding specificity and stability issues. Chicken immunization is emerging as an attractive mAb discovery method because it combines the benefits of both in vivo and in vitro display methods. Since chickens are phylogenetically separated from mammals, their proteins share less sequence homology with those of humans, so human proteins are often immunogenic and can readily elicit rodent cross-reactive clones, which are necessary for in vivo proof of mechanism studies. Here, we compare the binding characteristics of mAbs isolated from chicken immunization, mouse immunization, and phage display of human antibody libraries. Our results show that chicken-derived mAbs not only recapitulate the kinetic diversity of mAbs sourced from other methods, but appear to offer an expanded repertoire of epitopes. Further, chicken-derived mAbs can bind their native serum antigen with very high affinity, highlighting their therapeutic potential.     

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS?). In OCMS?, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS? demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. We produced and characterized OCMS?-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS? to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS? overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.     

Selection of human antibody fragments by phage display

Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Antigen-specific clones are enriched by binding to immobilized antigen, followed by elution and repropagation of phage. After multiple rounds of binding selection, specific clones are identified by ELISA. This article provides an overview of phage display and antibody technology, as well as detailed protocols for the immobilization of antigen, the selection of repertoires on purified or complex antigens and the identification of binders.     

Concerted Antibody and Antigen Discovery by Differential Whole-cell Phage Display Selections and Multi-omic Target Deconvolution

Monoclonal antibody (mAb)-based biologics are well established treatments of cancer. Antibody discovery campaigns are typically directed at a single target of interest, which inherently limits the possibility of uncovering novel antibody specificities or functionalities. Here, we present a target-unbiased approach for antibody discovery that relies on generating mAbs against native target cell surfaces via phage display. This method combines a previously reported method for improved whole-cell phage display selections with next-generation sequencing analysis to efficiently identify mAbs with the desired target cell reactivity. Applying this method to multiple myeloma cells yielded a panel of >50 mAbs with unique sequences and diverse reactivities. To uncover the identities of the cognate antigens recognized by this panel, representative mAbs from each unique reactivity cluster were used in a multi-omic target deconvolution approach. From this, we identified and validated three cell surface antigens: PTPRG, ICAM1, and CADM1. PTPRG and CADM1 remain largely unstudied in the context of multiple myeloma, which could warrant further investigation into their potential as therapeutic targets. These results highlight the utility of optimized whole-cell phage display selection methods and could motivate further interest in target-unbiased antibody discovery workflows.     

Selecting and screening recombinant antibody libraries

During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automation of the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.     

Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display

Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the surface of yeast and used to epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for epitope mapping. Using a library of yeast-displayed BoNT/A binding domain (H(C)) mutants and selecting for loss of binding, the fine epitopes of three neutralizing BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of H(C), with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of H(C), in an area not previously thought to be functionally important. Modeling the epitopes shows how all three mAbs could bind BoNT/A simultaneously and may explain, in part, the dramatic synergy observed on in vivo toxin neutralization when these antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody epitopes and the mapping results identify three neutralizing BoNT/A epitopes.     

Engineering Fully Human Monoclonal Antibodies from Murine Variable Regions

Fully human monoclonal antibodies (mAbs) derived from transgenic mice or human antibody libraries are the current state of the art for reducing the immunogenicity risk of antibody drugs. Here, we describe a novel method for generating fully human mAbs from nonhuman variable regions using information from the human germline repertoire. Central to our strategy is the rational engineering of residues within and proximal to CDRs and the V_H/V_L interface by iteratively exploring substitutions to the closest human germline sequences using semi-automated computational methods. Starting from the parent murine variable regions of three currently marketed mAbs targeting CD25, vascular endothelial growth factor, and tumor necrosis factor alpha, we have generated fully human antibodies with 59, 46, and 45 substitutions, respectively, compared to the parent murine sequences. A large number of these substitutions were in the CDRs, which are typically avoided in humanization methods. Antigen affinities of the fully human variants were comparable to the chimeric mAbs in each case. Furthermore, in vitro functional characterization indicated that all retain potency of the chimeric mAbs and have comparable activity to their respective marketed drugs daclizumab, bevacizumab, and infliximab. Based on local and global sequence identity, the sequences of our engineered mAbs are indistinguishable from those of fully human mAbs isolated from transgenic mice or human antibody libraries. This work establishes a simple rational engineering methodology for generating fully human antibody therapeutics from murine mAbs produced from standard hybridoma technology.     

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

ABSTRACT

Immunization of mice followed by hybridoma or B-cell screening is one of the most common antibody discovery methods used to generate therapeutic monoclonal antibody (mAb) candidates. There are a multitude of different immunization protocols that can generate an immune response in animals. However, an extensive analysis of the antibody repertoires that these alternative immunization protocols can generate has not been performed. In this study, we immunized mice that transgenically express human antibodies with either programmed cell death 1 protein or cytotoxic T-lymphocyte associated protein 4 using four different immunization protocols, and then utilized a single cell microfluidic platform to generate tissue-specific, natively paired immunoglobulin (Ig) repertoires from each method and enriched for target-specific binders using yeast single-chain variable fragment (scFv) display. We deep sequenced the scFv repertoires from both the pre-sort and post-sort libraries. All methods and both targets yielded similar oligoclonality, variable (V) and joining (J) gene usage, and divergence from germline of enriched libraries. However, there were differences between targets and/or immunization protocols for overall clonal counts, complementarity-determining region 3 (CDR3) length, and antibody/CDR3 sequence diversity. Our data suggest that, although different immunization protocols may generate a response to an antigen, performing multiple immunization protocols in parallel can yield greater Ig diversity. We conclude that modern microfluidic methods, followed by an extensive molecular genomic analysis of antibody repertoires, can be used to quickly analyze new immunization protocols or mouse platforms.     

A Sortase A Programmable Phage Display Format for Improved Panning of Fab Antibody Libraries

Phage display of combinatorial antibody libraries is a versatile tool in the field of antibody engineering, with diverse applications including monoclonal antibody (mAb) discovery, affinity maturation, and humanization. To improve the selection efficiency of antibody libraries, we developed a new phagemid display system that addresses the complication of bald phage propagation. The phagemid facilitates the biotinylation of fragment of antigen binding (Fab) antibody fragments displayed on phage via Sortase A catalysis and the subsequent enrichment of Fab-displaying phage during selections. In multiple contexts, this selection approach improved the enrichment of target-reactive mAbs by depleting background phage. Panels of cancer cell line-reactive mAbs with high diversity and specificity were isolated from a naïve chimeric rabbit/human Fab library using this approach, highlighting its potential to accelerate antibody engineering efforts and to empower concerted antibody drug and target discovery.     

Stability engineering of the human antibody repertoire

Human monoclonal antibodies often display limited thermodynamic and colloidal stabilities. This behavior hinders their production, and places limitations on the development of novel formulation conditions and therapeutic applications. Antibodies are highly diverse molecules, with much of the sequence variation observed within variable domain families and, in particular, their complementarity determining regions. This has complicated the development of comprehensive strategies for the stability engineering of the human antibody repertoire. Here we provide an overview of the field, and discuss recent advances in the development of robust and aggregation resistant antibody therapeutics.     

Comparing CDRH3 diversity captured from secondary lymphoid organs for the generation of recombinant human antibodies

The plasticity of natural immunoglobulin repertoires can be exploited for the generation of phage display libraries. Secondary lymphoid organs, such as the spleen and the lymph nodes, constitute interesting sources of diversity because they are rich in B cells, part of which can be affinity matured. These organs, however, differ in their anatomical structure, reflecting the different fluids they drain, which affects the B cell repertoires. The CDRH3 repertoires from these organs, extracted from naïve or immunized mice, were compared in the context of phage display libraries using human antibody framework families. Deep sequencing analysis revealed that all libraries displayed different CDRH3 repertoires, but the one derived from lymph nodes of naïve mice was the most diverse. Library performance was assessed by in vitro selection. For both organs, immunization increased substantially the frequency of molecules able to bind to the immunogen. The library derived from lymph nodes from naïve mice, however, was the most effective in generating diverse and high affinity candidates. These results illustrate that the use of a biased CDRH3 repertoire increases the performance of libraries, but reduces the clonal diversity, which may be detrimental for certain strategies.     

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)₂. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.     

Antibody repertoires of four- and five-feature translocus mice carrying human immunoglobulin heavy chain and kappa and lambda light chain yeast artificial chromosomes

We have produced mice that carry the human Ig heavy (IgH) and both kappa and lambda light chain transloci in a background in which the endogenous IgH and kappa loci have been inactivated. The B lymphocyte population in these translocus mice is restored to about one-third of normal levels, with preferential (3:1) expression of human lambda over human kappa. Human IgM is found in the serum at levels between 50 and 400 microg/ml and is elevated following immunization. This primary human Ab repertoire is sufficient to yield diverse Ag-specific responses as judged by analysis of mAbs. The use of DH and J segments is similar to that seen in human B cells, with an analogous pattern of N nucleotide insertion. Maturation of the response is accompanied by somatic hypermutation, which is particularly effective in the light chain transloci. These mice therefore allow the production of Ag-specific repertoires of both IgM,kappa and IgM,lambda Abs and should prove useful for the production of human mAbs for clinical use.     

Generation of “LYmph Node Derived Antibody Libraries” (LYNDAL) for selecting fully human antibody fragments with therapeutic potential

The development of efficient strategies for generating fully human monoclonal antibodies with unique functional properties that are exploitable for tailored therapeutic interventions remains a major challenge in the antibody technology field. Here, we present a methodology for recovering such antibodies from antigen-encountered human B cell repertoires. As the source for variable antibody genes, we cloned immunoglobulin G (IgG)-derived B cell repertoires from lymph nodes of 20 individuals undergoing surgery for head and neck cancer. Sequence analysis of unselected “LYmph Node Derived Antibody Libraries” (LYNDAL) revealed a naturally occurring distribution pattern of rearranged antibody sequences, representing all known variable gene families and most functional germline sequences. To demonstrate the feasibility for selecting antibodies with therapeutic potential from these repertoires, seven LYNDAL from donors with high serum titers against herpes simplex virus (HSV) were panned on recombinant glycoprotein B of HSV-1. Screening for specific binders delivered 34 single-chain variable fragments (scFvs) with unique sequences. Sequence analysis revealed extensive somatic hypermutation of enriched clones as a result of affinity maturation. Binding of scFvs to common glycoprotein B variants from HSV-1 and HSV-2 strains was highly specific, and the majority of analyzed antibody fragments bound to the target antigen with nanomolar affinity. From eight scFvs with HSV-neutralizing capacity in vitro, the most potent antibody neutralized 50% HSV-2 at 4.5 nM as a dimeric (scFv)₂. We anticipate our approach to be useful for recovering fully human antibodies with therapeutic potential.