Antibody Discovery Process Research Articles

High titer expression of antibodies using linear expression cassettes for early-stage functional screening.

Antibody discovery processes are continually advancing, with an ever-increasing number of potential binding sequences being identified out of in vivo, in vitro, and in silico sources. In this work we describe a rapid system for high yield recombinant antibody (IgG and Fab) expression using Gibson assembled linear DNA fragments (GLFs). The purified recombinant antibody yields from 1ml expression for this process are approximately five to ten-fold higher than previous methods, largely due to novel usage of protecting flanking sequences on the 5' and 3' ends of the GLF. This method is adaptable for small scale (1ml) expression and purification for rapid evaluation of binding and activity, in addition to larger scales (30ml) for more sensitive assays requiring milligram quantities of antibody purified over two columns (Protein A and size exclusion chromatography). When compared to plasmid-based expression, these methods provide nearly equivalent yield of high-quality material across multiple applications, allowing for reduced costs and turnaround times to enhance the antibody discovery process.

AB-Gen: Antibody Library Design with Generative Pre-trained Transformer and Deep Reinforcement Learning

Antibody leads must fulfill multiple desirable properties to be clinical candidates. Primarily due to the low throughput in the experimental procedure, the need for such multi-property optimization causes the bottleneck in preclinical antibody discovery and development, because addressing one issue usually causes another. We developed a reinforcement learning (RL) method, named AB-Gen, for antibody library design using a generative pre-trained transformer (GPT) as the policy network of the RL agent. We showed that this model can learn the antibody space of heavy chain complementarity determining region 3 (CDRH3) and generate sequences with similar property distributions. Besides, when using human epidermal growth factor receptor-2 (HER2) as the target, the agent model of AB-Gen was able to generate novel CDRH3 sequences that fulfill multi-property constraints. Totally, 509 generated sequences were able to pass all property filters, and three highly conserved residues were identified. The importance of these residues was further demonstrated by molecular dynamics simulations, consolidating that the agent model was capable of grasping important information in this complex optimization task. Overall, the AB-Gen method is able to design novel antibody sequences with an improved success rate than the traditional propose-then-filter approach. It has the potential to be used in practical antibody design, thus empowering the antibody discovery and development process. The source code of AB-Gen is freely available at Zenodo (https://doi.org/10.5281/zenodo.7657016) and BioCode (https://ngdc.cncb.ac.cn/biocode/tools/BT007341).

Abstract 5323: Accelerating the drug discovery process with an automated high-throughput protein production and characterization platform for AI-driven antibody development of immunotherapy

Abstract The demonstrated effectiveness and vast potential of large molecule therapeutics have driven biopharmaceutical companies to change the ways by which they discover and develop novel biologic therapies. While the conventional antibody discovery process via animal immunization and in vivo hypermutation can generate sufficient hits for functional screening, this approach can only cover a limited subset of sequence diversity. In silico, artificial intelligence (AI) driven methods, by contrast, have the potential to cover 2-3 orders of magnitude more sequence diversity compared to conventional methods and more rapidly produce a drug candidate with a superior safety and efficacy profile. However, this approach requires wet lab validation of predicted sequences as well as targeted data generation to improve AI-predicted rankings. Traditional wet lab methods for protein generation and characterization are expensive, time consuming, and prone to human error. These limitations restrict the overall potential benefit of the AI-driven antibody discovery process. To accelerate this process, which consists of a complex series of cycles spanning different functional teams, a substantial increase in the throughput of the end-to-end production and characterization workflow is established in-house such that 104 antibodies with property data can be generated and analyzed within a short period of time. BioMap has implemented a highly integrated and automated protein expression, purification, and characterization platform linked with a unified informatics system to eliminate as much manual operation as possible. This high-throughput robotic platform interweaves multiple procedures including plasmid preparation, cell dispensing, mammalian transfection, protein purification, and characterization, and it is coupled to our in-house informatics system and database. Empowered by state-of-the-art liquid handling system and well-established experimental protocols, the integrated facility is capable of the delivery of 103 protein samples from plasmids in ten days within a single batch. Benefiting from our robust transient expression platform, the production scale ranges from 0.5 to 30 mL with an average antibody yield of 300 mg/L. The high-throughput workflow brings a challenge to track the provenance of each protein and manage data flow across different stations. To address this bottleneck, a well-defined informatics platform and database have been developed to provide sample registration, interface with lab instruments, experimental process tracking, and automated data recording and analysis. The implementation of this system provides a massive amount of high-quality data available for AI training and validation with a very short turnover time and enables AI-driven development of next-generation biologics. Citation Format: Zhehao Xiong, Wei Jiang, Lijun Xia, Jianhua Huang, Cheng-chi Chao. Accelerating the drug discovery process with an automated high-throughput protein production and characterization platform for AI-driven antibody development of immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5323.

Antibody Isolation from Human Synthetic Libraries of Single-Chain Antibodies and Analysis Using NGS.

Antibody libraries came into existence 30years ago when the accumulating sequence data of immunoglobulin genes and the advent of PCR technology made it possible to clone antibody gene repertoires. Phage display (most common) and additional display and screening technologies were applied to pan out desired binding specificities from antibody libraries. As other antibody discovery tools, phage display is not an off-the-shelf technology and not offered as a kit but rather requires experience and expertise for making it indeed very useful.Next-generation sequencing (NGS) coupled with bioinformatics is a powerful tool for analyzing large amount of DNA sequence output of the panning. Here, we demonstrate how NGS analysis of phage biopanning (phage-Seq) of complex antibody libraries can facilitate the antibody discovery process and provide insights regarding the biopanning process (see Fig. 1).

A homogeneous bioluminescent immunoassay for parallel characterization of binding between a panel of antibodies and a family of Fcγ receptors

Fc engineering efforts are increasingly being employed to modulate interaction of antibodies with variety of Fc receptors in an effort to improve the efficacy and safety of the therapeutic antibodies. Among the various Fc receptors, Fc gamma receptors (FcγRs) present on variety of immune cells are especially relevant since they can activate multiple effector functions including antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). Depending on the desired mechanism of action (MOA) of the antibody, interactions between Fc domain of the antibody and FcγR (denoted as Fc/FcγR) may need to be enhanced or abolished. Therefore, during the antibody discovery process, biochemical methods are routinely used to measure the affinities of Fc/FcγR interactions. To enable such screening, we developed a plate based, simple to use, homogeneous immunoassays for six FcγRs by leveraging a luminescent protein complementation technology (NanoBiT). An added advantage of the NanoBiT immunoassays is their solution-based format, which minimizes well known surface related artifacts associated with traditional biosensor platforms (e.g., surface plasmon resonance and biolayer interferometry). With NanoBiT FcγRs assays, we demonstrate that assays are specific, report IgG subclass specific affinities and detect modulation in Fc/FcγR interactions in response to the changes in the Fc domain. We subsequently screen a panel of therapeutic antibodies including seven monoclonal antibodies (mAbs) and four polyclonal intravenous immunoglobulin (IVIg) products and highlight the advantages of parallel screening method for developing new antibody therapies.

Abstract 6055: Discovery of epitope-selective anti-CD25 targeting therapeutic antibodies for effective Treg cell depletion in cancer using a novel AI/ML based platform

Abstract Therapeutic antibodies have contributed significantly to better clinical outcomes across multiple tumor types. However, there is still unmet clinical need in cancers with an immunosuppressive and stroma rich tumor microenvironment (TME). One key limitation in early antibody discovery is immunodominance - an evolutionary bias toward specific epitopes that steer antibody selection away from potentially high-efficacy epitopes. Our integrated computational and laboratory approach to antibody discovery enabled us to build focused epitope embodiments of structural epitopes. These epitope embodiments, called Meso-scale Engineered Molecules (MEMs), are used in antibody selection to steer hits toward intended epitopes. CD25 (IL2Rα) is highly expressed on regulatory T-cells (Tregs) that drive immunosuppression in the TME. Depletion of Tregs can restore anti-tumor T effector (Teff) function in the TME, by increasing the Teff/Treg ratio, especially when combined with checkpoint-inhibitors. Using our epitope-selective antibody discovery platform, we built MEMs that embody eight CD25 structural epitopes outside the CD25:IL-2 interface. Through in vitro selection, several high-affinity (median KD = 25 nM) anti-CD25 antibodies from each of the eight intended CD25 epitopes were identified and confirmed to be on epitope by cross-blocking assays and in-epitope alanine mutations. Several conventional- and epitope-selective anti-CD25 clones were converted to human IgG1 and tested in in vitro assays. As a result, these antibodies bound specifically to CD25+ cells, preserve IL-2 signaling via pSTAT5 assays and elicited potent ADCC activity using human effector cells, superior to benchmark antibodies. These studies show that our integrated computational and laboratory platform improves the antibody discovery process, producing clones with biological function and improved therapeutic efficacy. Citation Format: Phung Gip, Bing Li, Angeles Estelles, Hanako Daino-Laizure, Oleg Gurinovich, Piotr Zalicki, Kevin Hauser, Alex Taguchi, Christian Barrett, Andrew Morin, Jordan Willis, Mohan Srinivasan, Isaac Bright, Matt Greving. Discovery of epitope-selective anti-CD25 targeting therapeutic antibodies for effective Treg cell depletion in cancer using a novel AI/ML based platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6055.

Abstract 5051: RenMab Mouse: A leading platform for fully human antibody generation

Abstract With the development of immune-oncology, therapeutic antibodies have been proven to be extraordinarily effective for cancer treatment. Conventional human antibody discovery process can be divided into steps including target selection, target validation, screening preparation, hits generation, leads selection, lead optimization, and clinical candidate selection. To accelerate antibody development process, Biocytogen has developed RenMab Mouse, a fully human antibody mouse whose entire variable regions were replaced by human Ig heavy chain and κ light chain through Biocytogen's unique Mb-scale chromosome engineering technology. RenMab Mouse provides a remarkably efficient therapeutic antibody discovery platform for fully human antibody generation, characterization, and rapid in vivo efficacy screening. We have characterized RenMab Mouse with the following features. 1. The entire mouse variable regions were replaced with complete human genome DNA in situ for complete human & mouse regulatory elements. The gene regulation of RenMab mouse is highly consistent with that of human. 2. Mouse constant region remains to ensure normal immune cell population, development and maturation. Immune system of RenMab Mouse has been proven to be almost identical to that of wild type mice. RenMab Mouse showed normal antibody immune responses to antigens. 3. RenMab Mouse generates a highly diverse repertoire of fully human antibody variable regions through V(D)J recombination. This capability can lead to improved hit rates in antibody drug discovery. In conclusion, RenMab Mouse, a fully human antibody mouse developed by Biocytogen's chromosome engineering technology, has been proven to exhibit normal immune cell profile and rapid immune response. Combined with existing inventory of single, double or triple target humanized mouse models and a world-leading proprietary gene editing platform, Biocytogen is dedicated to providing a one-stop solution for antibody discovery and development from target validation to IND application. Citation Format: Gary Ng, Huizhen Zhao, Yabo Zhang, Lili Liu, Hui Lu, Shuwen Huang, Yuelei Shen. RenMab Mouse: A leading platform for fully human antibody generation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5051.

Engineering Antibodies on the Surface of CHO Cells.

While antibody libraries are traditionally screened in phage, bacterial, or yeast display formats, they are produced in large scale for pharmaceutical and commercial use in mammalian cell lines. The simpler organisms used for screening have significantly different folding and glycosylation machinery than mammalian cells; consequently, clones resulting from these libraries may require further optimization for mammalian cell expression. To streamline the antibody discovery process, we developed a Chinese hamster ovary (CHO) cell-based selection system that allows for long-term display of antibody Fab fragments. This system is facilitated by a semi-stable Epi-CHO episomal platform to maintain antibody expression for up to 2months and is compatible with standard PCR-based mutagenesis strategies. This protocol describes the simple and accessible use of CHO display coupled with flow cytometry to enrich for antibody variants with increased ligand-binding affinity from large libraries of ~106 variants, using HER2-binding antibodies as an example.

Expediting Antibody Discovery with a Cell and Bead Multiplexed Competition Assay

With advances in molecular engineering and humanization, monoclonal antibodies are one of the fastest-growing classes of biopharmaceuticals. During antibody discovery, antibody from hybridoma or primary B-cell supernatants is screened for the desired binding characteristics, and secondary screens measure antibody function and concentration, identify immunoglobulin G (IgG) isotype, and assess cell health. In order to expedite the antibody discovery process, we developed a high-throughput, multiplexed cell and bead-based competition assay that identifies and quantitates mouse IgG isotypes and assesses cell health. No differences in assay performance were observed between single and multiplex formats. The linear range of the assay was from 0.5 to 50 µg/mL, and washing was not required, decreasing assay time and variability. Slight modifications to the protocol allowed quantification of dilute antibody supernatants (0.1–5 µg/mL). Using hybridoma cultures, we showed that cell viability measurements in the assay did not interfere with the bead-based IgG measurements. The assay described here is a simple mix-and-read, no-dilution screen that can reduce the time to antibody cloning and production. The high-content data can differentiate monoclonal and polyclonal wells, determine IgG quantity for downstream functional assays, provide isotype information, and monitor cell proliferation and viability.

Barcoded sequencing workflow for high throughput digitization of hybridoma antibody variable domain sequences

Since the invention of Hybridoma technology by Milstein and Köhler in 1975, its application has greatly advanced the antibody discovery process. The technology enables both functional screening and long-term archival of the immortalized monoclonal antibody producing B cells. Despite the dependable cryopreservation technology for hybridoma cells, practicality of long-term storage has been outpaced by recent progress in robotics and automations, which enables routine identification of thousands of antigen specific hybridoma clones. Such throughput increase imposes two nascent challenges in the antibody discovery process, namely limited cryopreservation storage space and limited throughput in conventional antibody sequencing. We herein provide a barcoded sequencing workflow that utilizes next generation sequencing to expand the conventional sequencing capacity. Accompanied with the bioinformatics tools we describe, the barcoded sequencing workflow robustly reports unambiguous antibody sequences as confirmed with Sanger sequencing controls. In complement with the commonly accessible recombinant DNA technology, the barcoded sequencing workflow allows for high throughput digitization of the antibody sequences and provides an effective solution to the limitations imposed by physical storage and sequencing capacity.

Antibody Screening Using High-Throughput Flow Cytometry.

Because molecular targets addressable with antibody therapeutics are present on the surface of cells or in circulation, they are ideal for screening by cell- or bead-based assays using flow cytometry, a powerful, high-content analysis technique for cells, beads, and other particles in suspension. The ability to analyze thousands of cells per second, combined with multiplexing capabilities, has made this technology indispensable for laboratories performing antibody development work. Advances in this field, particularly in the areas of plate-based sampling and high-throughput flow cytometry, are enabling the use of this technology earlier in the antibody development and discovery process.

Monitoring Phage Biopanning by Next-Generation Sequencing.

Phage display has enabled the rapid isolation of antigen-specific antibodies from combinatorial libraries of the variable heavy chain gene (VH) and variable light chain gene (VL). The method is based on genetic engineering of bacteriophages and repeated rounds of antigen-guided selection by phage biopanning.Next-Generation Sequencing (NGS) coupled with bioinformatics are powerful tools for analyzing the large number of DNA sequences present in an immune library.Here, we describe a method that demonstrates how NGS analysis enhances phage biopanning of complex antibody libraries as well as facilitates the antibody discovery process.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.

Antibody engineering and phage display technology

Abstract Here we provide our pipeline for antibody discovery process. Starting from de novo isolation from custom made phage display libraries to expression and downstream applications; it allows us to obtain recombinant antibodies with desired properties to vast set of antigens. We also developed the CYB5-fusion system for periplasmic expression of multimeric proteins with the possibility of process monitoring. The combination of redox dependent absorbance spectrum of red-colored cytochrome b5 with its high value molar extinction coefficient allows us to monitor antibody fusion proteins localization, redox state and quantify them in reliable way in turbid solutions. Moreover, it was revealed that due to outstanding stability and solubility, cytochrome b5 significantly enhances expression level of antibody fragments in Escherichia coli periplasm.

Promiscuity and Polyreactivity of Antibodies and their Binding Modes during B-Cell Differentiation

Many diversification mechanisms occurr during central tolerance leading to a wide array of antibodies (Abs) with a range of binding specificities, some of which lead to polyreactivity. The repertoire of preB cells (before central tolerance), immature B cells (during central tolerance) and naive B cells in the peripheral blood (after central tolerance) has been interrogated by high throughput sequencing of heavy and light chain Ig genes. It has been shown that older people display a less diverse repertoire of antibody-producing cells, and that this loss of diversity is associated with poor health. Within a population of responding cells there are favoured characteristics of a the Ab gene CDRH3 loop that is crucial in binding the foreign antigen. We hypothesise that favoured physico-chemical CDRH3 characteristics make a structure capable of binding to multiple foreign antigens (polyspecific).The ability to predict this promiscuous behaviour with respect to binding specificity would greatly improve the efficiency of the therapeutic antibody discovery process. From these data we want to extract the molecular determinants playing a role in polyreactivity by exploiting structural and dynamical information. We use statistical analyses to classify CDRH3 physico-chemical properties and Molecular Dynamics (MD) simulations at the atomistic and/or coarse grained levels to characterise flexibility requirements for binding multiplicity.Martin V, Wu YC, Kipling D, Dunn-Walters DK. Ann N Y Acad Sci. 2015 http://dx.doi.org/10.1111/nyas.12823.Fornili A, Pandini A, Lu HC, Fraternali F. J Chem Theory Comput. 2013 9:5127-5147.Marcatili P, et al. J Immunol. 2013 190:5771-8.

The evolution and development of the antibody repertoire.

The evolution and development of the antibody repertoire.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Deep sequencing of phage display libraries to support antibody discovery

The use of Next Generation Sequencing (NGS) for the analysis of antibody sequences both in phage display libraries and during in vitro selection processes has become increasingly popular in the last few years. Here, our methods developed for DNA preparation, sequencing and data analysis are presented. A key parameter has also been to develop new software designed for high throughput antibody sequence analysis that is used in combination with publicly available tools. As an example of our methods, we provide data from the extensive analysis of five scFv libraries generated using different heavy chain CDR3 diversification strategies. The results not only confirm that the library designs were correct but also reveal differences in quality not easily identified by standard DNA sequencing approaches. The very large number of reads permits extensive sequence coverage after the selection process. Furthermore, as samples can be multiplexed, costs decrease and more information is gained per NGS run. Using examples of results obtained post phage display selections against two antigens, frequency and clustering analysis identified novel antibody fragments that were then shown to be specific for the target antigen. In summary, the methods described here demonstrate how NGS analysis enhances quality control of complex antibody libraries as well as facilitates the antibody discovery process.

The Discovery, Engineering and Characterisation of a Highly Potent Anti-Human IL-13 Fab Fragment Designed for Administration by Inhalation

We describe the discovery, engineering and characterisation of a highly potent anti-human interleukin (IL)-13 Fab fragment designed for administration by inhalation. The lead candidate molecule was generated via a novel antibody discovery process, and the selected IgG variable region genes were successfully humanised and reformatted as a human IgG γ1 Fab fragment. Evaluation of the biophysical properties of a selection of humanised Fab fragments in a number of assays allowed us to select the molecule with the optimal stability profile. The resulting lead candidate, CA652.g2 Fab, was shown to have comparable activity to the parental IgG molecule in a range of in vitro assays and was highly stable. Following nebulisation using a mesh nebuliser, CA652.g2 Fab retained full binding affinity, functional neutralisation potency and structural integrity. Epitope mapping using solution nuclear magnetic resonance confirmed that the antibody bound to the region of human IL-13 implicated in the interaction with IL-13Rα1 and IL-13Rα2. The work described here resulted in the discovery and design of CA652.g2 human γ1 Fab, a highly stable and potent anti-IL-13 molecule suitable for delivery via inhalation.

Natural and man-made V-gene repertoires for antibody discovery

Antibodies are the fastest-growing segment of the biologics market. The success of antibody-based drugs resides in their exquisite specificity, high potency, stability, solubility, safety, and relatively inexpensive manufacturing process in comparison with other biologics. We outline here the structural studies and fundamental principles that define how antibodies interact with diverse targets. We also describe the antibody repertoires and affinity maturation mechanisms of humans, mice, and chickens, plus the use of novel single-domain antibodies in camelids and sharks. These species all utilize diverse evolutionary solutions to generate specific and high affinity antibodies and illustrate the plasticity of natural antibody repertoires. In addition, we discuss the multiple variations of man-made antibody repertoires designed and validated in the last two decades, which have served as tools to explore how the size, diversity, and composition of a repertoire impact the antibody discovery process.