High-efficiency antibody discovery achieved with multiplexed microscopy

Izquierdo, Shelley; Varela, S; Park, Minha; Collarini, Ellen J.; Lu, Daniel; Pramanick, Shreya; Rucker, Joseph; Lopalco, Lucia; Etches, Rob; Harriman, William

doi:10.1093/jmicro/dfw014

Cited by 30 publications

(37 citation statements)

References 64 publications

(45 reference statements)

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“…In the case of GIPR, chickens that were immunized and screened (in GEMs) 25 exclusively using human GIPR did indeed generate antibodies that were cross-reactive with murine GIPR, albeit at a relatively low frequency (∼5%), which can be considered a “baseline” cross-reactivity rate for this particular target. Protocol adjustments were made that included immunizing with murine GIPR (DNA) as well as screening in GEMs with mGIPR-expressing cells, which combined to significantly enhance the hit rate for human/murine cross-reactive mAbs.…”

Section: Discussionmentioning

confidence: 99%

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Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Könitzer

Pramanick²,

Pan

et al. 2017

mAbs

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Raising functional antibodies against G protein-coupled receptors (GPCRs) is challenging due to their low density expression, instability in the absence of the cell membrane's lipid bilayer and frequently short extracellular domains that can serve as antigens. In addition, a particular therapeutic concept may require an antibody to not just bind the receptor, but also act as a functional receptor agonist or antagonist. Antagonizing the glucose-dependent insulinotropic polypeptide (GIP) receptor may open up new therapeutic modalities in the treatment of diabetes and obesity. As such, a panel of monoclonal antagonistic antibodies would be a useful tool for in vitro and in vivo proof of concept studies. The receptor is highly conserved between rodents and humans, which has contributed to previous mouse and rat immunization campaigns generating very few usable antibodies. Switching the immunization host to chicken, which is phylogenetically distant from mammals, enabled the generation of a large and diverse panel of monoclonal antibodies containing 172 unique sequences. Three-quarters of all chicken-derived antibodies were functional antagonists, exhibited high-affinities to the receptor extracellular domain and sampled a broad epitope repertoire. For difficult targets, including GPCRs such as GIPR, chickens are emerging as valuable immunization hosts for therapeutic antibody discovery.

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Section: Discussionmentioning

confidence: 99%

“…We used a single lymphocyte screening and recovery method, the GEM assay, 25, 47 to isolate antigen-specific monoclonal antibodies from the GIPR-immunized chickens. The GEM assay involves placing single antibody-secreting lymphocytes in proximity with reporters (which can be cells or beads).…”

Section: Methodsmentioning

confidence: 99%

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Könitzer

Pramanick²,

Pan

et al. 2017

mAbs

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“…30,31 In total, eight immunized chickens produced high titer antibodies against SIRPα v1, v2 and mouse SIRPα (Table 2, Figure 2(b)) from which splenocytes were harvested, and single antibody- secreting B-lineage cells were screened for reactivity toward SIRPα using a gel-encapsulated microenvironment (GEM) assay. 32 The GEM assay consists of a single-antibody secreting B-cell encapsulated in a droplet containing up to three different fluorescent reporter beads, each coated with a target antigen of interest (Figure 2(a)). B-cell clones that produce antibodies that bind to the immobilized targets were detected with a secondary antibody against chicken IgY, visualized via fluorescent microscopy, isolated, and sequenced.…”

Section: Chicken Immunization and Gem Screen To Isolate Pan-allelic Amentioning

confidence: 99%

Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPα

Sim

Sockolosky

Sangalang

et al. 2019

mAbs

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Targeting the CD47-signal-regulatory protein α (SIRPα) pathway represents a novel therapeutic approach to enhance anti-cancer immunity by promoting both innate and adaptive immune responses. Unlike CD47, which is expressed ubiquitously, SIRPα expression is mainly restricted to myeloid cells and neurons. Therefore, compared to CD47-targeted therapies, targeting SIRPα may result in differential safety and efficacy profiles, potentially enabling lower effective doses and improved pharmacokinetics and pharmacodynamics. The development of effective SIRPα antagonists is restricted by polymorphisms within the CD47-binding domain of SIRPα, necessitating pan-allele reactive anti-SIRPα antibodies for therapeutic intervention in diverse patient populations. We immunized wild-type and human antibody transgenic chickens with a multi-allele and multi-species SIRPα regimen in order to discover pan-allelic and pan-mammalian reactive anti-SIRPα antibodies suitable for clinical translation. A total of 200 antibodies were isolated and screened for SIRPα reactivity from which approximately 70 antibodies with diverse SIRPα binding profiles, sequence families, and epitopes were selected for further characterization. A subset of anti-SIRPα antibodies bound to both human SIRPα v1 and v2 alleles with high affinity ranging from low nanomolar to picomolar, potently antagonized the CD47/SIRPα interaction, and potentiated macrophage-mediated antibody-dependent cellular phagocytosis in vitro. X-ray crystal structures of five anti-SIRPα antigen-binding fragments, each with unique epitopes, in complex with SIRPα (PDB codes 6NMV, 6NMU, 6NMT, 6NMS, and 6NMR) are reported. Furthermore, some of the anti-SIRPα antibodies cross-react with cynomolgus SIRPα and various mouse SIRPα alleles (BALB/c, NOD, BL/ 6), which can facilitate preclinical to clinical development. These properties provide an attractive rationale to advance the development of these anti-SIRPα antibodies as a novel therapy for advanced malignancies.

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“…FACS sorting of specific B cell subsets, plate‐based multiplex PCR, and recombinant antibody expression in suitable host cells have effectively recovered IgG sequences directly from select B cell subsets of both C57BL/6 wild‐type mice(Jin et al, ) and humans (Wrammert et al, ; X. Wu et al, ). Mettler‐Izquierdo et al () presented the gel encapsulated microenvironment (GEM) technique capable of interrogating up to 100 million B cells from hyperimmunized animals directly for antigen specificity. A relative of the fluorescent foci techniques (Babcook, Leslie, Olsen, Salmon, & Schrader, ; Tickle et al, ), the GEM technique encapsulates B cells of interesting in emulsion droplets using a mixture of agarose and dimethylpolysiloxane combined with either antigen‐coated beads and/or reporter cells expressing membrane antigens.…”

Section: Antibody Discoverymentioning

confidence: 99%

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring

Jorgolli

Nevill²,

Winters

et al. 2019

Biotech & Bioengineering

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The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high‐paced workflows necessary to support modern large molecule drug discovery. A high‐level aspiration is a true integration of “lab‐on‐a‐chip” methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light‐induced electrokinetics with micro‐ and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single‐cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low‐throughput bioprocess workflows in biopharma and life science research.

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High-efficiency antibody discovery achieved with multiplexed microscopy

Cited by 30 publications

References 64 publications

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Discovery of high affinity, pan-allelic, and pan-mammalian reactive antibodies against the myeloid checkpoint receptor SIRPα

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring

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