Site-directed neutralizing antibodies targeting structural sites on SARS-CoV-2 spike protein.

'Epivolve' (epitope evolution) is an innovative paratope-evolving technology using a haptenated peptide or protein immunogen as a means of directing the in vivo immune response to specifically targeted sites at a one amino acid residue resolution. Guided by protein structural analysis, Epivolve technology was tested to develop site-directed neutralizing antibodies (nAbs) in a systematic fashion against the SARS-CoV-2 Receptor Binding Domain (RBD). Thirteen solvent-exposed sites covering the ACE2 receptor-binding interface were targeted. Immunogens composed of each targeted site were used to immunize rabbits in separate cohorts. In vivo site-directed immune responses against all 13 targets were demonstrated by B cell secreted IgG and recombinant IgG testing. One site, SL13 (Y505) which mutates from tyrosine to histidine in the SARS-CoV-2 Omicron variant, was chosen as a proof-of-concept (PoC) model for further functional monoclonal antibody development. Epivolve technology demonstrated the capabilities of generating pan-variant antibodies and nAbs against the SARS-CoV-2 primary strain and the Omicron variant.

Use of Epivolve phage display to generate a monoclonal antibody with opsonic activity directed against a subdominant epitope on extracellular loop 4 of Treponema pallidum BamA (TP0326).

Introduction: Syphilis, a sexually transmitted infection caused by the spirochete Treponema pallidum (Tp), is resurging globally. Tp's repertoire of outer membrane proteins (OMPs) includes BamA (ß-barrel assembly machinery subunit A/TP0326), a bipartite protein consisting of a 16-stranded ß-barrel with nine extracellular loops (ECLs) and five periplasmic POTRA (polypeptide transport-associated) domains. BamA ECL4 antisera promotes internalization of Tp by rabbit peritoneal macrophages. Methods: Three overlapping BamA ECL4 peptides and a two-stage, phage display strategy, termed "Epivolve" (for epitope evolution) were employed to generate single-chain variable fragments (scFvs). Additionally, antisera generated by immunizing mice and rabbits with BamA ECL4 displayed by a Pyrococcus furiosus thioredoxin scaffold (PfTrxBamA/ECL4). MAbs and antisera reactivities were evaluated by immunoblotting and ELISA. A comparison of murine and rabbit opsonophagocytosis assays was conducted to evaluate the functional ability of the Abs (e.g., opsonization) and validate the mouse assay. Sera from Tp-infected mice (MSS) and rabbits (IRS) were evaluated for ECL4-specific Abs using PfTrxBamA/ECL4 and overlapping ECL4 peptides in immunoblotting and ELISA assays. Results: Each of the five mAbs demonstrated reactivity by immunoblotting and ELISA to nanogram amounts of PfTrxBamA/ECL4. One mAb, containing a unique amino acid sequence in both the light and heavy chains, showed activity in the murine opsonophagocytosis assay. Mice and rabbits hyperimmunized with PfTrxBamA/ECL4 produced opsonic antisera that strongly recognized the ECL presented in a heterologous scaffold and overlapping ECL4 peptides, including S2. In contrast, Abs generated during Tp infection of mice and rabbits poorly recognized the peptides, indicating that S2 contains a subdominant epitope. Discussion: Epivolve produced mAbs target subdominant opsonic epitopes in BamA ECL4, a top syphilis vaccine candidate. The murine opsonophagocytosis assay can serve as an alternative model to investigate the opsonic potential of vaccinogens. Detailed characterization of BamA ECL4-specific Abs provided a means to dissect Ab responses elicited by Tp infection.

Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols.

To develop reproducible results, it is critical that all reagents used in an experiment be validated in an alternative or independent method. We present two such independent methods for determining the specificity of antibodies: (1) "MILKSHAKE," which can be used to validate the liability and specificity of antibodies directed against post-translationally-modified epitopes, and (2) "Sundae," which is a more complete alanine-like scanning method that can be used to better understand the binding and bioactivity of specific residues of a protein. We apply both of these methods to the interaction between an antibody and its antigen.

Epivolve: A Protocol for Site-Directed Antibodies.

Researchers can often successfully generate antibodies to predicted epitopes. Especially when the epitopes are on the surface of a protein or in a hydrophilic loop. But it is difficult to direct recombinant antibodies to bind either to- or near a specific amino acid on a protein or peptide. We have developed a unique immune-targeting strategy, that we call "Epivolve," that enables us to make site-specific antibodies (Abs). Epivolve technology leverages a highly immunogenic modified amino acid that acts as a "pseudo-hapten" immuno-target and takes advantage of Ab affinity maturation technologies to make high-affinity site-specific antibodies. Epivolve functions by the evolution of an Ab paratope to either synonymous or especially non-synonymous amino acid (aa) binding. Here we describe the use of Epivolve technology in phage display and the protocols for developing site-specific antibodies.

Derivation of splice junction-specific antibodies using a unique hapten targeting strategy and directed evolution.

Alternative splicing of RNA occurs frequently in eukaryotic cells and can result in multiple protein isoforms that are nearly identical in amino acid sequence, but have unique biological roles. Moreover, the relative abundance of these unique isoforms can be correlative with diseased states and potentially used as biomarkers or therapeutic targets. However, due to high sequence similarities among isoforms, current proteomic methods are incapable of differentiating native protein isoforms derived from most alternative splicing events. Herein, a strategy employing a nonsynonymous, non-native amino acid (nnAA) pseudo-hapten (i.e. an amino acid or amino acid derivative that is different from the native amino acid at a particular position) as a targeting epitope in splice junction-spanning peptides was successful in directed antibody derivation. After isolating nnAA-specific antibodies, directed evolution reduced the antibody's binding dependence on the nnAA pseudo-hapten and improved binding to the native splice junction epitope. The resulting antibodies demonstrated codependent binding affinity to each exon of the splice junction and thus are splice junction- and isoform-specific. Furthermore, epitope scanning demonstrated that positioning of the nnAA pseudo-hapten within a peptide antigen can be exploited to predetermine the isolated antibody's specificity at, or near, amino acid resolution. Thus, this nnAA targeting strategy has the potential to robustly derive splice junction- and site-specific antibodies that can be used in a wide variety of research endeavors to unambiguously differentiate native protein isoforms.

Rational library design by functional CDR resampling.

Successful antibody discovery relies on diversified libraries, where two aspects are implied, namely the absolute number of unique clones and the percentage of functional clones. Instead of pursuing the absolute quantity thresholded by current display technology, we have sought to maximize the effective diversity by improving functional clone percentage. With the combined effort of bioinformatics, structural biology, molecular immunology and phage display technology, we devised a bioinformatic pipeline to construct and validate libraries via combinatorial assembly of sequences from a database of experimentally validated antibodies. Furthermore, we showed that the libraries constructed as such yielded a significantly increased success rate against different antigen types and generated over 20-fold more unique hits per targets compared with libraries based on traditional degenerate nucleotide methods. Our study indicated that predefined CDR sequences with optimized CDR-framework compatibility could be a productive direction of functional library construction for in vitro antibody development.

Platform for high-throughput antibody selection using synthetically-designed antibody libraries.

Synthetic humanized antibody libraries are frequently generated by random incorporation of changes at multiple positions in the antibody hypervariable regions. Although these libraries have very large theoretical diversities (>10(20)), the practical diversity that can be achieved by transformation of Escherichia coli is limited to about 10(10). To constrain the practical diversity to sequences that more closely mimic the diversity of natural human antibodies, we generated a scFv phage library using entirely pre-defined complementarity determining regions (CDR). We have used this library to select for novel antibodies against four human protein targets and demonstrate that identification of enriched sequences at each of the six CDRs in early selection rounds can be used to reconstruct a consensus antibody with selectivity for the target.

Generating Recombinant Antibodies to Membrane Proteins through Phage Display.

One of the most important classes of proteins in terms of drug targets is cell surface membrane proteins, and yet it is a challenging set of proteins for generating high-quality affinity reagents. In this review, we focus on the use of phage libraries, which display antibody fragments, for generating recombinant antibodies to membrane proteins. Such affinity reagents generally have high specificity and affinity for their targets. They have been used for cell staining, for promoting protein crystallization to solve three-dimensional structures, for diagnostics, and for treating diseases as therapeutics. We cover publications on this topic from the past 10 years, with a focus on the various formats of membrane proteins for affinity selection and the diverse affinity selection strategies used. Lastly, we discuss the challenges faced in this field and provide possible directions for future efforts.

In vivo elimination of parental clones in general and site-directed mutagenesis.

The Eco29k I restriction endonuclease is a Sac II isoschizomer that recognizes the sequence 5'-CCGCGG-3' and is encoded, along with the Eco29k I methylase, in the Escherichia coli strain 29k. We have expressed the Eco29k I restriction-methylation system (RM2) in E. coli strain TG1 to produce the strain AXE688. We have developed a directed molecular evolution (DME) mutagenesis method that uses Eco29k I to restrict incoming parental DNA in transformed cells. Using our DME method, we have demonstrated that our AXE688 strain results in mutated directed molecular evolution libraries with diversity greater than 10(7) from a single transformation and with greater than 90% recombinant clones.

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes.

Use of micro-emulsion technology for the directed evolution of antibodies.

Affinity reagents, such as antibodies, are needed to study protein expression patterns, sub-cellular localization, and post-translational modifications in complex mixtures and tissues. Phage Emulsion, Secretion, and Capture (ESCape) is a novel micro-emulsion technology that utilizes water-in-oil (W/O) emulsions for the identification and isolation of cells secreting phage particles that display desirable antibodies. Using this method, a large library of antibody-displaying phage will bind to beads in individual compartments. Rather than using biopanning on a large mixed population, phage micro-emulsion technology allows us to individually query clonal populations of amplified phage against the antigen. The use of emulsions to generate microdroplets has the promise of accelerating phage selection experiments by permitting fine discrimination of kinetic parameters for binding to targets. In this study, we demonstrate the ability of phage micro-emulsion technology to distinguish two scFvs with a 300-fold difference in binding affinities (100nM and 300pM, respectively). In addition, we describe the application of phage micro-emulsion technology for the selection of scFvs that are resistant to elevated temperatures.

Phage ESCape: an emulsion-based approach for the selection of recombinant phage display antibodies.

Antibody phage display technology is a well established method for selecting specific antibodies against desired targets. Although phage display is the most widely used method of generating synthetic antibodies, it is laborious to perform multiple selections with different antigens simultaneously using conventional manual methods. We have developed a novel approach to the identification and isolation of cells secreting phage encoding desirable antibodies that utilizes compartmentalization and Fluorescence Activated Cell Sorting (FACS). This method, termed Phage Emulsion, Secretion, and Capture (ESCape), allows us to individually query each phage against the antigen. Here, we demonstrate the ability of Phage ESCape to identify novel scFvs against a phosphopeptide epitope of the Her2 kinase from a phage display library containing approximately 10(8) synthetically diversified antibodies. Clones were analyzed by monoclonal phage ELISA against the Her2 phosphopeptide, and positive binders were identified as those showing a signal greater than 3-fold higher than the background signal against an irrelevant antigen. We isolated antibodies recognizing the phosphopeptide in a single round of selection by Phage ESCape, but the strength and specificity of the hits was substantially improved when the library was pre-enriched by a single round of biopanning. By minimizing the selection rounds required for phage display and using a FACS machine as a 'colony picker' equivalent, Phage ESCape has the potential to dramatically increase the throughput of in vitro screening methods.

Microdroplet-based PCR enrichment for large-scale targeted sequencing.

Targeted enrichment of specific loci of the human genome is a promising approach to enable sequencing-based studies of genetic variation in large populations. Here we describe an enrichment approach based on microdroplet PCR, which enables 1.5 million amplifications in parallel. We sequenced six samples enriched by microdroplet or traditional singleplex PCR using primers targeting 435 exons of 47 genes. Both methods generated similarly high-quality data: 84% of the uniquely mapping reads fell within the targeted sequences; coverage was uniform across approximately 90% of targeted bases; sequence variants were called with >99% accuracy; and reproducibility between samples was high (r(2) = 0.9). We scaled the microdroplet PCR to 3,976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample with both Illumina GAII and Roche 454, and obtained data with equally high specificity and sensitivity. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel enrichment of specific subsets of the human genome for targeted sequencing.

Molecular biomarkers in drug development.

Arguably, the most immediately promising reverberation of the genomics era has been the application of biomarkers to drug development. The promise of applying biomarkers to early drug development is that they might aid in preclinical and early clinical decisions such as dose ranging, definition of treatment regimen, or even a preview of efficacy. Later in the clinic, biomarkers could be used to facilitate patient stratification, selection and the description of surrogate endpoints. Information derived from biomarkers should result in a better understanding of preclinical and clinical data, which ultimately benefits patients and drug developers. If the promise of biomarkers is realized, they will become a routine component of drug development and companions to newly discovered therapies.