Antibody Discovery, Selection & Screening - Antibody Therapeutics eBook Series
This ebook presents a collection of industry insights, exploring the latest approaches and technologies for antibody drug discovery.
Antibody Discovery, Selection & Screening
Antibody Discovery, Selection & Screening
INTRODUCTION
What are the best steps in validating antibodies for use in therapeutics? How can researchers find the right antibody for new targets such as GPCRs? What is needed for fast and efficient development of anti-COVID-19 antibodies?
As common challenges in therapeutic antibody discovery are experienced across the field, companies are finding ways to innovate using new methods and technologies such as AI and machine learning.
The following pages present a collection of industry insights, exploring the latest approaches and technologies for therapeutic antibody discovery, alongside expert advice on tackling the key challenges along the way.
Jump to any article using the contents on the following page, or at any time using the Contents menu in the top left. There you can also download this eBook as a PDF.
Fully human antibody are the trend of therapeutic antibody. As of February 2021, the FDA has formally approved a cumulative total of 100 antibody drugs, of which 36 have been fully human antibody drugs.
GenScript ProBio provides a rapid solution to enable our clients to get fully human antibody sequences as fast as 1 month by combing Berkeley Lights BeaconTM single B cell screening platform with transgenic animals.
GenScript ProBio has established partnership with 3 transgenic animal platforms which are OmniAb® from Ligand in US, Alloy ATX-GKTM Mouse in US and CAMouseTM from CAMAB in China. We have been authorized to use their animals to provide antibody discovery service.
Moreover, GenScript ProBio offers a limited time 25% off discount for single B cell screening projects using transgenic animals.
Rapid Discovery of Antibodies to Integral Membrane Proteins: A CXCR5 Seven Transmembrane Receptor Case Study
Rapid Discovery of Antibodies to Integral Membrane Proteins: A CXCR5 Seven Transmembrane Receptor Case Study
At the Antibody Engineering & Therapeutics conference in December 2020, Dr Glanville, CEO of Distributed Bio and Centivax, presented his exciting work on anti-GPCR therapeutics. GPCRs are traditionally tough therapeutic targets because they are located in membranes, making targeting and modifications challenging.
Dr Glanville explained that the work of Distributed Bio is ideal for examining the potential for GPCRs as therapeutics. The company has in recent years created technology to generate a larger repertoire of human antibodies than previously possible, using natural CDRs, allowing bigger fitness than synthetic antibodies because they have already passed the natural selection process for antibodies in the human body.
In 2018, Distributed Bio released SuperHuman 2.0 computationally optimized antibody library. Because the library is more diverse than previous antibody libraries, this allowed the group to examine GPCRs as potential therapeutic targets by having enough antibodies to explore. Dr Glanville then explained that extraction of GPCRs and selection is very challenging because GPCRs are membrane-bound and will be located in the lipid fraction of an assay. Distributed Bio focused on CXCR5 as a target of interest.
Distributed Bio’s strategy for targeting GPCRs consisted of parallel panning of the same target against the same library, followed by NGS in an automated method. The company then looked at the individual clones among NGS-identified anti-CXCR5 specific clones. The anti-CXCR5 antibodies were then confirmed by FACS.
The anti-GPCR IgGs were tested for their specificity and thermostability through functional screens. The screens were successful, selecting three potential binders for CXCR5 GPCRs.
These IgGs were optimized through Tumbler Optimization Platform technology combining computational and wet-lab approaches to infer optimal theoretical sequence.
The Tumbler platform helped to improve the functional activity of the lead clone 70-fold, leading to affinity-matured fully human anti-CXCR5 antibody, confirmed with in vitro assays.
Switching gears, Dr Glanville presented Distributed Bio’s work on another anti-GPCR antibody provided by the industry. He demonstrated that the Tumbler platform can be used on several anti-GPCRs antibodies.
Dr Glanville concluded his talk demonstrated the versatility of SuperHuman 2.0 and Tumbler Platform approaches in the design of multi-specific, bi-specific antibodies, cell therapies, CAR-T therapies and IgG design.
GenScript ProBio’s ProSpeed™ accelerate anti-COVID-19 neutralizing antibody leads discovery with sub-nM IC50
Brought to you by Genscript ProBio
GenScript ProBio’s ProSpeed™ accelerates anti-COVID-19 neutralizing antibody leads discovery with sub-nM IC50
In December 2019, a third pathogenic human Coronaviruses, named 2019 novel coronavirus (2019-nCoV/SARS-CoV-2/COVID-19), was found in Wuhan, China. Until March, 2021 after it was identified, the total confirmed global cases have exceeded 120 million with more than 2.6 million deaths. The number of COVID-19 cases is still rising each day with an unprecedented speed and scale.
In the beginning of COVID-19 pandemic, vaccines as well as SARS-CoV-2 virus neutralizing antibodies which could be applied in treatment, prophylaxis, and detection are two major approaches to prevent virus transmission. GenScript ProBio has superior platforms in antibodies discovery, including hybridomas, phage displayed antibody library and single B cell screening platform. Single B cell screening and phage displayed libraries could screen antibody leads in short time and thus provide the best choice in this urgent moment.
Single B cell screening platform, a micro-chamber based platform, can be used to isolate, confine, and interrogate each individual B cells to maximize the antibody repertoires that we can access. GenScript ProBio has built our own single B cell platform, ProSpeed™ by using Beacon™, a micro-chamber based platform. To speed up the antibody discovery, each wild type mouse was received 7 doses of recombinant the receptor binding domain (RBD) of SARS-CoV-2 spike protein (S) in a total of 14 day duration.
After immunization, mice lymph nodes were harvested and processed to CD138+ cells enrichment. CD138+ antibody secreting cells (ASCs) were imported on chip for single B cell screening on Beacon™ (Berkeley Lights, CA) to identify positive single B cell clones that secrete RBD binding antibodies and block RBD binding ACE2 (Angiotensin-converting enzyme 2) overexpressing stable cell line at the same time. 93 RBD binders and blocker were identified and 65 recombinant IgG were successfully sequenced and produced. 65 leads were narrowed down to 23 clones with more than 40% FACS blocking ability. Then, 23 antibody leads were examined the SARS-CoV-2 pseudovirus neutralization ability (Figure 1).
Among those 23 antibody leads, all of them showed better potency than virus ligand ACE2-Fc fusion protein. The IC50 of SARS-CoV2 pseudovirus neutralization antibodies ranges from 70.6 pM to 2.729 nM (Figure 2).
Figure 2. Neutralizing capacity of SARS-CoV-2 neutralizing antibody leads screened by Genscript Probio’s ProspeedTM
Figure 1. SARS-CoV-2 neutralizing antibodies screening by GenScript ProBio’s ProspeedTM
In summary, through single B cell platform (GenScript ProBio’s ProSpeed™) and Express immunization, it took only 8 weeks from immunization to functional recombinant antibody protein. Unlike hybridomas and phage displayed library which screen binder and then test functions as well as sequencing, GenScript ProBio’s ProSpeed™ screens antibody leads by function in the front.
Combined with high-throughput sequencing by next generation sequencing, antibody leads for protein production could be narrowed down according to sequence clustering. To accelerate therapeutics development, human antibody transgenic animal combined with GenScript ProBio’s ProSpeed™ is highly recommended.
GenScript ProBio is now the certified CRO of Ligand’s Omni-rodent, Alloy Therapeutic’s ATX mice, and CAMouse in China. GenScript ProBio provides the best antibody discovery platforms and make therapeutics development faster and more efficient.
Service Highlights of ProSpeedTM Platform
Expedited timeline
- Screening completed in 1 day
- As fast as 1 month to get functional sequences
Screen the most B cells before losing them
- Minimal diversity loss, forward functional screening
Intrinsic advantage for challenging targets
- High hit rate: multiple screening/assay method to select functional leads at earlier stage
- Low hit rate: limited B cell repertoire due to fusion loss
Human Monoclonal Antibodies for SARS-CoV-2
by David Orchard-Webb
Human Monoclonal Antibodies for SARS-CoV-2
At the virtual Antibody Engineering & Therapeutics conference in December 2020, James Crowe Jr., Director at Vanderbilt Vaccine Center, discussed single cell technologies for the isolation of human monoclonals, the molecular basis for SARS-CoV-2 neutralization, and antibody synergistic exploration. Peripheral blood mononuclear cells (PBMCs) from survivors of COVID-19 disease were the starting material for this work.
The project started from a DARPA pandemic prevention platform. The original aspirational goal was to go from an outbreak to a cure in about 60 days. The New England Journal of Medicine released a case report on January 19, 2020 of the first contemporary COVID-19 patient in the US. Seven days later in Nashville, Dr. Crowe Jr. had received the first sample from that individual.
Chinese scientists released sequences very early, allowing Jason McLellan of the University of Texas at Austin and others to determine the structure of the Spike protein. Building on that structural data, Dr. Crowe’s team made antigens expressed in mammalian cell lines. They typically used antigen-coated beads or sometimes only the soluble antigens for antibody binding screens. Recombinant humanized FLAG-ACE2 was used for receptor blocking assays. By iterations of this, they were able to find antigen-specific B cells.
They used two different technologies in parallel to maximize chances of success; one being the sequence based 10x Genomics single cell platform, the other based on cell sorting by microfluidics and labelling with antibodies, a kind of specialized FACS, called Beacon, made by Berkeley Lights, Inc. The resulting B cells were sequenced. Dr. Crowe detailed the Berkeley Lights Beacon approach in the talk. The antibody sequences were cloned and synthesized by Twist Biosciences, ready for transfection by the Crowe lab.
The Crowe lab developed a novel single cell surrogate virus neutralization assay using the Beacon device, memory B cells, and combinations of the RBD virus antigen and ACE2 for blocking. The lab also developed a rapid real-time virus neutralization assay using an xCELLigence impedance device from ACEA Biosciences (now part of Agilent). Cytopathic effect (CPE) in a cell monolayer is detected as a loss of impedance. A chimaeric vesicular stomatitis virus/ SARS-CoV-2 hybrid was used for this purpose. Neutralizing antibodies prevent this loss of monolayer integrity and maintain impedance at control levels.
Therefore, by these two different methods they could determine neutralizing antibodies. Working with Mike Diamond lab at WUSTL, they tested neutralization against authentic SARS-CoV-2 virus and some of those antibodies were neutralizing with IC50 values close to 10-15 ng/ml.
They worked with the Baric and Diamond labs to make several different mouse models. For example, an adenovirus transduced model where humanized ACE2 was introduced into a mouse. Using anti-ifnar antibodies, virus, and treatment antibodies, they could protect against weight loss, and histopathology with a monotherapy of some of their antibodies. Other models were explored that also gave positive ‘go’ signals.
The selected antibody clones were passed on to five different companies. At the time of the presentation, the furthest along was their partner, AstraZeneca. A phase I started in August with the AZD7442 product and then it was announced in October that they were rolling out a phase III. Go to clinicaltrials.gov to see the various formats of these trials. The timeline from discovery to production at scale is remarkably rapid, even though it did not quite hit the aspiration of 60 days.
Moving forward, the Crowe lab are participating in a program called AHEAD100, which aims to make antibodies for the 100 most likely causes of the future pandemics. Dr. Crowe believes that antibodies are a big part of the future of epidemic control and COVID-19 is the first real pandemic where antibodies were brought to bear. He concluded that the faster they get at making antibodies and the farther ahead they plan, the more likely antibodies will be useful very early in epidemics.
Find out more about the 2020 Antibody Engineering & Therapeutics virtual event in our full overview here.
TARGATT™ Unique Technology for Antibody Discovery and Screening
Brought to you by Applied Stemcell, Inc.
TARGATTTM: Site-Specific Gene Integration Technology
Introduction
Applied StemCell’s (ASC) proprietary TARGATT™ gene editing technology allows for an irreversible, site-specific gene insertion at a safe-harbor locus. Because the insertion occurs at a defined safe-harbor locus, the cell lines are stable and there is consistent expression across constructs. The system permits large fragment knockin up to 20 kb, and the technology functions in non-dividing cells. TARGATT™ enables a single copy insertion, and we have observed high integration efficiencies and medium to high levels of protein expression.
ASC has used TARGATT™ to engineer CHO and HEK293 master cell lines for bioproduction, to establish a mammalian cell-based library for antibody screening, and to construct a naïve IgG library system.
Figure 1: TARGATT™ gene insertion schematic. The technology allows site-specific, irreversible gene insertion at a safe-harbor locus.
TARGATT™ CHO Master Cell Line: For Bioproduction
ASC engineered a TARGATT™ Master CHO Cell Line with a landing pad at a safe-harbor locus that permits a single copy insertion and expression of any gene of interest. The stable cell line grants high efficiency for gene insertion. We have measured the integration efficiency of the TARGATT™ Master CHO Cell Line and compared it to a control, random integration, using GFP as a reporter. Before drug selection, we observed a gene insertion efficiency ranging from 13-18%. A gene insertion efficiency of over 97% was reported post-selection.
Figure 2: GFP, western blot & flow cytometry analysis on polyclonal pools.
Confirming Single-Copy Insertion
Figure 3: Schematic of the single-copy insertion experimental design along with florescence imaging and flow cytometry confirming single-copy insertion.
To confirm single-copy insertion in the TARGATT™ CHO Master Cell Line, we designed an experiment with two different donor plasmids, one with a green florescence protein and the other with a red florescence marker, mCherry. A 1:1 mix of the donor plasmids was transfected into the CHO Master Cell Line, and distinct green and red cells were visualized without any overlap. This suggests that integration does in fact occur as a single copy.
Single-Copy Insertion at a Safe-Harbor Locus
Figure 4: The H11 locus and the animals that have an orthologous sequence of the H11 safe-harbor locus.
The single-copy, irreversible insertion results in the H11 safe-harbor locus that is identified in an intergenic region between two highly expressed genes. ASC has identified orthologous sequences of H11 in various cell lines (e.g., human cells, HEK293 cells, iPS cells, and CHO cells) and animals (e.g., mice, pigs, and rats). Insertion at the H11 site leads to little or no phenotypic change. Also, integration does not affect any endogenous gene expression or function, and medium to high levels of transgene expression have been observed.
Safe-Harbor Locus Case Study
In this case study, a collaboration partner collected expression data for the production of 2 antibodies using our TARGATT™ CHO Master Cell Line. A single copy of the transgene for antibody 1 and 2 was inserted into the H11 locus in the master cell line, and the cells were grown in a 17-day fed-batch shake-flask. We detected consistent production of greater than 0.5 g/l in bulk enriched pools with no optimization. These cells have the potential to generate stable CHO libraries for research-scale production.
Figure 5: Case study results depicting the increase of antibody (1 and 2) expression over a 17-day period.
TARGATT™ Library Screen Master Cell Lines: HEK293 & CHO
What do current library screening systems lack?
Traditionally, library screening is completed using bacteria or yeast phage display. Bacteria allow for the creation of large libraries in a short period of time while yeast provides a eukaryotic environment. These systems may be cost effective, but they lack post-translational modifications that mammalian cells permit.
Mammalian cells also offer a human like environment, but the current available systems are slow and laborious to work with at a high cost. The available mammalian library systems for screening may provide an environment closer to the human system, but the coverage they allow is very low compared to bacteria and yeast.
Applied StemCell’s Solution
Our goal was to engineer the TARGATT™ system into HEK293 & CHO cells in order to:
- Develop a mammalian display system with a higher efficiency
- Provide a mammalian display system that can reach E. coli and yeast library sizes
Figure 6: Comparison of the currently available library screening systems.
TARGATT™ Mammalian Display
Table 1: A comparison of the TARGATT™ mammalian display with available alternatives display systems.
To address the current library screening and size issues, Applied StemCell is using its TARGATT™ gene editing technology to develop a mammalian display system that can consistently hit within an order of magnitude typical for bacteria and yeast. When comparing the TARGATT™ system for mammalian display to other available systems, it is clear that the TARGATT™ system offers unique features including site-specific and single-copy gene insertion.
TARGATT™ Screen Master Cell Lines
The TARGATT™ Screen Master Cell Lines were engineered using a split-cassette selection/screen system. This allows us to obtain clean results with little background. We separated the promoter and the transgene. The promoter was inserted in the chromosome at a safe-harbor locus, and the transgene is carried by the donor plasmid. This system only allows expression of the insert if there is a site-specific gene insertion at the safe-harbor locus that contains the promoter. If random integration were to occur, the gene would not have a promoter and therefore would not be expressed.
Applied StemCell integrated this system into HEK293 and CHO cells. Both cell lines have reported high integration efficiencies and medium to high levels of protein expression.
Figure 7: Schematic of the split-cassette selection/screen system.
Integration Efficiencies Achieved by our TARGATT™ Master Cell Lines
TARGATT™ CHO Library Screen Master Cell Line:
Figure 8: The TARGATT™ HEK293 Library Screen Master Cell Line presented >40% integration efficiency without drug selection and >90% with drug selection.
Highest Efficiency Available
TARGATT™ HEK293 Library Screen Master Cell Line:
Figure 9: The TARGATT™ CHO Library Screen Master Cell Line presented an integration efficiency of ~18% without drug selection and >90% with drug selection.
TARGATT™ Screening System Advantages
Compared to the available screening systems, the TARGATT™ screening system has the potential to reach library sizes used in E.coli and surpass moderate yeast libraries with a much higher efficiency.
Figure 10: A size comparison between available screening systems and the TARGATT™ screening system.
- TARGATT™ efficiency: 1,600 – 4000x improvement over FlpIn (40% vs. 0.01% - 0.025%)
- If we match cell counts used in FlpIn projects, TARGATT™ HEK293 can reach library sizes used in E. coli
- Can surpass moderate yeast libraries
Figure 11: Applied StemCell’s platform for antibody discovery and screening.
Figure 12: Sequencing data that displays no repeat sequences for light (top row) and heavy (bottom row) chains.
TARGATT™ Naïve IgG Library System
Applied StemCell is taking the TARGATT™ system to construct a naïve IgG library system for antibody discovery and screening. ASC built the donor library by extracting RNA from a non-immunized human donor (spleen, lymph-node, or bone marrow), generating cDNA, and amplifying the heavy and light chains via PCR. The library was incorporated into a donor vector and Sanger sequencing and NGS was conducted for library QC.
ASC transfected the TARGATT™ HEK293 Library Screen Master Cell Line with the library donor DNA and the TARGATT™ integrase vector. Following a 40-hour period, drug selection was carried out followed by QC for library quality by Sanger sequencing and NGS. Our current data confirms we have high library diversity coverage and no repeat sequences.
Summary: TARGATT™ System Advantages
Table 2: A summary of the TARGATT™ system advantages compared to currently available systems.
Applied StemCell’s TARGATT™ technology allows faster and efficient site-specific integration of large DNA fragments. We incorporated our technology into CHO cells for bioproduction and HEK293/CHO cells for antibody screening and library construction.
While ASC offers TARGATT™ CHO and HEK293 stable cell lines, Applied StemCell can engineer the TARGATT™ system into your favorite cell line so you can use the technology for your specific research needs!
- TARGATT™ CHO Master Cell Lines
- Stable cell line
- Library screening
- TARGATT™ HEK293 Master Cell Lines
- Stable cell line
- Library screening
- TARGATT™ iPSC Master Cell Lines
- Differentiate to specific cell types: neurons, NK, T, RPE, etc.
- TARGATT™ Custom Master Cell Lines
- TARGATT™ Antibody Screening Library Construction Service
- Licensing and Evaluation Programs Available!
ASC Licenses Its TARGATT™ CHO Cell Technology for Biotherapeutics Development – Milpitas, California, January 19th, 2021
Read More Here
